title: RNA interference in meiosis I human oocytes: towards an understanding of human aneuploidy.
abstract: Although female meiosis I errors account for the majority of human aneuploidy, their molecular basis is largely unknown. By elucidating gene function, gene knockdown using RNA interference (RNAi) could shed light on this enigmatic process. In practice, however, the extreme paucity of immature human oocytes makes the evaluation of gene-targeting tools difficult. Here, we undertake RNAi in human oocytes and describe an approach employing mouse oocytes which could overcome the problem of limited biological material. We designed a short interfering RNA (siRNA) designated si539 to target the human mitotic arrest deficient 2 (hMad2) spindle checkpoint component. In human oocytes microinjected with si539, the hMad2 signal detected by Western blotting was 85-92% less intense than in oocytes injected with control siRNA indicating efficient silencing. Further examination of si539's targeting efficiency was undertaken using a green fluorescent protein (GFP)-tagged hMad2 mRNA construct in mouse oocytes. Consistent with Western blot analysis, si539 reduced hMad2-GFP expression in mouse oocytes by approximately 94% and relieved the meiosis I arrest otherwise induced by hMad2-GFP in mouse oocytes. By facilitating the investigation of candidate genes involved in regulating human female meiosis I, this approach can bring us closer to understanding the origins of aneuploidies such as Down's syndrome.
authors.affiliation: Newcastle Fertility Centre at Life, International Centre for Life, Times Square, Newcastle upon Tyne NE1, UK. h.a.homer@ncl.ac.uk
authors.forename: Hayden A
authors.initials: HA
authors.lastname: Homer
authors.forename: Alex
authors.initials: A
authors.lastname: McDougall
authors.forename: Mark
authors.initials: M
authors.lastname: Levasseur
authors.forename: Alison P
authors.initials: AP
authors.lastname: Murdoch
authors.forename: Mary
authors.initials: M
authors.lastname: Herbert
mesh_terms: D000782:Aneuploidy; D000818:Animals; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005260:Female; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D064246:Mad2 Proteins; D008540:Meiosis / Q000235:genetics*; D051379:Mice; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D012097:Repressor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins