Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism

Abstract

Background: A wider biological role of 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, in tissues not primarily related to mineral metabolism was suggested. Recently, we evidenced the ultrastructural localization the 1,25(OH)2D3 receptor in the human sperm. However, the 1,25(OH)2D3 action in human male reproduction has not yet been clarified.

Methods and results: By RT-PCR, Western blot and Immunofluorescence techniques, we demonstrated that human sperm expresses the 1,25(OH)2D3 receptor (VDR). Besides, 25(OH)D3-1 alpha-hydroxylase, evidenced by Western blot analysis, indicated that in sperm 1,25(OH)2D3 is locally produced, highlighting the potential for autocrine-paracrine responses. 1,25(OH)2D3 through VDR, increased intracellular Ca2+ levels, motility and acrosin activity revealing an unexpected significance of this hormone in the acquisition of fertilizing ability. In sperm, 1,25(OH)2D3 through VDR, reduces triglycerides content concomitantly to the increase of lipase activity. Rapid responses stimulated by 1,25(OH)2D3 have been observed on Akt, MAPK and GSK3 implying that this secosteroid is involved in different sperm signalling pathways.

Conclusion: Our data extended the role of 1,25(OH)2D3 beyond its conventional physiological actions, paving the way for novel therapeutic opportunities in the treatment of the male reproduction disorders.

Figure 1

VDR expression in human ejaculated spermatozoa. A: Reverse transcription-PCR analysis of human VDR, CD45 and c-Kit genes in percolled human spermatozoa (S1 and S2), negative control (no M-MLV reverse transcriptase added) (-), positive controls (MCF7 breast cancer cells for VDR, human germ cells for c-Kit and human leucocytes for CD45) (+), marker (lane M). Arrows indicated the expected size of the PCR products. B: Western blot of VDR protein in human sperm, expression in two samples of ejaculated spermatozoa from normal men (S1, S2). MCF-7 and LNCaP extracts were used as positive controls. C: VDR expression in severe oligoastenozoospermic patients. NC = Normal uncapacitated sample; P1, P2 = pathologic samples. D: Western blot of 1alpha--hydroxylase protein in human sperm, expression in two samples of ejaculated spermatozoa from normal men (S1, S2). MCF-7 extracts was used as positive control (+). The experiments were repeated at least four times for RT-PCR, seven times for Western blot and the autoradiographs of the figure show the results of one representative experiment.

Figure 2

Immunofluorescence localization of VDR in human ejaculated spermatozoa. A: VDR Immunolocalization; B: Sperm cells incubated without the primary Ab were utilized as negative control. C: Sperm cells incubated replacing the anti-VDR Ab by normal rabbit serum were utilized as negative control. The pictures shown are representative examples of experiments that were performed at least three times with reproducible results.

Figure 3

1,25(OH)2D3 increases intracellular Ca²⁺. Percoll-purified sperm washed spermatozoa were incubated in the unsupplemented Earle's medium for 30 min at 37°C and 5% CO₂, in the absence (NC) or in the presence of increasing 1,25(OH)2D3 concentrations (0.01 nM, 0.1 nM, 1 nM and 10 nM) or with 0.1 nM 1,25(OH)2D3 combined with anti-VDR Ab (AbVDR). Other samples were incubated in capacitating medium (Cap). Intracellular calcium was measured as reported in Materials and Methods. The calcium assay presented are representative examples of experiments that were performed at least seven times with repetitive results. Columns represent mean ± S.D. *P < 0.05 versus control, **P < 0.02, ***P < 0.01 versus control.

Figure 4

1,25(OH)2D3 effects on motility and acrosin activity are VDR-mediated. Percoll-purified sperm washed spermatozoa were incubated in the unsupplemented Earle's medium for 30 min at 37°C and 5% CO₂, in the absence (NC) or in the presence of increasing 1,25(OH)2D3 concentrations (0.01 nM, 0.1 nM, 1 nM and 10 nM) or with 0.1 nM 1,25(OH)2D3 combined with anti-VDR Ab (AbVDR). Other samples were incubated in capacitating medium (Cap). Sperm motility and acrosin activity were assessed as reported in Materials and methods. The sperm motility presented are representative examples of experiments that were performed at least five times with repetitive results while acrosin activity were performed at least seven times with repetitive results. Columns are mean ± S.D. Data are expressed as % for motility and as μIU acrosin/10⁶ sperms for acrosin activity. *P < 0.05 versus control; ** P < 0.02, ***P < 0.01 versus control.

Figure 5

1,25(OH)2D3 induces pERK1/2, AKT and GSK3 phosphorylations in human sperm through VDR. A: Percoll-purified sperm washed spermatozoa were incubated in the unsupplemented Earle's medium for 30 min at 37°C and 5% CO₂, in the absence (NC) or in the presence of increasing 1,25(OH)2D3 concentrations (0.01 nM, 0.1 nM, 1 nM and 10 nM) or with 0.1 nM 1,25(OH)2D3 combined with anti-VDR Ab (AbVDR). Other samples were incubated in capacitating medium (Cap). B: Time course study (0, 5, 10, 30 and 60 min) of ERK1/2, AKT and GSK3 phosphorylations treated with 0.1 nM 1,25(OH)2D3. Actin was used as loading control. On the side are reported the densitometric evaluations normalised against actin levels. The autoradiographs presented are representative examples of experiments that were performed at least seven times with repetitive results. *P < 0.05 versus control, **P < 0.01, ***P < 0.002 versus control.