Uterine microbial disease affects half of all dairy cattle after parturition, causing infertility by disrupting uterine and ovarian function. Infection with Escherichia coli , Arcanobacterium pyogenes , and bovine herpesvirus 4 causes endometrial tissue damage. Toll-like receptors on endometrial cells detect pathogen-associated molecules such as bacterial DNA, lipids, and lipopolysaccharide (LPS), leading to secretion of cytokines, chemokines, and antimicrobial peptides. Chemokines attract neutrophils and macrophages to eliminate the bacteria, although persistence of neutrophils is associated with subclinical endometritis and infertility. Cows with uterine infections are less likely to ovulate because they have slower growth of the postpartum dominant follicle in the ovary, lower peripheral plasma estradiol concentrations, and perturbation of hypothalamic and pituitary function. The follicular fluid of animals with endometritis contains LPS, which is detected by the TLR4/CD14/LY96 (MD2) receptor complex on granulosa cells, leading to lower aromatase expression and reduced estradiol secretion. If cows with uterine disease ovulate, the peripheral plasma concentrations of progesterone are lower than those in normal animals. However, luteal phases are often extended in animals with uterine disease, probably because infection switches the endometrial epithelial secretion of prostaglandins from the F series to the E series by a phospholipase A2-mediated mechanism, which would disrupt luteolysis. The regulation of endometrial immunity depends on steroid hormones, somatotrophins, and local regulatory proteins. Advances in knowledge about infection and immunity in the female genital tract should be exploited to develop new therapeutics for uterine disease.
Abstract The amnion is the inner of two membranes surrounding the fetus. That it arises from embryonic epiblast cells prior to gastrulation suggests that it may retain a reservoir of stem cells throughout pregnancy. We found that human amniotic epithelial cells (hAECs) harvested from term-delivered fetal membranes express mRNA and proteins present in human embryonic stem cells (hESCs), including POU domain, class 5, transcription factor 1; Nanog homeobox; SRY-box 2; and stage-specific embryonic antigen-4. In keeping with possible stem cell-like activity, hAECs were also clonogenic, and primary hAEC cultures could be induced to differentiate into cardiomyocytic, myocytic, osteocytic, adipocytic (mesodermal), pancreatic, hepatic (endodermal), neural, and astrocytic (neuroectodermal) cells in vitro, as defined by phenotypic, mRNA expression, immunocytochemical, and/or ultrastructural characteristics. However, unlike hESCs, hAECs did not form teratomas upon transplantation into severe combined immunodeficienc...
This study sought to determine the earliest response of the bovine uterine endometrium to the presence of the conceptus at key developmental stages of early pregnancy. There were no detectable differences in gene expression in endometria from pregnant and cyclic heifers on Days 5, 7, and 13 postestrus, but the expression of 764 genes was altered due to the presence of the conceptus at maternal recognition of pregnancy (Day 16). Of these 514 genes, MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD9, EIF4E, and IFIT2 increased to the greatest extent in pregnant endometria (>8-fold log2 fold change increase). The expression of OXTR, Bt.643 (unofficial symbol), and KCNMA1 was reduced the most, but short-term treatment with recombinant ovine interferon tau (IFNT) in vitro or in vivo did not alter their expression. In vivo intrauterine infusion of IFNT induced the expression of EIF4E, IFIT2, IFI44, ISG20, MX2, RSAD2, SAMD9, and USP18. These results revealed for the first time that changes that occur in the endometrial transcriptome are independent of the presence of a conceptus until pregnancy recognition. The differentially expressed genes (including MX2, BST2, RSAD2, ISG15, OAS1, USP18, IFI44, ISG20, SAMD, and EIF4E) are a consequence of IFNT production by the conceptus. The identified genes represent known and novel early markers of conceptus development and/or return to cyclicity and may be useful to identify the earliest stage at which the endometrial response to the conceptus is detectable.
The postovulatory rise in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our study objective was to determine how elevated P4 alters endometrial gene expression to advance conceptus development. Synchronized heifers were inseminated (Day 0) and randomly assigned to pregnant high P4 or to pregnant normal P4. All high P4 groups received a P4-release intravaginal device on Day 3 after insemination that increased P4 concentrations up to Day 7 ( P < 0.05). Tissue was collected on Day 5, 7, 13, or 16 of pregnancy, and endometrial gene expression was analyzed using the bovine Affymetrix (Santa Clara, CA) microarrays. Microarray analyses demonstrated that the largest number of P4-regulated genes coincided with the day when the P4 profiles were different for the longest period. Genes with the largest fold change increase (such as DGAT2 and MSTN [also known as GDF8 ]) were associated with triglyceride synthesis and glucose transport, which can be utilized as an energy source for the developing embryo. Temporal changes occurred at different stages of early pregnancy, with the greatest difference occurring between well-separated stages of conceptus development. Validation of a number of genes by quantitative real-time PCR indicated that P4 supplementation advances endometrial gene expression by altering the time ( FABP , DGAT2 , and MSTN ) or duration ( CRYGS ) of expression pattern for genes that contribute to the composition of histotroph.
ABSTRACT Preeclampsia affects 5%–8% of pregnancies and is the leading worldwide cause of maternal and fetal morbidity and mortality. Preeclampsia is associated with shallow trophoblast invasion and inadequate spiral artery remodeling, which are widely believed to lead to placental hypoxia, the putative culprit initiating the cascade of events that ultimately results in the maternal manifestations of the disease. Despite extensive research, however, the pathophysiology of this disease remains poorly understood, no effective prevention exists, and treatment is limited to symptomatic therapy. Recent research has introduced exciting new theories regarding the pathogenesis of preeclampsia. Clinical and experimental evidence implicating the circulating antiangiogenic molecules soluble Fms-like tyrosine kinase-1 (sFLT-1) and soluble endoglin (sENG), as well as endothelin-1 and the angiotensin II receptor type I autoimmune antibody (AT-1AA), have been especially promising. This review collates evidence for a role...
It is well established that cAMP signaling is an important regulator of the oocyte meiotic cell cycle. Conversely, the function of cGMP during oocyte maturation is less clear. Herein, we evaluated the expression of cGMP-hydrolyzing phosphodiesterases (PDEs) in the somatic and germ cell compartments of the mouse ovarian follicle and demonstrate that PDE5 is preferentially expressed in somatic cells. Cyclic GMP is a potent inhibitor of cAMP hydrolysis from oocyte extracts, with a 50% inhibitory concentration of 97 nM. Luteinizing hormone (LH) stimulation of cultured preovulatory follicles results in a marked decrease in cGMP content, and a nadir is reached in 1.5 h; similarly, oocyte cGMP levels decrease after gonadotropin stimulation in vivo. The LH-dependent decrease in cGMP requires activation of the epidermal growth factor network. Treatment of follicles with a PDE5 inhibitor increases cGMP in the follicle well above unstimulated levels. Although LH causes a decrease in cGMP in follicles preincubated with PDE5 inhibitors, the levels of this nucleotide remain above unstimulated levels. Under these conditions of elevated cGMP, LH stimulation does not cause oocyte maturation after 5 h of incubation. Microinjection of a cGMP-specific PDE into oocytes causes meiotic maturation of wild-type oocytes, suggesting that an intraoocyte pool of cGMP is involved in the maintenance of meiotic arrest. This effect is absent in PDE3A-deficient oocytes. Taken together, these findings provide evidence that cGMP and cAMP signaling cooperate in maintaining meiotic arrest via regulation of PDE3A and that a decrease in cGMP in the somatic compartment is one of the signals contributing to meiotic maturation.
The National Institutes of Health (NIH) miniature pig was developed specifically for xenotransplantation and has been extensively used as a large-animal model in many other biomedical experiments. However, the cloning efficiency of this pig is very low (<0.2%), and this has been an obstacle to the promising application of these inbred swine genetics for biomedical research. It has been demonstrated that increased histone acetylation in somatic cell nuclear transfer (SCNT) embryos, by applying a histone deacetylase (HDAC) inhibitor such as trichostatin A (TSA), significantly enhances the developmental competence in several species. However, some researchers also reported that TSA treatment had various detrimental effects on the in vitro and in vivo development of the SCNT embryos. Herein, we report that treatment with 500 nM 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (termed scriptaid ), a novel HDAC inhibitor, significantly enhanced the development of SCNT embryos to the blastocyst stage when NIH inbred fetal fibroblast cells (FFCs) were used as donors compared with the untreated group (21% vs. 9%, P < 0.05). Scriptaid treatment resulted in eight pregnancies from 10 embryo transfers (ETs) and 14 healthy NIH miniature pigs from eight litters, while no viable piglets (only three mummies) were obtained from nine ETs in the untreated group. Thus, scriptaid dramatically increased the cloning efficiency when using inbred genetics from 0.0% to 1.3%. In contrast, scriptaid treatment decreased the blastocyst rate in in vitro fertilization embryos (from 37% to 26%, P < 0.05). In conclusion, the extremely low cloning efficiency in the NIH miniature pig may be caused by its inbred genetic background and can be improved by alteration of genomic histone acetylation patterns.
Spermatogenesis is a temperature-dependent process, and increases in scrotal temperature can disrupt its progression. We previously showed that heat stress causes DNA damage in germ cells, an increase in germ cell death (as seen on TUNEL staining), and subfertility. The present study evaluated the stress response in mouse testes following a single mild transient scrotal heat exposure (40Â°C or 42Â°C for 30 min). We investigated markers of three types of stress response, namely, hypoxia, oxidative stress, and apoptosis. Heat stress caused an increase in expression of hypoxia-inducible factor 1 alpha ( Hif1a ) mRNA expression and translocation of HIF1A protein to the germ cell nucleus, consistent with hypoxic stress. Increased expression of heme oxygenase 1 ( Hmox1 ) and the antioxidant enzymes glutathione peroxidase 1 (GPX1) and glutathione S-transferase alpha (GSTA) was consistent with a robust oxidative stress response. Germ cell death was associated with an increase in expression of the effector caspase cleaved caspase 3 and a decrease in expression of the protein inhibitor of caspase-activated DNase (ICAD). Reduced expression of ICAD contributes to increased activity of caspase-activated DNase and is consistent with the increased rates of DNA fragmentation that have been detected previously using TUNEL staining. These studies confirmed that transient mild testicular hyperthermia results in temperature-dependent germ cell death and demonstrated that elevated temperature results in a complex stress response, including induction of genes associated with oxidative stress and hypoxia.
In addition to their contribution to the research on early human development, human embryonic stem (hES) cells may also be used for cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast feeder layers, which allow their continuous growth in an undifferentiated state. However, the use of hES cells in human therapy requires an animal-free culture system, in which exposure to mouse retroviruses is avoided. In this study we present a novel feeder layer-free culture system for hES cells, based on medium supplemented with 15% serum replacement, a combination of growth factors including transforming growth factor Î²1 (TGFÎ²1), leukemia inhibitory factor, basic fibroblast growth factor, and fibronectin matrix. Human ES cells grown in these conditions maintain all ES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of the three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. The culture system presented here has two major advantages: 1) application of a well-defined culture system for hES cells and 2) reduced exposure of hES cells to animal pathogens. The feeder layer-free culture system reported here aims at facilitating research practices and providing a safer alternative for future clinical applications of hES cells.
Methods to predict numbers of healthy oocytes in the ovaries of young adults could have important diagnostic relevance in family planning and animal agriculture. We have observed that peak antral follicle count (AFC) determined by serial ovarian ultrasonography during follicular waves is very highly reproducible within individual young adult cattle, despite 7-fold variation among animals. Herein, we tested the hypothesis that AFC is positively associated with the number of morphologically healthy oocytes and follicles in ovaries and with serum concentrations of anti-MÃ¼llerian hormone (AMH), an indirect marker for number of healthy follicles and oocytes in ovaries. In the present study, age-matched young adult cattle (12â18 mo old) were subjected to serial ultrasonography to identify animals with a consistently high (â¥25 follicles that were â¥3 mm in diameter) or low (â¤15 follicles) AFC during follicular waves. Differences in serum AMH concentrations, ovary weight, and number of morphologically healthy and atretic follicles and oocytes were determined. The phenotypic classifications of cattle based on AFC during follicular waves or AMH concentrations both predict reliably the relative number of morphologically healthy follicles and oocytes in ovaries of age-matched young adult cattle.