Prolactin mRNA Expression Research Articles

Differential involvement of cAMP/PKA-, PLC/PKC- and Ca2+/calmodulin-dependent pathways in GnRH-induced prolactin secretion and gene expression in grass carp pituitary cells.

Gonadotropin-releasing hormone (GnRH) is a key stimulator for gonadotropin secretion in the pituitary and its pivotal role in reproduction is well conserved in vertebrates. In fish models, GnRH can also induce prolactin (PRL) release, but little is known for the corresponding effect on PRL gene expression as well as the post-receptor signalling involved. Using grass carp as a model, the functional role of GnRH and its underlying signal transduction for PRL regulation were examined at the pituitary level. Using laser capture microdissection coupled with RT-PCR, GnRH receptor expression could be located in carp lactotrophs. In primary cell culture prepared from grass carp pituitaries, the native forms of GnRH, GnRH2 and GnRH3, as well as the GnRH agonist [D-Arg6, Pro9, NEt]-sGnRH were all effective in elevating PRL secretion, PRL mRNA level, PRL cell content and total production. In pituitary cells prepared from the rostral pars distalis, the region in the carp pituitary enriched with lactotrophs, GnRH not only increased cAMP synthesis with parallel CREB phosphorylation and nuclear translocation but also induced a rapid rise in cytosolic Ca2+ by Ca2+ influx via L-type voltage-sensitive Ca2+ channel (VSCC) with subsequent CaM expression and NFAT2 dephosphorylation. In carp pituitary cells prepared from whole pituitaries, GnRH-induced PRL secretion was reduced/negated by inhibiting cAMP/PKA, PLC/PKC and Ca2+/CaM/CaMK-II pathways but not the signalling events via IP3 and CaN/NFAT. The corresponding effect on PRL mRNA expression, however, was blocked by inhibiting cAMP/PKA/CREB/CBP and Ca2+/CaM/CaN/NFAT2 signalling but not PLC/IP3/PKC pathway. At the pituitary cell level, activation of cAMP/PKA pathway could also induce CaM expression and Ca2+ influx via VSCC with parallel rises in PRL release and gene expression in a Ca2+/CaM-dependent manner. These findings, as a whole, suggest that the cAMP/PKA-, PLC/PKC- and Ca2+/CaM-dependent cascades are differentially involved in GnRH-induced PRL secretion and PRL transcript expression in carp lactotrophs. During the process, a functional crosstalk between the cAMP/PKA- and Ca2+/CaM-dependent pathways may occur with PRL release linked with CaMK-II and PKC activation and PRL gene transcription caused by nuclear action of CREB/CBP and CaN/NFAT2 signalling.

Prolactin Mediates Long-Term, Seasonal Rheostatic Regulation of Body Mass in Female Mammals.

A series of well-described anabolic and catabolic neuropeptides are known to provide short-term, homeostatic control of energy balance. The mechanisms that govern long-term, rheostatic control of regulated changes in energy balance are less well characterized. Using the robust and repeatable seasonal changes in body mass observed in Siberian hamsters, this report examined the role of prolactin in providing long-term rheostatic control of body mass and photoinduced changes in organ mass (ie, kidney, brown adipose tissue, uterine, and spleen). Endogenous circannual interval timing was observed after 4 months in a short photoperiod, indicated by a significant increase in body mass and prolactin mRNA expression in the pituitary gland. There was an inverse relationship between body mass and the expression of somatostatin (Sst) and cocaine- and amphetamine-regulated transcript (Cart). Pharmacological inhibition of prolactin release (via bromocriptine injection), reduced body mass of animals maintained in long photoperiods to winter-short photoperiod levels and was associated with a significant increase in hypothalamic Cart expression. Administration of ovine prolactin significantly increased body mass 24 hours after a single injection and the effect persisted after 3 consecutive daily injections. The data indicate that prolactin has pleiotropic effects on homeostatic sensors of energy balance (ie, Cart) and physiological effectors (ie, kidney, BAT). We propose that prolactin release from the pituitary gland acts as an output signal of the hypothalamic rheostat controller to regulate adaptive changes in body mass.

FRI293 Phoenixin As A Novel Autocrine/Paracrine Factor Mediating Prolactin Regulation By Functional Interaction Of LH And IGF-I/-II In Carp Pituitary Cells

Abstract Disclosure: C. Ye: None. X. Qin: None. M. He: None. A.O. Wong: None. Phoenixin (PNX), a novel peptide identified by bioinformatics, is known to have pleiotropic functions mediated by its receptor GPR173. At present, very little is known for PNX regulation and the intracellular signaling for PNX expression is still unclear. Recently, PNX and GPR173 were cloned in grass carp and found to be co-expressed in carp lactotrophs. In carp pituitary cells, PNX was also confirmed to be a novel autocrine/paracrine factor maintaining/stimulating prolactin (PRL) synthesis & secretion. To unveil the functional role of PNX in PRL regulation, a systematic screening of >40 neuroendocrine factors/local growth factors was conducted in the same cell model to look for regulators with modulatory functions for both PNX and PRL in carp pituitary. Based on our study, IGF-I & -II were shown to elevate PNX mRNA level with parallel rise in PRL gene expression in carp pituitary cells whereas the opposite was true for the functional agonist of LH, namely hCG. In parallel experiment, PRL mRNA expression induced by IGF-I was negated by removing endogenous PNX using immunoneutralization with PNX antiserum. Besides, hCG inhibition on PNX & PRL gene expression could be mimicked by parallel treatment of LH but not FSH, whereas removal of endogenous LH by LH antiserum immunoneutralization could lead to the opposite effects. Of note, hCG co-treatment not only attenuated the PNX and PRL responses induced by IGF-I but also triggered notable suppression of IGF-I receptor (IGF1R) gene expression at pituitary cell level. In our study, IGF1R & insulin receptor (InsR) expressed in carp pituitary cells were both activated by IGF-I treatment, but IGF-I induced PNX and PRL mRNA expression could be blocked only by the inactivators for IGF1R but not InsR. Using a pharmacological approach, the gene expression of PNX and PRL induced by IGF-I was confirmed to be mediated through the PI3K/Akt and MEK1/2/ERK1/2 but not P38MAPK pathways. Our findings, as a whole, suggest that (i) IGF signals can induce PRL gene expression in carp pituitary via IGF1R coupled to PI3K/Akt & MAPK cascades and this stimulatory effect is mediated via local production of PNX, and (ii) LH released in the pituitary can act locally to inhibit both basal & IGF-induced PRL expression and these inhibitory actions are mediated by blocking pituitary expression of PNX & IGF1R. Presentation: Friday, June 16, 2023

P-760 Effect of relaxin on human endometrial stromal cell characteristics

Abstract Study question Does relaxin (RLX) regulate decidualization and pro- and antiangiogenic factors in primary human endometrial stromal cells (hESC), primary decidual cells (pD) and hTERT-immortalized hESC (T hESC)? Summary answer RLX treatment enhances decidualization and affects the mRNA expression levels of pro- and antiangiogenic factors. What is known already Patients undergoing frozen embryo transfer in an artificial cycle (AC-FET) are at a 2-fold increased risk for preeclampsia compared to embryo transfer in a natural cycle. This may be related to the absence of the corpus luteum in an artificial FET cycle, which produces important hormones such as estrogen and progesterone in early pregnancy. These hormones are supplied during AC-FET, whereas other products of the corpus luteum, such as RLX, are not. Low maternal RLX levels were found in women who developed preeclampsia, but the direct relationship between RLX and the occurrence of preeclampsia remains to be elucidated. Study design, size, duration Primary hESC were isolated from biopsies of women without endometrial pathology, pD cells from biopsies of the maternal side of the placenta of uncomplicated pregnancies and T hESC purchased from American Type Culture Collection. Cells were decidualized with either 0.5 mM cyclic adenosine monophosphate (cAMP) or a cocktail of 10 nM estradiol, 1 µM progesterone and 0.5 mM cAMP (EPC) and additionally treated with 0, 0.3 or 1 ng/ml RLX for twelve days. Participants/materials, setting, methods RNA was isolated on day zero and day twelve from all three cell types. mRNA expression level of decidualization markers (prolactin (PRL), insulin like growth factor binding protein 1 (IGFBP1)) as well as pro- and antiangiogenic factors (vascular endothelial growth factor (VEGF); placenta like growth factor (PlGF); soluble Fms-like tyrosine kinase-1 (sFlt-1); and endoglin (ENG)) were evaluated by quantitative real time polymerase chain reaction (qRT-PCR), N = 3 biological replicates. Main results and the role of chance Decidualization treatment of hESC with cAMP caused more noticeable changes to cell morphology than treatment with EPC. While there was no rise of decidualization markers PRL (p = 0.99) and IGFBP1 (p = 0.95) in the cAMP treatment group, hESC after EPC treatment showed a significant increase in both PRL (p = 0.0003) and IGFBP1 (p = 0.0001). RLX treatment in the EPC treated hESC and pD significantly increased the mRNA expression levels of PRL and IGFBP1 (both p < 0.05, respectively), as well as the expression of the proangiogenic marker VEGF (P = 0.05). A comparative analysis with T hESC showed a moderate effect on VEGF and PlGF. No effect of RLX was detected for IGFBP1 and PRL mRNA expression levels. The mRNA expression level of the antiangiogenic marker ENG increased in the EPC group in both, primary hESC (P = 0.04) and T hESC (P = 0.01) after treatment with 0.3 ng/ml RLX compared to control. ENG mRNA expression level increased in a dose dependent manner in pD but not in hESC and T hESC. RLX treatment with 1ng/ml of cAMP decidualized hESC significantly enhanced mRNA expression levels of VEGF (P = 0.002) and PlGF (P = 0.02) only in hESC but not in T hESC or pD. Limitations, reasons for caution Our pilot study indicates that RLX treatment improves decidualization and affects pro- and antiangiogenic factors in three different human endometrial stromal cell models. A wider approach is needed to investigate the effects of RLX in the decidualization process and pro- and antiangiogenic factors. Wider implications of the findings Our results provide the basis for further studies elucidating the relationship between low maternal RLX levels and the occurrence of preeclampsia. This could lead to the development of improved prediction models and new treatment options in assisted reproduction that reduce the risk of preeclampsia, e.g. in AC-FET. Trial registration number Not applicable

Estradiol and progesterone regulate NUCB2/nesfatin-1 expression and function in GH3 pituitary cells and THESC endometrial cells

ABSTRACT Estradiol (E2) and progesterone (P4) are essential sex steroid hormones that play critical roles in the pituitary gland and uterus. Recently, nesfatin-1, a polypeptide hormone that regulates appetite and energy homeostasis in the hypothalamus, was found to be expressed in the pituitary gland and uterus. In this study, we aimed to investigate the relationship between these two steroid hormones and the expression and function of nesfatin-1 in the pituitary gland and uterus using GH3 cells, a lacto-somatotroph cell line, and THESC cells, an endometrial stromal cell line. First, we verified the presence of nesfatin-1 and nesfatin-1 binding sites in GH3 and THESC cells. E2 increased the mRNA expression of NUCB2, the gene encoding the nesfatin-1 protein, in GH3 cells, while P4 had no significant effect. In THESC cells, NUCB2 mRNA expression was decreased by E2 but increased by P4. In addition, nesfatin-1 significantly increased growth hormone (GH) and prolactin (PRL) mRNA expression in GH3 cells, and E2 enhanced this effect. In THESC cells, nesfatin-1 significantly increased the mRNA expression of insulin-like growth factor binding protein 1 (IGFBP1) and PRL, which are decidualization marker genes, and P4 further enhanced this effect. These results suggest that nesfatin-1 may act as a local regulator of GH and PRL production in the pituitary gland and decidualization in the uterus, modulating its effects in response to E2 and P4.

Alterations in gonadotropin, prolactin, androgen and estrogen receptor and steroidogenesis-associated gene expression in gander testes in relation to the annual period

Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol–to–testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.

Bromocriptine inhibits proliferation in the endometrium from women with adenomyosis.

Bromocriptine treatment has been shown to reduce menstrual bleeding and pain in women with adenomyosis in a pilot clinical trial. The underlying mechanism contributing to the treatment effect is however unknown. The purpose of this study was to explore the effect of bromocriptine on the proliferation and migration properties of the endometrium in women with adenomyosis, by assessing cellular and molecular changes after six months of vaginal bromocriptine treatment. Endometrial specimens were collected during the proliferative phase from women with adenomyosis (n=6) before (baseline) and after six months of treatment with vaginal bromocriptine. Immunohistochemistry was used to determine changes in the protein expression of Ki67 in the endometrium of women with adenomyosis. Primary endometrial stromal cells isolated at baseline were expanded in vitro and exposed to different doses of bromocriptine to determine the optimal half-maximum inhibitory concentration (IC50) using CellTiter-Blue® Cell Viability Assay. Cell proliferation was assessed by bromodeoxyuridine ELISA assay and Ki67 gene expression was checked by real-time PCR. The migratory ability of endometrial stromal cells was determined by wound healing and transwell migration assays. Small RNA sequencing was applied on tissues collected from women with adenomyosis before and after bromocriptine treatment to identify differentially expressed microRNAs (miRNAs) after bromocriptine treatment. Bioinformatic methods were used for target gene prediction and the identification of biological pathways by enrichment procedures. Vaginal bromocriptine treatment reduced the Ki67 protein expression in the endometrium of women with adenomyosis and did not change the prolactin mRNA expression and protein concentration of prolactin in endometrial tissues. Bromocriptine significantly inhibited the proliferative and migrative abilities of endometrial stromal cells derived from women with adenomyosis in vitro. Moreover, small RNA sequencing revealed 27 differentially expressed miRNAs between the endometrium of women with adenomyosis before and after six months of vaginal bromocriptine treatment. KEGG pathway analysis on targeted genes of 27 miRNAs showed that several signaling pathways associated with cell proliferation and apoptosis were enriched after bromocriptine treatment. Bromocriptine treatment exhibits an anti-proliferative effect in the endometrium of women with adenomyosis in vivo and in vitro. Bromocriptine might inhibit the proliferation of endometrial tissue in adenomyosis in part through the regulation of dysregulated microRNAs and proliferation-associated signaling pathways.

Involvement of neurosteroids in the control of prolactin secretion in sheep under basal, stressful and pregnancy conditions

Prolactin (PRL) secretion by the anterior pituitary (AP) is responsive to changes in physiological conditions and many external factors that also affect brain neurosteroid levels. This study tested the hypothesis that neurosteroids can affect PRL secretion in sheep under basal, stressful and advanced pregnancy conditions. In Experiment 1, luteal-phase sheep were subjected to a three-day series of intracerebroventricular (icv.) control (n = 12) or allopregnanolone (AL, 4 × 15 μg/60 μL/30 min at 30-min intervals, n = 12) infusions. Acute stressful stimuli, isolation and partial movement restriction were applied on the third day of infusion to half of the animals in each group. In Experiment 2, pregnant sheep were subjected to a three-day series of icv. control (n = 6) or finasteride (4 × 25 μg/60 μL/30 min at 30-min intervals, n = 6) infusions during the 16th week of pregnancy. As a result, the relative abundance of PRL transcript increased in the AP of luteal-phase sheep treated with stress, AL and AL in combination with stress (P < 0.05 - P < 0.01) compared to controls. The level of PRL mRNA in stressed-AL-treated sheep was higher (P < 0.01) than in sheep only subjected to stress. The PRL protein content in the AP decreased in stressed-only sheep compared to controls (P < 0.05) and increased in stressed-AL-treated sheep compared to controls and other groups (P < 0.05 - P < 0.01). Plasma PRL concentration increased (P < 0.05 - P < 0.01) in stressed-only sheep compared to controls; AL infusion counteracted the stress-induced increase in PRL levels (P < 0.05 - P < 0.01) and had no effect in non-stressed animals. Inhibition of neurosteroid synthesis in the brain of pregnant sheep by finasteride caused transient increases (P < 0.05 - P < 0.001) in plasma PRL concentration compared to controls. In conclusion, the presented results indicate a bimodal effect of AL on PRL secretion in sheep: first at the molecular level – stimulation of PRL mRNA expression; second – inhibition of hormone release from pituitary lactotrophs. Both AL activities may involve various mechanisms regulating PRL secretion. In general, cerebral neurosteroids can affect the supply of pituitary PRL in the body under certain conditions, such as stress and pregnancy.

Metformin attenuates the production and proliferative effects of prolactin induced by medroxyprogesterone acetate during fertility-sparing treatment for endometrial cancer

BackgroundProgestin is used for fertility-sparing treatment in cases of endometrial cancer (EC). Progestin can induce hyperprolactinemia by increasing pituitary secretion and endometrial decidualization. However, progestin induces prolactin (PRL) secretion, which stimulates cell proliferation and deleteriously affects treatment. To date, the detrimental effect of PRL, the secretion of which is induced by medroxyprogesterone acetate (MPA) during fertility-sparing treatment, has not yet been fully elucidated. Therefore, we aimed to assess the effects of PRL on EC cells during combined treatment with progestin and metformin.MethodsIn total, 71 patients with EC/endometrial atypical hyperplasia who underwent fertility-sparing treatment at our institution from 2009–2019 were enrolled. Serum PRL levels were determined using enzyme immunoassays; mRNA levels in endometrial tissues were determined using quantitative reverse-transcription PCR. To evaluate MPA-induced decidualization, cancer-associated stromal cells were enzymatically released from surgically removed specimens of six patients with EC. To examine PRL-induced cell proliferation, the EC cell lines Ishikawa, HEC1B, and HEC265 were used. In vitro cell proliferation was evaluated using the WST assay; protein levels of signaling molecules were determined using western blotting.ResultsMPA administration significantly increased serum PRL levels at 3 and 6 months and upregulated IGFBP-1 and PRL mRNA expression in tissues at 3 months of fertility-sparing treatment. Metformin significantly reduced MPA-induced IGFBP-1 and PRL mRNA expression during fertility-sparing treatment and significantly inhibited the upregulation of IGFBP-1 and PRL mRNA and PRL levels due to decidualization induced by MPA and cAMP treatment in primary cultured EC stromal cells. In vitro, PRL increased cell proliferation and ERK1/2 phosphorylation levels, whereas metformin attenuated these increases.ConclusionsMPA upregulated PRL levels in serum and endometrial tissues during fertility-sparing treatment. Metformin co-administration reduced PRL production and attenuated PRL-induced cell-proliferation activity. This study may provide valuable insights on the application of metformin to improve the outcomes of fertility-sparing treatment.

Total Barley Maiya Alkaloids Prevent Increased Prolactin Levels Caused by Antipsychotic Drugs and Reduce Dopamine Receptor D2 via Epigenetic Mechanisms.

Background: The dopamine D2 receptor (DRD2) plays an important role in the increased prolactin (PRL) levels associated with the pathogenesis of antipsychotic drugs (ADs). Elevated prolactin levels can affect people’s quality of life. Maiya alkaloids has been used to treat diseases associated with high PRL levels. Maiya, is a processed product of the mature fruits of Hordeum vulgare L. (a gramineous plant) after sprouting and drying and also a common Chinese herbal drug used in the clinic, is traditionally used to treat abnormal lactation, and is currently used clinically for the treatment of abnormal PRL levels. Aims: Epigenetic mechanisms can be related to DRD2 expression. We investigated the role of DRD2 methylation in the induction of PRL expression by ADs and the mechanism underlying the effects of total barley maiya alkaloids (TBMA) on this induction. Methods: The methylation rate of DRD2 in 46 people with schizophrenia who took risperidone was detected by MassARRAY sequencing. Humans were long term users of Ris. Seventy Sprague Dawley female rats were divided into seven groups. A rat model of risperidone-induced PRL was established, and the potential protective effects of TBMA and its components [e.g., hordenine (Hor)] on these increased PRL levels were investigated. The PRL concentration was detected by Enzyme-linked immunosorbent assay. PRL, DRD2, and DNA methyltransferase (DNMT1, DNMT3α, and DNMT3β) protein and mRNA expression were detected by western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The positive rate of methylation in the DRD2 promoter region of rats was detected by MassARRAY sequencing. Results: Clinical studies showed that the positive rate of DRD2 methylation associated with increased PRL levels induced by ADs was significantly higher than in the normal prolactinemia (NPRL) group. In vivo and vitro, TBMA and Hor inhibited this induction of PRL expression and increased DRD2 expression by inhibiting the expression of the DNMTs. Conclusions: TBMA and hordenine increased DRD2 expression by inhibiting DNMT-dependent DRD2 methylation.

Ageing, a modulator of human endometrial stromal cell proliferation and decidualization: a role for implantation?

Research questionDoes ageing affect endometrial stromal cell function and gene expression involved in decidualization? DesignStromal cells were isolated and cultured (98% purity) from pipelle endometrial biopsies obtained from female donors, at the proliferative phase of the menstrual cycle. Initially, 28 samples were collected (age range 25–46 years). These samples were divided into two groups: 25–35 years and 36–46 years. Cell proliferation assays were carried out to determine changes in stromal cell proliferation, in vitro, between the two groups. In addition, mRNA and protein expression of decidualization factors, BMP2 and STAT3, were analysed for the same age groups and samples using real-time polymerase chain reaction and western blot analysis, respectively. Finally, another 12 pipelle endometrial biopsies (age range 25–46 years) were used in separate in-vitro decidualization experiments. The mRNA expression of decidualization markers, prolactin and IGFBP-1 were analysed in cultured stromal cells after adding 8-bromo-cAMP. ResultsA statistically significant decrease was observed in stromal cell proliferation with increasing age (P = 0.0006). Messenger RNA expression of BMP2 and STAT3 were found to decrease (P = 0.0167; P = 0.0037, respectively) in the older age group. In addition, BMP2 and STAT3 protein expression between the samples analysed changed significantly (P = 0.0085; P = 0.0463, respectively) with increasing age. In-vitro induced decidualization experiments showed significant changes in stromal cell mRNA expression of decidualization markers (IGFBP1 and prolactin) between different age groups. ConclusionAgeing affected endometrial cell function and gene expression. Changes in cell function and expression of associated molecules that differ in the ageing endometrium can help understand the causes of infertility.

Prolactin acts in the pathway of photoperiod regulating the reproduction of the Striped Hamsters

To study the effects of prolactin (PRL) in pituitary on the reproduction of the Striped Hamsters via the photoperiods. Mature female striped hamsters were raised under various photoperiods to study the differential expression pattern of PRL in pituitary, and prolactin receptor (PRLR) in the hypothalamus, pituitary and ovary. Photoperiods affect the pituitary PRL mRNA level and pituitary, hypothalamus and ovaries PRLR mRNA levels in striped hamsters. Positive correlations of PRL mRNA expression in the pituitary with that of PRLR in hypothalamus and ovaries, and that of GnRH in hypothalamus were determined. The serum concentration of PRL, FSH, LH and GnRH were significantly different among the striped hamsters from different photoperiods. Serum concentration of PRL is positively correlated with that of FSH, LH and GnRH of the striped hamsters. Photoperiod was important environment factor modulating the expression profile of PRL and PRLR in the striped hamsters.

Postpartum Fatigue and Inhibited Lactation

Background:Postpartum fatigue is a common disorder worldwide and affects both physical and mental functioning. In breastfeeding women, Prolactin (PRL) is not only involved in immunoregulation, but also responsible for lactation. Prolactin levels in women with chronic fatigue are higher than normal, but a chronic fatigue state inhibits postpartum lactation in humans. Objectives:This paper explored the inhibition mechanism of lactation by postpartum fatigue in rats.Methods:Postpartum fatigue models were built by forcing mother rats to stand in water and divided into 3-hour, 9-hour and 15-hour per day fatigue groups according to the underwater time. Mother rats and their offspring were reunited in a dry cage for 90 minutes every 3 hours for feeding. The expression of PRL, PRL receptor (PRLR), Janus Kinase 2 (JAK 2), and Signal transducers and activators of transcription 5 (STAT5) mRNA were analyzed and the microstructure of mammary gland were observed under light and electron microscopy. Results:The expression of pituitary PRL mRNA and its downstream signaling pathway JAK2 and STAT5 mRNA were down-regulated in the severe postpartum fatigue rats. PRL mRNA responses were dose-related to duration of fatigue. The expression of PRLR mRNA increased. Postpartum fatigue led to functional degeneration of mammary gland. The breast lobules were shrunk and the number of alveoli were decreased. Few milk protein granules and fat droplets were observed in the cytoplasm under transmission electron microscope. Conclusion:Postpartum fatigue inhibits the lactation by down-regulating the expression of PRL and PRL-dependent signaling pathway in rats.

Pituitary Actions of EGF on Gonadotropins, Growth Hormone, Prolactin and Somatolactins in Grass Carp

Simple SummaryIn mammals, the functions of epidermal growth factor (EGF) have been widely studied. However, little is known about the pituitary actions of EGF in teleost. Using primary cultured grass carp pituitary cells as model, we found that EGF could reduce pituitary luteinizing hormone β (LHβ) mRNA expression, but induce pituitary growth hormone (GH), prolactin (PRL) and somatolactins (SL) mRNA expression. Furthermore, we also found that NKB could suppress EGF-induced PRL mRNA expression in grass carp pituitary cells. These results suggested that EGF could directly regulate pituitary hormones expression in teleost.In mammals, epidermal growth factor (EGF) plays a vital role in both pituitary physiology and pathology. However, the functional role of EGF in the regulation of pituitary hormones has rarely reported in teleost. In our study, using primary cultured grass carp pituitary cells as an in vitro model, we examined the effects of EGF on pituitary hormone secretion and gene expression as well as the post-receptor signaling mechanisms involved. Firstly, we found that EGF significantly reduced luteinizing hormone (LHβ) mRNA expression via ErbB1 coupled to ERK1/2 pathway, but had no effect on LH release in grass carp pituitary cells. Secondly, the results showed that EGF was effective in up-regulating mRNA expression of growth hormone (GH), somatolactin α (SLα) and somatolactin β (SLβ) via ErbB1 and ErbB2 and subsequently coupled to MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways, respectively. However, EGF was not effective in GH release in pituitary cells. Thirdly, we found that EGF strongly induced pituitary prolactin (PRL) release and mRNA expression, which was mediated by ErbB1 and subsequent stimulation of MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways. Interestingly, subsequent study further found that neurokinin B (NKB) significantly suppressed EGF-induced PRL mRNA expression, which was mediated by neurokinin receptor (NK2R) and coupled to AC/cAMP/PKA signal pathway. These results suggested that EGF could differently regulate the pituitary hormones expression in grass carp pituitary cells.

A clinical and basic study of optimal endometrial preparation protocols for patients with infertility undergoing frozen‑thawed embryo transfer

The optimal protocol for endometrial preparation in patients with infertility remains unclear. Due to this, the current study retrospectively analyzed 1,589 patients with infertility and regular menstrual cycles to assess reproductive outcomes per embryo transferred and per embryo transfer (ET) cycle following the transfer of frozen-thawed embryos (FET) in a modified natural cycle (mNC) or hormone therapy cycle (HT) with or without gonadotropin-releasing hormone agonist (GnRHa)-induced pituitary suppression. The molecular mechanisms involved were also studied using tissues from endometrial biopsies. Patients who underwent FET were assigned to 5 groups as follows: Group A underwent a mNC (n=276); group B (n=338) received estradiol (E2) and progesterone (P4); group C received 1 cycle of GnRHa, E2 and P4 (n=323); group D received 2 cycles of GnRHa, E2 and P4 (n=329); and group E received 3 cycles of GnRHa, E2 and P4 (n=323). Tissues from endometrial biopsies of 91 patients performed on the day of ET were tested for endometrial receptivity marker mRNA expression and microRNA (miR)-223-3p mRNA. Furthermore, endometrial stromal cells (ESCs) were used for an in-depth study of the molecular mechanisms involved. Among the 5 groups of patients, implantation rates, clinical pregnancy rates and live birth rates were not significantly different. However, endometrial receptivity was enhanced in group E when compared with groups A-D, which was associated with endometrial leukemia inhibitory factor (LIF), osteopontin, vascular endothelial growth factor, integrin β3 and homeobox gene 10 and 11 mRNA upregulation, and miR-223-3p miRNA downregulation. Transfection of ESCs with an miR-223-3p mimic significantly reduced levels of LIF mRNA and protein. In addition, pre-treating ESCs with GnRHa upregulated mRNA and protein expression of the decidualization markers prolactin and insulin-like growth factor binding protein-1 in a time-dependent manner. In conclusion, these results indicated that HT with GnRHa may be a potential endometrial preparation protocol for FET.

The effect of parachlorophenylalanine treatment on the activity of gonadal and lactotrophic axes in native Polish crested chickens stimulated to broodiness

Brooding behavior, a common characteristic of native breeds of the domestic chicken, is marked by elevated prolactin (PRL) levels, which is necessary for incubation and connected with changes in hypothalamic–pituitary–gonadal axis activity. Evidence indicates the serotoninergic system is a potent modulator of PRL secretion. The objective of this study is to investigate whether blocking serotonin synthesis with parachlorophenylalanine (PCPA) prevents incubation behavior in native Polish crested chickens. In addition, we examined the effect of PCPA on the gene expression of the gonadal and lactotrophic axes. Birds were stimulated to broodiness by artificial eggs in nests. At 34 wk of age (April: spring period), the hens were divided into 2 groups (14 hens in each group): control and PCPA-treated (50 mg/kg BW) group. After 5 wk of treatment, the artificial eggs were removed from the nests. Egg production, incubation activity, and levels of plasma ovarian steroids progesterone (P4), testosterone (T), estradiol (E2), and PRL were examined. At the end of the experiment (45 wk of age, June: summer period), ovarian characteristics and mRNA gene expression of gonadal (gonadotropin-releasing hormone [GnRH] I, luteinizing hormone [LH] ß, follicle-stimulating hormone [FSH] ß) and lactotrophic (vasoactive intestinal peptide [VIP], PRL) axes were measured by quantitative real-time PCR. Incubation activity was observed in the hens of both groups but with lower frequency in PCPA-treated birds. Moreover, the PCPA group had a higher cumulative egg production than the controls. During the first six and 8 wk of the experiment, levels of P4 and E2, respectively, were similar in both groups, but all concentrations increased in the PCPA-treated hens after this period. In addition, increased GnRH-I, LHß, and FSHß and decreased VIP mRNA expression was observed in the PCPA group compared with the controls. There were no differences in PRL mRNA expression, the PRL level, and ovarian morphometry between the 2 groups. These results indicate that blockage of serotonin synthesis by PCPA does not effectively prevent incubation in native Polish crested chickens. However, treatment with PCPA increased gonadal axis activity and improved reproductive performance.

Potash mining effluents induce moderate effects on histopathological and physiological endpoints of adult zebrafish (Danio rerio)

Stress in fish can be caused by a variety of factors and has the potential to evoke stress responses leading to a reduction of physical condition and of health. The river Werra (Germany) presents a severe case of secondary salinisation caused by potash mining activities. The model organism Danio rerio was exposed to different ion-concentrations depicting current (HT) and future (LT) threshold values of the Werra, as well as to solutions with single-exceeding ions (Mg2+ + K+ (KMg), Mg2+ (Mg) and K+ (K)). After a six-week exposure period, cortisol levels, growth and weight were measured, gills and gonads were histologically analysed and mRNA expression of follicle stimulating hormone (FSH), luteinising hormone (LH), growth hormone (GH) and prolactin (PRL) were determined. Cortisol was still elevated in fish in the HT and K group, indicating moderate stress. However, gills revealed structural changes in zebrafish in all exposure groups, size of oocytes differed in the LT and K group, male FSH mRNA levels were elevated in the HT and LT group whereas PRL mRNA levels were lower in HT and LT for both, male and female fish.These results suggest that ion-stress induces moderate effects on a variety of biological parameters that mainly serve to adapt to elevated ion concentrations. For these reasons current and even future thresholds should be reconsidered, including thresholds for total as well as single ion concentrations. Future research looking at the effects on local fish species is needed, along with regular and long-term monitoring of environmental conditions, species abundance and diversity.

MON-485 Effects of Orexin A on Prolactin Production by Regulating BMP-4 Activity in Rat Pituitary Lactotorope Cells

Orexin A and B, are neuropeptides that are mainly produced in the hypothalamus. Orexins play important roles in food intake and in regulation of sleep and arousal. In the peripheral tissues, orexins also regulate various endocrine activities including the hypothalamic-pituitary-adrenal and -gonadal axes and growth hormone control. However, the effects of orexin on prolactin synthesis have not been clarified. Here, we focused on the role of bone morphogenetic protein (BMP)-4, which is involved in promoting PRL productivity and tumorigenesis of pituitary prolactinomas. We previously reported that BMP-4 induces prolactin secretion by rat lacto-somatotrope GH3 cells. In the present study, we studied the effects of orexin A and the interaction of orexin and BMP system on the regulatory role of prolactin synthesis by using rat lactotrope GH3 cells. Orexin type 1 receptor (OX1R), but not type 2 receptor (OX2R), was expressed in GH3 cells. Orexin A suppressed forskolin-induced, but not basal, prolactin mRNA expression without reducing cAMP levels. Of note, orexin A suppressed BMP-4-induced prolactin mRNA and cAMP synthesis. Interestingly, BMP-receptor signaling inhibitor reduced prolactin mRNA levels, which suggested that endogenous BMP action was to be involved in the activation of prolactin synthesis by GH3 cells. Orexin A suppressed Smad1/5/9 phosphorylation and Id-1 transcription induced by BMP-4, which was reversed by dual orexin-receptor antagonist, suggesting that the inhibitory effect of orexin A occurred via OX1R. Moreover, orexin A reduced BMP type I receptor, ALK-3 expression but increased inhibitory Smad6/7 expression, while BMP-4 treatment downregulated OX1R expression. These results indicated that orexin A has an inhibitory role in prolactin synthesis through suppression of endogenous BMP activity, suggesting that a new functional role of the interaction between orexin A and BMP-4 is modulation of prolactin levels in lactotrope cells.

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.

Impaired hypothalamic leptin sensitivity in pseudopregnant rats treated with chronic prolactin to mimic pregnancy.

Pregnancy in rodents is associated with hyperphagia, increased fat deposition, elevated leptin concentrations and insensitivity to the satiety action of leptin. To investigate the hormonal mechanisms involved in the development of this state of pregnancy-induced leptin resistance, we have used a pseudopregnancy rat model. We have previously demonstrated that pseudopregnant rats have a normal feeding response to leptin, although, if pseudopregnancy is extended using chronic i.c.v. ovine prolactin infusion along with progesterone implants, then leptin no longer suppresses food intake. The present study aimed to investigate the effect of chronically high lactogen levels, as seen in mid-pregnancy, on leptin-induced activation of hypothalamic Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal transduction and mRNA expression of leptin (LepR-B) and prolactin (Prlr-L) receptors, using pseudopregnant rats chronically infused with ovine prolactin. Groups of virgin (dioestrous) and pseudopregnant rats were treated with chronic i.c.v. infusion of either prolactin (2.5μgμL-1 h-1 for 5days) or vehicle (artificial cerebrospinal fluid [aCSF]) via a minipump connected to a cannula surgically implanted into the lateral ventricle. Rats were fasted overnight and then received an i.c.v. injection of leptin (400ng) or vehicle (aCSF) and were perfused 30minutes later. In chronic vehicle-infused pseudopregnant rats, i.c.v. leptin increased the number of phosphorylated STAT3 positive cells in the arcuate nucleus and ventromedial nucleus (VMH) of the hypothalamus, similar to all acute-leptin treated virgin groups. This effect of leptin, however, was not observed in the pseudopregnant rats that were chronically infused with prolactin. A quantitative polymerase chain reaction analysis also showed decreased expression of LepR-B in the arcuate and VMH nuclei, as well as decreased Prlr-L in the arcuate nucleus of prolactin-infused "extended pseudopregnancy" rats. These data suggest that the attenuation of the leptin-induced suppression of food intake caused by chronically high lactogen levels in pseudopregnant rats is associated with impaired leptin-induced activation of the JAK/STAT pathway in specific hypothalamic nuclei.