AMER. ZOOL., 15:523-551 (1975).
The Functional Anatomy of the Echinoderm Spermatozoon and its Interaction
with the Egg at Fertilization
ROBERT G. SUMMERS, BONNIE L. HYLANDER,
Department of Zoology, University of Maine, Orono, Maine 04473
LAURA H. COLWIN AND ARTHUR L. COLWIN
University of Miami, Rosensteil School ofAtmospheric and Marine Science, Miami, Florida
33149 and Marine Biological Laboratory, Woods Hole, Massachusetts 02543
SYNOPSIS. The ultrastructure of spermatozoa representing four echinoderm classes is presented. Sperm of these four classes are strikingly similar in their morphology, particularly
with regard to the acrosomal region. This region consists of two major components, an
acrosomal vesicle with a complete bounding membrane and the surrounding periacrosomal
material. The events of the acrosomal reaction in an echinoid and a holothurian are
described in detail and the functional role of the acrosomal region during fertilization is
presented. The role of extracellular coats in the establishment of gamete specificity is also
discussed with reference to echinoids. The later events of fertilization in echinoids, including sperm incorporation, pronuclear development, and pronuclear fusion are reviewed.
bined with the maternal genome, will constitute a diploid zygote. In order to accomplish the delivery of the paternal genetic material to the vicinity of the ovum, motility is generally a requisite. This is the function of the mitochondrial region or midpiece
and flagellum. The mitochondrial region is
located posterior to the nucleus and consists of a mitochondrion, macromolecular
reserves which provide the energy for
flagellar movement, and two centrioles
(one of which serves as the origin for the
SPERM MORPHOLOGY
flagellum). The anterior acrosomal region
enables the spermatozoon to gain access to
The spermatozoon is a uniquely the surface of and to establish contact with
specialized cell, for its differentiation and the ovum.
morphology most intimately reflect its
Two morphological types of echinoderm
functions during fertilization. The three
can be distinguished with the
spermatozoa
structural regions which comprise the
spermatozoon carry out these functions. light microscope based on The shape of the
Paternal hereditary material comprises the sperm head (see Dan, 1956). An elongate,
nuclear region or sperm nucleus. The nucleusconical head is characteristic of the
is generally composed of tightly packed or echinoids. A spheroidal head is typically
condensed chromatin which, when com- found in the asteroids, holothurians,
ophiuroids, and crinoids. It will be demonstrated that both types of sperm are conThe authors wish to acknowledge the assistance of structed along very similar lines, although
Drs. Paul Rusanowski and Richard Turner in various the shape and relative sizes of individual
aspects of the original investigations. The original components vary. We will present our obwork was supported by N.S.F. Grant GB 37967 and by
servations on the normal morphology of
N.S.F. Grant GV 24157 to Dr. John Dearborn.
This report is presented in two sections.
The first compares the ultrastructure of
spermatozoa representative of four
echinoderm classes: Echinoidea, Asteroidea, Ophiuroidea, and Holothuroidea. Emphasis is placed on the morphology
of the acrosomal region because of its
pivotal role in fertilization. The second part
examines the ultrastructural alterations of
the spermatozoon during fertilization.
523
524
SUMMERS, HYLANDER, COLWIN, AND COLWIN
spermatozoa of Echinarachnius parma
(Echinoidea), Ophiopholus aculeata (Ophiuroidea), Asteriasforbesi and Ctenodiscus crispatus (Asteroidea), and Thyone briareus
(Holothuroidea). It should be noted that
many of the morphological features of
these spermatozoa have been described
previously and that such cases will be so
indicated. The preparative methods for
specimens are described in detail by Summers and Hylander (1974), Colwin et al.
(1975) and Longo and Anderson (1968).
Echinoidea: Echinarachnius parma
The spermatozoon of Echinarachnius
parma (Fig. 1) has been described previously by Summers and Hylander (1974). In
its overall morphology it resembles the
spermatozoa of other echinoids (Afzelius,
1955; Bernstein, 1962; Dan et al. 1964;
Franklin, 1965; Longo and Anderson,
1969) and is typical of the class.
Acrosomal region. The structure of the acrosomal region of the living spermatozoon
is difficult to resolve with the light microscope, but with the electron microscope it
can be ascertained that the acrosome is a
bipartite complex which contains an acrosomal vesicle and periacrosomal material
(Figs. 2-6, 39A).
The acrosomal vesicle surmounts a cupshaped depression in the apex of the nucleus and is relatively spherical in shape
being slightly flattened at its base. The vesicle is completely contained within a membrane, the continuity of which is evident in
both longitudinal and transverse sections
(Summers and Hylander, 1974). The vesicle membrane is comprised of two morphologically distinct regions: the frequently crenulated anterior half, apposed
to the plasma membrane, and the more
FIG. 1. Diagrammatic representation of the spermatozoon of Echinarachnius parma. The anterior acrosomal region (AR) is comprised of a membrane
bounded acrosomal vesicle and the surrounding
periacrosomal material. The base of the nucleus (N)
lies within a doughnut-shaped mitochondrion (M).
Several lipid droplets (L) and two centrioles, one of
which serves as the origin of the flagellum, are encompassed by the mitochondrion. (From Summers and
Hylander, 1974).
AR
FIGS. 2-6. Echinarachnius parma. Acrosomal region.
The nearly spherical acrosomal vesicle is surrounded
by periacrosomal material. The acrosomal vesicle
membrane is continuous. The contents of the vesicle,
the acrosomal granule (Fig. 2,G) are homogeneous in
appearance. The granule is continuous with an elec-
tron dense layer at the base of the vesicle membrane
(Fig. 6, arrow) and separated from the vesicle membrane anteriorly by electron lucent region. The prospective "rim of dehiscence" can be identified (Figs. 2,
3, arrows). The majority of the periacrosomal material
is present as a subacrosomal specialization within a
526
SUMMERS, HYLANDER, COLWIN, AND COLWIN
fossa (Fig. 2, F) in the anterior of the nucleus (Fig. 2,
N). This material appears denser where it is apposed
to the nuclear envelope (Fig. 5, arrow). Periacrosomal
vesicles (Fig. 4, arrows) are present at the brim of the
subacrosomal fossa situated lateral to the base of the
vesicle. Fig. 2, x 60,000; Fig. 3, x 64,000; Fig. 4,
x 44,000; Fig. 5, x 60,000; Fig. 6, x 90,000. (From
Summers and Hylander, 1974.)
electron dense posterior half (Figs. 2, 5). It
will be demonstrated that these two regions
of membrane function differently during
fertilization.
The contents of the acrosomal vesicle are
relatively homogeneous in Echinarachnius
when compared with non-echinoid spermatozoa. The principal component within
the vesicle is a spheroidal acrosomal
granule (Fig. 2, G) which is separated from
the anterior half of the vesicle membrane
by an electron lucent region (see Longo and
Anderson, 1969). An electron dense component can be visualized between the base
of the granule and the posterior portion of
the vesicle membrane (Figs. 5,6). This basal
material, which stains preferentially with
uranyl acetate, may also be interpreted as a
coating which is applied to the inner surface of the acrosomal vesicle membrane.
Periacrosomal materials completely surround the acrosomal vesicle. The bulk of
periacrosomal material is contained within
an apical depression in the sperm nucleus
(subacrosomal fossa) and there is some
structural differentiation of this material
within the fossa (Fig. 5). An electron dense
portion is tightly applied to the nuclear envelope lining the fossa and a second region
of more loosely packed, reticulate material
is observed within the center of the fossa.
Periacrosomal material is also interposed
between the basolateral aspect of the acrosomal vesicle and the sperm plasmalemma, extending anteriorly between
the two membranes, and posteriorly for a
variable distance between the plasmalemma and nuclear envelope (Figs. 2-6).
Nuclear and mitochondrial regions. The
nucleus of the Echinarachnius spermatozoon is conical in shape (Fig. 1). Extending
from the nuclear envelope covering the
brim of the subacrosomal fossa are the
"horns" or "peg-like structures" (Dan et al.,
1964; Longo and Anderson, 1969) which
are characteristic of other echinoid sperm
(Fig. 4, H). The base of the nucleus lies
within a doughnut-shaped mitochondrion.
The base of the nucleus and the central
space within the mitochondrion form the
centriolar fossa which contains the proximal (flagellar) centriole. The distal centriole lies lateral to the proximal and is interposed between nuclear and mitochondrial membranes. For additional information on this region in echinoids, see Longo
and Anderson (1969), Summers and Hylander (1974), and Marshall and Luykx
(1973). The relationship of nucleus,
mitochondrion and centrioles in Echinarachnius is similar to that in most other
echinoderm spermatozoa with a few exceptions (see Chia et al., 1975).
Asteroidea: Asterias forbesi and Ctenodiscus
crispatus
The spheroidal spermatozoa of asteroids
(as well as those of holothurians and
ophiuroids) possess an acrosome which is
easily discernible with the light microscope,
although its individual constituents are not
so easily resolved. Spermatozoa from two
species will be described so that the extent
and nature of interspecific variation might
be appreciated. Previous electron microscopic studies on the normal morphology
of starfish sperm have been carried out by
Dan (1960), Dan et al. (1962), Hagiwara et
al. (1967), Dan and Hagiwara (1967),
Summers et al. (1971), and Tilney et al.
(1973).
Acrosomal region. In the spheroidal-type
spermatozoa the entire acrosomal complex
is embedded within the nucleus in an acrosomal fossa (Figs. 7,10, 14, 19). The fossa
is bounded anteriorly by the plasma membrane and postero-laterally by the nuclear
envelope. As in the echinoids, two components constitute the acrosome: an acrosomal vesicle and the surrounding
periacrosomal material. The vesicle in both
species is speroidal (Figs. 7-9, 10-13) in
shape but may be flattened or slightly in-
FIGS. 7-9. Asterias forbesi. FIG. 7. The entire ac- rosomal fossa in the nucleus (N). The mitochondrial
rosomal complex is embedded within the anterior ac- region is situated posterior to the nucleus. Within a
;
>5W^
hole in the single mitochondrion (M) lie the proximal
centriole (PC) and distal centriole (DC) which gives rise
to the flagellum. x 50,000. FIG. 8. The acrosomal
region. The junaion between the anterior portion of
the vesicle membrane and the posterior, more
electron-dense, portion is indicated by arrows.
x 56,000. FIG. 9. The vesicle membrane is indented
anteriorly where it is apposed to the plasmalemma
(arrow). Periacrosomal material surrounding the vesicle is homogeneous (Pt) except for a dense, clumped
subacrosomal specialization (P2) apposed to the base of
the vesicle, x 58,000.
528
SUMMERS, HYLANDER, COLWIN, AND COLWIN
FIGS. 10-13. Ctenodiscus crispatus. FIG. 10. The ac- anterior of the nucleus. A single mitochondrion (M) at
rosomal complex is contained within a fossa in the the base of the nucleus encompasses two centrioles (C);
FUNCTIONAL ANATOMY OF ECHINODERM SPERM
dented at its apex (Figs. 9, 12) where it is
juxtaposed to the plasmalemma. A slight
flattening or invagination is also evident at
the base of the vesicle. The acrosomal vesicle in both species is bounded by a single,
continuous membrane which displays regional differences in morphology. Anteriorly the membrane is less electron dense
and is frequently crenulated. In osmium
fixed material (OsO4 as a primary fixative)
this portion of the vesicle membrane displays frequent discontinuities (Fig. 13) (see
also Dan, 1960; Dan and Hagiwara, 1967;
Hagiwara et al., 1967; Summers et al.,
1971). The postero-lateral portion of the
bounding membrane is electron dense,
perhaps due to a densely staining coating
on its inner surface. The junction between
the anterior and posterior regions of acrosomal vesicle membrane is sharply demarcated in both species (Figs. 8, 11, arrows). The flattened or slightly invaginated
posterior portion of the membrane may be
recognized readily by its close association
with dense subacrosomal material (Figs. 9,
11, P2).
The acrosomal granule in Asterias displays a morphological differentiation of its
constituents whereas the vesicle contents in
Ctenodiscus are relatively homogeneous, although both species possess a disc-shaped
lucent region at the base of the vesicle
(compare Figs. 7-9 with Figs. 10-13). The
morphological differentiation of the acrosomal granule is a species variant; the
significance of such interspecific variation is
not clear in terms of acrosomal function
during fertilization.
The morphology of the periacrosomal
material of the two asteroid sperm is nearly
identical. In both, this material completely
surrounds the vesicle except, perhaps, at its
apex, where the acrosomal vesicle and
plasma membranes are tightly apposed. It
could not be ascertained that periacrosomal
the distal centriole serves as the origin of the flagellum
(F). x 22,000. FIG. 11. The acrosomal region. The
acrosomal vesicle membrane is continuous; the limit of
the electron-dense basal portion is indicated by arrows. The contents of the vesicle are homogeneous. A
subacrosomal specialization (P2) of the periacrosomal
529
material extends between the two membranes in the region of close apposition.
Periacrosomal material is homogeneous in
appearance except for a dense, clumped
subacrosomal specialization which is juxtaposed to the base of the acrosomal vesicle
(Figs. 9, 11). The dense specialization lies
within an invagination of the nucleus.
Membranous vesicles are frequently observed in the subacrosomal region (Fig. 7)
and are similar in size and appearance to
those which have been observed by Longo
and Anderson (1969) in echinoid sperm.
These are apparently portions of nuclear
envelope.
Nuclear and mitochondrial regions (Figs. 7,
10). The nucleoplasm is formed of coarse,
branching, or anastomosing strands of
chromatin which display no recognizable
order in their packing. The head is relatively spherical except for a flattened or
indented apical region where the acrosomal fossa is spanned by the plasmalemma. The nucleus may also be slightly
indented at its base where it is adjoined to
the proximal centriole. The arrangement
of the mitochondrion, centrioles, and flagellum is similar to that of the echinoid condition. The base of the nucleus lies within a
single doughnut-shaped mitochondrion.
Two centrioles, distal and proximal, are
present with their axes oriented perpendicularly (Figs. 7, 10). The flagellum originates from the distal centriole. Flagellar
and acrosomal axes lie at an angle of 35°
with relation to one another.
Ophiuroidea: Ophiopholus aculeata
The spheroidal spermatozoa of ophiuroids are constructed along lines most
similar to asteroids. There are no previous
ultrastructural descriptions of ophiuroid
spermatozoa.
Acrosomal region. The acrosome of the
material (Pi) is present apposed to the base of the
vesicle, x 58,000. FIG. 12. An anterior indentation
of the vesicle and sperm plasma membranes is indicated by an arrow, x 53,000. FIG. 13. Specimen
fixed with osmic acid only; discontinuities of the vesicle
membrane are indicated (arrows), x 46,000.
530
SUMMERS, HYLANDER, COLWIN, AND COLWIN
17
FIGS. 14-18. Ophiopholus aculeata. FIG. 14. Although the acrosomal fossa is shallower, the morphology of the Ophiopholus sperm is identical to that of the
asteroids. The mitochondrion has scroll-like cristae.
x 29,000. FIG. 15. Acrosomal region. The acrosomal vesicle is bounded by a complete membrane.
Scattered vacuosities are present within the granule
(G). x 50,000. FIG. 16. Two portions of vesicle
membrane can be distinguished by their differential
adherence to the granule (arrows), x 53,000.
FIG. 17. The subacrosomal specialization of periacrosomal material (arrow) is not as extensive as that of
the asteroids, x 100,000. FIG. 18. The differential
adhesion of the granule to the vesicle membrane is also
evident in oblique section (arrows). Note also that the
acrosomal vesicle membrane is complete in transverse
section, x 50,000.
FUNCTIONAL ANATOMY OF ECHINODERM SPERM
ophiuroid spermatozoon is also completely
embedded within the apex of the nucleus
(Fig. 14) although the flask-shaped acrosomal fossa of Ophiopholus is much shallower and occupies relatively less nuclear
volume than the fossa in either of the asteroid sperm examined. The two major
components which form the acrosomal
complex, an acrosomal vesicle and periacrosomal material, are also present in
Ophiopholus (Figs. 15-18).
The acrosomal vesicle is completely
bounded by a limiting membrane (Figs. 15,
16, 18). The membrane and the plasmalemma covering it are blebbed outward
(anteriorly) giving the vesicle a pyriform
shape. A similar situation is observed in the
holothurian Thyone (Colwin et al., 1975,
and this report, Fig. 19). The vesicle membrane is indented slightly at its base where it
overlies a dense tuft of subacrosomal material (Fig. 17, arrow). As in echinoids and
asteroids, two morphologically distinct regions of vesicle membrane may be distinguished, primarily by their differential
adherence to the acrosomal granule (Figs.
16, 18, arrows). The anterior half is separated from the granule by an electron lucent region whereas the posterior half is
tightly adherent to the basal half of the
granule. The posterior half of the membrane is also distinguished by the presence
of an inner coating of electron dense material. The acrosomal granule is homogeneous in appearance except for scattered electron lucent vacuosities and an electron lucent disc at its base (similar to that observed
in Asterias and Ctenodiscus).
Periacrosomal material surrounds the
acrosomal vesicle except perhaps for its anterior extremity in the region of tightly apposed vesicle and plasma membranes. The
electron dense differentiation of periacrosomal material observed in mid-sagittal
section (Figs. 15-17) is not as extensive as in
the asteroids. The subacrosomal specialization lies within a shallow invagination of the
nucleus.
In general, the ophiuroid acrosome most
closely resembles the asteroid condition
with regard to the arrangement of its constituents. However, the entire ophiuroid
531
acrosomal complex appears more flattened
apico-basally so as to appear wider and shallower in comparison.
Nuclear and mitochondrial regions. The
nucleus of the Ophiopholus spermatozoon is
an oblate spheroid containing uniformly
fine aggregates of chromatin (Fig. 14). The
posterior region of the nucleus is contained
within a single doughnut-shaped mitochondrion which possesses unusual scrolllike cristae (Fig. 14). The arrangement of
centrioles and the origin of the flagellum
within the centriolar fossa are identical to
their arrangement in the asteroids (above).
Holothuroidea: Thyone briareus
The acrosomal region of the spermatozoon of Thyone has been described with the
light microscope by Colwin and Colwin
(1956) and with the electron microscope by
Summers et al. (1971) and Colwin et al.
(1975). The sperm head is roughly spherical in shape and bears a prominent acrosome which is visible with the light microscope. The spermatozoon of a second
holothurian, Cucumaria frondosa, is nearly
identical to the spermatozoon of Thyone
with respect to the morphology of the acrosomal, nuclear and mitochondrial regions (unpublished observations).
Acrosomal region. The acrosomal region
(Figs. 19, 45/i) lies anteriorly within a
spheroidal or slightly ovoid depression in
the nucleus and is bounded anteriorly by
the plasmalemma and postero-laterally by
the nuclear envelope. The shape of the acrosomal fossa differs somewhat from the
flask or vase-shaped fossa of the asteroids
and ophiuroids. Two major components
again comprise the acrosomal complex: an
acrosomal vesicle with a completely bounding membrane and periacrosomal material.
The acrosomal vesicle is spherical except
for a shallow posterior invagination (Figs.
19, 20, 45/1) and a prominent bleb or protrusion at its apex which is also visible in the
living state (Figs. 19, 21). The limiting
membrane of the vesicle displays three regional differences with respect to associated
materials (Figs. 19, 45/4). The apical portion of the membrane is frequently crenu-
\,
20[
g
\
FIGS. 19-23. Thyone briareus. FIG. 19. Unreacted
acrosomal region. Acrosomal vesicle (a), its contents
(g) and periacrosomal material (Pi, P2) are embedded
within the nucleus (n). The subacrosomal specialization of periacrosomal material (P2) is conical in shape
s
g
and consists of fibrillar material. Note the three regions of the acrosomal vesicle membrane (a). The apical protrusion of acrosomal vesicle and its contents are
labeled (b). The prospective rim of dehiscence is indicated by arrows, x 56,000. FIG. 20. Posterior por-
FUNCTIONAL ANATOMY OF ECHINODERM SPERM
533
lated and is separated from the vesicle contents by a lucent zone of about 75 A in width.
The lateral two-thirds of vesicle membrane
is coated on its inner surface with electron
dense material and is also separated from
the vesicle contents by a lucent region. The
junction between the apical and lateral regions of membrane is sharply demarcated
(Fig. 19). The basal region of membrane is
indented slightly and is closely associated
with a dense, subacrosomal region of
periacrosomal material (Fig. 19). The contents of the acrosomal vesicle are regionally
differentiated with at least four separate
components being morphologically distinguishable (for details, see Colwin et al.,
1975). The major component, however, is a
homogeneous acrosomal granule.
Periacrosomal material surrounds the
acrosomal vesicle except for the apical
protrusion. The periacrosomal material
appears homogeneous and finely granular
except for a dense subacrosomal specialization, which in Thyone is conical in shape and
consists of longitudinally oriented fibrillar
material (Figs. 19, 20). Electron lucent
patches are occasionally observed in association with the subacrosomal specialization
of periacrosomal material.
Nuclear and mitochondrial regions. The
nucleus of the spermatozoon of Thyone is
almost perfectly spherical in shape but the
presence of the mitochondrion at its base
imparts an oblong appearance to the sperm
head (Fig. 22). The nucleoplasm is composed of coarse, branching and anastomosing strands of chromatin reminiscent of the
asteroid sperm nucleus. The arrangement
of mitochondrion, centrioles, and flagellum is similar to that in the other species
described above (Fig. 22). Centrioles are
oriented in a relatively parallel rather than
a perpendicular manner. The axis of the
proximal centriole is in line with the acrosomal axis and the central axis of the
distal centriole (and its flagellum) lies at an
angle of 35° to the proximal centriole.
tion of unreacted acrosomal region. The subacrosomal specialization of periacrosomal material (P2)
and the basal indentation of the acrosomal vesicle
membrane (t) are particularly evident. The contents of
the acrosomal vesicle are labeled (g). x 47,000.
FIG. 21. Anterior portion of unreacted acrosomal
region. The close apposition of sperm plasma and
acrosomal vesicle membranes is indicated. The acrosomal vesicle contents (g) and slight apical protrusion (b) are evident, x 67,000. FIG. 22. Mitochon-
drial region of the unreacted spermatozoon. Centrioles (C) are oriented at an angle of 35°. A single
doughnut-shaped mitochondrion (M) is present. A
portion of the acrosomal fossa (F) is included in the
micrograph, x 47,500. FIG. 23. Reacted spermatozoon. Acrosomal tubule (t) has been formed. Note the
microfilaments within its lumen. The arrows indicate
the morphological changes in the nucleus which occur
during the acrosomal reaction, x 28,000. (From Colwin et al., 1975).
GAMETE INTERACTION
Longo, in a recent review (1973), has
defined fertilization "as a multistep
phenomenon initiated with the interaction
and subsequent fusion of the gametes and
culminating in the association of the corresponding groups of chromosomes derived from two pronuclei, one of paternal
and the other of maternal origin." The
cytological events of fertilization generally
fall into four broad categories: (i) the reaction of the acrosomal region in proximity to
the egg, (ii) gamete membrane fusion, (iii)
reaction of the egg to membrane fusion,
and finally (iv) pronuclear association.
In the vicinity of the egg the sperm undergoes an acrosomal reaction which is induced by extracellular egg substances. This
reaction, which involves the formation of
an acrosomal tubule, is a prerequisite to
subsequent events (Dan, 1952). Cytoplasmic continuity between the two gametes is
then established by gamete membrane fusion
and the cytoplasmic constituents of the
spermatozoon are then incorporated into
the ooplasm, the sperm nucleus forms a
male pronucleus which becomes associated
with the female pronucleus.
These events of fertilization will be presented as we have observed them to occur in
representatives of the phylum Echinodermata. We have chosenEchinarachniusparma
(from Summers and Hylander, 1974) and
Arbacia punctulata (from Longo and Anderson, 1968; Longo, 1973), with elongate
spermatozoa, and Thyone briareus (from
Colwin et al., 1975), with a spheroidal
2A
•f: I
FIGS. 24-30. E. parma. Acrosomal reaction. The initial stage of the reaction involves three events (Fig. 24):
(i) unilateral fusion of the vesicle membrane with the
sperm plasma membrane (arrows), (ii) alteration in
appearance of acrosomal vesicle contents, and (iii) be-
ginning of tubule formation. Subsequently, the tubule
elongates; the former contents of the acrosomal vesicle
remain adherent to the exterior tubule membrane
(Figs. 25,26, 27). The junction of former vesicle membrane and sperm plasma membrane can be identified
FUNCTIONAL ANATOMY OF ECHINODERM SPERM
FIGS. 31-34. E. parma. Acrosomal reaction.
Ruthenium red staining only. The acrosomal granule
material (Fig. 31,R) has been densely stained, indicating its acid mucopolysaccharide nature. The acrosomal vesicle has opened in another plane of section.
During successive stages of tubule elongation, the
535
former contents of the acrosomal vesicle remain adherent to the exterior of the tubule membrane (Figs.
32-34). Fig. 31, x 50,000; Fig. 32, x 50,000; Fig. 33,
x 50,000; Fig. 34, x 50,000. (From Summers and
Hylander, 1974.)
spermatozoon. We wish to emphasize that
analogous morphological components are
present in the male gametes of both types
and that these sperm components function
similarly during interaction with the egg.
brane and the sperm plasma membrane
undergo unilateral fusion at a site (Figs. 24,
31, 33, 34, 395) along a predictable line
(Figs. 2, 3, 39/4, arrows). Fusion continues
around this line to circumscribe a "rim of
dehiscence" (Colwin and Colwin, 1961a)
Echinarachnius parma and Arbacia and ultimately the entire anterior half of
punctulata
the acrosomal vesicle membrane and the
overlying plasma membrane are lost as a
Acrosomal reaction. The first morphologi- result of this fusion (Figs. 25, 32, 39C.D).
cal indications that a spermatozoon has The detached byproduct of this dehiscence
begun the acrosomal reaction are is a rather large membranous vesicle and
threefold. First the acrosomal vesicle mem- there is an occasional indication that a sec-
in a late stage of tubule formation (Fig. 28, arrows).
Following eversion of the vesicle membrane (Fig. 29,
1°), a secondary elongation (Fig. 29, 2°) is evident.
Microfilaments of periacrosomal origin are apparent
within the tubule at all stages of formation. Occasionally, initiation of tubule formation appears to precede
membrane fusion (Fig. 30); this is attributable to the
plane of sectioning. Fig. 24, x 32,000; Fig. 25,
x 33,000; Fig. 26, x 47,000; Fig. 27, x 47,000; Fig.
28, x 58,000; Fig. 29, x 30,000; Fig. 30, x 30,000.
(From Summers and Hylander, 1974.)
536
SUMMERS, HYLANDER, COLWIN, AND COLWIN
m
V-jja;
\
.kt- i
;^>
R-
V
ik
n
u."V;A
"36
3
FIGS. 35 and 36. E. parma. Primary gamete binding. vitelline envelope (V), of the ovum. Fig. 35, x 62,000;
The extracellular coat on the tubule membrane (the Fig. 36, x 66,000. (From Summers and Hylander,
1974.)
former contents of the acrosomal vesicle) have formed
a morphological bond with the extracellular coat, and
ond smaller vesicle is produced. The more
posterior, electron dense portion of the acrosomal vesicle membrane is retained. Because of its electron density and inflexible
character, the posterior portion of vesicle
membrane can be distinguished from the
plasmalemma with which it has fused (Figs.
28, 39£, arrows) and thus the vesicle mem-
38
FIGS. 37 and 38. E. parma. Gamete membrane fusion. The tubule membrane and the egg plasma membrane have undergone fusion at the site of the arrows.
Fig. 37, x 53,000; Fig. 38, x 51,000. (From Summers
and Hylander, 1974.)
FUNCTIONAL ANATOMY OF ECHINODERM SPERM
337
FIG. 39. Schematic representation of the acrosomal
reaction. A, The unreacted acrosomal region. The
prospective "rim of dehiscence" is indicated by arrows.
B, Initiation of acrosomal reaction: unilateral membrane fusion of the acrosomal vesicle membrane with
the sperm plasma membrane, alteration in appearance of the acrosomal granule, and eversion of acrosomal vesicle membrane to form the acrosomal
tubule. Note microfilaments originating from periacrosomal material within the lumen of the tubule. C,
Fusion has progressed around the circumference of
the vesicle; the membranes have lifted from the surface of the acrosomal granule. D, Membrane fusion
has become complete and the overlying membrane has
been lost. E, Primary elongation has been completed.
Arrows indicate the juncture of former vesicle membrane and sperm plasmalemma. F, Completion of secondary elongation. Tubule has reached its maximum
length. G, Primary gamete binding. H, Gamete membrane fusion. (From Summers and Hylander, 1974.)
brane contribution to the acrosomal tubule
and the original site of fusion ("rim of dehiscence") can be identified throughout
fertilization. Apparently simultaneous with
opening of the acrosomal vesicle to the external environment, two additional morphological changes are observed: (i) contents of the acrosomal vesicle are altered in
their appearance (compare Figs. 2-6 with
24-26) and (ii) the acrosomal tubule begins
to form by eversion of the remaining basal
half of the acrosomal vesicle membrane
(Figs. 24-28, 31-34, S9B-F).
Initially the tubule is short (200 m/x) (Figs.
24, 395) but it elongates rapidly until the
remaining basal portion of the vesicle
membrane has completely everted. At this
stage, the tubule is 400 m/x, in length (Figs.
26, 27, 39£). Acrosomal vesicle materials
remain adherent to the exterior of the
tubule membrane throughout elongation
and subsequent gamete fusion (Figs. 2439). Such coating materials apparently contain an acid mucopolysaccharide since they
stain intensely with ruthenium red (Figs.
31 -36). Initially, these materials coat the en-
538
SUMMERS, HYLANDER, COLWIN, AND COLWIN
tire tubule, including its tip, but as elongation proceeds, the extracellular materials
are displaced from the tip of the tubule. In
later stages of elongation a secondary or
postacrosomal segment of acrosomal
tubule can be identified (Figs. 28, 29, 39F).
This segment is 100 m/x (20% of tubule
length) and lies posterior to that portion of
the tubule membrane which is of acrosomal
origin.
During elongation of the acrosomal
tubule, changes are observed within the
periacrosomal material, particularly in the
subacrosomal region. At a very early stage,
microfilaments are apparent within the
tubule (Fig. 26). The longitudinal axis of
such filaments conforms to the longitudinal
axis of the tubule. Similar filaments have
been reported in other echinoderm spermatozoa, both before and after the acrosomal reaction (Dan et al., 1964; Colwin
etal., 1975; Summers and Hylander, 1974)
and have been identified as actin (Tilney
et al., 1973; Jessen et al., 1973). Filaments
generally extend from periacrosomal
material at the brim of the fossa into the tip
of the tubule. Not all of the periacrosomal
material becomes filamentous during the
acrosomal reaction.
Gamete contact. Within 30 sec after insemination, spermatozoa reach the surface
of the ovum. The persistent extracellular
coating on the acrosomal tubule forms a
morphological complex with the vitelline
envelope of the ovum (Figs. 35, 36). This
stimulus of the spermatozoon, the ovum of
Echinarachnius undergoes the cortical reaction. Since the cortical reaction has been
studied in some detail by previous investigators (Anderson, 1968; Millonig, 1969;
Epel, 1975) and because our observations
corroborate the morphological findings, we
will not present the events of cortical reaction in great detail. In summary, the cortical granule membranes fuse with the
oolemma which overlies them. Such fusion
results in the release of granule contents
beneath the vitelline envelope which then
detaches from the oolemma (Fig. 46). The
elevated vitelline envelope is termed a fertilization envelope (activation calyx, by Anderson, 1968). During the course of its elevation, the fertilization envelope remains
intact and does not appear to be thinned or
degraded. In fact, cortical granule contents
become attached to the inner surface of the
envelope and become spread over it to
form a multilaminate structure (Inoue and
Hardy, 1971). Primary binding between
the acrosomal tubules of supernumerary
spermatozoa (which have not fused with
the oolemma) and the vitelline envelope
persists for a short period of time following
its elevation (Fig. 47). Subsequently, these
spermatozoa become detached (Vacquier,
etal., 1972; Aketaetal., 1972; Epel, 1975).
It is also apparent under the light microscope that additional sperm do not become
bound to the fertilization envelope after its
elevation. The possible mechanisms for
elevation of the fertilization envelope and
constitutes primary gamete binding. As will be the subsequent detachment of bound
seen below, the primary binding between spermatozoa are discussed below.
sperm and egg extracellular coatings is
quite persistent.
At the site of sperm entry, discontinuities
The second and final step in the process are present within the vitelline envelope
of sperm-egg binding is the establishment (Fig. 41, arrows). It is possible that these
of cytoplasmic continuity between sper- free edges will represent the edges of holes
matozoon and ovum. As in many other in the fertilization envelope after its elevaspecies (Colwin and Colwin, 1967) this is tion around the fertilization cone.
accomplished by membrane fusion. In
Development of the male pronucleus. The
Echinarachnius, the tip of the acrosomal most extensive investigations of pronuclear
tubule membrane and the oolemma fuse to formation and association in echinoderms
establish cytoplasmic confluence between have been carried out by Longo and Anthe gametes (Figs. 37, 38). It should be derson (1968) and we will summarize their
noted that the initial binding between observations in Arbacia. It should be noted
sperm and egg extracellular materials per- that our preliminary findings in Echinasists subsequent to membrane fusion.
rachnius generally support the findings of
Cortical reaction. In response to the these authors.
AC
FIG. 40. Arbacia punctulata. An early stage of the in- cleus; LB, lipid body; YB, yolk body; M, egg mitocorporation of the spermatozoon in Arbacia. AC, fer- chondrion; FC, fertilization cone, x 27,000. (From
tilization envelope (activation calyx); SM, sperm Longo, 1973.)
mitochondrion; SF, sperm flagellum; SN, sperm nu-
42
FUNCTIONAL ANATOMY OF ECHINODERM SPERM
Shortly after gamete membrane fusion
has occurred, ooplasm becomes interposed
between the former sperm plasma membrane and the cytoplasmic contents of the
spermatozoon. The contents of the spermatozoon and the ooplasm which envelopes them are collectively termed a fertilization or entry cone (Figs. 40, 41). The
spermatozoon contents, divested of plasma
membrane, pass from the cone into the cortical ooplasm and together undergo a 180°
rotation. The nuclear envelope begins to
break down by vesiculation in all regions
except for the small areas lining subacrosomal and centriolar fossae. Intact membrane persists in these regions throughout
pronuclear development. Following vesiculation of the nuclear envelope, dispersion
of the chromatin is initiated.
It is apparent that chromatin is first dispersed at the periphery of the nucleus and
that the process proceeds inwardly. In
more advanced stages, several distinct
zones of dispersion can be recognized (Fig.
42). At this stage the pronuclear envelope is
formed around the heart-shaped male
pronucleus (Fig. 42). Sperm organelles
(centriole, flagellum, and mitochondrion)
remain in the vicinity of the developing
pronucleus. The pronucleus then undergoes further chromatin dispersion, an increase in size and eventually assumes a
more spherical shape (Fig. 43). Male and
female pronuclei migrate to the central
ooplasm where they become closely apposed (Fig. 43). The pronuclear envelopes
meet and fuse forming a continuous zygote
nuclear envelope (Fig. 44); former pronuclear contents are then connected via a small
internuclear bridge which increases in
diameter until the zygote nucleus assumes a
more spheroidal appearance.
541
Thyone briareus
The spermatozoon of Thyone (unlike the
echinoid spermatozoon) does not enter the
thick jelly "hull" which surrounds the
ovum. The acrosomal reaction occurs at the
periphery of the jelly and a long acrosomal
tubule of up to 75/x in length extends
through the jelly to establish contact with
the ovum (Colwin and Colwin, 1956). This
observation also applies to the asteroids (see
Dan and Hagiwara, 1967). Our ultrastructural observations in Thyone will be concerned only with the early events of fertilization, namely acrosomal reaction and gamete contact. Later events, including pronuclear development and association remain to be investigated in the echinoderms
which possess a spheroidal spermatozoon.
Acrosomal reaction. The initial stage of the
acrosomal reaction in Thyone is the fusion of
the acrosomal vesicle membrane with the
overlying sperm plasma membrane along
an apical "rim of dehiscence" which lies at
the base of the apical protrusion. The contents of the acrosomal vesicle are rapidly
dispersed (Fig. 45B), except for a persistent
felt-like material of vesicle origin which
coats the anterior two-thirds of the acrosomal vesicle membrane. This material
also extends laterally for a short distance
along the sperm plasma membrane at the
mouth of the opened acrosomal vesicle
(Fig. 45B-C). Membranous vesicles, which
are most likely remnants of the fusion between acrosomal vesicle and plasma membranes, are observed at the opening of the
acrosomal vesicle during early stages (Fig.
45B). However, the felt-like coating and the
cluster of vesicles are not present in later
stages.
After the vesicle has opened, an ac-
nucleus delimited by a pronuclear envelope (PNE)
perforated by pores. The double- and single-stemmed
arrows denote the portions of the male pronuclear
envelope at the centriolar fossa and the apex of the
sperm nucleus, respectively. A layer of electronopaque material is associated with these regions. FDC,
finely dispersed chromatin; CDC, coarsely dispersed
chromatin; CC, condensed chromatin; SM, sperm
mitochondrion; SF, sperm flagellum. x 11,500.
FIG. 42. Arbacia punctulata. Heart-shaped male pro- (From Longo and Anderson, 1968.)
FIG. 41. E. parma. Early stage of sperm incorporation into the ooplasm. The tip of a sperm nucleus (SN)
is evident within the base of an obliquely sectioned
fertilization cone (FC). Arrows indicate the free edges
of the vitelline envelope. Note the lack of major cytoplasmic organelles within the ooplasm of the fertilization cone, x 38,500. (From Summers and Hylander,
1974.)
FIG. 43. Arbacia punctulata. Male and female pronu- male pronucleus; NL, nucleoli-like structures; SF,
clei prior to fusion. The sperm aster (arrows) has sperm flagellum. x 12,000. (From Longoand Andermerged with the ooplasmic region surrounding the son, 1968.)
female pronucleus. FPN, female pronucleus; MPN,
FIG. 44. Arbaciapunctulata. Completion of the fusion bridge (INB) connects the former male ("MPN") and
of the inner lamina of the male and female pronuclear female ("FPN") pronuclei. x 10,000. (From Longo,
envelopes in the Arbacia zygote. An internuclear 1973.)
rosomal tubule begins to form at its base
(Fig. 45C). The basal indentation of vesicle
membrane is the site of tubule initiation
and the indentation rapidly deepens to be-
come a long tubule, the lumen of which is
continuous with the periacrosomal material. The role of the dense, subacrosomal
specialization in tubule initiation and elon-
544
SUMMERS, HYLANDER, COLWIN, AND COLWIN
FIG. 45. Diagram of the acrosomal region of the
Thyone spermatozoon and its changes during fertilization. A, Unreacted acrosome: p, periacrosomal material; g, acrosomal granule; a, acrosomal vesicle membrane; ne, nuclear envelope; s, sperm plasma mem-
brane. B, Dehiscence of sperm apex and establishment
of acrosomal vesicle membrane and sperm plasma
membrane continuity: y, opened cavity of acrosomal
vesicle. C,D,E, Extension of the acrosomal tubule: c,
microfilaments within the lumen of the tubule; t,
FUNCTIONAL ANATOMY OF ECHINODERM SPERM
gation was not determined although it may
serve as a nucleating site for the polymerization of actin micro filaments (as suggested
by Colwin et al., 1975, and see below). Finally, the former acrosomal vesicle membrane, periacrosomal material, and underlying nuclear material evert to obliterate
the space formerly occupied by the acrosomal vesicle (Figs. 23, 45D). During this
process, marked changes in nuclear shape
occur in which nuclear material forming
the brim of the acrosomal fossa is displaced
laterally (Fig. 23, arrows).
At an early stage in its formation, microfilaments can be recognized within the acrosomal tubule (Fig. 45C). The longitudinal
axes of these filaments are oriented to conform with the longitudinal axis of the
tubule. Tilney et al. (1973) have provided
convincing biochemical evidence in Thyone
and several starfish that the filaments are
F-actin which have formed by the directional polymerization of the G-actin present in periacrosomal material. In addition,
membranous vesicles or groups of vesicles
are present within the tubule and its base
during the later phases of elongation (Fig.
45E, G-I). Such vesicles are helpful in recognizing portions of the tubule which have
interdigitated or fused with the egg.
The origin of the membranous covering
for the extremely long acrosomal tubule in
Thyone cannot be explained readily by the
simple eversion of the acrosomal vesicle
membrane or forward slippage of sperm
plasma membrane. The possible sources of
tubule membrane are discussed below.
Gamete contact. The spermatozoon of
Thyone establishes contact with the surface
of the ovum via the acrosomal tubule which
traverses the egg jelly. The tip of the tubule
fuses with the oolemma to establish cytoplasmic continuity between the gametes although the exact location at which membrane fusion occurs appears to be variable.
In one instance, fusion was observed at the
tubule membrane. Note the membranous vesicles
within the tubule and its base in E. F, Tip of acrosomal
tubule in proximity to the oolemma. G, Fusion of
tubule membrane (t) and oolemma (e) at egg surface.
H, Acrosomal tubule within an indentation corridor./.
Tubule membrane (s) and oolemma (e) are fused
545
immediate surface of the ovum, between
microvilli (Fig. 45G). In other cases, the
tubule extended into a depression or indentation corridor in the oolemma (Fig.
45//). The deepest of these depressions was
observed to be 17jn. Gamete membrane fusion is observed at various depths within the
indentation corridor (Fig. 45/,/) and there
is some indication that fusion may occur at
multiple loci within the corridor.
DISCUSSION
Ultrastructure of the acrosomal region
The first accurate analysis of the structure of the acrosomal region and its function during fertilization was provided by
the Colwins (I96la,b,c; I963a,b) for two invertebrates: Hydroides (a polychaete) and
Saccoglossus (a hemichordate). Since that
time, numerous observations of phyletically diverse invertebrates have demonstrated a remarkable unity of acrosomal
morphology and function (see reviews by
Colwin and Colwin, 1967, and Franklin,
1970). In most cases the acrosomal regions
contain two major components: a membrane bounded acrosomal vesicle and the
surrounding periacrosomal material.
One notable exception to this common
plan of acrosomal morphology has been
reported in several echinoderms by Dan
and her collaborators (Dan et al., 1962,
1964; Hagiwara et al., 1967; Dan and
Hagiwara, 1967; Dan, 1967, 1970). These
authors have maintained that the acrosomal vesicle membrane is discontinuous
(Fig. 48) and that the acrosomal reaction is
more complex than that of the generalized
Hydroides-Saccaglossus pattern. However,
other authors (Bernstein, 1962; Franklin,
1965; Longo and Anderson, 1969; Summers et al., 1971; Tilney et al., 1973) have
reported the presence of a complete acrosomal membrane in echinoids and as-
within a shallow indentation corridor.y, Fusion within
a deeper indentation corridor. Inset: K,L, Drawings of
spermatozoa in the living state. Note the apical protrusion in L. Drawings are not to scale. (From Colwin et
al., 1975.)
546
SUMMERS, HYLANDER, COLWIN, AND COLWIN
FIG. 47. and Inset. E. parma. The vitelline envelope
has elevated to become the fertilization envelope (FE).
The persistence of primary binding subsequent to
membrane elevation is apparent (arrows). The contents of a cortical granule (CC) can be seen on the inner
surface of the fertilization envelope. Fig. 47,
x 30,000; Inset, x 48,000. (From Summers and Hylander, 1974.)
teroids. Our findings confirm the presence
of a complete acrosomal vesicle membrane
in several echinoderm spermatozoa representing four classes. A possible explanation
of this discrepancy is that the use of osmic
acid as a primary fixative (as per Dan) has
been shown to result in discontinuities in
the acrosomal vesicle membrane (see Figs.
13, 48 this report). However, the use of
aldehydes as primary fixatives results in a
complete acrosomal vesicle membrane
(Summers et al., 1971). It is likely that the
acrosomal vesicle membrane is extremely
sensitive to fixation and that preservation
of membrane integrity depends on the
choice of a suitable fixative. Our observations thus indicate that the acrosomal region is constructed along lines similar to
Hydroides and Saccoglossus and therefore
could function similarly during fertilization.
Although we wish to stress qualitative
similarities in the structure of the acrosomal region, some quantitative differences should be noted. In the elongate
spermatozoon (echinoids) the acrosomal
FIG. 46. E. parma. Cortical reaction. An unreacted
cortical granule is present (CG). The contents of a
reacted cortical granule (CC) are adherent to the inner
surface of the elevating vitelline envelope (V).
x 34,000. (From Summers and Hylander, 1974.)
FUNCTIONAL ANATAOMY OF ECHINODERM SPERM
547
48
FIG. 48. Diagrammatic representation of the acrosomal region of spheroidal (A) and elongate (B)
echinoderm sperm with osmic acid fixation (after Dan,
1970). Note that the acrosomal vesicle membrane in
both types is indicated as being discontinuous.
vesicle lies anterior to the nucleus whereas
in the spheroidal spermatozoon the vesicle
is almost completely embedded in the acrosomal fossa within the nucleus. Although
the volume of the acrosomal vesicle is similar in both morphological types, the volume
of periacrosomal material is greater in the
spheroidal spermatozoon. Finally, in the
spheroidal spermatozoon, a prominent
subacrosomal density is present. While a
comparable structure was not noted in the
elongate spermatozoon (see Franklin,
1965; Longo and Anderson, 1969), filaments have been observed in the periacrosomal material of unreacted Strongylocentrotus droebachiensis (Hylander, unpublished) and Echinocardium (Jessen et al.,
1973) spermatozoa.
ment. In Thyone, only a small remnant of
the vesicle contents remains adherent to the
former vesicle membrane and these are dissipated at an early stage. In Echinarachnius
the majority of such materials remain
adherent to the exterior of the tubule
membrane and persist throughout the
reaction. The possible role of such materials in gamete contact in the echinoids is
discussed below.
The formation of an acrosomal tubule
begins upon or shortly after opening of the
acrosomal vesicle. A primary and consistent
difference between the two morphological
types of echinoderm spermatozoa appears
to be the length of the tubule formed (Dan,
1954, 1956; Afzelius and Murray, 1957;
Colwin and Colwin, 1956). The spheroidal
types form an extremely long tubule (25 to
90 p) in comparison to the elongate types
(0.5 to 3 yi). This is compatible with the
relative volumes of periacrosomal material
present in the two types. While tubule formation begins in Echinarachnius by simple
eversion of the remaining half of the acrosomal vesicle membrane, in Thyone the
tubule attains considerable length before
the acrosomal membrane is everted. This
poses a question as to the source of the
membrane which covers the tubule in
Thyone and other species possessing a long
tubule. According to Dan and Hagiwara
(1967), tubule membrane, in addition to
that derived from eversion of acrosomal
vesicle membrane, may be derived from
membrane precursors within the ac-
Acrosomal reaction
In both Echinarachnius and Thy one, the
initial step of the reaction is fusion of acrosomal vesicle membrane with sperm
plasma membrane. Fusion in both species
occurs along a "rim of dehiscence" (Colwin
and Colwin, 1961a, 1963a, 1964, 1967)
which is recognizable in the unreacted
spermatozoon. Membrane fusion in both
species results in incorporation of acrosomal membrane posterior to the "rim of
dehiscence" into the sperm plasma membrane. The apical fused portion of membrane is discarded as a vesicle or several
vesicles. Acrosomal vesicle contents are
thereby exposed to the external environ-
548
SUMMERS, HYLANDER, COLWIN, AND COLWIN
rosomal vesicle. However, our observations
oiThyone (Colwin et al., 1975) indicate that
the source of additional tubule membrane
is most likely periacrosomal material since
membranous vesicles, which are not apparent in the unreacted state, appear within
the lumen of the tubule and at its base.
Membrane material may be inserted directly into the lengthening tubule and/or
assembled into the membranous vesicles,
which might then be inserted into the
tubule membrane.
Finally, it should be pointed out that the
spheroidal spermatozoon oiThyone undergoes a striking change in nuclear morphology during the acrosomal reaction (see also
Dan, 1960; Dan and Hagiwara, 1967; Colwin et al., 1975) whereas that of the
echinoids does not. In the spheroidal
spermatozoon, nuclear and periacrosomal
materials are everted, eliminating the acrosomal fossa within the nucleus, although
such eversion contributes only a small
amount to the total length of the tubule
(Colwin et al., 1975).
Gamete contact
In Echinarachnius and Thyone, as in many
other vertebrates and invertebrates, gamete contact culminates in membrane fusion between the acrosomal tubule and the
oolemma, thus establishing cytoplasmic
confluence between the gametes (see reviews by Colwin and Colwin, 1967;
Franklin, 1970). In both species investigated here, the tubule membrane is of acrosomal origin. Our observations of
Echinarachnius provide some insight into
the way in which primary binding may
mediate gamete fusion in echinoids.
Several authors have stressed that the
covering of the acrosomal tubule is newly
exposed membrane, exposed as a result of
the acrosomal reaction (Colwin and Colwin
1963a, 1967; Franklin, 1965,1970). In light
of our observations, it is possible that this
membrane and perhaps more importantly,
its external coating contain specific substances necessary for species recognition
and membrane fusion. This idea is supported by the fact that it is the tubule which
first makes contact with the egg. In
echinoids, the acrosomal contents are not
completely dispersed, but remain adherent
to the newly exposed acrosomal vesicle
membrane which covers the tubule (although they are thinned from the tip of the
tubule). Such materials persist throughout
the acrosomal reaction and are still present
following fusion between the tip of the acrosomal tubule and the oolemma (see also
Franklin, 1965). The primary binding between materials on the acrosomal tubule
and the vitelline envelope of the ovum, as
observed in Echinarachnius, is apparent in
previously published micrographs of fertilization in other species of echinoids (Afzelius and Murray, 1957; Takashima and
Takashima, 1960; Pasteels, 1965; Franklin,
1965; Longo and Anderson, 1970). It has
been suggested that species-specific binding between sperm and egg occurs at the
level of the egg surface (Aketa, 1973) and
that substances within the vitelline envelope and on the surface of the spermatozoon are responsible for the recognition
and initial binding of gametes. The location
and morphology of such materials on the
surface of the spermatozoon were not presented by Aketa. It is our suggestion that: (i)
such specific materials are contained within
the acrosomal vesicle and (ii) these materials, which remain adherent to the surface
of the tubule, form a morphological bond
with the vitelline envelope prior to membrane fusion. The species-specificity of
such binding remains to be determined.
However, it is known that acrosomal vesicle
material is, at least in part, acid mucopolysaccharide as evidenced by its affinity for
ruthenium red. In light of the everincreasing body of literature which demonstrates the important role of mucopolysaccharide extracellular coats in cell recognition phenomena, our hypothesis seems a
reasonable one. It may be possible with
special techniques (such as lanthanum and
ruthenium red staining) to extend our observations to non-echinoid spermatozoa.
Contrary to the findings of Colwin and
Colwin (1960) in Hydroides or Austin and
Bishop (1958) in mammals, we found little
morphological evidence indicative of a lytic
function of acrosomal vesicle materials in
either Echinarachnius or Thyone. There is,
FUNCTIONAL ANATOMY OF ECHINODERM SPERM
however, preliminary evidence both for
(Stambaugh and Buckley, 1972) and
against (Longo and Schuel, 1973) the presence of trypsin-like enzyme in the acrosomes of echinoid spermatozoa. Nevertheless, it has been shown that successful fertilization occurs in the presence of trypsin
inhibitors (Hagstrom, 1956). Further discussion of the effects of trypsin inhibitor is
given by Epel (1975).
549
the surface of the ovum, remaining bound
to the elevating envelope. Thus, the possibility of supernumerary sperm undergoing
gamete membrane fusion is drastically reduced. These sperm are eventually released from the fertilization envelope,
perhaps as a secondary effect of the protease (Summers and Hylander, 1974). For
further details on echinoid sperm binding
and the cortical reaction, the reader is referred to Epel (1975).
Cortical reaction
The cortical reaction has received considerable attention in the echinoids (Endo,
1961; Anderson, 1968; Millonig, 1969), but
there has been little ultrastructural investigation of its morphology in any of the other
echinoderm classes. In Thyone (and
echinoderms possessing spheroidal spermatozoa) the cortical reaction is less dramatic than in the echinoids. Cortical granules
are present within the cortical ooplasm and
gradually become less numerous as a fertilization envelope is formed (Colwin et al.,
1975). Our ultrastructural observations in
Thyone (unpublished) generally confirm
this finding although the actual release of
cortical granule contents was not observed.
The cortical reaction has long been implicated in the establishment of a block to
polyspermy. A recent series of investigations has demonstrated that the cortical
granules of echinoids contain a trypsin-like
protease (Schuel et al., 1973) which is released during the reaction (Vacquier et al.,
1972; Epel, 1975). A variety of functions
has been suggested for the protease in preventing polyspermy. These include: (i) a
role in the discharge of cortical granules
(Schuel et al., 1973), (ii) release of the vitelline envelope from the oolemma (Vacquier
et al., 1972; Schuel et al., 1973) and (iii)
destruction of sperm binding sites on the
surface of the ovum (Tegner and Epel,
1973; Vacquier et al., 1973). Based on our
observations, we believe that the most significant effect of the tryptic enzyme must be
the release of the vitelline envelope from
the oolemma (Summers and Hylander,
1974). In this way, supernumerary sperm
which have undergone primary binding
(but not membrane fusion) are swept from
Note Added in Proof
Subsequent observations on four additional echinoid species have established
that primary gamete binding is an integral
step in the fertilization process. Ultrastructural observations of homologous and heterologous crosses of these echinoid gametes demonstrate that primary gamete binding is the species-exclusive step of echinoid
fertilization (Summers and Hylander,
Exp. Cell Res., In press).
Ultrastructural analysis of acrosomal
morphology and reaction in two ophiuroid
species (Hylander and Summers, 1975,
Cell Tissue Res. 158: 151-168) corroborate
our observation in Thyone that during acrosomal reaction, the acrosomal vesicle contents become redistributed from the acrosomal fossa over the anterior surface of
the spermatozoon to form a persistent extracellular coat. This extracellular coat is
established prior to tubule formation. The
acrosomal tubule does not become densely
coated as in the echinoids.
The apparent differences between extracellular coat formation in the echinoids
and non-echinoids can be reconciled in
light of the observed differences in mode
of gamete interaction. The elongate echinoid spermatozoa penetrate and undergo
the acrosomal reaction within the egg jelly,
producing a short tubule in the immediate
vicinity of the oolemma. Primary gamete
binding occurs between the vitelline envelope of the the ovum and the extracellular coat on the acrosomal tubule. However, the spherical non-echinoid sperm do
not enter the egg jelly but become positioned at its outer surface, producing a long
tubule which transverses the jelly layer to
550
SUMMERS, HYLANDER, COLWIN, AND COLWIN
contact the ovum. In these sperm, the extracellular coat formed on the anterior
sperm surface may serve to bind the sperm
to the jelly periphery, stabilizing and orienting the sperm during subsequent tubule
elongation. Membrane precursors are also
present within the periacrosomal material
of ophiuroid sperm and are incorporated
into the elongating tubule membrane.
Regarding the role of the cortical granule proteases in preventing polyspermy,
Longo et al. (1974, Develop. Biol. 41:193201) have demonstrated that it is detachment of the vitelline envelope from the
egg surface, rather than unbinding of
sperm, which functions to prevent supernumerary sperm entry. Therefore, unbinding of sperm must be of secondary importance. These observations are consistent with our findings (in seven echinoid
species) that sperm detachment does not
occur until after the vitelline envelope has
become elevated from the oolemma.
lytic activity of sperm extract and its significance in
relation to sperm entry in Hydroides hexagonus (Annelida). J. Biophys. Biochem. Cytol. 7:321-328.
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