The Effects of Platelet-Derived Growth Factor BB on Ovine Granulosa Cell Function.

Abstract

Platelet derived growth factors (PDGF) have been shown to modulate ovarian follicular development in some species. However, relatively little is known regarding the potential role of PDGF in regulating granulosa cell function in ruminants. Oocytes and granulosa cells in growing ovine follicles express mRNA encoding both PDGFA and PDGFB and follicular atresia appeared to cause decreased expression in the granulosa cells. However, with in situ hybridization, mRNAs for PDGF receptors (PDGFRA and PDGFRB) were not detectible in the granulosa cells. Thus, it is not clear whether PDGF can regulate granulosa cell function in vitro. Therefore, our aim was to determine the effects of PDGF on thymidine incorporation and progesterone production in sheep granulosa cells. Granulosa cells were collected from ovine follicles 1-2 mm in diameter. The incorporation of 3H-thymidine was determined during a 24 hour culture (20,000 viable cells per well) with M199 media. Secretion of progesterone was measured following culture of granulosa cells (100,000 viable cells per well) in an enriched McCoys 5a media with 30 ng/ml androstenedione, 3 ng/ml ovine FSH and 1 ng/ml IGF-1. Cells were cultured for 6 days with media being replaced every 48 hrs. The concentration of progesterone was measured by RIA in the media collected at the end of the culture period. The amount of DNA at the end of the culture period was also measured using binding of Hoechst 33258 dye. Triplicate wells were included in each assay and a minimum of 3 independent granulosa cell cultures were examined. The effects of human PDGFBB (10 ng/ml) was compared to vehicle treated control cells using paired t-tests. Addition of PDGFBB increased (P<0.05) 3H-thymidine incorporation 4.33 ± 1.10 fold compared to control cultures. Progesterone concentrations in PDGFBB treated granulosa cells decreased (P<0.01) to 0.33 ± 0.03 of those observed in control cultures with no effect on DNA content (1.06 ± 0.09 fold relative to control cultures). Given that PDGFBB regulated ovine granulosa cell function even though mRNA encoding PDGF receptors could not be localized to granulosa cells by in situ hybridization, RT-PCR was used to determine if culture of the granulosa cells might induce expression of PDGF receptors. Total cellular mRNA was extracted from freshly isolated granulosa cells or those cultured for 2 days (2 pools each). One pool of freshly isolated granulosa cells and both pools of cultured granulosa cells contained both PDGFRA and PDGFRB mRNA. Therefore, PDGFs are likely regulators of granulosa cell function in sheep although differences in receptor expression requires further exploration.