Expression and Regulation of Kit Ligand During Folliculogenesis in the Hen.

The Kit system, composed of Kit ligand (KL) and its cognate tyrosine kinase receptor, cKit, has been well studied in mammals, but its functions in reproduction of non-mammalian vertebrates remain largely unknown. Studies have shown that the Kit system is involved in signaling between the oocyte and somatic cells during the process of follicle maturation in mammals. In mammalian ovaries, KL is known to be present solely in the granulosa layer of ovarian follicles; cKit is present in both the theca layer and the oocyte. Although the pattern of KL expression during follicle development has been studied in mammals, this has not been examined in the hen. The unique hierarchical arrangement of follicles in the hen ovary makes it a good model for the study of folliculogenesis. The objective of our study was to characterize the expression pattern of KL during follicle maturation and to examine regulation of KL. The ovarian stroma and the granulosa layer from follicles of various sizes (1, 3, 5, 6-12 mm, F1) were collected from white Leghorn hens (n=4 replicates), and KL mRNA expression was assessed using quantitative PCR. All values were normalized to 18S values. We found that granulosa mRNA expression for KL decreased significantly (P <0.01) with increasing follicle development, with the highest expression found in 1 mm follicles and the lowest amount seen in the granulosa layer from 6-12 mm and F1 follicles. Additionally, western blot analysis confirmed the presence of cKit (at the expected size of 120 kDa) in the theca layer of 3-5 mm follicles and in a lysate of whole <1 mm follicles, suggesting oocyte expression of cKit. We also examined whether the oocyte (or oocyte factors) affected KL mRNA expression. In mammals, the oocyte-derived factors BMP15 and GDF9 have been found to influence KL expression. As a source of oocyte-derived factors, we used oocyte conditioned medium (OCM). OCM was produced by culturing small (<1 mm) follicles in M199 plus 0.1% BSA for three days, then collecting and filter sterilizing the supernatant. For these experiments, granulosa cells were collected from 6-8 mm follicles (n=4 replicates) and the cells were dispersed and plated in M199 plus 5% serum for 24 h. Medium was then replaced with M199 plus 0.1% BSA in the presence and absence of OCM (at 25% and 50% of volume). After another 24 h, the medium was removed, cells collected and RNA extracted. Quantitative real-time PCR was used to evaluate expression of KL mRNA, with all values normalized to 18S expression. OCM caused a dose-related increase (P < 0.05) in expression of KL. Western blot confirmed the presence of BMP15 and GDF9 in OCM, suggesting that one or both of these factors, possibly by signaling through cKit, may play a role in regulating KL expression in the hen. Supported by USDA-NRI-2008-35203-19097. (poster)