The vascular changes of the ovary were studied by scanning electron microscopy of vascular corrosion casts in estrous, pseudopregnant (stimulated with human chorionic gonadotropin - hCG) and pregnant rabbits. The results demonstrated that ovarian cycle and pregnancy may induce both structural and functional changes in the ovarian vessels. In fact the ovarian blood vessels adapted their structure to the temporary functional needs of the recruited follicles or corpora lutea. These changes involved both larger ovarian blood vessels (supplying the hilus and the medulla) and cortical blood vessels (supplying the luteo-follicular complexes). Hilar and medullary blood vessels also displayed morphological devices for the continuous control of the blood flow (spiral arteries) and for the local recirculation of endocrine products (artero-venous contacts). Such contacts, may likely sustain a "countercurrent mechanism" which was also shown in ovaries of other species. Scanning electron microscopy of vascular corrosion casts even demonstrated that cortical blood vessels are subjected to continuous remodeling. SEM showed different morphological types of vascular plexuses which supplied antral follicles (Types 1-2), atretic follicles (Types 3-4), peri-ovulatory follicles (Type 5), growing pseudopregnant corpora lutea (Type 6) regressing pseudopregnant corpora lutea (Type 7) and pregnant corpora lutea (Type 8). In estrous rabbit, growing to mature follicles (Types 1-2) showed a gradual enlargement and proliferation of the theca capillaries. These changes, associated with capillary hyper-permeabilization were observed in ovulatory and post-ovulatory follicles (Type 5), after hCG stimulation. The corpus luteum formation (Type 6 and 8) was accompanied by additional capillary dilation, diffuse angiogenetic sprouting and organization of conspicuous venous drainage which appeared more enhanced in pregnant (type 8) than in pseudopregnant (type 6) corpora lutea. The regression of the corpus luteum (Type 7) was characterized by the appearance of avascular areas within the gland and by regression of vascular dilatation. Finally, in estrous rabbit the atretic follicles (Types 3-4) wall showed large interruptions (avascular areas) and focal invasion of the central cavity by newly formed capillaries arranged randomly. The hCG stimulation did not affect consistently the interstitial microvasculature. It is concluded that the gradual increase of ovarian blood flow occuring during follicle growth and corpora lutea formation is provided by a functional adaptation of major ovarian vessels and by capillary functional (vasodilation) and structural (angiogenesis) support as well.

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We have previously shown that 48-h fasting profoundly suppresses pulsatile LH secretion in female rats in the presence of estrogen, and that the paraventricular nucleus (PVN) is an estrogen action site mediating this inhibition. The present study determined whether fasting suppresses pulsatile LH secretion in male rats in a testosterone-dependent manner, and whether the PVN is involved in the fasting-induced changes in pulsatile LH release. Mean LH concentrations and baseline levels of LH pulses were significantly reduced by 48-h fasting in castrated males implanted with various length (6, 12, and 24 mm) of subcutaneous testosterone implants. LH pulse frequency was significantly lower in fasted rats with a 24-mm testosterone implant compared with unfasted controls. On the contrary, mean LH levels and the amplitude and baseline of LH pulses were significantly increased by fasting in castrated rats without testosterone treatment. PVN lesions partially blocked the inhibitory effect of fasting on LH release in the testosterone-implanted rat model. In addition, local implantation of testosterone or estradiol into the PVN reversed fasting-induced increase in both mean LH levels and the pulse amplitude in castrated males, but did not cause a further suppression of these pulse parameters. These results suggest that testosterone is required to suppress LH pulses during fasting in the male rat. The PVN may mediate, at least partly, the fasting-induced inhibition of LH secretion, and is not the single steroid feedback site mediating the fasting-induced suppression of LH secretion in the male.

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The relationship of blood progesterone (P) and estradiol- 17β (E₂) concentrations to the pregnancy rates in Japanese Black beef cattle recipients was investigated by determining blood P and E₂ concentrations on the day before (day 6; day 0=onset of estrus) and the day of embryo transfer (day 7). Embryos were thawed by the stepwise method and transferred on day 7. The pregnancy rate showed a tendency toward increase with increasing P concentrations on both days 6 and 7 of embryo transfer. There was no significant correlation between the blood E₂ concentration on day 6 and the pregnancy rate, wthereas pregnancy rate tended to rise when the blood E₂ concentration on day 7 was low. The relationships of the blood E₂/P ratio (× 10³) on days 6 and 7 to the pregnancy rate were investigated. When the E₂/P ratio was ≥1.0, the pregnancy rate tended to decrease. The bovine recipients, which showed poorly development CL, high blood E₂/P ratio and coexistence of follicle ≥1.0 in diameter with CL, showed a tendency toward decrease in the pregnancy rate. From these observations, it is supposed to be important to select bovine recipient with the high bood P concentration and with the low blood E₂ concentration on days 6 and 7 as bovine recipients for improvement of the pregnancy rate in bovine frozen-thawed embryo transfer.

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It has been well demonstrated that the quantity and quality of the semen obtained from bulls improve with age. To understand the relationship between the semen characteristics and hormonal conditions in Holstein bulls, age-related changes in serum levels of inhibin, FSH, LH and testosterone were investigated. One hundred three Holstein bulls were divided into 11 age-groups as 2.5 (n=10), 6.5 (n=10), 12 (n=12), 24 (n=9), 36 (n=10), 48 (n=12), 60 (n=13), 72 (n=8), 84 (n=5), 96 (n=8) and 108 (n=6) months old. Serum levels of inhibin, FSH, LH and testosterone were measured by specific radioimmunoassays. Serum levels of inhibin were highest in bull calves before 12 months of age, and significantly decreased at 12 months old and remained low thereafter. In contrast to inhibin, serum levels of gonadotropins and testosterone were low in young bulls, and then increased with age. In summary, the concentrations of inhibin, gonadotropins and testosterone in serum changed with age in Holstein bulls.

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The requirement of mitogen-activated protein kinase (MAPK) for the initiation of meiotic maturation in porcine oocytes was examined using U0126, an inhibitor of MAPK activator. Immunoblot analysis and kinase assay showed that U0126 inhibited the phosphorylation and activation of MAPK incompletely in porcine oocytes. In this condition, maturation/M-phase promoting factor (MPF) activity was also inhibited and the rate of germinal vesicle breakdown (GVBD, 95%) was reduced to 42%. The classification of oocytes remaining at germinal vesicle stage (GV) and reached to second metaphase stage (M2) after 48h culture with U0126 revealed that GV oocytes had low MAPK and MPF activities while M2 oocytes had high activities in both kinases, suggesting the indispensability of MAPK activation for meiotic resumption of porcine oocytes. In mouse oocytes, however, the inhibition of MAPK activation by U0126 had no effect on GVBD nor MPF activation, consistent with previous reports using mos deficient mice. Additionally, we found that the inhibitory effects of U0126 needed the presence of cumulus cells because of the ineffectiveness of U0126 on denuded porcine oocytes. These results suggest that MAPK act as indispensable mediator of MPF activation and GVBD in porcine oocytes, like in Xenopus oocytes but not in mouse oocytes.

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Progesterone has been shown to inhibit the proliferative action of estrogen in the uterine epithelial cells. We have previously reported that the expression of cellular protooncogene, c-ski, is induced by estrogen treatment in the uterine epithelial cells of ovariectomized rats, and suggested its role in the proliferation of uterine epithelial cells. In the present study, we examined whether progesterone pretreatment would affect the estrogen-induced expression of c-ski mRNA in the uterine epithelial cells. In situ hybridization analysis revealed that estrogen-induced expression of c-ski mRNA was attenuated by progesterone pretreatment in the uterine luminal epithelial cells, accompanied by the decrease in the number of proliferating cell nuclear antigen-positive cells among the luminal epithelial cells. The results obtained further support our hypothesis that c-ski plays a role in proliferation of uterine epithelial cells and mediates the proliferative action of estrogen.

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The viability of reconstituted embryos derived from bovine somatic cells were evaluated. A total of 54 fetal somatic cell-derived embryos, in which some embryos were previously cryopreserved, were transferred to 12 recipient cattle (total 26 times). Three of 12 cattle that received fresh embryos became pregnant and delivered 3 calves (1 aborted at 223d and 2 live born), whereas none out of the 5 recipients became pregnant after transfer of embryos cryopreserved by slow cooling. Of 9 cattle that received embryos vitrificated 2 became pregnant. One of these two vitrification-derived embryos died during early pregnancy and another developed to term but was stillborn. These aborted fetuses, stillborn calf and two live born calves were identified as being derived from donor cells. The growth of live born calves showed no abnormalities when examined at 8 months of age. It was therefore concluded that: reconstituted embryos derived from fibroblast cells from female fetus could develop to term, that vitrification might be available to preserve these embryos and that transfer of these fresh embryos resulted in viable offspring with normal growth.

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