Na,K-ATPase alpha4 isoform is essential for sperm fertility.
ABSTRACT: Regulation of ion balance in spermatozoa has been shown to be essential for sperm motility and fertility. Control of intracellular ion levels requires the function of distinct ion-transport mechanisms at the cell plasma membrane. Active Na(+) and K(+) exchange in sperm is under the control of the Na,K-ATPase. Two molecular variants of the catalytic subunit of the Na,K-ATPase, ?1 and ?4, coexist in sperm. These isoforms exhibit different biochemical properties; however, their function in sperm fertility is unknown. In this work, we show that Na,K-ATPase ?4 is essential for sperm fertility. Knockout male mice lacking ?4 are completely sterile and spermatozoa from these mice are unable of fertilizing eggs in vitro. Furthermore, ?4 deletion results in severe reduction in sperm motility and hyperactivation typical of sperm capacitation. In addition, absence of ?4 causes a characteristic bend in the sperm flagellum, indicative of abnormal sperm ion regulation. Accordingly, ?4-null sperm present increased intracellular Na(+) and cell plasma membrane depolarization. These results are unique in demonstrating the absolute requirement of ?4 for sperm fertility. Moreover, the inability of ?1 to compensate for ?4 suggests that ?4 is the Na,K-ATPase-? isoform directly involved in sperm fertility. Our findings show ?4 as an attractive target for male contraception and open the possibility for the potential use of this Na,K-ATPase isoform as a biomarker for male fertility.
Project description:Most of our knowledge on the biological role of the testis-specific Na,K-ATPase alpha 4 isoform derives from studies performed in non-human species. Here, we studied the function of human Na,K-ATPase alpha 4 after its expression in transgenic mice. Using a bacterial artificial chromosome (BAC) construct containing the human ATP1A4 gene locus, we obtained expression of the human ?4 transgene specifically in mouse sperm testis and, in the sperm flagellum. The expressed human alpha 4 was active, and compared to wild-type sperm, those from transgenic mice displayed higher Na,K-ATPase alpha 4 activity and greater binding of fluorescently labeled ouabain, which is typical of the alpha 4 isoform. The expression and activity of endogenous alpha 4 and the other Na,K-ATPase alpha isoform present in sperm, alpha 1, remained unchanged. Male mice expressing the human ATP1A4 transgene exhibited similar testis size and morphology, normal sperm number and shape, and no changes in overall fertility compared to wild-type mice. Sperm carrying the human transgene exhibited enhanced total motility and an increase in multiple parameters of sperm movement, including higher sperm hyperactive motility. In contrast, no statistically significant changes in sperm membrane potential, protein tyrosine phosphorylation, or spontaneous acrosome reaction were found between wild-type and transgenic mice. Altogether, these results provide new genetic evidence for an important role of human Na,K-ATPase alpha 4 in sperm motility and hyperactivation, and establishes a new animal model for future studies of this isoform.
Project description:Na,K-ATPase ?4 is a testis-specific plasma membrane Na+ and K+ transporter expressed in sperm flagellum. Deletion of Na,K-ATPase ?4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase ?4 is characterized by a high affinity for the cardiac glycoside ouabain. With the goal of discovering selective inhibitors of the Na,K-ATPase ?4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue 25, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase ?4, with an outstanding ?4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca2+, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase ?4 and sperm function.
Project description:Calcium signalling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility and the acrosome reaction are all mediated by increases in intracellular Ca(2+). Cation channels of sperm proteins (CATSPERS1-4) form an alkalinization-activated Ca(2+)-selective channel required for the hyperactivated motility of spermatozoa and male fertility. Each of the CatSper1-4 genes encodes a subunit of a tetramer surrounding a Ca(2+)-selective pore, in analogy with other six-transmembrane ion channel ? subunits. In addition to the pore-forming proteins, the sperm Ca(2+) channel contains auxiliary subunits, CATSPER? and CATSPER?. Here, we identify the Tmem146 gene product as a novel subunit, CATSPER?, required for CATSPER channel function. We find that mice lacking the sperm tail-specific CATSPER? are infertile and their spermatozoa lack both Ca(2+) current and hyperactivated motility. We show that CATSPER? is an essential element of the CATSPER channel complex and propose that CATSPER? is required for proper CATSPER channel assembly and/or transport.
Project description:Mammalian spermatozoa become motile at ejaculation, but before they can fertilize the egg, they must acquire more thrust to penetrate the cumulus and zona pellucida. The forceful asymmetric motion of hyperactivated spermatozoa requires Ca2+ entry into the sperm tail by an alkalinization-activated voltage-sensitive Ca2+-selective current (ICatSper). Hyperactivation requires CatSper1 and CatSper2 putative ion channel genes, but the function of two other related genes (CatSper3 and CatSper4) is not known. Here we show that targeted disruption of murine CatSper3 or CatSper4 also abrogated ICatSper, sperm cell hyperactivated motility and male fertility but did not affect spermatogenesis or initial motility. Direct protein interactions among CatSpers, the sperm specificity of these proteins, and loss of ICatSper in each of the four CatSper-/- mice indicate that CatSpers are highly specialized flagellar proteins.
Project description:Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection. However, whether Chlamydia trachomatis has a negative impact on sperm quality and male fertility is still controversial. Herein, we report the effects on sperm quality of the in vitro exposure of spermatozoa to Chlamydia trachomatis, and also the effects of male genital infection on male fertility using an animal model. Human and mouse sperm were obtained from healthy donors and cauda epididimys from C57BL/6 mice, respectively. Highly motile human or mouse spermatozoa were in vitro exposed to C. trachomatis (serovar E or LGV) or C. muridarum, respectively. Then, sperm quality parameters were analyzed. Moreover, male fertility of Chlamydia muridarum infected male C57BL/6 mice was assessed. Human or murine sperm in vitro exposed to increasing bacterial concentrations or soluble factors from C. trachomatis or C. muridarum, respectively, did not show differences in sperm motility and viability, apoptosis, mitochondrial membrane potential, DNA fragmentation, ROS production and lipid peroxidation levels, when compared with control sperm (p?>?0.05). Moreover, no differences in fertility parameters (potency, fecundity, fertility index, pre- and post-implantation loss) were observed between control and infected males. In conclusion, our results indicate that Chlamydia spp. neither directly exerts deleterious effects on spermatozoa nor impairs male fertility.
Project description:Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = -0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = -0.49, P<0.006 and r = -0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1-3 vs. Bull 4-5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.
Project description:Aminopeptidase N (APN) is a naturally occurring ectopeptidase present in mammalian semen. Previous studies have demonstrated that APN adversely affects male fertility through the alteration of sperm motility. This enzyme constitutes 0.5 to 1% of the seminal plasma proteins, which can be transferred from the prostasomes to sperms by a fusion process. In the present study, we investigated the molecular mechanism of action of APN and its role in regulating sperm functions and male fertility. In this in vitro study, epididymal mouse spermatozoa were incubated in a capacitating media (pH 7) containing 20 ng/mL of recombinant mouse APN for 90 min. Our results demonstrated that the supplementation of recombinant APN in sperm culture medium significantly increased APN activity, and subsequently altered motility, hyperactivated motility, rapid and medium swimming speeds, viability, and the acrosome reaction of mouse spermatozoa. These effects were potentially caused by increased toxicity in the spermatozoa. Further, altered APN activity in sperm culture medium affected early embryonic development. Interestingly, the effect of elevated APN activity in sperm culture medium was independent of protein tyrosine phosphorylation and protein kinase A activity. On the basis of these results, we concluded that APN plays a significant role in the regulation of several sperm functions and early embryonic development. In addition, increased APN activity could potentially lead to several adverse consequences related to male fertility.
Project description:Conventional semen analyses are used to evaluate male factor fertility/infertility in humans and other animals. However, their clinical value remains controversial. Therefore, new tools that more accurately assess male fertility based on sperm function and fertilization mechanism are of interest worldwide. While protein markers in spermatozoa that might help differentiate fertile and infertile sperm have been identified, studies are in their infancy, and the markers require validation in field trials. In the present study, to discover more sensitive biomarkers in spermatozoa for predicting male fertility, we assessed protein expression in capacitated spermatozoa. The results demonstrated that cytochrome b-c1 complex subunit 2 (UQCRC2) was abundantly expressed in high-litter size spermatozoa (>3-fold). On the other hand, equatorin, beta-tubulin, cytochrome b-c1 complex subunit 1 (UQCRC1), speriolin, Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and seminal plasma sperm motility inhibitor were abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A and UQCRC1 expression negatively correlated with litter size, while UQCRC2 expression positively correlated with litter size. Finally, the putative biomarkers predicted litter size in field trials. Our study suggests that biomarkers present in spermatozoa after capacitation can help differentiate superior male fertility from below-average fertility with high sensitivity.
Project description:BACKGROUND: Male infertility is a major problem for mammalian reproduction. However, molecular details including the underlying mechanisms of male fertility are still not known. A thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder. RESULTS: Using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) and identified 125 putative biomarkers of fertility. We next used quantitative Systems Biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. Compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. Our data also suggests a hypothesis that low fertility sperm DNA integrity may be compromised because cell cycle: G2/M DNA damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa. CONCLUSION: This is the first comprehensive description of the bovine spermatozoa proteome. Comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype.
Project description:Gibberellin, a plant growth regulator, is widely used to increase the shelf life and quality of fruits and vegetables. In this study, human semen samples were exposed to different concentrations of gibberellin, which reduced spermatozoa motility in vitro. Gibberellin exposure also increased levels of reactive oxygen species and the protein levels of apoptosis markers in human sperm. Gibberellin inhibited the activity of Na+/K+-adenosine triphosphatase (ATPase) and Ca2+-ATPase, which maintain the stability of ions inside and outside the membranes of spermatozoa. Moreover, gibberellin exposure suppressed adenosine triphosphate production and reduced the protein levels of adenosine triphosphate synthases, which may have induced the protein expression of adenosine 5'-monophosphate-activated protein kinase (AMPK) and its phosphorylated form. These results suggest that gibberellin reduces human sperm motility in vitro by increasing reactive oxygen species levels and reducing ATPase activity, which may upregulate AMPK and consequently reduce the fertilization potential of spermatozoa.