The effect of porcine oocytes on the proliferation of rat granulosa cells was studied in a heterologous culture system. Morphologically high quality oocytes showed a more potent stimulatory effect on DNA synthesis in granulosa cells than low quality oocytes. This stimulatory effect of porcine oocytes was enhanced by FSH and cAMP, but was unaffected by both transforming growth factor-β and tumor necrosis factor-α. Maturation of porcine oocytes in vitro attenuated the stimulatory effect of FSH and cAMP on granulosa cell DNA synthesis. In addition, rat oocytes also exhibited a similar facilitatory effect on DNA synthesis in rat granulosa cells. These results suggest that a soluble factor(s) secreted from oocytes, the secretion of which is dependent on their meiotic progression, can stimulate proliferation of rat granulosa cells in a heterologous culture system. The molecular nature of the factor(s) remains to be elucidated.

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Species-specific differences in the process of apoptosis in granulosa and theca layers during follicular atresia in porcine and bovine ovaries were investigated by in situ DNA 3’end-labeling (TUNEL) at the level of individual cells. In porcine ovaries, granulosa cells located on the inner surface of the granulosa layer appeared to undergo apoptosis at the first stage, followed by neighboring granulosa cells. In the contrast, granulosa cells located on the middle region appeared to undergo apoptosis at the first stage in bovine ovaries. In both porcine and bovine ovaries, detachment and degeneration of the granulosa cell layer and fragmentation of basement membrane occurred in the follicles at advanced stage of atresia. In the ovaries of both species, theca interna cells were still intact at the stage of early atresia and then apoptotic cells appeared in the late to final stages of atresia, but no apoptotic cells were observed in theca externa layers during follicular atresia. The present findings indicated that apoptosis occurring in granulosa cells is an initial symptom of follicular atresia in the ovaries of both species, and that the apoptosis inducing factor(s) and/or survival factor(s) for the granulosa cells may be different between porcine and bovine ovaries.

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The present study was conducted to reveal the developmental changes in plasma IGF-1 concentrations in male and female Japanese macaques by a long-term longitudinal method from 3 to 6 years of age. The sexual maturation and reproductive seasonality in both genders were determined by plasma profiles of gonadal steroids, such as testosterone and progesterone. Since Japanese macaques have been known to have a rather unique characteristic of clear seasonality in reproduction, the present study introduced a time series analysis to extract two factors -seasonal and age changes- from the changes in plasma hormonal levels. Plasma IGF-1 concentrations showed seasonal and age-development changes in males. Plasma IGF-1 levels were high in summer and low in spring. Age-dependent changes would be superimposed on the seasonal change, because the IGF-1 levels of 4- and 5-year-old males were nearly twice as high as those of 3-year-old males in every corresponding season. Females, on the other hand, did not have clear age-dependent changes in the plasma IGF-1 level. The time series analysis suggests a close functional relationship between IGF-1 and testosterone secretion in regulating skeletal growth and seasonal changes in body weight.

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The present study was performed to clarify the synthesis of nitric oxide (NO) and its effect on the expression of transcription factor genes (c-fos, c-jun and ATF-4) during the differentiation of granulosa cells. Granulosa cells prepared from porcine ovarian follicles (1-4 mm diameter) were matured with FSH for 48 h. From 40 h to 48 h of ovine FSH stimulation, nitrite and nitrate increased by 2 folds, and this was accompanied by an increase of cyclic GMP. The cells were exposed to either NO scavenger (carboxy-PTIO: 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxy-3-oxide) or NO donor (NOC18: 2, 2'-(hydroxynitrosohydrazino)bis-ethanamine) before or after NO generation, and further stimulated with ovine LH until 48 h. Removal of endogenous NO (NO scavenger) induced a serious impairment in the LH-induced synthesis of progesterone, whereas NO donor had no significant effect on the synthesis. In the semi-quantitative reverse transcriptase-PCR of the transcription factor mRNAs, removal of endogenous NO resulted in a reduction of c-fos expression, an increase of c-jun and no changes in ATF-4 expression. In contrast, NO donor had no significant effects on the expression of these transcription factor genes. Consequently, the transient generation of NO may have critical roles in the LH-induced expression of transcription factor genes and thereafter transformation of the granulosa cell to the luteal cell.

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Exogenous genes (trial 1: lacZ/MiwZ and trial 2 : lacZ&GFP/pkkv4-lacZ) introduced into germinal crescent region (GCR) of chicken embryos were confirmed to be successfully transferred to the gonads via primordial germ cells (PGCs) and remained for a long time after hatching. The injected DNA was also successfully migrated and incorporated into next generation (F1). The F1 chickens were raised until sexual maturation, then the female F1 chicken and normal males, the male F1 chicken and normal females were subjected to artificial insemination according to the routine methods. The collected fertilized eggs were incubated for 72h to obtain F2 generation and the F2 embryos were examined for the detection of introduced DNA. In trial 1, the embryos obtained from F1 female chicken were examined by X-gal staining method, and showed the expression of the lacZ gene. Furthermore, the presence of lacZ gene in the extracts from the embryos was also determined by polymerase chain reaction (PCR) analysis. However fertilized eggs obtained from F1 male chicken showed no expression and presence of the DNA. In trial 2, the expression of green fluorescent protein (GFP) gene in the F2 embryos was not detected under the fluorescent microscope. The embryos were, then, examined by X-gal staining method and no detection was observed. Finally, the presence of GFP gene in the embryo extracts was determined by PCR analysis, indicating the presence only in the embryos obtained from male F1 chicken. These results suggest that the exogenously introduced DNA was successfully transferred to the F2 generation of chicken.

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The presence of viscous mucus in some mammalian oviducts has been related to the arrest of spermatozoa on their ascent to the site of fertilization. This study describes the distribution of intraluminal mucus in the porcine oviductal sperm reservoir during estrus, using histochemistry and electron microscopy, including high resolution cryo-scanning electron microscopy (Cryo-SEM) of frozen tissue samples. The luminal contents and apical epithelium stained with Alcian blue (AB) and periodic-acid Schiff (PAS), were most conspicuous in the lower isthmus of pre-ovulatory females. The SEM-observations of glutaraldehyde/AB-fixed specimens confirmed the presence of a conspicuous intraluminal material, most prominent in the sperm reservoir of pre- and peri-ovulatory animals. Such intraluminal material was consistently undetectable in glutaraldehyde-fixed, conventionally processed materials. In Cryo-SEM, a homogenous mass obliterated the lumen, especially in the pre-ovulatory UTJ/lower isthmus, diminishing towards the ampulla. Post-ovulation, the isthmic lumen appeared wider, showing only patches of amorphous material. If water sublimation was allowed during Cryo-SEM, the homogeneity of the luminal contents disappeared, leaving fibrillar remnants associated with the cilia and microvilli. The results clearly demonstrate the presence of luminal mucus in restricted areas of the pig oviduct, the pre-ovulatory sperm reservoir in particular, diminishing after ovulation. Its implication in the arrest and release of viable spermatozoa for fertilisation is discussed.

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Effects of protein kinase C (PKC) activators were investigated in mouse embryos during the developmental stage before compaction on cell division and subsequent development. Two-cell stage embryos were cultured for 24 h with phorbol 12-myristate 13-acetate (PMA; 0.1 or 1 nM) or 1 oleoyl 2-acetyl-sn-glycerol (OAG; 10 or 100 μM). PMA- and OAG-treatment inhibited cell division and embryos with binucleate blastomeres were produced in a dose dependent manner. The embryos in groups of 1 nM PMA and 100 μM OAG which were allowed to develop into the blastocyst stage had few nuclei and thin or no inner cell mass (ICM)-like structures. These blastocysts were transferred to the uterus of recipients. Consequently, some of the transferred embryos in groups of 1 nM PMA and 100 μM OAG implanted, but most of them had died by day 17.5 of pregnancy. In assessment of blastocyst outgrowth, the frequencies of outgrowths without ICM structure significantly increased in groups of 1 nM PMA and 100 μM OAG compared with the control. The present study indicates that application of PKC activators for 24 h from the 2-cell stage to mouse embryos leads to the production of tetraploid and tetraploid/diploid mosaic embryos, extreme reduction of embryonic cell number, and consequently the post-blastocyst development is obstructed in vivo and in vitro.

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Inhibin pro-αC is a precursor form of inhibin α-subunit. A large amount of inhibin pro-αC is secreted from testes in male animals, and it is considered to have an important role in the physiology of male reproduction. The purpose of this study was to determine the relationship between inhibin pro-αC and reproductive ability in the mature stallion. Peripheral blood samples were collected from normal stallions and anabolic steroid (nandrolone decanoate) treated stallions. Plasma concentrations of inhibin pro-αC were measured by an enzyme linked immunosorbent assay system. Circulating concentrations of inhibin pro-αC in normal stallions showed clear seasonal changes and the highest concentrations were observed in the breeding season (from March to June). After treatment with anabolic steroid, peripheral inhibin pro-αC levels rapidly decreased and reached significantly lower levels compared to the pre-treatment value (p<0.05). The variations of circulating inhibin pro-αC showed a high positive correlation with other testicular hormones. These results clearly demonstrate that circulating concentrations of inhibin pro-αC reflect spermatogenic ability due to seasonal change or treatment with anabolic steroid in the stallion, and it may be a useful new marker for testicular functions.

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