This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. Data Availability All relevant data are within the paper and its Supporting Information files.. DNA-containing cell population for the different times N-Dodecyl-β-D-maltoside presented as relative expression mean SEM of six experiments (E). *P<0.05 vs. control; **P<0.01 vs. control.(JPG) pone.0152162.s002.JPG (205K) GUID:?5759CE75-1830-483D-B18A-4BC8FBA5621C S3 Fig: Effect of selective DOR-agonist morphine on spermatogenic cell cycle. Flow cytometry histogram of spermatogenesis cell cycle measured by propidium iodide in control (green) and DPDPE-treated samples (red: 10?6 M) for short- (A) and long-term exposure (B). Representative plot from six experiments. Changes on percentages of 1c, 2c and 4c DNA-containing cell populations for short- (C) and long-term exposure (D) after DPDPE treatment. Percentage difference between treatment and control of the integrated area of the 1c, 2c and 4c DNA-containing cell population for the different times presented as relative expression mean SEM of six experiments (E). *P<0.05 vs. control; **P<0.01 vs. control.(JPG) pone.0152162.s003.JPG (194K) GUID:?738D3393-00C9-45F7-AFBD-D5A608844314 S4 Fig: Effect of selective KOR-agonist morphine on spermatogenic cell cycle. Flow cytometry histogram of spermatogenesis cell cycle measured by propidium iodide in control (green) and N-Dodecyl-β-D-maltoside "type":"entrez-nucleotide","attrs":"text":"U50488","term_id":"1277101","term_text":"U50488"U50488-treated samples (red: 10?6 M) for short- (A) and long-term exposure (B). Representative plot from six experiments. Changes on percentages of 1c, 2c and 4c DNA-containing Adamts5 cell populations for short- (C) and long-term exposure (D) after “type”:”entrez-nucleotide”,”attrs”:”text”:”U50488″,”term_id”:”1277101″,”term_text”:”U50488″U50488 treatment. Percentage difference between treatment and control of the integrated area of the 1c, 2c and 4c DNA-containing cell population for the different times presented as relative expression mean SEM of six experiments (E). *P<0.05 vs. control; **P<0.01 vs. control.(JPG) pone.0152162.s004.JPG (197K) GUID:?91BD0DD9-5D5E-4012-BFA6-B6762D497277 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract The presence of endogenous opioid peptides in different testicular cell types has been extensively characterized and provides evidence for the participation of the opioid system in the regulation of testicular function. However, the exact role of the opioid system during the spermatogenesis has remained controversial since the presence of the mu-, delta- and kappa-opioid receptors in spermatogenic cells was yet to be demonstrated. Through a combination of quantitative real-time PCR, immunofluorescence, immunohistochemistry and flow cytometry approaches, we report for the first time the presence of active mu-, delta- and kappa-opioid receptors in mouse male germ cells. They show an exposition time-dependent response to opioid agonist, hence suggesting their active involvement in spermatogenesis. Our results contribute to understanding the role of the opioid receptors in the spermatogenesis and could help to develop new strategies to employ the opioid system as a biochemical tool for the diagnosis and treatment of male infertility. Introduction Over 80 million people worldwide experience infertility and over one-third of infertility cases are due to male factors. Male infertility often reflects faults in spermatogenesis but little is known about the underlying causes, mostly because mechanisms and pathways involved in spermatogenesis remain unknown. The most well-known physiological effect associated with endogenous opioid peptides (EOPs) is their efficacy in pain reduction or analgesia, although their effect on a variety of other physiological functions has become apparent in recent years [1]. In particular, evidence of the widespread presence of EOPs and receptors in different organs and tissues of the male reproductive system indicates that EOPs likely participate in the regulation of male reproductive function [2]. EOPs are involved in cell communication and exert their action through G-protein-coupled opioid receptors. There are three principal types of opioid receptors: the mu-opioid (MOR), delta-opioid (DOR) and kappa-opioid (KOR) receptors [3]. Later, the orphanin 1 (ORL1) receptor (also known as the nociceptin receptor) was discovered and found to have high homology with opioid receptors [4].Our group described the presence of MOR, DOR and KOR and the other components of the opioid system in human sperm cells which seem to be functionally involved in control of human sperm motility [5C9]. However, the presence of MOR, DOR and KOR in male germ cells and their roles during spermatogenesis remain unknown. Spermatogenesis is a highly coordinated developmental process characterized by mitotic, meiotic and haploid differentiation phases. Spermatogenesis is initiated in the basal compartment of the seminiferous epithelium N-Dodecyl-β-D-maltoside by spermatogonial stem cells that proliferate and differentiate into type A1 spermatogonia. Type A1 spermatogonia undergo a series of synchronized mitotic divisions, giving rise to type B spermatogonia, which enter the meiotic phase of spermatogenesis as primary spermatocytes [10]. Meiosis is characterized.