Homologous and heterologous replacement of a small amount of cytoplasm or both pronuclei were performed in rat and mouse pronuclear eggs. Rat eggs after receiving rat or mouse cytoplast never developed beyond the 2-cell stage in vitro. On the other hand, the in-vitro development of mouse eggs that received rat cytoplasm was possible, but it was greatly inhibited compared with those that received mouse cytoplasm. None of enucleated rat eggs that received rat or mouse pronuclei also developed beyond the 2-cell stage. Although few enucleated mouse eggs after receiving rat pronuclei developed beyond the 2-cell stage, high proportion (64%) of those that received mouse pronuclei could develop to blastocysts in vitro. When 2-cell embryos obtained 18 h after fusing with donor karyoplast were transferred to pseudopregnant rats, 2 (9%) of 23 enucleated rat embryos and 1 (7%) of 15 enucleated mouse embryos each fused with heterologous karyoplast developed to early blastocyst 48-55 h after transfer. These results may indicate that the embryonic genome can be activated in the heterospecific cytoplasm.

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Seasonal changes in peripheral plasma androgen levels in male goats were studied in 3 Saanen goats (seasonal breeders) and 3 Shiba goats (continuous breeders). Hourly blood sampling was carried out for 24 h in May (spring) and November (autumn) for measurement of plasma concentrations of testosterone and androstenedione. There were episodic bursts of secretion of androstenedione and testosterone during the 24 h periods in both May and November in both breeds. The mean levels of testosterone were 7.0-9.1 fold higher in November than those in May in the Saanen goat, whereas 1.2-2.9 fold higher in the Shiba goat. The degree of changes in the peak magnitude and the base-line level of testosterone in each individual in November were also somewhat smaller in the Shiba goat than in the Saanen goat. The plasma androstenedione levels in both breeds showed a similar change to testosterone, but the magnitude of fluctuation was only 1-10% of that in testosterone. These results suggest that the pattern of androgen release in male goats is episodic and follows marked seasonal changes, which are more conspicuous in Saanen breed.

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Changes were demonstrated in isoelectric focusing (IEF) profiles of pituitary luteinizing hormone (LH) in the cynomolgus monkey during the menstrual cycle. The LH activity was determined through radioreceptorassay (RRA) and exhibited a heterogeneous IEF profile. Highly alkaline isohormones (pl ?? 8.6) were predominant in the pituitary collected from animals in the follicular phase. The IEF profile from periovulatory period animals showed a significant increase in relative amounts of lesser-alkaline (pl=8.3 and 7.5) and neutral (pl=7.0) isohormones. The IEF profile from luteal phase animals showed considerable LH activity from pH 8.3 to 9.1 with a narrow peak of LH at pH 7.5. Seven isohormone fractions (pl=7.0 to 11.5) were obtained from male monkeys. Serial dilution of the 7 isohormone fractions as well as human chorionic gonadotropin (hCG) and monkey pituitary fractions were tested in the RRA. There was a stepwise increase in the slopes of the dose-response curves with increasing pl values of the isohormones. The receptor-binding property of hCG and the highest alkaline isohormone is not affected by the presence or absence of NaCl, while the binding of cynomolgus monkey pituitary LH and other isohormones apparently decreases with the presence of NaCl. Receptor-binding properties of each LH-isohormone were analyzed.

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The elongated conceptuses were collected from pregnant sows on Day 14 by flushing uteri with PBS. Conceptuses cultured in vitro in M-199 at 37C, under an atmosphere of 5% CO₂ in air, formed trophoblastic vesicles by the following day. The trophoblastic vesicles were cultured continuously in vitro and subjected to successive subculture, where the free-floating trophoblastic cells again formed small trophoblastic vesicles. The trophoblastic vesicles of all stages of culture showed similar patterns of the protein synthesis. The elongated conceptus and trophoblastic vesicle synthesized α-fetoprotein and retinol-binding protein. While culturing trophoblastic vesicles in vitro, fairly large amounts of estradiol-17β (E₂) and progesterone were synthesized. Possibilities that E₂ acts as a signal for the recoginition of pregnancy were discussed.

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To evaluate the intrauterine environment on embryo development, we examined the effects of co-culture with uterine epithelial cells and of uterine fluids on mouse embryo development in vitro. In the co-cultures of mouse uterine epithelial cells and 8-cell embryos, the rate of expanded blastocyst after 24 h of co-culture and the rates of hatching blastocysts 48 h and 72h after the initiation of co-culture (29.4%, 56.7% and 78.9%, respectively) were significantly higher than those in the control (11.3%, 26.5% and 49.7%, respectively). The incorporation of L-[4, 5-³H] leucine by the morulae after 24 h of co-culture was significantly higher than that of the control (an increase of 50.1%). The similar results were obtained in the cultures of 8-cell embryos in the medium added with uterine fluids. Moreover, the stimulating effect was exerted not only by uterine fluids from random stages of the estrous cycle but also by the fluids collected in three different stages, suggesting that the effect observed in this study might be independent of the estrous cycle. These results suggest that the intrauterine environment promotes the embryo development throughout the estrous cycle.

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Freshly superovulated eggs of Jcl: ICR mature mice that had been freed from their cumulus oophorus with hyaluronidase were fertilized by epididymal spermatozoa preincubated for various periods in a modified Krebs-Ringer bicarbonate medium (TYH). Calcium concentration in the fertilization medium was elevated to the twice (3.42 mM) or to the 3 times (5.13 mM) of TYH (1.71 mM) and all incubations were performed at 37C under 5% CO₂ in air. Both the sperm preincubation time and the concentration of calcium in the fertilization medium were found to exert significant effects on fertilization of cumulus-free eggs in vitro, and the highest fertilization rate was obtained when the cumulus-free eggs were inseminated with 150 min-preincubated spermatozoa in a medium with 3.42 mM calcium.

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Frozen-thawed mouse preimplantation embryos were exposed to Sendai virus at the blastocyst and morula stages to determine if these embryos were susceptible to virus infection. It was also examined whether the frozen-thawed embryos would further develop into expanded blastocysts after in vitro culture. Approximately 50% of the frozen-thawed embryos at the blastocyst stage were susceptible to Sendai virus infection. On the other hand, frozen-thawed embryos exposed to Sendai virus at the morula stage were resistant to the infection. It was noted that most of the frozen-thawed embryos exposed to Sendai virus developed into expanded blastocysts. Significant differences in the development to expanded blastocysts were observed in the Sendai virus-exposed frozen-thawed blastocysts (57.0%) compared with the frozen-thawed control (82.1%) and unfrozen control (93.7%) blastocysts. However, development to expanded blastocysts of Sendai virus exposed frozen-thawed morulae (73.0%) was similar to the frozen-thawed control (81.0%) but significantly different with the unfrozen control (93.2%) morulae. These findings suggest that some changes in the structure of the zona pellucida may occur during freezing procedures making the frozen-thawed embryos susceptible to virus infection.

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A study was undertaken to examine the in vitro fertilization and development into the 2-cell stage of rat follicular oocytes matured in modified Krebs-Ringer solution (mKRB) supplemented with procine follicular fluid (pFF). The oocytes were recovered at 0, 4, or 8 hours (h) after hCG injection from follicles (F-0, F-4, or F-8 oocytes) of immature rats pretreated with PMSG. F-0 to F-8 oocytes were cultured in mKRB with or without 10% or 20% pFF for 12 to 4h. After maturation culture, these oocytes were transferred into sperm-suspended mKRB solution (4-14×10⁵ sperm/ml) and culture continued for 36 h. When F-0 to F-8 oocytes were matured in mKRB alone, the fertilization rates were low (28.2% of F-0 oocytes to 62.7% of F-8 oocytes). These rates markedly increased to 80.5 to 87.6% in oocytes matured in mKRB with pFF (P<0.01). Furthermore, a high incidence of 2-cell embryos was obtained in oocytes cultured in mKRB with pFF (75.8-78.2%, P<0.01). The maturation processes did not differ between oocytes cultured in mKRB with and without pFF, and those matured in vivo. These results indicate that addition of pFF to the maturation medium improves fertilization rates and development into the 2-cell stage of rat follicular oocytes. This pFF action appears to be non-specific for rat oocytes.

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Developmental changes in testicular size and plasma androgen levels in male Shiba goats were studied during 2-12 months of age. Scrotal circumference increased rapidly until 5 months of age (18.6±0.9 cm; mean±SEM). Plasma androstenedione and testosterone reached peak levels of 0.23-0.50 ng/ml and 5.47-9.69 ng/ml, respectively as testicular size increased, and then showed a tentative decline with nadirs of 0.01-0.04 ng/ml and 0.26-0.94 ng/ml, respectively after full growth in testicular size. The fluctuation of androstenedione was in parallel with that of testosterone but the androstenedione levels were only 1-10% those of testosterone. Thus, the results indicated that testosterone was the predominant androgen starting from 2-3 months of age through puberty in male Shiba goats.

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Granulosa cells(GC) collected from follicular fluids of the mature follicles of 9 estrous cows were cultured for 6, 12 or 24 h at 37C in a serum-free synthetic culture medium (SMF-101 "Nissui"). On completion of the culture periods, the concentrations of PGF_2α, in the culture supernatants were determined by radioimmunoassay. The following results were obtained. The PCF_2α concentrations in the culture supernatants were increased in 8 of the 9 samples as the culture elapsed. Statistic analysis was carried out by Tukey's q-test. Significant differences were found in the PGF_2α, concentrations in the supernatants between 6 and 24h culture. This suggested that GC derived from the mature follicles of the estrous stage of cows produced PGF_2α, in the medium.

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