Gilbert L. Hendricks

Gonadotropin-inhibitory hormone (GnIH) receptor gene is expressed in the chicken ovary: potential role of GnIH in follicular maturation

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 (E(2)) and/or progesterone (P(4)) 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 E(2) and/or P(4) 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.

Gonadotrophin‐Inhibitory Hormone Receptor Expression in the Chicken Pituitary Gland: Potential Influence of Sexual Maturation and Ovarian Steroids

Gonadotrophin‐inhibitory hormone (GnIH), a hypothalamic RFamide, has been found to inhibit gonadotrophin secretion from the anterior pituitary gland originally in birds and, subsequently, in mammalian species. The gene encoding a transmembrane receptor for GnIH (GnIHR) was recently identified in the brain, pituitary gland and gonads of song bird, chicken and Japanese quail. The objectives of the present study are to characterise the expression of GnIHR mRNA and protein in the chicken pituitary gland, and to determine whether sexual maturation and gonadal steroids influence pituitary GnIHR mRNA abundance. GnIHR mRNA quantity was found to be significantly higher in diencephalon compared to either anterior pituitary gland or ovaries. GnIHR mRNA quantity was significantly higher in the pituitaries of sexually immature chickens relative to sexually mature chickens. Oestradiol or a combination of oestradiol and progesterone treatment caused a significant decrease in pituitary GnIHR mRNA quantity relative to vehicle controls. GnIHR‐immunoreactive (ir) cells were identified in the chicken pituitary gland cephalic and caudal lobes. Furthermore, GnIHR‐ir cells were found to be colocalised with luteinising hormone (LH)β mRNA‐, or follicle‐stimulating hormone (FSH)β mRNA‐containing cells. GnIH treatment significantly decreased LH release from anterior pituitary gland slices collected from sexually immature, but not from sexually mature chickens. Taken together, GnIHR gene expression is possibly down regulated in response to a surge in circulating oestradiol and progesterone levels as the chicken undergoes sexual maturation to allow gonadotrophin secretion. Furthermore, GnIHR protein expressed in FSHβ or LHβ mRNA‐containing cells is likely to mediate the inhibitory effect of GnIH on LH and FSH secretion.

Adiponectin and its receptors are expressed in the chicken testis: influence of sexual maturation on testicular ADIPOR1 and ADIPOR2 mRNA abundance.

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.

Unique Profile of Chicken Adiponectin, a Predominantly Heavy Molecular Weight Multimer, and Relationship to Visceral Adiposity

Adiponectin, a 30-kDa adipokine hormone, circulates as heavy, medium, and light molecular weight isoforms in mammals. Plasma heavy molecular weight (HMW) adiponectin isoform levels are inversely correlated with the incidence of type 2 diabetes in humans. The objectives of the present study were to characterize adiponectin protein and quantify plasma adiponectin levels in chickens, which are naturally hyperglycemic relative to mammals. Using gel filtration column chromatography and Western blot analysis under nonreducing and non-heat-denaturing native conditions, adiponectin in chicken plasma, and adipose tissue is predominantly a multimeric HMW isoform that is larger than 669 kDa mass. Under reducing conditions and heating to 70-100 C, however, a majority of the multimeric adiponectin in chicken plasma and adipose tissue was reduced to oligomeric and/or monomeric forms. Immunoprecipitation and elution under neutral pH preserved the HMW adiponectin multimer, whereas brief exposure to acidic pH led to dissociation of HMW multimer into multiple oligomers. Mass spectrometric analysis of chicken adiponectin revealed the presence of hydroxyproline and differential glycosylation of hydroxylysine residues in the collagenous domain. An enzyme immunoassay was developed and validated for quantifying plasma adiponectin in chickens. Plasma adiponectin levels were found to be significantly lower in 8- compared with 4-wk-old male chickens and inversely related to abdominal fat pad mass. Collectively, our results provide novel evidence that adiponectin in chicken plasma and tissues is predominantly a HMW multimer, suggesting the presence of unique multimerization and stabilization mechanisms in the chicken that favors preponderance of HMW adiponectin over other oligomers.

The role of neuroendocrine immune interactions in the initiation of humoral immunity in chickens

The presence of neuroendocrine immune interaction in mammalian species has been studied extensively and has been established. However, such an interaction is not as well established in avian species. Furthermore, the role of such an interaction in the initiation of humoral immunity is not well understood. Therefore, the present studies were conducted to determine mechanisms involved in the initiation of humoral immunity in chickens. Cornell K-strain White Leghorn immature male chickens were used for all the experiments. Changes in hormonal and leukocyte profiles after antigen stimulation were studied. The ability of different leukocytes to produce ACTH was also investigated. It was concluded that the first step in the initiation of humoral immunity after antigen exposure is the release of interleukin-1 by macrophages, which in turn stimulates the production of CRF by hypothalamus and/or leukocytes. It is important to mention that CRF production could also be a direct effect of antigen stimulation. The CRF will then stimulate ACTH production by anterior pituitary and/or leukocytes. In addition, CRF will directly enhance lymphocyte activities in the spleen. Corticosteroid production will be stimulated by ACTH and will cause redistribution of lymphocytes from circulation to secondary lymphoid organs such as the spleen for antigen processing and eventual production of antibodies against the invading antigens. Finally, both ACTH and corticosteroids will later act in a negative feedback manner to regulate and control the process of antibody production by inhibiting lymphocyte activities and/or reducing the responsiveness to different stimuli.

NAMPT (visfatin) in the chicken testis: influence of sexual maturation on cellular localization, plasma levels and gene and protein expression

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.

Initiation of humoral immunity. I. The role of cytokines and hormones in the initiation of humoral immunity using T-independent and T-dependent antigens

The early events during the initiation of immune responses following the injection of T-independent (lipopolysaccharide, LPS) and T-dependent (bovine serum albumin, BSA) antigens were studied in immature male chickens. Specifically, the role of cytokines and hormones in the initiation of humoral immunity against these antigens was investigated. Both interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) increased significantly post-LPS but not post-BSA injection. While interleukin-2 (IL-2) significantly decreased post-LPS injection, IL-2 significantly increased post-BSA injection. Furthermore, corticosterone levels significantly increased and tri-iodothyronine (T(3)) levels significantly decreased post-LPS but not post-BSA injection. In this study, the results indicate that although LPS and BSA can induce a humoral antibody response in chickens, they activate different cytokines and neuroendocrine network systems.

Expression of adiponectin and its receptors in avian species.

Adipose tissue is a dynamic endocrine organ secreting a variety of hormones that affect physiological functions within the central nervous system, cardiovascular system, reproductive, and immune systems. The endocrine role of avian adipose tissue remains enigmatic as many of the classical hormones found in mammalian adipose tissue have not been found in avians. This mini-review summarizes our current knowledge on avian adiponectin, one of the most abundant adipose tissue hormones, and its receptors. We cloned the genes encoding chicken adiponectin and its receptors, AdipoR1 and AdipoR2. Using anti-chicken adiponectin antibody, we found that chicken adipose tissue and plasma predominantly contain a unique polymer of adiponectin with a mass greater than 669kDa, unlike mammalian adiponectin which is found as three distinct oligomers. Mass spectrometric analyses of chicken adiponectin revealed certain post-translational modifications that are likely to favor the unique multimerization of adiponectin in chickens. Unlike adiponectin, the nucleotide sequences of chicken AdipoR1- and AdipoR2 cDNA are highly similar to that of mammalian adiponectin receptors. Both adiponectin and adiponectin receptors are widely expressed in several tissues in the chicken. Herein, we review the unique biochemistry of adiponectin as well as expression of adiponectin and its receptors in the chicken. Future studies should focus on elucidating the role of adiponectin, AdipoR1, and AdipoR2 on metabolism, steroidogenesis, and adipose tissue remodeling during growth and reproduction in birds.

Initiation of humoral immunity. II. The effects of T-independent and T-dependent antigens on the distribution of lymphocyte populations

The effect of injecting T-independent (lipopolysaccharide, LPS) and T-dependent (bovine serum albumin, BSA) antigens on the redistribution of lymphocyte populations in immature male chickens was investigated. In the blood, percentages of total T-cells (CD3+), T-helper cells (CD4+), and T-cytotoxic/suppressor cells (CD8+) significantly decreased post-LPS injection (PLI) but not post-BSA injection (PBI), while percentages of monocytes/thrombocytes (K1+) significantly increased PLI. Interleukin-1 receptor expression on blood lymphocytes increased significantly PLI and PBI. In the spleen, the percentages of total T-cells (CD3+) increased significantly PLI and PBI, macrophage (K1+) percentages increased significantly PLI, while B-cell percentages decreased significantly PLI. These results indicate that following antigen injection, there is a redistribution of peripheral blood lymphocytes (specifically T-lymphocytes) to secondary lymphoid organs and the kinetics and magnitude of the changes can differ according to the type of antigen used.

Effects of corticotropin releasing factor on the production of adrenocorticotropic hormone by leukocyte populations

1. Corticotropin-releasing factor (CRF), a neuropeptide with immunomodulating properties, is known to stimulate avian splenic leukocytes to produce adrenocorticotropic hormone (ACTH). 2. The present study was to determine which avian splenic leukocyte subpopulation(s) produce ACTH in response to CRF stimulation. 3. Splenic leukocytes from 8-week-old male chickens were isolated on Histopaque 1077 and macrophages were separated from lymphocytes by adherence to a polystyrene surface. 4. Different concentrations of CRF (0, 5, 50, 500 or 1000 ng/m) were incubated with the different leukocyte populations, supernatants were collected and ACTH was measured using a radioimmunoassay. 5. Isolated macrophages, stimulated with CRF, produced significantly more ACTH than either unstimulated macrophages or CRF-stimulated lymphocytes, suggesting that ACTH may be produced by a particular subset of leukocytes, the macrophages (and monocytes), in response to CRF stimulation.