Cultured Preimplantation Embryos Research Articles

BET proteins are essential for the specification and maintenance of the epiblast lineage in mouse preimplantation embryos

BackgroundDuring mammalian preimplantation development, as the fertilized egg develops and differentiates, three cell lineages become specified: trophectoderm (TE), epiblast, and primitive endoderm (PrE). Through two steps of cell fate decisions, 16-cell blastomeres develop into TE and an inner cell mass (ICM), and thereafter, the latter differentiates into pluripotent epiblast and PrE. Although bromodomain and extra-terminal domain (BET) proteins, such as BRD4, are necessary for the transcriptional activation of genes involved in the maintenance of mouse embryonic stem cells by occupying their enhancers, their roles in the development of mouse preimplantation are unknown.ResultsTo evaluate the effect of BET protein deficiency on cell lineage formation, we cultured preimplantation embryos in the presence of JQ1, which blocks the binding of BET bromodomains to acetylated-histones. We found BET inhibition blocked the transcriptional activation of genes, such as Nanog, Otx2, and Sox2, important for the formation of the epiblast lineage in blastocysts. Expression studies with lineage-specific markers in morulae and blastocysts revealed BET proteins were essential for the specification and maintenance of the epiblast lineage but were dispensable for the formation of primarily extraembryonic TE and PrE lineages. Additional Ingenuity Pathway Analysis and expression studies with a transcriptionally active form of signal transducer and activator of the transcription 3 (STAT3) suggested BET-dependent activation was partly associated with the STAT3-dependent pathway to maintain the epiblast lineage. To identify BET proteins involved in the formation of the epiblast lineage, we analyzed mutant embryos deficient in Brd4, Brd2, and double mutants. Abolishment of NANOG-positive epiblast cells was only evident in Brd4/Brd2 double-deficient morulae. Thus, the phenotype of JQ1-treated embryos is reproduced not by a Brd4- or Brd2-single deficiency, but only Brd4/Brd2-double deficiency, demonstrating the redundant roles of BRD2 and BRD4 in the specification of the epiblast lineage.ConclusionsBET proteins are essential to the specification and maintenance of the epiblast lineage by activating lineage-specific core transcription factors during mouse preimplantation development. Among BET proteins, BRD4 plays a central role and BRD2 a complementary role in the specification and maintenance of epiblast lineages. Additionally, BET-dependent maintenance of the epiblast lineage may be partly associated with the STAT3-dependent pathway.

Raman spectroscopy analysis of differences in composition of spent culture media of in vitro cultured preimplantation embryos isolated from normal and fat mice dams

The aim of the present study was to compare overall patterns of metabolic activity of in vitro cultured preimplantation embryos isolated from normal and fat mice dams by means of non-invasive profiling of spent culture media using Raman spectroscopy.To produce females with two different types of body condition (normal and fat), a previously established two-generation model was used, based on overfeeding of experimental mice during prenatal and early postnatal development. Embryos were isolated from spontaneously ovulating and naturally fertilized dams at the 2-cell stage of development and cultured to the blastocyst stage in synthetic oviductal medium KSOMaa. Embryos from fat mice (displaying significantly elevated body weight and fat) showed similar developmental capabilities in vitro as embryos isolated from normal control dams (displaying physiological body weight and fat).The results show that alterations in the composition of culture medium caused by the presence of developing mouse preimplantation embryos can be detected using Raman spectroscopy. Metabolic activity of embryos was reflected in evident changes in numerous band intensities in the 1620–1690cm−1 (amide I) region and in the 1020–1140cm−1 region of the Raman spectrum for KSOMaa. Moreover, multivariate analysis of spectral data proved that the composition of proteins and other organic compounds in spent samples obtained after the culture of embryos isolated from fat dams was different from that in spent samples obtained after the culture of embryos from control dams.This study demonstrates that metabolic activity of cultured preimplantation embryos might depend on the body condition of their donors.

ICAM-1 Is Involved in Uterine Receptivity in Rats.

The uterine epithelium undergoes extensive molecular changes during early pregnancy to become receptive to an implanting embryo, and this is tightly regulated by ovarian hormones. Many adhesion molecules are known to reorganize during early pregnancy in order to facilitate embryo adhesion to the apical membrane of uterine epithelial cells and play an important role in uterine receptivity. This study examined the changes in distribution, expression and hormonal control of ICAM-1 in the rat uterus during early pregnancy, in ovariectomised rats, pseudo pregnant rats and cultured preimplantation embryos. The use of ICAM-1 specific vivo-morpholino antisense oligonucleotides was employed to discover the role of ICAM-1 in embryo development and implantation. ICAM-1 was visualized in the rat uterus using indirect immunofluorescence microscopy and was absent in the uterine epithelium on day 1 (pre-receptive) and day 3 of pregnancy. By day 6 (receptive) and day 7 of pregnancy at the time of implantation in the rat, ICAM-1 is present in the uterine epithelium and is localized to the apical region. ICAM-1 then disappears in uterine epithelial cells by day 9 (post receptive) of pregnancy. ICAM-1 thus is likely an important molecule in the adhesion of an implanting embryo to the apical membrane of uterine epithelial cells in rats. ICAM-1 is absent in the uterine epithelium of ovariectomised rats treated with oestrogen alone (non-receptive) and apical in the uterine epithelium of rats treated with progesterone alone, or combined with estrogen (receptive); and was apical in day 6 pseudo pregnant animals. Apical ICAM-1 staining in the uterine epithelium, on day 6 of pregnancy and in ovariectomised rats treated with progesterone and in pseudo pregnant rats always coincides with an increase in ICAM-1 monomeric expression on western blots. Dimeric expression of ICAM-1 did not alter from day 1 to day 6 of pregnancy and corresponds to the ICAM-1 protein found in endothelium. ICAM-1 was however present in endothelial cells of blood vessels found in the endometrial stroma at all stages of pregnancy. This indicates that ICAM-1 in uterine epithelial cells is under progesterone control and that endothelial ICAM-1 is not at all influenced by ovarian hormones. Previous studies have suggested that ICAM-1 might be up regulated by cytokines released by implanting embryos; however the use of pseudo pregnant and ovariectomised rats has shown that this is not the case. ICAM-1 is also present in pre-implantation embryos and may be necessary in facilitating embryo adhesion. Moreover, early day 3 embryos exposed to ICAM-1 vivo-morpholino antisense oligonucleotides fail to develop into blastocysts. In summary ICAM-1 is likely an important molecule in the receptivity of uterine epithelial cells and is under progesterone control. ICAM-1 is also a necessary molecule in embryo development. (platform)

Expression of adiponectin receptors and effects of adiponectin isoforms in mouse preimplantation embryos

Adiponectin, a pleiotropic hormone secreted from adipose tissue, can mediate some negative effects of obesity on female health, and can participate in the impaired reproductive performance of obese women. Using a mouse model, we investigated expression of adiponectin receptors in ovulated oocytes and in vivo derived preimplantation embryos, and tested effects of different adiponectin isoforms on development of preimplantation embryos in vitro. Using RT-PCR and immunohistochemistry, we found expression of adiponectin receptors AdipoR1 and AdipoR2, at the mRNA and protein level, in mouse ovulated oocytes and preimplantation embryos. Quantitative real-time RT-PCR analysis showed a decrease in the amount of AdipoR1 and AdipoR2 mRNA after fertilization, which was followed by an increase in mRNA at the morula and blastocyst stage; mRNA for adiponectin was detected only at the blastocyst stage. Administration of full-length adiponectin significantly changed the distribution in numbers of cells of cultured preimplantation embryos, increasing the proportion of embryos with high cell numbers (>128 cells) and decreasing the proportion of embryos with lower cell numbers (<65 cells). Blastocysts possessed significantly higher cell numbers after full-length adiponectin treatment. Mutated trimeric adiponectin had the opposite effect, a significant decrease in the proportion of embryos with higher cell numbers (>96 cells) and increase in the proportion of embryos with lower cell numbers (<65 cells). Trimeric adiponectin also significantly decreased the cell number and increased cell death in blastocysts. Truncated globular adiponectin had no significant effect on development of mouse preimplantation embryos. Our results indicate that adiponectin can directly influence the development of the preimplantation embryo, and the effects are isoform dependent.

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.

Alterations in Ultrastructural Morphology of Two-Cell Bovine Embryos Produced In Vitro and In Vivo Following a Physiologically Relevant Heat Shock1

Exposure of cultured preimplantation embryos to temperatures similar to those experienced by heat-stressed cows inhibits subsequent development. In this study, the effects of heat shock on the ultrastructure of two-cell bovine embryos were examined to determine mechanisms for inhibition of development. Two-cell embryos produced in vitro were harvested at approximately 28 h postinsemination and cultured for 6 h at one of three temperatures: 38.5 degrees C (cow body temperature), 41.0 degrees C (characteristic temperature for heat-stressed cows), or 43.0 degrees C (severe heat shock). Ultrastructural examinations revealed that both heat shocks resulted in the movement of organelles towards the center of the blastomere. In addition, heat shock increased the percentage of mitochondria exhibiting a swollen morphology. Distance between the membranes comprising the nuclear envelope was increased but only when embryos were treated at 43.0 degrees C. To determine whether ultrastructural responses to heat shock in culture were similar for embryos produced in vitro and in vivo, two-cell embryos were collected from superovulated Angus cows 48 h postinsemination and treated ex vivo for 6 h at 38.5 degrees C or 41.0 degrees C. Again, heat shock caused an increase in number of swollen mitochondria and movement of organelles away from the periphery of the blastomere. Exposure of two-cell bovine embryos to physiologically relevant elevated temperatures causes disruption in ultrastructural morphology that is inimical to development. The observation that overall morphology and response to heat was similar for embryos produced in vitro and in vivo implies that the former can be a good model for understanding embryonic responses to heat shock.

Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR

The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development. These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos. Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting. We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos. Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine embryos. These studies have demonstrated that culture conditions influence the amount of marker gene transcripts and downregulate or induce the expression of novel genes during early development. Optimized defined culture media maintain embryonic gene expression patterns closely resembling those displayed by embryos derived in vivo. Preimplantation mammalian embryos display an impressive capacity to respond to the pressures that suboptimal culture environments place upon them. However, this plasticity operates within a defined range of tolerances. Continued research using molecular techniques will lead to increased understanding of developmental mechanisms causing culture-related phenotypic abnormalities in post-implantation embryos.

Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR

The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development. These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos. Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting. We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos. Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine embryos. These studies have demonstrated that culture conditions influence the amount of marker gene transcripts and downregulate or induce the expression of novel genes during early development. Optimized defined culture media maintain embryonic gene expression patterns closely resembling those displayed by embryos derived in vivo. Preimplantation mammalian embryos display an impressive capacity to respond to the pressures that suboptimal culture environments place upon them. However, this plasticity operates within a defined range of tolerances. Continued research using molecular techniques will lead to increased understanding of developmental mechanisms causing culture-related phenotypic abnormalities in post-implantation embryos.

Insulin-like growth factors and their receptors in human fallopian tube in reproductive-age women

Insulin-like growth factors and their receptors in human fallopian tube in reproductive-age women

Effects of Uranium Poisoning on Cultured Preimplantation Embryos

The toxic effect of uranium in cultured preimplantation embryos of the mouse is presented. Embryos were obtained from hybrid females CBA x C57 BL following induction of superovulation and were incubated in M16 cultured medium. Two different experiments were performed. In one, embryos in a one-cell stage were placed in culture media with final concentrations of uranyl nitrate of 104 and 208 microg/mL during 120 h in the same dish. In the other experiment, embryos in a one-cell stage were placed in culture medium with uranyl nitrate with final U concentrations of 26, 52, 104, and 208 microg/mL. At 24 h, those embryos which had reached the two-cell stage were transferred to another culture dish to which fresh solutions with uranyl nitrate were added. The percentage of embryos in two-cell stage, morula, early blastocyst, expanded blastocyst, and hatched blastocyst were recorded at 24, 72, 96 and 120 h of culture. The results obtained showed that concentrations as from 26 microg U/mL induced the delay of embryo development and the impairment of blastomere proliferation. The toxic effect of uranium increased in those experiments in which the embryos were transferred to a new medium. This embryo-culture system appears to be appropriate to evaluate the toxic effect of uranium on embryos removed from maternal influences and represents a suitable test system for environmental pollutants.

Assessment of DNA damage in individual hamster embryos by comet assay.

DNA damage induced by either light exposure or oxidative stress likely contributes to the compromised development in vitro of cultured preimplantation embryos. Using the comet assay, which entails microgel electrophoresis that can readily detect single-strand breaks in DNA, a significant increase in DNA damage was detected in individual one-cell hamster embryos that were treated with either ultraviolet light or hydrogen peroxide. In addition, an increase in DNA damage also was observed following exposure of one-cell embryos to visible light. When the embryos were placed in drops of culture medium that were covered with mineral oil and the dishes then placed in a portable incubator containing 5% CO(2) in air at 37 degrees C, visible and UV light irradiation for 30 min still induced extensive DNA damage when compared to control embryos that were kept in the dark. In contrast, infrared irradiation did not induce an increase in DNA damage. DNA damage also was measured in individual one- and two-cell stage embryos developed in vivo or in vitro. The extent of DNA damage in the cultured embryos was significantly greater than in embryos that developed in vivo. These results highlight the usefulness of the comet assay to assess DNA damage in individual preimplantation embryos and how the assay can be used to monitor culture conditions in vitro.

DNA ploidy abnormalities in rabbit preimplantation embryos are not increased by conditions associated with in vitro culture.

Possible adverse effects of in vitro culture-associated physical factors were studied in 3- and 4-day-old rabbit embryos. Laboratory conditions were mimicked by exposure to visible light (320-740 nm, 1600 lx) or decreased temperature (22 +/- 1 degree C). Embryos were exposed for a 24-hr period followed by either immediate evaluation or an additional 24 hr of standard in vitro culture (darkness, 37 degrees C) and evaluation thereafter. Effects were assayed by cytophotometric measurement of the DNA content in Feulgen-stained cell nuclei and by cell number. The incidence of DNA aneuploid embryos and DNA aneuploid cell nuclei per embryo, as well as the average nuclear DNA content, was not significantly different between exposed embryos and controls. Both in vitro culture and reduced temperature caused a decrease in cell number. The temperature-induced cell number decrease was reversible within 24 hr after return to 37 degrees C. These results demonstrate that physical factors associated with in vitro culture do not increase DNA ploidy abnormalities in cultured preimplantation embryos.

Expression of leukaemia inhibitory factor receptor subunits LIFR beta and gp130 in human oocytes and preimplantation embryos.

The expression of both components of the high-affinity leukaemia inhibitory factor receptor, LIFR beta and glycoprotein 130 (gp130), was investigated in human oocytes and individual in-vitro cultured preimplantation embryos by reverse transcription-polymerase chain reaction (RT-PCR). Messenger RNA of both LIFR beta and gp130 was detected in as little as 1/30 and 1/12 sample equivalents of cDNA respectively, in oocytes (n = 4), 4-cell and expanded, blastacyst stage embryos. LIFR beta but not gp130 transcripts were detected at the 2-, 8- and 10-cell stages, and in cavitating and hatched blastocysts. In order to exclude a simian origin of these PCR products resulting from the Vero cell line that was used as a feeder during culture to the blastocyst stage, they were digested with restriction endonucleases Taql (LIFR beta) or Kpnl (gp130). Their human origin was confirmed. The results support an earlier finding of LIFR beta mRNA expression in human blastocysts, and extend these results to earlier stages and oocytes. This is the first report of LIFR beta and gp130 transcription in human oocytes. Taken together these results demonstrate that transcription of LIFR beta and gp130 takes place throughout human preimplantation development, and suggest that functional LIF receptors might be present at these stages. These results further confirm the feasibility of performing mRNA phenotyping of multiple genes with RNA derived from a single preimplantation stage embryo.

Expression of insulin-like growth factor-I (IGF-I) in the rat fallopian tube: possible autocrine and paracrine action of fallopian tube-derived IGF-I on the fallopian tube and on the preimplantation embryo

Recent studies have indicated that growth factors such as insulin-like growth factors (IGFs) increase the growth rate of cultured preimplantation embryos. We therefore hypothesized that the fallopian tube may produce IGFs which in turn participate in the regulation of preimplantation embryo development in vivo. In the present study we examined the expression of IGF-I in the fallopian tube. We demonstrated that IGF-I transcripts (7.0, 1.7, and 1.2-0.8 kilobases) were abundant in the fallopian tube. Immunoreactive IGF-I was most abundant in the epithelial cells in the fallopian tube, and IGF-I messenger RNA (mRNA) was detected in the luminal region of the fallopian tube. A solution hybridization assay was used to examine the regulation of IGF-I mRNA. The abundance of IGF-I transcripts changed markedly during the 4-day estrous cycle with the highest levels on the day of proestrus. The increase in IGF-I mRNA between the day of diestrus II and the day of proestrus was 4-fold (P < 0.01). The pattern of IGF-I mRNA expression in the fallopian tube resembled the pattern of ovarian estrogen production during the estrous cycle. The level of IGF-I mRNA decreased after hypophysectomy. The expression of IGF-I mRNA in the fallopian tube was dose-dependently regulated by estradiol, and a single sc injection of estradiol [5 micrograms/100 g body wt (BW)] increased the IGF-I mRNA in a time-dependent manner with a significant increase after 3 h (P < 0.01). The lowest estradiol dose tested (0.1 microgram/100 g BW) increased the expression after 6 h, whereas progesterone (5 micrograms/100 g BW) was ineffective. The presence of embryos in the fallopian tube did not statistically significantly influence the abundance of IGF-I transcripts as measured with a solution hybridization assay on RNA extracted from whole fallopian tubes. In order to determine possible targets for fallopian tube-derived IGF-I we examined the expression of IGF-I receptor mRNA. Northern blot analysis revealed that an 11-kilobase IGF-I receptor transcript was expressed in the fallopian tube. Using a reverse transcriptase-polymerase chain reaction, IGF-I receptor mRNA was also detected in the eight-cell but not two-cell preimplantation embryo. The present study demonstrates that IGF-I is produced in the fallopian tube and its expression is regulated by estradiol. Both the fallopian tube and the eight-cell preimplantation embryo express IGF-I receptors and are therefore potential target tissues.

Expression of insulin-like growth factor-I (IGF-I) in the rat fallopian tube: possible autocrine and paracrine action of fallopian tube-derived IGF-I on the fallopian tube and on the preimplantation embryo.

Recent studies have indicated that growth factors such as insulin-like growth factors (IGFs) increase the growth rate of cultured preimplantation embryos. We therefore hypothesized that the fallopian tube may produce IGFs which in turn participate in the regulation of preimplantation embryo development in vivo. In the present study we examined the expression of IGF-I in the fallopian tube. We demonstrated that IGF-I transcripts (7.0, 1.7, and 1.2-0.8 kilobases) were abundant in the fallopian tube. Immunoreactive IGF-I was most abundant in the epithelial cells in the fallopian tube, and IGF-I messenger RNA (mRNA) was detected in the luminal region of the fallopian tube. A solution hybridization assay was used to examine the regulation of IGF-I mRNA. The abundance of IGF-I transcripts changed markedly during the 4-day estrous cycle with the highest levels on the day of proestrus. The increase in IGF-I mRNA between the day of diestrus II and the day of proestrus was 4-fold (P < 0.01). The pattern of IGF-I mRNA expression in the fallopian tube resembled the pattern of ovarian estrogen production during the estrous cycle. The level of IGF-I mRNA decreased after hypophysectomy. The expression of IGF-I mRNA in the fallopian tube was dose-dependently regulated by estradiol, and a single sc injection of estradiol [5 micrograms/100 g body wt (BW)] increased the IGF-I mRNA in a time-dependent manner with a significant increase after 3 h (P < 0.01). The lowest estradiol dose tested (0.1 microgram/100 g BW) increased the expression after 6 h, whereas progesterone (5 micrograms/100 g BW) was ineffective. The presence of embryos in the fallopian tube did not statistically significantly influence the abundance of IGF-I transcripts as measured with a solution hybridization assay on RNA extracted from whole fallopian tubes. In order to determine possible targets for fallopian tube-derived IGF-I we examined the expression of IGF-I receptor mRNA. Northern blot analysis revealed that an 11-kilobase IGF-I receptor transcript was expressed in the fallopian tube. Using a reverse transcriptase-polymerase chain reaction, IGF-I receptor mRNA was also detected in the eight-cell but not two-cell preimplantation embryo. The present study demonstrates that IGF-I is produced in the fallopian tube and its expression is regulated by estradiol. Both the fallopian tube and the eight-cell preimplantation embryo express IGF-I receptors and are therefore potential target tissues.

Transient expression of a human β‐actin promoter/lacZ gene introduced into mouse embryos correlates with a low degree of methylation

We have analysed the correlation between expression and methylation for the human beta-actin promoter introduced into mouse embryos. The beta-actin promoter was fused to the reporter gene lacZ, and expression was analysed after pronuclear injection into fertilized mouse eggs. We analysed transient expression in in vitro cultured preimplantation embryos and expression after chromosomal integration in 5 independent lines of transgenic mice. The in vitro cultured preimplantation embryos expressed lacZ from the 2-cell to the blastocyst stages, and most abundantly at the morula stage. By increasing the amount of injected DNA, a larger proportion of embryos expressed lacZ. Embryos expressing lacZ in only a subset of the blastomeres were detected at all preimplantation stages. In contrast to the transient expression after injection, we have not detected lacZ expression in any of the 5 analysed lines of transgenic mice carrying the same construct. The lack of expression in transgenic mice correlates with hypermethylation of C residues in the vast majority of CG sequences in the integrated beta-actin/lacZ construct, whereas the injected construct was completely nonmethylated. We discuss methylation and other possible reasons for the observed differences in expression between injected and integrated copies of the beta-actin/lacZ construct and for lacZ expression in only a subset of blastomeres in preimplantation embryos.