Nitric oxide synthase and reproductive ageing

The excitatory amino acid receptors (NMDA receptors) are considered to be the main neurotransmitter receptors mediating fast synaptic excitation in the central nervous system (CNS) through the opening of Ca2+ channels triggering a series of neuronal cascades. One of the mechanistic pathways is the stimulation of neuronal nitric oxide synthase (nNOS) resulting in the synthesis of nitric oxide which has been presumed to be the mediator of some of the neuroendocrine effects of the excitatory amino acid glutamine specifically on GnRH and LH pulsatile secretions (Braun et al 1997). In the hypothalamus, nNOS is the predominant NOS isoform and has been demonstrated to colocalize with or adjacent to the NMDA receptors and GnRH neurons in the medial preoptic area (Grossman et al 1994). Thus in ageing, it is conceivable that the hypothalamic NMDA receptor/nNOS pathway may play a role in hypothalamic dysfunction. NMDA receptors might be decreased accompanied by a decrease in nNOS activity in the brain and other organs outside the CNS.

An alternative hypothesis is that ageing is associated with an excessive synthesis of NO resulting in the accumulation of its cytotoxic metabolites such as peroxynitrite, leading to neuronal apoptosis. The process might affect the hypothalamic neurons including those that secrete GnRH. Such cytotoxic effects ofexcessive levels ofNO could result in accelerated apoptosis ofother components of the reproductive axis such as the testes. The high NO levels in tissues of ageing animals may occur as a result of excessive stimulation of nNOS by activation of NMDA receptors or the spontaneous expression of inducible NOS (iNOS), the NOS isoform that is induced during autoimmunity, inflammation and degeneration. In normal physiological conditions iNOS is undetectable in the organs of adult laboratory animals and is expressed in high levels only after exogenous cytokine stimulation and in inflammatory or infectious processes. To study the role of NOS in reproductive ageing, our laboratories investigated the NMDA receptor content and binding, nNOS and iNOS levels, and activity in the brain and in the testes of the ageing BN rat.

The NMDA receptor binding activity in the hypothalamus of old rats was 66% lower than that of adult animals. The results of the binding activity were confirmed by the demonstration that the NMDA receptor content was also decreased by 34% compared to young animals. To our surprise, the decrease in NMDA receptors was associated with a 67% increase in NOS activity in the hypothalamus of old rats when compared to the adult animals, while nNOS content was not different between the two groups. In contrast, iNOS content in the hypothalamus of old rats were increased by 3.8-fold compared with adult animals (Fig. 5). The increase in iNOS content was demonstrated not only in the hypothalamus but also in the frontal and parietal cortex, and in the cerebellum (Vernet et al 1998). Our results showed that ageing in the BN rat was associated with high NO synthesis in the hypothalamus and other regions of the brain. This occurred independently of the NMDA receptors (which were decreased) and nNOS activity (which was unchanged). We thus concluded that increased iNOS might result in neurotoxicity which could be involved in the impaired GnRH pulsatile secretion and also a possible inducer of age-associated cell loss in the brain and other organs such as the testes.

We then proceeded to investigate the role of iNOS in male reproductive ageing in the hypothalamus and the end organ, the testis. Using immunohistochemistry we found significant increases in iNOS immunostaining in the supraoptic and paraventricular nucleus and the preoptic area of the hypothalamus in the old rats. Nitrotyrosine, a marker for peroxynitrite formation (a cytotoxic product of excess NO and reactive oxygen species interaction) was also elevated in the same areas of the hypothalamus of old rats. The accumulation of peroxynitrite was accompanied by an increase in the apoptotic index of neurons in the supraoptic, paraventricular and arcuate nucleii as well as in the preoptic area of the hypothalamus of old rats. Apoptosis of neurons is extremely rare in the hypothalamus and other areas of the brain of young animals. We thus hypothesized that neuronal apoptosis could be the cause of the reduction of GnRH neurons detected by in situ hybridization by Gruenewald et al (2000). In contrast, ageing did not affect nNOS expression. We further examined the anatomical relationship ofiNOS and GnRH-positive neurons in the hypothalamus using double immunofluorescence technique in combination with confocal laser scanning microscopy. The iNOS staining co-localized with GnRH staining in the same regions of the hypothalamus of the rat brain. These preliminary studies showed iNOS expression in the hypothalamus of the affected regions of the brain known to control the synthesis and release of GnRH, confirming our hypothesis that iNOS may indeed play a role in the reduction of GnRH pulsatile secretion resulting in reproductive dysfunctions such as lowered testosterone in the ageing males. Ongoing studies aim to demonstrate iNOS co-localization with apoptotic cells in the hypothalamus of old rats.

We also demonstrated that similar changes in NOS activity occurred in the testes of old BN rats. In the regressed testes of old animals NOS activity was increased

AGE (months)


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