Interspecies interaction of zona-free ova with spermatozoa in mouse,rat and hamster

Mouse, rat and hamster zona-free eggs were penetrated in vitro by spermatozoa of their own species and by alien spermatozoa of mouse, rat and hamster. The tested combinations showed very distinct differences in penetration ability. Mouse zona-free eggs were penetrated by spermatozoa of their own species only. Rat zona-free eggs were penetrated by their own and mouse spermatozoa. Hamster zona-free eggs were penetrated by their own, mouse and rat spermatozoa. Several proteolytic enzymes used for lysis of zona pellucida, time of sperm preincubation and sperm concentration did not affect sperm-egg interaction. It is concluded that the species specificity of egg plasma membrane in the rodents tested is probably based on some specific surface components.     

Difference in the Manner of Association of Acrosome-Intact and Acrosome-Reacted Hamster Spermatozoa with Egg Microvilli as revealed by Scanning Electron Microscopy

Scanning electron microscopy was employed to examine the manner of association between in vitro capacitated spermatozoa and zona-free eggs of the hamster. Spermatozoa with intact acrosomes, which were unable to fuse with eggs, were seen in general associated with egg microvilli in the region of the acrosomal cap. Acrosome-reacting spermatozoa were seen associated with egg microvilli with the dissociating acrosomal caps. Acrosome-reacted spermatozoa, which were able to fuse with eggs, generally associated with egg microvilli by the equatorial segment and the anterior portion of the postacrosomal region. It is inferred that the completion of the acrosome reaction signals changes in the plasma membrane over the equatorial segment of the acrosome and the anterior area of the postacrosomal region which give it a greater affinity to and fusibility with the oolemma.     

Scanning electron microscope study of in vitro prepenetration gamete interactions

Hamster and mouse capacitated spermatozoa were interacted in vitro with hamster and mouse eggs in homologous and heterologous combinations. Also, fertilized and trypsin treated unfertilized hamster eggs, and unfertilized rat eggs were made to interact with capacitated hamster spermatozoa. The surface of the zona pellucida was then examined with the scanning electron microscope. It was found that sperm attachment, followed by sperm binding and penetration through the zona pellucida, was observed only when homologous gamete combinations were used. Binding of the spermatozoa to the zona was evidenced by the lytic effect of the acrosomal enzymes on the zona substance. When fertilized eggs and trypsin-treated unfertilized hamster eggs were mixed with capacitated hamster spermatozoa as well as in the heterologous gamete combinations, we found that the spermatozoa were able to establish attachment but not binding. Under these conditions the outer surface of the zona pellucida was never found to have penetration tracks made by the spermatozoa. Failure of heterologous spermatozoa to cross the foreign zona pellucida is believed to be associated with the inability of the foreign spermatozoa to establish binding and to the inability of their acrosomal enzymes to digest the zona. A similar mechanism is believed to work in zona-reacted and in trypsin-treated hamster eggs inseminated in vitro with homologous spermatozoa.     

Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.     

Zona-induced acrosome reaction of hamster spermatozoa

It is well established that the zonae pellucidae of mature unfertilized eggs have the ability to induce the acrosome reaction of capacitated spermatozoa. To determine if this capacity of the zona is species-specific, hamster spermatozoa were allowed to attach to the zonae of homologous and heterologous eggs and examined for the acrosome reaction. The zonae of eggs from six different species were tested and the zona of hamster egg was found to have the strongest capacity to induce the acrosome reaction of hamster spermatozoa, followed by human and rat zonae. The zonae and mouse, guinea pig, and domestic fowl eggs were incapable of inducing the acrosome reaction of hamster spermatozoa. The acrosome reaction-inducing ability of the hamster zona was found to increase during maturation in the ovary. The zona of mature unfertilized hamster eggs maintained their acrosome reaction-inducing ability even after aldehyde fixation or storage in a highly concentrated solution of ammonium sulfate.     

In vitro studies of the golden hamster sperm acrosome reaction: completion on the zona pellucida and induction by homologous soluble zonae pellucidae

We have studied the occurrence of the golden hamster sperm acrosome reaction (AR) in vitro during interaction with the oocyte investments: the cumulus cell matrix and the zona pellucida. Hamster sperm were capacitated in a defined medium that does not induce the AR. These spermatozoa were allowed to interact with the ovum vestments, the events of which were recorded using high-speed videomicrography. Frame-by-frame analysis revealed that sperm did not complete the AR in the cumulus cell matrix, but did so on the zona pellucida. Furthermore, a higher percentage of sperm completed the AR on the zona pellucida of cumulus-invested than on cumulus-free eggs. We also investigated the effect of solubilized hamster and mouse zonae pellucidae on the hamster sperm AR. Addition of solubilized hamster zonae to capacitated sperm elicited the AR within 15 min. Solubilized mouse zonae were significantly less effective, indicating that the zona-induced AR in hamster sperm may be species specific. These results suggest that the hamster zona pellucida is an inducer of the AR in the intact or soluble form, and that the majority of spermatozoa traverse the cumulus cell matrix without completing the AR in our in vitro system.     

An investigation of the latency period between sperm oolemmal adhesion and oocyte penetration

In clinical studies of the ability of capacitated human sperm to penetrate zona-free hamster eggs, we have previously observed that the ratio of oolemmal adherent to penetrating sperm varied between men. Sperm incorporation did not occur immediately following gamete adhesion and not all adherent sperm penetrated the egg. To further investigate this phenomenon, comparisons were made of the kinetics of gamete adhesion, membrane fusion, and sperm incorporation of capacitated mouse and human spermatozoa by zona-free hamster eggs and of mouse sperm by zona-free mouse and hamster eggs. Eggs were inseminated with either capacitated human or mouse sperm or combinations of both, washed out of sperm suspension after initial gamete adherence, and further incubated in sperm-free medium. Gamete membrane fusion was judged by dye transfer of Hoechst 33342 and sperm entry of the cortical ooplasm by observation of expanded sperm heads within acridine orange stained eggs. Oolemmal adherent mouse and human sperm fused with and penetrated zona-free hamster eggs at different times whether eggs were inseminated in parallel or with combinations of sperm of both species. Oolemmal adherent mouse sperm penetrated zona-free hamster eggs prior to their penetration of zona-free mouse eggs. Ultrastructural studies of zona-free human eggs inseminated with human sperm confirmed prior observations with hamster eggs that only acrosome-reacted human sperm adhere to the oolemma. These results have lead us to postulate that sperm entry into the egg may occur through a "zipper" mechanism involving the ligation of local gamete receptors similar to the incorporation of target particles by phagocytes and suggest that not all oolemmal adherent human sperm are capable of being incorporated although they have undergone an acrosome reaction.     

Cross-fertilization between Syrian and Chinese hamsters

The role of the zona pellucida in the specificity of fertilization was studied by cross-inseminations between Syrian (Golden) and Chinese hamster gametes. Cumulus-enclosed eggs from both Syrian and Chinese hamsters were placed together in one dish and inseminated with spermatozoa from either one or the other species. Fertilization always took place between gametes of homologous species. Chinese hamster spermatozoa failed to bind to the zona pellucida of Syrian hamster eggs; hence, fertilization was never observed. However, Chinese hamster spermatozoa could fertilize zona-free Syrian hamster eggs. In the reciprocal cross, a large number of Syrian hamster spermatozoa could bind to and penetrate the zonae of Chinese hamster eggs. However, fusion of Syrian hamster spermatozoa with the vitellus of zona-intact Chinese hamster eggs was never observed. After removal of the zona pellucida, only a small percentage (31%) of Syrian hamster spermatozoa could fuse with Chinese hamster vitelli. Thus, in these species, the mechanisms of interspecific gamete recognition and the prevention of interspecies fertilization seem to differ according to the direction of the cross. In Syrian hamster eggs, the block to interspecies fertilization seems to exist at the level of the zona pellucida, while in Chinese hamster eggs the block is at the level of the egg plasma membrane. The implications of these results in analyses of the genetics of spermatozoa, the molecular basis of sperm-egg recognition, and mechanisms of reproductive isolation leading to speciation, are discussed.     

Comparative immunoreactivity of mouse and hamster sperm proteins recognized by an anti-P26h hamster sperm protein

The binding of the spermatozoon to the zona pellucida is a species-specific phenomenon. We have previously shown that the binding of hamster sperm to the homologous zona pellucida involves a sperm 26-kDa glycoprotein, the P26h, originating in the epididymis. In order to establish to what extent this sperm protein is involved in the species-specific recognition of the egg's extracellular coat, we have compared the inhibitory properties of anti-P26h antibodies in a sperm-zona pellucida assay using hamster and mouse gametes. Anti-P26h IgGs inhibit, in a dose-dependent manner, gamete interactions in both species, although in a less efficient manner in the mouse than in the hamster. While anti-26kDa Fab fragments are as efficient as the intact IgG to inhibit hamster sperm-zona pellucida binding, they have no effect on mouse gamete interaction. ELISA, Western blot, and immunohistochemical experiments have been performed in order to characterize the mouse antigen(s) recognized by the anti-P26h antiserum. ELISA and Western blots showed that this antiserum recognized two proteins on mouse spermatozoa that are less reactive than the hamster P26h. These antigens are localized in the acrosomal region of epididymal spermatozoa of both species. These results indicate that the hamster P26H involved in zona pellucida interaction has certain unique epitopes, while others are common to the sperm of both species. © 1995 Wiley-Liss, Inc.     

Human sperm-cervical mucus interaction and the ability of spermatozoa to fuse with zona-free hamster oocytes

Samples of semen and cervical mucus were provided by 18 couples. Cervical mucus was obtained for each day possible and stored at 4 degrees C until all the samples were collected. Flat capillary tubes were loaded with the mucous samples and spermatozoa from the husband's semen sample were allowed to migrate through the cervical mucus (3 cm column) into culture medium. The spermatozoa recovered after migration through cervical mucus were assayed in vitro with zona-free hamster oocytes. Control experiments were carried out using spermatozoa from the same semen sample but prepared by the swimming-up technique. Altogether, 557 eggs in the control group and 1236 eggs in the experimental group were analysed, and the results demonstrated that the % of sperm penetration, the mean number of sperm decondensations per penetrated egg and the mean number of spermatozoa adhering per egg all had higher values (P less than 0.05) for the control samples than for the experimental samples. We suggest that cervical mucus modifies human spermatozoa, as measured by their interaction with zona-free hamster oocytes.     

Time requirement for capacitation of boar spermatozoa assessed by their ability to penetrate the zona-free hamster egg.

Boar spermatozoa were preincubated for various times in the isolated uterus and oviduct from a maturing gilt and used to inseminate zona-free hamster eggs. The proportions of eggs penetrated and activated were increased, and the interval between insemination and sperm penetration was shortened when the spermatozoa were preincubated for 4--5.5 h instead of 2--.5 h. Overall penetration rates were higher and sperm penetration occurred about 1 h earlier when the eggs were inseminated with spermatozoa preincubated in the uterus than in the oviduct. It is concluded that the change in ability of boar spermatozoa to penetrate zona-free hamster eggs is due to capcitation which requires 4--4.5 h and 5--5.5 h of preincubation in the isolated uterus and oviduct, respectively.     

Human sperm chromosome complements after microinjection of hamster eggs

A technique was developed for microinjection of human spermatozoa into golden hamster (Mesocricetus auratus) eggs to obtain human pronuclear chromosome complements. Before microinjection the spermatozoa were treated by brief sonication or incubation in TEST-yolk buffer to reduce motility. Very few sperm chromosome complements developed after sperm treatment with sonication and the frequency of spermatozoa with structural chromosomal abnormalities was exceedingly high (91%). The majority of sperm chromosome complements analysed had multiple breaks and rearrangements. Sperm incubation in TEST-yolk buffer before microinjection provided more analysable sperm karyotypes with a significantly lower frequency of structural chromosomal abnormalities (39%, P less than 0.001). Our results therefore suggest that sonication induces structural chromosomal abnormalities in spermatozoa. Since the frequency of chromosomal abnormalities after microinjection was higher than after sperm fertilization of hamster eggs, it appears that microinjection per se may also increase the frequency of chromosomal abnormalities in spermatozoa. These results are based on small numbers and must be confirmed on larger sample sizes, but our study suggests that microinjection of spermatozoa into eggs should not be recommended for clinical use until fully evaluated.     

Inhibition of in-vitro fertilization of intact and denuded hamster eggs by univalent anti-sperm antibodies.

Univalent (Fab) rabbit anti-hamster sperm antibodies added to an in-vitro fertilization system did not interfere with the sperm acrosome reaction or motility, but inhibited cumulus dispersion by the spermatozoa, sperm binding to and passage through the zona pellucida as well as sperm-egg fusion. Addition of the Fab preparations to the capacitated spermatozoa at various times before or up to 40-45 min after the sperm-egg mixing prevented penetration of spermatozoa through the zona pellucida. Detachment of the spermatozoa already bound as well as those partly inside the zona pellucida was achieved by a late addition of antibodies. In experiments with zona-free hamster eggs, addition of the Fab antibodies to the spermatozoa 10 min to 5 h before the introduction of unfertilized eggs reduced the rate of adhesion and fertilization to very low levels. These antibodies were not absorbed on hamster ovary, liver or kidney and had no direct effect on the fertilizability of zona-intact or zona-free eggs.     

Comparison of the fertilizing ability of human spermatozoa preincubated in calcium- and strontium-containing media

Human spermatozoa freed from seminal plasma using a discontinuous Percoll gradient procedure were incubated overnight in modified Tyrode's media containing either CaCl2 (CA medium) or with SrCl2 instead of CaCl2 (SR medium). The following morning these spermatozoa were washed by centrifugation, resuspended in fresh CA medium and incubated for either 2 or 3 h with zona-free hamster eggs to test their fertilizing ability. Penetration rates were higher after 3 h of sperm-egg contact than after 2 h, and the spermatozoa preincubated in SR medium penetrated significantly more hamster oocytes than those preincubated in CA medium.     

Sperm entry into zona-free oocytes in the hamster oviduct: Implications for the mechanisms of acrosome reaction induction

The possible importance of the zona pellucida for induction of the acrosome reaction (AR) and establishment of sperm/egg associations in the fallopian tube was investigated by instilling zona-free eggs into the oviductal ampulla of hamsters that had been inseminated with epididymal spermatozoa 6–7 hours previously. The eggs were recovered only 60–90 minutes later because of increasing difficulty with time of collecting zona-free eggs from the oviduct. In the zona-free group, 41 (4%) of 1,101 transferred eggs were recovered, of which 20% contained spermatozoa with decondensing nuclei (mean 4.4/egg). A similar (22%) fertilization rate (mean 3.2 spermatozoa/egg) was found among intact (control) eggs recovered after instillation into the contralateral oviduct. Mammalian spermatozoa are not incorporated even into zona-free eggs before AR occurs. These results thus demonstrate that an AR in functional hamster spermatozoa in vivo and establishment of sperm/egg associations in vivo require no interaction with the zona pellucida nor with other products of ovulation.     

精卵相互作用：诱发仓鼠精子顶体反应的部位

65只成年仓鼠经超排卵后,于排卵前人工授精。授精后6一7小时,收集输卵管壶腹部液(AF)、活动精子和卵子包括卵丘细胞(CM)基质和透明带(ZP),以相差显微镜检查精子顶体帽状态。在AF中有65.3％精子顶体帽发生改变;顶体反应(AR)率随着精子穿过CM而增加(73.7％);当精子到达ZP后,97.1％精子完成AR。25只仓鼠体外授精表明,获能精子在CM内游动或穿过CM时,顶体帽发生改变,可达到71.4％,但AR百分率甚低。当精子到达ZP后约30分钟完成AR。虽然单独的CM不能诱发体外仓鼠精子发生AR,但它可引起精子AR的早期阶段发生改变,并协同ZP促进获能精子完成AR。同样地,可溶性CM和ZP及其复合物可明显地激发体外仓鼠精子AR。但豚鼠ZP则无此作用。这些结果提示:AF是AR早期阶段的发生部位,而CM和ZP是体内仓鼠精子AR的主要部位;CM和ZP复合物是体外精子AR的诱导者。     

Mouse sperm capacitation assessed by kinetics and morphology of fertilization in vitro

Epididymal mouse spermatozoa were preincubated for periods of 5-120 min and then tested for their ability to penetrate freshly ovulated eggs synchronously and rapidly. When zona-intact eggs were used, only suspensions preincubated for 120 min gave consistently high rates of fertilization, but suspensions preincubated for 30 min were functionally equivalent to those incubated for 120 min when used with zona-free eggs; the only major observable differences were a 15-min lag in sperm-egg interaction and an increased incidence of asynchrony with multiple sperm penetrations. A morphological study of sperm-egg interactions using zona-intact eggs indicated that, within 35 min of gamete mixing, egg microvilli could be detected by SEM in association with the fertilizing sperm head. Using conventional light microscopic examination of fixed and stained preparations, initial stages of sperm head decondensation could be detected in the majority of eggs after 45-60 min and the process was essentially completed, with the egg at the telophase-second polar body stage of meiosis II, after 75 min. Similar kinetics were observed with sperm concentrations of 10(5) and 10(6)/ml. The time required for penetration by capacitated sperm suspensions is therefore relatively short and the most accurate information regarding state of capacitation and rate of sperm penetration can be obtained by choosing an appropriately short interval for sperm-egg interaction before assessment.     

Sperm-egg interaction in the mouse using live and glutaraldehyde-fixed eggs

A study of interaction between gametes in the mouse, using live and glutaraldehyde-fixed eggs, showed that, while in live eggs the binding is a two-step process (“early” and “late” binding), the process is one step when the spermatozoa interact with fixed eggs. The second point that emerged from this study is that uncapacitated and capacitated spermatozoa bind the zonae pellucidae of live and fixed eggs in the same way, but only the capacitated spermatozoa bound to live eggs undergo a complete acrosome reaction and penetrate the zona pellucida. Moreover, it has been shown that binding to fixed eggs, just as to live eggs, is Ca²⁺-dependent, and it can be reversed by EGTA. Fixed eggs thus are a good model to study only one step of the sperm-egg interaction removed from all the other events of the fertilization process.     

Thermostability of sperm nuclei assessed by microinjection into hamster oocytes

Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronuclei. Mature, mammalian sperm nuclei, which are stabilized by protamine disulfide bonds, were moderately heat resistant. For example, they remained capable of pronucleus formation even after pretreatment for 30 min at 90 degrees C. Indeed, a temperature of 125 degrees C (steam) was required to inactivate hamster sperm nuclei completely. On the other hand, nuclei of rooster and tilapia spermatozoa and those of immature hamster and mouse spermatozoa, which are not stabilized by protamine disulfide bonds, were sensitive to heating; although some of them decondensed after exposure to 90 degrees C, none formed male pronuclei. Furthermore, nuclei of mature hamster sperm became heat labile when they were pretreated with dithiothreitol to reduce their protamine disulfide bonds. These observations suggest that the thermostability shown by the nuclei of mature spermatozoa of eutherian mammals is related to disulfide cross-linking of sperm protamines.     

Role for fucose-sulfate-rich carbohydrates in the penetration of zona-pellucida-free hamster eggs by hamster spermatozoa

The interaction of acrosome-reacted hamster spermatozoa and zona-pellucida-free hamster eggs was investigated by incubating gametes in the presence of a variety of simple and complex carbohydrates. Significant inhibition of gamete fusion was achieved only in the presence of fucoidan and ascophyllin, two algal polysaccharides containing fucose sulfate. These compounds did not interfere with sperm motility, capacitation, or acrosome reactions. It is concluded that these two compounds share common structural features with putative cell-surface carbohydrates involved in sperm-oolemmal interaction.