Effects of inducible nitric oxide synthase (iNOS) deficiency in mice on Sertoli cell proliferation and perinatal testis development.

Nitric oxide (NO) plays crucial roles in several physiological and pathological conditions. The iNOS isoform produces high levels of NO independent of intracellular calcium and, in the testis, which is expressed in Sertoli (SC), Leydig (LC) and germ cells. The testicular roles of NO are unclear, but it can inhibit LC testosterone production. Our aim was to evaluate the effects of iNOS deficiency on testis development in mice from late fetal life through early puberty. Therefore, testes from wild type (C57BCL/6) and iNOS(-/-) mice (B6.129P2- Nos2(tm1Lau) /J) were sampled at various ages between e18.5 and Pnd20 and evaluated by histological and stereological analyses; proliferating cells were labelled with (3)H-thymidine. At all ages, testis weight and anogenital index, a measure of fetal androgen exposure, were greater in iNOS-deficient mice than in wild type mice. At all ages after birth, iNOS-deficient mice exhibited increased (p < 0.05) SC number per testis, and this was accounted for by a higher SC proliferation index (p < 0.05) in iNOS-deficient mice, especially on Pnd1 and Pnd5. Similarly, LC number per testis was higher (p < 0.05) in iNOS(-/-) mice than in wild type at all post-natal ages. Highly positive and significant correlations were observed between the proliferation index for SC, LC and peritubular myoid cells on e18.5 and post-natally. Although lumen formation was slightly advanced in iNOS(-/-) mice, no obvious other effects on pubertal testis development were observed. These results imply that NO may normally constrain testis somatic cell development, especially SC, perhaps by limiting testosterone production. Removal of this constraint results in normal, but larger, testes with greater sperm production. Our data pinpoint the window of iNOS (NO) action on SC proliferation and raise the possibility that experimental manipulation of NO in early post-natal life could be used to enhance SC proliferation if this was deficient for any reason.

Sertoli cell numbers and spermatogenic efficiency are increased in inducible nitric oxide synthase mutant mice.

Nitric oxide (NO) is produced via oxidation of l-arginine by nitric oxide synthases (NOSs), and is known as inducible (iNOS), neuronal, endothelial or testis-specific. Suggesting important functions for NOS in the normal rat and mouse testis, iNOS is reported to be constitutively expressed in Leydig cells (LC), Sertoli cells (SC) and germ cells. In our study, we sought to provide further insights into the roles of iNOS in the adult mouse testis using iNOS(-/-) mice. Perfusion-fixed testes from wild type (WT) and iNOS(-/-) mice were used for histological and stereological evaluations. Some of the mice had been injected with (3) H-thymidine to label proliferating cells and to determine the duration of spermatogenesis that was unaffected in iNOS(-/-) mice. Both LC nuclear volume and individual cell size were significantly decreased in iNOS(-/-) mice, but the total number of LC per testis was increased (p < 0.05) by approximately 16%. The number of SC per testis was strikingly increased (approximately twofold) in iNOS(-/-) mice, and testis weight and DSP per gram of testis (spermatogenic efficiency) were similarly increased. The anogenital distance was also significantly increased in iNOS(-/-) mice, and this key endpoint suggests that the augmentation observed for the SC number may be related to increased foetal T-exposure during the masculinization programming window. Compared with WT testes, the numbers of spermatocytes and spermatids and SC per tubule cross sections were significantly increased in iNOS(-/-) mice. Except for stages V-VI and VII-VIII, iNOS(-/-) mice exhibited approximately 3.5-fold fewer apoptotic germ cells than in WT mice. Taken together, our results provide new evidence that iNOS plays an important role in numerical and functional regulation of key somatic cells in the testis, which in turn impacts on germ cells and their survival and thus on daily sperm production.