Cytochrome P450arom, androgen and estrogen receptors in pig sperm

Abstract

Background: Androgens and estrogens are crucial for mammalian sperm differentiation but their role in biology of mature male gamete is not still defined. The expression of proteins involved in the biosynthesis and action of these steroid hormones has been demonstrated in human spermatozoa, but very few data have been reported in mature sperm from non human species. The purpose of the current study was to investigate the expression of aromatase (P450arom), estrogen (ERalpha/ERbeta) and androgen (AR) receptors in ejaculated spermatozoa of pig.

Methods: The immunfluorescence experiments were carried out treating pig sperm with anti-P450arom, anti-ERalpha, anti-ERbeta and anti-AR as primary antibodies, while Texas-Red/FITC conjugated IgG were applied as secondary antibodies. Furthermore, Western blot analysis was performed on sperm lysates.

Results: Aromatase was immunolocalized in the sperm tail, ERalpha and AR were localised in the sperm midpiece, while ERbeta was confined in the acrosomal region of the male gamete. Immunoblots detected a ~52 kDa aromatase band, a ~110 kDa AR band, a ~67 kDa ERalpha and two ERbeta bands, at ~50 kDa and ~59 kDa.

Conclusion: This is the first report demonstrating that pig ejaculated spermatozoa express aromatase, estrogen and androgen receptors with a differential intra-cellular localization revealing a species-specific expression pattern. Therefore, pig sperm could be considered as a potential estrogen source while the different hormone cellular sites suggest distinct roles of androgens and estrogens in pig sperm physiology.

Figure 1

Immunofluorescence labelling of androgen and estrogen receptors in pig spermatozoa: A) AR red fluorescence in sperm proximal mid-piece. B) P450arom red brilliant light in the proximal tail of sperm with a diffuse labelling in the distal tail. C) ERα red fluorescence in the sperm mid-piece, together with a faint labelling in the tail. D) ERβ green intense light in the sperm acrosomal region. Inserts: immunonegative absorption controls. Scale bars: 5 μm.

Figure 2

Western blotting analysis of AR, P450arom, ERα and ERβ of pig sperm extracts. Individual sperm samples: lanes 1–3. LNCaP extracts were used as positive controls for AR and ERβ (lanes C+) while MCF7 extracts were used as positive controls for P450 arom and ERα (lanes C+). No bands in negative controls (lanes C-). β actin served as loading control. Molecular weights (KD) were indicated on the right of the blot.