The Regulation of Anti-Mullerian Hormone Expression in Granulosa Cells of the Hen.

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Anti-Mullerian Hormone (AMH) plays a role in the regression of mullerian ducts in mammalian and avian male embryos and aids in the regression of the right mullerian duct in avian female embryos. AMH has also been shown to be expressed in the granulosa cells of adult mammals and hens although mechanisms regulating AMH are not well understood. We have shown that AMH is expressed in a defined pattern during follicle maturation in White Leghorn hens, suggesting a high level of regulation. It is likely that AMH is regulated by paracrine factors, some of which may originate in the oocyte, as it has been shown that there is communication between the oocyte and surrounding granulosa cells. We previously showed that a heat labile substance (not GDF9) found in oocyte-conditioned medium (OCM) inhibits AMH expression in a dose-related way. OCM was produced by incubation of small oocytes (<1 mm) in M199 + 5% serum for 72 h. Medium was collected, filter-sterilized and used in cell cultures. A protein band from the lysate of small oocytes (at approximately 38kDa) was demonstrated by Western blot and the binding could be competed off by pre-incubation of the antiserum with BMP15 peptide. The objective of this experiment was to evaluate the effects of several factors, including BMP15, on AMH expression in chicken granulosa cells. We hypothesized that the oocyte factor BMP15 may decrease AMH expression. To test our hypothesis, granulosa cells from 6-8 mm follicles (n=4 experiments) were plated in M199 + 5% serum for 24 h. At the end of this time, the medium was removed and OCM + 0.1%BSA (at doses of 25% and 50%) was added in the presence or absence of BMP15 antibody and cultured for an additional 24 h. Cells were harvested and quantitative real-time PCR was used to assess AMH expression using Taqman primers and probes. All values were normalized to 18S reactions. There was a significant (p<0.01) dose-related effect of OCM on AMH expression as we had previously shown, and this inhibition was not blocked by pre-incubation of the OCM with BMP15 antibody. Epidermal Growth Factor (EGF) and Insulin Growth Factor-1 (IGF-1) are regulators of cell growth and differentiation, and have also been found to be produced by the oocyte. Granulosa cells were cultured (as above) with EGF (0-200 ng/ml; n=4 experiments) and IGF-1 (0-200 ng/ml; n=4 experiments). Quantitative PCR was used to determine AMH expression in granulosa cells. No significant effect of EGF or IGF-1 was found on AMH expression. Our data suggest the BMP15, EGF and IGF-1 do not directly regulate AMH expression in granulosa cells from 6-8 mm follicles of the hen. Supported by USDA-NRI-2008-35203-19097. (poster)