Expression and localization of relaxin family peptide receptor 4 in human spermatozoa and impact of insulin-like peptide 5 on sperm functions.

Insulin-like peptide 5 (INSL5) is a member of the insulin superfamily peptide that interacts with the relaxin family peptide receptor 4 (RXFP4). Numerous recent studies have focused on the functional effects of INSL5 on fat and glucose metabolism. Although there is no evidence that the human sperm may be a candidate target of INSL5, it has been detected in mice testis and sperm. Therefore, the present study sought to analyze the localization and expression of RXFP4 on human sperm and determine the efficiency of INSL5 in human sperm. Normal semen samples were incubated in different doses and exposure time periods of INSL5. We analyzed sperm motility by computer-assisted sperm analysis (CASA) and ROS levels by flow cytometry using the MitoSOX™ Red probe. Localization and expression of RXFP4 were assayed by immunofluorescence and RT-PCR, respectively. The results confirmed the presence of RXFP4 in human spermatozoa, which localized in the neck and midpiece of sperm. Nested PCR showed the expression of RXFP4 in human sperm. INSL5 could attenuate generation of mitochondrial ROS at the 1, 10, 30, and 100nmol/L doses. This result was particularly noted in the 30nmol/L treated samples after 4h incubation. Total motility of sperm was significantly preserved in the 100nmol/L after 2h and in 30nmol/L after 4h incubation period. This study, for the first time, clarified the expression and localization of RXFP4 on human sperm and revealed the role of INSL5 in sperm motility and mitochondrial ROS generation in a dose-dependent manner.