Partial cDNA sequence of a relaxin‐like factor (RLF) receptor, LGR8 and possible existence of the RLF ligand‐receptor system in goat testes

Abstract

Relaxin-like factor (RLF), also known as insulin-like factor 3 (INSL3), is produced by testicular Leydig cells, but its specific receptor LGR8 (leucine-rich repeat family of G-protein-coupled receptor 8) has not been identified in goats. This study aimed to identify complementary DNA (cDNA) sequences of goat LGR8, and characterize the expression of both RLF and LGR8 in goat testes by RT-PCR and immunohistochemistry. Testes were collected from immature (3-month-old) and mature (24-month-old) Saanen goats, and partial cDNA sequences of the goat homologue of human LGR8 were identified. The sequence encoded a reduced peptide sequence of 167 amino acids, which corresponded to transmembrane regions 2 through 5, followed by the beginning of intracellular loop 3 of human LGR8. Expression of both LGR8 and RLF genes was drastically increased in mature testes compared with immature ones. Although RLF protein was restricted to Leydig cells, LGR8 protein was detected in both Leydig cells and seminiferous epithelial cells (possibly germ cells and Sertoli cells). These results reveal a possible existence of the RLF-LGR8 ligand-receptor system within the goat testis, suggesting that RLF may play a role in testicular function through LGR8 on Leydig cells and seminiferous epithelial cells in an autocrine and/or paracrine manner.

Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars

“…This finding is in good agreement with that of human RLF/INSL3, as shown by Ferlin and Foresta [31], who revealed that RLF/INSL3 is an endocrine factor. In contrast, receptor RXFP2 mRNA and/or protein have been shown to be expressed in Leydig cells and seminiferous tubule epithelial cells in adult testes of some species, including humans [5,17,32,33]. There is also evidence that RLF/INSL3 suppresses germ cell apoptosis in the rat testis [5], whereas the treatment of mouse Leydig TM3 cells with RLF/INSL3 in vitro induces cell proliferation [33].…”

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

“…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 .…”

The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells

“…The INSL3 secreted into the circulation is likely to function as an endocrine factor (Ferlin and Foresta , 2005 ), although its extra-testicular targets are undetermined. In contrast, receptor RXFP2 mRNA and/or protein have recently been shown to be expressed in Leydig cells and seminiferous germ cells in adult testes of some species (Kawamura et al , 2004 ;Anand -Ivell et al, 2006 ;Feng et al , 2007 ;Filonzi et al , 2007 ;Koeva et al , 2008 ), including goats (Siqin et al , 2010b ). Taken together with previous findings, single-chain INSL3 secreted into body fluids may function as an autocrine and/or paracrine factor in Leydig cells and seminiferous germ cells in goat testes.…”