Identification and Characterization of Functional Nongenomic Progesterone Receptors on Human Sperm Membrane1

Abstract

The presence of functional nongenomic progesterone (P) receptors in human spermatozoa has been investigated by equilibrium binding studies in intact spermatozoa, ligand blot and Western blot analysis of sperm lysates, as well as determination of the effects of the steroid on sperm intracellular Ca 2ϩ concentrations. Binding experiments were performed using progesterone-11␣-glucuronide-[125 I]iodotyramine as tracer. Computer analysis of competition curves using different steroids as competitors indicated the presence of two distinct binding sites for P. The high affinity site (K d in the nanomolar range) appears to be specific for P, whereas the low affinity one (K d in the micromolar range) binds with equal affinity 11␤-hydroxyprogesterone (11␤OHP) and 17␣-hydroxyprogesterone (17␣OHP). A significant correlation exists among affinity constants (as determined by binding studies) and EC 50 values for the effects of P, 11␤OHP, and 17␣OHP on intracellular Ca 2ϩ in fura-2-loaded spermatozoa, strongly indicating the involvement of P-binding sites in the biological effect of the steroid. In particular, dose-response curves for P were biphasic, with an EC 50 in the nanomolar range and another in the micromolar range. Conversely, curves for 11␤OHP and 17␣OHP were monophasic, with an EC 50 just in the micromolar range. Ligand blot analysis of sperm total lysates performed with peroxidase-conjugated P revealed the presence of two binding proteins of 54 and 57 kDa that were specific for P. Indeed, peroxidase-conjugated P binding was blocked by the simultaneous presence of the unconjugated steroid. Using ␣c262 antibody, which is directed against the P-binding domain of genomic receptor, we detected two proteins of similar molecular mass (54 and 57 kDa), whereas using antibodies directed against the DNA-binding and N-terminal domains of the genomic P receptors, the two proteins were not detected. In addition, p54 and p57 appear to be mostly localized in sperm membranes and virtually absent in the cytoplasm. The involvement of these proteins in the biological effects of P is indicated by the strong inhibitory effect of ␣c262 on P-induced acrosome reaction of capacitated human spermatozoa. (J Clin Endocrinol Metab 83: [877][878][879][880][881][882][883][884][885] 1998) A CROSOME reaction (AR) is an essential event of the process of fertilization. In mammals, physiological AR occurs after interaction with the zona pellucida, the glycoprotein coat overlying the egg plasma membrane (1), and is mediated by specific sperm receptor (2). During the process of fertilization, AR can also be induced by progesterone (P), which is present in high concentrations in the cumulus matrix that surrounds the oocyte and in the follicular fluid (3, 4). In particular, in human spermatozoa, P induces a rapid influx of calcium, calcium-dependent phosphoinositide hydrolysis, an increase in tyrosine phosphorylation of sperm proteins, and chloride efflux, ultimately leading to stimulation of AR (for review, see Ref. 5). In addition, in vitro st...

The Novel Isoform of the Progesterone Receptor cDNA in the Human Testis and Detection ofIts mRNA in the Human Uterine Endometrium

“…However, all of the proteins which are possibly translated from the PR isoform S mRNA have various lengths of the carboxy terminal part of the progesterone-binding domain of the iPR and lack the amino terminal and DNA-binding domains. This putative structure of the proteins encoded by the PR isoform S mRNA correspond to that of the mPR in the Sp as indicated in previous reports which revealed that the sperm mPR possessed the carboxy terminal part of the progesterone-binding domain but not the amino terminal- and DNA-binding domains [7, 9, 10]. On the other hand, the calculated molecular weight of the longest protein encoded by the isoform S transcript, which utilizes codon 691 (indicated by the double underlining in fig.…”

“…1) of iPR as the start codon, is approximately 29 kD. The weight was lower than that in the two previous reports; Sabeur et al [7]indicated approximately 54 and 57 kD [7], and Luconi et al [9]reported approximately 50 and 52 kD [9]. Therefore, we cannot conclude that the protein(s) encoded by the PR isoform S mRNA is the sperm mPR at the present time.…”

“…It has been demonstrated that the transient increase in the intracellular calcium ion triggers the acrosome reaction [3, 4], and that progesterone can induce calcium transit in vitro via the cell surface membrane progesterone receptor (mPR) in the human Sp [5, 6, 7, 8, 9]. Previous reports have shown that the mPR on the Sp can be recognized by the antibody (C-262) against the carboxy terminal region of the progesterone-binding domain of the intracellular PR (iPR) [7, 9], but not by the antibody against the amino-terminal and DNA-binding domains of the iPR [10]. Therefore, it is considered that the mPR in the Sp possesses the same structure as the progesterone-binding domain of the iPR.…”

Detection of progesterone receptors in human spermatozoa and their correlation with morphological and functional properties

“…Currently, three types of receptors present in the human spermatozoa have been described to mediate the action of P in these cells: (1) a GABA A -like receptor Winstrom & Meizel (1993); (2) a receptor with partial homology to the intracellular progesterone receptor Luconi et al (1998), and (3) a receptor with partial homology to the liver porcine endomembrane receptor Buddhikot et al (1999). Binding of P to its receptor stimulates the AR through a mechanism involving a rapid and transient increase in intracellular Ca 2+ concentration, because of Ca 2+ in¯ux from the extracellular medium Blackmore et al (1990), Thomas & Meizel (1989).…”

Binding of progesterone to cell surfaces of human granulosa-lutein cells

“…In human decidua two bands were observed, at 45 and 36 kDa corresponding to different isoforms of the progesterone receptor [51]. A non-classical form of PR involved in the opening of calcium channels in the mitochondrial and cellular membranes has been characterized in the cytoplasmic membranes of human sperm [52,53] with molecular weights ranging from 25 to 85 kDa [49]. The well established membrane progesterone receptor found in fish was cloned and found to have a molecular weight of 40 kDa [54].…”