Melanin-concentrating hormone stimulates the release of luteinizing hormone-releasing hormone and gonadotropins in the female rat acting at both median eminence and pituitary levels.

The purpose of this study was to investigate whether melanin-concentrating hormone (MCH) acts directly on the median eminence and on the anterior pituitary of female rats regulating LHRH and gonadotropin release. In addition, immunohistochemistry was used to examine the density and distribution of MCH-immunoreactive fibers in the median eminence of proestrous rats. MCH-immunoreactive fibers were found in both the internal and external layers of the median eminence and in close association with hypophysial portal vessels. In the first series of in vitro experiments, median eminences and anterior pituitaries were incubated in Krebs-Ringer bicarbonate buffer containing two MCH concentrations (10(-10) and 10(-8) M). The lowest MCH concentration (10(-10) M) increased (P < 0.01) LHRH release only from proestrous median eminences. Anterior pituitaries incubated with both MCH concentrations also showed that 10(-10) M MCH increased gonadotropin release only from proestrous pituitaries. In the second series of experiments, median eminences and pituitaries from proestrous rats were incubated with graded concentrations of MCH. MCH (10(-10) and 10(-9) M) increased (P < 0.01) LHRH release from the median eminence, and only 10(-10) M MCH increased (P < 0.01) LH and FSH release from the anterior pituitary. The effect of MCH on the stimulation of both gonadotropins from proestrous pituitaries was similar to the effect produced by LHRH. Simultaneous incubation of pituitaries with MCH and LHRH did not modify LH but increased the FSH release induced by LHRH. The present results suggest that MCH could be involved in the regulation of preovulatory gonadotropin secretion.

Neural control of the compensatory increase of testosterone in hemicastrated adult male rats.

It is unclear why the concentration of testosterone increases in the testicular vein after hemicastration without corresponding alteration in gonadotropins. The present work was undertaken to examine whether the testosterone levels could be modified by denervation of the testis in adult rats. Both systemic and testicular blood samples were collected either immediately before or 6 and 24 hours after hemicastration from the rats two weeks after denervation of either inferior spermatic nerves (ISN) or ISN plus superior spermatic nerves (ISN-SSN). Increase of testicular testosterone induced by hemicastration was significantly (P<0.05) inhibited in these rats, as compared with the sham animals (at 6 and 24 hours, ISN vs sham: 16.00+/-3.35 vs 42.72+/-13.85 and 26.93+/-8.68 vs 71.16+/-13.30 whilst ISN-SSN vs sham: 31.63+/-7.92 vs 60.61+/-18.11 and 27.70+/-8.93 vs 93.92+/-19.73 ng/ml, respectively), whereas no significant change in LH was observed in all the experimental groups. FSH underwent no alteration in all the ISN denervation groups, but a significant elevation was observed in the ISN-SSN denervation groups (P<0.05) before hemicastration. Therefore, it appears that the change in FSH is not the cause of the inhibition of testosterone increase in the hemicastrated rats after testicular denervation and that ISN plays an active role in regulation of testosterone increase induced by hemicastration.

Differential effects of superior and inferior spermatic nerves on testosterone secretion and spermatic blood flow in cats.

It has been postulated that testosterone secretion is partially regulated by signals from the spermatic nerves. To further examine this hypothesis in vivo, the superior (SSN) or the inferior (ISN) spermatic nerves were stimulated electrically (varying intensity, 25 Hz, 0.2 msec, 10 min) in anesthetized cats, determining the testosterone concentration and the blood flow in the spermatic vein. In some additional experiments arterial blood was sampled, and norepinephrine (NE) output was calculated. Stimulation of the SSN (25-35 V) increased the testosterone concentration in spermatic vein blood (P < 0.01 compared with prestimulation levels). The response varied among animals, reaching a 50-100% increase in some animals, whereas in others it ranged from almost undetectable to more than 10 ng/100 g x min. Under the same experimental conditions, the NE output increased from 135.4 +/- 99 to 1614.2 +/- 347 pg/ml (P < 0.01), and spermatic blood flow decreased from 24.1 +/- 1.42 to 20.2 +/- 1.65 ml/min x 100 g (P < 0.05) during nerve stimulation. By contrast, stimulation of the ISN (25-35 V) modified neither the testosterone concentration, the NE output, nor the blood flow in the spermatic vein. High intensity stimulation (36-70 V) of each spermatic nerve evoked different vascular and hormonal effects. SSN activation induced a marked decrease in spermatic blood flow during stimulation and an increase in the testosterone response, whereas ISN activation resulted only in an enhanced spermatic blood flow. Our results suggest that testosterone secretion, although mainly dependent on gonadotropin secretion, could be further regulated by neural inputs from the SSN acting directly or alternatively through changes in blood flow. It would appear that the SSN mainly supplies the vasoconstrictor fibers to the testis, whereas the ISN provides vasodilator fibers.

[Vasoactive intestinal polypeptide induced prolactin release is model dependent in free-moving rats].

Our previous experiments in vitro showed that the stimulating effects of vasoactive intestinal polypeptide (VIP) on pituitary (PRL) depended on the endocrinal status of the animals. The present investigation was to determine whether the effect of VIP varied in vivo with changes of different physiological conditions. For infusion of VIP (5 micrograms/100 g body weight) and collection of blood sample, all the animals were cannulated with silicon tube into jugular vein 2-3 d before the experiments. The results showed that VIP concentration in blood was increased rapidly after the infusion (maximum: 21.32 +/- 2.33 ng/ml at 10 min and lasting more than 30 min). The concentration of PRL in blood of all the animals tested was increased significantly (P < 0.05) after VIP infusion. The increase rate of PRL induced by VIP was higher in male rats (158.04 +/- 37.06), but lower in the female (Diestrus: 50.42 +/- 16.44, Proestrus: 62.67 +/- 21.34) and in Suckling-depended lactating ones (Suckled 90.00 +/- 36.00 vs. Separated 31.05 +/- 4.42). The above observations suggest that the VIP action in vivo depends on the endocrinal and/or neural status of the animals.

Testicular denervation-induced nuclear changes in Leydig cell of hemicastrated adult rats.

In the adult male rat, hemicastration (HC) induces a remarkable enhancement of testosterone secretion from the Leydig cells of the remaining testis. We have observed previously that the denervation of inferior spermatic nerves inhibits this enhancement. The present experiments were designed to assess morphometrically whether or not this change had a cytological correlate in Leydig cells. At least five testes from each group (denervation plus HC, sham denervation plus HC and intact rats) were prepared for both optical and electron microscopy studies. The results showed that after 24 h of denervation, the nuclear volume of the Leydig cells in denervation plus HC rats was smaller (p < 0.01) than those of sham denervation plus HC (196.56 +/- 16.53 vs. 280.71 +/- 13.37), whereas both the volume density of heterochromatin (19.84 +/- 3.14 vs. 10.03 +/- 2.47%) and the heterochromatin index (expressed as periphery heterochromatin area divided by nuclear perimeter, 0.149 +/- 0.046 vs. 0.094 +/- 0.026) were significantly (p < 0.01) higher in denervation plus HC rats than in its sham groups. No changes in Leydig cell numbers or cytoplasmic organelles were detected. The results suggest that some nuclear and heterochromatin-associated cellular activity might be inhibited by testicular denervation in hemicastrated rats.

Monoaminergic and peptidergic contributions of the superior and the inferior spermatic nerves to the innervation of the testis in the rat.

Sections of the rat testis and whole-mounts of the testicular capsule were studied microscopically using the glyoxylic acid-induced fluorescence method, to detect monoamines, and immunohistochemical procedures for the detection of immunoreactivities to protein gene-product 9.5 (PGP 9.5), the C-terminal accompanying peptide of neuropeptide Y (CPON), and vasoactive intestinal polypeptide (VIP). Monoaminergic nerves were only observed around the intracapsular blood vessels: the initial segment of the testicular artery and the superior venous plexus, and in the anterior aspect of the upper and lower testicular poles. These capsular nerve networks were associated with the superior and inferior ligaments of the testis. Nerves displaying PGP 9.5 and CPON immunoreactivity appeared in the same sites and followed the same distribution as monoaminergic nerves. By contrast, VIP-immunoreactive fibers were only found in the nerve network of the lower pole. Observations done after different surgical denervation procedures demonstrated that the superior spermatic nerve was the source of fibers for testicular vessels and for the nerve network of the upper pole. On the other hand, fibers from the inferior spermatic nerve were restricted to the nerve network of the lower pole.

Serotonin, a neurotransmitter involved in the regulation of luteinizing hormone release.

The involvement of serotonin (5-HT) in the regulation of LH secretion is discussed on the basis of experimental and physiological models. The role of 5-HT on low amplitude pulsatile LH release in male rats is not yet clear, in spite of the fact that recent results suggest a weak permissive role. In ovariectomized rats, 5-HT expresses a negative influence on the increased rate of LH release, which is converted into a stimulatory effect by pretreatment of the animals with E2. Moreover, in castrated female rats there is a morning/afternoon oscillatory pattern in 5-HT metabolism of brain areas associated with the control of LH secretion including the hypothalamus. In this area, the fluctuation is modulated by E2 in such a way that the peak of 5-HT neural activity occurs simultaneously with the induced afternoon discharge of LH. On the other hand, P enhances the amplitude of the oscillation of hypothalamic 5-HT metabolism concomitantly with a potentiation of the induced LH surge. This facilitatory role of 5-HT upon phasic LH discharge is also evident in intact female rats. The preovulatory surge of LH is accompanied by an increased hypothalamic and, more precisely, ME 5-HT turnover. Furthermore, 5-HT stimulates in vitro LHRH release from the ME. Serotonergic nuclei located in the brain stem seem to mediate this effect.

Effect of bilateral denervation of the immature rat testis on testicular gonadotropin receptors and in vitro androgen production.

We have studied the effect of superior spermatic nerve (SSN) section on testicular gonadotropin receptors and in vitro androgen production by immature rat testis. Bilateral testicular denervation had no effect on testicular weight, serum androgens, LH, FSH and PRL levels. Denervation resulted in a significant inhibition of hCG stimulated in vitro androgen production. A reduction in the number of testicular LH receptors was observed after SSN section, while FSH binding sites remained unchanged. These results indicate that the number of LH receptors and testicular steroidogenic response to hCG are influenced by nerves reaching the testis.

Epidermal growth factor stimulates thyrotropin secretion in the rat.

The present work studied the effects of epidermal growth factor (EGF) on the release of thyrotropin (TSH) and prolactin (PRL) from perifused pituitary glands of 200-gram male Wistar rats. Each pituitary gland, cut into halves, was placed in a chamber of a perifusion system connected to a peristaltic pump which conveyed the perifusion medium (Medium 199, pH 7.3, Gibco, USA) from a reservoir to a chamber at a flow rate of 100 microliters/min. Each tightly closed chamber contained one pituitary gland and 600 microliters medium and it was placed in a water bath at 37 degrees C throughout the experiment. One milliliter samples of effluent were collected every 10 min for 60 min to obtain baseline values of TSH and PRL. Thereafter, TSH-releasing hormone (TRH) 10(-8) M or EGF (10(-11), 10(-10), 10(-9) or 10(-8) M) were added to individual chambers and the 10-min sampling of effluent continued for 60 min. EGF 10(-11) M elicited no TSH response, but 10(-10) and 10(-9) M doses induced significant increases in TSH secretion (p < 0.01) with a peak at 10 min after addition of EGF. In another experiment, EGF 10(-8) M or TRH 10(-8) M significantly elevated TSH secretion (p < 0.01). However, TRH, but not EGF, stimulated PRL secretion (p < 0.01). In the in vivo studies, the intravenous administration of EGF 10(-5) M or TRH 10(-5) M both induced significant elevation of TSH release at 10 min after the injection (p < 0.02 for EGF and p < 0.01 for TRH).(ABSTRACT TRUNCATED AT 250 WORDS)

Suckling-induced changes of vasoactive intestinal peptide concentrations in hypothalamic areas implicated in the control of prolactin release.

The present study was designed to investigate whether the vasoactive intestinal peptide (VIP) concentration in hypothalamic nuclei, dorsal raphe nucleus (DR) and pituitary lobes of lactating rats changes in physiological situations when prolactin (PRL) secretion is stimulated (suckling) or inhibited (pup separation). In addition VIP levels in blood plasma were determined in both situations. Acute suckling induced changes in VIP concentration only in the rostral part of the anterior hypothalamic (rAHN) and the paraventricular (PVN) nuclei of all the brain areas examined. VIP concentration in the rAHN increased at 5 min from 3.52 +/- 0.30 (mean +/- SEM) to 8.67 +/- 1.91 ng/mg protein (p less than 0.05) but fell to baseline values after 30 min suckling (p less than 0.05; 5 vs. 30 min). Although changes in VIP concentration in the suprachiasmatic nucleus (SCN) did not attain statistical significance, they followed the same trends as the changes of VIP in the rAHN. The opposite pattern of changes was observed in the PVN with a decrease in VIP concentration following 5 min suckling (p less than 0.01). At 30 min the VIP values showed a trend towards 0-min values. Pup removal did not affect VIP concentrations in the rAHN, PVN, SCN, median eminence, supraoptic nucleus and DR. VIP values were not detectable in the arcuate nucleus in any of the experimental situations examined. Lactation increased VIP concentration only in the rAHN and PVN when lactating rats with their pups were compared with virgin female diestrous rats. VIP concentration in the anterior lobe of the pituitary from lactating rats did not change with pup separation or suckling.(ABSTRACT TRUNCATED AT 250 WORDS)

Catecholamine distribution in adult rat testis.

Catecholamine distribution in the adult rat testis was examined using a sensitive radioenzymatic method. Norepinephrine was present in the capsule and the interstitial fluid, in higher concentrations than dopamine, while in the interstitial cell preparations only norepinephrine was found. Epinephrine was undetectable in all testicular compartments investigated. No catecholamines were found in the seminiferous tubules. Testicular denervation caused a significant decrease in capsular catecholamines, confirming the neural origin of these amines.

Serotonergic innervation of the rat testis.

The presence of 5-hydroxytryptamine (5-HT) was determined by h.p.l.c. in perchloric extracts of each isolated compartment of the adult rat testis. The testicular capsule, interstitial cells and interstitial fluid contained 5-HT, but 5-HT was not detected in the tubular compartment. In a group of adult rats, one testis was unilaterally denervated, and the contralateral testis used as control. The superior spermatic nerve, arising from the renal plexus, was excised and 1 week after surgery 5-HT content was measured in the capsule and interstitial fluid of both testes. Denervation caused a significant fall (34%) in 5-HT content. These results indicate that at least part of the testicular 5-HT derives from a serotonergic innervation of the gonad.

Dorsal raphe lesion alters the estrous cycle and the preovulatory gonadotropin release.

The purpose of this study was to examine the role that the dorsal raphe (DR)-median eminence (ME) serotonergic projection may have in the proestrous gonadotropin and prolactin (PRL) release. DR electrolytic lesions were performed in cycling rats during the first day of diestrus. The effect of DR lesions after 48-72 h of survival (short-term lesioned animals) or after 35-40 days of survival (long-term lesioned animals) on estrous cyclicity, preovulatory gonadotropin and PRL releasing pattern, ovulation and serotonin (5-HT) content of the ME were studied. Following DR lesions the estrous cycle became irregular, remaining in the diestrus phase for several days. Preovulatory gonadotropin release in short-term lesioned animals was increased; on the contrary, in long-term lesioned rats a delay in the surge of these two hormones and a decrease in LH secretion were detected. Long-term lesioned animals also showed a diminished secretion of PRL. The number of ova did not differ between control and lesioned animals. DR lesions in both short- and long-term lesioned rats reduced 5-HT levels in the ME by about 50% and nullified the normal 5-HT decline during the afternoon of proestrus. Our results suggest that the DR exerts a stimulatory influence on the preovulatory gonadotropin release by means of its 5-HT projection to the ME. As the pattern of hormonal secretion in lesioned animals remains similar to that of controls, it may be suggested that this pathway acts as a fine modulator of the mechanisms involved in the regulation of LH and FSH release in cycling rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonergic terminals in the anterior hypothalamic nucleus involved in the prolactin release during suckling.

The present studies were designed to localize within the hypothalamus and neighboring areas the serotonergic terminals which are implicated in suckling-induced PRL release. The initial experiments were performed to characterize the circulating hormone profile induced by suckling in lactating rats, previously separated from their pups. Five minutes of suckling induced an increase in serum PRL only. During these 5 min, 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid concentrations were determined in the pars nervosa of the pituitary gland, hypothalamic nuclei, dorsal, and median raphe nuclei. An increase by 80% (P less than 0.01) in 5-HT concentration was found only in the rostral part of the anterior hypothalamic nucleus (rNHA). In order to investigate causal effect between the altered 5-HT neuronal activity in the rNHA and the suckling-induced PRL release, serotonergic neurotoxin was bilaterally injected in the rNHA on day 1 of lactation. Litters were adjusted to eight pups each and weighed daily to determine litter growth rates. On day 8 of lactation, litters were separated from their mothers for 4 h and allowed to suckle for 5 or 15 min after which the mothers were decapitated. Litters from lesioned animals grew at a lower rate (P less than 0.0001) than control and sham-operated animals. Serum PRL increased with suckling in animals bearing the correct rNHA lesions, but the values were lower than in control and sham-operated animals after 5 (P less than 0.05) and 15 (P less than 0.01) min. Therefore we postulate that the rNHA is the site of termination of a stimulatory serotonergic pathway on PRL release induced by suckling.

Serotonin induces gonadotrophin release through stimulation of LH-releasing hormone release from the median eminence.

The effects of serotonin (5-HT) on the release of gonadotrophins and LH-releasing hormone (LHRH) were examined in an in-vitro perifusion system using median eminences and/or anterior pituitaries obtained from male or pro-oestrous female rats. Animals were killed by decapitation between 12.00 and 13.00 h. A serial double-chamber perifusion system was employed. Three types of experiments were performed. In the first, median eminences were placed in the first chamber and one anterior pituitary in the second chamber. In the second group, only the anterior pituitary was perifused. In the third group, only five median eminences were perifused. In the first and second experiments, LH, FSH and prolactin were determined in the perifusion efflux by radio-immunoassay (RIA). In the third experiment, LHRH was determined by RIA. Addition of 5-HT (final concentrations 0.06, 0.6 and 6.0 mumol/l) into the first chamber containing the median eminences stimulated the release of LH and FSH from the pituitary, but did not affect the levels of prolactin in the effluent in the same experiment (pro-oestrous rats). The stimulatory effect of 5-HT was blocked by the addition of cyproheptadine (l mumol/l) in the perifusion fluid. The introduction of 5-HT (0.6 mumol/l) into the tube connecting the first and second chambers did not modify the release of LH, nor did 5-HT added to the pituitaries perifused alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin stimulates gonadotrophin release by acting directly on the median eminence.

The study examined the effect of serotonin (5-HT) on gonadotrophin release and its tissue site of action using a sequential double chamber perifusion system. Cycling female rats were killed between 12.00 and 13.00 h on the day of proestrus. Median eminences and anterior pituitaries were removed and transferred to perifusion chambers. Two types of experiments were performed: a) five median eminences (ME) were placed in the first chamber and one anterior pituitary in the second chamber. b) In the second group, only the anterior pituitary was perifused. The effluent from the first chamber perifused the second chamber. The effluent from the second chamber was collected for hormone assays. Addition of serotonin (final concentration 0.06, 0.6 and 6.0 uM) stimulated the release of LH and FSH into the perifusion fluid draining the pituitary in series with the median eminences. Pretreatment with the 5-HT receptor blocker cyproheptadine (1 uM) completely inhibited the stimulatory effect of 5-HT. Serotonin was ineffective in stimulating gonadotrophin release when injected into the tube connecting the first and the second chambers (50 ul of 0.6 uM solution) or when the anterior pituitary was perifused alone. Serotonin did not affect the prolactin release in any of the experimental conditions studied. These observations demonstrate that serotonin stimulated the gonadotrophin release by acting on serotoninergic receptors at the level of the median eminence.

Dopamine enters lactotrophs and reaches their secretory granules.

The presence of dopamine in the lactotroph cell, as well as in isolated prolactin secretory granules, was demonstrated by means of an histochemical reaction for electron microscopy. Biochemical assays further confirmed the presence of dopamine in the secretory granules. Autoradiographic preparations examined by light microscopy showed dopamine internalization in dispersed anterior pituitary cells. Isolated anterior pituitary lactotroph cells incorporated more [3H]dopamine than a fraction containing other anterior pituitary cells.

Serotonin stimulates gonadotrophin release by acting directly on the median eminence.

The study examined the effect of serotonin (5-HT) on gonadotrophin release and its tissue site of action using a sequential double chamber perifusion system. Cycling female rats were killed between 12.00 and 13.00 h on the day of proestrus. Median eminences and anterior pituitaries were removed and transferred to perifusion chambers. Two types of experiments were performed: a) five median eminences (ME) were placed in the first chamber and one anterior pituitary in the second chamber. b) In the second group, only the anterior pituitary was perifused. The effluent from the first chamber perifused the second chamber. The effluent from the second chamber was collected for hormone assays. Addition of serotonin (final concentration 0.06, 0.6 and 6.0 uM) stimulated the release of LH and FSH into the perifusion fluid draining the pituitary in series with the median eminences. Pretreatment with the 5-HT receptor blocker cyproheptadine (1 uM) completely inhibited the stimulatory effect of 5-HT. Serotonin was ineffective in stimulating gonadotrophin release when injected into the tube connecting the first and the second chambers (50 ul of 0.6 uM solution) or when the anterior pituitary was perifused alone. Serotonin did not affect the prolactin release in any of the experimental conditions studied. These observations demonstrate that serotonin stimulated the gonadotrophin release by acting on serotoninergic receptors at the level of the median eminence.

Origin and termination of dorsal raphe-median eminence projection.

The origin and termination of the dorsal raphe (DR)-median eminence (ME) projection were studied by combining well-limited small electrolytic lesions of different parts of the DR, the axonal degeneration and the serotonin content in the median eminence. Our results show that the rostral third of the DR is a source of serotonergic innervation to the ME and that the rest of the nucleus does not contribute to this projection. The light microscopy study revealed that degenerating fibers occupy the internal layer of the ME. These results were confirmed by electron microscopy which in addition showed degenerating terminals.