Identification and Characterization of a Novel Functional Estrogen Receptor on Human Sperm Membrane That Interferes with Progesterone Effects

Abstract

The presence of a novel functional estrogen receptor on the human sperm surface has been demonstrated by using different experimental approaches. Ligand blot analysis of sperm lysates, using peroxidase-conjugated estradiol as probe, identified a specific estradiol-binding protein of approximately 29-kDa apparent molecular mass. The same protein band was also revealed by using αH222 antibody, which is directed against the steroid binding domain of the genomic estrogen receptor. The biological effects of estrogen receptor were investigated by analyzing calcium fluxes, tyrosine phosphorylation, and acrosome reaction (AR) in response to 17β-estradiol (17βE2) and by measuring the steroid influence on calcium and AR in responses to progesterone (P), a well-known physiological stimulus for human spermatozoa. Our results demonstrate that 17βE2 induces a rapid and sustained increase of intracellular calcium concentrations ([Ca2+]i). This effect is totally dependent on the presence of extracellular calcium, because it is completely abolished in a calcium-depleted medium. The dose-response curve for calcium increase to 17βE2 is biphasic with a first component in the nanomolar range (effective concentration 50 = 0.60 ± 0.12 nmol/L) and a second component in the micromolar range (EC50 = 3.80 ± 0.26 μmol/L). 17βE2 stimulates tyrosine phosphorylation of several sperm proteins, including the 29-kDa protein band, and determines a reduction of calcium response to P, finally resulting in inhibition of P-stimulated sperm AR. Conversely, no direct effect of 17βE2 is observed on AR. 17βE2 effects on calcium are clearly mediated by a membrane receptor, because they are reproduced by the membrane-impermeable conjugate of the hormone BSA-E2 and reduced by sperm preincubation with αH222 antibody. Taken together, our results clearly show the presence of a functional surface estrogen receptor, of 29 kDa, on human spermatozoa. This receptor may play a role in the modulation of nongenomic action of P in these cells during the process of fertilization.

Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells

“…In addition, both ER-a and ER-b membrane receptors could Using a ligand-blot approach, binding of E 2 b-POD to a 67-kDa band is likewise found to be enriched in plasma membranes (PM) and in nuclear (N) fractions. E 2 b-POD binding is shown in the absence (none) and presence (E 2 b) of free estradiol-17b at a 10-fold molar excess in order to assess speci®c steroid binding (Luconi et al, 1999) signal to MAPK which was necessary for activating DNA synthesis (Razandi et al, 1999).…”

“…To characterize putative estrogen receptor forms associated with PM fractions, samples were subjected to Western blot analysis, and blots were probed either with anti-ER antibody Ab2 or with E 2 b-POD (Luconi et al, 1999). PM puri®ed from MCF-7 cells show signi®cant enrichment of a primary 67-kDa protein that reacts strongly with antibody Ab2 to LBD of nuclear ER-a (Figure 4a).…”

“…To characterize putative estrogen receptor forms associated with PM fractions, membrane proteins were separated by SDS ± PAGE and then transferred to a nitrocellulose membrane for immunodetection by Western blot. The blots were probed as before (Pietras et al, 1995) with ER-a antibodies, Ab1 (Upstate Biotechnology) or Ab2 (Neomarkers), ER-b antibody (PA1-310B; Anity Bioreagents, Golden, CO, USA) or E 2 b-POD (Luconi et al, 1999).…”

The role of aromatase inhibitors in ameliorating deleterious effects of ovarian stimulation on outcome of infertility treatment

“…Non-genomic effects of estrogens have also been reported in several cell types including the spermatozoa [158]. Immunohistochemical detection of ER has been reported for human spermatozoa, with distribution in both the head and flagellum [159].…”

Of Oestrogens and Sperm: A Review of the Roles of Oestrogens and Oestrogen Receptors in Male Reproduction

“…This protein was described as a novel oestrogen receptor that may mediate oestrogen effects in the sperm [122]; however, no further studies have investigated this in more detail.…”