Putative role of an SLC45 H+/sugar cotransporter in mammalian spermatozoa

Abstract

In the present study, we describe the detection and analysis of a novel type of sugar transporter in mammalian spermatozoa. This transporter belongs to the SLC45 family for which two features are remarkable and distinguish it from other known families of sugar transporters. Firstly, SLC45 transporters recognise not only the monosaccharides glucose or fructose but also the disaccharide sucrose as a substrate. Secondly, the uptake of sugars is coupled to a proton gradient. Uptake experiments using radioactively labelled sucrose indicated a functional transporter of the SLC45 family in bull spermatozoa. Real-time PCR as well as Western blots demonstrated the occurrence of the SLC45 member A4 in mouse testis and sperms. Furthermore, immunocytochemical analysis of mouse tissues revealed that the signal of SLC45A4 was mainly located in the principle piece of spermatozoa. We postulate that the SLC45A4 transporter plays an important role in nutrition of spermatozoa during their maturation in the epididymis. Moreover, we suggest that knowledge about the presence of the SLC45A4 may be useful also for the methodical improvement of cryopreservation of mammalian spermatozoa.

Keywords: Proton-coupled sugar transporter; SLC45A4; Spermatozoa maturation.

Fig. 1

Proton-dependent sucrose uptake in bull spermatozoa. Spermatozoa were purified from fresh bull ejaculate by centrifugation in a Percoll gradient, washed three times with corresponding uptake buffer and used for uptake experiments with ¹⁴C-sucrose. Uptake of ¹⁴C-sucrose by bull spermatozoa was investigated at 37 °C in the presence of 50 μM CCCP or with DMSO as a control. a Time dependence. Uptake of 10 mM ¹⁴C–sucrose was performed in 40 mM potassium phosphate, 150 mM NaCl, pH 6.0. The CCCP inhibitable part of uptake is shown. b Coupling to a proton gradient. Uptake was performed for 3 min in three buffer systems as follows: Na pH 6.0: 40 mM potassium phosphate, 150 mM NaCl, pH 6.0; Li pH 6.0: 40 mM potassium phosphate, 150 mM LiCl, pH 6.0; Na pH 7.5: 40 mM potassium phosphate, 150 mM NaCl, pH 7.5. c Substrate specificity. Uptake was performed for 3 min in 40 mM potassium phosphate, 150 mM NaCl, pH 6.0 with 10 mM ¹⁴C-sucrose alone (none) or in the presence of 40 mM of one of the putative competitors. The figure presents the CCCP inhibitable part of uptake. Data points of a, b and c correspond to the means ± s.d. of three independent experiments

Fig. 2

Expression of SLC45 family members in mouse testis and spermatozoa. The amount of mRNA was analysed by real-time PCR and expressed as the ratio of the sample to ribosomal phosphoprotein P0, a housekeeping gene. Data correspond to the means ± s.d. of three independent tissue preparations, and all PCRs were run in triplicates. a mRNA isolated from testis parenchyma. b mRNA isolated from spermatozoa. Normalised to housekeeping gene expression, expression of slc45a4 gene was taken as 100% and was compared to normalised expression of slc45a1, slc45a2 or slc45a3

Fig. 3

Detection of SLC45A4 in testis and spermatozoa by Western blot. Approximately 50 μg protein per gel lane was separated by SDS-PAGE, transferred onto nitrocellulose membranes and probed with an antibody against the SLC45A4 transporter. Membranes probed with antibody which had been pre-incubated for 1 h with a tenfold excess of antigen peptide were used as controls for specificity of the antibody. The primary antibody was visualised with anti-rabbit antibody conjugated to alkaline phosphatase. a Mouse tissues. St—Thermo Scientific PageRuler™ Prestained Protein Ladder, 1—testis crude extract, 2—membrane fraction of testis crude extract, 3—crude extract of spermatozoa isolated from epididymis. b Crude extract of spermatozoa isolated from bull ejaculate. St—Thermo Scientific PageRuler™ Prestained Protein Ladder. Figures show representative views of three independent preparations

Fig. 4

Localisation of SLC45A4 transporter in mouse testis. Six micrometres of cryosections of mouse testis were probed with an antibody against the SLC45A4 transporter. Primary antibody was visualised with anti-rabbit antibody conjugated to Cy3 (yellow). Cell nuclei were stained with DAPI (blue). Sections were analysed by a laser scanning microscope, and pictures present 1-μm optical sections. a Overview of a control section probed by primary antibody after its saturation with antigenic peptide. b Overview of an examined section. c Detailed view of b. I SLC45A4 signal, II composite of SLC45A4 signal with brightfield and DAPI staining. Figures show representative views of three independent experiments. Overall, roughly 170 examined and 110 control cryosections were analysed. L lumen of a seminiferous tubule; arrows point to tails of spermatids; arrow heads indicate spermatogonia

Fig. 5

Localisation of SLC45A4 transporter in mouse spermatozoa. Spermatozoa from mouse epididymis were probed with an antibody against the SLC45A4 transporter and analysed by a laser scanning microscope. Primary antibody was visualised with anti-rabbit antibody conjugated to Cy3 (yellow). Cell nuclei were stained with DAPI (blue). a Overview of a control preparation probed by primary antibody after its saturation with antigenic peptide. b Overview of an examined preparation. c Detailed view of b. d Detailed view of a single sperm as a 0.5-μm optical section. I SLC45A4 signal, II composite of SLC45A4 signal with brightfield and DAPI staining; III DAPI staining, IV brightfield, H head, M midpiece, P principal piece of spermatozoon. Figures show representative views of 18 examined and 12 control samples from three independent tissues preparations