The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells

Abstract

Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is accumulating, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 72% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was 6 amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.

International Union of Basic and Clinical Pharmacology. XCV. Recent Advances in the Understanding of the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors 1–4, the Receptors for Relaxin Family Peptides

“…Similar processing has been demonstrated for porcine relaxin-3 (Liu et al, 2003b) but not yet for INSL5. INSL3 has been isolated as both a prohormone form in the goat (Siqin et al, 2013) and boar (Minagawa et al, 2012) and as a processed form in the bovine (Bullesbach and Schwabe, 2002). Importantly, and as for relaxin, both processed and unprocessed forms of INSL3 are bioactive, although relaxin-3 must be processed to be active at RXFP3 (Ganella et al, 2013a).…”

Functional insulin‐like factor 3 (INSL3) hormone‐receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility

“…; Siqin et al . ). Although INSL3 is essential for testicular descent in fetal males (Nef & Parada ; Zimmermann et al .…”

“…In fact, we have demonstrated in male goats that INSL3 produced in Leydig cells is secreted not only into the blood circulation but also into the lumen fluid of seminiferous tubules (Siqin et al . ), where RXFP2 receptors are expressed on germ cells (Siqin et al . ; Pitia et al .…”

Morphological changes in testicular tissue in infertility

“…Трансформирующие факторы роста α и β, синтезируемые клетками Сертоли, подав-ляют стероидогенез в клетках Лейдига, воздействуя на экспрессию рецепторов лютеинизирующего гормо-на [3,9,10]. В то же время образование трансформи-рующих факторов роста в клетках Сертоли тормозится фолликулостимулирующим гормоном [6].…”