Long Form Of Prolactin Receptor Research Articles

Prolactin enhances T regulatory cell promotion of breast cancer through the long form prolactin receptor

Previous work has shown systemic knockdown of the long form prolactin receptor (LFPRLR) in vivo markedly reduced metastasis in mouse models of breast cancer, but whether this translated to prolonged survival was unknown. Here we show that LFPRLR knockdown in the highly metastatic, immunocompetent 4T1 model prolonged survival and reduced recruitment of T regulatory cells (Tregs) to the tumor through effects on the production of CCL17. For the Tregs still recruited to the primary tumor, LFPRLR knockdown both directly and indirectly reduced their ability to promote tumor parenchymal epithelial to mesenchymal transition. Importantly, effects of prolactin on expression of mesenchymal genes by the tumor parenchyma were very different in the absence and presence of Tregs. While systemic knockdown of the LFPRLR downregulated transcripts important for immune synapse function in the remaining tumor Tregs, splenic Tregs seemed unaffected by LFPRLR knockdown, as demonstrated by their continued ability to suppress anti-CD3/CD28-stimulated effector cell proliferation at 1–5 months. These results demonstrate that knockdown of the LFPRLR achieves intra-tumor immunotherapeutic effects and suggest this occurs with reduced likelihood of peripheral inflammatory/autoimmune sequelae.

Expression of long-form prolactin receptor is associated with lower disease-free and overall survival in node-negative breast cancer patients

Breast cancer is the most frequent female malignancy in Thailand. Prolactin (PRL) and prolactin receptor (PRLR) play an important role in normal breast development and carcinogenesis of breast cancer. There are two major isoforms of PRLR, consisting of long-form (LF-PRLR) and short-form (SF-PRLR) that stimulate different signaling pathways. This study aims to explore the associations between all PRLR isoforms (all-PRLR) and LF-PRLR with clinicopathological parameters in breast cancer patients. A total of 340 patients were recruited from January 2009 to December 2015. Expressions of PRLR in breast cancer tissue were determined by immunohistochemistry using specific antibodies that recognize different domains of PRLR (B6.2 for all-PRLR and H-300 for LF-PRLR). The associations between all-PRLR and LF-PRLR expressions with clinicopathological parameters were evaluated. Expression of all-PRLR was observed in 86.2% of all patients while LF-PRLR expression was observed in 54.4%. All-PRLR was co-expressed with estrogen receptor (ER) and progesterone receptor (PR). LF-PRLR expression was associated with high grade tumor and human epidermal growth factor receptor-2 (HER2) overexpression (P=0.010 and <0.001, respectively). Subgroup analysis revealed that LF-PRLR expression was the independent predictor for lower disease-free survival (DFS) in node-negative breast cancer patients with high expression of all-PRLR [hazard ratio (HR): 5.224, 95% confidence interval (CI): 1.089-25.064, P=0.039]. The presence of LF-PRLR in the patients with high expression of all-PRLR was associated with adverse outcome. Evaluation of all-PRLR and LF-PRLR might be used as novel prognosticators in node-negative breast cancers.

Prolactin regulation of the HPA axis is not mediated by a direct action upon CRH neurons: evidence from the rat and mouse.

Centrally acting prolactin has been shown to have anti-stress effects by modulating the activity of the hypothalamic-pituitary-adrenal axis. We tested the hypothesis that prolactin directly targets hypothalamic corticotropin-releasing hormone (CRH) neurons. In situ hybridisation confirmed expression of mRNA encoding the long, but not the short, isoform of the prolactin receptor (PRLR) within the paraventricular nucleus (PVN) of the virgin rat; however, only 6% of CRH neurons expressed long-form Prlr mRNA. Examination of the functional response of CRH neurons to intracerebroventricular prolactin (500ng) showed that these neurons did not respond with activation of phosphorylated signal transducer and activator of transcription 5 (pSTAT5), a marker of long-form PRLR activation. However, as only a subset of neurons expressing Crh mRNA could be detected using immunohistochemistry, we utilised a transgenic mouse model to label CRH neurons with a fluorescent reporter (CRH-Cre-tdTomato). In lactating animals, chronically elevated prolactin levels resulted in significantly increased pSTAT5 expression in the PVN. Overall, few tdTomato-labelled CRH neurons were double-labelled, although a small subset of CRH neurons in the caudal PVN were pSTAT5 positive (approximately 10% of tdTomato neurons at this level, compared to 1% in the rostral PVN). These data suggest that most CRH neurons do not respond directly to prolactin. To confirm that prolactin was not activating another signalling pathway, we used a transgenic mouse line to label PRLR-expressing neurons with Cre-dependent green fluorescent protein (GFP) expression (CRH-Cre-Prlr lox/lox ). No GFP-expressing cells were evident in the PVN, indicating that in the mouse, as in the rat, the CRH neurons do not express either PRLR isoform. Together these data showed that the anti-stress effects of prolactin are not the result of prolactin directly regulating CRH neurons.

Prolactin and its receptors in the chronic mild stress rat model of depression

Prolactin (PRL) exhibits many physiological functions with wide effects on the central nervous system including stress responses. Our study aimed to investigate the effect of chronic unpredictable mild stress (CMS) – which is a good animal model of depression – on PRL receptor (PRLR) expression in the rat brain. Rats were exposed to CMS for two weeks and subsequently to CMS in combination with imipramine (IMI) treatment for five consecutive weeks. Behavioral deficit measured in anhedonic animals is a reduced intake of sucrose solution. Two weeks of CMS procedure allowed the selection of animals reactive to stress and displaying anhedonia, and the group which is considered as stress-non-reactive as far as behavioral measures are concerned. In this group the elevated level of PRL in plasma was observed, decrease in dopamine release in the hypothalamus, increase in [125I]PRL binding to PRLR in the choroid plexus, increase of mRNA encoding the long form of PRLR in the arcuate nucleus and the decrease of mRNA encoding its short form, and decrease in the mRNA encoding dopamine D2 receptor. All these alterations indicate these parameters as involved in the phenomenon of stress-resilience. The prolongation of the CMS procedure for additional five weeks shows the form of habituation to the stressful conditions. The most interesting result, however, was the up-regulation of PRLR in the choroid plexus of rats subjected to full CMS procedure combined with treatment with IMI, which may speak in favor of the role of this receptor in the mechanisms of antidepressant action.

Abstract 2444: Anticancer effects of antimaia in a highly metastatic, orthotopic, immunologically competent model of breast cancer.

Abstract Traditional testing of candidate therapeutics has examined the ability of drugs to shrink pre-formed, primary xenografts in immunocompromised animals. While shrinkage of primary tumors can be crucial for inoperable tumors, the vast majority of primary tumors are surgically excised. Moreover, patient death results from cells that escaped the primary tumor. In new environments, these metastatic escapees may respond entirely differently to the drug, especially in the presence of an intact immune system. To test a novel anti-breast cancer drug, we have therefore used a highly metastatic, syngeneic model (4T1) that develops in an immunologically and endocrinologically normal animal. The drug, for brevity named Antimaia, is ∼10kDa, readily synthesized, and designed to inhibit expression of the long form (LF) of the prolactin receptor (PRLR). Studies in vivo demonstrated inhibited expression of LF PRLR without any effect on expression of the short forms of the PRLR or the growth hormone receptor in normal liver and ovary and the primary tumor. Treatment of 4T1 cells in vitro inhibited the ability of cells within the population to form mammospheres (100/well in controls and 0/well in Antimaia-treated, when defining a mammosphere as a colony &amp;gt;50μm) indicative of an effect on stem-like cells within the population. This anti-stem cell effect was duplicated when stem cells were identified on the basis of cell markers (Lin-CD29hiCD24+ AldH+), but stem cell elimination was still ongoing at 15 days. With the 4T1 model, orthotopic injection of 1x106 cells produces multiple, grossly-visible metastases in the lung and liver by 14 days. Antimaia treatment (100 pmoles/h/mouse via Alzet minipump) at the time of orthotopic injection reduced both gross and microscopic metastases without effect on primary tumor weight. However, histological examination of the primary tumors nevertheless showed only a rim of viable cells in the Antimaia-treated group. Morphometric analysis quantified the area of central tumor death as 3 fold that seen in the two control groups. Since in vitro stem cell death was still ongoing at 15 days, the next goal was to examine the effect of longer term treatment in vivo. To do this, it was necessary to initiate primary tumors with fewer cells to be compliant with standards of animal care. When 1x 103 cells were orthotopically injected and treatment continued for 29 days, 4 of 6 primary tumors in the Antimaia group (and 0 of 6 in the control group) showed a flattened growth curve after initial tumor formation. Micrometastasis histological analyses are ongoing, but ex vivo analysis of immune cells from the livers has shown an increase in tumor-specific IFNγ+ CD4+ cells that correlates with effects of treatment on final tumor weight. Analysis of liver enzymes at 14 and 29 days showed no evidence of toxicity. We conclude that Antimaia holds great promise as a breast cancer therapeutic. CBCRP 171B-0053. Citation Format: Tomohiro Yonezawa, KaunHui E. Chen, David Duong, Lisa Ma, Mrinal Ghosh, Mitsumori Kawaminami, Ameae M. Walker. Anticancer effects of antimaia in a highly metastatic, orthotopic, immunologically competent model of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2444. doi:10.1158/1538-7445.AM2013-2444

Kisspeptin Cells in the Ovine Arcuate Nucleus Express Prolactin Receptor but not Melatonin Receptor

Melatonin is secreted at night by the pineal gland and governs the reproductive system in seasonal breeders, such as sheep. The mechanism by which melatonin regulates reproduction is not known. The circannual rhythmicity of other factors, including prolactin, is also regulated by photoperiod via changes in melatonin secretion. In sheep, plasma prolactin levels are higher in the nonbreeding season than the breeding season. Kisspeptin, synthesised by neurones in the ovine arcuate nucleus (ARC) and preoptic area, is a key regulator of reproduction through stimulation of gonadotrophin-releasing hormone secretion and its expression in the ARC is reduced during the nonbreeding season. We hypothesised that kisspeptin expression is directly, or indirectly, regulated by melatonin and/or prolactin. We first examined the expression of melatonin receptor (MTNR1A) in kisspeptin (Kiss1 mRNA) neurones in the ARC of ovariectomised (OVX) sheep using double-label in situ hybridisation. MTNR1A mRNA was not expressed by kisspeptin neurones, whereas strong expression was detected in the pars tuberalis. We then examined the expression of the long-form prolactin receptor (PRLR-L) in ARC kisspeptin neurones. In OVX ewes, approximately 60% of kisspeptin neurones expressed PRLR-L mRNA at similar levels in the breeding and nonbreeding seasons. We then aimed to determine whether prolactin treatment during the breeding season regulates kisspeptin expression in the ARC. Continuous central infusion of prolactin (20 μg/h for 7 days) in oestradiol-treated OVX sheep did not alter Kiss1 mRNA expression or luteinising hormone secretion, although it induced substantial phosphorylated signal transducer and activator of transcription 5-immunoreactive nuclei staining in the mediobasal hypothalamus. We conclude that the seasonal change in kisspeptin neurones cannot be regulated directly by melatonin, although it may be a result of changes in prolactin levels. Despite this, kisspeptin expression was unchanged after exogenous prolactin treatment in breeding season ewes.

Differences in Prolactin Receptor (PRLR) in Mouse and Human Fallopian Tubes: Evidence for Multiple Regulatory Mechanisms Controlling PRLR Isoform Expression in Mice1

The anterior pituitary-derived hormone prolactin (PRL) signals through the PRL receptor (PRLR) and is important for female reproductive function in mammals. In contrast to the extensive studies of PRLR expression and regulation in human and mouse ovary and uterus, the mechanisms controlling the regulation of PRLR isoform expression in the fallopian tube are poorly understood. Because dynamic interaction of hormonal signaling in gonadal tissue and the pituitary or in gonadal tissues themselves in mammals suggests endocrine or paracrine regulation of PRLR expression, we questioned whether differential regulation of PRLR isoforms by PRL ovarian-derived estrogen (E(2)) and progesterone (P(4)) exists in the fallopian tube and pituitary of prepubertal female mice. Western blot analysis showed distinct molecular separation of PRLR isoforms in mouse and human fallopian tubes, and cellular localization was found in mouse and human tubal epithelia but not in mouse tubal smooth muscle cells. These data support the concept of an isoform- and cell type-specific expression of PRLR in human and mouse fallopian tubes. Moreover, expression of the long form of PRLR decreased after PRL treatment and increased after blockage of endogenous PRL secretion by bromocriptine (an inhibitor of PRL secretion) in a time-dependent manner in mouse fallopian tube. The opposite regulation was observed in the pituitary. Treatment with exogenous E(2) or P(4) led to changes in PRLR expression in the fallopian tube similar to those of PRL treatment. However, E(2) and P(4) did not affect PRLR expression in the pituitary. Estrogen had no effect on the long form of PRLR expression, whereas P(4) regulated the long form of PRLR in the fallopian tube, as did PRL. Taken together, the data from our comparative study provide evidence that PRLR can be regulated by an interplay of two different mechanisms, PRL or ovarian steroid hormones independently or in combination in a tissue-specific manner. Furthermore, we found that ovarian steroid hormones selectively suppress the expression of PRLR isoforms in mouse fallopian tubes. These findings may contribute to our understanding of the mechanisms controlling PRLR isoform expression in the fallopian tube (in addition to ovary and uterus), with implications for female reproduction.

Prolactin stimulates ubiquitination, initial internalization, and degradation of its receptor via catalytic activation of Janus kinase 2

Prolactin (PRL) activates its receptor to initiate signal transduction pathways (including activation of Janus kinases, Jak) but also stimulates downregulation of this receptor to limit the magnitude and duration of signaling. Degradation of the long form of PRL receptor (PRLr) depends on its phosphorylation on Ser349 that is required to facilitate PRLr ubiquitination. Signaling events that mediate PRL-induced degradation of PRLr remain to be elucidated. Here, we investigated the role of Jak2 activity in ligand-triggered increase of PRLr phosphorylation on Ser349, PRLr ubiquitination, endocytosis, and degradation. Using Jak2 reconstitution in Jak2-null cells as well as pharmacologic approaches, we found that treatment with PRL (but not with PRLr antagonist) promotes phosphorylation of PRLr on Ser349 and accelerates endocytosis of PRLr. Furthermore, PRL-stimulated PRLr phosphorylation, endocytosis, and degradation in Jak2-null cells reconstituted with wild type but not with catalytically inactive Jak2. We discuss how Jak2-mediated signaling might be transduced into Ser349 phosphorylation of PRLr as well as its ubiquitination and endocytosis.

Mammary Response to Exogenous Prolactin or Frequent Milking During Early Lactation in Dairy Cows

Frequent milking of dairy cows during early lactation results in a persistent increase in milk yield; however, the mechanism underlying this effect is unknown. We hypothesized that increased exposure of the mammary gland to prolactin (PRL) mediates the milk yield response. Fifteen multiparous Holstein cows were assigned to 3 treatments for the first 3 wk of lactation: twice daily milking with (2× + PRL) or without (2×) supplemental exogenous PRL, or 4 times daily milking (4×). Mammary biopsies were obtained at 7 DIM, and rates of [3H]thymidine incorporation into DNA in vitro were determined. Mammary expression of suppressors of cytokine signaling (SOCS)-1, -2, and -3; the long form of PRL-receptor; and α-lactalbumin mRNA was measured by real-time reverse-transcription PCR. Incorporation of [3H]thymidine into DNA was not affected by frequent milking or PRL treatment; however, analysis of autoradiograms revealed that stromal cell proliferation was greater in 4× cows. Mammary expression of SOCS-1 was not affected by milking frequency or PRL treatment. Expression of SOCS-2 mRNA was increased with frequent milking or PRL treatment, whereas expression of SOCS-3 mRNA was reduced by frequent milking or exogenous PRL. Abundance of PRL-receptor mRNA was reduced, whereas α-lactalbumin mRNA was increased with PRL treatment. These results demonstrate that the bovine mammary gland is responsive to exogenous PRL during early lactation. In addition, differences in the response to frequent milking or exogenous PRL during early lactation indicate distinct effects of PRL and milk removal on the mammary function of dairy cows.

Reproductive Experience Increases Prolactin Responsiveness in the Medial Preoptic Area and Arcuate Nucleus of Female Rats

The experience of pregnancy plus lactation produces long-term enhancements in maternal behavior as well as reduced secretion of prolactin, a key hormone for the initial establishment of maternal care. Given that prolactin acts centrally to induce maternal care as well as regulate its own secretion, we tested whether prolactin receptors in brain regions known to regulate behavioral and neuroendocrine processes were up-regulated and more responsive to prolactin in reproductively experienced females. Diestrous primiparous (8 wk after weaning) and age-matched virgin rats were treated with 250 microg ovine prolactin sc or vehicle and the brains collected 2 h later for measurement of mRNA for genes involved in prolactin signaling. Reproductively experienced rats had lower serum prolactin concentrations, compared with virgin rats, suggesting enhanced prolactin feedback on the arcuate neurons regulating prolactin secretion. In the medial preoptic area and arcuate nucleus (regions involved in regulating maternal behavior and prolactin secretion, respectively), the level of long-form prolactin receptor mRNA was higher in primiparous rats, and prolactin treatment induced a further increase in receptor expression in these animals. In the same regions, suppressors of cytokine signaling-1 and -3 mRNA levels were also markedly increased after prolactin treatment in reproductively experienced but not virgin rats. These results support the idea that reproductive experience increases central prolactin responsiveness. The induction of prolactin receptors and enhanced prolactin responsiveness as a result of pregnancy and lactation may help account for the retention of maternal behavior and shifts in prolactin secretion in reproductively experienced females.

Expression of the long form of the prolactin receptor in magnocellular oxytocin neurons is associated with specific prolactin regulation of oxytocin neurons

Magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) show considerable plasticity during pregnancy and lactation. Prolactin receptors (PRL-R) have been identified in both these nuclei. The aim of this study was to investigate the cell type(s) expressing mRNA for the long form of prolactin receptor (PRL-R(L)) and to determine whether patterns of expression change during pregnancy and lactation. In addition, we examined effects of prolactin on excitability of oxytocin and vasopressin neurons. Sections from brains of nonpregnant, pregnant, and lactating rats were hybridized with an 35S-labeled probe to label PRL-R(L) mRNA together with digoxigenin-labeled probes to detect either oxytocin or vasopressin mRNA. In the SON, PRL-R(L) mRNA was predominantly colocalized with oxytocin mRNA, with over 80% of oxytocin neurons positive for PRL-R(L) mRNA. Very few (<10%) vasopressin neurons expressed PRL-R(L) mRNA. In the PVN, PRL-R(L) mRNA was also predominantly found in oxytocin neurons, and the proportion of PRL-R(L)-positive oxytocin neurons increased significantly during pregnancy and lactation. As in the SON, relatively few vasopressin cells contained PRL-R(L) mRNA. For in vivo electrophysiology, nonpregnant rats were anesthetized, and then extracellular single neuron activity was recorded in identified oxytocin and vasopressin neurons. After a period of baseline recording, the effect of prolactin (1 microg i.c.v.) on firing rate was examined. Prolactin treatment of nonpregnant rats induced a significant decrease in firing rates of oxytocin neurons. There was no effect of prolactin on the activity of vasopressin neurons. Together, these data provide strong evidence that prolactin directly and specifically regulates activity of oxytocin neurons.

Expression of mRNA for Prolactin Receptor (Long Form) in Dopamine and Pro‐Opiomelanocortin Neurones in the Arcuate Nucleus of Non‐Pregnant and Lactating Rats

Under most conditions, prolactin secretion from the pituitary gland is subject to negative-feedback regulation. Prolactin stimulates dopamine release from tuberoinfundibular (TIDA) neurones in the arcuate nucleus of the hypothalamus, which in turn suppresses the production of prolactin. However, during late pregnancy and continuing into lactation, this feedback mechanism becomes less responsive to prolactin and, as a result, a hyperprolactinaemic state develops. We investigated whether long-form prolactin receptor (PRL-R(L)) mRNA is present on TIDA neurones in nonpregnant and lactating rats. In addition, we examined whether PRL-R(L) mRNA is colocalized on hypothalamic pro-opiomelanocortin (POMC) neurones. Dual-label in situ hybridizations using an (35)S-labelled cRNA probe specific for long-form PRL-R, together with a digoxigenin-labelled RNA probe that encoded either tyrosine hydroxylase (TH) or POMC mRNA, were performed on brain sections. In both nonpregnant and lactating rats, the majority of TH mRNA-positive cells (> 90%) were found to express long-form PRL-R mRNA. In sections from nonpregnant rats, few non-TH positive cells expressed PRL-R(L) mRNA. By contrast, during lactation, the proportion of PRL-R(L) mRNA-positive cells that were not TH mRNA-positive increased to approximately 70%. Only a small number of neurones in this subpopulation of PRL-R(L) mRNA-positive neurones were found to be positive for POMC mRNA. These data show that the loss of responsiveness to prolactin occurring during lactation is not due to down regulation of long-form PRL-R gene expression on TIDA neurones. Moreover, the persistent expression of PRL-R(L) in arcuate neuroendocrine circuits suggests that PRL-R-mediated signalling continues to be important in these neurones during lactation.

Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation.

PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation.

Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation.

Prolactin receptor (PRL-R) expression in the brain is increased in lactating rats compared with non-pregnant animals. The aim of the present study was to determine the time-course of changes in PRL-R mRNA levels during pregnancy and/or lactation, and to determine relative levels of the two forms (short and/or long form) of receptor mRNA in specific brain regions. Brains were collected from female rats on dioestrus, days 7, 14 or 21 of pregnancy, day 7 of lactation or day 7 post-weaning. Frozen, coronal sections were cut (300 microm) and specific hypothalamic nuclei and the choroid plexus were microdissected using a punch technique. Total RNA was extracted and reverse transcribed, then first strand cDNA was amplified using quantitative real-time PCR. Results showed an up-regulation of long-form PRL-R mRNA in the choroid plexus by day 7 of pregnancy compared with dioestrus, which further increased on days 14 and 21 of pregnancy and day 7 of lactation, and then decreased to dioestrous levels on day 7 post-weaning. Short-form PRL-R mRNA levels increased on day 14 of pregnancy relative to dioestrus, increased further on day 7 of lactation and decreased on day 7 post-weaning. Changes in mRNA were reflected in increased levels of PRL-R immunoreactivity in the choroid plexus during pregnancy and lactation, compared with dioestrus. In the arcuate nucleus, long-form PRL-R mRNA was increased during pregnancy. In contrast to earlier work, no significant changes in short- or long-form PRL-R mRNA expression were detected in several other hypothalamic nuclei, suggesting that changes in hypothalamic mRNA levels may not be as marked as previously thought. The up-regulation of PRL-R mRNA and protein expression in the choroid plexus during pregnancy and lactation suggest a possible mechanism whereby increasing levels of peripheral prolactin during pregnancy may have access to the central nervous system. Together with expression of long-form PRL-R mRNA in specific hypothalamic nuclei, these results support a role for prolactin in regulating neuroendocrine and behavioural adaptations in the maternal brain.

Visualization of gene expression of prolactin-receptor (PRL-R) by in situ hybridization in reproductive organs of Typhlonectes compressicauda, a gymnophionan amphibian.

The expression of the long form of prolactin receptor (PRL-R) mRNAs was studied in reproductive organs from both male and female Typhlonectes compressicauda, an amphibian, by quantitative in situ hybridization. These PRL-R mRNAs were expressed in all the organs studied. In ovarian tissue, mRNAs coding for the long form of PRL-R were strongly expressed in ovaries with compact and vascularized corpora lutea, i.e., during the middle and the end of pregnancy. During the other periods of cycle, sexual rest, vitellogenesis and beginning of pregnancy, expression of these mRNAs was lower than in other periods. In the testis, mRNAs coding for the long form of PRL-R were expressed in germ cells as well as in Leydig and Sertoli cells. In müllerian glands, important variations of expression in these mRNAs have been observed with a large increase during the breeding period and a decrease during sexual rest. Variations of gene expression of the long form of PRL-R for all tissues studied are correlated to synthesis of PRL in the pituitary in both male and female Typhlonectes compressicauda.

Prolactin and Its Receptor Are Expressed in Murine Hair Follicle Epithelium, Show Hair Cycle-Dependent Expression, and Induce Catagen

Here, we provide the first study of prolactin (PRL) and prolactin receptor (PRLR) expression during the nonseasonal murine hair cycle, which is, in contrast to sheep, comparable with the human scalp and report that both PRL and PRLR are stringently restricted to the hair follicle epithelium and are strongly hair cycle-dependent. In addition we show that PRL exerts functional effects on anagen hair follicles in murine skin organ culture by down-regulation of proliferation in follicular keratinocytes. In telogen follicles, PRL-like immunoreactivity was detected in outer root sheath (ORS) keratinocytes. During early anagen (III to IV), the developing inner root sheath (IRS) and the surrounding ORS were positive for PRL. In later anagen stages, PRL could be detected in the proximal IRS and the inner layer of the ORS. The regressing (catagen) follicle showed a strong expression of PRL in the proximal ORS. In early anagen, PRLR immunoreactivity occurred in the distal part of the ORS around the developing IRS, and subsequently to a restricted area of the more distal ORS during later anagen stages and during early catagen. The dermal papilla (DP) stayed negative for both PRL and PRLR throughout the cycle. Telogen follicles showed only a very weak PRLR staining of ORS keratinocytes. The long-form PRLR transcript was shown by real-time polymerase chain reaction to be transiently down-regulated during early anagen, whereas PRL transcripts were up-regulated during mid anagen. Addition of PRL (400 ng/ml) to anagen hair follicles in murine skin organ culture for 72 hours induced premature catagen development in vitro along with a decline in the number of proliferating hair bulb keratinocytes. These data support the intriguing concept that PRL is generated locally in the hair follicle epithelium and acts directly in an autocrine or paracrine manner to modulate the hair cycle.

Influence of cortisol on adipose tissue development in the fetal sheep during late gestation.

The present study examined the extent to which the late gestation rise in fetal plasma cortisol influenced adipose tIssue development in the fetus. The effect of cortisol on the abundance of adipose tIssue mitochondrial proteins on both the inner (i.e. uncoupling protein (UCP)1) and outer (i.e. voltage-dependent anion channel (VDAC)) mitochondrial membrane, together with the long and short forms of the prolactin receptor (PRLR) protein and leptin mRNA was determined. Perirenal adipose tIssue was sampled from ovine fetuses to which (i) cortisol (2-3 mg/day for 5 days) or saline was infused up to 127-130 days of gestation, and (ii) adrenalectomised and intact controls at between 142 and 145 days of gestation (term=148 days). UCP1 protein abundance was significantly lower in adrenalectomised fetuses compared with age-matched controls, and UCP1 was increased by cortisol infusion and with gestational age. Adrenalectomy reduced the concentration of the long form of PRLR, although this effect was only significant for the highest molecular weight isoform. In contrast, neither the short form of PRLR, VDAC protein abundance or leptin mRNA expression was significantly affected by gestational age or cortisol status. Fetal plasma triiodothyronine concentrations were increased by cortisol and with gestational age, an affect abolished by adrenalectomy. When all treatment groups were combined, both plasma cortisol and triiodothyronine concentrations were positively correlated with UCP1 protein abundance. In conclusion, an intact adrenal is necessary for the late gestation rise in UCP1 protein abundance but cortisol does not appear to have a major stimulatory role in promoting leptin expression in fetal adipose tIssue. It remains to be established whether effects on UCP1 protein are directly regulated by cortisol alone or mediated by other anabolic fetal hormones such as triiodothyronine.

Sex difference and estrous cycle: expression of prolactin receptor mRNA in rat brain

The short and long forms of prolactin receptor (PRL-R) mRNA have been detected in the female rat brain. The present study aimed to investigate: (1) if the PRL-R mRNA is expressed in the male rat brain; (2) if expression levels in the female brain vary during the estrous cycle. All animals were sacrificed between 12:00 and 14:00 h. Radioactive RNase protection assay was used to measure mRNA levels. The results showed that both forms of PRL-R mRNA were expressed to varying degrees in the choroid plexus (ChP), preoptic area (POA), mediobasal hypothalamus (MBH), cerebral cortex (CTX) and pons-medulla PON) in both male and female rats. The average amount of both forms of PRL-R mRNA in the ChP, POA, MBH of cycling females was significantly higher than in the male rat. Among cycling female rats, the expression levels of both forms of PRL-R mRNA in the ChP, POA and MBH during proestrous were significantly greater than during diestrous or estrous. In proestrous females, the ChP expressed the highest levels of mRNA whereas the CTX contained the lowest. The ratios of short:long form mRNA were not significantly changed according to sex, estrous stage or brain regions although a slightly higher amount of the short form was observed. The detection of PRL-R mRNA in the male rat implicates that PRL may be involved in regulation of brain function in the male subject. The higher levels of PRL-R mRNA in female rats on proestrous suggest that PRL-R may be regulated by PRL or steroid hormones that show a surge on this day.

Fetal endocrinology and development--manipulation and adaptation to long-term nutritional and environmental challenges.

This article reviews the fetal endocrine system in sheep, a species that has a long gestation and primarily produces a singleton fetus. Attention is focused on information that is applicable to humans. The endocrinology of metabolic homeostasis in sheep fetuses is well adapted to respond to a range of metabolic challenges, including placental restriction and maternal undernutrition. A small placenta results in hypoxaemia, hypoglycaemia, reduced abundance of anabolic hormones, and fetal growth restriction. Fetuses with restricted growth are characterized by tissue-specific reductions in hormone receptor mRNA, for example mRNA for the long form of prolactin receptor is reduced in adipose tissue. In contrast, the adipose tissue of fetuses with accelerated growth, stimulated by increasing maternal nutrition in the second half of gestation, has more protein for the long form of the prolactin receptor and more uncoupling protein 1, by which large amounts of heat are generated at birth. Maternal undernutrition in early gestation, coinciding with the period of rapid placental growth, initially restricts placental growth, but when mothers are fed to requirements, a longer fetus results with a disproportionately large placenta. This nutritional manipulation replicates, in part, epidemiological findings from the Dutch famine of 1944-1945, for which the offspring are at increased risk of adult obesity.

Role of protein kinases in the prolactin-induced intracellular calcium rise in Chinese hamster ovary cells expressing the prolactin receptor.

There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx.