Effect of follicle size and atresia grade on mitochondrial membrane potential and steroidogenic acute regulatory protein expression in bovine granulosa cells

During follicular development, granulosa cells undergo functional and structural changes affecting their steroidogenic activity. Estrogen synthesis mainly occurs in the endoplasmic reticulum and relies on aromatase activity to convert androgens arose from theca cells. In the present study, indicators of mitochondria-related steroidogenic capacity, as steroidogenic acute regulatory (StAR) protein expression and mitochondrial membrane potential (MMP), have been evaluated in bovine granulosa cells (GC) and related to follicle growth and atresia. Atresia was estimated by morphological examination of follicle wall and cumulus-oocyte complex (COC) and assessed by TUNEL assay for apoptosis detection. Bovine ovarian follicles were macroscopically classified according to their atresia grade and grouped in small, medium and large follicles. After follicle opening, the COC was morphologically classified for follicle atresia estimation and the GC were collected. GC were fixed for immunofluorescence (IF) and TUNEL, frozen for western blot (WB) or freshly maintained for mitochondrial membrane potential (MMP) analyses. StAR protein expression was assessed by using both IF and WB analyses. Follicle atresia grade could be efficiently discriminated based on either follicle wall or COC morphological evaluations. GC collected from small non-atretic follicles showed a higher (P<0.01) MMP and WB-based StAR protein expression than small atretic follicles. At IF analysis, StAR protein expression in large atretic follicles was higher (P<0.05) than in large non-atretic follicles. These results suggest a role played by mitochondria in GC steroidogenic activity, which declines in healthy follicles along with their growth. In large follicles, steroidogenic activity increases with atresia occurrence possibly associated with progesterone production.