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).

Do some viruses use growth hormone, prolactin and their receptors to facilitate entry into cells?: Episodic evolution of hormones and receptors suggests host-virus arms races; related placental lactogens may provide protective viral decoys.

The molecular evolution of pituitary growth hormone and prolactin in mammals shows two unusual features: episodes of markedly accelerated evolution and, in some species, complex families of related proteins expressed in placenta and resulting from multiple gene duplications. Explanations of these phenomena in terms of physiological adaptations seem unconvincing. Here, I propose an alternative explanation, namely that these evolutionary features reflect the use of the hormones (and their receptors) as viral receptors. Episodes of rapid evolution can then be explained as due to "arms races" in which changes in the hormone lead to reduced interaction with the virus, and subsequent changes in the virus counteract this. Placental paralogues of the hormones could provide decoys that bind viruses, and protect the foetus against infection. The hypothesis implies that the extensive changes introduced into growth hormone, prolactin and their receptors during the course of mammalian evolution reflect viral interactions, not endocrine adaptations.

Actions of DKK1 on the preimplantation bovine embryo to affect pregnancy establishment, placental function, and postnatal phenotype†.

One mechanism by which the maternal environment regulates the early embryo is by secretion of cell-signaling molecules. One of these is dickkopf WNT signaling pathway inhibitor 1. Objectives were to (A) resolve discrepancies in the literature regarding effects of dickkopf WNT signaling pathway inhibitor 1 in the bovine embryo on development of trophectoderm and competence to establish pregnancy after embryo transfer and (B) determine whether there are long-term consequences of dickkopf WNT signaling pathway inhibitor 1 on placental function and postnatal phenotype. Embryos produced in vitro were cultured with vehicle or 100 ng/mL recombinant human dickkopf WNT signaling pathway inhibitor 1 from Days 5 to 7.5 of development (i.e., the morula and blastocyst stages of development). dickkopf WNT signaling pathway inhibitor 1 increased the number of cells positive for the trophectoderm marker CDX2 at Day 7.5 of development while having no effect on number of cells positive for the inner cell mass marker SOX2. There was no effect of dickkopf WNT signaling pathway inhibitor 1 on pregnancy or calving rate after transfer of blastocysts produced with Y-sorted semen to either lactating dairy cows or suckling beef cows. Treatment with dickkopf WNT signaling pathway inhibitor 1 at the morula-to-blastocyst stages programmed placental function, as measured by an effect of dickkopf WNT signaling pathway inhibitor 1 on plasma concentrations of pregnancy associated glycoproteins and placental lactogen at Day 160 of gestation (although not on other days examined). dickkopf WNT signaling pathway inhibitor 1 treatment also resulted in calves that were heavier at birth as compared to calves derived from control embryos. After birth, dickkopf WNT signaling pathway inhibitor 1 calves grew slower than controls. Results confirm that dickkopf WNT signaling pathway inhibitor 1 alters the developmental program of the bovine embryo to affect both prenatal and postnatal phenotypes.

Impact of chorionic somatomammotropin RNA interference on uterine blood flow and placental glucose uptake in the absence of intrauterine growth restriction.

Chorionic somatomammotropin (CSH) is one of the most abundantly produced placental hormones, yet its exact function remains elusive. Near-term [135 days of gestational age (dGA)], CSH RNA interference (RNAi) results in two distinct phenotypes: 1) pregnancies with intrauterine growth restriction (IUGR), and 2) pregnancies with normal fetal and placental weights. Here, we report the physiological changes in CSH RNAi pregnancies without IUGR. The trophectoderm of hatched blastocysts (9 dGA) were infected with lentiviral-constructs expressing either a scrambled control (Control RNAi) or CSH-specific shRNA (CSH RNAi), prior to transfer into synchronized recipient ewes. At 126 dGA, Control RNAi (n = 6) and CSH RNAi (n = 6) pregnancies were fitted with maternal and fetal catheters. Uterine and umbilical blood flows were measured at 132 dGA and nutrient uptakes were calculated by the Fick's principle. Control RNAi and CSH RNAi pregnancies were compared by analysis of variance, and significance was set at P ≤ 0.05. Absolute (mL/min) and relative (mL/min/kg fetus) uterine blood flows were reduced (P ≤ 0.05) in CSH RNAi pregnancies, but umbilical flows were not impacted. The uterine artery-to-vein glucose gradient (mmol/L) was significantly (P ≤ 0.05) increased. The uteroplacental glucose uptake (µmoL/min/kg placenta) was increased (P ≤ 0.05), whereas umbilical glucose uptake (µmoL/min/kg fetus) was reduced. Our results demonstrate that CSH RNAi has significant physiological ramifications, even in the absence of IUGR, and comparing CSH RNAi pregnancies exhibiting both IUGR and non-IUGR phenotypes may help determine the direct effects of CSH and its potential impact on fetal development.

CSH RNA Interference Reduces Global Nutrient Uptake and Umbilical Blood Flow Resulting in Intrauterine Growth Restriction.

Deficiency of the placental hormone chorionic somatomammotropin (CSH) can lead to the development of intrauterine growth restriction (IUGR). To gain insight into the physiological consequences of CSH RNA interference (RNAi), the trophectoderm of hatched blastocysts (nine days of gestational age; dGA) was infected with a lentivirus expressing either a scrambled control or CSH-specific shRNA, prior to transfer into synchronized recipient sheep. At 90 dGA, umbilical hemodynamics and fetal measurements were assessed by Doppler ultrasonography. At 120 dGA, pregnancies were fitted with vascular catheters to undergo steady-state metabolic studies with the 3H2O transplacental diffusion technique at 130 dGA. Nutrient uptake rates were determined and tissues were subsequently harvested at necropsy. CSH RNAi reduced (p ≤ 0.05) both fetal and uterine weights as well as umbilical blood flow (mL/min). This ultimately resulted in reduced (p ≤ 0.01) umbilical IGF1 concentrations, as well as reduced umbilical nutrient uptakes (p ≤ 0.05) in CSH RNAi pregnancies. CSH RNAi also reduced (p ≤ 0.05) uterine nutrient uptakes as well as uteroplacental glucose utilization. These data suggest that CSH is necessary to facilitate adequate blood flow for the uptake of oxygen, oxidative substrates, and hormones essential to support fetal and uterine growth.

Persistent Human KIT Receptor Signaling Disposes Murine Placenta to Premature Differentiation Resulting in Severely Disrupted Placental Structure and Functionality.

Activating mutations in the human KIT receptor is known to drive severe hematopoietic disorders and tumor formation spanning various entities. The most common mutation is the substitution of aspartic acid at position 816 to valine (D816V), rendering the receptor constitutively active independent of ligand binding. As the role of the KIT receptor in placental signaling cascades is poorly understood, we analyzed the impact of KITD816V expression on placental development using a humanized mouse model. Placentas from KITD816V animals present with a grossly changed morphology, displaying a reduction in labyrinth and spongiotrophoblast layer and an increase in the Parietal Trophoblast Giant Cell (P-TGC) layer. Elevated differentiation to P-TGCs was accompanied with reduced differentiation to other Trophoblast Giant Cell (TGC) subtypes and by severe decrease in proliferation. The embryos display growth retardation and die in utero. KITD816V-trophoblast stem cells (TSC) differentiate much faster compared to wild type (WT) controls. In undifferentiated KITD816V-TSCs, levels of Phosphorylated Extracellular-signal Regulated Kinase (P-ERK) and Phosphorylated Protein Kinase B (P-AKT) are comparable to wildtype cultures differentiating for 3-6 days. Accordingly, P-TGC markers Placental Lactogen 1 (PL1) and Proliferin (PLF) are upregulated as well. The results reveal that KIT signaling orchestrates the fine-tuned differentiation of the placenta, with special emphasis on P-TGC differentiation. Appropriate control of KIT receptor action is therefore essential for placental development and nourishment of the embryo.

Chromosomal architecture and placental expression of the human growth hormone gene family are targeted by pre-pregnancy maternal obesity.

The human (h) placental lactogenic hormone chorionic somatomammotropin (CS) is highly produced during pregnancy and acts as a metabolic adaptor in response to maternal insulin resistance. Maternal obesity can exacerbate this "resistance", and a >75% decrease in CS RNA levels was observed in term placentas from obese vs. lean women. The genes coding for hCS ( hCS-A and hCS-B) and placental growth hormone ( hGH-V) as well as the hCS-L pseudogene and pituitary growth hormone (GH) gene ( hGH-N) are located at a single locus on chromosome 17. Three remote hypersensitive sites (HS III-V) located >28 kb upstream of hGH-N as well as local hCS gene promoter and enhancer regions are implicated in hCS gene expression. A placenta-specific chromosomal architecture, including interaction between HS III-V and hCS but not hGH gene promoters, was detected in placentas from lean women (BMI <25 kg/m2) by using the chromosome conformation capture assay. This architecture was disrupted by pre-pregnancy maternal obesity (BMI >35 kg/m2), resulting in a predominant interaction between HS III and the hGH-N promoter, which was also observed in nonplacental tissues. This was accompanied by a decrease in hCS levels, which was consistent with reduced RNA polymerase II and CCAAT/enhancer-binding protein-ß association with individual hCS promoter and enhancer sequences, respectively. Thus, pre-pregnancy maternal obesity disrupts the placental hGH/CS gene locus chromosomal architecture. However, based on data from obese women who develop GDM, insulin treatment partially recapitulates the chromosomal architecture seen in lean women and positively affects hCS production, presumably facilitating prolactin receptor-related signaling by hCS.

Nuclear transfer alters placental gene expression and associated histone modifications of the placental-specific imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2) in cattle.

Abnormal placental development is frequent in nuclear transfer (NT) pregnancies and is likely to be associated with altered epigenetic reprogramming. In the present study, fetal and placental measurements were taken on Day 60 of gestation in cows with pregnancies produced by AI, IVF and NT. Placentas were collected and subjected to histological evaluation, the expression of genes important in trophoblast differentiation and expression of the placental imprinted gene pleckstrin homology-like domain, family A, member 2 (PHLDA2), as well as chromatin immunoprecipitation (ChIP) for histone marks within the promoter of PHLDA2. Fewer binucleated cells were observed in NT cotyledons, followed by IVF and AI cotyledons (P<0.05). Expression of heart and neural crest derivatives expressed 1 (HAND1), placental lactogen (PL), pregnancy-associated glycoprotein 9 (PAG-9) and PHLDA2 was elevated in NT cotyledons compared with AI cotyledons. Expression of PHLDA2 was higher in IVF than AI samples (P<0.05). ChIP revealed an increase in the permissive mark dimethylation of lysine 4 on histone H3 (H3K4me2), surprisingly associated with the silent allele of PHLDA2, and a decrease in the inhibitory mark H3K9me2 in NT samples. Thus, genes critical for placental development were altered in NT placentas, including an imprinted gene. Allele-specific changes in the permissive histone mark in the PHLDA2 promoter indicate misregulation of imprinting in clones. Abnormal trophoblast differentiation could have resulted in lower numbers of binucleated cells following NT. These results suggest that the altered expression of imprinted genes associated with NT are also caused by changes in histone modifications.

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.

Tissue specific CTCF occupancy and boundary function at the human growth hormone locus.

The robust and tissue-specific activation of the human growth hormone (hGH) gene cluster in the pituitary and placenta constitutes an informative model for analysis of gene regulation. The five-gene hGH cluster is regulated by two partially overlapping sets of DNase I hypersensitive sites (HSs) that constitute the pituitary (HSI, II, III and V) and placental (HSIII, IV, and V) locus control regions (LCRs). The single placenta-specific LCR component, HSIV, is located at -30 kb to the cluster. Here we generate a series of hGH/BAC transgenes specifically modified to identify structural features of the hGH locus required for its appropriate placental expression. We find that placental specificity is dependent on the overall multigene configuration of the cluster whereas the distance between the cluster and its LCR impacts the level of placental expression. We further observe that a major function of the placental hGH LCR is to insulate the transgene locus from site-of-integration effects. This insulation activity is linked to placenta-specific occupancy of the chromatin architectural protein, CTCF, at HSIV. These data reveal a remarkable combination of structural configurations and regulatory determinants that must work in concert to insure robust and tightly controlled expression from a complex multigene locus.

Alternative splicing of placental lactogen (CSH2) in somatotroph pituitary adenomas.

OBJECTIVES: Somatotroph adenomas secrete supraphysiological amounts of GH, causing acromegaly. We have previously shown epithelial splicing regulator 1 (ESRP1) to play a role in epithelial mesenchymal transition (EMT) progression in these adenomas and account for poor treatment response. We evaluated if the mRNA levels of the GH/CSH gene cluster in somatotroph adenomas are associated with an epithelial phenotype and response to SA treatment. METHODS: We investigated the associations between ESRP1 and the growth hormone/chorionic somatomammotropin (GH/CSH) gene cluster by RNA sequencing (RNAseq). CSH2 isoform 3 mRNA was further evaluated in 65 somatotroph adenomas and associations with disease severity and treatment response. RESULTS: mRNA for all genes in the GH/CSH cluster were expressed, however, only chorionic somatomammotropin 2/placental lactogen 2 (CSH2) displayed an alternative splicing pattern. CSH2 isoform 3 was associated with a dense granulation pattern and an epithelial phenotype with high levels of ESRP1 and E-cadherin expression. Further, CSH2 isoform 3 was associated with reduced serum GH and IGF-I levels after somatostatin analog treatment. CONCLUSIONS: Attenuated CSH2 isoform 3 was associated with mesenchymal phenotype and a blunted clinical response to somatostatin analog treatment in patients with acromegaly.

Predominantly placenta-expressed mRNAs in maternal plasma as predictive markers for twin-twin transfusion syndrome.

OBJECTIVE: This study aimed to identify a set of predominantly placental (PP) mRNAs, which are associated with later-developing twin-to-twin transfusion syndrome (TTTS). METHOD: First, out of 50 PP mRNAs we previously reported, we select target mRNAs that are ordinarily detectable in maternal plasma. Plasma concentrations of these PP mRNAs were measured in monochorionic diamniotic twin (MCDA-T) pregnancies complicated by TTTS later (n = 11) and in uncomplicated MCDA-T pregnancies (n = 17). Finally, the diagnostic values of the PP mRNAs in plasma were evaluated. RESULTS: From 50 PP mRNAs, nine [human placental lactogen (hPL); pregnancy-specific glycoproteins 2 (PSG2); human pregnancy-specific glycoproteins 3 (PSG3); syncytin; syncytin 2; retinoic acid-induced 14; A disintegrin and metalloproteinase domain-containing protein 12 (ADAM12); chorionic glycoprotein hormones, alpha polypeptide; and chorionic glycoprotein hormones, and beta polypeptide] were selected as target mRNAs. Changes in six PP mRNAs [increased hPL, PSG2, and PSG3 and decreased syncytin, syncytin2, and ADAM12] in maternal plasma were detected in MCDA-T pregnant women who subsequently developed TTTS. Finally, mRNA signatures gave elevated AUCs (hPL/PSG2: 0.8717; hPL/PSG3: 0.8449; hPL/ADAM12: 0.8396) compared with single hPL mRNA. CONCLUSION: Quantitative aberration of plural cell-free PP mRNAs in maternal plasma precedes the appearance of clinically apparent TTTS. This suggests that pathophysiological changes in the placenta are associated with morbid conditions of TTTS.

Heart and extra-embryonic mesodermal defects in mouse embryos lacking the bHLH transcription factor Hand1.

The basic helix-loop-helix (bHLH) transcription factors, Hand1 and Hand2 (refs 1,2), also called eHand/Hxt/Thing1 and dHand/Hed/Thing2 (refs 3,4), respectively, are expressed in the heart and certain neural-crest derivatives during embryogenesis. In addition, Hand1 is expressed in extraembryonic membranes, whereas Hand2 is expressed in the deciduum. Previous studies have demonstrated that Hand2 is required for formation of the right ventricle of the heart and the aortic arch arteries. We have generated a germline mutation in the mouse Hand1 gene by replacing the first coding exon with a beta-galactosidase reporter gene. Embryos homozygous for the Hand1 null allele died between embryonic days 8.5 and 9.5 and exhibited yolk sac abnormalities due to a deficiency in extraembryonic mesoderm. Heart development was also perturbed and did not progress beyond the cardiac-looping stage. Our results demonstrate important roles for Hand1 in extraembryonic mesodermal and heart development.

What can we learn from rodents about prolactin in humans?

Prolactin (PRL) is a 23-kDa protein hormone that binds to a single-span membrane receptor, a member of the cytokine receptor superfamily, and exerts its action via several interacting signaling pathways. PRL is a multifunctional hormone that affects multiple reproductive and metabolic functions and is also involved in tumorigenicity. In addition to being a classical pituitary hormone, PRL in humans is produced by many tissues throughout the body where it acts as a cytokine. The objective of this review is to compare and contrast multiple aspects of PRL, from structure to regulation, and from physiology to pathology in rats, mice, and humans. At each juncture, questions are raised whether, or to what extent, data from rodents are relevant to PRL homeostasis in humans. Most current knowledge on PRL has been obtained from studies with rats and, more recently, from the use of transgenic mice. Although this information is indispensable for understanding PRL in human health and disease, there is sufficient disparity in the control of the production, distribution, and physiological functions of PRL among these species to warrant careful and judicial extrapolation to humans.

Crystal structure of an affinity-matured prolactin complexed to its dimerized receptor reveals the topology of hormone binding site 2.

We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL.PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural components of PRL site 2 ("three-pin plug"): the conserved glycine 129 of helix alpha3, the hydrogen bond network involving surrounding residues (glycine cavity), and the N terminus. The model provides a molecular basis for the properties of the different PRL analogs designed to date, including PRLR antagonists. Finally, comparison of our 1:2 PRL.PRLR(2) structure with those of free PRL and its 1:1 complex indicates that the structure of PRL undergoes significant changes when binding the first, but not the second receptor. This suggests that the second PRLR moiety adapts to the 1:1 complex rather than the opposite. In conclusion, this structure will be a useful guiding tool for further investigations of the molecular mechanisms involved in PRLR dimerization and activation, as well as for the optimization of PRLR antagonists, an emerging class of compounds with high therapeutic potential against breast and prostate cancer.

Obligate ordered binding of human lactogenic cytokines.

Class 1 cytokines bind two receptors to create an active heterotrimeric complex. It has been argued that ligand binding to their receptors is an ordered process, but a structural mechanism describing this process has not been determined. We have previously described an obligate ordered binding mechanism for the human prolactin/prolactin receptor heterotrimeric complex. In this work we expand this conceptual understanding of ordered binding to include three human lactogenic hormones: prolactin, growth hormone, and placental lactogen. We independently blocked either of the two receptor binding sites of each hormone and used surface plasmon resonance to measure human prolactin receptor binding kinetics and stoichiometries to the remaining binding surface. When site 1 of any of the three hormones was blocked, site 2 could not bind the receptor. But blocking site 2 did not affect receptor binding at site 1, indicating a requirement for receptor binding to site 1 before site 2 binding. In addition we noted variable responses to the presence of zinc in hormone-receptor interaction. Finally, we performed Förster resonance energy transfer (FRET) analyses where receptor binding at subsaturating stoichiometries induced changes in FRET signaling, indicative of binding-induced changes in hormone conformation, whereas at receptor:hormone ratios in excess of 2:1 no additional changes in FRET signaling were observed. These results strongly support a conformationally mediated obligate-ordered receptor binding for each of the three lactogenic hormones.

Plasmin generates vasoinhibin-like peptides by cleaving prolactin and placental lactogen.

Vasoinhibin is an antiangiogenic, profibrinolytic peptide generated by the proteolytic cleavage of the pituitary hormone prolactin by cathepsin D, matrix metalloproteinases, and bone morphogenetic protein-1. Vasoinhibin can also be generated when placental lactogen or growth hormone are enzymatically cleaved. Here, it is investigated whether plasmin cleaves human prolactin and placental lactogen to generate vasoinhibin-like peptides. Co-incubation of prolactin and placental lactogen with plasmin was performed and analyzed by gel electrophoresis and Western blotting. Mass spectrometric analyses were carried out for sequence validation and precise cleavage site identification. The cleavage sites responsible for the generation of the vasoinhibin-like peptides were located at K170-E171 in prolactin and R160-T161 in placental lactogen. Various genetic variants of the human prolactin and placental lactogen genes are projected to affect proteolytic generation of the vasoinhibin-like peptides. The endogenous counterparts of the vasoinhibin-like peptides generated by plasmin may represent vasoinhibin-isoforms with inhibitory effects on vasculature and coagulation.

Adiponectin Promotes Maternal ß-Cell Expansion Through Placental Lactogen Expression.

Hypoadiponectinemia is a risk factor of gestational diabetes mellitus (GDM). Our previous study reported that adiponectin gene knockout mice (Adipoq -/- ) develop GDM due to insulin insufficiency. The main objective of this study was to elucidate the underlying mechanism through which adiponectin controls islet expansion during pregnancy. A significant reduction in ß-cell proliferation rates, ß-cell areas, and blood insulin concentrations was detected in Adipoq -/- mice at midpregnancy. Surprisingly, conditionally knocking down adiponectin receptor 1 (AdipoR1) or AdipoR2 genes in ß-cells during pregnancy did not reduce ß-cell proliferation rates or blood insulin concentrations. In vitro adiponectin treatment also failed to show any effect on ß-cell proliferation of isolated pancreatic islets. It was reported that placental lactogen (PL) plays a crucial role in pregnancy-induced maternal ß-cell proliferation. A significant decrease in phosphorylation of signal transducer and activator of transcription 5, a downstream molecule of PL signaling, was observed in islets from Adipoq -/- dams. The mRNA levels of mouse PL genes were robustly decreased in the placentas of Adipoq -/- dams. In contrast, adiponectin treatment increased PL expression in human placenta explants and JEG3 trophoblast cells. Most importantly, bovine PL injection restored ß-cell proliferation and blood insulin concentrations in Adipoq -/- dams. Together, these results demonstrate that adiponectin plays a vital role in pregnancy-induced ß-cell proliferation by promoting PL expression in trophoblast cells.

Forensic pregnancy diagnostics with placental mRNA markers.

Current methods for pregnancy diagnostics are based on immunodetection of pregnancy-specific proteins and in a forensic context suffer from sensitivity and specificity issues. Here, we applied reverse transcriptase polymerase chain reaction (RT-PCR) technology to 11 genes previously reported with placental mRNA circulating in maternal blood. We found two genes, hPL and betahCG, with pregnancy-specific expression in whole blood samples. RT-PCR detection of hPL was positive in all samples tested throughout the pregnancy, whereas betahCG was detectable until half of the second trimester but not at later gestation ages. For hPL, in vitro stability of the transcript was demonstrated until 2 months of age, and the hPL-specific RT-PCR assay applied was highly sensitive with reliable detection from down to 0.25 cm(2) dried bloodstain. We therefore suggest hPL-specific RT-PCR as a new molecular tool for forensic pregnancy diagnostics from dried blood stains. Moreover, our results indicate that the time-wise reverse expression of hPL and betahCG during pregnancy may allow an RT-PCR-based estimation of the gestational age from blood stains, adding to the value of forensic pregnancy diagnosis for crime scene investigations.

Identification of universal mRNA markers for noninvasive prenatal screening of trisomies.

OBJECTIVE: The discovery of placental transcripts in peripheral blood of pregnant women prompted us to investigate which was the most appropriate biological specimen, between plasma and serum, to easily detect them and to exploit hPL (human placental lactogen), betahCG (human chorionic gonadotrophin beta-subunit), LOC90625, and TFPI2 (tissue factor pathway inhibitor 2) levels in order to establish whether an abnormal variation degree of presence of these placental transcripts are likely to be associated to specific fetal trisomies. METHOD: RNA was extracted from plasma and serum samples of 255 pregnant women bearing euploid fetuses, 17 bearing fetuses affected by trisomy 21 and 10 with fetuses affected by trisomy 18. Placental transcript analysis was performed by real time RT-PCR using relative quantification. RESULTS: Results obtained from euploid samples showed that fetal transcripts were more abundant in plasma than in serum samples. Euploid samples had a placental transcript abundance distinguishable from those with trisomy 21 but not from those with trisomy 18. In particular, high betahCG abundance and advanced maternal age were significantly associated with trisomy 21 pregnancy. CONCLUSION: Plasma was the most suitable tool to be employed in the detection and dosage of placental transcripts. betahCG transcript together with maternal age could be a potential marker for noninvasive prenatal screening of fetal trisomy 21.