This experiment was designed to evaluate the ability of 4 different types of somatic cells to promote development of early cleavage in rat embryos. Embryos were collected from Wistar-Imamichi rats. Four and 8-cell rat embryos were co-cultured with rat granulosa, oviductal, uterine and kidney monolayer cells. The culture was performed in TCM 199 supplemented with 10% fetal calf serum, sodium lactate and pyruvate alone (m199FCS). Among the co-culture systems, morula and blastocyst development was better with granulosa, oviductal and uterine cells (98.6, 95.6 and 94.1%, respectively) than with m199FCS alone and kidney cells (77.6 and 73.7%, respectively). Similarly, 4-cell rat embryo development also followed the same trend and the development percentages with granulosa, oviductal, uterine, kidney cells and m199FCS alone were 32.0, 30.7, 29.3, 0 and 2.7%, respectively. Nevertheless, 8-cell embryos were developed to hatched blastocysts in co-culture with oviductal, uterine cells and in the conditioned medium of oviductal cells, 21.0, 19.0 and 2.1%, respectively. But co-culture with granulosa, oviductal and uterine cells represented the best physiologic approach and showed superiority to the other cell types. Forty-four of 184 1-cell embryos co-cultured with oviduct cells developed to the morula/blastocyst stage. The co-culture of early rat embryos in a medium with oviductal explants can support further development.

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This experiment was designed to evaluate the ability of Estradiol-17β (E₂) and progesterone (P₄) to promote development of rat blastocysts. Eight-cell rat embryos were co-cultured for 5 days with rat uterine epithelium (UE) cells with inclusion of E₂ and/or P₄. Embryos were collected from mature Wistar-Imamichi rats. UE cells were recovered from the same washed with modified TCM199 (m 199FCS) and placed in 4-well Multi-dishes. UE cells of monolayers were developed to 70-80% confluence prior to initiation of the embryo culture. Embryos were recovered, selected and randomly placed in one of the four steroid hormone dose concentration media. They were evaluated every 24 h to determine their stage of development. More (p<0.05) embryos were developed to both hatched and post-hatched blastocysts in E₂ with a concentration of 3.5 × 10^-5, 10^-4 M and P₄ with a concentration of 3.5 × 10 ^-6, 10^-5,10^-4 M. The combination of E₂ (3.5 × 10^-5M) and P₄ (3.5 × 10^-6, 10^-5, 10^-4 M) also improved (p<0.05) both hatched and post-hatched blastocysts. These finding showed that the presence of ovarian steroid hormones in a primary culture of UE cells promotes in vitro development of post-blastocysts in rat embryos.

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The effects of caffeine on the motility of frozen-thawed bull spermatozoa from 4 different bulls and their ability to fertilize in vitro matured oocytes was assessed. Frozen-thawed semen was divided into two groups. One group was incubated for 6 h in BO solution either with or without 2.5 mM caffeine. During the incubation period, the motility for each bull was observed at 0.5 h intervals. The other group was used to inseminate in vitro matured bovine oocytes. The addition of caffeine to the BO solution did not stimulate the motility of all bulls. The percentage of embryos that cleaved by the third day after insemination varied between bulls. In one bull, the percentage of cleaved embryos was significantly lower (P<0.05) than for the other bulls in the presence of caffeine. This percentage was also significantly lower (P<0.05) than that in the absence of caffeine in the same bull. These results indicated that caffeine do not stimulate the motility of frozen-thawed bovine spermatozoa in the BO medium, and that caffeine is not necessarily useful for the induction of in vitro sperm capacitation.

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It is well known that hamster embryos cannot complete the second cleavage division in vitro (2-cell block), if cultured in glucose and inorganic phosphate (Glu/Pi) containing medium. It is unclear how the block to development in vitro is caused in the presence of Glu/Pi. In the present study, the ATP in hamster embryos at the 2-cell block in M199 supplemented with 5% heated calf serum was examined and compared to the ATP in 2-cell embryos just recovered from oviducts and preimplantation hamster embryos developing in vitro from 2-cell to blastocyst stages in Glu/Pi-free medium (HECM3). ATP in hamster embryos was measured by an ATP-dependent luciferin - luciferase bioluminescence assay. The average ATP content and SEM was 44.5 ± 7.4 fmol/embryo at the 2-cell stage, 13.1 ± 1.2 fmol/embryo at the 4-cell stage, 72.3 ± 21.0 fmol/embryo at the 8-cell stage and 22.7 ± 6.6 fmol/embryo at the blastocyst stage. A significant decrease in ATP content was observed at the 4-cell stage and blastocyst stage as compared to the 8-cell stage (P<0.05). The other hand, the average amount of ATP and SEM in the 2-cell block embryos was 98.3 ± 17.1 fmol/embryo and was much higher than that in the preimplantaion embryos developing in vitro. We therefore suggest that the 2-cell block of hamster embryos may be due, in part to the high ATP content, that is, low levels of cytochrome activity and low levels of ATP synthesis.

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The purpose of the present experiment was to determine the fertilizability of bovine oocytes frozen in various cryoprotectants: 1.6 M 1,2-propanediol (PROH)+0.2 M sucrose, 1.6 M dimethylsulfoxide (DMSO)+0.2 M sucrose, 1.6 M glycerol (GL)+0.2 M sucrose, 0.8 M PROH+0.8 M DMSO, and 0.8 M PROH+0.8 M GL. The incidence of morphologically normal oocytes was significantly higher in the 0.8 M PROH+0.8 M DMSO group (56.0%) than in other groups (P<0.05). At 48 h after insemination, the number of eggs that cleaved into the 2-cell stage, in both the 0.8 M PROH+0.8 M DMSO and 1.6 M PROH+0.2 M sucrose groups, was significantly greater than in other groups (P<0.05). By analyzing the chromosomes of first-cleavage eggs, which were fertilized in vitro after freezing and thawing, it was found that the incidences of polyploids were higher in all of the frozen-thawed groups than in the control group, but among them there was no significant difference. The frequency of eggs with chromosomes having structural aberrations did not increase in any of the treated groups.

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We analyzed the expression of mRNAs for growth factor receptors with receptor tyrosine kinases [ErbB3 (a member of the EGF-R subfamily), insulin-like growth factor-I receptor (IGF-I-R), basic fibroblast growth factor receptor (bFGF-R) and platelet-derived growth factor receptor α (PDGF-Rα)] by reverse transcription- polymerase chain reaction during in vitro development (10 to 168 h of in vitro insemination: zygotes to blastocysts) of porcine ova matured and fertilized in vitro. Transcripts for IGF-I-R, bFGF-R and PDGF-Rα were detectable in ova at all stages of in vitro development. Transcripts for ErbB3 were present in embryos from the late pronucleate stage onward. The results provide evidence that several growth factor receptor mRNAs are present in porcine ova during development in vitro.

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Surface characteristics of the bovine cumulus-oocyte complex (COC) during maturation in vivo versus in vitro were compared by scanning electron microscopy (SEM). Oocyte diameter changes during maturation were also analyzed prior to oocyte fixation. In vivo matured oocytes were retrieved from live super-ovulated cows by transvaginal ultrasound-guided aspiration of all visible ovarian follicles (≥2 mm in diameter) 0, 12 and 24 h (n=85, 38 and 39, respectively) after hCG administration. In vitro matured (IVM) oocytes refer to those recovered from small antral follicles (2-6 mm in diameter) of slaughterhouse ovaries and then cultured in standard maturation conditions for 0 h (n=188), 12 h (n=138) or 24 h (n=228). SEM analysis showed that immature oocytes of both origins manifested compact COCs with smooth cumulus surface and minimal intercellular spaces among the cumulus cell mass. At 24 h of maturation, more IVM oocytes manifested cumulus expansion compared to in vivo matured oocytes (100%, n=228 vs 44%, n=39), but cumulus expansion was found much more prominently in the cumulus-expanded in vivo matured oocytes than in the IVM oocytes. The zona pellucida showed a fibrous, open mesh-like structure, irrespective of the maturation stage and the source of oocytes. The vitelline surface of the immature oocytes was characterized by distribution of large cellular tongue-shaped protrusions (TSPs) varying in density. These TSPs structures gradually changed to microvilli (MV)-predominant structures upon maturation of the oocytes. At 12 h of maturation, significantly more oocytes (p<0.05) manifested cumulus expansion and transition from TSPs- to MV-predominant vitelline surface structures in the in vitro group (100%, n=106) compared to only 11% (n=38) in the in vivo group. Oocyte diameter decreased gradually in the oocytes maturing in vitro tween 0 and 24 h of incubation, being 127 ± 1, 122 ± 1 and 116 ± 1 μm at 0, 12 and 24 h of incubation, respectively. The corresponding values for the oocytes maturing in vivo were 121 ± 2, 129 ± 2, and 101 ± 1 μm, respectively. In conclusion, we found in our study conditions that initiation of oocyte maturation in vivo after hCG administration seemed to be slower than in vitro after maturation culture. But, at 24 h after the onset of maturation more prominent and complete cumulus expansion and more dramatic volumetric changes were observed in the in vivo matured oocytes than in the IVM oocytes.

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The aim of this study was to examine the effect of the endocrine environment of dominant follicles on the competence of bovine oocytes to develop from small antral follicles 2-5 mm in diameter. Twenty-seven pairs of ovaries with mature corpora lutea were obtained from a local abattoir. The mean diameter and wet weight of the corpora lutea of individual cows were 23.3 ± 2.7 mm and 4.7 ± 1.0 g, respectively. Follicular fluid was aspirated from large follicles more than 6 mm in diameter, and estradiol (E₂) and progesterone (P₄) concentrations were determined by a RIA. Ovaries were classified into each group of E₂-active-dominant (E₂-A-dom; E₂≥50 ng/ml and E₂:P₄ ratio>1); 12 pairs of ovaries, E₂-dominant (E₂-dom; E₂<50 ng/ml and E₂:P₄ ratio>1); 6 pairs of ovaries or P₄-dominant (P₄-dom; E₂:P₄ ratio<1); 9 pairs of ovaries. The concentrations of E₂ and P₄ in E₂-A-dom were 132.5 ± 97.6 ng/ml and 8.4 ± 3.5 ng/ml, in E₂-dom were 27.7 ± 9.7 ng/ml and 9.2 ± 3.7 ng/ml, and in P₄-dom were 4.2 ± 4.7 ng/ml and 98.5 ± 208.4 ng/ml, respectively. The mean number of small antral follicles, the mean number of oocytes fertilized, the rate of cleavage and of blastocysts in E₂-A-dom were 44.4 ± 20.3, 24.2 ± 10.9, 68.2%, 26.0%, in E₂-dom were 48.5 ± 11.9, 28.7 ± 8.6, 70.4%, 20.2%, in P₄-dom were 41.7 ± 24.0, 23.0 ± 15.3, 70.9%, 20.0%, respectively. There were no significant differences in the number of small antral follicles, the number of oocytes fertilized and developmental capacity of the oocytes among the groups. Results of the present study indicate that in cattle E₂ and P₄ in a dominant follicle do not affect the competence of the development of oocytes derived from small antral follicles.

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Porcine cumulus oocyte complexes (COCs) were cultured in an inhibitor free-medium for 24 h and then some of them were denuded. The COCs and denuded oocytes (DOs) were further cultured for 24 h in the presence of Phosphatidylinositol 3-kinase (PI 3-kinase) specific inhibitors, wortmannin and LY294002. In COCs and DOs, wortmannin (10^-8, 10^-7 M) and LY294002 (5.0 × 10^-5 M) significantly increased the proportion of oocytes arrested at M I and decreased that of oocytes reaching M II. Nevertheless, in DOs the differences between in the presence of the inhibitors and the control in the proportion of oocytes reaching M II substantially diminished as compared to those in the case of COCs. Cumulus cell expansion was significantly suppressed by wortmannin and LY294002. It is concluded that PI 3-kinase in cumulus cells plays a regulatory role in meiotic progression beyond M I and cumulus cell expansion in porcine COCs.

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Cryopreservation of mouse spermatozoa has recently become available for use. In this study, we attempted to apply this technique to the maintenance of gene disrupted mouse lines. Cauda epididymides that were taken from Et1^tm1Csk and Gk^tm1Csk mice were minced in 100 μl of a cryopreservation solution which consisted of 18% raffinose and 3% skim milk. Sperm suspensions in plastic straws were cooled rather rapidly by being placed in the gas phase above liquid nitrogen, and then were stored at -196°C. The frozen straws were thawed rapidly being by immersing in a water bath at 30°C. For in vitro fertilization, 1 μl of thawed sperm suspensions from each mouse strain was added directly to oocytes contained in 200 μl of medium. Following pre-incubation of the frozen-thawed spermatozoa, ICR oocytes were introduced into the medium containing the frozen-thawed spermatozoa. The fertilization rates of the oocytes inseminated with Et1^tm1Csk and Gk^tm1Csk frozen-thawed spermatozoa were 71% and 77%, respectively. The development rates into young after embryo transfer were in a range of 32-65%. Progeny tests revealed that the mutated locus/loci were transmitted to the next generation according to the Mendelian law of the inheritance of characteristics. These results indicate that cryopreservation of spermatozoa provides an effective alternative to embryo freezing for maintenance of gene disrupted mouse strains.

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In this study, the activity of mouse phosphoglycerate kinase promoter (PGK) and murine embryonic stem cell virus promoter (MESV), enhanced by the connection of the R-segment and part of the U5 sequence (RU5) of the long terminal repeat of human T-cell leukemia virus type 1, was compared with that of simian virus 40 early promoter (SV40) and human cytomegalovirus early promoter (CMV). Escherichia coli β-galactosidase (LacZ) reporter gene connected to these promoters was microinjected into pronuclei of mouse zygotes, and their expression of developing embryos during the preimplantation period was evaluated histochemically with X-gal. No difference was observed in the proportion of embryos which developed into the morula stage among the promoter sequences at 96 h after hCG injection, but the expression of LacZ gene connected to MESV-RU5 (MESV-LacZ) was lower than that to PGK-RU5 (PGK-LacZ), CMV (CMV-LacZ) and SV40 (SV40-LacZ) in the morula stage embryos (P<0.05). In another experiment, more than 50% of embryos microinjected with PGK- and CMV-LacZ responded positively to X-gal staining at 48 h after hCG injection and the activity of these promoters continued at nearly the same rate from there onwards. However, the rate of expression of SV40- and MESV-LacZ was lower than that of PGK- and CMV-LacZ at 48 h after hCG injection (P<0.05). Although expression of MESV-LacZ was consistently low in proportion and weak in intensity, that of SV40-LacZ was high at 72 h after hCG injection and was equivalent to that of PGK- and CMV-LacZ at 96 h after hCG injection. Regardless of the promoters used, the expression of LacZ gene in the embryos showed various intensities of blue staining. The frequency of mosaic patterns and a weak intensity of gene expression in morphologically normal embryos had a tendency to be higher than in degenerated embryos or those whose development had been arrested.

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For the development of a safe contraceptive vaccine, it is necessary to define B cell epitopes which could induce fertilization-blocking autoantibodies. In this study, an 18mer peptide (CTYILDPEKLTLRVPYKA) of rabbit ZPA which was recognized by a fertilization-blocking monoclonal antibody was chemically synthesized and conjugated with diphtheria toxoid (DT). The conjugate was injected into rabbits i.d. with complete Freund's adjuvant. As a control, DT was injected into rabbits in the same way. Mice were also immunized with the same synthetic peptide to induce heteroantibodies. Antisera from both rabbits and mice reacted not only with the cognate peptide but also with the isolated intact zona pellucida of rabbits. The immunohistochemical study showed that the antibodies had remained bound to the zona pellucida of the immunized rabbits in vivo. It is therefore concluded that the synthetic peptide could induce autoantibodies reactive to the auto-antigen of the zona pellucida in rabbits.

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The authors examined the effects of RU486 on the In Vitro fertilization (IVF) of hamsters. Six groups of medium containing 1, 5, 10, 20, 40 and 60 μM RU486 were prepared. In the control group without RU486, the fertilization rate was 97.3%. In the 1 μM RU486 group, the fertilization rate was 94.6%, and the difference between the control and 1 μM RU486 groups was not significant. In the remaining 5, 10, 20, 40 and 60 μM RU486 groups, the fertilization rate decreased in a concentration-dependent manner, and in the 60 μM RU486 group, it was reduced to 56.3% (P<0.001). These results showed that RU486, an antagonist of progesterone, inhibited IVF of hamsters in a concentration-dependent manner.

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The present study was designed to investigate the physiological significance of epidermal growth factor (EGF) receptors expressed on rat uterine epithelial cells during embryo implantation. The number of uterine EGF receptor sites, as determined with ¹²⁵I-labelled EGFas a ligand, progressively increased from the 3rd to the 7th day of pregnancy. Analysis of the EGF receptor by Scatchard transformation of EGF-binding data and affinity labelling with ¹²⁵I-labelled EGF indicated that EGF receptor proteins were noticeably expressed during postimplantation. Determination for EGF receptor sites was further carried out in the uterine tissues of rats, which were superovulating after transplantation of pituitary tissue into a capsule of kidney or subcutaneous injection of 50 IU pregnant mare serum gonadotropin (PMSG) and 50 IU human chorionic gonadotropin (hCG). The transplantation caused an increase in the number of implantation sites (24.5 ± 3.9, n=4) as compared to control rats (14.0 ± 0.6, n=7). Nevertheless, PMSG- and hCG-primed rats totally failed to initiate the implantation process, whereas the number of EGF receptor sites significantly increased during preimplantation (3rd day). In pituitary-transplanted rats on the 7th day, the number of EGF receptor sites was increased as observed in control rats (30.0 ± 0.5 fmol/mg proteins). In PMSG-and hCG-primed rats, however, the EGF receptor was noticeably reduced (7.3 ± 0.4 fmol/mg proteins). These results strongly suggest that functional EGF receptor proteins were noticeably expressed during postimplantation and that its drastic enhancement depends upon the interaction of blastocysts with uterine epithelial cells.

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