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The follicular hierarchy in broiler breeder chicken ovary is often deranged due to excessive ovarian follicular recruitment, resulting in a condition that resembles polycystic ovary syndrome (PCOS) in women. Metformin is widely prescribed to correct PCOS and has been shown to affect granulosa cell functions in humans and rodent models. The objectives of this study are to determine the effects of metformin on signal transduction pathways, gene expression related to steroidogenesis, and progesterone secretion from granulosa cells isolated from the most recently recruited preovulatory and prehierarchical follicles of broiler breeder chickens. Granulosa cells were treated with 0, 1, 10, or 20 mM of metformin in the presence of FSH. The abundance of pAMPK, pACC, pERK, and pAkt was determined by Western blotting. The expression of genes related to progesterone biosynthesis was quantified by qPCR. Progesterone concentrations in culture media were quantified by ELISA. Metformin treatment did not have an effect on the abundance of pAMPK and pACC in prehierarchical follicles but significantly decreased the abundance of pERK and pAkt in a dose-dependent manner in preovulatory and prehierarchical follicles. The expression of genes related to steroidogenesis such as FSHR, STAR, CYP11A1, HSD3B, and progesterone secretion was significantly decreased in response to metformin treatment in a dose-dependent manner. Our data suggest that metformin treatment attenuates progesterone secretion via AMPK-independent pathways in granulosa cells of prehierarchical and preovulatory follicles of broiler breeder hens. Further studies are required to determine if metformin administration could ameliorate ovarian dysfunction in obese broiler breeder hens.

Adiponectin, an adipokine hormone, influences glucose utilization, insulin sensitivity and energy homeostasis by signaling through two distinct receptors, ADIPOR1 and ADIPOR2. We previously reported that adiponectin and its receptors are expressed in several organs, including testes in chicken. We report herein that adiponectin gene is expressed exclusively in theca layer while ADIPOR1 and ADIPOR2 genes are expressed in granulosa and theca layers of all preovulatory and prehierarchical follicles of the chicken ovary. Estradiol and/or progesterone treatment of sexually immature chickens significantly altered expression of adiponectin and ADIPOR1 in the ovary. Using anti-chicken adiponectin-, ADIPOR1-, or ADIPOR2- antibodies, adiponectin-immunoreactive (ir) cells were found exclusively in the theca layer, and ADIPOR1-ir and ADIPOR2-ir cells were found both in theca and granulosa layers. Theca layer cells dispersed from preovulatory and prehierarchical follicles were found to synthesize and secrete a 720 kDa heavy molecular weight (HMW) isoform of adiponectin in vitro. Recombinant chicken adiponectin (rcADN) expressed in eukaryotic cells under serum-free conditions comprised primarily of the HMW isoform. Treatment of granulosa cells dispersed from 9 to 12 mm preovulatory follicle and 6 to 8 mm prehierarchical follicle with rcADN or an adiponectin receptor agonist, adipoRon, increased pERK and pACC abundance. In addition, both rcADN and adipoRon were found to significantly decrease the expression of steroidogenic acute regulatory protein gene expression in granulosa cells of preovulatory and prehierarchical follicles. In conclusion, adiponectin secreted by theca cell layer is identical in mass to circulating adiponectin. Systemic and/or theca-derived adiponectin is likely to affect proliferation, metabolism, and steroidogenesis of ovarian follicular cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is a cytokine hormone and rate-limiting enzyme involved in production of NAD and therefore affects a variety of cellular functions requiring NAD. Spermatogenesis and testicular steroidogenesis are likely to depend on NAD-dependent reactions and may therefore be affected by changes in testicular NAMPT expression. The objectives of the present study are to investigate testicular NAMPT expression as well as plasma NAMPT levels in prepubertal and adult chickens. By RT-PCR, NAMPT cDNA expression was detected in prepubertal and adult chicken testes. Using immunohistochemistry, NAMPT was predominantly localized in the nucleus of myoid cells, Sertoli cells, and Leydig cells in the prepubertal chicken testis. In adult chickens, however, NAMPT-immunostaining was observed in the cytoplasm of Leydig cells, Sertoli cells, primary spermatocytes, secondary spermatocytes, round spermatids, and elongated spermatids, but not in the spermatogonial cells. Using real-time quantitative PCR, adult chicken testis was found to contain fourfold greater NAMPT mRNA quantity compared with prepubertal chickens. Testicular NAMPT protein quantities determined by western blotting were not significantly different between adult and prepubertal chicken testes. Using immunoblotting, NAMPT was detected in the seminal plasma and sperm protein extracts obtained from chicken semen. Plasma NAMPT levels, determined by enzyme immunoassay, were at least 28-fold higher in the adult chickens compared with prepubertal male chickens. Taken together, sexual maturation is associated with several changes in testicular NAMPT expression indicating that NAMPT is likely to play a significant role in testicular functions such as spermatogenesis and steroidogenesis.

Adiponectin is an adipokine hormone that influences glucose utilization, insulin sensitivity, and energy homeostasis by signaling through two distinct receptors, ADIPOR1 and ADIPOR2. While adipose tissue is the primary site of adiponectin expression in the chicken, we previously reported that adiponectin and its receptors are expressed in several other tissues. The objectives of the present study are to characterize adiponectin, ADIPOR1, and ADIPOR2 expressions in the chicken testis and to determine whether sexual maturation affects the abundance of testicular adiponectin, ADIPOR1, and ADIPOR2 mRNAs. By RT-PCR and nucleotide sequencing, testicular adiponectin, ADIPOR1, and ADIPOR2 mRNAs were found to be identical to that expressed in the abdominal fat pad. Using anti-chicken adiponectin, ADIPOR1, or ADIPOR2 antibodies and immunohistochemistry, adiponectin-immunoreactive (ir) and ADIPOR1-ir cells were found exclusively in the peritubular cells as well as in Leydig cells. However, ADIPOR2-ir cells were found in the adluminal and luminal compartments of the seminiferous tubules as well as in interstitial cells. In particular, Sertoli cell syncytia, round spermatids, elongating spermatids, spermatozoa, and Leydig cells showed strong ADIPOR2 immunoreactivity. Using quantitative real-time PCR analyses, testicular ADIPOR1 and ADIPOR2 mRNA abundance were found to be 8.3- and 9-fold higher (P<0.01) in adult chickens compared with prepubertal chickens respectively, suggesting that sexual maturation is likely to be associated with an up-regulation of testicular ADIPOR1 and ADIPOR2 gene expressions. Collectively, our results indicate that adiponectin and its receptors are expressed in the chicken testis, where they are likely to influence steroidogenesis, spermatogenesis, Sertoli cell function as well as spermatozoa motility.

Gonadotropin-inhibitory hormone (GnIH), an RFamide peptide, has been found to inhibit pituitary LH secretion in avian and mammalian species. The gene encoding a putative receptor for GnIH (GnIHR) was recently identified in the chicken and Japanese quail brain and pituitary gland. GnIHR appears to be a seven-transmembrane protein belonging to a family of G-protein-coupled receptors. In the present study, we have characterized the expression of GnIHR mRNA in the chicken ovary and demonstrate that GnIHR may exert an inhibitory effect on ovarian follicular development. By RT-PCR, we detected GnIHR mRNA in the chicken testis and in the ovary, specifically both thecal and granulosa cell layers. Real-time quantitative PCR analysis revealed greater GnIHR mRNA quantity in theca cells of prehierarchial follicles compared with that of preovulatory follicles. GnIHR mRNA quantity was significantly decreased in sexually mature chicken ovaries versus ovaries of sexually immature chickens. Estradiol (E₂) and/or progesterone (P₄) treatment of sexually immature chickens significantly decreased ovarian GnIHR mRNA abundance. Treatment of prehierarchial follicular granulosa cells in vitro with chicken GnIH peptide significantly decreased basal but not FSH-stimulated cellular viability. Collectively, our results indicate that the ovarian GnIHR is likely to be involved in ovarian follicular development. A decrease in ovarian GnIHR mRNA abundance due to sexual maturation or by E₂ and/or P₄ treatment would implicate an inhibitory role for GnIHR in ovarian follicular development. Furthermore, GnIH may affect follicular maturation by decreasing the viability of prehierarchial follicular granulosa cells through binding to GnIHR.