Tex101 is essential for male fertility by affecting sperm migration into the oviduct in mice.
نویسندگان
چکیده
Dear Editor, Sperm transport in the female genital tract is physiologically important for mammalian fertilization. The female reproductive system contains multiple natural selective barriers, such as successful uterotubal junction (UTJ) migration and zona pellucida (ZP) binding, to ensure sperm with normal motility and morphology to transmit into oviduct for fertilization (Yanagimachi, 1994; Ikawa et al., 2010). Tex101 is a glycosylphosphatidyl inositol (GPI)-anchored glycoprotein identified as a molecular marker of germ cells (Kurita et al., 2001). Although there have been indications that the malfunction of Tex101 may affect male fertility (Yin et al., 2009), little is known about its exact physiological function and the underlying molecular mechanisms. Recently, a study showed that Tex101 gene knockout sperm were unable to pass through UTJ or bind to ZP, which led to male infertility (Fujihara et al., 2013). Here, we independently generated Tex101 knockout mice and confirmed the infertile phenotype caused by UTJ migration defect. We also found that Tex101 knockout sperm lost the adhesive ability to the surface of female genital tract. Several members of a disintegrin and metalloprotease (ADAM) transmembrane protein family with cell adhesion ability, including ADAM3, ADAM4, ADAM5, and ADAM6, were lost in Tex101 knockout epididymal sperm. These observations may shed new light on the diagnosis of male infertility and development of contraceptive methods in human. High abundant Tex101 protein was only detected in the testis of male mice (Supplementary Figure S1A). To investigate the function of Tex101 in vivo, we generated Tex101 gene knockout mice (Supplementary Figure S1). During the 2-year observation period, neither Tex101 heterozygous mutant (Tex101+/2 ) nor Tex101 homozygous mutant (Tex101 ) mice (over 30 mice per group) showed any overt developmental abnormalities. However, although with normal mating ability, male Tex101 mice could not produce offspring, which confirmed the infertile defect of Tex101 deletion (Supplementary Table S1) (Fujihara et al., 2013). We next characterized the defects of Tex101 sperm causing male infertility. The histology and weight of testis from wildtype (Tex101+/+ ) and Tex101 male mice exhibited no identifiable difference (Supplementary Figure S2). In addition, no difference in sperm count, sperm viability, or motility parameters was observed (Supplementary Table S2). However, none of oocytes from females mated with Tex101 mice was fertilized at 18 h after mating plug formation (Supplementary Figure S3), suggesting that sperm from Tex101 mice were either unable to reach the fertilization place or unable to fertilize the oocytes. We then counted sperm collected from the oviducts of mated females. Large amounts of sperm were found in female mice mated with Tex101+/+ males (323 + 84, n 1⁄4 8), yet no sperm (0, n 1⁄4 24) was recovered from females mated with Tex101 males (Figure 1A). Similarly, sperm were only observed in the UTJ lumen of female mice mated with Tex101+/+ males but not those mated with Tex101 males (Supplementary Figure S4). These results demonstrated that Tex101 sperm were unable to pass through the UTJ of female genital tract. However, Tex101 sperm still fertilized oocytes (Figure 1B) at a lower rate compared with Tex101+/+ sperm (Figure 1C, 40% vs. 58%, P 1⁄4 0.048) in in vitro fertilization (IVF) assays. Moreover, among 24 in-tubal inseminated (ITI) female mice, four were successfully pregnant and produced 12 healthy offspring, indicating that Tex101 sperm were still capable to fertilize oocytes in vivo when the UTJ transportation was avoided (Figure 1D, E, and Supplementary Table S3). In contrast, in intra-uterine insemination (IUI) assays, no offspring was produced in the Tex101 group (Supplementary Table S3), further confirming that the male infertility defect of Tex101 mice was primarily caused by the UTJ migration defect of sperm. We noticed that Tex101 sperm seldom bound to dissected epithelium and ZP in the computer-assisted sperm analysis and IVF experiments. To further assess the membrane adhesive ability of Tex101 sperm, different cells inside the female genital tract, including the epithelium of UTJ and isthmus oviduct, cumulus cells, and oocytes, were dissected out and incubated separately in vitro with Tex101+/+ and Tex101 sperm. After incubation for 30 min, Tex101+/+ sperm adhered to all types of epithelium cells robustly, whereas Tex101 sperm were rarely attached (Figure 1F and G). These results demonstrated that sperm of Tex101 mice had lost their adhesive ability, thus failed to bind to the surface of cells in female genital tract. To investigate the functional mechanisms of Tex101, we used mass spectrometry to characterize the differentially expressed proteins between Tex101+/+ and Tex101 cauda epididymal sperm. A total of 30 proteins were identified with .1.5-fold expression changes, including two ADAM protein family members, ADAM5 and ADAM6 (Supplementary Table S4). Previous studies showed that ADAM3 but not other ADAM proteins played a key role in causing the infertile phenotypes (Ikawa et al., 2010; Fujihara et al., 2013); therefore, we detected the expression of all ADAM family proteins with predominant expression in testis by western blot. All examined proteins had no observable expression difference in testicular sperm between Tex101+/+ and Tex101 mice. However, in cauda doi:10.1093/jmcb/mjt031 Journal of Molecular Cell Biology (2013), 5, 345–347 | 345 Published online August 22, 2013
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عنوان ژورنال:
- Journal of molecular cell biology
دوره 5 5 شماره
صفحات -
تاریخ انتشار 2013