Sperm capacitation and gamete interaction.
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Animals↗
Biomedical subjects
R Yanagimachi
Publications and source records attributed to R Yanagimachi.
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.
Acrosome↗
Acrosome-reacted guinea pig spermatozoa become fusion competent in the presence of extracellular potassium ions.
Guinea pig spermatozoa are able to undergo capacitation and the acrosome reaction in a K+-free (-deficient) medium. However, they are unable to fuse with eggs unless they are exposed to a millimolar concentration of extracellular K+ during or after the acrosome reaction. Apparently, the plasma membrane over the equatorial segment gains the ability to fuse with eggs in the presence of K+ during and/or after the acrosome reaction. Once it becomes fusible, the membrane retains its fusibility even in a K+-deficient medium. Rb+ is almost as effective as K+ in rendering the sperm membrane fusible. Li+ and Cs+ are less effective. The molecular mechanism by which K+ renders acrosome-reacted spermatozoa fusion competent is unknown, but it may involve K+-mediated efflux of H+ from the spermatozoa.
Acrosome↗
A glycoprotein of oviductal origin alters biochemical properties of the zona pellucida of hamster egg.
Eggs were isolated from ovaries and oviducts of the golden hamster and the components of zonae pellucidae were examined using density gradient SDS-polyacrylamide electrophoresis. Zonae of ovarian eggs (ZP-OVA) had three major components corresponding to the so-called ZP-1, ZP-2, and ZP-3. Zonae of recently ovulated eggs collected from oviducts (ZP-OVI) had a 200-240 K component (ZP-O) in addition to the three components present in ZP-OVA. When ovarian and oviductal eggs were stained with FITC-conjugated B. simplicifolia-1 lectin (BS-1), which specifically binds to alpha-D-galactose- or alpha-N-acetyl-D-galactosamine-like terminal saccharide residues, ZP-OVI was intensely stained, while ZP-OVA was not. ZP-OVA gained the ability to bind to BS-1 after a brief treatment with oviduct extracts. These results suggest that biochemical properties of hamster zonae change after transport of eggs from ovary to the oviduct. The addition of the 200-240 K component of oviductal origin to preexisting zona components seems to be responsible for this change.
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Synthetic organic pH buffers can support fertilization of guinea pig eggs, but not as efficiently as bicarbonate buffer.
Bicarbonate ions (HCO3-) in the medium are not absolutely essential for the fertilization of guinea pig eggs. However, fertilization takes place most efficiently in HCO3- -buffered medium. Capacitation, the acrosome reaction, and zona penetration by spermatozoa can occur in HCO3- -free media with synthetic organic buffers (i.e., MOPS, TES, HEPES, Tris, and TAPSO) but not as efficiently as in the HCO3- -buffered medium. It appears that HCO3- functions as more than just a pH-buffering molecule.
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Quantitative comparison of the passage of homologous and heterologous spermatozoa through the uterotubal junction of the golden hamster.
A quantitative method was used to determine whether the spermatozoa of foreign species could pass through the uterotubal junction (UTJ) of the hamster as efficiently as homologous (hamster) spermatozoa. Estrous female hamsters were artificially inseminated with epididymal spermatozoa of homologous and heterologous (foreign) species. The number and distribution of spermatozoa in the oviduct were determined several hours after insemination (shortly before ovulation). The passage of immotile (dead) hamster spermatozoa through the UTJ was also examined. It was found that the spermatozoa of all foreign species tested (rat, mouse, guinea pig, and rabbit), as well as immotile hamster spermatozoa, could pass through the UTJ but did so in much smaller numbers compared to live hamster spermatozoa. This was not specifically due to poor survival of foreign spermatozoa in the hamster uterus, as the viability of all inseminated spermatozoa (including hamster spermatozoa) was considerably reduced by 1 h after insemination. While a large number of live hamster spermatozoa were distributed throughout the caudal isthmus at the time of examination, none or only a very few foreign spermatozoa had advanced this far. The few foreign and immotile spermatozoa that reached the caudal isthmus were confined to the first ascending loop of this segment. Some possible causes for the small number and retarded advance of foreign spermatozoa in the hamster oviduct were discussed.
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Human sperm nuclei can transform into condensed chromosomes in Xenopus egg extracts.
The conditions necessary for inducing pronucleus formation and chromosome condensation of human sperm nuclei were studied using Xenopus egg extracts. When lysolecithin (LC)-and dithiothreitol (DTT)-treated spermatozoa were incubated with egg extract prepared with an extraction medium containing 10 mM EGTA, sperm nuclei transformed into chromosomes, bypassing the pronuclear stage. On the contrary, LC-DTT-treated spermatozoa incubated in egg extract without EGTA mimicked LC-permeabilized Xenopus spermatozoa in that they underwent pronucleus formation accompanied by DNA synthesis and subsequent chromosome condensation in the correct chronological order. These observations suggest the importance of intracellular calcium for regulating the states of nuclear chromatin. LC-permeabilized human spermatozoa were not responsive to the egg extract without any additives, but formed pronuclei when incubated with the egg extract supplemented with 5-10 mM DTT. However, subsequent chromosome condensation of sperm nucleus did not occur in the DTT extract. This suggests that disulfide-reducing agent blocks transformation of sperm pronuclei into condensed chromosomes.
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Zonae pellucidae of salt-stored hamster and human eggs: their penetrability by homologous and heterologous spermatozoa.
Zonae pellucidae of unfertilized hamster and human eggs remain penetrable by homologous spermatozoa when they are stored in highly concentrated salt solutions [Yanagimachi et al., 1979]. Neutralization of salt solutions with the appropriate pH buffers is recommended for long-term storage of the eggs (zonae), because zonae may gradually swell or even dissolve in acidic or alkaline solutions. At least for the hamster, 2 M ammonium sulfate solution containing 40 mM Hepes and 0.5% dextran (pH 7.0) seems to serve the purpose satisfactorily. Thorough rinsing of salt-stored zonae prior to insemination is important, but inclusion of protease inhibitors in the rinsing medium does not appear to be essential. A dialysis method is recommended when a large number of salt-stored eggs must be rinsed at one time. Salt-stored hamster zonae, like fresh (control) zonae, permit attachment and penetration of hamster spermatozoa, but not human spermatozoa. Salt-stored human zonae, on the other hand, allow penetration by not only human spermatozoa, but also hamster spermatozoa.
Ammonium Sulfate↗
Ovarian histopathology of bitches immunized with porcine zonae pellucidae.
The ovarian histopathology of bitches immunized with crude (cPZP) or partially purified (pPZP) porcine zona pellucida proteins was examined in order to determine the cause of abnormal estrous cycles. The majority of immunized bitches had ovarian cytes. Those immunized with cPZP had follicular cysts lined with a thin layer of granulosa cells, while in those immunized with pPZP, the cysts were lined by a basement membrane with a clump of luteinized cells. In two bitches immunized with cPZP, oocytes were present only in primordial follicles. Similar abnormalities were not found in a bitch immunized with human serum albumin or in 12 untreated bitches. Oocytes flushed from the oviducts of mated, immunized bitches were degenerating, which may have been a primary cause of infertility in such bitches. Ovaries studied 2-6 weeks after immunization showed no loss of gap junctional communication between oocytes and granulosa cells, nor was any inflammatory reaction seen. IgG was bound to the zona as revealed by fluoresceinated protein A staining of frozen sections of those ovaries. Abnormal estrous cycles in PZP-immunized bitches appear to result from follicular dysgenesis or cyst formation, but the etiology of these conditions is unresolved.
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Effects of agents used for zona pellucida removal on hamster oocyte penetration by human spermatozoa.
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Animals↗
Superovulation in immature and mature Chinese hamsters.
Mature female Chinese hamsters ovulate an average of 8.8 +/- 1.0 (mean +/- SD) eggs per female in each estrous cycle. Superovulation can be induced in both immature and mature females by subcutaneous or intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and either human chorionic gonadotropin (hCG) or pituitary luteinizing hormone (PLH). The best superovulation in immature females was induced by the administration of 15 IU of PMSG followed 72 hr later by injection of 15 IU of hCG (about 25 eggs per female) or 0.2 mg (200 IU) PLH (about 46 eggs per female). Ovulation started about 13-15 hr after administration of hCG (or PLH) and was completed during the next 5-6 hr. Superovulation in mature females could be induced by injecting PMSG any day of the estrous cycle, but the best superovulation (about 39 eggs per female) was induced by injecting 15 IU of PMSG on day 1 (day of ovulation) followed by the injection of 0.4 mg of PLH 72 hr later. When immature females treated with the best superovulatory protocol were mated on the evening of PLH injection, only 5% of the eggs were found fertilized 50 hr after PLH administration. On the other hand, about 60% of the eggs were found fertilized in mature females mated following treatment with the best superovulatory protocol. The majority (83-85%) of superovulated eggs obtained from both immature and mature females were normally fertilized in vitro.
Aging↗
Sperm chromosome analysis as a new system to test human X- and Y-sperm separation.
The correlation between the percentage of F-body-positive spermatozoa (F-sperm) and that of Y-chromosome-bearing spermatozoa (Y-sperm) determined by human sperm chromosome analysis was examined after spermatozoa were separated by an albumin column method [Ericsson et al, Nature 246:421-424:1973]. The percentages of F-sperm were 43.1 +/- 4.8 (36.1-48.8) and 45.8 +/- 8.3 (35.3-66.5) before and after separation, respectively. A slight but significant (p less than 0.05) increase in F-sperm after the separation was observed in two semen donors. A total of 570 sperm karyotypes was analyzed and the percentage of Y-sperm ranged from 36.0 to 59.1 with a mean of 47.3. In one semen donor, the percentage of Y-sperm (45.6) was significantly (p less than 0.05) higher than that of F-sperm (35.3) after separation. In the remaining nine semen donors, the results obtained by chromosome analysis corresponded well with those obtained by F-body scoring. The validity of the albumin column method in isolating a Y-sperm-rich sperm population is discussed.
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Distribution and number of spermatozoa in the oviduct of the golden hamster after natural mating and artificial insemination.
A group of female hamsters was mated with males of proven fertility either several hours before or during ovulation. Another group of females was artificially inseminated several hours before ovulation. Females were killed at various times after the onset of mating or artificial insemination, oviducts were fixed and sectioned serially, and spermatozoa were counted individually as to their location in the oviduct. Regardless of the type or time of insemination, the vast majority of spermatozoa that entered the oviduct remained in the lower segments of the isthmus (the intramural and caudal isthmus) without ascending to the ampulla. The lower segments of the oviduct, particularly the caudal isthmus, appeared to be acting as a "sieve" and/or "sperm reservoir." In females mated or artificially inseminated prior to ovulation, virtually no spermatozoa reached the cephalic isthmus or ampulla until the commencement of ovulation. Although a few spermatozoa reached the ampulla by 1 h after the onset of mating, they were the exception rather than the rule. When females were mated during ovulation, spermatozoa spent a minimum of about 3 h in the caudal isthmus before ascending to the ampulla. The number of spermatozoa that entered the oviduct after artificial insemination was considerably lower than in naturally mated animals, but this low number was apparently large enough to ensure complete fertilization.
Animals↗
Alpha-L-fucosidase from bull seminal plasma: its purification and acrosome reaction promoting property.
Alpha-L-fucosidase was purified from the bull seminal plasma by chromatography on DEAE-disk, octyl sepharose hydrophobic column and HPLC. The enzyme appeared to be pure as judged by the polyacrylamide gel electrophoresis both under the nondenaturing and denaturing conditions. The pure enzyme promoted the acrosome reaction of guinea pig spermatozoa in vitro. This is the first report showing that an acrosomal enzyme induces acrosome reaction which is an essential pre-requisite for the gamete interaction and fertilization.
Acrosome↗
Development of pronuclei from human spermatozoa injected microsurgically into frog (Xenopus) eggs.
Human spermatozoa were demembranated with Triton X-100 (TX) and injected into the mature eggs of Xenopus laevis. The nuclei of these spermatozoa decondensed and developed into pronuclei. Chromosomes did not appear in the eggs until the end of a 5-hr incubation period. When the demembranated human spermatozoa were further treated with dithiothreitol (DTT) before they were injected into the eggs, the sperm nuclear decondensation and pronuclear development took place considerably faster than in spermatozoa treated with the detergent alone. By the end of the 5-hr incubation period, decondensed chromatin threads or chromosome-like structures appeared, but none of the eggs cleaved. When human spermatozoa were injected into full-grown ovarian oocytes with intact germinal vesicle (GV) or oocytes which had matured without GV, the nuclei of a proportion of TX-treated and all TX-DTT-treated sperm decondensed but showed no sign of developing into pronuclei. Sperm nuclei injected into maturing oocytes formed condensed chromatin fragments as long as the oocytes were not activated, but they transformed into pronuclei when the oocytes were stimulated with electric shock. These results indicate that the cytoplasmic factors responsible for the decondensation of human sperm nuclei are present in egg cytoplasm independent of GV-materials. We also suggest that the factors controlling development of decondensed sperm nuclei into pronuclei are dependent on GV materials.
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Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status.
Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the head alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems.
Acrosome↗
Normal development of hamster and rabbit eggs fertilized by spermatozoa labelled with the fluorescent thiol alkylating agent, monobromobimane.
Cauda epididymal spermatozoa of the golden hamster were labelled with the thiol-alkylating reagent, monobromobimane (MB). Female hamsters underwent uterine insemination with labelled spermatozoa at laparotomy under metofane anesthesia. All 12 females examined between 5 and 54 h postinsemination yielded a total of 83/100 (83%) eggs in the process of fertilization or embryos. Under ultraviolet (UV) exposure all exhibited a fluorescent tail which, in the 4- and 8-cell embryos, could be seen to be fraying or disintegrating. As cleavage progressed, labelled tail components came to be restricted among the blastomeres such that at the 4- and 8-cell stage the tail could be seen in only one to three blastomeres. To study complete development and pregnancy another 12 females received uterine insemination. After recovery from anesthesia (approximately equal to 4 h) these females were mated with a vasectomized male bearing a dominant genetic marker (black eyes) to allow unequivocal determination of paternity in the fetuses and young produced. Seven became pregnant with one female losing her pregnancy about Day 7 of gestation. Two females sacrificed on Day 13 produced 17 normal fetuses and one resorption. Four females delivered 16 young, all normal at birth and in subsequent growth and fertility. In addition, insemination of female rabbits with MB-labelled spermatozoa yielded normal embryos (50/52 96%) from 3 does on Day 2 and (38/64 60%) from 4 does on Days 4 or 5. Two normal litters (9 bunnies) have delivered from 3 does allowed to carry to term.(ABSTRACT TRUNCATED AT 250 WORDS)
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Acrosome reaction in human spermatozoa.
Human spermatozoa were allowed to undergo the acrosome reaction in vitro by incubation in media with or without reagents known to accelerate the onset of the acrosome reaction. The first observable change before the acrosome reaction was a partial decondensation of the acrosomal matrix. This was followed by invaginations of the outer acrosomal membrane alone or of both the plasma and outer acrosomal membranes, which resulted in formation of many vesicles within the acrosomal cap. Subsequently, the plasma and outer acrosomal membranes fused, but the fusion was seldom seen in the acrosomal cap region. On the other hand, fusion of the two membranes was observed consistently at the anterior end of the equatorial segment of the acrosome. Soon after the membranes over the acrosomal cap disappeared, many vesicles, which were originally within the cap, were seen on or in the vicinity of the inner acrosomal membrane. These vesicles dispersed eventually, leaving the inner acrosomal membrane completely exposed. Thus the acrosome reaction in human spermatozoa seems to proceed in a way somewhat different from that in spermatozoa of most other species, although the end result of the reaction is the same.
Acrosome↗