Effects of food restriction on spermatogenesis were examined. A total of 20 male Slc: SD rats were used. Food intake was restricted to 25, 50 and 75% of ad libitum consumpti on over 4 weeks. Body weights in 25, 50 and 75% groups showed 54, 72 and 90% of control group, respectively. Although a significant decrease in testicular weights was recorded in 25% group, there was no significant difference bet ween food restricted and control groups in sperm head count analysis. Histologically, degenerated spermatocytes were detected in the stage 7 tubules, however, the number of the affected cells was quite a few. The incidence (%) of seminiferous tubule containing degenerated spermatocytes was 1.2 in control, 2.2 in 75% group, 2.2 in 50% group and 19.8 in 25% group. These results suggest that nutritional deficiency associated with marked reduction of body weight induced the mild damage of spermatocytes.

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Intracellular changes of calcium ion [Ca²⁺] _i induced by sperm (in vitro insemination) or three artificial activations were studied using in vitro matured bovine oocytes. Bovine oocytes were matured in TCM-l99 containing 10%(v/v) FCS, 2.5 μg/ml FSH, 2.5 μg/ml LH, 1μg/ml estradiol-17β and 2 × 10⁶ granulosa cells/ml for 2l-23 h, and the oocytes were transferred into the medium containing fluorescent Ca²⁺ indicator Indo-l acetoxymetylester for loading into the oocytes. Immediately after sperm attachment, the increase of [Ca²⁺]_i began from near the sperm attachment site and the Ca²⁺ rise spread over the entire cytoplasm. The peak Ca²⁺ value by sperm stimulation was 788 ± 80 nM. Activation treatments with ethanol, Ca-ionophore or electrical stimulation induced an acute rise of Ca²⁺ beginning at the superficial area of the oocyte, and finally spread over the entire egg. The peak Ca²⁺ values of Ca²⁺ elevation induced by ethanol, Ca-ionophore and electrical stimulation were 1158 ± 93, 1048 ± 79 and 719 ± 9 nM, respectively. The peak values of Ca²⁺ elevation by ethanol and Ca-ionophore were significantly higher (p<0.05) than that with sperm. Among the activation treatments tested in the present study, electrical stimulation showed the most similar pattern of Ca²⁺ change inducing by sperm. The present results indicate that changes of intracellular Ca²⁺ l evels are different in sperm and three artificial activation treatments, and electrical stimulation may be an effective method for parthenogenetic activation of bovine oocytes.

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To monitor the feto-placental function, blood samples were collected by jugular venipuncture from 54 dams at 257 (4 weeks before due date: -4W) and 271 (2 weeks before due date: -2W) days of gestation. Serum progesterone levels (P) were measured by enzymeimmunoassay. Free estrone(E₁), estradiol-17β (E₂) and estrone sulfate(E₁S) in the serum were measured by radioimmunoassay. After delivery, 54 dams were divided into two groups: a group of 41 dams with a newborn calf over 20 kg body weight (normal birth weight group; NBW), another group of 13 dams with a calf of 20 kg or less (low birth weight group; LBW). From -4W to -2W, slight decrease in P, significant increases in E_l and E₂ were observed, but no significant differences were present in those steroids between LBW and NBW. E₁S was significantly lower in LBW than in NBW (P<0.01) and correlated positively with the birth weight of calves (r=0.50, P<0.01) at -4W and -2W, respectively. In the 13 LBW calves, eight showed neonatal weakness and two of them died, while the other 5 calves did not show the weakness. Eight LBW calves with neonatal weakness had a relatively lower maternal E₁S level than the other 5 viable calves at -4W, and it remained at the same level at-2W. In contrast, the 5 vital calves with LBW showed a significant increase of maternal E₁S from -4W to -2W. These results suggest that maternal serum E₁S level provides useful and practical information for monitoring the feto-placental function and for predicting the maturity and viability of newborn calves.

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A morphometric study of gonadal interstitial cells in 16 equine fetuses, ranging in age from 120 to 330 days of fetal age, was done to assess the possible relationship of fetal gonads to high plasma concentrations of immunoreactive inhibin in pregnant mares during the second half of pregnancy with its peak at about 280 days of gestation, which has been reported recently. Interstitial cells of fetal gonads underwent progressive hypertrophy to reach a maximum size around 250 days of gestation, and subsequent degenerative changes in these cells resulted in a rapid decrease in the volume density and size of the interstitial cells. Thus, the morphometric change in the interstitial cells was almost synchronous with high concentrations of immunoreactive inhibin in the plasma of pregnant mares. This observation, taken in conjunction with the fact that inhibin α-chain (1-30) was detected immunohistochemically in the cytoplasm of the interstitial cells, appears to provide morphometric evidence that the interstitial cells in fetal gonads are closely related to inhibin production.

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A total of 311 recipient cows received one frozen-thawed embryo non-surgically using a vaginal speculum. and an embryo transfer gun. Group A(n=153)receiving the embryo using the gun without a protective sheath showed a pregnancy rate of 56.9%(87/153), while the rate in Group B (n=158) using the gun covered with the protective sheath was 54.4%(86/158). The results indicate the usefulness of the vaginal speculum for non-surgical embryo transfer with an embryo transfer gun without a protective sheath.

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In the present study, a regulation mechanism of porcine oocyte maturation was examined with special focus on maturation promoting factor (MPF), a crucial regulator of cell division cycle composed of catalytic subunit, p34^cdc2 and regulatory subunit, cyclin B, and Mitogen-activated protein kinase (MAPK). At first, an in vitro maturation system using porcine follicular fluid as maturation medium was established in order to mature the oocytes physiologically normal. The oocytes in the germinal vesicle stage (GV) had a significant amount of p34^cdc2 and MAPK, which concentrations were not changed throughout maturation. An extremely small amount of cyclin B was present in GV oocytes and cyclin B was not synthesised during GV. The extremely small amount of cyclin B associated p34^cdc2 was inactivated by tyrosine phosphorylation and MAPK is also inactive during GV. Germinal vesicle breakdown (GVBD) was induced by tyrosine dephosphorylation of p34^cdc2. After GVBD, cyclin B synthesis was started and the amount of cyclin B associated p34^cdc2 and MPF activity were increased dramatically. MAPK was also activated by phosphorylation and maintained the high activity until the second metaphase (Met2). MPF activity was decreased transiently after the first metaphase (Merl) then reactivated at Met2 without tyrosine dephosphorylation. The amount of cyclin B associated p34^cdc2 in Met2 was greater than that in Metl and most of them were stored as pre-MPF. These results suggest that the regulation of porcine oocyte maturation is essentially similar with other reported species but have some modifications specific for porcine oocytes.

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There are many types of chromosome abnormalities in domestic animals and these abnormalities are closely related to the reproductive disorders. From the results reported, the author proposes that chromosome abnormalities are divided into four Groups: Group A is made up of cases that have abnormal chromosomal structures(translocation, inversion)and show relative sterility; Group B contains cases that have normal chromosomal karyotypes(XY female, intersex)and show absolute sterility; Group C contains cases with an increasing or decreasing number of chromosomes(trisomy, monosomy)and show absolute sterility; Group D contains cases with sex chromosomal chimerism(freemartin)as well as absolute sterility. Because the cases from these groups are accompanied by characteristic gene abnormalities, these cases would be useful model in order to resolve the mechanism of gene function in domestic animals.

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Fas antigen (Fas) is a cell-surface receptor molecule that mediates apoptosis-inducing signals by stimulation with Fas ligand (FasL) or agonistic anti-Fas antibodies. It has been shown that Fas and FasL play important roles in the immune system. Although significant amounts of Fas and FasL mRNAs can be detected in the non-immune tissues including the reproductive organs, their roles are still unclear in these tissues. We have investigated the role of the Fas system in the cell death occurred in the reproductive organs. In mouse ovary, Fas-expressing granulosa and luteal cells were detected. Intraperitoneal administration with an agonistic anti-Fas antibody enhanced follicular atresia and luteolysis. Furthermore, mature ovaries from lpr mice carrying the mutant Fas gene were histologically abnormal in terms of follicular development. These results indicated that the Fas system is involved in the physiological cell death in the ovary. In the testis, although Fas protein is rarely expressed on testicular cells, transcripts for FADD and FLICE molecules that are specifically involved in the Fas-mediated apoptosis were detected. This result suggests that the Fas system may function in the testis. We propose that the Fas system plays important roles in the reproductive organs as well as in immune tissues.

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Apoptosis is a fundamental process in embryogenesis, immune system and tissue homeostasis. Its disregulation may have important implications for congenital anomalies, immune system disorders or tumorigenesis. So, elucidating the molecular mechanism of apoptosis may be important not only in basic science but also for treatment of a range of human diseases. I will briefly review the studies of developmental cell death with special emphasis in cell biological analysis of apoptosis in limb morphogenesis. At first, we have revealed that treatment of embryos with BrdU, a differentiation inhibitor, specifically modified the developmental fate of cells destined to die. Cells in inter-digital necrotic zone (INZ) were rescued from death. Cell cycle and cell death were closely related events and there existed a critical S-phase, which corresponded to the most sensitive time for inhibition of cell death by BrdU. Biochemical examination of DNA of INZ cells revealed the existence of DNA ladders, indicating that dying cells in limb undergo classical apoptosis. The use of microfluorometry coupled with TUNEL method enabled us to detect DNA breaks in individual nuclei very early in the death process. Finally, I will mention the molecules involved in death triggering during morphogenesis.

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The porcine antral follicles, 4-6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, apoptotic cells were observed histochemically among granulosa cells only. In atretic follicles, high activity of neutral Ca²⁺/Mg²⁺-dependent endonuclease by which the chromatin DNA is degraded at internucleosomal sites in the cells undergoing apoptosis was noted only in granulosa cells but not in cumulus cells. Low activity of the Ca²⁺/Mg²⁺-dependent endonuclease was observed in both types of cells obtained from healthy follicles. Low activities of neutral Ca²⁺-dependent endonuclease, neutral Mg²⁺-dependent endonuclease and acidic cation-independent endonuclease were demonstrated in all cases. A good correlation between the Ca²⁺/Mg²⁺-dependent endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle, which provides an index of follicular atresia in pig ovaries, was found. In vitro study showed that the specific inhibitors for interleukin-β converting enzyme (ICE) like cysteine proteases could stop the apoptotic process induced by specific monoclonal antibody to granulosa cell membrane protein in primary cultured granulosa cells. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs, and ICE like protease (s) is involved in intracellular signal transmission of granulosa cell apoptosis of the atretic antral follicles in pigs.

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The mammary gland is an unique organ present only in the mammal. The mammary gland, grown and differentiated during pregnancy, begins to synthesize milk following parturition. The lactating mother continues actively to supply milk to the young for a certain period, but milk synthesis terminates at the late stage of lactation. In various species, the changing profile of milk production is known as the lactation curve. The increase or decrease in milk production has merits for both the young and the mother. At the termination of milk synthesis, the mammary gland undergoes regression and most mammary epithelial cells disappear from the gland through the process of apoptosis. The mammary gland returns the state identical to that before pregnancy. It is well known that prolactin regulates mammary gland functions; growth, differentiation, milk synthesis and regression. The mammary gland modulates sensitivity to prolactin by changing the number of prolactin receptors of the mammary epithelial cell. As described here, milk production is regulated primarily by the level of the prolactin receptor gene expression. It is believed that as a result of the law of the survival of the fittest, the mammary gland acquired the ability of regulating its own functions by modulating the prolactin receptor gene expression. Milk production of dairy cattle has been improved during the last two centuries. By manipulating prolactin receptor gene expression, it will be possible to further improve milk production in dairy cattle.

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Germ cell deletion during normal spermatogenesis has been estimated to result in the loss of up to 75% of potential numbers of mature sperm cells in the adult testis. During neonatal and pubertal development, the incidence of testicular germ cell degeneration varies. Morphological analysis of rat indicated that germ cell demise is most commonly found in spermatogonia and is present throughout testis development. Recently, biochemical evidence was presented that links testicular cell death to increased internucleosomal DNA fragmentation, a hallmark of apoptosis. Gonadotropins and sex steroids can act as survival factors in the testis of immature rats. There are age-dependent change of apoptosis in testis of developing rats. Apoptotic DNA degradation was found only in germ cells of selected phases of spermatogenesis. Besides, testicular cell apoptosis is affected by varying photoperiod length or by temperature elevation induced by cryptorchidism. Analysis of regulation of testicular apoptosis should provide new insight on the physiology and pathophysiology of testis.

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