Abstract

In this study oxytocin (OT) receptors have been characterized and localized in the testis of the rat using the radioiodinated OT receptor antagonist ¹²⁵I-labelled d(CH₂)⁵ [Tyr(Me)²,Thr⁴,Tyr⁹-NH₂]-vasotocin (OTA). Receptor density and localization have been compared with the rat testis arginine vasopressin (AVP) receptor using the radioiodinated AVP V_1a receptor antagonist ¹²⁵I-labelled d(CH₂)₅Sar⁷-AVP and the radioiodinated linear AVP V_1a antagonist ¹²⁵I-labelled [(C₆H₅-CH₂CO)-O-methyl-d-Tyr-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH₂]. ¹²⁵I-labelled OTA bound with high affinity to membrane fractions of the rat testis (K _a = 13·8 ± 1·25 litres/nmol), mammary tissue (K _a=20·3± 4·36 litres/nmol) and uterus (K _a=27·8±0·74 litres/nmol). Competition studies with various OT and AVP receptor agonists and antagonists confirmed that the binding was to OT receptors. AVP receptors in the testis were found to be identical to AVP V_1a receptors in the liver. The AVP receptor density in the testis was much higher than the OT receptor density (109 ±12·3 vs 5·2 ±0·79 (mean ± s.e.m.) fmol/mg protein). Autoradiographical localization showed that both OT and AVP receptors were present in the interstitial spaces in the testis consistent with binding to Leydig cells. AVP receptors were also localized on the epithelial surfaces of the seminiferous tubules and on testicular blood vessels. This study has, for the first time, found OT receptors in the testis of the rat which have similar ligand-binding characteristics to mammary and uterine OT receptors. The receptor localizations are consistent with binding to Leydig cells.

Journal of Endocrinology (1994) 141, 343–352

Abstract

In this study arginine vasopressin (AVP) and oxytocin (OT) receptors have been characterized in the brushtail possum. AVP receptors were characterized using [³H]AVP and the radioiodinated AVP V_1a receptor antagonist ¹²⁵I-labelled [(C₆H₅-CH₂CO)-O-methyl-d-Tyr-Phe-Gln-Asn-Arg-Pro-Arg-Tyr- NH₂] while OT receptors were characterized using the radioiodinated OT receptor antagonist ¹²⁵I-labelled d(CH₂)₅[Tyr(Me)²,Thr⁴,Orn⁸, Tyr-NH₂ ⁹]-vasotocin. The receptor affinities and densities have been compared with the rat AVP and OT receptors. Low densities of OT receptors were present in the possum ovary and kidney. High densities of AVP-binding sites were found in the possum adrenal, testis, mesenteric artery, ovary and renal medulla and lower densities in the possum liver. The AVP-binding sites showed marked differences in ligand-binding characteristics from the rat AVP V_1a and V₂ receptors. Receptor affinities were similar between tissues, except for a distinctly lower value in the renal medulla. It is concluded that the brushtail possum expresses AVP receptors with distinct ligand specificities from those of the rat AVP V_1a and V₂ receptors.

Journal of Endocrinology (1995) 144, 19–29

Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the precise roles of this receptor in many of these tissues are unknown. In an effort to better understand INSL3–LGR8 systems in the rat, we cloned the full-length Lgr8 cDNA and investigated the presence and cellular localization of Lgr8 mRNA expression in adult and developing rat kidney. On the basis of these findings, we investigated the presence and distribution of renal ¹²⁵I-labelled human INSL3-binding sites and the nature of INSL3–LGR8 signalling in cultured renal cells. Thus, using in situ hybridization histochemistry, cells expressing Lgr8 mRNA were observed in glomeruli of renal cortex from adult rats and were tentatively identified as mesangial cells. Quantitative, real-time PCR analysis of the developmental profile of Lgr8 mRNA expression in kidney revealed highest relative levels at late stage gestation (embryonic day 18), with a sharp decrease after birth and lowest levels in the adult. During development, silver grains associated with Lgr8 mRNA hybridization were observed overlying putative mesangial cells in mature glomeruli, with little or no signal associated with less-mature glomeruli. In adult and developing kidney, specific ¹²⁵I-INSL3-binding sites were associated with glomeruli throughout the renal cortex. In primary cultures of glomerular cells, synthetic human INSL3 specifically and dose-dependently inhibited cell proliferation over a 48 h period, further suggesting the presence of functional LGR8 (receptors) on these cells (mesangial and others). These findings suggest INSL3–LGR8 signalling may be involved in the genesis and/or developmental maturation of renal glomeruli and possibly in regulating mesangial cell density in adult rat kidney.