ABSTRACT
Recent studies disclosed a cross talk between testis and bone. By the action of LH, Leydig cells are able to modulate bone metabolism through testosterone and insulin-like factor 3. Moreover, LH modulates the Leydig expression of CYP2R1, the key enzyme involved in vitamin D (Vit D) 25-hydroxylation. However, pathways regulating CYP2R1 expression have been poorly investigated. The cross talk from the bone to the testis of the vitamin D 25-hydroxylase CYP2R1 involves osteocalcin (OC), which is produced by the osteoblasts and stimulates the production of testosterone by the Leydig cells through its putative receptor GPRC6A, a cation-sensing G-protein-coupled receptor. The aim of this study was to investigate the possible action of OC on CYP2R1 expression and 25-hydroxy Vit D (25-OH Vit D) production in a mouse Leydig cell line (MA-10). After confirmation of the expression of GPRC6A by MA-10, we found that stimulation with either human chorionic gonadotropin or uncarboxylated-OC (ucOC) increases CYP2R1 protein expression in a dose-dependent manner and, in turn, increases the release of 25-OH Vit D in culture medium. This effect was abolished by receptor blockade with, respectively, anti-LH receptor and anti-GPRC6A antibodies. Moreover, both agonists converged to phosphorylation of Erk1/2 by a likely differential action on second messengers. Human chorionic gonadotropin induced slow "tonic" increase of intercellular calcium and accumulation of cAMP, whereas ucOC mainly induced phasic increase of cell calcium. Supporting these findings, we found that serum ucOC positively correlated with 25-OH Vit D levels in 40 overweight male patients and 21 controls. Altogether, our results suggest that OC contributes with LH to 25-OH Vit D production by Leydig cells.
Subject(s)
Leydig Cells/drug effects , Osteocalcin/pharmacology , Receptors, G-Protein-Coupled/physiology , Vitamin D/analogs & derivatives , Adult , Animals , Carbon Dioxide , Case-Control Studies , Cell Line , Cholestanetriol 26-Monooxygenase/genetics , Cholestanetriol 26-Monooxygenase/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Humans , Leydig Cells/metabolism , Male , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Mice , Middle Aged , Osteocalcin/blood , Osteocalcin/chemistry , Vitamin D/blood , Vitamin D/metabolismABSTRACT
STUDY QUESTION: What are the dynamics of zinc (Zn) trafficking in sperm, at the testicular, epididymal and ejaculate levels? SUMMARY ANSWER: Zn transporters are peculiarly expressed in the cells of the germ line and Zn uptake is maximal at the post-epididymal phase, where Zn is involved in the regulation of sperm functions. WHAT IS KNOWN ALREADY: Zn is known to influence several phases of sperm life, from germ cell development to spermiation. Zn trafficking across the membrane is allowed by specific families of transporters known as the ZnTs, which are involved in effluent release, and the Zips, which mediate uptake. STUDY DESIGN, SIZE, DURATION: We enrolled 10 normozoospermic healthy participants in an infertility survey programme, as well as 5 patients affected by testicular germ cell cancer, and 18 patients presenting with obstructive azoospermia, without mutations of the CFTR gene, and undergoing assisted reproductive technologies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The research study was performed at our University Clinic. Semen samples, or biopsies or fine needle aspirates from the testis or epididymis, were obtained from each of the participants. Protein expression of main members of the ZnT and Zip families of Zn transporters was examined in human testis and epididymis samples by immunofluorescence. Quantification of sperm Zn content was performed by flow cytometry, atomic absorption spectrometry (AA) and autometallography. MAIN RESULTS AND THE ROLE OF CHANCE: Intratubular cells of the germ line displayed a high redundancy of Zip family members involved in Zn uptake, while ZnT transporters were more represented in epididymis. Testicular and epididymal spermatozoa contained less Zn than ejaculated spermatozoa (2.56 ± 0.51 and 12.58 ± 3.16 versus 40.48 ± 12.71 ng Zn/10(6)cells, respectively). Gain of hypermotility and acrosomal reaction were significantly linked to the loss of Zn content in ejaculated spermatozoa. LIMITATIONS, REASONS FOR CAUTION: This was an ancillary study performed on a small cohort of normozoospermic subjects. Although these results clarify the Zn trafficking during different phases of sperm life, no conclusive information can be drawn about the fertilizing potential of sperm, and the overall pregnancy outcomes, after Zn supplementation. WIDER IMPLICATIONS OF THE FINDINGS: Our data disclose the dynamics of Zn trafficking during over the sperm lifespan. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought or obtained for this study. No conflict of interest is declared.
Subject(s)
Carrier Proteins/metabolism , Infertility, Male/metabolism , Spermatozoa/metabolism , Zinc/metabolism , Adolescent , Adult , Humans , Male , Sperm Retrieval , Young AdultABSTRACT
Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17betaE(2)) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17betaE(2) induced a rapid increase of intracellular calcium (Ca(2+)) concentrations dependent on an influx of Ca(2+) from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17betaE(2) showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca(2+) in the extracellular medium since it was absent in Ca(2+) free-medium. When sperm were pre-incubated in the presence of the K(+) channel inhibitor tetra-ethylammonium, the 17betaE(2) induced plasma membrane hyperpolarization was blunted suggesting the involvement of K(+) channels in the hyperpolarizing effects of 17betaE(2). Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17betaE(2) inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17betaE(2) were specific since its inactive steroisomer 17alphaE(2) was inactive. Furthermore the effects of 17betaE(2) were not inhibited by tamoxifen, an antagonist of the classic 17betaE(2) intracellular receptor.
