Effects Of Prolactin Research Articles

Abstract 22: Differential IGF-II response of AA and CA normal breast cell lines to prolactin treatment

Abstract African-American (AA) breast cancer patients have lower survival compared with Caucasian-American (CA) patients. This survival disparity is significant in view of the lower incidence of breast cancer among AA. Lower incidence of breast cancer is associated with parity and lactation in young women. Lactation's protective effect is mediated by fatty-acid binding protein 3 (FABP3), a protein that promotes differentiation of breast epithelial cells. We have demonstrated that AA normal women below 50 years have lower FABP3 levels as compared to CA women (p = 0.030). FABP3 is a protective tumor suppressor that increases during pregnancy and lactation, in response to the lactation hormone prolactin (PRL). PRL effects on the breast are mediated by IGF-II. Thus, we propose that PRL treatment of normal epithelial breast cell lines would increase IGF-II to promote differentiation, increase FABP3, and decrease FABP5. FABP5 is associated with metastasis and may compensate for FABP3 decrease. We treated MCF10A (CA) and AG11132 (AA) normal breast epithelial cell lines with recombinant human PRL (0-200 ng/ml) for 24 hours. Cell lysates were examined for protein and mRNA levels of IGF-II, FABP3, and FABP5. In MCF10A cells, PRL stimulated an increase in protein levels of IGF-II (p=0.044), and a decrease in protein levels of FABP5 (p=0.044). Protein levels of FABP3 significantly decreased with increasing PRL concentration (p=0.030). IGF-II mRNA levels also increased at 50 and 200 ng/ml PRL (2.59 and 2.58 fold, respectively). In AG11132 cells, protein levels of FABP3 significantly increased with increasing PRL concentration (p=0.016). IGF-II and FABP5 mRNA levels increased significantly at 10 ng/ml PRL (49.91 and 2.13 fold, respectively); however, no corresponding changes were seen in protein levels. Though both cell lines respond to PRL treatment, only the MCF10A line displays increased IGF-II and decreased FABP5, which are protective changes in normal mammary cells. FABP3 levels decreased in MCF10A cells at higher PRL concentrations; this may be due to untreated MCF10A cells expressing significantly more FABP3 and prolactin, relative to AG11132 cells. The higher levels of PRL and FABP3 in untreated MCF10A cells indicate that they are more differentiated and thus require less PRL stimulation, relative to the AG11132 cells, to achieve a differentiated state. The dissimilar responsiveness of these cell lines to PRL may suggest decreased protection against breast carcinogenesis in AA patients, and may help us to further understand this health disparity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 22. doi:1538-7445.AM2012-22

Photoperiodic Modulation of the Suppressive Actions of Prolactin and Dopamine on the Pituitary Gonadotropin Responses to Gonadotropin-Releasing Hormone in Sheep1

In a variety of species, the LH-secretory response to gonadotropin-releasing hormone (GnRH) is completely suppressed by the combined actions of prolactin (PRL) and dopamine (DA). In sheep, this effect is only observed under long days (nonbreeding season [NBS]). To investigate the level at which these mechanisms operate, we assessed the effects of PRL and bromocriptine (Br), a DA agonist, on the gonadotropin-secretory and mRNA responses to GnRH in pituitary cell cultures throughout the ovine annual reproductive cycle. As expected, the LH-secretory response to GnRH was only abolished during the NBS following combined PRL and Br application. Conversely, the LHB subunit response to GnRH was reduced during both the BS and NBS by the combined treatment and Br alone. Similar results were obtained in pars distalis-only cultures, indicating that the effects are pars tuberalis (PT)- independent. Further signaling studies revealed that PRL and Br alter the LH response to GnRH via convergence at the level of PLC and PKC. Results for FSH generally reflected those for LH, except during the BS where removal of the PT allowed PRL and Br to suppress the FSH-secretory response to GnRH. These data show that suppression of the LH-secretory response to GnRH by PRL and DA is accompanied by changes in mRNA synthesis, and that the photoperiodic modulation of this inhibition operates primarily at the level of LH release through alterations in PKC and PLC. Furthermore, the suppressive effects of PRL and DA on the secretion of FSH are photoperiodically regulated in a PT-dependent manner.

Site-specific regulation of ion transport by prolactin in rat colon epithelium

The effect of prolactin (PRL) on ion transport across the rat colon epithelium was investigated using Ussing chamber technique. PRL (1 μg/ml) induced a sustained decrease in short-circuit current (I(sc)) in the distal colon with an EC(50) value of 100 ng/ml and increased I(sc) in the proximal colon with an EC(50) value of 49 ng/ml. In the distal colon, the PRL-induced decrease in I(sc) was not affected by Na(+) channel blocker amiloride or Cl(-) channel blockers, NPPB, DPC, or DIDS, added mucosally. However, the response was inhibited by mucosal application of K(+) channel blockers glibenclamide, quinidine, and chromanol 293B, whereas other K(+) channel blockers, Ba(2+), tetraethylammonium, clotrimazole, and apamin, failed to have effects. The PRL-induced decrease in I(sc) was also inhibited by Na(+)-K(+)-2Cl(-) transporter inhibitor bumetanide, Ba(2+), and chromanol 293B applied serosally. In the transverse and proximal colon, the PRL-induced increase in I(sc) was suppressed by DPC, glibenclamide, and bumetanide, but not by NPPB, DIDS, or amiloride. The PRL-induced changes in I(sc) in both distal and proximal colon were abolished by JAK2 inhibitor AG490, but not BAPTA-AM, the Ca(2+) chelating agent, or phosphatidylinositol 3-kinase inhibitor wortmannin. These results suggest a segment-specific effect of PRL in rat colon, by activation of K(+) secretion in the distal colon and activation of Cl(-) secretion in the transverse and proximal colon. Both PRL actions are mediated by JAK-STAT-dependent pathway, but not phosphatidylinositol 3-kinase pathway or Ca(2+) mobilization. These findings suggest a role of PRL in the regulation of electrolyte transport in mammalian colon.

Chondroregulatory action of prolactin on proliferation and differentiation of mouse chondrogenic ATDC5 cells in 3-dimensional micromass cultures

A recent investigation in lactating rats has provided evidence that the lactogenic hormone prolactin (PRL) increases endochondral bone growth and bone elongation, presumably by accelerating apoptosis of hypertrophic chondrocytes in the growth plate and/or subsequent chondrogenic matrix mineralization. Herein, we demonstrated the direct chondroregulatory action of PRL on proliferation, differentiation and apoptosis of chondrocytes in 3-dimensional micromass culture of mouse chondrogenic ATDC5 cell line. The results showed that ATDC5 cells expressed PRL receptor (PRLR) transcripts, and responded typically to PRL by downregulating PRLR expression. Exposure to a low PRL concentration of 10ng/mL, comparable to the normal levels in male and non-pregnant female rats, increased chondrocyte viability, differentiation, proteoglycan accumulation, and mRNA expression of several chondrogenic differentiation markers, such as Sox9, ALP and Hspg2. In contrast, high PRL concentrations of ⩾100ng/mL, comparable to the levels in pregnancy or lactation, decreased chondrocyte viability by inducing apoptosis, with no effect on chondrogenic marker expression. It could be concluded that chondrocytes directly but differentially responded to non-pregnant and pregnant/lactating levels of PRL, thus suggesting the stimulatory effect of PRL on chondrogenesis in young growing individuals, and supporting the hypothesis of hypertrophic chondrocyte apoptosis in the growth plate of lactating rats.

Prolactin and blood vessels: A comparative endocrinology perspective

The hormone prolactin (PRL), fundamental for lactation in mammals, is known to exert a wide diversity of actions in the various vertebrate groups. Blood vessels are surfacing as important PRL targets, contributing to these hormonal functions. PRL promotes the growth of new blood vessels (angiogenesis) and is proteolytically cleaved to vasoinhibins, a family of peptides (including 16-kDa PRL) with potent antiangiogenic and blood vessel regression effects. These opposing actions point to the regulation of the proteases responsible for PRL cleavage as an efficient way to balance blood vessel growth and involution. This review briefly summarizes the effects of PRL and vasoinhibins on blood vessels in mammals and discusses whether similar vascular actions could contribute to the effects of PRL on the development, growth, and reproduction of lower vertebrates. A comparative study in diverse species may lead to a better understanding of blood vessels as a driving force for the biological actions of PRL.

Prolactin regulates luminal bicarbonate secretion in the intestine of the sea bream (Sparus auratus L.)

The pituitary hormone prolactin is a pleiotropic endocrine factor that plays a major role in the regulation of ion balance in fish, with demonstrated actions mainly in the gills and kidney. The role of prolactin in intestinal ion transport remains little studied. In marine fish, which have high drinking rates, epithelial bicarbonate secretion in the intestine produces luminal carbonate aggregates believed to play a key role in water and ion homeostasis. The present study was designed to establish the putative role of prolactin in the regulation of intestinal bicarbonate secretion in a marine fish. Basolateral addition of prolactin to the anterior intestine of sea bream mounted in Ussing chambers caused a rapid (<20 min) decrease of bicarbonate secretion measured by pH-stat. A clear inhibitory dose-response curve was obtained, with a maximal inhibition of 60-65% of basal bicarbonate secretion. The threshold concentration of prolactin for a significant effect on bicarbonate secretion was 10 ng ml(-1), which is comparable with putative plasma levels in seawater fish. The effect of prolactin on apical bicarbonate secretion was independent of the generation route for bicarbonate, as shown in a preparation devoid of basolateral HCO(3)(-)/CO(2) buffer. Specific inhibitors of JAK2 (AG-490, 50 μmol l(-1)), PI3K (LY-294002, 75 μmol l(-1)) or MEK (U-012610, 10 μmol l(-1)) caused a 50-70% reduction in the effect of prolactin on bicarbonate secretion, and demonstrated the involvement of prolactin receptors. In addition to rapid effects, prolactin has actions at the genomic level. Incubation of intestinal explants of anterior intestine of the sea bream in vitro for 3 h demonstrated a specific effect of prolactin on the expression of the Slc4a4A Na(+)-HCO(3)(-) co-transporter, but not on the Slc26a6A or Slc26a3B Cl(-)/HCO(3)(-) exchanger. We propose a new role for prolactin in the regulation of bicarbonate secretion, an essential function for ion/water homeostasis in the intestine of marine fish.

Expression of prolactin receptors in normal canine mammary tissue, canine mammary adenomas and mammary adenocarcinomas

BackgroundMammary tumors represent the most common neoplastic disease in female dogs. Recently, the promoting role of prolactin (PRL) in the development of human breast carcinoma has been shown. Possible proliferative, anti-apoptotic, migratory and angiogenic effects of PRL on human mammary cancer cells in vitro and in vivo were suggested. The effects of PRL are mediated by its receptor, and alterations in receptor expression are likely to play a role in tumor development. Currently, not much data is available about prolactin receptor (PRLR) expression in canine mammary tumors. To set the basis for investigations on the role of PRL in mammary tumorigenesis in this species, prolactin receptor expression was evaluated by semi-quantitative real time PCR and immunohistochemistry on 10 formalin-fixed, paraffin-embedded samples each of canine non-neoplastic mammary tissue, mammary adenomas and adenocarcinomas.ResultsThe highest PRLR expression levels were found in normal mammary tissue, while adenomas, and to an even higher degree adenocarcinomas, showed a significant decrease in prolactin receptor expression. Compared to normal tissue, PRLR mRNA was reduced 2.4 fold (p = 0.0261) in adenomas and 4.8 fold (p = 0.008) in adenocarcinomas. PRLR mRNA expression was significantly lower in malignant than in benign lesions (p = 0.0165). Immunohistochemistry demonstrated PRLR expression in all three tissue types with signals mostly limited to epithelial cells.ConclusionsMalignant transformation of mammary tissue was associated with a decline in prolactin receptor expression. Further studies are warranted to address the functional significance of this finding.

TRIENNIAL LACTATION SYMPOSIUM: Prolactin: The multifaceted potentiator of mammary growth and function1,2

At face value there are clear and established roles for prolactin (PRL) in the regulation of mammary gland growth, lactogenesis, and galactopoiesis. These actions of PRL do not occur in isolation; rather, they are finely attuned to and coordinated with many local, reproductive, and metabolic events in the female. Hence, to understand PRL action at the level of the mammary gland is to understand the systemic and local contexts in which it acts and functions. Herein we review the functions of PRL, its receptors, and the pathways leading to the phenotypes it evokes within the mammary glands, including growth and lactation, across a variety of species. At one level, the actions of PRL are mediated by several PRL receptor (PRLR) isoforms, including its long form and various short PRLR variants that are generated by alternative splicing in a species- and tissue-dependent manner. In turn, these PRLR activate a variety of intracellular signaling cascades. We also focus on how PRL coordinates with other endocrine cues to impart its effects on the mammary glands, where the ovarian hormones can independently and substantially modulate PRL action. Many of these effects of PRL are also realized at the local level of the mammary gland, either through the autocrine or paracrine synthesis of a multitude of molecules and transcription factors or through its effects on adjacent supporting tissues, including the mammary vasculature. Taken together, it is clear that PRL directs a variety of mechanisms during growth and function of the mammary gland and is deserving of its classification as the master hormone.

Prolactin increases SMN expression and survival in a mouse model of severe spinal muscular atrophy via the STAT5 pathway

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease that is characterized by the loss of motor neurons, resulting in progressive muscle atrophy. It is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. A potential treatment strategy for SMA is to upregulate levels of SMN protein. Several agents that activate STAT5 in human and mouse cell lines enhance SMN expression from the SMN2 gene and can compensate, at least in part, for the loss of production of a functional protein from SMN1. Here, we have shown that prolactin (PRL) increases SMN levels via activation of the STAT5 pathway. PRL increased SMN mRNA and protein levels in cultured human and mouse neuronal cells. Administration of STAT5-specific siRNA blocked the effects of PRL, indicating that the PRL-induced transcriptional upregulation of the SMN-encoding gene was mediated by activation of STAT5. Furthermore, systemic administration of PRL to WT mice induced SMN expression in the brain and spinal cord. Critically, PRL treatment increased SMN levels, improved motor function, and enhanced survival in a mouse model of severe SMA. Our results confirm earlier work suggesting STAT5 pathway activators as potential therapeutic compounds for the treatment of SMA and identify PRL as one such promising agent.

Prolactin (PRL) induction of cyclooxygenase 2 (COX2) expression and prostaglandin (PG) production in hamster Leydig cells

Serum prolactin (PRL) variations play a crucial role in the photoperiodic-induced testicular regression–recrudescence transition in hamsters. We have previously shown that cyclooxygenase 2 (COX2), a key enzyme in the biosynthesis of prostaglandins (PGs), is expressed mostly in Leydig cells of reproductively active hamsters with considerable circulating and pituitary levels of PRL. In this study, we describe a stimulatory effect of PRL on COX2/PGs in hamster Leydig cells, which is mediated by IL-1β and prevented by P38-MAPK and JAK2 inhibitors. Furthermore, by preparative isoelectric focusing (IEF), we isolated PRL charge analogues from pituitaries of active [isoelectric points (pI): 5.16, 4.61, and 4.34] and regressed (pI: 5.44) hamsters. More acidic PRL charge analogues strongly induced COX2 expression, while less acidic ones had no effect. Our studies suggest that PRL induces COX2/PGs in hamster Leydig cells through IL-1β and activation of P38-MAPK and JAK2. PRL microheterogeneity detected in active/inactive hamsters may be responsible for the photoperiodic variations of COX2 expression in Leydig cells.

In vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian, pituitary, and pineal function in pigs

The aims of the study were: (1) to examine 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and/or prolactin (PRL) effects on in vitro secretion of progesterone (P4) and estradiol (E2) by luteinized granulosa and theca cells from porcine preovulatory follicles; and (2) to determine the effects of TCDD on PRL, luteinizing hormone (LH), and melatonin luteal phase in pigs. We found that TCDD itself did not affect progesterone secretion, but it abolished the stimulatory effect of PRL in the follicular cells. TCDD stimulated PRL secretion during the luteal phase and inhibited during the follicular phase. Moreover, TCDD increased luteinizing hormone secretion by pituitary cells during the follicular phase. In contrast to protein and steroid hormones, melatonin secretion in vitro was not affected by TCDD. In conclusion, it was found that the pituitary-ovarian axis in pigs is sensitive to TCDD, and the dioxin exhibited a profound ability to disrupt the ovarian actions of prolactin.

Prolactin differentially modulates the macrophage activity of lactating rats: possible role of reproductive experience

Reproductive experience (i.e., pregnancy and lactation) induces physiological changes in mammals. We recently showed that a previous reproductive experience can modulate the activity of dopaminergic hypothalamic systems while decreasing serum prolactin (PRL) levels and oxidative burst activity in peritoneal macrophages. Dopamine receptor antagonists increase serum PRL levels, and both PRL and dopamine receptors might be involved in the modulation of macrophage activity, providing a means of communication between the nervous and immune systems. The present study evaluated the in vitro effects of PRL and the dopamine receptor D2 antagonist domperidone (DOMP) on the peritoneal activity of macrophages from primiparous and multiparous female rats during lactation. Oxidative bursts and phagocytosis in peritoneal macrophages were evaluated by flow cytometry. Primiparous and multiparous Wistar rats, during the period of lactation (i.e., days 5-7 after parturition) were used. Samples of peritoneal fluid from these rats were first incubated with PRL (10 and 100 nM) for different periods of time. The same procedure was repeated to evaluate the effects of DOMP (10 and 100 nM). Our results showed that macrophages from multiparous rats respond more effectively to in vitro incubation with PRL, especially with regard to oxidative bursts and the percentage of phagocytosis. Additionally, these effects were more pronounced after 30 min of incubation. These data suggest that reproductive experience is associated with a reduction in serum PRL levels, and cells in experienced female animals, including their macrophages, become more sensitive to the effects of PRL.

Differential and Complementary Effects of Glucose and Prolactin on Islet DNA Synthesis and Gene Expression

The mechanisms by which lactogenic hormones promote β-cell expansion remain poorly understood. Because prolactin (PRL) up-regulates β-cell glucose transporter 2, glucokinase, and pyruvate dehydrogenase activities, we reasoned that glucose availability might mediate or modulate the effects of PRL on β-cell mass. Here, we used male rat islets to show that PRL and glucose have differential but complementary effects on the expression of cell cyclins, cell cycle inhibitors, and various other genes known to regulate β-cell replication, including insulin receptor substrate 2, IGF-II, menin, forkhead box protein M1, tryptophan hydroxylase 1, and the PRL receptor. Differential effects on gene expression are associated with synergistic effects of glucose and PRL on islet DNA synthesis. The effects of PRL on gene expression are mirrored by β-cell overexpression of signal transducer and activator of transcription 5b and are opposed by dexamethasone. An ad-small interfering RNA specific for cyclin D2 attenuates markedly the effects of PRL on islet DNA synthesis. Our studies suggest a new paradigm for the control of β-cell mass and insulin production by hormones and nutrients. PRL up-regulates β-cell glucose uptake and utilization, whereas glucose increases islet PRL receptor expression and potentiates the effects of PRL on cell cycle gene expression and DNA synthesis. These findings suggest novel targets for prevention of neonatal glucose intolerance and gestational diabetes and may provide new insight into the pathogenesis of β-cell hyperplasia in obese subjects with insulin resistance.

Establishing a relationship between prolactin and altered fatty acid β-Oxidation via carnitine palmitoyl transferase 1 in breast cancer cells

BackgroundMammary carcinomas have been associated with a high-fat diet, and the rate of breast cancer in overweight post-menopausal women is up to 50% higher than in their normal-weight counterparts. Epidemiological studies suggest that prolactin (PRL) plays a role in the progression of breast cancer. The current study examined breast cancer as a metabolic disease in the context of altered fatty acid catabolism by examining the effect of PRL on carnitine palmitoyl transferase 1 (CPT1), an enzyme that shuttles long-chain fatty acids into the mitochondrial matrix for β-oxidation. The effect of PRL on the adenosine 5'-monophosphate-activated protein kinase (AMPK) energy sensing pathway was also investigated.MethodsMCF-7 and MDA-MB-231 breast cancer cells and 184B5 normal breast epithelial cells treated with 100 ng/ml of PRL for 24 hr were used as in vitro models. Real-time PCR was employed to quantify changes in mRNA levels and Western blotting was carried out to evaluate changes at the protein level. A non-radioactive CPT1 enzyme activity assay was established and siRNA transfections were performed to transiently knock down specific targets in the AMPK pathway.ResultsPRL stimulation increased the expression of CPT1A (liver isoform) at the mRNA and protein levels in both breast cancer cell lines, but not in 184B5 cells. In response to PRL, a 20% increase in CPT1 enzyme activity was observed in MDA-MB-231 cells. PRL treatment resulted in increased phosphorylation of the α catalytic subunit of AMPK at Thr172, as well as phosphorylation of acetyl-CoA carboxylase (ACC) at Ser79. A siRNA against liver kinase B1 (LKB1) reversed these effects in breast cancer cells. PRL partially restored CPT1 activity in breast cancer cells in which CPT1A, LKB1, or AMPKα-1 were knocked down.ConclusionsPRL enhances fatty acid β-oxidation by stimulating CPT1 expression and/or activity in MCF-7 and MDA-MB-231 breast cancer cells. These PRL-mediated effects are partially dependent on the LKB1-AMPK pathway, although the regulation of CPT1 is also likely to be influenced by other mechanisms. Ultimately, increased CPT1 enzyme activity may contribute to fueling the high energy demands of cancer cells. Targeting metabolic pathways that are governed by PRL, which has already been implicated in the progression of breast cancer, may be of therapeutic benefit.

Prolactin as a biomarker for treatment response and tardive dyskinesia in schizophrenia subjects: old thoughts revisited from a genetic perspective

Previous studies investigated whether prolactin (PRL) serum level was a biomarker of antipsychotic response, schizophrenia symptomatology, and tardive dyskinesia. Most of the findings support that antipsychotic drugs modulate PRL levels but PRL is not a steady indicator. Recent results suggest a genetic effect of PRL and PRL receptor (PRLR) polymorphisms in PRL levels indicating that independently of antipsychotic therapy subjects could have altered PRL levels due to their genetic background.We evaluated whether PRL and PRLR variants were associated with treatment outcome and tardive dyskinesia. We observed no association of PRL/PRLR polymorphism with treatment response (best genotypic results include PRL rs849885 and PRLR rs4703509 permuted p=0.326). Regarding tardive dyskinesia, the major allele of PRL rs37364 was nominally associated with risk for tardive dyskinesia in the European ancestry sub-sample (permuted p=0.183). Although we reported no significant associations, it is definitely worthy of investigation to see if together (genetic variants in the PRL system and PRL serum measures) could be a reliable biomarker for antipsychotic response and TD prevalence. Our results suggest that more studies in this context are required to shed light in the molecular mechanisms underlying antipsychotic response and tardive dyskinesia occurrence.

Seasonal effects of central leptin infusion and prolactin treatment on pituitary SOCS-3 gene expression in ewes

Suppressors of cytokine signalling (SOCS) negatively regulate cytokine-induced signalling pathways and may be involved in leptin and prolactin (PRL) interactions. Herein, we examined the effect of PRL on SOCS-3 mRNA expression in pituitary explants and investigated whether leptin could modify the expression of SOCS-3 mRNA in pituitary explants. In the first experiment, we used pituitaries isolated from 16 ewes decapitated in March, May, July and October (four per month). Tissues were cut into 50 mg explants, which were treated with control or medium containing PRL (100 or 300 ng/ml). Incubation was maintained for different time intervals: 0, 60, 120, 180, 240 or 300 min. Real-time PCR was used to measure SOCS-3 mRNA levels. In the second study, we used 24 ewes surgically fitted with third ventricle cannulas (12 were used during the long-day period, and 12 were used during the short-day (SD) period). Each ewe was administered an i.c.v. injection of Ringer-Locke buffer or leptin (0.5 or 1.0 μg/kg body weight). Explants of anterior pituitaries were collected and snap frozen 1 h after injection. Semi-quantitative expression of SOCS-3 mRNA was performed using reverse transcription-PCR. PRL stimulated SOCS-3 expression in the pituitaries collected in March (P<0.05) and May (P<0.01 and P<0.05 for lower and higher doses respectively), inhibited SOCS-3 expression in pituitaries collected in July (P<0.01) and had no effect in pituitaries collected in October. Treatment with leptin increased SOCS-3 expression during the SDs in a dose-dependent manner (P<0.01). The results demonstrated that photoperiod may be involved in leptin and PRL effects on SOCS-3 expression in sheep.

Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei

Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction.

A Novel Antagonistic Effect of the Bone Morphogenetic Protein System on Prolactin Actions in Regulating Steroidogenesis by Granulosa Cells

To investigate the mechanism by which prolactin (PRL) regulates follicular steroidogenesis in the ovary, we examined the functional roles of PRL in steroidogenesis using rat oocyte/granulosa cell coculture and focusing on the bone morphogenetic protein (BMP) system. The expression of long and short forms of PRL receptor (PRLR) were detected in both oocytes and granulosa cells, and PRL effectively up-regulated PRLR expression in granulosa cells in the presence of FSH. PRL suppressed FSH-induced estradiol production and increased FSH-induced progesterone production in granulosa cells. The PRL effects on FSH-induced progesterone were blocked by coculture with oocytes, implying roles of oocyte-derived factors in suppression of progesterone production in PRL-exposed granulosa cells. In accordance with the data for steroids, FSH-induced aromatase expression was suppressed by PRL, whereas FSH-induced steroidogenic acute regulatory protein, P450scc (P450 side-chain cleavage enzyme), and 3β-hydroxysteroid dehydrogenase type 2 levels were amplified by PRL. However, forskolin- and N(6),O(2)-dibutyryl cAMP-induced steroid levels and FSH- and forskolin-induced cAMP were not affected by PRL, suggesting that PRL action on FSH-induced steroidogenesis was not due to cAMP-protein kinase A regulation. Treatment with a BMP-binding protein, noggin, facilitated PRL-induced estradiol reduction, and noggin increased PRL-induced progesterone production in FSH-treated granulosa cells cocultured with oocytes, suggesting that endogenous BMPs reduce progesterone but increase estradiol when exposed to high concentrations of PRL. PRL increased the expression of BMP ligands in oocyte/granulosa cell coculture and augmented BMP-induced phosphorylated mothers against decapentaplegic 1/5/8 signaling by reducing inhibitory phosphorylated mothers against decapentaplegic 6 expression through the Janus kinase/signal transducer and activator of transcription (STAT) pathway. In addition to STAT activation, PRL enhanced FSH-induced MAPK phosphorylation in granulosa cells, in which ERK activation was preferentially involved in suppression of FSH-induced estradiol. Furthermore, noggin treatment enhanced PRLR signaling including MAPK and STAT. Considering that BMPs suppressed PRLR in granulosa cells, it is likely that the BMP system in growing follicles plays a key role in antagonizing PRLR signaling actions in the ovary exposed to high concentrations of PRL.

Characterization of the Effects of Prolactin in Gonadotroph Target Cells1

Hyperprolactinemia is a major cause of infertility, brought about by inhibition of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus and impairment of luteinizing hormone (LH) output from the pituitary gland. However, whereas the actions of prolactin (PRL) within the brain have been investigated extensively, its specific effects at the level of pituitary gonadotroph target cells remain unclear. Here, we provide evidence that the actions of PRL within the gonadotroph are more complex than originally envisaged. Using a gonadotroph cell monoculture, the first series of experiments showed that PRL is, paradoxically, a potent stimulator of LH release, with a three- to fourfold increase in LH values at hyperprolactinemic concentrations of PRL. Conversely, PRL dose-dependently modulated the LH secretory response to GnRH in a biphasic manner, with classical suppression of LH output only detected under a narrow dose range. In contrast, at all doses tested, PRL blocked the LHB mRNA response to the secretagogue. Subsequent studies revealed that the stimulatory effects of PRL on LH release are not mediated by the conventional cytokine receptor pathways but, rather, by a novel JAK2-PIK3-PKC-dependent signaling cascade. Moreover, the experiments showed that these actions of PRL within gonadotroph cells are controlled by dopamine, the main hypothalamic inhibitory regulator of PRL release in vivo. Our findings have unraveled specific actions of PRL within the gonadotroph and the cell-signaling interactions that ultimately underlie hyperprolactinemia-induced infertility.

Prolactin Supplementation to Culture Medium Improves β-Cell Survival

Recent studies demonstrated that prolactin (PRL) has beneficial effects on beta cells for islet transplantation. We examined the effect of human recombinant PRL (rhPRL) supplementation to the culture media to determine its potential use in the context of clinical islet transplantation. Each human islet isolated from 14 deceased multiorgan donors was cultured in Miami modified media-1 supplemented with or without rhPRL (500 microg/L) for 48 hr. beta-Cell survival and proliferation (BrdU and Ki-67) were determined by laser scanning cytometry. The cytoprotective effects of rhPRL against noxious stimuli were assessed by flow cytometry (tetramethylrhodamine ethyl ester). Cytokine/chemokine and tissue factor productions were measured in vitro, and islet potency was assessed in vivo in diabetic immunodeficient mice. beta-Cell survival during culture was 37% higher in the rhPRL group than in control (P=0.029). rhPRL protected beta cells in vitro from cytokines, Nitric oxide donor, and H2O2. The exposure to rhPRL did not affect human beta-cell proliferation with our protocol. rhPRL treatment did not alter cytokine/chemokine and tissue factor production in vitro or affected human islet functionality in vivo: recipient mice achieved normoglycemia with a comparable tempo, whereas loss of graft function was observed in two of the seven mice in the control group and in none of the rhPRL group (p=n.s.). rhPRL supplementation to islet culture media improved human beta-cell-specific survival without altering islet quality. Addition of rhPRL to cultured islets may grant a more viable beta-cell mass in culture. The development of beta-cell cytoprotective strategies will be of assistance in improving islet transplantation outcomes.