Investigations of the function of AMH in granulosa cells in hens.

Anti-mullerian hormone (AMH) plays a crucial role in follicle regulation in mammals by preventing premature primordial follicle activation and restricting follicle development through reduction of FSH sensitivity and inhibition of FSH-induced increase of steroidogenic enzymes. AMH is produced by granulosa cells from growing follicles and expression declines at the time of selection in both mammalian and avian species. The role of AMH in chicken granulosa cells remains unclear, as research is complicated because mammalian AMH is not bioactive in chickens and there is a lack of commercially available chicken AMH. In the current experiments, we used RNA interference to study the role of AMH on markers of follicle development in the presence and absence of FSH. Cultured chicken granulosa cells from 3-5 mm follicles and 6-8 mm follicles, the growing pool from which follicle selection is thought to occur, were used. Transfection with an AMH-specific siRNA significantly reduced AMH mRNA expression in granulosa cells from 3-5 mm and 6-8 mm follicles. Genes of interest were only measured in granulosa cells of 3-5 mm follicles due to low expression of AMH mRNA at the 6-8 mm follicle stage. Knockdown of AMH mRNA did not affect markers of follicle development (follicle stimulating hormone receptor, FSHR; steroidogenic acute regulatory protein, STAR; cytochrome P450 family 11 subfamily A member 1, CYP11A1; bone morphogenetic protein receptor type 2, BMPR2) or FSH responsiveness in granulosa cells from 3-5 mm follicles, indicating that AMH does not regulate follicle development directly by affecting markers of steroidogenesis, FSHR or BMPR2 at this follicle stage in chickens.

Effect of IGF1 and FSH on the function of granulosa cells from prehierarchal follicles in chickens†.

Insulin-like growth factor 1 (IGF1) is an essential regulator of mammalian follicle development and synergizes with follicle-stimulating hormone (FSH) to amplify its effects. In avian preovulatory follicles, IGF1 increases the expression of genes involved in steroidogenesis and progesterone and inhibin A production. The role of IGF1 in prehierarchal follicles has not been well studied in chickens. The aim of this study was to investigate the role of IGF1 in granulosa cells from prehierarchal follicles and to determine whether IGF1 and FSH synergize to promote follicle development. Granulosa cells of 3-5 and 6-8 mm prehierarchal follicles were cultured with IGF1 (0, 10, 100 ng/mL) in the presence or absence of FSH (0, 10 ng/mL). Cell proliferation, expression of genes important in follicle development (FSHR, IGF1R, AMH, STAR, CYP11A1, INHA, and INHBA), and progesterone production were evaluated following treatment. IGF1 treatment alone significantly increased STAR, CYP11A1, and INHBA mRNA expression and cell proliferation in granulosa cells of 6-8 mm follicles. IGF1 and FSH synergized to increase STAR mRNA expression in 6-8 mm follicles. IGF1 and FSH co-treatment were necessary to increase INHA mRNA expression in 6-8 mm follicles. Although IGF1 significantly increased the expression of genes involved in steroidogenesis, progesterone production in granulosa cells of 6-8 mm follicles was not affected. IGF1 did not affect AMH mRNA expression, although FSH significantly decreased AMH expression in granulosa cells of 3-5 mm follicles. These results suggest that IGF1 may act with FSH to promote follicle selection at the prehierarchal follicle stage.

Feeding level is associated with altered liver transcriptome and follicle selection in hen†.

Genetic selection for particular traits in domestic animals may have altered the optimal feedback regulation among systems regulating appetite, growth, and reproduction. Broiler breeder chickens have been selected for fast and efficient growth and, unless feed restricted, consume excessively resulting in poor reproductive efficiency. We examined the effect of dietary treatment in full-fed and restricted-fed broiler breeder hens on ovarian responses, liver morphology, and transcriptome associated with reproductive function. Although full-fed broiler breeder hens had lower egg production (P < 0.01), the total number of ovarian follicles >8 mm (P < 0.01), 6-8 mm (P < 0.03), and 3-5 mm (P < 0.04) were greater in full-fed hens compared to restricted-fed hens. There was a large amount of lipid accumulation in the liver of full-fed hens and differential gene analysis yielded 120 genes that were differentially expressed >2-fold in response to feeding level (P < 0.01; false discovery rate < 0.05). Elevated T3 may indicate that general metabolism was affected by diet and GHR (P < 0.01) and insulin like growth factor 1 (IGF1) (P < 0.04) mRNA expression were both greater in the liver of full-fed hens as compared to restricted-fed hens. It is likely that selection for increased growth, associated with enhanced activity of the IGF1 system, has altered nutritional coupling of feed intake to follicle development.

Ad Libitum Feeding in Broiler Breeder Hens Alters the Transcriptome of Granulosa Cells of Pre-Hierarchal Follicles.

Intense selective breeding of chickens has resulted in suboptimal egg production in broiler breeder hens. This reproductive phenotype is exacerbated by ad libitum feeding, which leads to excessive and disorganized follicular growth. One strategy used to improve broiler breeder hens' reproductive efficiency is restricted feeding. In this study, we sought to identify transcriptional changes, which translate the level of dietary intake into increased follicle selection. Broiler breeder hens (n = 16 per group) were raised according to commercial guidelines until 28 weeks of age and then randomly assigned to an ad libitum diet (FF) or continued on a restricted diet (RF) for 6 weeks. Following dietary treatment, FF hens (n = 2) with excessive follicle selection and RF hens (n = 3) with normal follicle selection were selected for RNA-sequencing. Transcriptomes of granulosa cells from 6-8-mm follicles were sequenced to identify transcriptional differences in the follicle population from which selection was made for the preovulatory stage. Differential expression analysis identified several genes known to play a role in follicle development (CYP11A1, STAR, INHA, and INHBB) that are upregulated in FF hens. These changes in gene expression suggest earlier granulosa cell differentiation and steroidogenic competency in the granulosa layer from FF hens.