Christian Klausen

Signal transduction in multifactorial neuroendocrine control of gonadotropin secretion and synthesis in teleosts-studies on the goldfish model.

In teleosts, gonadotropin (GTH) secretion and synthesis is controlled by multiple neuroendocrine factors from the hypothalamus, pituitary and peripheral sources. Pituitary gonadotropes must be able to differentiate and integrate information from these regulators at the cellular and intracellular level. In this article, the intracellular signal transduction mechanisms mediating the actions of some of these regulators, including GTH-releasing hormones, pituitary adenylate cyclase-activating polypeptide, dopamine, ghrelin, sex steroids, activin, and follistatin from experiments with goldfish are reviewed and discussed in relation with recent findings. Information from other teleost models is briefly compared. Goldfish gonadotropes possess multiple pharmacologically distinct intracellular Ca2+ stores that together with voltage-sensitive Ca2+ channels, Na+/H+ exchangers, protein kinase C, arachidonic acid, NO, protein kinase A, ERK/MAPK, and Smads allows for integrated control by different neuroendocrine factors.

The effect of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the pituitary of goldfish, Carassius auratus.

The goldfish brain contains at least two forms of gonadotropin-releasing hormone (GnRH): sGnRH and cGnRH-II. In goldfish sGnRH and cGnRH-II are present both in the brain and pituitary, and exert direct effects via specific GnRH receptors stimulating growth hormone (GH) and gonadotropin hormone (GtH) synthesis and secretion. In this study, we investigated the effects of sGnRH and cGnRH-II on GtH subunit (alpha, FSH-beta and LH-beta) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 microg/fish) stimulated GtH-alpha, FSH-beta and LH-beta mRNA levels after 24 h. For in vitro studies, goldfish pituitary fragments were treated continuously for 12 h with 10(-7) M sGnRH or cGnRH-II. Both sGnRH and cGnRH-II stimulated GtH-alpha, FSH-beta, LH-beta and GH mRNA levels, however, cGnRH-II appeared to have a more pronounced effect. Similar experiments were carried out using cultured dispersed goldfish pituitary cells. In this study, treatments for 12 h with 10(-7) M sGnRH or cGnRH-II also stimulated GtH and GH gene expression. The present results provide a basis for the investigation of the signal transduction pathways that mediate GnRH-induced changes in GtH subunit and GH mRNA levels in the goldfish pituitary.

A gonadotropin-releasing hormone insensitive, thapsigargin-sensitive Ca2+ store reduces basal gonadotropin exocytosis and gene expression: comparison with agonist-sensitive Ca2+ stores.

We examined whether distinct Ca2+ stores differentially control basal and gonadotropin (GTH‐II)‐releasing hormone (GnRH)‐evoked GTH‐II release, long‐term GTH‐II secretion and contents, and GTH‐II‐β mRNA expression in goldfish. Thapsigargin (Tg)‐sensitive Ca2+ stores mediated neither caffeine‐evoked GTH‐II release, nor salmon (s)GnRH‐ and chicken (c)GnRH‐II‐stimulated secretion; the latter responses were previously shown to involve ryanodine (Ry)‐sensitive Ca2+ stores. Surprisingly, Tg decreased basal GTH‐II release. This response was attenuated by prior exposure to sGnRH and caffeine, but was insensitive to the phosphatase inhibitor okadaic acid, the inhibitor of constitutive release brefeldin A and cGnRH‐II. GTH‐II‐β mRNA expression was decreased at 24 h by 2 µm Tg, and by inhibiting (10 µm Ry) and stimulating (1 nm Ry) Ry receptors. Transient increases in GTH‐II‐β mRNA were observed at 2 h and 12 h following 10 µm and 1 nm Ry treatment, respectively. Effects of Tg, Ry and GnRH on long‐term GTH‐II secretion, contents and apparent production differed from one another, and these changes were not well correlated with changes in GTH‐II‐β mRNA expression. Our data show that GTH‐II secretion, storage and transcription can be independently controlled by distinct Ca2+ stores.

Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish

The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4beta-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4alpha-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish.