title: Melatonin improves the fertilization ability of post-ovulatory aged mouse oocytes by stabilizing ovastacin and Juno to promote sperm binding and fusion.
abstract: What are the underlying mechanisms of the decline in the fertilization ability of post-ovulatory aged oocytes?
  
  
  
  Melatonin improves the fertilization ability of post-ovulatory aged oocytes by reducing aging-induced reactive oxygen species (ROS) levels and inhibiting apoptosis and by maintaining the levels and localization of the fertilization proteins, ovastacin and Juno.
  
  
  
  Following ovulation, the quality of mammalian metaphase II oocytes irreversibly deteriorates over time with a concomitant loss of fertilization ability. Melatonin has been found to prevent post-ovulatory oocyte aging and extend the window for optimal fertilization in mice.
  
  
  
  Mouse oocytes were randomly assigned to three groups and aged in vitro for 0, 6, 12 and 24 h, respectively. Increasing concentrations of melatonin (10-9 M, 10-7 M, 10-5 M and 10-3 M) were added to the 24 h aging group.
  
  
  
  Sperm binding assays, in-vitro fertilization, immunofluorescent staining and western blotting were performed to investigate key regulators and events during fertilization of post-ovulatory aged mouse oocytes.
  
  
  
  We found that the actin cap which promotes a cortical granule (CG) free domain is disrupted with a re-distribution of CGs in the subcortex of aged oocytes. Ovastacin, a CG metalloendoprotease, is mis-located and prematurely exocytosed in aged oocytes with subsequent cleavage of the zona pellucida protein ZP2. This disrupts the sperm recognition domain and dramatically reduces the number of sperm binding to the zona pellucida. The abundance of Juno, the sperm receptor on the oocyte membrane, also is reduced in aged oocytes. Exposure of aged oocytes to melatonin significantly elevates in-vitro fertilization rates potentially by rescuing the above age-associated defects of fertilization, and reducing ROS and inhibiting apoptosis.
  
  
  
  N/A.
  
  
  
  We explored the mechanisms of the decline in fertilization ability decline in aged mouse oocytes, in vitro but not in vivo.
  
  
  
  Our findings may contribute to the development a more efficient method, involving melatonin, for improving IVF success rates.
  
  
  
  This study was supported by the National Natural Science Foundation (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose.
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Xiaoxin
authors.initials: X
authors.lastname: Dai
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Lu
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Mianqun
authors.initials: M
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Yilong
authors.initials: Y
authors.lastname: Miao
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Changyin
authors.initials: C
authors.lastname: Zhou
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Zhaokang
authors.initials: Z
authors.lastname: Cui
authors.affiliation: College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xiong
mesh_terms: D000818:Animals; D005260:Female; D005306:Fertilization / Q000187:drug effects* / Q000502:physiology; D008297:Male; D008550:Melatonin / Q000494:pharmacology*; D045726:Metalloproteases / Q000378:metabolism*; D051379:Mice; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013084:Sperm-Ovum Interactions / Q000187:drug effects* / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism; D015044:Zona Pellucida / Q000187:drug effects / Q000378:metabolism
chemical_list: D011956:Receptors, Cell Surface; C543634:folate receptor 4, mouse; C487827:Astl protein, mouse; D045726:Metalloproteases; D008550:Melatonin
keywords: juno; melatonin; ovastacin; post-ovulatory aging; zp2 cleavage