Italian National Research Council

Targeted Project on Ageing

Research Unit Director: MARRAMA Paolo

Address: Università di Modena - Dipartimento di Medicina Interna - Facoltà Medicina e Chirurgia
Via del Pozzo, 71 - 41100 - Modena
tel.: 059-422429 - fax: 059-422672

Scientific informations

Subproject

3

Ageing and endocrine-metabolic diseases: pathogenesis

Title of research

The aging male: biochemical and morphological studies of the mechanism controlling hormone secretions in the hyphotalamus and in the testis.

Keywords
hypothalamus	neuropeptides	testosterone	corticosterone

Aims

The interest of our research unit has been focused on changes in neuropeptide levels in the mediobasal hypothalamus and related areas as function of aging processes. In particular, we have addressed our attention to the role of the hypothalamic-pituitary-adrenal axis and repeated stress as possible factors that, during normal aging, can favour neuronal death or decrease of neuropeptide levels, as previously shown by our laboratory. Lines of evidence suggest that glucocorticoid plasma levels increase during aging in rodents and man. Thus, it has been advanced the hypothesis that glucocorticoids may affect aging-related cellular alterations in tissues of the whole body (Selye H and Tuchweber B, 1976, in: Hypothalamus, pituitary and aging. Everitt AF and Burgess JA (eds) CH Thomas, Springfield: pp 553-569; Landfield PW, Baskin RK and Pitler TA, 1981, Science 214: 581-584). In aged rats, an impairment of the negative feedback regulation controlling the neuroendocrine response to stress was observed (Salposky RM, Krey LC and Mc Ewen, 1986, Endocr Rev 7: 284-301). Several authors have addressed the question of the possible anatomical site involved in the impairment of this feedback regulation, but evidence is still unconclusive. Moreover, it was suggested that glucocorticoids can enhance the lesioning effects of neurotoxic agents. Our research was aimed to investigate these aspects of the relationship between stress, glucocorticoids and neurodegeneration as far as regards the neurobiology of aging.
One aspect of male aging is the decreased performance of sexual life. This phenomenon can be related to the neuroendocrine control or to aging of the male gonad. The hypothalamic-pituitary-testicular axis presents thus changes specular to those of the hypothalamic-pituitary-adrenal axis. It is possible that both phenomena can be related or have a common cause. In fact, the exposure to repeated stress during life can lead to an impairment of the glucocorticoid negative feedback on the adrenocortical activity which may be responsible for a decline of male sexual performance. On the other hand, the decrease in sexual hormone plasma levels can lead to a higher vulnerability of nervous tissue to noxae (Gould E, Woolley CS, Frankfurt M and McEwen BS, 1990, J Neurosci 10: 1286-1291). These hypotheses guided our research activity toward the study of alterations in male sexual performance and their neuroendocrine correlates in men and animals.

Scientific activity

STUDIES ON THE EXPERIMENTAL ANIMAL:


1) CHANGES IN NEUROPEPTIDE LEVELS AND CELL NUMBER OF THE MEDIOBASAL HYPOTHALAMUS AND NEOSTRIATUM DURING AGING. OBSERVATIONS ON PUTATIVE GLUCOCORTICOID NEUROTOXICITY

a) Age-dependent changes in neuropeptide levels of the hypothalamus and related areas

- Changes in galanin immunoreactivity

- Changes in neuropeptide Y immunoreactivity and the control of food intake

- Alterations of tanycytes

b) Neurochemical alterations but not cell loss in striatum of the male rat during aging

c) Investigation on the putative site of lesion controlling glucocorticoid secretion in the old rat

d) Studies on glucocorticoid neurotoxicity

- In neonatal rats

- In aged rats

e) Effects of adrenalectomy in a rat model of Alzheimer's disease

2) EFFECTS OF TREATMENT WITH L-DEPRENYL, A MAO-B INHIBITOR, ON ASTROCYTE ACTIVATION AFTER NEOSTRIATAL LESION IN YOUNG AND OLD RATS

3) PROTECTIVE EFFECTS OF MODAFINIL OR THE GANGLIOSIDE AGF2 IN ANIMAL MODELS OF NEURODEGENERATION OF THE CENTRAL NERVOUS SYSTEM

- Effects in ischemia models

- Effects in models of Parkinson's disease

4) BENZODIAZEPINE PERIPHERAL RECEPTORS IN TESTIS OF YOUNG AND AGED RATS: MODULATION BY GLUCOCORTICOID TREATMENT

5) CENTRAL MECHANISMS INVOLVED IN THE CONTROL OF SEXUAL ACTIVITY

- Effects of neuropeptides

- Effects of nitric oxide (NO)



STUDIES ON MAN:

6) STUDIES ON PENILE ERECTILE RESPONSE AND ENDOCRINE FUNCTIONS IN THE AGED MAN

a) Studies on nocturnal penile tumescence

b) studies on endocrine functions in health aged or demented men

6) DEPENDANCE OF THE CIRCADIAN RHYTHM OF PLASMA TESTOSTERONE FROM THE PULSATILE RELEASE OF GnRH IN A MODEL OF HYPOGONADISM

7) HYPOTHESIS ON THE RELATIONSHIP BETWEEN NEURONAL PLASTICITY AND NEURODEGENERATION IN THE AGING BRAIN

Scientific results

AGE-RELATED CHANGES IN NEUROPEPTIDE LEVELS AND FUNCTIONS IN THE HYPOTHALAMUS

- Changes in galanin immunoreactivity
The peptide galanin has been suggested to be involved in the control of food intake, increasing it,, and adenohypophyseal hormone releasing factors. In particular, after intracerebroventricular injection, a marked, dose related, increase of growth hormone, luteotropic hormone and prolactin was found. Galanin also inhibits dopamine and corticotropin releasing hormone release and utilization in rat median eminence. By means of semiquantitative immunocytochemistry the distribution of galanin like-immunoreactivity (LI) in various diencephalic and subcortical telencephalic areas of young (3 months), adult (12 months) and aged (24 months) rats was evaluated. Sampled aeras were: arcuate nuclei, central nuclei of the amygdala, hypothalamic dorsomedial nuclei, median eminence, thalamic paraventricular nuclei, periarcuate regions and hypothalamic paraventricular nuclei. A significant decrease in galanin LI was observed in all the analysed areas of the aged rats when compared with the young ones. In the adult animals a decrease in the microdensitometrical values with respect to the young rats was present only in the periventricular hypothalamic nucleus. A disappearance of nerve cell bodies was also observed in the central amygdaloid nucleus of the aged animals. The present data show a widespread age-related decrease of galanin LI in discrete diencephalic and subcortical telencephalic areas and suggest an involvement of this peptide in the multiple impairments of neuroendocrine and autonomic functions related to aging.

- Changes in neuropeptide Y immunoreactivity and food intake
Neuropeptide Y (NPY) is a peptide of the pancreatic polypeptide family, localized also in the mediobasal hypothalamus, where appears to regulate the secretion of luteotropic hormone, growth hormone, adrenocorticotropic hormone, tireotropic hormone and prolactin. Aged rodents develop anorexia and a modified light/dark daily cycle pattern of feeding moreover it has been demonstrated that several NPY-containing systems are altered with advancing age. NPY exerts a potent stimulatory actions on eating when injected into the paraventricular hypothalamic nucleus (PVN) in rats, thus it can be interesting to study changes in hypothalamic NPY systems linked to aging. In the present study eating and drinking behaviour in satiated and fasted young and aged rats have been investigated after NPY injection into the PVN. The levels of NPY IR in PVN were also evaluated by means of semiquantitative immunocytochemistry. NPY injections into PVN (0.01, 0.05, 0.1, 1 mmol/50 µl) improved food and water consumption in both young and aged satiated rats. However, the feeding response in satiated rats elicited by 0.5-1.0 nmol of NPY injected into the PVN was attenuated in 24-month-old rats when compared with the values obtained in 3-month-old rats. This difference was observed at short term after NPY injection (30, 90 and 240 min after injection), but not at 1320 min. An attenuated feeding response was also observed in aged rats after 24 h of food and water deprivation, an effect again not present at 1320 min. When injected with saline, aged rats showed a food consumption which was significantly lower than that of young rats in spite of the higher body weight, suggesting a possibly reduced appetite. These results and the severe reduction of NPY IR levels in PNV nerve terminals of aged rats, suggest that NPY deficiency may be a factor responsible for anorexia in the aged.

- Changes in tanycytes
Tanycytes are specialized glial cells lining the inferolateral portion of the III ventricle. They are known to interact with neuroendocrine structures of the mediobasal hypothalamus, thus contributing to the endocrine functions of this region. In addition, they represent a preferential way of
communication between the hypothalamus and the cerebrospinal fluid of the III ventricle. By means of semi-quantitative immunocytochemistry, possible age-related changes in dopamine and cyclic AMP-regulated phosphoprotein mr 32 (DARPP-32) and glial fibrillary acidic protein (GFAP) immunoreactivities (IR) were investigated in tanycytes of the arcuate nucleus.These two markers showed opposite changes during aging. DARPP-32 IR decreased by around 50%, whereas GFAP IR increased by around 300% in 24-month-old vs 3-month-old rats. No significant age-related change was instead observed either in GFAP IR in astrocytic populations of the mediobasal hypothalamus or in tyrosine hydroxylase IR in dopaminergic neurons of the dorsal arcuate nucleus. These observations imply that an impairment in the intracellular signalling cascade linked to DARPP-32 is present in the aged tanycytes. However, the increase in GFAP IR suggests that aged tanycytes are not reduced in number but rather become hypertrophic. These changes may be related to the alterations in the neuroendocrine systems known to occur during aging.

AGE-RELATED NEUROCHEMICAL CHANGES, BUT NOT CELL LOSS, IN RAT STRIATUM
Numerical changes in the overall neostriatal neuronal population have been investigated by morphometric analysis of Nissl stained and glucocorticoid receptor (GR) immunoreactive neurons. Number and staining intensity of various chemically-identified nerve cell populations were analyzed by means of immunocytochemistry coupled with computer-assisted image analysis (IBAS I-II, Zeiss-Kontron). Three and 24 month-old male Sprague Dawley rats were used. No change in the number of Nissl stained, glucocorticoid receptor-, dopamine and adenosine 3':5' monophosphate regulated phosphoprotein- and enkephalin-immunoreactive neurons and a 50% decrease of neuropeptide Y-immunoreactive neurons were observed in the aged rat. In our preparations, the glucocorticoid receptor antibody stains around 90% of the neostriatal neurons, the dopamine and adenosine 3':5' monophosphate regulated phosphoprotein and enkephalin antibodies label 25-35% and the neuropeptide Y antibody stains only 1% of neostriatal neurons. In the same preparations a significant decrease in the intensity of immunostaining was observed for enkephalin-, dopamine and adenosine 3':5' monophosphate regulated phosphoprotein- and neuropeptide Y-immunoreactive neuronal cell bodies and tyrosine hydroxylase-immunoreactive nerve terminals in the aged rat. In the case of neuropeptide Y- and dopamine and adenosine 3':5' monophosphate regulated phosphoprotein-immunoreactive neurons, the changes in the intensity of
immunostaining were differentially compartmentalized within neostriatum, suggesting selective vulnerability of striatal subregions to aging processes. In conclusion, these data indicate that no significant age-related neuronal cell loss occurs in neostriatum. On the other hand, a generalized decrease in the levels of peptide transmitters and molecules related to dopamine transmission is observed in aged rat neostriatum, possibly resulting in the known age-related deficits of
neostriatally-controlled behaviours.

CHANGES IN GLUCOCORTICOID RECEPTOR IMMUNOREACTIVITY IN DIFFERENT CEREBRAL AREAS DURING AGING
Hippocampus is involved in the negative feedback regulation of corticosterone after stress through activation of glucocorticoid receptors (GR). In the present study the possible changes in GR immunoreactivity (IR) in various tel- and diencephalic regions of the aged rat have been investigated using immunocytochemistry coupled with computer-assisted image analysis. In particular, we have evaluated changes in the number of positive cells and in the intensity of staining per cell. A Nissl staining was also performed to allow automatic cell count. Male Sprague Dawley rats of 3, 12 and 24 months of age were used (n = 5/group). A selective decrease of GR IR was observed in the CA1 hippocampal field and central amygdaloid nucleus of the 24 month-old with respect to both 3 and 12 month-old rats. While in the former region GR IR decrease was paralleled by a decrease of IR field area, no age-related decrease of GR IR profile number was detected in central amygdaloid nucleus. A significant decrease of GR IR and IR field area was also observed in the dentate gyrus of 24 vs 12 month-old rats but not vs 3 month-old rats. The analysis of adjacent sections stained with cresyl violet showed a pattern of age-related changes (decrease of neuronal profiles in CA1 field pyramidal layer and dentate gyrus granular layer, and no change in the central amygdaloid nucleus of aged rats) which paralleled the observed changes in GR IR in the same areas. This study provides evidence that GR are selectively decreased in the hippocampal formation and in the central amygdaloid nucleus of the aged rat. However, only in the aged hippocampal formation GR decrease seems to be due to a loss of GR-containing neurons. The absence of age-related changes in the number of GR target neurons in all the other areas analyzed (even neurons which contain high levels of GR) suggests that the activation of the mineralocorticoid receptors (MR), which are selectively concentrated in the hippocampal formation, or possibly the combined activation of MR and GR, may be responsible for corticosterone cytotoxicity.

PERIPHERAL BENZODIAZEPINE RECEPTOR LEVELS IN TESTIS OF YOUNG AND OLD RATS: EFFECT OF GLUCOCORTICOID ADMINISTRATION
A number of evidence have recently been collected about a possible role for the peripheral benzodiazepine receptor (PBR) in the regulation of steroid synthesis process in Leydig cells of rat testis. Autoradiographic studies using 3H-RO 54864, a benzodiazepine showing high and specific affinity to PBR, have revealed an intense signal in interstitial tissue of rat testis, whereas a lower one was detected in tubular epithelium. In addition, RO 54864 increases testosterone release from rat testicular interstitial cells suspensions. On the basis of the well known inhibitory effect of glucocorticoids on testicular steroidogenesis and of the reported reduction of fertility and steroidogenesis by aging in the rat, we studied the effect of the repeated administration of corticosterone (40 mg/kg/day, suspended in sesame oil, for 7 days s.c.) in rats (n = 36) of various age on PBR expression in the testis. For this purpose we performed autoradiography using 3H-PK 11195 on testis slices (14 µm) obtained from three groups of rats, respectively aged 3, 12 and 24 months. Half of the animals from each of the three groups (n=6/group) received corticosterone treatment, whereas control rats were treated with vehicle. Autoradiographic films were analyzed using IBAS I-II image analyzer (Zeiss Kontron, Munich, Germany). The microdensitometric
measurements performed indicated that the area covered by specific 3H-PK 11195 binding was significantly reduced in rats treated with corticosterone. No difference was found between the vehicle-treated rats of different age groups. On the basis of these results, a reduction of PBR in interstitial cells could be viewed as one of the possible mechanisms for the demonstrated
inhibitory effects of glucocorticoid on testicular steroidogenesis, whereas aging effects on the testis did not seem to affect PBR density in the interstitial compartment.

CORTICOSTERONE TREATMENT ENHANCES ENDOTHELIN-1 INDUCED LOCAL CEREBRAL ISCHEMIA IN YOUNG BUT NOT IN AGED RATS
Some evidence point to glucocorticoids and their interaction with neurotoxins as possible mechanisms responsible for neuronal damage in the central nervous system. Seric concentration of glucocorticoids increases during aging and this phenomenon is even more evident in Alzheimer's disease. Thus, it is conceivable that during aging some pathological conditions may be potentated in the presence of an increased adrenal activity. One of this conditions is cerebral ischemia. In an experimental model of cerebral ischemia it was shown that corticosterone (CORT) aggravated the neuronal damage in hippocampus and striatum of young rats (Sapolsky and Pulsinelli, 1985, Science 229: 1397-1400). No data are currently available about the effect of glucocorticoids on cerebral ischemia induced in aged rats, thus our aim was to study, in a model of local cerebral ischemia, the effect of CORT administration on the extension of the lesioned area in rats of different age. Ischemia was induced in the striatal region by local injection of endothelin-1 (ET-1), an endothelium-derived 21-amino acid residue which possesses a powerful and long-lasting vasoconstrictor action. The administration of ET-1 into the rat striatum produces a lesion that is similar to that obtained in the Pulsinelli's model of transient forebrain ischemia (Fuxe et al., 1991, Acta Physiol. Scand. 137: 155-156). Thirty six rats of the Sprague-Dawley strain (n = 6 per group) 3, 12, 24 month aged were used. Starting 5 days before ET-1 administration, they were injected with CORT (40 mg/kg/ml s.c.) or vehicle (sesame oil). Then, the rats were halothane anesthetized, mounted in a stereotaxic frame and implanted with a steel cannula (32 gauge) 1 mm anterior and 2.5 mm lateral to the bregma. ET-1 (0.8 µg/0.8 µl) was injected in the right striatum 5.5 mm below the skull bone. After surgical procedure rats were injected with CORT or vehicle until sacrifice, 72 hours later, by transcardiac cold saline perfusion. Then, the brains were rapidly dissected and stored to -80 °C. Eventually, cryostat cutted 14 µm sections were processed for the analysis of the lesion by means of Nissl staining, autoradiography with a selective ligand of the peripheral benzodiazepine receptor, 3H-PK 11195 (considered to be a marker for ischemic lesions) and in situ hybrydization with sulphated glycoprotein 2 (SGP2) mRNA labelling, a protein which is putatively involved in programmed cell death. The loss of neuronal cells, assessed histologically, was mainly
localized in the striatum and in the surrounding cortex. The lesioned area, studied by means of computerized image analysis (IBAS I-II, Zeiss Kontron) resulted significantly larger in 3 month aged CORT-treated rats. Twelve and 24 month aged rats did not significantly differ from young control rats. 3H-PK 11195 and SGP2 specific signal, sorrounding the necrotic area, were discriminated interactively choosing a mean gray tone value distant two standard deviation from that one of the background. The specific signal was significantly higher in 3 month CORT-treated rats versus all the other experimental groups. These results confirm previous data supporting a neurotoxic role of glucocorticoids in potentating striatal ischemic lesion in young rats but we also show an age dependent reactivity of the tissue to the lesion. These results seem not to be related to age dependent variation of glucocorticoid receptor immunoreactivity, which is not modified in striatum (Zoli et al. 1991, Brain Res. 545: 199-207) and are probably related to other mechanisms, like a difference of blood vessel reactivity to ET-1 or a reduced susceptibility of striatum to toxic lesions with aging (Finn et al., 1991, Brain Res. 562:276-280).

CHRONIC ADMINISTRATION OF L-SULPIRIDE AT NON-NEUROLEPTIC DOSES REDUCES THE DURATION OF IMMOBILITY IN EXPERIMENTAL MODELS OF "DEPRESSION-LIKE" BEHAVIOR
It has been shown that long-term administration of l-sulpiride induces a down-regulation of b-receptor-associated adenylate cyclase activity in the frontal cortex of rats, an adaptive response that is typically associated with the chronic administration of antidepressants. Here we show that in two animal models of "depression-like behavior" (forced swim in rats and tail suspension in mice), the long-term (21 days) administration of l-sulpiride at non-neuroleptic dose (2 mg/kg IP twice a day) significantly decreases the duration of immobility, the effect being similar to that of desipramine (20 mg/kg IP twice a day). The same dose (2 mg/kg) of l-sulpiride, acutely administered, has no effect at all. On the other hand, either chronic (21 days) or acute administration of neuroleptic doses of l-sulpiride have an opposite effect, and indeed increase the duration of immobility. These results are in vivo support to the in vitro findings suggesting that low doses of l-sulpiride may have antidepressant-like activity.

AGED RATS ARE STILL RESPONSIVE TO THE ANTIDEPRESSANT AND MEMORY-IMPROVING EFFECTS OF OXYTOCIN
Oxytocin, intraperitoneally injected to 26 month-old male rats 60 min before testing, significantly improved social memory (at doses of 3 and 6 ng/kg) and reduced the duration of immobility in the behavioral despair test (at doses of 50 and 500 mg/kg). These results are in agreement with previous data obtained in adult rats and indicate that aging does not compromise the social memory improving and antidepressant-like activities of oxytocin.

OLD RATS ARE UNRESPONSIVE TO THE BEHAVIORAL EFFECTS OF ADRENOCORTICOTROPIN
In 28 month-old male rats, the i.c.v. injection of adrenocorticotropin (ACTH-(1-24)) (4 m/rat) did not induce the typical behavioral syndrome (excessive grooming, stretching, yawning, penile erections). This indicates that the behavioral effect of melanocortins are age-dependent, suggesting either an aging-linked impairment of the nervous circuitries involved or a reduction of the number (or affinity or both) of the brain melanocortin receptors in the elderly.

NITRIC OXIDE IS INVOLVED IN THE ACTH-INDUCED BEHAVIORAL SYNDROME
In many animal species, the intracerebroventricular (i.c.v.) injection of ACTH and of several shorter sequences of the ACTH molecule (melanocortin peptides) induces a peculiar behavioral syndrome mainly characterized by excessive grooming and by repeated acts of stretching and yawning. In adult males, spontaneous penile erections with ejaculation are also induced. We have studied the effect of NO synthase inhibition on this behavioral syndrome. The intraperitoneal (i.p.) injection of the NO synthase inhibitor L-NG nitroarginine methyl ester (NAME) significantly prevented - at the doses of 50 and 100 mg/Kg - all the above behavioral symptoms induced by the i.c.v. administration of ACTH-(1-24) L-NG nitroarginine methyl ester (NAME)(4 mg/rat). On the other hand, the i.c.v. injection of NAME (up to 300 mg/rat) had no influence on the ACTH-induced excessive grooming and stretching, while significantly inhibited the display of yawnings and penile erections. These data indicate that brain NO synthase is involved in the mechanism of ACTH-induced yawning and penile erections, while peripheral NO synthase is involved in the induction of stretching and grooming.

NITRIC OXIDE IS INVOLVED IN MALE SEXUAL BEHAVIOR OF RATS
In male rats, either sexually-experienced or sexually-naive, the intraperitoneal administration of L-arginine (the natural substrate for NO synthase)(10, 25, 50 mg/kg) both increased the percentage of copulating in sexually-naive rats and improved the indexes of sexual performance in sexually-experienced rats, whereas the intraperitoneal administration of NG-nitro-L-arginine methyl ester (NAME)(a potent inhibitor of NO synthase)(10, 25, 50 mg/kg) had opposite effects. On the other hand, after intracerebroventricular administration, L-arginine (25, 50, 100 mg/rat) had no effect - either in naive or in experienced rats - whereas NAME completely prevented ejaculation in naive rats, at the dose of 100 mg/rat, but had no effect at all in experienced rats, up to the dose of 300 mg/rat. Finally, a direct relationship seems to exist between male copulatory performance and NO synthase activity in a discrete and defined brain area, the paraventricular nucleus of the hypothalamus (PVN): indeed, the NO synthase mRNA expression in this nucleus in sexually potent rats is about twice that in sexually impotent rats. It is concluded that NO synthase is involved in the expression of male sexual activity, in spite of some inconsistencies of not easy interpretation.

PROLACTIN AND TESTOSTERONE: THEIR ROLE IN MALE SEXUAL FUNCTION
The role of androgens in the sexuality of men is still not completely clear. Men with severe hyperprolactinaemia frequently show mild hypogonasism, and many complain of loss of libido and penile erectile dysfunction (ED). We studied the night-sleep related erections and the penile response to visual erotic stimuli (VES) in 44 men: 13 with severe hypogonadism (Group 1; serum testosterone < 1.4 ng/ml), 10 with mild hypogonadism (Group 2; serum testosterone 2-3.5 ng/ml), nine with severe hyperprolactinaemia and mild hypogonadism (Group 3) and 12 control men (Group 4). All of the patients complained of loss of libido and ED. Group 1 showed significantly impaired night erections when compared with any of the other three groups, but no differences were detected between Groups 2, 3 and 4. The penile response to VES did not show any significant difference between the four groups, but was lower in Group 1 than in Group 4. These data confirm that night erections are androgen-dependent, but also suggest that there are two thresholds for serum testosterone: one below which sexual behavior is impaired with normal night erections, and a lower threshold below which night erections are also impaired. The penile response to VES was confirmed as being only partially androgen-independent. Furthermore, hyperprolactinaemia does not affect night erections or the penile response to VES, suggesting that its effect on libido and sexual behavior is due mainly to modulation of the psychological pattern of the patient.

THE EFFECTS OF TESTOSTERONE REPLACEMENT ON NOCTURNAL PENILE TUMESCENCE AND RIGIDITY AND ERECTILE RESPONSE TO VISUAL EROTIC STIMULI IN HYPOGONADAL MEN.
Nocturnal penile tumescence (NPT) and erectile response to visual erotic stimuli (VES) were measured, by means of a Rigiscan device, in nine hypogonadal men, and repeated after 3 months of androgen replacement. The same assessments were carried out once in 12 eugonadal controls. The number of satisfactory NPT responses, in terms of both circumference increase and rigidity, were less in the hypogonadal men than the controls and were significantly increased by androgen replacement, confirming the results of earlier studies. In terms of circumferenze increase, erectile response to VES did not differ between the hypogonadal men and the controls, and did not increase with androgen replacement. In terms of rigidity, the erectile response to VES did not differ between hypogonadal men and controls. However, in terms of both duration and maximum level of rigidity, there was a significant increase following androgen replacement in the hypogonadal men. These new findings, in relation to rigidity, require a modification of the earlier formulation, which saw as androgen dependent and erectile response to VES as androgen independent. NPT, and possibly spontanous erections at other times, clearly involve an androgen sensitive system. Erectile response to VES predominantly involves an androgen independent system but may also be influenced by androgen sensitive mechanisms.