Thyroid hormones participate in regulating growth and homeostatic processes in vertebrates, including development and adult functioning of the reproductive system. Here we report a new stimulatory role of thyroid hormone on the proliferation of Sertoli cells (SCs) and single, type A undifferentiated spermatogonia (A(und)) in adult zebrafish testes. A role for T3 in zebrafish testis is suggested by in situ hybridization studies, which localized thyroid receptor α (thrα) in SCs and the β (thrβ) mRNA in Sertoli and Leydig cells. Using a primary zebrafish testis tissue culture system, the effect of T3 on steroid release, spermatogenesis, and the expression of selected genes was evaluated. Basal steroid release and Leydig cell gene expression did not change in response to T3. However, in the presence of FSH, T3 potentiated gonadotropin-stimulated androgen release as well as androgen receptor (ar) and 17α-hydroxylase/17,20 lyase (cyp17a1) gene expression. Moreover, T3 alone stimulated the proliferation of both SCs and A(und), potentially resulting in newly formed spermatogonial cysts. Additional tissue culture studies demonstrated that Igf3, a new, gonad-specific member of the IGF family, mediated the stimulatory effect of T3 on the proliferation of A(und) and SCs. Finally, T3 induced changes in connexin 43 mRNA levels in the testis, a known T3-responsive gene. Taken together, our studies suggest that T3 expands the population of SCs and A(und) involving Igf signaling and potentiates gonadotropin-stimulated testicular androgen production as well as androgen sensitivity.

INSL3 (insulin-like peptide 3) is a relaxin peptide family member expressed by Leydig cells in the vertebrate testis. In mammals, INSL3 mediates testicular descent during embryogenesis but information on its function in adults is limited. In fish, the testes remain in the body cavity, although the insl3 gene is still expressed, suggesting yet undiscovered, evolutionary older functions. Anti-Müllerian hormone (Amh), in addition to inhibiting spermatogonial differentiation and androgen release, inhibits the Fsh (follicle-stimulating hormone)-induced increase in insl3 transcript levels in zebrafish testis. Therefore, the two growth factors might have antagonistic effects. We examine human INSL3 (hINSL3) effects on zebrafish germ cell proliferation/differentiation and androgen release by using a testis tissue culture system. hINSL3 increases the proliferation of type A undifferentiated (Aund) but not of type A differentiating (Adiff) spermatogonia, while reducing the proliferation of Sertoli cells associated with proliferating Aund. Since the area occupied by Aund decreases and that of Adiff increases, we conclude that hINSL3 recruits Aund into differentiation; this is supported by the hINSL3-induced down-regulation of nanos2 transcript levels, a marker of single Aund spermatogonia in zebrafish and other vertebrates. Pulse-chase experiments with a mitosis marker also indicate that hINSL3 promotes spermatogonial differentiation. However, hINSL3 does not modulate basal or Fsh-stimulated androgen release or growth factor transcript levels, including those of amh. Thus, hINSL3 seems to recruit Aund spermatogonia into differentiation, potentially mediating an Fsh effect on spermatogenesis.

Follicular atresia in fish ovary provides an interesting model for studying autophagy and apoptosis. In order to improve knowledge of the mechanisms regulating ovarian regression, we investigated the immunolocalisation of various proteins involved in the complex network of autophagy and apoptosis. Females of three species of freshwater fish maintained in captivity were sampled after the reproductive period and the main events of follicular atresia were assessed by histology: splits in the zona radiata, yolk degradation and reabsorption, hypertrophy of the follicular cells, accumulation of autophagic vacuoles, closing of the follicular lumen and thickening of the theca. The interplay of apoptosis and autophagy was analysed by TUNEL in situ and by immunocytochemistry for caspase-3, bax, bcl-2, beclin-1 and cathepsin-D. During early and advanced stages of follicular regression, the actin cytoskeleton was well developed and labelling for bcl-2 and cathepsin-D were pronounced in the follicular cells at a stage when they were intensively involved in yolk phagocytosis. Immunofluorescence for beclin-1 was prevalent in the follicular cells, punctate labelling often surrounding autophagic vacuoles during the advanced stage of follicular regression, a critical step towards cell death. TUNEL-positive reaction and immunostaining for bax and caspase-3 demonstrated the participation of apoptosis in late follicular regression. Overall, this study provides evidence that autophagic and apoptotic proteins are activated in a coordinated fashion depending on the stage of follicular regression, with interplay between autophagy and apoptosis being essential in determining the fate of the cell during follicular atresia in fish ovary.

IGF binding proteins (IGFBPs) modulate the availability of IGFs for their cognate receptors. In zebrafish testes, IGF3 promotes the proliferation and differentiation of type A undifferentiated (A) spermatogonia, and igf3 expression is strongly elevated by FSH but also responds to T. Here we report the effects of FSH and T on igfbp transcript levels in adult zebrafish testis. We then examined T and FSH effects on zebrafish spermatogenesis and explored the relevance of IGFBPs in modulating these T or FSH effects, using a primary tissue culture system for adult zebrafish testis. T up-regulated igfbp1a and igfbp3 expression, whereas FSH reduced igfbp1a transcript levels. To quantify effects on spermatogenesis, we determined the mitotic index and relative section areas occupied by A, type A differentiating, or type B spermatogonia. In general, T and FSH stimulated spermatogonial proliferation and increased the areas occupied by spermatogonia, suggesting that both self-renewal and differentiating divisions were stimulated. Preventing IGF/IGFBP interaction by NBI-31772 further increased T- or FSH-induced spermatogonial proliferation. However, under these conditions the more differentiated type A differentiating and B spermatogonia occupied larger surface areas at the expense of the area held by A spermatogonia. Clearly decreased nanos2 transcript levels are in agreement with this finding, and reduced amh expression may have facilitated spermatogonial differentiation. We conclude that elevating IGF3 bioactivity by blocking IGFBPs shifted T- or FSH-induced signaling from stimulating spermatogonial self-renewal as well as differentiation toward predominantly stimulating spermatogonial differentiation, which leads to a depletion of type A spermatogonia.

Fsh-mediated regulation of zebrafish spermatogenesis includes modulating the expression of testicular growth factors. Here, we study if and how two Sertoli cell-derived Fsh-responsive growth factors, anti-Müllerian hormone (Amh; inhibiting steroidogenesis and germ cell differentiation) and insulin-like growth factor 3 (Igf3; stimulating germ cell differentiation), cooperate in regulating spermatogonial development. In dose response and time course experiments with primary testis tissue cultures, Fsh up-regulated igf3 transcript levels and down-regulated amh transcript levels; igf3 transcript levels were more rapidly up-regulated and responded to lower Fsh concentrations than were required to decrease amh mRNA levels. Quantification of immunoreactive Amh and Igf3 on testis sections showed that Fsh increased slightly Igf3 staining but decreased clearly Amh staining. Studying the direct interaction of the two growth factors showed that Amh compromised Igf3-stimulated proliferation of type A (both undifferentiated [A] and differentiating [A]) spermatogonia. Also the proliferation of those Sertoli cells associated with A spermatogonia was reduced by Amh. To gain more insight into how Amh inhibits germ cell development, we examined Amh-induced changes in testicular gene expression by RNA sequencing. The majority (69%) of the differentially expressed genes was down-regulated by Amh, including several stimulators of spermatogenesis, such as igf3 and steroidogenesis-related genes. At the same time, Amh increased the expression of inhibitory signals, such as inha and id3, or facilitated prostaglandin E (PGE) signaling. Evaluating one of the potentially inhibitory signals, we indeed found in tissue culture experiments that PGE promoted the accumulation of A at the expense of A and B spermatogonia. Our data suggest that an important aspect of Fsh bioactivity in stimulating spermatogenesis is implemented by restricting the different inhibitory effects of Amh and by counterbalancing them with stimulatory signals, such as Igf3.

No abstract

In fish ovaries, postovulatory follicles (POFs) are key biomarkers of breeding and provide an interesting model for studying the relationship between autophagy and apoptosis. In this study, we investigated the immunohistochemical expression of autophagic and apoptotic proteins to improve the knowledge on the mechanisms regulating ovarian remodeling after spawning. Females from three neotropical fish species kept in captivity were submitted to hormonal induction. After ova stripping, ovarian sections were sampled daily until 5 days postspawning (dps). Similar events of POF regression were detected by histology, terminal transferase-mediated dUTP nick-end labeling (TUNEL), and electron microscopy in the three species: follicular cells hypertrophy, progressive disintegration of the basement membrane, gradual closing of the follicular lumen, theca thickening, and formation of large autophagic vacuoles preceding apoptosis of the follicular cells. Autophagic and apoptotic proteins were assessed by immunohistochemistry. Morphometric analysis of the immunolabeling revealed a more intense reaction for bcl-2 and beclin-1 (BECN1) in POFs at 0 to 1 dps and for bax at 2 to 3 dps (P < 0.001), the later period being the peak of apoptosis of the follicular cells. The immunostaining for cathepsin-D was more elevated until 2 to 3 dps and decreased significantly at 4 to 5 dps, when the POFs were in late stage of regression. Double labeling for BECN1 and caspase-3 indicated a shift in the relationship between autophagy and apoptosis at 2 to 3 dps, a critical period in determining the fate of follicular cells in POFs. Together, these results indicate that the bcl-2 family, BECN1, and cathepsin-D can be involved in the regulation of ovarian remodeling in teleost fish.

Fish embryos are particularly vulnerable to temperature changes, with the effects varying with developmental stage. The major aim of the present study was to analyse the relationship between apoptosis and heat shock protein (HSP) 70 during embryo development under thermal stress conditions. To this end, Prochilodus lineatus embryos at the blastopore closure stage were subjected to one of three thermal treatments for 1h (Group 1, 25°C (control); Group 2, 20°C; Group 3, 30°C) and then examined at 0, 4 and 8h posttreatment (h.p.t.). The viability of embryos was highest in Group 1 (81.33±16.65%), followed by Group 3 and Group 2 (75.33±12.10% and 68.67±16.86% respectively), with significant difference between Groups 1 and 2 (P&lt;0.05). At 0h.p.t., embryos subjected to thermal stress (Group 3) had a significantly higher number of terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL)- and caspase-3-labelled cells, and a lower number of HSP70-positive cells than those in the control group. At 4h.p.t., there was a decrease in the TUNEL reaction and an increase in HSP70 in embryos in Group 3. At 8h.p.t., the size of Group 3 embryos was significantly smaller than that of Group 1 embryos. The results indicate a cytoprotective role for HSP70, regulating caspase-3-mediated apoptosis during embryo development of P. lineatus; however, this mechanism is not effective in controlling embryo viability and larval malformations.