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

The micropyle: a 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 Inn Road, London, WC1X 8LD, UK

Summary

The micropylar region of the Rosy barb, Barbus concho- guidance role of the micropylar region was calculated to nius, egg consists of 7-10 grooves and ridges, which enhance chances of egg penetration/fertilization by as drain directly into a funnel-shaped vestibule, the only much as 99.7 % once sperm were within the micropylar point on the chorion through which sperm-egg contact region, possibly in response to some form of chemo- is achieved during fertilization. Results of time-lapse attractant(s) from the egg. Sperm agglutination post- video microscope study and computer-aided analysis of fertilization was also found to occur preferentially along sperm motility pattern in the micropylar region snowed the grooves. Results of our in vitro fertilization exper- that the fertilizing sperm, usually the first to enter the iments showed association between point of sperm entry micropylar region, always travelled preferentially along and blastodisc formation: the blastodisc formed directly the grooves into the micropylar pit. Subsequently, 86 % beneath the micropyle in all undisturbed eggs. of sperm arriving the micropylar region within 30 s travelled preferentially along the grooves into the im- Key words: micropyle, sperm guidance, fertilization, mediate vicinity of the micropylar pit. The sperm teleost.

Introduction squeezing through one funnel-shaped micropylar open- ing on the tough protective multilayered egg coat, the A wide range of behaviourial, morphological and chorion. In order to increase the chances of sperm-egg physiological strategies have evolved to ensure success- contact, considerable reproductive effort and invest- ful sperm-egg contact and fertilization in different ment are made in the production of vast numbers of species. In where copulation and internal gametes, which are shed synchronously in close proxim- fertilization bring gametes into close proximity, recep- ity, often following an elaborate species-specific court- tor-mediated gamete recognition and adhesion is ship behaviour in response to physiological and/or known to occur (see recent review by Wassarman, environmental cues. 1990). In addition to the large numbers of gametes In his sperm redundancy theory, Cohen (1975, 1977) usually produced, sperm also undergo acrosome reac- argues that only a very small proportion of the large tions in order to release sufficient enzyme needed to numbers of sperm generally introduced into the female breakdown the zona pellucida thereby making it poss- tract are suitable for fertilization. This is understand- ible for the fertilizing sperm to penetrate the egg at able as a proportion of every sperm sample analyzed is virtually any point on the zona pellucida. usually morphologically abnormal and has been shown In many non-mammalian aquatic and marine organ- to be selectively eliminated by the human cervical isms, the primitive and wasteful condition of external mucus (Barros et al. 1984). Assuming that Cohen's fertilization prevails. It is most likely that gamete hypothesis is also true in the teleost where fertilization recognition at the molecular level is also a common is external, it seems highly unlikely (even with the vast feature in these organisms. In the , for numbers of sperm produced) that contact between,the example, sperm have been shown to possess a protein few 'suitable' sperm and eggs should be entirely random molecule, bindin, which interacts with a glycoprotein in view of the added problems of sperm penetration receptor in the egg vitelline envelope in a species- occurring only at a single point on the egg chorion. specific manner (Vacquier and Moy, 1977). Furthermore, the relatively short time within which The teleost sperm, unlike the mammalian sperm, fertilization must occur after gametes are in contact lacks an acrosome. Sperm penetration is, therefore, with water presents further constraint. entirely mechanical and dependent on the sperm 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 the development of the micropyle increases the chances of successful fertilization once some form of species- specific chemoattraction brings the sperm into close proximity with the egg.

Materials and methods

Rosy Barb, Barbus conchonius, adults were obtained from local aquarists and sexes maintained separately under tropical aquarium conditions on a 12 h light: 12 h dark cycle. Prior to collecting gametes, 2 males and 3 females were put into different compartments of a breeding trap approximately 16 h before the next light period. The transparent barrier separating the sexes was removed about 5min after the onset of the light period and adults exhibiting prolonged mating behaviour were individually anaesthetized in ethyl m-aminobenzoate, MS222 (Sigma, England) at finalconcentration s of 80-100 ppm in tank water until ventilation movement of the operculum stopped and the fish was completely immobilised in 3-7 min. Fish were wiped dry of water and anaesthetics, held in damp tissue and stripped of gametes by the application of gentle pressure to the lower abdomen. The stripping process usually took less than 30 s and stripped fish almost always showed complete recovery on return to aerated tank water. Fig. 1. A diagrammatic illustration of trajectories of Sperm were stripped into capillary tubes. The undiluted 'guided' (g) and 'unguided' (u) sperm in the micropylar sperm retained normal motility for up to 2 h at room tempera- region of a Rosy Barb egg. mp=micropylar pit, stippled ture but were always used within 10 min. The behaviour of region=ridges. Details of sperm morphology are omitted sperm during in vitro fertilization was studied in fifteen for reasons of clarity. batches of eggs (10-20 eggs per batch) which were stripped directly into specially designed microscope observation chambers where they were brought in contact with diluted sperm are shown in Fig. 1. A mathematical model, based on suspension of sperm (20-25000 in 500^1 of tank water). observed fertilization events, was developed demonstrating the role of the micropyle in achieving successful fertilization Materials for scanning electron microscopy (SEM) were by increasing chances of sperm-egg contact. prepared according to standard procedures. Specimens were fixed for 24h in freshly prepared 0.1M sodium cacodylate containing 2% glutaraldehyde at 4°C, rinsed twice in 0.1M cacodylate buffer at ph 7.2 and then postfixed for 2h in 1 % Results osmium tetroxide in cacodylate buffer. Dehydration was gradual in graded alcohol over the range of 30-100%. The Rosy Barb egg has a maximum chorion diameter of Samples were critical point dried at 31.5°C and then coated approximately 100 microns and a large surface area of with gold-palladium in an SEM-PREP sputter coater and about 31,428 square microns. The micropylar region scanned using a Hitachi (F800) scanning electron microscope. consists of a non-sticky sperm catchment area (SCA) of Still 35 mm photographs were taken with an Olympus camera approximately 20 microns in diameter and surface area (C-35AD-2) coupled to an Olympus photomicroscope (BHS/ of 314 square microns. At the centre of the SCA is a PM-10AD) using Kodak Technical Pan black and white film. funnel-shaped vestibule with a maximum diameter of Sperm count (1.2bn per ml of undiluted sample) was done 4.5 microns at the micropylar entrance and less than a on digitised video images of sperm samples on a haemocyt- micron at the bottom of the micropylar pit. The SCA is ometer using a counting application on a VIDS IV computer a system of 7-10 micropylar grooves and ridges which image analysis package (Analytical Measuring Systems, Cam- bridge England). Time-lapse video (TLV) recordings of drain directly into the vestibule (Fig. 2). The Rosy Barb sperm motility patterns and early fertilization events were sperm head has a diameter of 1.0-1.3 microns, giving a made using a high-resolution charge coupled device (CCD) maximum cross-sectional area of approximately one colour camera coupled to an Olympus photomicroscope square micron, maximum head and mid-section lengths (BHS/PM-10AD) and a VHS recorder (JVC model BR-9000 of 2 microns and an overall length of 13-17 microns, UEK). Sperm behaviour was analyzed visually and by com- respectively. The anterior end is rounded and lacks an puter-aided image analysis of traces of actual recordings. A acrosome. Both the structure and size of the Rosy Barb sperm was considered 'guided' if it travelled along a micropy- sperm are comparable to the published data (head lar groove into the immediate vicinity of the micropylar pit diameter of 1.8^m, fiagellum length of 30ftm) for the which is the only point from which egg penetration could be closely related Zebra fish, Brachydanio rerio (Wolenski achieved. On the other hand, sperm that transversed ridges and grooves, or travelled almost entirely on ridges were and Hart, 1987). considered 'unguided' even if they eventually arrived at the Analysis of sperm movement in the vicinity of the immediate vicinity of the micropylar pit. Sample trajectories, micropylar region of a total of over 60 unfertilized eggs based on actual motility patterns of 'guided' and 'unguided' showed that the fertilizing sperm, usually the first sperm Micropylar sperm guidance system 497

Fig. 2. SEM micrograph of the micropylar region of an unfertilized Rosy Barb egg showing micropylar ridges and grooves. Bar=4/jm. to arrive, always travelled preferentially along the showed 'searching' behaviour once in the im- grooves. Using our criteria of guidance described above mediate vicinity of the micropylar pit: sperm repeatedly (see Materials and methods), 86% of sperm entering returned to the region of the micropylar pit after the vicinity of the micropylar region within the first 30 s moving away from it (Fig. 4). Pattern of sperm aggluti- of sperm-egg contact (mean=100) were guided along nation in the micropylar region showed highly signifi- the micropylar groove into the immediate vicinity of the cant differential distribution between micropylar ridges micropylar pit, giving a guidance efficiency (GE) of 0.86 and grooves: almost all the sperm agglutinated along (100% guidance as shown by the fertilizing sperm=GE the grooves (Fig. 5). Interestingly, sperm were not of 1.0). observed to agglutinate on any other area of the chorion However, the behaviour of sperm coming into the other than the micropylar region. micropylar region of water-hardened or already ferti- The egg showed considerable increase in volume as a lized eggs showed a temporal reduction in guidance direct result of elevation of the fertilization membrane efficiency, with only 50 % of sperm showing directional following sperm penetration or water activation. Such guidance behaviour 80s postfertilization (Fig. 3). A physical changes were also found to alter the physical small proportion (20%) of such postfertilization guided structure of the micropylar region: the ridges and grooves became less distinct as a result of the egg becoming more rounded. In our in vitro fertilization system where eggs were stripped directly onto fertilization chambers and ferti- lized in situ, preliminary observations consistently showed association between the point of sperm entrance and blastodisc formation: the blastodisc formed directly underneath the micropyle in all cases where eggs remained undisturbed following sperm penetration as typically represented in Fig. 6. However, if the eggs were disturbed then egg rotation within the chorion occurred and the relationship between the micropyle and blastodisc was lost.

ll Discussion Al F«rt 30 50 SO Time (leci) Po»t Ferllllraiion. There are various biochemical, biophysical, morpho- Fig. 3. Showing temporal changes in sperm guidance logical and physiological factors that affect directiona- efficiency. 100% Guidance Efficiency refers to the lity in sperm motility and ensure sperm-egg contact in fertilizing sperm, usually the first sperm to enter the almost all studied models, especially those micropylar region. 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 in previously fertilized eggs. Arrow indicates direction of sperm motility. Outline of ridges and grooves is omitted in panels B and C for clarity.

fluid current and ciliary movement within, and contrac- tion of, the reproductive tract (Hawk, 1983; Fujihara et al. 1983), diffusible components from the egg (Rossig- nol and Lennarz, 1983) and various other biophysical and biochemical factors in the immediate environment of the sperm (Katz et al. 1989). A significant proportion of these mechanisms are maternal contributions of evolutionary significance in that they ensure species- specific fertilization. In this study, we have presented the first evidence of sperm guidance in an externally fertilized system, and one where sperm entry into the egg is only achieved through a specialized and predeter- mined point, the micropyle. Heterogeneity, with respect to sperm reception and penetration, of the egg surface has been demonstrated in a wide range of and vertebrate animal models 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 (Discoglossus pictus, Talevi and Campanella, 1988). Furthermore, our results, which are also consistent with results of the above studies on point of sperm entry, showed a correlation between point of sperm entry and formation of the blastodisc in undisturbed eggs. It would appear that a specialized point of sperm entry is a retained primitive feature which, in addition to admitting the fertilising sperm and preventing polyspermy, also con- tributes to some major morphogenetic decisions such as determination of the animal hemisphere (Speksnijder et

Fig. 5. Differential Interference Contrast (DIC) microscope picture showing preferential distribution of agglutinated sperm along the grooves with little or no sperm on the ridges. Sperm suspension was introduced from the top left hand corner of the photograph. Note that the ridges have become less morphologically distinct following increase in egg volume postfertilization. Micropylar sperm guidance system 499

Fig. 6. Differential Interference Contrast (DIC) microscope Fig. 7. SEM micrograph of a fertilized Rosy Barb egg picture showing the formation of a blastodisc directly showing the micropylar pit plugged by three late-arriving beneath the micropyle in a typical undisturbed Rosy Barb sperm (arrowed). Bar=i/un. egg fertilized in vitro. tant was, however, not the main focus of the study and al. 1989) and axis formation (Danilchik and Black, therefore not investigated experimentally. 1988) in animal development. Our morphometric measurements of the micropylar Based on theoretical calculations and assuming a pit and sperm head agree with the earlier view (Hart sperm head cross-sectional area the same size as the and Donovan, 1983) that polyspermy in the closely micropylar pit, the probability (Prandom) of any single related Zebra fish was prevented by the physical plug- sperm randomly migrating through the micropyle in the ging of the micropyle by the fertilizing sperm head and absence of sperm guidance is given by the equation: other non-fertilizing sperm arriving postfertilization; thus the micropyle becomes the primary physical bar- P '_ area of micropylar pit _ n fWVV^ ^(random) ~Z Z U.UUUlOZ rier against polyspermy. Furthermore, it is plausible surface area of egg that the observed decrease in guidance efficiency and With guidance, however, the probability of a sperm physical morphological changes in the egg postfertiliz- migrating through the micropyle once it was within the ation have some contributory roles to preventing poly- sperm catchment area would be: spermy. _ area of sperm catchment area _ The molecular and ultrastructural basis of sperm -z z guidance and motility along the micropylar grooves surface area of egg remain unclear and analysis of the mechanism a logical Difference between the probability of 'guided' and sequel to this study. 'unguided' sperm penetrating the micropylar pit P(deita) is attributed to sperm guidance role of the micropylar We would like to express our gratitude to Peter Thorogood for making available research facilities and laboratory space region and given by the equation: without which the work would not have been possible, and for P(delto) = P(guided) ~~ P(random) = 0.009959 his guidance and encouragement in the preparation of the Given the observed sperm guidance efficiency of 1.0 manuscript. We also thank Norman Maclean, David Penman (for the fertilizing sperm) to 0.86 within the first critical and Gary Carvalho for their helpful discussions and sugges- tions at various stages of the work. Heather Caldwell helped 30 s, and assuming that all sperm are equally capable of with photography. D.A. was supported by a Wellcome grant fertilizing an egg, our results suggest that the sperm to Peter Thorogood. catchment area enhances the chances of fertilization by as much as 99.7% once sperm were in the immediate vicinity of the micropylar region. In the absence of References sperm guidance, it would require an enormous increase in sperm number in order to achieve the same level of BARROS, C, VIGIL, P., HERRERA, E., ARGUELLO, B. AND WALKER, fertilization. R. (1984). Selection of morphologically abnormal sperm by human cervical mucus. Arch. Androl. 12 Suppl, 95-107. We suggest that some form of chemical attractant COHEN, J. (1975). Gamete redundancy - wastage or selection? In emanates from the micropylar pit. The searching be- Gamete Competition in Plants and Animals (ed. D. L. Mulcahy), haviour around the micropylar entrance may, there- pp. 99-112. Amsterdam, North Holland. fore, result from traces of such a chemical attractant COHEN, J. (1977). Reproduction, pp. 356. London, Butterworth. even after the fertilization membrane has elevated and DANILCHIK, W. V. AND BLACK, S. D. (1988). The first cleavage plane and the embryonic axis are determined by separate the micropylar entrance blocked by non-fertilizing mechanisms in Xenopus laevis. I. Independence in undisturbed sperms (Fig. 7). The presence of any chemical attrac- embryos. Devi Biol. 128, 58-64. 500 D. Amanze and A. Iyengar

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