Cortisol is the major endocrine factor mediating the inhibitory effects of stress on vertebrate reproduction. It is well known that cortisol affects reproduction by interacting with the hypothalamic–pituitary–gonads axis, leading to downstream inhibitory and stimulatory effects on gonads. However, the mechanisms are not fully understood. In this study, we provide novel data demonstrating the stimulatory effects of cortisol on spermatogenesis using an ex vivo organ culture system. The results revealed that cortisol treatment did not modulate basal androgen production, but it influenced transcript levels of a selected number of genes involved in the zebrafish testicular function ar (androgen receptor), star (steroidogenic acute regulatory), cyp17a1 (17α-hydroxylase/17,20 lyase/17,20 desmolase), cyp11a2 (cytochrome P450, family 11, subfamily A, polypeptide 2), hsd11b2 (11-beta hydroxysteroid dehydrogenase), cyp2k22 (cytochrome P450, family 2, subfamily K, polypeptide 22), fkbp5 (FKBP prolyl isomerase 5), grα (glucocorticoid receptor alpha), and grβ (glucocorticoid receptor beta) in a short-term culture. We also showed that cortisol stimulates spermatogonial proliferation and differentiation in an androgen independent manner as well as promoting meiosis and spermiogenesis by increasing the number of spermatozoa in the testes. Moreover, we demonstrated that concomitant treatment with RU 486, a potent glucocorticoid receptor (Gr) antagonist, did not affect the cortisol effects on spermatogonial differentiation but blocked the induced effects on meiosis and spermiogenesis. Supporting the Gr-mediated effects, RU 486 nullified the cortisol-induced expression of sycp3l (synaptonemal complex protein 3), a marker for the meiotic prophase that encodes a component of the synaptonemal complex. This is consistent with in silico analysis that found 10 putative GREs (glucocorticoid response elements) upstream of the zebrafish sycp3l. Finally, we also showed that grα mRNA is expressed in Sertoli and Leydig cells, but also in several types of germ cells, including spermatogonia and spermatocytes. Altogether, this evidence indicates that cortisol exerts paracrine roles in the zebrafish testicular function and spermatogenesis, highlighting its effects on spermatogonial differentiation, meiosis, and spermiogenesis.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor α1-GFRα1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in mammalian testes. In nonmammalian species, Gdnf and Gfrα1 orthologs have been found but their functions remain poorly investigated in the testes. Considering this background, this study aimed to understand the roles of the Gdnf-Gfrα1 signaling pathway in zebrafish testes by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibit two paralogs for Gndf (gdnfa and gdnfb) and its receptor, Gfrα1 (gfrα1a and gfrα1b), in accordance with a teleost-specific third round of whole genome duplication. Expression analysis further revealed that both ligands and receptors were expressed in zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfrα1a/Gfrα1b was detected in early spermatogonia (mainly in types A_und and A_diff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating the proliferation of both type A_und spermatogonia and their surrounding Sertoli cells but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation, as shown by the decrease in type B spermatogonia and down-regulation of dazl in a co-treatment with Fsh. Altogether, our data revealed that a germ cell-derived factor is involved in maintaining germ cell stemness through the creation of new available niches, supporting the development of spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine- and paracrine-dependent manners.

Megaleporinus macrocephalus (piauçu) is a Neotropical fish within Characoidei that presents a well-established heteromorphic ZZ/ZW sex determination system and thus constitutes a good model for studying W and Z chromosomes in fishes. We used PacBio reads and Hi-C to assemble a chromosome-level reference genome for M. macrocephalus. We generated family segregation information to construct a genetic map, pool sequencing of males and females to characterize its sex system, and RNA sequencing to highlight candidate genes of M. macrocephalus sex determination. The reference genome of M. macrocephalus is 1,282,030,339 bp in length and has a contig and scaffold N50 of 5.0 Mb and 45.03 Mb, respectively. In the sex chromosome, based on patterns of recombination suppression, coverage, F_ST, and sex-specific SNPs, we distinguished a putative W-specific region that is highly differentiated, a region where Z and W still share some similarities and is undergoing degeneration, and the PAR. The sex chromosome gene repertoire includes genes from the TGF-β family (amhr2, bmp7) and the Wnt/β-catenin pathway (wnt4, wnt7a), some of which are differentially expressed. The chromosome-level genome of piauçu exhibits high quality, establishing a valuable resource for advancing research within the group. Our discoveries offer insights into the evolutionary dynamics of Z and W sex chromosomes in fish, emphasizing ongoing degenerative processes and indicating complex interactions between Z and W sequences in specific genomic regions. Notably, amhr2 and bmp7 are potential candidate genes for sex determination in M. macrocephalus.

Germ cell transplantation in fish is a promising technique for surrogate broodstock parents with broader application in aquaculture and conserving endangered and valuable genetic resources. Herein, we describe the establishment of an intrapapillary xenogeneic transplant of germ cells from sexually mature goldfish (C. auratus) males into common carp (C. carpio) males cytoablated with a thermochemical treatment (two doses of busulfan at 40 mg/kg at 35°C). To analyze the presence and development of donor germ cells in recipient testes, donor germ cells were labeled with PKH26, a fluorescent cell membrane dye, before transplantation. Our results demonstrated that thermochemical treatment caused effective spermatogenesis suppression and pronounced germ cell loss. Moreover, transplanted spermatogonial cells were able to colonize the recipients’ testes, resume spermatogenesis, and generate spermatozoa within eight weeks after germ cell transplantation. These findings suggested that recipient testes provided suitable conditions for the survival, colonization, proliferation, and differentiation of donor spermatogonia from a related species. This study indicated that recipients’ testes exhibited a high degree of plasticity to accept and support xenogeneic donor germ cells, which were able to form sperm in a short time frame. This approach has significant implications for assisted animal reproduction, biotechnology, conservation, and the production of valuable genetic resources and endangered fish species.