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Identification, Characterization, and Functional Analysis of Sp1 Transcript Variants Expressed in Germ Cells During Mouse Spermatogenesis¹

Department of Anatomy and Neurobiology,³ Department of Obstetrics and Gynecology,⁴ Cooperative Reproductive Science Research Center,⁵ Morehouse School of Medicine, Atlanta, Georgia 30310-1495 Department of Cell Biology and Neuroscience,⁶ University of South Carolina School of Medicine, Columbia, South Carolina 29208 Department of Biology,⁷ University of Texas, San Antonio, Texas 78249 Department of Cell Biology and Physiology,⁸ University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 Department of Pharmaceutical Sciences,⁹ University of Pittsburgh School of Pharmacology, Pittsburgh, Pennsylvania 15261

The SP family of zinc-finger transcription factors are important mediators of selective gene activation during embryonic 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 3 full-length Sp1 transcripts (4.1, 3.7, and 3.2 kilobases [kb]) and an additional 1.4-kb 5'-truncated Sp1 transcript that are temporally expressed during spermatogenesis. Quantitative real-time polymerase chain reaction studies verified that the highest levels of Sp1 transcript expression of 4.1, 3.7, and 3.2 kb 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, or 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-specific and cell type-specific gene expression during mouse spermatogenesis.

¹ Supported by grant HD41749 from the National Institute for Child Health and Human Development, by Minority Biomedical Research Support grant SO6GM08248 from the National Institute of General Medical Sciences, and by Research Centers in Minority Institutions grant RRAI03034 from the National Institutes of Health.

² Correspondence: Kelwyn Thomas, Department of Anatomy and Neurobiology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, Georgia 30310-1495. FAX: 404 752 1028; kethomas{at}msm.edu

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