Autocrine/paracrine proliferative effect of ovarian GH and IGF-I in chicken granulosa cell cultures.

It is known that growth hormone (GH) and its receptor (GHR) are expressed in granulosa cells (GC) and thecal cells during the follicular development in the hen ovary, which suggests GH is involved in autocrine/paracrine actions in the female reproductive system. In this work, we show that the knockdown of local ovarian GH with a specific cGH siRNA in GC cultures significantly decreased both cGH mRNA expression and GH secretion to the media, and also reduced their proliferative rate. Thus, we analyzed the effect of ovarian GH and recombinant chicken GH (rcGH) on the proliferation of pre-hierarchical GCs in primary cultures. Incubation of GCs with either rcGH or conditioned media, containing predominantly a 15-kDa GH isoform, showed that both significantly increased proliferation as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, proliferating cell nuclear antigen (PCNA) quantification and ((3)H)-thymidine incorporation ((3)H-T) assays in a dose response fashion. Both, locally produced GH and rcGH also induced the phosphorylation of Erk1/2 in GC cultures. Furthermore, GH increased IGF-I synthesis and its release into the GC culture incubation media. These results suggest that GH may act through local IGF-I to induce GC proliferation, since IGF-I immunoneutralization completely abolished the GH-induced proliferative effect. These data suggest that GH and IGF-I may play a role as autocrine/paracrine regulators during the follicular development in the hen ovary at the pre-hierarchical stage.

Development of a microplate duplex immunoassay to simplify detection of growth hormone doping: Proof of concept.

The World Anti-Doping Agency (WADA) prohibits athletes from using recombinant growth hormone (GH). The validated method used in antidoping laboratories for the direct detection of exogenous GH in serum requires two immunoluminometric assays (ILMAs): The first mainly measures the concentration of the full-length (22 kDa) form of GH (recGH), and the second measures concentrations of multiple GH fragments produced by the pituitary gland (22 kDa, 20 kDa and other forms) (pitGH). The tube-by-tube analysis is laborious. A recent development opened new possibilities to simplify the detection of recGH in serum: multiplexed immunoassays that detect multiple targets in a single well of a 96-well plate using an ELISA-like procedure with high sensitivity. Our aim was to evaluate this technology by developing a customized assay for GH detection. One pair of antibodies with specificities similar to those of the recGH assay and one pair of antibodies compatible with pitGH detection were selected for a single duplex assay. Forty-eight serum samples (negative athlete samples and positive samples following GH administration) were analyzed using the two methods. The microplate duplex assay discriminated between the negative athlete samples and the positive controls, although the rec/pit ratios from the duplex assay were lower than those obtained with the ILMAs. This new assay would offer a modern alternative to ILMAs, with fewer analytical steps and a smaller sample volume. However, an adaptation of the decision limits seems mandatory.

Constitutively Active STAT5b Feminizes Mouse Liver Gene Expression.

STAT5 is an essential transcriptional regulator of the sex-biased actions of GH in the liver. Delivery of constitutively active STAT5 (STAT5CA) to male mouse liver using an engineered adeno-associated virus with high tropism for the liver is shown to induce widespread feminization of the liver, with extensive induction of female-biased genes and repression of male-biased genes, largely mimicking results obtained when male mice are given GH as a continuous infusion. Many of the STAT5CA-responding genes were associated with nearby (< 50 kb) sites of STAT5 binding to liver chromatin, supporting the proposed direct role of persistently active STAT5 in continuous GH-induced liver feminization. The feminizing effects of STAT5CA were dose-dependent; moreover, at higher levels, STAT5CA overexpression resulted in some histopathology, including hepatocyte hyperplasia, and increased karyomegaly and multinuclear hepatocytes. These findings establish that the persistent activation of STAT5 by GH that characterizes female liver is by itself sufficient to account for the sex-dependent expression of a majority of hepatic sex-biased genes. Moreover, histological changes seen when STAT5CA is overexpressed highlight the importance of carefully evaluating such effects before considering STAT5 derivatives for therapeutic use in treating liver disease.

Ghrelin: ghrelin as a regulatory Peptide in growth hormone secretion.

BACKGROUND: Ghrelin is a type of growth hormone (GH) secretagogue that stimulates the release of GH. It is a first hormone linking gastrointestinal-pituitary axis. OBJECTIVE: This review highlights the interaction of ghrelin with GHRH and somatostatin to regulate the secretion of GH and intends to explore the possible physiological role of the ghrelin-pituitary-GH axis linkage system. OBSERVATION: Ghrelin is highly conserved among species and is classified into octanoylated (C8:0), decanoylated (C10:0), decenoylated (C10:1) and nonacylated,ghrelin. Acylated ghrelin is the major active form of human ghrelin. The primary production site of ghrelin is the stomach, and it interacts with stomach ghrelin as well as hypothalamic GHRH and somatostatin in the regulation of pituitary GH secretion. Ghrelin stimulate GH release through the GHS receptor to increase intracellular Ca2+ ([Ca2+] levels via IP3 signal transduction pathway. Ghrelin is a specific endogenous ligand for the GHS receptor and provides a definitive proof of the occurance of a GHS-GHS receptor signalling system in the regulation of GH secretion. CONCLUSION: Studies suggests that ghrelin is a powerful pharmacological agent that exerts a potent, time-dependent stimulation of pulsatile secretion of GH.