Placental leukocyte infiltration accompanies gestational changes induced by hyperthyroidism.

In brief: The endocrine and immunological disruption induced by hyperthyroidism could alter gestation, placenta, and fetal development. This study suggests an immunological role of thyroid hormones in gestation. Abstract: Thyroid dysfunctions lead to metabolic, angiogenic, and developmental alterations at the maternal-fetal interface that cause reproductive complications. Thyroid hormones (THs) act through their nuclear receptors that interact with other steroid hormone receptors. Currently, immunological regulation by thyroid status has been characterized to a far less extent. It is well known that THs exert regulatory function on immune cells and modulate cytokine expression, but how hyperthyroidism (hyper) modulates placental immunological aspects leading to placental alterations is unknown. This work aims to throw light on how hyper modulates immunological and morphological placental aspects. Control and hyper (induced by a daily s.c. injection of T4 0.25 mg/kg) Wistar rats were mated 8 days after starting T4 treatment and euthanized on days 19 (G19) and 20 (G20) of pregnancy. We removed the placenta to perform qPCR, flow cytometry, immunohistochemistry, Western blot and histological analysis, and amniotic fluid and serum to evaluate hormone levels. We observed that hyper increases the fetal number, fetal weight, and placental weight on G19. Moreover, hyper induced an endocrine imbalance with higher serum corticosterone and changed placental morphology, specifically the basal zone and decidua. These changes were accompanied by an increased mRNA expression of glucocorticoid receptor and monocyte chemoattractant protein-1, an increased mRNA and protein expression of prolactin receptor, and an increase in CD45+ infiltration. Finally, by in vitro assays, we evidenced that TH induced immune cell activation. In summary, we demonstrated that hyper modulates immunological and morphological placental aspects and induces fetal phenotypic changes, which could be related to preterm labor observed in hyper.

Alterations in Hypothalamic Mechanisms Associated with Reduced Suckling-Induced Prolactin Secretion in the Lactation-Deficient OFA hr/hr Rat.

INTRODUCTION: OFA hr/hr rats have deficient lactation with impaired suckling-induced PRL release. Unlike their background strain, Sprague-Dawley (SD) rats, OFA rats display abnormal mediobasal hypothalamus (MBH) dopaminergic tone during late pregnancy and lactation. We explored if the expression of MBH components, including various receptors (R) and proteins that regulate the dopaminergic system, is altered in mid-lactating OFA compared to SD rats, which may be associated with the abnormality. METHODS: Four groups of mid-lactating rats were used: continuous lactation; pups separated overnight; 30-min suckling (S); and 2 h or 4 h S after separation. Mothers were sacrificed to obtain serum for PRL RIA and MBHs to determine tyrosine hydroxylase (TH), PRL-R, PRL signaling molecules (activator: STAT5b; inhibitors: SOCS1, SOCS3, CIS), opioids (PENK, PDYN), and µ- and κ-opioid R (MOR, KOR) mRNA expression by qPCR and phospho-TH (p-TH) and TH proteins by Western blot. RESULTS: Suckling-induced PRL was lower in OFA and p-TH expression diminished in both strains. Separation increased TH mRNA and protein in SD, which decreased after 4 h S, but OFA protein levels remained unchanged. Separation of pups also resulted in decreased PRL-R and CIS expression in SD but increased PRL-R and SOCS3 in OFA. Despite the lower PRL-R, STAT5b, SOCS1, and SOCS3 levels in OFA compared to SD, suckling diminished them further. We observed subtle changes in SD opioids and their R, but in OFA, suckling decreased PENK, KOR, and MOR. CONCLUSION: The different patterns of TH, opioids, their R, and PRL signaling inhibitor expression with conserved TH activation by suckling may disturb the balance between stimulation and inhibition of PRL release resulting in impaired suckling-induced PRL secretion in OFA rats.

Differential effects of hypo- and hyperthyroidism on remodeling of contacts between neurons expressing the neuropeptide EI and tyrosine hydroxylase in hypothalamic areas of the male rat.

The Neuropeptide EI (NEI, glutamic acid- isoleucine amide) participates in neuroendocrine function. Previously we demonstrated that NEI concentration is regulated by thyroid hormones in discrete hypothalamic areas in rats. We observed that the thyroid status affects the dopaminergic regulation of the pituitary hormones. In this study we explored possible interactions between NEI and tyrosine hydroxylase (TH) containing elements in selected hypothalamic areas of male rats. Neuronal somas, terminals and boutons were assessed by confocal microscopy, in hypo- and hyperthyroid animals. We observed a remodeling of the contacts between the TH and NEI immunoreactive elements in the incerto-hypothalamic area (IHy, also known as rostromedial zona incerta) according to thyroid function. However, in the dorsolateral zone of the peduncular part of the lateral hypothalamus (DL-PLH) the thyroid hormones affect the dendritic trees of the neurons without perturbing the overall NEI/TH contacts. Also, we demonstrated that TRH Receptor 1 (TRH-R1) is colocalized in NEI immunoreactive neurons in the peduncular part of the lateral hypothalamus (PLH) and NEI precursor mRNA expression increased by hypothyroidism indicating that NEI neurons are responsive to the feedback mechanisms of the Hypothalamic Pituitary-Thyroid Axis (HPT). In conclusion, the hypothyroid status seems to increase the interactions between the NEI neurons and the dopaminergic pathways while hyperthyroidism either decreases or displays no effects. Altogether these observations support the participation of the IHy and PLH NEI as a modulating component of the HPT suggesting that altered neuroendocrine, behavioral and cognitive dysfunctions induced by dysthyroidism could be in part mediated by NEI.

Role of Oxytocin in Prolactin Secretion during Late Pregnancy.

BACKGROUND/AIMS: During late pregnancy, the blockade of progesterone action by mifepristone (Mp) treatment induces a dopaminergic tone fall that enables naloxone (NAL) administration to release pituitary prolactin (PRL). We determined whether oxytocin (OT), which stimulates PRL secretion acting directly on anterior pituitary lactotrophs, mediates the stimulatory action of Mp and NAL on PRL secretion during late pregnancy. METHODS: On day 19 of pregnancy, circulating and pituitary OT and PRL levels were measured by radioimmunoassay, 10, 20, and 30 min after NAL (given at 17:30 h) in rats pretreated with Mp (at 08:00 h). Pituitary OT receptor (OTR) expression in Mp-treated rats was evaluated by RT-PCR. Activation of OT neurons in Mp-NAL-treated rats was measured counting double immunoreactive neurons for Fos and OT (Fos-OT-ir) in supraoptic nuclei (SON), and medial (PaMM) and lateral magnocellular divisions of paraventricular nuclei. RESULTS: Elevated serum OT and decreased pituitary OT were observed 10 min after NAL administration in both vehicle- and Mp-treated rats. This PRL increase was prevented by previous i.p. administration of an OTR antagonist, but intracerebroventricular OT administration was ineffective. Mp increased pituitary OTR expression at 18:00 h. Only Mp-NAL increased Fos-OT-ir neurons in the PaMM and SON. CONCLUSIONS: These findings suggest that PRL secretion induced by Mp-NAL treatment is preceded by OT release. These results, together with the activation of hypothalamic OT neurons and the higher expression of pituitary OTR, support the hypothesis that, during late pregnancy, OT may act at the pituitary level to facilitate PRL secretion if the inhibitory action of progesterone is blocked.

Effect of hyperthyroidism on circulating prolactin and hypothalamic expression of tyrosine hydroxylase, prolactin signaling cascade members and estrogen and progesterone receptors during late pregnancy and lactation in the rat.

Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth.

Role of Estradiol in the Regulation of Prolactin Secretion During Late Pregnancy.

Estrogen action is necessary for evidencing the stimulatory action of mifepristone and naloxone on prolactin (PRL) secretion during late pregnancy. Our aim is to determine the mechanism mediating this facilitator action of estrogens. To investigate the hypothalamic mechanisms involved in estrogen actions in PRL secretion at the end of pregnancy, we measured the effect of pretreatment with the estrogen antagonist tamoxifen on the expression of tyrosine hydroxylase (TH), hormone receptors (ERα and ß, PRs, PRLR(long)), and µ- and κ- opioid receptors (ORs) at mRNA (by semiquantitative RT-PCR) and protein (by western blot for TH, PRLR(long), ERα, PRs, µ- and ORs) levels in extracts of medial basal hypothalamus (MBH) and serum PRL, E2 and P4 levels (by RIA) in mifepristone- and naloxone-treated rats. Tamoxifen administration partially prevented PRL release induced by the combined treatment. TH expression diminished and ERα expression increased in mifepristone-treated rats at mRNA and protein levels and tamoxifen partially prevented these changes with no effect on PRs expression. Mifepristone increased PRLR(long) mRNA levels; this increase was blocked by tamoxifen. Combined tamoxifen and mifepristone treatment decreased µ- and k-ORs mRNA but not protein levels. In conclusion, E2 induces neuroadaptive mechanisms necessary to facilitate PRL release preceding delivery. Acting through ERα, E2 modulates hypothalamic dopaminergic neurons activity, regulating TH, µ- and κ-ORs and PRLR(long) expression, and is necessary for evidencing the effects of P4 withdrawal. Its presence on days 14 and 15 of pregnancy is crucial to facilitate the opioid system modulation of PRL secretion at the end of pregnancy in the rat.

Opioid modulation of prolactin secretion induced by stress during late pregnancy. Role of ovarian steroids.

BACKGROUND: The opioid system modulates prolactin release during late pregnancy. Its role and the participation of ovarian hormones in this modulation are explored in ether stress-induced prolactin release. METHODS/RESULTS: Estrous, 3-day and 19-day pregnant rats were used. We administered the antagonist mifepristone (Mp) and tamoxifen to evaluate progesterone and estradiol action in naloxone (NAL, opioid antagonist) or saline treated rats. Ether stress had no effect on serum prolactin levels in controls but increased prolactin release in NAL-treated rats. Prolactin response to stress in NAL-treated rats was blocked by l-DOPA administration. Mp treatment on day 18 of pregnancy increased prolactin levels after stress without alterations by NAL. Tamoxifen on days 14 and 15 of pregnancy completely blocked Mp and NAL effects on prolactin release at late pregnancy. In contrast, stress significantly increased prolactin levels in estrous rats and pretreatment with NAL prevented this. On day 3 of pregnancy, at 6.00 p.m., stress and NAL treatment inhibited prolactin levels in saline-treated rat. No effect of stress or NAL administration was detected on day 3 of pregnancy at 9.00 a.m. icv administration of specific opioids antagonist, B-Funaltrexamine but not Nor-Binaltorphimine or Naltrindole, caused a significant increase in stress-induced prolactin release. CONCLUSIONS: Opioid system suppression of prolactin stress response during late pregnancy was observed only after progesterone withdrawal, involving a different opioid mechanism from its well-established stimulatory role. This mechanism acts through a mu opioid receptor and requires estrogen participation. The opioid system and progesterone may modulate stress-induced prolactin release, probably involving a putative prolactin-releasing factor.

Effects of thyroid status on NEI concentration in specific brain areas related to reproduction during the estrous cycle.

We previously showed that short-term hypo- and hyperthyroidism induce changes in neuropeptide glutamic-acid-isoleucine-amide (NEI) concentrations in discrete brain areas in male rats. To investigate the possible effects of hypo- and hyperthyroidism on NEI concentrations mainly in hypothalamic areas related to reproduction and behavior, female rats were sacrificed at different days of the estrous cycle. Circulating luteinizing hormone (LH), estradiol and progesterone concentrations were measured in control, hypothyroid (hypoT, treated with PTU during 7-9 days) and hyperthyroid (hyperT, l-T4 during 4-7 days) animals. Both treatments blunted the LH surge. Hypo- and hyperthyroidism increased estradiol concentrations during proestrus afternoon (P-PM), although hypoT rats showed lower values compared to control during proestrus morning (P-AM). Progesterone levels were higher in all groups at P-PM and in the hyperT during diestrus morning (D2). NEI concentrations were lower in hypoT rats during the estrous cycle except in estrus (E) in the peduncular part of the lateral hypothalamus (PLH). They were also reduced by both treatments in the perifornical part of the lateral hypothalamus (PeFLH) during P-PM. Hypothyroidism led to higher NEI concentrations during P-PM in the organum vasculosum of the lamina terminalis and anteroventral periventricular nucleus (OVLT+AVPV). The present results indicate that NEI concentration is regulated in a complex manner by hypo- and hyperthyroidism in the different areas studied, suggesting a correlation between NEI values and the variations of gonadal steroid levels during estrous cycle. These changes could be, in part, responsible for the alterations observed in the hypothalamic-pituitary-gonadal axis in these pathologies.

17ß-Estradiol modulates the prolactin secretion induced by TRH through membrane estrogen receptors via PI3K/Akt in female rat anterior pituitary cell culture.

Considering that estradiol is a major modulator of prolactin (PRL) secretion, the aim of the present study was to analyze the role of membrane estradiol receptor-α (mERα) in the regulatory effect of this hormone on the PRL secretion induced by thyrotropin-releasing hormone (TRH) by focusing on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway activation. Anterior pituitary cell cultures from female rats were treated with 17ß-estradiol (E(2), 10 nM) and its membrane-impermeable conjugated estradiol (E(2)-BSA, 10 nM) alone or coincubated with TRH (10 nM) for 30 min, with PRL levels being determined by RIA. Although E(2), E(2)-BSA, TRH, and E(2)/TRH differentially increased the PRL secretion, the highest levels were achieved with E(2)-BSA/TRH. ICI-182,780 did not modify the TRH-induced PRL release but significantly inhibited the PRL secretion promoted by E(2) or E(2)-BSA alone or in coincubation with TRH. The PI3K inhibitors LY-294002 and wortmannin partially inhibited the PRL release induced by E(2)-BSA, TRH, and E(2)/TRH and totally inhibited the PRL levels stimulated by E(2)-BSA/TRH, suggesting that the mER mediated the cooperative effect of E(2) on TRH-induced PRL release through the PI3K pathway. Also, the involvement of this kinase was supported by the translocation of its regulatory subunit p85α from the cytoplasm to the plasma membrane in the lactotroph cells treated with E(2)-BSA and TRH alone or in coincubation. A significant increase of phosphorylated Akt was induced by E(2)-BSA/TRH. Finally, the changes of ERα expression in the plasmalemma of pituitary cells were examined by confocal microscopy and flow cytometry, which revealed that the mobilization of intracellular ERα to the plasma membrane of lactotroph cells was only induced by E(2). These finding showed that E(2) may act as a modulator of the secretory response of lactotrophs induced by TRH through mER, with the contribution by PI3K/Akt pathway activation providing a new insight into the mechanisms underlying the nongenomic action of E(2) in the pituitary.

Effect of progesterone withdrawal on hypothalamic mechanisms related to prolactin release in late pregnant rats.

BACKGROUND/AIMS: Progesterone (P(4)) fall provoked by spontaneous or prostaglandin F2α (PGF2α)-induced luteolysis in late pregnant rats triggers a prolactin (PRL) surge 12-24 h later. METHODS: To investigate the hypothalamic mechanism mediating this response, we determined expression of tyrosine hydroxylase (TH), PRL receptors (long form, PRLR(long)), estrogen-α (ERα) and ERß, P(4) (PR) A and B receptors, and STAT5a, STAT5b, suppressors of cytokine signaling 1 (SOCS1), SOCS3 and CIS at mRNA (by semiquantitative and real-time RT-PCR) and protein (by Western blot only for TH, ERα and PRs) levels, and dopamine and DOPAC (by high-performance liquid chromatography) contents in the mediobasal hypothalamus (MBH) 24 h after luteolysis induced by a PGF2α analogue (cloprostenol, 25 µg/rat s.c. at 8 and 12 h on day 19 of pregnancy). RESULTS: PGF2α treatment decreased circulating P(4) and estradiol and increased PRL and the estradiol/P(4) ratio. MBH DOPAC and DOPAC/dopamine ratio fell, indicating decreased dopaminergic transmission. PRLR(long), PRB and ERα mRNA increased. ERα and PR proteins were not modified. However, TH protein and mRNA did not change. PRA, the small PR isoform, was much more abundant than PRB, the isoform considered to mediate P(4) genomic actions. STAT5a, SOCS1 and SOCS3 mRNA were also increased. CONCLUSION: The P(4) fall induced by PGF2α treatment induces PRL release through diminution in MBH dopaminergic transmission without change in TH expression. The increased PRLR along with elevated circulating PRL may be responsible for maintaining high TH expression through activation of short-loop feedback mechanisms, counteracting the effect of the fall in circulating P(4). In parallel, SOCS expression contributes to limit PRL signaling.

Hypo- and hyperthyroidism affect NEI concentration in discrete brain areas of adult male rats.

To date, there has been only one in vitro study of the relationship between neuropeptide EI (NEI) and the hypothalamic-pituitary-thyroid (HPT) axis. To investigate the possible relationship between NEI and the HPT axis, we developed a rat model of hypothyroidism and hyperthyroidism that allows us to determine whether NEI content is altered in selected brain areas after treatment, as well as whether such alterations are related to the time of day. Hypothyroidism and hyperthyroidism, induced in male rats, with 6-propyl-1-thiouracil and l-thyroxine, respectively, were confirmed by determination of triiodothyronine, total thyroxine, and thyrotropin levels. All groups were studied at the morning and the afternoon. In rats with hypothyroidism, NEI concentration, evaluated on postinduction days 7 and 24, was unchanged or slightly elevated on day 7 but was decreased on day 24. In rats with hyperthyroidism, NEI content, which was evaluated after 4 days of l-thyroxine administration, was slightly elevated, principally in the preoptic area in the morning and in the median eminence-arcuate nucleus and pineal gland in the afternoon, the morning and afternoon NEI contents being similar in the controls. These results provide the bases to pursue the study of the interaction between NEI and the HPT axis.

Epidermal growth factor induces a sexually dimorphic proliferative response of lactotroph cells through protein kinase C-ERK1/2-Pit-1 in vitro.

Lactotroph cells display morphological and functional heterogeneity, a feature which is closely related to the oestrogenic environment. In this study, we focused on sex-related differences linked to the proliferative and secretory responses of lactotrophs exposed to EGF in vitro. Furthermore, we addressed the involvement of the PKCε/ERK1/2 signalling pathway and the contribution of Pit-1 in the EGF actions in primary pituitary cultures from male and female rats. EGF promoted a differential proliferative activity on PRL cells, which was closely associated to the sex, as revealed by the uptake of bromodeoxyuridine (BrdU). In females, the mitogenic activity was up to nine times greater, whereas in males, the number of BrdU-labelled PRL cells was only doubled compared to control. However, in both models, EGF had a similar effectiveness in promoting PRL secretion. EGF also induced a significant increase in the PKCε, P -ERK 1/2, and Pit-1 protein levels, which were higher in females than in males. Pre-incubation with BIM blocked EGF-induced ERK 1/2 activation and Pit-1 expression. These results suggest a sexually dimorphic response of lactotroph cells to the proliferative effects of EGF, with the PKCε/ERK1/2 Pit-1 pathway being involved in this action.

Estrogen inhibits tuberoinfundibular dopaminergic neurons but does not cause irreversible damage.

Dopaminergic neurons of the hypothalamic tuberoinfundibular dopaminergic (TIDA) system exert a tonic inhibitory control on prolactin (PRL) secretion whereas estrogen, known to inhibit TIDA neuron function, has been postulated to be toxic to TIDA neurons when it is chronically high. In order to determine whether estrogen in high doses can cause permanent damage to TIDA function, we submitted young female rats to continue high doses of estrogen administered, either centrally (intrahypothalamic estrogen implants) or peripherally (subcutaneous estrogen implants or weekly intramuscular (i.m.) injections for 7 weeks), subsequently withdrawing the steroid and observing the evolution of lactotrophes, serum PRL and TIDA neurons. Serum PRL was measured by radioimmunoassay whereas tyrosine hydroxylase positive (TH+) neurons and PRL cells were morphometrically assessed in sections of fixed hypothalami and pituitaries, respectively. After 30 days, hypothalamic estrogen implants induced a significant increase in serum PRL, whereas TH+ neurons were not detectable in the arcuate-periventricular hypothalamic (ARC) region of estrogen-implanted rats. Removal of implants on day 30 restored TH expression in the ARC and brought serum PRL back to basal levels 30 days after estrogen withdrawal. Subcutaneous or i.m. administration of estrogen for 7 weeks induced a marked hyperprolactinemia. However, 30 weeks after estrogen withdrawal, TH neuron numbers in the ARC were back to normal and serum PRL returned to basal levels. After peripheral but not central estrogen withdrawal, pituitary weight and lactotrophic cell numbers remained slightly increased. Our data suggest that estrogen even at high doses, does not cause permanent damage to TIDA neurons.

Neuropeptide glutamic-isoleucine (NEI) specifically stimulates the secretory activity of gonadotrophs in primary cultures of female rat pituitary cells.

The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1-400x10(-8)M) and time periods (1-5h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100x10(-8)M) and GnRH (0.1 and 1x10(-9)M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4h of NEI incubation. NEI (100x10(-8)M) induced a significant LH secretion after 2h of stimulus, reaching a maximum response 4h later. A rapid and remarkable LH release was induced by NEI (400x10(-8)M) 1h after stimulus, attaining its highest level at 2h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10x10(-8)M), caused no action when used alone, its co-incubation with GnRH (1x10(-9)M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.

Pituitary changes involved in prolactin secretion induced by mifepristone and naloxone during late pregnancy.

BACKGROUND/AIMS: The antiprogesterone mifepristone facilitates prolactin release, an effect enhanced by administration of the opioid antagonist naloxone. The present study explores ultrastructural changes in lactotropes after mifepristone and naloxone administration, correlating them with the expression of pituitary prolactin. METHODS/RESULTS: Rats were sacrificed at 18:00 h on day 19 of pregnancy. Prolactin immunoelectron microscopy of lactotropes from control rats showed characteristics of quiescent cells with numerous small and spherical secretory granules. Naloxone administration did not modify lactotrope morphology or prolactin expression in terms of mRNA or protein abundances. Mifepristone treatment induced lactotrope activation with development of the rough endoplasmic reticulum and Golgi complex with prolactin immunoreactive small newly formed and large mature secretory granules. Mifepristone increased prolactin mRNA and protein expression. Naloxone administration to mifepristone-treated rats potentiated lactotrope activation compared with mifepristone alone showing exocytotic images of prolactin granules and some cells with evident signs of involution. CONCLUSIONS: (1) Blockade of progesterone action by mifepristone activated the lactotrope, increased significantly prolactin mRNA and protein expression and prepared the pituitary for naloxone action. (2) The high serum prolactin levels induced by mifepristone and naloxone may regulate negatively lactotrope activity as suggested by the presence of regressing cells neighboring the actively secreting cells.

Estradiol interacts with insulin through membrane receptors to induce an antimitogenic effect on lactotroph cells.

The signaling mechanisms of estrogens interact with those of growth factors to control the pituitary gland functions. The contribution of the membrane bound estrogen receptor in these actions is not fully understood. In this study, we focused on the regulatory action of estradiol in interaction with insulin on the secretory and proliferative lactotroph cell activities from primary pituitary cell cultures. Furthermore, we studied the involvement of ERK1/2, PKC epsilon and Pit-1 in these actions. In serum free conditions, estradiol and estradiol-BSA promoted a differential secretory activity on PRL cells but were unable to induce lactotroph cell proliferation. However, both free and conjugated estradiol were competent arresting the mitogenic activity promoted by insulin. Estradiol, estradiol-BSA and insulin stimuli increased the PKC epsilon, phosphorylated ERK 1/2 and Pit-1 expression, although combined treatments with estradiol/insulin or estradiol-BSA/insulin induced a significant reduction in these levels, in close correlation with the decrease of lactotroph cell proliferation. The pre-treatment with PKC inhibitor BIM significantly inhibited the ERK activation promoted by insulin without modifying the ERK expression levels induced by estradiol or estradiol-BSA. By immuno-electron-microscopy the alpha nuclear estrogen receptor was localized in the plasma membrane of lactotroph cells. These findings suggest that the membrane bound ER participates modulating lactotroph cells proliferation via PKC epsilon, ERK1/2 and Pit-1. The interactions between estradiol and growth factors, inducing both mitogenic and antimitogenic effects, could provide glandular plasticity preventing an over-proliferation induced by growth factors.

Dopaminergic mechanisms involved in prolactin release after mifepristone and naloxone treatment during late pregnancy in the rat.

BACKGROUND/AIMS: During late pregnancy, the antiprogesterone mifepristone facilitates prolactin release. This effect is enhanced by administration of the opioid antagonist naloxone, suggesting an inhibitory-neuromodulatory role of the opioid system. Since hypothalamic dopamine (DA) is the main regulator of prolactin release, in this study we explored the role of DA on prolactin release induced by mifepristone and naloxone treatment. METHODS/RESULTS: Rats on day 19 of pregnancy were used. Naloxone treatment did not modify the 3,4-dihydroxyphenylacetic acid/DA (DOPAC/DA) ratio or serum prolactin concentration in control rats. After mifepristone treatment, DA activity diminished significantly without modifying serum prolactin levels. Naloxone administration to antiprogesterone-treated rats did not change the DOPAC/DA ratio but increased serum prolactin. Tyrosine hydroxylase (TH) expression in medial basal hypothalamus (MBH) protein extracts was lowered by pretreatment with mifepristone, with no additional effect of naloxone. While mifepristone decreased the intensity of TH immunoreactivity in the arcuate and periventricular nuclei and in fibers of the median eminence, naloxone treatment had no further effect. CONCLUSIONS: (1) A reduction of tuberoinfundibular dopaminergic (TIDA) neuron activity is suggested by the fall of the DOPAC/DA ratio and the low expression of MBH TH; (2) this reduction facilitates prolactin secretion by naloxone, indicating that progesterone stimulates DA neurons to maintain low serum prolactin; (3) naloxone action seems to depend on a previous decrease of DA tone induced by mifepristone, without involve a direct effect on neuronal DA activity, and (4) endogenous opioids may inhibit prolactin secretion through a non-dopaminergic neuronal system that regulates prolactin secretion in which as yet undetermined prolactin-releasing factors may participate.

Neurotransmitters involved in the opioid regulation of prolactin secretion at the end of pregnancy in rats.

Using a pharmacological approach, we explored potential mechanisms for the regulation of prolactin secretion by opioid peptides at the end of pregnancy in rats. On day 19 of pregnancy, intracereboventricular administration of the mu-opioid receptor agonist (D-Ala2, NMe-Phe4, Gly-ol5)-enkephalin (DAMGO) or beta-endorphin (beta-END) induced a dose-related increase in serum prolactin levels 30 min later. Pretreatment with the opioid antagonist naloxone abolished the increase induced by DAMGO injection. At lower doses, DAMGO and beta-END did not modify the 3,4-dihydroxyphenylacetic acid/dopamine ratio, but at higher doses, the mu-agonists evoked a significant increase of the dopaminergic activity as compared with saline control. The time course of the effects of beta-END (2.5 microg/rat) showed a higher increase in serum prolactin levels at 15 min than at 30 min after treatment. The 3,4-dihydroxyphenylacetic acid/dopamine ratio increased 15 min after beta-END administration and was even higher 30 min later. Neither the selective kappa-agonist U50,488H nor the selective delta-agonist (D-Pen2, D-Pen5)- enkephalin were able to modify the serum prolactin levels at the doses studied. To evaluate potential neurotransmitters involved in the regulation of prolactin secretion at the end of pregnancy, we combined the administration of serotoninergic or GABAergic antagonists with the opioid agonist DAMGO. The serotonin 5-HT2 receptor antagonist ketanserin increased the serum prolactin levels and potentiated the effect of DAMGO. The intracerebroventricular administration of SR-95531 did not modify the serum prolactin concentration under basal conditions, but partially prevented the increase induced by DAMGO injection. The intracerebroventricular administration of the GABA(B) receptor antagonist phaclofen had no effect on the serum prolactin levels either in naive or DAMGO-treated rats. The present results support the proposal that activation of mu-opioid receptors stimulates prolactin secretion at the end of pregnancy. Although the exact mechanisms by which the opioid system modulates prolactin secretion at the end of pregnancy are unclear, these results suggest an interaction of the opioidergic system with serotoninergic and GABAergic systems, without ruling out a direct or indirect action on dopaminergic neurons. In conclusion, the opioid system may regulate prolactin secretion at the end of pregnancy through either stimulatory (present results) or inhibitory actions previously described.