Aging-related increased expression of inducible nitric oxide synthase and cytotoxicity markers in rat hypothalamic regions associated with male reproductive function

We have previously demonstrated that the inducible nitric oxide synthase (i NOS) protein and total NOS activity increase in the hypothalamus and other regions of the male rat brain during aging. We have now tested the hypothes is that increased iNOS results in excessive nitric oxide (NO) and peroxynit rite production, and leads to increased apoptosis in CNS cells, including t he GnRH and oxytocin hypothalamic neurons involved in the control of male r eproductive function. Young (3-month-old) and old (24-month-old) male Brown Norway rats (n = 6) were perfused with 4% formalin. Adjacent coronal paraf fin-embedded sections (5 mum) of preoptic area (POA), supraoptic nucleus (S ON), paraventricular nucleus (PVN), and arcuate nucleus (ARC) of the hypoth alamus were immunostained with antibodies for iNOS, neuronal NOS (nNOS), an d nitrotyrosine (a marker of peroxynitrite formation). The intensity of imm unostaining was measured using a densitometric image analysis system. Apopt osis was determined by the TUNEL assay. Double immunofluorescence staining with confocal laser scanning microscopy was used for co-localization studie s. A significant increase in the iNOS immunostaining measured as optical de nsity (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 a ffect nNOS expression. Nitrotyrosine was elevated in the hypothalamic regio ns 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 v s. 0.03 +/- 0.003, p < 0.001). Increased nitrotyrosine was accompanied by a n 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 ma le rat affects regions known to control the synthesis and release of GnRH ( POA, ARC) and oxytocin (PVN, SON), and the factors regulating penile erecti on (POA, and PVN). These observations suggest that iNOS may play a role in the reduction in GnRH and oxytocin neuronal secretion resulting in reproduc tive dysfunctions such as lowered serum testosterone, hypospermatogenesis, and diminished copulatory function in the aging male animal. Copyright (C) 2001 S. Karger AG, Basel.