Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat

A new member of the insulin gene family (INSL6) was identified from an Expressed Sequence Tag database through a search for proteins containing the insulin family B-chain cysteine motif. Human and rat INSL6 encoded polypeptides of 213 and 188 amino acids, respectively. These orthologous sequences contained the B-chain, C-peptide, and A-chain motif found in other members of the insulin family. Human INSL6 was 43% identical to human relaxin H2 in the B- and A-chain regions. As with other family members, human and rat INSL6 had predicted dibasic sequences at the junction of the C-peptide and A-chain. Human INSL6 sequence had an additional dibasic site near the C-terminus of the A-chain. The presence of a single basic residue at the predicted junction of the B-chain and C-peptide suggests that multiple prohormone convertases are required to produce the fully mature hormone. INSL6 was found to be expressed at high levels in the testis as determined by Northern blot analysis and specifically within the seminiferous tubules in spermatocytes and round spermatids as detected by in situ hybridization analysis. Radiation hybrid mapping placed the human INSL6 locus at chromosome 9p24 near the placenta insulin-like homologue INSL4 and the autosomal testis-determining factor (TDFA) locus. 
Lok S, et al. Biol Reprod. 2000 Jun;62(6):1593-9.

Pair-wise alignment between human and rat INSL6. The two polypeptides exhibited 47% identity. ":" Denotes a conserved residue. "." Denotes a conservative substitution. The nucleotide sequences encoding human and rat INSL6 are available through GenBank accession nos. AF156094 and AF159506, respectively. The predicted signal peptide sequences are underlined. The B- and A-domain regions are in shaded boxes. Lok S, et al. Biol Reprod. 2000 Jun;62(6):1593-9.

Multiple alignment of insulin gene family members with human and rat INSL6 over the B- and A-domains. Below the alignment are the conserved B- and A-domain motifs. "h" Denotes a conserved hydrophobic residue. The lengths of the nonconserved sequences linking the B- and A-domains are indicated. Regions of conserved amino acids are in bold. Domain boundaries for INSL3, IINSL4, INSL5, and INSL6 are predicted

Northern blot analysis of total INSL6 transcript. RNA isolation, fractionation, and hybridization to 32P-labeled INSL6 cDNA are described in Materials and Methods. a) Human multiple-tissue mRNA blot (Clontech). Each lane contained approximately 2 µg of poly(A)+ RNA from the specified tissues. Hybridization of the blot to a human clathrin probe to confirm the integrity of the mRNA is shown at the bottom. b) Rat mRNA blots. Each lane represents 10 µg of total RNA from the indicated tissues. The locations of the 28S and 18S ribosomal RNAs are indicated. Hybridization of the blots to a cyclophilin probe to confirm the integrity of the mRNA is shown in the lower panels

In situ hybridization distribution of INSL6 in emulsion-dipped tissue sections of rat testis. a) Lightfield micrograph of a rat testis section labeled with [33P]INSL6 rat antisense RNA. Signal was detected in seminiferous tubules but not in the interstitium. b) Corresponding darkfield micrograph of section shown in a. Signal from INSL6-labeled probe could be seen primarily over pachytene spermatocytes and round spermatids. c) High-magnification lightfield micrograph of a rat testis section labeled with [33P]INSL6 rat antisense RNA. d) Corresponding darkfield micrograph of section shown in c. e) Lightfield micrograph of a rat testis section labeled with [33P]INSL6 rat sense RNA. All detected signals were considered nonspecific. f) Corresponding darkfield micrograph of section shown in e.