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

Aquaculture 270 (

Sperm maturation and capacitation in the open thelycum shrimpLitopenaeus (Crustacea: Decapoda: Penaeoidea)

Jorge Alfaro a,⁎, Karol Ulate a, Maribelle Vargas b

a Estación de Biología Marina, Escuela de Ciencias Biológicas, Universidad Nacional, Puntarenas, Costa Ricab 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 nucleusand the hemispherical cap, develops differently in these three closely related species. In L. vannamei, the FM is synthesized in themale reproductive system, but seems to complete its formation after mating. In L. stylirostris, the FM region was not present inspermatophores collected from males or in sperm from the thelycum. In L. occidentalis, the FM region is fully developed in malesperm. It is suggested that completion of the FM is required for acrosome maturation, and the process continues after mating insome species of Litopenaeus. In vitro induction of the acrosome reaction in sperm from males and females of L. occidentalisdemonstrated for the first time that reactivity is significantly superior in sperm cells that have been attached to the open thelycumfor some hours, as compared to sperm in males (prior to transfer). This finding suggests that matured sperm cells of L. occidentalisbecome 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 ofthe cell head, and it consists of three elements in theclosed thelycum species Sicyonia ingentis: membranepouches, anterior granule, and spike (Clark et al., 1981).The acrosome contains enzymes that function in bothexocytosis and sperm penetration of the eggs duringfertilization (Gwo, 2000); therefore, sperm undergo anacrosome reaction induced by egg factors.

The primary binding between the sperm spike andthe 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 acrosomereaction in vivo (Clark et al., 1981). Cortical rodsemerge within 60 s after seawater contact in Litope-naeus (Rojas and Alfaro, 2007); therefore, in vivoacrosome reaction is a fast event. The reaction includesthe release of acrosomal contents to aid in penetratingegg investment coats, exposure of inner sperm surfacesfor binding to the egg, and in some cases the formationof an acrosome filament to facilitate sperm entry (Griffinand Clark, 1990; Lindsay and Clark, 1992). Griffin et al.(1987) developed a technique to induce the acrosomereaction in vitro: the egg water technique. Egg water(EW) is seawater collected at the time of spawning,containing the egg-derived inducers of the spermacrosome reaction.

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In S. ingentis, sperm cells mature in the female'sthelycum, where further ultrastructural developmentwithin the cells takes place (Clark et al., 1984; Shigekawaand Clark, 1986). It has also been demonstrated for Tra-chypenaeus byrdi andXiphopenaeus riveti (closed thelycashrimps) that only sperm cells from females, but not frommales, react against conspecific egg water, indicating thatfurther maturation or capacitation is required in seminalreceptacles (Alfaro et al., 2003).

Sperm of the closed thelyca shrimps Farfantepe-naeus aztecus and S. ingentis undergo a capacitationprocess, as in mammalians, where matured sperm cellsexperience physiological changes to facilitate theirreactivity. Sperm must be transferred and stored withinthe seminal receptacle of a female for a period of timebefore they achieve the ability to fertilize (Clark et al.,1984; Clark and Griffin, 1988, 1993; Griffin and Clark,1990). Uncapacitated sperm of S. ingentis haveextremely low Ca2+ levels, which increase duringcapacitation (Lindsay and Clark, 1992).

The general ultrastructure of Litopenaeus sperm hasbeen described by Dougherty and Dougherty (1989),who reported on the pathology of melanized spermato-phores of pond-cultured Litopenaeus vannamei. Spermcells consist of a spike (S) and hemispherical cap ofmoderate electron density (C), a nucleus (N) containinga network of chromatin threads, a filamentous mesh-work (FM) between the nucleus and hemispherical cap,and a hemispherical rim of cytoplasmic particles (CP).In L. stylirostris, spike elongation takes place in thedescending medial vas deferens of the reproductivesystem (Alfaro, 1994). The Litopenaeus acrosomeseems to be formed by sections S, C, and FM.

In open thelycum shrimps, it has been assumed thatspermatophores within terminal ampoules contain fullymatured and capacitated sperm, but no scientific observa-tions have been published to improve our knowledge onthis crucial topic. It has been stated that sperm of openthelyca penaeoideans do not appear to undergo capacita-tion after transfer to the female (Clark and Griffin, 1993),but recently, it has been proposed that final spermmaturation on the external surface of the thelycum maybe required for fertilization in open thelyca shrimps (Alfaroet al., 2003). This hypothesis was based on experimentalobservations of in vitro fertilizations (Alfaro et al., 1993;Misamore and Browdy, 1997), and the low spermactivation obtained with the EW technique applied tosperm removed from spermatophores (Alfaro et al., 2003).

This research was designed to compare the ultrastruc-ture of sperm cells from male's spermatophores andfemale's thelyca of the three Litopenaeus species from thePacific coast of the Americas: L. vannamei, L. stylirostris

and L. occidentalis. In addition, in vitro induction ofacrosome reaction was evaluated comparing spermresponse before and after mating in L. occidentalis.

2. Materials and methods

2.1. Animals

Specimens of L. vannamei were selected beforeharvesting at 90 days of commercial semi-intensive culturein earthen ponds at Asociación de Camaronicultores de laPenínsula de Nicoya, Costa Rica. Individuals weremaintained at Estación de Biología Marina (EBM) in ashaded external tank (18 m2; N=250 animals). Waterexchange was kept at 48% per day, using new waterpretreated by high pressure silica sand filtration andsedimentation. Animals were fed a commercial dry food at3% bodyweight (b.w.) daily and fresh frozen sardine at 1%b.w. Mature animals of L. stylirostris and L. occidentaliswere collected from Golfo de Nicoya.

2.2. Sperm ultrastructure

For transmission electron microscopy (TEM), sper-matophores were collected by manual ejaculation fromwild specimens of L. stylirostris and L. occidentalis,and cultured specimens of L. vannamei held undercontrolled reproduction conditions (water temper-ature=28 °C) at EBM, as previously described (Alfaroet al., 2004). Additionally, naturally inseminatedfemales of L. stylirostris and L. occidentalis from thewild, and of L. vannamei from maturation tanks wereisolated in spawning tanks for a few hours beforeremoving the attached sperm mass.

Sperm masses were expelled from male spermato-phores before fixation. Spermatophores from thelycawere in an advanced stage of reaction from the time ofmating judging by its distinctive compact shape. In openthelycum shrimps, females retain sperm cells for a fewhours (6–7 h) until spawning. Compact spermatophoreswere fixed as a whole. The fixative used was a solutionreported by Ro et al. (1990) for marine shrimp re-productive systems, which consists of paraformalde-hyde (2.0%), glutaraldehyde (2.5%), and sucrose (5.0%)in 0.1 M sodium cacodylate buffer at pH 7.4. While infixative, samples were observed under light microscopyto identify sperm masses and eliminate unnecessarymaterial. After repeated washing in fresh 0.1 M ca-codylate buffer, samples were postfixed in 1% OsO4 in0.1 M cacodylate buffer, pH 7.4, at room temperature for2 h. Samples were then rinsed with cacodylate bufferand distilled water several times, dehydrated in an

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. Thefollowing regions are named based onDougherty andDougherty (1989): S = spike, C = hemispherical cap, N = nucleus containing a network of chromatinthreads, FM = filamentous meshwork between the nucleus and the hemispherical cap, CP = cytoplasmic particles forming a hemispherical rim.

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ascending series of ethanol, then embedded, viapropylene oxide, in Spurr's resin. Ultrathin sectionswere cut with an ultramicrotome, picked up on coppergrids, stained sequentially with uranyl acetate and leadcitrate, and examined under a transmission electronmicroscope. Some samples were processed in CostaRica (Unidad de Microscopia Electrónica, Universidadde Costa Rica) and others in Germany (Institute forAnimal Ecology and Cell Biology, University ofVeterinary Medicine Hannover).

2.3. In vitro sperm activation

A modified EW technique was used to induce theacrosome reaction in sperm of L. occidentalis. A

positive reaction under light microscopy is characterizedby the loss of the spike followed by eversion of cellcontents, which leaves a distinctive rounded mark at theacrosome region. The technique developed by Griffinet al. (1987) was modified as reported by Alfaro et al.(2003). Basically, at the time of spawning, females werecollected and allowed to spawn over 100 ml glassbeakers containing filtered (1 μm) and UV-treatednatural 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 homogenizingspermatophores from males and compacted spermmasses from females in 3 ml of NSW. Two drops ofsperm suspension were mixed with six drops of EW in a

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

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vial. As negative controls, sperm suspensions weremixed with NSW. The rate of activation was measuredover a microscope slide, cataloging the number ofreacted and nonreacted cells for 100 sperm cells (threereplications per trial).

Three experiments were conducted. Experiment 1evaluated EW batch 1 in sperm masses removed fromthree females of L. occidentalis; sperm activation wasmonitored at 0, 15, 30, and 45 min from EW exposure.Experiment 2 evaluated EW batch 2 in sperm from amale and a female for the same time interval. These twoexperiments were intended to define the optimum timeresponse for in vitro sperm activation, since it has beenobserved that the reaction occurs slowly under in vitroconditions. Experiment 3 evaluated sperm activation at45 min from EW exposure, using a mixed-effect blockdesign (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 = thelycumsperm in NSW (control-thelycum), and T4 = thelycumsperm in EW. Blocks 1 and 2 used two and threesamples per treatment, respectively.

2.4. Statistics

Percentages of sperm reactivity were transformedusing arcsine of squared root to make the varianceindependent of the mean (Ott, 1984). The block designwas analyzed using the General Linear Model of theMinitab 13.2 software program. Tukey simultaneoustests were applied for pairwise comparisons amongtreatments. Alpha level was set at 0.05. Untransformeddata are presented as mean±standard deviation (s.d.).

3. Results

Fig. 1 (micrographs 1 and 2) shows the ultrastructureof sperm removed from spermatophores of males ofL. vannamei (body diameter=3–4 μm). Micrographs 3and 4 (Fig. 1) were taken from a sperm mass attached tothe thelycum of a female L. vannamei. The generalmorphology is similar before and after mating; spermcells show the different regions previously described byDougherty and Dougherty (1989), but the degree ofdevelopment of the FM region seems to be moreadvanced in sperm cells from the thelycum. Micrograph4 shows a detail of the FM region from a thelycumsperm cell, and filamentous elements are still prolifer-ating into the empty anterior space.

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

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

Fig. 3. Electron micrographs of sperm from male spermatophores(plate 8) and thelycum (plate 9) of L. occidentalis.

Table 1Induced acrosome reaction in Litopenaeus occidentalis sperm beforeand after insemination

Treatments Blocks a Reactivity(%)±s.d.

Tukeytest b

(Pb0.05)EWbatch 3

EWbatch 4

n n

T1: control-male 2 3 13.0±6.5 aT2: male-EW 2 3 17.0±3.6 aT3: control-thelycum 2 3 12.6±5.9 aT4: thelycum-EW 2 3 33.6±2.5 ba 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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Fig. 2, micrograph 5) show no development of the FMregion, however, the space where it should developappears empty; every sperm cell observed presented thispattern. Another distinctive feature observed betweenthese two species was the thickness of the cytoplasmicparticles rim (CP), which is remarkably thicker for

L. stylirostris. Sperm removed from intact spermatophorestaken from females' thelycum (early stage of spermato-phore reaction; Fig. 2, micrographs 6 and 7), show somedegree of development of the FM region. However, theregion was still not as developed as in L. vannamei.

The morphology of L. occidentalis sperm cells (bodydiameter=4 μm) is also similar to the other two species,but the FM region seems to be fully developed in male'sspermatophores (Fig. 3, micrograph 8). The morphologyof 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 inL. occidentalis with the EW technique (Fig. 4) wasapplied to three sperm masses from thelyca (experiment1). Data clearly indicate a positive response of spermcells to conspecific EW. Sperm cells from thelyca showa pattern characterized by a progressive increase ofreacted cells as time advances until reaching a finalvalue of 58.8%±5.4 after 45 min exposure time. On thecontrary, sperm cells from a male show no increase in

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reactivity after 45 min when exposed to EW (experi-ment 2). Reactivity measured in experiment 2 for spermcells from thelyca was lower (25.3%) than the reactivityobtained 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 incontrol-thelycum (T3), control-male (T1), and male-EW (T2, Pb0.05).

4. Discussion

The transmission electron microscopy of sperm frommales' spermatophores and females' thelyca of the threespecies evaluated reveals a similar morphology amongspecies, but a different pattern in the process ofmaturation. Our observations indicate that the regionbetween the nucleus and the hemispherical cap of thesperm cell accumulates filamentous materials (FM),which seems to involve an active synthesis before andafter mating, depending on the species.

Our observations on L. vannamei, as well as a previousreport by Dougherty and Dougherty (1989), clearlyindicate that this species initiates the synthesis of theFM in the male reproductive system; however, it seemsthat more material is still accumulating after mating.On the contrary, the male reproductive system ofL. stylirostris does not appear to activate the synthesisof the FM. It seems that after transfer of spermatophores tothe thelycum, the synthesis of the FM is activated;however, this statement requires further confirmation. Wehave not been capable of observing sperm ofL. stylirostris with a similar degree of development ofthe FM as in L. vannamei and L. occidentalis, indicatingthat the process is slow or that the amount or compositionof FM is different between these three closely relatedpenaeoid species. It seems unlikely that such a largecellular region remains empty (in L. stylirostris), when itis occupied by a filamentous meshwork in L. vannameiand L. occidentalis.

The FM region of L. occidentalis seems to be fullydeveloped before mating; this was the only speciesshowing a compartment fully occupied by this filamentousmeshwork. However, a previous report has shown thateggs of this species spawn in vitro into a sperm suspensiononly generate massive primary bindings between vitellineenvelopes and sperm spikes (Alfaro et al., 1993; Rojas andAlfaro, 2007), without detectable acrosome reactions.

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

(Clark et al., 1980). In sperm cells from the distal vasdeferens of the male reproductive system of T. byrdi(closed thelycum), the filamentous material is not pre-sent (Alfaro, 1994). It is proposed that the FM region isan essential part of the acrosome, that continues itsformation after mating in some species of Litopenaeus.

For the first time, data are presented from an openthelycum shrimp (L. occidentalis) that confirm thelycumsperm react to EW at higher rates than male sperm.Reactivity rates at 45 min from EW exposure for thelycasperm were 58.8%±5.4, 25.3%, and 33.6%±2.5 forexperiments 1, 2, and 3, respectively. The variability withinreplicates for experiments 1 and 3 was very low; however,between experiments rates were variable, suggesting thateach EWbatch induced a different homogeneous reactivitylevel. Reactivity rates for male sperm were 6.7% and17.0%±3.6 for experiments 2 and 3, respectively. Aprevious study alsomeasured a low percentage of reactivityforL. occidentalismale sperm cells incubatedwith differentconspecific 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 inL. vannamei (37.4%±18.5). This value indicates thatsome degree of capacitation was acquired in the malereproductive system for this species, but the responsewas highly variable. This pattern is in accordance withour ultrastructural observations, and indicates that eachmale may show a different degree of sperm maturation/capacitation before mating. This hypothesis requiresfurther confirmation by analyzing the reactivity ofthelycum sperm.

From ultrastructural observations no differenceswere detected between male and thelycum sperm ofL. occidentalis, but in vitro reactivity against egg watersuggests some physiological changes took place withinsperm cells after mating. It seems that sperm cells fromL. occidentalis become competent in the femalethelycum. Sperm capacitation in other species of Lito-penaeus must be investigated to get a better understand-ing of this process in open thelycum shrimps.

Completion of other cellular regions may still berequired for sperm maturation after mating; however,under our sampling protocol we did not detect thedevelopment of any other structural changes during thefew hours after natural spermatophore transfer to thethelycum. Spermatophores experience a gradual reaction assoon as they are transferred to the thelycum, leading to therelease of the sperm mass and its attachment around thegonopores. We have not yet analyzed sperm from the finalreactive stage of spermatophores from L. stylirostris. Suchobservations would certainly contribute to our understand-ing of sperm maturation/capacitation in Litopenaeus.

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Based on the evidence presented, sperm maturationin Litopenaeus requires the completion of the FMregion, which is synthesized differently in L. stylirostrisas compared to L. vannamei and L. occidentalis. Aftermating, sperm cells from L. occidentalis are subjected tophysiological changes, which improve their capacity toreact against conspecific egg water. These findings willimprove our understanding on fertilization in penaeoidshrimps, and serve as a basis towards defining newapproaches for the in vitro fertilization of open thelycumshrimps.

Acknowledgements

The authors wish to thank the staff of Estación deBiología Marina for their permanent cooperation.Special thanks to Dr. Koenneman from the Universityof Veterinary Medicine Hannover, for his cooperationwith TEM. This research was supported by Ley de Pescafrom the Government of Costa Rica.

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