C Jean1,2, F Haghighirad3,4, Y Zhu3,4,5, M Chalbi6, A Ziyyat2, E Rubinstein3,4, C Gourier6, P Yip7, J P Wolf1,2, J E Lee7, C Boucheix3,4, V Barraud-Lange1,2. 1. University Paris Descartes, Sorbonne Paris Cité, Faculty of Medicine, Assistance Publique-Hôpitaux de Paris, University Hospital Paris Centre, CHU Cochin, Laboratory of Histology Embryology Biology of Reproduction, 123 boulevard de Port Royal, Paris, France. 2. Department of Genetic, Development and Cancer, Cochin Institute, Inserm U1016, Team Genomic Epigenetic and Physiopathology of Reproduction, University Paris Descartes, Sorbonne Paris Cité, 22 rue Méchain, Paris, France. 3. Inserm, UMR-S 935, SFR André Lwoff, 12 Avenue Paul Vaillant Couturier, Villejuif, France. 4. University Paris-Sud 11, UFR Medicine, 63 rue Gabriel Péri, Le Kremlin Bicêtre, France. 5. Department of Oncology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei Province, China. 6. Laboratory of Physic Statistic, Ecole Normale Superieure/PSL Research University, UPMC University Paris 6, University Paris Diderot, CNRS, 24 rue Lhomond, Paris, France. 7. Faculty of Medicine, Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Room 6314, Toronto, ON, Canada.
Abstract
STUDY QUESTION: Is JUNO protein present at the surface membrane of human oocytes and involved in the fertilisation process? SUMMARY ANSWER: JUNO protein is expressed on the plasma membrane of human oocytes and its inhibition by a monoclonal antibody completely blocks gamete fusion. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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). MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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.
STUDY QUESTION: Is JUNO protein present at the surface membrane of human oocytes and involved in the fertilisation process? SUMMARY ANSWER: JUNO protein is expressed on the plasma membrane of human oocytes and its inhibition by a monoclonal antibody completely blocks gamete fusion. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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). MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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.
Authors: Tyler D R Vance; Patrick Yip; Elisabet Jiménez; Sheng Li; Diana Gawol; James Byrnes; Isabel Usón; Ahmed Ziyyat; Jeffrey E Lee Journal: Commun Biol Date: 2022-09-17
Authors: Avinash S Gaikwad; Amanda L Anderson; D Jo Merriner; Anne E O'Connor; Brendan J Houston; R John Aitken; Moira K O'Bryan; Brett Nixon Journal: BMC Biol Date: 2019-10-31 Impact factor: 7.431