The role of adrenomedullin in regulating human sperm motility

Abstract

INTRODUCTION: Adrenomedullin (ADM) is a highly conserved member of the calcitonin/calcitonin-gene-related peptide (CRGP)/amylin peptide family. In reproduction, ADM is known to regulate uterine contraction, fetal development and human placentation. ADM acts through a dimeric G-protein-coupled receptor system consisting of a calcitonin-receptor-like receptor (CRLR) and a receptor-activity-modifying protein (RAMP). CRLR coupled to RAMP2 or RAMP3 binds ADM, while CRLR-RAMP1 complex binds more to CGRP than ADM. We hypothesized that ADM modulated human sperm motility. This is based on the observations: (1) the concentration of ADM in seminal plasma is 10-fold higher than that in the systemic circulation; (2) the up-regulation of oviductal ADM expression in mice by spermatozoa; and (3) the existence of ADM in the female reproductive tract through which the spermatozoa must pass before fertilization. MATERIALS AND METHODS: Spermatozoa were obtained from men attending the infertility clinic. A routine semen analysis was performed according to World Health Organization (WHO, 1999). Samples with normal semen parameters were processed by Percoll density gradient centrifugation. The high and low motility fractions were obtained from the 90 and 45% Percoll layers respectively. The binding of Alexa Fluor-555-conjugated ADM to spermatozoa was visualized by cytochemical staining. Localization of ADM receptor components in spermatozoa was demonstrated by immunostaining and western blotting. Hobson Sperm Tracker System was used to study the sperm motility parameters after ADM treatment. Since meta-analysis had shown that the spermatozoa-zona pellucida (ZP) binding had a high predictive power for fertilization outcome, therefore, ZP binding capacity will also be studied by hemizona binding assay. RESULTS: ADM significantly (P<0.05) enhanced the flagellar beating and ZP-binding capacity of Percoll-processed spermatozoa. At the concentration found in the seminal plasma, it increased the average path velocity, mean straight line velocity, head beat cross frequency, progressive motility and hemizona binding index of human spermatozoa when compared to the control. This action of ADM was completely blocked by the administration of 10× human ADM22-52 (antagonist of ADM receptors), but not by CGRP8–37 (antagonist of CGRP receptors), suggesting that the ADM receptor plays a predominant role in mediating the observed effects. Alexa fluor-555-labeled ADM bound to 91.2±2.1% of the Percoll-processed spermatozoa. Strong ADM staining was observed over the neck and the midpiece of spermatozoa. Western blotting and immunohistochemical staining demonstrated for the first time the presence of CRLR and RAMP1-3 on both the neck and the midpiece in human spermatozoa. Compared with the high-motility fraction, the low-motility fraction after Percoll processing had significantly lower sperm motility and normal form. They also had significantly (P<0.05) lower level of ADM binding and CRLR, RAMP1 and RAMP3 expressions. Significant (P<0.05) decrease in ADM binding and CRLR and RAMP3 expressions were also observed in spermatozoa from semen sample with ≤30% progressive motile spermatozoa (WHO, grade A + grade B) when compared to those with ≥50% progressive motile spermatozoa. CONCLUSION: Sperm motility is one of the most important predictors of fertility. It has also been found to be highly predictive of intrauterine insemination success and in vitro/in vivo fertilization rates. The present results suggest that ADM is a factor in the male sex-accessory gland stimulating the progressive motility of human spermatozoa via its classical receptor, CRLR/RAMP. The sperm-ZP binding capacity, an indication of successful fertilization, is also increased by ADM treatment. A significantly negative correlation between ADM binding and sperm motility was consistent with the results. The present investigation provides a physiological basis on possible use of ADM for enhancing sperm motility and the fertilization success. In addition, ADM receptor abundance could be a possible indicator of infertility.