Development 109, 495-500 (1990)
Printed in Great Britain © T h e 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,
SO9 3TU, UK
Southampton,
* 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 conchonius, egg consists of 7-10 grooves and ridges, which
drain directly into a funnel-shaped vestibule, the only
point on the chorion through which sperm-egg contact
is achieved during fertilization. Results of time-lapse
video microscope study and computer-aided analysis of
sperm motility pattern in the micropylar region snowed
that the fertilizing sperm, usually the first to enter the
micropylar region, always travelled preferentially along
the grooves into the micropylar pit. Subsequently, 86 %
of sperm arriving the micropylar region within 30 s
travelled preferentially along the grooves into the immediate vicinity of the micropylar pit. The sperm
guidance role of the micropylar region was calculated to
enhance chances of egg penetration/fertilization by as
much as 99.7 % once sperm were within the micropylar
region, possibly in response to some form of chemoattractant(s) from the egg. Sperm agglutination postfertilization was also found to occur preferentially along
the grooves. Results of our in vitro fertilization experiments showed association between point of sperm entry
and blastodisc formation: the blastodisc formed directly
beneath the micropyle in all undisturbed eggs.
Introduction
squeezing through one funnel-shaped micropylar opening on the tough protective multilayered egg coat, the
chorion. In order to increase the chances of sperm-egg
contact, considerable reproductive effort and investment are made in the production of vast numbers of
gametes, which are shed synchronously in close proximity, often following an elaborate species-specific courtship behaviour in response to physiological and/or
environmental cues.
In his sperm redundancy theory, Cohen (1975, 1977)
argues that only a very small proportion of the large
numbers of sperm generally introduced into the female
tract are suitable for fertilization. This is understandable as a proportion of every sperm sample analyzed is
usually morphologically abnormal and has been shown
to be selectively eliminated by the human cervical
mucus (Barros et al. 1984). Assuming that Cohen's
hypothesis is also true in the teleost where fertilization
is external, it seems highly unlikely (even with the vast
numbers of sperm produced) that contact between,the
few 'suitable' sperm and eggs should be entirely random
in view of the added problems of sperm penetration
occurring only at a single point on the egg chorion.
Furthermore, the relatively short time within which
fertilization must occur after gametes are in contact
with water presents further constraint.
In this study, we present the first evidence of a sperm
A wide range of behaviourial, morphological and
physiological strategies have evolved to ensure successful sperm-egg contact and fertilization in different
species. In mammals where copulation and internal
fertilization bring gametes into close proximity, receptor-mediated gamete recognition and adhesion is
known to occur (see recent review by Wassarman,
1990). In addition to the large numbers of gametes
usually produced, sperm also undergo acrosome reactions in order to release sufficient enzyme needed to
breakdown the zona pellucida thereby making it possible for the fertilizing sperm to penetrate the egg at
virtually any point on the zona pellucida.
In many non-mammalian aquatic and marine organisms, the primitive and wasteful condition of external
fertilization prevails. It is most likely that gamete
recognition at the molecular level is also a common
feature in these organisms. In the sea urchin, for
example, sperm have been shown to possess a protein
molecule, bindin, which interacts with a glycoprotein
receptor in the egg vitelline envelope in a speciesspecific 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
Key words: micropyle, sperm guidance, fertilization,
teleost.
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 speciesspecific 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) atfinalconcentrations 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.
Sperm were stripped into capillary tubes. The undiluted
sperm retained normal motility for up to 2 h at room temperature but were always used within 10 min. The behaviour of
sperm during in vitro fertilization was studied in fifteen
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
suspension of sperm (20-25000 in 500^1 of tank water).
Materials for scanning electron microscopy (SEM) were
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 %
osmium tetroxide in cacodylate buffer. Dehydration was
gradual in graded alcohol over the range of 30-100%.
Samples were critical point dried at 31.5°C and then coated
with gold-palladium in an SEM-PREP sputter coater and
scanned 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 done
on digitised video images of sperm samples on a haemocytometer using a counting application on a VIDS IV computer
image analysis package (Analytical Measuring Systems, Cambridge England). Time-lapse video (TLV) recordings of
sperm motility patterns and early fertilization events were
made 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-9000
UEK). Sperm behaviour was analyzed visually and by computer-aided image analysis of traces of actual recordings. A
sperm was considered 'guided' if it travelled along a micropylar groove into the immediate vicinity of the micropylar pit
which is the only point from which egg penetration could be
achieved. On the other hand, sperm that transversed ridges
and grooves, or travelled almost entirely on ridges were
considered 'unguided' even if they eventually arrived at the
immediate 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 micropylar
region of a Rosy Barb egg. mp=micropylar pit, stippled
region=ridges. Details of sperm morphology are omitted
for reasons of clarity.
sperm are shown in Fig. 1. A mathematical model, based on
observed fertilization events, was developed demonstrating
the role of the micropyle in achieving successful fertilization
by increasing chances of sperm-egg contact.
Results
The Rosy Barb egg has a maximum chorion diameter of
approximately 100 microns and a large surface area of
about 31,428 square microns. The micropylar region
consists of a non-sticky sperm catchment area (SCA) of
approximately 20 microns in diameter and surface area
of 314 square microns. At the centre of the SCA is a
funnel-shaped vestibule with a maximum diameter of
4.5 microns at the micropylar entrance and less than a
micron at the bottom of the micropylar pit. The SCA is
a system of 7-10 micropylar grooves and ridges which
drain directly into the vestibule (Fig. 2). The Rosy Barb
sperm head has a diameter of 1.0-1.3 microns, giving a
maximum cross-sectional area of approximately one
square micron, maximum head and mid-section lengths
of 2 microns and an overall length of 13-17 microns,
respectively. The anterior end is rounded and lacks an
acrosome. Both the structure and size of the Rosy Barb
sperm are comparable to the published data (head
diameter of 1.8^m, fiagellum length of 30ftm) for the
closely related Zebra fish, Brachydanio rerio (Wolenski
and Hart, 1987).
Analysis of sperm movement in the vicinity of the
micropylar region of a total of over 60 unfertilized eggs
showed that the fertilizing sperm, usually thefirstsperm
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
grooves. Using our criteria of guidance described above
(see Materials and methods), 86% of sperm entering
the vicinity of the micropylar region within the first 30 s
of sperm-egg contact (mean=100) were guided along
the micropylar groove into the immediate vicinity of the
micropylar pit, giving a guidance efficiency (GE) of 0.86
(100% guidance as shown by the fertilizing sperm=GE
of 1.0).
However, the behaviour of sperm coming into the
micropylar region of water-hardened or already fertilized eggs showed a temporal reduction in guidance
efficiency, with only 50 % of sperm showing directional
guidance behaviour 80s postfertilization (Fig. 3). A
small proportion (20%) of such postfertilization guided
Al F«rt
30
ll
50
sperms showed 'searching' behaviour once in the immediate vicinity of the micropylar pit: sperm repeatedly
returned to the region of the micropylar pit after
moving away from it (Fig. 4). Pattern of sperm agglutination in the micropylar region showed highly significant differential distribution between micropylar ridges
and grooves: almost all the sperm agglutinated along
the grooves (Fig. 5). Interestingly, sperm were not
observed to agglutinate on any other area of the chorion
other than the micropylar region.
The egg showed considerable increase in volume as a
direct result of elevation of the fertilization membrane
following sperm penetration or water activation. Such
physical changes were also found to alter the physical
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 fertilized 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.
Discussion
SO
Time (leci) Po»t Ferllllraiion.
Fig. 3. Showing temporal changes in sperm guidance
efficiency. 100% Guidance Efficiency refers to the
fertilizing sperm, usually the first sperm to enter the
micropylar region.
There are various biochemical, biophysical, morphological and physiological factors that affect directionality in sperm motility and ensure sperm-egg contact in
almost all studied animal models, especially those
animals 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 contraction of, the reproductive tract (Hawk, 1983; Fujihara et
al. 1983), diffusible components from the egg (Rossignol 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 speciesspecific 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 predetermined point, the micropyle.
Heterogeneity, with respect to sperm reception and
penetration, of the egg surface has been demonstrated
in a wide range of invertebrate and vertebrate animal
models such as ascidians (Phallusis mammillata, Speksnijder et al. 1989), freshwater bivalves (Unio elongatulus, 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 contributes 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
Fig. 6. Differential Interference Contrast (DIC) microscope
picture showing the formation of a blastodisc directly
beneath the micropyle in a typical undisturbed Rosy Barb
egg fertilized in vitro.
al. 1989) and axis formation (Danilchik and Black,
1988) in animal development.
Based on theoretical calculations and assuming a
sperm head cross-sectional area the same size as the
micropylar pit, the probability (Prandom) of any single
sperm randomly migrating through the micropyle in the
absence of sperm guidance is given by the equation:
P
' _ area of micropylar pit _ n f W V V ^
~Z
Z
U.UUUlOZ
surface area of egg
With guidance, however, the probability of a sperm
migrating through the micropyle once it was within the
sperm catchment area would be:
^(random)
_ area of sperm catchment area _
-z
z
surface area of egg
Difference between the probability of 'guided' and
'unguided' sperm penetrating the micropylar pit P(deita)
is attributed to sperm guidance role of the micropylar
region 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 critical
30 s, and assuming that all sperm are equally capable of
fertilizing an egg, our results suggest that the sperm
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
sperm guidance, it would require an enormous increase
in sperm number in order to achieve the same level of
fertilization.
We suggest that some form of chemical attractant
emanates from the micropylar pit. The searching behaviour around the micropylar entrance may, therefore, result from traces of such a chemical attractant
even after the fertilization membrane has elevated and
the micropylar entrance blocked by non-fertilizing
sperms (Fig. 7). The presence of any chemical attrac-
499
Fig. 7. SEM micrograph of a fertilized Rosy Barb egg
showing the micropylar pit plugged by three late-arriving
sperm (arrowed). Bar=i/un.
tant was, however, not the main focus of the study and
therefore not investigated experimentally.
Our morphometric measurements of the micropylar
pit and sperm head agree with the earlier view (Hart
and Donovan, 1983) that polyspermy in the closely
related Zebra fish was prevented by the physical plugging of the micropyle by the fertilizing sperm head and
other non-fertilizing sperm arriving postfertilization;
thus the micropyle becomes the primary physical barrier against polyspermy. Furthermore, it is plausible
that the observed decrease in guidance efficiency and
physical morphological changes in the egg postfertilization have some contributory roles to preventing polyspermy.
The molecular and ultrastructural basis of sperm
guidance and motility along the micropylar grooves
remain unclear and analysis of the mechanism a logical
sequel to this study.
We would like to express our gratitude to Peter Thorogood
for making available research facilities and laboratory space
without which the work would not have been possible, and for
his guidance and encouragement in the preparation of the
manuscript. We also thank Norman Maclean, David Penman
and Gary Carvalho for their helpful discussions and suggestions at various stages of the work. Heather Caldwell helped
with photography. D.A. was supported by a Wellcome grant
to Peter Thorogood.
References
BARROS, C , VIGIL, P., HERRERA, E., ARGUELLO, B. AND WALKER,
R. (1984). Selection of morphologically abnormal sperm by
human cervical mucus. Arch. Androl. 12 Suppl, 95-107.
COHEN, J. (1975). Gamete redundancy - wastage or selection? In
Gamete 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 separate
mechanisms in Xenopus laevis. I. Independence in undisturbed
embryos. Devi Biol. 128, 58-64.
500
D. Amanze and A. Iyengar
FOCARELLI, R., RENIERI, T. AND ROSATI, F. (1988). Polanzed site of
sperm entrance in the egg of a freshwater bivalve, Unio
elongatulus. Devi. Biol. 127, 443-451.
FUJIHARA, N., KOGA, O. AND NISHIYAMA, H. (1983). Possible
participation of oviducal movement in spermatozoal transport in
domestic fowl. Poult. Sci. 62, 1101-1103.
HART, D. AND DONOVAN, M. (1983). The structure of the chorion
and 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 female
reproductive tract. J. Dairy Sci. 66, 2645-2660.
KATZ, D. F., DROBNIS, E. Z. AND OVERSTREET, J. W. (1989).
Factors regulating mammalian sperm migration through the
female reproductive tract and oocyte vestments. Gamete Res. 22,
443-469.
ROSSIGNOL, D. P. AND LENNARZ, W. J. (1983). The molecular basis
of 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 distinct
regions of the dimple and occurrence of a late stage of sperm
penetration. Devi Biol. 130, 524-535.
VACQUIER, V. D. AND MOY, G. (1977). Isolation of bindin: the
protein 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 electron
microscope studies of sperm incorporation into the Zebra fish
(Brachydanio) egg. J. exp. Zool. 243, 259-273.
(Accepted 2 April 1990)