Sperm-egg ratios and the site of the acrosome reaction during in vivo fertilization in the hamster

Little is known about the timing of the mammalian sperm acrosome reaction during fertilization in vivo. To study this problem, female hamsters were inseminated at about the time of ovulation, and the contents of the ampullary regions of their oviducts were subsequently examined at various intervals. No living spermatozoa were recovered from ampullae earlier than 4 hr after insemination. The first appearance of living spermatozoa coincided closely with the first appearance of fertilized eggs in the same oviduct. The total numbers of living spermatozoa did not start to exceed the number of eggs in the same ampulla, until after 50% or more of the eggs had been fertilized. Hamster spermatozoa are highly efficient at making contact with eggs, and the fertilizing spermatozoon probably spends no more than 2½ –5½ min in penetrating the cumulus oophorus. Spermatozoa that enter the ampulla appear to be ready to undergo the acrosome reaction, and complete it while they are passing through the cumulus or shortly before, or after, contacting the surface of the zona pellucida.     

The oocyte of a new world marsupial, Monodelphis domestica: structure, formation, and function of the enveloping mucoid layer

Ovulated oocytes of the gray short-tailed opossum Monodelphis domestica are surrounded by a thin zona pellucida and are devoid of a cumulus oophorus. In the ampulla of the oviduct, oocytes acquire a thick mucoid layer composed of concentrically arranged fibrillar material. Exocytosis by the secretory cells of the oviductal epithelium occurs in the region of the oviduct adjacent to the egg. This suggests that the oocyte-zona-mucus layer complex may influence the oviductal epithelium to secrete. During secretion, fibrillar contents of the secretion granules appear to be transformed into membranous material which presumably becomes fibrillar again as it is incorporated into the forming mucoid layer. Spermatozoa (which are known to pair in the cauda epididymis) are found in pairs and with intact acrosomes in the mucoid layer of fertilized eggs. This suggests that spermatozoa of Mondelphis remain paired until they reach the zona pellucida and that the acrosome functions in zona binding and/or penetration.     

Ultrastructural observations on gamete interactions using micromanipulated mouse oocytes

Cumulus-free mouse oocytes were subjected to zona opening by cracking with microhooks (ZC) or acid drilling (ZD) and fixed 30–90 min after insemination (10⁵ pre-capacitated motile sperms/ml). Ultrastructural observations were made on serially thin-sectioned oocytes: 15 ZC and 12 ZD. The zona lesion in ZC oocytes was a clean cut, whereas in ZD oocytes it formed a patchy area of partial zona loss, with reduced microvillar height on the underlying oocyte surface. Spermatozoa were observed within the perivitelline space and partially fusing with the oocyte after 30 min in both situations. Only acrosome-reacted sperm heads were observed to fuse: acrosome intact forms were generally in contact with the zona pellucida, either with the inner or outer surface. Acrosome-intact spermatozoa were also observed deeply embedded in the zona matrix, possibly indicating surface enzyme activity preceding the membrane fusion events of the acrosome reaction proper. The observations are consistent with the need for spermatozoa to make contact preferentially with the zona pellucida during the course of the acrosome reaction.     

Spermatozoa of the shrew, Suncus murinus, undergo the acrosome reaction and then selectively kill cells in penetrating the cumulus oophorus

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 oophorus-associated glycodelin-C displaces sperm-bound glycodelin-A and -F and stimulates spermatozoa-zona pellucida binding

Spermatozoa have to swim through the oviduct and the cumulus oophorus before fertilization in vivo. In the oviduct, spermatozoa are exposed to glycodelin-A and -F that inhibit spermatozoa-zona pellucida binding. In this study, we determined whether these glycodelins would inhibit fertilization. The data showed that the spermatozoa without previous exposure to glycodelin-A and -F acquired glycodelin immunoreactivity during their passage through the cumulus oophorus. On the other hand, when glycodelin-A or -F-pretreated spermatozoa were exposed to the cumulus oophorus, the zona pellucida binding inhibitory activity of glycodelin-A and -F was not only removed, but the spermatozoa acquired enhanced zona pellucida binding ability. These actions of the cumulus oophorus were due to the presence of a cumulus isoform of glycodelin, designated as glycodelin-C. The cumulus cells could convert exogenous glycodelin-A and -F to glycodelin-C, which was then released into the surrounding medium. The protein core of glycodelin-C was identical to that in other glycodelin isoforms, as demonstrated by mass spectrum, peptide mapping, and affinity to anti-glycodelin antibody recognizing the protein core of glycodelin. In addition to having a smaller size and a higher isoelectric point, glycodelin-C also had lectin binding properties different from other isoforms. Glycodelin-C stimulated spermatozoazona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound glycodelin-A and -F. In conclusion, the cumulus cells transform glycodelin-A and -F to glycodelin-C, which in turn removes the spermatozoazona binding inhibitory glycodelin isoforms and enhances the zona binding capacity of spermatozoa passing through the cumulus oophorus.     

Status of the rat acrosome during sperm-zona pellucida interactions

Over the past 40 years evidence from many sources has indicated that the mammalian acrosome reaction occurs within or near the cumulus oophorus. Recently, however, workers investigating in vitro fertilization in the mouse have concluded that in this system the acrosome reaction takes place on the surface of the zona pellucida. We have investigated the interaction of rat spermatozoa and the zona pellucida by using the scanning electron microscope (SEM) and two monoclonal antibodies which are directed to antigens of the rat sperm acrosome. When in vitro inseminated eggs from which the cumulus has been removed are viewed with the SEM some sperm heads on the surface of the zona pellucida appear unaltered whereas others appear to be undergoing changes. In vivo, all displayed altered head morphology. Using immunogold labeling we found that the two antibodies employed, 2C4 and 5B1, were directed to acrosomal content and vesiculating acrosomal membranes. Immunofluoresence staining of zonae pellucidae in in vitro fertilization studies revealed numerous small positive regions. These were presumably acrosomal content and membranes which had been left on the zona surface by spermatozoa which had been associated with the zona surface. Our results suggest that the rat acrosome interacts with the zona pellucida. During this interaction some acrosomal content and membranes detach from the spermatozoon and remain on the surface of the zona pellucida.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

Prolonged survival of the Graafian follicle and fertilization in the Japanese greater horseshoe bat, Rhinolophus ferrumequinum nippon

After the mating season of the Japanese greater horseshoe bat in mid- or late October, only the right ovary maintained a single Graafian follicle throughout hibernation until early April. During this time the ovum was in prophase of meiosis I (resting stage) with many large lipid droplets as a nutrient source. In synchrony with stigma formation, there was resumption of meiotic activity, separation of the cumulus oophorus from the granulosa layer and dispersion of the follicle cells just before ovulation in spring. The block to polyspermy seemed to reside in the zona pellucida, because no spermatozoa could be detected in the perivitelline space of the 6 fertilized ova examined, although a second spermatozoon was recognized in the zona pellucida of 3 ova.     

Effect of oocyte-sperm co-incubation on acrosome reaction in the goat

The induction of acrosome reaction of goat spermatozoa was investigated. The acrosomal status of spermatozoa was determined by a triple-staining technique. The effect of the presence of goat oocytes on the proportion of acrosome-reacted spermatozoa was also determined. Ovulated oocytes were obtained from superstimulated adult goats. Other sources of oocytes were adult and prepubertal goats; oocytes from both sources were maturated in vitro. There was an increase in the percentage of acrosome-reacted spermatozoa from 4% +/- 0.98 to 9% +/- 1.41 when oocytes from adult females were used. Similar induction rates were measured with prepubertal and adult oocytes maturated in vitro (10.4% +/- 2.06 and 8.75% +/- 1.06, respectively). The influence of several qualities of cumulus oophorus as well as the presence of zona pellucida was also investigated. No significant differences were obtained with any cumulus oophorus or zona pellucida oocyte complexes. Although oocyte quality is important for high fertilization rates, it does not seem to be crucial for the induction of acrosome reaction.     

Sperm: Egg ratios and putative molecular signals to modulate gamete interactions in polytocous mammals

Reasoning from the premises that 1) the sperm:egg ratio at the time of activation of the secondary oocyte in mammals is close to unity under conditions of spontaneous mating, 2) a majority of eggs within the cumulus oophorus of a polytocous species is fertilized in a reasonably short interval of time, and 3) spermatozoa would find it difficult to reverse their approach to the zona pellucida, it is proposed that molecular gradients exist to divert spermatozoa penetrating the cumulus mass away from eggs already activated and towards eggs as yet unfertilised. Possible sources of such molecular cues are considered, as is the event that triggers their release. © 1993 Wiley-Liss, Inc.     

The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo

Adult female golden hamsters were induced to superovulate. When they were mated several hours prior to ovulation or artificially inseminated about the time of ovulation, nearly 100% of their eggs were subsequently fertilized monospermically. During the progression of fertilization when the eggs were still surrounded by compact cumulus oophorus, the contents of the ampullary region of the oviducts were collected and spermatozoa moving in the ampullary fluid, within the cumulus and on/in the zonae pellucidae of unfertilized eggs, were examined by light and electron microscopy to evaluate the status of their acrosomal caps. Most spermatozoa swimming in the ampullary fluid had apparently intact acrosomal caps, while the vast majority moving within the cumulus had distinctly modified acrosomal caps. Most spermatozoa that had passed through the cumulus and reached the zona surfaces had remnants of their acrosomal caps (“acrosomal ghosts”). When the ghosts were present around the sperm heads on the zona, the heads pivoted about a point roughly corresponding to the places where the ghosts were located. The ghosts seemed to firmly attach to the zona surfaces, then were split open by the sperm heads and left behind as the sperm heads advanced into the zona. A few spermatozoa on the zona surfaces had no acrosomal ghosts (at least not detectable by light microscopy). In this case, the sperm head pivoted about either the inner acrosomal membrane or the equatorial segment of the acrosome. In no instance were spermatozoa with intact acrosomal caps found on zona surfaces. We infer from these observations that most spermatozoa in vivo initiate their acrosome reactions while they are advancing through the cumulus. When they arrive at the zona surfaces, acrosomal ghosts are generally present on the sperm heads. These ghosts appear to hold sperm heads to zona surfaces as well as to restrict the direction of advancement of sperm head through the zona. In a minority of cases, ghostless spermatozoa reach the zona surfaces. As these spermatozoa appear to be able to penetrate the zona successfully, structures other than the acrosomal ghost (ie, the inner acrosomal membrane and the plasma membrane over the equatorial segment of the acrosome) may also attach to zona surfaces before spermatozoa penetrate into the zona.     

Ultrastructural localization of labeled acrosomal glycoproteins during in vivo fertilization in the rabbit

Rabbit spermatozoa were labeled predominantely in their acrosomal glycoproteins by 1-³H-glucosamine during spermiogenesis. Ova fertilized in vivo by spermatozoa labeled 22 days earlier were analyzed by fine-structure autoradiography for the localization of the label. The latter was found associated with 1) the fused membranes of the acrosomal cap remaining on the zona pellucida surface, 2) the material released on the zona surface after the acrosome reaction and possibly detectable after tannic acid fixation, 3) the equatorial segment of the sperm head and the preequatorial swellings, and 4) other sperm components, eg, the sperm tail. No labeling, on the other hand, was detected on the denuded leading edge of spermatozoa found either in the penetration slit or in the perivitelline space. Our observations suggest the involvement of acrosomal glycoproteins in different mechanisms of sperm/zona pellucida interaction but are not in favor of a major role of (enzymatic) glycoproteins bound to the inner acrosomal membrane during the penetration of the zona pellucida.     

An ultrastructural study of epididymal mouse spermatozoa binding to zonae pellucidae in vitro: sequential relationship to the acrosome reaction

Mouse sperm bind to the zona pellucida of the egg prior to penetration of the zona and entry into the perivitelline space. The question then arises: when does the acrosome reaction occur relative to these processes? An ultrastructural study of mouse epididymal sperm bound to the surface of the zona and in the privitelline space was undertaken to clarify this point. Cumulus-free mouse eggs were inseminated in either a complete defined culture medium capable of supporting in vitro fertilization or in Tris/NaCl buffer containing Ca+2. Both media support sperm binding to the zona to the same extent; binding is complete in 15 minutes. Unbound sperm were removed by a step gradient density centrifugation to yield a preparation of eggs with sperm firmly bound. All sperm in the perivitelline space had undergone the acrosome reaction. Sperm bound at the surface of the zonae pellucidae of eggs recovered at ten minutes after insemination all had intact acrosomes. At 40 minutes after insemination, half of the sperm were intact; the other half were in the initial stages of the acrosome reaction. At 90 minutes after insemination, 12% of the sperm had undergone the full acrosome reaction and were starting to penetrate the zona; of the balance, half were in various stages of the acrosome reaction, while half were still intact. These findings support the hypothesis that the sequence of the early reactions leading to fertilization in the mouse is: intact sperm binding to zona; acrosome reaction at the zona surface; penetration of the zona.     

Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization—by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin—of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the anti-acrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa. © 1994 Wiley-Liss, Inc.     

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.     

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。     

Motility of rat spermatozoa at the site of fertilization

This study was undertaken to examine the factors that may affect the numbers and motility patterns of spermatozoa at the site of fertilization. The contents of the oviductal ampullae of previously mated cycling or superovulated immature rats were examined microscopically. We determined whether spermatozoa were free or associated with cells and whether they exhibited hyperactivated motility, forward progressive motility, or were immotile. These data were correlated with the percentage of fertilized eggs. In addition, the beat pattern of hyperactivated spermatozoa was characterized by using high-speed video microscopy. At the time when half of the eggs were fertilized, ampullae of cycling rats contained an average of less than one motile spermatozoon per ampulla. Most of these motile spermatozoa were hyperactivated. About half of these were free in the ampulla and about half were in the cumulus or zona pellucida. Hyperactivated spermatozoa displayed a nonprogressive whiplash wave form with a high amplitude recovery stroke similar to that described in hamster and guinea pig spermatozoa capacitated in vitro. In addition to motile spermatozoa, we counted about three immotile spermatozoa for each motile spermatozoon. In superovulated, immature female rats, we found about ten times as many spermatozoa in each category as in cycling rats. From our observations, it is clear that very few spermatozoa reach the ampulla of the oviduct. Furthermore our observations suggest that in cycling rats progressively swimming spermatozoa may become hyperactivated shortly after entering the ampulla of the oviduct. They probably enter the cumulus mass within a short time or become immotile.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.     

Acrosomal status and motility of guinea pig spermatozoa during in vitro penetration of the cumulus oophorus

Previous studies have suggested that both acrosome-intact and acrosome-reacted guinea pig sperm are capable of binding to the zona pellucida of cumulus-free oocytes, but the acrosomal status of guinea pig sperm during penetration of the cumulus has not been reported. We made video recordings of the interaction between capacitated guinea pig sperm and cumulus-invested guinea pig oocytes. The videotapes were analysed to identify sperm with hyperactivated motility and to classify the acrosomal status of sperm during penetration of the cumulus and after binding to the zona pellucida. The resolution of the video recordings was not sufficient to recognise sperm with swollen acrosomes. However, sperm that had completed the acrosome reaction were easily identified. Acrosome-reacted sperm were found adherent to the outer boundary of the cumulus, but were never observed to penetrate the cumulus. The percentage of acrosome-intact, hyperactivated sperm was higher in the cumulus oophorus than in culture medium, suggesting that changes in motility were elicited in response to contact with the cumulus. Fully acrosome-reacted sperm were found adherent to the zona pellucida, and solubilised guinea pig zona pellucida was capable of inducing acrosome reactions in capacitated guinea pig sperm. Acrosome-intact sperm were also observed on the zona, but they were not tightly bound and did not have hyperactivated motility, suggesting that these sperm were not functionally capacitated. Our observations demonstrate that guinea pig sperm penetrate the cumulus matrix in an acrosome-intact state. Although we did not observe sperm undergoing the acrosome reaction, our observations and experimental data suggest that the acrosome reaction of guinea pig sperm is completed on or near the surface of the zona pellucida.     

The penetrability of rabbit ova treated with enzymes or anti-progesterone antibody: a probe into the nature of a mammalian fertilizin.

The acrosome reaction of rabbit spermatozoa, an essential prerequiste for penetration of the zona, occurs usually in the vicinity of the egg, suggesting that the rabbit may produce a factor akin to the 'fertilizin' of some invertebrates. Specific inactivation of such a factor should render eggs impenetrable and possibly point to the nature of a 'fertilizin' in mammals. Rabbit eggs with granulosa cells removed were treated for different periods with trypsin, chymotrypsin, neuraminidase or anti-progesterone antiserum, and then transferred alone, or together with control eggs (one group labelled with fluorescein isothiocyanate), to the oviducts of inseminated recipients. Three hours later the eggs were recovered and the experimental and control groups were compared for penetration of the vitellus and for numbers of spermatozoa within the perivitelline space or in the zona pellucida. None of these treatments affected the penetrability of the zona pellucida significantly since the number of spermatozoa within treated eggs in any one experiment was always comparable to that of untreated eggs exposed to the same fertilization environment. If there is a specific substance emanating from or present on the surface of the rabbit egg which induces the acrosome reaction, its activity seems unaffected by trypsin or chymotrypsin; the charged radicals of N-acetyl neuraminic acid or local concentrations of progesterone do not appear to be involved.