Decreased STAT3 in human idiopathic fetal growth restriction contributes to trophoblast dysfunction.

Abnormal trophoblast function is associated with fetal growth restriction (FGR). The JAK-STAT pathway is one of the principal signalling mechanisms by which cytokines and growth factors modulate cell proliferation, differentiation, cell migration and apoptosis. The expression of placental JAK-STAT genes in human idiopathic FGR is unknown. In this study, we propose the hypothesis that JAK-STAT pathway genes are differentially expressed in idiopathic FGR-affected pregnancies and contribute to abnormal feto-placental growth by modulating the expression of the amino acid transporter SNAT2, differentiation marker CGB/human chorionic gonadotrophin beta-subunit (ß-hCG) and apoptosis markers caspases 3 and 8, and TP53. Expression profiling of FGR-affected placentae revealed that mRNA levels of STAT3, STAT2 and STAT5B decreased by 69, 52 and 50%, respectively, compared with gestational-age-matched controls. Further validation by real-time PCR and immunoblotting confirmed significantly lower STAT3 mRNA and STAT3 protein (total and phosphorylated) levels in FGR placentae. STAT3 protein was localised to the syncytiotrophoblast (ST) in both FGR and control placentae. ST differentiation was modelled by in vitro differentiation of primary villous trophoblast cells from first-trimester and term placentae, and by treating choriocarcinoma-derived BeWo cells with forskolin in cell culture. Differentiation in these models was associated with increased STAT3 mRNA and protein levels. In BeWo cells treated with siRNA targeting STAT3, the mRNA and protein levels of CGB/ß-hCG, caspases 3 and 8, and TP53 were significantly increased, while that of SNAT2 was significantly decreased compared with the negative control siRNA. In conclusion, we report that decreased STAT3 expression in placentae may contribute to abnormal trophoblast function in idiopathic FGR-affected pregnancies.

Energy and lipid metabolism gene expression of D18 embryos in dairy cows is related to dam physiological status.

We analyzed the change in gene expression related to dam physiological status in day (D)18 embryos from growing heifers (GH), early lactating cows (ELC), and late lactating cows (LLC). Dam energy metabolism was characterized by measurement of circulating concentrations of insulin, glucose, IGF-1, nonesterified fatty acids, ß-hydroxybutyrate, and urea before embryo flush. The metabolic parameters were related to differential gene expression in the extraembryonic tissues by correlation analysis. Embryo development estimated by measuring the length of the conceptuses and the proportion of expected D18 gastrulating stages was not different between the three groups of females. However, embryo metabolism was greatly affected by dam physiological status when we compared GH with ELC and GH with LLC but to a lesser extent when ELC was compared with LLC. Genes involved in glucose, pyruvate, and acetate utilization were upregulated in GH vs. ELC conceptuses (e.g., SLC2A1, PC, ACSS2, ACSS3). This was also true for the pentose pathway ( PGD, TKT), which is involved in synthesis of ribose precursors of RNA and DNA. The pathways involved in lipid synthesis were also upregulated in GH vs. ELC. Despite similar morphological development, the molecular characteristics of the heifers' embryos were consistently different from those of the cows. Most of these differences were strongly related to metabolic/hormone patterns before insemination and during conceptus free-life. Many biosynthetic pathways appeared to be more active in heifer embryos than in cow embryos, and consequently they seemed to be healthier, and this may be more conducive to continue development.

Pre- and post-partum mild underfeeding influences gene expression in the reproductive tract of cyclic dairy cows.

Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post-partum. This study investigated the effects of pre- and post-partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post-partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post-partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi- and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR) < 0.10 and 1370 DEGs vs 3, respectively, under FDR < 0.15. Additionally, we used dedicated statistics (regularized canonical correlation analysis) to correlate the post-partum patterns of six plasma metabolites and hormones related to energy metabolism measured weekly between calving and D80 with gene expression. High correlations were observed between post-partum patterns of IGF-1, insulin, ß-hydroxybutyrate and the expression in the oviduct of genes related to reproductive system disease, connective tissue disorders and metabolic disease. Moreover, we found special interest in the literature to retinoic acid-related genes (e.g. FABP5/CRABP2) that might indicate abnormalities in post-partum tissue repair mechanisms. In conclusion, this experiment highlights relationships between underfeeding and gene expression in the oviduct and endometrium after ovulation in cyclic Holstein cows. This might help to explain the effect of mild undernutrition on fertilization failure and early embryonic mortality in post-partum dairy cows.

Drug transporter expression during in vitro differentiation of first-trimester and term human villous trophoblasts.

Drug transporters interfere with drug disposition during pregnancy by actively transporting drugs from mother to fetus, and vice versa. Data on their placental expression are scarce, especially during the first trimester of pregnancy. The aim of our study was to assess mRNA expression of more than 80 drug transporters by using an RT-qPCR array in primary cytotrophoblastic cells isolated from first-trimester and term human placentas and cultured for 72 h to form syncytiotrophoblasts. This original expression panel of human placental drug transporters should help to understand transplacental drug transfer and to ensure more rational drug use during pregnancy.

Transcervical collection of bovine embryos up to Day 21: an 8-year overview.

Transcervical embryo collection is used routinely in the bovine species throughout the world to collect Day 6 to Day 9 embryos (early embryos) for genetic selection. For research purposes, however, the collection of embryos at later stages of pregnancy, i.e., Days 12 to 21 (late embryos), is needed. So far, for the recovery of late embryos, females are euthanized and embryo collection is performed after recovery of the genital tract. To reduce the number of animals used and still provide valuable material for embryo research, we have therefore developed a transcervical technique to collect late embryos. The objective of this study was to compare embryo recovery results at early and late stages within our laboratory. Altogether, 232 cows were used for this study. One hundred forty-five flushes were performed to collect embryos from Days 6 to 9, and 251 flushes were performed to collect embryos from Days 12 to 21. For the early embryos, a classical three-way collection equipment was used. To collect the late embryos, the same equipment was used, but the extensible flexible catheter that goes inside the external rigid catheter was removed, so that larger embryos could be collected through the remaining larger hole (two-way collection). All females were submitted to ovum pick up to remove the dominant follicle and were subsequently superovulated with FSH. Luteolysis was induced 48 hours before artificial insemination. Two artificial inseminations were performed with frozen semen, 48 and 56 hours after PGF2α injection. Before embryo collection, cows were treated with an epidural injection of a local anesthetic drug. The presence of CL was checked, and they were counted by rectal palpation. For all collections, the cervix was prepared with the initial introduction of a dilator. Then, the catheter was introduced in one horn, and the cuff was inflated as low as possible. For the collection of late embryos, the flushing solution (30 mL) was injected slowly twice to suspend the embryos before flushing the horn with 500 mL, and the same operation was performed on the second horn. There was no significant difference in the number of embryos collected per flush in the early- and late-stage (758 embryos collected, 5.22 ± 6.02 per flush vs. 1238 embryos collected, 4.93 ± 5.07 per flush, respectively). The number of embryos collected per CL, however, was significantly lower in the early versus late group (0.39 ± 0.32% vs. 0.44 ± 0.34%, respectively). The late collection allowed the retrieval of full conceptuses (embryonic and extraembryonic tissues), even at very late stages such as Days 18 to 21. Careful collection is needed, however, so that conceptuses are not damaged or torn: the horn must be massaged gently and the flush should be ideally recovered in one single flow. This technique is a powerful tool to collect the late-stage embryos for research purposes. Because it is not traumatic, animals can be used again for the same procedure.

Review: Annexin-A5 and cell membrane repair.

Annexins are soluble proteins that bind to biological membranes containing negatively charged phospholipids, principally phosphatidylserine, in a Ca(2+)-dependent manner. Annexin-A5 (AnxA5), the smallest member of the annexin family, presents unique properties of membrane binding and self-assembly into ordered two-dimensional (2D) arrays on membrane surfaces. We have previously reported that AnxA5 plays a central role in the machinery of membrane repair by enabling rapid resealing of plasma membrane disruption in murine perivascular cells. AnxA5 promotes membrane repair via the formation of a protective 2D bandage at membrane damaged site. Here, we review current knowledge on cell membrane repair and present recent findings on the role of AnxA5 in membrane resealing of human trophoblasts.

Iodide transporters expression in early human invasive trophoblast.

CONTEXT: The placenta plays an essential role in the fetomaternal exchanges of iodine and thyroid hormones. Propylthiouracil (PTU) is presently considered to be the treatment of choice for hyperthyroidism during the first trimester of pregnancy. Little is known on the expression of iodide transporters in invasive human trophoblast and the possible effect of PTU on this early phase of human placental development. OBJECTIVE: To analyze during early pregnancy expression of sodium/iodide symporter (NIS) and pendrin at the feto-maternal interface in situ in first trimester placentas, in vitro during human trophoblastic cell differentiation in presence or not of PTU. DESIGN: NIS and pendrin immunodetection were performed on 8-10 WG placental tissue sections and in primary cultures of first trimester placenta trophoblastic cells, which differentiate in vitro into syncytiotrophoblast or invasive extravillous cytotrophoblasts (EVCT). The effect of PTU (1 mM) was tested in EVCT on iodide transporters expression, cell invasion, and hCG secretion. RESULTS: NIS and pendrin were present in early human trophoblast at the maternofetal interface. Their expression was modulated with in vitro trophoblast differentiation. Early invasive EVCT were characterized by higher expression of NIS than pendrin. In vitro PTU did modify significantly neither EVCT iodide transporters expression nor EVCT biological functions: i.e. invasive properties and hCG secretion. CONCLUSION: This study reveals that NIS is highly expressed in early human trophoblast at the feto-maternal interface. PTU has no effect on early human trophoblast invasion.

Expression and localization of DLX3, PPARG and SP1 in bovine trophoblast during binucleated cell differentiation.

The differentiation of the trophoblast is marked in ruminants by the formation of binucleated cells (BNC). They appear from pre-implantation onwards but the molecular mechanisms underlying their differentiation remain largely unexplored. Taking advantage of our recent data, we analyzed the expression pattern of DLX3 and PPARG that are known regulators of early placenta formation and extended our analysis to one of their potential regulators, SP1. Our study is the first to demonstrate the co-expression of DLX3, PPARG and SP1 in bovine BNC nuclei. This suggests a possible role of these transcription factors through BNC specific genes at the time of pre-placental differentiation.

Abnormal expression of the imprinted gene Phlda2 in cloned bovine placenta.

Cloning in mammals suffers from high rates of pregnancy losses associated with abnormal placentation, mainly placentomegaly, leading to fetal death. Placental growth is dependent on the regulated expression of many genes of which imprinted genes play a fundamental role. Among them, the Phlda2 gene is expressed from the maternal allele and acts to limit placental growth in mouse and human. Here we used Northern blots, quantitative RT-PCR and in situ hybridization to analyze the expression patterns of bovine PHLDA2 and to compare its expression levels in normal and somatic cell nuclear transfer (SCNT) placentas over a range of gestational stages. PHLDA2 is not expressed in extra-embryonic tissues before d32 of gestation but the level of expression increases throughout pregnancy until term in the placental villi collected from pregnancy obtained by artificial insemination (AI). At all stages of pregnancy, PHLDA2 mRNA are specifically localized in the trophoblast mononucleated cells contrasting with lack of expression in the binucleated cells and uterine tissues. In SCNT placentas, a similar pattern of expression was observed during early pregnancy. In contrast the level of expression is significantly reduced around d200 of gestation in the placental villi from pathological clones. The reduced expression of PHLDA2 was obvious particularly in the placental villi anchored within the uterine crypts with expression confined to the trophoblast of the chorionic plate. Altogether, these results highlight a similarity in expression patterns for PHLDA2 bovine and human where expression is localized to the trophoblast throughout pregnancy and parallels the continuous growth of the placenta. Moreover, the lack of expression in the fetal villi from oversized bovine cloned placenta is consistent with the function of PHLDA2 in restraining placental growth and underlines an aberrant expression of this gene after somatic cloning.

Gene expression in elongating and gastrulating embryos from ruminants.

In ruminants, more than 30% of the embryonic losses observed after artificial insemination (AI) have an early origin, coincident with a marked elongation of the trophoblast which occurs before implantation. Several observations provide clear evidence that early elongation of the conceptus relies on cell multiplication, cell growth and cell shape remodeling. Recent results indicating an intense multiplication of a non-fully differentiated trophoblast, which still expresses some epiblast genes, has to be considered at the onset of elongation. It has also been shown in the last two years that general metabolism and protein trafficking are characteristic of the onset of elongation whereas cellular interactions, cell to cell signaling and cell adhesion become predominant at the end of elongation. Accordingly, expression of most of the single genes identified so far increases during elongation and is related to the establishment of embryo-maternal exchanges before implantation. However, not much is known of what controls the induction of the elongation process or the coordinated development of the embryonic and extra-embryonic tissues. This review highlights new information on this developmental phase and summarizes the views on the complex cross-talk among molecules which might govern conceptus development and lead to successful implantation.

A cDNA macroarray resource for gene expression profiling in ruminant tissues involved in reproduction and production (milk and beef) traits.

cDNA arrays have proven to be useful tools to screen gene expression in many animal species including livestock species. A collaborative program was launched to construct a ruminant cDNA collection, representative of three tissues: Muscle, Embryo and Mammary gland, named MEM. This collection gathers clones mainly arising from 3 non-normalised cDNA libraries: a directed bovine muscle library, a 14-day-old bovine embryo library and a goat lactating mammary library. It is made up of 1896 clones (637 muscle, 882 embryo and 377 mammary cDNAs), selected after sequencing and bioinformatic analyses. Amplification products yielded from these clones as well as controls were printed onto Nylon membranes to generate macroarrays. Hybridisation with relevant cDNA targets allowed checking the location of about 50 cDNAs and the specificity of each sub-set of the repertoire. Macroarrays were hybridised with radiolabelled cDNA complex targets from five different tissues (muscle, embryo, mammary gland, adipose tissue and oocyte). Both somatic and germinal complex targets gave valid hybridisation signals with 45 to 80% of the printed probes. This specific cDNA collection now provides a powerful tool for transcriptomic studies with the ultimate objective to better understand physiological and metabolic functions in ruminants. It will be subsequently included into a forthcoming larger collection.