Sperm maturation and capacitation

Aquaculture 270 (2007) 436 – 442 www.elsevier.com/locate/aqua-online

Sperm maturation and capacitation in the open thelycum shrimp Litopenaeus (Crustacea: Decapoda: Penaeoidea) Jorge Alfaro a,⁎, Karol Ulate a , Maribelle Vargas b b

a Estación de Biología Marina, Escuela de Ciencias Biológicas, Universidad Nacional, Puntarenas, Costa Rica Unidad de Microscopia Electrónica, Universidad de Costa Rica, San Pedro de Montes de Oca, San José, Costa Rica

Received 16 February 2007; received in revised form 11 May 2007; accepted 14 May 2007

Abstract Transmission electron microscopy was applied to sperm removed from males and females belonging to Litopenaeus vannamei, L. stylirostris and L. occidentalis. It was discovered that a region named filamentous meshwork (FM), located between the nucleus and the hemispherical cap, develops differently in these three closely related species. In L. vannamei, the FM is synthesized in the male reproductive system, but seems to complete its formation after mating. In L. stylirostris, the FM region was not present in spermatophores collected from males or in sperm from the thelycum. In L. occidentalis, the FM region is fully developed in male sperm. It is suggested that completion of the FM is required for acrosome maturation, and the process continues after mating in some species of Litopenaeus. In vitro induction of the acrosome reaction in sperm from males and females of L. occidentalis demonstrated for the first time that reactivity is significantly superior in sperm cells that have been attached to the open thelycum for some hours, as compared to sperm in males (prior to transfer). This finding suggests that matured sperm cells of L. occidentalis become capacitated to react against egg water after mating. © 2007 Elsevier B.V. All rights reserved.

Keywords: Acrosome reaction; Sperm ultrastructure; Dendrobranchiata; Penaeoid; Shrimp

1. Introduction The sperm acrosome is located at the anterior part of the cell head, and it consists of three elements in the closed thelycum species Sicyonia ingentis: membrane pouches, anterior granule, and spike (Clark et al., 1981).

The acrosome contains enzymes that function in both exocytosis and sperm penetration of the eggs during fertilization (Gwo, 2000); therefore, sperm undergo an acrosome reaction induced by egg factors.

The primary binding between the sperm spike and the vitelline envelope, being the first contact between ⁎ Corresponding author. Tel.: +506 277 3324; fax: +506 237 6427.

E-mail address: jalfarom@una.ac.cr (J. Alfaro).

0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.05.011

gametes, seems to be the initiator of the acrosome reaction in vivo (Clark et al., 1981). Cortical rods emerge within 60 s after seawater contact in Litopenaeus (Rojas and Alfaro, 2007); therefore, in vivo acrosome reaction is a fast event. The reaction includes the release of acrosomal contents to aid in penetrating egg investment coats, exposure of inner sperm surfaces for binding to the egg, and in some cases the formation of an acrosome filament to facilitate sperm entry (Griffin and Clark, 1990; Lindsay and Clark, 1992). Griffin et al.

(1987) developed a technique to induce the acrosome reaction in vitro: the egg water technique. Egg water (EW) is seawater collected at the time of spawning, containing the egg-derived inducers of the sperm acrosome reaction.

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In S. ingentis, sperm cells mature in the female's thelycum, where further ultrastructural development within the cells takes place (Clark et al., 1984; Shigekawa and Clark, 1986). It has also been demonstrated for Trachypenaeus byrdi and Xiphopenaeus riveti (closed thelyca shrimps) that only sperm cells from females, but not from males, react against conspecific egg water, indicating that further maturation or capacitation is required in seminal receptacles (Alfaro et al., 2003).

Sperm of the closed thelyca shrimps Farfantepenaeus aztecus and S. ingentis undergo a capacitation process, as in mammalians, where matured sperm cells experience physiological changes to facilitate their reactivity. Sperm must be transferred and stored within the seminal receptacle of a female for a period of time before they achieve the ability to fertilize (Clark et al., 1984; Clark and Griffin, 1988, 1993; Griffin and Clark, 1990). Uncapacitated sperm of S. ingentis have extremely low Ca2+ levels, which increase during capacitation (Lindsay and Clark, 1992).

The general ultrastructure of Litopenaeus sperm has been described by Dougherty and Dougherty (1989), who reported on the pathology of melanized spermatophores of pond-cultured Litopenaeus vannamei. Sperm cells consist of a spike (S) and hemispherical cap of moderate electron density (C), a nucleus (N) containing a network of chromatin threads, a filamentous meshwork (FM) between the nucleus and hemispherical cap, and a hemispherical rim of cytoplasmic particles (CP).

In L. stylirostris, spike elongation takes place in the descending medial vas deferens of the reproductive system (Alfaro, 1994). The Litopenaeus acrosome seems to be formed by sections S, C, and FM. In open thelycum shrimps, it has been assumed that spermatophores within terminal ampoules contain fully matured and capacitated sperm, but no scientific observations have been published to improve our knowledge on this crucial topic. It has been stated that sperm of open thelyca penaeoideans do not appear to undergo capacitation after transfer to the female (Clark and Griffin, 1993), but recently, it has been proposed that final sperm maturation on the external surface of the thelycum may be required for fertilization in open thelyca shrimps (Alfaro et al., 2003). This hypothesis was based on experimental observations of in vitro fertilizations (Alfaro et al., 1993; Misamore and Browdy, 1997), and the low sperm activation obtained with the EW technique applied to sperm removed from spermatophores (Alfaro et al., 2003).

This research was designed to compare the ultrastructure of sperm cells from male's spermatophores and female's thelyca of the three Litopenaeus species from the Pacific coast of the Americas: L. vannamei, L. stylirostris

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and L. occidentalis. In addition, in vitro induction of acrosome reaction was evaluated comparing sperm response before and after mating in L. occidentalis.

2. Materials and methods 2.1. Animals Specimens of L. vannamei were selected before harvesting at 90 days of commercial semi-intensive culture in earthen ponds at Asociación de Camaronicultores de la Península de Nicoya, Costa Rica. Individuals were maintained at Estación de Biología Marina (EBM) in a shaded external tank (18 m2; N = 250 animals). Water exchange was kept at 48% per day, using new water pretreated by high pressure silica sand filtration and sedimentation. Animals were fed a commercial dry food at 3% body weight (b.w.) daily and fresh frozen sardine at 1% b.w. Mature animals of L. stylirostris and L. occidentalis were collected from Golfo de Nicoya.

2.2. Sperm ultrastructure For transmission electron microscopy (TEM), spermatophores were collected by manual ejaculation from wild specimens of L. stylirostris and L. occidentalis, and cultured specimens of L. vannamei held under controlled reproduction conditions (water temperature = 28 °C) at EBM, as previously described (Alfaro et al., 2004). Additionally, naturally inseminated females of L. stylirostris and L. occidentalis from the wild, and of L. vannamei from maturation tanks were isolated in spawning tanks for a few hours before removing the attached sperm mass.

Sperm masses were expelled from male spermatophores before fixation. Spermatophores from thelyca were in an advanced stage of reaction from the time of mating judging by its distinctive compact shape. In open thelycum shrimps, females retain sperm cells for a few hours (6–7 h) until spawning. Compact spermatophores were fixed as a whole. The fixative used was a solution reported by Ro et al. (1990) for marine shrimp reproductive systems, which consists of paraformaldehyde (2.0%), glutaraldehyde (2.5%), and sucrose (5.0%) in 0.1 M sodium cacodylate buffer at pH 7.4. While in fixative, samples were observed under light microscopy to identify sperm masses and eliminate unnecessary material. After repeated washing in fresh 0.1 M cacodylate buffer, samples were postfixed in 1% OsO4 in 0.1 M cacodylate buffer, pH 7.4, at room temperature for 2 h. Samples were then rinsed with cacodylate buffer and distilled water several times, dehydrated in an

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Fig. 1. Electron micrographs of sperm from male spermatophores (plate 1 = male 1, plate 2 = male 2) and thelycum (plates 3–4) of L. vannamei. The following regions are named based on Dougherty and Dougherty (1989): S = spike, C = hemispherical cap, N = nucleus containing a network of chromatin threads, FM = filamentous meshwork between the nucleus and the hemispherical cap, CP = cytoplasmic particles forming a hemispherical rim.

ascending series of ethanol, then embedded, via propylene oxide, in Spurr's resin. Ultrathin sections were cut with an ultramicrotome, picked up on copper grids, stained sequentially with uranyl acetate and lead citrate, and examined under a transmission electron microscope. Some samples were processed in Costa Rica (Unidad de Microscopia Electrónica, Universidad de Costa Rica) and others in Germany (Institute for Animal Ecology and Cell Biology, University of Veterinary Medicine Hannover).

2.3. In vitro sperm activation A modified EW technique was used to induce the acrosome reaction in sperm of L. occidentalis. A

positive reaction under light microscopy is characterized by the loss of the spike followed by eversion of cell contents, which leaves a distinctive rounded mark at the acrosome region. The technique developed by Griffin et al. (1987) was modified as reported by Alfaro et al.

(2003). Basically, at the time of spawning, females were collected and allowed to spawn over 100 ml glass beakers containing filtered (1 μm) and UV-treated natural seawater (NSW) at ambient temperature (26 °C). EW was used immediately after collection; therefore, no centrifugation or storage was applied.

Sperm suspensions were prepared by homogenizing spermatophores from males and compacted sperm masses from females in 3 ml of NSW. Two drops of sperm suspension were mixed with six drops of EW in a

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vial. As negative controls, sperm suspensions were mixed with NSW. The rate of activation was measured over a microscope slide, cataloging the number of reacted and nonreacted cells for 100 sperm cells (three replications per trial).

Three experiments were conducted. Experiment 1 evaluated EW batch 1 in sperm masses removed from three females of L. occidentalis; sperm activation was monitored at 0, 15, 30, and 45 min from EW exposure.

Experiment 2 evaluated EW batch 2 in sperm from a male and a female for the same time interval. These two experiments were intended to define the optimum time response for in vitro sperm activation, since it has been observed that the reaction occurs slowly under in vitro conditions. Experiment 3 evaluated sperm activation at 45 min from EW exposure, using a mixed-effect block design (Ott, 1984), with two EW batches (3, 4; blocks: random factor) and four treatments (fixed factor).

Treatments were as follows: T1 = male sperm in NSW (control-male), T2 = male sperm in EW, T3 = thelycum sperm in NSW (control-thelycum), and T4 = thelycum sperm in EW. Blocks 1 and 2 used two and three samples per treatment, respectively.

2.4. Statistics Percentages of sperm reactivity were transformed using arcsine of squared root to make the variance independent of the mean (Ott, 1984). The block design was analyzed using the General Linear Model of the Minitab 13.2 software program. Tukey simultaneous tests were applied for pairwise comparisons among treatments. Alpha level was set at 0.05. Untransformed data are presented as mean ± standard deviation (s.d.).

3. Results Fig. 1 (micrographs 1 and 2) shows the ultrastructure of sperm removed from spermatophores of males of L. vannamei (body diameter = 3–4 μm). Micrographs 3 and 4 (Fig. 1) were taken from a sperm mass attached to the thelycum of a female L. vannamei. The general morphology is similar before and after mating; sperm cells show the different regions previously described by Dougherty and Dougherty (1989), but the degree of development of the FM region seems to be more advanced in sperm cells from the thelycum. Micrograph 4 shows a detail of the FM region from a thelycum sperm cell, and filamentous elements are still proliferating into the empty anterior space.

In contrast with L. vannamei, sperm removed from spermatophores of L. stylirostris (body diameter = 6.5 μm;

Fig. 2. Electron micrographs of sperm from male spermatophores (plate 5) and thelycum (plates 6–7) of L. stylirostris.

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Fig. 4. Time response over induced acrosome reaction in sperm from thelyca (experiments 1 and 2) and male (experiment 2) of L. occidentalis.

L. stylirostris. Sperm removed from intact spermatophores taken from females' thelycum (early stage of spermatophore reaction; Fig. 2, micrographs 6 and 7), show some degree of development of the FM region. However, the region was still not as developed as in L. vannamei.

The morphology of L. occidentalis sperm cells (body diameter = 4 μm) is also similar to the other two species, but the FM region seems to be fully developed in male's spermatophores (Fig. 3, micrograph 8). The morphology of the region is different, being smaller and compact, with no empty space. Sperm cells from the thelycum (Fig. 3, micrograph 9) present a similar morphology.

In vitro induction of acrosome reaction in L. occidentalis with the EW technique (Fig. 4) was applied to three sperm masses from thelyca (experiment 1). Data clearly indicate a positive response of sperm cells to conspecific EW. Sperm cells from thelyca show a pattern characterized by a progressive increase of reacted cells as time advances until reaching a final value of 58.8% ± 5.4 after 45 min exposure time. On the contrary, sperm cells from a male show no increase in

Table 1 Induced acrosome reaction in Litopenaeus occidentalis sperm before and after insemination Treatments Fig. 3. Electron micrographs of sperm from male spermatophores (plate 8) and thelycum (plate 9) of L. occidentalis.

Fig. 2, micrograph 5) show no development of the FM region, however, the space where it should develop appears empty; every sperm cell observed presented this pattern. Another distinctive feature observed between these two species was the thickness of the cytoplasmic particles rim (CP), which is remarkably thicker for

T1: control-male T2: male-EW T3: control-thelycum T4: thelycum-EW

Blocks a EW batch 3

EW batch 4

n

n

2 2 2 2

3 3 3 3

Reactivity (%) ± s.d.

Tukey test b (P b 0.05)

13.0 ± 6.5 17.0 ± 3.6 12.6 ± 5.9 33.6 ± 2.5

a a a b

a A mixed-effect block design: treatments as fixed factor and two EW batches as random factor. b Different letters indicate statistically significant differences.

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reactivity after 45 min when exposed to EW (experiment 2). Reactivity measured in experiment 2 for sperm cells from thelyca was lower (25.3%) than the reactivity obtained in experiment 1, using a different EW batch.

Table 1 summarizes the results of the third experiment, which indicates that sperm reactivity at 45 min in T4 (thelycum-EW) was significantly superior than in control-thelycum (T3), control-male (T1), and maleEW (T2, P b 0.05).

4. Discussion The transmission electron microscopy of sperm from males' spermatophores and females' thelyca of the three species evaluated reveals a similar morphology among species, but a different pattern in the process of maturation. Our observations indicate that the region between the nucleus and the hemispherical cap of the sperm cell accumulates filamentous materials (FM), which seems to involve an active synthesis before and after mating, depending on the species.

Our observations on L. vannamei, as well as a previous report by Dougherty and Dougherty (1989), clearly indicate that this species initiates the synthesis of the FM in the male reproductive system; however, it seems that more material is still accumulating after mating.

On the contrary, the male reproductive system of L. stylirostris does not appear to activate the synthesis of the FM. It seems that after transfer of spermatophores to the thelycum, the synthesis of the FM is activated; however, this statement requires further confirmation. We have not been capable of observing sperm of L. stylirostris with a similar degree of development of the FM as in L. vannamei and L. occidentalis, indicating that the process is slow or that the amount or composition of FM is different between these three closely related penaeoid species. It seems unlikely that such a large cellular region remains empty (in L. stylirostris), when it is occupied by a filamentous meshwork in L. vannamei and L. occidentalis.

The FM region of L. occidentalis seems to be fully developed before mating; this was the only species showing a compartment fully occupied by this filamentous meshwork. However, a previous report has shown that eggs of this species spawn in vitro into a sperm suspension only generate massive primary bindings between vitelline envelopes and sperm spikes (Alfaro et al., 1993; Rojas and Alfaro, 2007), without detectable acrosome reactions.

In naturally spawned eggs from F. aztecus, the FM named as electron-dense material, always appears along the periphery of the activated surface of the sperm as well as in association with the opposed egg membranes

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(Clark et al., 1980). In sperm cells from the distal vas deferens of the male reproductive system of T. byrdi (closed thelycum), the filamentous material is not present (Alfaro, 1994). It is proposed that the FM region is an essential part of the acrosome, that continues its formation after mating in some species of Litopenaeus.

For the first time, data are presented from an open thelycum shrimp (L. occidentalis) that confirm thelycum sperm react to EW at higher rates than male sperm.

Reactivity rates at 45 min from EW exposure for thelyca sperm were 58.8% ± 5.4, 25.3%, and 33.6% ± 2.5 for experiments 1, 2, and 3, respectively. The variability within replicates for experiments 1 and 3 was very low; however, between experiments rates were variable, suggesting that each EW batch induced a different homogeneous reactivity level. Reactivity rates for male sperm were 6.7% and 17.0%± 3.6 for experiments 2 and 3, respectively. A previous study also measured a low percentage of reactivity for L. occidentalis male sperm cells incubated with different conspecific EW batches: 4.2%± 2.1, 3.0% ± 0.9, 16.8% ± 0.4 (Alfaro et al., 2003).

Wang et al. (1995) measured male sperm reactivity in L. vannamei (37.4% ± 18.5). This value indicates that some degree of capacitation was acquired in the male reproductive system for this species, but the response was highly variable. This pattern is in accordance with our ultrastructural observations, and indicates that each male may show a different degree of sperm maturation/ capacitation before mating. This hypothesis requires further confirmation by analyzing the reactivity of thelycum sperm.

From ultrastructural observations no differences were detected between male and thelycum sperm of L. occidentalis, but in vitro reactivity against egg water suggests some physiological changes took place within sperm cells after mating. It seems that sperm cells from L. occidentalis become competent in the female thelycum. Sperm capacitation in other species of Litopenaeus must be investigated to get a better understanding of this process in open thelycum shrimps.

Completion of other cellular regions may still be required for sperm maturation after mating; however, under our sampling protocol we did not detect the development of any other structural changes during the few hours after natural spermatophore transfer to the thelycum. Spermatophores experience a gradual reaction as soon as they are transferred to the thelycum, leading to the release of the sperm mass and its attachment around the gonopores. We have not yet analyzed sperm from the final reactive stage of spermatophores from L. stylirostris. Such observations would certainly contribute to our understanding of sperm maturation/capacitation in Litopenaeus.

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Based on the evidence presented, sperm maturation in Litopenaeus requires the completion of the FM region, which is synthesized differently in L. stylirostris as compared to L. vannamei and L. occidentalis. After mating, sperm cells from L. occidentalis are subjected to physiological changes, which improve their capacity to react against conspecific egg water. These findings will improve our understanding on fertilization in penaeoid shrimps, and serve as a basis towards defining new approaches for the in vitro fertilization of open thelycum shrimps.

Acknowledgements The authors wish to thank the staff of Estación de Biología Marina for their permanent cooperation.

Special thanks to Dr. Koenneman from the University of Veterinary Medicine Hannover, for his cooperation with TEM. This research was supported by Ley de Pesca from the Government of Costa Rica.

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