男性不妊の疾患モデルと遺伝子工学的治療法　　３．精巣特異的細胞質ポリＡポリメラーゼＴＰＡＰによる精子形態形成制御

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  • 柏原 真一
    筑波大学大学院生命環境科学研究科応用生物化学系
  • 馬場 忠
    筑波大学大学院生命環境科学研究科応用生物化学系

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タイトル別名
  • Regulation of Spermatogenesis by Testis-specific, Cytoplasmic Poly (A) Polymerase, TPAP

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Poly (A) tails of eukaryotic mRNAs are implicated in various aspects of mRNA metabolism, including transport into the cytoplasm, stability, and translational control. Thus, the control of poly (A) tail length is one of the posttranscriptional regulations of gene expression. Spermatogenesis is a highly specialized process of differentiation of male germ cells to produce spermatozoa. This differentiation process requires a controlled program of stage-specific gene expression, which is precisely regulated at the transcriptional, posttranscriptional, and translational levels. We have identified a novel, testis-specific cytoplasmic poly (A) polymerase, TPAP (PAPβ), as a candidate molecule responsible for additional extension of poly (A) tails of specific mRNAs in haploid, round spermatids. The TPAP gene was most abundantly expressed coincident with the additional elongation of mRNA poly (A) tails in round spermatids. TPAP shared a high degree of amino acid sequence identity (86%) with a constitutively expressed nuclear poly (A) polymerase, PAPII. Despite the sequence conservation of functional elements, including three catalytic Asp residues, an ATP-binding site, and an RNA-binding domain, TPAP lacked an approximately 100-residue C-terminal sequence carrying one of two bipartite-type nuclear localization signals, and part of a Ser/Thr-rich domain found in PAPII. Indeed, TPAP was localized in the cytoplasm of spermatogenic cells as an enzymatically active 70-kDa protein. To elucidate the role of TPAP in spermatogenesis, we produced mutant mice lacking the functional TPAP gene, using homologous recombination in embryonic stem cells. The absence of TPAP resulted in the arrest of spermiogenesis at step 7. TPAP-deficient mice displayed impaired expression of haploid-specific genes that are required for the morphogenesis of spermatozoa. The TPAP deficiency also caused incomplete elongation of poly (A) tails of specific transcription factor mRNAs. Although the overall cellular level of the transcription factor TAF10 was unaffected, TAF10 was insufficiently transported into the nucleus of germ cells. These data demonstrate that TPAP affects the transport of at least TAF10 in the nucleus, possibly by additional polyadenylation-dependent translational activation of dormant mRNA encoding a transporter protein or possibly by TPAP itself. Our study shows a direct link between the deficiency of a cytoplasmic poly (A) polymerase, TPAP, and the arrest of mouse spermiogenesis, providing new insights into the regulation of haploid-specific gene expression by cytoplasmic polyadenylation in male germ cells.

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