The role of IGF3 in zebrafish spermatogenesis
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Spermatogenesis is the process in which haploid flagellated spermatozoa arise from undifferentiated spermatogonial stem cells. This process takes place in the testes and is well regulated by the pituitary hormones, luteinizing hormone (LH) and follicle-stimulation hormone (FSH). In mammals, FSH targets the Sertoli cells, which support the germ cells during development, and LH targets the steroid producing Leydig cells. In fish, FSH is able to stimulate the production of steroids by the Leydig cells too. FSH is suspected to be the gonadotropin that regulates the early stages of spermatogenesis. It stimulates the spermatogenesis in several ways. Firstly, FSH stimulates the production is 11-KT, the main androgen in fish. This androgen stimulates the proliferation and differentiation of the spermatogonia via the Sertoli cells. FSH further down-regulates the release of anti-Müllerian hormone (AMH), an Sertoli cells derived growth factor that inhibits spermatogenesis. Next to the stimulation of androgen production and the down-regulated of AMH, FSH stimulates spermatogenesis in another way. A possible mediator of this stimulation is insulin-like growth factor 3 (IGF3). This factor is a fish-specific IGF and is only expressed in the gonads, which suggests an important role in spermatogenesis. It was already found that igf3 expression is stimulated by gonadotropins and that igf3 is expressed in Sertoli cells. Further, it was found that IGF1 stimulates the spermatogenesis, which indicates that IGF3 can stimulate spermatogenesis too. Therefore, it is quite possible that IGF3 is a mediator of the effect of FSH on spermatogenesis. Different techniques were used in this master project to test this hypothesis on different levels and to find out more about the role and regulation of IGF3 in the testis. Firstly, a gene expression analysis was done to examine the response in igf3 and amh expression to different doses of FSH. This analysis confirms that FSH stimulates igf3 expression and down-regulates amh expression. Clear dose response curves were found for both genes. Second, a fish injection experiment was done in order to determine the effect of FSH on IGF3 protein level. This experiment indicates that FSH injection causes an increase in IGF3 protein level in the testes, but the experiment needs to be repeated. Lastly, NVP-AEW541, an inhibitor of the IGF1R, was tested for its capability to inhibit the IGF3-mediated stimulation of spermatogenesis. This inhibitor caused a clear reduction in spermatogonial proliferation in the testes. Therefore, this inhibitor can be used to test whether IGF3 is mediating the androgen-independent stimulation by FSH.