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.
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.
Background
Oocyte components are maternally provided, solely determine oocyte quality, and coordinately determine embryo quality with zygotic gene expression. During oocyte maturation, maternal ...organelles are drastically reorganized and specialized to support oocyte characteristics. A large number of maternal components are actively degraded after fertilization and gradually replaced by zygotic gene products. The molecular basis and the significance of these processes on oocyte/embryo quality are not fully understood.
Methods
Firstly, recent findings in organelle characteristics of other cells or oocytes from model organisms are introduced for further understanding of oocyte organelle reorganization/specialization. Secondly, recent progress in studies on maternal components degradation and their molecular mechanisms are introduced. Finally, future applications of these advancements for predicting mammalian oocyte/embryo quality are discussed.
Main findings
The significance of cellular surface protein degradation via endocytosis for embryonic development, and involvement of biogenesis of lipid droplets in embryonic quality, were recently reported using mammalian model organisms.
Conclusion
Identifying key oocyte component characteristics and understanding their dynamics may lead to new applications in oocyte/embryo quality prediction and improvement. To implement these multidimensional concepts, development of new technical approaches that allow us to address the complexity and efficient studies using model organisms are required.
During oocyte maturation, maternal organelles are drastically reorganized and specialized to support oocyte characteristics, whereas large amounts of maternal components are actively degraded after fertilization and gradually replaced by zygotic gene products. The significance of cellular surface protein degradation via endocytosis for embryonic development, and involvement of biogenesis of lipid droplets in embryonic quality, were recently reported using mammalian model organisms. Identifying key oocyte component characteristics and understanding their dynamics may lead to new applications in oocyte/embryo quality prediction and improvement.
Fertilization triggers a process called maternal-to-zygotic transition, in which the oocyte undergoes oocyte-to-embryo transition, leading to massive intracellular remodeling toward early ...embryogenesis. This transition requires the degradation of oocyte-derived components; however, the significance and mechanism of degradation of cell surface components remain unknown. In this review, we focused on the dynamics of plasma membrane proteins and investigated the relationship between embryonic development and endocytosis. Our survey of the extant literature on the topic led to the conclusion that clathrin-mediated endocytosis is essential for the progression of early embryogenesis and selective degradation of oocyte-derived plasma membrane proteins in mouse embryos, as reported by studies analyzing maternal cellular surface proteins, including a glycine transporter, GlyT1a. Evaluation of such endocytic activity in individual embryos may allow the selection of embryos with higher viability in assisted reproductive technologies, and it is important to select viable embryos to increase the rates of successful pregnancy and live birth. Although the early embryonic developmental abnormalities are mainly accompanied with chromosomal aneuploidy, other causes and mechanisms remain unclear. This review summarizes molecular biological approaches to early embryonic developmental abnormalities and their future prospects.
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 oocyte's 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.
Many factors are involved in acrosome biogenesis in order for appropriate acrosome formation to occur. Here, we demonstrate that IZUMO family member 3, IZUMO3, plays an important role in acrosome ...biogenesis, as proven by gene disruption experiments. A loss of IZUMO3 in round spermatids affects acrosomal granule positioning due to lack of acrosomal granule contact with the inner acrosomal membrane, leading to the formation of grossly malformed spermatozoa associated with male subfertility. Thus, we suggest that mammalian spermiogenesis needs an elaborate acrosome biogenesis through IZUMO3 involvement.
Many investigators maintain that spermatozoa that have initiated the acrosome reaction (AR) before reaching the surface of the egg's 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.
ABSTRACT
Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the ...degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.
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