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FRIMEDBIO-Fri prosj.st. med.,helse,biol

Maturation of the electrophysiological and intracellular signaling systems in teleost gonadotropes during puberty

Awarded: NOK 6.6 mill.

The main goal for this project is to assess how the regulating mechanism of the hormone producing gonadotrope cells in the pituitary mature during puberty, focusing on components that participate in electrophysiological signaling triggered by the releasin g hormone GnRH. We use Atlantic cod and medaka (Japanese rice fish) as model organisms, because the two relevant pituitary hormones are produced by two separate cell types in fish, opposite to in mammals. Thus, we can study the regulation of these two hor mones (FSH and LH) separately. We have shown that GnRH initiates relatively similar responses in fish as in mammals. But, interestingly, we do see some differences in the electrophysiological response between LH- and FSH-producing cells in cod. Furtherm ore, we have shown that the electrophysiological properties are regulated according to breeding season, and that the seasonal variations are oppositely directed in the two cell types. We have also found some small differences in the GnRH response between cod and medaka. For medaka, we have in addition compared the response to three different forms of GnRH, and we find moderate differences that we would like to study in more detail. These differences may imply that the different GnRH forms use partly dif ferent intracellular signaling pathways in the same cell type. This is not well described in any species. We have also shown that there are several different GnRH receptors expressed in the pituitary, and even specifically in LH-producing cells. This may be part of the explanation for the observed differences in GnRH response. Particularly interesting is the fact that the receptor type we found to be most highly expressed in the pituitary is the form most recently discovered, and so far only describes as a gene sequence. For medaka, we have also mapped the transcriptome of the LH-producing cells. This means that we have measured all genes that are expressed, and how much they are expressed, using RNA sequencing technology. We have then compared the tran scriptome from immature and sexually mature females, to search for gene products that are heavily regulated by puberty. Already now we find that some of the components of the electrophysiological signaling that varies with breeding season in cod also are strongly regulated through puberty in medaka. An exciting side project that has been dominating the last part of the project period is the study of long, neurite-like extensions seen on many LH-producing cells. We are still working on mapping the appeara nce of these extensions in both cell cultures and intact pituitaries, and study whether they are affected by the presence of GnRH. Furthermore we would like to test if the extensions make active contact points between cells, but this must be a topic for a later project. Finally, we also work on an additional side project, where we study how the expression of central genes (for LH, FSH etc) is affected by different steroids as well as certain hormone-mimicking environmental toxins.

Puberty and sexual maturation in vertebrates are regulated through increased activity in the brain-pituitary-gonad axis. Stimulatory and inhibitory inputs merge in the forebrain on neuroendocrine neurons that produce gonadotropin-releasing hormones (GnRHs ). Upon stimulation, FSH and LH are released independently of each other into the circulation, and each stimulates different stages of gonadal development. The key factors in pubertal development probably include increased release of GnRH and maturation o f gonadotropes, e.g. adequate expression of GnRH receptors and components of intracellular signaling pathways. However, how this is regulated and the triggering mechanism(s) for puberty are not known. The proposed experiments may answer these questions in fish, and imply similar answers for vertebrates in general. We believe that teleost fish are useful models, since they have separate cells producing FSH and LH. We have developed relevant methodologies, like primary pituitary cultures suitable for electr ophysiological recordings. This project aims to use transgenic medaka lines that express GFP in gonadotropes as a new model system. The transgenic fish will enable us to identify and isolate FSH- and LH-producing gonadotropes, respectively. In parallel, w e will perform similar studies on Atlantic cod, a species we already have started to characterize. In this species, we will be able to identify FSH- and LH-producing cells through single-cell PCR. The identified cells will be used in electrophysiological analyses as well as Ca2+-imaging studies to investigate properties of the two different gonadotropes at various stages of sexual maturation and upon exposure to various endogenous factors. We will simultaneously monitor changes in both membrane properties and [Ca2+]i during the response to different versions of the GnRH peptide, activators of putative messengers, like PKA, PKC and cAMP, and agonists/antagonists to the GnRH-R(s).

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FRIMEDBIO-Fri prosj.st. med.,helse,biol

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