Fertilization Of Mouse Oocytes Research Articles

Enhancing in vitro fertilization of mouse oocytes by partial zona pellucida digestion.

This work was undertaken in order to evaluate the effect of partial zona digestion on fertilization in vitro of mouse oocytes and assess zona surface changes induced by the procedure. Three hundred forty-six oocytes allocated for treatment were exposed to Ham's F-10 medium supplemented with 0.5% Pronase for either 3 min (188 oocytes) or 5 min (158 oocytes); 324 oocytes served as controls. Oocyte losses incurred as a result of the procedure were small (15 oocytes; 4.3%). Control and Pronase-treated oocytes were each divided into four subgroups and inseminated with 5 x 10(5), 5 x 10(4), 5 x 10(3), or 5 x 10(2) sperm cells/ml. Fertilization was assessed 8 hr following insemination by the appearance of two pronuclei and development to the two- to four-cell stage the following day. The morphology of the zona pellucida following Pronase treatment was assessed by phase-contrast and scanning electron (SEM) microscopies performed immediately after treatment. Fertilization rate of control oocytes was 80% at a sperm concentration of 500,000/ml and gradually declined to approximately 30% at 500 cells/ml. In contrast, treated oocytes inseminated with 500 sperm cells/ml demonstrated a normal rate of fertilization. At this low sperm concentration the longer Pronase treatment was significantly (P less than 0.05) more efficient in enhancing fertilization (69 and 88% for 3 and 5 min of Pronase treatment, respectively). Polyspermic fertilization was not observed in any of the subgroups. Phase-contrast microscopic examination of oocytes at the time of Pronase treatment showed an initial swelling of the zona pellucida for 30-60 sec with a time-dependent increase in its transparency.

Influence of male sexual rest and oocyte aging on parthenogenesis frequency in mice: cytogenetic analysis after in vitro fertilization.

This study was conducted to evaluate the influence of male sexual rest and oocyte aging on fertilization rate and parthenogenesis frequency after in vitro fertilization of mouse oocytes. We used a comparison between cleavage rates and fertilization rates according to chromosomal analysis of oocytes to estimate the parthenogenesis frequency. Fertilization rate was not impaired by male sexual rest. Parthenogenesis frequency was increased by male sexual rest. This effect was enhanced by a concomitant moderate oocyte aging. It is concluded that cleavage rate could not be considered as a reliable test of fertilization after attempted in vitro fertilization in such conditions.

Assisted fertilization of mouse oocytes and preliminary results for human oocytes using zona drilling

The zona-drilling procedure was investigated in mouse oocytes prior to a study on human oocytes. The procedure involved the injection of 5-nl volumes of acidic Hepes-buffered medium at pH 2.5 using a microinjection instrument. Zona-drilled mouse oocytes had significantly higher rates of fertilization (60/99; 61%) than zona-intact oocytes (6/103; 6%) at an insemination concentration of 1 x 10(4) sperm/ml (P less than 0.001). The procedure did not induce parthenogenetic activation of oocytes and more than 97% of zygotes developed to the blastocyst stage. A similar rate of live progeny was observed when zona-drilled (38.0%) and control embryos (38.5%) were transferred to pseudopregnant recipients. Chromosome analyses were performed on zona-intact, zona-free, and zona-drilled oocytes inseminated with varying concentrations of sperm and analysed at the first cleavage division. Zona-free oocytes had high rates of polyploidy (greater than or equal to 40%) with varying insemination numbers but the zona-drilled oocytes did not reveal significant increases in the rate of polyploidy or aneuploidy when compared to controls. In the human studies, zona-drilled oocytes achieved higher rates of fertilization than zona-intact oocytes, with sperm numbers as low as 1 x 10(4)/ml (6/8; 75%). Polyspermic fertilization was observed in 1/2 and 2/6 of fertilized oocytes inseminated with 1 x 10(5) and 1 x 10(4) sperm/ml, respectively. With the low sperm concentration 2/4 of those which were normally fertilized developed to healthy blastocysts. These studies suggest that the zona-drilling technique as described can be performed without apparent harm to oocytes and generate normal embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine

A monoclonal antibody, HS-63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human. The corresponding sperm antigen designated as MSA-63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits. The cDNA fragments of MSA-63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA-63. Using the established cDNA clone as a probe, the gene encoding for MSA-63 protein was found to be conserved among different mammalian species. Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues. The recombinant fusion protein containing MSA-63 protein fragment was produced in Escherichia coli and used to immunize female mice. Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes. The results of this preliminary study suggest that it is feasible to mass produce sperm-specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen-based immunocontraceptive vaccines.

Follicular fluid Lidocaine levels during transvaginal oocyte retrieval

Lidocaine has been shown to have adverse effects on mouse oocyte fertilization and embryo development. We have demonstrated the presence of pharmacologic levels of lidocaine in human serum and follicular fluid obtained during ultrasound guided transvaginal oocyte retrieval. The significance of this finding is unclear, as four of the eight patients studied became pregnant, including the patient with the highest follicular fluid lidocaine levels. Further evaluation of the effect of lidocaine on human embryos is warranted.

Effects of platelet activating factor on mouse oocyte fertilization in vitro

Platelet activating factor is rapidly gaining acceptance as a potent mediator in many reproductive processes. This study presents data that indicate a direct role of platelet activating factor in fertilization. Platelet activating factor was shown to significantly increase (p less than 0.001) the fertilization rate of mouse oocytes in vitro. Furthermore, CV3988, an inhibitor of platelet activating factor, was noted to significantly decrease in vitro fertilization rates at 10(-5) and 10(-4) mol/L concentrations.

Quality control in the IVF laboratory: in-vitro and in-vivo development of mouse embryos is unaffected by the quality of water used in culture media.

We have examined the effect of media made with tap water or with various purified waters on the fertilization of mouse oocytes, their development to blastocysts, their rate of hatching in vitro and their survival after transfer to recipients. Zona-intact and zona-free embryos, as well as cell clusters from 8-cell stage embryos, were also used. The macromolecular composition of the media was varied. We were unable to find any adverse effect of tap water under any condition examined. The implications of these findings for quality control in IVF units are discussed.

Sperm antibodies induced by anti-idiotype antibodies: A strategy in development of immunocontraceptive vaccines

The potential of using anti-idiotype antibodies as immunocontraceptive vaccines is evaluated in this study. Two sperm monoclonal antibodies, HS 63 and MS 204, which have significant inhibitory effect on the in vitro and in vivo fertilization of mouse were selected to elicit heterologous anti-idiotype antibodies. Rabbit antisera against HS 63 or MS 204 were collected after the third immunization. After the removal of anti-Fc fragments with an irrelevant mouse IgG immunoadsorbent column, anti-idiotype antibodies (anti-Id HS 63 or anti-Id MS 204) were purified from the antisera with affinity chromatography. HS 63 or MS 204 was used as respective ligand. The purified anti-idiotype antibodies conjugated with hemocyanin were used to immunize female CD-1 mice as the experimental group. Another group of female CD-1 mice were immunized with adjuvant only as the control group. Each group had three mice. The immune responses varied significantly among individual mice. The antisera could stain the acrosomal region of sperm in the indirect immunofluorescent assay in a way which is similar to the original monoclonal antibodies (HS 63 and MS 204). The inhibitory effect of antisera on the sperm fertilization of mouse oocytes in vitro was significant in both cases. In the case of antisera against anti-Id HS 63, the control group showed 81.8% and 49.3% of fertilization rates, whereas the corresponding rates of the experimental group were only 35.7% and 20.5%, respectively. Similarly, for antisera against anti-Id MS 204, the experimental group also revealed lower fertilization rates as compared with those of the controls (50.0% vs. 95.8% and 36.8% and 81.3%). The results of this study suggest that anti-idiotype antibodies against HS 63 and MS 204 contain the internal image of the sperm antigen and they could elicit an immune response with a significant antifertility effect. Therefore, they might not only be contributory to further study and understanding of the original antigens in structure and function but also be used as an alternative in the development of immunocontraceptive vaccines.

Preimplantation development following in vitro fertilization of mouse oocytes: effects of timing of superovulation and preincubation in vitro.

The early embryonic development of in vitro fertilized oocytes was assessed following superovulation in F1 hybrid (C57BL/6 X CBA/Ca) mice. Decreasing the time interval between the administration of constant doses of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) resulted in decreases in the frequency of development to the blastocyst stage but had no significant effect on development to the two-cell stage. Preincubation of postovulatory oocytes in vitro prior to insemination did not compensate for the reduced preovulatory development in vivo but resulted in decreases in the frequency of development to the blastocyst stage. The results indicate that inadequate preovulatory development of superovulated mouse oocytes can adversely affect the preimplantation development of in vitro fertilized embryos in the absence of a visible inhibitory effect on development to the two-cell stage and also that preincubation of postovulatory oocytes in vitro prior to fertilization reduces subsequent developmental capacity.

Inhibitory effects of monoclonal sperm antibodies on the fertilization of mouse oocytes in vitro and in vivo

The inhibitory effects of monoclonal antibodies on the fertilization of mouse oocytes were evaluated in vitro and in vivo. Among the 40 sperm-specific monoclonal antibodies that had been examined, nine showed significant inhibition of the fertilization of mouse oocytes in vitro. MS 204 was shown to cause a high incidence of penetration of the zona pellucida of mouse eggs by multiple sperm, when the sperm concentration for insemination exceeds 1 × 10 5/ml. This antibody prevented further penetration of the vitelline membrane by sperm. On the other hand, sperm penetration to zona was inhibited in the presence of MS 207. However, neither MS 204 nor MS 207 caused significant inhibition of penetration of zona-free mouse or hamster eggs by sperm. MS 204 and MS 207 were also found to inhibit the fertilization of mouse oocytes in vivo and embryo development in vitro, when superovulated mice were injected intraperitoneally with given doses of antibodies prior to the mating. Further in vitro culture of the recovered 2-cell oocytes revealed little or no further embryo development beyond two- to four-cell stages. In the controls, > 80% of the retrieved oocytes were fertilized and successively developed to the blastocyst stages in vitro when the ascites fluid from NS-1 cells was administered. Two of the monoclonal antibodies generated against human sperm antigens. HS11 and HS 63, were shown to cross-react specifically with mouse sperm acrosomal antigens and also inhibited the fertilization of mouse oocytes in vitro and in vivo. The results of this study suggest that some monoclonal antibodies to sperm acrosomal antigens exhibit strongly inhibitory effects on the in vitro and in vivo fertilization of mouse oocytes as well as subsequent development of early embryos. As a comparative control, rabbit antisera against sperm-specific enzymes, lactate dehydrogenase-X and 3-phosphoglycerate kinase-2, showed little or no inhibition on the fertilization of mouse oocytes in vitro or in vivo or the subsequent embryo development.

The cytochemistry of glycoconjugates in the zona pellucida of murine ovarian oocytes and two-cell embryos

Changes in glycoconjugates of the zona pellucida induced by maturation, ovulation and fertilization of mouse oocytes have been studied by means of light microscopic methods of cytochemistry. These methods consisted of periodic acid-Schiff (PAS), Alcian Blue pH 1.0 and pH 2.5, and peroxidase-labelled lectin diaminobenzidine (PO-LT-DAB) procedures in combination with the digestion technique with neuraminidase. According to the results obtained, glycoconjugates of the zona pellucida of fertilized eggs contained a smaller amount of sulphate groupings than that in ovarian oocytes, whereas their reactions for sialic acid and fucose residues were significantly stronger in intensity in the former, as compared with those in the latter. The cytophysiological significance of such cytochemical changes in glycoconjugates of the zona pellucida has been discussed with special reference to its functional alterations following maturation, ovulation and fertilization.

Action of phorbol myristate acetate (PMA) at fertilization of mouse oocytes in vitro

Phorbol ester (PMA) in concentration 5 and 10 ng ml-1 blocks cytokinesis of the second maturation division in mouse oocytes. Karyokinesis is not impaired and digynic triploid oocytes are obtained which undergo first cleavage division. Effectiveness of blocking cytokinesis is dependent on the timing of exposure of oocytes to PMA action. When oocytes are subjected to PMA at the onset of the second maturation division only 14.5% of eggs are triploid. PMA present during fertilization in vitro (about 1 h exposure to PMA) induces triploidy in 40% eggs. Extending the time of exposure of oocytes to 2 h produces 76% tripronucleate eggs. Applicability of PMA is compared with the use of cytochalasin B to induce triploidy in the mouse.

PARTIAL REVERSAL OF ETHANOL OF ETHANOL-INDUCED MALE REPRODUCTIVE PATHOLOGY FOLLOWING ABSTINENCE

To date, no longitudinal studies have been carried out to determine the recovery of ethanol-related reproductive failure subsequent to moderate periods of abstinence. An animal model (C57B1 mouse) was utilized to examine the effectiveness of abstinence for reversal of ethanol-induced reproductive failure. After treatment with either a 5% (v/v) ethanol diet (10 weeks) or a 6% (v/v) ethanol diet (5 weeks), ethanol-treated animals and their pair-fed controls were electroejaculated and hemicastrated (right testis and accessory organs); their reproductive tracts and epididymal spermatozoa were examined. Ingestion of the 5% and 6% ethanol diets resulted in significantly (P less than 0.05) decreased weights of testes (24% and 28%, respectively) and seminal vesicles/prostate (20%, 6% diet only), increased frequencies of germ cell desquamation (480% and 400%), inactive seminiferous tubules (186% and 567%), sperm dysmorphology (31% and 119%) and inhibition of in vitro fertilization of mouse oocytes by epididymal spermatozoa (26% and 62%), as compared to their respective pair-fed control values. Also observed were significant decreases in epididymal sperm content (72%, 6% diet only), total motile spermatozoa (85%, 6% diet only) and seminal vesicles epithelial cell height (13% and 29%). No abnormal semen parameters were observed after treatment with the 5% ethanol diet; however, in animals treated with the 6% diet, significant decreases were noted in coagulum weight (50%), sperm count (85%) and acid phosphatase content (53%). Improvement in all parameters was observed in the contralateral half of the reproductive tract subsequent to 10 weeks abstinence. Only germ cell desquamation remained significantly elevated (100% as compared to control) in animals that ingested the 5% diet. In contrast, significant abnormalities persisting 10 weeks after treatment with the 6% ethanol diet included increased germ cell desquamation (200%), inactive seminiferous tubules (157%) and sperm dysmorphology (39%) and decreased forward progression (17%) of epididymal sperm, as compared to values. These findings are discussed in relation to the influence of alcohol consumption on male reproductive function in man.

Spontaneous recovery from ethanol-induced male infertility

Chronic ethanol ingestion by the male is associated with reproductive impairment. No longitudinal studies have been carried out to determine the recovery of ethanol-related reproductive failure subsequent to moderate periods of abstinence. An animal model (C57B1 mouse) was utilized to examine the effectiveness of abstinence for reversal of ethanol-induced reproductive failure. After treatment with either a 5% ethanol diet (10 weeks) or a 6% ethanol diet (5 weeks), animals were hemicastrated (right testis and accessory organs); their reproductive tracts and epididymal spermatozoa were examined. Ingestion of the 5% and 6% ethanol diets resulted in significantly ( p<0.05) decreased testicular weights (24% and 28%, respectively) and seminal vesicle/prostate weights (20%, 6% diet only), increased frequencies of germ cell desquamation (480% and 400%), inactive seminiferous tubules (186% and 567%) and inhibition of in vitro fertilization of mouse oocytes by epididymal spermatozoa (26% and 62%), as compared to their respective pair-fed control values. Also observed was a significant decrease in total motile spermatozoa (85%, 6% diet only). Improvement in all parameters was observed in the contralateral reproductive organs subsequent to ten weeks abstinence. Only germ cell desquamation remained significantly elevated (100% as compared to control) in animals that ingested the 5% diet. In contrast, significant abnormalities persisted ten weeks after treatment with the 6% ethanol diet, including increased germ cell desquamation (200%) and inactive seminiferous tubules (157%); decreased forward progression (17%) of epididymal sperm also persisted. If these data can be applied clinically, male alcoholic patients with reproductive disorders could have a prognosis of at least partial recovery following moderate periods of abstinence from ethanol.

Generation of mouse oocyte monoclonal isoantibodies: their effects and those of antisperm monoclonal antibodies on in vitro fertilization

Monoclonal isoantibodies to mouse oocyte antigens were generated by modified hybridoma techniques similar to those described for mouse sperm monoclonals. Following isoimmunization with mouse oocytes and cell fusion, hybrid cells were cultured initially in a semi-solid medium containing methylcellulose. Seven to ten days after cell fusion about 350 hybrid clones were recovered for subculture. By an indirect immunofluorescence assay using frozen or fresh mouse oocytes, twenty hybridomas were shown to produce antibodies that bind to various oocyte components including antigens of the zona pellucida. However, they did not cross-react with mouse spermatozoa or lymphocytes. A system was established to evaluate whether monoclonal antibodies to gamete-specific antigens have any inhibitory effects on the fertilization of mouse oocytes in vitro. A monoclonal antibody against zona antigen(s), ME 56, was shown to block fertilization of mouse oocytes via the inhibition of sperm binding to the zona pellucida. On the other hand, three out of four antibodies reacting with mouse sperm acrosomes were also inhibitory to mouse in vitro fertilization, perhaps mainly due to the inhibition of sperm acrosomal reactions. Using a sodium dodecylsulfate gel/protein blot radioimmunobinding method, the molecular weight of zona antigen(s) that react with ME 56 was determined to be in the range of 95,000, whereas that of the acrosomal antigen(s) reacting with the fertilization-inhibiting antibody, MS 207, was about 30,000. The results of this preliminary study suggest that monoclonal antibodies to certain gamete antigens can be a valuable tool for the analysis of sperm-egg interactions during the fertilization processes.

Reacting mouse sperm with monoclonal H-Y antibodies does not influence sex ratio of eggs fertilized in vitro

The fertility of Y-bearing mouse sperm was examined after reacting cauda epididymal sperm with monoclonal H-Y antibodies and protein A sensitized sheep red blood cells. Treated sperm were used for the in vitro fertilization of mouse oocytes which subsequently produced live offspring. There was no significant shift in the sex ratio in favor of females, suggesting that X- and Y-bearing sperm may share the surface antigen. Additional studies were directed toward ascertaining whether haploid expression, as measured by the presence of H-Y antigen, occurs in epididymal sperm or during their capacitation in vitro. Ligation of the corpus epididymus, preventing subsequent transport of sperm to the cauda region, resulted in a linear decrease in H-Y positive cauda sperm. By 17 days after ligation, no positively reacting sperm were observed. Incubation of cauda epididymal sperm for 3 h in capacitating medium eliminated positive reaction by the capacitated sperm to the H-Y antiserum. Furthermore, the percentage of H-Y-positive sperm from different regions of the male reproductive tract appeared to decrease during their transport from the testis to the epididymus and vas deferens. We suggest that H-Y antigen appears on the sperm surface during association with testicular constituents and is removed during epididymal transport and capacitation. No evidence of haploid expression by epididymal mouse sperm was found.