Characterization Of Two Factors With Opposing Regulatory Actions On Follicle Development In The Hen Ovary: Kit Ligand And Anti-MÜLlerian Hormone

Abstract

In two separate studies, the present research investigated two factors noted to have opposing regulatory actions on follicle activation: KL, which is thought to promote follicle activation, and AMH, known to inhibit follicle activation. With respect to regulation and function, there has been little study of either factor in the hen ovary. The domestic hen ovary, with its highly regulated follicle hierarchy, is a useful animal model for study of folliculogenesis. Our goal was to characterize the mRNA expression pattern of kit ligand (KL) throughout follicle development, as well as examine regulation and function of the Kit system in the hen ovary. Additionally, we examined the effects of bioactive chicken AMH on granulosa cell mRNA expression of FSHR and steroid production. In the first study, the highest levels of steady-state KL mRNA expression were seen in the very small ([LESS THAN] 1 mm) follicles and 3-5 mm follicle granulosa cells, with lower levels seen in the larger, selected follicles (p [LESS THAN] 0.001). There was no difference seen in KL mRNA expression between granulosa cells from the germinal disc region versus the non-germinal disc region. With regard to regulation, KL mRNA expression was examined following various treatments of granulosa cell culture. KL expression in granulosa cells from 6-8 mm follicles was shown to be increased by treatment with oocyte-conditioned medium (OCM), vitamin D3, and FSH. KL mRNA expression was unaffected by treatment with testosterone, progesterone and estradiol. With respect to function, the Kit system appears to regulate granulosa cell progesterone production from the large follicles, but has no influence on theca estradiol production or on granulosa cell proliferation. Expression of cKit protein was observed in [LESS THAN] 1 mm follicles, theca tissue, and ovarian body, but was not seen in granulosa cells. In the second study, we examined effects of bioactive chicken AMH (in the form of TCM, testis conditioned media) on granulosa cell mRNA expression of FSHR. TCM consistently decreased granulosa cell FSHR mRNA expression in 3-5 mm and 9-16 mm follicles, but we were unable to determine the specificity of this effect using an AMH antibody. TCM increased granulosa cell proliferation, indicating downregulation of FSHR mRNA was not due to adverse effects on the granulosa cells. TCM treatment significantly decreased granulosa cell progesterone production, but this was not blocked by AMH antibody. Our results show that TCM contains a bioactive substance which regulates granulosa cell function, and suggest that this substance is not AMH. However, it is possible that the antibody against AMH did not completely immunoneutralize the AMH present in TCM, so we are hesitant to rule out the possibility that our results are due to bioactive AMH.