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.     

In vitro fertilization of two species of deer mouse eggs by homologous or heterologous sperm and penetration of laboratory mouse eggs by deer mouse sperm

Newly ovulated eggs from immature deer mice (Peromyscus maniculatus and P. polionotus) and mature laboratory mice (Mus musculus) treated with PMSG and HCG were inseminated in vitro with spermatozoa recovered from the cauda epididymidis of mature males. The time required for capacitation of deer mouse sperm in culture was estimated to be about two to five hours based on the dispersal of sperm agglutination and increase of sperm motility. The rate of sperm penetration through the zona pellucida of deer mouse eggs by homologous or heterologous sperm was relatively high (72-91%) but that of laboratory mouse eggs by deer mouse sperm was low (20-21%). After penetration through the zona pellucida, a high proportion of deer mouse eggs (79-93%) were fertilized by homologous or heterologous deer mouse sperm but no laboratory mouse eggs were fertilized by sperm of two species of deer mice. The zona pellucida was dissolved in a higher proportion of laboratory mouse eggs cultured with P. maniculatus (45%) than with P. polionotus sperm (3.4%), but this did not happen by incubation of deer mouse eggs with homologous or heterologous sperm. It seems that there is little difference in sperm penetration and fertilization between these two closely related species of deer mice but the reactions between the mouse eggs and deer mouse sperm are quite different.     

Differences in sperm function in vitro but not in vivo between inbred and random-bred mice

Genetic variation in spermatozoa was used to examine mechanisms important for fertilization in the mouse. A significantly greater proportion of cauda epididymal sperm from C57BL/6 (inbred) males were motile than from random-bred (CFW) males. Random-bred sperm, however, were able to fertilize a significantly greater percentage of eggs in vitro than were inbred sperm. When sperm of these two genotypes were used for insemination in vivo, and the penetrated eggs cultured through the first cleavage, the levels of cleavage were similar, suggesting that neither levels of sperm motility nor sperm penetration in vitro accurately reflect the ability of the same sperm populations to penetrate eggs in vivo.     

In vivo and in vitro fertilization of hamster, rat and mouse eggs after treatment with anti-hamster ovary antiserum.

Rabbit antiserum against hamster ovary was examined on agargel diffusion plates against several hamster tissues, and also against rat and mouse ovarian extracts. Unabsorbed anti-hamster ovary antiserum showed eight to nine precipitin bands for hamster ovary and four to eight bands for other tissue extracts, but no bands against sperm antigens. Anti-hamster ovary antiserum also showed three to four bands for rat and one to two bands for mouse ovarian extracts. The present experiments confirmed previous reports for the hamster and mouse that treatment of eggs with anti-ovary antiserum blocked in vitro fertilization and that the extent of the inhibition was related to the formation of a precipitate on the zone pellucida. A single injection of anti-hamster ovary antiserum inhibited fertilization in mice but not in rats. In vitro fertilization of mouse eggs was also inhibited in the presence of such antiserum.     

Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes

Pig follicular oocytes cultured in a defined medium for 28-29 h were inseminated in vitro by epididymal or ejaculated boar spermatozoa that were preincubated in a modified KRB solution at various sperm concentrations for 4 h at 37 degrees C. Sperm concentration at insemination was 2 X 10(6) cells/ml. When epididymal spermatozoa were preincubated at concentrations of 4-16 X 10(8) cells/ml, 71-75% of oocytes were penetrated. In contrast, preincubation at a low concentration (0.8 X 10(8) cells/ml) resulted in a low penetration rate (11%). Epididymal spermatozoa preincubated at a concentration of 4 X 10(8) cells/ml could also penetrate denuded oocytes. None of the oocytes were penetrated by epididymal spermatozoa that were exposed to seminal plasma before preincubation or by ejaculated spermatozoa. After preincubation, whiplash motility was observed in the epididymal spermatozoa, but not in the ejaculated spermatozoa.     

In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars

The objective of this study was to compare the in vitro fertilizing capacity of porcine spermatozoa from fresh and frozen-thawed semen and frozen-thawed epididymal spermatozoa obtained from identical boars. Prior to IVF, fresh spermatozoa were capacitated in TCM 199. Frozen semen samples were stored in 0.25-ml plastic straws using a lactose/glycerol/orvus-es-paste extender. Cumulus-oocyte-complexes (COC) obtained from superovulated prepuberal gilts were fertilized in vitro within 2 h after aspiration with one of the semen samples. After final dilution for IVF, frozen-thawed epididymal semen samples showed motility rates (72.2 +/- 5.6%) similar to those of spermatozoa in fresh semen (76.4 +/- 4.5%), while sperm motility decreased in frozen-thawed ejaculated semen (40.2 +/- 9.4%). Considerable individual differences in sperm motility between boars were observed for ejaculated semen but not for epididymal semen. Enhanced fertilizing capacity of frozen-thawed epididymal spermatozoa was confirmed by pronucleus formation and cleavage rates, with significantly more embryos developing to the 2- and 4-cell stages compared with the groups fertilized with fresh or with frozen-thawed ejaculated semen (59.7 vs 14.6 and 16%). In conclusion, consistent in vitro fertilization rates with minimal semen variability are obtained using frozen-thawed epididymal semen. Following a modified freezing protocol, epididymal spermatozoa can easily be frozen in small containers for IVF, with higher resultant motility and fertilization rates than with ejaculated semen.     

Ejaculated Mouse Sperm Enter Cumulus-Oocyte Complexes More Efficiently In Vitro than Epididymal Sperm

The mouse is an established and popular animal model for studying reproductive biology. Epididymal mouse sperm, which lack exposure to secretions of male accessory glands and do not precisely represent ejaculated sperm for the study of sperm functions, have been almost exclusively used in studies. We compared ejaculated and epididymal sperm in an in vitro fertilization setting to examine whether ejaculated sperm enter cumulus-oocyte complexes more efficiently. In order to prepare sperm for fertilization, they were incubated under capacitating conditions. At the outset of incubation, ejaculated sperm stuck to the glass surfaces of slides and the incidences of sticking decreased with time; whereas, very few epididymal sperm stuck to glass at any time point, indicating differences in surface charge. At the end of the capacitating incubation, when sperm were added to cumulus-oocyte complexes, the form of flagellar movement differed dramatically; specifically, ejaculated sperm predominantly exhibited increased bending on one side of the flagellum (a process termed pro-hook hyperactivation), while epididymal sperm equally exhibited increased bending on one or the other side of the flagellum (pro-hook or anti-hook hyperactivation). This indicates that accessory sex gland secretions might have modified Ca²⁺ signaling activities in sperm, because the two forms of hyperactivation are reported to be triggered by different Ca²⁺ signaling patterns. Lastly, over time, more ejaculated than epididymal sperm entered the cumulus oocyte complexes. We concluded that modification of sperm by male accessory gland secretions affects the behavior of ejaculated sperm, possibly providing them with an advantage over epididymal sperm for reaching the eggs in vivo.     

Time and process of sperm penetration into cumulus-free mouse eggs fertilized in vitro

Sperm penetration through the zona pellucida and fusion of the sperm head with the vitellus were observed continuously and filmed under phase optics in cumulus-free living mouse eggs inseminated in vitro with capacitated epididymal sperm. Most spermatozoa penetrated the zona pellucida, traversed the perivitelline space, and fused with the vitellus at an angle nearly perpendicular to the surface. The mean duration required for sperm to penetrate the zona pellucida was 20 minutes with a range of 15–26 minutes. Sperm traversed the perivitelline space in less than one second. The initial contact of sperm with the vitellus generally took place at the tip of the sperm head. When the tip of the sperm head contacted the vitellus there was an immediate reduction in the rate of flagellation, followed by the gradual sinking of the sperm head into the vitellus.     

Sperm concentration and the fertilization of human eggs in vitro

The effect of sperm concentration on the fertilization of preovulatory and immature human eggs was studied in the context of an ongoing in vitro fertilization-embryo transfer (IVF-ET) program. Fertilization success was independent of the follicular recruitment protocol used, and with preovulatory eggs, was inversely related to sperm concentration over the range of 2.5 - 50 X 10(4) motile sperm/ml. Maximum fertilization (80.8%) occurred at a concentration of 2.5 X 10(4) motile sperm/ml. The incidence of polyspermic fertilization was directly related to the sperm concentration, decreasing from 5.5% at 10 X 10(4) to 0% at 1-2.5 X 10(4) motile sperm/ml. Immature eggs cultured in vitro, then inseminated, also demonstrated an inverse relationship between fertilization and sperm concentration with a maximum fertilization rate of 66.6% at 5 X 10(4) motile sperm/ml. The percentage of motile sperm in the inseminating population had no influence on fertilization rates unless the value dropped below 40%. Fertilization success using sperm from oligospermic and polyzoospermic males was also examined. In contrast to males with normal semen parameters, oligospermic males demonstrated highest fertilization success at 50 X 10(4) motile sperm/ml. The IVF of preovulatory eggs using sperm from polyzoospermic males was comparable to that for males with normal semen parameters at equivalent sperm concentrations. The implications of these findings to the application of IVF-ET technology to the infertile couple is discussed.     

Antigens on rat spermatozoa with a potential role in fertilization

Eight monoclonal antibodies (McAbs), directed against antigens on rat cauda epididymal spermatozoa, were tested for their capacity to interfere with fertilization in vitro as a means of identifying molecules with a potential role in sperm-egg recognition and fusion. Antigens recognized by the McAbs were visualized on live spermatozoa by indirect immunofluorescence (IIF) and characterized by immunoblotting. Five McAbs (designated 1B5, 2C4, 4B5, 5B1, and 8C4) recognized antigens specifically on the sperm acrosome and three (designated 2B1, 2D6, and 6B2) bound to the flagellum. Of the eight McAbs investigated, three (2B1, 2C4, and 6B2) were effective in blocking fertilization in vitro when added as culture supernatants to mixtures of sperm and eggs. McAb 6B2 was inhibitory due to its ability to agglutinate spermatozoa. McAbs 2B1 and 2C4 did not agglutinate capacitated spermatozoa, had no observable effect on motility, and yet blocked fertilization in a dose-dependent manner. McAb 2C4 did not give a reaction on immunoblots, but the 2B1 antigen was identified as an Mr 40 kD glycoprotein. McAb 2B1 appeared to block fertilization at the level of zona binding, whereas the effects of 2C4 were directed more against zona penetration and/or fusion with the vitellus. When sperm-egg complexes were stained with 2C4 or 2B1 McAbs and viewed by IIF, all spermatozoa that were attached to the zona showed fluorescence on the head. These results suggest that different antigens on the rat sperm head participate in different aspects of the fertilization process and that during capacitation there is either exposure of these antigens or else they migrate to their site of action from the flagellum.     

The effect of semen extension, cAMP and caffeine on in vitro fertilization of bovine oocytes

A previously reported in vitro system that used epididymal spermatozoa for fertilizing bovine follicular oocytes (1) has been expanded to include ejaculated semen as the sperm source. Frequency of fertilization was higher when semen was extended 1:1 prior to transport to the laboratory rather than transport as neat semen. Pretreatment of spermatozoa with cAMP, caffeine or both prior to insemination of oocytes did not increase frequency of either acrosome reactions or fertilization after sperm/oocyte incubation.     

Full-term development of rats from oocytes fertilized in vitro using cryopreserved ejaculated sperm

For preservation of rat spermatozoa, the general-purpose method requires that the male be sacrificed for collection of spermatozoa from the epididymides. However, it would be highly useful if the ejaculated spermatozoa could be successfully cryopreserved and the frozen–thawed spermatozoa used for in vitro fertilization, since this would allow the genetically valuable rats to be maintained alive rather than sacrificed. The aim of the present study was to clarify whether ejaculated rat spermatozoa could be successfully cryopreserved and fertilized in vitro. The motility and viability of frozen–thawed ejaculated spermatozoa were similar to those of frozen–thawed epididymal spermatozoa (around 10%). The percentage of acrosomal integrity in epididymal spermatozoa was significantly higher than that in ejaculated spermatozoa after freezing/thawing. The level of capacitation-associated protein tyrosine phosphorylation in frozen–thawed ejaculated sperm was slightly increased at 5 h. When the frozen–thawed ejaculated spermatozoa were used for in vitro fertilization, the percentages of fertilization, pronuclear formation, and development to the 2-cell stage (26.5%, 23.0%, and 91.0%, respectively) were similar to those of frozen–thawed epididymal spermatozoa (19.4%, 15.0%, and 84.1%, respectively). However, the rate of blastocyst formation in the ejaculated group was significantly lower than that in the epididymal group (12.0% vs 43.2%). Results from the embryo transfer experiment showed that the proportions of embryos developed to term were similar between the ejaculated (47.7%) and epididymal groups (53.7%). We showed here for the first time that ejaculated spermatozoa can be cryopreserved and the frozen–thawed sperm could be developed to term via in vitro fertilization in rats.     

Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture

Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozenthawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca²⁺. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine-containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs-free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca²⁺ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca²⁺ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca²⁺ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2. These results indicate that CPPs promote Ca²⁺ uptake by spermatozoa and are effective for capacitation and/or acrosome reaction of spermatozoa leading to sperm penetration when caffeine is present in the medium and that the effect is reduced by dephosphorylation of CPPs. © 1994 Wiley-Liss, Inc.     

小鼠卵子在不同条件下的体外受精

本实验比较了小鼠精、卵细胞在不同生理状态下体外的受精能力。结果表明,体内受精率明显地高于体外(p<0.05),自发排出的卵子比超数排出的卵子受精率高(p<0.05),体外获能的附睾精子比体内获能的子宫精子受精率高(p<0.01)。唯独用超数排出的卵子和体外获能的附辜精子体外受精时,其受精率和体内相似。     

Ejaculated and epididymal mouse spermatozoa are different in their susceptibility to nuclease-dependent DNA damage and in their nuclease activity

Ejaculated mouse sperm retrieved from the uteri are more susceptible to DNA damage during freeze-drying and freezing without cryoprotection than epididymal sperm. This prompted us to speculate that a factor present in the uterus after mating, either male or female derived, was responsible for increased susceptibility of ejaculated sperm to DNA damage during preservation and that the differences between epididymal and ejaculated mouse sperm in response to stress originated from varying nuclease activity. We first exposed epididymal sperm to the uterine content from females mated to vasectomized males (UCSP), to the uterine content from unmated females in estrus (UC), and to the seminal vesicle fluid (SVF) and examined sperm chromosomes after intracytoplasmic sperm injection (ICSI). We found an increased incidence of chromosome breaks and extremely severe DNA breakage after exposure to UCSP and SVF, respectively, but the chromosomes were normal in sperm exposed to UC. Comet assay results verified that DNA damage after exposure to SVF was present in sperm before fertilization. Next, we examined nuclease activity in sperm and their associated components with a plasmid digestion assay. Nuclease activity was detected in isolated epididymal and ejaculated sperm, as well as in epididymal fluid and seminal plasma, and was much more pronounced in all samples originating from ejaculate. The combined results from the present study imply that there are intrinsic differences between the epididymal and ejaculated mouse sperm preparations in their susceptibility to nuclease-dependent DNA damage that originates from their nuclease activity. This nuclease activity was detected both in the sperm-free fraction of preparations and isolated sperm.     

Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion

CRISP2, originally known as Tpx-1, is a cysteine-rich secretory protein specifically expressed in male haploid germ cells. Although likely to be involved in gamete interaction, evidence for a functional role of CRISP2 in fertilization still remains poor. In the present study, we used a mouse model to examine the subcellular localization of CRISP2 in sperm and its involvement in the different stages of fertilization. Results from indirect immunofluorescence and protein extraction experiments indicated that mouse CRISP2 is an intraacrosomal component that remains associated with sperm after capacitation and the acrosome reaction (AR). In vitro fertilization assays using zona pellucida-intact mouse eggs showed that an antibody against the protein significantly decreased the percentage of penetrated eggs, with a coincident accumulation of perivitelline sperm. The failure to inhibit zona pellucida penetration excludes a detrimental effect of the antibody on sperm motility or the AR, supporting a specific participation of CRISP2 at the sperm-egg fusion step. In agreement with this evidence, recombinant mouse CRISP2 (recCRISP2) specifically bound to the fusogenic area of mouse eggs, as previously reported for rat CRISP1, an epididymal protein involved in gamete fusion. In vitro competition investigations showed that incubation of mouse zona-free eggs with a fixed concentration of recCRISP2 and increasing amounts of rat CRISP1 reduced the binding of recCRISP2 to the egg, suggesting that the proteins interact with common complementary sites on the egg surface. Our findings indicate that testicular CRISP2, as observed for epididymal CRISP1, is involved in sperm-egg fusion through its binding to complementary sites on the egg surface, supporting the idea of functional cooperation between homologous molecules to ensure the success of fertilization.     

Fertilization of rat eggs in vitro at various times before and after ovulation with special reference to fertilization of ovarian oocytes matured in culture.

Oocytes recovered at various times from immature rats treated with PMSG and HCG were incubated with capacitated epididymal spermatozoa of mature rats. In the presence of follicular cells, sperm penetration was not observed 4 hr after incubation in the oocytes at stages from the intact germinal vesicle to the chromatin mass, but 7 to 55% of oocytes were penetrated at stages from the condensed germainal vesicle to metaphase II. After the removal of follicular cells, 15 to 72% of the oocytes at any stage were penetrated. After further incubation for 15 hr, the proportion of penetrated oocytes increased from 8 to 98% from early to late stages and that of penetrated oocytes with a male and female pronucleus increased from 9 to 100% as maturation progressed. Although the average number of spermatozoa/oocyte was not correlated with its maturation, transformation of the sperm head into a male pronucleus was retarded or failed, especially in the younger oocytes. Following incubation in a defined medium for 13 hr, 85% of oocytes at the intact germinal vesicle stage matured to the stage of the first polar body formation, but only 18 to 22% of these mature oocytes were penetrated by spermatozoa and only a few of the penetrated oocytes cleaved into normal two-cell eggs. When eggs recovered from oviducts 14 to 20 hr after ovulation were exposed to capacitated spermatozoa, the proportion of penetrated eggs (86 to 98%) and that of polyspermic eggs (11 to 27%) were not related to the ages of the eggs, but failure of transformation of the sperm head and the proportion of abnormal eggs increased 14 to 20 hr after ovulation.     

Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro

Glycosaminoglycans (GAGs) were reported to induce acrosome reactions (AR) in epididymal and ejaculated bovine sperm (4,5). The GAGs chondroitin sulfate A (CS-A) and heparin were tested on ejaculated bovine sperm for their ability to increase in vitro fertilization (IVF) frequencies. Regardless of treatment, a sperm-egg incubation time of 18 hr was sufficient to achieve maximal rates of fertilization. The IVF frequency of sperm incubated 6 hr with 10 mug/ml heparin (116 173 , 67%) was increased (P<0.05) above control levels (56 181 , 31%); however, 10 mug/ml CS-A (56 164 , 34%) was without effect (P>0.05). In contrast to previous reports, CS-A did not (P>0.05) induce AR in ejaculated (9.5-hr incubation) or epididymal sperm (22.5-hr incubation). Linear increases in fertilization frequency (40% to 81%; P=0.001) and AR (9% to 32%; P相似文献   

Fertilization of hamster eggs in vitro at sperm:egg ratios close to unity

Hamster epididymal spermatozoa were washed and preincubated at extremely low sperm concentrations (100/ml or less) in a culture medium containing naturally-occurring sperm motility-stimulating substances. These substances were partially purified "sperm motility factor" (SMF) derived from hamster adrenal glands and catecholamines (epinephrine or isoproterenol). After preincubation for three hours, a small number (5 or less) of washed, cumulus-free hamster eggs was added to each sperm suspension. Many of these eggs were undergoing fertilization when examined two to three hours later. Fertilization was accomplished in vitro at sperm:egg ratios approaching 1:1, a situation comparable to that believed to exist in vivo. It appears that this demonstration will considerably enhance the potential of in vitro fertilization studies for providing useful information on mammalian gamete interactions.     

Stimulation of hamster sperm capacitation and acrosome reaction in vitro by glucose and lactate and inhibition by the glycolytic inhibitor α-chlorohydrin

Studies were made of the effects of D(+)-glucose, L-lactate and pyruvate on in vitro capacitation and acrosome reactions (AR) of hamster sperm using a more “defined” medium that that used in previous similar studies. In the absence of glucose or lactate, sperm underwent very few AR and activation (whiplash-like motility characteristic of capacitated hamster sperm) was reduced compared to those events in sperm preincubated in the presence of glucose plus lactate plus pyruvate. Glucose and pyruvate supported more AR than glucose alone, but less than glucose, lactate, and pyruvate. The glycolytic inhibitor α-chlorohydrin (10 μm) inhibited AR by 50% and reduced activation by less. When glucose was added to sperm incubated 2 hr with pyruvate and lactate, the number of AR observed after 4 hr was the same as that obtained when glucose was present throughout the incubation. When glucose was added after 3.5 hr, AR were delayed for 1 hr and lower numbers of sperm underwent AR. In the presence of lactate and pyruvate, 0.38 mM glucose was able to support activation and AR as well as 3.24 mM glucose. These results indicate that exogenous glucose and lactate are necessary for in vitro capacitation and AR of hamster sperm; only low levels of exogenous glucose are required; exogenous glucose is not required during the first 2 hr of capacitation; and glycolytic activity is necessary for capacitation and the AR.