LH receptor (LHR), Steroidogenic Acute Regulatory protein (StAR) and Mitochondrial Membrane Potential (MMP) in bovine granulosa cells are related to follicular size and atresia.

Granulosa cells (GC) play a key role in creating a suitable environment for the growth and the maturation of the oocyte as well as in determining the proper endocrine conditions for breeding and fertilization. However, only the GC of the ovulatory follicle have the opportunity to fully provide these tasks. Cumulus-oocyte complexes (COCs) used for in vitro embryo production (IVEP) are collected from follicles whose GC have not fully acquired or have lost these functions. This may be a reason of low IVEP efficiency. LH receptor (LHR), Steroidogenic Acute Regulatory protein (StAR) and Mitochondrial Membrane Potential (MMP) are direct or indirect markers of endocrine functions and putative candidates for evaluation of follicle quality. This study is aimed at evaluating the quality of GC of bovine ovarian follicles, classified according to their size and atresia grade, in order to provide new information to clarify the poor IVEP success. Bovine ovaries were collected from abattoir and transported to the lab at 4°C. Follicles were dissected, measured and classified according to their atresia grade (Kruip and Dieleman, 1982). The collected COCs were morphologically classified according to criteria related to follicular atresia (Boni et al., 2002). GC were obtained by scraping the follicular wall and filtered on a 50 μm nylon mesh. For each follicle, a part of GC was fixed with 2% paraformaldehyde for 1h. The remaining part was incubated with 5μM JC1 for 30 min followed by washing and reading with a spectrofluorometer (ex. 490 nm, em. 510 to 650 nm). Negative control samples were treated with 2 μM CCCP for 1 h before reading. The MMP values were expressed as the ratio between the fluorescence peaks at ~595 and ~525 nm. In fixed cells, immunofluorescence was carried out after treatment with blocking solution (20% Sea Block blocking buffer in PBS) with either anti-LH receptor antibodies (K-15) or anti-StAR antibodies (K-20) at 1:200 dilution for 90 min. After washing twice with TPBS (PBS + 0.05% Tween), the samples were incubated with secondary FITC-conjugated anti-goat antibodies. The samples were read at fluorescence microscope and the fluorescence intensity analyzed by ImageJ. Statistical analysis was carried out by ANOVA (Systat 11.0). Follicle size negatively affected (P< 0.01) the MMP as well as the expression of both LHR and StAR. Also the atresia grade, when evaluated on the basis of COC morphology, negatively influenced (P< 0.01) the expression of both LHR and StAR but positively influenced (P< 0.01) MMP. The evaluation of atresia grade on the basis of follicle morphology did not show significant effects on both LHR and StAR expression. These results highlight a discrepancy between the morphological characteristics of the follicle/COC and functionality of the GC, as previously demonstrated between COC quality and IVEP efficiency (Boni et al., 2002). Whereas the evaluated parameters represent markers of the steroidogenic activity, it is likely that the mechanisms of follicular regression pass through an upregulation of the GC metabolic activity.