PRL mRNA Research Articles

Regulation of Prolactin Release at the End Stage of Chicken Embryogenesis.

Difference of onset of increase of PRL content in the anterior pituitary gland and plasma PRL concentration during the late stage of chicken embryogenesis is well known. To investigate the disagreement, changes in PRL content and PRL mRNA levels, and the effects of vasoactive intestinal polypeptides (VIP) on PRL release and PRL mRNA expression were examined using western blot analysis and real-time PCR quantification. Changes in SPRL content were strongly correlated with PRL mRNA levels. The increase in PRL content on day 17 of incubation may be caused by the increase in PRL mRNA levels on day 16 of incubation. Additionally, the effects of VIP on PRL release from the embryonic anterior pituitary gland were not observed until day 18 of embryogenesis. These results suggest that increased levels of PRL mRNA and PRL content in the anterior pituitary gland are closely correlated. However, the increased expression of PRL mRNA observed on day 17 and the initiation of PRL release from the anterior pituitary gland on day 19 were differentially regulated. According to the results of western blot analysis, the proportion of glycosylated PRL (G-PRL) and non-glycosylated PRL (NG-PRL) in the anterior pituitary gland at the end stage of development differed from the proportion of PRL released from the anterior pituitary gland. According to the results of two-dimensional western blot analysis, no isoforms with different isoelectric points were detected in the culture medium on days 19 and 20. These data suggest that the peptide chains of G-PRL and NG-PRL were not modified. In conclusion, the differentiation of PRL-producing cells and the maturation of the hypothalamus and anterior pituitary gland were completed at the end stage of incubation, and that different factors regulated the initiation of PRL mRNA expression before day 18 of incubation.

A positive COX-2/IL-1β loop promotes decidualization by upregulating CD82.

A successful pregnancy requires sufficient decidualization of endometrial stromal cells (ESCs). CD82, a metastasis suppressor, is a critical regulator for trophoblast invasion but the effect in decidualization was largely unknown. Here we reported that there was a high level of CD82 in DSC by the immunohistochemistry staining and flow cytometer analysis. Stimulation with prostaglandin E2 (PGE2) elevated the expression of CD82 in ESCs. In contrast, celecoxib, a selective COX-2 inhibitor, significantly downregulated the expression of CD82 in decidual stromal cells (DSCs). Bioinformatics analysis and further research showed that recombinant human interleukin (IL)-1β protein (rhIL-1β) upregulated CD82 in ESCs. Of note, blocking IL-1β signaling with anti-human IL-1β neutralizing antibody could reverse the stimulatory effect of PGE2 on CD82 in ESCs. Silencing CD82 resulted in the decease of the decidualization markers PRL and IGFBP1 mRNA levels in DSCs. More importantly, we observed rhIL-1β also upregulated the expression of COX-2, and the upregulation of PRL and IGFBP1 induced by rhIL-1β could be abolished by celecoxib in ESCs or CD82 deficiency in DSCs. This study suggests that CD82 should be a novel promotor for decidualization under a positive regulation of the COX-2/PGE2/IL-1β positive feedback loop.

Metabolic signalling to somatotrophs: Transcriptional and post-transcriptional mediators.

In normal individuals, pituitary somatotrophs optimise body composition by responding to metabolic signals from leptin. To identify mechanisms behind the regulation of somatotrophs by leptin, we used Cre-LoxP technology to delete leptin receptors (LEPR) selectively in somatotrophs and developed populations purified by fluorescence-activated cell sorting (FACS) that contained 99% somatotrophs. FACS-purified, Lepr-null somatotrophs showed reduced levels of growth hormone (GH), growth hormone-releasing hormone receptor (GHRHR), and Pou1f1 proteins and Gh (females) and Ghrhr (both sexes) mRNAs. Pure somatotrophs also expressed thyroid-stimulating hormone (TSH) and prolactin (PRL), both of which were reduced in pure somatotrophs lacking LEPR. This introduced five gene products that were targets of leptin. In the present study, we tested the hypothesis that leptin is both a transcriptional and a post-transcriptional regulator of these gene products. Our tests showed that Pou1f1 and/or the Janus kinase/signal transducer and activator of transcription 3 transcriptional regulatory pathways are implicated in the leptin regulation of Gh or Ghrhr mRNAs. We then focused on potential actions by candidate microRNAs (miRNAs) with consensus binding sites on the 3' UTR of Gh or Ghrhr mRNAs. Somatotroph Lepr-null deletion mutants expressed elevated levels of miRNAs including miR1197-3p (in females), miR103-3p and miR590-3p (both sexes), which bind Gh mRNA, or miRNA-325-3p (elevated in both sexes), which binds Ghrhr mRNA. This elevation indicates repression of translation in the absence of LEPR. In addition, after detecting binding sites for Musashi on Tshb and Prl 3' UTR, we determined that Musashi1 repressed translation of both mRNAs in in vitro fluc assays and that Prl mRNA was enriched in Musashi immunoprecipitation assays. Finally, we tested ghrelin actions to determine whether its nitric oxide-mediated signalling pathways would restore somatotroph functions in deletion mutants. Ghrelin did not restore either GHRH binding or GH secretion in vitro. These studies show an unexpectedly broad role for leptin with respect to maintaining somatotroph functions, including the regulation of PRL and TSH in subsets of somatotrophs that may be progenitor cells.

MON-459 ONO-5788, a Novel Oral Small Molecule Somatostatin Receptor Type-2 (SST2) Agonist, Attenuates GH Hypersecretion in Human GH-Secreting, Pituitary Adenoma-Derived Cells

Somatostatin receptor ligands (SRL) and GHR antagonists are used for persistent or recurrent acromegaly following noncurative surgery and as primary therapy for patients unsuitable for surgery. We tested in vitro action of a novel oral SRL compound ONO-5788 and its active metabolite (ONO-ST1-641) which signals via SST2. With IRB approval and informed consent, 12 human somatotroph adenomas, obtained at transsphenoidal resection were cultured. In the 1st cohort, 8 somatotroph adenomas were treated with ONO-ST1-641 (0, 100, 1000 nM) and octreotide (OCT, 100 nM) for 24 h, and media collected at 2, 6, and 24 h respectively to assess GH and PRL concentrations by ELISA. Cells harvested at 24 h were used to measure mRNA levels of SST2 and SST5, hormone genes and cell cycle genes CDKN1A, CDKN1B by qPCR. Three of 8 tumors (38%) responded to ONO-ST1-641 and OCT as early as 2 and 6 h. GH was significantly reduced by OCT (18% to 58% reduction, P<0.05) and similarly attenuated by ONO-ST1-641 (28 to 59% reduction, P<0.05). GH secretion in 6 of 8 tumors (75%) was decreased at 24 h, showing reduction by OCT and ONO-ST1-641 by up to 48% (P<0.01) and 50% (P<0.01), respectively. PRL levels were undetectable, very low or unchanged by treatments. Related compound ONO-5788 showed similar responses. SST2 and SST5 expression was unchanged during treatments in the 6/8 responders as assessed by TaqMan qPCR using HPRT1 and GAPDH as controls. Neither was GH, PRL, CGA, CDKN1A and CDKN1B mRNA expression altered by ONO-ST1-641 or OCT for 24 h, as assessed by SYBR qPCR using RPL13A, GAPDH and 18S as controls, suggesting that ONO-ST1-641 did not affect these hormone genes and cell cycle regulator mRNA levels. In the 2nd cohort, pretreatment baseline SST2 and SST5 expression were assessed in 4 cultured tumors treated with ONO-ST1-641 (0, 100, 1000 nM) or OCT (100 nM) for 6 h. Untreated tumor cell mRNA was extracted at baseline to assess SST2 and SST5 expression and GH and PRL measured at 6 h. Two of 4 tumors were unresponsive to both ONO-ST1-641 and OCT, 1 showed mild GH reduction by ONO-ST1-641 (15%) but not by OCT. Notably, 1 specimen expressing highly abundant baseline SST2 exhibited marked GH suppression by ONO-ST1-641 and OCT at 6 h (63 and 62%, respectively, P<0.01). High baseline SST2 expression further validated by Western blot and confocal immunofluorescence, imply that SST receptor expression associates with ONO-ST1-641 responsiveness. Available SRLs and GHR antagonists are parenteral, requiring lifelong injection, with variable and suboptimal efficacy, necessitating development of novel oral small molecule drugs for acromegaly. Our study provides preclinical evidence that ONO-5788 and its active metabolite attenuate GH hypersecretion in primary somatotroph adenoma cells cultures, suggesting the potential application of ONO-5788 for acromegaly patients.

MON-434 Familial Alactogenesis Associated with a Prolactin Mutation

BACKGROUND: Isolated prolactin deficiency is a rare disorder manifesting as absence of puerperal lactation. We identified a family with multiple members who had alactogenesis and examined genetic causes. SUBJECTS: Three women in one family (proband, sister and niece) reported puerperal alactogenesis, i.e. no milk production postpartum. All the women had regular menstrual cycles during their reproductive years. The proband, currently 66 yrs, had 7 pregnancies and 2 live births. Prolactin level was 1.16 ng/mL (2.8-29.2 ng/mL) at the age of 47 yrs. The sister, currently 73 yrs, had 3 children. Her prolactin level was 1.4 ng/mL (2.8-29.2 ng/mL) at the age of 64 yrs. Both women had menopause before age 45 years. The niece, currently 43 years old, had infertility of unknown cause and conceived 2 children with fertility treatment. She had prolactin levels of 1.13 ng/mL, 0.618 ng/mL and 0.759 ng/mL at the age of 34-36 yrs. METHODS: DNA and mRNA were extracted from blood of all three women and a fertile control with a history of normal lactation. PRL exons 1-6 were Sanger sequenced from the DNA. cDNA was subjected to RT-PCR with primers spanning intron 5 and the terminal portion of exon 6. RT-PCR was analyzed using the 2-ΔΔCT method. Prolactin mRNA expression was compared among the affected women and the control subject. RESULTS: We identified a heterozygous mutation (c.658C>T) changing CGA (arginine) to TGA (stop codon) (p.Arg220Ter) in exon 6 of the prolactin gene. PRL mRNA expression (0.011±0.003 vs. 1.0±0.0; p<0.001) and terminal exon 6 expression (0.0027±0.0028 vs. 0.99±0.05; p<0.001) was decreased compared to control mRNA expression. DISCUSSION: We have identified a genetic cause of puerperal alactogenesis resulting from a stop-gain mutation in the prolactin gene (PRL). PRL mRNA expression is decreased in these subjects. These findings suggest that the mutated PRL may undergo nonsense decay. The low circulating prolactin resulted in inability to breastfeed. It may also be the cause of infertility and early menopause, but further studies are needed. CONCLUSION: We report a stop-gain mutation in exon 6 of the prolactin gene (PRL) resulting in familial puerperal alactogenesis.

Effect and molecular regulatory mechanism of monochromatic light colors on the egg-laying performance of Yangzhou geese

Photoperiodic control is essential for manipulating the reproductive performance of avian species. This study was conducted to assess the neuroendocrine mechanisms that regulate reproductive functions of Yangzhou geese when there are different monochromatic light colors from light emitter diode (LED) sources. A flock of geese was divided into four groups with white, red, blue, and green light treatments being imposed. The results indicated that peak laying rates and reproductive performance were greater in geese treated with white or red as compared with blue or green light treatments. The fertilization rate of eggs and hatchability of fertilized eggs were greater with the white or red as compared with blue or green light treatments. There was a greater abundance of OPN5, Dio2, c-Fos, and GnRH-I mRNA in the hypothalamus earlier in the treatment period and abundances of these hypothalamic factors were greater with the white or red light treatments. Abundances of pituitary LH beta and FSH beta mRNA increased at a lesser rate with the blue or green light treatments and were in greater abundances with the white or red light treatments. The lighting regimen also resulted in photo-refractoriness with there being greater abundances of GnIH, VIP, and PRL mRNA with the use of white or red light treatments. The results indicate that the use of white or red monochromatic lights while imposing a long photoperiod of 11 h daily could result in sustaining functions of the reproductive system of Yangzhou geese for considerably longer times, thus, resulting in greater egg-laying performance.

Forms of selenium in vitamin-mineral mixes differentially affect the expression of genes responsible for prolactin, ACTH, and α-MSH synthesis and mitochondrial dysfunction in pituitaries of steers grazing endophyte-infected tall fescue.

The goal of this study was to test the hypothesis that sodium selenite (inorganic Se, ISe), SEL-PLEX (organic forms of Se, OSe), vs. a 1:1 blend (MIX) of ISe and OSe in a basal vitamin-mineral (VM) mix would differentially alter pituitary transcriptome profiles in growing beef steers grazing an endophyte-infected tall fescue (E+) pasture. Predominately Angus steers (BW = 183 ± 34 kg) were randomly selected from fall-calving cows grazing E+ pasture and consuming VM mixes that contained 35 ppm Se as ISe, OSe, or MIX forms. Steers were weaned, depleted of Se for 98 d, and subjected to summer-long common grazing of a 10.1 ha E+ pasture containing 0.51 ppm ergot alkaloids. Steers were assigned (n = 8 per treatment) to the same Se-form treatments on which they were raised. Selenium treatments were administered by daily top-dressing 85 g of VM mix onto 0.23 kg soyhulls, using in-pasture Calan gates. As previously reported, serum prolactin was greater for MIX (52%) and OSe (59%) steers vs. ISe. Pituitaries were collected at slaughter and changes in global and selected mRNA expression patterns determined by microarray and real-time reverse transcription PCR analyses, respectively. The effects of Se treatment on relative gene expression were subjected to one-way ANOVA. The form of Se affected the expression of 542 annotated genes (P < 0.005). Integrated pathway analysis found a canonical pathway network between prolactin and pro-opiomelanocortin (POMC)/ACTH/α-melanocyte-stimulating hormone (α-MSH) synthesis-related proteins and that mitochondrial dysfunction was a top-affected canonical pathway. Targeted reverse transcription-PCR analysis found that the relative abundance of mRNA encoding prolactin and POMC/ACTH/α-MSH synthesis-related proteins was affected (P < 0.05) by the form of Se, as were (P ≤ 0.05) mitochondrial dysfunction-related proteins (CYB5A, FURIN, GPX4, and PSENEN). OSe steers appeared to have a greater prolactin synthesis capacity (more PRL mRNA) vs. ISe steers through decreased dopamine type two receptor signaling (more DRD2 mRNA), whereas MIX steers had a greater prolactin synthesis capacity (more PRL mRNA) and release potential by increasing thyrotropin-releasing hormone concentrations (less TRH receptor mRNA) than ISe steers. OSe steers also had a greater ACTH and α-MSH synthesis potential (more POMC, PCSK2, CPE, and PAM mRNA) than ISe steers. We conclude that form of Se in VM mixes altered expression of genes responsible for prolactin and POMC/ACTH/α-MSH synthesis, and mitochondrial function, in pituitaries of growing beef steers subjected to summer-long grazing an E+ pasture.

Body condition of late pregnant gilts affects the expression of selected adipokines and their receptors in mammary fat and backfat tissues.

The impact of body condition in late gestating gilts on gene expression of selected adipokines and their receptors in backfat and mammary fat tissues was studied. The presence of associations between mammary gland composition variables and the mRNA abundance of selected genes and serum concentrations of adiponectin and leptin was also investigated. A total of 45 gilts were selected at mating based on their backfat depth and were allocated to three groups: (1) low backfat (LBF; 12-15 mm; n = 14), (2) medium backfat (MBF; 17-19 mm; n = 15), and (3) high backfat (HBF; 22-26 mm; n = 16). Gilts were fed different amounts of a conventional diet to maintain differences in backfat depth throughout the gestation period. Blood samples were collected at day 109 of gestation to measure adiponectin and leptin serum concentrations. Gilts were slaughtered on day 110 of gestation, and mammary glands were collected to determine mammary composition. Mammary fat and backfat tissues were also sampled to measure the mRNA abundance of selected genes. In mammary fat tissue, there was an effect of body condition on the prolactin (PRL; P = 0.01), adiponutrin (PNPLA3; P < 0.10), and prolactin receptor long form (PRLR-LF; P < 0.10) genes. There was a greater PRL mRNA abundance in mammary fat tissue from HBF than LBF or MBF gilts (P < 0.05). The PNPLA3 mRNA abundance was lower in HBF than in MBF gilts (P < 0.05), and that of PRLR-LF was lower in LBF than in HBF gilts (P < 0.05). In backfat, body condition affected the mRNA abundance of leptin (P < 0.05) and PNPLA3 (P < 0.01), with the greatest expression levels being observed in HBF gilts for both genes. Association analyses suggest a detrimental effect of high circulating leptin concentrations on gilts mammary development, as reflected by the negative correlations between serum leptin and protein percent (r = -0.66, P < 0.01), and concentrations of DNA (r = -0.62, P < 0.01) and RNA (r = -0.60, P < 0.01) in mammary parenchyma. Current results show that body condition of gilts at the end of gestation can affect the expression of adipokines in mammary fat and backfat tissues, with a different regulation of transcript abundance being observed in these two fat depots. Results also suggest that circulating leptin is strongly associated with mammary gland composition of late pregnant gilts, whereas locally synthesized leptin from mammary fat tissue is not.

MicroRNA-9 regulates fetal alcohol-induced changes in D2 receptor to promote prolactin production.

Fetal alcohol exposure (FAE) is known to increase prolactin (PRL) secretion from the pituitary lactotropes. In this study, we determined whether microRNAs (miRs) are involved in FAE-induced alteration in PRL release. We employed a rat animal model of FAE involving feeding pregnant Fisher 344 rats with a liquid diet containing 6.7% alcohol between gestational days 7-21 (AF). Both cyclic and estradiol-implanted FAE females showed increased levels of plasma PRL and pituitary Prl mRNA but reduced levels of pituitary dopamine D2 receptor (D2r) and its short spliced form (D2s). FAE increased the expression levels of miR-9 and miR-326 and did not produce any significant changes in miR-153 or miR-200a levels in the pituitary. Effects of FAE on miR-9 and miR-326 were associated with reduced levels of D2r and D2s, increased levels of Prl in the pituitary, and in plasma. These effects of FAE on D2r, D2s and Prl were enhanced following estradiol treatment. In PRL-producing MMQ cells, ethanol increased miR-9 but not miR-326, reduced levels of D2r and D2s and increased levels of Prl Treatment of MMQ cells with an anti-miR-9 oligo reduced ethanol effects on miR-9, D2r, D2s and Prl miR-9 mimic oligos reduced the luciferase activity of reporter vector containing D2r 3'UTR, but failed to reduce the mutant luciferase activity. These data suggest that FAE programs the pituitary to produce increased amounts of miR-9 expression that represses the D2r gene and its spliced variant D2s by targeting its 3'UTR leading to an increase in PRL production and secretion.

Mechanisms for PACAP-induced prolactin gene expression in grass carp pituitary cells.

In mammals, pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone with diverse functions but its role in prolactin (PRL) regulation is highly controversial. To shed light on Prl regulation by PACAP in fish model, grass carp pituitary cells was used as a model to examine the receptor specificity and signal transduction for PACAP modulation of prl gene expression in the carp pituitary. Using RT-PCR, PACAP-selective PAC1 receptor was detected in carp lactotrophs. In carp pituitary cells, nanomolar doses of PACAP, but not VIP, could elevate Prl secretion and protein production with concurrent rise in prl mRNA and these stimulatory effects were blocked by PACAP antagonist but not VIP antagonist. PACAP-induced prl mRNA expression could be mimicked by activating adenylate cyclase (AC), increasing cAMP level by cAMP analog, or increasing intracellular Ca2+ ([Ca2+]i) by Ca2+ ionophore/voltage-sensitive Ca2+ channel (VSCC) activator. PACAP-induced prl gene expression, however, was attenuated/abolished by suppressing cAMP production, inhibiting PKA activity, blocking [Ca2+]i mobilization and VSCC activation, calmodulin (CaM) antagonism, and inactivation of JNK and CaM Kinase II (CaMK-II). Similar sensitivity to CaM, JNK, and CaMK-II blockade was also noted by substituting cAMP analog for PACAP as the stimulant for prl mRNA expression. These results, as a whole, provide evidence for the first time that (i) PACAP activation of PAC1 receptor expressed in carp lactotrophs could induce Prl synthesis and secretion, and (ii) Prl production induced by PACAP was mediated by upregulation of prl gene expression, presumably via functional coupling of cAMP/PKA-, Ca2+/CaM-, and MAPK-dependent cascades.

Molecular performance of Prl and Gh/Igf1 axis in the Mediterranean meager, Argyrosomus regius, acclimated to different rearing salinities.

Aquaculture industry in the Mediterranean region exhibits a growing interest for the Mediterranean meager Argyrosomus regius. Some preliminary works showed a good growth performance of the species in nearly isosmotic salinities. However, the patterns of alteration of prolactin (Prl) as well as growth hormone (Gh)/insulin growth factor-1 (Igf1) axis at the molecular level are not yet described in this species. Therefore, we cloned and sequenced partial cDNAs for pituitary prolactin (prl) and growth hormone (gh), hepatic insulin-like growth factor (igf1), and β-actin (actb). Expression patterns of these transcripts were tested in juveniles of A. regius acclimated to four different environmental salinities: (1) 5‰ (hyposmotic); (2) 12‰ (isosmotic); (3) 38‰ (hyperosmotic; seawater control); and (4) 55‰ (extremely hyperosmotic). All investigated transcripts shared high sequence identities with their counterparts in other perciformes. prl mRNA levels showed inverse pattern with increasing salinities. gh mRNA enhanced significantly in both 12 and 55‰ salinity groups in comparison with the control group, while igf1 showed its maximum expression levels under the nearly isosmotic environment. The results indicated clear sensitivity of prl, gh and igf1 to changes in environmental salinity, which can possibly control the euryhalinity capacity of this species.

Effect of breeding stage and photoperiod on gonadal and serotonergic axes in domestic ganders

Reduction in reproductive potential of ganders with progress in seasonal breeding is a known problem in commercial geese production. The role of changes in hypothalamic–pituitary–gondal axis and testis functions in this process is not clear. This article presents studies on the hypothalamic (GnRH-I, vasoactive intestinal peptide), pituitary (LHβ, prolactin [PRL], PRL receptor [PRLR]), testis (PRLR) axis messenger RNA (mRNA) expression during different stages of the breeding period and photoperiodic conditions. Testis mass; histologic and functional (testosterone [T]) parameters; and plasma concentrations of T, LH, and PRL were evaluated. We collected (six times) samples from 2-year-old ganders (n = 48) maintained in short day (10L:14D) during the period from November to July. Moreover, in the peak of sexual activity (March), an additional group was on exposure (6 weeks) to long day (LD; 16L:8D). During the first half of reproduction (January, March; photosensitive period), GnRH-I (1.9 vs. 0.3 relative quantity [RQ]) and LHβ (3.0 vs. 0.7 RQ) mRNA transcript expression and concentrations of T (1.9 - 2.9 vs. 0.3 ng/mL), LH (13.6 - 7.4 vs. 0.7 ng/mL) were found to be higher (P < 0.05) than at the end of breeding (July). With progress in breeding, marked elevation (P < 0.05) in PRL (22.0 - 387.1 ng/mL) concentration related to similar changes in vasoactive intestinal peptide (0.9 - 3.0 RQ) and PRL mRNA abundance (1.3 - 11.5 RQ; May, July) was observed. However, testis PRLR mRNA increased (P < 0.05) only at the end of reproduction (1.2 RQ) compared to the peak of sexual activity (0.4 RQ; March). Furthermore, changes in mRNA transcript expression of the lactotrophic axis were accompanied with reduction of testis weight (left: 11.1 - 5.8 g), spermatogenesis (spermatogenic index: 5.4 - 3.0), and steroidogenesis (T: 24.8 - 1.3 ng/g testis), which may suggest their pivotal inhibitory modulation role in the regression of seasonal reproductive activity in ganders. The LD conditions (similar to spring-summer) resulted in earlier peripheral changes in T (0.9 vs. 1.8 ng/mL), LH (1.1 vs. 3.8 ng/mL), and PRL (296.1 vs. 161.2 ng/mL) concentrations than in short day, and this may be related to the advance in the timing of the sexual activity failure observed under natural light regimes. The lack of differences in gonadal and lactotrophic axis mRNA expression after LD treatment suggested a regulation based on the posttranslational mechanisms or modification of transcript or protein.

Characterization and Expression of Turkey Prolactin Regulatory Element Binding in the Anterior Pituitary Gland and Pancreas During Embryogenesis.

The PRL regulatory element-binding (PREB) protein is a transcription factor that was originally cloned from the rat anterior pituitary gland and characterized as a regulator of the PRL promoter. It is also strongly expressed in several extrapituitary tissues; however, its functional role is not well understood to date. In this study, we aimed to clone and characterize the turkey PREB gene and investigate its mRNA expression in the anterior pituitary gland and pancreas during embryogenesis. Based on the conserved sequence of chicken and mammalian PREB cDNAs, a turkey PREB cDNA fragment was obtained, and after sequencing of the fragment, the 5′-and 3′-ends of mRNA were amplified and determined. To identify the PREB gene structure, polymerase chain reaction (PCR) amplification was performed. The turkey PREB gene consists of 9 exons and 8 introns, and it encodes a 411-amino-acid protein. The expression of PREB mRNA in the anterior pituitary gland was measured during embryogenesis. Levels of PREB mRNA significantly increased at embryonic day 22, with maximum levels being detected on day 25 of ontogeny, which correlated with similar changes in levels of PRL mRNA. The highest level of PREB mRNA was detected on day 19 in the pancreas. However, the highest level of insulin mRNA was detected at embryonic day 25. These results indicate that PREB may be involved in the expression of PRL mRNA in the anterior pituitary gland, whereas insulin mRNA may be expressed independently of the expression of PREB mRNA in the pancreas during embryogenesis.

The acute salinity changes activate the dual pathways of endocrine responses in the brain and pituitary of tilapia

To analyze and compare the stress and osmoregulatory hormones and receptors in pituitary during acute salinity changes, the expression patterns of corticotropin releasing hormone (crh) in hypothalamus, prolactin (prl) releasing peptide (pRrp) in telencephalon and diencephalon, glucocorticoid receptors 2 (gr2), and mineralocorticoid receptor (mr), crh-r, pro-opiomelanocorticotropin (pomc), pRrp, prl, dopamine 2 receptor (d2-r), growth hormone (gh), gh-receptor (gh-r) and insulin-like growth hormone (igf-1) transcripts in pituitary were characterized in euryhaline tilapia. The results indicate that the crh transcripts increased in the hypothalamus and rostral pars distalis of the pituitary after the transfer of fish to SW. Similarly, the pRrp transcripts were more abundant in SW acclimated tilapia forebrain and hypothalamus. The crh-r, gr2 and mr transcripts were more expressed in rostral pars distalis and pars intermedia of pituitary at SW than FW tilapia. The data indicate that the SW acclimation stimulates these transcripts in the specific regions of the brain and pituitary which may be related to the activation of the hypothalamic–pituitary–interrenal (HPI)-axis. The results of dual in situ hybridization reveal that the transcripts of crh-r, gr2 and mr with pomc are highly co-localized in corticotrophs of pituitary. Furthermore, we demonstrate high expression of pRrp in the brain and low expression of pRrp and prl transcripts in the pituitary of SW fish. No crh-r and corticosteroid receptors were co-localized with prl transcripts in the pituitary. The gh-r and igf-1 mRNA levels were significantly increased in SW acclimated tilapia pituitary whereas there was no difference in the gh mRNA levels. The data suggest that the locally produced pRrp and d2-r may control and regulate the expression of prl mRNA in pituitary. Therefore, the dual roles of pRrp are involved in the stress (via brain-pituitary) and osmoregulatory (via pituitary) pathways in tilapia exposed to acute salinity changes.

Abstract 4409: The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro.

Abstract Tamoxifen is the most widely used agent in the world for antihormonal therapy of Estrogen Receptor (ER)-positive breast cancer for both pre- and postmenopausal patients. Tamoxifen is a pro-drug and needs to be metabolically activated to a more potent antiestrogen with a higher affinity for the ER. Initially 4-hydroxytamoxifen (4OHT) was the active metabolite of interest, but more recently 4-hydroxy-N-desmethyltamoxifen (endoxifen) is also of significant interest. The role of endoxifen in therapy remains controversial, however both 4OHT and endoxifen exist in two isomeric forms- cis and trans. Previously, it was shown that cis-isomers of tamoxifen metabolites have weak anti-estrogenic properties using in vitro models available (Jordan VC et al, Endocrinology, 1987, 122(4); 1449-54). However, studies show that an increase of cis-isomers of 4OHT in animals and patients may contribute to tamoxifen resistance (Osborne CK et al, JNCI, 1991, 83; 1477-82; Osborne CK et al, J Clin Oncol, 1992, 10; 304-10). Thus, it is important to know the estrogenic properties of clinically relevant tamoxifen metabolites. To document the anti-estrogenic profiles of both geometric isomers of two major hydroxylated metabolites of tamoxifen, isomerically stable fixed-ring derivatives of 4OHT and endoxifen were synthesized. We have assessed the anti-estrogenic properties of these compounds using cell proliferation assays in selectively cloned hypersensitive ER-positive human breast cancer cell lines MCF-7:WS8 and T47D:A18. Also to assess to estrogenic/anti-estrogenic properties of synthesized compounds on transcriptional level, we have used real-time PCR of rat Prolactin (Prl) mRNA isolated from GH3 rat pituitary cell line. Our results indicate that the fixed-ring trans-4OHT and trans-Endoxifen are potent antiestrogens and equivalent to each other, and to their non-fixed-ring counterparts in both cell proliferation assays and Prl gene activity, as they do not possess any agonistic activities by themselves and are able to inhibit the estrogenic actions of 0,1nM 17β-estradiol (E2) completely (IC50 1nM). However, cis-isomers of the fixed-ring compounds are very weakly estrogenic by themselves but are very weak anti-estrogens in combination with 0,1nM E2 in all assays. Conclusions: the weak estrogen-like activity and low potency of the cis-isomers is unlikely to influence the growth of breast cancer during long-term therapy. The high potency of trans-endoxifen and 4OHT will dominate the control of the tumor growth. Support: This study (PYM) was supported by Susan G. Komen for the Cure International Postdoctoral Fellowship under award number SAC100009 (VCJ). Citation Format: Philipp Y. Maximov, Cynthia Myers, Daphne J. Fernandes, V. Craig Jordan. The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro. [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 4409. doi:10.1158/1538-7445.AM2013-4409

Anterior Pituitary Morphology and Hormone Production During Sustained Critical Illness in a Rabbit Model

Prolonged critical illness is hallmarked by striking alterations in the somatrope, thyrotrope, and lactotrope axes, the severity of which is associated with the risk of morbidity and mortality. The exact role of the pituitary gland in these alterations is unknown. We studied the impact of sustained critical illness on pituitary morphology and hormone production in a standardized rabbit model of prolonged (7 days) burn injury-induced critical illness. In healthy and prolonged critically ill rabbits, we determined pituitary weight, size, morphology and orientation of the somatrope, lactotrope and thyrotrope cells and the pituitary expression of GH, PRL, and TSH at gene and protein level. The weight of the pituitary gland was unaltered by 7 days of critical illness. Also, spatial orientation and morphology of the GH, PRL, and TSH producing cells remained normal. In prolonged critically ill rabbits GH mRNA levels were higher and PRL mRNA levels were lower than in healthy controls, whereas TSH mRNA was not affected. The sizes of GH, PRL, or TSH producing cells and the pituitary content of GH, PRL, and TSH proteins were unaltered. In conclusion, in this rabbit model of prolonged critical illness, the morphology of the pituitary gland and the pituitary GH, PRL, and TSH content was normal. The alterations in pituitary hormone mRNA levels with sustained critical illness are compatible with altered hypothalamic and peripheral regulation of pituitary hormone release as previously suggested indirectly by responses to exogenous releasing factors.

Aryl‐hydrocarbon receptor activity modulates prolactin expression in the pituitary

Pituitary tumors account for 15% of intracranial neoplasms, however the extent to which environmental toxicants contribute to the proliferation and hormone expression of pituitary cells is unknown. Aryl-hydrocarbon receptor (AhR) interacting protein (AIP) loss of function mutations cause somatotrope and lactotrope adenomas in humans. AIP sequesters AhR and inhibits its transcriptional function. Because of the link between AIP and pituitary tumors, we hypothesize that exposure to dioxins, potent exogenous ligands for AhR that are persistent in the environment, may predispose to pituitary dysfunction through activation of AhR. In the present study, we examined the effect of AhR activation on proliferation and endogenous pituitary hormone expression in the GH3 rat somatolactotrope tumor cell line and the effect of loss of AhR action in knockout mice. GH3 cells respond to nM doses of the reversible AhR agonist β-naphthoflavone with a robust induction of Cyp1a1. Although mRNA levels of the anti-proliferative signaling cytokine TGFbeta1 are suppressed upon β-naphthoflavone treatment, we did not observe an alteration in cell proliferation. AhR activation with β-naphthoflavone suppresses Ahr expression and impairs expression of prolactin (PRL), but not growth hormone (GH) mRNA in GH3 cells. In mice, loss of Ahr similarly leads to a reduction in Prl mRNA at P3, while Gh is unaffected. Additionally, there is a significant reduction in pituitary hormones Lhb and Fshb in the absence of Ahr. Overall, these results demonstrate that AhR is important for pituitary hormone expression and suggest that environmental dioxins can exert endocrine disrupting effects at the pituitary.

Differences in retinoid uptake and metabolism causes altered paracrine signaling in endometriosis

Decidualization alters molecular pathways in the endometrium to permit successful embryo implantation. Paracrine factors, including retinoids, secreted from progesterone-treated endometrial stromal cells (HESC), act on epithelium to alter steroid hormone response. These factors are absent in endometriotic stromal cells (OSIS). We noted significant differences in retinoid uptake, metabolism and action in endometriotic tissue and stromal cells compared to normal endometrium. Here we investigate retinoid signaling during decidualization in HESC and OSIS. Basic science. Primary HESC and OSIS were isolated, cultured and treated with decidualization cocktail over 14d. Protein and mRNA expression of genes involved in retinoid metabolism and trafficking (STRA6, CRBP1, ALD1A2, CRABP2, FABP5, RARα, RXRα, PPARβ/δ and CYP26B1) were examined with RT-PCR and western blotting. Decidualization markers (PRL and IGFBP1) were examined with RT-PCR and ELISA. RBP4 secretion was measured with ELISA. siRNA was used to silence genes. HESC and OSIS expressed genes involved in retinoid uptake and metabolism (n=5). Decidualization significantly reduced expression of genes responsible for RA uptake and shuttling to the nucleus (P<0.05). However, mRNA and protein expression of CRBP1, a retinol intracellular carrier protein, increased, as did RBP4 mRNA. Secreted RBP4 was detected by ELISA in media from decidualized HESC. In OSIS, there was neither an increase in CRBP1 mRNA nor protein expression with decidualization, and secreted RBP4 was not detected. Interestingly, silencing CRBP1 in HESC caused an increase in PRL mRNA expression and secretion. Decidualization alters RA trafficking to favor paracrine over intacrine signaling. RBP4 secretion may be an important paracrine signal between stroma and nearby epithelial cells. This signaling is blunted in endometriosis and may help explain its altered decidualization.

Somatostatin analogs and chimeric somatostatin–dopamine molecules differentially regulate human growth hormone and prolactin gene expression and secretion in vitro

We tested effects of selective somatostatin receptor 2 (SST2) agonist BIM-23120, SST5 agonist BIM-23206 and chimeric somatostatin–dopamine molecules (SRIF/DA) BIM-23A760 and BIM-23A761 on GH and PRL secretion and gene expression in human GH/PRL-secreting pituitary tumors in vitro.In “responders” group BIM-23120 suppressed GH levels by 26±4%, BIM-23206 by 31±5%, BIM-23A760 by 23±4%, BIM-23A761 by 39±8% and D2-dopamine agonist BIM-53097 by 31±5%. Using real-time PCR we demonstrated that GH inhibition was not accompanied by decreased GH mRNA levels. PRL secretion was inhibited by BIM-23A760 (29±5%), BIM-23A761 (34±4%), BIM-23206 (26±4%) and BIM-53097 (36±2%). SRIF/DA and BIM-53097 also suppressed PRL mRNA levels.Concluding, SST2 and SST5 agonists and SRIF/DA inhibit GH secretion, but do not suppress GH gene transcription. SRIF/DA and BIM-53097 inhibit both PRL secretion and PRL gene expression. SST5 agonist inhibits PRL secretion, but does not suppress PRL gene expression. D2 affinity is crucial in SRIF/DA action on PRL gene expression.

Cyclic Changes in the Expression of p57kip2 in Human Endometrium and its Regulation by Steroid Hormones in Endometrial Stromal Cells In Vitro

We investigated the expressions of p57(kip2) and p27(kip) and its regulation by steroids in the normal and abnormal human endometrium. Endometrial p27(kip1) and p57(kip2) messenger RNA (mRNA) were markedly increased in the secretory phase. P57(kip2) protein was absent in proliferative phase but appeared in glandular epithelium together with early- to mid-secretory phase stromal cells. During the late secretory phase, strong P57(kip2) protein immunoreactivity was found in the stromal cells. In both endometrial hyperplasia and cancer, the expression of P57(kip2) protein was low. In cultured human endometrial stromal cells (ESCs), p27(kip1) mRNA levels were increased together with the decidual marker prolactin (prl), following treatment with 17β-estradiol (E2) and progesterone (P4). At 1 nmol/L, the glucocorticoid receptor (GR) agonist dexamethasone (DEX) induced prl, p57(kip2) , and p27(kip1) mRNA in ESCs. Taken together, upregulation of p57(kip2) may play an important role in the decidual differentiation by P4 and growth inhibition of malignant cells in human endometrium.