Luteinizing Hormone effect on the GDF-9 and BMPR-1a Expression of Bovine Granulosa Cells culture

Abstract

Growth Differentiation Factor-9 (GDF-9) and Bone Morphogenetic Protein Receptor 1a (BMPR-1a) was a member of the transforming growth factor-β (TGF-β) superfamily known to regulated ovarian functions in mammals. In addition, Luteinizing Hormone (LH) also has an important role in ovarian function. The objectives of this research are to observe effect of LH on the GDF-9 and BMPR-1a expression in granulosa cells (GCs) culture. Bovine ovaries were obtained from a slaughterhouse and carried to the laboratory within 30 min in a container kept at 37 °C in saline containing gentamicin and amphotericin. The ovaries were washed with pre-warmed PBS supplemented with gentamicin and amphotericin. Granulosa cells were collected from follicles by aspiration using a syringe with needle (20G). The cells were centrifuged for 1 min at 800g at room temperature and washed twice in DMEM/F12 medium and filtered through a stainless steel filter (100 um) to get the GCs then seeded in 6 well culture plates. The cells were cultured at 37 °C in a 5% CO2 atmosphere for 24 h and then the wells were washed with PBS to remove unattached cells. The culture medium was replaced with serum-free medium supplemented with LH at 100 ng/mL and cultured for 24 h. Total RNA was extracted from GCs using TRIsure. The RNA quality and quantity were estimated by using spectrophotometer at 260/280 nm. Aliquots (1 ug) of total RNA from each pool of GCs were independently reverse-transcribed to cDNA. The mRNA expression of GDF-9 and BMPR-1a was estimated by the quantitative real-time PCR method. The results showed that LH added to the culture medium can increase the expression of GDF9 and BMPR-1a of granulosa cells.