The main function of the corpus luteum (CL) is production of progesterone (P4). Adequate luteal function to secrete P4 is crucial for determining the physiological duration of the estrous cycle and for achieving a successful pregnancy. The bovine CL grows very fast and regresses within two days at luteolysis. The mechanism controlling the development and secretory function of the bovine CL may involve many factors that are produced both within and outside the CL. Some of these regulators seem to be prostaglandins (PGs), oxytocin (OT) and adrenergic factors such as noradrenaline (NA). Recently, there has been some evidence that P4 acts within the bovine CL as an autocrine and/or paracrine regulator. Each of these factors may act on the CL independently or may modify the actions of others. The purpose of this paper is to review and discuss the possible roles of OT, PGs, P4 and NA as luteotropic auto/paracrine regulators in the bovine CL. Furthermore, intra-luteal mechanisms controlling the sensitivity of the CL to extragonadal PGF_2α are discussed.

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Uniparental mouse embryos are generally used in the study of imprinting mechanisms as models to determine parental expression of imprinted genes. In this study, we used the real-time RT-PCR method to carry out a quantitative analysis of the expression of the insulin-like growth factor II (Igf2) gene, which is imprinted and expressed solely from the paternal allele, in androgenetic and parthenogenetic fetuses at day 9.5 of gestation. The mean expression, relative to that of control biparental fetuses, was detected to be 319% (90 to 585%) in androgenetic fetuses and 5.9% (3.7 to 9.5%) in parthenogenetic fetuses. The present results confirm that parental-specific expression of the Igf2 gene is maintained in uniparental fetuses, and also show that the real-time RT-PCR procedure is an effective method for quantitative analysis of gene expression using amounts of mRNA that are too small for other methods to detect.

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The aim of the present experiment was to investigate the effect of follicular size on oocyte cumulus-expansion, in vitro fertilization and subsequent developmental competence. After maturation culture, the rates of porcine oocytes with expanded cumulus derived from antral follicles ≥5 mm, 4-4.9 mm, 3-3.9 mm and 2-2.9 mm in diameter were 90.5%, 89.7%, 85.4% and 67.4%, respectively. After in vitro fertilization, the developmental competence of oocytes dependently increased with follicular size. Higher cleavage rates and higher proportions of 3-4-cell embryos were obtained from ≥4 mm follicles compared to 2-2.9 mm follicles (P<0.05). Although the proportions of 6-8-cell and 12-16-cell embryos obtained from ≥5 mm, 4-4.9 mm, 3-3.9 mm and 2-2.9 mm follicles showed no significant differences, embryos obtained from 2-2.9 mm follicles showed a complete failure to develop beyond the 8-cell stage, embryos obtained from 3-3.9 mm follicles failed to develop beyond the 16-cell stage and none of embryos developed beyond the early stage of morula. The percentages of expanded cumulus-oocyte complexes (COCs) and 2-cell cleavage rates of oocytes derived from 2-2.9 mm follicles were not significantly different when the oocytes were matured for 36 h , 42 h and 48 h in vitro. The exposure to 5% or 15% porcine follicular fluid (PFF) from the large follicles during in vitro maturation (IVM) had no significant effect on the cleavage and subsequent developmental rates of porcine oocytes compared to PFF from small follicles.

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The senescence accelerated mouse prone (SAMP) mice with a shortened life span show accelerated changes in many of the signs of aging and have a shorter reproductive life span than SAM resistant (SAMR) controls. The reproductive senescence of SAMP is more accelerated than that of SAMR. In the present study, we found abnormal accumulation of luteal bodies (LBs) only in the ovaries of SAMP mice and, we examined the mechanism of abnormalities in luteal cell regression in the ovaries of SAMP mice. No significant changes were detected in serum progesterone or 17β-estradiol levels during estrus cycle between SAMP and SAMR mice. In abnormally accumulated LBs of SAMP mice, extremely high levels of activity of 20α-hydroxysteroid dehydrogenase, which catalyzes conversion of progesterone to the inactive form, 20α-hydroxyprogesterone were demonstrated histochemically. However, no marked differences in activity of 17β-hydroxysteroid dehydrogenase were detected histochemically. Moreover, no apoptotic cells were detected in the abnormally accumulated LBs of SAMP mice. Extremely weak immunohistochemical reactivity for endothelial nitric oxide synthase (eNOS) was noted in the abnormally accumulated LBs of SAMP mice, but no differences were seen in inducible NOS (iNOS). No marked differences in expression of eNOS- or iNOS-mRNA were seen during the estrus cycle in the ovaries of SAMP mice. However, high levels of expression of eNOS-mRNA at estrus stage and of iNOS-mRNA at estrus and metestrus stages were noted in the ovaries of SAMR mice. SAMP mice may be a useful model in which to examine the mechanism of regression of LBs in mammalian ovaries.

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A multicellular mass (spheroid) was developed from bovine endometrial stromal cells with non-adherent culture plates. Cells cultured in a spheroid-plate^® were spontaneously aggregated and formed a cell mass rough in appearance 24 h after the start of culture. After 3 days of culture, the cell mass became oval-shaped in the range 375 to 900 μm in diameter. The size of the spheroid depended on the number of cells plated in each well. Histological examination indicated that each spheroid was covered with a single layer of squamous cells, and cuboidal cells occupied the inside of the spheroid. The size of the spheroid decreased gradually and the inner cells of the spheroid became sparse and irregular in size after culture for more than 7 days. ProMMP-2 mRNA was found both in monolayer culture cells and the spheroid, but the active form of MMP-2 protein was found only in the condition medium of the spheroid. The present study demonstrated that the spheroid was easily generated from bovine endometrial stromal cells by using a non-adherent plate. Expression of the active form MMP-2 indicates that the cells in the spheroid changed their character not only morphologically but also functionally. These results indicate that the spheroid developed by the present method could provide a new tool for analyzing the functions of bovine endometrium.

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To analyze the function of heat shock protein (HSP) in spermatogenesis, rat suppressor of potassium transport defect 3 (SKD3) cDNA, a novel member of the HSP family, was cloned in rat testis, and its expression and localization was investigated in rat testis at the mRNA and protein levels. The cDNA and predicted amino acid sequence of rat SKD3 showed approximately 95.6% and 97.0% homology, respectively, with mouse SKD3. All motifs recognized in mouse were conserved in rat. SKD3 mRNA (2.3 kbs) was restricted to the testis among the tissues investigated. SKD3 mRNA was detected by in situ hybridization in spermatogonia, spermatocytes, Leydig cells, and Sertoli cells. Immunoreactive SKD3 from whole rat testis and a Leydig cell line (LC540) was seen at 76 kDa by Western blotting. SKD3 immunoreactive cells were Leydig and Sertoli cells. Although the cellular localizations of SKD3 mRNA and protein were somewhat inconsistent, rat SKD3 cDNA was characterized and various expression patterns of SKD3 mRNA and protein in rat testis were revealed in the present study.

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The objective of this study was to establish a serum-free culture system for bovine endometrial epithelial cells, and to identify the function of these cells. Epithelial cells were separated from uterine endometrium at Days 5-10 of the estrous cycle by 0.76% EDTA-PBS and collagenase treatment. After histological observation, the epithelium was completely separated from the basement membrane by EDTA treatment. The separated epithelial cells were then dispersed by collagenase and cultured in Dulbecco's Modified Eagle's Medium/Ham's F-12 1:1 supplemented with insulin, transferrin, sodium-selenite, hydrocortisone, retinol, ascorbic acid and antibiotics on collagen-coated culture dishes. The cells attached to the substrate and reached confluency after 5-7 days of culture. An immunocytochemical study revealed that the cultured epithelial cells were positive to cytokeratin, but not vimentin. The production of prostaglandin (PG) F_{2 α} was significantly higher (p<0.001) in the presence of 100 nM oxytocin (OT; 2.81 fold greater than the concentration in the control) than in the absence of OT. These results indicate that it is possible to culture bovine endometrial epithelial cells in the serum-free culture system, and that cultured cells have some typical characteristics of endometrial epithelial cells.

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