Study On Mechanisms Of Progestin (DHP)Actions In The Meiotic Initiation In The Teleosts

Generally, there are mainly two types of cells, i.e. somatic cells and germ cells in the gonad in vertebrates.Therefore, sex determination/differentiationin vertebrates indicates sex determination/differentiation of somatic cells and germ cells, the latter of which refers to meiotic initiationof germ cells. It was well know that meiosis of germ cell in female initiated much earlier that of male. In mammalian species, the level of retinoic acid (RA) controlled by Cyp26bl played a central role in regulating the sex-specific timing of meiotic initiation, while it remained to be controversial in other reports. So far, whether RA is required for meiotic initiation or not in non-mammalian species has not been proved. Therefore, the molecular mechanism of meiotic initiation in germ cellsin non-mammalian species needs further investigations. Fish progestin (DHP) was capable of inducing gametes maturation and ovulation (spermiation). Since the gamete maturation process is the second division of meiosis, we speculate that DHP might also play an essential role in the initiation of meiosis.Therefore, we focus on the role of DHP in germ cell meiotic initiation and the possible mechanismsin teleost. In this study, DHP nuclear receptor (Pgr) gene was cloned and Pgr antagonist treatment experiments were carried out.The main results are listed as follows:Firstly, we cloned DHP nuclear receptor gene Pgr from the gonad of tilapia. Multiple amino acids alignments showed that tilapia Pgr has the conversed domains as Pgrs in other vertebrates, including the N-terminal transactivation domain, DNA-binding domain (DBD) and the ligand binding domain (LBD). Phylogenetic analysis revealed that tilapia Pgr clustered with other teleosteanPgrs, while tetrapods Pgrs formed a distinct clade.Secondly, real-time PCR revealed that tilapia Pgr was mainly expressed in gonads. Ontogenic analysis demonstrated that tilapia pgr was significantly up-regulated in the critical period of the meiotic initiation and maintained at a high level until adulthood in both XX and XY fish gonads. Immunohistochemistry demonstrated that tilapia Pgr was expressed in different cell types during the development process. In female fish, Pgr was expressed in interstitial cells in the2-month old ovary, and was detected in follicle cells in the adult ovary. In male fish, Pgr was mainly expressed in sertoli cells in the testis of one-month old tilapia,while it was found in cells surrounding mature spermatids. The expression profiles indicated that Pgr-mediated signaling pathway might be involved in meiotic initiation in tilapia.Thirdly, continuous treatment exceeding4mah severely disrupted spermatogenesis and led to depletion of germ cells. Continuous treatment by RU486for two months and then fed normally for another two months, didn’t affect spermatogonia proliferation and spermatogenesis. The co-treatment of DHP and RU486showed that DHP could rescue the disruption by RU486treatment and restored spermatogenesis, but combination of DHP and11-KT failed to do so. Continuous treatmentwith RU486for four months resulted in the down-regulation of germ cell related genes of vasa, sycp3,piwil, dazl, but not the somatic cell related genes of amh, dmrtl, gsdf and steroidogenic enzymes genes.The down-regulation of Cypl7a2, which is essential for the production of DHP, demonstrate that RU486might inhibit the synthesis of DHP. Furthermore, EIA measurement showed that DHP treatment couldn’t alter the serum level of11-KT. Taken together, RU486treatment, by binding to Pgr competitively, inhibited spermatogonia proliferation and spermatogenesis. Therefore, Pgr mediated DHP pathway might play a pivotal role in the spermatogonia proliferation and meiotic initiation. Meanwhile, RU486treatment blocked spermatogenesis without affecting11-KT level, therefore, DHP regulated though completely different pathway from that of11-KT.Fourthly, in female fish, continuous RU486treatment exceeding4mah severely disrupted the initiation of meiosis.Continuous RU486treatmentled to masculinizationof XX somatic cells with development of Sertoli cells (Dmrt1and Gsdf positive cells) and Leydig cells (Star and Cyp11b2positive cells). Moreover, RU486treatment for3months and normally feeding for another one month, resulted in sex reversal and reactive spermatogenesis with different phase of spermgenic cells, including spermatogonia, spermatocytes and spermatids, in XX gonad. Our data showed that DHP was essential for the oogonia proliferation and meiotic initiation of germ cells in XX gonads. In the XX fish, germ cells meiosis is a prerequisite in the ovarian differentiation and maintenance.In conclusion, the dysfunction of DHP could block the mitosis and meiotic initiation in germ cells, which in turn affected the direction of sex determination and differentiation.