Regulation of gonadotropin-releasing hormone (GnRH)-receptor gene expression in tilapia: effect of GnRH and dopamine.

The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5-50 microg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 microg/kg and at 36 h. At the higher dose of sGnRHa (50 microg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1-100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Pituitary adenylate cyclase activating polypeptide and neuropeptide Y regulation of gonadotropin subunit gene expression in tilapia: role of PKC, PKA and ERK.

There is ample information on the hypophysiotropic function of pituitary adenylate cyclase-activating polypeptide (PACAP) and neuropeptide Y (NPY) in fish as in mammals, although evidence as to their direct effects on gonadotropic cells is scarce. We have previously reported that NPY and PACAP38 augment gonadotropin-releasing hormone (GnRH)-induced expression of glycoprotein alpha (alpha) subunit gene in the teleost fish, tilapia. The aim of the present study was to elucidate possible direct effects of these peptides on gonadotropin subunit gene expression in culture of tilapia pituitary cells, as well as the transduction pathways involved. Both NPY and PACAP38 (0.001-10 nM) increased the level of phosphorylated extracellular signal-regulated kinase (pERK) dose-dependently, reaching a peak at 0.1 and 0.01 nM, respectively. Inhibition of protein kinase C (PKC) by GF109203X (GF; 0.01-10 nM) suppressed NPY-stimulated pERK levels and its effect on alpha and luteinizing hormone (LH) beta subunit mRNA levels. However, NPY had no effect on follicle stimulating hormone (FSH) beta mRNA levels. NPY-elevated alpha, LHbeta mRNA and pERK levels were also attenuated by inhibition of protein kinase A (PKA) with H89 (0.01-10 nM). Exposure of the cells to the MAPK kinase (MEK) inhibitor (PD98059; PD 10, 25 and 50 microM) completely blocked NPY-induced ERK activity. In addition, this inhibitor abated the alpha and LHbeta mRNA responses to NPY. Similar experiments conducted to elucidate PACAP38 signaling revealed that PACAP38 (0.01 nM) elevated all three-gonadotropin subunit gene expression via both PKC-ERK and PKA-ERK cascades. It is suggested that both NPY and PACAP38 act directly on gonadotropes to elevate gonadotropin subunit gene expression. Whereas the expression of alpha and LHbeta subunit genes is regulated by both NPY and PACAP, the effect on the FSHbeta transcript is elicited only by PACAP38. NPY and PACAP38 stimulatory actions are mediated via protein kinase C (PKC) and protein kinase A (PKA), converging at the MEK-ERK cascade. These findings represent one of the fine tuning levels that differentially regulates gonadotropin subunit gene expression.