Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor‐mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318‐null male mice exhibited infertility, whereas Zfp318‐null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318‐null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.

Semenogelins (SEMGs) are major components of human seminal vesicle secretions. Due to SEMG’s sperm-motility inhibitor, a significant negative correlation between sperm motility and the proportion of SEMG-bound spermatozoa (SEMG+) was found in asthenozoospermic patients. SEMGs also show intrinsic inhibitory capability for sperm capacitation; however, studies on actual clinical specimens have not been conducted. To reveal the relationship between SEMGs and the fertilizing capacity of sperm from male infertile patients who are not restricted to asthenozoospermia, we measured the proportion of SEMG+ in the spermatozoa of 142 male infertile patients. The pregnancy outcomes in partners of these patients were retrospectively analyzed using questionnaires. Among examined semen parameters, only the total SEMG-unbound sperm count showed a tendency to be different between the spontaneous pregnancy or intra-uterine-insemination-pregnancy groups and in-vitro-fertilization- or intracytoplasmic-sperm-injection-pregnancy groups. It was elevated in the former group, which includes patients who used in vivo fertilization. The total SEMG-unbound sperm count would be a relevant parameter for in vivo fertilization. This result suggests that SEMGs inhibit ectopic capacitation before sperm reach the fertilization site and that the number of total SEMG-unbound sperm is a parameter directly linked to the possibility of in vivo fertilization.

Semenogelin 1 (SEMG1), a main component of human seminal plasma, is a multi-functional protein involved in the regulation of sperm motility and fertility. SEMG1 is orthologous to mouse seminal vesicle secretion 2 (SVS2), required for sperm survival in the female reproductive tract after copulation; however, its in vivo function remains unclear. In this study, we addressed this issue by examining the effect of recombinant SEMG1 on intrauterine mouse sperm survival. SEMG1 caused a dose-dependent decrease in mouse sperm motility, similar to its effect on human sperm, but SVS2 had no effect on mouse sperm motility. Mouse epididymal sperm in the presence of 100 µM SEMG1, a concentration that does not affect mouse sperm motility, were injected into the mouse uterus (intrauterine insemination, IUI). IUI combined with SEMG1 significantly increased the survival rate of intrauterine mouse sperm. The effect of SEMG1 on intrauterine sperm survival was comparable with that of SVS2. For clinical applications, three potentially sperm-protecting polypeptides that are easy to handle were designed from SEMG1, but their individual use was unable to mimic the ability of SEMG1. Our results indicate that SEMG1 has potential clinical applications for effective IUI and thereby for safe, simple, and effective internal fertilization.

MAMLD1 has been implicated in testicular function in both human and mouse fetuses. Although three patients with MAMLD1 mutations were reported to have hypergonadotropic hypogonadism in their teens, the functional significance of MAMLD1 in the postnatal testis remains unclear. Here, we analyzed the phenotype of Mamld1 knockout (KO) male mice at reproductive ages. The reproductive organs of KO male mice were morphologically unremarkable, except for relatively small testes. Seminiferous tubule size and number of proliferating spermatogonia/spermatocytes were reduced in the KO testis. Daily sperm production of KO mice was mildly attenuated, whereas total sperm counts in epididymal semen remained normal. Sperm motility and morphology, as well as androgen levels in serum and testicular tissues and the number of pups born from cross-mated wildtype (WT) female mice, were comparable between WT and KO male mice. These results indicate that MAMLD1 contributes to the maintenance of postnatal testicular growth and daily sperm production but is dispensable for androgen biosynthesis and fertility. MAMLD1 likely plays supporting roles in multiple and continuous steps of male reproduction.

Multiple genes, whose functions or expression are overlapping, compensate for the loss of one gene. A gene cluster in the mouse genome encodes five seminal vesicle proteins (SVS2, SVS3, SVS4, SVS5, and SVS6). These proteins are produced by male rodents and function in formation of the copulatory plug following mating. SVS2 plays an essential role in the successful internal fertilization by protecting the sperm membrane against a uterine immune attack. We hypothesized that the four remaining seminal vesicle proteins (SVPs) of this gene cluster may partially/completely compensate for the deficiency of SVS2. For confirming our hypothesis, we generated mice lacking the entire SVP-encoding gene cluster and compared their fecundity with Svs2-deficient (Svs2^−/−) mice; that is, mice deficient in Svs2 alone. A single loxP site remained after the deletion of the Svs2 gene. Therefore, we inserted another loxP site by combining the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides (ssODN). Male mice lacking the entire SVP-encoding gene cluster (Svs2–6^−/− mice) and thereby all five SVP proteins, generated by the deletion of 100kbp genomic DNA, showed low fecundity. However, the fecundity level was comparable with that from Svs2^−/− male mice. Our results demonstrate that SVS3, SVS4, SVS5, and SVS6 do not function in the protection of sperm against a uterine immune attack in the absence of SVS2. Thus, Svs2 is the critical gene in the SVP gene cluster.

Introduction: Sperm motility, including chemotactic behavior, is regulated by changes in the intracellular Ca²⁺ concentration, and the sperm-specific Ca²⁺ channel CatSper has been shown to play an important role in the regulation of intracellular Ca²⁺. In particular, in mammals, CatSper is the only functional Ca²⁺ channel in the sperm, and mice deficient in the genes comprising the pore region of the Ca²⁺ channel are infertile due to the inhibition of sperm hyperactivation. CatSper is also thought to be involved in sea urchin chemotaxis. In contrast, in ascidian Ciona intestinalis, SAAF, a sperm attractant, interacts with Ca²⁺/ATPase, a Ca²⁺ pump. Although the existence of CatSper genes has been reported, it is not clear whether CatSper is a functional Ca²⁺ channel in sperm.Results: We showed that CatSper is present in the sperm flagella of C. intestinalis as in mammalian species, although a small level of gene expression was found in other tissues. The spermatozoa of CatSper3 KO animals were significantly less motile, and some motile sperms did not show any chemotactic behavior. These results suggest that CatSper plays an important role in ascidians and mammals, and is involved in spermatogenesis and basic motility mechanisms.