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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
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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
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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
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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.
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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.
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(Accepted 2 April 1990)