Identification and localization of molecular sequences associated with regulation of Human Acrosin expression during Spermatogenesis.

VAZQUEZ-LEVIN, MH; GHIRINGHELLI, DP; ZAHN, A; RIVAROLA V; CHARREAU, EH

Boston - Estados Unidos

Congreso; 52nd Annual Meeting of the American Society for Reproductive Medicine; 1996

American Society for Reproductive Medicine

Objective: Acrosin, a testis-specific serine proteinase localized on the sperm acrosome of all mammalian species studied including humans, is involved in sperm penetra-tion´of the zona pellucida during fertilization. During spermatogenesis, human acrosin is íirst detected in midpachitene primary spermatocytes (Escalier et al, Development, 1991). The purpose of the present study was to perform a molecular analysis on the nucleotide sequence of the 5´ untranslated región (5´UTR) of the human acrosin gene, in an attempt to identify and localize specific sequences associated with the regulation of acrosin expression during spermatogenesis. Design/Materials and Methods: Sequence analysis was performed on ~2.0 kb of the 5´UTR of the human acrosin gene, using a clone previously characterized (PGH9, Vasquez et al, Eur. J. Biochem., 1992) and the Sequenase kit (US Biochemical Corp., USA). Results: A previous analysis of the first 500 nucleotides (nt) of the 5´UTR of the human acrosin gene had revealed the presence of a group of sequences highly homologous to regulatory elements of gene expression of protamine Pl and P2, rat Histone Hit, and human phosphoglycerate kinase-2 genes. This initial analysis failed identify the presence of conventional CAAT and TATA boxes (Keinie et al, 1990, Vazquez-Levin et al, 1992). The present analysis identiíied a TATA sequence at positions —629 and —1,024 from the initiation codon, and a CAAT sequence was local-ized at position -742; other sequences related were found at more distal positions of the 5´UTR. Localization of these sequences was similar in rat (TATA:-607, CAAT:-678) and mouse (TATA:-588; CAAT:-656) acrosin 5´UTRs. In addition, complementary sequences to ACGTCA were lo-calized at positions —332 and —521. This sequence has been described as the binding site for a testis specific tran-scription factor of Histone H2B. Moreover, the sequence GACTTCAGAA was identified at position -1288. This sequence is present in the mouse and rat 5´UTRs, and is identical in 9 of 10 nucleotides at the binding sequence of the Tet-1 testis specific factor. Finally, the analysis showed the presence of the sequence TGAGGTCA at position -39. This sequence is present in the mouse and rat acrosin 5´UTRs, and is highly conserved in several promot-ers for testis-specific genes; in particular this sequence is highly homologous (7/8 nt) to a consensus sequence of the CREM (cAMP responsive element); CREM is highly ex-pressed in pachitene spermatocytes. Conclusions: Nucleotide sequence analysis has allowed the identification and localization of specific elements in the human acrosin 5´UTR, highly conserved in the rat and mouse acrosin genes, as well as in other testis specific genes. These sequences have been shown to participate in the regulation of gene expression during spermatogenesis, and are probably responsible for acrosin expression in midpachitene spermatocyte and spermatids.