Aging-Related Increased Expression of Inducible Nitric Oxide Synthase and Cytotoxicity Markers in Rat Hypothalamic Regions Associated with Male Reproductive Function

Abstract

We have previously demonstrated that the inducible nitric oxide synthase (iNOS) protein and total NOS activity increase in the hypothalamus and other regions of the male rat brain during aging. We have now tested the hypothesis that increased iNOS results in excessive nitric oxide (NO) and peroxynitrite production, and leads to increased apoptosis in CNS cells, including the GnRH and oxytocin hypothalamic neurons involved in the control of male reproductive function. Young (3-month-old) and old (24-month-old) male Brown Norway rats (n = 6) were perfused with 4% formalin. Adjacent coronal paraffin-embedded sections (5 µm) of preoptic area (POA), supraoptic nucleus (SON), paraventricular nucleus (PVN), and arcuate nucleus (ARC) of the hypothalamus were immunostained with antibodies for iNOS, neuronal NOS (nNOS), and nitrotyrosine (a marker of peroxynitrite formation). The intensity of immunostaining was measured using a densitometric image analysis system. Apoptosis was determined by the TUNEL assay. Double immunofluorescence staining with confocal laser scanning microscopy was used for co-localization studies. A significant increase in the iNOS immunostaining measured as optical density (OD) was found in the old compared to the young animals (SON: 0.32 ± 0.02 vs. 0.23 ± 0.03, p < 0.05; PVN: 0.34 ± 0.03 vs. 0.07 ± 0.05, p < 0.001; POA: 0.18 ± 0.02 vs. 0.01 ± 0.02, p < 0.001). Aging did not affect nNOS expression. Nitrotyrosine was elevated in the hypothalamic regions of old compared to young rats (SON: 0.32 ± 0.05 vs. 0.10 ± 0.04, p < 0.05; PVN: 0.32 ± 0.04 vs. 0.13 ± 0.03, p < 0.01; POA: 0.72 ± 0.06 vs. 0.03 ± 0.003, p < 0.001). Increased nitrotyrosine was accompanied by an elevation of the apoptotic index in the old rats (SON: 11.01 ± 3.33 vs. 0.57 ± 0.50, p < 0.001; PVN: 3.08 ± 1.12 vs. 0.42 ± 0.32; POA: 6.60 ± 1.93 vs. 0.18 ± 0.17, p < 0.01; ARC: 0.001 ± 0.0001 vs. 4.33 ± 2.33). iNOS staining co-localized with GnRH and oxytocin staining. In conclusion: The aging-related iNOS increased expression in the hypothalamus of the male rat affects regions known to control the synthesis and release of GnRH (POA, ARC) and oxytocin (PVN, SON), and the factors regulating penile erection (POA, and PVN). These observations suggest that iNOS may play a role in the reduction in GnRH and oxytocin neuronal secretion resulting in reproductive dysfunctions such as lowered serum testosterone, hypospermatogenesis, and diminished copulatory function in the aging male animal.