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.     

Relationship between the length of sperm preincubation and zona penetration in the golden hamster: A scanning electron microscopy study

Hamster spermatozoa are able to fertilize a high percentage of zona-intact hamster oocytes when they are preincubated for 2 hr in a chemically defined medium. From this time on, the longer the preincubation time the lower the percentage penetration. Spermatozoa preincubated for 6 or more hr are unable to cross the zona pellucida, retaining however their ability to fuse with zona-free hamster oocytes. Zona-intact hamster oocytes, as described above, were observed with the scanning electron microscope. When the oocytes were inseminated with spermatozoa preincubated for 1 to 5 hr the outer surface of the zona showed the penetrating spermatozoa and the sperm tracks made by those that failed to cross it. With longer preincubation times no penetrating spermatozoa were observed, and very few sperm tracks were present on the outer surface of the zona. Control experiments showed that neither eggs, spermatozoa, nor fertilization were affected by the medium recovered after long preincubations. These results show that care should be taken regarding the preincubation time when using the in-vitro fertilization technique.     

Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis. © 1993 Wiley-Liss, Inc.     

Ethanol inhibits human and hamster sperm penetration of eggs

The effect of alcohol on the fertilizing ability of both human and hamster spermatozoa was examined by an in vitro fertilization assay using hamster ova. Spermatozoa were incubated in capacitating media for 3 hr (hamster sperm) and 4 hr (human sperm). Hamster ova were inseminated with preincubated sperm and were examined after 2 to 3 hr. Ethanol was added to the capacitating media at concentrations of 25, 50, 100, 200, and 400 mg%. Fertilization of zona-free hamster eggs by human spermatozoa was reduced from 49.6% in no alcohol to 16.7% in 400 mg% ethanol. Fertilization of hamster eggs by hamster sperm revealed a reduction from 63.6% to 33.7% in cumulus-intact eggs and from 65.8% to 10.8% in cumulus-free eggs in the presence of ethanol at 400 mg%. Hamster sperm acrosome reaction was reduced from 47% to 12%. When these hamster sperm with reduced acrosome reaction were placed with zona-free hamster eggs, the 100% fertilization rate was not reduced; however, the fertilization index, which reflects the number of swelling sperm heads per egg, was reduced from 8.5 to 1.8. This suggests that as little as 12% of the sperm with an acrosome reaction is sufficient to fertilize 100% of the zona-free eggs. If ethanol was added to the insemination media only, there was no inhibition of fertilization by human sperm or hamster sperm that had been previously capacitated in an ethanol-free media. Removal of the ethanol from the preincubated sperm produced fertilization at control levels; thus the inhibitory effect is reversible. These results indicate that ethanol may affect fertilization by an inhibition of the capacitation and/or acrosome reaction process.     

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.     

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.     

精胺抑制人精子的体外受精能力

以精子穿透去透明带仓鼠卵试验(SPA)为模型,评价了精胺对人精子体外受精能力的影响。精胺(0.25—8.0mmol/L)可抑制人精子体外获能和受精,其抑制作用与精胺浓度呈正相关,此种抑制作用是可逆的。用 HPLC 测定精子精胺含量表明,精子获能后精胺含量明显下降。dbcAMP(0.5—1.0mmol/L)或咖啡因(10mmol/L)可拮抗精胺抑制人精子体外获能。其拮抗作用随 dbcAMP 浓度而增强。钙离子载体 A 23187 2/μmol/L 或胰蛋白酶0.05%均可拮抗精胺抑制人精子穿卵率。上述结果提示,精胺可能通过降低精子 cAMP 含量和抑制钙内流或顶体酶活性,从而阻止人精子体外获能和受精。     

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.     

Sperm penetration in vitro of pig follicular oocytes matured in culture.

Boar ejaculated and epididymal spermatozoa were preincubated in modified KRB or the isolated oviduct and uterine horn of an oestrous sow for 4.5-5 h at 37 degrees C before introduction into medium containing ovarian oocytes previously cultured for 24 h. At examination 17-20 h after insemination 60.6% of the total oocytes had reached at least the 2nd metaphase. The proportions of oocytes penetrated (i.e. enlarged sperm head or male pronucleus and corresponding sperm tail) were 0, 10.0 and 16.7% with ejaculated spermatozoa, and 3.3, 19.6 and 26.4% with epididymal spermatozoa preincubated in modified KRB, oviduct and uterus, respectively. Although the proportion of oocytes with morphologically normal male and female pronuclei was low (10/36 = 27.8%), the results suggest that boar spermatozoa can be capacitated in the isolated genital tract of an oestrous sow and that capacitation of epididymal is better than that of ejaculated spermatozoa.     

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.     

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.     

Evidence suggesting a role for sperm metalloendoprotease activity in penetration of zona-free hamster eggs by human sperm

It has been reported that metalloendoprotease (MEP) activity is involved in somatic cell membrane fusion events and in the sea urchin sperm acrosome reaction (AR). MEP activity also has been demonstrated in human and other mammalian sperm. The present study was concerned with investigating whether a human sperm MEP is important in membrane events necessary for sperm egg fusion. Ejaculated human sperm were washed, capacitated in vitro, and preincubated with the competitive MEP inhibitors phosphoramidon (50 microM) or CBZ-L-phenylalanine (1 mM), with 100 microM diethylenetriaminepentaacetic acid (DTPA), a heavy metal chelator, or as controls, with the appropriate solvents. The AR was initiated in vitro with preovulatory human follicular fluid and the sperm washed to dilute inhibitors and then coincubated with zona-free golden hamster eggs (zonae and cumuli removed with trypsin and hyaluronidase, respectively). Eggs were washed after 0.5 h, and the number of sperm remaining bound was counted. After 2.5 h further incubation, the eggs were stained with acetolacmoid or acetoorcein and penetration was assayed by counting the number of decondensed sperm heads per egg (penetration index) and the percent of penetrated eggs. The inhibitor treatments did not decrease the percentage of penetrated eggs (range 80-90%), but a significant reduction in the penetration index was observed. Phosphoramidon reduced the penetration index by 45%, CBZ-L-phenylalanine by 57%, and DTPA by 56%. None of the inhibitors decreased the penetration index or the percentage of penetrated eggs when added directly to suspensions of acrosome-reacted sperm and zona-free eggs at the diluted levels that would have been present after washing inhibitor-treated sperm.(ABSTRACT TRUNCATED AT 250 WORDS)     

γ-氨基丁酸诱发人精子顶体反应及其受精能力

本文的目的是为了探讨γ－氨基丁酸是否可激发人精子顶体反应,受主其可能的作用方式。实验采用金霉素荧光染色法,胞内游离Ｃ６２＋测定和精子穿透去透明带仓鼠卵试验,分别评价ＧＡＢＡ诱发人精子ＡＲ及其穿卵能力。结果表明;ＧＡＢＡ可诱发人获能精子发生ＡＲ,且随精子获得进程而显著增加,并存在明显的量效关系,该作用可被Ｐ４加强。     

Penetration of zona-free hamster eggs by liposome-treated sperm from the bull, ram, stallion, and boar

Spermatozoa from each of four rams, four stallions, and three boars (six semen samples) were treated with dilauroylphosphatidylcholine (PC12) liposomes and compared with control bull sperm to induce the acrosome reaction (AR) and study possible penetration of the sperm into zona-free hamster eggs. Diluted sperm were incubated with several concentrations of PC12 for 7 min at 39 degrees C prior to insemination of the hamster eggs in vitro. The sperm from the bull were diluted to 10(6) cells/ml, as previously studied. Sperm from the ram, stallion, and boar were diluted to 6 X 10(6) and 20 X 10(6) cells/ml. After addition to the eggs, the sperm concentration was reduced by 75 percent. Inseminated eggs were incubated with sperm for 3 h at 39 degrees C prior to being fixed, stained, and observed for sperm penetration. At an initial concentration of 6 X 10(6) cells/ml, bull sperm treated with 36.7 microM PC12 achieved an egg penetration rate of 92%, whereas under nearly identical conditions stallion spermatozoa achieved only 54% egg penetration. Under similar conditions, ram spermatozoa failed to penetrate eggs, but when the initial sperm concentration was increased to 20 X 10(6) cells/ml, sperm incubated with 51.1 microM PC12 achieved 52% egg penetration. Boar spermatozoa treated with PC12 at either sperm concentration failed to exhibit an AR or penetrate hamster eggs. In general, as PC12 concentration increased the percentage of sperm with an AR increased and sperm motility decreased. It is concluded that 1) PC12 liposomes are effective in inducing the AR in sperm from the bull, ram, and stallion, but under conditions tested are ineffective with boar sperm;(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

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.     

A quantitative comparison of the passage of capacitated and uncapacitated hamster spermatozoa through the uterotubal junction.

Female hamsters were artificially inseminated at the time of ovulation with an equal concentration and volume of capacitated sperm suspension in one uterus and uncapacitated sperm suspension in the contralateral uterus. When oviducts were examined 3.5-4.0 h after insemination, a significantly (paired t-test, p less than 0.05) lower number of spermatozoa were found in the oviduct from the side inseminated with capacitated sperm suspension compared to the side inseminated with uncapacitated sperm suspension. The reduction in the number of spermatozoa entering the oviduct on the side inseminated with capacitated sperm suspension was particularly evident when nearly all the spermatozoa in the suspension were hyperactivated. These results suggest that hamster spermatozoa require a progressive linear type of motility pattern to pass efficiently through the uterotubal junction and that under normal conditions in vivo, fertilizing spermatozoa initiate hyperactivated motility after entering the oviduct.     

Assessment of human sperm function after recovery from the female reproductive tract

The physiology and fertile life of human spermatozoa in the female reproductive tract have received little previous attention. A technique was developed for recovering spermatozoa from human cervical mucus at various intervals after artificial insemination. The functions of these cells as measured by penetration of the human zona pellucida and fusion with the zona-free hamster oocytes were examined. Penetration into the zona pellucida was consistently observed when sperm were recovered from 1 to 80 h after insemination. Penetration through the zona into the perivitelline space (PVS) was seen from 1 to 72 h after insemination. Fusion of human sperm with zona-free hamster oocytes was observed from 1 to 48 h after insemination. Motile sperm were recovered 112 and 120 h after insemination with swimming speeds comparable to freshly capacitated spermatozoa. Concentrations of recovered sperm at these longer intervals from insemination were insufficient for sperm-oocyte assays. These studies demonstrate that human spermatozoa aged in vivo may be recovered from cervical mucus for physiologic study, and suggest that the fertile life of human sperm may be 80 h or more.     

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.     

Capacitation status of hamster spermatozoa in the oviduct at various times after mating

Female hamsters were mated shortly after the onset of oestrus or immediately after ovulation. At various times after mating, spermatozoa were flushed from the isthmus of the oviduct using a modified Tyrode's medium supplemented with 20% hamster serum. Cumulus oophorus-free eggs were introduced into the suspensions of isthmic spermatozoa. Some eggs were removed every 30 min and examined for evidence of fertilization. For females mated shortly after the onset of oestrus, spermatozoa recovered from the oviducts 8 h after mating (about 1.5 h after ovulation) could penetrate eggs within 30 min and were considered fully capacitated. When spermatozoa were recovered at earlier times (1, 2, 4 and 6 h after mating) they required additional time (2, 1.5, 1 and 1 h respectively) in vitro before penetrating eggs. Therefore, when mating occurs shortly after the onset of oestrus, spermatozoa in the oviduct do not appear to become fully capacitated until about the time of ovulation. For females mated immediately after ovulation, spermatozoa recovered from the oviducts at 4 h after mating could penetrate eggs within 30 min. Spermatozoa recovered at 1 and 3 h after mating required 2 and 1 h respectively in vitro before penetrating eggs. These results suggest that sperm capacitation proceeds at a faster rate when mating occurs after ovulation.