The Insulin-Like Factor 3 (INSL3)-Receptor (RXFP2) Network Functions as a Germ Cell Survival/Anti-Apoptotic Factor in Boar Testes

• Published in: Endocrinology (Philadelphia) 2015-04, Vol.156 (4), p.1523-1539
• Main Authors: Sagata, Dai, Minagawa, Itaru, Kohriki, Hiroshi, Pitia, Ali Mohammed, Uera, Naoto, Katakura, Yuta, Sukigara, Hiroyuki, Terada, Kei, Shibata, Masatoshi, Park, Enoch Y, Hasegawa, Yoshihisa, Sasada, Hiroshi, Kohsaka, Tetsuya
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Apoptosis; Apoptosis - genetics; BAX protein; Bcl-2 protein; bcl-2-Associated X Protein - genetics; bcl-2-Associated X Protein - metabolism; Caspase 3 - genetics; Caspase 3 - metabolism; Caspase-3; Cell survival; Cell Survival - genetics; Down-Regulation; Fetuses; Gene expression; Germ cells; Germ Cells - metabolism; HEK293 Cells; Humans; Immunization; Immunology; Insulin; Insulin - genetics; Insulin - metabolism; Leydig cells; Male; Meiosis; mRNA; Neutralization; Proteins - genetics; Proteins - metabolism; Receptors; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Relaxin; Relaxin-like factor; Signal Transduction - genetics; Sperm; Survival; Sus scrofa; Testes; Testis - metabolism; Up-Regulation; XIAP protein
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2014-1473

Relaxin-like factor, commonly known as insulin-like factor (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. The study aimed to identify a relaxin family peptide receptor 2 (RXFP2)–specific antibody suitable for immunological approaches, analyze which testicular germ cell types express RXFP2, and clarify its expression dynamics in the boar testis. In addition, the function of INSL3-RXFP2 signaling on the germ cells was explored by neutralizing INSL3 using long-term active immunization. Samples were collected from Duroc boars, and a commercially available RXFP2-specific antibody directed against the human RXFP2 endodomain was identified by characterizing its specificity in HEK-293 cells expressing mouse RXFP2, and by demonstrating the suitability for analyzing RXFP2 expression in porcine tissues. RXFP2 mRNA and protein were both localized mainly in meiotic and post-meiotic germ cells, but not in Leydig cells. Functional RXFP2, which enables INSL3 to bind, was detected as an ∼85-kDa band, which increased in intensity from the pubertal stage onward. Interestingly, INSL3 immunization significantly reduced testis weight and induced a 4-fold increase in the frequency of apoptotic germ cells, which was associated with the up-regulation of pro-apoptotic caspase-3 (CASP3) and BAX, and the down-regulation of anti-apoptotic XIAP and BCL2, and a substantial reduction in sperm concentration. These results revealed that RXFP2 was expressed in boar meiotic and post-meiotic germ cells, where INSL3 neutralization led to increased germ cell apoptosis and reduced sperm output, suggesting that INSL3 acts as a survival/anti-apoptotic factor in maintaining sperm production.