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.
Obese pregnant women have increased levels of proinflammatory cytokines in maternal circulation and placental tissues. However, the pathways contributing to placental inflammation in obesity are largely unknown. We tested the hypothesis that maternal body mass index (BMI) was associated with elevated proinflammatory cytokines in maternal and fetal circulations and increased activation of placental inflammatory pathways. A total of 60 women of varying pre-/early pregnancy BMI, undergoing delivery by Cesarean section at term, were studied. Maternal and fetal (cord) plasma were collected for analysis of insulin, leptin, IL-1beta, IL-6, IL-8, monocyte chemoattractant protein (MCP) 1, and TNFalpha by multiplex ELISA. Activation of the inflammatory pathways in the placenta was investigated by measuring the phosphorylated and total protein expression of p38-mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase (JNK)-MAPK, signal transducer-activated transcription factor (STAT) 3, caspase-1, IL-1beta, IkappaB-alpha protein, and p65 DNA-binding activity. To determine the link between activated placental inflammatory pathways and elevated maternal cytokines, cultured primary human trophoblast (PHT) cells were treated with physiological concentrations of insulin, MCP-1, and TNFalpha, and inflammatory signaling analyzed by Western blot. Maternal BMI was positively correlated with maternal insulin, leptin, MCP-1, and TNFalpha, whereas only fetal leptin was increased with BMI. Placental phosphorylation of p38-MAPK and STAT3, and the expression of IL-1beta protein, were increased with maternal BMI; phosphorylation of p38-MAPK was also correlated with birth weight. In contrast, placental NFkappaB, JNK and caspase-1 signaling, and fetal cytokine levels were unaffected by maternal BMI. In PHT cells, p38-MAPK was activated by MCP-1 and TNFalpha, whereas STAT3 phosphorylation was increased following TNFalpha treatment. Maternal BMI is associated with elevated maternal cytokines and activation of placental p38-MAPK and STAT3 inflammatory pathways, without changes in fetal systemic inflammatory profile. Activation of p38-MAPK by MCP-1 and TNFalpha, and STAT3 by TNFalpha, suggests a link between elevated proinflammatory cytokines in maternal plasma and activation of placental inflammatory pathways. We suggest that inflammatory processes associated with elevated maternal BMI may influence fetal growth by altering placental function.
Human endometrium is a highly regenerative tissue undergoing more than 400 cycles of growth, differentiation, and shedding during a woman's reproductive years. Endometrial regeneration is likely mediated by adult stem/progenitor cells. This study investigated key stem cell properties of individual clonogenic epithelial and stromal cells obtained from human endometrium. Single-cell suspensions of endometrial epithelial or stromal cells were obtained from hysterectomy tissues from 15 women experiencing normal menstrual cycles, and were cultured at clonal density (10 cells/cm 2 ) or limiting dilution. The adult stem cell propertiesâself-renewal, high proliferative potential, and differentiation of single epithelial and stromal cellsâwere assessed by harvesting individual colonies and undertaking serial clonal culture, serial passaging, and culture in differentiation-induction media, respectively. Lineage differentiation markers were examined by RT-PCR, immunocytochemistry, and flow cytometry. Rare single human endometrial EpCAM + epithelial cells and EpCAM â stromal cells demonstrated self-renewal by serially cloning >3 times and underwent >30 population doublings over 4 mo in culture. Clonally derived epithelial cells differentiated into cytokeratin + gland-like structures in three dimensional culture. Single stromal cells were multipotent, as their progeny differentiated into smooth muscle cells, adipocytes, chondrocytes, and osteoblasts. Stromal clones expressed mesenchymal stem cell (MSC) markers ITGB1 (CD29), CD44, NT5E (CD73), THY1 (CD90), ENG (CD105), PDGFRB (CD140B), MCAM (CD146) but not endothelial or hemopoietic markers PECAM1 (CD31), CD34, PTPRC (CD45). Adult human endometrium contains rare epithelial progenitors and MSCs, likely responsible for its immense regenerative capacity, which may also have critical roles in the development of endometriosis and endometrial cancer. Human endometrium may provide a readily available source of MSCs for cell-based therapies.
In this study, we performed small RNA library sequencing using human placental tissues to identify placenta-specific miRNAs. We also tested the hypothesis that human chorionic villi could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation. By small RNA library sequencing, most placenta-specific miRNAs (e.g., MIR517A ) were linked to a miRNA cluster on chromosome 19. The miRNA cluster genes were differentially expressed in placental development. Subsequent validation by real-time PCR and in situ hybridization revealed that villous trophoblasts express placenta-specific miRNAs. The analysis of small RNA libraries from the blood plasma showed that the placenta-specific miRNAs are abundant in the plasma of pregnant women. By real-time PCR, we confirmed the rapid clearance of the placenta-specific miRNAs from the plasma after delivery, indicating that such miRNAs enter into maternal circulation. By using the trophoblast cell line BeWo in culture, we demonstrated that miRNAs are indeed extracellularly released via exosomes. Taken together, our findings suggest that miRNAs are exported from the human placental syncytiotrophoblast into maternal circulation, where they could target maternal tissues. Finally, to address the biological functions of placenta-specific miRNAs, we performed a proteome analysis of BeWo cells transfected with MIR517A . Bioinformatic analysis suggests that this miRNA is possibly involved in tumor necrosis factor-mediated signaling. Our data provide important insights into miRNA biology of the human placenta.
Oocyte and embryo metabolism are closely linked with their subsequent developmental capacity. Lipids are a potent source of cellular energy, yet little is known about lipid metabolism during oocyte maturation and early embryo development. Generation of ATP from lipids occurs within mitochondria via beta-oxidation of fatty acids, with the rate-limiting step catalyzed by carnitine palmitoyl transferase I (CPT1B), a process also requiring carnitine. We sought to investigate the regulation and role of beta-oxidation during oocyte maturation and preimplantation development. Expression of Cpt1b mRNA, assessed by real-time RT-PCR in murine cumulus-oocyte complexes (COCs), increased following hormonal induction of oocyte maturation and ovulation in vivo with human chorionic gonadotropin (5 IU) and in embryos reaching the blastocyst stage. Beta-oxidation, measured by the production of (H2O)-H-3 from [H-3]palmitic acid, was significantly increased over that in immature COCs following induction of maturation in vitro with epidermal growth factor (3 ng/ml) and follicle-stimulating hormone (50 mlU/ml). The importance of lipid metabolism for oocyte developmental competence and early embryo development was demonstrated by assessing the rate of embryo development following inhibition or upregulation of beta-oxidation with etomoxir (an inhibitor of CPT1B) or L-carnitine, respectively. Inhibition of beta-oxidation during oocyte maturation or zygote cleavage impaired subsequent blastocyst development. In contrast, L-carnitine supplementation during oocyte maturation significantly increased beta-oxidation, improved developmental competence, and in the absence of a carbohydrate energy supply, significantly increased 2-cell cleavage. Thus, carnitine is an important cofactor for developing oocytes, and fatty acids are an important energy source for oocyte and embryo development.
In cattle, elevated concentrations of circulating progesterone (P4) in the immediate postconception period are associated with advanced conceptus development, while low P4 is implicated as a causative factor in low pregnancy rates observed in dairy cows. This study aimed to: 1) describe the transcriptional changes that occur in the bovine endometrium during the estrous cycle, 2) determine how elevated P4 affects these changes, 3) identify if low P4 alters the expression of these genes, and 4) assess the impact that low P4 has on conceptus development. Relatively few differences occurred in endometrial gene expression during the early luteal phase of the estrous cycle (Day 5 vs. 7), but comparison of endometria from more distant stages of the luteal phase (Day 7 vs. 13) revealed large transcriptional changes, which were significantly altered by exogenous supplementation of P4. Induction of low circulating P4 altered the normal temporal changes in gene expression, and these changes were coordinate with a delay in the down-regulation of the PGR from the LE and GE. Altered endometrial gene expression induced by low P4 was associated with a reduced capacity of the uterus to support conceptus development after embryo transfer on Day 7. In conclusion, the present study provides clear evidence that the temporal changes in the transcriptome of the endometrium of cyclic heifers are sensitive to circulating P4 concentrations in the first few days after estrus. Under low P4 conditions, a suboptimal uterine environment with reduced ability to support conceptus elongation is observed.
Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differs in exosomes isolated from follicular fluid in young compared to old mares. We demonstrate the presence of vesicles resembling microvesicles and exosomes in ovarian follicular fluid using transmission electron microscopy and CD63-positive and RNA containing vesicles using flow cytometry. Moreover, proteomics analysis reveals that follicular fluid-isolated exosomes contain both known exosomal proteins and proteins not previously reported in isolated exosomes. MicroRNAs were detected in microvesicle and exosomes preparations isolated from follicular fluid by real-time PCR analysis. Uptake of fluorescent-labeled microvesicles by granulosa cells was examined using in vitro and in vivo approaches. MicroRNA expression profiling reveals that miRNAs in microvesicle and exosome preparations isolated from follicular fluid also are present within surrounding granulosa and cumulus cells. These studies revealed that cell communication within the mammalian ovarian follicle may involve transfer of bioactive material by microvesicles and exosomes. Finally, miRNAs present in exosomes from ovarian follicular fluid varied with the age of the mare, and a number of different miRNAs were detected in young vs. old mare follicular fluid.
Recent evidence has linked human phthalate exposure to abnormal reproductive and hormonal effects. Phthalates are plasticizers that confer flexibility and transparency to plastics, but they readily contaminate the body and the environment. In this study, timed pregnant CD1 outbred mice were treated with di-(2-ethylhexyl) phthalate (DEHP) from Embryonic Day 7 (E7) to E14. The subsequent generation (F1) offspring were then bred to produce the F2, F3, and F4 offspring, without any further DEHP treatment. This exposure scheme disrupted testicular germ cell association and decreased sperm count and motility in F1 to F4 offspring. By spermatogonial transplantation techniques, the exposure scheme also disrupted spermatogonial stem cell (SSC) function of F3 offspring. The W/W-V recipient testes transplanted with F3 offspring germ cells from the DEHP-treated group had a dramatically lower percentage of donor germ cell-derived spermatogenic recovery in seminiferous tubules when compared to the recipient testes transplanted with CD1 control germ cells. Further characterization showed that the major block of donor germ cell-derived spermatogenesis was before the appearance of undifferentiated spermatogonia. Interestingly, the testes transplanted with the F3 offspring germ cells from the DEHP-treated group, when regenerated, replicated testis morphology similar to that observed in the testes from the F1 to F3 offspring of the DEHP-treated group, suggesting that the germ cell disorganization phenotype originates from the stem cells of F3 offspring. In conclusion, embryonic exposure to DEHP was found to disrupt testicular germ cell organization and SSC function in a transgenerational manner.
Infection of the bovine endometrium with Gram-negative bacteria commonly causes uterine disease. Toll-like receptor 4 (TLR4) on cells of the immune system bind Gram-negative bacterial lipopolysaccharide (LPS), stimulating the secretion of the proinflammatory cytokines interleukin 1B (IL1B) and IL6, and the chemokine IL8. Because the endometrium is the first barrier to infection of the uterus, the signaling cascade triggered by LPS and the subsequent expression of inflammatory mediators were investigated in endometrial epithelial and stromal cells, and the key pathways identified using short interfering RNA (siRNA) and biochemical inhibitors. Treatment of endometrial cells with ultrapure LPS stimulated an inflammatory response characterized by increased IL1B, IL6, and IL8 mRNA expression, and IL6 protein accumulation in epithelial cells, and by increased IL1B and IL8 mRNA expression, and IL6 and IL8 protein accumulation in stromal cells. Treatment of endometrial cells with LPS also induced the degradation of IKB and the nuclear translocation of NFKB, as well as rapid phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) and MAPK14. Knockdown of TLR4 or its signaling adaptor molecule, myeloid differentiation factor 88 (MYD88), using siRNA reduced the inflammatory response to LPS in epithelial and stromal cells. Biochemical inhibition of MAPK3/1, but not JNK or MAPK14, reduced LPS-induced IL1B, IL6, and IL8 expression in endometrial cells. In conclusion, epithelial and stromal cells have an intrinsic role in innate immune surveillance in the endometrium, and in the case of LPS this recognition occurs via TLR4- and MYD88-dependent cell signaling pathways.
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.
Embryo implantation into receptive endometrium requires synergistic endometrial-blastocyst interactions within the uterine cavity and is essential for establishing pregnancy. We demonstrate that exosomes (40-150 nm nanovesicles) released from endometrial epithelial cells are an important component of these interactions. We defined the proteome of purified endometrial epithelial-derived exosomes (Exos) influenced by menstrual cycle hormones estrogen (E; proliferative phase) and estrogen plus progesterone (EP; receptive phase) and examined their potential to modify trophoblast function. E-/EP-Exos were uniquely enriched with 254 and 126 proteins, respectively, with 35% newly identified proteins not previously reported in exosome databases. Importantly, EP-Exos protein cargo was related to fundamental changes in implantation: adhesion, migration, invasion, and extracellular matrix remodeling. These findings from hormonally treated ECC1 endometrial cancer cells were validated in human primary uterine epithelial cell-derived exosomes. Functionally, exosomes were internalized by human trophoblast cells and enhanced their adhesive capacity, a response mediated partially through active focal adhesion kinase (FAK) signaling. Thus, exosomes contribute to the endometrialembryo interactions within the human uterine microenvironment essential for successful implantation.
Mammalian mitochondrial DNA (mtDNA) is a small, maternally inherited genome that codes for 13 essential proteins in the respiratory chain. Mature oocytes contain more than 150 000 copies of mtDNA, at least an order of magnitude greater than the number in most somatic cells, but sperm contain only approximately 100 copies. Mitochondrial oxidative phosphorylation has been suggested to be an important determinant of oocyte quality and sperm motility; however, the functional significance of the high mtDNA copy number in oocytes, and of the low copy number in sperm, remains unclear. To investigate the effects of mtDNA copy number on fertility, we genetically manipulated mtDNA copy number in the mouse by deleting one copy of Tfam, an essential component of the mitochondrial nucleoid, at different stages of germline development. We show that males can tolerate at least a threefold reduction in mtDNA copy number in their sperm without impaired fertility, and in fact, they preferentially transmit a deleted Tfam allele. Surprisingly, oocytes with as few as 4000 copies of mtDNA can be fertilized and progress normally through preimplantation development to the blastocyst stage. The mature oocyte, however, has a critical postimplantation developmental threshold of 40 000-50 000 copies of mtDNA in the mature oocyte. These observations suggest that the high mtDNA copy number in the mature oocyte is a genetic device designed to distribute mitochondria and mtDNAs to the cells of the early postimplantation embryo before mitochondrial biogenesis and mtDNA replication resumes, whereas down-regulation of mtDNA copy number is important for normal sperm function.
This study was designed to isolate, characterize, and culture human spermatogonia. Using immunohistochemistry on tubule sections, we localized GPR125 to the plasma membrane of a subset of the spermatogonia. Immunohistochemistry also showed that MAGEA4 was expressed in all spermatogonia (A dark , A pale , and type B) and possibly preleptotene spermatocytes. Notably, KIT was expressed in late spermatocytes and round spermatids, but apparently not in human spermatogonia. UCHL1 was found in the cytoplasm of spermatogonia, whereas POU5F1 was not detected in any of the human germ cells. GFRA1 and ITGA6 were localized to the plasma membrane of the spermatogonia. Next, we isolated GPR125-positive spermatogonia from adult human testes using a two-step enzymatic digestion followed by magnetic-activated cell sorting. The isolated GPR125-positive cells coexpressed GPR125, ITGA6, THY1, and GFRA1, and they could be cultured for short periods of time and exhibited a marked increase in cell numbers as shown by a proliferation assay. Immunocytochemistry of putative stem cell genes after 2 wk in culture revealed that the cells were maintained in an undifferentiated state. MAPK1/3 phosphorylation was increased after 2 wk of culture of the GPR125-positive spermatogonia compared to the freshly isolated cells. Taken together, these results indicate that human spermatogonia share some but not all phenotypes with spermatogonial stem cells (SSCs) and progenitors from other species. GPR125-positive spermatogonia are phenotypically putative human SSCs and retain an undifferentiated status in vitro. This study provides novel insights into the molecular characteristics, isolation, and culture of human SSCs and/or progenitors and suggests that the MAPK1/3 pathway is involved in their proliferation.
MicroRNAs (miRNAs), a class of small noncoding RNAs that regulate gene expression, have fundamental roles in biological processes, including cell differentiation and proliferation. These small molecules mainly direct either target messenger RNA (mRNA) degradation or translational repression, thereby functioning as gene silencers. Epithelial cells of the uterine lumen and glands undergo cyclic changes under the influence of the sex steroid hormones estradiol-17beta and progesterone. Because the expression of miRNAs in human endometrium has been established, it is important to understand whether miRNAs have a physiological role in modulating the expression of hormonally induced genes. The studies herein establish concomitant differential miRNA and mRNA expression profiles of uterine epithelial cells purified from endometrial biopsy specimens in the late proliferative and midsecretory phases. Bioinformatics analysis of differentially expressed mRNAs revealed cell cycle regulation as the most significantly enriched pathway in the late proliferative-phase endometrial epithelium (P = 5.7 x 10(-15)). In addition, the WNT signaling pathway was enriched in the proliferative phase. The 12 miRNAs (MIR29B, MIR29C, MIR30B, MIR30D, MIR31, MIR193A-3P, MIR203, MIR204, MIR200C, MIR210, MIR582-5P, and MIR345) whose expression was significantly up-regulated in the midsecretory-phase samples were predicted to target many cell cycle genes. Consistent with the role of miRNAs in suppressing their target mRNA expression, the transcript abundance of predicted targets, including cyclins and cyclin-dependent kinases, as well as E2F3 (a known target of MIR210), was decreased. Thus, our findings suggest a role for miRNAs in down-regulating the expression of some cell cycle genes in the secretory-phase endometrial epithelium, thereby suppressing cell proliferation.
DNA damage in human spermatozoa has been associated with a range of adverse clinical outcomes, including infertility, abortion, and disease in the offspring. We have advanced a two-step hypothesis to explain this damage involving impaired chromatin remodeling during spermiogenesis followed by a free radical attack to induce DNA strand breakage. The objective of the present study was to test this hypothesis by determining whether impaired chromatin protamination is correlated with oxidative base damage and DNA fragmentation in human spermatozoa. DNA fragmentation, chromatin protamination, mitochondrial membrane potential, and formation of the oxidative base adduct, 8-hydroxy-2'-deoxyguanosine (8OHdG), were monitored by flow cytometry/fluorescence microscopy. Impairment of DNA protamination during late spermatogenesis was highly correlated (P < 0.001) with DNA damage in human spermatozoa. The disruption of chromatin remodeling also was associated with a significant elevation in the levels of 8OHdG (P < 0.001), and the latter was itself highly correlated with DNA fragmentation (P < 0.001). The significance of oxidative stress in 8OHdG formation was demonstrated experimentally using H2O2/Fe2+ and by the correlation observed between this base adduct and superoxide generation (P < 0.001). That 8OHdG formation was inversely associated with mitochondrial membrane potential (P < 0.001) suggested a possible role for these organelles in the creation of oxidative stress. These results clearly highlight the importance of oxidative stress in the induction of sperm DNA damage and carry significant implications for the clinical management of this condition.
In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development, leading to an increased appreciation of the importance of RNA interference pathways, and in particular miRNAs, as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon, we employed next-generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the posttesticular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively, between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Because sperm are not capable of de novo transcription, these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to influence the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring.
The embryonic origins of ovarian granulosa cells have been a subject of debate for decades. By tamoxifen-induced lineage tracing of Foxl2-expressing cells, we show that descendants of the bipotential supporting cell precursors in the early gonad contribute granulosa cells to a specific population of follicles in the medulla of the ovary that begin to grow immediately after birth. These precursor cells arise from the proliferative ovarian surface epithelium and enter mitotic arrest prior to upregulating Foxl2. Granulosa cells that populate the cortical primordial follicles activated in adult life derive from the surface epithelium perinatally, and enter mitotic arrest at that stage. Ingression from the surface epithelium dropped to undetectable levels by Postnatal Day 7, when most surviving oocytes were individually encapsulated by granulosa cells. These findings add complexity to the standard model of sex determination in which the Sertoli and granulosa cells of the adult testis and ovary directly stem from the supporting cell precursors of the bipotential gonad.
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 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...
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 (R-2 = 0.90), rapid motility (R-2 = 0.89), average path velocity (VAP; R-2 = 0.59), and curvilinear velocity (VCL; R-2 = 0.66). Similarly, lipid peroxidation was positively correlated with total motility (R-2 = 0.46), rapid motility (R-2 = 0.51), average path velocity (R-2 = 0.62), and VCL (R-2 = 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.