CRISPR/Cas9 system has opened a new era for reverse genetics. Taking advantage of the CRISPR/Cas9-mediated approaches, our laboratory has knocked out 421 male reproductive organ-specific genes and ...analysed their phenotypes in vivo. Surprisingly, even with the highly testis-specific genes (TPM>10 and 10 times higher expression than any other non-reproductive tissues), only one-third is individually essential for male fertility (37/120=30.8%), suggesting that the tissue-specific expression does not necessarily mean essential for physiological functions ( 1 ). Nevertheless, this KO screening approach has allowed us to identify 116 genes essential for male reproductive function. Among them, we recently elucidated the long-sought lumicrine system. Testicular NELL2/NICOL goes through the efferent duct’s lumen and stimulates the ROS1 signalling pathway in the epididymal epithelial cells. Thus, differentiated epididymal epithelial cells secrete proteases such as OVCH2 that mediate sperm ADAM3 trimming and sperm maturation ( 2 - 3 ). Finally, I would like to introduce recent findings on the molecular mechanism of mammalian fertilization ( 4 - 6 ).
In sexually reproducing organisms, the genetic information is transmitted from one generation to the next via the merger of male and female gametes. Gamete fusion is a two-step process involving ...membrane recognition and apposition through ligand-receptor interactions and lipid mixing mediated by fusion proteins. HAP2 (also known as GCS1) is a bona fide gamete fusogen in flowering plants and protists. In vertebrates, a multitude of surface proteins have been demonstrated to be pivotal for sperm-egg fusion, yet none of them exhibit typical fusogenic features. In this Cell Science at a Glance article and the accompanying poster, we summarize recent advances in the mechanistic understanding of gamete fusion in eukaryotes, with a particular focus on mammalian species.
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) contain a covalently linked GPI anchor located on outer cell membranes. GPI-APs are ubiquitously conserved from protozoa to vertebrates and ...are critical for physiological events such as development, immunity, and neurogenesis in vertebrates. Both membrane-anchored and soluble GPI-APs play a role in regulating their protein conformation and functional properties. Several pathways mediate the release of GPI-APs from the plasma membrane by vesiculation or cleavage. Phospholipases and putative substrate-specific GPI-AP-releasing enzymes, such as NOTUM, glycerophosphodiesterase 2, and angiotensin-converting enzyme, have been characterized in mammals. Here, the protein modifications resulting from the cleavage of the GPI anchor are discussed in the context of its physiological functions.
Tetraspanins play critical roles in various physiological processes, ranging from cell adhesion to virus infection. The members of the tetraspanin family have four membrane-spanning domains and short ...and large extracellular loops, and associate with a broad range of other functional proteins to exert cellular functions. Here we report the crystal structure of CD9 and the cryo-electron microscopic structure of CD9 in complex with its single membrane-spanning partner protein, EWI-2. The reversed cone-like molecular shape of CD9 generates membrane curvature in the crystalline lipid layers, which explains the CD9 localization in regions with high membrane curvature and its implications in membrane remodeling. The molecular interaction between CD9 and EWI-2 is mainly mediated through the small residues in the transmembrane region and protein/lipid interactions, whereas the fertilization assay revealed the critical involvement of the LEL region in the sperm-egg fusion, indicating the different dependency of each binding domain for other partner proteins.
Ovulation in the mouse and other mammals is controlled by hormones secreted by the hypothalamo-pituitary-ovarian axis. We describe anovulation and infertility in female mice lacking the microRNAs ...miR-200b and miR-429. Both miRNAs are strongly expressed in the pituitary gland, where they suppress expression of the transcriptional repressor ZEB1. Eliminating these miRNAs, in turn, inhibits luteinizing hormone (LH) synthesis by repressing transcription of its β-subunit gene, which leads to lowered serum LH concentration, an impaired LH surge, and failure to ovulate. Our results reveal roles for miR-200b and miR-429, and their target the Zebl gene, in the regulation of mammalian reproduction. Thus, the hypothalamo-pituitary-ovarian axis was shown to require miR-200b and miR-429 to support ovulation.
The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used
Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for ...target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.
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.
Gene disruption experiments have proven that the acrosomal protein IZUMO1 is essential for sperm-egg fusion in the mouse. However, despite its predicted function, it is not expressed on the surface ...of ejaculated spermatozoa. Here, we report the dynamics of diffusion of IZUMO1 from the acrosomal membrane to the sperm surface at the time of the acrosome reaction, visualized using a fluorescent protein tag. IZUMO1 showed a tendency to localize in the equatorial segment of the sperm surface after the acrosome reaction. This region is considered to initiate fusion with the oolemma. The moment of sperm-egg fusion and the dynamic movements of proteins during fusion were also imaged live. Translocation of IZUMO1 during the fertilization process was clarified, and a fundamental mechanism in mammalian fertilization is postulated.
About 10% of male infertile patients show abnormalities in spermatogenesis. The microdeletion of azoospermia factor a (AZFa) region of the Y chromosome is thought to be a cause of spermatogenic ...failure. However, candidate gene responsible for the spermatogenic failure in AZFa deleted patients has not been elucidated yet. Using mice, we explored the function of Ddx3y, a strong candidate gene in the Azfa region, and Ddx3x, a Ddx3y paralog on the X chromosome, in spermatogenesis. We first generated Ddx3y KO male mice using CRISPR/Cas9 and found that the Ddx3y KO male mice show normal spermatogenesis, produce morphologically normal spermatozoa, and sire healthy offspring. Because Ddx3x KO males were embryonic lethal, we next generated chimeric mice, which contain Ddx3x and Ddx3y double KO (dKO) germ cells, and found that the dKO germ cells can differentiate into spermatozoa and transmit their mutant alleles to offspring by normal mating. We conclude that Ddx3x and Ddx3y are dispensable for spermatogenesis at least in mice. Unlike human, mice have an additional Ddx3y paralog D1pas1, that has been reported to be essential for spermatogenesis. These findings suggest that human and mouse DDX3 related proteins have distinct differences in their functions.
The mitochondrial sheath is composed of mitochondria that coil tightly around the midpiece of sperm flagellum. These mitochondria are recruited from the cytoplasm to the flagellum late in ...spermatogenesis. Initially, recruited mitochondria are spherical-shaped but then elongate laterally to become crescent-like in shape. Subsequently, crescent-like mitochondria elongate continuously to coil tightly around the flagellum. Recently, disorganization of the mitochondrial sheath was reported in Glycerol kinase 2 (Gk2) disrupted mice. To analyze the disorganization of the mitochondrial sheath further, we generated Gk2-deficient mice using the CRISPR/Cas9 system and observed sperm mitochondria in testis using a freeze-fracture method with scanning electron microscopy. Gk2-disrupted spermatids show abnormal localization of crescent-like mitochondria, in spite of the initial proper alignment of spherical mitochondria around the flagellum, which causes abnormal mitochondrial sheath formation leading to exposure of the outer dense fibers. These results indicate that GK2 is essential for proper arrangement of crescent-like mitochondria to form the mitochondrial sheath during mouse spermatogenesis.
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