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Analysis of Germ Cell Nuclear Factor Transcripts and Protein Expression During Spermatogenesis¹

The Laboratories for Reproductive Biology⁴ Department of Pediatrics,⁵ University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 State Key Laboratory of Molecular Biology⁶ , Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science, Chinese Academy of Sciences, Shanghai, 200031 China Curriculum in Genetics and Molecular Biology,⁷ University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 Laboratory of Pulmonary Pathobiology,⁸ Cell Biology Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Department of Cell and Developmental Biology,⁹ University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Germ cell nuclear factor (GCNF), an orphan receptor in the nuclear receptor superfamily, is expressed predominantly in developing germ cells in the adult mouse. Two Gcnf transcripts (7.4 and 2.1 kilobase [kb]) encoded by a single copy gene are expressed in the testis of several mammalian species. To identify features that regulate Gcnf expression, we characterized the structure and sequence of the mouse gene and its two transcripts and determined the expression profile of the GCNF protein during spermatogenesis. Genomic fragments spanning part of the 5'-untranslated region (UTR), the coding sequence, and the complete 3'-UTR (80 kb) were isolated and sequenced. The 3'-UTRs of the two transcripts are quite distinct. The 7.4 kb transcript, which appears earlier in spermatogenesis, has a very long 3'-UTR of 4451 nucleotides. In contrast, the 2.1 kb transcript, which is expressed predominantly during the haploid phase of spermatogenesis, has a 3'-UTR that is only 202 nucleotides in length. Additional analyses indicate that both transcripts share the same coding region and are associated with polysomes. A single GCNF protein band was detected in testis extracts by Western blotting with a specific antiserum. Immunohistochemical analysis showed that GCNF is localized in the nuclei of pachytene spermatocytes and round spermatids. GCNF is first detectable in early pachytene spermatocytes (stage II) and is continuously expressed until spermatids begin to elongate in stage IX. Although GCNF is generally distributed throughout the nucleus, it is particularly prominent in heterochromatic regions at some stages and in condensed chromosomes undergoing the meiotic divisions. This expression profile suggests that GCNF plays a role in transcriptional regulation during meiosis and the early haploid phase of spermatogenesis.

¹ Supported by NIH grants TW/HD00627, CA16086 (UNC Lineberger Comprehensive Cancer Center), and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproductive Research. Funding was also provided by the Chinese National Natural Sciences Foundation 39893320 and the Chinese 95 Yu-Pan-deng Plan funding project 1997-066.

² Correspondence: Deborah A. O'Brien, Department of Cell and Developmental Biology, CB# 7090, 214A Taylor Hall, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7090. FAX: 919 966 1856; dao{at}med.unc.edu

³ Current address: Vanderbilt-Ingram Cancer Center, 512 Preston Research Building, Vanderbilt University, Nashville, TN 37232-6838

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