Expression analyses of insulin-like peptide 3, RXFP2, LH receptor, and 3β-hydroxysteroid dehydrogenase in testes of normal and cryptorchid dogs

Insulin-like peptide 3 (INSL3) plays a key role in testicular descent in rodents, whereas in domestic animals, many aspects of the roles of INSL3 in reproductive organs after puberty are still unknown. This study was undertaken to (1) determine the quantitative changes of gene expression of testicular INSL3, its receptor (RXFP2), LH receptor, and 3β-hydroxysteroid dehydrogenase during and after puberty in normal male dogs; (2) compare the expressions of these substances in normal and cryptorchid dogs; and (3) localize the cells expressing INSL3 in normal and retained canine testes. Testes were obtained from small-breed normal male dogs (n = 56) and cryptorchid dogs (n = 22). Normal scrotal testes from the normal dogs (normal testes), retained testes from both the unilateral and bilateral cryptorchid dogs (retained testes), and scrotal testes of the unilateral cryptorchid dogs (cryptorchid scrotal testes) were used. We measured the concentrations of these testicular messenger RNAs (mRNAs) by quantitative real-time reverse transcription polymerase chain reaction, and an enzyme immunoassay was used for measuring INSL3 peptide. Immunohistochemistry for INSL3 peptide was done in paraformaldehyde-fixed frozen testicular tissue. In the normal dogs, total amount of INSL3 mRNA per testis tended to decrease (P = 0.05) from pubertal (6-12 months) to postpubertal (1-5 years) and decreased (P < 0.01) to middle age (5-10 years), but total amount of INSL3 peptide per testis did not change among age groups. Concentrations of INSL3 mRNA were higher (P < 0.01) in retained testes than those in the normal testes and cryptorchid scrotal testes, and similar differences were observed for INSL3 peptide. Reversely, total amounts of INSL3 mRNA and peptide per retained testis were lower (P < 0.01) than those per normal testis because of smaller weight of retained testes. Concentrations and total amount of RXFP2 mRNA in the retained testes were almost nil and lower (P < 0.01) than those in the normal testes and in the cryptorchid scrotal testes. Total amount of LH receptor mRNA per retained testis was lower (P < 0.01) than that per normal testis. The immunohistochemical analysis revealed that INSL3 was expressed only in Leydig cells of both the normal and retained canine testes. These results suggest that INSL3 in retained testes of cryptorchid dogs is substantially expressed per unit-weight basis but may be produced with lower amount as a whole testis. Also, this study provides findings that RXFP2 gene is expressed scarcely in the retained testes but normally in cryptorchid scrotal testes.