Excitatory aminoacids as modulators of gonadotropin secretion

The effects of quinolinic acid (QUIN) and quisqualate (QA) on the secretion of GnRH from MBH and LH and FSH from AP of 50 day old male rats have been evaluated by means of an 'in vitro' perifusion technique. QUIN (100 \u3bcM) is able to increase GnRH secretion with an action mediated by an NMDA receptor type, as shown by the inhibitory effect exerted by both a competitive (AP-5) and a non-competitive (MK-801) specific antagonist. QA 'per se' at the concentrations tested (1-100 \u3bcM) does not modify GnRH and gonadotropin secretion, but in the presence of a specific KA/QA receptor antagonist (DNQX) exerts a stimulatory effect at both levels. This observation might indicate that of the two QA receptor subtypes (ionotropic and metabotropic), this agonist binds to the metabotropic one with very low affinity: thus it is likely that a higher dose is required in order to have any effect on gonadotropin secretion. However, in the presence of DNQX, which binds to the ionotropic receptor, all the available QA can bind to the metabotropic one and can exert its action at MBH AP levels

Testosterone and the control of hypothalamic GnRH

The aim of the present study was to test the hypothesis that the restoring effect of testosterone on hypothalamic GnRH stores in orchidectomized rats might be linked to the intrahypothalamic transformation of the hormone into estrogenic metabolites. To this purpose, advantage has been taken of the availability of the potent antiestrogen, tamoxifen (TMX). Different groups of adult male rats castrated since 4 weeks were submitted to a 6-day treatment with testosterone propionate (TP, 2 mg/rat daily); TMX (50 or 200 micrograms/rat daily); or TP (2 mg/rat daily) plus TMX (50 or 200 micrograms/rat daily). The animals were sacrificed 24 h after the last injection, and hypothalamic GnRH content was measured by radioimmunoassay. The results have confirmed the ability of TP to counteract the decreasing effect of orchidectomy on hypothalamic GnRH stores, and have shown that TMX does not have any intrinsic activity on this parameter. Furthermore, TMX at either dose used in the present experiments, was found not to be able to abolish the restoring effect of TP on MBH-GnRH stores. It is concluded that the action of testosterone on hypothalamic GnRH does not require the conversion of the hormone into estrogens

The anterior pituitary gland as a possible site of action of kainic acid

The purpose of the present study was to analyze the direct effect of kainic acid (KA), an agonist of L-Glutamate, on the secretion of LH and FSH from anterior pituitary (AP) of male rats perifused in vitro. At low concentrations (1 microM), KA was able to stimulate the release of both gonadotropins from AP of 50-day-old male rats, but the response to subsequent stimuli was markedly impaired. This, however, was not due to a neurotoxic action of KA, but seemed rather suggestive of a down-regulation or desensitization of KA receptors. The stimulatory action of KA on LH and FSH secretion was age-dependent, since the agonist was completely ineffective on the AP of 75-day- and 18-month-old male rats. DNQX (6,7-dinitroquinoxaline-2,3-dione), a specific antagonist of the KA receptor subtype, was able to block the KA-induced gonadotropin secretion; similarly, AP-5 (2-amino-5-phosphonovalerate), a competitive NMDA receptor antagonist, prevented the stimulatory effect of KA on LH and FSH release. An interaction between the opiatergic and the excitatory aminoacid (EAA) systems emerged from the observation that pulses of KA applied to AP of 50-day-old male rats during a continuous perifusion with a medium containing morphine (5 microM) failed to increase gonadotropin secretion. These results indicate that KA can, at low concentrations, directly stimulate LH and FSH secretion by acting at AP level; this effect disappears with progression of age, and might be exerted both through NMDA and non-NMDA receptor subtypes. Finally, the results provide evidence that opioids and excitatory aminoacids might influence gonadotropin secretion from AP by acting in opposite directions

Transforming growth factor-beta and astrocytic conditioned medium influence luteinizing hormone-releasing hormone gene expression in the hypothalamic cell line GT1

On the basis of our previous observations indicating that a principle [possibly transforming growth factor-beta1 (TGFbeta1)] secreted by type 1 astrocytes may increase the release of LHRH in the GT1 cell line, it was deemed of interest to analyze whether TGFbeta1 might influence LHRH gene expression in addition to LHRH release in GT1-1 neurons. The effects of TGFbeta1 on the levels of LHRH messenger RNA (mRNA) present in GT1-1 cells have been compared to those found after either coculture of these cells with type 1 astrocytes or exposure of GT1-1 cells to the conditioned medium in which type 1 astrocytes were grown for 24 h. The data obtained indicate that 1) TGFbeta1 increases LHRH mRNA levels 1 and 6 h after the beginning of treatment; longer exposures (24 h) bring about a decrease in LHRH gene expression; 2) a significant stimulatory effect of TGFbeta1 (1 and 6 h of exposure) is also evident on LHRH release; 3) the exposure to the conditioned medium of type 1 astrocytes is able to increase LHRH gene expression in GT1-1 cells at 1 h; LHRH mRNA levels show a small decrease after 6 h of exposure, which becomes more evident at 24 h; and 4) the coculture of GT1-1 cells with type 1 astrocytes is not able to modify LHRH mRNA levels at any time considered. The present data support the concept that glial cells are able to control, possibly through the release of TGFbeta, the gene expression of LHRH in hypothalamic neurons

Aging and the hypothalamo-pituitary-testicular axis in the rat

Several experiments have been performed in order to clarify the mechanisms through which aging in male rats brings about profound modifications of the neuroendocrine system (reduced pulsatile secretion of LH and FSH, decreased serum levels of gonadotropins and testosterone, etc.). (1) It has been found that the number of mu opioid receptors decreases significantly in the hypothalami of old male rats; the substitution therapy with testosterone is ineffective in increasing the number of mu opioid receptors. These data suggest that the decrease of hypothalamic mu opioid receptors is not due to a decline of serum testosterone levels, but appears to be an independent phenomenon. (2) K opioid receptors increase significantly in the amygdala and in the thalamus of old male rats. These results show that aging, in addition to mu receptors, affects also the number of K receptors in selected areas of the brain. The increase of the number of K receptors in the amygdala might have some bearing on the decrease of serum gonadotropins observed in aged rats, since the amygdala is involved in the nervous circuitry influencing the hypothalamo-pituitary-gonadal axis. (3) The study of the release of LHRH from the hypothalamus of old male rats with an in vitro perfusion system shows that the release of the hormone is comparable in young and old animals, both in basal and in K+ stimulated conditions. These results indicate that the hypothalamus of old male rats retains the capacity of releasing LHRH both in basal and in stimulated conditions. (4) It has been observed that the number of LHRH receptors at the level of the anterior pituitary is significantly reduced in old male rats. This finding might explain the low serum levels of gonadotropins and testosterone in aged rats, due to a lack of an adequate response of the pituitary to hypothalamic LHRH

Differential effects of low- and high-dose estrogen treatments on vascular responses in female rats

In an attempt to study the mechanisms by which estrogens affect vascular responses, we utilized aortic preparations from intact and ovariectomized female rats receiving low- and high-dose subcutaneous estrogen treatments. Oil-treated, as well as male rats, served as controls. In ovariectomized females, low-dose 17-beta-estradiol injections (5 microg/kg daily for two days) affected the basal release of nitric oxide, as evaluated by concentration-related curves to superoxide dismutase and N(G)-Methyl-L-arginine acetate, which was found to be greater in 17-beta-estradiol-treated females compared to oil-treated females or males. Conversely, the nitric oxide-related vascular relaxation evoked by acetylcholine and sodium nitroprusside was unchanged. Prostacyclin production was also evaluated. Aortic rings from ovariectomized 17-beta-estradiol-treated females released significantly more prostacyclin than those from oil-treated females. These results point out a possible role for nitric oxide and prostacyclin in the vascular protection brought about by physiological levels of estrogens. When intact females were treated with high doses of ethynilestradiol (100 microg/Kg daily for one month), a component of contraceptive pills, either the basal release of nitric oxide, or acetylcholine-induced relaxation underwent a significant decrease. Likewise, the relaxant responses to sodium nitroprusside were impaired in the aortic rings obtained from ethynilestradiol-treated animals when compared to controls. Similarly, the amount of prostacyclin released from aortic tissues obtained from ethynilestradiol-treated animals was significantly reduced. These results may provide a possible explanation for the higher incidence of cardiovascular disease in women who take contraceptive preparations containing high doses of estrogens

Expression of a leptin receptor in immortalized gonadotropin-releasing hormone-secreting neurons.

Leptin is secreted by adipocytes and regulates food intake and energy balance through the activation of specific receptors (OB-R). Recent evidence suggests that it is also involved in the control of reproductive processes, by possibly acting on central and peripheral targets. In particular, it has been shown that leptin may indirectly stimulate GnRH release from hypothalamic fragments by acting on interneurons impinging on GnRH-secreting neurons. The possibility that leptin might additionally modulate the activity of GnRH-secreting neurons in a direct way has been addressed in the present study, by using the immortalized GnRH-secreting cell line GT1-7. The presence of OB-R messenger RNA (mRNA) (long form) was detected by RT-PCR analysis of total RNA from GT1-7 cells. An OB-R protein is also expressed in these cells; as shown by immunochemistry and by Western blot analysis. The latter has revealed the presence of a single immunoreactive OB-R with an approximate size of 130 kDa. To study the functionality of these receptors, the effect of leptin treatment on GnRH secretion and gene expression in GT1-7 cells were evaluated. Under static conditions, GnRH release was stimulated by exposure to low concentrations of leptin (10-12 M after 30 min; 10-10 M after 60 min). The 10-12 M dose was selected for studying the effect of leptin on GnRH secretion under dynamic conditions. To this purpose, GT1-7 cells were placed in a perifusion system; treatment with leptin (10-12 M) for 60 min stimulated GnRH release with no changes of pulse frequency. On the contrary, exposure to leptin (10-12-10-10 M) for 1, 3, 6, and 24 h did not affect GnRH gene expression in GT1-7 cells. The present results indicate that GT1-7 cells possess OB-Rs and that leptin may directly affect their function. Taken together with the available reports, these findings suggest that leptin might participate in the regulation of reproductive processes by acting at multiple levels, both centrally and peripherally