Completely everted uterine horns of proestrous mice were treated with trypsin at 4°C and with a minimum mechanical agitation, and their surface epithelia were separated from the stroma and uterine glands with a minimum contamination of non-epithelial cells. The epithelial cell fraction was further purified by collagenase-dispase treatment, Percoll gradient separation and short term cultures on the collagen gel (5 min×3). Immunocytochemical check of cytokeratin networks in the cytoplasm proved well the purity of the cell fraction finally obtained.
The epithelial cells could proliferate well in the medium composed of a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle medium containing 50 μM of Ca²⁺ and supplemented with epidermal growth factor (EGF), insulin, transferrin, bovine serum albumin (BSA), vitamin A and hydrocortisone, when seeded on the collagen gel at a density higher than 2×10⁵/well (ca. 1, 000 cells/mm²). Deletion of anyone of insulin, transferrin and BSA from the culture medium suppressed severely the cell proliferation, while an only partial suppression was caused by the omission of EGF. Either single or simultaneous removal of vitamin A and hydrocortisone did not affect the proliferation at all. No stimulating effect of estradiol-17β on the proliferation was observed.

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Effect of the contact between embryos and cumulus cell monolayers on the development of 8-16 cell bovine embryos to blastocysts was investigated in IVF system. In vitro matured oocytes were inseminated by frozen-thawed sperm capacitated with heparin. After 72 hr of the insemination, oocytes were removed from cumulus cells and embryos developed to 8-16 cell stage were cultured for eight days after insemination under three different conditions; i.e. (i) direct contact: the embryos were directly placed on the cumulus cell monolayers, (ii) non-contact: a permeable filter was placed between embryos and the cumulus cell monolayers, (iii) without cumulus cells: the embryos were cultured without cumulus cell monolayers. Frozen-thawed semen from two bulls were used. The proportion of embryos developed to blastocyst stage (no. of blastocysts/no. of 8-16 cell embryos) were 32.9 (23/70)36.9 (24/65), 25.7 (18/70)44.1 (30/68) and 7.4 (5/68)9.2% (6/65), respectively. The result showed that the direct contact of embryos with cumulus cell monolayers was not necessary for the expression of the co-culture effect on the development to blastocyst stage and suggested that the effect was due to diffusable factors.

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This study was conducted to examine the possibility of performing nuclear transplanta-tion among different developmental stage embryos in cattle. The embryos, which were recovered nonsurgically from the uteri of superovulated cattle on day 5 to 8, were used as nuclear donors. The unfertilized eggs matured in vitro were used for recipient cytoplasm after removal of chromosomes at the second meiotic metaphase. Each enucleated egg was fused with a blastomere from donor embryos by electrofusion. The developmental ability of reconstituted eggs were examined by in vitro culture method.
The proportions of the enucleated eggs, which fused successfully with a blastomere from the 8-cell and the 16-cell stage embryos, were significantly higher (67/79, 102/121) than those obtained with a blastomere from the 32-cell or more advanced stage embryos (32-cell, 73/109; compact morula, 15/42; blastocyst, 0/56). Eleven and 20% of the reconstituted eggs fused with a blastomere from the 8-cell and 16-cell stage embryos developed in vitro to the 8-16-cell stage. None or very few reconstituted from the 32-cell or more advanced stage embryos developed to the 8-16-cell stage.

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The response of serum testosterone to human chorionic gonadotropin (hCG, 10000 IU) given as a single intra-muscular injection was evaluated in middle age (n=3; 8, 9 and 10 years old) and old (n=2; 19 and 25 years old) stallions in nonbreeding (September) season. A sharp rise of testosterone after 2 hr was observed only in middle age stallions, but not in old stallions. However, 4872 hr after hCG administration, a clear and sharp increase was demonstrated in all stallions regardless of age. The different response of serum testosterone to hCG according to age should prompt a reevaluation for understanding of endocrine function of horse testis using this agent.

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Eighteen suffolk ewes were devided into 3 groups and treated with intravaginal sponges impregnated with varing doses of progesterone (250, 500 or 750 mg) for 9 days during anestrous season (mid May). They received a single injection of PMSG (500 iu) 2 days before sponge removal. The induction rates of estrus were 0, 66.7 and 83.3% in ewes treated with 250, 500 and 750 mg progesterone, respectively. The ewes which manifested estrus showed serum progesterone higher than those did not (1.07 vs 0.58 ng/ml, p<0.01) during the treatment period; behavioral estrus was observed in all the 7 ewes which maintained serum progesterone levels greater than 0.8ng/ml. These results suggest that sustained rise in circulating progesterone (probably>0.8 ng/ml) is necessary for the PMSG induced ovulation to be accompanied by behavioral estrus in seasonally anestrous ewes.

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Twenty-nine postpartum Japanese Black cows were divided into 5 groups based on expected dates of estrus, at 4249 d (Group 1), 6370 d (Group 2), 8491 d (Group 3), 105112 d (Group 4), and 126133 d (Group 5), to determine the optimum time to initiate superovulation treatment. They were superovulated with FSH-P on Day 10 of the estrous cycle. The concentrations of plasma estradiol-17β and progesterone in groups 1, 3, and 5 were determined on the following periods: before superovulation treatment, at standing estrus, and at embryo recovery. The mean numbers of recovered and transferable embryos were 12.8±11.6 and 3.0±2.5; 6.5±2.5 and 4.0±2.0; 25.4±12.3 and 12.0±6.5; 6.5±0.5 and 3.5±0.5; and 12.0±6.5 and 3.0±3.4, for groups 1, 2, 3, 4, and 5, respectively. Group 3 had a significantly higher number of recovered and transferable embryos as compared to the other groups (P<0.05). The concentrations of estradiol-17β at standing estrus in groups 3 and 5 were similar. On the other hand, the progesterone concentration at embryo recovery was higher in group 5, but more embryos were recovered from group 3. It is concluded that the optimum time to initiate superovulation treatment using FSH-P, regardless of the number of estrus after parturition, is 94101 d postpartum (10 d following estrus at 8491 d postpartum).

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The establishment of new embryonic stem (ES) cell lines (A3-1, A3-2) from the blastocysts of 129/SvJ mice is described. The cells can be maintained in the undifferentiated state when grown on inactivated primary mouse embryonic fibroblast feeder layers. In suspension culture they spontaneously differentiated into embryoid bodies of increasing complexity which contained a variety of tissues including embryonic ectoderm and myocardium. The majority of the cells have kept normal set of chromosomes in Giemsa staining. Between 1 and 10 ES cells (A3-1) were introduced into the blastocoelic cavity of host-fertilized blastocysts homozygous for the dominant black locus. Eleven re-expanded blastocysts were transferred to the uterus of a recipient on the second day of pseudopregnancy, resulting in the birth of a phenotypically male chimera (agouti/albino). After mating with virgin IQI and ICR females homozygous for the albino locus, the male chimera has produced 19 agouti and 23 albino young, thus demonstrating that the transferred ES cells have c/c genotype and have colonized in the germ line.

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The localization of α-fetoprotein (AFP) in the bovine placentome was shown by the immunohistochemical method using Avidin-Biotin Peroxidase Complex (ABC). The intense staining specific to AFP was recognized in the fetal part of the placentome, especially capillary of the villus and wall of blood vessel in the allanto-chorion. Trophoblastic cells such as binucleate giant and mononucleate cells were negative for AFP. In contrast to the fetal parts of the placentome, no specific staining to AFP was found in the maternal component such as the cryptal epithelia, blood vessels in the maternal septa and the pedicle of the placentome. The localization of AFP was restricted in the fetal component of the placentome. Therefore, it is suggested that AFP may not transfer from fetus to the maternal blood circulation across the placental barrier.

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Changes in pituitary prolactin (PRL) content were examined in pseudopregnant rats. Blood and anterior pituitary glands were harvested every 3 hr on day 6 of pseudopregnancy. There were 2 plasma PRL surges with peaks at 18:00 hr (diurnal surge) and 3:00 hr (nocturnal surge). Pituitary PRL contents, assessed by RIA of pituitary homogenate made in phosphate buffered saline with teflon glass homogenizer, showed single peaked variation with a peak at midnight and a nadir at the time of the nocturnal surge. The same pituitary homogenate samples were assessed for PRL again after 4 months' frozen storage. The value by this 2nd RIA was dramatically increased in samples obtained around the time of the diurnal surge (12:0021:00 hr) and the peak shifted from midnight to 15:00 hr. This change between 2 assessments was reproduced by the experiment in which the PRL content was assessed after 4 cycles of freezing and thawing homogenate sample. Since almost all PRL of pituitary homogenate was seen in 105, 000 × g pellet fraction, shown by the result of basic ethanol extraction, the observed effect of freezing is thought to be the result of rupturing undestroyed membrane particle existed in homogenate sample.
These data show the change in pituitary PRL content in pseudopregnant rats and suggest the diurnal variation in the activity of PRL synthesis, which accompanies the change in the distribution of PRL among intracellular compartments and the peak of which is assumed to be just prior to the diurnal surge.

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The effect of the reduction of prolactin (PRL) release by the bromocriptine (CB-154) treatment on suppression of luteinizing hormone (LH) pulses by the suckling stimulus was examined in ovariectomized lactating rats at mid-lactation. Litter size was adjusted to 8 on day 1 (day 0=the day of parturition). Postpartum rats deprived of their pups on day 2 served as non-lactating controls. All rats were ovariectomized on day 2. CB-154 (0.6 mg/day) or saline was injected daily into both lactating and non-lactating rats from day 2. Ovine PRL (0.3 mg/day) was infused with a mini-osmotic pump into the half of the animals treated with CB-154. Litters were rotated every day among a CB-154 treated mother, 2 intact mothers and a saline-injected mother to ensure the similar strength of the suckling stimulus. Blood samples were taken at 6-min intervals for 3 hr on day 7 or 8. Pulsatile LH secretion was strongly suppressed in all lactating rats in spite of the treatment of CB-154. Frequent LH pulses were observed in all non-lactating animals, suggesting that CB-154 or ovine PRL did not directly affect LH secretion at the doses employed. These results suggest that PRL does not mediate the suppressing effect of the suckling stimulus on pulsatile LH secretion in rats at mid-lactation.

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