Expression of cyclooxygenase-1 (COX-1) and COX-2 in human male gametes from normal patients, and those with varicocele and diabetes: a potential molecular marker for diagnosing male infertility disorders

Abstract

Rising rates of varicocele and diabetes mellitus (DM) pose a significant problem to human fertility. Recent studies have pointed out the impact of cyclooxygenase (COX) in the regulation of testicular function and male fertility. Prominent COX-2 expression has been described recently in the testes of infertile patients, but little is known about the role and identity of COX isoforms in human sperm under certain disease states such as varicocele and DM. We therefore examined the expression profile and ultrastructural localization of COX-1 and COX-2 concomitantly in semen samples from healthy donors, and patients with varicocele and DM. Using Western blotting assay, 'varicocele' and 'diabetic' sperm showed enhanced COX isoforms expression with respect to the 'healthy' sperm. Immunogold labeling revealed human sperm anatomical regions containing COX-1 and COX-2, confirming their increased expression in pathological samples. Our data demonstrate that both COX isoforms are upregulated in the spermatozoa of varicocele and diabetic patients, suggesting the harmful effect of the diseases also at the sperm molecular level, going beyond the abnormal morphology described to date. In conclusion, COX enzymes may possess a biological relevance in the pathogenesis and/or maintenance of male factor infertility associated with varicocele and DM, and may be considered additional molecular markers for the diagnosis of male infertility disorders.

Fig. 1

COX-1 and COX-2 expression is enhanced in varicocele and diabetes mellitus (DM) sperm. Extracts of pooled purified ejaculated spermatozoa were subjected to electrophoresis on 11% sodium dodecyl sulfate–polyacrylamide gels, blotted onto nitrocellulose membranes, and probed with rabbit polyclonal Ab to human COX-1 (a) or with rabbit polyclonal Ab to human COX-2 (b). (a) MCF7, human breast cancer cells, used as positive control. Norm, expression of COX-1 in ejaculated sperm from normal men. V, expression of COX-1 in ejaculated sperm from varicocele men. DM, expression of COX-1 in ejaculated sperm from patients with diabetes. (a1) Immunoblot of the negative control (membrane incubated with normal rabbit serum). (b) Norm, expression of COX-2 in ejaculated sperm from normal men. V, expression of COX-2 in ejaculated sperm from varicocele men. DM, expression of COX-2 in ejaculated sperm from patients with diabetes. Immunoblot of the negative control (membrane incubated with normal rabbit serum). The number on the left corresponds to molecular masses (kd) of the marker proteins. (b1) Immunoblot of the negative control (membrane incubated with normal rabbit serum). The experiments were repeated at least six times, and the autoradiographs of the figure show the results of one representative experiment.

Fig. 2

Immunoelectron microscopic localization of COX-1 in normal human spermatozoa. Sperm were collected and prepared as described in Materials and methods. (A–F) Micrographs of sections from ejaculated sperm of normal patients probed with rabbit polyclonal Ab to human COX-1. In all cases, a secondary anti-rabbit antibody conjugated to 10-nm colloidal gold particles was used for labeling. (A, B) Sections through the head; (C, D): longitudinal sections and cross-sections of the midpiece of the flagellum; (E, F) longitudinal sections and cross-sections of the principal and the end piece of the flagellum. Representative of three similar experiments. Scale bars: 0.25 μm (A–C, F); 0.2 μm (D, E).

Fig. 3

Immunoelectron microscopic localization of COX-2 in normal human spermatozoa. Sperm were collected and prepared as described in Materials and methods. (A–F) Micrographs of sections from ejaculated sperm of normal patients probed with rabbit polyclonal Ab to human COX-2. In all cases, a secondary anti-rabbit antibody conjugated to 10-nm colloidal gold particles was used for labeling. (A, B) Sections through the head; (C, D) longitudinal sections and cross-sections of the midpiece of the flagellum; (E, F) longitudinal sections and cross-sections of the principal and the end piece of the flagellum. Representative of three similar experiments. Scale bars: 0.3 μm (A); 0.25 μm (B, D, F); 0.2 μm (C, E).

Fig. 4

Immunoelectron microscopic localization of COX-1 in varicocele spermatozoa. Sperm were collected and prepared as described in Materials and methods. (A–F) Micrographs of sections from ejaculated sperm of varicocele patients probed with rabbit polyclonal Ab to human COX-1. In all cases, a secondary anti-rabbit antibody conjugated to 10-nm colloidal gold particles was used for labeling. (A, B) Sections through the head; (C, D) longitudinal sections and cross-sections of the midpiece of the flagellum; (E, F) longitudinal sections and cross-sections of the principal and the end piece of the flagellum. Representative of three similar experiments. Scale bars: 0.2 μm (A, E); 0.25 μm (B, D); 0.3 μm (C); 0.15 μm (F).

Fig. 5

Immunoelectron microscopic localization of COX-2 in varicocele spermatozoa. Sperm were collected and prepared as described in Materials and methods. (A–F) Micrographs of sections from ejaculated sperm of varicocele patients probed with rabbit polyclonal Ab to human COX-2. In all cases, a secondary anti-rabbit antibody conjugated to 10-nm colloidal gold particles was used for labeling. (A, B) Sections through the head; (C, D) longitudinal sections and cross-sections of the midpiece of the flagellum; (E, F) longitudinal sections and cross-sections of the principal and the end piece of the flagellum. Representative of three similar experiments. Scale bars: 0.25 μm (A); 0.2 μm (B, C, E, F).

Fig. 6

Immunoelectron microscopic localization of COX-1 in DM spermatozoa. Sperm were collected and prepared as described in Materials and methods. (A–F) Micrographs of sections from ejaculated sperm of patients with DM probed with rabbit polyclonal Ab to human COX-1. In all cases, a secondary anti-rabbit antibody conjugated to 10-nm colloidal gold particles was used for labeling. (A, B) Sections through the head; (C, D) longitudinal sections and cross-sections of the midpiece of the flagellum; (E, F) longitudinal sections and cross-sections of the principal and the end piece of the flagellum. Representative of three similar experiments. Scale bars: 0.2 μm.

Fig. 7

Immunoelectron microscopic localization of COX-2 in DM spermatozoa. Sperm were collected and prepared as described in Materials and methods. (A–F) Micrographs of sections from ejaculated sperm of patients with DM probed with rabbit polyclonal Ab to human COX-2. In all cases, a secondary anti-rabbit antibody conjugated to 10-nm colloidal gold particles was used for labeling. (A, B) Sections through the head; (C, D) longitudinal sections and cross-sections of the midpiece of the flagellum; (E, F) longitudinal sections and cross-sections of the principal and the end piece of the flagellum. Representative of three similar experiments. Scale bars: 0.3 μm (A); 0.25 μm (B, D, F); 0.2 μm (C, E).