The immunotoxin targeting PRLR increases tamoxifen sensitivity and enhances the efficacy of chemotherapy in breast cancer.

BACKGROUND: Though tamoxifen achieves success in treating estrogen receptor α (ERα)-positive breast cancer, the followed development of tamoxifen resistance is a common challenge in clinic. Signals downstream of prolactin receptor (PRLR) could synergize with ERα in breast cancer progression. However, the potential effect of targeting PRL-PRLR axis combined with tamoxifen has not been thoroughly investigated. METHODS: High-throughput RNA-seq data obtained from TCGA, Metabric and GEO datasets were analyzed to explore PRLR expression in breast cancer cell and the association of PRLR expression with tamoxifen treatment. Exogenous or PRL overexpression cell models were employed to investigate the role of activated PRLR pathway in mediating tamoxifen insensitivity. Immunotoxin targeting PRLR (N8-PE24) was constructed with splicing-intein technique, and the efficacy of N8-PE24 against breast cancer was evaluated using in vitro and in vivo methods, including analysis of cells growth or apoptosis, 3D spheroids culture, and animal xenografts. RESULTS: PRLR pathway activated by PRL could significantly decrease sensitivity of ERα-positive breast cancer cells to tamoxifen. Tamoxifen treatment upregulated transcription of PRLR and could induce significant accumulation of PRLR protein in breast cancer cells by alkalizing lysosomes. Meanwhile, tamoxifen-resistant MCF7 achieved by long-term tamoxifen pressure exhibited both upregulated transcription and protein level of PRLR. Immunotoxin N8-PE24 enhanced sensitivity of breast cancer cells to tamoxifen both in vitro and in vivo. In xenograft models, N8-PE24 significantly enhanced the efficacy of tamoxifen and paclitaxel when treating PRLR-positive triple-negative breast cancer. CONCLUSIONS: PRL-PRLR axis potentially associates with tamoxifen insensitivity in ERα-positive breast cancer cells. N8-PE24 could inhibit cell growth of the breast cancers and promote drug sensitivity of PRLR-positive breast cancer cells to tamoxifen and paclitaxel. Our study provides a new perspective for targeting PRLR to treat breast cancer.

Significance of Duodenal Prolactin Receptor Modulation by Calcium and Vitamin D in Sulpiride-Induced Hyperprolactinemia.

Background and Objectives: Hyperprolactinemia, as a potential side-effect of some antipsychotic medications, is associated with decreased bone density and an increased risk of fractures. This study investigates whether calcium and vitamin D supplementation affects prolactin receptor (Prlr) gene expression in the duodenum, vertebrae, and kidneys of female rats with sulpiride-induced hyperprolactinemia. Materials and Methods: Twenty-one-week-old female Wistar rats were assigned to three groups: Group S consisted of ten rats who received sulpiride injections (10 mg/kg) twice daily for 6 weeks; Group D (10 rats) received daily supplementation of 50 mg calcium and 500 IU vitamin D along with sulpiride for the last 3 weeks; and Group C consisting of seven age-matched nulliparous rats serving as a control group. Real-time PCR was used to assess Prlr gene expression in the duodenum, vertebrae, and kidneys. Results: In Group S, Prlr gene expression was notably decreased in the duodenum (p < 0.01) but elevated in the vertebrae and kidneys compared to Group C. Conversely, Group D exhibited significantly increased Prlr expression in the duodenum (p < 0.01) alongside elevated expression in the vertebrae and kidneys. Conclusions: In sulpiride-induced hyperprolactinemia, decreased Prlr gene expression in the duodenum may lead to reduced intestinal calcium absorption. Consequently, prolactin may draw calcium from the skeletal system to maintain calcium balance, facilitated by increased Prlr gene expression in the vertebrae. However, vitamin D supplementation in sulpiride-induced hyperprolactinemia notably enhances Prlr gene expression in the duodenum, potentially ameliorating intestinal calcium absorption and mitigating adverse effects on bone health.

Prolactin receptor potentiates chemotherapy through miRNAs-induced G6PD/TKT inhibition in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a notoriously dismal prognosis. As a competitive inhibitor of DNA synthesis, gemcitabine is the cornerstone drug for treating PDAC at all stages. The therapeutic effect of gemcitabine, however, is often hindered by drug resistance, and the underlying mechanisms remain largely unknown. It is unclear whether their response to chemotherapeutics is regulated by endocrine regulators, despite the association between PDAC risk and endocrine deregulation. Here, we show that prolactin receptor (PRLR) synergizes with gemcitabine in both in vitro and in vivo treatment of PDAC. Interestingly, PRLR promotes the expression of miR-4763-3p and miR-3663-5p, two novel miRNAs whose functions are unknown. Furthermore, the analysis of transcriptome sequencing data of tumors from lactating mouse models enriches the PPP pathway, a multifunctional metabolic pathway. In addition to providing energy, the PPP pathway mainly provides a variety of raw materials for anabolism. We demonstrate that two key enzymes of the pentose phosphate pathway (PPP), G6PD and TKT, are directly targeted by miR-4763-3p and miR-3663-5p. Notably, miR-4763-3p and miR-3663-5p diminish the nucleotide synthesis of the PPP pathway, thereby increasing gemcitabine sensitivity. As a result, PRLR harnesses these two miRNAs to suppress PPP and nucleotide synthesis, subsequently elevating the gemcitabine sensitivity of PDAC cells. Also, PDAC tissues and tumors from LSL-KrasG12D/+, LSL-Trp53R172H/+, and PDX1-cre (KPC) mice exhibit downregulation of PRLR. Bisulfite sequencing of PDAC tissues revealed that PRLR downregulation is due to epigenetic methylation. In this study, we show for the first time that the endocrine receptor PRLR improves the effects of gemcitabine by boosting two new miRNAs that block the PPP pathway and nucleotide synthesis by inhibiting two essential enzymes concurrently. The PRLR-miRNAs-PPP axis may serve as a possible therapeutic target to supplement chemotherapy advantages in PDAC.

Involvement of somatotrophic hormones in the postpartum regulation of ovarian activity in mares.

Mares resume ovarian activity rapidly after foaling. Besides follicle-stimulating hormone (FSH) and luteinizing hormone (LH), the pituitary synthesizes prolactin and growth hormone which stimulate insulin-like growth factor (IGF) synthesis in the liver. We tested the hypothesis that follicular growth is initiated already antepartum, mares with early and delayed ovulation differ in IGF-1 release and that there is an additional IGF-1 synthesis in the placenta. Plasma concentrations of LH, FSH, IGF-1, IGF-2, activin and prolactin. IGF-1, IGF-2, prolactin and their receptors in placental tissues were analyzed at the mRNA and protein level. Follicular growth was determined from 15 days before to 15 days after foaling in 14 pregnancies. Mares ovulating within 15 days postpartum formed group OV (n=5) and mares not ovulating within 15 days group NOV (n=9). Before foaling, follicles with a diameter >1 cm were present in all mares and their number increased over time (p<0.05). Follicle growth after foaling was more pronounced in OV mares (day p<0.001, group p<0.05, day x group p<0.05) in parallel to an increase in LH concentration (p<0.001, day x group p<0.001) while FSH increased (p<0.001) similarly in both groups. Plasma concentrations of IGF-1 and prolactin peaked one day after foaling (p<0.001). The IGF-1 mRNA abundance was higher in the allantochorion but lower in the amnion of OV versus NOV mares (group p=0.01, localization x group p<0.01). The IGF-1 receptor mRNA was most abundant in the allantochorion (p<0.001) and IGF-1 protein was expressed in placental tissue without differences between groups. In conclusion, follicular growth in mares is initiated before foaling and placental IGF-1 may enhance resumption of ovulatory cycles.

The Human Intermediate Prolactin Receptor I-tail Contributes Breast Oncogenesis by Targeting Ras/MAPK Pathway.

Prolactin and its receptor (PRLr) in humans are significantly involved in breast cancer pathogenesis. The intermediate form of human PRLr (hPRLrI) is produced by alternative splicing and has a novel 13 amino acid tail ("I-tail") gain. hPRLrI induces significant proliferation and anchorage-independent growth of normal mammary epithelia in vitro when coexpressed with the long form hPRLr (hPRLrL). hPRLrL and hPRLrI coexpression is necessary to induce the transformation of mammary epithelia in vivo. The I-tail is associated with the ubiquitin-like protein neural precursor cell expressed developmentally downregulated protein 8. Treatment with the neural precursor cell expressed developmentally downregulated protein 8-activating enzyme inhibitor pevonedistat resulted in increased hPRLrL and the death of breast cancer cells. The goal of this study was to determine the function of the hPRLrI I-tail in hPRLrL/hPRLrI-mediated mammary transformation. hPRLrL/hPRLrI and hPRLrL/hPRLrIΔ13 (I-tail removal mutant) were delivered to MCF10AT cells. Cell proliferation was decreased when hPRLrI I-tail was removed. I-tail deletion decreased anchorage-independent growth and attenuated cell migration. The I-tail was involved in Ras/MAPK signaling but not PI3K/Akt signaling pathway as shown by western blot. I-tail removal resulted in decreased hPRLrI stability. RNA-sequencing data revealed that I-tail removal resulted in differential gene expression induced by prolactin. Ingenuity Pathway Analysis revealed that the activity of ERK was attenuated. Treatment of breast cancer cells with ERK1/2 inhibitor ulixertinib resulted in decreased colony-forming ability and less proliferation. These studies suggest that the hPRLrI I-tail contributed to breast oncogenesis and may be a promising target for the development of new breast cancer therapies.

The prolactin receptor: A cross-species comparison of gene structure, transcriptional regulation, tissue-specificity, and genetic variation.

The conserved and multifaceted functions of prolactin (PRL) are coordinated through varied distribution and expression of its cell-surface receptor (PRLR) across a range of tissues and physiological states. The resultant heterogeneous expression of PRLR mRNA and protein across different organs and cell types supports a wide range of PRL-regulated processes including reproduction, lactation, development, and homeostasis. Genetic variation within the PRLR gene also accounts for several phenotypes impacting agricultural production and human pathology. The goal of this review is to highlight the many elements that control differential expression of the PRLR across tissues, and the various phenotypes that exist across species due to variation in the PRLR gene.

Dopamine D2 receptor antagonist counteracts hyperglycemia and insulin resistance in diet-induced obese male mice.

Obesity leads to insulin resistance (IR) and type 2 diabetes. In humans, low levels of the hormone prolactin (PRL) correlate with IR, adipose tissue (AT) dysfunction, and increased prevalence of T2D. In obese rats, PRL treatment promotes insulin sensitivity and reduces visceral AT adipocyte hypertrophy. Here, we tested whether elevating PRL levels with the prokinetic and antipsychotic drug sulpiride, an antagonist of dopamine D2 receptors, improves metabolism in high fat diet (HFD)-induced obese male mice. Sulpiride treatment (30 days) reduced hyperglycemia, IR, and the serum and pancreatic levels of triglycerides in obese mice, reduced visceral and subcutaneous AT adipocyte hypertrophy, normalized markers of visceral AT function (PRL receptor, Glut4, insulin receptor and Hif-1α), and increased glycogen stores in skeletal muscle. However, the effects of sulpiride reducing hyperglycemia were also observed in obese prolactin receptor null mice. We conclude that sulpiride reduces obesity-induced hyperglycemia by mechanisms that are independent of prolactin/prolactin receptor activity. These findings support the therapeutic potential of sulpiride against metabolic dysfunction in obesity.

Prolactin promotes the recruitment of main olfactory bulb cells and enhances the behavioral exploration toward a socio-sexual stimulus in female mice.

Olfactory communication is triggered by pheromones that profoundly influence neuroendocrine responses to drive social interactions. Two principal olfactory systems process pheromones: the main and the vomeronasal or accessory system. Prolactin receptors are expressed in both systems suggesting a participation in the processing of olfactory information. We previously reported that prolactin participates in the sexual and olfactory bulb maturation of females. Therefore, we explored the expression of prolactin receptors within the olfactory bulb during sexual maturation and the direct responses of prolactin upon pheromonal exposure. Additionally, we assessed the behavioral response of adult females exposed to male sawdust after prolactin administration and the consequent activation of main and accessory olfactory bulb and their first central relays, the piriform cortex and the medial amygdala. Last, we investigated the intracellular pathway activated by prolactin within the olfactory bulb. Here, prolactin receptor expression remained constant during all maturation stages within the main olfactory bulb but decreased in adulthood in the accessory olfactory bulb. Behaviorally, females that received prolactin actively explored the male stimulus. An increased cFos activation in the amygdala and in the glomerular cells of the whole olfactory bulb was observed, but an augmented response in the mitral cells was only found within the main olfactory bulb after prolactin administration and the exposure to male stimulus. Interestingly, the ERK pathway was upregulated in the main olfactory bulb after exposure to a male stimulus. Overall, our results suggest that, in female mice, prolactin participates in the processing of chemosignals and behavioral responses by activating the main olfactory system and diminishing the classical vomeronasal response to pheromones.

Mechanisms of adverse mammary effect induced by olanzapine and therapeutic interventions in rat model.

BACKGROUND: Olanzapine antagonizes dopamine receptors and is prescribed to treat multiple psychiatric conditions. The main side effect of concern for olanzapine is weight gain and metabolic syndrome. Olanzapine induces hyperprolactinemia, however its effect on the mammary gland is poorly documented. METHODS: Rats received olanzapine by gavage or in drinking water at 1, 3, and 6 mg/kg/day for 5-40 days or 100 days, with and without coadministration of bromocriptine or aripiprazole and using once daily or continuous administration strategies. Histomorphology of the mammary gland, concentrations of prolactin, estradiol, progesterone, and olanzapine in serum, mammary gland and adipose tissue, and mRNA and protein expressions of prolactin receptors were analyzed. RESULTS: In adult and prepubescent female rats and male rats, olanzapine induced significant development of mammary glands in dose- and time-dependent manners, with histopathological hyperplasia of mammary ducts and alveoli with lumen dilation and secretion, marked increase of mammary prolactin receptor expression, a marker of breast tissue, and with mild increase of circulating prolactin. This side effect can be reversed after medication withdrawal, but long-term olanzapine treatment for 100 days implicated tumorigenic potentials indicated by usual ductal epithelial hyperplasia. Olanzapine induced mammary development was prevented with the coaddition of the dopamine agonist bromocriptine or partial agonist aripiprazole, or by continuous administration of medication instead of a once daily regimen. CONCLUSIONS: These results shed light on the previously overlooked effect of olanzapine on mammary development and present experimental evidence to support current clinical management strategies of antipsychotic induced side effects in the breast.

PRL-mediated STAT5B/ARRB2 pathway promotes the progression of prostate cancer through the activation of MAPK signaling.

Previous study showed that higher expression of prolactin (PRL) was found in CRPC samples compared with hormone-naive prostate cancer (HNPC) and benign prostatic hyperplasia (BPH) samples. We further investigate the function of PRL in prostate cancer (PCa) and explored its downstream effects. We found heterogeneous expression of the PRLR in clinical prostate samples. The VCaP and 22Rv1 cells exhibited PRLR expression. Among the downstream proteins, STAT5B was the dominant subtype in clinical samples and cell lines. Human recombinant PRL stimulation of PCa cells with PRLR expression resulted in increased phosphorylation of STAT5B(pSTAT5B) and progression of PCa in vitro and in vivo, and STAT5B knockdown can suppress the malignant behavior of PCa. To understand the mechanism further, we performed Bioinformatic analysis, ChIP qPCR, and luciferase reporter gene assay. The results revealed that ARRB2 was the transcription target gene of STAT5B, and higher expression of ARRB2 was related to higher aggression and poorer prognosis of PCa. Additionally, Gene set enrichment analysis indicated that higher expression of ARRB2 was significantly enriched in the MAPK signaling pathway. Immunohistochemistry (IHC) demonstrated elevated pSTAT5B, ARRB2, and pERK1/2 expression levels in CRPC tissues compared to HNPC and BPH. Mechanically, ARRB2 enhanced the activation of the MAPK pathway by binding to ERK1/2, thereby promoting the phosphorylation of ERK1/2 (pERK1/2). In conclusion, our study demonstrated that PRL stimulation can promote the progression of PCa through STAT5B/ARRB2 pathway and activation of MAPK signaling, which can be suppressed by intervention targeting STAT5B. Blockade of the STAT5B can be a potential therapeutic target for PCa.

Current Insights in Prolactin Signaling and Ovulatory Function.

Prolactin (PRL) is a pleiotropic hormone released from lactotrophic cells of the anterior pituitary gland that also originates from extrapituitary sources and plays an important role in regulating lactation in mammals, as well as other actions. Acting in an endocrine and paracrine/autocrine manner, PRL regulates the hypothalamic-pituitary-ovarian axis, thus influencing the maturation of ovarian follicles and ovulation. This review provides a detailed discussion of the current knowledge on the role of PRL in the context of ovulation and ovulatory disorders, particularly with regard to hyperprolactinemia, which is one of the most common causes of infertility in women. Much attention has been given to the PRL structure and the PRL receptor (PRLR), as well as the diverse functions of PRLR signaling under normal and pathological conditions. The hormonal regulation of the menstrual cycle in connection with folliculogenesis and ovulation, as well as the current classifications of ovulation disorders, are also described. Finally, the state of knowledge regarding the importance of TIDA (tuberoinfundibular dopamine), KNDγ (kisspeptin/neurokinin B/dynorphin), and GnRH (gonadotropin-releasing hormone) neurons in PRL- and kisspeptin (KP)-dependent regulation of the hypothalamic-pituitary-gonadal (HPG) axis in women is reviewed. Based on this review, a rationale for influencing PRL signaling pathways in therapeutic activities accompanying ovulation disorders is presented.

Codonopsis lanceolata polysaccharide ameliorates high-fat diet induced-postpartum hypogalactia via stimulating prolactin receptor-mediated Jak2/Stat5 signaling.

This study aims to investigate the ameliorative effect of Codonopsis lanceolata polysaccharide (PCL) on mice with hypogalatia induced by a high-fat diet (HFD) and the potential underlying mechanism. We found that oral administration of PCL demonstrated significant benefits in countering the negative effects of HFD, including weight gain, hepatic steatosis, mesenteric adipocyte hypertrophy, and abnormal glucose/lipid metabolism. In addition, PCL improved mammary gland development and enhanced lactogenesis performance. Histologically, PCL ameliorated the retardation of ductal growth, reduced mammary fat pad thickness, improved the incomplete linear encapsulation of luminal epithelium and myoepithelium, and increased the proliferation of mammary epithelial cells. Flow cytometry analysis showed that PCL mitigated the detrimental effects of HFD on mammary gland development by promoting the proliferation and differentiation of mammary epithelial cells. Mechanistic studies revealed that PCL upregulated the levels of prolactin (PRL) and its receptor (PRLR) in the mammary gland, activated JAK2/STAT5 signaling pathway, and increased the expression of p63, ERBB4, and NRG1. Overall, PCL can ameliorate HFD-induced hypogalactia by activating PRLR-mediated JAK2/STAT5 signaling. Our findings offer a methodological and theoretical foundation for investigating the functional constituents of traditional Chinese medicine in the treatment of hypogalactia.

A Novel Mechanism of hPRL-G129R, a Prolactin Antagonist, Inhibits Human Breast Cancer Cell Proliferation and Migration.

Prolactin (PRL) and its receptor, PRLR, are closely related to the occurrence and development of breast cancer. hPRL-G129R, an hPRLR antagonist, has been found to induce apoptosis in breast cancer cells via mechanisms currently unknown. Recent studies have indicated that PRLR exhibits dual functions based on its membrane/nucleus localization. In that context, we speculated whether hPRL-G129R is a dual-function antagonist. We studied the internalization of the hPRLR-G129R/PRLR complex using indirect immunofluorescence and Western blot assays. We found that hPRL-G129R not only inhibited PRLR-mediated intracellular signaling at the plasma membrane, but also blocked nuclear localization of the receptor in T-47D and MCF-7 cells in a time-dependent manner. Clone formation and transwell migration assays showed that hPRL-G129R inhibited PRL-driven proliferation and migration of tumor cells in vitro. Further, we found that increasing concentrations of hPRL-G129R inhibited the nuclear localization of PRLR and the levels of signal transducer and activator of transcription (STAT) 5 in tumor-bearing mice and hPRL-G129R also exerted an antiproliferative effect in vivo. These results indicate that hPRL-G129R is indeed a dual-function antagonist. This study lays a foundation for exploring and developing highly effective agents against the proliferation and progression of breast malignancies.

High fat diet-induced maternal obesity in mice impairs peripartum maternal behaviour.

Obesity during pregnancy represents a significant health issue and can lead to increased complications during pregnancy and impairments with breastfeeding, along with long-term negative health consequences for both mother and offspring. In rodent models, diet-induced obesity (DIO) during pregnancy leads to poor outcomes for offspring. Using a DIO mouse model, consisting of feeding mice a high fat diet for 8 weeks before mating, we recapitulate the effect of high pup mortality within the first 3 days postpartum. To examine the activity of the dam around the time of birth, late pregnant control and DIO dams were recorded in their home cages and the behaviour of the dam immediately before and after birth was analysed. Prior to giving birth, DIO dams spent less time engaging in nesting behaviour, while after birth, DIO dams spent less time in the nest with their pups compared to control dams, indicating reduced pup-engagement in the early postpartum period. We have previously reported that lactogenic hormone action, mediated by the prolactin receptor, in the medial preoptic area of the hypothalamus (MPOA) is critical for the onset of normal postpartum maternal behaviour. We hypothesized that DIO dams may have lower lactogenic hormone activity during late pregnancy, which would contribute to impaired onset of normal postpartum maternal behaviour. Day 16 lactogenic activity, transport of prolactin into the brain, and plasma prolactin concentrations around birth were all similar in control and DIO dams. Moreover, endogenous pSTAT5, a marker of prolactin receptor activity, in the MPOA was unaffected by DIO. Overall, these data indicate that lactogenic activity in late pregnancy of DIO dams is not different to controls and is unlikely to play a major role in impaired onset of normal postpartum maternal behaviour.

The prolactin receptor gene (PRLR) is linked and associated with the risk of polycystic ovarian syndrome.

The prolactin receptor gene (PRLR) may contribute to polycystic ovarian syndrome (PCOS) since it plays important roles in physiological ovarian functions. PRLR-knockout mice have irregular cycles and subfertility and variants in or around the PRLR gene were associated in humans with female testosterone levels and recurrent miscarriage. We tested 40 variants in the PRLR gene in 212 Italian families phenotyped by type 2 diabetes (T2D) and PCOS and found two intronic PRLR-variants (rs13436213 and rs1604428) significantly linked to and/or associated with the risk of PCOS. This is the first study to report PRLR as a novel risk gene in PCOS. Functional studies are needed to confirm these results.

Astragalus polysaccharide promotes autophagy and alleviates diabetic nephropathy by targeting the lncRNA Gm41268/PRLR pathway.

BACKGROUND: Astragalus polysaccharide (APS) is a major bioactive component of the Chinese herb astragalus, with well-established protective effects on the kidney. However, the effect of APS on diabetic nephropathy (DN) is unclear. METHODS: Long non-coding RNA (lncRNA) expression profiles in kidney samples from control, db/db, and APS-treated db/db mice were evaluated using RNA high-throughput sequencing techniques. Additionally, rat renal tubular epithelial (NRK-52E) cells were cultured in high glucose (HG) media. We inhibited the expression of Gm41268 and prolactin receptor (PRLR) by transfecting NRK-52E cells with Gm41268-targeting antisense oligonucleotides and PRLR siRNA. RESULTS: We found that APS treatment reduced 24-h urinary protein levels and fasting blood glucose and improved glucose intolerance and pathological renal damage in db/db mice. Furthermore, APS treatment enhanced autophagy and alleviated fibrosis in the db/db mice. We identified a novel lncRNA, Gm41268, which was differentially expressed in the three groups, and the cis-regulatory target gene PRLR. APS treatment induced autophagy by reducing p62 and p-mammalian target of rapamycin (mTOR) protein levels and increasing the LC3 II/I ratio. Furthermore, APS alleviated fibrosis by downregulating fibronectin (FN), transforming growth factor-ß (TGF-ß), and collagen IV levels. In addition, APS reversed the HG-induced overexpression of Gm41268 and PRLR. Reduction of Gm41268 decreased PRLR expression, restored autophagy, and ameliorated renal fibrosis in vitro. Inhibition of PRLR could enhance the protective effect of APS. CONCLUSIONS: In summary, we demonstrated that the therapeutic effect of APS on DN is mediated via the Gm41268/PRLR pathway. This information contributes to the exploration of bioactive constituents in Chinese herbs as potential treatments for DN.

Temperature modulates the osmosensitivity of tilapia prolactin cells.

In euryhaline fish, prolactin (Prl) plays an essential role in freshwater (FW) acclimation. In the euryhaline and eurythermal Mozambique tilapia, Oreochromis mossambicus, Prl cells are model osmoreceptors, recently described to be thermosensitive. To investigate the effects of temperature on osmoreception, we incubated Prl cells of tilapia acclimated to either FW or seawater (SW) in different combinations of temperatures (20, 26 and 32 °C) and osmolalities (280, 330 and 420 mOsm/kg) for 6 h. Release of both Prl isoforms, Prl188 and Prl177, increased in hyposmotic media and were further augmented with a rise in temperature. Hyposmotically-induced release of Prl188, but not Prl177, was suppressed at 20 °C. In SW fish, mRNA expression of prl188 increased with rising temperatures at lower osmolalities, while and prl177 decreased at 32 °C and higher osmolalities. In Prl cells of SW-acclimated tilapia incubated in hyperosmotic media, the expressions of Prl receptors, prlr1 and prlr2, and the stretch-activated Ca2+ channel, trpv4,decreased at 32 °C, suggesting the presence of a cellular mechanism to compensate for elevated Prl release. Transcription factors, pou1f1, pou2f1b, creb3l1, cebpb, stat3, stat1a and nfat1c, known to regulate prl188 and prl177, were also downregulated at 32 °C. Our findings provide evidence that osmoreception is modulated by temperature, and that both thermal and osmotic responses vary with acclimation salinity.

Seasonal patterns of prolactin, prolactin receptor, and STAT5 expression in the ovaries of wild ground squirrels (Citellus dauricus Brandt).

Prolactin (PRL) is a hormone crucial for normal reproduction, functioning as an autocrine, paracrine, and endocrine factor. This study aimed to examine the immunolocalization and expression patterns of PRL, prolactin receptor (PRLR), and signal transducer and activator of transcription 5 (STAT5) in the ovaries of wild ground squirrels during both breeding and non-breeding periods. Significant seasonal variations were observed in ovarian weights, with higher values during the breeding season and relatively lower values during the nonbreeding season. PRL, PRLR, STAT5, and p-STAT5 were immunolocalized in granulosa cells and luteal cells during the breeding season, whereas they were exclusively found in granulosa cells during the non-breeding season. The mRNA expression levels of Prl, Prlr, and Stat5 were increased in ovarian tissues during the breeding season compared to the non-breeding season. Moreover, the mean mRNA levels of Prl, Prlr, and Stat5 exhibited a positive correlation with ovarian weights. Both circulating PRL and ovarian PRL concentrations were significantly elevated during the breeding season. Additionally, transcriptomic analysis of ovarian tissues revealed differentially expressed genes possibly associated with ovarian function and mammary gland development, including ovarian follicle development, steroid synthesis, and regulation of reproductive process. These findings suggest that PRL might play an essential endocrine, autocrine, or paracrine role in the regulation of seasonal changes in the ovarian functions in wild ground squirrels.

High Prolactin Concentration Induces Ovarian Granulosa Cell Oxidative Stress, Leading to Apoptosis Mediated by L-PRLR and S-PRLR.

High prolactin (PRL) concentration has been shown to induce the apoptosis of ovine ovarian granulosa cells (GCs), but the underlying mechanisms are unclear. This study aimed to investigate the mechanism of apoptosis induced by high PRL concentration in GCs. Trial 1: The optimal concentration of glutathion was determined according to the detected cell proliferation. The results showed that the optimal glutathione concentration was 5 µmol/mL. Trial 2: 500 ng/mL PRL was chosen as the high PRL concentration. The GCs were treated with 0 ng/mL PRL (C group), 500 ng/mL PRL (P group) or 500 ng/mL PRL, and 5 µmol/mL glutathione (P-GSH group). The results indicated that the mitochondrial respiratory chain complex (MRCC) I-V, ATP production, total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and thioredoxin peroxidase (TPx) in the C group were higher than those in the P group (p < 0.05), while they were lower than those in the P-GSH group (p < 0.05). Compared to the C group, the P group exhibited elevated levels of reactive oxygen species (ROS) and apoptosis (p < 0.05) and increased expression of ATG7 and ATG5 (p < 0.05). However, MRCC I-V, ATP, SOD, A-TOC, TPx, ROS, and apoptosis were decreased after the addition of glutathione (p < 0.05). The knockdown of either L-PRLR or S-PRLR in P group GCs resulted in a significant reduction (p < 0.05) in MRCC I-V, ATP, T-AOC, SOD and TPx, while the overexpression of either receptor showed an opposite trend (p < 0.05). Our findings suggest that high PRL concentrations induce apoptotic cell death in ovine ovarian GCs by downregulating L-PRLR and S-PRLR, activating oxidative stress and autophagic pathways.

Discovery and characterization of prolactin neutralizing monoclonal antibodies for the treatment of female-prevalent pain disorders.

Prolactin (PRL) has recently been demonstrated to elicit female-selective nociceptor sensitization and increase pain-like behaviors in female animals. Here we report the discovery and characterization of first-in-class, humanized PRL neutralizing monoclonal antibodies (PRL mAbs). We obtained two potent and selective PRL mAbs, PL 200,031 and PL 200,039. PL 200,031 was engineered as human IgG1 whereas PL 200,039 was reformatted as human IgG4. Both mAbs have sub-nanomolar affinity for human PRL (hPRL) and produce concentration-dependent and complete inhibition of hPRL signaling at the hPRL receptor (hPRLR). These two PRL mAbs are selective for hPRL as they do not inhibit other hPRLR agonists such as human growth hormone or placental lactogen. They also cross-react with non-human primate PRL but not with rodent PRL. Further, both mAbs show long clearance half-lives after intravenous administration in FcRn-humanized mice. Consistent with their isotypes, these mAbs only differ in binding affinities to Fcγ receptors, as expected by design. Finally, PL 200,019, the murine parental mAb of PL 200,031 and PL 200,039, fully blocked stress-induced and PRL-dependent pain behaviors in female PRL-humanized mice, thereby providing in vivo preclinical proof-of-efficacy for PRL mAbs in mechanisms relevant to pain in females.