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General Comment |
Molecular architecture of the human sperm IZUMO1 and egg JUNO fertilization complex. Aydin H et al. (2016) Fertilization is an essential biological process in sexual reproduction and comprises a series of molecular interactions between the sperm and egg. The fusion of the haploid spermatozoon and oocyte is the culminating event in mammalian fertilization, enabling the creation of a new, genetically distinct diploid organism. The merger of two gametes is achieved through a two-step mechanism in which the sperm protein IZUMO1 on the equatorial segment of the acrosome-reacted sperm recognizes its receptor, JUNO, on the egg surface. This recognition is followed by the fusion of the two plasma membranes. IZUMO1 and JUNO proteins are indispensable for fertilization, as constitutive knockdown of either protein results in mice that are healthy but infertile. Despite their central importance in reproductive medicine, the molecular architectures of these proteins and the details of their functional roles in fertilization are not known. Here we present the crystal structures of human IZUMO1 and JUNO in unbound and bound conformations. The human IZUMO1 structure exhibits a distinct boomerang shape and provides structural insights into the IZUMO family of proteins. Human IZUMO1 forms a high-affinity complex with JUNO and undergoes a major conformational change within its N-terminal domain upon binding to the egg-surface receptor. Our results provide insights into the molecular basis of sperm-egg recognition, cross-species fertilization, and the barrier to polyspermy, thereby promising benefits for the rational development of non-hormonal contraceptives and fertility treatments for humans and other mammals.//////////////////
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Comment |
JUNO, the receptor of sperm IZUMO1, is expressed by the human oocyte and is essential for human fertilisation. Jean C et al. (2018) Is JUNO protein present at the surface membrane of human oocytes and involved in the fertilisation process? JUNO protein is expressed on the plasma membrane of human oocytes and its inhibition by a monoclonal antibody completely blocks gamete fusion. Fusion of gamete membranes is the culminating event of the fertilisation process, but its molecular mechanisms are poorly understood. Until now, three molecules have been shown to be essential: CD9 tetraspanin in the oocyte, Izumo1 protein on the sperm and Juno, its corresponding receptor on the oocyte. Oocyte CD9 and sperm IZUMO1 have been identified in human gametes and their interaction is also well-conserved among several mammalian species. The presence of JUNO on human oocytes, however, has not yet been reported, nor has its role in fertilisation been investigated. We selected an anti-human JUNO antibody in order to investigate the presence of JUNO on the oocyte membrane surface and studied its potential involvement in gamete membrane interaction during fertilisation. Monoclonal antibodies against human JUNO (anti-hJUNO mAb) were produced by immunisation of mice with HEK cells transfected with the putative human JUNO sequence (HEK-hJUNO). These antibodies were used for immunostaining experiments and in vitro fertilisation assays with human gametes (GERMETHEQUE Biobank). Three hybridoma supernatants, verified by immunostaining, revealed specifically HEK-hJUNO cells. The three purified monoclonal antibodies, FJ2E4 (IgG1), FJ8E8 (IgG1) and FJ4F5 (IgG2a), recognised the soluble recombinant human JUNO protein and, in a western blot of HEK-hJUNO extracts, a protein with an expected MW of 25 kDa. In addition, soluble recombinant human IZUMO protein inhibited the binding of anti-hJUNO mAbs to cells expressing hJUNO. Using these anti-hJUNO mAbs in immunostaining, we identified the presence of JUNO protein at the plasma membrane of human oocytes. Furthermore, we revealed a progressive expression of JUNO according to oocyte maturity. Finally, we showed that human zona-free oocytes, inseminated in the presence of anti-hJUNO mAb, were not fertilised by human sperm. These results suggest that, as seen in the mouse, JUNO is indeed involved in human gamete membrane fusion during fertilisation. N/A. In accordance with French bioethics laws, functional tests were performed using zona-free oocytes, which of course does not fully encompass all normal in vivo physiological conditions. However, these in vitro tests do provide direct information regarding sperm-oocyte membrane interactions. Mechanisms of gamete fusion appear to be homologous between mice and humans. However, some differences do exist and analysing the human mechanisms is essential. In fact, this is the first report describing the presence of JUNO on human oocytes and its involvement in human fertilisation. This discovery allows further examination of the understanding of molecular mechanisms that drive gamete fusion: a crucial challenge at a time when infertility affects 16% of reproductively active couples. This work was supported by the Agence Nationale pour la Recherche, Grant no. ANR-13-BVS5-0004, and by Association Institut du Cancer et d'Immunogénétique (ICIG). There are no competing interests.//////////////////
Location and expression of Juno in mice oocytes during maturation. Suzuki B et al. (2017) Oocyte-sperm interaction is the essential step in fertilization. Juno, which has been known as Folate receptor 4, is the Izumo1 receptor expressed on the oocyte membrane. This study aims to investigate the location and expression of Juno in mice oocytes during maturation. To confirm the stage at which Juno expression begins in the mice oocytes and its location pattern, we performed immunostaining methods. Next, we evaluated Juno mRNA expression by a half quantitative RT-PCR. Juno knockdown oocytes were generated by microinjecting siRNA into the germinal vesicle (GV) stage oocytes, and analyzed the maturation rate. Our results showed that Juno was expressed on the surface of the oocyte cytoplasmic membrane at the GV stage and it continues to be expressed at similar levels in the metaphase II (MII) stages of oocytes maturation. Interestingly, Juno is also expressed on the first polar body membrane at the MII stage. Fluorescence showing Juno expression was decreased in the oolemma of siRNA injected oocytes, but it was not completely disappearing in knock down oocytes. MII stage-rates of siRNA injected oocytes were not significantly different from sham controls. Juno was expressed in oocytes at the GV stage and it continues to be expressed at similar levels in later stages of oocytes maturation. Juno accumulation in oolemma during oocyte maturation is essential for fertilization, such as membrane recognition of both gametes.//////////////////
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Mutations |
1 mutations
Species: mouse
Mutation name: None
type: null mutation
fertility: infertile - ovarian defect
Comment: Juno is the egg Izumo receptor and is essential for mammalian fertilization. Bianchi E 2014 et al.
Fertilization occurs when sperm and egg recognize each other and fuse to form a new, genetically distinct organism. The molecular basis of sperm-egg recognition is unknown, but is likely to require interactions between receptor proteins displayed on their surface. Izumo1 is an essential sperm cell-surface protein, but its receptor on the egg has not been described. Here we identify folate receptor 4 (Folr4) as the receptor for Izumo1 on the mouse egg, and propose to rename it Juno. We show that the Izumo1-Juno interaction is conserved within several mammalian species, including humans. Female mice lacking Juno are infertile and Juno-deficient eggs do not fuse with normal sperm. Rapid shedding of Juno from the oolemma after fertilization suggests a mechanism for the membrane block to polyspermy, ensuring eggs normally fuse with just a single sperm. Our discovery of an essential receptor pair at the nexus of conception provides opportunities for the rational development of new fertility treatments and contraceptives.
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