256 ROLE OFTHE BOVINE OOCYTE-SPECIFIC PROTEIN JY-1 IN REGULATION OF MESSENGER RNA ABUNDANCE FOR GENES LINKED TO CUMULUS EXPANSION

Abstract

We have previously demonstrated a requirement of the oocyte-specific protein JY-1 for oocyte and early embryonic development in cattle. Microin- jection of JY-1 siRNA into cumulus-enclosed germinal vesicle-stage oocytes impedes progression to metaphase II and cumulus expansion during in vitro maturation and limits subsequent embryonic development following in vitro fertilization. Negative effects of siRNA-mediated reduction in JY-1 on oocyte maturation, cumulus expansion, and initial cleavage divisions following in vitro fertilization can be rescued bysupplementation with recom- binant JY-1 protein during oocyte culture. However, the mechanisms involved in JY-1 regulation of above developmental endpoints are unknown. The objective of the current study was to determine whether JY-1-induced regulation of cumulus expansion during meiotic maturation is linked to alterations in mRNA abundance for genes that promote formation and stabilization of the mucified extracellular matrix characteristic of an expanded cumulus layer. Cumulus-enclosed germinal vesicle stage-bovine oocytes were microinjected with JY-1 siRNA, subjected to negative control siRNA microinjection or served as uninjected controls (n = 10 oocytes/treatment; n = 5 replicates), and cultured for 48 h in maturation medium containing 50 μM S-roscovitine to block spontaneous germinal vesicle breakdown. Cumulus-oocyte complexes 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 cumulus-enclosed oocytes were cultured as described earlier but in the presence of 1 ng/mL of recombinant JY-1 protein (n = 10 oocytes/treatment; n = 5 replicates). Dose of recombinant JY-1 protein utilized was previously shown to reverse inhibitory effects of JY-1 siRNA injection on cumulus expansion. After in vitro maturation, cumulus cells were harvested and RNA isolated and subjected to reverse transcription. Real-time PCR analysis was then conducted to determine the effect of treatments on cumulus-cell mRNA abundance for HAS2, HAS3, PTX3, and TNFAIP6. Effects of JY-1 supplementation or depletion (siRNA injection) on cumulus-cell mRNA abundance for HAS2 and HAS3 (enzymes involved in hyaluronan synthesis) were not observed. However, abundance of mRNA for TNFAIP6 and PTX3 (molecules implicated in stabilization of the hyaluronan-rich extracellular matrix) was reduced (relative to uninjected and negative control siRNA groups) in response to JY-1 siRNA injection (P < 0.05) and effects of JY-1 siRNA were rescued by JY-1 protein treatment (P < 0.05). Effects of JY-1 protein supplementation on cumulus-cell TNFAIP6 and PTX3 mRNA in uninjected controls were not observed. Results support a requirement of the oocyte-specific protein JY-1 for regulation of expression of genes functionally linked to stabilization of the hyaluronan-rich extracellular matrix and cumulus expansion.

This research was supported by USDA 2008-35203-19094 to G. W. Smith.