Detection of estrogen receptors ER-alpha and ER-beta in human ejaculated immature spermatozoa with excess residual cytoplasm

Abstract

Background: A key role of estrogens in human sperm biology has been recently suggested by aromatase and estrogen receptor detection in human testicular germ cells and ejaculated spermatozoa. However, the involvement of these hormones in the sperm maturation process is still not defined. The aim of this work was to investigate the expression of estrogen receptors, ER-alpha and ER-beta, in human ejaculated immature spermatozoa with excess residual cytoplasm.

Methods: Immunofluorescence labelling has been used to localize ER-alpha and ER-beta proteins in immature spermatozoa isolated by Percoll gradient, while Western blot analysis was carried out on sperm protein extracts.

Results: Both estrogen receptors were localized in excess residual cytoplasm of immature sperm, while sperm tails showed only ER-beta. Furthermore, in the same cells, immunoblots detected the presence of the full-length (approximately 67 kDa) ER-alpha and (approximately 59 kDa) ER-beta proteins, together with a approximately 50 kDa ER-beta species, lacking in mature sperm.

Conclusion: The present investigation demonstrated ER-alpha and ER-beta presence in excess residual cytoplasm of human abnormal sperm cells, suggesting the hypothesis that both the 'classical' ERs could be able to mediate estrogen action in spermatogenetic cells. Furthermore, the presence of the short ER-beta form in abnormal germ cells and its disappearance in mature sperm, support estrogen modulation via different ER forms during sperm maturation.

Figure 1

ERα and ERβ immunofluorescence labelling of morphologically normal spermatozoa and spermatozoa carrying superfluous cytoplasm A: ERα red brilliant light (Texas-Red) in excess residual cytoplasm of immature spermatozoa. A1: ERα fluorescence in mid-piece regions of normal sperm. B: ERβ green intense light (FITC) in excess residual cytoplasm and tails of immature spermatozoa. B1: ERβ fluorescence in mid-piece regions and tails of normal sperm. Scale bars 5 μm.

Figure 2

Western blotting analysis of ERα and ERβ in human immature and mature ejaculated spermatozoa. A. Immunoblots of sperm extracts by using anti-ERα: a 67 kDa band has been detected in positive control (MCF-7 extract) (lane C+), normal sperm (lane 1) and immature spermatozoa (lines 2–5); the band was lacking in negative control (lane C-). B. Immunoblots of sperm extracts by using anti-ERβ: two bands, 59 and 50 kDa, have been revealed in positive control (LNCaP extract) (lane C+) and immature spermatozoa (lanes 2–5), while normal sperm have shown only the 59 kDa band (lane 1); both the bands were absent in negative control (lane C-). β-actin (in A and B) serves as a loading control.