Handelsman: Can I draw you out on one aspect you didn't mention, which is the opioid control of GnRH neurons. As far as I understand it, what you are working on is within the GnRH neuron itself. Is there any relationship between opioid effects and iNOS?

Wang: We haven't studied this. Opiates exert negative effects on GnRH hormones. We don't know whether the iNOS is important as the mechanism of action, but this is what we are pursuing. We are looking at both the brain and the testis.

Müller: You have shown an increase of iNOS in both the testis and the brain. In the brain iNOS increases in many areas beside the hypothalamus. Can you speculate on this finding a little bit? Could it be that NOS increase is not only a marker of decreased fertility but also a general phenomenon? We also have some indirect data in this respect as far as growth hormone (Rigamonti et al 1999), and sexual function (Melis et al 2001) are concerned.

Wang: There are data even in the human brain showing that iNOS activity may be increased in older subjects, but these data are not as solid as the rat data. The NMDA receptor has also been implicated in a stress-related increase in cortisol, causing damage to the brain, especially in rat models.

Björntorp: I am going to ask a terribly ignorant question. How do you imagine that the NOS is acting here?

Wang: We think that ageing causes decreasing blood flow and increased ROS generation, including iNOS. I believe there is also an increase in cytokines in the brain. The peroxynitrite products of iNOS are cytotoxic and kill the neurons that secrete GnRH and oxytocin, which causes some of the changes we observed. We also have evidence that the iNOS is colocalized in cells undergoing apoptosis.

Laron: You said that this is the best model for ageing studies in humans. We learned earlier about the characteristics of ageing in humans. What are the characteristics of ageing in the rat?

Wang: I think osteoporosis is present. I have no idea about muscle. The only reason we think this is a good model is that the testosterone is low, caused by both testicular and hypothalamic-pituitary dysfunction.

Morley: You are looking at 50 year olds. These could very well be human data, but you would have to go to 27-28 month old rats to be looking at the equivalent of 'old' subjects. The use of 'old' is a misnomer in this situation.

Wang: Initially we did experiments on 30 month old rats, but in these rats the testis is so small we cannot do many experiments. The supply ofvery old rats can be limited and 50% of the rats die by the age of 30 months. With the iNOS knockout mice we wish to study them when they are very young and then sacrifice them at different ages until old age.

Morley: You also didn't look at 12 month old rats. In our paper we found that some of those increases were present at 12 months (Morley et al 1996). It really is a maturational change with the iNOS. We have found a reduction of NOS mRNA in older animals.

Wang: We have looked at 3, 6, 9,12 and 18 month old animals, because we want to characterize the changes so that we can do interventions. This is why we need to know when they start to change with ageing.

Handelsman: A point of clarification. You mentioned transgenics in your paper. Are you planning to do knockouts in the mouse? If so, is the mouse the same as the Brown Norway rat?

Wang: We plan to use iNOS knockout mice. We have studied both the testis and the brain of the mouse, we can see an increase in iNOS with ageing. We have not done the pulsatile LH secretion; this is very difficult in the mouse.

Brabant: With the maturational change of iNOS decreasing in the old animals, it would be interesting to know what happens if you castrate young animals.

Wang: We haven't done castration experiments, although we really should. Al Matsumotos's group did castration experiments in the young and old rats, and compared the GnRH content in hypothalamic neurons (Gruenewald & Matsumoto 1991, Gruenewald et al 1994, 2000). They showed that GnRH mRNA and content are decreased in castrated ageing BN rats. They did not measure iNOS. We also haven't done testosterone replacement experiments in these rats.

Veldhuis: There may be a slightly different question here: in the female rat, dozens of reports have shown that high dose oestradiol is neurotoxic to certain of these centres, while at the same time being protective against brain ischaemia and hypoxia. Georg Brabant is raising an interesting point: could you prevent ageing of the GnRH neurons by castration?

Wang: Castration has not been shown to be protective in the hypothalamus. There was a recent paper in which researchers gave contraceptive doses of testosterone into the old BN rat, which caused the suppression of testosterone and spermatogenesis (Chen & Zirkin 1999). The treatment caused the Leydig cells to go into what they called 'hibernation'. If this was done for 6 months and then the treatment was stopped, the testosterone production in the young and old rats became similar.

Veldhuis: What makes you say that the upstream activators of this iNOS are probably cytokines? What evidence do you have that cytokines are increased in the ageing male rat in these areas?

Wang: There is scattered information about increased cytokine levels, both in the rat brain and the human brain. iNOS is traditionally induced by inflammatory responses or cytokines.

Veldhuis: So the neuronal death could be a glial event? Wang: Yes.

Laron: What do you know about the influence of insulin-like growth factor (IGF)1 on the ageing brain? A few months ago I went through the data on IGF1 in the brain, and it seems to be anti-apoptotic. You showed increased apoptosis. Wang: I don't know.

Veldhuis: Is this the same model in which a single group has reported that fetal neuronal transplantation has restored potency in the old male rat? This raises the possibility that there is structural loss of GnRH neurons, and it is not just functional. I didn't believe that this was the case. I had always preferred the hypothesis that there was functional loss of input signals to the GnRH ensemble, or loss of coordinate secretion. Your data are suggesting that there is increased cell death.

Prior: Tomorrow we will be talking about the perimenopause in women. To use similar language and call it the 'andropause' is to make light of the huge number of changes that occur over 5—10 years in women, versus the very gradual, slow changes that occur in men. I wish we could take away that word and not use it. Perhaps the 'andropause' applies to the Norway rat, but I don't think it applies to human males.

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