Yuhkoh Satouh

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm–oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm–zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocytes plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.

Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.

Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs

Many investigators maintain that spermatozoa that have initiated the acrosome reaction (AR) before reaching the surface of the eggs zona pellucida (ZP) are unable to bind and penetrate the ZP. A recent study has revealed that most fertilizing mouse spermatozoa initiate the AR before contacting the ZP. We found that acrosome-reacted spermatozoa collected from the perivitelline space of Cd9-null mice (whose egg plasma membranes are incapable of fusing with spermatozoa) were able to pass through both the cumulus and ZP of WT mouse eggs and produced live offspring. This means that the spermatozoa we used had the ability to pass through the ZP at least twice. Apparently, some spermatozoa that had undergone the AR long before contact with the ZP remained capable of crossing the ZP and fertilizing eggs. Thus, the concept that acrosome-reacted spermatozoa are unable to bind to the ZP and have lost their fertilizing capacity must be reconsidered.

Calsperin Is a Testis-specific Chaperone Required for Sperm Fertility

Calnexin (CANX) and calreticulin (CALR) are homologous lectin chaperones located in the endoplasmic reticulum and cooperate to mediate nascent glycoprotein folding. In the testis, calmegin (CLGN) and calsperin (CALR3) are expressed as germ cell-specific counterparts of CANX and CALR, respectively. Here, we show that Calr3−/− males produced apparently normal sperm but were infertile because of defective sperm migration from the uterus into the oviduct and defective binding to the zona pellucida. Whereas CLGN was required for ADAM1A/ADAM2 dimerization and subsequent maturation of ADAM3, a sperm membrane protein required for fertilization, we show that CALR3 is a lectin-deficient chaperone directly required for ADAM3 maturation. Our results establish the client specificity of CALR3 and demonstrate that the germ cell-specific CALR-like endoplasmic reticulum chaperones have contrasting functions in the development of male fertility. The identification and understanding of the maturation mechanisms of key sperm proteins will pave the way toward novel approaches for both contraception and treatment of unexplained male infertility.

SPACA1-deficient male mice are infertile with abnormally shaped sperm heads reminiscent of globozoospermia

SPACA1 is a membrane protein that localizes in the equatorial segment of spermatozoa in mammals and is reported to function in sperm-egg fusion. We produced a Spaca1 gene-disrupted mouse line and found that the male mice were infertile. The cause of this sterility was abnormal shaping of the sperm head reminiscent of globozoospermia in humans. Disruption of Spaca1 led to the disappearance of the nuclear plate, a dense lining of the nuclear envelope facing the inner acrosomal membrane. This coincided with the failure of acrosomal expansion during spermiogenesis and resulted in the degeneration and disappearance of the acrosome in mature spermatozoa. Thus, these findings clarify part of the cascade leading to globozoospermia.

Sperm equatorial segment protein 1, SPESP1, is required for fully fertile sperm in mouse

Mammalian fertilization is a multistep process that culminates in the fusion of the sperm and egg plasma membrane. It is widely accepted that the equatorial segment of the acrosome-reacted sperm is important in initiating fusion with the egg plasma membrane during fertilization. There are various proteins known to be distributed only in the equatorial segment of sperm. The role of these proteins must be clarified to understand the membrane fusion process. We produced a mouse line that lacked SPESP1 (sperm equatorial segment protein 1) and analyzed the fertilizing ability of the sperm. The average number of pups that were fathered by Spesp1+/− and Spesp1−/− males was significantly lower than that of wild-type fathers. In these mouse lines, fewer sperm were found to migrate into oviducts and fewer eggs were fertilized. The Spesp1+/− and Spesp1−/− sperm showed a lower fusing ability compared with the wild-type sperm. The disruption of Spesp1 was shown to cause an aberrant distribution of various sperm proteins. Moreover, scanning electron microscopy revealed that the membrane in the equatorial segment area, which usually forms an acrosomal sheath, disappears after acrosome reaction in Spesp1-deficient mice. It was demonstrated that SPESP1 is necessary to produce the fully ‘fusion competent’ sperm.

Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes

The recombinant clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system has opened a new era for mammalian genome editing. Here, we constructed pX330 plasmids expressing humanized Cas9 (hCas9) and single guide RNAs (sgRNAs) against mouse genes and validated them both in vitro and in vivo. When we randomly chose 291 target sequences within protein coding regions of 73 genes, an average number of off‐target candidates (exact match 13 nucleotides from 3′ target and NGG) found by Bowtie software was 9.2 ± 21.0 (~1.8 times more than the estimated value, 5.2). We next validated their activity by observing green fluorescence reconstituted by homology dependent repair (HDR) of an EGFP expression cassette in HEK293T cells. Of the pX330 plasmids tested, 81.8% (238/291) were found to be functional in vitro. We finally injected the validated pX330 plasmids into mouse zygotes in its circular form against 32 genes (including two genes previously tested) and obtained mutant mice at a 52.9 ± 22.3% (100/196) mutation frequency. Among the pups carrying mutations on the autosomes, 43.6% (47/96) carried the mutations in both alleles. When off‐target candidate sites were examined in 63 mutant mice, 0.8% (3/382) were mutated. We conclude that our method provides a simple, efficient, and cost‐effective way for mammalian gene editing that is applicable for large scale mutagenesis in mammals.

Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive

Mouse work may lead to male contraceptive Unintended pregnancies are a major health issue worldwide. Although oral contraceptives were developed decades ago for use in women, there are no male oral contraceptives. Miyata et al. show that genetic deletion or drug inhibition of sperm-specific calcineurin enzymes in mice cause male sterility (see the Perspective by Castaneda and Matzuk). Although calcineurin inhibitors resulted in male infertility within 2 weeks, fertility recovered 1 week after halting drug administration. Because the sperm-specific calcineuin complex is also found in humans, its inhibition may be a strategy for developing reversible male contraceptives. Science, this issue p. 442, see also p. 385 Inhibiting the function of a protein involved in sperm maturation may help in the development of future male contraceptives. [Also see Perspective by Castaneda and Matzuk] Calcineurin inhibitors, such as cyclosporine A and FK506, are used as immunosuppressant drugs, but their adverse effects on male reproductive function remain unclear. The testis expresses somatic calcineurin and a sperm-specific isoform that contains a catalytic subunit (PPP3CC) and a regulatory subunit (PPP3R2). We demonstrate herein that male mice lacking Ppp3cc or Ppp3r2 genes (knockout mice) are infertile, with reduced sperm motility owing to an inflexible midpiece. Treatment of mice with cyclosporine A or FK506 creates phenocopies of the sperm motility and morphological defects. These defects appear within 4 to 5 days of treatment, which indicates that sperm-specific calcineurin confers midpiece flexibility during epididymal transit. Male mouse fertility recovered a week after we discontinued treatment. Because human spermatozoa contain PPP3CC and PPP3R2 as a form of calcineurin, inhibition of this sperm-specific calcineurin may lead to the development of a reversible male contraceptive that would target spermatozoa in the epididymis.

Sperm Postacrosomal WW Domain-Binding Protein Is Not Required for Mouse Egg Activation

ABSTRACT To begin embryonic development, the zygote must resume the cell cycle correctly after stimulation by sperm-borne oocyte-activating factors (SOAFs). The postacrosomal WW domain-binding protein (PAWP) is one of the strongest SOAF candidates and is widely conserved among eutherian mammals. It has been reported that the microinjection of recombinant PAWP protein can trigger not only Ca2+ oscillations in mammalian eggs but also intracellular Ca2+ release in amphibian eggs. It was also suggested that PAWP is involved in the formation of high-quality spermatozoa. On the other hand, negligible SOAF activity for PAWP cRNA has also been reported. In this study, we generated PAWP null mice and examined the fertilizing ability of male mice. Electron microscopy showed no aberrant morphology in spermatogenesis. Intracytoplasmic injection of a single spermatozoon from the null mouse line showed that depletion of PAWP elicited no quantitative differences in Ca2+ oscillations or in subsequent development of the embryos. We conclude that PAWP does not play an essential role in mouse fertilization.

Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice.

Significance In the mouse genome, thousands of genes are predominantly expressed in the testis, where these genes are thought to play important roles in spermatogenesis and fertilization. However, in this study, we report that 54 evolutionarily conserved and testis-enriched genes are not essential individually for male mouse fertility. Because the recent development of the CRISPR/Cas9 system has made it faster and easier to produce knockout mice, our results suggest that one should determine whether a gene of interest is essential for male fertility in vivo before spending significant effort to analyze the molecular function of the gene in vitro. Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201–12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract–enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the “gold standard” to determine whether a gene’s function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.