Evidence Supporting a Role for the Novel Bovine Oocyte-Specific Protein JY-1 in Regulation of Cumulus Cell Steroidogenesis.

We have previously demonstrated pleiotropic effects of the novel bovine oocyte-specific protein JY-1 during oocyte maturation and early embryogenesis. Microinjection of JY-1 siRNA into cumulus enclosed germinal vesicle stage oocytes (COCs) impedes nuclear maturation and expansion of surrounding cumulus cell layer and limits embryonic development following in vitro fertilization. Negative effects of JY-1 depletion on nuclear maturation, cumulus expansion and early embryogenesis can be rescued by recombinant JY-1 (rJY-1) protein supplementation during oocyte/embryo culture. In addition, pharmacological effects of rJY-1 treatment of bovine granulosa cells in vitro suggest that JY-1 may help promote luteinization. However, a requirement for endogenous (oocyte-derived) JY-1 in regulation of steroidogenesis has not been established. Therefore, we hypothesized that oocyte-derived JY-1 is a positive regulator of cumulus cell progesterone production during the periovulatory period and tested the effects of JY-1 ablation/replacement on cumulus cell progesterone production and expression of mRNA for STAR, HSD3B1 and CYP11A1 during in vitro maturation. Cumulus enclosed germinal vesicle stage bovine oocytes were microinjected with previously validated JY-1 siRNA, subjected to negative control siRNA injection or served as uninjected controls and cultured for 48 h in maturation medium containing 50 µM S-roscovitine to block spontaneous germinal vesicle breakdown. Oocytes were then washed and in vitro matured for an additional 24 h in maturation medium minus S-roscovitine. Additional JY-1 siRNA injected and uninjected COCs were cultured as described above, but in the presence of dose of rJY-1 protein (1 ng/ml) previously shown to reverse above described effects of oocyte JY-1 depletion. Effects of treatment of uninjected COCs with rJY-1 protein were not observed. However, concentrations of progesterone in maturation media (ng/ml/10,000 cells) and cumulus cell abundance of mRNA for STAR, HSD3B1 and CYP11A1 were reduced in JY-1 siRNA injected COCs relative to controls and such effects of endogenous JY-1 depletion were reversed by rJY-1 protein supplementation during in vitro maturation. To determine whether observed effects of oocyte-derived JY-1 on progesterone production are mediated in combination with additional oocyte secreted factors, COCs were subjected to oocytectomy (evacuation of oocyte cytoplasm and removal of all secreted factors including JY-1). Oocytectomized COCs were then cultured as described above in the presence or absence of 1 ng/ml JY-1 protein, with untreated, intact COCs used as controls. Concentrations of progesterone in culture media (ng/ml/10,000 cells) were reduced for oocytectomized COCs relative to intact COCs. Effects of oocytectomy on cumulus cell progesterone production were totally reversed by rJY-1 protein supplementation. Collectively, results established a regulatory role for oocyte-derived JY-1 in control of cumulus cell steroidogenesis (progesterone production) and expression of STAR, HSD3B1 and CYP11A1 mRNAs. In contrast to regulatory effects of oocyte-derived JY-1 on cumulus cell expansion, results suggest that JY-1 regulation of cumulus cell progesterone production during in vitro maturation is not dependent on presence of additional oocyte secreted factors. Supported by National Research Initiative Competitive Grant no. 2008-35203-19094 from the USDA National Institute of Food and Agriculture (GWS).

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