INSL3 stimulates spermatogonial differentiation in testis of adult zebrafish (Danio rerio)

Abstract

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.

Fsh Stimulates Spermatogonial Proliferation and Differentiation in Zebrafish via Igf3

“…To estimate the relative mRNA expression levels of a number of selected genes (Supplemental Table 1) by validated real-time, quantitative PCR (qPCR) assays, samples were processed as previously described (35,36). To visualize the cellular expression sites of igf3 mRNA in zebrafish testis, in situ hybridization (ISH) was carried out as described previously (37), using DIG-labeled sense and antisense cRNA synthesized from a igf3-specific PCR product generated with primers 2878 -2879 (Supplemental Table 2). …”

A short evolutionary history of FSH-stimulated spermatogenesis

“…20 Another potential Leydig cell factor stimulating spermatogenesis could be insulin-like factor 3 (INSL3), though it has been shown to be dispensable for mouse spermatogenesis. 21 However, recent studies have shown that INSL3 can promote spermatogonial differentiation in zebrafish testis, 22,23 providing another example of evolutionary divergence in the hormonal regulation of gametogenesis. The spermatogenic effect of INSL3 thus seems to have been lost in mammals during evolution.…”

Functional insulin‐like factor 3 (INSL3) hormone‐receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility

“…), and as a germ cell proliferation factor in zebrafish (Assis et al . ). In addition, INSL3 possibly plays a role in sperm function as suggested by the detection of its receptor, relaxin family peptide receptor 2 (RXFP2) messenger RNA (mRNA) or protein in spermatozoa of rodents (Filonzi et al .…”