Prolactin Gene Research Articles

Indel variants within the PRL and GHR genes associated with sheep litter size.

Growth hormone and prolactin belong to the class of peptide hormones that have a wide range of regulatory functions. In this study, polymorphisms of growth hormone receptor (GHR) and prolactin (PRL) genes were analysed as candidate genes, which are responsible for the litter size in Australian White (AUW) sheep. According to the statistical analyses results, the polymorphism information content (PIC) values of the PRL-P1-ins-23bp, GHR-P2-del-23bp and GHR-P8-del-23bp were 0.371, 0.366 and 0.375, respectively, which indicates the high genetic polymorphism in AUW sheep. Moreover, all indel loci are not conformed to the HWE (p<.05). Further, our findings revealed that the PRL-P1-ins-23bp polymorphism in the ovine PRL gene was significantly related to the first parity litter size (p=.001) and the DD genotype displaying the highest genotypic mean. Meanwhile, the GHR-P2-del-23bp and GHR-P8-23bp indels in the ovine GHR gene were significantly correlated with first parity litter size (p<.05), and the individuals with the genotype II showed significantly higher litter size than others. Collectively, these results demonstrated that our findings could be useful for future sheep breeding strategies based on the molecular-assisted selection (MAS).

Diverse effects of phytoestrogen biochanin A on rat pituitary tumor cells

Biochanin A, a nonsteroidal estrogen present in plants, has been suggested in epidemiological studies to be associated with a reduced risk of hormone-related cancers. In this study, we examined the effects of biochanin A on the cell growth and gene expression of an estrogen-responsive pituitary cell line GH3. Results showed that biochanin A inhibited growth of GH3 cells in a dose-dependent manner and at physiologically relevant concentrations. At concentrations of 1 and 5 μg/mL, inhibition of cell growth was observed. The gene expressions of estrogen receptor (ER) mRNA and an estrogen responsive gene, the progesterone receptor (PR), were down-regulated by the biochanin A treatment. In addition, co-administration of antiestrogen ICI182,780 blocked this biochanin A induced down-regulation of the ER and PR mRNA level. However, the expression level of rat prolactin (rPRL) gene, one of the typical estrogen responsive genes, was not regulated by biochanin A. These results indicated that biochanin A at physiologically achieveable concentrations was able to inhibit cell growth of estrogen-responsive rat pituitary cells. In addition, dietary-derived biochanin A compounds exhibit diverse estrogenic and antiestrogenic effects on estrogen-related gene expression, suggesting gene-specific regulation.

Obesity and regulation of human placental lactogen production in pregnancy.

The four genes coding for placental members of the human (h) growth hormone (GH) family include two that code independently for placental lactogen (PL), also known as chorionic somatomammotrophin hormone, one that codes for placental growth hormone (PGH) and a pseudogene for which RNA but no protein product is reported. These genes are expressed preferentially in the villus syncytiotrophoblast of the placenta in pregnancy. In higher primates, the placental members, including hPL and PGH, are the result of multiple duplication events of the GH gene. This contrasts with rodents and ruminants, where PLs result from duplication of the prolactin (PRL) gene. Thus, unlike their mouse counterparts, the hPL and PGH hormones bind both lactogenic and somatogenic receptors with varying affinity. Roles influenced by nutrient availability in both metabolic control in pregnancy and maternal behaviour are supported. However, the effect maternal obesity has on the activation of placental members of the hGH gene family, particularly the expression and function of those genes, is poorly understood. Evidence from partially humanised hGH/PL transgenic mice indicates that both the remote upstream hPL locus control region (LCR) and more gene-related regulatory regions are required for placental expression in vivo. Furthermore, a specific pattern of interactions between the LCR and hPL gene promoter regions is detected in term placenta chromatin from women with a normal body mass index (BMI) in the range 18.5-25kgm-2 by chromosome conformation capture assay. This pattern is disrupted with maternal obesity (class II BMI>35kgm-2 ) and associated with a >40% decrease in term hPL RNA levels, as well as serum hPL but not PRL levels, during pregnancy. The relative importance of the chromosomal architecture and predicted properties for transcription factor participation in terms of hPL production and response to obesity are considered, based on comparison with components required for efficient human pituitary GH gene expression.

PCR- RFLP Polymorphism at Exon 3 and Promoter Region of Prolactin Gene in Gaolao Cattle

The present study was planned to investigate significant polymorphism in exon3 and promoter regions of prolactin gene in Gaolao cattle and its association (if any) with milk components. A total of 50 randomly selected Gaolao cows were genotyped for PRLGI-RsaI and PRLGIII-Tsp45I loci using PCR-RFLP technique. The PRLGI-RsaI locus was polymorphic and PRLGIII-Tsp45I locus was monomorphic indicating genetic variation at PRLGI-RsaI locus in Gaolao cattle. The allele frequency for the PRLA alleles was found 0.933 and for allele PRLB was 0.067 at PRLGI–RsaI locus. The result of analysis variance showed significant (PÂ0.05) effect of AA genotype on fat % as compared to AB genotype; however non-significant effect of these genotypes was found on protein %, SNF % and lactose % which may be due to small sample resulting in higher error variance. The results of present study indicated that identified prolactin gene polymorphism at PRLGI-RsaI in exon 3 after validation in large breeding populations having precise data records may prove as important candidate gene marker for selection and breeding decisions in genetic improvement of indigenous cattle for milk productivity.

Prolactin gene polymorphism in crossbred cattle of Assam and its association with productive and reproductive traits

The present investigation was designed to study the performance of crossbred cows as well as to identify polymorphism in genomic sequences of PRL gene and its association with various reproductive and productive traits of crossbred cows maintained at the Bull Mother Farm, Barapeta, Assam. A total of 40 cows were utilized to study the polymorphism of the PRL gene, the results of which showed polymorphic banding pattern in most of the samples of crossbred cows with respect to the gene under study. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism studies on PRL gene in crossbred cows, using Rsa I restriction enzyme revealed three types of fragment pattern, arbitrarily designated as AA (156 bp), AB (74, 82 and 156 bp), BB (74 and 82 bp) genotype. The frequencies of A and B alleles were calculated as 0.534 and 0.466 and those of AA, AB and BB genotypes as 0.334, 0.400 and 0.266 respectively. The prolactin genotypes were found to be significantly related to age at sexual maturity, age at first calving, first lactation milk yield. The crossbred cattle with AB genotype showed better performance than those with AA and BB genotype in the population studied.

Genotypic confirmation of non-broody behaviour of white leghorn by 24bp indel(s) polymorphism at the promoter region of prolactin gene

Broodiness is a sex-linked observable trait in most of the domestic hens but in case of White Leghorn breed this trait was found to be seldom. Prolactin hormone plays a major role in broodiness or incubation behaviour in birds. The main aim of the present study was genotypic confirmation of non-broody behaviour of White Leghorn by identifying a 24bp insertion-deletion (indel)s at the promoter region of prolactin gene. A total of 200 birds of White Leghorn were randomly selected from All India Co-ordinated Research Project (AICRP) on Poultry Improvement, Mannuthy, Thrissur, Kerala. Blood samples were collected from the selected birds and isolation of genomic DNA was done. Polymerase chain reaction (PCR) assay was carried out to identify a 24bp indel(s). Based on the polymorphic patterns, birds were categorized to three different genotype groups namely, II, ID and DD. Association of non-broody behaviour and egg production with 24bp insertion polymorphism was analyzed in White Leghorn. Our findings support the view that loss of maternal incubation behaviour in the White Leghorn breed is the result of 24bp insertion polymorphism and intensive selection which improves the egg laying persistency.

Прогностическая модель риска развития гиперпролактинемии у пациентов с шизофренией на фоне антипсихотической терапии

Использование антипсихотических средств ассоциировано с повышенным уровнем пролактина. Лекарственно-индуцированная гиперпролактинемия (ГП) имеет не только краткосрочные, но и долгосрочные последствия, которые могут серьезно повлиять на качество жизни пациента. Клинические проявления ГП включают гинекомастию, галакторею, нарушения менструального цикла, сексуальные дисфункции, бесплодие, а также значимый рост вероятности развития остеопороза и онкологических заболеваний. В развитии данного побочного эффекта антипсихотической терапии участвуют не только экзогенные, но и генетические факторы. Целью данной работы было исследование клинических и молекулярно-генетических факторов развития лекарственно-индуцированной ГП и создание прогностической модели риска развития ГП, которая в перспективе может быть использована для оптимизации и персонализации назначаемой терапии при лечении шизофрении. Исследование проведено в выборке русских, проживающих в Западно-Сибирском регионе России, больных шизофренией (n=446). Средний возраст пациентов составил 41,5 ± 13,4 года (возрастной диапазон - от 18 до 65 лет). Клиническая симптоматика оценивалась по шкалам позитивных и негативных синдромов (Positive and Negative Syndrome Scale - PANSS), общего клинического впечатления (Clinical Global Impression - CGI), оценки побочного действия (Udvalg for Kliniske Undersogelser Scale - UKU), на всех пациентов заполнялся модифицированный вариант карты стандартизированного описания больного шизофренией. На основании содержания гормона пролактина в сыворотке крови были выделены подгруппы пациентов с ГП (n=227) и с нормальным уровнем пролактина (n=219). Выполнен молекулярно-генетический анализ 88 полиморфных вариантов генов серотониновых (HTR2C, HTR3A, HTR3B, HTR6, HTR2A, HTR1A, HTR1B) и дофаминовых (DRD1, DRD2, DRD2/ANKK1, DRD3, DRD4) рецепторов, дофаминового транспортера SLC6A3, переносчика норадреналина SLC6A2, системы ферментов цитохромов P450 (CYP1A2*1F, CYP2D6*3, CYP2D6*4, CYP2C19*3, CYP2C19*17, CYP2C19*2), катехол-О-метилтрансферазы СОМТ, p-глутатион S-трансферазы GSTP1, ATXN1, KREMEN1 и пролактина PRL. Получена прогностическая модель с включением генетических маркеров «rs1176744» (HTR3B), «rs10042486» (HTR1A), «rs936461» (DRD4), «rs179997» (ATXN1), «rs1076562» (DRD2), «rs3773678» (DRD3), «rs167771» (DRD3), «rs1587756» (DRD3), «rs134655» (KREMEN1),»rs3892097» (CYP2D6*4), «rs1341239» (PRL), «rs4975646» (SLC6A3), «rs13333066» (SLC6A2), а также негенетических факторов, таких как пол, возраст и доза антипсихотика в хлорпромазиновом эквиваленте (CPZeq). Разработанную фармакогенетическую панель можно рассматривать в качестве биологического предиктора развития антипсихотик-индуцированной ГП при шизофрении до назначения фармакотерапии. The use of antipsychotics is associated with elevated prolactin levels. Drug-induced hyperprolactinemia (HP) has not only short-term, but also long-term consequences, which can seriously affect the patient’s quality of life. Clinical manifestations of HP include gynecomastia, galactorrhea, menstrual irregularities, sexual dysfunctions, infertility, as well as a significant increase in the likelihood of developing osteoporosis and cancer. Not only exogenous, but also genetic factors are involved in the development of this side effect of antipsychotic therapy. The aim of this work was to study the clinical and molecular genetic factors of the development of drug-induced HP and the creation of a prognostic model of the risk of developing HP, which in the future can be used to optimize and personalize the prescribed therapy in the treatment of schizophrenia. The study was conducted in populations of russians with schizophrenia (n = 446), living in the West Siberian region of Russia. The average age of patients was 41,5 ± 13,4 years (18 - 65 age range). Clinical symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS), the Clinical Global Impression scale (CGI), the side effect rating scale (Udvalg for Kliniske Undersogelser Scale - UKU), and a modified version of the card of a standardized description of patient with schizophrenia. Based on the results on the serum prolactin level patients were divided into subgroups: with HP (n = 227) and with normal prolactin levels (n = 219). Molecular genetic analysis of 88 polymorphic variants of serotonin receptors genes (HTR2C, HTR3A, HTR3B, HTR6, HTR2A, HTR1A, HTR1B) and dopamine receptors genes (DRD1, DRD2, DRD2 / ANKK1, DRD3, DRD4), dopamine transporter gene SLC6A3, noradrenaline transporter gene SLC6A2, genes of the cytochrome P450 enzymes (CYP1A2*1F, CYP2D6*3, CYP2D6*4, CYP2C19*3, CYP2C19*17, CYP2C19*2), gene of catechol-O-methyltransferase COMT, gene of glutathione S-transferase P (GSTP1), genes ATXN1, KREMEN1 and prolactin gene PRL was performed. A prognostic model was obtained with the inclusion of the genetic markers “rs1176744” (HTR3B), “rs10042486” (HTR1A), “rs936461” (DRD4), “rs179997” (ATXN1), “rs1076562” (DRD2), “rs3773678” (DRD3), “rs167771” (DRD3), “rs1587756” (DRD3), “rs134655” (KREMEN1), “rs3892097” (CYP2D6*4), “rs1341239” (PRL),”rs4975646” (SLC6A3),”rs1333302” (SLC6A2), as well as non-genetic factors such as gender, age and dose of antipsychotic in chlorpromazine equivalent (CPZeq). The developed pharmacogenetic panel can be considered as biological predictors of the development of antipsychotic-induced HP in schizophrenia before the appointment of pharmacotherapy.

Activin-inhibitory action on lactotrophs is decreased in lactotroph hyperplasia.

Among all the hormone-secreting pituitary tumours, prolactinomas are the most frequently found in the clinic. Since dopamine is the primary inhibitor of lactotroph function, dopamine agonists represent the first-line therapy. However, a subset of patients exhibits resistance to these drugs, and therefore, alternative treatments are desired. As activins inhibit prolactin gene expression through the inhibition of Pit-1 involving the p38MAPK pathway, in the present work, we studied the local activin system as an alternative inhibitory system for lactotroph hyperplasia treatment. We used two different mouse models of prolactinoma: transgenic mice with overexpression of the human chorionic gonadotropin β-subunit (hCGβ) and mice lacking dopamine receptor type 2. In both models, females, but not males, develop lactotroph hyperplasia from the fourth month of life. We found reduced expression of pituitary activin subunits and activin receptors in hyperplastic pituitaries from both models compared with wild-type counterparts. Consequently, hyperplastic pituitaries presented a reduced activin-inhibitory action on prolactin secretion. Additionally, while female wild-type lactotrophs presented high levels of phospho-p38MAPK, it was lost in prolactinomas, concomitant with decreased activin expression, increased Pit-1 expression and tumour development. In contrast, male pituitaries express higher mRNA levels of activin subunits βA and βB, which would suggest a stronger activin inhibitory function on lactotrophs, protecting this sex from tumour development, despite genotype. The present results highlight the importance of the activin inhibitory action on lactotroph function and place the local activin system as a new target for the treatment of dopamine agonist-resistant prolactinomas.

Cloning and molecular characterization of PRL and PRLR from turbot (Scophthalmus maximus) and their expressions in response to short-term and long-term low salt stress.

The pituitary hormone prolactin (PRL) regulates salt and water homeostasis by altering ion retention and water uptake through peripheral osmoregulatory organs. To understand the role of PRL and its receptor (PRLR) in hypoosmoregulation of turbot (Scophthalmus maximus), we characterized the PRL and PRLR gene and analyzed the tissue distribution of the two genes and their gene transcriptional patterns in the main expressed tissues under long-term and short-term low salt stress. The PRL cDNA is 1486bp in length, incorporating an ORF of 636bp with a putative primary structure of 211 residues. And the PRLR cDNA is 2849bp in length, incorporating an ORF of 1944bp with a putative primary structure of 647 residues. The deduced amino acid sequences of these two genes shared highly conserved structures with those from other teleosts. Quantitative real-time PCR results showed that PRL transcripts were strongly expressed in the pituitary and very weakly in brain, but were hardly expressed in other tissues. PRLR transcripts were most abundant in the kidney, to a lesser extent in the gill, intestine, brain, and spleen, and at low levels in the pituitary and other tissues examined. The expression of PRL in the pituitary increased after short-term or long-term low salt stress, and the highest expression level appeared 12h after stress (P < 0.05). And there is no significant difference between both low salt group (5ppt and 10ppt) at each sampling point. The variation of PRLR expression in gill under short-term low salt stress is similar to that of PRL gene in pituitary, with highest value in 12h (P < 0.05). However, the expression under long-term low salt stress was significantly higher than control group even than 12h group under 5ppt (P < 0.05). The expression of PRLR in the kidney increased first and then decreased after low salt stress, and the highest value also appeared in 12h after stress and there was no significant difference between the salinity groups. After long-term low salt stress, the expression level also increased significantly (P < 0.05), but it was flat with 24h, which was lower than 12h. The variation of PRLR expression in the intestine was basically consistent with that in the kidney. The difference was that the expression level of 24h after stress in the 5ppt group was significantly higher than that of the 10ppt group (P < 0.05). After a comprehensive analysis of the expression levels of the two genes, it can be found that the expression level increased and peaked at 12h after short-term low salt stress, indicating that this time point is the key point for the regulation of turbot in response to low salt stress. This also provides very important information for studying the osmotic regulation of turbot. In addition, our results also showed that the expression of PRLR was stable in the kidney and intestine after long-term low salt stress, while the expression in the gill was much higher than short-term stress. It suggested that PRL and its receptors mainly exert osmotic regulation function in the gill under long-term low salt stress. At the same time, such a result also brings a hint for the low salt selection of turbot, focusing on the regulation of ion transport in the gill.

Short Communication: Prolactin and x-collagen genes polymorphism in Central Javanese local ducks, Indonesia

Abstract. Susanti R, Yuniastuti A. 2020. Short Communication: Prolactin and x-collagen genes polymorphism in Central Javanese local ducks, Indonesia. Biodiversitas 21: 605-610. Genetic conservation is an effective approach to preserve unique and protected species in Indonesia, including indigenous duck (Anas platyrhynchos Linnaeus, 1758). One of the most important things is an identification effort by studying genetic characteristics to support genetic resource conservation programs. This study aim was to analyze the genetic polymorphisms of Central Javanese local duck based on prolactin (PRL) and collagen-x (COLX) genes. A total of 35 local ducks in Central Java was collected as samples, the duck’s calamus feathers were taken for tissue DNA isolation. Polymorphism analysis of PRL and COLX genes were performed using single nucleotide polymorphism (SNP) markers and PCR-RFLP method. The amplicons were then digested with DraI restriction enzyme (AhaIII) (5'TTT/ AAA) for PRL gene, and HaeIII (BsuRI) enzyme (5'GG/ CC-3 ') for COLX gene. The genotype was determined based on RFLP PCR amplicon band results from each target gene. For each gene, data on genotype frequencies, allele frequencies, expected heterozygosities (HE), and Hardy-Weinberg equilibrium were analyzed using PopGene32 software. Based on the PRL gene, there were 25 ducks (71.43%) with BB genotype and 10 ducks (28.57%) with AB genotype. While based on the COLX gene, as many as 25 ducks (71.43%) had TT genotypes, 9 ducks (25.71%) had TC types and 1 duck (1.86%) had CC types. Both PRL and COLX genotypes were clustered the Central Javanese local ducks into 5 haplotypes (A-E). The high allele frequency has belonged to B allele (85.71%) of the PRL gene and T allele (84.25%) of the COLX gene that showed high egg production and quality. Allele B (PRL gene) and T allele (COLX gene) could be predicted as weight eggs’ alleles marker.

Prolactin gene polymorphisms and associations with reproductive traits in Indonesian local ducks.

Background and Aim:Reproductive traits play an important role in population increases and the egg production (EP) abilities of Indonesian local ducks (ILD). The prolactin (PRL) gene is a single chain polypeptide hormone belonging to a family of growth hormone genes that are mainly synthesized in the anterior pituitary gland in all vertebrates. It has a significant effect on reproductive traits and EP. Single nucleotide polymorphisms (SNPs) present in PRL are a useful molecular marker for EP. This study aimed to identify the PRL polymorphisms based on these SNPs and to uncover the associations with reproductive traits in ILD.Materials and Methods:A total of 280 ILDs consisting of Tegal and Magelang (F0) ducks and their reciprocal crosses, namely, Gallang (F1) and Maggal (F1), were maintained and specific variables were recorded, that is, age at first egg, body weight at first egg, first egg weight, and EP, for 90 days. Allele and genotype frequencies were used to determine the Hardy-Weinberg (H-W) equilibrium. The association between the SNP genotypes of PRL and reproductive traits was analyzed using one-way analysis of variance, following the GLM procedure of SAS. The genotypic effects on the reproductive traits were determined using regression analysis.Results:This study successfully amplified a polymerase chain reaction product of 190 bp, which was used to identify the SNP. Results indicated that PRL in ILDs is polymorphic. A SNP was found at position 164 nt (c.164G >A), consisting of three different genotypes, namely, GG, GA, and AA. The genotypes of Tegal and Magelang (F0), and Gallang (F1) populations were not in H-W equilibrium. The Maggal population (F1) was in H-W equilibrium. Significant associations were detected between the genotypes and EP in all ILDs (p<0.01), following a regression line of y=2.337x+64.605, with a determination coefficient of 0.0188 (r=0.14).Conclusion:PRL can be recommended as a candidate gene for reproductive traits in ILD, especially EP.

Genetic structure features of cattle populations of Ukrainian selection by polymorphism of loci, associated with milk productivity traits

Aim. To study the genetic structure of cattle populations of Ukrainian selection by polymorphism of functional genes (PRL, PL) and microsatellites (BM027, RM185). Methods. The study was conducted using the method of polymerase chain reaction (PCR) and restriction analysis in case of loci PRL and PL, and using classic PCR with subsequent electrophoresis in polyacrylamide gel to analyze microsatellite variability. Results. The results of the studies demonstrated that the locus of prolactin by RsaI-polymorphism in the fourth exon was polymorphic in both experimental populations (Ukrainian Black-and-White and Ukrainian Red-and-White dairy breeds of cattle). The mutation (Indel) was fi rst determined in the fourth exon of prolactin gene, the variants of which correlated with some alleles of the locus by RsaI-polymorphism. The locus of placental lactogen by RsaI-polymorphism in the fi fth exon was monomorphic in both experimental populations. Microsatellite locus RM185 was polymorphic in both groups of animals, whereas BM027 – only in the Black-and-White dairy breed. Conclusions. The specifi cities of the genetic structure of the Ukrainian Black-and-White and Red-andWhite dairy breed populations by polymorphism of functional genes and microsatellite loci were determined. The locus of placental lactogen by RsaI-polymorphism in the fi fth exon cannot be used in further studies due to the absence of alternative variants of the gene in both studied populations of animals. The analysis of the distribution of haplotype frequencies demonstrated the absence of deviation from the equilibrium state by linkage for each of the investigated markers which makes their use impossible in the breeding programs as a separate functional unit.

Changes in PRL Gene Expression During Upstream Movement of the Japanese Eel, Anguilla japonica.

Changes in mRNA levels of prolactin (PRL) during upstream movement were examined in juvenile Japanese eels, Anguilla japonica. Glass eels and elvers were collected from 2007 to 2009 near the entrance of Hamana Lake, and in a small inflowing stream, the Egawa River. Quantification of mRNA was performed by real-time PCR and expressed as whole-body content. PRL mRNA levels of glass eels caught in the coastal zone and tidal area were low. Eels that moved downward in the tidal zone and migrated upstream to enter into freshwater showed increased levels of PRL mRNA. These changes suggest the importance of up-regulation of PRL gene expression in juvenile eels during their upstream movement from seawater to fresh water, particularly in relation to hyperosmoregulation.

The effect of alleles of the prolactin gene of cows of the black-motley holstein breed on the treatment of mastitis

The effect of alleles of the prolactin gene of cows of the black-motley holstein breed on the treatment of mastitis

Identification of single nucleotide polimorphisms and restriction enzym on prolactin gene in Alabio and Mojosari duck

Prolactin plays important roles in avian reproduction as it induces broody behavior and regulates follicular development. The objectives of this study were to detect Single Nucleotide Polymorphisms (SNPs) of prolactin gene in Alabio and Mojosari ducks and determine the restriction enzymes for genotyping. Genomic DNA was isolated from 50 blood samples per each Alabio and Mojosari ducks. PCR amplification and sequencing were carried out to identify the SNP. In this study, SNP T-6068C was found based on three GenBank sequences alignment. Two SNPs (C-5796A and T-5817C) in intron 4 region were detected based on sequences of Alabio and Mojosari PCR products. Enzymes Fok1, BtsCI, BsrI were detected to recognize the SNP C-5796A. SNP T-6068C can be digested by enzym TspDTI. However no enzym was detected to recognize SNP T-5817C. In conclusion the SNPs detected from this study may be used in future studies to investigate the association of prolactin gene and egg production traits in Alabio and Mojosari ducks.

MiR-1299 promotes the synthesis and secretion of prolactin by inhibiting FOXO1 expression in drug-resistant prolactinomas

Prolactinoma is a clinically common intracranial tumor. When serum prolactin levels are not controlled despite administration of a dopamine agonist, the condition is referred to as drug-resistant prolactinoma. The mechanism underlying persistent prolactin secretion in drug-resistant prolactinoma remains unclear. MicroRNAs play an important role in tumorigenesis and development as well as chemotherapeutic resistance. This study was conducted to investigate the mechanism by which miRNA regulates prolactin secretion in drug-resistant prolactinoma. We first found that miR-1299 was elevated in drug-resistant prolactinoma and inhibited FOXO1 in a targeted manner through miRNA sequencing and luciferase assays. We then confirmed that FOXO1 binds to the promoter of the prolactin gene to inhibit its expression through chromatin immunoprecipitation-quantitative PCR and cytological experiments. Finally, inhibition or overexpression of miR-1299 in primary tumor cells confirmed that drug-resistant prolactinoma promoted prolactin secretion by promoting miR-1299 expression and reducing intracellular FOXO1. These results indicate that FOXO1 and miR-1299 are potential therapeutic targets for drug-resistant prolactinoma as well as other pituitary diseases.

GH3 and RC-4BC cell lines are not suitable as in vitro models to study prolactin modulation and AHR responsiveness in rat pituitary

Some environmental contaminants and pharmaceuticals increase the incidence of uterine tumors in toxicological studies with rats. These tumors can result from a hormonal imbalance due to rat-specific disrupted pituitary prolactin regulation, and are therefore of questionable relevance for humans. In this study we compared in vitro prolactin regulation in rat primary pituitary cells to that in pituitary cell lines, GH3 and RC-4BC. Moreover, we assessed the potential effects of aryl hydrocarbon receptor (AHR) activation on prolactin regulation by using two different AHR agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and DELAQ, the N-deethylated minor metabolite of the pharmaceutical laquinimod.In rat primary pituitary cells, known prolactin stimulant thyrotropin-releasing hormone (TRH) marginally increased prolactin secretion (1.2-fold) and gene expression (1.3-fold). In contrast, synthetic dopamine receptor agonist quinpirole, a known inhibitor of prolactin release, significantly inhibited prolactin secretion (2.6-fold) and gene expression (3.6-fold). In GH3 cells, TRH strongly increased prolactin secretion (6.8-fold) and gene expression (30.8-fold), whereas quinpirole did not affect prolactin secretion nor gene expression. In RC-4BC cells, both TRH and quinpirole did not modulate prolactin secretion nor gene expression. Prolactin secretion and gene expression did not significantly change upon exposure to TCDD or DELAQ. However, DELAQ, but not TCDD, attenuated quinpirole-inhibited prolactin gene expression by 51% in primary pituitary cells.This study shows that pituitary prolactin regulation in rat primary pituitary cells in vitro is distinctly different from rat pituitary cell lines GH3 and RC-4BC. Therefore, effects on pituitary prolactin regulation in vitro should best be performed using rat primary pituitary cells. Additionally, AHR ligands may interact with rat pituitary prolactin regulation, but this appears to depend on the ligand and constitutive prolactin secretion. However, interpretation of the in vitro results with respect to occurrence of uterine tumors in rats should take the complex regulation of prolactin release in the pituitary into account as well as the in vivo hypothalamus-pituitary-gonadal (HPG) axis and its feedback loops.

Evaluation of screening for drug use using postmortem prolactin levels in serum and cerebrospinal fluid.

Prolactin (PRL) levels can usually be controlled by PRL-inhibiting psychiatric drugs that include anti-dopamine agents. However, the use of dopamine (DA) antagonists may lead to hyperprolactinemia under certain clinical conditions. The aim of this study was to investigate postmortem PRL levels as potential markers of drug abuse, especially that of DA antagonists, in autopsy cases. We examined 121 autopsy cases, excluding cases involving acute hypoxia/ischemia, such as asphyxia, because PRL concentrations are reportedly increased under acute hypoxic conditions. Detected drugs were classified as either DA antagonists, stimulants, psychotropic drugs other than DA antagonists, or other non-psychotropic drugs, and many cases had no detected drugs. Samples comprised blood collected from the right heart chamber and cerebrospinal fluid (CSF). PRL protein level was measured by chemiluminescent immunoassay, and PRL gene expression in the anterior pituitary of autopsy cases was analyzed by reverse transcription-polymerase chain reaction. The PRL-positive cell ratio in the anterior pituitary gland was also measured by immunohistochemical analysis. The results indicated that PRL levels in the serum and CSF were higher in DA antagonist cases than in other cases. PRL levels in the serum and CSF also correlated with the PRL gene expression in cases with abuse of DA antagonists. However, no significant difference in the PRL-positive cell ratio in the anterior pituitary gland was evident between any of the classes of drug-detected and drug-undetected cases. These results suggest that postmortem measurements of PRL transcription levels may be useful for diagnosing cases of DA antagonist use.

Inhibition of epidermal growth factor receptor stimulates prolactin expression in primary culture of the mouse pituitary gland.

The roles of epidermal growth factor (EGF) in the regulation of prolactin (PRL) gene expression in the normal pituitary gland remain poorly understood. In the present study, the effects of EGF and an inhibitor of the EGF receptor, erlotinib, on PRL gene expression were examined both in the pituitary tumour cell line GH3 and in a primary culture of the mouse pituitary gland under similar experimental conditions. The results showed that EGF stimulated PRL expression in GH3 cells, but not in normal cells. Erlotinib was found to counteract EGF in GH3 cells inhibiting the PRL expression enhanced by EGF. By contrast, erlotinib induced an elevation in the PRL mRNA levels in the primary culture of the adult pituitary gland and the initiation of PRL production in the culture of the foetal pituitary gland in which PRL production had not yet occurred. Western blot analyses showed that EGF induced and erlotinib inhibited the activation of extracellular regulated protein kinase equally in GH3 and normal cells. These results suggest that the consequences of EGF receptor activation in normal PRL cells contradict those in adenomatous PRL cells.

Polymorphism of prolactine genes and its association with body weight in Bayang ducks, local duck from West Sumatera, Indonesia

This study aims to determine the polymorphism of Prolactin (PRL | XbaI and PRL | DraI) genes in Bayang ducks using the PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) technique and its association with weight of ducks aged 1-10 weeks. This study used 200 Bayang duck blood samples consisting of 102 male ducks (♂) and 98 female ducks (♀). DNA from blood samples was isolated using the Genomic DNA Purifification Kit (Promega) using protocol from the manucfacture. The DNA was then amplified using two primers with F: 5′-AAA TTC CCT CTC ACA GTT ACA-3′ and R: 5′-GAT GCA GAG ACA AGT TTC ACC-3′ and F: 5′-GAATAGAACACTTGACCCTG-3′ and R: 5′-TAGAGGAGGCAAGCATAG-3′ which produces fragments with a length of 416 bp and 566 bp. Restriction with XbaI enzymes that recognized the binding site (5-TT CTAGA-3′) resulted 3 genotypes: homozygote (+/+), heterozygote (+/−) and homozygote (−/−) with frequencies 0.455, 0.495 and 0.050 respectively and with frequency allel (+) 0.702 and frequency allel (−) 0.297. While the results of the restriction with enzyme DraI found three types of genotypes, namely (+/+), (+/−) and (−/−) with frequencies of 0.64, 0.35 and 0.01 respectively with frequency allel (+) 0.82 and allele frequencies (−) 0.18. From the results of the analysis, it was found that there was no relationship between these two diversity and weight ducks of duck.