Sorbitol can fuel mouse sperm motility and protein tyrosine phosphorylation via sorbitol dehydrogenase

Abstract

Energy sources that can be metabolized to yield ATP are essential for normal sperm functions such as motility. Two major monosaccharides, sorbitol and fructose, are present in semen. Furthermore, sorbitol dehydrogenase (SORD) can convert sorbitol to fructose, which can then be metabolized via the glycolytic pathway in sperm to make ATP. Here we characterize Sord mRNA and SORD expression during mouse spermatogenesis and examine the ability of sorbitol to support epididymal sperm motility and tyrosine phosphorylation. Sord mRNA levels increased during the course of spermatogenic differentiation. SORD protein, however, was first detected at the condensing spermatid stage. By indirect immunofluorescence, SORD was present along the length of the flagella of caudal epididymal sperm. Furthermore, immunoelectron microscopy showed that SORD was associated with mitochondria and the plasma membranes of sperm. Sperm incubated with sorbitol maintained motility, indicating that sorbitol was utilized as an energy source. Sorbitol, as well as glucose and fructose, were not essential to induce hyperactive motility. Protein tyrosine phosphorylation increased in a similar manner when sorbitol was substituted for glucose in the incubation medium used for sperm capacitation. These results indicate that sorbitol can serve as an alternative energy source for sperm motility and protein tyrosine phosphorylation.

FIG. 1.

SORD is present in sperm but not in SDS-insoluble accessory structures of tails. Proteins (10 μg per lane) from kidney (Kd), epididymal sperm (Sp), and SDS-insoluble accessory structures of tails (Ta) were prepared and processed for immunoblot analysis. An immunoreactive band of the predicted size for SORD was present in both kidney and sperm samples. The SORD signal was not detected in SDS-insoluble tails. A) Immunoblot probed with anti-SORD polyclonal antibody. B) Normal goat IgG. C) Anti-SORD polyclonal antibody. Numbers to the left of the figure represent molecular weights of standard proteins (×10⁻³).

FIG. 2.

Sord mRNA and its encoded protein are highly expressed at the end stage of spermiogenesis; SORD protein becomes more abundant in condensing spermatids and epididymal sperm. A) Quantitative RT-PCR of Sord from pachytene spermatocytes (PS), round spermatids (RS), and condensing spermatids (CS). Results were normalized to mRNA corresponding to ribosomal protein S16. B) Immunoblot analysis of SORD (arrow) from pachytene spermatocytes (PS), round spermatids (RS), condensing spermatids (CS), and epididymal sperm (Sp). a) Anti-SORD polyclonal antibody. b) Normal goat IgG. The arrow indicates the specific SORD band. Please note: to see the weak signal of SORD in condensing spermatids, the film is overexposed. Therefore, some weak background bands appeared (also present in control), compared with Figure 1. Numbers to the left of the figure represent molecular weights of standard proteins (×10⁻³).

FIG. 3.

SORD is present along the entire length of sperm flagellum, but does not show the same distribution pattern as α-tubulin. Indirect immunofluorescence of sperm probed with anti-SORD antibody (A), normal goat IgG (B), monoclonal antibody to α-tubulin (C), and normal mouse IgG (D). E) Merged image of A and C. F) Merged image of B and D. G) Corresponding Nomarski differential interference contrast image of test group. H) Corresponding Nomarski image of control group. Bar = 10 μm.

FIG. 4.

SORD is associated with mitochondria and near the plasma membrane of the sperm flagellum. Immunoelectron microscopy of cross sections of sperm (left column) and longitudinal sections (right column) of similar regions. A, B, E, and F are views of the midpiece. C, D, G, and H are sections of the principal piece. Sperm sections in A–D were probed with normal goat IgG. Sections in E–H were probed with anti-SORD polyclonal antibody. Bar = 0.5 μm.

FIG. 5.

Sorbitol helps maintain sperm motility during incubation. Motility of sperm incubated in PBS with NaCl (control), sorbitol, fructose, or glucose was measured by computer-assisted semen analysis (CASA) at different time points. Three experiments were performed, and the results were reproducible. This figure demonstrates the changes over the course of incubation: A) The progressive sperm percentage of total sperm (n = 3; *P < 0.05). B) ACH effect on sperm motility maintained by sorbitol (n = 3; *P < 0.05, **P < 0.01).

FIG. 6.

Sperm were incubated in PBS with an additional 2.5 mM NaCl (control) or with 5 mM sorbitol added (experimental). Sperm progressive motility patterns were measured by CASA and recorded as videos. This image is a still from a composite video (see Supplemental Movie available online at http://www.biolreprod.org) of the various treatments presented in the following sequence: control at 1 h, experimental at 1 h, control at 4 h, and experimental at 4 h.

FIG. 7.

Sorbitol can substitute for glucose or fructose in capacitating media. Sperm were incubated in noncapacitating medium (ModW with monosaccharide), regular capacitating medium (ModW with monosaccharide, sodium bicarbonate, 2-OH-β-CD [labeled as 2-OH-b-CD]), or regular capacitating medium lacking monosaccharide for 1.5 h. A) Sperm proteins were extracted, subjected to SDS-PAGE, immunoblotted, and probed with anti-phosphotyrosine antibody. This experiment was repeated twice with similar results. Numbers to the left of the figure represent molecular weights of standard proteins (×10⁻³). B) Hyperactive motility was measured among the different groups above. Increased hyperactive motility was normalized by subtracting baseline hyperactive motility (at time point 0). The sperm were incubated in noncapacitating media (with glucose [a], fructose [b], sorbitol [c]) or in capacitating medium (with no added sugar [d], glucose [e], fructose [f], sorbitol [g]) (n = 3).

FIG. 8.

Sorbitol can act as an alterative energy source for sperm motility and signal transduction in proposed metabolic pathway. ACH blocks glyceraldehyde 3-phosphate dehydrogenase. DAP, dihydroxyacetone phosphate; F-1-P, fructose 1-phosphate; F-6-P, fructose 6-phosphate; GAP, glyceraldehyde 3-phosphate; GLUT, glucose transporter; HK, hexokinase; PFK, phosphofructokinase; PGI, phosphoglucose isomerase; SORD, sorbitol dehydrogenase; TCA, citric acid cycle; TK, triokinase.