Prolactin Receptor mRNA Research Articles

Effects of metoclopramide-induced hyperprolactinemia on the prolactin receptor of murine endometrium

To evaluate the effects of metoclopramide-induced hyperprolactinemia on the prolactin receptor of murine endometrium. Experimental study using the RNA extraction to detect tissue prolactin receptor isoforms by reverse-transcriptase polymerase chain reaction (RT-PCR). University-based laboratory. Seventy-two female swiss albino mice (Mus musculus), approximately 100 days old, were divided into six 12-animal groups: (GI) nonoophorectomized mice given vehicle; (GII) nonoophorectomized mice treated with metoclopramide; (GIII) oophorectomized mice treated with metoclopramide; (GIV) oophorectomized mice treated with metoclopramide and 17beta-estradiol; (GV) oophorectomized mice treated with metoclopramide and micronized progesterone; (GVI) oophorectomized mice treated with metoclopramide and a solution of 17beta-estradiol and micronized progesterone. Drugs were administered for 50 days. Following euthanasia, the middle portions of the uterine horns were removed, sectioned, and immediately frozen for RT-PCR procedures. Blood was collected for the dosage of prolactin and serum estrogen and progesterone using radioimmune assay. Identification of uterine prolactin receptor isoforms. The PRL receptor and its isoform L were identified only in GI (control group) and GII (metoclopramide), the two groups with nonoophorectomized animals. The amount of PRL receptor mRNA and that of its isoform L from GII were the largest. No other isoforms of the prolactin receptor were identified in any of the groups. Our results suggest that replacement of estrogen and progestin may not increase the mRNA of endometrial PRL receptor in metoclopromide-induced hyperprolactinemia in rats after castration.

Prolactin receptor mRNA is upregulated in discus fish (Symphysodon aequifasciata) skin during parental phase

Prolactin (PRL) has been shown to directly influence parental-care associated behavior in many vertebrate species. The discus fish (Symphysodon aequifasciata) displays extensive parental care behavior through utilization of epidermal mucosal secretion to raise free-swimming fry. Here, we cloned the full-length cDNA sequence of the S. aequifasciata prolactin receptor (dfPRLR) and investigated the mRNA expression pattern in several adult tissues. Bioinformatic analysis showed the dfPRLR shared rather high identity (79 and 67%) with the Nile tilapia PRLR 1 and black seabream PRLR 1, respectively. The presence of dfPRLR in several osmoregulatory tissues including kidney, gill and intestine is consistent with the known role of PRL in mediating hydromineral balance in teleosts. In addition, upregulated expression of PRLR mRNA was observed in skin of parental fish compared to non-parental fish, indicating possibility of a role of the PRL hormonal signaling in regulation of mucus production in relation to parental care behaviour.

Differences in adrenocortical secretory and gene expression responses to stimulation in vitro by ACTH or prolactin between high- and low-avoidance Hatano rats

Rats of the Hatano high-avoidance (HAA) and low-avoidance (LAA) strains have been genetically selected on the basis of their two-way active avoidance behavior, and have different endocrine responses to stress. The present study focused on the adrenal steroid hormone responses of the Hatano strains and identifies differences in regulation of the adrenal cortex in vitro of HAA and LAA rats. Although incubation with prolactin (PRL) and/or adrenocorticotrophic hormone (ACTH) resulted in a dose-dependent increase of corticosterone and progesterone release by adrenal cells from both HAA and LAA male rats, the responses were markedly increased for adrenal cells from LAA rats as compared with HAA rats. This finding suggested that adrenal glands of HAA rats are less sensitive to PRL and/or ACTH than adrenals from LAA rats. Several possible intra-adrenal regulators were investigated. The basal level of expression of steroidogenic acute regulatory protein (StAR) and the long form of the PRL receptor (PRLR-L) mRNAs was higher in adrenals of LAA rats. ACTH treatment of adrenal cells from HAA rats resulted in statistically significant increases in melanocortin receptor 2 (MC2R) mRNA expression, while neither ACTH nor PRL altered MC2R mRNA expression in adrenal cells of LAA rats. Conversely, the increase in PRLR-L mRNA expression induced by PRL was observed only in adrenal cells from LAA rats. Treatment of adrenal cells with PRL and/or ACTH increased the expression of StAR and CYP11A1 mRNAs for both Hatano strains. However, the induction of StAR mRNA expression was higher in LAA rats, but the CYP11A1 response was lower. These findings indicate that adrenal cells of the LAA strain have higher sensitivity to secretagogues than those of the HAA strain. These results suggest that PRL may also be important in stimulating secretion of adrenal steroid hormones.

249 THE DIRECT AND CUMULUS-MEDIATED EFFECTS OF PROLACTIN ON BOVINE OOCYTES

Prolactin (PRL) is an important regulator of female reproduction, as indicated using PRL receptor knockout mice (Bole-Feysot C et al. 1998 Endocrinol. Rev. 19, 225–268). The presence of PRL receptors or their mRNA has been shown in different follicular compartments of various mammalian species, including oocytes of sheep and mice (Picazo RA et al. 2004 Reproduction 128, 545–553; Kiapekou E et al. 2005 Reprod. Biomed. Online 10, 339–346). The aim of the present study was to characterize the direct and cumulus-mediated pathways of PRL signaling into bovine oocytes. The expression of PRL receptor mRNA in follicular cells was detected by reverse transcription polymerase chain reaction (RT-PCR) analysis. For oocytes, nested PCR was used. In addition, a total of 707 COC and 689 denuded oocytes (DO) from follicles 2 to 8 mm in diameter were cultured for 6 h (COC), 8 h (DO), or 24 h (COC and DO) in the presence or absence of 50 ng mL–1 bovine PRL (20 IU mg–1; Research Center for Endocrinology, Moscow, Russia). The following systems for COC and DO culture were used: (1) TCM-199 containing 10% fetal calf serum (system 1) and (2) DMEM containing 10 U mL–1 pregnant mare’s serum gonadotropin (PMSG), 5 U mL–1 hCG (Intervet Scandinavia, Copenhagen, Denmark), and 5% estrous cow serum (system 2). The nuclear status of oocytes was evaluated by Tarkowski’s cytogenetic method. After IVM in system 2, a portion of the oocytes (348 COC and 311 DO) underwent IVF and IVC, and the embryo development was tracked until the blastocyst stage. All treatments were repeated 4 to 7 times. Results were expressed as mean ± SEM. Arcsine-transformed data were analyzed by two-way ANOVA. Messenger RNA expression of long and short isoforms of PRL receptor was revealed in both bovine oocytes and cumulus cells. In system 1, PRL raised the rate of DO remaining at the diplotene stage by 8 h of culture (from 32.6 ± 3.0 to 47.5 ± 4.7%, P < 0.05), whereas there was no effect of PRL on the meiotic resumption in COC. By contrast, the hormone exerted a stimulatory action on the meiotic progression in the presence of cumulus cells by increasing the proportion of oocytes reaching the telophase I or metaphase II stages during 24 h of maturation (from 61.9 ± 1.2 to 73.5 ± 0.9%, P < 0.05). In system 2, PRL did not affect nuclear maturation of either cumulus-enclosed or cumulus-free oocytes, with the maturation rate varying between 93.9 ± 2.7 and 95.2 ± 2.4% (COC) and between 85.6 ± 3.0 and 83.0 ± 4.0% (DO). When added to IVM system 2, PRL raised the cleavage and blastocyst rates only in the case of COC (from 72.2 ± 2.4 to 80.2 ± 2.1%, P < 0.05, and from 20.5 ± 3.6 to 40.9 ± 4.2%, P < 0.01, respectively). These findings suggest functioning of the direct and cumulus-mediated pathways of PRL signaling into bovine oocytes, with the hormone affecting meiosis via both pathways. We thank A. D. Roed for expert technical assistance with IVF and IVC. This research was supported in part by RFBR (project no. 07-04-01485). I.L. was the recipient of an Erasmus Mundus fellowship.

Physiological responses and expression of arginine vasotocin receptor, prolactin and prolactin receptor mRNA in olive flounder Paralichthys olivaceus during osmotic stress

We cloned complementary DNA (cDNA) encoding the arginine vasotocin receptor (AVT-R) from the kidney of olive flounder (Paralichthys olivaceus). Olive flounder AVT-R cDNA consists of 1155 bp, and encodes a protein of 384 amino acids. To investigate the stress responses and osmoregulatory abilities of olive flounder in changing salinities (35, 17.5, 8.75, 4, and 0 psu), we examined the expression of AVT-R, prolactin (PRL), and PRL receptor (PRL-R) messenger RNA (mRNA) in osmoregulatory organs using quantitative real time PCR (QPCR). AVT-R and PRL-R were expressed in the gill, kidney, and intestine, whereas PRL mRNA was expressed only in the pituitary gland. In addition, we measured the levels of plasma glucose, cortisol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). The mRNA expression and plasma parameters increased in hypoosmotic environments. Furthermore, osmolality decreased significantly according to salinity concentration decreased. These results suggest that AVT-R, PRL, and PRL-R mRNA play important roles in the hormonal regulation of osmoregulatory organs, thereby improving the osmoregulatory ability of olive flounder in hypoosmotic environments.

Short-Day Photoperiod Increases Milk Yield in Cows with a Reduced Dry Period Length

Exposure of cows to a short-day photoperiod (SDPP; 8h light:16h dark) during a 60-d dry period increases milk yield in the subsequent lactation compared with cows exposed to a long-day photoperiod (LDPP; 16h light:8h dark). Whereas the traditional recommendation for dry period length is 60 d, recent studies indicate that the dry period length can be reduced without depressing the yield in the next lactation. However, the optimal duration of the dry period appears to be between 40 and 60 d, because fewer than 30 d could result in a significant loss of milk production. Our main objective was to determine whether treatment with SDPP combined with a reduced dry period length of 42 d would increase milk yield in the next lactation relative to treatment with LDPP, even though SDPP exposure was limited to 42 d. Multiparous Holstein cows (n=40) were randomly assigned to 1 of 2 treatments during the dry period: LDPP or SDPP. Each treatment group (n=20) was balanced according to the previous 305-d mature equivalent milk yield. To quantify plasma prolactin (PRL) concentration, blood samples were collected weekly during the dry period. Dry matter intake (DMI) was recorded during the dry period. Health was monitored weekly during the dry period and at calving. During lactation, milk yield and DMI were recorded for 120 and 42 d, respectively. Cows exposed to SDPP calved 4.8 d earlier than cows exposed to LDPP and days dry averaged 37 and 42 d for cows exposed to SDPP and LDPP, respectively. Cows on SDPP consumed more dry matter (17.0±1.1kg/d) during the dry period than did cows on LDPP (15.9±1.1kg/d), but DMI after parturition did not differ. In the first 42 d of lactation, cows exposed to SDPP and LDPP consumed 18.0 and 17.7±1.4kg/d, respectively. The periparturient PRL surge was greater in cows exposed to LDPP (22.6±3.2ng/mL) than in those exposed to SDPP (17.1±4.1ng/mL). Milk yield was inversely related to the magnitude of the periparturient PRL surge, but was directly related to the expression of PRL-receptor mRNA in lymphocytes during the dry period. Through 120 d of lactation, cows exposed to SDPP when dry produced more milk (40.4±1.1kg/d) than cows exposed to LDPP (36.8±1.1kg/d). These results support the concept that SDPP, combined with a targeted 42-d dry period, increases milk yield in the subsequent lactation, relative to a 42-d dry period combined with LDPP, and that exposure to 42 d of SDPP in the dry period is sufficient to increase milk yield in the next lactation.

Circulating prolactin, MPOA prolactin receptor expression and maternal aggression in lactating rats

Maternal aggression is most intense in lactating rats from the 3rd to the 12th day postpartum. The purpose of this study was to determine if plasma prolactin (PRL) and prolactin receptor (PRL-R L) mRNA expression in the medial preoptic area (MPOA) of lactating rats are altered in association with maternal aggression. Lactating Sprague Dawley rats were divided into five groups and exposed for 10 min to an intruder male or to an object on postpartum day 8. Trunk blood and the brain of the dams were collected 30 or 240 min after exposure and from a non-exposed group. Lower levels of prolactin were found 30 min after the aggression test. No change was detected in the number of cells expressing PRL-R L mRNA by in situ hybridization histochemistry (ISHH) as a function of testing. However, the correlation between plasma PRL and PRL-R L mRNA expression in the mothers changed from positive in control females to negative in intruder exposed animals. These data support the concept that a maternal aggressive experience, while acutely altering PRL secretion, fails to affect PRL-R L mRNA expression.

Cortisol and prolactin modulation of caudal neurosecretory system activity in the euryhaline flounder Platichthys flesus

Previous studies have shown roles for cortisol and prolactin in osmoregulatory adaptation to seawater and freshwater, respectively, in euryhaline fish. This study of the European flounder investigated the potential for these hormones to modulate activity of the caudal neurosecretory system (CNSS), which is thought to be involved in physiological adaptation to changing external salinity. Superfusion of isolated CNSS with either cortisol or prolactin (10 μM; 15 min) led to changes in firing activity in neuroendocrine Dahlgren cells, recorded extracellularly. Cortisol evoked a modest increase in overall firing activity, with the response delayed by 4 h after treatment. The response to prolactin was short latency, continued to build up over the subsequent 4-h wash period, and comprised increased firing activity together with recruitment of previously silent Dahlgren cells. Immunoreactivity for glucocorticoid and prolactin receptors was localised to Dahlgren cells. The CNSS expression level for glucocorticoid-2 receptor mRNA, measured by Q-PCR, was significantly lower in fish fully acclimated to freshwater, compared to seawater. No differences were seen between these two states for prolactin receptor mRNA expression. These results provide evidence for a modulatory action of both hormones on the neurosecretory function of the CNSS.

Direct effects of prolactin and dopamine on the gonadotroph response to GnRH

The intrapituitary mechanisms underlying the inhibitory actions of hyperprolactinaemia on the reproductive axis remain unclear. Previous work on primary pituitary cultures revealed combined suppressive effects of prolactin (PRL) and dopamine on the gonadotrophin response to GnRH. However, whether these effects occur directly at the level of the gonadotroph and are accompanied by changes in gene expression is still unresolved. Here, alphaT(3)-1 and LbetaT2 cells were used to investigate the effects of PRL and dopamine on gonadotrophin synthesis and release in gonadotroph monocultures under basal and GnRH-stimulated conditions. PRL receptor and dopamine receptor mRNA expressions were first determined by RT-PCR in both cell lines. Then, PRL and the dopamine agonist bromocriptine (Br), alone or in combination, were shown to block the maximal alpha-subunit and LHbeta-subunit mRNA responses to a dose-range of GnRH. The LH secretory response was differentially affected by treatments. GnRH dose-dependently stimulated LH release, with a 4-5 fold increase at 10(-8) M GnRH. Unexpectedly, PRL or Br stimulated basal LH release, with PRL, but not Br, enhancing the LH secretory response to GnRH. This effect was, however, completely blocked by Br. These results reveal direct effects of PRL and dopamine at the level of the gonadotroph cell, and interactions between these two hormones in the regulation of gonadotrophin secretion. Moreover, uncoupling between LH synthesis and release in both the basal and the GnRH-stimulated responses to PRL and dopamine was clearly apparent.

Hypothalamic Prolactin Receptor Messenger Ribonucleic Acid Levels, Prolactin Signaling, and Hyperprolactinemic Inhibition of Pulsatile Luteinizing Hormone Secretion Are Dependent on Estradiol

Hyperprolactinemia can reduce fertility and libido. Although central prolactin actions are thought to contribute to this, the mechanisms are poorly understood. We first tested whether chronic hyperprolactinemia inhibited two neuroendocrine parameters necessary for female fertility: pulsatile LH secretion and the estrogen-induced LH surge. Chronic hyperprolactinemia induced by the dopamine antagonist sulpiride caused a 40% reduction LH pulse frequency in ovariectomized rats, but only in the presence of chronic low levels of estradiol. Sulpiride did not affect the magnitude of a steroid-induced LH surge or the percentage of GnRH neurons activated during the surge. Estradiol is known to influence expression of the long form of prolactin receptors (PRL-R) and components of prolactin's signaling pathway. To test the hypothesis that estrogen increases PRL-R expression and sensitivity to prolactin, we next demonstrated that estradiol greatly augments prolactin-induced STAT5 activation. Lastly, we measured PRL-R and suppressor of cytokine signaling (SOCS-1 and -3 and CIS, which reflect the level of prolactin signaling) mRNAs in response to sulpiride and estradiol. Sulpiride induced only SOCS-1 in the medial preoptic area, where GnRH neurons are regulated, but in the arcuate nucleus and choroid plexus, PRL-R, SOCS-3, and CIS mRNA levels were also induced. Estradiol enhanced these effects on SOCS-3 and CIS. Interestingly, estradiol also induced PRL-R, SOCS-3, and CIS mRNA levels independently. These data show that GnRH pulse frequency is inhibited by chronic hyperprolactinemia in a steroid-dependent manner. They also provide evidence for estradiol-dependent and brain region-specific regulation of PRL-R expression and signaling responses by prolactin.

Genital sensory stimulation shifts estradiol intraoviductal signaling from nongenomic to genomic pathways, independently from prolactin surges.

Estradiol (E2) accelerates oviductal egg transport through nongenomic pathways involving oviductal protein phosphorylation in non-mated rats, and through genomic pathways in mated rats. Here we investigated the ability of cervico-vaginal stimulation (CVS) to switch the mode of action of E2 in the absence of other male-associated components. Pro-estrous rats were subjected to CVS with a glass rod and 12 hours later were injected subcutaneously with E2 and intrabursally with the RNA synthesis inhibitor Actinomycin D or the protein phosphorylation inhibitor H-89. The number of eggs in the oviduct, assessed 24 h later, showed that Actinomycin D, but not H-89 blocked the E2-induced egg transport acceleration. This clearly indicates that CVS alone, without other mating-associated signals, is able to shift E2 signaling from nongenomic to genomic pathways. Since mating and CVS activate a neuroendocrine reflex that causes iterative prolactin (PRL) surges, the involvement of PRL pathway in this phenomenon was evaluated. Prolactin receptor mRNA and protein expression in the rat oviduct was demonstrated by RT-PCR and Western blot, but their levels were not different on day 2 of the cycle (C2) or pregnancy (P2). Activated ST AT 5a/b (phosphorylated) was detected by Western blot on P2 in the ovary, but not in the oviduct, showing that mating does not stimulate this PRL signalling pathway in the oviduct. Other rats subjected to CVS in the evening of pro-estrus were treated with bromoergocriptine to suppress PRL surges. In these rats, H-89 did not block the E2-induced acceleration of egg transport suggesting that PRL surges are not essential to shift E2 signaling pathways in the oviduct. We conclude that CVS is one of the components of mating that shifts E2 signaling in the oviduct from nongenomic to genomic pathways, and this effect is independent of PRL surges elicited by mating.

Expression Patterns of the Prolactin Receptor Gene in Chicken Lymphoid Tissues During Embryogenesis and Posthatch Period

Prolactin (PRL) is a pituitary hormone with multiple homeostatic roles among vertebrates. Although it has mainly been studied in relation to its role during the initiation and maintenance of incubation behavior in avian species, it has also been shown to act on the immune system. In this study, levels of PRL receptor (PRLR) mRNA were quantified by real-time PCR, and tissue expression was localized by in situ hybridization in primary and secondary lymphoid organs. Prolactin receptor was shown to be expressed in the bursa follicles, thymus lobules, and splenic pulp at all stages of development examined. Levels of PRLR expression were consistently higher in the bursa of Fabricius when compared with other lymphoid organs, suggesting that PRL acts primarily on bursal development. Furthermore, levels of PRLR mRNA appeared to fluctuate during embryogenesis, with a significant increase observed at embryonic day 19 in the bursa, at 7 d of age in the thymus, and on hatching day in the spleen. Thus, PRL might play an important role during the development of the immune system in chickens.

Corticosterone has direct inhibitory effect on the expression of peptide hormone receptors, 11β-HSD and glucose oxidation in cultured adult rat Leydig cells

The present study was designed to investigate the dose-dependent direct effect of corticosterone on the expression of peptide hormone receptors, 11β-hydroxysteroid dehydrogenase (11β-HSD) and glucose oxidation in cultured adult rat Leydig cells. Leydig cells were isolated from the testis of normal adult male albino rats, purified on discontinuous Percoll gradient and plated in culture plates/flasks overnight at 34 °C in a CO 2 incubator under 95% air and 5% CO 2 using DME/F12 medium containing 1% fetal bovine serum. After the attachment of cells, serum containing medium was removed and cells were exposed to different doses (0, 50, 100, 200, 400 and 800 nM) of corticosterone using serum-free fresh medium for 24 h at 34 °C. At the end of exposure period, cells were utilized for the quantification of cell-surface LH, prolactin, insulin receptors and their mRNA expression, the activity and mRNA expression of 11β-HSD and glucose oxidation. Testosterone production was estimated in cell pellets and in culture media. At all doses employed, corticosterone significantly decreased the production of testosterone by Leydig cells. The concentration of cell-surface LH and prolactin receptors were significantly reduced after corticosterone exposure whereas the concentration of insulin receptor was diminished only at 200–800 nM doses of corticosterone. The levels of LH and prolactin receptor mRNAs were significantly decreased after corticosterone (100–800 nM) exposure whereas the mRNA level of insulin receptor was significantly reduced only at 800 nM dose of corticosterone. 11β-HSD mRNA expression as well as the activity was significantly inhibited by corticosterone treatment. Glucose oxidation was markedly inhibited by corticosterone exposure in a dose-dependent manner. It is concluded from this in vitro study that corticosterone induces steroidogenic lesion in testicular Leydig cells by decreasing the number of cell-surface LH, prolactin and insulin receptors, the activity of 11β-HSD and their mRNA levels and glucose oxidation.

Endocrine systems in juvenile anadromous and landlocked Atlantic salmon ( Salmo salar): Seasonal development and seawater acclimation

The present study compares developmental changes in plasma levels of growth hormone (GH), insulin-like growth factor I (IGF-I) and cortisol, and mRNA levels of their receptors and the prolactin receptor (PRLR) in the gill of anadromous and landlocked Atlantic salmon during the spring parr-smolt transformation (smoltification) period and following four days and one month seawater (SW) acclimation. Plasma GH and gill GH receptor (GHR) mRNA levels increased continuously during the spring smoltification period in the anadromous, but not in landlocked salmon. There were no differences in plasma IGF-I levels between strains, or any increase during smoltification. Gill IGF-I and IGF-I receptor (IGF-IR) mRNA levels increased in anadromous salmon during smoltification, with no changes observed in landlocked fish. Gill PRLR mRNA levels remained stable in both strains during spring. Plasma cortisol levels in anadromous salmon increased 5-fold in May and June, but not in landlocked salmon. Gill glucocorticoid receptor (GR) mRNA levels were elevated in both strains at the time of peak smoltification in anadromous salmon, while mineralocorticoid receptor (MR) mRNA levels remained stable. Only anadromous salmon showed an increase of gill 11β-hydroxysteroid dehydrogenase type-2 (11β-HSD2) mRNA levels in May. GH and gill GHR mRNA levels increased in both strains following four days of SW exposure in mid-May, whereas only the anadromous salmon displayed elevated plasma GH and GHR mRNA after one month in SW. Plasma IGF-I increased after four days in SW in both strains, decreasing in both strains after one month in SW. Gill IGF-I mRNA levels were only increased in landlocked salmon after 4 days in SW. Gill IGF-IR mRNA levels in SW did not differ from FW levels in either strain. Gill PRLR mRNA did not change after four days of SW exposure, and decreased in both strains after one month in SW. Plasma cortisol levels did not change following SW exposure in either strain. Gill GR, 11β-HSD2 and MR mRNA levels increased after four days in SW in both strains, whereas only the anadromous strain maintained elevated gill GR and 11β-HSD2 mRNA levels after one month in SW. The results indicate that hormones and receptors of the GH and cortisol axes are present at significantly lower levels during spring development and SW acclimation in landlocked relative to anadromous salmon. These findings suggest that attenuation of GH and cortisol axes may, at least partially, result in reduced preparatory upregulation of key gill ion-secretory proteins, possibly a result of reduced selection pressure for marine adaptations in landlocked salmon.

Effects of short day photoperiod on prolactin signaling in dry cows: A common mechanism among tissues and environments?1,2

Photoperiodic manipulation has dramatic physiological and production effects in dairy cows. During lactation, exposure to long day photoperiod (LDPP) increases milk yield and circulating IGF-I and prolactin (PRL) concentrations. Conversely, cows housed under a short day photoperiod (SDPP) during the dry period produce more milk in the subsequent lactation than cows exposed to LDPP or natural photoperiod. Exposure to SDPP depresses PRL secretion but increases PRL receptor mRNA levels in mammary, immune, and hepatic tissues. In dry cows under SDPP, PRL signaling is a potential mechanism to drive more extensive mammary cell differentiation and growth relative to LDPP. In mammary biopsies taken during the dry period and into lactation, the amount of IGF-II mRNA was greater in SDPP vs. LDPP cows during the dry period, whereas IGFBP-5 mRNA increased in both groups during lactation even though photoperiodic treatments ended at parturition and all cows were on an ambient lighting schedule when lactating. Levels of IGF-I mRNA did not differ over time or between treatments; however, during the dry period, lower IGFBP-5 and increased IGF-II expression in SDPP cows may enhance mammary cell growth and survival. Key among the potential modulators of PRL signaling is the suppressors of cytokine signaling (SOCS) family. Mammary transcription of mRNA for SOCS proteins was low during the dry period but increased in lactation. During the dry period, SOCS mRNA level in the mammary gland of cows on SDPP was reduced compared with cows on LDPP, which may enhance PRL-induced proliferation and subsequent milk production. However, improved mammary capacity and immune function alone are likely insufficient to support increased milk yield. Using improved milk yield as a functional indicator of greater animal well-being during the transition, it is clear that some metabolic accommodation is necessary for expression of that capacity. Emerging evidence supports a link between PRL signaling and hepatic lipid metabolism, with decreases in PRL being beneficial to lipid metabolism. Extending that concept to broad environmental responses, it can be speculated that altered PRL signaling impairs lipid metabolism, mammary growth, and immune function under conditions of stress (e.g., heat stress) also. Thus, shifts in gene expression related to PRL signaling may provide an environmentally mediated mechanism to alter production and health in cows as they transition into lactation.

Region‐Specific Expression and Hormonal Regulation of the First Exon Variants of Rat Prolactin Receptor mRNA in Rat Brain and Anterior Pituitary Gland

Recent studies have revealed the occurrence of five first exon variants of the rat prolactin receptor mRNA, suggesting that multiple promoters direct prolactin receptor transcription in response to different regulatory factors. In the present study, regional expression of these first exon variants, as well as two prolactin receptor subtypes generated by alternative splicing, was examined in the brains and anterior pituitary glands of female rats. Expression of the long-form was detected in the choroid plexus, hypothalamus, hippocampus, cerebral cortex and anterior pituitary gland, whereas the short form was detected only in the choroid plexus. E1-3 mRNA, a first exon variant, was detected in the choroid plexus, hypothalamus, and anterior pituitary gland, whereas E1-4 was detected only in the choroid plexus. Other variants were not detectable by the polymerase chain reaction protocol employed in this study. Ovariectomy increased the short form in the choroid plexus and the E1-3 expression in the choroid plexus and pituitary gland, but changes in the long-form and E1-4 expression were minimal. Replacement of oestrogens and prolactin suggest that oestrogens down-regulate E1-3 expression in the choroid plexus and pituitary gland, and that the negative effect of oestrogen is mediated by prolactin in the pituitary gland. The present results revealed the region-specific promoter usage in prolactin receptor mRNA transcription, as well as the involvement of oestrogens in the regulation of E1-3 mRNA expression in the brain and pituitary gland.

Prolactin Regulation of Gonadotropin-Releasing Hormone Neurons to Suppress Luteinizing Hormone Secretion in Mice

Hyperprolactinemia causes infertility, but the mechanisms involved are not known. The present study aimed to determine whether and how prolactin may influence LH secretion in the adult female mouse. Using ovariectomized, estrogen-treated (OVX+E) mice, we found that 7 d of intracerebroventricular prolactin potently suppressed serum LH levels (P < 0.05). To examine whether this central action of prolactin may involve the GnRH neurons, the effects of acute and chronic prolactin on cAMP response element-binding protein phosphorylation (pCREB) in GnRH neurons were examined using dual-label immunocytochemistry. In diestrous and OVX+E mice, a single sc injection of ovine prolactin resulted in a significant (P < 0.05) doubling of the number of GnRH neurons expressing pCREB. OVX+E mice treated with five injections of ovine prolactin over 48 h showed a 4-fold increase in the number of GnRH neurons with pCREB. To determine whether GnRH neurons might be regulated directly by prolactin, we examined prolactin receptor (PRL-R) mRNA expression in green fluorescent protein-tagged GnRH neurons by single-cell RT-PCR. As a positive control, PRL-R mRNA was measured in arcuate dopaminergic neurons obtained from green fluorescent protein-tagged tyrosine hydroxylase neurons. Three of 23 GnRH neurons (13%) were identified to express PRL-R transcripts, whereas nine of 11 arcuate dopaminergic neurons (82%) were found to coexpress PRL-R mRNA. These data demonstrate that prolactin suppresses LH levels in the mouse, as it does in other species, and indicate that it acts centrally to regulate intracellular signaling within GnRH neurons. This is likely to occur, at least in part, through the direct regulation of a subpopulation of GnRH neurons.

Ontogenesis of the Expression of Prolactin Receptor Messenger Ribonucleic Acid During Late Embryogenesis in Turkeys and Chickens

Changes in circulating levels of prolactin (PRL) and tissue content of PRL receptor (PRLR) messenger RNA (mRNA) in the liver, pancreas, kidney, and gonads (testis/ovary) were measured in turkey and chicken embryos from embryonic day (ED) 21 or ED15, respectively, to 1 d after hatch by real-time PCR. There were no differences between the sexes in chickens or turkeys. Both species had very similar patterns of PRL release and expression of PRLR mRNA, and no major differences were observed between turkey or chicken embryos. Plasma levels of PRL increased from low levels during the last week of embryonic development and were at significantly higher levels (about 4-fold) by 1 d after hatch. Similarly, in all tissues the content of PRLR mRNA was minimal at the outset and increased to reach maxima about the time of hatch. In both species, the highest levels of transcript were observed in the kidney followed by the gonad, liver, and pancreas. The tissue content of PRLR was correlated (0.6 to 0.8 dependent on the tissue) to circulating levels of PRL, which suggested that PRL may be associated with an increase in its receptor number around the time of hatch. Because levels of PRL and tissue content of PRLR mRNA increased around the time of hatch, this suggests that these tissues may be targets for PRL and may be involved in the physiologic changes occurring in embryos around the time of hatching.

Prolactin receptor and proliferating/apoptotic cells in esophagus of the Mozambique tilapia ( Oreochromis mossambicus) in fresh water and in seawater

We have previously shown that esophageal epithelium of a euryhaline goby displays elevated cell proliferation in freshwater (FW) fish, but undergo apoptosis during seawater (SW) acclimation. Prolactin (PRL) injection into the goby induced the cell proliferation, whereas cortisol treatment stimulated the cell proliferation and apoptosis [Takahashi, H., Sakamoto, T., Narita, K., 2006a. Cell proliferation and apoptosis in the anterior intestine of an amphibious, euryhaline mudskipper ( Periophthalmus modestus). J. Comp. Physiol. B 176, 463–468, 2006). In the euryhaline tilapia ( Oreochromis mossambicus), the dynamics of changes in cortisol-glucocorticoid receptors (GR) during acclimation to different salinities also suggests a role for glucocorticoid signaling in the esophageal cell turnover, but the mode of PRL action remains largely unclear. In the present study, we report on effects in the tilapia esophagus that result from changes in environmental salinity. Specifically, we assessed: (1) mRNA expression of PRL receptor (PRLR) using quantitative real-time RT-PCR; (2) esophageal cell proliferation and apoptosis, using immunohistochemistry of proliferating cells nuclear antigen (PCNA) and in situ nick end-labeling of genomic DNA (TUNEL); (3) the possible localization of immunoreactive PRLR on proliferating/apoptotic cells. Plasma PRL increased after FW acclimation; PRLR mRNA levels in the esophagus of FW fish were significantly higher than those in SW-acclimated fish. Cell proliferation was induced randomly throughout the esophageal epithelium after FW acclimation, while cell division and increased apoptosis were concentrated at the tips of esophageal epithelial folds in SW-acclimated fish. Immunoreactive PRLR appeared to be localized at proliferating cells and at certain apoptotic cells, whereas immunoreactive GR was observed over the whole epithelium including the apoptotic/proliferating cells. Thus, PRL appears to affect cell turnover directly in the esophageal epithelium of the euryhaline tilapia.

Developmental Changes in The Expression Levels of Alternative First Exons of Prolactin Receptor Gene in Rat Brain

One of the alternative first exons, E14, of the rat prolactin receptor (PRLR) gene was identified 5.4 kb downstream of exon 2 by sequence analysis of a rat genomic clone. In female and male rat brains, expression levels of E14-containing PRLR mRNA increased remarkably between 2 and 4 weeks of age during postnatal development, whereas the levels of PRLR mRNAs containing other first exons, E13 and E15, did not change throughout the development. The levels of E14-containing PRLR mRNA in the female rats at 8 weeks of age decreased by ovariectomy, and recovered by the administration of 17β-estradiol, whereas castration and following testosterone treatment showed no effect on the levels of E14-containing PRLR mRNA in the male rats. The levels of E13- and E15-containing PRLR mRNAs were not affected by gonadectomy and following sex steroid hormone treatments in both sexes. These results indicate that expression of PRLR gene in the female and male rat brains increases during postnatal development due to the transcriptional activation of the E14 first exon.