Development 109, 495-500 (1990)Printed in Great Britain ©The Company of Biologists Limited 1990

495

The micropyle: a sperm guidance system in teleost fertilization

DIALA AMANZE* and ARATIIYENGAR

Department of Biology, University of Southampton, Medical and Biological Sciences Building, Bassett Crescent East, Southampton,SO9 3TU, UK

* Present address for all correspondence: Division of Oral Biology, Institute of Dental Surgery, Eastman Dental Hospital, 256, Gray's InnRoad, London, WC1X 8LD, UK

Summary

The micropylar region of the Rosy barb, Barbus concho-nius, egg consists of 7-10 grooves and ridges, whichdrain directly into a funnel-shaped vestibule, the onlypoint on the chorion through which sperm-egg contactis achieved during fertilization. Results of time-lapsevideo microscope study and computer-aided analysis ofsperm motility pattern in the micropylar region snowedthat the fertilizing sperm, usually the first to enter themicropylar region, always travelled preferentially alongthe grooves into the micropylar pit. Subsequently, 86 %of sperm arriving the micropylar region within 30 stravelled preferentially along the grooves into the im-mediate vicinity of the micropylar pit. The sperm

guidance role of the micropylar region was calculated toenhance chances of egg penetration/fertilization by asmuch as 99.7 % once sperm were within the micropylarregion, possibly in response to some form of chemo-attractant(s) from the egg. Sperm agglutination post-fertilization was also found to occur preferentially alongthe grooves. Results of our in vitro fertilization exper-iments showed association between point of sperm entryand blastodisc formation: the blastodisc formed directlybeneath the micropyle in all undisturbed eggs.

Key words: micropyle, sperm guidance, fertilization,teleost.

Introduction

A wide range of behaviourial, morphological andphysiological strategies have evolved to ensure success-ful sperm-egg contact and fertilization in differentspecies. In mammals where copulation and internalfertilization bring gametes into close proximity, recep-tor-mediated gamete recognition and adhesion isknown to occur (see recent review by Wassarman,1990). In addition to the large numbers of gametesusually produced, sperm also undergo acrosome reac-tions in order to release sufficient enzyme needed tobreakdown the zona pellucida thereby making it poss-ible for the fertilizing sperm to penetrate the egg atvirtually any point on the zona pellucida.

In many non-mammalian aquatic and marine organ-isms, the primitive and wasteful condition of externalfertilization prevails. It is most likely that gameterecognition at the molecular level is also a commonfeature in these organisms. In the sea urchin, forexample, sperm have been shown to possess a proteinmolecule, bindin, which interacts with a glycoproteinreceptor in the egg vitelline envelope in a species-specific manner (Vacquier and Moy, 1977).

The teleost sperm, unlike the mammalian sperm,lacks an acrosome. Sperm penetration is, therefore,entirely mechanical and dependent on the sperm

squeezing through one funnel-shaped micropylar open-ing on the tough protective multilayered egg coat, thechorion. In order to increase the chances of sperm-eggcontact, considerable reproductive effort and invest-ment are made in the production of vast numbers ofgametes, which are shed synchronously in close proxim-ity, often following an elaborate species-specific court-ship behaviour in response to physiological and/orenvironmental cues.

In his sperm redundancy theory, Cohen (1975, 1977)argues that only a very small proportion of the largenumbers of sperm generally introduced into the femaletract are suitable for fertilization. This is understand-able as a proportion of every sperm sample analyzed isusually morphologically abnormal and has been shownto be selectively eliminated by the human cervicalmucus (Barros et al. 1984). Assuming that Cohen'shypothesis is also true in the teleost where fertilizationis external, it seems highly unlikely (even with the vastnumbers of sperm produced) that contact between,thefew 'suitable' sperm and eggs should be entirely randomin view of the added problems of sperm penetrationoccurring only at a single point on the egg chorion.Furthermore, the relatively short time within whichfertilization must occur after gametes are in contactwith water presents further constraint.

In this study, we present the first evidence of a sperm

496 D. Amanze and A. Iyengar

guidance system in a teleost. We propose that thedevelopment of the micropyle increases the chances ofsuccessful fertilization once some form of species-specific chemoattraction brings the sperm into closeproximity with the egg.

Materials and methods

Rosy Barb, Barbus conchonius, adults were obtained fromlocal aquarists and sexes maintained separately under tropicalaquarium conditions on a 12 h light: 12 h dark cycle. Prior tocollecting gametes, 2 males and 3 females were put intodifferent compartments of a breeding trap approximately 16 hbefore the next light period.

The transparent barrier separating the sexes was removedabout 5min after the onset of the light period and adultsexhibiting prolonged mating behaviour were individuallyanaesthetized in ethyl m-aminobenzoate, MS222 (Sigma,England) at final concentrations of 80-100 ppm in tank wateruntil ventilation movement of the operculum stopped and thefish was completely immobilised in 3-7 min.

Fish were wiped dry of water and anaesthetics, held indamp tissue and stripped of gametes by the application ofgentle pressure to the lower abdomen. The stripping processusually took less than 30 s and stripped fish almost alwaysshowed complete recovery on return to aerated tank water.Sperm were stripped into capillary tubes. The undilutedsperm retained normal motility for up to 2 h at room tempera-ture but were always used within 10 min. The behaviour ofsperm during in vitro fertilization was studied in fifteenbatches of eggs (10-20 eggs per batch) which were strippeddirectly into specially designed microscope observationchambers where they were brought in contact with dilutedsuspension of sperm (20-25000 in 500^1 of tank water).

Materials for scanning electron microscopy (SEM) wereprepared according to standard procedures. Specimens werefixed for 24h in freshly prepared 0.1M sodium cacodylatecontaining 2% glutaraldehyde at 4°C, rinsed twice in 0.1Mcacodylate buffer at ph 7.2 and then postfixed for 2h in 1 %osmium tetroxide in cacodylate buffer. Dehydration wasgradual in graded alcohol over the range of 30-100%.Samples were critical point dried at 31.5°C and then coatedwith gold-palladium in an SEM-PREP sputter coater andscanned using a Hitachi (F800) scanning electron microscope.Still 35 mm photographs were taken with an Olympus camera(C-35AD-2) coupled to an Olympus photomicroscope (BHS/PM-10AD) using Kodak Technical Pan black and white film.

Sperm count (1.2bn per ml of undiluted sample) was doneon digitised video images of sperm samples on a haemocyt-ometer using a counting application on a VIDS IV computerimage analysis package (Analytical Measuring Systems, Cam-bridge England). Time-lapse video (TLV) recordings ofsperm motility patterns and early fertilization events weremade using a high-resolution charge coupled device (CCD)colour camera coupled to an Olympus photomicroscope(BHS/PM-10AD) and a VHS recorder (JVC model BR-9000UEK). Sperm behaviour was analyzed visually and by com-puter-aided image analysis of traces of actual recordings. Asperm was considered 'guided' if it travelled along a micropy-lar groove into the immediate vicinity of the micropylar pitwhich is the only point from which egg penetration could beachieved. On the other hand, sperm that transversed ridgesand grooves, or travelled almost entirely on ridges wereconsidered 'unguided' even if they eventually arrived at theimmediate vicinity of the micropylar pit. Sample trajectories,based on actual motility patterns of 'guided' and 'unguided'

Fig. 1. A diagrammatic illustration of trajectories of'guided' (g) and 'unguided' (u) sperm in the micropylarregion of a Rosy Barb egg. mp=micropylar pit, stippledregion=ridges. Details of sperm morphology are omittedfor reasons of clarity.

sperm are shown in Fig. 1. A mathematical model, based onobserved fertilization events, was developed demonstratingthe role of the micropyle in achieving successful fertilizationby increasing chances of sperm-egg contact.

Results

The Rosy Barb egg has a maximum chorion diameter ofapproximately 100 microns and a large surface area ofabout 31,428 square microns. The micropylar regionconsists of a non-sticky sperm catchment area (SCA) ofapproximately 20 microns in diameter and surface areaof 314 square microns. At the centre of the SCA is afunnel-shaped vestibule with a maximum diameter of4.5 microns at the micropylar entrance and less than amicron at the bottom of the micropylar pit. The SCA isa system of 7-10 micropylar grooves and ridges whichdrain directly into the vestibule (Fig. 2). The Rosy Barbsperm head has a diameter of 1.0-1.3 microns, giving amaximum cross-sectional area of approximately onesquare micron, maximum head and mid-section lengthsof 2 microns and an overall length of 13-17 microns,respectively. The anterior end is rounded and lacks anacrosome. Both the structure and size of the Rosy Barbsperm are comparable to the published data (headdiameter of 1.8^m, fiagellum length of 30ftm) for theclosely related Zebra fish, Brachydanio rerio (Wolenskiand Hart, 1987).

Analysis of sperm movement in the vicinity of themicropylar region of a total of over 60 unfertilized eggsshowed that the fertilizing sperm, usually the first sperm

Micropylar sperm guidance system 497

Fig. 2. SEM micrograph of themicropylar region of anunfertilized Rosy Barb eggshowing micropylar ridges andgrooves. Bar=4/jm.

to arrive, always travelled preferentially along thegrooves. Using our criteria of guidance described above(see Materials and methods), 86% of sperm enteringthe vicinity of the micropylar region within the first 30 sof sperm-egg contact (mean=100) were guided alongthe micropylar groove into the immediate vicinity of themicropylar pit, giving a guidance efficiency (GE) of 0.86(100% guidance as shown by the fertilizing sperm=GEof 1.0).

However, the behaviour of sperm coming into themicropylar region of water-hardened or already ferti-lized eggs showed a temporal reduction in guidanceefficiency, with only 50 % of sperm showing directionalguidance behaviour 80s postfertilization (Fig. 3). Asmall proportion (20%) of such postfertilization guided

l lAl F«rt 30 50 SO

Time (leci) Po»t Ferllllraiion.

Fig. 3. Showing temporal changes in sperm guidanceefficiency. 100% Guidance Efficiency refers to thefertilizing sperm, usually the first sperm to enter themicropylar region.

sperms showed 'searching' behaviour once in the im-mediate vicinity of the micropylar pit: sperm repeatedlyreturned to the region of the micropylar pit aftermoving away from it (Fig. 4). Pattern of sperm aggluti-nation in the micropylar region showed highly signifi-cant differential distribution between micropylar ridgesand grooves: almost all the sperm agglutinated alongthe grooves (Fig. 5). Interestingly, sperm were notobserved to agglutinate on any other area of the chorionother than the micropylar region.

The egg showed considerable increase in volume as adirect result of elevation of the fertilization membranefollowing sperm penetration or water activation. Suchphysical changes were also found to alter the physicalstructure of the micropylar region: the ridges andgrooves became less distinct as a result of the eggbecoming more rounded.

In our in vitro fertilization system where eggs werestripped directly onto fertilization chambers and ferti-lized in situ, preliminary observations consistentlyshowed association between the point of spermentrance and blastodisc formation: the blastodiscformed directly underneath the micropyle in all caseswhere eggs remained undisturbed following spermpenetration as typically represented in Fig. 6. However,if the eggs were disturbed then egg rotation within thechorion occurred and the relationship between themicropyle and blastodisc was lost.

Discussion

There are various biochemical, biophysical, morpho-logical and physiological factors that affect directiona-lity in sperm motility and ensure sperm-egg contact inalmost all studied animal models, especially thoseanimals where fertilization is internal. These include

498 D. Amanze and A. Iyengar

Fig. 4. Showing representative traces of sperm exhibiting'searching behaviour' around the micropylar region inpreviously fertilized eggs. Arrow indicates direction ofsperm motility. Outline of ridges and grooves is omitted inpanels B and C for clarity.

fluid current and ciliary movement within, and contrac-tion of, the reproductive tract (Hawk, 1983; Fujihara etal. 1983), diffusible components from the egg (Rossig-nol and Lennarz, 1983) and various other biophysicaland biochemical factors in the immediate environmentof the sperm (Katz et al. 1989). A significant proportionof these mechanisms are maternal contributions ofevolutionary significance in that they ensure species-specific fertilization. In this study, we have presentedthe first evidence of sperm guidance in an externallyfertilized system, and one where sperm entry into theegg is only achieved through a specialized and predeter-mined point, the micropyle.

Heterogeneity, with respect to sperm reception andpenetration, of the egg surface has been demonstratedin a wide range of invertebrate and vertebrate animalmodels such as ascidians (Phallusis mammillata, Speks-nijder et al. 1989), freshwater bivalves (Unio elongatu-lus, Focarelli el al. 1988), teleosts (Brachydanio,Wolenski and Hart, 1987) and anurans (Discoglossuspictus, Talevi and Campanella, 1988). Furthermore, ourresults, which are also consistent with results of theabove studies on point of sperm entry, showed acorrelation between point of sperm entry and formationof the blastodisc in undisturbed eggs. It would appearthat a specialized point of sperm entry is a retainedprimitive feature which, in addition to admitting thefertilising sperm and preventing polyspermy, also con-tributes to some major morphogenetic decisions such asdetermination of the animal hemisphere (Speksnijder et

Fig. 5. Differential InterferenceContrast (DIC) microscopepicture showing preferentialdistribution of agglutinatedsperm along the grooves withlittle or no sperm on the ridges.Sperm suspension wasintroduced from the top lefthand corner of the photograph.Note that the ridges havebecome less morphologicallydistinct following increase in eggvolume postfertilization.

Micropylar sperm guidance system 499

Fig. 6. Differential Interference Contrast (DIC) microscopepicture showing the formation of a blastodisc directlybeneath the micropyle in a typical undisturbed Rosy Barbegg fertilized in vitro.

al. 1989) and axis formation (Danilchik and Black,1988) in animal development.

Based on theoretical calculations and assuming asperm head cross-sectional area the same size as themicropylar pit, the probability (Prandom) of any singlesperm randomly migrating through the micropyle in theabsence of sperm guidance is given by the equation:

P ' _ area of micropylar pit _ n f W V V^^(random) ~Z Z U.UUUlOZ

surface area of egg

With guidance, however, the probability of a spermmigrating through the micropyle once it was within thesperm catchment area would be:

_ area of sperm catchment area _-z z

surface area of eggDifference between the probability of 'guided' and'unguided' sperm penetrating the micropylar pit P(deita)is attributed to sperm guidance role of the micropylarregion and given by the equation:

P(delto) = P(guided) ~~ P(random) = 0 .009959

Given the observed sperm guidance efficiency of 1.0(for the fertilizing sperm) to 0.86 within the first critical30 s, and assuming that all sperm are equally capable offertilizing an egg, our results suggest that the spermcatchment area enhances the chances of fertilization byas much as 99.7% once sperm were in the immediatevicinity of the micropylar region. In the absence ofsperm guidance, it would require an enormous increasein sperm number in order to achieve the same level offertilization.

We suggest that some form of chemical attractantemanates from the micropylar pit. The searching be-haviour around the micropylar entrance may, there-fore, result from traces of such a chemical attractanteven after the fertilization membrane has elevated andthe micropylar entrance blocked by non-fertilizingsperms (Fig. 7). The presence of any chemical attrac-

Fig. 7. SEM micrograph of a fertilized Rosy Barb eggshowing the micropylar pit plugged by three late-arrivingsperm (arrowed). Bar=i/un.

tant was, however, not the main focus of the study andtherefore not investigated experimentally.

Our morphometric measurements of the micropylarpit and sperm head agree with the earlier view (Hartand Donovan, 1983) that polyspermy in the closelyrelated Zebra fish was prevented by the physical plug-ging of the micropyle by the fertilizing sperm head andother non-fertilizing sperm arriving postfertilization;thus the micropyle becomes the primary physical bar-rier against polyspermy. Furthermore, it is plausiblethat the observed decrease in guidance efficiency andphysical morphological changes in the egg postfertiliz-ation have some contributory roles to preventing poly-spermy.

The molecular and ultrastructural basis of spermguidance and motility along the micropylar groovesremain unclear and analysis of the mechanism a logicalsequel to this study.

We would like to express our gratitude to Peter Thorogoodfor making available research facilities and laboratory spacewithout which the work would not have been possible, and forhis guidance and encouragement in the preparation of themanuscript. We also thank Norman Maclean, David Penmanand Gary Carvalho for their helpful discussions and sugges-tions at various stages of the work. Heather Caldwell helpedwith photography. D.A. was supported by a Wellcome grantto Peter Thorogood.

References

BARROS, C , VIGIL, P., HERRERA, E., ARGUELLO, B. AND WALKER,R. (1984). Selection of morphologically abnormal sperm byhuman cervical mucus. Arch. Androl. 12 Suppl, 95-107.

COHEN, J. (1975). Gamete redundancy - wastage or selection? InGamete Competition in Plants and Animals (ed. D. L. Mulcahy),pp. 99-112. Amsterdam, North Holland.

COHEN, J. (1977). Reproduction, pp. 356. London, Butterworth.DANILCHIK, W. V. AND BLACK, S. D. (1988). The first cleavage

plane and the embryonic axis are determined by separatemechanisms in Xenopus laevis. I. Independence in undisturbedembryos. Devi Biol. 128, 58-64.

500 D. Amanze and A. Iyengar

FOCARELLI, R., RENIERI, T. AND ROSATI, F. (1988). Polanzed site ofsperm entrance in the egg of a freshwater bivalve, Unioelongatulus. Devi. Biol. 127, 443-451.

FUJIHARA, N., KOGA, O. AND NISHIYAMA, H. (1983). Possible

participation of oviducal movement in spermatozoal transport indomestic fowl. Poult. Sci. 62, 1101-1103.

HART, D. AND DONOVAN, M. (1983). The structure of the chorionand site of sperm entry in the egg of Brachydanio. J. exp. Zool.227, 277-2%.

HAWK, H. W. (1983). Sperm survival and transport in the femalereproductive tract. J. Dairy Sci. 66, 2645-2660.

KATZ, D. F., DROBNIS, E. Z. AND OVERSTREET, J. W. (1989).

Factors regulating mammalian sperm migration through thefemale reproductive tract and oocyte vestments. Gamete Res. 22,443-469.

ROSSIGNOL, D. P. AND LENNARZ, W. J. (1983). The molecular basisof sperm-egg interaction in the sea urchin. Ciba. Found. Symp.98, 268-296.

SPEKSNUDER, J. E., JAFFE, L. F. AND SARDET, C. (1989). Polarity of

sperm entry in the ascidian egg. Devi Biol. 133, 180-184.TALEVI, R. AND CAMPANELLA, C. (1988). Fertilization in

Discoglossuspictus (Anura). I. Sperm-egg interactions in distinctregions of the dimple and occurrence of a late stage of spermpenetration. Devi Biol. 130, 524-535.

VACQUIER, V. D. AND MOY, G. (1977). Isolation of bindin: theprotein responsible for adhesion of sperm to sea urchin eggs.Proc. natn. Acad. Sci. USA. 74, 2456-2460.

WASSARMAN, P. M. (1990). Profile of a mammalian sperm receptor.Development 108, 1-17.

WOLENSKI, J. S. AND HART, D. (1987). Scanning electronmicroscope studies of sperm incorporation into the Zebra fish(Brachydanio) egg. J. exp. Zool. 243, 259-273.

(Accepted 2 April 1990)