Novel insights on the role of selenoprotein p in sperm viability

Abstract

Selenium (Se) is a micronutrient essential for life in many organisms. Selenium is incorporated into selenoproteins as the twenty-first amino acid, selenocysteine (Sec), and has antioxidant properties. One member of the family of twenty-five selenoproteins in humans is Selenoprotein P (Sepp1). This protein is synthesized primarily in liver and is proposed to transport selenium throughout the body, particularly to the brain and testes. Sepp1 knockout (KO) mice on (normal) diets without selenium supplementation have decreased selenoprotein expression in brain and testes. Previous studies have suggested that Sepp1 male KO mice are infertile due to kinks in the flagellum of spermatozoa, greatly reducing sperm motility, therefore leading to dramatically decreased fertility. In this two-part study, our first objective was to further understand the role of Sepp1 on sperm viability. We hypothesize that Sepp1 plays a critical role in sperm DNA viability independent of motility, potentially through modulating glutathione peroxidase 4 (GPx4) biosynthesis. GPx4 is another selenoprotein known to protect cells from membrane lipid peroxidation and has been implicated in development and fertility [71]. The second objective of this study was to introduce a novel application to rescue Sepp1 global expression in knockout animals (Sepp1r/r CMV+) using Cre recombinase transgenic mice. We addressed the role of Sepp1 in sperm DNA viability with intracytoplasmic spermatozoa injections (ICSI) of Sepp1 KO sperm into wild type oocytes. Surrogate female mice carrying embryos resulting from injection of Sepp1-/-sperm resulted in a 72.3% reduction in live pups born compared to Sepp1 heterozygous control sperm. Our results from the ICSI experiments, in which sperm were directly injected into oocytes without flagella, suggest that Sepp1 is critical for sperm DNA viability independent of motility. We show through western blot analysis that GPx4 levels are significantly decreased in the testes and epididymides of Sepp1 KO mice, whereas Sepp1r/r CMV+ rescue mice had restored expression levels comparable to the Sepp1 wild type (Sepp1 WT). Immunohistochemistry studies using an antibody against GPx4 further confirmed that GPx4 levels were undetectable in fresh Sepp1 KO mouse sperm, while GPx4 levels in Sepp1r/r CMV+ rescue mice were similar to those of wild type controls. Cre-Lox recombination is a commonly used genetic tool for site-specific gene deletion. However, we demonstrate that this system can be used to rescue gene expression as well, restoring the expression of Sepp1 in KO mice. We show that this approach produced viable progeny of the systemic Sepp1r/r CMV+ (rescue) mice that express the CMV-Cre driven Sepp1 gene in all tissues. We confirmed through the Morris Water Maze (MWM) and other behavior assays that in contrast to Sepp1 KO mice, Sepp1r/r CMV+ mice had normal neuromotor function and memory compared to Sepp1+/+. Successful implementation of this method can further be utilized to restrict gene expression to specific cells. Our study presents new data showing that Sepp1 is crucial for viability of sperm DNA, potentially through regulation of GPx4 levels. Furthermore, we demonstrate an innovative method for restoration of gene expression using the Cre recombinase transgenic system, which can be applied to restrict gene expression to specific cells.