New insights into the evolution, hormonal regulation, and spatiotemporal expression profiles of genes involved in the Gfra1/Gdnf and Kit/Kitlg regulatory pathways in rainbow trout testis.

The present study aimed to investigate whether the Gfra1/Gdnf and/or Kit/Kitlg regulatory pathways could be involved in the regulation of spermatogonial cell proliferation and/or differentiation in fish. Homologs of the mammalian gfra1, gdnf, kitr, and kitlg genes were identified in gnathostomes and reliable orthologous relationships were established using phylogenetic reconstructions and analyses of syntenic chromosomal fragments. Gene duplications and losses occurred specifically in teleost fish and members of the Salmoninae family including rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Some duplicated genes exhibited distinct spatiotemporal expression profiles and were differently regulated by hormones in rainbow trout. Undifferentiated type A spermatogonia expressed higher levels of kitrb and kitra2 making them possible target cells for the gonadal kitlgb and somatic kitlga before the onset of spermatogenesis. Interestingly, gdnfa and gdnfb ohnologous genes were poorly expressed before the onset of spermatogenesis. The expression level of gdnfb was correlated with that of the vasa gene suggesting that the late increased abundance of gdnfb during spermatogenesis onset was due to the increased number of spermatogonial cells. gfra1a2 was expressed in undifferentiated type A spermatogonia whereas gfra1a1 was mainly detected in somatic cells. These observations indicate that the germinal gdnfb ligand could exert autocrine and paracrine functions on spermatogonia and on testicular somatic cells, respectively. Fsh and androgens inhibited gfra1a2 and gdnfb whereas gfra1a1 was stimulated by Fsh, androgens, and 17α, 20ß progesterone. Finally, our data provide evidences that the molecular identity of the male germ stem cells changes during ontogenesis prior to spermatogenesis onset.

The nanos1 gene was duplicated in early Vertebrates and the two paralogs show different gonadal expression profiles in a shark.

Nanos are RNA-binding proteins playing crucial roles in germ cell development and maintenance. Based on phylogenetic and synteny analyses, this study reveals that nanos1 gene has undergone multiple duplications and gene copies losses in Vertebrates. Chondrichthyan species display two nanos1 genes (named nanos1A/1B), which were both retrieved in some Osteichthyes at basal positions in Sarcopterygii and Actinopterygii lineages. In contrast, Teleosts have lost nanos1A but duplicated nanos1B leading to the emergence of two ohnologs (nanos1Ba/1Bb), whereas Tetrapods have lost nanos1B gene. The two successive nanos gene duplications may result from the second and third whole genome duplication events at the basis of Vertebrates and Teleosts respectively. The expression profiles of nanos1A and nanos1B paralogs were characterized in the dogfish, Scyliorhinus canicula. Nanos1A was strongly expressed in brain and also localized in all germ cell types in the polarized testis. In contrast, nanos1B was detected in testis with the highest expression in the germinative zone. In addition, Nanos1B protein was predominantly located in the nuclei of male germinal cells. In the ovary, both paralogs were detected in germinal and somatic cells. Our study opens new perspectives concerning the complex evolution of nanos1 paralogs and their potential distinct roles in Vertebrates gonads.

Gdnf-Gfra1 pathway is expressed in a spermatogenetic-dependent manner and is regulated by Fsh in a fish testis.

What makes the spermatogonial stem cells (SSCs) self-renew or differentiate to produce spermatozoa is barely understood, in particular in nonmammalian species. Our research explores possible regulations of the SSC niche in teleost, locally by paracrine factors and peripherally by hormonal regulation. In the present study, we focus on the Gdnf-Gfra1 pathway that plays a major role in the regulation of SSC self-renewal in mammals. We describe a complex evolution of the genes encoding for Gdnf and Gfra1 proteins in trout with the emergence of three gdnf and two gfra1 paralogs. Using quantitative PCR measurements in isolated testicular cell populations, the gdnfb paralog was found expressed in A-spermatogonia and probably in another testicular cell type. In contrast, the transcript of gfra1a, the Gdnf receptor, was preferentially expressed in a population of undifferentiated A-spermatogonia (und A-Spg) separated by centrifugal elutriation. These und A-Spg also demonstrated high stemness potential in transplantation studies and preferentially expressed nanos2, a putative SSC marker in trout (Bellaiche et al., Biol Reprod 2014; 90:79). Flow cytometer experiments demonstrate that only a subfraction of und A-Spg express Gfra1. In trout, spermatogenesis develops along a strict annual cycle, and gdnfb and its receptor were expressed in a spermatogenetic activity-dependent manner. In particular, a dramatic increase of the gdnfb transcript coincided with the progressive cessation of rapid spermatogonial proliferation and of meiosis toward the end of the reproductive cycle. Together these results suggest that, in trout, Gdnfb is involved in the repression of und A-Spg differentiation. Fsh is an endocrine regulator of SSCs self-renewal through the up-regulation of Gdnf in rodents. We demonstrate that in trout, in vitro Fsh treatment stimulated the expression of the gfra1a1 but not of its ligand, gdnfb. Fsh treatment also stimulated the proliferation of und A-Spg cocultured with testicular somatic cells. Based on those results, the Gfra1-positive cells could correspond to the putative SSCs in rainbow trout, and we propose that the balance between SSC self-renewal and differentiation during the trout spermatogenetic cycle is under paracrine regulation by Gdnfb, which represses, and under peripheral regulation by Fsh via the control of gfra1a1 expression.

Spermatogonial stem cell quest: nanos2, marker of a subpopulation of undifferentiated A spermatogonia in trout testis.

Continuous or cyclic production of spermatozoa throughout life in adult male vertebrates depends on a subpopulation of undifferentiated germ cells acting as spermatogonial stem cells (SSCs). What makes these cells self-renew or differentiate is barely understood, in particular in nonmammalian species, including fish. In the highly seasonal rainbow trout, at the end of the annual spermatogenetic cycle, tubules of the spawning testis contain only spermatozoa, with the exception of scarce undifferentiated spermatogonia that remain on the tubular wall and that will support the next round of spermatogenesis. Taking advantage of this model, we identified putative SSCs in fish testis using morphological, molecular, and functional approaches. In all stages, large spermatogonia with ultrastructural characteristics of germinal stem cells were found, isolated or in doublet. Trout homologues of SSC and/or immature progenitor markers in mammals-nanos2 and nanos3, pou2, plzf, and piwil2-were preferentially expressed in the prepubertal testis and in the undifferentiated A spermatogonia populations purified by centrifugal elutriation. This expression profile strongly suggests that these genes are functionally conserved between fish and mammals. Moreover, transplantation into embryonic recipients of the undifferentiated spermatogonial cells demonstrated their high "stemness" efficiency in terms of migration into gonads and the ability to give functional gametes. Interestingly, we show that nanos2 expression was restricted to a subpopulation of undifferentiated spermatogonia (less than 20%) present as isolated cells or in doublet in the juvenile and in the maturing trout testis. In contrast, nanos2 transcript was detected in all the undifferentiated spermatogonia remaining in the spawning testis. Plzf was also immunodetected in A-Spg from spawning testis, reinforcing the idea that these cells are stem cells. From those results, we hypothesize that the subset of undifferentiated A spermatogonia expressing nanos2 transcript are putative SSC in trout.