Characterization of organic cation/carnitine transporter family in human sperm

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Abstract

Spermatozoan maturation, motility, and fertility are, in part, dependent upon the progressive increase in epididymal and spermatozoal carnitine, critical for mitochondrial fatty acid oxidation, as sperm pass from the caput to the cauda of the epididymis. We demonstrate that the organic cation/carnitine transporters, OCTN1, OCTN2, and OCTN3, are expressed in sperm as three distinct proteins with an expected molecular mass of 63 kDa, using Western blot analysis and our transporter-specific antibodies. Carnitine uptake studies in normal control human sperm samples further support the presence of high-affinity (OCTN2) carnitine uptake (Km of 3.39 ± 1.16 μM; Vmax of 0.23 ± 0.14 pmol/min/mg sperm protein; and mean ± SD; n=12), intermediate-affinity (OCTN3) carnitine uptake (Km of 25.9 ± 14.7 μM; Vmax of 1.49 ± 1.03 pmol/min/mg protein; n=26), and low-affinity (OCTN1) carnitine uptake (Km of 412.6 ± 191 μM; Vmax of 32.7 ± 20.5 pmol/min/mg protein; n=18). Identification of individuals with defective sperm carnitine transport may provide potentially treatable etiologies of male infertility, responsive to l-carnitine supplementation.

Section snippets

Materials and methods

Sperm preparation and analysis. All studies were performed with the approval of the Institutional Review Board of the Hospital for Sick Children, Toronto. Residual unused portions of semen samples were obtained, with informed consent, from men, with normal semen parameters, who were donating samples for artificial insemination at the Sunnybrook and Women’s College Hospital Fertility Centre (Toronto, Canada). Samples were examined and selected according to World Health Organization [41]

Western blot of OCTN1, OCTN2, and OCTN3

To date, three proteins have been shown to transport l-carnitine, namely OCTN1, OCTN2, and OCTN3. Importantly, we have demonstrated the expression of all three, each with an expected molecular weight of ∼63 kDa, in human sperm by Western blot analysis using our anti-murine transporter-specific antibodies (Fig. 1).

Carnitine uptake in sperm

Carnitine uptake studies in human sperm from normal control samples support the presence of a high-affinity transport process with an apparent Km of 3.39 ± 1.16 μM and a maximal velocity

Acknowledgements

This work was supported in part by a grant from the Physicians’ Services Incorporated Foundation of Ontario and the Heart and Stroke Foundation of Ontario NA # 4964. We thank Nelson Palomaris, Domingo Molingbayan, Jennifer Willis, and Dr. Wei Qiu for their helpful assistance.

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