In humans, primitive fetal nucleated red blood cells (FNRBCs) are thought to be as vital for embryonic life as their counterpart, adult red blood cells (adult RBCs) are in later-gestation fetuses and adults. Unlike adult RBCs, the identity and functions of FNRBC proteins are poorly understood owing to a scarcity of FNRBCs for proteomic investigations. The study aimed to investigate membrane proteins of this unique cell type. We present here, the first report on the membrane proteome of human primitive FNRBCs investigated by two-dimensional liquid chromatography coupled with mass-spectrometry (2D-LCMS/MS) and bioinformatics analysis. A total of 273 proteins were identified, of which 133 (48.7%) were membrane proteins. We compared our data with membrane proteins of adult RBCs to identify common, and unique, surface membrane proteins. Twelve plasma membrane proteins with transmembrane domains and eight proteins with transmembrane domains but without known sub-cellular location were identified as unique-to-FNRBCs. Except for the transferrin receptor, all other 19 unique-to-FNRBC membrane proteins have never been described in RBCs. Reverse-transcriptase PCR (RT-PCR) and immunocytochemistry validated the 2D-LCMS/MS data. Our findings provide potential surface antigens for separation of primitive FNRBCs from maternal blood for noninvasive prenatal diagnosis, and to understand the biology of these rare cells.

Caffeine is the psychostimulant most consumed worldwide. However, little is known about its effects during fetal brain development. In this study, adult female Wistar rats received caffeine in drinking water (0.1, 0.3 and 1.0 g/L) during the active cycle in weekdays, two weeks before mating and throughout pregnancy. Cerebral cortex and hippocampus from embryonic stages 18 or 20 (E18 or E20, respectively) were collected for immunodetection of the following synaptic proteins: brain-derived neurotrophic factor (BDNF), TrkB receptor, Sonic Hedgehog (Shh), Growth Associated Protein 43 (GAP-43) and Synaptosomal-associated Protein 25 (SNAP-25). Besides, the estimation of NeuN-stained nuclei (mature neurons) and non-neuronal nuclei was verified in both brain regions and embryonic periods. Caffeine (1.0 g/L) decreased the body weight of embryos at E20. Cortical BDNF at E18 was decreased by caffeine (1.0 g/L), while it increased at E20, with no major effects on TrkB receptors. In the hippocampus, caffeine decreased TrkB receptor only at E18, with no effects on BDNF. Moderate and high doses of caffeine promoted an increase in Shh in both brain regions at E18, and in the hippocampus at E20. Caffeine (0.3g/L) decreased GAP-43 only in the hippocampus at E18. The NeuN-stained nuclei increased in the cortex at E20 by lower dose and in the hippocampus at E18 by moderate dose. Our data revealed that caffeine transitorily affect synaptic proteins during fetal brain development. The increased number of NeuN-stained nuclei by prenatal caffeine suggests a possible acceleration of the telencephalon maturation. Although some modifications in the synaptic proteins were transient, our data suggest that caffeine even in lower doses may alter the fetal brain development.

As depots for neutral lipids, lipid storage droplets (LDs) accumulate with advancing gestation within the fetal membranes. Little is currently known about the proteins associated with the LDs of these cells. The PAT family [perilipin, adipose differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47)] represents a unique group of proteins thought to contribute to LD formation and function. We examined the association of each of the PAT proteins with LDs of term fetal membranes. We found that large LDs of amnion epithelial cells were reactive for neutral lipid stains and simultaneously encoated with ADRP and TIP47, but not perilipin. Within the remaining cell types, LDs were frequently co-labeled with antibodies recognizing ADRP and TIP47; however, in cells harboring only small LDs, the majority of TIP47 labeling was cytoplasmic. Structures labeled with perilipin antibodies were present only in chorion laeve trophoblasts. Gene and protein expression analyses suggested this to be a small molecular weight perilipin isoform, such as that seen in steroidogenic cells. We conclude that LDs are heterogeneous among differing cell types of the fetal membranes. Subclassification of LDs based on associated proteins suggests that these organelles may serve specialized functions within individual cells.

Intrauterine growth rate is associated with body distribution in adulthood suggesting differential response of fetal fat depots to nutritional modifications. We hypothesize that there is regional differences in fetal adipogenesis, in part, due to depot-specific regulation of the availability of insulin growth factors. In near-term baboon fetuses (n=3-5), the subcutaneous abdominal vs. omental preadipocytes had (1) more extensive lipid accumulation as assessed by BODIPY (lipid staining) to DAPI (nuclei) absorbance ratios (mean+/-SEM; 0.51+/-0.21, 0.35+/-0.09, p<0.05), (2) lower (p<0.05) secretion of IGF-binding protein 4 (9.6+/-1.2 vs. 17.4+/-2.8 ng/ml) and its protease pregnancy associated plasma protein A (24.6+/-1.9 vs. 39.1+/-6.3 microIU/ml), (3) lower protein expression of IGF2 "clearance" receptor in cell lysate (0.28+/-0.03 vs. 0.53+/-0.02 OD U/mm(2), p<0.05); all variables were intermediate in femoral preadipocytes. The regional variation of the adipogenesis and the IGF regulatory pathway set the stage for differential responsiveness of fat depots to external signals.

Transcripts of alpha-fetoprotein (Afp), H19, and insulin-like growth factor 2 (Igf2) genes are highly expressed in mouse fetal liver, but decrease drastically during maturation. While transcriptional regulation of these genes has been well studied, the post-transcriptional regulation of their developmental decrease is poorly understood. Here, we show that shortening of poly(A) tails and subsequent RNA decay are largely responsible for the postnatal decrease of Afp, H19, and Igf2 transcripts in mouse liver. IGF2 mRNA binding protein 1 (IMP1), which regulates stability and translation efficiency of target mRNAs, binds to these fetal liver transcripts. When IMP1 is exogenously expressed in mouse adult liver, fetal liver transcripts show higher expression and possess longer poly(A) tails, suggesting that IMP1 stabilizes them. IMP1 declines concomitantly with fetal liver transcripts as liver matures. Instead, RNA-binding proteins (RBPs) that promote RNA decay, such as cold shock domain containing protein E1 (CSDE1), K-homology domain splicing regulatory protein (KSRP), and CUG-BP1 and ETR3-like factors 1 (CELF1), bind to 3' regions of fetal liver transcripts. These data suggest that transitions among RBPs associated with fetal liver transcripts shift regulation from stabilization to decay, leading to a postnatal decrease in those fetal transcripts.

In mammal circulation, ferritin-binding proteins (FBPs) are thought to be involved in clearance of circulating ferritin after complex formation with it through receptor-mediated uptake. However, there is no report on fetal FBP in fetal circulation. Although iron concentrations of fetal horse plasma were higher than those of adult horse plasma, plasma ferritin concentrations and ferritin-binding activities were found to be significantly lower in fetus than in adult. FBPs were purified from fetal or adult horse plasma on horse spleen ferritin-Sepharose 4B affinity column. Partially affinity-purified fetal horse plasma FBPs were mainly separated into 65 and 41 kDa bands in addition to minor bands with higher molecular masses ranged from 102 to 140 kDa on SDS-PAGE under reducing condition. The adult horse plasma FBPs were separated into 74, 54 and 28 kDa bands, and the 74 and 54 kDa bands reacted with antibodies specific for horse IgM and IgG heavy chains, respectively, by immunoblotting analyses. On the other hand, no antibodies to horse immunoglobulin classes detected any bands in fetal horse plasma FBPs. The affinity-purified adult and fetal horse plasma FBPs did not contain fibrinogen as a plasma specific FBP, probably due to its lower affinity to the ligand ferritin. These results demonstrate the presence of FBPs which are different from adult horse plasma FBPs including anti-ferritin autoantibodies in fetal plasma.

Introduction: Antenatal steroid improves respiratory distress syndrome in preterm infants. The molecular mechanism of the process is not well established. The aim of this study is to investigate the possible association between antenatal steroid and fetal Forkhead box M1(Foxm1) expression. Materials and methods: An animal study using mated pregnant New Zealand white rabbits and their fetuses was designed. Fourteen mother rabbits were assigned to four groups to undergo a cesarean section. In groups 1, 2, and 3, preterm pups were harvested on day 27 of gestation. In group 4, term pups were harvested on day 31. Antenatal maternal intramuscular injection was performed in groups 2 (normal saline) and 3 (betamethasone). Using qRT-PCR and Western blot, mRNA transcription and protein expression of surfactant protein (SP) A, B, C, and Foxm1 were compared between the pups of those four groups. Results: Sixty two fetal rabbits were harvested. One-way ANOVA test showed higher mRNA transcription of SPs in groups 3 and 4 than groups 1 and 2. Significantly lower Foxm1 mRNA transcription and protein expression were observed in group 3 or 4 compared with group 1 or 2. Conclusion: Decreased Foxm1 expression was associated in an antenatal betamethasone animal model.

Betamethasone has been used extensively to accelerate fetal lung maturation, yet little is known of its effects on neuronal morphogenesis in the developing fetus. Microtubule-associated proteins (MAPs) are a diverse family of cytoskeletal proteins that are important for brain development and the maintenance of neuroarchitecture. Vehicle (n = 7) or betamethasone (10 ug h-1, n = 7) was infused I.V. to fetal sheep over 48 h beginning at 0.87 of gestation (128 days of gestation), producing fetal plasma betamethasone concentrations resembling those to which the human fetus is exposed during antenatal glucocorticoid therapy. Paraffin sections of the left hemisphere were stained with monoclonal antibodies against MAP1B and the MAP2 isoforms MAP2a,b,c and MAP2a,b. The level of the juvenile isoform MAP2c was determined by comparison of the two MAP2 immunostainings. We were able to detect MAP1B and MAP2 immunoreactivity (IR) in the fetal sheep brain. MAP2c was the major MAP2, constituting 90.2 % of the total MAPBetamethasone exposure diminished MAP1B IR in the frontal cortex and caudate putamen (P < 0.05) but not in the hippocampus. A decrease of MAP2 IR was found in the frontal cortex, hippocampus and caudate putamen (P < 0.05). Loss of MAP2 IR was mainly due to the loss of MAP2c IR. Haematoxylin-eosin staining did not demonstrate irreversible neuronal damage. Regional cerebral blood flow determined using coloured microspheres was significantly decreased by 28 % in the frontal cortex and by 36 % in the caudate putamen but not in the hippocampus 24 h after the onset of betamethasone exposure (P < 0.05). The loss of MAP1B and MAP2a,b,c IR showed a significant correlation to the cerebral blood flow decrease only in the frontal cortex (P < 0.05). These data suggest that mechanisms other than metabolic insufficiency caused by the decreased cerebral blood flow may contribute to the loss of MAPs. The results suggest that clinical doses of betamethasone may have acute effects on cytoskeletal proteins in the fetal brain.

A sequential dissociative extraction scheme is described in which tooth matrix proteins are extracted first in 4 M guanidine HCl, pH 7.4, and then in 4 M guanidine HCl, 0.5 M EDTA, pH 7.4, both with protease inhibitors present. The latter step dissolves the mineralized portion of the tissue and extracts noncollagenous proteins closely associated with hydroxyapatite crystallites in the mineralized matrix. In fetal bovine enamel, the initial dissociative extraction step completely removes proline-rich amelogenins from the tissue without dissolving the enamel apatite. The amelogenin proteins consist of several species on polyacrylamide gel electrophoresis with sodium dodecyl sulfate, but display anomalous migration behavior relative to conventional marker proteins in this technique. Subsequent extraction of fetal bovine enamel with guanidine HCl/EDTA removes matrix enamelins, acidic glycoproteins that are tightly bound to the enamel hydroxyapatite. This latter fetal protein type has not been isolated previously. The enamelins are adsorbed strongly by DEAE-cellulose in 7 M urea and totally adsorb to synthetic apatite, even in 4 M guanidine HCl. The enamelins display normal behavior on polyacrylamide gels and stain positively for sialic acid/phosphate and carbohydrate. With advancing tooth maturation, amelogenins disappear while enamelins are conserved. Gel filtration chromatography in 4 M guanidine HCl showed amelogenin components at apparent molecular weights of approximately 25,000, 15,000, 9,500, 7,500, and 6,000, while the enamelins eluted at Mr positions of approximately 72,000, 56,000, 42,000, 30,000, 21,000, 13,000, and 8,000. The gel filtration data showed a clear shift in molecular size population from higher to lower components for both amelogenins and enamelins with progressive enamel maturation.

In mammals, the timing of meiosis entry is regulated by signals from the gonadal environment. All-trans retinoic acid (ATRA) signaling is considered the key pathway that promotes Stra8 (stimulated by retinoic acid 8) expression and, in turn, meiosis entry. This model, however, is debated because it is based on analyzing the effects of exogenous ATRA on ex vivo gonadal cultures, which not accurately reflects the role of endogenous ATRA. Aldh1a1 and Aldh1a2, two retinaldehyde dehydrogenases synthesizing ATRA, are expressed in the mouse ovaries when meiosis initiates. Contrary to the present view, here, we demonstrate that ATRA-responsive cells are scarce in the ovary. Using three distinct gene deletion models for Aldh1a1;Aldh1a2;Aldh1a3, we show that Stra8 expression is independent of ATRA production by ALDH1A proteins and that germ cells progress through meiosis. Together, these data demonstrate that ATRA signaling is dispensable for instructing meiosis initiation in female germ cells.

In developing countries, maternal undernutrition is the major intrauterine environmental factor contributing to fetal development and adverse pregnancy outcomes. Maternal nutrition restriction (MNR) in gestation has proven to impact overall growth, bone development, and proliferation and metabolism of mesenchymal stem cells in offspring. However, the efficient method for elucidation of fetal bone development performance through maternal bone metabolic biochemical markers remains elusive.

Reactivation of fetal-specific genes and isoforms occurs during heart failure. However, the underlying molecular mechanisms and the extent to which the fetal program switch occurs remains unclear. Limitations hindering transcriptome-wide analyses of alternative splicing differences (i.e. isoform switching) in cardiovascular system (CVS) tissues between fetal, healthy adult and heart failure have included both cellular heterogeneity across bulk RNA-seq samples and limited availability of fetal tissue for research. To overcome these limitations, we have deconvoluted the cellular compositions of 996 RNA-seq samples representing heart failure, healthy adult (heart and arteria), and fetal-like (iPSC-derived cardiovascular progenitor cells) CVS tissues. Comparison of the expression profiles revealed that reactivation of fetal-specific RNA-binding proteins (RBPs), and the accompanied re-expression of 1,523 fetal-specific isoforms, contribute to the transcriptome differences between heart failure and healthy adult heart. Of note, isoforms for 20 different RBPs were among those that reverted in heart failure to the fetal-like expression pattern. We determined that, compared with adult-specific isoforms, fetal-specific isoforms encode proteins that tend to have more functions, are more likely to harbor RBP binding sites, have canonical sequences at their splice sites, and contain typical upstream polypyrimidine tracts. Our study suggests that compared with healthy adult, fetal cardiac tissue requires stricter transcriptional regulation, and that during heart failure reversion to this stricter transcriptional regulation occurs. Furthermore, we provide a resource of cardiac developmental stage-specific and heart failure-associated genes and isoforms, which are largely unexplored and can be exploited to investigate novel therapeutics for heart failure.

In early pregnancy, fetal skin wounds can heal quickly and undergo a transition period from scarless healing to scar formation. The aim of the present study was to identify potential biomarkers associated with scarless repair of cleft lips, in order to determine the intrinsic factors leading to scar formation in embryonic tissue. A stable model of cleft lip was established using microsurgery by constructing a wedge‑shaped cleft lip‑like defect in fetal rats at gestational age (GA) 16.5 and GA18.5. The GA16.5 and GA18.5 groups were used to model scarless healing and scar formation, respectively. The fetuses were returned to the uterus following surgery, then removed 72 h after the procedure. Macroscopic observation of the cleft defect and histological examination were carried out. Reverse transcription‑quantitative (RT‑q) PCR and parallel reaction monitoring (PRM) were used to detect mRNA and protein expression levels, respectively. The upper‑left lip completely healed 72 h after surgery in the GA16.5 group of fetal rats. However, this was not the case in the GA18.5 group. Histological examination indicated new follicles visible under the epidermis of the scarless group (GA16.5). Scarring was visible on the upper‑left cleft lip wound of the fetal rats in the GA18.5 group. The expression of some growth and pro‑inflammatory factors, including TNF‑α, were also different between two groups. Label‑free quantification was used to identified differentially expressed proteins and five differentially expressed proteins (Smad4, Fabp5, S100a4, S100a8 and S100a9) were identified. The relative expression of these molecules at the mRNA and protein levels were measured using RT‑qPCR and PRM. These molecules may represent potential biomarkers for the scarless repair of fetal rat cleft lip wounds.

The syncytiotrophoblast, a key barrier between the mother and fetus, is a polarized epithelium composed of a microvillus and basal membrane (BM). We sought to characterize BM proteins of BeWo cells in relation to hypoxia and to investigate their expression in placentas from pregnancies complicated by fetal growth restriction (FGR).

The contribution of peripheral immunity to autism spectrum disorders (ASDs) risk is debated and poorly understood. Some mothers of children with ASD have autoantibodies that react to fetal brain proteins, raising the possibility that a subset of ASD cases may be associated with a maternal antibody response during gestation. The mechanism by which the maternal immune system breaks tolerance has not been addressed. We hypothesized that the mechanism may involve decreased expression of the MET receptor tyrosine kinase, an ASD risk gene that also serves as a key negative regulator of immune responsiveness. In a sample of 365 mothers, including 202 mothers of children with ASD, the functional MET promoter variant rs1858830 C allele was strongly associated with the presence of an ASD-specific 37+73-kDa band pattern of maternal autoantibodies to fetal brain proteins (P=0.003). To determine the mechanism of this genetic association, we measured MET protein and cytokine production in freshly prepared peripheral blood mononuclear cells from 76 mothers of ASD and typically developing children. The MET rs1858830 C allele was significantly associated with MET protein expression (P=0.025). Moreover, decreased expression of the regulatory cytokine IL-10 was associated with both the MET gene C allele (P=0.001) and reduced MET protein levels (P=0.002). These results indicate genetic distinction among mothers who produce ASD-associated antibodies to fetal brain proteins, and suggest a potential mechanism for how a genetically determined decrease in MET protein production may lead to a reduction in immune regulation.

1. The purpose of this study was to examine the changes in circulating concentrations of insulin-like growth factor (IGF)-I, IGF-II, IGF-binding protein (IGFBP)-1, IGFBP-2 and insulin following asphyxia in utero. 2. Fetal sheep at 90-93 days gestation underwent either sham occlusion (n = 7) or asphyxia (n = 6) induced by complete umbilical cord occlusion for 30 min. Fetal blood samples were taken before occlusion and 4, 6, 24, 48 and 72 h post-occlusion. 3. During the early phase of recovery there was a substantial fall (80 %) in circulating plasma IGF-I concentrations by 6 h post-asphyxia (P < 0.001). This was associated with a rapid rise in IGFBP-1 (P < 0.001), but no change in IGF-II or IGFBP-2. Insulin was significantly reduced at 4 h (P < 0.001) and glucose slightly elevated (P < 0.05), but insulin values returned to baseline by 6 h. Between 24 and 72 h of recovery, IGF-I gradually increased, IGFBP-1 returned to control values, and there was an increase in IGFBP-2 after 24 h (P < 0.05) and in IGF-II by 72 h (P < 0.05) after asphyxia. 4. These data demonstrate a differential effect of asphyxia on the IGF axis of the premature fetal sheep. A key finding was the large fall in circulating IGF-I, but not IGF-II, during the early phase of recovery. IGF-I bioavailability was, in part, regulated by IGFBP-1, but maximal changes in IGF-I and IGFBP-1 were independent of plasma insulin and glucose.5. The impact of this substantial change in circulating IGF-I on the fetus is unknown. It may facilitate metabolic requirements by promoting catabolism. Alternatively, as IGFs play a role in wound repair, the acute changes in IGF-I and IGFBP-1 may reflect transport of IGF-I from the circulatory pool to injured tissues to promote wound repair.

Many subclinical diseases associated with inflammation occur in sheep during the periparturient period. Clinical symptoms are usually detected at an advanced stage of the disease; therefore, there are considerable risks of permanent health disorders in fetuses or dams. Determination of acute phase proteins (APPs) as markers of inflammation may allow for an earlier diagnosis and effective treatment. Furthermore, multi-fetus pregnancies are often associated with hematological disturbances. The study objective was to compare plasma concentrations of serum amyloid A (SAA), haptoglobin (Hp), fibrinogen (Fb), and cortisol in ewes bearing one and two fetuses in the period from 2 weeks before to 2 weeks after parturition as well as to determine hematological parameters in peripheral blood. There was an important effect of fetal number on APP and cortisol concentrations in periparturient ewes. There was a greater concentration of SAA, Hp, Fb, and cortisol in ewes bearing two fetuses compared with those bearing one fetus. Profiles for APP and cortisol concentrations and hematological parameters were similar for ewes bearing one and two fetuses, and trends were within normal reference ranges for the periparturient period. Furthermore, there were no differences in values for hematological variables between ewes bearing one and two fetuses. with there being no ewes with anemia. In summary, separate determination of the previously undefined physiological ranges of APPs and cortisol for ewes bearing one and two fetuses may facilitate diagnosis of subclinical disorders and enable comparison of laboratory test results with different reference values for ewes bearing different numbers of fetuses.

Background and Objectives: Fetal growth restriction (FGR) is associated with fetal mortality and is a risk factor for cerebral palsy and future lifestyle-related diseases. Despite extensive research, no effective treatment strategy is available for FGR. Mammalian target of rapamycin (mTOR) signaling is important for the growth of fetal organs and its dysregulation is associated with miscarriage. Here, we focused on mTOR signaling and investigated how the activities of phospho-ribosomal protein S6 (rps6) and phospho-eukaryotic translation initiation factor 4E (eIF-4E), which act downstream of mTOR signaling in the human placenta, change following treatment of FGR with tadalafil and aimed to elucidate the underlying mechanism of action. Placental hypoxia was investigated by immunostaining for hypoxia-inducible factor (HIF)-2α. Materials and Methods: Phosphor-rps6 and phosphor-eIF4E expression were examined by Western blotting and enzyme-linked immunosorbent assay, respectively. Results: HIF-2α expression significantly increased in FGR placenta compared with that in the control placenta but decreased to control levels after tadalafil treatment. Levels of phospho-rps6 and phospho-eIF-4E were significantly higher in FGR placenta than in control placenta but decreased to control levels after tadalafil treatment. Conclusions: Tadalafil restored the levels of HIF-2α, phospho-rps6, and eIF-4E in FGR placenta to those observed in control placenta, suggesting that it could be a promising treatment strategy for FGR.

Human fetal membranes (FM) at term have been shown to contain a weak zone in the region overlying the cervix which exhibits characteristics of increased collagen remodeling and apoptosis. It has been hypothesized that the FM rupture initiation site is within this weak zone. Although the FM weak zone has been partially characterized, it is unclear what structural differences in the extracellular matrix result in its decreased rupture strength. A screen for differentially expressed proteins in the amnion of the weak zone versus other FM areas demonstrated that fibulin 1 was decreased. We investigated potential regional differences in all fibulin protein family members.

During pregnancy, the immune system is modified to allow developmental tolerance of the semi-allogeneic fetus and placenta to term. Pregnant women suffering from stress, anxiety, and depression show dysfunctions of their immune system that may be responsible for fetal and/or newborn disorders, provided that placental gene regulation is compromised. The present study explored the effects of maternal chronic self-perceived stress, anxiety, and depression during pregnancy on the expression of immune-related genes and pathways in term placenta. Pregnancies were clinically monitored with the Beck Anxiety Inventory (BAI) and Edinburgh Postnatal Depression Scale (EPDS). A cutoff threshold for BAI/EPDS of 10 divided patients into two groups: Index group (>10, n = 11) and a Control group (<10, n = 11), whose placentae were sampled at delivery. The placental samples were subjected to RNA-Sequencing, demonstrating that stress, anxiety, and depression during pregnancy induced a major downregulation of placental transcripts related to immune processes such as T-cell regulation, interleukin and cytokine signaling, or innate immune responses. Expression differences of main immune-related genes, such as CD46, CD15, CD8α & β ILR7α, and CCR4 among others, were found in the Index group (P < 0.05). Moreover, the key immune-like pathway involved in humoral and cellular immunity named "Primary immunodeficiency" was significantly downregulated in the Index group compared with Controls. Our results show that mechanisms ruling immune system functions are compromised at the maternal-fetal interface following self-perceived depressive symptoms and anxiety during pregnancy. These findings may help unveil mechanisms ruling the impact of maternal psychiatric symptoms and lead to new prevention/intervention strategies in complicated pregnancies.