OOCYTE SECRETED FACTORS REGULATE FSHR AND AMH mRNA LEVELS IN CUMULUS CELLS

Abstract

Considering that (1) FSH activity decreases cumulus-oocyte communication via retraction of transzonal processes (TZP), (2) AMH inhibits FSH signalling by decreasing FSHR expression and adenylyl cyclase activation, and (3) the oocyte is an active player in the cumulus-oocyte complex (COC) regulating cumulus cell metabolism and gene expression via oocyte-secreted factors (OSF), we tested the hypothesis that the oocyte decreases mRNA abundance of FSHR while increasing that of AMH in cumulus cells through OSF, as part of an effort to preserve its communication with cumulus cells thus safeguarding its developmental competence. The relative mRNA abundance of FSHR and AMH in cumulus cell was compared among 3 treatment-groups: (1) Intact COC subjected to in vitro maturation (IVM); (2) oocytectomized COC (OOX) subjected to IVM and (3) OOX subjected to IVM with the addition of denuded oocytes (OOX+DO; 1 DO/μL). Four culture replicates including all three treatment-groups were performed. COC were aspirated from 3-8mm follicles of bovine ovaries. Oocytectomy was achieved by aspiration of the ooplasm with a micromanipulator while preserving the structure of the COC. COC and OOX underwent IVM in pools of 20 for 22 hours. IVM was performed in 100 μL of TCM199 with Earl’s salts supplemented with 1μg/mL FSH, 0.4% BSA, 22 μg/mL sodium pyruvate, 75 μg/mL of amikacin at 38,5°C and 5.5% CO2 in humid atmosphere. Abundance of mRNA encoding FSHR and AMH in cumulus cells was assessed by real-time RT-PCR using Power SybrGreen (LifeTech®) and RPL15 (ribosomal protein 15) as the reference gene. Effects of treatments were tested by ANOVA and groups were compared with the Tukey test. Statistically significant differences were those with p<0.05, while p values >0.05 and <0.1 were considered as tendency of difference. Oocyctectomy decreased AMH mRNA levels (p=0.005) while tended to increase FSHR mRNA abundance (p=0.077) in cumulus cells. The addition of denuded oocytes (DO) reversed the effects of oocytectomy on both genes. Relative AMH mRNA levels (AMH/RPL15 mRNA abundance, mean ± EPM) were 0.875 ± 0.1, 0.3675 ± 0.02 and 0.7825 ± 0.1 for COC, OOX and OOX+DO, respectively. For FSHR, the respective values (FSHR/RPL15 mRNA abundance) were 1.495 ± 0.54, 2.575 ± 0.52 and 0.875 ± 0.29. Oocyte secreted factors seem to reduce FSHR expression while enhancing AMH expression in cumulus cells. We speculate that this is part of an effort of the oocyte to restrain FSH activity in the COC in order to preserve its communication with cumulus cells thus favouring developmental competence.