Development of real-time PCR assays in the study of gonadotropin subunits, follistatin and prolactin genes expression in the porcine anterior pituitary during the preovulatory period.

OBJECTIVES: Neural control of the anterior pituitary function consists of the interplay of neuropeptides action, gonadal steroid hormones and many other factors. The physiological effect of this regulatory action is the release and synthesis of protein hormones in the precise time and quantity. The main factor responsible for the gonadotropins release and synthesis is the gonadotropin-releasing hormone (GnRH). We must still study the modulation of the synthesis of the gonadotropins subunits - LHbeta, FSHbeta and alpha subunit by different forms of GnRH and by its analogs, in order to better understand the regulation of gonadotropin release and synthesis. THE AIM of this study was to develop real-time PCR assays of five candidate reference genes for normalization purposes in order to quantify target transcripts in anterior pituitary cells during the preovulatory period. Moreover, we focused on the influence of GnRH receptor antagonist (antide) treatment on mRNA expression levels of GPalpha, LHbeta, FSHbeta, FST(follistatin) and PRL(prolactin) genes in these cells. MATERIAL AND METHODS: Anterior pituitary cells were obtained from pituitary glands of four mature pigs at the preovulatory phase. Cells were incubated with or without antide and relative mRNA level of target genes was measured using the Applied Biosystems 7500 Real Time System. For an exact comparison of mRNA quantity, the stability of five reference genes, ACTB, B2M, GAPDH, RPL1, and TOP2B was evaluated to choose the most appropriate reference gene for qRT-PCR normalization in the pituitary cells. Expression stability of reference genes was calculated using the geNorm application. The developed method of PCR assay was applied to study gene expression in pig pituitary cells in short culture. RESULTS: The most stably expressed genes in the pituitary cells were GAPDH and TOP2B. The expression of ACTB, B2M and RPL1 appeared to be highly unstable. After normalization to the GAPDH/TOP2B, results showed that the mRNA expression of the FSHbeta gene was highest in comparison with LHbeta, GPalpha, FST and PRL genes (p<0.005). Pre-treatment of cells by the antide resulted in lower mRNA expression of these genes, while FSHbeta mRNA had a significantly lower expression (p<0.05) in comparison with control. CONCLUSIONS: Real-time PCR analysis of the expression of LHbeta, FSHbeta, alpha subunit, follistatin and prolactin genes in porcine anterior pituitary cells during the preovulatory period is suitable for the study of modulatory action of metal complexes with GnRH on the expression of these genes.

LH release by Cu and Ni salts and metal-GnRH complexes, in vitro.

The present studies were undertaken to examine the effect of copper and nickel salts and their complexes with GnRH on LH release from the pig anterior pituitary cells in vitro. The potency of Cu-GnRH and Ni-GnRH binding to GnRH receptors with iodinated GnRH as a radioactive tracer was also verified. The incubation of pig pituitary cells with Cu and Ni acetate salts showed no effect of the studied ions on LH release at any concentration used. However, nickel salt at a lower dose (10(-10) and 10(-9) M) tended to decrease LH output. By contrast, the native GnRH as well as its metal complexes significantly stimulated LH release after three hours of treatment and Cu-GnRH was found to be the most effective. The results showed that Cu and Ni complexes with GnRH but not their acetate salts are effective in LH release from pig pituitary cells collected from adult female pigs.

Structural analysis and sheep pituitary receptor binding of GnRH and its complexes with metal ions.

Binding of GnRH and its metal complexes to a sheep pituitary receptor have been investigated showing that Cu(II)-GnRH complex is more effectively bound to the receptor than the metal-free ligand, while Ni(II) and Co(II) complexes are less effective than the metal-free GnRH. Earlier studies have explained reasonably well the complex formation with cupric ion, while in this work extensive 1H NMR measurements have been performed for free gonadotropin-releasing hormone (GnRH) and its complexes with Ni(II) in DMSO (dimethyl sulfoxide) solution. This study shows the high order of organization of the metal-free peptide in DMSO solution with two structured 'domains' whose relative orientation is modulated by the mobility of the central glycine. Furthermore, theoretical calculations were performed for the Ni(II)-GnRH complex. The data obtained in this work supports previous studies on the co-ordination of Ni(II) ions with GnRH in aqueous solutions at high pH [J. Inorg. Biochem. 33 (1988) 11] and suggest an experimental procedure to reproduce high pH in DMSO solution. In the Ni(II) complex, the metal ion was found to co-ordinate with four nitrogen atoms inducing a well definite arrangement of aromatic side-chains and a rigid backbone structure.