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Division of Metabolism, Endocrinology, and Diabetes
Department of Internal Medicine
University of Michigan
Ann Arbor, Michigan United States
Robert Barbieri, MD
Kate Macy Ladd Distinguished Professor
Obstetrics, Gynecology and Reproductive Biology
Harvard Medical School; Chair, Emeritus
Department of Obstetrics and Gynecology
Brigham and Women’s Hospital Boston, Massachusetts United States
Misty Blanchette Porter, MD, FAIUM
Vice Chair for Education and Faculty Affairs
Obstetrics, Gynecology and Reproductive Sciences
Larner College of Medicine Burlington, Vermont United States
Kassie Bollig, MD
Assistant Professor Department of Obstetrics and Gynecology
Penn State College of Medicine and Penn State Health
Hershey, Pennsylvania United States
Paula C. Brady, MD
Assistant Professor
Obstetrics and Gynecology
Columbia University Irving Medical Center
New York, New York United States
Robert Brannigan, MD
Professor and Vice Chair of Clinical Urology
Director of Andrology Fellowship Urology
Northwestern University, Feinberg School of Medicine
Chicago, Illinois United States
Leah H. Bressler, MD
Department of Obstetrics and Gynecology
University of North Carolina—Chapel Hill Chapel Hill, North Carolina United States
Serdar E. Bulun, MD Chair
Obstetrics and Gynecology
Northwestern University Feinberg School of Medicine
Chicago, Illinois United States
Enrico Carmina, MD
Professor of Endocrinology
Endocrine Unit
University of Palermo School of Medicine Palermo, Italy
Alice Y. Chang, MD Endocrinology
Mayo Clinic
Jacksonville, Florida
United States
R. Jeffrey Chang, MD Professor Emeritus
Obstetrics, Gynecology and Reproductive Sciences
University of California—San Diego La Jolla, California United States
John A. Cidlowski, PhD
Senior Investigator Laboratory of Signal Transduction
National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina United States
Amander T. Clark, PhD Professor
Molecular, Cell and Developmental Biology
UCLA Center for Reproductive Science, Health and Education
Broad Stem Cell Research Center
University of California—Los Angeles Los Angeles, California United States
Emmanuèle C. Dêlot, PhD Research Professor
George Washington University Center for Genetic Medicine Research Children’s National Research and Innovation Campus Washington, Washington DC United States
James A. Dias, BS, MS, PhD
Vice President for Research Emeritus State University of New York; Professor Emeritus Department of Biomedical Science
Albany, New York United States
Anuja Dokras, MD, MHCI, PhD Professor
Obstetrics and Gynecology
University of Pennsylvania Philadelphia, Pennsylvania United States
Daniel A. Dumesic, MD
Professor
Department of Obstetrics and Gynecology
University of California—Los Angeles Los Angeles, California United States
Francesca E. Duncan, PhD
Assistant Professor Obstetrics and Gynecology
Northwestern University Chicago, Illinois United States
Andrea G. Edlow, MD, MSc
Staff Physician, Obstetrics and Gynecology, Division of Maternal-Fetal Medicine
Massachusetts General Hospital; Associate Professor Obstetrics, Gynecology and Reproductive Biology
Harvard Medical School
Boston, Massachusetts United States
Dieter Egli, PhD
Associate Professor
Department of Pediatrics and Obstetrics & Gynecology
Columbia Stem Cell Initiative
Columbia University Irving Medical Center
New York, New York United States
William S. Evans, MD
Professor Emeritus of Medicine
School of Medicine
University of Virginia
Charlottesville, Virginia United States
Bart C.J.M. Fauser, MD, PhD
Professor Emeritus
Reproductive Medicine
University of Utrecht and University Medical Center Utrecht, The Netherlands
Jill P. Ginsberg, MD
Professor Pediatrics
Perelman School of Medicine
Director, Cancer Survivorship
Pediatric Oncology
Children’s Hospital of Philadelphia
Philadelphia, Pennsylvania
United States
Elizabeth S. Ginsburg, MD
Professor, Harvard Medical School
Obstetrics and Gynecology
Brigham and Women’s Hospital
Boston, Massachusetts
United States
Linda C. Giudice, MD, PhD
Distinguished Professor
Obstetrics, Gynecology and Reproductive Sciences
University of California—San Francisco
San Francisco, California
United States
Sierra Goldsmith, MS
Infertility Center of St. Louis
St. Lukes Hospital
St. Louis, Missouri
United States
Steven Robert Goldstein, MD
Professor
Obstetrics and Gynecology
New York University Grossman School of Medicine
New York, New York
United States
Clarisa R. Gracia, MD, MSCE Professor Director
Reproductive Endocrinology and Infertility
Department of Obstetrics and Gynecology
University of Pennsylvania Philadelphia, Pennsylvania
United States
Janet E. Hall, MD, MSc
Clinical Director and Senior Investigator
National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina
United States
Eduardo Hariton, MD, MBA
Reproductive Endocrinology and Infertility Physician
Reproductive Science Center of the San Francisco Bay Area
VP of Strategic Initiatives and Managing Director of the USF Innovation Fund
US Fertility
Oakland, California
United States
Katsuhiko Hayashi, PhD Professor
Stem Cell Biology and Medicine
Faculty of Medical Sciences
Kyushu University
Fukuoka, Japan
Xin He, MD Professor
Division of Metabolism, Endocrinology, and Diabetes
Department of Internal Medicine
University of Michigan
Ann Arbor, Michigan
United States
Rinath Jeselsohn, MD
Assistant Professor Medical Oncology
DFCI
Boston, Massachusetts
United States
Daniel J. Kaser, MD
Physician, Director of Third Party
Reproduction and LGBTQ+ Care
Reproductive Endocrinology & Infertility
Reproductive Medicine Associates of Northern California
San Francisco, California
United States
Andrew M. Kelleher, PhD Department of Obstetrics
Gynecology and Women’s Health University of Missouri Columbia, Missouri
United States
Laxmi A. Kondapalli, MD, MSCE Physician
Reproductive Endocrinology and Infertility
Colorado Center for Reproductive Medicine
Lone Tree, Colorado
United States
William Hanna Kutteh, MD, PhD, HCLD
Clinical Professor of Obstetrics and Gynecology
Vanderbilt University Medical Center
Managing Partner
Fertility Associates of Memphis Memphis, Tennessee
United States
Monica M. Laronda, PhD
Assistant Professor
Pediatrics
Lurie Children’s Hospital
Northwestern University Chicago, Illinois
United States
Richard S. Legro, MD Chair
Department of Obstetrics and Gynecology; Professor
Obstetrics and Gynecology and Public Health Sciences
Penn State College of Medicine and Penn State Health
Hershey, Pennsylvania
United States
Peter Y. Liu, MBBS, PhD
Professor
David Geffen School of Medicine
University of California – Los Angeles; Investigator
The Lundquist Institute at Harbor-UCLA Medical Center
Los Angeles, California
United States
Roger A. Lobo, MD
Professor
Obstetrics and Gynecology
Columbia University College of Physicians & Surgeons
New York, New York
United States
Thanh-Ha Luu, MD
Invia Fertility
Chicago, Illinois
United States
Philip Marsh, MS, TS Embryologist
Obstetrics and Gynecology
University of California—San Francisco
San Francisco, California
United States
John C. Marshall, MD, PhD
Andrew D Hart Professor of Medicine
Emeritus Division of Endocrinology and Metabolism
Department of Medicine
University of Virginia
Charlottesville, Virginia
United States
Christopher R. McCartney, MD
Professor of Medicine
Department of Medicine
Division of Endocrinology and Metabolism
Center for Research in Reproduction
University of Virginia School of Medicine
Charlottesville, Virginia
United States
Melissa Menezes, MD
Assistant Clinical Professor of Pediatrics
Division of Adolescent Medicine
Department of Pediatrics
Children’s Hospital at Montefiore Bronx, New York
United States
Sam Mesiano, PhD
William H Weir MD Professor of Reproductive Biology
Department of Reproductive Biology
Case Western Reserve University; Vice Chair for Research
Department of Obstetrics and Gynecology
University Hospitals of Cleveland Cleveland, Ohio
United States
Diana Monsivais, PhD Department of Pathology & Immunology
Baylor College of Medicine Houston, Texas United States
Jerrine Morris, MD, MPH Third Year Clinical Fellow REI Obstetrics, Gynecology and Reproductive Sciences
University of California—San Francisco San Francisco, California United States
Prema Narayan, PhD Associate Professor Physiology
Southern Illinois University School of Medicine Carbondale, Illinois United States
Ralf Nass, MD
Assistant Professor of Medicine School of Medicine University of Virginia Charlottesville, Virginia United States
Kathleen O’Neill, MD, MSTR Assistant Professor Obstetrics and Gynecology University of Pennsylvania Philadelphia, Pennsylvania United States
Sharon E. Oberfield, MD Professor of Pediatrics and Division Director Pediatric Endocrinology, Diabetes and Metabolism
Columbia University Medical Center New York, New York United States
Takehiko Ogawa, MD, PhD Professor Department of Regenerative Medicine Graduate School of Medicine Yokohama City University Yokohama, Japan
Giovanna Olivera, MS, TS Senior Embryologist Obstetrics and Gynecology/REI University of California—San Francisco San Francisco, California United States
Kyle E. Orwig, PhD
Professor Obstetrics, Gynecology and Reproductive Sciences
Magee-Womens Research Institute
University of Pittsburgh School of Medicine
Pittsburgh, Pennsylvania United States
Stephanie A. Pangas, BA, MS, PHD Associate Professor Pathology & Immunology
Baylor College of Medicine Houston, Texas United States
Alex J. Polotsky, MD
Medical Director
SGF Colorado; Professor
Obstetrics and Gynecology, Reproductive Endocrinology and Infertility Division and Fellowship; Director
University of Colorado School of Medicine
Greenwood Village, Colorado United States
Molly Quinn, MD
Assistant Professor
Department of Obstetrics and Gynecology
Division of Reproductive Endocrinology and Infertility
Keck School of Medicine of the University of Southern California Los Angeles, California United States
Catherine Racowsky, PhD Professor Emeritus
Obstetrics, Gynecology and Reproductive Biology
Brigham & Women’s Hospital and Harvard Medical School Boston, Massachusetts United States
Lauren Kendall Rauchfuss, MD
Obstetrics and Gynecology
Mayo Clinic Rochester, Minnesota United States
Salustiano Ribeiro, MS, TS, ELS/ALS Embryologist
Obstetrics, Gynecology and Reproductive Sciences
University of California—San Francisco San Francisco, California United States
Jessica Rieder, MD, MS Associate Clinical Professor of Pediatrics Division of Adolescent Medicine Department of Pediatrics
Children’s Hospital at Montefiore Bronx, New York United States
Tamar Reisman, MD
Assistant Professor of Medicine
Division of Endocrinology and Center for Transgender Medicine and Surgery
Icahn School of Medicine at Mount Sinai New York, New York
United States
Andrea H. Roe, MD, MPH
Assistant Professor
Department of Obstetrics and Gynecology
Perelman School of Medicine
University of Pennsylvania
Cassandra Roeca, MD
Assistant Professor
Obstetrics and Gynecology
University of Colorado School of Medicine
Shady Grove Fertility
Denver, Colorado United States
Andrew Runge, BS, TS
Senior Embryologist
Obstetrics, Gynecology and Reproductive Sciences
University of California—San Francisco San Francisco, California United States
Mitchell Rosen, MD, HCLD Professor
Obstetrics, Gynecology and Reproductive Sciences
University of California—San Francisco
San Francisco, California United States
Joshua D. Safer, MD, FACP, FACE
Executive Director
Center for Transgender Medicine and Surgery
Mount Sinai Health System; Professor of Medicine
Icahn School of Medicine at Mount Sinai
New York, New York
United States
Nanette Santoro, MD
Professor and Chair
Obstetrics and Gynecology
University of Colorado School of Medicine
Aurora, Colorado United States
Karen Schindler, PhD
Associate Professor Department of Genetics
Human Genetics Institute of NJ Rutgers
The State University of New Jersey New Brunswick, New Jersey United States
Peter N. Schlegel, MD
James J. Colt Professor Urology
Weill Cornell Medicine
New York, New York United States
Courtney A. Schreiber, MD, MPH
Stuart and Emily B.H. Mudd Professor of Human Behavior & Reproduction
Chief, Division of Family Planning Department of Obstetrics and Gynecology
Executive Director, FOCUS on Health and Leadership for Women
Perelman School of Medicine
University of Pennsylvania
Philadelphia, Pennsylvania
Danny J. Schust, MD
Professor and Vice Chair for Research Director, Duke Reproductive Endocrinology and Infertility Fellowship Training Program
Department of Obstetrics and Gynecology
Duke University
Durham, North Carolina
United States
Thalia R. Segal, MD
Assistant Professor
Obstetrics, Gynecology, and Reproductive Sciences
University of California—San Francisco San Francisco, California United States
Molly R. Siegel, MD
Clinical and Research Fellow
Obstetrics and Gynecology
Massachusetts General Hospital Boston, Massachusetts
United States
Sherman J. Silber, MD Director
Infertility Center of St. Louis St. Lukes Hospital St. Louis, Missouri
United States
Peter J. Snyder, MD Professor of Medicine
Medicine
University of Pennsylvania Philadelphia, Pennsylvania
United States
Aviva B. Sopher, MD, MS
Associate Professor Department of Pediatrics
Columbia University Irving Medical Center
New York, New York
United States
Thomas E. Spencer, PhD Professor
Obstetrics Gynecology and Women’s Health
University of Missouri Columbia, Missouri
United States
Frank Z. Stanczyk, PhD
Professor
Department of Obstetrics and Gynecology
Department of Population and Public Health Sciences
Keck School of Medicine
University of Southern California
Los Angeles, California
United States
Aleksandar K. Stanic, MD, PhD
Associate Professor
Department of Obstetrics and Gynecology
Division of Reproductive Endocrinology and Infertility, Reproductive Sciences
University of Wisconsin-Madison Madison, Wisconsin
United States
Elizabeth A. Stewart, MD
Professor and Consultant, Obstetrics and Gynecology
Mayo Clinic
Rochester, Minnesota
United States
Jerome F. Strauss III, MD, PhD
Professor Emeritus
Department of Obstetrics and Gynecology
Perelman School of Medicine, University of Pennsylvania
Philadelphia, Pennsylvania
United States
Yousin Suh, PhD
Charles and Marie Robertson Professor of Reproductive Sciences
Professor of Genetics and Development Director of Reproductive Aging Program
Columbia University Vagelos College of Physicians & Surgeons
New York, New York
United States
Alok K. Tewari, MD, PhD
Physician
Medical Oncology
Dana-Farber Cancer Institute; Instructor in Medicine
Harvard Medical School
Boston, Massachusetts
United States
Nicholas A. Tritos, MD, DSc, MMSc
Staff Neuroendocrinologist
Neuroendocrine Unit
Massachusetts General Hospital; Associate Professor of Medicine, Medicine
Harvard Medical School
Boston, Massachusetts
United States
Jenna Turocy, MD
Assistant Professor
Obstetrics and Gynecology
Columbia University Irving Medical Center
New York, New York
United States
Alfredo Ulloa-Aguirre, MD, DSc
Scientific Director
RAI, Instituto Nacional de Ciencias Médicas y Nutrición SZ-Universidad Nacional Autónoma de México
Mexico DF, Mexico
Eric Vilain, MD, PhD
Associate Vice Chancellor
Clinical and Translational Science
Professor
Department of Pediatrics
University of California
Irvine, California
United States
Shannon Whirledge, PhD, MHS
Associate Professor
Department of Obstetrics, Gynecology and Reproductive Sciences
Yale School of Medicine
New Haven, Connecticut
United States
Carmen J. Williams, MD, PhD
Senior Investigator
Reproductive & Developmental Biology
Laboratory
National Institute of Environmental Health Sciences
Research Triangle Park, North Carolina
United States
Zev Williams, MD, PhD
REI Division Chief
Obstetrics and Gynecology
Columbia University Medical Center
New York, New York
United States
Selma Feldman Witchel, MD
Professor of Pediatrics
Division of Pediatric Endocrinology, Department of Pediatrics
UPMC Children’s Hospital of Pittsburgh, University of Pittsburgh
Pittsburgh, Pennsylvania
United States
Tracey J. Woodruff, PhD, MPH
Professor and Director
Program on Reproductive Health and the Environment Department of Obstetrics, Gynecology, and Reproductive Sciences
Institute for Health Policy Studies
University of California
San Francisco, California
United States
Steven L. Young, MD, PhD
Professor
Reproductive Endocrinology and Infertility, Obstetrics, and Gynecology
University of North Carolina School of Medicine
Chapel Hill, North Carolina
United States
Marya G. Zlatnik, MD, MMS
Professor
Obstetrics, Gynecology and Reproductive Sciences
University of California—San Francisco
San Francisco, California
United States
Preface
The first edition of Yen and Jaffe, Reproductive Endocrinology was published in 1978, the year of the first birth conceived through in-vitro fertilization (IVF). The foreword to the edition was written by Roger Guillemin MD PhD, who received the 1977 Nobel Prize in Physiology or Medicine for contributions to the discovery of thyrotropin releasing hormone and gonadotropin releasing hormone. In the foreword, Dr Guillemin highlighted the importance of advances in neuroendocrinology to our understanding of reproduction. For the fifth edition of the text, published in 2004, the founding editors transitioned editorial responsibility to us. At the time, the importance of the field of reproductive endocrinology and infertility was firmly established. Scientific advances were rapidly occurring in gamete biology and the genetics of reproduction, and IVF was established as a highly effective and safe treatment for infertility. Acknowledging the importance of IVF and the field of reproductive endocrinology, Robert G Edwards PhD was awarded the 2010 Nobel Prize in Physiology or Medicine for his contributions to the development of IVF.
The publication of the ninth edition of Yen & Jaffe marks another milestone in the history of this text, which has achieved a prominent place in the field of reproductive medicine. We were entrusted with the future of the book by its originators, who were giants in our field. Having done our best to meet their expectations, we now prepare for another transition in editorship of the text. With the publication of the ninth edition, we will be transitioning editorial responsibilities to Anuja Dokras, MD, MHCI, PhD, Carmen J. Williams, MD, PhD, and Zev Williams, MD, PhD, our co-editors of this edition. Collectively, their expertise covers the breadth of science underpinning reproductive
endocrinology and infertility, and we are confident that they will ensure that the text will continue to be comprehensive and authoritative.
We are extremely grateful to our many colleagues who authored chapters in the five editions we have overseen. We are especially appreciative of the efforts of those who contributed chapters to the ninth edition in the face of challenges resulting from a pandemic that disrupted clinical care, academic pursuits, and training. The assistance we have received from the professional staff at Elsevier, who facilitated the publication process for the 20 years during which we served as editors, has been invaluable, and we want to thank Nancy Duffy, Joanie Milnes, Nadhiya Sekar, Manikandan Chandrasekaran for their outstanding support in the preparation of the ninth edition.
Yen & Jaffe has always been an evolving text, and that will continue to be one of its hallmarks. The ninth edition incorporates new chapters that cover advances in basic and clinical science that have enriched the field since publication of the previous edition, including emerging technologies involving gamete production and maturation, genetic testing and genetic manipulation, and uterus transplantation. Other chapters have been extensively revised to cover contemporary practices in assisted reproduction, fertility preservation, and ovulation induction.
We have been honored to carry on the work initiated by Sam Yen and Bob Jaffe and will always be mindful of their substantive scientific contributions and the opportunity they gave to us to carry forward their tradition of excellence.
Jerome F. Strauss III, MD, PhD Robert Barbieri, MD
Robert B. Jaffe, MD 1933–2020
In 2020, endocrinology in general—and reproductive endocrinology in particular—lost a giant in basic and translational reproductive endocrinology research. Robert B. Jaffe MD was a visionary leader whose contributions were essential to the development of the field of reproductive endocrinology. Following training at the University of Michigan, the University of Colorado, and the Karolinska Institute with Egon Diczfalusy, Dr Jaffe joined the Michigan faculty, rising to professor over a nine-year tenure in Ann Arbor. Drs Jaffe and Samuel S.C. Yen began a productive collaboration during this time. Dr Yen would drive from Cleveland, where he was on the Case Western faculty, to Ann Arbor with biological samples in the trunk of his car and have them analyzed for myriad reproductive hormones in Dr Jaffe’s laboratory. Following Dr Yen’s move to San Diego in 1970 and Dr Jaffe’s move to San Francisco in 1973, the two maintained a close relationship. In 1976, they were both Visiting Scholars of the Rockefeller Foundation working at the Villa Serbelloni, Lake Como, Italy. There, they conceived the idea of Yen & Jaffe’s Reproductive Endocrinology, first published in 1978.
The book became one of the pillars of the field of reproductive medicine. It is often referred to as “The Bible,” with the ninth edition in preparation. To fully appreciate the impact of the visionary thinking that gave birth to this textbook, one has to consider its temporal context. Reproductive medicine was very much in its infancy in the mid-1970s. At that time, the development of methods to quantify physiological levels of proteins and steroid hormones,
the elucidation of mechanisms of hormone action, and advances in reproductive biology were transforming the science of endocrinology. Innovation in surgical and medical approaches to the diagnosis and treatment of infertility—and the broader availability of hormones to regulate fertility and stimulate the gonads—were establishing the foundations of a new clinical discipline. The first edition of Yen & Jaffe assembled and synthesized the core knowledge upon which this field, Reproductive Endocrinology and Infertility, would rapidly grow, both as a science and as a subspecialty of Obstetrics and Gynecology.
Dr Jaffe was an indefatigable leader and champion for academic reproductive endocrinology. As an example, he founded and was the principal investigator for 25 years of the innovative NIH Reproductive Scientist Development Program (RSDP). The RSDP trained dozens of academic leaders in our field and continues to actively support the research career development of early career investigators in reproduction. Dr Jaffe was an exceptional collaborator. He developed a journal club focused on ovarian biology and cancer at MD Anderson Cancer Center, regularly flying from San Francisco to Houston to help lead the discussion of seminal publications. This remarkable collaboration resulted in an NIH-funded research project involving both UCSF and MD Anderson.
Dr Jaffe received many honors and awards, including the Distinguished Scientist Award from the American Society for Reproductive Medicine and the Sydney H. Ingbar Distinguished Service Award from the Endocrine Society. He was a President of the Endocrine Society’s Hormone Foundation and a member of the National Academies of Sciences, Engineering, and Medicine. Dr Jaffe will be greatly missed by his many friends and colleagues throughout the world. His legacy will live on through his many trainees and this book.
Video Contents xv
PART 1
The Fundamentals of Reproduction 1
1. Neuroendocrinology of Reproduction 1
Christopher R McCartney and John C Marshall
2. The Gonadotropin Hormones and Their Receptors 23
Prema Narayan, Alfredo Ulloa-Aguirre, and James A Dias
3. Prolactin in Human Reproduction 56
Nicholas A Tritos
4. Steroid Hormones and Other Lipid Molecules Involved in Human Reproduction 73
Jerome F Strauss III, Emanuela Ricciotti, and Garret A FitzGerald
5. Steroid Hormone Action 110
Shannon Whirledge and John A Cidlowski
6. Growth Factors and Reproduction 125
Diana Monsivais and Stephanie A Pangas
7. Neuroendocrine Control of the Menstrual Cycle 142
Janet E Hall
8. The Ovarian Life Cycle 158
Jerome F Strauss III and Carmen J Williams
9. Meiosis, Fertilization, and Preimplantation Embryo Development 188
Carmen J Williams and Karen Schindler
10. Structure, Function, and Evaluation of the Female Reproductive Tract 217
Andrew M Kelleher, Leah H Bressler, Steven L Young, and Thomas E Spencer
11. Endocrinology of Human Pregnancy and Fetal-Placental Neuroendocrine Development 254
Sam Mesiano
12. The Breast 277
Robert Barbieri
13. The Hypothalamo-Pituitary Unit, Testis, and Male Accessory Organs 285
Peter Y. Liu
14. Menopause and Aging 300
Roger A Lobo and Yousin Suh
15. Male Reproductive Aging 338
Peter J. Snyder
16. Immunology and Reproduction 345
Aleksandar K Stanic, William Hanna Kutteh, Kassie Bollig, and Danny J Schust
PART 2
Pathophysiology and Therapy: Pediatric, Adolescent, and Adult 365
17. Differences of Sex Development 365
Emmanuèle C Délot and Eric Vilain
18. Puberty: Gonadarche and Adrenarche 395
Aviva B Sopher, Sharon E Obereld, and Selma Feldman Witchel
19. Nutrition and Reproduction 449
Nanette Santoro, Alex J Polotsky, Thanh-Ha Luu, Melissa Menezes, Jessica Rieder, Laxmi A Kondapalli, and Cassandra Roeca
20. Environmental Factors and Reproduction 461
Linda C Giudice, Marya G Zlatnik, Thalia R Segal, and Tracey J Woodruff
21. Physiological and Pathophysiological Alterations of the Neuroendocrine Components of the Reproductive Axis 475
Ralf Nass and William S Evans
22. Polycystic Ovary Syndrome and Hyperandrogenic States 516
R Jeffrey Chang, Anuja Dokras, and Daniel A Dumesic
17.1 Developmental anomalies of the uterus and vagina: classification and treatment
26.1 Overview of the FIGO classification of uterine leiomyoma and the various operative techniques used for myomectomy, including hysteroscopic myomectomy, laparoscopic myomectomy, roboticassisted myomectomy, and open myomectomy
26.2 Overview of different endometrial pathologies and the various hysteroscopic techniques employed for their resection, including hysteroscopic myomectomy, hysteroscopic metroplasty, and hysteroscopic lysis of adhesions
27.1 Descriptions of lesion types, how to report endometriosis findings, and the various surgical management options for each endometriosis scenario
33.1 Sonohysterography (SHG) dynamic imaging of performance of an SHG with a polyp or myoma
33.2 Hysterosalpingo-contrast sonography video
33.3 Dynamic ultrasound demonstrating anterior and posterior cul-de-sac adhesions relative to deeply invasive endometriosis (sliding sign)
36.1 The IVF laboratory: management and technologies
37.1 Mini laparotomy, ovarian tissue preparation on the bench prior to vitrification, and ovarian tissue transplantation
OUTLINE
The Fundamentals of Reproduction
Neuroendocrinology of Reproduction
Christopher R. McCartney and John C. Marshall
CENTRAL CONTROL OF REPRODUCTION
NEUROENDOCRINOLOGY: THE INTERFACE BETWEEN NEUROBIOLOGY AND ENDOCRINOLOGY
Anatomy of the Reproductive Hypothalamic-Pituitary Axis
Gonadotropin-Releasing Hormone: The Final Common Pathway for the Central Control of Reproduction
Neuronal Inputs Into Gonadotropin-Releasing Hormone Neurons
Gonadotropin-Releasing Hormone Pulse Generator
Gonadotropin-Releasing Hormone Secretion During Development and in Adulthood
Physiologic Development of Reproductive Neuroendocrine Function
Patterns of Pulsatile Gonadotropin-Releasing Hormone Secretion in Adults
Feedback Regulation of Gonadotropin-Releasing Hormone and Gonadotropin Secretion
Reproductive Neuroendocrine Adaptations in Settings of Reduced Energy Availability, Stress, and Lactation
Interface Between Reproductive Neuroendocrine Function and Energy Availability
Impact of Stress on Reproductive Neuroendocrine Function
Lactation and Reproductive Neuroendocrine Function
Miscellaneous Physiologic Influences on GonadotropinReleasing Hormone Secretion
CENTRAL CONTROL OF REPRODUCTION
Successful reproduction is essential to the survival of a species. The reproductive system represents a highly complex functional organization of diverse tissues and signaling pathways that, when properly functioning, ensures a number of key endpoints. The most important of these are the adequate production and development of gametes (ova and sperm), successful delivery of gametes for fertilization, and physiologic preparation for possible pregnancy in women. Neuroendocrine systems are the principal drivers of reproductive function in both men and women. In particular, hypothalamic gonadotropin-releasing hormone (GnRH) is the primary—if not exclusive—feedforward stimulatory signal to gonadotrope cells of the anterior pituitary, which induces the synthesis and secretion of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Together, these two gonadotropins direct the primary functions of the reproductive axis: gamete development and gonadal sex steroid synthesis. Given its critical importance to a species, the reproductive system must be robust, continuing to function properly in the
face of various physiologic perturbations. In contrast, in settings of marked physiologic stress (e.g., significantly reduced energy availability), mechanisms that temporarily limit fertility—the teleological outcome of which is metabolically expensive in women—are biologically advantageous for the individual and, ultimately, the species. Appropriate function (or quiescence) of the reproductive system is governed by a number of intricate relationships. For example, feedback signals from the gonads (e.g., circulating sex steroid concentrations) communicate the status of gonadal function to the hypothalamic-pituitary axis; these signals in turn influence GnRH and gonadotropin output, rendering a coordinated and tightly regulated feedback system that maintains gonadal function within narrow limits. The reproductive system also has extensive interactions with other neuroendocrine systems, such as those involved with energy balance and adaptations to stress. The reproductive neuroendocrine system integrates these myriad feedback signals, and the GnRH-secreting neuronal network represents the final common pathway for the central control of reproduction. Thus the regulation of GnRH secretion represents a major focus of reproductive neuroendocrinology.
Much of our understanding of reproductive neuroendocrinology has emerged from the study of rodents, ruminants, and nonhuman primates, largely reflecting the ethical boundaries inherent to human research. Because many neurobiological principles are similar among all mammals, these animal studies continue to be indispensable. Nonetheless, certain aspects of reproductive neuroendocrinology may differ markedly among species. Thus, when available, human data will be prioritized throughout this chapter, but animal studies will also be discussed when appropriate—emphasizing nonhuman primate studies when available— recognizing that specific findings may or may not be generalizable to humans. The reader is referred to Chapters 2, 7, 13, 18, and 21 for additional discussion of neuroendocrine physiology and pathophysiology related to reproduction.
NEUROENDOCRINOLOGY: THE INTERFACE BETWEEN NEUROBIOLOGY AND ENDOCRINOLOGY
Endocrinology is the study of cell-to-cell signaling that occurs via specific chemicals (hormones) traveling through the bloodstream to influence remote targets. The term “neuroendocrinology” refers to the involvement of the central nervous system (CNS) in this process, particularly the hypothalamus. This field of study has traditionally focused on hypothalamic neuron-derived factors that influence various target tissues either directly, as with the hormones of the neurohypophysis, or indirectly, as with hypothalamic releasing factors that control anterior pituitary hormone secretion. Neuroendocrine systems direct a wide variety of critical biologic processes, such as growth and development, energy and fluid homeostasis, responses to stress, and reproduction.
Neurons are highly specialized, morphologically diverse cells that transmit information via electrical impulses called action potentials. Neurons have a cell body (perikaryon) containing the
cell nucleus, mitochondria, and synthetic organelles. Neurons also have cell processes that participate in the reception and delivery of electrical impulses (Fig. 1.1). Dendrites are short processes—often extensively branched to increase surface area—that typically receive information by way of afferent electrical impulses from other neurons. The axon is a single neuronal extension that generally transmits efferent electrical impulses away from the cell body via a process called neuronal firing. However, GnRH neuron fibers extending from the cell body to the median eminence (the location of GnRH release) in mice demonstrate characteristics of both axons and dendrites and thus have been called dendrons.1
In unstimulated neurons, the inner portion of the neuronal membrane is negatively charged compared with the outer membrane surface; this resting membrane potential is typically between −50 and −75 mV in GnRH neurons. Such electrical polarization reflects transmembrane ionic differences, which are maintained by protein channels that govern transmembrane passage of specific ions (e.g., sodium, potassium, chloride). Regulated changes of transmembrane ion differences may cause the membrane potential to become more or less negative (hyperpolarization and depolarization, respectively). Depolarization to a certain threshold results in a rapid and temporary reversal of membrane potential—an action potential—which is propagated along the neuronal membrane. Notably, the amplitude of the action potential does not vary with the strength of stimulation; instead, once a threshold is reached, a full action potential occurs (the so-called all-or-none phenomenon). However, the degree of neuronal stimulation can alter the frequency of action potentials generated. In this way, neurons transmit information to other neurons and effector tissue cells.
Neuronal signals are transferred across neuron-to-neuron connections (synapses) via chemical neurotransmitters. This process begins with bursts of neuronal firing, which result in the opening of voltage-gated calcium channels at the axon terminal. The influx of calcium promotes exocytosis of neurotransmittercontaining synaptic vesicles, releasing neurotransmitters into the synaptic cleft. Neurotransmitters then activate specific liganddependent ion channels in the postsynaptic membrane, which can stimulate an action potential in the postsynaptic neuron. A wide variety of factors serve as neurotransmitters, including amino acids (e.g., acetylcholine, glutamate, γ-aminobutyric acid [GABA]), biogenic amines (e.g., norepinephrine, epinephrine, dopamine, serotonin), and neuropeptides (e.g., kisspeptin, neurokinin B [NKB], dynorphin, β-endorphin, somatostatin, proopiomelanocortin [POMC], neuropeptide Y [NPY]).
Bursts of neuronal firing can also elicit release of neuronal products into the bloodstream to influence remote targets (i.e., neurosecretion of neurohormones). Hypophysiotropic neurons are specialized hypothalamic neurons that secrete peptidereleasing factors (GnRH, corticotropin-releasing hormone [CRH], thyrotropin-releasing hormone [TRH], and growth hormone–releasing hormone [GHRH]) into the hypophyseal por tal circulation. These releasing factors in turn stimulate specific
anterior pituitary cell populations. In contrast, hypothalamic release of dopamine into the portal circulation provides tonic inhibition of pituitary prolactin secretion. Hypothalamic neurosecretion of vasopressin and oxytocin, which are released directly into the systemic circulation, alter the function of distant targets such as the renal tubules and uterus, respectively.
Neuroglial cells (e.g., astrocytes, ependymal cells, oligodendrocytes, microglia) represent approximately 90% of cells in the CNS. Neuroglia do not conduct action potentials, but they perform critical supportive functions. For example, astrocytes form the supportive framework of the CNS, help isolate synaptic junctions to prevent nonspecific spread of neuronal impulses, facilitate nutrient delivery to neurons, and contribute to the blood-brain barrier. Of interest, astrocytes have been implicated in the control of GnRH secretion and the mechanisms underlying pubertal onset.2 For example, astrocytes may impact neuronal activity via secretion of numerous growth factors, and astrocytes abundantly appose GnRH neurons; these contacts can influence synaptic input and may be influenced by estrogen in both rodents and nonhuman primates. Similarly, specialized ependymal cells (tanycytes) in the median eminence appear to modify access of GnRH neuron terminals to the hypophyseal portal system.
Anatomy of the Reproductive Hypothalamic-Pituitary Axis
• GnRH neuronal cell bodies are located in the infundibular (arcuate) nucleus and the medial preoptic area of the hypothalamus.
• GnRH neurons extend processes to the median eminence, where GnRH gains access to the hypophyseal portal system.
• The hypophyseal portal circulation represents the functional connection between hypothalamic GnRH neurons and the gonadotropes of the anterior pituitary.
Portions of the hypothalamus and the anterior pituitary gland constitute the primary effector arm of the central reproductive axis. In particular, hypothalamic neural systems regulate GnRH release into the hypophyseal portal veins, with GnRH being the signal to gonadotropes (anterior pituitary) to secrete LH and FSH. In turn, these gonadotropins direct gonadal (ovarian and testicular) function.
Hypothalamus
The hypothalamus is located at the base of the brain (Fig. 1.2). Although small (approximately 10 g, less than 1% of total brain weight), it performs critical functions for maintenance of wholeorganism homeostasis, including regulation of hunger and body weight, growth, various aspects of metabolism, thirst and renal water handling, body temperature, autonomic function, sleep, circadian rhythms, and emotion. Importantly, the hypothalamus is also a primary control center for reproduction and influences sexual behavior.
As an anatomic structure, the hypothalamus does not have discrete borders, but it generally forms the floor and inferior-lateral walls of the third ventricle (Fig. 1.3). The medial portions of the hypothalamus are primarily made up of cell bodies, whereas the lateral portions are mostly composed of neuron fibers (axons), such as those connecting the medial hypothalamus to other areas of the brain. By convention, closely associated collections of neuron cell bodies are called nuclei; and the paraventricular, dorsomedial, ventromedial, and infundibular nuclei contain a majority of the neurons that secrete hypophysiotropic hormones into the portal circulation. (The human infundibular nucleus is the analogue to the arcuate nucleus in lower mammalian species.) GnRH cell bodies do not form discrete nuclei but are instead diffusely located throughout the preoptic area and the mediobasal
Axon ter minals
Axon
Dendrites
Cell body (perikar yon)
Fig. 1.1 Morphologic components of a neuron.
Corpus callosum Thalamus
Fornix
Hypothalamus
Anterior commissure
Lamina terminalis
Optic chiasm
Pituitary in fossa of sphenoid bone
Mammillary body Median eminence
Paraventricular nucleus Dorsal hypothalamic area
Anterior hypothalamic area
Preoptic area
Supraoptic nucleus
Dorsomedial nucleus
Pineal gland attached to epithalamus
Midbrain colliculi
Midbrain
Medulla Pons
Fig. 1.2 Cross-sectional representation of the human brain (sagittal plane), including hypothalamus, median eminence, and pituitary gland. (Modified from Johnson MH, Everitt BJ. Essential Reproduction, ed 5, Blackwell Science; 2000:Fig. 6.1.)
Lateral
Anterior hypothalamic area
Preoptic area
Suprachiasmatic nucleus
Optic chiasm
Cerebral peduncle
Mammillothalamic tract
Median eminence
Posterior hypothalamic nucleus
Posterior hypothalamic
Premammillary nucleus
Ventromedial nucleus
Infundibular (arcuate) nucleus
Pituitary gland
Paraventricular
Fornix III Ventricle
Ventromedial nucleus
Lateral
Mammillary nuclear complex
Fig. 1.3 Nuclei and areas of the hypothalamus. (A) By custom, the nuclei and areas of the hypothalamus are often divided into three groups according to their location along the anteroposterior plane: the anterior group, the tuberal group, and the posterior (or mammillary) group. The anterior group is formed by the paraventricular, supraoptic, and suprachiasmatic nuclei, along with the anterior hypothalamic and preoptic areas. The tuberal group—so-called because of its position above the tuber cinereum, from which the infundibulum or pituitary stalk extends, contains the dorsomedial, ventromedial, and infundibular (arcuate) nuclei along with the median eminence. Along with the paraventricular nucleus, the nuclei of the tuberal group contain a majority of the neurons that secrete hypophysiotropic hormones (i.e., hypothalamic hormones regulate hormone synthesis and release from cells in the anterior pituitary). Finally, the posterior group includes the posterior hypothalamic nucleus and mammillary nuclei. (B) Cross-sectional representations (coronal planes) of the rostral (1), mid (2), and caudal (3) portions of the human hypothalamus. ([B] Modified from Johnson MH, Everitt BJ. Essential Reproduction, ed 5, Blackwell Science; 2000:Fig. 6.3.)
Preoptic area
Optic chiasm
Superior hypophyseal artery
Hypophyseal portal system
Adenohypophysis
Vein
Mammillary body
Infundibular (arcuate)
Fig. 1.4 Anatomic relationship between hypothalamic gonadotropin-releasing hormone (GnRH) neurons and their target cell populations in the adenohypophysis (anterior pituitary). The majority of GnRH neuron cell bodies are located in the infundibular (arcuate) nucleus and the medial preoptic area. GnRH neuron projections terminate at the median eminence, where GnRH is secreted into the hypophyseal portal system. (Modified from Johnson MH, Everitt BJ. Essential Reproduction, ed 5. Blackwell Science; 2000:Fig. 6.4.)
hypothalamus (Fig. 1.4); the latter is situated caudal to the preoptic area, extending from the retrochiasmatic area (i.e., the area located behind the optic chiasm) to the mammillary bodies, and including both the infundibular (arcuate) nucleus and the median eminence.
Median Eminence
Positioned at the base of the third ventricle, the median eminence is part of the anatomic link between the hypothalamus and anterior pituitary. The internal zone of the median eminence is located along the ventral floor of the third ventricle and is largely composed of axonal fibers from both magnocellular neurons (larger neurons that secrete vasopressin and oxytocin) and hypophysiotropic neurons as they travel from hypothalamic nuclei/areas to their final destinations; the neurohypophysis (posterior pituitary) and the external zone of the median eminence, respectively (Fig. 1.5). The external zone contains hypophysiotropic neuron terminals, which release hypophysiotropic hormones into an extensive capillary plexus (the proximal end of the hypophyseal portal system). Some nerve terminals in this zone act on other nerve terminals to influence hormone release (e.g., kisspeptin neurosecretion at GnRH neuron terminals influences GnRH release).
The ependymal layer lining the third ventricle includes a population of specialized ependymal cells called tanycytes, which have a short process extending toward the ventricular surface and a long process extending into the median eminence toward areas around portal capillaries. The latter tanycyte projections envelop or retract from GnRH nerve terminals during episodes of low and high GnRH neuronal activity, respectively. Thus, tanycytes may influence GnRH secretion via the regulated process of physically isolating GnRH neuron terminals from portal capillaries.3 Tanycytes may also represent a link between cerebrospinal fluid and the external zone of the median eminence (e.g., by transport ing substances from the third ventricle to portal blood).
Third ventricle floor
Supraopticohypophyseal fibers
Adrenergic/peptidergic axon
GnRH axon
Portal capillary plexus
Tanycytes Portal capillary loop
INTERNAL ZONE
EXTERNAL ZONE
Fig. 1.5 Diagram of the median eminence.
The median eminence is among the so-called circumventricular organs, which lie adjacent to the ventricular system and represent openings in the blood-brain barrier. Although lipid-soluble molecules can diffuse in and out of the CNS relatively easily, and cellular transport mechanisms allow selective entry of ions, the blood-brain barrier functions to protect certain regions of the brain and hypothalamus from larger charged molecules, with physical protection provided by (1) tight junctions between endothelial cells and (2) neuron-capillary separation by both astrocyte foot processes and microglia. However, the CNS requires feedback signals, including hormonal, metabolic, and toxic cues via macromolecules of peripheral origin that would otherwise be excluded by the blood-brain barrier. Accordingly, capillaries of the circumventricular organs are fenestrated and permit transcapillary exchange of larger charged molecules (e.g., proteins, peptide hormones). Thus the median eminence represents a key access point for central sensing of peripheral cues. Similarly, fenestrated vessels readily allow entry of hypothalamic-releasing factors into portal blood.
Hypophyseal Portal Circulation
No direct neuronal connections exist between the hypothalamus and the anterior pituitary. However, the hypophyseal portal circulation (hypothalamic-hypophyseal portal system, pituitary portal system) represents the functional connection between the median eminence and the anterior pituitary (see Fig. 1.4). The superior hypophyseal artery—a branch of the internal carotid artery—subdivides to form an extensive capillary network in the external zone of the median eminence, with loops that reach into the inner zone. Capillary blood then drains into sinusoids that converge into the hypophyseal portal veins. Traversing the pituitary stalk, the hypophyseal portal system forms the primary blood supply of the anterior pituitary. The direction of blood flow is primarily, but not exclusively, from the hypothalamus to the anterior pituitary; some retrograde flow allows for short-loop hypothalamic feedback.
Pituitary Gland (Hypophysis)
The pituitary gland appears as an extension at the base of the hypothalamus and resides cradled within the sella turcica, a saddle-like structure of the sphenoid bone (see Fig. 1.2). The adenohypophysis (anterior pituitary) is of ectodermal origin, derived from an upward invagination of pharyngeal epithelium (Rathke pouch) during embryologic development. The adenohypophysis is composed of primarily the anterior lobe (pars distalis), which contains specialized cell populations that produce specific hormones: gonadotropes (the gonadotropins LH and FSH), mammotropes (prolactin), corticotropes (adrenocorticotropic
hormone [ACTH]), thyrotropes (thyroid-stimulating hormone [TSH]), and somatotropes (growth hormone). The intermediate lobe is vestigial in adult humans but includes a small population of cells (e.g., POMC cells) in contact with the posterior lobe; the pars tuberalis is a slender layer of tissue (e.g., LH-producing cells and TSH-producing cells) surrounding the infundibulum (the funnel-shaped connection between the hypothalamus and the posterior pituitary) and pituitary stalk.
In contrast to the adenohypophysis, the neurohypophysis (posterior pituitary) is composed of neural tissue and forms as a downward extension of neuroectodermal tissue from the infundibulum during embryologic development. It is thus a direct extension of the hypothalamus. The neurohypophysis includes the infundibular stalk and the pars nervosa (posterior lobe of the pituitary). The supraoptic and paraventricular nuclei include magnocellular neurons that produce oxytocin and arginine vasopressin (AVP; also known as antidiuretic hormone [ADH]), respectively; these axons project to the posterior lobe of the pituitary, where oxytocin and AVP are secreted into a capillary network that drains into the hypophyseal veins (i.e., directly into the systemic circulation). The posterior lobe also includes specialized glial cells called pituicytes, which envelop or retract from magnocellular nerve terminals during episodes of low and high neuronal activity, respectively.
Gonadotropin-Releasing Hormone: The Final Common Pathway for the Central Control of Reproduction
• Pulsatile GnRH secretion is the proximate stimulus for LH and FSH synthesis and secretion by pituitary gonadotropes.
• Although numerous internal and external factors influence gonadotropin secretion via numerous neuronal pathways, GnRH is the final common pathway for the stimulation of LH and FSH release.
GnRH, previously called luteinizing hormone–releasing hormone (LHRH), is synthesized and released by a relatively small population of specialized hypothalamic neurons. GnRH was initially isolated from porcine hypothalami and shown to stimulate pituitary gonadotropin release.4 Although the primary function of GnRH is to regulate pituitary gonadotropin secretion, GnRH also appears to have autocrine and paracrine functions in diverse tissues (e.g., ovary, placenta).5
The regulation of GnRH secretion is complex and involves overlapping pathways, which likely increases the robustness of
central reproductive function. However, there are no known parallel or backup pathways for the stimulation of gonadotropin secretion. Thus natural fertility is absolutely dependent on appropriate GnRH secretion. For example, mice with loss-of-function variants of the GnRH-1 gene are hypogonadal, but reproduction can be restored via GnRH-1 gene therapy6 or transplantation of fetal GnRH neurons.7 Similarly, a variety of human conditions associated with absent (or near-absent) GnRH secretion lead to pubertal failure, hypogonadotropic hypogonadism, and infertility, all of which can be fully reversed with exogenous GnRH therapy.8
GnRH secretion is influenced by numerous factors, including sex steroids, energy availability, and stress. In some mammalian species, GnRH secretion is also affected by circadian rhythms, photoperiod (e.g., seasonal breeders such as sheep), social cues, and pheromones.
Gonadotropin-Releasing Hormone Structure
GnRH (GnRH-1 in particular) is a decapeptide, with the amino acid structure (pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-ArgPro-Gly-NH2. The amino acid structure of GnRH is identical in essentially all mammalian species; with the exception of the central Tyr-Gly-Leu-Arg segment, the amino acids of GnRH are highly conserved among vertebrate species.9 The GnRH-1 gene (GNRH1) is located on human chromosome 8 (8p11.2-p21) and produces a 92–amino acid precursor peptide called preproGnRH, which includes a signal sequence (23 amino acids), GnRH (10 amino acids), a proteolytic processing site (3 amino acids), and GnRH-associated peptide (56 amino acids) (Fig. 1.6). The latter peptide can stimulate gonadotropin secretion and inhibit prolactin secretion, although its precise physiologic role, if any, remains unclear. The actions of GnRH are mediated through the GnRH type I receptor.
Another form of GnRH (GnRH-2) and its receptor have been identified in a variety of animal species, including humans. 10 GnRH- 2 is a decapeptide with a similar structure to GnRH- 1: (pyro)Glu- His- Trp- Ser- His - Gly- Trp - Tyr - ProGly- NH2 (italicized amino acids denote differences compared with GnRH- 1). However, the gene for GnRH-2 is located on human chromosome 20 (20p13). GnRH-2 is widely expressed in the CNS and extra-CNS tissues, and it may contribute to reproductive behavior regulation in some species. In lower animals, GnRH- 2 can act via its own receptor, which is structurally and functionally distinct from the GnRH type I receptor. Although a homologue of the GnRH- 2 receptor gene has
Fig. 1.6 Schematic of gonadotropin-releasing hormone (GnRH) synthesis. (A) Representation of prepro-GnRH, including a 23–amino acid signal sequence, GnRH, a proteolytic processing site (Gly-Lys-Arg), and GnRH-associated peptide. The arrow indicates the site of proteolytic cleavage and C-amidation. (B) Schematic of neuronal GnRH synthesis and secretion.
been detected in humans, it includes a frameshift and premature stop codon. Thus the physiologic role of GnRH- 2 in humans remains unclear.
Anatomy of Gonadotropin-Releasing Hormone-Secreting Neurons
GnRH neurons are a heterogeneous population of hypothalamic neurons. They are relatively few, numbering approximately 7000 in humans, and the majority of GnRH neuronal cell bodies are located in the infundibular (arcuate) nucleus—part of the mediobasal hypothalamus—and the medial preoptic area.11,12 GnRH neurons in the infundibular (arcuate) nucleus appear to be requisite for gonadotropin secretion. For example, selective radiofrequency ablation of the arcuate nucleus in adult female monkeys obliterates gonadotropin secretion.13 Although GnRH neurons are rather loosely affiliated anatomically, they are functionally integrated and form a complex, interconnected network with extensive connections to other neuronal populations. GnRH neurons extend projections through the tuberoinfundibular tract to the median eminence, where neuron terminals gain access to the hypophyseal portal system.
Recent work in mice suggests that the GnRH neuron fibers extending from the cell body to the median eminence are morphologically atypical: although they do not exhibit many of the molecular markers classically associated with axons or dendrites, they demonstrate morphologic and functional characteristics of both axons and dendrites, including functional synaptic inputs along the fiber.1,14 Accordingly, the term dendron has been used for such projections.1 In mice, the distal portion of such dendrons exhibit a particularly high density of dendritic spines and synaptic inputs, beyond which the dendron branches into multiple short axons at the median eminence.15
The physiologic function of other GnRH neurons, which arise from the anterior and posterior hypothalamus and project to the limbic system and posterior pituitary, respectively, remains unclear, although some of these circuits may possibly be involved with various behavioral responses.
Embryologic Development of the Gonadotropin-Releasing Hormone Neuronal Network
The ontogeny of GnRH neurons in vertebrate species is unique among neuronal systems of the CNS: nascent GnRH neurons are initially identified outside of the CNS in the nasal placode (sometimes called the olfactory placode). However, GnRH cells migrate during embryologic development, as directly observed in embryonic nasal explant cultures16 and in murine embryonic head slices.17 The specific migratory pathway of GnRH neurons was first demonstrated in mice by documenting the presence of GnRH-immunoreactive cells in different areas at different stages of embryonic development (Fig. 1.7).18–20 Specifically, GnRH expression is first observed within the nasal placode circa embryonic day 10 or 11. By embryonic day 13, GnRH-expressing cells are primarily located around the cribriform plate, and GnRHexpressing cells begin to reach the hypothalamus by embryonic day 14, approaching their final positions around embryonic day 16. This migratory pathway has been confirmed in both nonhuman primates21 and humans.22
Successful migration of GnRH neurons is inextricably intertwined with olfactory system development, perhaps reflecting the close functional relationship between reproduction and the olfactory system (e.g., pheromones) in mammalian phylogeny. The nasal placode gives rise to nasal epithelium and olfactory sensory neurons, the latter of which extends axonal projections to the olfactory bulb. Vomeronasal neurons are a subset of olfactory neurons believed to be involved with pheromone detection; these axons originate in the vomeronasal organ and largely extend to the accessory olfactory bulb. At the level of the cribriform plate, some olfactory (vomeronasal) axons separate and form a branch that extends caudally into the forebrain. Of great importance, migrating GnRH neurons maintain adhesion to these axons; thus, these olfactory neurons form a critical guidance track for GnRH neuronal migration across the nasal epithelium and through the forebrain toward the hypothalamus.23,24 After reaching the hypothalamus, GnRH neurons detach from olfactory nerve axons and may disperse further before resting. A critical step is the extension
Fig. 1.7 Gonadotropin-releasing hormone (GnRH) neuron migration during embryogenesis. (A)
Location of GnRH-immunoreactive cells (red circles) as a function of embryologic age (mouse). On embryologic day 11 (11E), GnRH cells are located in the nasal (olfactory) placode and presumptive vomeronasal organ (vno). GnRH cells migrate across the cribriform plate toward the olfactory bulb (ob). GnRH neurons then follow the caudal branch of the vomeronasal nerve toward the forebrain and hypothalamus. By day 16 (16E), GnRH neurons largely reside in the preoptic area (poa) of the hypothalamus. (B) Sagittal brain slice (mouse, embryonic day 15) demonstrating the migratory route of GnRH-immunoreactive cells. Staining is for GnRH and peripherin (a neuronal intermediate filament). BF, Basal forebrain; CP, cribriform plate; gt, ganglion terminale; OB, olfactory bulb; OP/VNO, olfactory placode-vomeronasal organ. ([A] Modified from Schwanzel-Fukuda M, Pfaff DW. Origin of luteinizing hormone-releasing hormone neurons. Nature. 1989;338:161–164; and [B] Modified from Wierman ME, Pawlowski JE, Allen MP, et al. Molecular mechanisms of gonadotropin-releasing hormone neuronal migration. Trends Endocrinol Metab 2004;15:96–102.)
of GnRH neuronal projections to the median eminence, where GnRH gains access to the hypophyseal portal system.
The dependence of GnRH neuronal migration on normal olfactory system development is exemplified by Kallmann syndrome, a form of isolated hypogonadotropic hypogonadism accompanied by an absent or reduced sense of smell (anosmia and hyposmia, respectively).25 In this syndrome, faulty development of the olfactory system renders an inadequate guidance infrastructure for migrating GnRH neurons, leading to absent or incomplete GnRH neuron migration to the hypothalamus. The first identified cause of Kallmann syndrome was a deletion of ANOS1 (formerly the Kallmann syndrome 1 sequence [KAL1] gene), which is located on the X chromosome (Xp22.3) and encodes anosmin-1, a secreted matrix glycoprotein expressed in the presumptive olfactory bulb. Although precise mechanisms are unclear, anosmin-1 is believed to be important for the formation of olfactory elements that provide migratory guidance to GnRH neurons as they move out of the nasal placode. Evaluation of a 19-week-old human fetus with X-linked Kallmann syndrome demonstrated GnRH-immunoreactive cells within a tangle of olfactory and vomeronasal nerves at the dorsal surface of the cribriform plate, along with the absence of olfactory tracts and bulbs.26 In a second human fetus (16 weeks) with X-linked Kallmann syndrome, GnRH was detected along terminal nerve fascicles in the nasal mucosa only.27 This form of Kallmann syndrome illustrates that without the guidance framework provided by the olfactory neuronal system, GnRH neurons do not appropriately migrate into the hypothalamus and thus cannot release GnRH into the hypophyseal portal system.
A number of additional single-gene defects have been associated with Kallmann syndrome,25 including variants in the genes for prokineticin 2 (PROK2) and its receptor (PROKR2), 28 fibroblast growth factor 8 (FGF8) and its receptor fibroblast growth factor receptor 1 (FGFR1), 29,30 NMDA receptor synaptonuclear signaling and neuronal migration factor (NSMF; formerly nasal embryonic LH-releasing hormone factor [NELF]),31 chromodomain helicase DNA binding protein 7 (CHD7),32 semaphorin 3A (SEMA3A),33 and SRY-box 10 (SOX10).34 The importance of these genes in GnRH neuronal development is corroborated by mouse studies. For example, in fetal mice lacking either Prok2 or Prokr2, GnRH neurons are trapped in a tangled web of olfactory/ vomeronasal axons, with few, if any, reaching the forebrain.35 Although such gene products are clearly important for GnRH neuron migration, their precise roles remain unclear.
Of interest, patients with specific Kallmann syndromerelated gene variants can exhibit variable penetrance and different phenotypic expressions (even within families), suggesting the importance of other factors such as gene-gene interactions (oligogenicity).36 Some Kallmann syndrome-related gene variants (e.g., PROK2, PROKR2, FGF8, FGFR1, NSMF, CHD7) are also associated with normosmic hypogonadotropic hypogonadism.25 In addition, a minority of patients with some Kallmann syndromerelated gene variants (e.g., ANOS1, PROKR2, FGFR1, NSMF, or CHD7) may demonstrate partial or full recovery of reproductive function (reversal of hypogonadotropic hypogonadism) in later life,37,38 suggesting plasticity of the GnRH neuronal network and, perhaps, gene-environment interactions. Mechanisms accounting for such phenomena remain unclear.
Gonadotropin-Releasing Hormone Neuronal Firing and Gonadotropin-Releasing Hormone Secretion
The activity of many GnRH neurons is marked by bursts of action potentials (burst firing), the patterns and rates of which change across time. Such changes in GnRH neuron firing rates may relate to changes in pulsatile GnRH release at the median eminence, although this hypothesis has not been experimentally verified. Variable firing rate patterns (e.g., times of high and low
firing rates) appear to be intrinsic to GnRH neurons, but they can also be altered by neurotransmitters and neuromodulators (e.g., glutamate, GABA, kisspeptin). Although sex steroids can markedly influence GnRH neuronal firing rates, GnRH neurons lack the primary receptors mediating sex steroid feedback (i.e., estrogen receptor alpha, progesterone receptor, androgen receptor). However, many studies suggest that sex steroid actions on GnRH neuronal activity are mediated primarily via afferent neurons (e.g., those secreting glutamate, GABA, kisspeptin).
GnRH neuron cell bodies are relatively scattered across the mediobasal hypothalamus and preoptic area, yet GnRH is secreted into the hypophyseal portal system in a coordinated, pulsatile fashion. Specifically, GnRH secretion is marked by episodic bursts of hormone release into the portal system, as demonstrated in rats,39 sheep,40 and monkeys.41 After being released into the portal vascular compartment, GnRH is rapidly degraded via enzymatic proteolysis, and the half-life of GnRH in the blood is very short—approximately 2 to 4 minutes. Thus GnRH presentation to gonadotrope cells is intermittent.
Pulsatile GnRH secretion is an absolute requirement for longterm stimulation of gonadotropin synthesis and secretion, and there is a relatively narrow window of GnRH pulse frequency and amplitude that will optimally stimulate gonadotropin secretion. Intermittent GnRH stimulation of gonadotrope cells can increase (or maintain) GnRH receptors on gonadotropes (the self-priming or autopriming effect). Thus intermittent GnRH stimulation facilitates or maintains gonadotrope responsiveness to GnRH. However, more frequent exposure to GnRH pulses can reduce gonadotropin responses to GnRH.42 In classic experiments involving rhesus monkeys with hypothalamic lesions that abolished GnRH secretion, once-hourly exogenous GnRH administration restored pituitary gonadotropin secretion. However, changing from once-hourly pulses to twice-hourly pulses reduced LH and FSH secretion by 50% to 60%, while 3 to 5 pulses per hour profoundly suppressed plasma LH and FSH concentrations.42 Marked desensitization of gonadotropin release is also observed when changing from once hourly (intermittent) to continuous GnRH administration (Fig. 1.8).43 Although reduced GnRH receptor expression on gonadotropes (i.e., receptor downregulation) plays a role in desensitization, additional mechanisms contribute to the uncoupling of GnRH receptor agonism and gonadotropin synthesis.44
Fig. 1.8 The influence of pulsatile versus continuous gonadotropin-releasing hormone (GnRH) administration to GnRHdeficient monkeys. Intermittent exogenous GnRH administration reconstitutes normal gonadotropin secretion. However, continuous GnRH infusion leads to a marked reduction (downregulation) of luteinizing hormone (LH; green) and follicle-stimulating hormone (FSH; purple) concentrations. Resumption of pulsatile GnRH administration restores LH and FSH secretion. (Modified from Belchetz PE, Plant TM, Nakai Y, et al. Hypophysial responses to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science
D-amino acid substitution in antagonists
A B
Fig. 1.9 Structure of gonadotropin-releasing hormone (GnRH) and GnRH receptor agonists and antagonists. (A) Schematic of GnRH-1 in its folded conformation. Folding around the glycine in position 6 enhances GnRH receptor binding. Substitution of the glycine in position 6 with D-amino acids stabilizes the molecule in the folded conformation, which increases affinity for the GnRH receptor and reduces metabolic clearance. The amino-terminal (red) is involved with receptor binding and activation, and GnRH antagonists involve modifications of these residues that prevent receptor activation. The carboxyl-terminal (green) participates in receptor binding, but not in activation. Substitution at position 10 (e.g., replacement of glycinamide by ethylamide) can increase binding affinity. (B) Amino acid structure of GnRH along with selected GnRH receptor agonists and antagonists. Solid blue circles represent amino acids that are unchanged compared with native GnRH. (From Millar RP, et al. Gonadotropin-releasing hormone receptors. Endocr Rev 2004;25:235–275.)
The foregoing phenomenon can be exploited therapeutically with the use of long-acting GnRH receptor agonists. Such agonists are peptides with structures very similar to that of GnRH but with amino acid substitutions that enhance receptor binding affinity, increase resistance to proteolytic degradation, or both (Fig. 1.9), thus providing continuous GnRH receptor stimulation. Although initial GnRH receptor agonism temporarily (for 1 to 2 weeks) increases gonadotropin release (gonadotropin “flare”), continued agonism leads to desensitization of gonadotropin secretion with accompanying reductions of gonadal sex steroid concentrations to castrate levels (“medical oophorectomy,” “medical castration,” “pseudomenopause”), typically over several weeks. These agents can be useful in the therapy of gonadotropin-dependent disorders such as central precocious puberty, endometriosis, and prostate cancer.
Peptide GnRH receptor antagonists are also available for clinical use. These antagonists reversibly bind to, but do not stimulate, the GnRH receptor (i.e., competitive antagonism). Thus these agents do not initially stimulate gonadotropin release, and they reduce gonadotropins more rapidly than GnRH agonists— usually within 24 to 72 hours.
Gonadotropin-Releasing Hormone Stimulation of Gonadotrope Cells
The specialized cells that synthesize and secrete gonadotropins (i.e., gonadotropes) are located mainly in the lateral portions of the anterior pituitary gland and constitute approximately 10% of the adenohypophysis cell population. GnRH action at the pituitary gonadotrope begins with GnRH binding to the GnRH type I receptor on the plasma membrane.9 The GnRH type I receptor is a member of the seven-transmembrane receptor family, a G protein–coupled receptor, and encoded on chromosome 4. GnRH receptor density varies in different physiologic conditions and exhibits a positive correlation with gonadotrope responsiveness to GnRH (e.g., both are high in rodents during preovulatory gonadotropin surges45). GnRH receptor density appears to be modulated primarily by GnRH, with intermittent GnRH stimu lation leading to increased GnRH receptor expression; this is a
central facet of the self-priming effect of GnRH and an important mechanism by which GnRH action is modulated in different physiologic states.
A majority of gonadotropes synthesize and secrete both LH and FSH. A detailed description of the intracellular mechanisms of GnRH action on the gonadotrope is provided in Chapter 2 Briefly, GnRH receptor binding activates the guanosine triphosphate (GTP)-binding protein Gq/11, leading to an increase in second messengers inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). Further intracellular signaling involves increased intracellular calcium and activation of various protein kinase C (PKC) isoforms, mitogen-activated protein kinases (e.g., extracellular signal–regulated kinase [ERK], c-Jun NH2-terminal kinase [JNK], p38), and calcium/calmodulin-dependent kinase II (Ca/CaMK II). A pathway involving adenylate cyclase, cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element binding proteins (CREBs) also plays a role.
Each gonadotropin consists of two protein subunits, α and β. The 92-amino acid α-subunit is common to both LH and FSH, in addition to human chorionic gonadotropin (hCG) and TSH. The β-subunits for LH (LHβ) and FSH (FSHβ) are 121 and 111 amino acids in length, respectively, and account for the biologic specificity of these two hormones. GnRH stimulates gene expression of LHβ, FSHβ, and α-subunit; the latter noncovalently dimerizes with either LHβ or FSHβ to form LH or FSH, respectively. Gonadotropin subunits also undergo variable posttranslational modification, primarily glycosylation (addition of oligosaccharide moieties to specific asparagine residues); such modifications appear to facilitate gonadotropin assembly and influence gonadotropin bioactivity and elimination half-life.46 The gonadotropins are then packaged into secretory granules for eventual secretion.
Although GnRH is the primary stimulus for LH and FSH synthesis and release from a common cell type, concentrations of these two gonadotropins vary differentially throughout ovulatory cycles, with FSH predominance in the early follicular phase and LH predominance in the late follicular phase. This sequential pattern of FSH and LH predominance is important
Fig. 1.10 Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations in gonadectomized (but sex steroid–replaced) monkeys after arcuate nucleus ablation—a model of isolated GnRH deficiency. Exogenous GnRH administered in a pulsatile fashion every hour reconstituted LH and FSH secretion. Changing GnRH pulse administration from a relatively high frequency (hourly) to a relatively low frequency (every 3 hours) resulted in decreased LH but increased FSH secretion. (Modified from Wildt L, et al. Frequency and amplitude of gonadotropin-releasing hormone stimulation and gonadotropin secretion in the rhesus monkey. Endocrinology. 1981;109:376–385.)
for normal follicular maturation, ovarian steroid production, and subsequent ovulation. At least two mechanisms govern differential gonadotropin secretion throughout ovulatory cycles. First, both estradiol and inhibins selectively inhibit FSH release from gonadotropes during the mid- and late follicular phase in addition to the luteal phase.47,48 Second, different patterns of pulsatile GnRH release differentially affect gonadotropin synthesis and secretion. Specifically, high-frequency GnRH pulses favor LH production, whereas low-frequency GnRH pulses favor FSH production. For example, studies in ovariectomized, GnRH-deficient monkeys reveal that a decrease in the frequency of exogenously administered GnRH pulses from one pulse per hour to one pulse every 3 hours results in a 65% increase in plasma FSH, despite a 50% decrease in LH (Fig. 1.10).42 Similar findings have been described in sheep49 and humans.50,51,27 Detailed studies in rats demonstrate that rapid GnRH pulse stimulation favors α-subunit and LHβ mRNA expression, whereas slow GnRH pulses favor FSHβ mRNA expression.52 The mechanisms effecting differential LH and FSH expression in response to changes in GnRH pulse frequency are complex53–55 but include variations of GnRH receptor number on the gonadotrope cell surface56 and alterations of gonadotrope activin βB and follistatin expression.57
A pulse of GnRH release stimulates a pulse of LH release on a one-to-one basis, and LH (or α-subunit) pulse patterns, as assessed by frequent sampling of peripheral blood, accurately mirror GnRH pulse patterns in animal studies (Fig. 1.11).40,58 Similarly, exogenous GnRH pulses elicit corresponding LH pulses in GnRH-deficient patients. Because measurable GnRH is effectively confined to the hypophyseal portal system, which is inaccessible in humans, GnRH pulse frequency is inferred from LH pulse frequency (or α-subunit pulse frequency59,60) in human studies. Although pulses of GnRH stimulate pulsatile release of FSH, the longer serum half-life of FSH renders FSH pulses more difficult to identify via frequent sampling of peripheral blood. In addition, although short-term LH secretion is very closely tied to continued GnRH stimulation, FSH secretion is less acutely dependent on GnRH stimulation.61,62 For example, with GnRH antagonism, the percentage reduction in LH exceeds that of FSH.
2 Hours 34 5
Fig. 1.11 Close temporal relationship between pulses of luteinizing hormone (LH) (jugular vein) and gonadotropin-releasing hormone (GnRH) (pituitary portal system) in the sheep model. (Modified from Moenter SM, et al. Dynamics of gonadotropin-releasing hormone release during a pulse. Endocrinology. 1992;130:503–510.)
Neuronal Inputs Into Gonadotropin-Releasing Hormone Neurons
• Normal pulsatile GnRH secretion is dependent on complex interactions among numerous afferent neuronal inputs, including those expressing kisspeptin, neurokinin B, and dynorphin.
• According to current models, kisspeptin stimulates GnRH release, whereas neurokinin B and dynorphin modulate GnRH release primarily by stimulation and suppression, respectively, of kisspeptin release.
The governance of GnRH neurons is highly complex and involves numerous interacting neural systems involving various neurotransmitters and neuromodulators. The neuronal populations upstream of the GnRH neuron play key roles in puberty and are important mediators of sex steroid feedback and the influence of nutritional cues and stress on GnRH secretion. Numerous neurotransmitters appear to be involved in the regulation of GnRH secretion—including dopamine, norepinephrine, glutamate, GABA, and nitric oxide—and the recent discovery of several neuronal populations upstream of the GnRH neuron (e.g., kisspeptin neurons) has markedly enhanced our understanding of reproductive neuroendocrinology.
Kisspeptin
Kisspeptin-secreting neurons appear to be requisite for normal GnRH secretion, serving as a “gatekeeper” of puberty and helping to mediate the effects of sex steroids and metabolic cues on GnRH secretion. Kisspeptin was originally called metastin because of its ability to suppress metastatic spread of human melanomas and breast carcinomas. However, in recognition of its discovery at Pennsylvania State University in Hershey, Pennsylvania, it was later named kisspeptin after Hershey’s chocolate KISSES. Herein we will use the following abbreviations64: KISS1 and Kiss1, the human and nonhuman kisspeptin genes, respectively; KISS1R (Kiss1R) and KISS1R (Kiss1R), the human (nonhuman) kisspeptin receptor genes and gene products, respectively.
The KISS1 gene product is a 154–amino acid precursor protein (kisspeptin 1-145). Variable proteolytic modification yields kisspeptins of different lengths: kisspeptin-54, -14, -13, and -10, with the numbers referring to the amino acid length of bioactive kisspeptin fragments (Fig. 1.12). Importantly, functional native kisspeptins terminal (kisspeptin
GnRH
Fig. 1.12 Schematic of the precursor kisspeptin-145 and the functional kisspeptin (Kp) fragments, including size and cleavage sites. Note that all functional Kp fragments maintain amino acids 112 to 121 (red) SP, Signal peptide. (Modified from Roseweir AK, Millar RP. The role of kisspeptin in the control of gonadotrophin secretion. Hum Reprod Update. 2009;15:203–212.)
amino acids 112 to 121), which are important for receptor binding and function. Kisspeptin is the natural ligand of KISS1R—formerly known as the G protein–coupled receptor 54 (GPR54)—a seven–transmembrane domain, G protein–coupled receptor.
The importance of the kisspeptin system in reproduction was initially revealed by members of two consanguineous families with loss-of-function KISS1R variants leading to pubertal failure and normosmic hypogonadotropic hypogonadism.65,66 Inactivating KISS1 variants leading to pubertal failure and normosmic hypogonadotropic hypogonadism have also been described in four sisters.67 Murine Kiss1R and Kiss1 knockout models exhibit hypogonadotropic hypogonadism with impaired sexual maturation, reduced gonadal size, failure of estrous cyclicity in females, impaired spermatogenesis in males, and infertility.66,68,69 In contrast, gain-of-function KISS1R and KISS1 variants may cause precocious puberty.70,71 KISS1R and KISS1 variants neither interrupt GnRH neuron migration to the hypothalamus nor impair GnRH synthesis.
Single boluses of kisspeptin markedly stimulate LH release in rodents, sheep, monkeys, and humans. This effect of kisspeptin is mediated by stimulation of GnRH neurons as supported by the following: kisspeptin fibers form synaptic contacts with GnRH neurons,72,73 which is observable in utero74,75; the kisspeptin receptor is expressed by a majority of GnRH neurons76–78; kisspeptin directly depolarizes GnRH neurons79,80; a kisspeptin antagonist inhibits murine GnRH neuron firing rates and reduces pulsatile GnRH release in female pubertal monkeys81; and kisspeptin stimulation of gonadotropin secretion is completely blocked by GnRH antagonists.64,76,82 However, kisspeptin also appears to work indirectly because kisspeptin can increase GABAergic and glutamatergic postsynaptic currents onto GnRH neurons.83 Kisspeptin does not stimulate LH secretion in Kiss1R knockout mice,69,84 suggesting that kisspeptin acts exclusively through its cognate receptor. Moreover, mice with GnRH neuron-specific Kiss1R knockout exhibit hypogonadotropic hypogonadism and infertility85,86; but in global Kiss1R knockout mice, restoration of GnRH neuron-specific Kiss1R expression restores normal reproductive function.85,86 These findings suggest that kisspeptin action at GnRH neurons is critical for reproductive function.
In addition to acting upon GnRH neuron cell bodies,87 kisspeptin neurons extensively synapse with GnRH neuron terminals in the external zone of the median eminence,88 where kisspeptin stimulates GnRH release (exocytosis) into the hypothalamic portal system.78,89 Although kisspeptin may have direct effects on gonadotropes, available data suggest that this does not play a major role in kisspeptin’s ability to stimulate gonadotropin secretion. For example, pulsatile GnRH can restore normal reproductive function in patients with loss-of-function KISS1R variants.90
In primates (including humans), the majority of the kisspeptin cell bodies reside in the infundibular (arcuate) nucleus.91–93 In con trast, rodents have two primary populations of kisspeptinneurons in the hypothalamus: one in the arcuate nucleus (mediobasal
hypothalamus), and the other in the rostral periventricular area of the third ventricle (RP3V) of the preoptic area, which includes the anteroventral periventricular nucleus (AVPV).94 Of interest, kisspeptin expression in the AVPV is much higher in female compared with male rodents, which appears to reflect organizational effects of sex steroids during early development95,96; and kisspeptin neurons in the AVPV appear to be specifically important for LH surge generation in rodents. Sexual dimorphism of kisspeptin expression has also been described in sheep97 and humans.92 Although two studies suggest that adult women exhibit kisspeptin neurons in the rostral hypothalamus,91,98 it remains unclear whether or not such kisspeptin neurons are homologous to those in the rodent AVPV/RP3V.
Kisspeptin’s ability to stimulate LH release in women may vary according to cycle phase or hormonal milieu. For example, although bolus kisspeptin administration consistently increases LH release in women studied in the luteal and preovulatory phases, its effects are less consistent when administered in the early to mid-follicular phase.99–101 Such differences may reflect the observation in some studies that LH responses to kisspeptin positively correlate with circulating estradiol concentrations.102,103 However, compared with findings in cycling women studied during the follicular phase, acute LH responses to bolus kisspeptin administration appear to be more pronounced in women with functional hypothalamic amenorrhea.104 Moreover, one study suggested that LH responses to bolus kisspeptin are more pronounced in postmenopausal women,105 although another study suggested that 24-hour kisspeptin infusions do not increase LH release in hypoestrogenic postmenopausal women.103
Kisspeptin and its analogues may hold therapeutic utility in the future.106 Kisspeptin has been investigated in several disorders marked by impaired GnRH secretion and low gonadotropins: functional hypothalamic amenorrhea,104,107,108 hypogonadism associated with obesity and diabetes,109 and hyperprolactinemia.110 Rapid proteolytic degradation of kisspeptin may limit its therapeutic utility, however. Although long-acting KISS1R agonists are actively being developed, the precise effects of longterm KISS1R agonism on gonadotropin release remain unclear; in this regard, desensitization to kisspeptin may represent a practical challenge. Kisspeptin has been evaluated as a trigger for final oocyte maturation and ovulation in women at risk for ovarian hyperstimulation syndrome.111,112 In this scenario, rapid proteolytic degradation may be advantageous as compared to hCG. Finally, when complete gonadal steroid suppression is not required (e.g., endometriosis), KISS1R antagonists may permit partial inhibition of gonadotropin production.
Neurokinin B
Neurokinin B—a decapeptide (Asp-Met-His-Asp-Phe-Phe-Val) product of the tachykinin 3 gene (TAC3)—is a member of the tachykinin family, which also includes substance
-Neoendorphin
Prodynorphin
Proenkephalin POMC
-MSH
Leu Enkephalin
Met Enkephalin
-MSH
OctaPeptide
-LPH
-LPH Dynorphin A Dynorphin B
Fig. 1.13 Schematic of endogenous opiate precursors. ACTH, Adrenocorticotropic hormone; CLIP, corticotropin-like intermediate lobe peptide; LPH, lipotropin; MSH, melanocyte-stimulating hormone; POMC, proopiomelanocortin. (Modified from Akil H, et al. Endogenous opioids: overview and current issues. Drug Alcohol Depend. 1998;51:127–140.)
P (SP) and neurokinin A (NKA), which are products of the TAC1 gene. There are several neurokinin receptors (NK1R, NK2R, NK3R), and although NKB can produce some agonism at NK1R and NK2R, NKB binds preferentially to and acts primarily via its cognate receptor NK3R (encoded by the TACR3 gene).113 Studies of patients with idiopathic hypogonadotropic hypogonadism from consanguineous families revealed that homozygous loss-offunction variants of either TAC3 or TACR3 can cause pubertal failure and severe hypogonadotropic hypogonadism, highlighting the importance of NKB in human reproduction.114,115
The role of NKB in central reproductive function is complex and appears to vary according to species, sex, and sex steroid milieu.116,117 The selective NK3R agonist senktide can stimulate LH secretion—albeit not as potently as kisspeptin—in rats,118 sheep,119 and monkeys.120 Such stimulation of LH secretion by NKB is mediated by GnRH secretion, and GnRH receptor antagonism abolishes LH responses to senktide in the monkey.120
Although it remains unclear to what degree NKB may have direct actions on GnRH neurons,121–123 a number of observations suggest that NKB primarily influences pulsatile GnRH secretion indirectly by stimulating kisspeptin release. For example, kisspeptin neurons express NK3R, and senktide increases kisspeptin neuronal activity.118 LH responses to senktide are either absent or markedly reduced in Kiss1R knockout mice,124 in the presence of Kiss1R antagonism,125 or after Kiss1R desensitization.126 Moreover, continuous kisspeptin infusion can restore pulsatile LH secretion in patients with loss-of-function variants of TAC3 or TACR3. 127
In contrast to Kiss1 and Kiss1R knockout mice, Tacr3 knockout mice remain fertile, although they can demonstrate reproductive defects.128,129 This apparent discordance may reflect redundancy in the roles of SP-NK1R, NKA-NK2R, and NKB-NK3R in rodents. For example, SP, NKA, and NKB can all activate kisspeptin neurons in mice,130,131 and antagonism of NK1R, NK2R, and NK3R is required to block NKB activation of murine kisspeptin neurons.130 In contrast, less redundancy is evident in ruminants and primates.132
Regarding the therapeutic potential of NKB analogues, two studies in women with polycystic ovary syndrome (PCOS, a
HeptaPeptide
-Endorphin
disorder marked by persistently high GnRH pulse frequency, LH excess, and hyperandrogenemia) suggested that selective NK3R antagonism for 7 days reduces LH (GnRH) pulse frequency, LH area under the curve, serum LH concentrations, basal (nonpulsatile) LH secretion, and total testosterone concentrations, with essentially no change in estradiol concentrations.133,134 Short-term studies also suggest that selective NK3R antagonists may be useful in disorders requiring only partial reductions in gonadotropins and gonadal steroids (e.g., endometriosis).135–138 Such potential uses require further study, however, as the impact of chronic NK3R antagonism has not yet been assessed in premenopausal women.
Also of interest, accumulating data suggests that increased NKB signaling plays a role in the vasomotor symptoms associated with estrogen deficiency,139 and early clinical trials suggest that NK3R antagonism ameliorates menopausal hot flashes.140–142
Endogenous Opioid Peptides
Endogenous opioid peptides (EOPs), which include endorphins, enkephalins, and dynorphins, participate in myriad processes such as motor activity, cognitive functions, water and food intake, and regulation of neuroendocrine function.143 Most active EOPs share a common sequence (Tyr-Gly-Gly-Phe-[Met or Leu]) at the amino-terminal, although endorphins, enkephalins, and dynorphins are derived from different precursor proteins that undergo regulated posttranslational processing (Fig. 1.13).144 Endorphins such as β-endorphin are products of the precursor protein POMC. POMC can be preferentially processed to produce ACTH and β-lipotropin, as occurs in corticotropes (adenohypophysis) under the control of CRH. However, in the hypothalamus, POMC processing primarily yields β-endorphin and α-melanocyte-stimulating hormone. Hypothalamic βendorphin participates in the regulation of reproduction, temperature, and cardiovascular and respiratory functions, and acts opioid receptors. Enkephalins are derived from proenkephalin, and their primary functions appear to relate to
CLIP
ACTH
Peptide F
autonomic nervous system modulation, mainly via δ-receptor activation. Dynorphins are products of the precursor prodynorphin and act chiefly at κ-opioid receptors (KORs). Importantly, although β-endorphin, enkephalins, and dynorphins act primarily via μ, δ-, and κ-opioid receptors, respectively, each can act as agonists at more than one receptor subtype.
Numerous studies provide evidence that hypothalamic opiates partly mediate sex steroid negative feedback on GnRH release. For example, GnRH neurons express few, if any, progesterone receptors, whereas β-endorphin concentrations increase in hypophyseal blood during the luteal phase in monkeys (when progesterone suppresses GnRH pulse frequency).145,146 Moreover, naloxone and naltrexone (opiate receptor antagonists acting primarily at μ- and κ-opioid receptors) increase LH (GnRH) pulse frequency when administered to luteal phase women147 or progestin-treated postmenopausal women.148 Similarly, morphine suppresses GnRH secretion from mediobasal hypothalami isolated from fetal and adult humans—an effect that is reversed by naloxone—and chronic high-dose opiate administration can cause hypogonadotropic hypogonadism by suppressing GnRH and LH secretion.143,149
Several animal studies implicate dynorphin as a principal mediator of progesterone negative feedback on GnRH pulse frequency in females.87 Progesterone treatment in ewes increases dynorphin A concentrations in third ventricle cerebrospinal fluid,150 and central infusion of dynorphin in goats reduces volleys of multiple-unit activity in the mediobasal hypothalamus and reduces LH pulses.151 In luteal phase ewes, specific κ-opioid receptor antagonists—but not antagonists to δ- or μ-opioid receptors—reverse progesterone inhibition of LH secretion and LH pulse frequency when locally administered into the mediobasal hypothalamus.152 Dynorphin may exert its mediating actions directly on GnRH neurons: for example, dynorphin neurons in the arcuate nucleus colocalize with progesterone receptors in ewes,153 and dynorphin-containing varicosities are closely associated with GnRH neuron cell bodies in the mediobasal hypothalamus.152,154 As described in more detail below, dynorphin also appears to influence kisspeptin release.155 In addition, other EOPs (e.g., β-endorphin) in other hypothalamic areas may also be involved in the control of GnRH release. For example, in the aforementioned study,152 κ- and μ-receptor antagonists locally administered into the preoptic area increased LH and LH pulse frequency.
In the arcuate nucleus, kisspeptin, NKB, and dynorphin are frequently coexpressed in the same neuron. For example, kisspeptin neurons in the arcuate nucleus have been found to coexpress NKB and dynorphin in rodents,156,157 goats,151 and sheep.158 For convenience, and as a nod to kisspeptin (namesake of Hershey’s chocolate KISSES), such neurons are often called KNDy neurons (Kisspeptin, Neurokinin B, Dynorphin; pronounced candy).97 KNDy neurons in the arcuate nucleus form an extensively interconnected network.156,159,160 KNDy axons also project to the internal zone of the median eminence where they are in close proximity to GnRH fibers.121,161 As with kisspeptin neurons, KNDy neuron neuroanatomy exhibits sexual dimorphism, possibly related to perinatal sex steroid exposure.97 In addition, robust experimental data in rodents and ruminants suggest that KNDy neurons are intimately involved with sex steroid feedback on GnRH secretion, and general consensus holds that a subpopulation of KNDy neurons represents a fundamental component of the GnRH pulse generator in these species.87,162
Corresponding data in humans are limited. In one autopsy study, 77% of kisspeptin cell bodies (and 56% of kisspeptin axon fibers) in the infundibular nucleus coexpressed preproNKB, and 95% of preproNKB-
kisspeptin.92 However, the degree of colocalization in humans appears to differ according to sex and age. For example, one autopsy study suggested that only 10% and 26% of kisspeptincontaining afferent contacts onto GnRH neurons coexpressed preproNKB in older men and women, respectively163; another autopsy study in young men suggested that 75% of infundibular kisspeptin-containing cell bodies also contained NKB, 33% of NKB-containing cell bodies also contained kisspeptin, and colocalization with dynorphin was uncommon.164 Although these small studies suggested limited colocalization in humans, it is unclear to what degree postmortem degradation may have influenced these findings. Regardless, it remains well accepted that kisspeptin, NKB, and EOPs (e.g., dynorphin)—released from neurons that do or do not colocalize with the other peptides— substantially influence GnRH neuronal function in humans.
Gonadotropin-Inhibitory Hormone and RFamide-Related Peptides
The roles of gonadotropin-inhibitory hormone (GnIH) and its mammalian orthologues, RFamide-related peptides (RFRPs), in the central control of reproduction have been recently reviewed.165,166 Briefly, RFRP-immunoreactive cells have been identified in hypothalami of a number of species, including RFRP-1 and RFRP-3 in humans.167 RFRP-immunoreactive fibers project to GnRH neurons in the median eminence in rhesus macaques and humans,167,168 in addition to a subset of arcuate kisspeptin neurons in mice.169 RFRP-3 can reduce GnRH neuronal firing rates in mice170; RFRP-3 inhibits pituitary gonadotropin release from cultured ovine pituitary cells171; and intravenous RFRP-3 administration suppresses LH pulse amplitude in ovariectomized ewes.172 One study revealed reduced RFRP expression in the preovulatory period in ewes, suggesting a reciprocal relationship with GnRH release, and infusion of GnIH blocked the estrogen-induced LH surge.173 In contrast, RFRP precursor expression did not decrease in the late follicular phase in a study of rhesus macaques, suggesting the possibility of species differences in this regard.174 GnIH and RFRPs have been implicated in the regulation of gonadal function (decrease), food intake (increase), and sexual motivation (decrease), in addition to mediating the inhibitory influence of stress on reproduction.165,166 Although a growing body of data suggests that RFRPs are important factors controlling GnRH and gonadotropin secretion in a number of mammalian species, an understanding of their precise role(s) in humans awaits further investigation.
Gonadotropin-Releasing Hormone Pulse Generator
• Discrete, intermittent bursts of coordinated GnRH neuron activity lead to pulsatile release of GnRH into the hypophyseal portal system.
• Although pulsatility is an intrinsic property of GnRH neurons, afferent inputs (e.g., neurons expressing kisspeptin, NKB, and/ or dynorphin) are required for normal GnRH pulse generation and appear to represent integral components of the GnRH pulse generator.
As described previously, intermittent GnRH receptor stimulation is an absolute requirement for physiologic maintenance of gonadotropin secretion. Although the precise basis of pulsatile GnRH release remains unclear, a number of observations strongly support the concept that neuronal systems within the mediobasal hypothalamus effect pulsatile release of GnRH into the hypophyseal portal system. In animal models, volleys of multiple unit electrical activity (i.e., detection of activity in multiple neurons near an electrode) in the area of the mediobasal hypothalamus coincide with the initiation of LH pulses (Fig. 1.14).175,176 Similarly, electrical stimulation via electrodes placed in the mediobasal
Fig. 1.14 Temporal association between volleys of multiple unit activity (MUA) in the hypothalamus and luteinizing hormone (LH) pulses (green) detected in peripheral blood in an ovariectomized monkey. (Modified from Knobil E. The electrophysiology of the GnRH pulse generator in the rhesus monkey. J Steroid Biochem. 1989;33:669–671.)
hypothalamus stimulates GnRH release into the hypophyseal portal system in monkeys.177 Mediobasal hypothalami isolated from both fetal (20 to 23 weeks’ gestation) and adult humans release GnRH in discrete pulses with a frequency approximating one pulse per 60 to 100 minutes,149 and mediobasal hypothalami separated from the remainder of the brain can maintain pulsatile LH secretion in monkeys.178 These data suggest that the mediobasal hypothalamus houses all requisite components for GnRH pulse generation (i.e., the GnRH pulse generator) and that pulsatile GnRH release does not require innervation from outside of the mediobasal hypothalamus. Nonetheless, mechanisms underlying episodic GnRH pulse generation, and what neuroanatomic components constitute the GnRH pulse generator, remain uncertain.
Several studies suggest that pulsatility is an intrinsic property of GnRH neurons. For example, pulsatile GnRH release is exhibited by immortalized GnRH-secreting neurons179,180 and by cultured GnRH neurons obtained from fetal rats, sheep, and monkeys.181–183 If GnRH pulse generation reflects an intrinsic property of GnRH neurons, then coordination of GnRH release could be facilitated by cell-to-cell interconnections among GnRH neurons.14,184
It is well accepted that afferent inputs (e.g., kisspeptin neurons) are important for normal GnRH secretion, and accumulating research supports the hypothesis that kisspeptin (KNDy) neurons represent a fundamental component of the GnRH pulse generator, in essence orchestrating coordinated GnRH neuronal activity and GnRH secretion accordingly.87,162,185 As described previously, LH pulses are temporally associated with volleys of multiunit activity in the arcuate nucleus, which contains both GnRH and kisspeptin (KNDy) neurons.186 In addition, kisspeptin release at the median eminence appears to be pulsatile: although kisspeptin pulses were not clearly coincident with peripheral LH pulses in ovariectomized ewes,187 kisspeptin pulses corresponded to GnRH pulses 75% of the time in midpubertal rhesus monkeys.188 Moreover, a GnRH neuron cell culture study suggests that pulsatile kisspeptin administration entrains synchronous cycles of GnRH gene transcription and pulsatile GnRH secretion.189 Similarly, a recent in vivo mouse study indicates that episodic increases in arcuate kisspeptin neuron activity correlate very highly with LH pulse generation, and brief optogenetic activation of arcuate kisspeptin neurons generated LH pulses.190 However, work in sheep suggests that additional elements (e.g.,
upstream glutamate-secreting neurons) may also be important in this regard.191 Therefore, although kisspeptin neurons appear to be an important mediator of GnRH pulse secretion, the fundamental nature of the GnRH pulse generator remains unclear.
Human studies also imply that kisspeptin plays a role GnRH pulse generation. For example, in men, continuous intravenous infusion of a relatively low dose of kisspeptin can increase LH pulse frequency192; and a single injection of kisspeptin may reset the GnRH pacemaker.193 (In the latter study, the interval between the kisspeptin-induced LH pulse and the immediately preceding endogenous LH pulse was variable but on average shorter than the normal LH interpulse interval; in contrast, the interval between the kisspeptin-induced LH pulse and the subsequent endogenous LH pulse was similar to normal interpulse intervals [approximately 2 hours], suggesting that kisspeptin administration reset the hypothalamic GnRH clock.) Parallel results in women are mixed: although bolus kisspeptin administration did not appear to reset the GnRH pacemaker in women,101 singledose subcutaneous kisspeptin administration during the follicular phase has been reported to increase LH pulse frequency.194
NKB and dynorphin may also play important roles in the coordination of pulsatile GnRH release. This notion is consistent with a number of experimental observations. For example, KNDy neurons exhibit both NK3R and κ-opioid receptors87,195; murine kisspeptin neuron firing rates are increased by NK3R agonists and reduced by κ-opioid receptor agonists130,196—effects that appear to be modulated by gonadal steroids.196,197 In addition, central administration of dynorphin in goats inhibits both multiple unit activity (MUA) volleys in the mediobasal hypothalamus and pulsatile LH release, whereas NKB provokes MUA volleys.151 Studies using microimplants in the arcuate nucleus of ewes revealed consistent findings: LH pulse frequency was decreased by an NK3R antagonist, whereas LH pulse frequency was increased by either NKB or a κ-opioid receptor antagonist.198
Fig. 1.15 depicts a working model proposed by Moore et al., primarily based on experiments performed in sheep.155 According to this model, KNDy neurons signal to other KNDy neurons— and perhaps to other neurons within the arcuate nucleus—with NKB release stimulating kisspeptin secretion, which in turn initiates GnRH pulse secretion. Subsequent dynorphin release then inhibits kisspeptin secretion, effecting GnRH pulse termination. Importantly, this KNDy hypothesis of pulsatile GnRH secretion is largely based on detailed studies in rodents and ruminants (sheep, goats), and a full understanding of how these data relate to humans awaits further research.132,162
Some data suggest that kisspeptin may not be required for GnRH pulse generation. In particular, frequent sampling studies reveal that humans with loss-of-function KISSR variants demonstrate pulsatile LH release, albeit at low amplitude.66,199 Similarly, a study suggested that puberty occurs and fertility is preserved in female mice with either (1) congenital absence of kisspeptin neurons or (2) congenital absence of neurons expressing Kiss1R.200 When taken as a whole, available data imply that kisspeptin action may not be an absolute requirement for pulsatile GnRH secretion, but it is clear that kisspeptin is required for normal GnRH pulse secretion and normally exerts a profound influence on GnRH pulse generation.
Gonadotropin-Releasing Hormone Secretion During Development and in Adulthood
• Gonadotropin secretion is robust during fetal development and early infancy but quiescent during childhood; puberty represents the reemergence and amplification of gonadotropin secretion, which stimulates gametogenesis, gonadal sex steroid secretion, and the physical manifestations of puberty.
• GnRH pulse frequency changes across the normal menstrual cycle, being highest in the late follicular phase and lowest in the luteal
Fig. 1.15 Working model regarding how KNDy neurons may participate in gonadotropin-releasing hormone (GnRH) pulse generation and termination. By this model, neurokinin B (NKB; green) stimulates and dynorphin (Dyn; red) suppresses kisspeptin (Kp; blue) release, with kisspeptin stimulating GnRH release from GnRH neurons (gray). The onset of a GnRH pulse is triggered by an initial increase in NKB release, which increases kisspeptin output. Kisspeptin may also stimulate interneurons (orange) that support/strengthen NKB stimulation of KNDy neurons. After a short period of time, an increase in DYN release from KNDy neurons suppresses kisspeptin release from KNDy neurons in addition to acting directly on GnRH neurons to inhibit GnRH release. Dynorphin may also inhibit interneurons (orange) that support and strengthen kisspeptin release from KNDy neurons. Note that the neuronal process and terminal color denotes the active neuropeptide at the relevant synapse; it does not indicate selective neuropeptide transport through the neuronal process. The dashed circle represents the arcuate nucleus (ARC). RDyn, kappa opioid receptor; RKp, kisspeptin receptor; RNKB, neurokinin 3 receptor. (Modified from Moore AM, et al. KNDy cells revisited. Endocrinology 2018;159:3219–3234.)
phase; these day-to-day changes primarily reflect the imposition or removal of progesterone negative feedback, and they contribute to the normal cyclic patterns of LH and FSH secretion.
• Men demonstrate consistent day-to-day GnRH pulse patterns, with GnRH pulse frequency approximating one pulse every 2 hours.
Physiologic Development of Reproductive Neuroendocrine Function
Patterns of GnRH secretion change markedly across human development. Reproductive neuroendocrine events throughout early maturation, including both before and during the establishment of reproductive competence, are discussed in detail in Chapter 18. Briefly, GnRH and gonadotropin secretion is robust in utero, peaking in midgestation. In males, gonadotropin secretion markedly stimulates testicular androgen secretion, which is important for normal genital differentiation. The gestational increase in sex steroid (e.g., estradiol) production from the fetoplacental unit provides negative feedback to limit fetal GnRH and gonadotropin secretion. Birth is followed by a marked but transient (3- to 9-month) increase in GnRH and gonadotropin secretion (the “minipuberty of infancy”), perhaps related to the withdrawal of fetoplacental sex steroids. A marked sex difference of gonadotropin release is evident at this time, with LH concentrations being higher in males and FSH levels higher in females. The possibility that kisspeptin is important for the minipuberty of infancy is suggested by a patient with a compound heterozygote loss-of-function KISSR variant, who had micropenis, undescended testes, and undetectable serum gonadotropins at 2 months of age—a time usually marked by robust gonadotropin secretion.201
By late infancy or early childhood (earlier in boys than in girls), GnRH and gonadotropin secretion markedly decreases, leading to a hypogonadotropic phase of childhood marked by low sex steroid concentrations—the juvenile pause. Studies of gonadotropin secretion in children reveal low LH and FSH concentrations, a high FSH-to-LH ratio, and low LH pulse amplitude and frequency.202 Mechanisms accounting for low GnRH secretion during this time appear to include inhibition of the GnRH pulse generator (neurobiologic brake) by higher-order neuronal systems (e.g., involving GABA- and NPY-secreting neurons) and a developmental removal of stimulation (e.g., involving neurons secreting glutamate and norepinephrine).
Near the close of the first decade, a marked nocturnal amplification of pulsatile LH secretion indicates the neuroendocrine initiation of puberty. A majority of studies suggest that early pubertal subjects demonstrate sleep-entrained increases in LH (GnRH) pulse frequency and amplitude.203 Gonadotropin concentrations rise across puberty,204,205 stimulating gametogenesis, gonadal sex steroid secretion, and the development of secondary sexual characteristics. Mechanisms underlying puberty are poorly understood, but they likely reflect developmental remodeling of inhibitory and stimulatory neural circuits in the hypothalamus. For example, puberty has been associated with reductions of GABAergic inhibitory neurotransmission and an increase in excitatory neurotransmitters such as glutamate. Kisspeptin and NKB also appear to play critically important roles in human puberty because inactivating variants of KISS1, KISS1R, TAC3, or TACR3 result in pubertal failure. Conversely, central precocious puberty has been associated with gain-of-function KISS1R variants70 and KISS1 variants that may impair kisspeptin degradation.71 In addition, loss-of-function variants in the maternally imprinted genes
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Arad was undeniably frightened. Although he might explain the fact of his opening Don’s letter as eminently proper, to himself, he well knew that he could not make these friends of his nephew see it in the same light.
“I—I—it came arter Brandon went away,” he gasped in excuse.
“It did, hey?” exclaimed Caleb suspiciously.
Mr Pepper took the envelope again and examined the postmark critically.
“Hum—um,” he said slowly, “postmarked in New York on the third; received on the afternoon of the fourth at the Chopmist post office.
I’m afraid, my dear sir, that that yarn won’t wash.”
Uncle Arad was speechless, and looked from one to the other of the stern faced men in doubt.
“He—he was my nevvy; didn’t I hev a right ter see what he had written ter him?”
“You can bet ye didn’t,” Caleb declared with confidence, and with a slight wink at Adoniram. “Let me tell ye, Mr. Tarr, that openin’ other folks’ correspondence is actionable, as the lawyers say. I reckon that you’ve laid yourself li’ble to gettin’ arrested yourself, old man.”
“Ye—ye can’t do it,” sputtered Arad.
“If that monkey of a sheriff finds Brandon (w’ich same I reckon he won’t), we’ll see if we can’t give you a taste of the same medicine.”
The old man was undeniably frightened and edged towards the door.
“I guess I better go,” he remarked hesitatingly “I dunno as that officer’ll be able ter ketch thet reskil.”
“No, I don’t b’lieve he will myself,” Caleb declared. “And if you want to keep your own skin whole, you’d best see that he doesn’t touch the lad.”
Old Arad slunk out without another word, and the two friends allowed him to depart in contemptuous silence.
When he had disappeared Adoniram turned to the sailor at once.
“Where has Don gone, Caleb?” he asked anxiously
“You’ve got me. He told me he was goin’ to skip, and for us to go ahead with the preparations for getting off next week, just the same. He’d lay low till the old scamp had given it up, and then slip aboard the steamer. Oh, the boy’s all right.”
“He is, if that sheriff doesn’t find him,” said the merchant doubtfully.
“I’ll risk that,” responded Caleb, who had vast confidence in Brandon’s ability to take care of himself.
But Adoniram shook his head.
“New York is a bad place for a boy to be alone in. Where will he go?”
“Down to the pier, I reckon, and hide aboard the steamer. I’ll agree to put him away there so that no measly faced sheriff like that fellow can find him.”
“It’s a bad business,” declared Mr. Pepper, shaking his head slowly.
“If he hadn’t run off there might have been some way of fixing it up so that he wouldn’t have had to go back to Rhode Island, and thus delay the sailing of the steamer. We might have scared the uncle out of prosecuting him. He was badly frightened as it was.”
Caleb gazed at his friend for several moments with a quizzical smile upon his face.
“Do you know, Adoniram,” he said at length, “I b’lieve you’re too innocent for this wicked world.”
“How do you mean?” asked the merchant, flushing a little, yet smiling.
“Well, you don’t seem to see anything fishy in all this.”
“Fishy?”
“Yes, fishy,” returned Caleb, sitting down and speaking confidentially.
“Several things make me believe that you (and me, too) haven’t been half awake in this business.”
“I certainly do not understand you,” declared Adoniram.
“Well, give me a chance to explain, will you?” said the sailor impatiently. “You seem to think that this old land shark of an uncle of the boy’s is just trying to get him back on the farm, and has hatched up this robbery business for that purpose? I don’t suppose you think Don stole any money from him, do you?” he added.
“Not for an instant!” the merchant replied emphatically.
“That’s what I thought. Well, as I say, you suppose he wants Brandon back on the farm—wants his work, in fact?”
“Ye—es.”
“Well, did it ever strike you, ’Doniram,” Caleb pursued, with a smile of superiority on his face—“did it ever strike you that if he was successful in proving Brandon guilty, the boy would be locked up and then nobody would get his valuable services—nobody except the State?”
“Why, that’s so.”
“Of course it’s so.”
“Then, what is his object in persecuting the poor lad? Is he doing it just out of spite?”
“Now, see here; does that look reasonable? Do you think for a moment that an old codger like him—stingy as they make ’em—d’ye think he’d go ter the expense o’ comin ’way down here to New York out of revenge simply? Well, I guess not!”
“Then, what is he up to?” demanded Adoniram, in bewilderment.
“Well, of that I’m not sure, of course; but,” said Caleb, with vehemence, “I’m willing to risk my hull advance that he’s onter this di’mond business.
“Why, Pepper, how could he help being? Didn’t he get that letter of mine, an’ didn’t I give the hull thing away in it, like the blamed idiot I was? Man alive, a sharper like that feller would sell his immortal soul for a silver dollar. What wouldn’t he for a big stake like this?”
“But—” began Adoniram.
“Hold on a minute and let me finish,” urged Caleb. “That scoundrel Leroyd was up to Chopmist, mind ye. Who knows but what he an’ old Arad Tarr have hitched hosses and gone inter this together? I haven’t told either you or Brandon, for I didn’t want to worry you, but I learned yesterday that Jim is tryin’ ter charter a craft of some kind— you an’ I know what for.
“He’s got no money; what rascal of a sailor ever has? He must have backing, then. And who is more likely to be the backer than the old sharper who’s just gone out of here! I tell ye, ’Doniram, they’re after them di’monds, and it behooves us ter git up an’ dust if we want ter beat ’em.”
The ship owner shook his head unconvinced.
“You may be right, of course, Caleb; I don’t say it is an impossibility. But it strikes me that your conclusions are rather far fetched. They are not reasonable.”
“Well, we’ll see,” responded the old seaman, pursing up his lips. “I shall miss Brandon’s help—a handier lad I never see—but he will have to lay low till after the whaleback sails.”
He went back to the work of getting the steamer ready for departure, expecting every hour that Brandon would appear. But the captain’s son did not show up that day, nor the next.
Monday came and Number Three was all ready for sailing. Her crew of twenty men, beside the officers, were aboard.
The first and third mates were likewise present, the former, Mr. Coffin, being a tall, shrewd looking, pleasant faced man, who eternally chewed on the end of a cigar (except when eating or sleeping) although he was never seen to light one; and Mr. Bolin, the third, a keen, alert little man who looked hardly older than Brandon himself.
But Brandon did not come. The new captain of the whaleback, and the owner himself, were greatly worried by the boy’s continued absence.
They had already set on foot inquiry for the youth’s whereabouts, but nothing had come of it.
They did discover that Uncle Arad had gone back to Rhode Island, and gone back alone. The “scaly” ward politician who held the onerous position of deputy sheriff, and who had sought to arrest the boy, had not been successful, Brandon’s friends knew, for the man haunted the pier at which the whaleback lay, day and night.
“If he don’t come tonight, Adoniram,” Caleb declared, “we shall sail in the morning, just the same—and that by the first streak of light, too. You will be here, and I can trust you to look out for the lad. I must be away after those di’monds. Don’ll turn up all right, I know right well; and we mustn’t let them swabs get ahead of us, and reach the brig first.”
He had taken the precaution ere this to have his own and Brandon’s effects brought down to the boat. He was ready, in fact, to cast off and steam away from the dock at a moment’s notice.
As the evening approached Caleb ordered the fires built under the boilers, and everything to be made ready for instant departure. Adoniram Pepper came down after dinner and remained in the whaleback’s cabin, hoping to see Brandon once again before the steamer sailed.
Caleb was too anxious to keep still at all, but tramped back and forth, occasionally making trips to the wheelman’s turret in which he had stationed Mr. Coffin and one of the sailors, so as to have no delay in starting, no matter what should happen.
“By Jove, this beats blockade running at Savannah in the sixties,” muttered the first mate, after one of his commander’s anxious trips to the forward turret to see that all was right. “This youngster they’re taking all this trouble for must be a most remarkable boy.”
“There’s two fellows watching the steamer from the wharf,” Caleb declared, entering the cabin again.
Just then there was a sound outside, and a heavy knock sounded at the cabin door. Caleb pulled it open in an instant.
Without stood three burly police officers.
“Well, well!” exclaimed Mr. Pepper, in wonder.
“What do you want?” Caleb demanded, inclined to be a little combative.
“Beg pardon, sir,” said the spokesman of the two, nodding respectfully to Mr. Pepper, “but we’ve been sent to search the steamer for a boy against whom this man holds a warrant,” and the officer motioned to a third individual who stood without. It was the deputy sheriff.
“Very well,” said Mr. Pepper quietly.
“Search and be hanged,” growled Caleb, glowering at the man with the warrant. “If you can find him you’ll have better luck than we.”
He refused to assist them in any way, however, and Mr. Bolin politely showed the party over the whole steamer. But of course, they found not a sign of Brandon.
After nearly an hour’s search the officers gave it up and departed, Caleb hurling after them several sarcastic remarks about their supposed intellectual accomplishments—or rather, their lack of such accomplishments.
The deputy sheriff, whose name was Snaggs, by the way, would not give it up, however, but still remained on the wharf.
Mr. Coffin, who had begun to take a lively interest in the proceedings, was pacing the inclined deck of the whaleback on the side furtherest from the pier, a few minutes past midnight (everybody on board was still awake at even this late hour) when his ear caught the sound of a gentle splash in the black waters just below him.
He stopped instantly and leaned over the rail.
“Hist!” whispered a voice out of the darkness. “Toss me a rope. I want to come aboard.”
Mr. Coffin was not a man to show his emotions, and therefore, without a word, he dropped the end of a bit of cable into the water, just where he could see the faint outlines of the owner of the voice.
Hidden by the wheelhouse from the view of anybody who might be on the wharf, he assisted the person aboard, and in a minute the mysterious visitor stood upon the iron plates at Mr. Coffin’s side.
CHAPTER XXVIII
THE DEPARTURE OF THE WHALEBACK, NUMBER THREE
N emergency was ever too great for Lawrence Coffin. The appearance of the stranger whom he had lifted over the rail to the steamer’s deck may have surprised him; but he gave no visible sign. The instant the fellow was on his feet, Mr. Coffin slid open the door of the wheelhouse and pushed the newcomer in.
“Jackson,” he said sharply, to the man inside, “go for Captain Wetherbee.”
Then he turned to the dripping figure that stood just within the door of the turret.
The stranger was a youth of fifteen or sixteen, with a sharp, intelligent face, and his saturated clothing was little more than rags.
“Hullo!” said the mate, “you’re not Brandon Tarr, I take it.”
“You kin bet on that, mister,” responded the youth grinning. “An’ you, I reckon, ain’t Cale Wetherbee. He’s got a wooden leg.”
“I’ve sent for Mr. Wetherbee,” replied Mr. Coffin. “What do you want?”
“I’ll tell th’ boss, wot I was told ter see,” declared the fellow shrewdly. The youth was evidently of that great class of individuals known as “street gamins” who, in New York City, are numbered by the thousand.
He was thin and muscular, quick in his movements, and his eyes were shifty and uneasy, not from any lack of frankness or honesty, perhaps, but because his mode of life forced him to be ever on the watch for what might “happen next.”
Mr Coffin had hardly made this mental inventory of the fellow, when Caleb, accompanied by Mr. Pepper, came forward. The strange youth evidently recognized the captain of the whaleback at once as the individual he wished to see.
“You’re Captain Wetherbee,” he said quickly fumbling in the inside of his coarse flannel shirt (the shirt and trousers were all he had on) “I got somethin’ fur you from Brandon Tarr.”
“Where is he?” cried Mr. Pepper, in great excitement.
“He’s gone to sea, boss,” responded the boy calmly.
“Hey!” roared Caleb, and then the messenger brought forth that which he was fumbling for—a little waterproof matchbox.
“Gone to sea?” repeated Adoniram, in bewilderment.
“Dat’s it,” said the boy. “He went day ’fore yest’day mornin’ in de Success.”
But Caleb had opened the matchbox and drawn forth the folded paper it contained.
“It’s a letter—the young rascal! Why didn’t he come himself?”
“Didn’t I tell ye he’d gone ter sea?” demanded the youth in disgust.
“Listen to this,” exclaimed Caleb, paying not the least attention to the messenger’s words, and he read the closely written page aloud:
“D C —Swivel is going to make a break with this letter for me, although the Success sails, we understand, in an hour or two. He can tell you how I came aboard here, so I won’t stop to do that.
“What I want to say is, that Leroyd is aboard and that the brig will touch at Savannah for Mr Pepper’s old clerk, Mr Weeks, who is in the plot to find the Silver Swan, too. I shall leave her at Savannah if it is a possibility.
“If you get into Savannah while she is there, however, and I don’t appear, try to find some way of getting me out. I’m
afraid of Leroyd—or, that is, I should be if he knew I was here.
“I’ve got enough to eat and drink to last me a long time and am comfortable. I can make another raid on the pantry, too, if I run short.
“Look out for Swivel; he’s a good fellow. He can tell you all that I would like to, if space and time did not forbid.
“Yours sincerely, “B T .
“P. S. We’ll beat these scamps and get the Silver Swan yet.”
“Well, well!” commented Mr. Pepper, in amazement. “What will that boy do next?”
“The young rascal!” Caleb exclaimed in vexation. “What does he mean by cutting up such didoes as this? Aboard the very vessel the scoundrels have chartered, hey?”
“But how did he get there?” cried Adoniram wonderingly.
“This young man ought to be able to tell that,” suggested Mr. Coffin, referring to the dripping youth.
Caleb looked from the open letter to the boy.
“So you’re Swivel, eh?” he demanded.
The lad grinned and nodded.
“Well, suppose you explain this mystery.”
But here Adoniram interposed.
“Let us take him to the cabin, and give him something dry to put on. He’ll catch his death of cold here.”
“’Nough said. Come on,” said Caleb leading the way.
Fifteen minutes later the youth who rejoiced in the name of Swivel was inside of warm and dry garments, several sizes too large for him, and was telling his story to a most appreciative audience.
I will not give it in detail, and in Swivel’s bad grammar; a less rambling account will suffice.
When Brandon Tarr had made his rapid retreat from the office of Adoniram Pepper and Co. he had run across the street, dodged around the first corner, and then walked hastily up town. He determined to keep away from the office for the remainder of the day, hoping to tire out both Uncle Arad and the deputy sheriff.
Finally he took a car and rode over to Brooklyn, and it was there that he fell in with Swivel, who was a veritable street gamin—a “wharf-rat” even—though a good hearted and not an altogether bad principled one.
It being a time in the day when there were no papers to sell, Swivel (wherever the boy got the name he didn’t know, and it would have been hard to trace its origin) was blacking boots, and while he shined Brandon’s the two boys scraped up an acquaintance.
Fearing that Uncle Arad or the officer, or perhaps both, would be on the watch about the shipping merchant’s office, or the steamer dock, Brandon decided that Swivel would be a good one to have along with him to send ahead as “scout,” and for a small sum the gamin agreed.
Brandon was a country boy, and was unfamiliar with city ways or city conveniences. It never crossed his mind to use the telephone communicating with his friends, and Swivel knew very little about telephones, any way.
So they waited until toward evening and then came back to New York.
Water Street and its vicinity, and the docks, were as familiar to Swivel as were the lanes and woods of Chopmist to Brandon. By devious ways the gamin led the captain’s son to the ship owner’s office, but it was quite dark by that time and the place was closed.
So they went to the pier at which the whaleback lay, and here Swivel showed that he was of great use to Brandon, for had it not been for him, his employer would have run straight into a trap. The deputy
sheriff, Snaggs, was watching the steamer, and no less a person than Mr. Alfred Weeks himself, was talking with him.
By careful maneuvering the two boys got into a position from which they could hear some of the conversation of the two rascals; but the way to the steamer was right under Snaggs’ eye, and Brandon dared not attempt it.
By intently listening, the captain’s son heard several important items of news, and, greatly to his astonishment, discovered that Uncle Arad, Leroyd, and Mr. Weeks himself were playing right into each other’s hands, and that their object was to keep Brandon from getting back to his friends, and thus delay the sailing of the whaleback so that the craft on which the plotters expected to sail might get away first.
Snaggs was to keep a sharp lookout from the shoreward side of the whaleback and there was already a man in a boat patroling the riverside that Brandon might not return from that direction, and a third person was “shadowing” Adoniram Pepper’s residence. The ship owner’s office would be watched during the day.
As soon as Brandon made his appearance he was to be seized at once on the strength of the Rhode Island warrant and sent back to Chopmist. This, the conspirators hoped, would keep Caleb Wetherbee from sailing for several weeks, and by that time Leroyd and the ex-clerk expected to overhaul the Silver Swan—that is, this is what Weeks and Leroyd themselves were planning to do; but the former took care to say nothing about the Silver Swan to the deputy sheriff.
Finding that there was no chance to get aboard the whaleback just then, and having heard Weeks say that he was going to meet Leroyd and that they two were to go that night and see the vessel and her commander, Brandon decided to follow them, and find out the name of the craft and where she lay, believing that the information would be of value to himself and to his friends.
Piloted by Swivel, Brandon followed “Sneaky Al” to the New England Hotel and while the ex-clerk went inside for Leroyd the two boys
waited without, and then took up the trail again when the two conspirators appeared.
The sailor and Weeks went over to Brooklyn and after two hours’ dodging and running and hiding, they tracked the rascals to the brig Success, lying at a Brooklyn wharf.
Brandon decided that it would never do to be so near and not hear the plans the villains made with the captain of the Success, so he rashly crept aboard and listened to the conversation at the cabin skylight. And this was when he got into trouble.
He heard the two plotters agree with the captain of the vessel (who was not in the scheme at all) to pay two hundred dollars for six week’s use of the brig, providing the Success put to sea at once.
She already had a very fair cargo for Savannah, and the agreement was that she should put in at that port for the time necessary for the cargo to be landed.
Thus, of course, the captain, who was the owner as well, was going to make a very good thing out of it, indeed. He asked no questions as to what use the brig was to be put to; and he agreed to allow Leroyd to accompany him to Savannah, where Weeks would meet them.
Brandon made a shrewd guess that the ex-clerk was to remain in New York until he was certain of his capture and incarceration; then he would reach Savannah by steamer.
It was quite evident that the two rascals had managed to “boil” more money out of old Arad Tarr than they had first expected, and could afford to be more lavish with their funds.
But, as I said, the boys, by venturing aboard the Success, got into trouble. Somebody came aft while they were listening to the conference below, and to escape discovery, they dodged down the after hatch.
The crew of the Success were already aboard, and the two men who constituted the “anchor watch” remained near the open hatchway
(the other hatches were battened down), and the two boys were unable to leave the hold.
Morning came, and found them still there. The cargo was nearly all in, and the crew went to work to finish the lading by daylight. Brandon and Swivel retreated into the bows of the vessel, and managed to remain hidden all day.
They did not dare leave the hold, although they suffered extremely from lack of food and water, for Leroyd had come aboard to superintend the work, and would have seen them.
At evening the hatches were battened down, and the unintentional stowaways were left in darkness. But Swivel, who a shrewd and sharp eyed lad, had noticed a small door in the cabin bulkhead by which the cook doubtless entered the hold for provisions from time to time.
With their pocket knives they forced the fastenings of this door and Swivel made a raid into the pantry, which was left unguarded, and returned laden with provisions enough to last them a week if need be. He secured a big “beaker” of water, too.
Brandon also discovered the ship’s provisions stored near the bows, and was sure that he could stand a siege.
Leroyd, they ascertained, hardly ever left the cabin or deck of the Success, and Brandon dared not venture out. At last, after talking the whole matter over, Swivel agreed to take the risk of giving himself up as a stowaway, and thus get put ashore before the brig started.
Then he was to make his way to the whaleback and explain Brandon’s situation to Caleb.
The captain’s son wrote his letter and placed it in the matchbox, which Swivel in turn had hidden in the breast of his shirt. Then the gamin pounded on the hatch until the crew heard him and let him out.
Naturally the captain of the Success was angry enough, for the brig was already to sail, and they were getting the lines cast off, so he
summoned a night watchman from the dock, who took the unlucky Swivel in charge and handed him over to a policeman.
This was a phase of the situation which neither of the boys had considered. But there was no way out of it, and the gamin spent the day in the police station, for it was Sunday.
He was brought before the magistrate the next morning, but of course there was nobody to appear against him, so he was discharged with a reprimand. The police captain, however, kept him busy about the station until late in the afternoon, before he would let him go.
“He kep’ me jugglin’ wid er mop er wipin’ up de floor,” as the gamin expressed it to his hearers.
As soon as he was free he had hurried to the New York side; but upon reaching the vicinity of the whaleback he discovered that the “patrol line” was drawn even closer than before.
Snaggs and two of his friends were on duty, for as the time approached for the sailing, they decided that if Brandon came back he would do so very soon.
Swivel had seen the raid the policemen made under the deputy’s instigation, and after the bluecoats were safely out of the way, he had slipped into the water and made for the steamer.
“An’ here I is,” he said, in conclusion. “Dey didn’t ketch me, nor dat Brandon Tarr, nuther. We’s too fly for ’em.”
“Of all the scrapes I ever heard of, this is the worst,” Adoniram exclaimed in comment.
But Caleb, now that his fears for Don’s safety were somewhat allayed, seemed rather to enjoy the situation.
“Oh, that boy’s smart,” he declared, with a chuckle. “I’ll risk him even if he is in that vessel’s hold. Leroyd won’t get the best of him. Probably, too, the captain of the Success is not a bad sort of a fellow, an’ he won’t see the boy maltreated.
“I feel better, ’Doniram, and with your permission we’ll get under way at once.”
“But what shall we do with this lad?” asked the little merchant, nodding and smiling at Swivel. “He’s deserving of much praise for his honesty and faithfulness.”
“Oh, take me along, will yer?” exclaimed the gamin, with eagerness. “I’ll work hard ef ye will! I jest wanter see dis thing out, I do! I like dat Brandon Tarr, an’ I wanter see him git the di’monts wot he said was on dat wreck yer arter. Say, lemme go, will yer?”
Caleb looked at the ship owner in perplexity.
“Oh, take him, Caleb,” said Adoniram quickly. “It may be the making of the lad to get him off the city streets. He deserves it.”
“So be it then,” said Caleb, rising. “Now, Mr. Coffin and Mr. Bolin—to work! You’ll have to go ashore at once, Adoniram. I shall have Number Three out of her berth in half an hour.”
Steam was got up, the crew flew about their several duties under the energetic commands of the officers, and within a short time the whaleback, to the manifest disappointment of Mr Snaggs, who watched proceedings from the shadow of the wharf, cast off her lines and steamed down the bay into the darkness of the night.
Thus did she begin the voyage whose object was the finding of the wreck of the Silver Swan.
CHAPTER XXIX
THE STOWAWAY ABOARD THE SUCCESS
A we know, Brandon Tarr had no intention of remaining long away from his friends when he slipped out of Adoniram Pepper’s office to escape arrest on the fraudulent charge of robbery, concocted by Uncle Arad.
The events which followed, however, made it necessary for him to remain away, and, finally, to go to sea as a stowaway in the hold of the Success, the vessel chartered by the conspirators to make search for the Silver Swan.
After the friendly street gamin, Swivel, left him in the hold, in the early hours of Sunday morning, Brandon of course had no means of knowing what had become of him—whether he had accomplished his purpose of getting away from the brig before she sailed, or whether, because she was short handed, the captain of the Success had retained him.
After Swivel was let up on deck, and the hatch closed, however, Brandon heard nothing further, except the heavy tramping of the sailors, the creaking of the ropes, and the hoarse roars of command from the officers.
The work of getting the Success away from the dock went rapidly on.
Quite fortunately for the stowaway, the hold of the Success was little more than two thirds filled with Savannah goods. In the bows, beside a great many bags and boxes and barrels of provisions for the use of the crew, there were likewise spare sails, cordage, etc.
It would be a very easy matter indeed for him to hide among the stuff if any one came into the hold.
The scent of bilge water was not at all strong, for the Success was a comparatively new vessel and had evidently been recently pumped
out.
Brandon judged her to be about the size of the Silver Swan, much the same sort of craft in fact, and, like his father’s vessel, the Success was a “tramp.”
It was night—or at least a gloomy twilight—at all times in the hold; but Brandon thought that it was surely daylight by the time the brig was under way.
She was taken down the river by a fussy little steam tug and then, meeting the swells of the Atlantic, and a brisk gale springing up, she shook out her sails and dropped the tug astern.
Brandon was fearful that he might be sick, for he had never really been to sea and the brig pitched not a little in the waves of the ocean.
To reduce the possibility of this misfortune to a minimum, he ate but sparingly the first day or two out, and by that time all “squeamish” feelings passed away
It was dreadfully dull in the dark hold, however. Of food and water he had a sufficiency, although the latter was warm and brackish; but there was absolutely nothing for him to do to pass away the time. There was not even the spice of danger about his situation, for nobody came into the hold.
He dared not explore much at first, for he was afraid that he might be heard from the cabin or forecastle.
During a slight blow which came up the fourth day, however, while the spars and cordage were creaking so that all other sounds were drowned, he felt perfectly safe in moving about. If he could not hear what went on outside, nobody outside would be likely to hear him.
On this day, however, he received several tumbles, for the ship occasionally pitched so suddenly that he was carried completely off his feet. Nothing worse happened to him, though, than the barking of his elbows and knees.
Gaining confidence in his ability to get around without being discovered, he changed his position more frequently after this. The
weather remained fair for some time following this small blow, and Brandon hung about the cabin bulkhead, striving to hear more of Leroyd’s plans, if possible.
It was plain that the captain of the brig knew nothing of the real plans of the conspirators. They had told him what they pleased, and he was to ask no questions.
It was not long, however, before the stowaway discovered something which was quite a surprise to him. There was a woman on board the brig; he heard the rustle of her garments, and occasionally the tones of a female voice.
At first he thought her to be the captain’s wife, but because of the youthfulness of her tones and some words which the captain addressed to her, he changed this opinion, and decided that she was his daughter.
Brandon was quite interested in her, for a girl on a sailing vessel was certainly a novelty. He was sure she must be a “jolly one,” as he expressed it, to sail with her father on a merchantman. Not many girls would have the pluck to do that.
As the days passed by, and the Success fled on before the favoring gales, drawing nearer and nearer to Savannah, Brandon became correspondingly worried over the obstructions to a safe escape from the brig, which were presented to his mind.
Once the brig reached port and the hatches were opened, it would be “all day” with him. Nothing but a miracle would save him from falling into the hands of Jim Leroyd, and he didn’t like to think of that. He had good reason to believe that the rascally sailor would not hesitate to injure him in any way possible.
Naturally his mind reverted to the trap in the cabin bulkhead by which Swivel had gained access to the cook’s galley, as a possible means of escape before the hatches were removed. If the brig reached Savannah late in the day, doubtless the hatches would remain battened down till the next morning. In that case the trap might be his salvation.
Several times during the voyage the steward, sometimes with a seaman with him, entered the hold by this door, for something among the stores. At such times Brandon “laid low” and his presence was not discovered.
What little food he had purloined from the stores was not noticed either.
Therefore, as the brig drew nearer to her destination Brandon set about studying the topography of the cabin—its entrances and exits —and how he could best pass through it and reach the deck without attracting the attention of anybody on board.
All this scouting had to be done at night, of course, and many were his narrow escapes while engaged in this most perilous undertaking.
“Nothing ventured, nothing gained,” was the motto of the Tarrs, father and son. In Captain Tarr’s case, and in that of his brother Anson, it had been, as a usual thing, a good deal of venture and little gain.
The same motive, however, was predominant in Brandon’s nature, and he took many risks in thus scouting about the brig’s cabin that almost any other boy would not have taken.
One night he had cautiously set the narrow door leading into the steward’s pantry ajar, and sat just under it in the darkness of the hold, trying to discover if all but the officers, excepting the one in command of the watch, had turned in.
There was a light in the outer cabin, but he could not see into the room from where he sat, and he dared not enter the pantry until he was sure that the cabin was unoccupied. Occasionally a sound of low conversation would reach his ears from the deck, but otherwise all was still.
