Dynamics of insulin-like factor 3 and its receptor expression in boar testes

Abstract

Relaxin-like factor (RLF), now mainly known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. As a major step toward understanding the as-yet-unknown function of INSL3 in boars, this study aimed to develop a time-resolved fluoroimmunoassay for boar INSL3, characterize the dynamics of INSL3 expression during development, and demonstrate the expression of the INSL3 hormone-receptor system in the testis. All samples were collected from Duroc boars. The sensitivity of the assay system established was 8.2 pg/well (164 pg/ml), and no cross-reactivity with other hormones, such as porcine relaxin, was observed. Circulating INSL3 was shown to increase progressively during development. INSL3 secreted from the Leydig cells was released not only into the blood circulation but also into the interstitial and seminiferous compartments in sufficient concentrations. A testicular fractionation study revealed that its receptor RXFP2 transcripts were expressed mainly in testicular germ cells. In addition, INSL3 bound to the germ cell membranes in a hormone-specific and saturable manner. These results reveal that INSL3 secreted into the interstitial compartment from the Leydig cells is transported into the seminiferous compartments, where its receptor RXFP2 is expressed mainly in the germ cells to which INSL3 binds, suggesting that INSL3 functions as a paracrine factor on seminiferous germ cells.

Expression profiles of relaxin family peptides and their receptors indicate their influence on spermatogenesis in the domestic cat (Felis catus)

“…A significant difference between prepuberty and the onset of puberty was detected by Minagawa et al for porcine testis. During further development, a slight but not statistical decrease was observed in the boar [55]. This was not confirmed in cats, in which we observed large variability within each postpubertal group; consequently, the values of postpubertal groups did not differ from puberty or from prepuberty.…”

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

“…Morphometric analyses for INSL3‐ or RXFP2‐expressing cells in fertile and subfertile bulls were performed according to our previously employed method (Minagawa et al . ; Pitia et al ). Briefly, images of at least 20 different fields per animal (fertile, n = 2; subfertile, n = 2) were obtained using a BZ‐9000 microscope (Keyence, Tokyo, Japan).…”

“…Synthetic human INSL3 peptide (Phoenix Pharmaceuticals) was labeled with 4′‐(4′‐(4,6‐dichloro‐1,3,5‐triazin‐2‐ylamino)‐4‐biphenylyl)‐2,2′:6′,2″‐terpyridine‐6,6″‐diylbis (methyliminodiacetato) (DTBTA)‐europium (Eu) (Tokyo Chemical Industry, Tokyo Japan) according to Minagawa et al (). Its bioactivity was confirmed by a cell‐based bioassay as previously described (Minagawa et al .…”

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

“…We also showed that receptor RXFP2 is expressed in meiotic and post‐meiotic germ cells, where INSL3 neutralisation by active immunisation with INSL3 increased germ cell apoptosis and reduced sperm output and quality, suggesting that INSL3 functions as a germ cell survival/anti‐apoptotic factor in maintaining sperm production (Sagata et al., ). However, a study using conditional mutant male mice reported that INSL3 was dispensable for germ cell survival (Huang, Rivas, & Agoulnik, ), which is in stark contrast to findings in rat (Del Borgo et al., ; Kawamura et al., ), pig (Minagawa et al., ; Sagata et al., ) and fish (Assis et al., ) models.…”

“…Boars differ from other mammals in terms of testicular morphology and function, with a higher volume percentage of Leydig cells per whole testis (Peyrat, Meusy‐Dessolle, & Garnier, ) than rodents (Ewing et al., ; Mendis‐Handagama, ) and a higher level of oestrogen production (Allrich, Christenson, Ford, & Zimmerman, ; Minagawa et al., ). We previously demonstrated that boar INSL3, like rat homolog (Anand‐Ivell, Heng, Hafen, Setchell, & Ivell, ), is secreted into the circulation and interstitial compartment and then transported into the seminiferous compartments by unknown mechanisms (Minagawa et al., , ). We also showed that receptor RXFP2 is expressed in meiotic and post‐meiotic germ cells, where INSL3 neutralisation by active immunisation with INSL3 increased germ cell apoptosis and reduced sperm output and quality, suggesting that INSL3 functions as a germ cell survival/anti‐apoptotic factor in maintaining sperm production (Sagata et al., ).…”