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Reproductive products in the adult snow crab
(Chionoecetes opilio). I. Observations on
spermiogenesis and spermatophore formation in
the vas deferens
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Guy Sainte-Marie and Bernard Sainte-Marie
Abstract: Some of the events unfolding in the vas deferens of the adult snow crab (Chionoecetes opilio) were
examined by means of light microscopy. Sperm cells entered the vas deferens as precursors of immature spermatids
and developed into immature or mature spermatids within it. However, spermatozoa were not observed in the male
reproductive tract. Two types of amorphous matter were added successively to sperm cells in the vas deferens. The
first type was periodic acid – Schiff (PAS)-positive and apparently induced spermiogenesis when present in a
sufficiently large amount. However, a smaller amount of this amorphous matter was sufficient to form the basal pellicle
of spermatophores. The second type was PAS-negative and thickened the wall of spermatophores. Immature and mature
spermatids were usually enclosed within distinct spermatophores. Enclosed mature spermatids were connected together
by bridges formed by Feulgen-positive spikes coated and extended by PAS-positive amorphous matter. Once broken,
the bridges appeared as arms that radiated from a spermatid. Peripheral mature spermatids were furthermore linked to
the spermatophore wall by threads of PAS-positive amorphous matter. The bridges and threads may form a pathway for
the diffusion of extraneous substances through the spermatophore wall to the innermost cells.
Résumé : Certains des événements se déroulant dans le vas deferens du Crabe des neiges (Chionoecetes opilio) adulte
ont été examinés par microscopie photonique. Les cellules spermiques entrent dans le vas comme précurseurs de la
spermatide immature et se développent en spermatides immatures ou matures à l’intérieur du vas. Cependant, aucun
spermatozoïde n’a été observé dans le tractus reproducteur mâle. Deux types de matière amorphe s’ajoutent aux
cellules spermiques du vas. Le premier type réagit positivement à l’acide périodique Schiff (APS) et déclenche
apparemment la spermiogenèse lorsque présent en quantité importante. Cependant, une plus petite quantité de cette
matière amorphe suffit à former la pellicule basale des spermatophores. Le deuxième type réagit négativement à l’APS
et est responsable de l’épaississement de la paroi du spermatophore. Les spermatides immatures et matures sont
enfermées dans des spermatophores distincts. Les spermatides matures enfermées sont reliées les unes aux autres par
des ponts formés de pointes qui réagissent positivement au Feulgen et sont recouvertes et prolongées de matière
amorphe réagissant positivement à l’APS. Une fois brisés, ces ponts apparaissent comme des bras qui rayonnent à
partir d’une spermatide. Les spermatides matures périphériques sont en outre rattachées à la paroi du spermatophore
par des fils de matière amorphe réagissant positivement à l’APS. Il se peut que ce réseau de ponts et de fils serve à la
diffusion de substances externes vers les cellules centrales à travers la paroi du spermatophore.
Sainte-Marie and
Sainte-Marie:
I 450
Introduction
Some aspects of the morphology of the internal reproductive organs and of spermatogenesis have been described for
males of the snow crab, Chionoecetes opilio (Brachyura:
Majidae). Each of the paired internal reproductive tracts of
Received May 21, 1998. Accepted November 17, 1998.
G. Sainte-Marie. Département de pathologie et biologie
cellulaire, Université de Montréal, C.P. 6128, Succursale
Centre-Ville, Montréal, QC H3C 3J7, Canada.
B. Sainte-Marie.1 Division des invertébrés et de la biologie
expérimentale, Institut Maurice-Lamontagne, Ministère des
pêches et des océans, 850 route de la Mer, C.P. 1000,
Mont-Joli, QC G5H 3Z4, Canada.
1
Author to whom all correspondence should be addressed
(e-mail: [email protected]).
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the male consists of a testis connected via tubules with a socalled vas deferens, which passes its contents to the exterior
by way of an ejaculatory duct (Kon and Honma 1970). The
vas deferens has further been tentatively divided into two or
three parts, based on morphological features and contents.
Kon and Honma (1970) distinguished an anterior part consisting of short wart-like sacs and a posterior part consisting
of long caeca. Sapelkin and Fedoseev (1981) called the vas
deferens an “appendage” in which they recognized an anterior
“spermatophorogenous tubule,” a middle “spermatophorotheca,” and a posterior “rosette.” Spermatogenesis reportedly
unfolds in the testis, and sperm cells leaving the testis are
considered to be spermatozoa that become enclosed in spermatophores in the anterior part of the vas deferens (Kon and
Honma 1970; Sapelkin and Fedoseev 1981; Beninger et al.
1988; Chiba et al. 1992). The middle part of the vas deferens
apparently serves to store spermatophores, while the posterior part, or rosette, may be devoid of spermatophores and
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Sainte-Marie and Sainte-Marie: I
may have only a secretory role (Sapelkin and Fedoseev
1981; Beninger et al. 1988).
As part of a broader investigation of the mating system of
the snow crab, we initiated a light-microscopy study of the
internal reproductive organs of the female (Sainte-Marie and
Sainte-Marie 1998). During that study, we noted that inseminated females stored three hitherto unreported types of spermatophores containing either immature spermatids, mature
spermatids, or spermatozoa (Sainte-Marie and Sainte-Marie
1999). This unexpected finding prompted us to reexamine
the vas deferens of males. In the present paper, we show that
spermiogenesis, the differentiation of immature spermatids
into mature spermatids, does not occur in the testis but instead can occur in the vas deferens. We also reveal the existence in the male of different spermatophore types enclosing
either immature spermatids, mature spermatids, or cell forms
intermediate between these two types.
Materials and methods
We examined the vasa deferentia from 10 males collected in
Baie Sainte-Marguerite, eastern Canada (ca. 50°06′N, 66°35′W),
during late April 1996. The males were adults (Sainte-Marie et al.
1995) with the following combinations of carapace width (mm)
and chela height (mm): 114/29, 109/27, 105/29, 102/27, 101/21,
100/24, 93/21, 92/20, 92/19, and 90/19. Males had exoskeletons in
the intermediate condition, i.e., a hard shell with light epibiont
fouling (Sainte-Marie et al. 1995).
The dorsal carapace of males was raised to expose the internal
organs, and their vasa deferentia were prefixed in situ for about an
hour with Bouin’s solution containing 1.5% acetic acid and 1.5%
trichloracetic acid added immediately prior to use. The vasa deferentia were then dissected out and fixed in the same solution for
2 d while being continuously and gently agitated, and then were
washed for 4 months in renewed 70% ethanol baths until most of
the picric acid was gone. Segments of each vas deferens were
paraffin-embedded and cut serially in 7- µm-thick longitudinal or
transverse sections. One out of 10 tissue sections from each segment of the vas deferens was mounted and stained using the
Dominici, Feulgen, periodic acid – Schiff (PAS), or PAS–methyl
(PAS-M) method (Luna 1968). Note that fixed vas deferens contents were very brittle, rendering manipulations difficult, as remarked upon by Beninger et al. (1988). Thus, organ contents or
walls were often damaged.
Results
A transverse tissue section through the vas deferens
showed several similarly sized rounded structures containing
sperm cells, each representing an oblique to cross cut of the
apparently coiled or ramified vas deferens. We made no attempt to clarify the tridimensional organization of the vas
deferens, as this was beyond the scope of the present study.
For sake of simplicity, we refer to each sectioned structure
as a “cut” of the vas deferens. There was little heterogeneity
in the contents of neighboring cuts of the vas deferens present in any given tissue section, but the contents of cuts observed in distant segments of the organ differed greatly.
Based on observations of longitudinal as well as transverse
tissue sections, processes unfolding in the vas deferens appeared to change as the distance from the testis increased,
according to the following sequence: (i) introduction of
young sperm cells and amorphous matter, (ii) development
441
of immature spermatids, (iii) differentiation of spermatids,
(iv) formation and storage of spermatophores, and (v) accumulation of amorphous matter. The observations reported
below are from transverse tissue sections of the vas deferens
stained by the Dominici method, unless stated otherwise.
Introduction of young sperm cells and amorphous
matter
Most cuts of the vas deferens in tissue sections taken
nearest to the testis were loaded with a dense and homogeneous population of “young sperm cells” (Fig. 1A). These
cells were rounded, pinkish, and very pale except for a blue
“dark ring” present at the surface of one cell pole (Fig. 1B).
The ring delimited a minute intrusive colorless area that apparently corresponded to a shallow cell-surface invagination.
Other nearby cuts showed comparable but slightly more
evolved cells whose dark ring was even more prominent
(Fig. 1C). In this case, one half of the ring appeared to be
homogeneously dark, whereas the other half appeared as a
row of small and slightly lighter dots (Fig. 1D).
Cuts taken farther along the vas deferens revealed additional changes to sperm cells that took place before the enclosure of cells in spermatophores (Figs. 1E–1F). Initially,
under the dark ring of the more evolved young sperm cells
there appeared a small, light brownish “hemispherical component,” below which a thin, colorless crescentic area was
detected (Fig. 1G). The cell invagination extending within
this hemispherical component became more obvious, while
the ring, hemispherical component, and invagination widened and the related cell pole became flatter. Simultaneously, the cell margin at the opposite cell pole thickened
and darkened somewhat, forming a pale blue “chromatin
cap” as demonstrated by its Feulgen-positive reaction. The
central part of the cap often was produced into a “spike-like
structure” (Fig. 1G). With further development of a cell, the
hemispherical component appeared to elongate and give rise
to a short, narrower, and somewhat tubular element that
joined the chromatin cap at the base of its spike (Fig. 1H).
Completion of these changes resulted in the presence of a
brownish “funnel-like apparatus” crossing the center of the
cell, with two tiny colorless rounded areas (possibly centrioles) appearing at the junction of the wide and narrow parts
of the apparatus (Fig. 1I). The resulting cell had a hexagonal
shape that was best seen in polar view (Fig. 1J). Overall,
such a cell was still pink and its funnel-like apparatus was
less evident than its dark ring. Cells at this developmental
stage will be tentatively referred to as “immature spermatids.”
In the anterior part of the vas deferens, besides the many
sperm-loaded cuts described above, one or a few peculiar
structures were also present. These structures were very
large relative to sperm-loaded cuts of the vas deferens and
were filled only with dark blue matter (Fig. 2A), which
we call “type I amorphous matter” (IAM). This matter was
PAS-positive and seemed to be homogeneous at low magnification, but higher magnification revealed that it was actually composed of little spheres. There was no evidence that
IAM was secreted locally by the wall of the vas deferens.
Indeed, sperm-loaded cuts of the vas deferens closest to the
testis contained no detectable or only tiny amounts of IAM
that were restricted to one side of the periphery of a cut, of© 1999 NRC Canada
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Fig. 1. (A) A transverse tissue section of the vas deferens of male 6, nearest to the testis. In this cut of the vas deferens, the lumen is
loaded only with rounded “young sperm cells,” which are very pale except for a “dark ring” at one cell pole. Dominici’s stain. About
×825. (B) Part of A, enlarged. The pale area within a dark ring (arrows) corresponds to a slight cell invagination. About ×1650.
(C) Another cut of the vas deferens in the tissue section shown in A. The young sperm cells are somewhat more evolved, their dark
rings being better developed and obvious. About ×825. (D) Enlargement of cells shown in C. One half of each dark ring looks
homogeneous, the other half is visible as a row of lighter small dots (arrow). About ×1650. (E) Another cut of the vas deferens near
that shown in A; however, unenclosed cells have darkened upon exposure to blue IAM and have become spermatids (see G–H). About
×1650. (F) Cells enclosed in this spermatophore are more evolved spermatids (see I–J). The arrow indicates the spermatophore wall.
About ×1650. (G) Part of E enlarged. Cells have developed a small dark “hemispherical component” beneath their dark ring. This
component, in turn, is underlined by a thin, pale crescentic area. At the opposite cell pole, a dark “chromatin cap” is present and is
often produced into a “spike” (arrow). About ×3200. (H) Part of E enlarged. In some cells the hemispherical component is followed by
a narrow part (arrow), which appears to join the spike of the chromatin cap. About ×3200. (I) Part of F enlarged. A central “funnellike apparatus” extends from the dark ring to the chromatin cap of a cell. The part of the apparatus near the dark ring is widemouthed. At its margin with the narrow part of the apparatus, there are two small colorless rounded areas (arrow). Overall, the cells
are hexagonal (arrowhead). About ×3200. (J) Part of F enlarged. The hexagonal shape of spermatids is more obvious where cells are
seen in polar view (arrowhead). About ×3200.
ten occurring in the form of a rounded mass or a single
strand (Figs. 2B–2D). Other vas deferens cuts close to the
testis were partitioned into two very unequal parts by a fine
membrane (Fig. 2C). The large part contained only a dense
and homogeneous population of pinkish young sperm cells,
while the small part contained a little IAM. Farther along
the vas deferens, the small part widened and IAM became
more abundant and split into droplets. Where the partitioning membrane faded, IAM remained restricted to the periphery, and most often only to one side, of the vas deferens.
With increasing distance from the testis, the quantity of IAM
in all cuts progressively increased and IAM mixed with the
population of young sperm cells to varying degrees. In some
cuts, IAM was mixed with young sperm cells only in a
restricted peripheral site; in other cuts, IAM had spread
broadly and mixed with a large portion, or all, of the cell
population (Fig. 2E). In cuts of the vas deferens where the
distribution of IAM was restricted, young sperm cells developed into immature spermatids only where the amorphous
matter was present (Fig. 2F).
Spermatid differentiation
The subsequent differentiation of immature spermatids
into mature spermatids unfolded wherever a substantial
quantity of IAM was mixed with the cell population. IAM
appeared to form a thin veil at the surface of each cell, and
an initially pink cell acquired a light blue hue. Simultaneously, the cell’s chromatin cap and funnel-like apparatus
became more conspicuous, but the apparatus was thereafter
progressively masked as a cell turned a darker blue. The detected chromatin was concentrated in the chromatin cap,
which appeared to cover about half of the cell opposite to its
flat pole. Faintly visible Feulgen-positive spikes radiated
from the angles of such a hexagonal cap, and under favorable conditions a similarly slightly Feulgen-positive spike
could be observed protruding outward at the narrow end of
the funnel-like apparatus. The most striking visible change
that took place during spermatid differentiation was the formation of the acrosome, revealed in tissue sections stained
with PAS, which typically colors the acrosome purple. The
immature spermatids were PAS-negative, while the mature
spermatids stained dark purple. Therefore, in mature spermatids the funnel-like apparatus was detected only as a
lighter area in equatorial view or as a colorless core in a polar view of the flat cell pole (see Fig. 2D in Sainte-Marie
and Sainte-Marie 1999). Differentiated cells with these new
characteristics will be referred to as “mature spermatids.” It
is noteworthy that the latter were the most developed form
of sperm cell observed in the vasa deferentia of all examined
adult males.
Spermatophore formation and storage
Both immature and mature spermatids were enclosed in
spermatophores. The early stage of spermatophore formation
was most easily observed in areas where pink immature
spermatids became enclosed, their light color contrasting
with the dark basal pellicle outlining nascent spermatophores. Indeed, a spermatophore wall first consisted of a
thin pellicle of blue IAM. In places where such pellicles
formed, the luminal surface of the epithelium of the vas deferens was wavy, the distance between wave crests approximating the diameter of the spermatophores (Figs. 3A–3B).
Each crest was capped with a small triangle of IAM. Thin
projections of IAM extended between the tips of neighboring triangles, from the tips of the triangles to the tips of
stellate masses of IAM that were present deeper in the lumen of the vas deferens (Fig. 3B), and between the tips of
neighboring stellate masses. These IAM projections delimited clumps of sperm cells and formed the basal pellicle of
nascent spermatophores, this pellicle initially being common
to contiguous spermatophores. There was relatively little
IAM inside spermatophores, but important amounts were
present between spermatophores in the form of the abovementioned stellate masses (Figs. 3A–3B). In occasional
places where there were no sperm cells, projections of IAM
still formed a reticulum similar to the basal pellicle of spermatophores.
Farther along in the lumen of the vas deferens, the late
stage of spermatophore formation was preceded by the introduction of a second type of amorphous matter. “Type II
amorphous matter” (IIAM) was PAS-negative and stained
pink–red with the Dominici method and, as was the case for
IAM, was not observed to be secreted locally by the vas
deferens wall. After IIAM appeared, the blue basal pellicle
of IAM that delimited contiguous clumps of sperm cells
divided and a relatively thick band of IIAM spread in be© 1999 NRC Canada
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Fig. 2. (A) The tissue section from male 6 seen in Fig. 1, showing a large structure filled only with dark blue IAM. About ×35.
(B) Tissue section near that seen in Fig. 1A, showing part of a IAM-loaded structure in the upper right-hand corner. The smaller pale
structures below are cross to oblique cuts of the vas deferens filled with pale young sperm cells as shown in Figs. 1A and 1C. A tiny
amount of dark IAM occurs at a restricted site at the periphery of most cuts of the vas deferens (arrowheads). About ×20. (C) Tissue
section seen in A, showing a cut of the vas deferens filled with pale young sperm cells. The arrowheads point to drops of IAM
present in spaces within the epithelium of the vas deferens. The arrows indicate a small area at the periphery of the lumen of the vas
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deferens that is delimited by a very fine membrane and contains tiny dark droplets of IAM. About ×210. (D) Tissue section seen in A.
The lumen of this cut of the vas deferens is loaded with immature spermatids that are darker than the young sperm cells shown in C.
Beneath the epithelium, a canal (arrows) carries dark IAM. Below, a peripheral part of another cut of the vas deferens is present. The
latter contains pale young sperm cells, few of which are seen here. To the right of the right-hand arrow, a rather large drop of IAM
occurs at the inner surface of the epithelium of the vas deferens. About ×80. (E) A cut of the vas deferens from the tissue section seen
in A, shows the mixing of dark droplets of IAM (arrow) with darkened immature spermatids unenclosed in a spermatophore. About
×825. (F) A cut of the vas deferens from the tissue section seen in A. The bottom part contains pale young sperm cells that have not
yet mixed with dark IAM. The upper part contains dark immature spermatids that have mixed with drops of IAM (arrow). About × 825.
tween, coating the two IAM pellicles that resulted. Spermatophore walls then appeared to be multilayered (Fig. 3C).
Moreover, spheres of IIAM were gradually incorporated
within the stellate masses of IAM that occurred between
spermatophores and, there, eventually predominated over IAM.
No IIAM was observed inside spermatophores.
Spermatophores generally sheltered a homogeneous population of either pink immature spermatids or blue mature
spermatids, but some contained a mixture of the two cell
types (Fig. 3D). Spermatophores sheltering different populations of spermatids tended to be topographically segregated
within a given cut of the vas deferens (Figs. 3A and 3D).
Corresponding spatial segregation of immature and mature
spermatids was also seen inside heterogeneously populated
spermatophores, which occurred at the interface between
patches of spermatophores with distinct cell types.
As spermatophore formation progressed, the remaining
droplets of IAM in spermatophores enclosing mature spermatids appeared to form fine bridges that linked individual
cells (Fig. 4A). As was the case for IAM projections forming the basal pellicles of spermatophores, the bridges exhibited scattered droplets of IAM (Figs. 4A–4C). Except for
these bridges, the intercellular space appeared to be devoid
of amorphous matter and was colorless. Actually, the delicate IAM bridges were not readily observable; they were
more easily detected where individual cells were at a
distance from one another. Furthermore, obtaining photographic evidence of these bridges required that micrographs
be overexposed (Figs. 4A–4E). The bridges were, in fact,
extensions of IAM that coated the Feulgen-positive spikes
protruding from the angles of the hexagonal spermatids. The
bridges varied in length as a function of intercellular distance, and where broken, because of cutting or tissue damage, they appeared as arms that radiated from individual
cells. Moreover, IAM threads linked the peripheral spermatids to the spermatophore wall, where they appeared to be
anchored in the thicker IIAM coat of the wall by a terminal
droplet of IAM (Figs. 4D–4E). Finally, just as a reticulum of
fine IAM projections could form in a vas deferens site lacking sperm cells, a network of IAM threads could also form
at a peripheral spermatophore site without sperm cells.
Accumulation of amorphous matter
A short distance before the rosette, the vas deferens contents changed from a dense accumulation of spermatophores
interspersed mostly with IIAM to a mixture of both types of
amorphous matter lacking, or containing only a scattering
of, spermatophores. The rosette formed a rounded cluster of
elongate caeca and was up to a few times wider than the preceding portion of the vas deferens. From the center of the
posterior end of the rosette emerged a narrow ejaculatory
duct. The caeca of the rosette had a markedly thinner epithelium than the anterior and middle parts of the vas deferens
and they were filled with either IAM or IIAM or a mixture
of the two. Caeca with a similar content appeared to be topographically segregated. As a rule, the rosette’s caeca were
devoid of spermatophores, but one male had a few caeca
containing scattered spermatophores.
Discussion
Site of spermiogenesis
Our observations reveal that sperm cells which leave the
testis and enter the vas deferens are not spermatozoa. The
initial network of saccular or tubular structures with a thin
epithelium, which might represent the terminal segments of
testicular tubules extending into the intricate anterior mass
of the vas deferens, contained only variably developed precursors of the immature spermatid (Figs. 1A–1B). Further
development of these cells proceeded within the vas deferens, as was shown by profound changes in sperm cell
morphology and staining properties over some distance from
the testis. In this respect, it is interesting to note that in the
case of the snow crab, Sapelkin and Fedoseev (1981) hinted
that sperm cells continue to develop in the vas deferens,
while Fedoseev (1988) commented that various staining techniques revealed a difference in the degree of development
between so-called spermatozoa in the testis and in spermatophores of the middle part of the vas deferens. The differentiation of spermatids in the vas deferens has been documented
in other crustacean decapods, such as the shrimp genera
Penaeus (King 1948) and Sicyonia (Shigekawa and Clark
1986; Subramoniam 1995), but to our knowledge this is the
first report for a brachyuran crab.
Morphology of sperm cells
Given that most of our observations on the morphology of
sperm cells in the vas deferens agree with those presented in
earlier reports (Sapelkin and Fedoseev 1981; Beninger et al.
1988; Chiba et al. 1992), and that Sainte-Marie and SainteMarie (1999) observed in the spermathecae of inseminated
females a form of sperm cell still more evolved than the mature spermatid described here, it appears that cells previously
interpreted as spermatozoa were in fact spermatids. A preliminary description of the snow crab spermatozoon and a
discussion concerning the site of transformation of mature
spermatids into spermatozoa is presented in Sainte-Marie
and Sainte-Marie (1999).
The present study provides additional information on
some controversial aspects of the morphology of sperm cells
found in the vas deferens of the snow crab, regardless of
their proper denomination. Sapelkin and Fedoseev (1981) re© 1999 NRC Canada
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Fig. 3. (A) A transverse tissue section of the vas deferens of male 6, farther from the testis than the section seen in Fig. 2. The cut of
the vas deferens is loaded with nascent spermatophores. The palest spermatophores contain pale young sperm cells; the darkest ones
contain dark spermatids. Spermatophores with a mixed cell population occur. The arrowheads indicate triangles of dark IAM covering
epithelial crests. The arrow points to a site enlarged in B. Dominici’s stain. About ×80. (B) Part of A enlarged. A thin projection of
IAM forms the basal pellicle of the nascent spermatophore. Inset: Enlargement of the pointed site. The IAM projection extends from
the tip of a peripheral triangle of IAM (lower arrowhead) to the tip of a deep interspermatophoral, grossly stellate mass of IAM (upper
arrowhead). Little IAM is present in the spermatophores. (C) Tissue section of the vas deferens more distant from the testis than the
section seen in A. The vas deferens contains better developed spermatophores with a thick band of pale IIAM between the thin and
darker basal IAM pellicle of neighboring spermatophores whose wall is double- (or triple-) layered, as can be seen enlarged in the
inset. Both arrows point to the same site. About ×825. (D) The same tissue section as in A, showing round to kidney-shaped cross
cuts of the vas deferens. Each cut contains a variable proportion of spermatophores loaded with pale immature or dark mature
spermatids. Spermatophores with an intermediate cell population occur. Spermatophores containing either cell type are topographically
segregated within individual cuts of the vas deferens. The cut indicated by the arrow is that shown enlarged in A. About ×20.
Fig. 4. (A) Overexposed micrograph of a section from male 6, showing the reticulum of fine IAM bridges that link the spermatids
together in a spermatophore of the vas deferens. Tiny droplets of IAM are often scattered along individual bridges (arrow at left).
Dominici’s stain. About ×800. (B and C) Same section as seen in A. Both spermatophores are sectioned close to their inner surface,
revealing the density of the network of IAM bridges and threads at the periphery of spermatophore’s contents. (D) Same section as
seen in A, showing the anchoring of IAM threads in the relatively thick coat of paler IIAM of a spermatophore wall. Near the bottom,
the arrow points to a portion of the thin layer of IAM constituting the basal pellicle of a nascent spermatophore. A still darker print
would better show the many (here hardly detectable) threads linking the spermatophore wall to peripheral sperm cells. (E) The arrow
indicates a particularly thick IAM thread linking the spermatophore wall to a partially visible cell. Note the density of the superficial
IAM reticulum above this thread.
ported that so-called spermatozoa had a hemispherical, hollow capsule, with the chromatin forming a narrow ring at the
margin of the capsule. This ring is illustrated in their schematic Fig. 5 and is also detectable with some difficulty in
smeared cells in their Fig. 4. The smearing method they
used appears to have artifactually caused the detachment of
the ring from the wider extremity of the central funnel-like
apparatus and partial extrusion of the apparatus from the
cell. Beninger et al. (1988) did not observe such a ring, but
suggested that it may have been a structure analogous to the
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448
“granular belt” described for spermatids of Cancer spp. (see
Langreth 1969). The ringlike structure described by Sapelkin
and Fedoseev (1981) seems to correspond to the dark ring
on the flat cell pole at the margin of the central funnel-like
apparatus of spermatids in the present study (Fig. 1D). However, the dark ring in the snow crab (this study), like the
granular belt of Cancer spp. (Langreth 1969), does not appear to contain chromatin.
The presence of “radial arms” on snow crab sperm cells
was not mentioned by Sapelkin and Fedoseev (1981), but
was reported by Beninger et al. (1988) and Chiba et al.
(1992). The two last teams of investigators concluded that
the arms were nuclear in nature; our observations indicate
that they result from the coating of fine Feulgen-positive
spikes by IAM. Chiba et al. (1992) found that the arms numbered 4–10 per sperm cell, and examination of their Fig. 2
suggests that one of these processes may represent the spikelike structure seen projecting from the chromatin cap at the
end of the funnel-like apparatus in the present study
(Fig. 1G). We further observed that the arms are, in fact, the
remnants of broken intercellular bridges that form a complex
network with connections to the spermatophore’s wall (this
study; Sainte-Marie and Sainte-Marie 1999).
The most conspicuous and a key diagnostic feature of
the mature spermatid of the snow crab was its acrosome,
composed of a PAS-positive vesicle surrounding the PASnegative funnel-like apparatus. This apparatus was termed a
central stem by Sapelkin and Fedoseev (1981) and an acrosomal tubule by Beninger et al. (1988) and Chiba et al. (1992),
and has been given a variety of names in other brachyuran
species (e.g., Krol et al. 1992). While we found this structure to be PAS- and Feulgen-negative, it was also slightly
acidophilic, suggesting a core comprising proteins.
Origins and roles of amorphous matter
Amorphous matter is reportedly secreted by the epithelium of the vas deferens in the male snow crab (Beninger et
al. 1988) and males of several other crustacean decapods
(e.g., Krol et al. 1992). However, based on the present findings, it is clear that the introduction of amorphous matter
into the sperm-loaded cuts of the snow crab vas deferens is
not a topographically pervasive process, and various clues
indicate instead that the source(s) of amorphous matter is
(are) external to the vas deferens. First, large homogeneous
accumulations of IAM or IIAM were observed only in distinct structures neighboring sperm-loaded vas deferens cuts
(this study) and in the posterior vas deferens (Beninger et al.
1988; this study), or rosette. Fedoseev (1988) considered
that the rosette is not a part of the vas deferens, but rather an
accessory gland. Similarly, in the ghost crab, Ocypode
ceratophthalmus, there is a large coral-shaped accessory
gland toward the end of the vas deferens (Sudha Devi and
Adiyodi 1995) that apparently corresponds in topographical
location and in function to the snow crab’s rosette. Second,
our observations revealed point-source introduction (Figs. 2A–
2B) followed by uneven distribution and progressive spreading of IAM in some sperm-loaded cuts of the anterior vas
deferens (Figs. 2C and 3A). Such a pattern would not be expected if secretions were produced broadly by the epithelium of the vas deferens.
Can. J. Zool. Vol. 77, 1999
There is obviously a need for more detailed histological
studies to ascertain the origin, and more histochemical analyses to determine the nature, of the various types of amorphous matter in the male snow crab. Nevertheless, it is clear
that IAM and IIAM fulfill critical roles in the reproductive
process. Overall, it appears that the amorphous matter in
males is involved in mid and possibly late sperm development, as well as in spermatophore formation.
A primer role for IAM in sperm-cell development was revealed by the observation that young sperm cells began to
transform into immature spermatids only upon exposure to
IAM and that spermiogenesis unfolded only where immature
spermatids mixed extensively with large amounts of IAM.
Therefore, the observation that the introduction and distribution of IAM into sperm-loaded cuts of the anterior vas
deferens are uneven suggests controlled production of populations of different types of sperm cells for enclosure into
spermatophores. The frequent occurrence of spermatophores
enclosing only immature spermatids implies that the amount
of IAM necessary to form the spermatophore wall is, however, too small to induce differentiation of spermatids. As to
the occurrence of spermatophores containing immature and
mature spermatids, it can be explained by the formation of
some spermatophores at the interface between contiguous
populations of the two cell types. Thus, the uneven distribution of IAM in cuts of the vas deferens can explain both the
segregated distribution of spermatophores with different
spermatid populations, and the corresponding segregation of
immature and mature spermatids within individual spermatophores bearing both cell types.
The contribution of amorphous matter to the formation of
spermatophores in brachyuran crabs has been known for
some time (e.g., Spalding 1942; Ryan 1967). Beninger et al.
(1988) stated that the snow crab’s spermatophore wall is
formed of a layer of PAS-negative secretion, but they also
reported a weak PAS-positive reaction of the wall. We found
instead that the formation of the spermatophore wall proceeds in two steps: first, a basal pellicle of PAS-positive
IAM that segregates clumps of sperm cells is formed; second, a thick coating of PAS-negative IIAM appears. We suggest that the coating by, and some progressive blending
with, the abundant PAS-negative IIAM partially or largely
masks the PAS-positive reaction of the fine basal pellicle,
rendering its observation difficult. In fact, the process of
spermatophore formation in snow crab is somewhat similar
to that described in other brachyuran crabs. In the majids
Libinia emarginata and L. dubia, a substance initially separates the sperm into discrete clumps before the reportedly
single-layered wall is formed (Hinsch and Walker 1974). In
the portunids Scylla serrata and Portunus pelagicus, the
spermatophore wall is composed of a thin inner layer and a
thick outer layer (Uma and Subramoniam 1979; El-Sherief
1991). Double-layered spermatophore walls are also a feature of species of the geryonid Chaceon (as Geryon in
Hinsch 1988), the ocypodid Uca pugilator (Becker 1983,
cited in Hinsch 1991) and the raninid Ranina ranina
(Minagawa et al. 1994). In the last species, the inner spermatophore layer is reportedly PAS-negative, while the outer
layer is PAS-positive, contrary to the present observations
on the snow crab. Our observation that the wall of the snow
crab’s vas deferens is wavy at sites of spermatophore formation
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suggests that contraction of the vas deferens contributes to
molding sperm cells into discrete clumps, as is proposed
for other crustacean decapods (e.g., Spalding 1942; Dudenhausen and Talbot 1983).
Function of spermatophores
The function of spermatophores in the reproductive process of brachyuran crabs, during which the male passes his
reproductive products internally to the female, is still debated. Some investigators view the brachyuran spermatophore as a vestigial structure whose original function was to
protect the sperm cells against a hostile environment in species where the male transfers his reproductive products externally to the female. The brachyuran spermatophore would
now serve merely to package sperm cells for transfer to the
female (Spalding 1942; Hinsch 1988; Subramoniam 1993).
For example, spermatophores of L. emarginata reportedly all
dehisce shortly after being passed to the female (Hinsch
1986). However, Beninger et al. (1988, 1993) found that in
the snow crab, some spermatophores dehisced much more
rapidly than others under experimental conditions, a finding
that they attributed to variations in the state of the spermatophore wall. Therefore, they proposed that a function of the
snow crab spermatophore is to facilitate or delay mobilization of sperm cells so as to favor either immediate use or
long-term storage, and thus reduce sperm wastage.
In light of the present observation that spermatophores
can enclose sperm cells at different stages of development
and are passed along to females as such (Sainte-Marie and
Sainte-Marie 1999), we formulate the following proposal.
The spermatophore wall may serve to protect the enclosed
sperm cells from substances in the spermatheca that can induce their full development before they are needed for fertilization, and this protection increases their potential for
survival in long-term storage. When needed, modification of
the internal environment of the spermatheca could alter the
spermatophore wall, thus allowing diffusion of substances
that could lead to further development of the stored cells. Indeed, we found that peripheral enclosed cells had a polarized
orientation with respect to a spermatophore wall (SainteMarie and Sainte-Marie 1999) and were connected to the
wall and to other cells by a network of IAM bridges and
threads (this study). This suggests that the peripheral cells
are “feeding” at the spermatophore surface and that substances penetrating the spermatophore wall from the exterior
can diffuse even to the innermost stored cells. Additionally,
there may be a correlation between the type of enclosed
sperm cell and the dehiscence characteristics of a spermatophore.
Diffusion of substances into a spermatophore likely depends on the nature and organization of the constituents of
its wall. In the snow crab, however, the exact nature of these
constituents is uncertain, and permeability to chemical
substances remains undetermined. Sapelkin and Fedoseev
(1981) reported that the wall is composed, at least in part, of
proteins and polysaccharides. By contrast, Beninger et al.
(1988) concluded that the pellicle is not rich in proteins and
suggested that is chitinous. In S. serrata, both spermatophore layers are rich in proteins and polysaccharides, and
the outer layer is apparently composed of chitin. The inner
layer is structurally weak, while the outer layer is structur-
449
ally resistant but may nonetheless be permeable to low molecular weight substances (Uma and Subramoniam 1979).
Observations in the present study and in Sainte-Marie and
Sainte-Marie (1999) shed no light on the elemental composition of the spermatophore wall of the snow crab. However,
they do strongly indicate that the wall is not inert and that its
organization and properties change during ontogeny and reproductive events in response to internal and external factors, so as to delay or promote the development and release
of enclosed sperm cells.
Vas deferens or epididymis?
In accordance with the current trend in crustacean literature, we have used the term vas deferens to designate the
part of the male reproductive tract in which sperm cells are
mixed with amorphous matter, partly mature, and become
enclosed in spermatophores. However, the nature and complexity of the events unfolding in this organ lead us to question the appropriateness of the term vas deferens. A similar
organ is the site of similar processes in other animal taxa,
such as sharks (e.g., Jones et al. 1984) and the better studied
mammals (e.g., Weiss 1988, although spermatophores do not
form), yet there is a major and irreconcilable difference between the names that are used to designate this organ. Based
on its roles and topographical relationship to the testis, the
organ commonly termed vas deferens in crabs corresponds
to the epididymis of sharks and mammals. Epididymis, etymologically meaning an organ resting on the testis, seems
to be more appropriate than vas deferens to designate the
duct receiving cells from the testis. This view was first
propounded for the snow crab by Sapelkin and Fedoseev
(1981), who noted that the term vas deferens more properly
applies to a conduit that carries the mature ejaculatory products to the exterior, i.e., the present ejaculatory duct. We also suggest that the term vas deferens as it is applied to
brachyuran crabs should be reconsidered.
Acknowledgments
We thank M. Pelletier and F. Pothier for discussion on the
morphology of sperm cells and the male reproductive tract,
G. Guay for preparation of tissue sections, and J. Léveillé
for photographic work. Financial support for micrography
was provided by the Department of Fisheries and Oceans,
Government of Canada.
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