Fsh stimulates Leydig cell Wnt5a production, enriching zebrafish type A spermatogonia

in Journal of Endocrinology
Correspondence should be addressed to R W Schulz: r.w.schulz@uu.nl
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Follicle-stimulating hormone (Fsh) modulates vertebrate spermatogenesis by regulating somatic cell functions in the testis. We have found previously that zebrafish Fsh stimulated the differentiating proliferation of type A undifferentiated spermatogonia (Aund) in an androgen-independent manner by regulating the production of growth factors and other signaling molecules in both Sertoli (SCs) and Leydig cells (LCs). For example, Fsh triggered the release of Igf3 that subsequently activated β-catenin signaling to promote the differentiating proliferation of Aund. In the present study, we report that Fsh moreover uses the non-canonical Wnt pathway to promote the proliferation and accumulation of Aund. Initially, we found that the stimulatory effect of Fsh on the proliferation activity of Aund was further strengthened when β-catenin signaling was inhibited, resulting in an accumulation of Aund. We then showed that this Fsh-induced accumulation of Aund was associated with increased transcript levels of the non-canonical Wnt ligand, wnt5a. In situ hybridization of insl3 mRNA, a gene expressed in LCs, combined with Wnt5a immunocytochemistry identified LCs as the cellular source of Wnt5a in the adult zebrafish testis. Addition of an antagonist of Wnt5a to incubations with Fsh decreased both the proliferation activity and the relative section area occupied by Aund, while an agonist of Wnt5a increased these same parameters for Aund. Taken together, our data suggest that Fsh triggered LCs to release Wnt5a, which then promoted the proliferation and accumulation of Aund. Hence, Fsh uses non-canonical Wnt signaling to ensure the production of Aund, while also triggering β-catenin signaling via Igf3 to ensure spermatogonial differentiation.

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  • Supplemental figure 1. (A-E) Immunocytochemical detection of BrdU in sections of zebrafish testis incubated in the presence of 100 ng/mL Fsh (A) or 100 ng/mL Fsh and XAV939 (10 µM) (B) or 100 ng/mL Fsh, 10 µM XAV939 and 50 µM IWP-12 (C) or 100 ng/mL Fsh and 50 µM IWP-12 (D) or under basal condition, presence of 10 µM XAV939 or 50 µM IWP-12 (E) for 5 days, showing BrdU-positive (+) and BrdU-negative (-) Aund and Adiff spermatogonia. (K-I) Whole-mount co-localization of Wnt5a by immunocytochemistry and insl3 mRNA by in situ hybridization in Leydig cells of adult zebrafish testis.

 

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    Inhibiting canonical β-catenin signaling increases Fsh-stimulated self-renewal of Aund spermatogonia but compromises the production of Adiff spermatogonia. (A) BrdU-labeling indices of type Aund and type Adiff spermatogonia in the presence of Fsh alone (100 ng/mL; dotted line; control condition) or in combination with 10 µM XAV393, an inhibitor of β-catenin signaling (represented by bars; n = 7). (B) Proportion of section surface area occupied by cysts containing type Aund and type Adiff spermatogonia in the presence of Fsh alone (100 ng/mL; dotted line; control condition) or in combination with 10 µM XAV393 (represented by bars; n = 7). (C) Gene expression analysis of germ cell markers in adult zebrafish testis after 5 days of tissue culture in the presence of Fsh (100 ng/mL; dotted line; control condition) or in combination with 10 µM XAV393 (represented by bars; n = 7). The quantification cycles (Cq) of the reference gene (elf1a) are shown in the insert. Results are presented as fold changes with respect to the control group (100 ng/mL Fsh). Asterisks indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001) between groups.

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    Fsh modulates the proliferation activity of type A spermatogonia via non-canonical Wnt ligands. (A) Gene expression analysis in adult zebrafish testis after 5 days of tissue culture in the presence of Fsh (100 ng/mL; dotted line; control condition) or in combination with 10 µM XAV393 (represented by bars; n = 7). (B) BrdU-labeling indices of type Aund under control (dotted line) or experimental (represented by a bars) conditions after 5 days of incubation: Fsh (n = 8); Fsh+XAV (n = 7); Fsh + XAV + IWP-12 (n = 8); Fsh + IWP-12 (n = 8); XAV (n = 5); IWP-12 (n = 6). (C) BrdU-labeling indices of type Adiff under control (dotted line) or experimental (represented by a bars) conditions after 5 days of incubation: Fsh (n = 8); Fsh + XAV (n = 7); Fsh + XAV + IWP-12 (n = 8); Fsh + IWP-12 (n = 8); XAV (n = 5); IWP-12 (n = 6). Results are presented as fold changes with respect to the control group (see ‘Material and methods’ section). Asterisks indicate significant differences (*P < 0.05; **P < 0.01) between groups and different letters indicate significant differences (P < 0.05) between groups.

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    Endocrine regulation of Wnt5a protein production and cellular localization of Wnt5a.(A) Transcript levels of wnt5a and wnt11 in basal conditions (dotted line; control condition) or in the presence of Fsh (100 ng/mL; n = 8; represented by black bars), 11-KT (200 nM; n = 6; represented by gray bars) or Lh (500 ng/mL; n = 9; represented by white bars) in adult zebrafish testis. The quantification cycles (Cq) of the reference gene (elf1a) are shown in the insert. Results are presented as fold changes with respect to the control group (basal). (B) Western blot and (C) densitometric analysis of Wnt5a in zebrafish testis under basal conditions or in response to Fsh (100 ng/mL) after 5 days of incubation (n = 4). Asterisks indicate significant differences (*P < 0.05; **P < 0.01) between basal or control and experimental group. (D, E, F, G, H and I) Immunofluorescent detection of Wnt5a in paraffin sections of zebrafish testis. White dotted lines delimitate the spermatogenic tubules compartment. White arrows indicate the interstitial compartment. (G, H and I) Wnt5a antibody (Ab) preabsorbed with blocking peptide (B.P.) in paraffin sections of zebrafish testis.

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    Effects of the Wnt5a antagonist (WAIIIB5) and the Wnt5a agonist (Foxy-5) on type A spermatogonia. (A) BrdU-labeling indices of type Aund and type Adiff spermatogonia in the presence of Fsh (100 ng/mL; dotted line; control condition) or in combination with WAIIIB5 (100 µM; n = 7; represented by bars). (B) Proportion of section surface area occupied by cysts containing type Aund and type Adiff spermatogonia in the presence of Fsh (100 ng/mL; dotted line; control condition) or in combination with WAIIIB5 (100 µM; n = 7; represented by bars). (C) Gene expression analysis of selected genes in adult zebrafish testis after 5 days of tissue culture in the presence of Fsh (100 ng/mL; dotted line; control condition) or in combination with WAIIIB5 (100 µM; n = 9; represented by bars). The quantification cycles (Cq) of the reference gene (elf1a) are shown in the insert. (D) BrdU-labeling indices of type Aund and type Adiff spermatogonia under basal conditions (dotted line; control condition) or in the presence of Foxy-5 (100 µM; n = 5; represented by bars). (E) Proportion of section surface area occupied by cysts containing type Aund and type Adiff spermatogonia under basal conditions (dotted line; control condition) or in the presence of Foxy-5 (100 µM; n = 5; represented by bars). (F) Gene expression analysis of selected genes in adult zebrafish testis after 5 days of tissue culture under basal conditions (dotted line; control condition) or in the presence of Foxy-5 (100 µM; represented by bars; n = 7). The quantification cycles (Cq) of the reference gene (elf1a) are shown in the insert. Results are presented as fold changes with respect to the control group (basal). Asterisks indicate significant differences (*P < 0.05; **P < 0.01) between control and experimental group.

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    Transcript levels of retinoic acid-related genes in response to Fsh and the Wnt5a antagonist (WAIIIB5) or in response to the Wnt5a agonist (Foxy-5) after 5 days of incubation. Transcript levels are expressed as fold-change compared to control condition represented by a dotted line (100 ng/mL Fsh or basal conditions), as induced by Fsh (100 ng/mL) and WAIIIB5 (100 µM; n = 8; represented by bars) or Foxy-5 (100 µM; n = 7; represented by bars). Asterisks indicate significant differences (*P < 0.05) between basal and experimental group.

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    Effect of Fsh in basal conditions or in the presence of inhibitors or Foxy-5 on Sertoli cell proliferation contacting type A spermatogonia. Number of BrdU-positive ‘free’ Sertoli cells, Sertoli cells contacting BrdU-negative and BrdU-positive Aund, and Sertoli cells contacting BrdU-negative and BrdU-positive Adiff in the presence of: (A) 25 µg/mL trilostane (dotted line; control condition) or in the presence of Fsh (100 ng/mL; n = 8; represented by bars), (B) in the presence of Fsh (100 ng/mL; dotted line; control condition) or in combination with XAV393 (10 µM; n = 7; represented by bars), (C) in the presence of Fsh (100 ng/mL, dotted line; control condition) or in combination with WAIIIB5 (100 µM; n = 7; represented by bars) and (D) in basal conditions or in the presence of Foxy-5 (100 µM; n = 5). Results are presented as fold changes with respect to the control group. Asterisks indicate significant differences (*P < 0.05; **P < 0.01) between control and experimental group.

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    Schematic representation of the effects of Fsh on spermatogenesis by modulating canonical and β-catenin signaling. Fsh stimulates the production of the β-catenin-independent ligand Wnt5a in LCs, which modulates the transcription of genes important for spermatogenesis, to stimulate the accumulation of type Aund spermatogonia and the production of new cysts (left). Fsh also stimulates differentiating divisions of spermatogonia by increasing Igf3 release from Sertoli cells (Nóbrega et al. 2015), which activates β-catenin-dependent signaling (Safian et al. 2018). A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0447.

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