Journal of Peptide Science J. Peptide Sci. 7: 495–501 (2001) DOI: 10.1002/psc.344 Chemical Synthesis and Biological Activity of Rat INSL3 KATHRYN J. SMITH a , JOHN D. WADE a , ANTONIA A. CLAASZ a , LASZLO OTVOS Jr b , CATHERINE TEMELCOS c , YOSHIHIRO KUBOTA c , JOHN M. HUTSON c , GEOFFREY W. TREGEAR a and ROSS A. BATHGATE a, * a The Howard Florey Institute, University of Melbourne, Victoria, Australia b The Wistar Institute, Philadelphia, PA, USA c F. Douglas Stephens’ Surgical Research Laboratory, Murdoch Children’s Research Institute, Parkville, Victoria, Australia Received 30 April 2001 Accepted 7 May 2001 Abstract: The recently identified protein, insulin 3 (INSL3), has structural features that make it a bona fide member of the insulin superfamily. Its predicted amino acid sequence contains the classic two-peptide chain (A- and B-) structure with conserved cysteine residues that results in a disulphide bond disposition identical to that of insulin. Recently, the generation of insl3 knockout mice has demonstrated that testicular descent is blocked due to the failure of a specific ligament, the gubernaculum, to develop. The mechanism by which INSL3 exerts its action on the gubernaculum is currently unknown. The purpose of this study was to, for the first time, synthesize rat INSL3 and test its action on organ cultures of foetal rat gubernaculum. INSL3 also contains a cassette of residues Arg-X-X-X-Arg within the B-chain, a motif that is essential for characteristic activity of another related member of the superfamily, relaxin. Hence, the relaxin activity of rat INSL3 was also tested in two different relaxin bioassays. The primary structure of rat INSL3 was determined by deduction from its cDNA sequence and successfully prepared by solid phase peptide synthesis of the two constituent chains followed by their combination in solution. Following confirmation of its chemical integrity by a variety of analytical techniques, circular dichroism spectroscopy confirmed the presence of high  -turn and  -helical content, with a remarkable spectral similarity to the synthetic ovine INSL3 peptide and to synthetic rat relaxin. The synthetic rat INSL3 bound with very low affinity to rat relaxin receptors and had no activity in a relaxin bioassay. Furthermore, it did not augment or antagonize relaxin activity. The rat INSL3 did however induce growth of foetal rat gubernaculum in whole organ cultures demonstrating that INSL3 has a direct action on this structure. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. Keywords: insulin 3; relaxin-like factor; testis descent; gubernaculum; solid phase peptide synthesis INTRODUCTION Insulin 3 (INSL3; also called the relaxin-like factor, RLF; or Leydig cell insulin-like peptide, Ley-I-L) is a recently identified member of the insulin-relaxin superfamily of peptide hormones. It is primarily expressed in the Leydig cells of the testis and thecal cells of the ovary [1]. Recently, two independent groups developed knockout mice for INSL3 [2,3]. The major phenotype in these animals was the lack of testicular descent in the male due to the failure to develop of a specific ligament, the genito-inguinal ligament or gubernaculum. During normal testicular descent, the caudal end of the gubernaculum, the gubernacular bulb, enlarges or ‘swells’ and this process is disrupted in the INSL3 knockout. The mechanism(s) by which INSL3 in- duces this ‘swelling’ of the gubernacular bulb is unknown. * Correspondence to: The Howard Florey Institute, University of Melbourne, Victoria 3010, Australia; e-mail: r.bathgate@ hfi.unimelb.edu.au Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.