Development 109, 495-500 (1990) 495 Printed in Great Britain ©The Company of Biologists Limited 1990 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 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 mammals 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 sea urchin, 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 final concentrations 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 sperms showed 'searching' behaviour once in the im- grooves.