360 P. ESPONDA THE JOURNAL AND M.OF MORENO EXPERIMENTAL ZOOLOGY 282:360–366 (1998) Acrosomal Status of Mouse Spermatozoa in the Oviductal Isthmus PEDRO ESPONDA* AND MARTA MORENO Centro de Investigaciones Biológicas, CSIC, 28006 Madrid, Spain ABSTRACT The principal purpose of this study was to establish the acrosomal status of mouse spermatozoa stored in the isthmus of the oviduct after natural mating. Scanning electron microscopy of oviducts fixed about 6 hr before the estimated ovulation time showed numerous acrosome-intact spermatozoa attached to the mucosal surface of the oviduct, or trapped in the oviductal crypts. Nevertheless, an unambiguous assessment of the state of the acrosome requires transmission electron microscopy. Using this method, it was observed that the acrosome was intact in the 81% of spermatozoa attached to the mucosal surface but in only 31% of spermatozoa that were free in the lumen. Most of the free spermatozoa showed swelling or disruption of the acrosome. This result might indicate that the in vivo spermatozoonoviductal mucosa interaction maintains the acrosome intact. Alternatively, it could mean that only ejaculated spermatozoa with a normal acrosome can establish such a mucosal relationship. J. Exp. Zool. 282:360366, 1998. © 1998 Wiley-Liss, Inc. In most vertebrates with internal fertilization, sperm storage occurs in the female genital tract to a variable degree. In some reptiles, fertile spermatozoa are stored there for years (Howarth, ’74; Gist and Jones, ’87). In birds, according to species, this may occur for days or weeks in the uterovaginal glands of the female tract (Howarth, ’74; Baskt, ’87). Among mammals, the female tract of some marsupials can store spermatozoa for periods of several days (Rodger and Bedford, ’82; Taggart and Temple-Smith, ’91), and in some bats this can extend for months (Racey, ’79). In shrews, spermatozoa are sequestered for a short period by either isthmic crypts or the “bubble-like” ciliated crypts in the oviduct ampulla without attachment to the epithelium (Bedford et al., ’97a,c). However, for many mammals, a short interval of sperm storage in the isthmus region of the oviduct seems to be the rule (Harper, ’94), a phenomenon that has been preferentially examined in the hamster (Smith et al., ’87; Smith and Yanagimachi, ’90, ’91), the rat (Shalgi and Kraicer, ’78), sheep (Hunter et al., ’82; Hunter and Nichol, ’83), rabbit (Overstreet and Cooper, ’79), cow (Hunter and Wilmut, ’84), sow (Fléchon and Hunter, ’81), mare (Parker et al., ’75; Bader, ’82), human (Croxatto et al., ’75), and mouse (Suarez, ’87). In this group, in general, soon after mating a few thousand spermatozoa pass through the uterotubal junction and remain confined to the isthmus of the oviduct. At around the time of ovulation, a very small proportion of these spermato© 1998 WILEY-LISS, INC. zoa ascend to the site of fertilization in the ampulla (Harper, ’94). After their arrival in the isthmus, some spermatozoa lie free in the lumen, but others attach to the epithelium of the isthmus crypts. In the hamster, the percentage of living spermatozoa appears to be higher in the fraction attached to the isthmic epithelium than in that free in the lumen (Smith and Yanagimachi, ’90). Furthermore, some data suggest that sperm capacitation may be completed during storage in the isthmus (Smith and Yanagimachi, ’91). On the other hand, numerous in vitro experiments, using monolayers of oviduct epithelial cells, have shown that the attachment to the oviductal epithelium promotes the hyperactivated motility and the fertilizing ability of spermatozoa (Pollard et al., ’91; Anderson and Killian, ’94; Chian and Sirard, ’95; Dobrinski et al., ’97; Smith and Nothnick, ’97). In vitro experiments have also established that the spermatozoon-epithelium association is related to, or would induce, sperm capacitation (Anderson and Killian, ’94; Chian and Sirard, ’95; Lefebvre and Suarez, ’96). These results suggest that the phenomenon of sperm storage in the mammalian oviduct represents an Grant sponsor: DGICYT (Spain); Grant number: PB93-0117. *Correspondence to: Dr. Pedro Esponda, C. Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain. E-mail: [email protected] Received 23 September 1997; Accepted 16 March 1998 ACROSOME IN OVIDUCTAL SPERM important step for the achievement of mammalian fertilization. The acrosomal status is a significant factor when considering the fertilizing capacity of mammalian spermatozoa. In the mouse, only acrosomeintact spermatozoa are able to fertilize the egg (Wassarman, ’87; Yanagimachi, ’94), and several reports have pointed out that the acrosomal reaction phenomenon occurs only after the spermatozoa contact the zona pellucida, which contains factors to initiate the reaction (Bleil and Wassarman, ’83; Yanagimachi, ’94). Moreover, in numerous mammals it has been shown that the acrosomal reaction seems to be a prerequisite for the spermatozoa to penetrate the zona (Yanagimachi, ’94). Thus, the acrosomal status would appear to be an important correlate of fertilizing capacity. In an attempt to clarify the functional status of the two sperm populations in the oviduct isthmus, we have analyzed the acrosomal status of those spermatozoa free in the lumen and those attached to the epithelium of the isthmus at various times after natural mating. MATERIAL AND METHODS Swiss albino mice housed in the animal facilities of C. Investigaciones Biológicas (Madrid, Spain) were fed with food and water ad libitum and were maintained at a constant temperature and with a light-dark cycle of 12 hr–12 hr. Males of proved fertility and virgin females were used. Females were injected with 4 IU of PMSG and 44 hours later with 5 IU of human chorionic gonadotropin (HCG). After HCG injection, each female was placed with a male, and the presence of a vaginal plug was checked. Mated females were killed by ether at different times after HCG injection, and the uterus and oviduct were promptly fixed for light or electron microscopy. Fixation was conducted at 37°C in order to avoid muscle contractions of the female tract, which can displace spermatozoa. Females were anesthetized, and before they died, the genital tract was gently exposed. The fixing solution was spread on the abdominal cavity, and then part of the uterus and the whole oviduct were sectioned and immersed in the fixative. After 30 min, the uterine end and the complete oviduct were isolated and fixed during other 30 min. For light microscopy, samples were fixed in 2% p-formaldehyde in phosphate buffer (pH 7.3), dehydrated, and embedded in paraffin. Sections stained with hematoxylin-eosin were studied us- 361 ing bright- or dark-field microscopy. For electron microscopy, tissues were fixed in a solution of 2% glutaraldehyde, 1% p-formaldehyde, and 0.1 M sucrose in 0.5 M cacodylate buffer (pH 7.3). Samples were thoroughly washed with the buffer and post-fixed in 2% osmium tetroxide in cacodylate buffer. Then they were treated in two ways. First, for scanning electron microscopy, pieces of oviducts were dehydrated in graded series of alcohols, dried with CO2 in a critical-point drying apparatus and coated with gold. For transmission electron microscopy, samples were dehydrated in alcohol and embedded in an epoxy resin. Semithin sections were stained with 0.2% toluidine blue and observed under a light microscope in order to locate spermatozoa in the oviductal isthmus. Ultrathin sections cut in an LKB ultratome, and stained with uranyl acetate and lead citrate, were studied in either a Hitachi Scanning S-2100 or a Philips 400 electron microscope. The acrosomal characteristics (acrosome intact or modified) of spermatozoa were checked and recorded under transmission electron microscopy, and the twotailed Fisher’s exact probability test applied to these data. RESULTS Hematoxylin-eosin-stained sections showed that spermatozoa invade the lower oviduct region soon after coitus. Under bright-field microscopy, the recognition of spermatozoa was difficult (Fig. 1), but in dark field, the sperm nuclei appeared strongly brilliant and were easily identifiable (Fig. 2). This clearly demonstrated the presence of sparse spermatozoa inside the oviductal isthmus from 6 hr before the estimated time of ovulation (Fig. 2), when many spermatozoa were still present in the uterus. The scarcity and location of spermatozoa observed in these sections coincide with the results of previous articles on spermatozoon transport in the mouse (Austin, ’60; Harper, ’94). The use of scanning electron microscopy clearly demonstrated the characteristics of the oviductal spermatozoa attached to the mucosal surface. In these preparations, those with an intact acrosome were clearly distinguishable from spermatozoa with modified acrosomes (Fig. 3). The intact state of the acrosome was defined by the presence of the acrosomal ridge and the arched profile of the apical region of the spermatozoon head (Figs. 3–6). By contrast, modified acrosomes show an irregular surface (Fig. 3). Observations of oviducts at different time periods before ovulation showed that numerous spermatozoa attached to the mucosal surface 362 P. ESPONDA AND M. MORENO Fig. 1. Section of the caudal region of an oviduct fixed 2 hr before ovulation. Two crypts are indicated (C) in which sperm heads are not clearly distinguished. Hematoxylin-eosin staining. ×425. Fig. 2. Same as Fig. 1 but observed under dark-field microscopy. Sperm heads can be seen clearly because their nuclei are strongly refringent. Fig. 3. Scanning electron microscopy of an oviduct fixed 4 hr before ovulation. A spermatozoon shows an intact acrosome (arrowhead); the other two exhibit modified acrosomes. ×4,350. Figs. 4 and 5. Oviducts fixed 4 hr before ovulation. Acrosome-intact spermatozoa are in close relation with the numerous microvilli (mv) present in the crypts. 4: ×5,200; 5: ×4,900. Fig. 6. Scanning electron microscopy of an oviduct fixed 1 hr before ovulation. Several spermatozoa lying on the oviductal surface can be seen. All show intact acrosomes (arrowheads). ×6,900. ACROSOME IN OVIDUCTAL SPERM displayed intact acrosomes (Figs. 4–6). The attachment of the spermatozoon head to the microvilli of the oviductal crypts was sometimes particularly noticeable, and in these cases spermatozoa also showed intact acrosomes (Figs. 4 and 5). Because scanning electron microscopy did not permit an accurate analysis of the free spermatozoa or the beginning of acrosome modifications, transmission electron microscopy was used to analyze the morphology of isthmic spermatozoa. This revealed that the 81.08% of spermatozoa associated with the oviductal epithelial surface had an intact acrosome (Table 1) (Figs. 7 and 8), whereas only 31.25% of luminal spermatozoa had intact acrosomes (Table 1). This differential was seen at all times before ovulation. However, spermatozoa attached to the mucosa showed a slight increase of modified acrosomes when they were close to the time of ovulation (Table 1). Attached spermatozoa appeared to be embraced by the abundant microvilli and in contact with numerous lucid vacuolar structures in the crypt lumen (Fig. 8). These vacuoles undoubtedly represent the serous material secreted by isthmus oviductal cells described in other mammals (Harper, ’94). Modification of acrosome structure was usually first reflected in a swallowing (Fig. 11), with some disruptions of the plasma and external acrosomal membranes (Figs. 9 and 10), and with a nonhomogeneous acrosomal content (compare Figs. 10 and 11 with Figs. 7 and 8). DISCUSSION The acrosomal characteristics of spermatozoa stored in the lower oviduct have been analyzed in very few mammals. In the Dasyurid marsupial Sminthopsis, transmission electron microscopy techniques have demonstrated that spermatozoa stored in the crypts of the oviductal isthmus had intact acrosomes at ovulation time (Breed et al., ’89), a fact that also was evident in some Insecti- 363 vora spermatozoa stored in the oviduct ampullar crypts (Bedford et al., ’97a,c). Light microscopy studies in the rabbit have shown that 36% and 66% of oviductal spermatozoa had modified acrosomes when they were collected 1.5 and 12 hr postcoitus, respectively (Overstreet and Cooper, ’75). In contrast, spermatozoa collected from the uterus at the same time periods showed that only 7.8 to 13.9% of them exhibited modified or reacted acrosomes (Overstreet and Cooper, ’75). An accurate analysis of rabbit spermatozoa collected solely from the oviductal ampulla at fertilization time demonstrated that 98% possessed intact acrosomes (Suarez et al., ’83). Scanning electron microscopy of bovine and pig spermatozoa in the lower oviduct demonstrated that the majority were acrosome intact when observed before ovulation, but many observed shortly before or after ovulation displayed vesiculation of the acrosomal surface (Hunter et al., ’87, ’91). Mouse spermatozoa from the entire oviduct analyzed by light microscopy showed that 43% of them presented modified acrosomes (Klemm and Engel, ’91). These results suggest that the lower oviduct produces a detrimental effect on the acrosome stability of some spermatozoa (Overstreet and Cooper, ’75). This study indicates that the spermatozoon-epithelium relationship protects the acrosome in the mouse. The 81% of spermatozoa attached to the oviductal epithelium exhibited intact acrosomes, whereas almost 70% lying free in the isthmus lumen showed modified acrosomes. How the epithelial relationship supports acrosomal integrity is not known, though Hunter (’94) has commented that some oviductal factors, for example, glycoproteins, could be acting in this way. The in vivo site of the acrosomal reaction is still a matter of controversy (Wassarman, ’87). This fact is probably due to the rare in vivo ultrastructural analyses assayed. In this sense, transmission electron microscopy is probably TABLE 1. Transmission electron microscopical observations of the acrosomal status of spermatozoa stored in the oviductal isthmus at different times before ovulation* Hours before ovulation 0–2 2–3 3–6 % N 5† 6 5 Sperm attached to the epithelium IA MA 11 10 9 81.08 4 2 1 18.92 *N = number of females analyzed; IA = intact acrosomes; MA = modified acrosomes. † Two females were used at ovulation time. 1 P = Two tailed Fisher’s exact probability test. Sperm free in the lumen IA MA P1 2 2 4 31.25 9 7 8 68.75 0.0154 0.0092 0.0150 3 × 10–6 364 P. ESPONDA AND M. MORENO Fig. 7. Transmission electron microscopy of an oviductal crypt fixed 3 hr before ovulation. A spermatozoon head with an intact acrosome (a) is closely connected with the numerous microvilli of the crypt. (n: sperm nucleus). ×22,500. Fig. 8. A similar image to that shown in Fig. 7, but from an oviduct fixed at ovulation time. The intact acrosome (arrowhead) is clearly seen. Note the numerous microvilli and clear vacuoles, which engulfed the spermatozoon. n, sperm nucleus. ×15,200. Figs. 9–11. Figs. 9 and 11 show two spermatozoa free in the oviductal lumen displaying modified acrosomes (arrow head in 9 and a in 11). Fig. 10 shows a higher magnification of the acrosome of Fig. 9. In both cases, acrosomes appeared swollen, with some membrane disruptions (arrows in Fig. 10) and containing a nonhomogeneous material. Fixation 2 hr before ovulation. Fig. 9, ×11,000; Fig. 10, ×34,000; Fig. 11, ×25,200. ACROSOME IN OVIDUCTAL SPERM the only method that permits discriminating plasma and acrosomal membrane characteristics. In the shrew Suncus murinus, an in vivo analysis has shown that all the spermatozoa in the cumulus oophorus had reacted, suggesting that the cumulus would stimulate the reaction (Bedford et al., ’97b). In the hamster, in vivo and in vitro experiments have shown that in some spermatozoa among the cumulus cells, the acrosomes were swollen, wrinkled, or totally absent (Yanagimachi and Noda, ’70). Also in the rabbit, electron microscopical analysis of oocytes fertilized in vivo indicated that many spermatozoa lying between the granulosa cells already displayed initial stages of the acrosomal reaction (Bedford, ’68). In the cow, an electron microscopical study showed that the acrosomal reaction probably occurs when spermatozoa contact the zona pellucida (Crozet, ’84). In the mouse, transmission electron microscopy of in vivo fertilized oocytes leads to the assumption that the acrosomal reaction occurs outside the zona pellucida (Stefanini et al., ’69; Zamboni, ’72), and similar descriptions were made using oocytes fertilized in vitro (Saling et al., ’79; Wassarman, ’87). In general, in the mouse it is accepted that only acrosome-intact spermatozoa can bind to the zona, and that is the zona that stimulates the acrosomal reaction (Wassarman, ’87). In mammals the site of acrosomal reaction could vary to some extent among species. Nevertheless, the general picture is that spermatozoa must face egg vestments with an intact acrosome and must complete the acrosomal reaction before entering the zona (Yanagimachi, ’88, ’94). In this regard, our results show that oviductal storage maintains the acrosome intact, contributes to the maintenance of fertilization ability, and operates as a sort of selective mechanism for the gametes attached to the isthmus epithelium. On the other hand, our observations reinforce the belief that the spermatozoon-epithelium relationship is a phenomenon related to sperm-fertilizing ability (Smith and Yanagimachi, ’90, ’91). Spermatozoon storage in the oviduct represents another important phase in the events occurring to the spermatozoon before fertilization. Initially, the epididymis induces spermatozoon maturation. During ejaculation spermatozoa interact with macromolecules secreted by the male accessory glands and must ascend through the uterus. In general, a few spermatozoa are able to reach the oviduct, and some of them are engulfed by the crypts where sperm capacitation probably occurs. 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