322 LEPTIN TREATMENT DURING BOVINE OOCYTE MATURATION AFFECTS mRNA LEVELS OF APOPTOSIS-RELATED GENES

Abstract

The series of events associated with oocyte growth and maturation determines its ability to undergo successful fertilization, cleavage, and embryonic development. Among the molecules involved in these events, leptin has been identified as a modulator of oocyte function. The latest studies have focused on long-term effects of leptin during maturation of bovine oocytes on apoptosis and gene expression in in vitro-produced blastocysts. Briefly, blastocysts originating from leptin-treated oocytes exhibited decreased transcript levels of BCL2 associated X-protein (BAX), but increased mRNA concentrations for leptin receptor (LEPR), signal transducer and activator of transcription 3 (STAT3), and baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4, also known as XIAP) (Boelhauve et al. 2005 Biol. Reprod. 73, 737-744). In the present study, we analyzed single oocytes and their surrounding cumulus cells matured in the presence of 0, 1, and 10 ng/mL leptin. Transcript levels of LEPR, STAT3, BAX, BIRC4, FASLG (encoding Fas ligand), and FAS (encoding Fas receptor) were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of matured oocytes and cumulus cells. Four IVM replicates with four individual samples were collected 22 h after the start of IVM. Total RNA was isolated using a modified TriZol protocol, and reverse transcribed using the enzyme Superscript II-RT and random hexamer primers. Quantitative PCR was conducted with SYBR-Green as a double-stranded DNA-specific fluorescent dye in an ABI 7000 SDS apparatus. Treatment of COCs with 1 or 10 ng/mL leptin increased transcript levels of LEPR (2-fold; P < 0.001), STAT3 (2-fold; P < 0.001), BAX (2-fold; P < 0.001) and FAS (2-fold; P < 0.001) in cumulus cells. Interestingly, the transcript level of the well-known inhibitor of apoptosis, BIRC4, was increased about 4-fold (P < 0.001). In oocytes, leptin treatment increased the mRNA abundance of STAT3 (10 ng/mL; P < 0.05), FAS (1 ng/mL; P < 0.05 and 10 ng/mL; P < 0.001), and FASLG (10 ng/mL; P < 0.05). In conclusion, physiological doses of leptin during maturation of COCs have effects on the expression of developmentally important and apoptosis-associated genes in the oocyte and surrounding cumulus cells. The higher level of BIRC4 mRNA in leptin-treated cumulus cells was associated with a reduced proportion of cumulus cell apoptosis, which might explain the positive and long-lasting effects of leptin on the developmental competence of bovine oocytes.