Guadalberto Hernández

Gonadal steroid modulation of neuroendocrine transduction: A transynaptic view

Summary1. Steroid hormones act on neuronal communication through different mechanisms, ranging from transynaptic modulation of neurotransmitter synthesis and release to development and remodeling of synaptic circuitry. Due the wide distribution of putative brain targets for steroid hormones, acute or sustained elevations of their circulating levels may affect, simultaneously, a variety of neuronal elements. In an elementary mode of interaction, steroids are able to modulate both the synthesis and release of a neurotransmitter at a particular synapse, and the response of its target postsynaptic cells. Using two neuroendocrine transducing systems—the rat pineal gland and the GT1–7 cell line—we have examined these interactions and the following findings are discussed in this article.2. In the rat, pineal melatonin production is partially controlled by gonadal hormones. In females, melatonin synthesis and secretion is reduced during the night of proestrus, apparently as a consequence of elevated estradiol and progesterone levels. In males, circulating testosterone seems to be necessary to maintain the amplitude of the nocturnal melatonin peak.3. Some gonadal effects on pineal activity are exerted on its noradrenergic input, since changes in circulating steroid hormone levels are able to induce acute modifications of tyrosine hydroxylase activity in pineal sympathetic nerve terminals.4. Gonadal steroids are also able to regulate the response of pineal cells to adrenergic stimulation, since in vivo treatment of both male and female rats with steroid hormone blockers induces profound modifications in adrenergically-induced accumulation of cyclic AMP (cAMP) in dispersed pinealocytes.5. Direct exposure of pineal cells from gonadectomized female and male rats to estradiol (E2) or testosterone (T), respectively, potentiates pinealocyte response to adrenergic activation. In addition, short-term (15 min) exposure to either progesterone (Pg) or progesterone coupled to bovine serum albumin (P-3-BSA) suppresses the E2-dependent potentiation of adrenergic response in female rat pinealocytes.6. Exposure of GT1–7 cells to E2 completely blocked the norepinephrine (NE)-induced elevation of cAMP content. In E2-treated GT1–7 cells, additional exposure (15 min) to either Pg or P-3-BSA abolished E2-dependent inhibition of NE responsiveness. In addition, P-3-BSA alone increased basal cAMP levels in GT1–7 cells, regardless previous exposure to E2.7. In conclusion, there are evidences, both from the current literature and from the present results, supporting the view that in some neuroendocrine systems gonadal hormones modulate neurotransmission by acting, simultaneously, at pre- and postsynaptic sites. The models presented here constitute appropriate examples of this transynaptic mode of steroid action and, therefore, may offer a useful approach to investigate steroid hormone actions on the brain.

Ovarian steroids block the isoproterenol-induced elevation of pineal melatonin production in the female rat

Changes in pineal indole metabolism during the estrous cycle, as well as in response to estrogen administration, were studied in female rats. Tyrosine hydroxylase (TH) activity, norepinephrine (NE) and indoleamine levels were determined by high-performance liquid chromatography (HPLC). Pineal melatonin was measured by radioimmunoassay (RIA). Both 5-hydroxytryptamine (5-HT) and melatonin levels, but not TH activity or NE content, were reduced during proestrus. Ovarian hormones blocked the isoproterenol-induced elevation of pineal melatonin, and reduced 5-HT levels in ovariectomized rats. These results suggest that an estrogen-induced inhibition of the pineal response to adrenergic stimulation, could be responsible for the reduction in pineal melatonin production occurring during the proestrus stage of the rat estrous cycle.

Tamoxifen but Not Other Selective Estrogen Receptor Modulators Antagonizes Estrogen Actions on Luteinizing Hormone Secretion while Inducing Gonadotropin-Releasing Hormone Self-Priming in the Rat

Selective estrogen receptor modulators (SERMs) are compounds which may function as agonists or antagonists depending upon the target tissue. This study compares the actions of different SERMs on luteinizing hormone (LH) secretion, and on gonadotropin-releasing hormone (GnRH) self-priming in the rat. To do this, 4-day cyclic rats were injected twice, on day 2 (metestrus) and day 3 of the estrous cycle, with one of the following SERMs: 0.25 mg ICI 182,780, 3 mg tamoxifen (TX), LY139481-HCl or LY117018-HCl, or 0.5 mg RU58668. Control rats were given subcutaneous injections of 0.2 ml oil. On the morning of day 4 (proestrus in controls), rats from each group were either injected intraperitoneally with pentobarbital (40 mg/kg) for in vivo study or decapitated and their pituitaries collected for incubation (in vitro study). Additionally, pituitaries taken on each day of the estrous cycle from control rats as well as on day 4 from SERM-treated rats were processed for immunohistochemical determination of the estrogen receptor-α (ERα) gonadotrope. The plasma concentration or accumulation of LH in the medium was determined after 1 h (basal secretion). Thereafter, an intravenous bolus of GnRH (50 ng/0.5 ml/100 g BW) or 10–8M GnRH was injected or added to the medium, respectively. After 1 h of GnRH exposure, blood or medium were taken, and another challenge of GnRH was made. At the end of the 3rd h of the experiment, blood or medium samples were taken again and the LH plasma concentration or accumulation in the medium were determined. All SERM treatments reduced uterus weight and decreased basal and stimulated LH secretion. Also, on day 4, rats treated with any SERM other than TX showed vaginal smears infiltrated by leukocytes and a reduction in GnRH self-priming. TX-treated rats exhibited cornified vaginal smears and an estrogenic effect on GnRH self-priming. Moreover, 15-min exposure to two consecutive GnRH (10–8M) challenges 1 h apart in incubated pituitaries with estradiol (E2, 10–8M), TX (10–7M), E2 + TX, or medium alone form ovariectomized rats injected for 3 days with estradiol benzoate (25 µg), TX (3 mg), estradiol benzoate + TX, or 0.2 ml oil, respectively, showed that TX increased GnRH self-priming, as did E2, whereas it reduced the E2-sensitizing effect on GnRH-stimulated LH secretion and cancelled the E2-dependent GnRH self-priming. All SERMs prevented the physiological nucleocytoplasmic shuttling of ERα exhibited during proestrus in control rats, and TX, in addition, induced a significantly larger number of gonadotropes displaying strong cytosolic immunosignals corresponding to ERα than the rest of the experimental groups. Overall, data from this study indicated that, in contrast to the general antagonistic effect of the tested SERMs, TX seemed to display both selective agonist and antagonist activity at the gonadotrope level and on GnRH self-priming of LH secretion respectively.

Day‐Night Rhythm of Rat Pineal Tyrosine Hydroxylase Activity as Determined by HPLC with Amperometric Detection

Abstract: Tyrosine hydroxylase activity in the rat pineal gland was measured by means of HPLC determination of the amount of l‐3,4‐dihydroxyphenylalanine formed. Enzyme activity showed a clear day‐night rhythm, paralleling that of plasma melatonin levels in the same animals, with values being high during the dark period apparently because of changes in Vmax. In animals maintained under constant illumination for 3 days, tyrosine hydroxylase activity and plasma melatonin level rhythms were completely abolished, a result indicating that both are under photoperiodic control.

Estrogen modulates norepinephrine-induced accumulation of adenosine cyclic monophosphate in a subpopulation of immortalized luteinizing hormone-releasing hormone secreting neurons from the mouse hypothalamus

A subpopulation of luteinizing hormone-releasing hormone (LHRH)-producing cells that express the intermediate filament protein vimentin and the neuronal marker neurofilament 145, but not neurofilament 200 nor glial fibrillary acidic protein, has been isolated from GT1-7 cultures. These cells express the mRNA encoding estrogen receptor alpha (ERalpha) and respond to physiological concentrations of 17beta-estradiol (E2) by reducing the accumulation of cyclic adenosine monophosphate induced by norepinephrine, but not that induced by direct activation of adenylate cyclase. These results indicate that the activity of LHRH-producing neurons may be directly modulated by estrogen. In addition, they are suggestive of an estrogen-dependent desensitization of the beta-adrenoceptor in these cells.

Determination of Pineal Melatonin by High-Performance Liquid Chromatography With Electrochemical Detection: Application for Rhythm Studies and Tissue Explants

High‐pressure liquid chromatography with electrochemical detection has been used for the determination of melatonin in rat pineal glands and tissue explants. Rat pineal melatonin content was measured at 2‐hr intervals during a 2.t‐hr period by direct injection of supernatants from centrifuged pineal homogenates into a reversed‐phase C18 column. The lower limit of sensitivity was 32 pg per gland for a 2:1 signal‐to‐noise ratio, which allows melatonin measurements during daytime. Both absolute values and circadian rhythmicity obtained with the present method are in agreement with previously validated rddioimmunoassays. In addition, isoproterenolinduced release of melatonin from individual rat pineal explants was measured after chloroform extraction of the incubation medium.

Reproductive hormones control striatal tyrosine hydroxylase activity in the male rat

The effects of castration, hypophysectomy and testosterone treatment on striatal tyrosine hydroxylase (TOH) activity were examined in male rats. Enzyme activity was measured by means of high-performance liquid chromatography (HPLC) determination of L-3,4-dihydroxphenylalanine (DOPA) formed. Serum levels of both testosterone and luteinizing hormone (LH) were measured by radioimmunoassay (RIA). Castration, but not hypophysectomy, reduced TOH activity in the striatum. The administration of testosterone propionate (TP) to castrated animals in a dose of 10 micrograms/100 g b.wt. during the two days previous to sacrifice, completely prevented the castration-induced reduction of striatal TOH activity. In orchidectomized rats treated with different doses of testosterone propionate (TP) up to 20 micrograms/100 g b.wt., the levels of striatal TOH activity were apparently related to either the dose-related increase of serum testosterone or the decrease of serum LH. Higher doses of the androgen failed to further modify striatal TOH activity, in spite of the dose-related elevation of serum testosterone concentration. These results suggest that circulating levels of gonadal and/or pituitary hormones partially control dopaminergic synthesis in striatal terminals, which in turn may account for some behavioral effects of reproductive hormones.

Luteinizing hormone secretion elicited in a ligand-independent activation of progesterone receptor manner at pituitary level in the rat: differential effect of two selective estrogen receptor modulators.

In the absence of progesterone, RU486 reduced basal and luteinizing hormone-releasing hormone (LHRH)-stimulated LH secretion in pituitaries from proestrous rats, a fact which evidences a ligand-independent activation of progesterone receptors (LIAPR) at pituitary level. This was also observed in pituitaries from rats treated with tamoxifen, and absent in glands from either ovariectomized or raloxifene-treated animals. Both ovariectomy or raloxifene treatment reduced the stimulatory effect of LHRH on LH secretion, while tamoxifen induced an even higher response. Prolactin (PRL) secretion was unaffected by either RU486 or LHRH, nor it was influenced by ovariectomy or raloxifene treatment. However, treatment with tamoxifen elevated PRL in all groups. These findings indicate that LIAPR is an estrogen-dependent phenomenon at the anterior pituitary of the female rat, and that tamoxifen and raloxifene present agonist and antagonist estrogen activity, respectively, at this level.

Developmental Expression of Neurotensin in Thyrotropes and Gonadotropes of Male and Female Rats

Besides its potential roles as a central neuromodulator or a hypothalamic neurohormone, neurotensin (NT) may also have endocrine function in the anterior pituitary of mammals. We previously found that NT immunoreactivity is present in the secretory granules of gonadotropes and thyrotropes in both male and female rats, where its levels of expression are under the control of sex steroids. In this work, using immunocytochemistry and in situ hybridization, we have studied the postnatal development of NT-like immunoreactivity (NTir) and the mRNA encoding NT (mRNA-NT) in specific anterior pituitary cells of both male and female rats. NT expression starts after birth and displays an identical pattern in both sexes until sexual maturity, with mRNA-NT being detected from day 2 of postnatal life in thyrotropes localized in the central portion of the anterior lobe. This pattern of expression develops progressively throughout the 2nd and 3rd weeks in both sexes. By the beginning of the 3rd week, mRNA-NT can also be detected in gonadotropes localized in the periphery of the gland coinciding with a rise in serum estradiol concentrations in both sexes, and by day 21, mRNA-NT is extensively present in both the periphery and the central region. NTir is observed from days 5–6 in thyrotropes predominantly localized in the central portion of the anterior lobe, and by day 21, NTir is also detected in gonadotropes localized in the periphery of the gland. This pattern remains similar in both sexes until the time of puberty, when female rats start displaying plastic changes in NT expression according to the stage of the estrous cycle. These findings indicate that NT expression in the rat anterior pituitary is cell specific, and develops from birth to adulthood under the control of sex steroid hormones. In addition, preliminary data showing the presence of NT receptors in rat pituitary cells support the hypothesis of a paracrine or an autocrine role for this peptide within the pituitary.

Castration Reduces the Nocturnal Rise of Pineal Melatonin Levels in the Male Rat by Impairing its Noradrenergic Input

The effects of castration and testosterone treatment on pineal day‐night rhythms were studied in male rats. Bilateral gonadectomy was performed at 21 days of age. Testosterone propionate was given subcutaneously to castrated animals in a dose of 10 μg/100 g body weight during two consecutive days before sacrifice. Animals were killed 40 days after gonadectomy at four different times of a 12:12 h light‐dark cycle (1600, 2400, 0400 and 0800h). Tyrosine hydroxylase activity was measured in individual pineals by means of high‐performance liquid chromatography determination of L‐DOPA formed. Pineal levels of norepinephrine, dopamine, 5‐hydroxytryptamine and 5‐hydroxyindole acetic acid were determined by high‐performance liquid chromatography with amperometric detection, while pineal melatonin content was measured by radioimmunoassay. Castration abolished the day‐night rhythms of pineal tyrosine hydroxylase activity and norepinephrine content, both by elevating their daytime levels and by blocking their nocturnal rise. In addition, gonadectomy drastically modified pineal indoleamine metabolism by increasing daytime levels of both 5‐hydroxytryptamine and 5‐hydroxyindole acetic acid, and by reducing the nocturnal elevation of pineal melatonin content. Testosterone treatment was unable to prevent the effect of orchidectomy on pineal rhythms of tyrosine hydroxylase activity, 5‐hydroxytryptamine or 5‐hydroxyindole acetic acid content, however it partially restored the day‐night pineal rhythms of both norepinephrine and melatonin content. These results are indicative of a possible participation of reproductive hormones in the control of pineal rhythmic activity in the male rat. Apparently, since gonadectomy abolished the nocturnal rise of both pineal tyrosine hydroxylase activity and norepinephrine content, the primary site of action of reproductive hormones could be at the level of the superior cervical ganglion.