The Journal of Reproduction and Development Supplement

The improvement of in-vitro growth culture (IVG) system for bovine oocytes derived from early antral follicles (EAFs, 0.5 to 1 mm) is required for production and research purposes. In experiment 1, growing oocytes (95 μm in diameter) collected from EAFs were submitted to 12-day IVG. After IVG, oocytes with normal appearance were submitted to 0-, 10- or 20-h pre-IVM with 3-isobutyl-1-methylxanthine (IBMX) followed by 22-h IVM (0-, 10- and 20-h pre-IVM group, respectively). Diameter and nuclear maturation (M II) rate of IVG oocytes were examined after IVM. Mean diameters were similar in all group (about 115 μm), but they were smaller than that of in-vivo grown oocytes derived from antral follicles (2 to 8 mm). M II rate in 10-h pre-IVM group (90%) was higher than those in 0-h (55%) and 20-h pre-IVM groups (73%), and similar to that of in-vivo grown oocytes (94%). In experiment 2, IVG oocytes in 0-, 10- and 20-h pre-IVM groups were submitted to IVF and their developments were examined. Cleavage (80%) and blastocyst rates (41%) in 10-h pre-IVM group were higher than those in 0-h (46 and 13%, respectively) and 20-h pre-IVM groups (63 and 26%, respectively), and similar to that of in-vivo grown oocytes (83 and 42%, respectively). To clarify the cause of difference in maturational and developmental competences, mitochondrial activity of IVG oocytes was examined by JC-1 staining after 0-, 10- and 20-h pre-IVM. Mitochondrial activity of IVG oocytes after 10-h pre-IVM was higher than those after 0- and 20-h pre-IVM. In conclusion, 10-h pre-IVM culture with IBMX improves maturational and developmental competences of bovine IVG oocytes, and it seems to be related to the activation of mitochondria.

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This study was conducted to establish the method for preserving chicken primordial germ cells (PGCs) that enables long-term storage in liquid nitrogen for preservation of species. The purpose of this study is to clarify the effects of fetal bovine serum (FBS) or chicken serum (CS) treatment on viability of cryopreserved PGCs in Korean Native Chicken (Ogye). PGCs separated from a germinal gonad of an early embryo of 5.5–6 day (stage 28) are suspended in a freezing medium containing a freezing and protecting agents (e.g. DMSO or ethylene glycol). After the tube was preserved in liquid nitrogen for 1 month at least, the viability of PGCs after freeze-thaw via 0, 5, 10 and 15% EG plus FBS treatment were 22.36, 40.12, 42.96, 64.36 and 55.36%, respectively. Viability assays were conducted on both the frozen group (~20ml cell suspension), and on the control group (~20mL cell suspensions of 100 PGCs in modified buffer). 0.4% Trypan blue solution (10 mL) was then added to each drop of PGCs suspension and the mixture incubated for 2 min at room temperature. These values of the 0, 5, 10 and 15% DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39 and 48.36%, respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% EG+FCS treatment (p<0.05) (64.36% vs 50.66%). This study established a method for preserving chicken PGC that enables systematic storage and labeling of cryopreserved PGC in liquid N at a germplasm repository and ease of entry into a database. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

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Background: P450scc, 3βHSD, P450c17 and P450arom are responsible for steroid biosynthesis, which are mainly produced in Leydig cells in testes. Testes are also the source of many cell growth factors, including inhibins and activins. In mammalian species, apart from their action on FSH secretion, inhibins and activins have been shown to exert paracrine/autocrine effects within the gonads. The purpose of this study was to investigate the cellular immunolocalization of P450scc, 3βHSD, P450c17, P450arom and inhibin/activin subunits in the adult male African elephant testis. Materials and Methods: Hematoxylin-eosin (HE) staining was used to observe testicular tissues of the African elephant. The sections of testis were immunostained using polyclonal antisera raised against bovine adrenal P450scc, human placental 3βHSD, porcine testicular P450c17, human placental P450arom, porcine inhibin α, porcine inhibin/activin β_A and β_B. Results: Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were found in African elephant testis. P450scc, 3βHSD, P450c17 and P450arom were all detected in cytoplasm of Leydig cells. In addition, positive immunostaining for inhibin a and inhibin/activin (β_A and β_B) subunits was also observed in Sertoli and Leydig cells in the African elephant testis. Conclusions: These results suggest that Leydig cells of adult African elephant testis have the ability to synthesize progestin, androgen, estrogen, and Sertoli and Leydig cells are the major source of inhibin secretion in male African elephants.

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Background: Expression and the function of genes generating circadian rhythms in the ovary have not been fully examined from proestrous stage to estrous stage in spite of the interest for the involvement of the circadian clock on ovulation. Here, the circadian clock network and the involvement of one of its regulators, RevErba, in the expression of Cox2, the inducer of ovulation in ovaries, were examined. Results: Coordinated, rhythmic expression was found for main circadian regulatory genes, suggesting that the ovarian circadian clock is functional from proestrous to estrous stage. Because the expression level of RevErba was the lowest at the time of occurrence of LH surge but increased immediately after that, we hypothesized that the duration of the transient rise of Cox2 expression induced by LH surge is adjusted by the suppressive effect of REVERBa on Cox2 expression. Using cultured granulosa cells and a linker-scan mutagenesis screen of the potential regulatory region of Cox2, we identified REVERBa binding sites (RORE). Luciferase reporter vectors containing the promoter region of Cox2 with and without RORE (pGLR and pGLdR) were transfected into cultured mouse granulosa cells. Agreeing with our hypothesis, dose-dependent expression of RevErba into the culture cells suppressed the induction of pGLR by forskolin, which mimicks the LH surge in vitro. Contrarily, over-expression of RevErba did not affect expression of pGLdR. Finally, binding of REVERBa was detected to this regulatory region in phase of the beginning repression of endogenous Cox2. Conclusion: Our results suggest that RevErba is involved in fine-tuning of Cox2 expression, which is important for the efficiency of ovulation.

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