Molecular Cloning and Functional Characterization of a Zebrafish Nuclear Progesterone Receptor1

Abstract

Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, including teleost fish. To further our understanding of how progesterone modulates testis functions in fish, we set out to clone a progesterone receptor (pgr) cDNA exhibiting nuclear hormone receptor features from zebrafish testis. The open reading frame of pgr consists of 1854 bp, coding for a 617-amino acid-long protein showing the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that zebrafish Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency. Expression of pgr mRNA: 1) appeared in embryos at 8 h after fertilization; 2) was significantly higher in developing ovary than in early transforming testis at 4 wk of age but vice versa in young adults at 12 wk of age, and thus resembling the expression pattern of the germ cell marker piwil1; and, 3) was restricted to Leydig and Sertoli cells in adult testis. Zebrafish testicular explants released DHP concentration dependently in response to high concentrations of recombinant zebrafish gonadotropins. In addition, DHP stimulated 11-ketotestosterone release from zebrafish testicular explants, but only in the presence of its immediate precursor, 11 beta-hydroxytestosterone. This stimulatory activity was blocked by a Pgr antagonist (RU486), suggesting that 11 beta-hydroxysteroid dehydrogenase activity was stimulated by DHP via Pgr. Our data suggest that DHP contributes to the regulation of Leydig cell steroidogenesis, and potentially--via Sertoli cells--also to germ cell differentiation in zebrafish testis.

Blocking of progestin action disrupts spermatogenesis in Nile tilapia (Oreochromis niloticus)

“…An in vitro assay carried out using mammalian cell culture has proved that a functional progestin receptor (Pgr) can transactivate target genes in a DHP-dependent manner, supporting the view that DHP is the major native ligand of nuclear Pgr in fish (Chen et al 2010). Using a testicular incubation system, Chen et al (2010) also demonstrated that DHP stimulates the production of 11-KT in a Pgr-dependent manner, while RU486 (a synthetic Pgr antagonist) could inhibit its action by suppressing Pgr-mediated transactivation. Therefore, it has been proposed that Pgr might mediate the actions of DHP in spermatogonial cell proliferation, meiosis initiation, and spermatogenesis in fish.…”

“…However, it has been shown that DHP is produced by both mature and immature testes at different stages, indicating its diverse roles before and during spermatogenesis in rainbow trout (Oncorhynchus mykiss; Depeche & Sire 1982). Recent reports have revealed that DHP might play critical roles in the initiation of meiosis in Japanese eel (Anguilla japonica) and in the early stages of spermatogenesis in Atlantic salmon (Salmon salar), cod (Gadus morhua), and zebrafish (Danio rerio) (Chen et al 2010(Chen et al , 2011(Chen et al , 2012(Chen et al , 2013. Furthermore, it has been proven in in vitro experiments that DHP can induce the conversion of 11b-hydroxytestosterone to 11-ketotestosterone (11-KT, the main androgen in fish) by stimulating the activity of 11b-hydroxysteroid dehydrogenase (encoded by cyp11b2) in Japanese eel and zebrafish , Chen et al 2010.…”

“…Recent reports have revealed that DHP might play critical roles in the initiation of meiosis in Japanese eel (Anguilla japonica) and in the early stages of spermatogenesis in Atlantic salmon (Salmon salar), cod (Gadus morhua), and zebrafish (Danio rerio) (Chen et al 2010(Chen et al , 2011(Chen et al , 2012(Chen et al , 2013. Furthermore, it has been proven in in vitro experiments that DHP can induce the conversion of 11b-hydroxytestosterone to 11-ketotestosterone (11-KT, the main androgen in fish) by stimulating the activity of 11b-hydroxysteroid dehydrogenase (encoded by cyp11b2) in Japanese eel and zebrafish , Chen et al 2010. In contrast, it has been shown in adult zebrafish that 17b-estradiol (E 2 )-mediated suppression of spermatogenesis could be successfully restored by DHP treatment without increasing 11-KT production (Chen et al 2013).…”

“…RU486, an orally active compound for absorption used in FDA-approved medical treatment, binds to the progesterone receptor with twofold higher affinity than progesterone (Heikinheimo et al 2003). So far, nuclear progestin receptors have been identified and characterized in several fish species, with variable expression profiles being detected in Sertoli cells, Leydig cells, and germ cells (Chen et al 2010, 2011, Hanna et al 2010). An in vitro assay carried out using mammalian cell culture has proved that a functional progestin receptor (Pgr) can transactivate target genes in a DHP-dependent manner, supporting the view that DHP is the major native ligand of nuclear Pgr in fish (Chen et al 2010).…”

“…So far, nuclear progestin receptors have been identified and characterized in several fish species, with variable expression profiles being detected in Sertoli cells, Leydig cells, and germ cells (Chen et al 2010, 2011, Hanna et al 2010). An in vitro assay carried out using mammalian cell culture has proved that a functional progestin receptor (Pgr) can transactivate target genes in a DHP-dependent manner, supporting the view that DHP is the major native ligand of nuclear Pgr in fish (Chen et al 2010). Using a testicular incubation system, Chen et al (2010) also demonstrated that DHP stimulates the production of 11-KT in a Pgr-dependent manner, while RU486 (a synthetic Pgr antagonist) could inhibit its action by suppressing Pgr-mediated transactivation.…”