Testis

Identification, Characterization, and Functional Analysis of Sp1 Transcript Variants Expressed in Germ Cells During Mouse Spermatogenesis

Abstract
The SP-family of zinc-finger transcription factors are important mediators of selective gene activation during development and cellular differentiation. SP-binding GC-box domains are common cis-regulatory elements present in the promoters of several genes expressed in a developmentally-specific manner in differentiating mouse germ cells. Four Sp1 cDNAs were isolated from a mouse pachytene spermatocyte cDNA library and characterized by DNA sequence analysis. Northern Blot studies revealed that these cDNAs corresponded to three full-length Sp1 transcripts (4.1, 3.7, and 3.2 kb) and an additional 1.4 kb 5'-truncated Sp1 transcript that are temporally expressed during spermatogenesis. Quantitative real-time PCR studies verified that the highest levels of expression of 4.1, 3.7 and 3.2 kb Sp1 transcripts occur in the primary spermatocytes. The spatial and temporal expression patterns of these Sp1 transcripts and their encoded 60 kDa and 90 kDa Sp1 proteins were demonstrated using in situ hybridization and immunohistochemical analyses. To assess the transcriptional properties of these Sp1 transcription factors, SP-deficient Drosophila SL2 cells were stably transfected with the respective Sp1 cDNA expression vectors and cotransfected with either Ldh2, Ldh3 and Creb promoter/Luciferase reporter constructs. The levels of SP-mediated luciferase expression observed depended on the structure of the glutamine-rich transactivation domains and the number of GC-box elements present in the respective promoters. The alterations observed in germ cells in the patterns of expression of the Sp1 transcripts encoding the 60 kDa and 90 kDa SP1 isoforms suggest that these SP1 factors may be involved in mediating stage and cell-type-specific gene expression during mouse spermatogenesis.

Key words: Male Reproductive Tract • Testis • Developmental biology • Sperm motility and transport • Spermatogenesis

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