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Isolation and Expression Analysis of the Canine Insulin-Like Factor 3 Gene¹

Department of Obstetrics and Gynecology,³ Baylor College of Medicine, Houston, Texas 77030 Department of Animal Science,⁴ Swiss Federal Institute of Technology and Faculty of Veterinary Medicine, University of Zürich, CH-8092 Zürich, Switzerland Institute of Animal Genetics, Nutrition, and Housing,⁵ University of Berne, CH-3012 Berne, Switzerland

The insulin-like factor 3 (INSL3 or relaxin-like factor) is a hormone produced mainly in gonadal tissues in males and females. Deletion of INSL3 or its receptor in male mice leads to the undescended testes, or cryptorchidism. Here we describe an isolation and analysis of full-length canine INSL3 gene. The INSL3 gene is composed of two exons within a small genomic region. Putative translation of the isolated cDNA yields 132 amino acid preproINSL3 that has the domain structure characteristic for the insulin-relaxin peptide superfamily with a well-conserved receptor-binding domain. Northern blot hybridization showed stronger expression of INSL3 in testis than in ovary. Reverse transcription-polymerase chain reaction analysis of the INSL3 expression revealed a minor splice variant of INSL3 potentially encoding 105 amino acids peptide. We established that the medium, conditioned with recombinant canine INSL3, produced from the full-length cDNA, but not from the minor splice variant, activated human GREAT/LGR8 receptor in vitro. In addition to the functional allele of INSL3, genomic DNA of one of the analyzed dogs contained an intronless nonexpressed pseudogene of INSL3. We isolated canine INSL3 promoter and showed that its activity was strongly mediated by steroidogenic factor-1 in vitro. Using site-specific mutagenesis, we identified a well-conserved steroidogenic factor-1 binding site within canine INSL3 promoter.

¹ This work was supported by Grant R01 HD37067 from the National Institutes of Health (to A.I.A.).

² Correspondence: Alexander Agoulnik, Department of Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin St., Suite 861, Houston, TX 77030. FAX: 713 798 5074; agoulnik{at}bcm.tmc.edu

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