Prolactin Family Research Articles

Conditionally mutant animal model for investigating the invasive trophoblast cell lineage.

Placental development involves coordinated expansion and differentiation of trophoblast cell lineages possessing specialized functions. Among the differentiated trophoblast cell lineages are invasive trophoblast cells, which exit the placenta and invade the uterus, where they restructure the uterine parenchyma and facilitate remodeling of uterine spiral arteries. The rat exhibits deep intrauterine trophoblast cell invasion, a feature shared with human placentation, and is also amenable to gene manipulation using genome-editing techniques. In this investigation, we generated a conditional rat model targeting the invasive trophoblast cell lineage. Prolactin family 7, subfamily b, member 1 (Prl7b1) is uniquely and abundantly expressed in the rat invasive trophoblast cell lineage. Disruption of Prl7b1 did not adversely affect placental development. We demonstrated that the Prl7b1 locus could be effectively used to drive the expression of Cre recombinase in invasive trophoblast cells. Our rat model represents a new tool for investigating candidate genes contributing to the regulation of invasive trophoblast cells and their roles in trophoblast-guided uterine spiral artery remodeling.

Plasma prolactin axis shift from placental to pituitary origin in late prepartum mice.

The placenta secretes a prolactin (PRL)-like hormone PRL3B1 (placental lactogen II), a luteotropic hormone essential for maintaining pregnancy until labor in mice. A report from 1984 examined the secretion pattern of PRL3B1 in prepartum mice. In the current study, we found contradictory findings in the secretion pattern that invalidate the previous report. By measuring maternal plasma PRL3B1 and PRL every 4 hrs from gestational day 17 (G17), we newly discovered that maternal plasma PRL3B1 levels decrease rapidly in prepartum C57BL/6 mice. Interestingly, the onset of this decline coincided with the PRL surge at G18, demonstrating a plasma prolactin axis shift from placental to pituitary origin. We also found that maternal plasma progesterone regression precedes the onset of the PRL shift. The level of Prl3b1 mRNA was determined by RT-qPCR in the placenta and remained stable until parturition, implying that PRL3B1 peptide production or secretion was suppressed. We hypothesized that production of the PRL family, the 25 paralogous PRL proteins exclusively expressed in mice placenta, would decrease alongside PRL3B1 during this period. To investigate this hypothesis and to seek proteomic changes, we performed a shotgun proteome analysis of the placental tissue using data-independent acquisition mass spectrometry (DIA-MS). Up to 5,891 proteins were identified, including 17 PRL family members. Relative quantitative analysis between embryonic day 17 (E17) and E18 placentas showed no significant difference in the expression of PRL3B1 and most PRL family members except PRL7C1. These results suggest that PRL3B1 secretion from the placenta is suppressed at G18 (E18).

A possible function of Nik-related kinase in the labyrinth layer of delayed delivery mouse placentas.

In mice and humans, Nik-related protein kinase (Nrk) is an X-linked gene that encodes a serine/threonine kinase belonging to GCK group 4. Nrk knockout (Nrk KO) mice exhibit delayed delivery, possibly due to defective communication between the Nrk KO conceptus and its mother. However, the mechanism of delayed labor remains largely unknown. Here, we found that in pregnant mothers with the Nrk KO conceptus, the serum progesterone (P4) and placental lactogen (PL-2) concentrations in late pregnancy were higher than those in the wild type. Moreover, we demonstrated that Nrk is expressed in trophoblast giant cells (TGCs) and syncytiotrophoblast-2 (SynT-2) in the labyrinth layer of the mouse placenta. In the human placenta, NRK is also expressed in Syn-T in villi. Both human Syn-T and mouse TGCs of the labyrinth layer are present within fetal tissues that are in direct contact with the maternal blood. The labyrinth layer of the Nrk KO conceptus was gigantic, with enlarged cytoplasm and Golgi bodies in the TGCs. To investigate the function of Nrk in the labyrinth layer, a differentially expressed gene (DEG) analysis was performed. The DEG analysis revealed that labor-promoting factors, such as prostaglandins, were decreased, and pregnancy-maintaining factors, such as the prolactin family and P4 receptor, were increased. These findings suggest that the Nrk KO mice exhibit delayed delivery owing to high P4 concentrations caused by the hypersecretion of pregnancy-maintaining factors, such as PL-2, from the placenta.

Discovery of prolactin-like in lamprey: Role in osmoregulation and new insight into the evolution of the growth hormone/prolactin family

We used a representative of one of the oldest extant vertebrate lineages (jawless fish or agnathans) to investigate the early evolution and function of the growth hormone (GH)/prolactin (PRL) family. We identified a second member of the GH/PRL family in an agnathan, the sea lamprey (Petromyzon marinus). Structural, phylogenetic, and synteny analyses supported the identification of this hormone as prolactin-like (PRL-L), which has led to added insight into the evolution of the GH/PRL family. At least two ancestral genes were present in early vertebrates, which gave rise to distinct GH and PRL-L genes in lamprey. A series of gene duplications, gene losses, and chromosomal rearrangements account for the diversity of GH/PRL-family members in jawed vertebrates. Lamprey PRL-L is produced in the proximal pars distalis of the pituitary and is preferentially bound by the lamprey PRL receptor, whereas lamprey GH is preferentially bound by the lamprey GH receptor. Pituitary PRL-L messenger RNA (mRNA) levels were low in larvae, then increased significantly in mid-metamorphic transformers (stage 3); thereafter, levels subsided in final-stage transformers and metamorphosed juveniles. The abundance of PRL-L mRNA and immunoreactive protein increased in the pituitary of juveniles under hypoosmotic conditions, and treatment with PRL-L blocked seawater-associated inhibition of freshwater ion transporters. These findings clarify the origin and divergence of GH/PRL family genes in early vertebrates and reveal a function of PRL-L in osmoregulation of sea lamprey, comparable to a role of PRLs that is conserved in jawed vertebrates.

Diversity Outbred and Collaborative Cross mouse models identify and validate host genetic loci associated with Immune Checkpoint Inhibitor response

Abstract Immune checkpoint inhibitors (ICI) for cancer therapy have improved outcomes for a variety of malignancies, however many patients fail to benefit. While tumor-intrinsic mechanisms are involved in therapy resistance, it is unclear to what extent host genetic background plays in response. To investigate this in a pre-clinical setting, we utilized C57BL/6 (B6) F1 crosses of the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. We find a wide variation in response to anti-PD-1/anti-CTLA-4 ICI in B16F0-bearing DOB6F1 mice (n=142) with ~30% showing partial response and ~13% showing complete tumor elimination. Genetic linkage analysis identified several genomic loci associated with therapeutic outcomes. Of the associated loci, the region within chromosome 13 was further validated in CC mice bearing the positive (NZO) versus negative (C57BL/6) driver genotypes. ICI response was detected in 2/3 CCB6F1 predicted responder models, with no response in predicted non-responders. This locus contains the murine prolactin family which is a known immunomodulating cytokine with association to various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Combined ICI with prolactin shows an interaction effect against B16F0, with 5/8 mice exhibiting slowed tumor growth. Analysis of CD8 infiltrates by IHC demonstrate that dual treatment of prolactin and ICI results in a significant increase of CD8 infiltrates over other groups. Collectively, this study shows that host genetics play a role in ICI response and that prolactin may be a positive regulator of ICI efficacy. Supported by grants from NIH (R37 CA220482, T32-CA009531, P30 CA022453), the Pardee Foundation, and the DMC Foundation

Inserting Cre recombinase into the Prolactin 8a2 gene for decidua-specific recombination in mice.

An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here, we describe a new decidua-specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG/+ or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions ofgenetic networks and cellular dynamics associated with decidualization and infertility.

A diversity outbred F1 mouse model identifies host-intrinsic genetic regulators of response to immune checkpoint inhibitors

ABSTRACT Immune checkpoint inhibitors (ICI) have improved outcomes for a variety of malignancies; however, many patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent host genetic background influences response. To investigate this, we utilized the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are an outbred stock generated by crossbreeding eight inbred founder strains, and CC mice are recombinant inbred mice generated from the same eight founders. We generated 207 DOB6F1 mice representing 48 DO dams and demonstrated that these mice reliably accept the C57BL/6-syngeneic B16F0 tumor and that host genetic background influences response to ICI. Genetic linkage analysis from 142 mice identified multiple regions including one within chromosome 13 that associated with therapeutic response. We utilized 6 CC strains bearing the positive (NZO) or negative (C57BL/6) driver genotype in this locus. We found that 2/3 of predicted responder CCB6F1 crosses show reproducible ICI response. The chromosome 13 locus contains the murine prolactin family, which is a known immunomodulating cytokine associated with various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Prolactin augmented ICI response against B16F0, with increased CD8 infiltration and 5/8 mice exhibiting slowed tumor growth relative to controls. This study highlights the role of host genetics in ICI response and supports the use of F1 crosses in the DO and CC mouse populations as powerful cancer immunotherapy models.

The Influence of the Prolactins on the Development of the Uterus in Neonatal Mice.

The endometrial gland is one of the most important components of the mammalian uterus. However, few studies have been conducted on the regulatory mechanisms of adenogenesis during the development of endometrium. In the present study, we detected the genes expression of 35 different prolactin family members (PRLs) together with the prolactin receptor (PRL-R) in the endometrium of neonatal mice along with the adenogenesis process, to address which prolactin-like genes play a key role during gland development in mice. We found that: (1) The expression of Prl1a1, Prl3d1, Prl5a1, Prl7a1, Prl7a2, Prl7d1, Prl8a6, Prl8a8, and Prl8a9 genes were significantly increased along with the development of uterine glands. Prl7c1 and Prl8a1 were observably up-regulated on Postnatal day 5 (PND5) when the uterine glandular bud invagination begins. Prl3a1, Prl3b1, and Prl7b1 suddenly increased significantly on PND9. But, Prl3c1 and Prl8a2 were markedly down-regulated on PND5 and the expression of Prl6a1 and Prlr were stable extremely. (2) After continuous injection of Progesterone (P4), a well-known method to suppress the endometrial adenogenesis, the expression of Prl1a1, Prl3d1, Prl5a1, Prl7a1, Prl7a2, Prl7d1, Prl8a6, Prl8a8, Prl8a9, and Prlr were suppressed on PND7. And on PND9, Prl1a1, Prl3d1, Prl8a6, Prl8a8, and Prl8a9 were significantly inhibited. (3) Further analysis of the epithelial and stroma showed that these PRLs were mainly expressed in the endometrial stroma of neonatal mice. Our results indicate that multiple PRLs are involved in uterine development and endometrial adenogenesis. Continued progesterone therapy may alter the expression pattern of these PRLs in endometrial stromal cells, thereby altering the interaction and communication between stroma and epithelium, and ultimately leading to complete suppression of endometrial adenogenesis.

Abstract P060: Identification of host-intrinsic resistance mechanisms to immune checkpoint inhibitors (ICI) in Diversity Outbred mice

Abstract The advent of immune checkpoint inhibitors (ICI) for cancer therapy has improved outcomes for a variety of malignancies, however many a significant proportion of patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent genetic heterogeneity of the host plays in response. To investigate this in a pre-clinical setting, we utilize the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are genetically distinct animals generated by cross-breeding 8 inbred founder strains, and CC mice are recombinant inbred mice generated from the same 8 founders as DO. We find a wide variation in response to anti-PD-1/anti-CTLA-4 ICI in B16F0-bearing (C57BL/6xDO)F1 mice (n=142) with ~30% showing partial response with delayed tumor onset and ~13% showing complete tumor elimination with protection against subsequent contralateral rechallenge. Genetic linkage analysis using the R/qtl2 package identifies genomic loci associated with therapeutic outcomes. Importantly, the MHC locus was not significantly associated with response. Of the strongly associated loci, the region within chromosome 13 was further validated in CC mice bearing the positive (NZO) versus negative (C57BL/6) driver genotypes at this locus. ICI response was detected in 2/3 (C57BL/6xCC)F1 predicted responder models, with no response in predicted non-responders. This locus solely contains the murine prolactin family. Prolactin is a known immunomodulating cytokine with association to various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice, which notoriously fail to respond to ICI alone against B16F0 tumors, with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Combined ICI with prolactin shows an interaction effect against B16F0, with 5/8 mice exhibiting slowed tumor growth relative to controls. To test whether this host-derived phenomenon applies to additional tumor models, we tested ICI response to Lewis Lung Carcinoma (LLC) in a (C57BL/6xCC)F1 model where response to B16F0 was observed. Relative to inbred C57BL/6 mice, where no response to ICI is observed, this (C57BL/6xCC)F1 model shows a similar response to ICI against LLC as was detected for B16F0. Collectively, this study shows that host genetics play a role in ICI response and that prolactin may be a positive regulator of ICI efficacy. Citation Format: Justin Hackett, James Glassbrook, Maria Muniz, Heather M. Gibson. Identification of host-intrinsic resistance mechanisms to immune checkpoint inhibitors (ICI) in Diversity Outbred mice [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P060.

The Prolactin Family of Hormones as Regulators of Maternal Mood and Behavior.

Transition into motherhood involves profound physiological and behavioral adaptations that ensure the healthy development of offspring while maintaining maternal health. Dynamic fluctuations in key hormones during pregnancy and lactation induce these maternal adaptations by acting on neural circuits in the brain. Amongst these hormonal changes, lactogenic hormones (e.g., prolactin and its pregnancy-specific homolog, placental lactogen) are important regulators of these processes, and their receptors are located in key brain regions controlling emotional behaviors and maternal responses. With pregnancy and lactation also being associated with a marked elevation in the risk of developing mood disorders, it is important to understand how hormones are normally regulating mood and behavior during this time. It seems likely that pathological changes in mood could result from aberrant expression of these hormone-induced behavioral responses. Maternal mental health problems during pregnancy and the postpartum period represent a major barrier in developing healthy mother-infant interactions which are crucial for the child's development. In this review, we will examine the role lactogenic hormones play in driving a range of specific maternal behaviors, including motivation, protectiveness, and mother-pup interactions. Understanding how these hormones collectively act in a mother's brain to promote nurturing behaviors toward offspring will ultimately assist in treatment development and contribute to safeguarding a successful pregnancy.

Sex Differences in Embryonic Gonad Transcriptomes and Benzo[a]pyrene Metabolite Levels After Transplacental Exposure.

Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are generated during incomplete combustion of organic materials. Prior research has demonstrated that BaP is a prenatal ovarian toxicant and carcinogen. However, the metabolic pathways active in the embryo and its developing gonads and the mechanisms by which prenatal exposure to BaP predisposes to ovarian tumors later in life remain to be fully elucidated. To address these data gaps, we orally dosed pregnant female mice with BaP from embryonic day (E) 6.5 to E11.5 (0, 0.2, or 2 mg/kg/day) for metabolite measurement or E9.5 to E11.5 (0 or 3.33 mg/kg/day) for embryonic gonad RNA sequencing. Embryos were harvested at E13.5 for both experiments. The sum of BaP metabolite concentrations increased significantly with dose in the embryos and placentas, and concentrations were significantly higher in female than male embryos and in embryos than placentas. RNA sequencing revealed that enzymes involved in metabolic activation of BaP are expressed at moderate to high levels in embryonic gonads and that greater transcriptomic changes occurred in the ovaries in response to BaP than in the testes. We identified 490 differentially expressed genes (DEGs) with false discovery rate P-values < 0.05 when comparing BaP-exposed to control ovaries but no statistically significant DEGs between BaP-exposed and control testes. Genes related to monocyte/macrophage recruitment and activity, prolactin family genes, and several keratin genes were among the most upregulated genes in the BaP-exposed ovaries. Results show that developing ovaries are more sensitive than testes to prenatal BaP exposure, which may be related to higher concentrations of BaP metabolites in female embryos.

Uterine Notch2 facilitates pregnancy recognition and corpus luteum maintenance via upregulating decidual Prl8a2.

The maternal recognition of pregnancy is a necessary prerequisite for gestation maintenance through prolonging the corpus luteum lifespan and ensuring progesterone production. In addition to pituitary prolactin and placental lactogens, decidual derived prolactin family members have been presumed to possess luteotropic effect. However, there was a lack of convincing evidence to support this hypothesis. Here, we unveiled an essential role of uterine Notch2 in pregnancy recognition and corpus luteum maintenance. Uterine-specific deletion of Notch2 did not affect female fertility. Nevertheless, the expression of decidual Prl8a2, a member of the prolactin family, was downregulated due to Notch2 ablation. Subsequently, we interrupted pituitary prolactin function to determine the luteotropic role of the decidua by employing the lipopolysaccharide-induced prolactin resistance model, or blocking the prolactin signaling by prolactin receptor-Fc fusion protein, or repressing pituitary prolactin release by dopamine receptor agonist bromocriptine, and found that Notch2-deficient females were more sensitive to these stresses and ended up in pregnancy loss resulting from abnormal corpus luteum function and insufficient serum progesterone level. Overexpression of Prl8a2 in Notch2 knockout mice rescued lipopolysaccharide-induced abortion, highlighting its luteotropic function. Further investigation adopting Rbpj knockout and DNMAML overexpression mouse models along with chromatin immunoprecipitation assay and luciferase analysis confirmed that Prl8a2 was regulated by the canonical Notch signaling. Collectively, our findings demonstrated that decidual prolactin members, under the control of uterine Notch signaling, assisted pituitary prolactin to sustain corpus luteum function and serum progesterone level during post-implantation phase, which was conducive to pregnancy recognition and maintenance.

Genetic linkage analysis using Diversity Outbred mice identifies prolactin as a host-intrinsic regulator of immune checkpoint inhibitor response

Abstract The use of immune checkpoint inhibitors (ICI) targeting CTLA-4 and PD-1 have greatly improved patient survival in metastatic melanoma, but a significant proportion of patients fail to respond to therapy. Incorporating what has been learned in autoimmune conditions, we hypothesize the host genetic background plays a significant role in determining response to immunotherapy. To implement genetic diversity, Diversity Outbred (DO) mice were crossed with C57BL/6 to generate (C57BL/6xDO)F1 mice that reliably develop B16F0 tumors after subcutaneous inoculation. (C57BL/6xDO)F1 mice (n=142) treated with combined anti-PD1/anti-CTLA-4 on days 3, 6, and 10 after inoculation show wide variation in tumor latency up to a maximum of 65 days, with 19 mice never developing tumor. Genetic linkage analysis using the R/qtl2 package shows a suggestive association within the murine prolactin family on chromosome 13. Visualization of QTL effects by DO founder identifies the NZO mouse genotype as a driver of increased tumor latency. Validation was performed using Collaborative Cross (CC) mouse lines that were either the NZO or C57BL/6 genotype within this locus. (C57BL/6xCC)F1 mice containing the NZO genotype have lower tumor burden by day 15 compared to mice with the C57BL/6 genotype, validating the locus as influential on the response to ICI. Combination of systemic prolactin treatment and ICI shows evidence of an interaction effect. Mice treated with both prolactin and ICI show lower tumor burden by day 19 versus single therapy or placebo controls. These results show that the host’s genetic background significantly contributes to response to ICI, and modulation of prolactin expression may augment ICI activity.

Comparative Analysis of Mouse Decidualization Models at the Molecular Level.

The mouse is widely used to study decidualization and there are three well-established mouse models of decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). However, the extent of similarity and difference between these models at the molecular level remains largely unknown. Here, we performed a comparative analysis using the RNA-seq approach. In the NPD model, which is thought to be the golden standard of mouse decidualization, we found a total of 5277 differentially expressed genes, with 3158 genes being up-regulated and 2119 genes being down-regulated. A total of 4294 differentially expressed genes were identified in the AD model: 1127 up-regulated genes and 3167 down-regulated genes. In comparison to NPD, 1977 genes were consistently expressed, whereas only 217 genes were inconsistently expressed, indicating that AD is a reliable model for mouse decidualization. In the IVD model, RNA-seq analysis revealed that 513 genes were up-regulated and 988 genes were down-regulated. Compared to NPD, 310 genes were consistently expressed, whereas 456 genes were inconsistently expressed. Moreover, although the decidualization marker Prl8a2 (prolactin family 8 subfamily a member 2) was up-regulated, the widely-used marker Alpl (alkaline phosphatase liver/bone/kidney) was down-regulated in the IVD model. Therefore, we suggest that the IVD model should be optimized to mimic NPD at the transcriptomic level. Our study contributes to an increase in the knowledge about mouse models of decidualization.

CGMP-dependent protein kinase II determines β-catenin accumulation that is essential for uterine decidualization in mice.

In the early phase of pregnancy, decidualization is an indispensable event after mammal embryo implantation, accompanied by proliferation and differentiation of uterine stromal cells. Type II cGMP-dependent protein kinase (Prkg2) belongs to the family of serine/threonine kinase, which plays multiple roles in cellular signaling pathways to control proliferation and differentiation. However, the regulatory function and molecular mechanism of Prkg2 in decidualization are still unknown. In this study, we show that Prkg2 has a gradually increased expression pattern during peri-implantation and artificial decidualization, and the expression of Prkg2 is induced by estrogen and progesterone in the ovariectomized mouse uteri and primary cultured uterine stromal cells, the process of which is blocked by treating with estrogen receptor (ER) antagonist (ICI-182,780) and progesterone receptor (PR) antagonist (RU-486). Inhibition of Prkg2 activity by HA-100 promotes uterine stromal cell proliferation but compromises decidualization with decreased expression of prolactin family 8, subfamily a, member 2. In addition, the functional regulation of decidualization by Prkg2 is accomplished by its induced phosphorylation of glycogen synthase kinase-3β (GSK-3β) at serine-9, which results in accumulation of β-catenin in the decidual cells. Taken together, our findings demonstrate that estrogen and progesterone upregulate the expression of Prkg2 in uterine stromal cells depending on ER and PR; Prkg2 promotes phosphorylation of GSK-3β at serine-9 and inactivates it, leading to the accumulation of β-catenin and promoting the process of decidualization. In addition to revealing the regulatory mechanism of Prkg2 that ensures the success of uterine decidualization, our findings will contribute to the understanding in the maintenance of early pregnancy.

Deletion of Prl7d1 causes placental defects at mid-pregnancy in mice.

Prolactin family 7, subfamily d, member 1 (Prl7d1), a member of the expanding prolactin family, is mainly expressed in the placental junctional zone (including trophoblast giant cells and spongiotrophoblast cells) with peak expression observed at 12 days postcoitum (dpc) in mice. Previous studies have shown that PRL7D1 is a key mediator of angiogenesis in vitro; however, its physiological roles in placental development in vivo have not been characterized. To address this issue, we deleted Prl7d1 in mice and demonstrated that its absence results in reduced litter size and fertility. Histologically, Prl7d1 mutants exhibited striking placental abnormalities at 12.5 dpc, including a reduction in the proportion of labyrinth layers and a significant increase in decidual natural killer cells, glycogen trophoblasts, and trophoblast giant cells in the junctional zone. Moreover, placentas from Prl7d1-null mice displayed a thickened decidual spiral artery. Notably, these negative effects were more pronounced in male fetuses. Further RNA-sequencing analysis showed that Prl7d1 deletion results in significant differences in the placental transcriptome profile between the two sexes of fetuses. Together, this study demonstrates that Prl7d1 possesses antiangiogenic properties in deciduas and inhibits the development of junctional zone, which potentially alters the functional capacity of the placenta to support optimal fetal growth. Moreover, of note, the role of Prl7d1 in the placenta is regulated in a fetal sex-specific manner.

High Mobility Group Box 1 Regulates Uterine Decidualization through Bone Morphogenetic Protein 2 and Plays a Role in Kruppel-Like Factor 5-Induced Stromal Differentiation

Background/Aims: High mobility group box 1 (Hmgb1) is associated with a variety of physiological processes including embryonic development, cell proliferation and differentiation, but little information is available regarding its biological role in decidualization. Methods: In situ hybridization, real-time PCR, RNA interference, gene overexpression and MTS assay were used to analyze the spatiotemporal expression of Hmgb1 in mouse uterus during the pre-implantation period, and explore its function and regulatory mechanisms during uterine decidualization. Results: Hmgb1 mRNA was obviously observed in uterine epithelium on day 2 and 3 of pregnancy, but its expression was scarcely detected on day 4 of pregnancy. With the onset of embryo implantation, abundant Hmgb1 expression was noted in the subluminal stromal cells around the implanting blastocyst at implantation sites. Meanwhile, the accumulation of Hmgb1 mRNA was visualized in the decidual cells. Hmgb1 advanced the proliferation of uterine stromal cells and induced the expression of prolactin family 8, subfamily a, member 2 (Prl8a2), a reliable differentiation marker for decidualization. In uterine stromal cells, cAMP analogue 8-Br-cAMP up-regulated the expression of Hmgb1, but the up-regulation was abrogated by protein kinase A (PKA) inhibitor H89. Silencing of Hmgb1 by specific siRNA impeded the induction of 8-Br-cAMP on Prl8a2. Further analysis evidenced that Hmgb1 was a critical mediator of Kruppel-like factor 5 (Klf5) function in stromal differentiation. Knockdown of bone morphogenetic protein 2 (Bmp2) prevented the up-regulation of Prl8a2 elicited by Hmgb1 overexpression, whereas addition of exogenous recombinant Bmp2 protein (rBmp2) reversed the repression of Hmgb1 siRNA on Prl8a2 expression. Conclusion: Hmgb1 may play an important role during mouse uterine decidualization.

Hypoxic Stress Forces Irreversible Differentiation of a Majority of Mouse Trophoblast Stem Cells Despite FGF4.

Hypoxic, hyperosmotic, and genotoxic stress slow mouse trophoblast stem cell (mTSC) proliferation, decrease potency/stemness, and increase differentiation. Previous reports suggest a period of reversibility in stress-induced mTSC differentiation. Here we show that hypoxic stress at 0.5% O2 decreased potency factor protein by ∼60%–90% and reduced growth to nil. Hypoxia caused a 35-fold increase in apoptosis at Day 3 and a 2-fold increase at Day 6 above baseline. The baseline apoptosis rate was only 0.3%. Total protein was never less than baseline during hypoxic treatment, suggesting 0.5% O2 is a robust, nonmorbid stressor. Hypoxic stress induced ∼50% of trophoblast giant cell (TGC) differentiation with a simultaneous 5- to 6-fold increase in the TGC product antiluteolytic prolactin family 3, subfamily d, member 1 (PRL3D1), despite the presence of fibroblast growth factor 4 (FGF4). Hypoxia-induced TGC differentiation was also supported by potency and differentiation mRNA marker analysis. FGF4 removal at 20% O2 committed cell fate towards irreversible differentiation at 2 days, with similar TGC percentages after an additional 3 days of culture under potency conditions when FGF4 was readded or under differentiation conditions without FGF4. However, hypoxic stress required 4 days to irreversibly differentiate cells. Runted stem cell growth, forced differentiation of fewer cells, and irreversible differentiation limit total available stem cell population. Were mTSCs to respond to stress in a similar mode in vivo, miscarriage might occur as a result, which should be tested in the future.

Nm23 regulates decidualization through the PI3K-Akt-mTOR signaling pathways in mice and humans.

Does nm23 have functional significance in decidualization in mice and humans? nm23 affects decidualization via the phosphoinositide 3 kinase/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathways in mouse endometrial stromal cells (ESCs; mESCs) and human ESCs. The function of nm23 in suppressing metastasis has been demonstrated in a variety of cancer types. nm23 also participates in the control of DNA replication and cell proliferation and differentiation. We first analyzed the expression profile of nm23 in mice during early pregnancy (n=6/group), pseudopregnancy (n=6/group) and artificial decidualization (n=6/group) and in humans during the menstrual cycle phases and the first trimester. We then used primary cultured mESCs and a human ESC line, T-HESC, to explore the hormonal regulation of nm23 and the roles of nm23 in in vitro decidualization, and as a possible mediator of downstream PI3K-Akt-mTOR signaling pathways. We evaluated the dynamic expression of nm23 in mice and humans using immunohistochemistry, western blot and real-time quantitative RT-PCR (RT-qPCR). Regulation of nm23 by steroid hormones was investigated in isolated primary mESCs and T-HESCs by western blot. The effect of nm23 knockdown (using siRNA) on ESC proliferation was analyzed by 5-ethynyl-2'-deoxyuridine staining (EdU) and proliferating cell nuclear antigen protein (PCNA) expression. The influence of nm23 expression on the differentiation of ESCs was determined by RT-qPCR using the mouse differentiation markers decidual/trophoblast PRL-related protein (dtprp, also named prl8a2) and prolactin family 3 subfamily c member 1 (prl3c1) and the human differentiation markers insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). The effects of nm23 siRNA (si-nm23) and the PI3K inhibitor LY294002 on the downstream effects of nm23 on the PI3K-Akt-mTOR signaling pathway were estimated by western blot. NM23-M1 was specifically expressed in the decidual zone during early pregnancy and in artificially induced deciduoma, and NM23-H1 was strongly expressed in human first trimester decidua. The expression of nm23 was upregulated by oestradiol and progesterone (P<0.05 versus control) in vitro in mESCs and T-HESC, and this was inhibited by their respective receptor antagonists, ICI 182,780 and RU486. Mouse and human nm23 knockdown decreased ESC proliferation and differentiation (P<0.05 versus control). The PI3K-Akt-mTOR signaling pathways were downstream mediators of nm23 in mESCs and T-HESCs decidualization. Whether the nm23 regulates decidualization via the activation of AMPK, RAS, PKA, STAT3 or other signaling molecules remains to be determined. The role of nm23 in decidualization was tested in vitro only. Results demonstrate that nm23 plays a vital role in decidualization in mice and humans and that nm23 gene expression is hormonally regulated. The downregulation of nm23 in decidua during the first trimester may be associated with infertility in women. This study was supported by the National Natural Science Foundation of China (grant nos. 81370731, 31571551 and 31571190), the Science and Technology Project of Chongqing Education Committee (KJ130309), open funding by the Chongqing Institute for Family Planning (1201) and the Excellent Young Scholars of Chongqing Medical University (CQYQ201302). The authors have no conflicts of interest to declare.

Germline recombination in a novel Cre transgenic line, Prl3b1-Cre mouse.

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1-cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL-2) protein in placenta along with increased expression toward the end of pregnancy. PL-2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1-cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1-cre;R26GRR mice revealed that tdsRed-positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1-cre;R26GRR testes suggested that Cre-mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1-cre mice line provides a unique resource to understand testicular germ-cell development. genesis 54:389-397, 2016. © 2016 Wiley Periodicals, Inc.