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ria
a Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
b Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, New York 10021
ABSTRACT
In the musk shrew, Suncus murinus (and other shrews), the cumulus oophorus is ovulated as a discrete, compact, matrix-free ball of cells linked by specialized junctions. In examining how they penetrate the cumulus, Suncus spermatozoa were observed to first bind consistently by the ventral face over the acrosomal region to the exposed smooth surface of a peripheral cumulus cell. This was apparently followed by point fusions between the plasma and outer acrosomal membranes. Thereafter, spermatozoa without acrosomes were observed within cumulus cells that displayed signs of necrosis, as did some radially neighboring cumulus cells linked by zona adherens and gap junctions. Eventually, penetration of spermatozoa as far as the perizonal space around the zona pellucida left linear tracks of locally necrotic cells flanked by normal cumulus cells. Based on these and previous observations, we conclude that the acrosome reaction in Suncus is always induced by cumulus cells, and that reacted spermatozoa penetrate the cumulus by selective invasion and killing of cumulus cells along a linear track. Loss of the acrosome also exposes an apical body/perforatorium that is covered with barbs that appear to assist reacted fertilizing spermatozoa in binding to the zona pellucida. Because fertilized eggs displayed no other spermatozoa within or bound to the zona, an efficient block to polyspermy must prevent such binding of additional spermatozoa.
cumulus cells, fallopian tubes, sperm, sperm capacitation/acrosome reaction, sperm motility and transport
INTRODUCTION
In eutherian mammals the egg vestments present an unusually formidable barrier to the fertilizing spermatozoon. In contrast to the single generally thin (0.5–1.0 µm) egg coat seen in most biological groups, the newly ovulated eutherian egg is enveloped by a resilient zona pellucida that is about 8–15 µm thick according to species, and outside this by several layers of cells that constitute the cumulus oophorus. There have been relatively few in vivo studies of the timing and pattern of the changes that occur in the eutherian acrosome as spermatozoa penetrate the cumulus oophorus and then bind to the robust zona pellucida. Most recent analyses, conducted in vitro and generally in the mouse, have led to a concept that mammalian spermatozoa pass through the cumulus and bind to the surface of the zona pellucida with the acrosome generally still intact, then undergo the acrosome reaction in response to one zona component, ZP3 (e.g., [1]). On the other hand, it is clear that some spermatozoa may undergo the membrane fusion step of the acrosome reaction already within the cumulus, in rabbit and man and the hamster for instance [2–4], though in these and other mammals used commonly for fertilization research, fertilization can occur in the absence of the cumulus.
As a variant to the mammals that have been studied most, recent investigations of gamete function in putatively more primitive mammals, the Insectivora, suggest that the cumulus has an essential role for fertilization, at least in the Soricidae (shrews). In shrews the cumulus appears as a compact hyaluronidase-insensitive ball of cells devoid of intercellular matrix [5], in contrast to the often-diffuse hyaluronic acid matrix-rich structure ovulated in many mammals. In one soricine shrew tested, Cryptotis parva, eggs were not fertilizable in the absence of the cumulus, and most acrosomes were lost in its cumulus that undergoes a late mucification in the oviduct [6]. In another (crocidurine) shrew, Suncus murinus, the giant form of the acrosome made it possible to see that this fan-like organelle is lost in all spermatozoa present within the cumulus around unfertilized and fertilized eggs, whether motile or not, and in any spermatozoa binding to the zona surface of unfertilized eggs [5, 7]. The same was true for Crocidura russula [8], another crocidurine shrew with a similarly large acrosome [9]. The evidence suggests, therefore, that the cumulus around the eggs of shrews has an essential role in fertilization—most likely as the inducer of the acrosome reaction.
In the perifertilization period in the crocidurine shrews, S. murinus and C. russula, acrosome-free spermatozoa within the cumulus typically congregate within the perizonal space—a cavity brought by withdrawal of the otherwise intact cumulus from around the zona pellucida [7, 8]. However, in reaching that space, it is not clear how the spermatozoa penetrate a barrier of cumulus cells linked at multiple sites by specialized cell-cell junctions or at what point in its interaction with the cumulus the acrosome of Suncus begins to react; and finally, whether the reaction involves the point fusions between the plasma and outer acrosomal membranes seen in other mammals.
In the present study, we have addressed these questions by means of electron microscopy and immunocytochemistry performed on cumulus-oocyte complexes during the fertilization period, in the oviduct of naturally mated females, or in vitro. At the same time we have obtained further evidence for the disposition of the saw-toothed apical body or perforatorium seen in crocidurine spermatozoa [9, 10] (and in modified form in other insectivores [11]), as acrosome-free spermatozoa interact with the zona pellucida.
MATERIALS AND METHODS
Investigations were conducted in accordance with the National Research Council (NRC) publication Guide for Care and Use of Laboratory Animals (copyright 1996, National Academy of Science). Shrews of 3 mo or more, originating from stocks caught in Katmandu (KAT) and in Bangladesh (BK), were raised at Kyushu University or at the Laboratory of Animal Management and Resources, Graduate School of Bioagriculture Sciences, Nagoya University. The shrews were housed individually on wood chips in plastic cages of 39 by 21 by 15 cm for males, and of 35 by 20 by 12 cm for the smaller females, with a 12L:12D photoperiod, at 20–25°C. Water and food were available ad libitum, the food being a commercial chow for trout, Hipro 5p (Nippon Formula Feed Manuf. Co., Ltd., Yokohama, Japan). In the mating experiments copulation was observed when a receptive female was introduced into the cage of a fertile male. Such males were used no more than once every 4 days.
Spermatozoa
To obtain spermatozoa, the cauda epididymidis was removed from a male killed with ether. Some of the spermatozoa emerging from an incision were placed on a slide beneath a coverglass, in Krebs Ringer Bicarbonate medium (mKRB) (Sigma Chemical Co., St. Louis, MO) containing NaHCO3, CaCl2, minimum essential medium (MEM) amino acids solution, and MEM nonessential amino acids solution (Life Technologies, Inc., Rockville, MD). These spermatozoa were then studied with differential interference contrast (DIC) optics. In addition, small pieces of intact cauda epididymidis were fixed in 3% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.4), washed and postfixed in 1.3% osmium tetroxide, dehydrated with alcohols, and embedded in epoxy resin. Thin sections of 
60 nm, cut with a diamond knife, were mounted on copper grids, stained with uranyl and lead acetates, and examined in an electron microscope (Hitachi H-600A, Tokyo, Japan) at 75 kV.
Cumulus Oophorus
Ampullae containing cumulus-oocyte complexes were fixed as above between 15.5 and 26.5 h after coitus, and thick sections (1.5 µm) cut with glass knives were mounted on glass slides, stained with toluidine blue, and studied in a light microscope. In addition, cumuli released by stripping the ampulla with fine forceps some 15.5–16 h after coitus were fixed in 1% paraformaldehyde-3% glutaraldehyde [12], embedded in 1.5% agar, postfixed in 1.3% osmium tetroxide, and embedded for electron microscopy.
In exploring the mode of association between the cumulus cells, the localization of adherens junctions was demonstrated by immunocytochemistry using anti-zonal occludens (anti-ZO-1) antibody. Ovulation was induced by i.p. injection of 30 U of eCG (Teikoku-zoki Pharmaceutical Co., Ltd., Tokyo, Japan), and cumuli were collected 20–21.5 h later. These were fixed in 6% (w/v) paraformaldehyde dissolved in PBS and placed in 0.2% Triton X-100. After blocking in 0.2% gelatin-1% BSA, the samples were stained immunocytochemically with anti-ZO-1 and with Texas red-conjugated anti-rabbit IgG, and then examined in a confocal laser scanning microscope (Olympus LSM-GB 200, Tokyo, Japan).
In order to explore the permeability of the intercellular spaces, cumuli were collected 18.5–20 h after hCG and incubated with wheat germ agglutinin peroxidase (WGA-HRP–50 µg/ml) (EY Labs., Inc., San Mateo, CA) in M199 at 4°C for 20 min. After incubation, cumuli were washed in buffer, fixed in 3% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.4), washed again, and processed in 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB) for 20 min, then in 0.05% DAB-0.01% H2O2 for 15 min. Finally, the cells were washed in buffer, embedded in 1.5% agar, dehydrated in alcohols, and embedded in epoxy resin. Thin sections, cut with a diamond knife, were stained with lead acetate and examined in an electron microscope.
Sperm-Cumulus Relationship
To clarify the relationship between spermatozoa and the cumulus in vivo, ampullae were collected between 21.5 and 29 h after coitus (6–13.5 h after ovulation). Then, segments of ampulla including cumuli in the lumen were fixed in 1% paraformaldehyde-3% glutaraldehyde, washed in buffer, postfixed in osmium, embedded, and sections were examined with the light and the electron microscope. In addition, extruded cumulus-oocytes complexes mounted on glass slides in mKRB were examined directly by DIC. For examination of spermatozoa that had penetrated the cumulus and were lying in the perizonal space, these were extruded under observation from cumuli fractured with a needle, into a drop of mKRB on a coverglass. The live spermatozoa adherent to the surface of the coverglass were then fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, washed in buffer, dehydrated with alcohol, and freeze-dried with t-butanol, sputtered with gold, and examined in a JEOL JSM-5200 scanning electron microscope.
Finally, in order to study the acrosome reaction in vitro, culture dishes containing 0.4 ml mKRB (pH 7.4) under mineral oil were equilibrated in 5% CO2 at 37°C. A sperm bolus oozing from the cauda epididymidis was placed in equlibrated mKRB and incubated at 37°C in 5% CO2 for 2.5 h (5 x 105/ml). Cumulus-oocyte complexes collected 22 h after hCG were cocultured with 20 µl of such a sperm suspension for a further 3 h in equilibrated mKRB. These complexes were then prepared as described above and studied in the light and the electron microscope.
RESULTS
Spermatozoa
The sperm nucleus and a large fan-shaped acrosome were clearly evident in the light microscope (Fig. 1). As reported previously, electron microscopy revealed that the apical body or perforatorium has a saw-tooth configuration (Fig. 2). The matrix of this near to the nuclear membrane was composed of fine granules and presented a higher electron density than that within the barbs. In an arrangement that seems significant for the tail beat pattern, the outer dense fibers 5 and 6 were relatively large.
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Cumulus Oophorus
About 15.5 h after mating or hCG, eggs are ovulated within a mass of cumulus cells that are closely apposed to each other, consist of about six layers, and are insensitive to hyaluronidase [5]. Within the cumulus, a perizonal space began to form immediately before ovulation, growing bigger in the first hours after ovulation (Fig. 3), but the cumulus remained intact and maintained a compact spherical shape. The cumulus persisted as such during its penetration by spermatozoa that occurs only several hours after ovulation and for several hours after fertilization (Figs. 3 and 4). The cells of the outermost layer adhered to one another through adherans and gap junctions at several sites (Fig. 4, and inset). As can be seen in Figure 3, the outer surface of the cumulus was generally flat without gaps between the outer cells, and the periphery of the cells contained a rich population of microfilaments beneath the plasma membrane. Immunostaining showed the presence of ZO-1 distributed over the periphery of the cumulus cells (Fig. 5). The external surface of the cumulus revealed a strong affinity for WGA-HRP, some of which passed through the cell-to-cell junction between the cells of the outermost layer and permeated the intercellular spaces of the middle layer (Fig. 6). Experiments using lucifer yellow confirmed that this marker had indeed infiltrated into the perizonal space (unpublished observation).
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While the cells of the deeper region of the cumulus had small populations of microvilli and cytoplasmic processes extending radially, it is important to note, as mentioned earlier, that the inner layer—the corona radiata—had withdrawn from the zona pellucida to leave a perizonal space (Fig. 3). Although the appearance and surface associations of the cumulus cells varied somewhat according to their position in the mass, the ultrastructure of the individual cells was similar—they contained a well-developed endoplasmic reticulum, many rod-like mitochondria of high electron density, large round nuclei, small Golgi complexes, enormous microfilaments (Fig. 4), and glycogen particles. Characteristically, glycogen deposits were present within the cells comprising the central and inner layers of the cumulus.
Sperm-Cumulus Relationship
In mated animals about five eggs were ovulated, and no more than an average total of about 38 spermatozoa ever reached both ampullae. Some 21.5–24 h after mating (and so 6–8.5 h after ovulation), and 3–5.5 h after insemination in vitro, occasional intact spermatozoa had bound to the cumulus surface (Fig. 7) by the ventral face of the large fan-like acrosome (Fig. 8), over which face the plasma membrane is more loosely associated with the outer acrosomal membrane. At this point of contact the surfaces of the cell and sperm head were closely associated and the sperm plasma membrane and outer acrosomal membrane appeared to have vesiculated in places (Fig. 9). Within the cytoplasm of the cumulus cell immediately underlying this close attachment, there were gatherings of microtubules and signs of degeneration as reflected in swollen endoplasmic reticulum (Fig. 8).
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Typically, there were only a few spermatozoa within cumuli collected about 6–11 h after ovulation (21.5–26.5 h after mating). However, whether motile or not, all the spermatozoa within the cumulus lacked an acrosome, and where spermatozoa were present within individual cells a locally degenerative state in these cells was very obvious (Fig. 10). Characteristically, these cells displayed many vesicles, markedly swollen mitochondria with degenerate cristae, and atrophic nuclei. In particular, material of high electron density, numerous vesicles, and abundant microfilaments surrounded the sperm head (Fig. 11). Swollen mitochondria, a reduction in endoplasmic reticulum, and an increased complement of microfilaments were evident also in cumulus cells central to the one penetrated by a spermatozoon, especially near to the contact region. Between degenerate cells containing a spermatozoon and laterally adjacent cells there were few surface contact sites, in contrast to the remainder of the cumulus. Where the spermatozoon had passed with only the mainpiece of the tail remaining, a track constituted by necrotic cumulus cells was now evident in the cumulus (Fig. 12). Notably, the necrotic process was confined to the line of cells within the slit, with no such effect expressed by neighboring cells.
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In this particular series, from one to four spermatozoa occupied each perizonal space, all lacking the acrosome (Fig. 13), and motile to a variable degree with a circular motion observed in some. These spermatozoa released from the perizonal space had apical barbs exposed over the leading edge (Fig. 14), and one such motile spermatozoon was observed adhering to the zona pellucida of an unfertilized egg (Fig. 13). Finally, we note that many vesicles of unknown significance were present around the heads and tails of spermatozoa in the perizonal space (Fig. 15).
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DISCUSSION
Regardless of egg size, the follicular (cumulus) cells are shed before ovulation in nonmammalian vertebrates and even in marsupial mammals. Thus, the cumulus oophorus is a structure unique to eutherian mammals. However, there is no consensus as to the adaptive or functional significance of this eutherian feature, not least because cumulus-free eggs of most mammals can be fertilized and develop normally in vitro and in vivo.
In considering the significance of the cumulus oophorus, it is interesting that this structure in shrews differs in several ways from that of the more commonly studied mammals, including man. In most Eutheria, a gonadotropin-stimulated secretion of glycosaminoglycans by cumulus cells some hours before ovulation [13] ensures expansion of the now matrix-rich cumulus; and as a consequence, all or most of the mature cumulus oophorus is readily dispersed by hyaluronidase. In addition, the mature cumulus is generally rather diffuse and irregular, and in polytocous species, such as rabbits and rats, several cumuli coalesce after ovulation to form one mass. Finally, as mentioned earlier, in many mammals the zona-invested egg nevertheless remains quite fertilizable after loss of the cumulus. By contrast, the soricid cumulus is ovulated as a discrete compact ball that remains as such for many hours. It has no matrix and so is resistant to hyaluronidase, with neighboring cumulus cells being associated rather by structural junctions. Finally and most importantly, the soricid cumulus appears to play an essential role in the fertilizability of the egg within it. In the least shrew, C. parva, eggs were not fertilizable in vitro in the absence of an investing cumulus [6]; and in Suncus, as observed here too, all spermatozoa within the intact cumulus lack an acrosome, whether motile or not [5, 7]. These observations suggest that the cumulus rather than the zona is responsible for induction of the acrosome reaction in shrews.
In shedding new light here on the soricid cumulus and the way that spermatozoa respond to it, the present study focuses on several intriguing points. In particular, there is the relationship between neighboring cumulus cells, the induction and the mode of the acrosome reaction, an unusual type of cumulus penetration, and subsequently of sperm binding to the zona pellucida.
The compact nature of the cumulus ball in Suncus is maintained optimally in the presence of Ca2+ (unpublished observations), but in experiments here using WGA (molecular weight [MW] about 36 000)-HRP (MW about 44 000), nevertheless some reaction product was formed between the cells of the central cumulus layers, indicating that some cell-cell associations are permeable to molecules of at least 80 000 MW. While gap junctions have been identified between cumulus cells previously in S. murinus [5], some intercellular junction points were reactive throughout to anti-ZO-1 antibody (Fig. 5). ZO-1 is a support protein binding at the COOH-termini of claudins that are one class of tight junction transmembrane proteins [14]. However, in nonepithelial cells ZO-1 is believed to bind with cadherin through 
-catenin [15, 16], and the dot-like staining for ZO-1 within the cumulus in Suncus points to the presence of adherens junctions there. Moreover, because ZO-1 is involved in signal transmission and stabilization by connection of junctional components with elements of the cytoskeleton [17], ZO-1 may have a role in ordaining cumulus permeability as well as the formation and breakdown of junctions within the cumulus. Such a permeability may allow communication between the egg and cumulus after fertilization, because the cumulus of Suncus dissociates and is lost several hours earlier from around the fertilized than from the unfertilized egg [7].
Previous studies have shown that all spermatozoa within the Suncus cumulus have undergone the acrosome reaction [5, 7] but without any clue as to how and where this reaction occurs. Here, in vitro and in vivo, Suncus spermatozoa first adhered to the flat external surface of an outer cumulus cell by the plasma membrane on one specific side of the head—previously designated as ventral, according to a different binding pattern of cationized ferric oxide colloid [18]. It is very possible that one face of the sperm head functions preferentially at fertilization in several other mammals. The flatter or slightly concave side of the sperm head is apposed to the zona at the onset of successful penetration in the pig, horse, cow, sheep, and rabbit [19, 20] (P.J. Dziuk, personal communication to J.M.B.). Nonetheless, the distinction in molecular character between opposing surfaces of the Suncus sperm head, revealed by the binding patterns of cationic ferric oxide colloid [18], appears to represent the only direct demonstration of different surface properties over one side of the acrosomal region versus the other, in mammals at least. It is striking, therefore, that after insemination of isolated cumuli in vitro, Suncus spermatozoa could adhere only to the outer surface and not to the perizonal or inner surface of the cumulus mass. This suggestion of a specificity in initial interaction of the sperm head with the cumulus cell surface is of interest also in view of the fact that WGA showed a strong and specific affinity here for the external surface of the cumulus. Such an affinity of this lectin suggests the presence of abundant N-acetylglucosamine-like moieties in the surface glycocalyx of the outer cumulus cells. In view of the possible role of this molecule as a sperm receptor on the zona of, for example, the hamster [21], it could have such a role also in Suncus but at the surface of the cumulus rather than of the zona. However, this possibility remains only as speculation, because it is not yet clear that the periacrosomal surface specifically binds N-acetylglucosamine or a similar moiety. Nor is it clear whether it is this or some other cumulus cell-related molecule that is responsible for induction of the acrosome reaction. Further specific studies are underway to examine these critical points.
Electron microscopy of the site of adhesion of the sperm plasma membrane to an outer cumulus cell surface (Fig. 9) suggests that fusion between the sperm plasma and underlying outer acrosomal membrane is beginning, with vesicles being formed. If this interpretation is correct, a similar picture is provided for the acrosome reaction occurring on the zona pellucida, in many common mammals. Microtubules and table-like structures were gathered in the submembranous region of the cumulus cell beneath the sperm adhesion site. In addition, some degenerate-appearing vesicles derived from swollen endoplasmic reticulum had gathered locally in the cytoplasm. Whether or not due to some local influence of acrosomal enzymes, this development would seem to be a forerunner of a more extensive similar reaction associated with the presence of sperm heads once inside the cumulus cell. These not only lacked the acrosome but had many vesicles around the head, some probably originating from mitochondria and from endoplasmic reticulum and perhaps even from the reacting acrosome.
It is important to note that such necrotic cells were not evident in cumuli of similar age collected from hCG-injected nonmated animals and so were only associated with the presence of spermatozoa. Moreover, we consider this to represent necrosis rather than apoptosis because the degenerative changes in the organelles in these cells are not anticipated by nuclear change. It must be emphasized also that this picture of specific cumulus cell necrosis associated with an invading spermatozoon stands in marked contrast to cases of spermatozoa seen within occasional cumulus cells of the rabbit [2], pig [22], and man [23, 24]. In those, the presence of an internalized spermatozoon had no transforming effect on the ultrastructure of the host cumulus cell or of adjacent cumulus cells.
As Suncus spermatozoa advance within one necrotized cell toward the egg, necrosis appears very locally also in a radially adjacent cell (Fig. 11). In the latter, mitochondria and the endoplasmic reticulum already had begun to degenerate (as judged by swelling) before entry of the sperm head, and because the lateral association between cumulus cells is relatively loose, it is possible that some necrotizing factor may transmit information radially through gap junctions. Such pictures therefore indicate that Suncus spermatozoa create a path through the intact cumulus not by squeezing through the interstices or dissociating intercellular junctions but by killing a series of cells to create a track of dead cells flanked by cumulus cells of normal ultrastructure. The sequence of this process is depicted in Figure 16. Although one or more such tracks of necrotic cells were seen in a single cumulus, only one spermatozoon per slit was ever observed.
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Finally, pictures such as that in Figures 10, 11, 14, and 15 confirm that a series of exposed barbs adorn the rostrum of the head in reacted Suncus spermatozoa. The timing of acrosome loss in Suncus means that its fertilizing spermatozoa lack the receptors for the zona that are located on the plasmalemma of intact and early-reacting spermatozoa in most mammals studied. However, these recurved barbs that decorate the perforatorium present an alternative mechanism, acting to hook into and so to link the acrosomeless fertilizing spermatozoon to the zona surface [7]. Allied to this, the strong lateral beat of the sperm tail in this species, reflective of the arrangement of the dense fibers, ensures a vigorous oscillation in the flat plane of the head that could favor such binding, as well as determine the success of spermatozoa in penetrating the zona pellucida [25]. Given this unusual scenario, an unanswered question concerns the mechanism of the block to polyspermy. This block appears to operate at the zona surface in Suncus, as no additional spermatozoa are bound to or seen within the zona of fertilized eggs, despite the common presence of several active spermatozoa moving in the perizonal space. The zona pellucida of Suncus does appear to have a relatively soft character in that it is punctured more easily than that of Mus or Microtus (unpublished observations). However, whether its physical character is changed at fertilization to a consistency incompatible with attachment by the rostral barbs is a possibility that remains to be investigated.
In conclusion, spermatozoa of Suncus exhibit an alternative mode of penetration of its typically dense matrix-free cumulus oophorus—one that involves first a cumulus-induced reaction and loss of the acrosome, followed by sperm invasion and killing of a discrete line of cumulus cells en route to a perizonal space within the cumulus. In considering the significance of the cumulus around the ovulated egg, a structure unique to eutherian mammals, its apparently essential role for fertilization in the few shrews studied thus stands in contrast to that in most mammals, in which the matrix-rich cumulus probably serves primarily as an egg-associated trap for the very few spermatozoa present in the ampulla as fertilization begins [26, 27]. The ability to induce an acrosome reaction persists in the cumulus of some higher mammals too (e.g., rabbit [2], Syrian hamster [28–30], Chinese hamster [31], man [23, 32, 33]), but in these cumulus-free eggs are fertilized readily because the zona pellucida clearly can and often does induce the acrosome reaction. Thus, present evidence suggests two possibilities as to the significance of the cumulus—that its essential role in shrews and perhaps some other insectivores is a derived character; or, if shrews really represent the primitive situation, that its function has evolved from one role to another during the later evolution of many extant eutherian groups. Finally, loss of the acrosome before reaching the zona means that shrews cannot utilize receptors on the periacrosomal plasmalemma as the means of binding to the zona. Present observations reinforce the conclusion that this is achieved via barbs on the perforatorium that are exposed by prior loss of the acrosome. Because such barbs decorate the perforatorium in certain other groups that include different megachiroptera [34] (unpublished observations), elephant shrews [35], and all other Insectivora and even canids [11], the cumulus may prove to have a similar key role in fertilization in several other eutherian groups, in addition to the Soricidae.
ACKNOWLEDGMENTS
The authors are very grateful to Dr. S. Oda of the Laboratory of Animal Management and Resources, Graduate School of Bioagriculture Sciences, Nagoya University, for supplying the musk shrew and to Dr. Y. Kawaguchi of the Laboratory of Genetics and Plant Breeding, Graduate School of Agriculture, Kyushu University, for placing the scanning electron microscope at our disposal.
FOOTNOTES
First decision: 30 January 2001.
1 This work was supported by a grant from the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. 
2 Correspondence: Takayuki Mri, Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Higashiku Hakozaki 6-10-1, Fukuoka
812-8581, Japan. FAX: 092 642 2804; tmohri{at}agr.kyushu-u.ac.jp
Accepted: April 3, 2001.
Received: December 19, 2000.
REFERENCES
ri T, Arai S, Shiraishi S, Uchida TA. Ultrastructural observations on spermatozoa of the Soricidae, with special attention to a subfamily revision of the Japanese water shrew Chimarrogale himalayica. J Mammal Soc Jpn 1991; 16:1-12 catenin. J Biol Chem 1999; 274:5981-5986-catenin required for the strong state of cadherin-based cell adhesion. J Cell Biol 1999; 144:1311-1322This article has been cited by other articles:
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  G. E. Olson, V. P. Winfrey, M. Bi, D. M. Hardy, and S. K. NagDas Zonadhesin Assembly into the Hamster Sperm Acrosomal Matrix Occurs by Distinct Targeting Strategies During Spermiogenesis and Maturation in the Epididymis Biol Reprod, October 1, 2004; 71(4): 1128 - 1134. [Abstract] [Full Text] [PDF]
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