Introduction: This study investigates Insulin-like 3 (INSL3), also known as the Leydig cell-specific insulin-like peptide (LEY I-L), within the framework of evolutionary biology. It examines key aspects of gene structure, such as the dynamics and intronic phase, and employs bioinformatics tools to explore both the ancestral and modern functional roles of Insl3.

Insulin-like 3 (INSL3) belongs to a superfamily of peptides, growth factors, and hormones that include insulin, IGF, and relaxin. INSL3 is primarily produced as a pre-prohormone in somatic cells of the gonads, particularly in the Leydig cells of the testes and theca cells of ovarian follicles. cDNA sequences of INSL3 have been isolated from several vertebrate species. The structure of the INSL3 gene is similar to that of insulin and relaxin, consisting of two exons and one intron.

Investigation Methods: We used in silico analyses to determine and compare the structure of the Insl3 coding gene across vertebrate, invertebrate and ancestral chordate genomes.

Results: Bioinformatics analyses presented here and performed on nearly all known insl3 genes and cDNA sequences from the database reveal that the insl3 gene structure is conserved among fish and various non-mammalian vertebrate species, although intron lengths differ.

The in silico results also provide evidence for the gain-of-function role of a single copy of the insl3 gene in mammalian testis development: zebrafish insl3 proximal promoters reveal the presence of the same point mutation in the binding site of a key transcription factor that affects the promoter activity of mouse and human INSL3. This mutation negatively impacts the transcription of the insulin-like 3 gene in Leydig cells. Since the gene for the binding factor is present in the zebrafish genome, the proposed hypothesis suggests that Insl3's new key role in testicular descent and in testis development might also arise from a point mutation in its DNA binding site on the promoter.