The cisgenic and transgenic capabilities of the CRISPR/Cas9 system provides important applications to create animal models and revolutionize all aspects of science in agriculture and human health.

Progesterone plays an essential role in the maintenance of the endometrium; it prepares the endometrium for pregnancy, promotes decidualization, and inhibits estrogen-dependent proliferation. Progesterone function is often dysregulated in endometrial disease states. In addition, the PI3K/AKT signaling pathway is often overactive in endometrial pathologies and promotes the survival and proliferation of the diseased cells. Understanding how AKT influences progesterone action is critical in improving hormone-based therapies in endometrial pathologies. Here, we summarize recent studies investigating the crosstalk between the AKT pathway and progesterone receptor function in endometriosis and endometrial cancer.

Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent.

Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.

Physiology of the adult can be modified by alterations in prenatal development driven by the maternal environment. Developmental programming, which can be established before the embryo implants in the uterus, can affect females differently than males. The mechanism by which sex-specific developmental programming is established is not known. Here we present evidence that maternal regulatory signals change female embryos differently than male embryos. In particular, actions of the maternally derived cytokine CSF2 from Day 5 to Day 7 of development affected characteristics of the embryo at Day 15 differently for females than males. CSF2 decreased length and IFNT secretion of female embryos but increased length and IFNT secretion of male embryos. Analysis of a limited number of samples indicated that changes in the transcriptome and methylome caused by CSF2 also differed between female and males. Thus, sex-specific programming by the maternal environment could occur when changes in secretion of maternally derived regulatory molecules alter development of female embryos differently than male embryos.

Macrophages are abundant in the uterine stroma and are intimately juxtaposed with other cell lineages comprising the uterine epithelial and stromal compartments. We postulated that macrophages may participate in mediating or amplifying the effects of ovarian steroid hormones to facilitate the uterine remodeling that is a characteristic feature of every estrus cycle and is essential for pregnancy. Using the Cd11b-Dtr transgenic mouse model with an ovariectomy and hormone replacement strategy, we depleted macrophages to determine their role in hormone-driven proliferation of uterine epithelial and stromal cells and uterine vascular development. Following diphtheria toxin (DT) administration, approximately 85% of EMR1-positive (EMR1 ) macrophages, as well as 70% of CD11C dendritic cells, were depleted from Cd11b-Dtr mice. There was no change in bromodeoxyuridine incorporation into epithelial cells induced to proliferate by administration of 17beta-estradiol (E2) to ovariectomized mice or into stromal cells induced to proliferate in response to E2 and progesterone (P4), and the resulting sizes and structures of the luminal epithelial and stromal cell compartments were not altered compared with those of leukocyte replete controls. Depletion of CD11B myeloid cells failed to alter the density or pattern of distribution of uterine blood vessels, as identified by staining PECAM1-positive endothelial cells in the uterine stroma of E2- or E2 combined with P4 (E2P4)-treated ovariectomized mice. These experiments support the interpretation that macrophages are dispensable to regulation of proliferative events induced by steroid hormones in the cycling and early pregnant mouse uterus to establish the epithelial, stromal, and vascular architecture which is critical for normal reproductive competence.

The establishment of a receptive uterus is the prime requirement for embryo implantation. In mice, the E₂-induced cytokine leukemia inhibitory factor (LIF) is essential in switching the uterine luminal epithelium (LE) from a nonreceptive to a receptive state. Here we define the LIF-mediated switch using array analysis and informatics to identify LIF-induced changes in gene expression and annotated signaling pathways specific to the LE. We compare gene expression profiles at 0, 1, 3, and 6 h, following LIF treatment. During the first hour, the JAK-STAT signaling pathway is activated and the expression of 54 genes declines, primarily affecting LE cytoskeletal and chromatin organization as well as a transient reduction in the progesterone, TGFbetaR1, and ACVR1 receptors. Simultaneously 256 genes increase expression, of which 42 are transcription factors, including Sox, Kfl, Hes, Hey, and Hox families. Within 3 h, the expression of 3987 genes belonging to more than 25 biological process pathways was altered. We confirmed the mRNA and protein distribution of key genes from 10 pathways, including the Igf-1, Vegf, Toll-like receptors, actin cytoskeleton, ephrin, integrins, TGFbeta, Wnt, and Notch pathways. These data identify novel LIF-activated pathways in the LE and define the molecular basis between the refractory and receptive uterine phases. More broadly, these findings highlight the staggering capacity of a single cytokine to induce a dynamic and complex network of changes in a simple epithelium essential to mammalian reproduction and provide a basis for identifying new routes to regulating female reproduction.

Expression and function of the follicle-stimulating hormone receptor (FSHR) in females were long thought to be limited to the ovary. Here, however, we identify extragonadal FSHR in both the human female reproductive tract and the placenta, and test its physiological relevance in mice. We show that in nonpregnant women FSHR is present on: endothelial cells of blood vessels in the endometrium, myometrium, and cervix; endometrial glands of the proliferative and secretory endometrium; cervical glands and the cervical stroma; and (at low levels) stromal cells and muscle fibers of the myometrium. In pregnant women, placental FSHR was detected as early as 8–10 wk of gestation and continued through term. It was expressed on: endothelial cells in fetal portions of the placenta and the umbilical cord; epithelial cells of the amnion; decidualized cells surrounding the maternal arteries in the maternal decidua; and the stromal cells and muscle fibers of the myometrium, with particularly strong expression at term. These findings suggest that FSHR expression is upregulated during decidualization and upregulated in myometrium as a function of pregnancy. The presence of FSHR in the placental vasculature suggests a role in placental angiogenesis. Analysis of genetically modified mice in which Fshr is lacking in fetal portions of the placenta revealed adverse effects on fetoplacental development. Our data further demonstrate FSHB and CGA mRNAs in placenta and uterus, consistent with potential local sources of FSH. Collectively, our data suggest heretofore unappreciated roles of extragonadal FSHR in female reproductive physiology.

5-Methylcytosine is one of the most important epigenetic modifications and has a profound impact on embryonic development. After gamete fusion, there is a widespread and rapid active demethylation process of sperm DNA, which suggests that the paternal epigenome has an important role during embryonic development. To better understand the epigenome of sperm DNA and its possible involvement in a developing embryo, we determined epigenetic marks in human sperm DNA and in surrogate somatic tissue leukocytes; the analyzed epigenetic modifications included 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, and 5-hydroxymethyl-2′-deoxyuridine. For absolute determination of the modification, we used liquid chromatography with UV detection and tandem mass spectrometry techniques with isotopically labeled internal standards. Our analyses demonstrated, for the first time to date, that absolute global values of 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, and 5-hydroxymethyl-2′-deoxyuridine in sperm are highly statistically different from those observed for leukocyte DNA, with respective mean values of 3.815% versus 4.307%, 0.797 versus 2.945 per 10⁴ deoxynucleosides, and 5.209 versus 0.492 per 10⁶ deoxynucleosides. We hypothesize that an exceptionally high value of 5-hydroxymethyluracil in sperm (>10-fold higher than in leukocytes) may play a not yet recognized regulatory role in the paternal genome.

Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols.

The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca² imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during, and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome-reacted spermatozoa, which was confirmed by scanning electron microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca² indicator. These AR and intracellular Ca² ([Ca² ]i) recordings revealed that the presence of [Ca² ]i oscillations, both spontaneous and progesterone induced, prevents AR in human spermatozoa. Notably, the progesterone-induced AR is preceded by a second [Ca² ]i peak and ∼40% of reacting spermatozoa also manifest a slow [Ca² ]i rise ∼2 min before AR. Our findings uncover new AR features related to [Ca² ]i.

Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.

The relationship between stallion fertility and oxidative stress remains poorly understood. The purpose of this study was to identify criteria for thoroughbred fertility assessment by performing a logistical regression analysis using “dismount” sperm parameters as predictors and weekly per-cycle conception rate as the dependent variable. Paradoxically, positive relationships between fertility and oxidative stress were revealed, such that samples that produced pregnancies exhibited higher rates of 8-hydroxy-2′-deoxyguanosine release (1490.2% vs. 705.5 pg/ml/24 h) and lower vitality (60.5% vs. 69.6%) and acrosome integrity (40.2% vs. 50.1%) than those that did not. We hypothesized that the most fertile spermatozoa exhibited the highest levels of oxidative phosphorylation (OXPHOS), with oxidative stress simply being a by-product of intense mitochondrial activity. Accordingly, an experiment to investigate the relationship between oxidative stress and motility was conducted and revealed positive correlations between mitochondrial ROS and total motility (R² = 0.90), rapid motility (R² = 0.89), average path velocity (VAP; R² = 0.59), and curvilinear velocity (VCL; R² = 0.66). Similarly, lipid peroxidation was positively correlated with total motility (R² = 0.46), rapid motility (R² = 0.51), average path velocity (R² = 0.62), and VCL (R² = 0.56), supporting the aforementioned hypothesis. The relative importance of OXPHOS in supporting the motility of equine spermatozoa was contrasted with human spermatozoa, which primarily utilize glycolysis. In this study, mitochondrial inhibition significantly reduced the velocity (P < 0.01) and ATP (P < 0.05) content of equine, but not human, spermatozoa, emphasizing the former's relative dependence on OXPHOS. The equine is the first mammal in which such a positive relationship between oxidative stress and functionality has been observed, with implications for the management of stallion fertility in vitro and in vivo.

Accumulating data suggest that adrenomedullin (ADM) regulates the trophoblast cell growth, migration, and invasion. However, the effect of ADM on trophoblast differentiation is poorly understood. In this study, we hypothesized that ADM promotes the differentiation of trophoblast stem cells (TSCs) into trophoblast giant cells (TGCs). Using rat TSCs, Rcho-1 cells, we investigated the effect of ADM on TSC differentiation into TGCs in differentiation or stem cell media, respectively, and explored the effect of ADM on the mechanistic target of rapamycin (MTOR) signaling in trophoblast cell differentiation. The results include: 1) in the presence of differentiation medium, 10⁻⁷ M ADM, but not lower doses, elevated (P < 0.05) Prl3b1/Esrrb (i.e., the ratio of mRNA levels) by 1.7-fold compared to that in control; 2) the supplementation of ADM antagonist, regardless of the concentration of ADM, reduced (P < 0.05) Prl3b1/Esrrb by 2-fold, compared to control group, while the supplementation of CGRP antagonist, regardless of the concentration of ADM, did not change Prl3b1/Esrrb; 3) in the presence of stem cell medium, ADM did not alter the expression of TSC and TGC marker genes, however, the ratio of Prl3b1/Esrrb was reduced (P < 0.05) by ADM antagonist compared to that in control; and 4) ADM increased (P < 0.05) phosphorylated MTOR proteins and the ratio of phosphorylated to total MTOR proteins by 2.0- and 1.7-fold, respectively. The results indicate that ADM promotes but does not induce the differentiation of TSCs to TGCs in a dose-dependent manner and MTOR signaling may play a role in this process.

Previously, we showed luteal stage-specific regulation of angiogenesis-modulating factors by prostaglandin F2 alpha (PGF2alpha). Fibroblast growth factor 2 (FGF2) and thrombospondins (THBSs) exhibited the most divergent profile of induction by PGF2alpha. We therefore examined the transcriptional regulation and roles of THBSs in luteal cells and studied their interaction with FGF2. THBSs and their receptors exhibited cell-specific expression: THBS1 was the predominant form in luteal endothelial cells (LEC), whereas luteinized granulosa cells (LGC) expressed mostly THBS2. CD36 was confined to LGC, but CD47 did not exhibit preferential expression between LEC and LGC. THBS1 and THBS2 were both stimulated in vitro by PGF2a and its analog in LGC. In contrast, luteinizing signals (LH and insulin) decreased the expression of THBS1, THBS2, and CD36. Importantly, LH increased FGF2 expression, suggesting that THBSs and FGF2 are conversely regulated. We found that FGF2 inhibited THBS1 and vice versa, and that THBS1 treatment decreased FGF2 expression, suggesting reciprocal inhibition. In agreement, ablation of THBS1 by specific small interference RNAs elevated FGF2 levels. THBS1 reduced LEC numbers and promoted apoptosis by activation of caspase-3. In contrast, FGF2 reduced basal and THBS1-induced caspase-3 levels. Consistent with these findings, small interference RNA silencing of THBS1 in luteal cells reduced the levels of active caspase-3 and improved the survival of cells when challenged with staurosporine. Taken together, these studies suggest that THBSs are suppressed during luteinization but are induced by PGF2alpha in luteolysis. THBS1 has antiangiogenic, proapoptotic properties; these, together with its ability to inhibit FGF2 expression and activity, can promote luteolysis.

Primordial follicle assembly is essential for reproduction in mammalian females. Oocytes develop in germ cell cysts that in late fetal development begin break down into individual oocytes and become surrounded by pregranulosa cells, forming primordial follicles. As they separate, many oocytes are lost by apoptosis. Exposure to steroid hormones delays cyst breakdown, follicle formation, and associated oocyte loss in some species. One model for regulation of follicle formation is that steroid hormones in the maternal circulation keep cells in cysts and prevent oocyte death during fetal development but that late in pregnancy hormone levels drop, triggering cyst breakdown and associated oocyte loss. However, herein we found that, while maternal circulating levels of progesterone drop during late fetal development, maternal estradiol levels remain high. We hypothesized that fetal ovaries were the source of hormones and that late in fetal development their production stops. To test this, mRNA and protein levels of steroidogenic enzymes required for estradiol and progesterone synthesis were measured. We found that aromatase and 3-beta-hydroxysteroid dehydrogenase mRNA levels drop before cyst breakdown. The 3-beta-hydroxysteroid dehydrogenase protein levels also dropped, but we did not detect a change in aromatase protein levels. The steroid content of perinatal ovaries was assayed, and both estradiol and progesterone were detected in fetal ovaries before cyst breakdown. To determine the role of steroid hormones in oocyte development, we examined the effects of blocking steroid hormone production in organ culture and found that the number of oocytes was reduced, supporting our model that steroid hormones are important for fetal oocyte survival.

Peroxisome proliferator-activated receptor delta (Ppard) activation has been implicated in regulating a multitude of biological processes in placental development. In this study, we employed the UPLC-ESI-TOFMS approach to investigate the metabolic traits in placenta from GW501516-treated mice at Embryonic Day 10.5. By analyzing the mass spectrum data, ions with the most significant differences between control and GW501516-treated groups were identified. Among these metabolites, the fatty acids, phospholipids, and sterol lipids were dramatically increased. Ingenuity Pathway Analysis (IPA) showed that phosphatidylethanolamine biosynthesis and glycolysis were the top two altered metabolic pathways involved in carbohydrate metabolism, energy production, and lipid metabolism. Subsequent immunoblotting experiments provided evidence for positive correlation of PPARD level and AKT and ERK signaling pathways upon GW501516 treatment. Furthermore, the stimulation of GW501516 increased trophoblast cell fusion gene syncytin-A (Syna), but not syncytin-B (Synb), expression, suggesting a potential role of Ppard activation in promoting cytotrophoblast differentiation. Our results reveal that Ppard activation elicits dramatic changes of the metabolic activities in placenta, which is correlated to AKT and ERK signaling.

Nitric oxide (NO) is a gaseous molecule that regulates angiogenesis and vasodilation via activation of the cGMP pathway. However, functional roles of NO during embryonic development from spherical blastocysts to elongated filamentous conceptuses (embryo and extraembryonic membrane) during the peri-implantation period of pregnancy have not been elucidated in vivo. In order to assess roles of NO production in survival and development of the ovine conceptus, we conducted an in vivo morpholino antisense oligonucleotide (MAO)-mediated knockdown trial of nitric oxide synthase-3 (NOS3) mRNA, the major isoform of NO synthase, in ovine conceptus trophectoderm (Tr). Translational knockdown of NOS3 mRNA results in small, thin, and underdeveloped conceptuses, but normal production of interferon-tau, the pregnancy recognition signal in sheep. MAO-NOS3 knockdown in conceptuses decreased the abundance of NOS3 (72%, P < 0.05) and the arginine transporter SLC7A1 proteins in conceptus Tr. Furthermore, the amounts of ornithine and polyamines were less (P < 0.01) in uterine fluid, whereas the amounts of arginine (58%, P < 0.01), citrulline (68%, P < 0.05), ornithine (68%, P < 0.001), glutamine (78%, P < 0.001), glutamate (68%, P < 0.05), and polyamines (P < 0.01) were less in conceptuses, which likely accounts for the failure of MAO-NOS3 conceptuses to develop normally. For MAO-NOS3 conceptuses, there were no compensatory increases in the expression levels of either nitric oxide synthase-1 (NOS1) or nitric oxide synthase-2 (NOS2) or in expression of enzymes for synthesis of polyamines (ornithine decarboxylase, arginine decarboxylase, agmatinase) from arginine or ornithine with which to rescue development of MAO-NOS3 conceptuses. Thus, the adverse effect of MAO-NOS3 to reduce NO generation and the transport of arginine and ornithine into conceptuses is central to an explanation for failure of normal development of MAO-NOS3, compared to control conceptuses. The study, for the first time, created an NO-deficient mammalian conceptus model in vivo and provided new insights into the orchestrated events of conceptus development during the peri-implantation period of pregnancy. Our data suggest that NOS3 is the key enzyme for NO production by conceptus Tr and that this protein also regulates the availability of arginine in conceptus tissues for synthesis of polyamines that are essential for conceptus survival and development.

Increasing evidence indicates that endoplasmic reticulum (ER) stress is involved in various diseases. In nongestational tissues, several markers of the unfolded protein response (UPR) have been shown to regulate the inflammatory response. Thus, the aim of this study was to determine the effect of human labor on markers of ER stress in fetal membranes and myometrium. In addition, the effect of ER stress inhibition on the expression and secretion of proinflammatory and prolabor mediators was also assessed. The markers of ER stress, GRP78, IRE1, and spliced XBP1 (XBP1s), were significantly increased in fetal membranes and myometrium after term and preterm labor compared to nonlaboring samples. Given that inflammation is considered to be one of the leading causes of spontaneous preterm birth, here we used bacterial endotoxin lipopolysaccharide (LPS) as a model for infection-induced preterm birth. In term nonlabored fetal membranes and myometrium, LPS induced UPR activation as evidenced by a significant increase in the expression of GRP78, IRE1, and XBP1s in fetal membranes and myometrium. The use of the chemical chaperones 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA) alleviated ER stress induced by LPS. 4-PBA and TUDCA also ameliorated the increase in LPS-induced prolabor mediators. Our data suggest that the UPR may regulate the inflammatory responses associated with labor or infection in fetal membranes and myometrium of pregnant term and preterm women. Thus, the use of ER stress inhibitors, in particular 4-PBA or TUDCA, may be a potential therapeutic strategy for the prevention of infection-mediated spontaneous preterm birth.

EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors.

MicroRNAs are expressed abundantly in the placenta throughout pregnancy. We have previously reported that microRNA (miR)-378a-5p promoted trophoblast migration and invasion. To further understand the role of miR-378a-5p during placental development, we investigated whether it may regulate the differentiation of syncytiotrophoblast (STB). Using a choriocarcinoma cell line, BeWo, we found that miR-378a-5p was down-regulated during forskolin-induced STB differentiation. Transfection of a miR-378a-5p mimic into BeWo cells decreased the formation of multinucleated STB, increased E-cadherin, and decreased the expression level of STB marker genes. On the other hand, transfection of anti-miR-378a-5p resulted in an increase in formation of multinucleated STB and expression of STB marker genes, as well as the loss of E-cadherin. Bioinformatic analysis revealed that miR-378a-5p has four potential binding sites at the 3′ untranslated region (UTR) of cyclin G2 (CCNG2). Using luciferase reporter assays, we showed that miR-378a-5p decreased the luciferase activity of reporter constructs that contain CCNG2 3′ UTR. In addition, miR-378a-5p decreased, whereas anti-miR-378a-5p increased, CCNG2 mRNA levels. Overexpression of CCNG2 increased the expression of syncytin-1 and fusion index and reversed the inhibitory effects of miR-378a-5p. In contrast, silencing of CCNG2 using siRNA increased E-cadherin and decreased syncytin-1 levels. These findings provide initial evidence that CCNG2 promotes STB differentiation and suggest that miR-378a-5p exerts an inhibitory role in STB differentiation, in part, by down-regulating CCNG2 expression, in the BeWo cell model.

Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca² -free CZB medium containing 10 mM SrCl₂) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF.

Cardiovascular dysfunction and remodeling have been found in some children conceived by in vitro fertilization (IVF). However, the underlying mechanisms remain unclear. In this study, the retrospective investigation showed that the blood pressure of IVF-conceived Chinese children was higher than that of naturally conceived (NC) children at ages 3–13 yr. We analyzed the expression profile of proteins in the umbilical veins of IVF and NC newborns by proteomic techniques. Using iTRAQ (isobaric tags for relative and absolute quantitation), 47 differentially expressed proteins (DEPs) were identified by feature selection in IVF umbilical veins compared with NC. Ingenuity Pathway Analysis, which is used to explore the signaling pathways of DEPs, revealed that these DEPs played important roles in vascular system development and carbon metabolism, implying that these DEPs might be potential candidates for further exploration of the mechanism(s) of vascular dysfunction in IVF children. We found that the serum estradiol (E₂) level in the cord blood of IVF newborns was significantly higher than that of NC newborns. High concentrations of E₂ induced alteration of lumican and vimentin expression in human umbilical vein endothelial cells, which was consistent with the proteomic results. These findings suggested that abnormal expression of proteins in umbilical veins might be related to the cardiovascular dysfunction and remodeling in IVF offspring. In conclusion, our data for the first time reveal the protein expression profile in blood vessels of IVF offspring and provide information for further mechanism study and evaluation of risks of cardiovascular abnormality in IVF children.

We mapped global changes in miRNA and mRNA profiles spanning the first wave of spermatogenesis using prepubertal (Postnatal Day 8 [P8]), pubertal (P16), and adolescent (P24) Mus musculus testes and identified the differential expression of 67 miRNAs and 8226 mRNAs. These two data sets were integrated into miRNA-dependent regulatory networks based on miRWalk predictions. In a network representing the P8 to P16 transition, downregulation of four miRNAs and upregulation of 19 miRNAs were linked with 81 upregulated target mRNAs and 228 downregulated target mRNAs, respectively. Furthermore, during the P16 to P24 transition, two miRNAs were downregulated, and eight miRNAs were upregulated, which linked with 64 upregulated mRNAs and 389 downregulated mRNAs, respectively. Only three of the miRNAs present in the network (miR-34b-5p, miR-34c, and miR-449a) showed a progressive increase from P8 through P16 to P24, while the remaining miRNAs in the network showed statistically significant changes in their levels either during the P8 to P16 transition or during the P16 to P24 transition. Analysis of the chromosomal location of these differentially expressed miRNAs showed that 14 out of 25 miRNAs upregulated from P8 to P16, and 18 out of 40 miRNAs upregulated from P8 to P24 were X-linked. This is suggestive of their escape from meiotic sex chromosome inactivation and postmeiotic sex chromatin. This integrated network of miRNA-level and mRNA-level changes in mouse testis during the first wave of spermatogenesis is expected to build a base for evaluating the role of miRNA-mediated gene expression regulation in maturing mammalian testis.

Fetal exposure to environmental endocrine disruptors (EDs) is thought to contribute to reported idiopathic increases in adult male reproductive abnormalities. Although humans are exposed to myriad EDs from conception to adulthood, few studies have evaluated the effects of combined EDs on male reproduction. In the present study, we demonstrate that simultaneous gestational exposure to the phytoestrogen genistein and the antiandrogenic plasticizer di-(2-ethyhexyl) phthalate (DEHP) induces long-term alterations in testis development and function. Pregnant Sprague Dawley rats were gavaged from Gestational Day 14 to birth with corn oil, genistein, DEHP, or their mixture at 10 mg/kg/day, a dose selected from previous dose-response studies using single chemicals for its lack of long-term testicular effects. Hormonal and testicular end points were examined in adult male offspring. Serum testosterone levels were unchanged. However, significant increases were observed in testis weight and in the expression of mast cell markers in testes from adult rats exposed gestationally to combined compounds. The ED mixture also altered the mRNA expression of Sertoli cell makers Wt1 and Amh and germ cell markers cKit and Sox17, measured by quantitative real-time PCR (qPCR), suggesting long-term disruption in testis function and spermatogenesis. Alterations in germ cell markers might reflect direct effects on fetal gonocytes or indirect effects via primary targeting of somatic cells, as suggested by differentially regulated Leydig cell associated genes (Hsd3b, Anxa1, Foxa3, and Pdgfra), determined by gene expression array, qPCR, and protein analyses. The two chemicals, when given in combination, induced long-term reproductive toxicity at doses not previously reported to produce any conspicuous long-term effects. Our study therefore highlights a need for a more comprehensive evaluation of the effects of ED mixtures.