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

Abstract

Insulin-like factor 3 (INSL3) is essential for fetal testis descent, and has been implicated in the testicular and sperm functions in adult males; however, similar functions in domestic ruminants remain largely unknown. This study investigated the functional INSL3 hormone-receptor system in adult ruminant testes and spermatozoa, and explored its potential to diagnose the fertility of sires. Testes and spermatozoa were obtained from fertile bulls, rams and he-goats, whereas subfertile testes and spermatozoa were obtained only from bulls. As expected, INSL3 was visualized in Leydig cells, while we clearly demonstrated that the functional receptor, relaxin family peptide receptor 2 (RXFP2), enabling INSL3 to bind was identified in testicular germ cells and in the sperm equatorial segment of bulls, rams and he-goats. In comparison to fertile bulls, the percentage of INSL3- and RXFP2-expressing cells and their expression levels per cell were significantly reduced in the testes of subfertile bulls. In addition, the population of INSL3-binding spermatozoa was also significantly reduced in the semen of subfertile bulls. These results provide evidence for a functional INSL3 hormone-receptor system operating in ruminant testes and spermatozoa, and its potential to predict subfertility in sires.

Recent Advances in Research on the Hormone INSL3 in Male Goats

“…( Figure 8B and C). Especially, RXFP2 expression in meiotic and postmeiotic germ cells in goats are fundamentally consistent with our studies in boars [9,35], bulls, and rams [36], as well as other reports in humans [37] and rats [8].…”

“…With the help of immunofluorescence using RXFP2-specific antibody, we found that RXFP2 was expressed in the equatorial segment and midpiece of goat spermatozoa (Figure 10A). Similar findings are also found in bull and ram spermatozoa [36].…”

“…Furthermore, we investigated the functionality of RXFP2 in these spermatozoa using in situ ligand binding assay with DTBTA-Eu-INSL3 and clearly showed the presence of functional RXFP2 enabling INSL3 to bind with the equatorial segment of he-goat (Figure 10B), bull, and ram spermatozoa, suggesting that a functional INSL3 hormone-receptor system operates in the equatorial segment of ruminant spermatozoa [36]. It has been reported to date that RXFP2 mRNA is detected in the spermatozoa of rats [41] and boars [42] and its protein is distributed throughout the entire spermatozoon in boars [43].…”

Characterizing the reproductive transcriptomic correlates of acute dehydration in males in the desert-adapted rodent,Peromyscus eremicus

“…Indeed, recent research has investigated the utility of Insl3 as an indicator of mammalian fertility (e.g. in humans: [110]; in bulls: [111]). Insl3 is also critical for the first phase of testicular descent, the transabdominal phase, which occurs during fetal development in rodents; but Insl3 does not appear to be involved in the inguinoscrotal phase which happens in sexually immature or inactive male rodents (reviewed in [112]).…”

Evidence for the role of INSL3 on sperm production in boars by passive immunisation

“…Deparaffinised sections (4 μm) were immunostained with anti‐RXFP2 antibody (5 μg ml −1 ; GTX108235; GeneTex, Alton Parkway Irvine, Ca, USA) or antiserum pre‐absorbed with recombinant RXFP2 endodomain (400 μg ml −1 ; Kato et al., ) followed by peroxidase labelled polymer conjugated to goat anti‐rabbit IgG (Dako, Carpinteria, CA), according to our method (Minagawa et al., ). Anti‐RXFP2 antibody was directed against human RXFP2 endodomain, and its specificity has been described elsewhere (Pitia et al., , ; Sagata et al., ). Signals were visualised using diaminobenzidine (Dako), and the sections were counterstained with haematoxylin and examined under a BX50 Olympus microscope equipped with a CCD camera and Instudio V105 software (Pixera).…”