RNA interference during spermatogenesis in mice.
Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.
Shoji
Masanobu
M
Department of Development and Differentiation, Institute for Frontier Medical Science, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Chuma
Shinichiro
S
Yoshida
Kayo
K
Morita
Takashi
T
Nakatsuji
Norio
N
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D018274:Electroporation; D005434:Flow Cytometry; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008297:Male; D051379:Mice; D009687:Nuclear Proteins; D028044:Phosphate-Binding Proteins; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013091:Spermatogenesis / Q000235:genetics* / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism*; D013737:Testis / Q000378:metabolism*
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C513032:Dmc1 protein, mouse; D009687:Nuclear Proteins; D028044:Phosphate-Binding Proteins; D034741:RNA, Small Interfering