Expression of insulin-like factor 3 hormone-receptor system in the reproductive organs of male goats

Abstract

Relaxin-like factor (RLF), generally known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development. However, its role in adult males is not fully understood. We investigate the function of INSL3 in male Saanen goats by identifying cell types expressing its receptor, relaxin/insulin-like family peptide receptor (RXFP)2 and by characterizing the developmental expression pattern of INSL3 and RXFP2 and the binding of INSL3 to target cells in the male reproductive system. A highly specific RXFP2 antibody that co-localizes with an anti-FLAG antibody in HEK-293 cells recognizes RXFP2-transcript-expressing cells in the testis. INSL3 and RXFP2 mRNA expression is upregulated in the testis, starting from puberty. INSL3 mRNA and protein expression has been detected in Leydig cells, whereas RXFP2 mRNA and protein localize to Leydig cells, to meiotic and post-meiotic germ cells and to the epithelium and smooth muscle of the cauda epididymis and vas deferens. INSL3 binds to all of these tissues and cell types, with the exception of Leydig cells, in a hormone-specific and saturable manner. These results provide evidence for a functional intra- and extra-testicular INSL3 ligand-receptor system in adult male goats.

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 goat (Pitia et al . ) tissues. In contrast, the INSL3 antibody (RLF‐A‐Ab808) was generated in our laboratory in New Zealand White rabbits against a synthetic peptide of 15 amino acid residues of the A‐domain, which shared 100% amino acid homology among porcine, bovine, ovine and caprine INSL3 complementary DNAs (cDNAs).…”

“…In contrast, the INSL3 antibody (RLF‐A‐Ab808) was generated in our laboratory in New Zealand White rabbits against a synthetic peptide of 15 amino acid residues of the A‐domain, which shared 100% amino acid homology among porcine, bovine, ovine and caprine INSL3 complementary DNAs (cDNAs). The specificity of this antibody has been described elsewhere (Siqin et al 2010a,b, 2013; Minagawa et al ; Pitia et al ).…”

“…Immunohistochemistry was performed using the Vectastain Universal avidin‐biotin complex (ABC) Elite kit (Vector Laboratories, Burlingame, CA, USA) with the biotin blocking kit (Biotin Blocking System; DakoCytomation, Carpinteria, CA, USA) as previously described (Siqin et al 2010a,b, 2013; Pitia et al ). Briefly, deparaffinized 4‐μm testicular sections were incubated with anti‐INSL3 immunoglobulin G (IgG) (0.5 μg/mL), anti‐RXFP2 IgG (5 μg/mL) or antisera preabsorbed with synthetic human INSL3 (hINSL3) (25 μg/mL) (6292 Da; Phoenix Pharmaceuticals, Burlingame, CA, USA) for anti‐INSL3 (Siqin et al ) or recombinant RXFP2 endodomain peptide (400 μg/mL) for anti‐RXFP2 (Pitia et al ; Sagata et al . ), followed by biotinylated secondary antibody and ABC solution.…”

“…; Pitia et al . ), suggesting that intra‐testicular INSL3 hormone‐receptor system operates in goat germ cells (Pitia et al . ).…”

“…), suggesting that intra‐testicular INSL3 hormone‐receptor system operates in goat germ cells (Pitia et al . ). However, it is presently unknown whether RXFP2 exists in the testicular germ cells in bulls and rams, despite the fact that Leydig cells are the sole source of INSL3 mRNA and protein in these ruminants (Bathgate et al .…”