| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 95 === Infertility affects 10–15% of all couples, and impaired sperm production accounts for about half of these cases. Deleted in Azoospermia (DAZ) gene was originally isolated from the AZFc region on the long arm of the human Y chromosome that is frequently deleted in infertile men. DAZ-like (DAZL), an autosomal homolog located on chromosome 3p24, are expressed exclusively in the germ cells, and their protein products contain a highly conserved RNA-binding motif and a unique DAZ repeat. DAZL gene is essential for the differentiation of germ cells. Loss of germ cells was observed in Dazl knockout mice. And the effect of DAZL regulate substrate is on the translational control. We have previously found that the DAZL transcript amounts were lower in men with spermatogenic failure. To investigate the role of the DAZL gene in human spermatogenesis, DAZL was genotyped in 231 infertile Taiwanese men presenting with severe oligozoospermia and nonobstructive azoospermia. We identified two polymorphisms－A to G transition at nucleotide 386 in exon 3 (T54A), and nucleotide 260 in exon 2 (T12A); as well as two mutations: T to G transversion at nucleotide 382 in exon 3 (D52E) and A to C transversion at nucleotide 427 in exon 3 (K67N). The frequency of T54A allele was found to be more prevalent in patients than in controls (P=0.0004). To characterize the functional significance of DAZL polymorphisms or mutations, we identified the mRNA substrates that are specifically bound by human DAZL protein. We found that SDAD1, STK31, TPX1, MVH were the substrates of DAZL by the tether function assay analysis. Meanwhile, T12A and T54A were associated with decreased translational efficiency for TXP1. T12A, D52E, and K67N of DAZL were associated with decreased protein expression of STK31. All polymorphisms and mutations decreased protein expression of MVH. These results suggest that different polymorphisms may exert different effects on different genes during spermatogenesis. By using RNA-EMSA, we confirmed that TPX1 and STK31 were indeed DAZL substrates. In addition, by using RNA-EMSA, we define 3’UTR 1bp-101bp and 301-414bp of TPX1 interacts with DAZL. However, the variant DAZL proteins also bind to TPX1 and STK31. These findings suggest mutant proteins regulate substrates translation not directly through their binding activity. Other DAZL interacting partners may be involved in pathogenesis of these genetic variants and polymorphisms.
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