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Shanequa I. Smith

Bone Morphogenetic Protein 6 (BMP6) Expression and function in ovarian granulosa cells from the laying hen

Department of Poultry Science, The Pennsylvania State University
North Carolina Agricultural and Technical State University

Currently: Second year graduate student at University of North Carolina working on masters in Biomedical Science and Pharmaceutical Science, Graduating December 2013

Purpose or objective:

  • To determine the function and expression of BMP6 in granulosa cells from the laying hen
  • Establish BMP6 is expression within the prehierarchal follicles
  • Determine the cell signaling pathway of BMP6
  • Determine if BMP6 from ovarian follicles blocks TGF and FSH-induced FSHR mRNA expression


Significance:
The domestic hen represents a unique and important animal model system for such studies due to the ability to reliably identify a small cohort of prehierarchal follicles from which a single follicle per day is recruited into the preovulatory hierarchy. Understanding the specific cell signaling will allow chickens to be used as a universal model for reproduction and possibly eliminate infertility across species as well as find cures and preventions against various reproductive conditions (i.e. Menopause).


Prior research in mammals has shown that BMP 6 (Bone Morphogenetic Protein 6) is expressed in oocytes and is thought to regulate ovarian folliculogenesis. In mammals, BMP6 has been shown to inhibit FSHr mRNA expression levels, adenylyl cyclase coupling, and increase progesterone production. An increase in progesterone suggests that the cells are undifferentiated. 


To date, the role for Bone Morphogenetic Protein 6 (BMP6) in regulating the differentiation of granulosa cells has not been evaluated in any Avian species. In this study, prehierarchal follicles were collected from laying hens and granulosa cells were cultured. Cells were treated with FSH, TGF beta (transforming growth factor b) and/or BMP6 then cultured for an additional 3 to 24 h. Cellular ribonucleic acid (RNA) was analyzed by quantitative polymerase chain reaction (qPCR) for FSHr, and an Enzyme-linked immunosorbent assay (ELISA) was used to measure P4 production.

Methodology:
Mature female Leghorn laying hens were sacrificed using cervical dislocation. Granulosa cells were cultured from follicles. For these studies, cells were treated for 3 hours with different concentrates of BMP6 (excluding the control). RNA was extracted from the cells and was used in reverse transcription to make mRNA. Real-time quantitative PCR and PCR were executed. Protein was also extracted and a western blot was conducted and treated with two different antibodies to establish total and phosphorylated SMAD.

Status of Project:
BMP6 is expressed in the prehierarchal granulosa cells and activates pSMAD 1/5/8 to mediate activities of BMP ligands. The inhibitory effect(s) of BMP6 on the FSHr is under investigation.     

Conclusion:

  • BMP6 is expressed in prehierarchal 6-8 mm and F1 granulosa and theca cells
  • BMP6 activates (phosphorylates) SMAD 1/5/8 to mediate activities of BMP ligands.
  • BMP6 effects on FSHr receptor expression are currently under investigation.


Future Studies:

  • Determine BMP6’s effects on:Female fertility and FSH signaling in the ovary
  • FSHr mRNA expression in undifferentiated granulosa cells from hen pre-hierarchal follicles;
  • Progesterone production (a marker of differentiation in avian granulosa);
  • Noggin mRNA expression and activity in granulosa and theca tissues;
  • ID1, ID2, ID3 and ID4 mRNA expression.
 

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