title: The insulin-like factor 3 (INSL3)-receptor (RXFP2) network functions as a germ cell survival/anti-apoptotic factor in boar testes.
abstract: 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.
authors.affiliation: Laboratory of Animal Reproduction and Physiology (D.S., I.M., H.K., A.M.P., N.U., Y.K., H.Su., T.K.), Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan; Division of Animal Resource production (I.M., A.M.P., T.K.), The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan; Shizuoka Swine and Poultry Experimental Station (K.T., M.S.), Kikugawa 439-0037, Japan; Laboratory of Biotechnology (E.Y.P.), Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan; Animal Science High-Tech Research Center (Y.H.), School of Veterinary Science, Kitasato University, Towada 034-8628, Japan; and Laboratory of Animal Reproduction (H.Sa.), School of Veterinary Science, Kitasato University, Towada 034-8628, Japan.
authors.forename: Dai
authors.initials: D
authors.lastname: Sagata
authors.forename: Itaru
authors.initials: I
authors.lastname: Minagawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kohriki
authors.forename: Ali Mohammed
authors.initials: AM
authors.lastname: Pitia
authors.forename: Naoto
authors.initials: N
authors.lastname: Uera
authors.forename: Yuta
authors.initials: Y
authors.lastname: Katakura
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sukigara
authors.forename: Kei
authors.initials: K
authors.lastname: Terada
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Shibata
authors.forename: Enoch Y
authors.initials: EY
authors.lastname: Park
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sasada
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kohsaka
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics*; D015536:Down-Regulation; D005854:Germ Cells / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D008297:Male; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D034421:Sus scrofa; D013737:Testis / Q000378:metabolism*; D015854:Up-Regulation; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; D043562:Receptors, G-Protein-Coupled; D051028:bcl-2-Associated X Protein; D053148:Caspase 3