Mouse Embryo Development Research Articles

Cell Specific Expression of Vascular Endothelial Growth Factor Receptor-2 (Flk-1/KDR) in Developing Mice Embryo and Supporting Maternal Uterine Tissue during Early Gestation (D4-D7)

Cell Specific Expression of Vascular Endothelial Growth Factor Receptor-2 (Flk-1/KDR) in Developing Mice Embryo and Supporting Maternal Uterine Tissue during Early Gestation (D4-D7)

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis.

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis. The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed. This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

Investigation of PRDM10 and PRDM13 Expression in Developing Mouse Embryos by an Optimized PACT-Based Embryo Clearing Method.

Recent developments in tissue clearing methods have significantly advanced the three-dimensional analysis of biological structures in whole, intact tissue, providing a greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, limiting the wide application of these techniques to fragile tissues such as developing embryos. Here, we present an optimized passive tissue clearing technique (PACT)-based embryo clearing method, initial embedding PACT (IMPACT)-Basic, that improves tissue rigidity without compromising optical transparency. We also present IMPACT-Advance, which is specifically optimized for thin slices of mouse embryos past E13.5. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PR domain (PRDM) proteins, PRDM10 and PRDM13, in intact cleared mouse embryos at various stages of development. We observed strong PRDM10 and PRDM13 expression in the developing nervous system and skeletal cartilage, suggesting a functional role for these proteins in these tissues throughout embryogenesis.

Effects of Insulin-Like Growth Factor 1 on the In Vitro Development of Mouse Embryos after Cryopreservation

Cryopreservation of gametes and embryos is widely used for mammalian genome resource banking. Effects of insulin-like growth factor-1 (IGF-1) on the in vitro development of cleavage stage CD1 mouse embryos after their cryopreservation were studied. The mouse embryos developed in vivo up to the 4–6-cell stage were frozen in a programmable freezer according to standard protocol with 10% propylene glycol as a cryoprotectant and thereafter were thawed and cultured in vitro in KSOM for 48 h. The percentage of embryos developed up to blastocyst stage in culture medium supplemented with IGF-1 (40 ng/mL) was higher as compared to controls (78.9 and 47.4%, respectively, p < 0.01). Moreover, blastocysts developed in the presence of IGF-1 contained more cells as compared to controls (79.8 ± 3.9 and 58.6 ± 3.6 cells, respectively, p < 0.001). However, the index of nuclear fragmentation did not differ between these groups. Experimental results presented here revealed the stimulating effects of IGF-1 on the mouse preimplantation embryos after their cryopreservation.

Effects of maternal diazinon exposure on frontal cerebral cortical development in mouse embryo

Background Diazinon including organophosphate (OP) that is widely used in agriculture and animal husbandry industry and the risk of human infection with the toxin and their toxicity. Methods Pregnant balb/c mice (30–35 g) were randomly divided into five groups of five: the control group (no intervention), two sham groups (emulsifier 0.52, and 5.2 liters/volume). From the seventh to the eighteenth day of pregnancy, two experimental groups received diazinon inhaled 1.3 (EXP1) and 13 liters/volume (EXP2) for 40 min every other day, respectively. On the 18th day of pregnancy, the animals were killed and their embryos were removed to appraisal the growth of fetus and development of the frontal cortex. A computer-assisted morphometric quantitative images analysis were performed on the frontal cerebral cortex (FCC) including cortical plate (CP), intermediate zone (IZ) and matrix (proliferative) zone (MZ) of the mouse embryos. Findings The average of crown-rump length and weight of the embryos in the experimental groups were increased without any significant difference. The mean fetal FCC thickness in the EXP2 group was significantly reduced compared to the control group, CP thickness was remarkably increased in fetuses exposed to diazinon. Comparing the mean thickness of MZ and IZ in EXP groups with the sham and control groups indicated a significant decrease. The positive K-67 cells in the FCC of the EXP2 group were significantly reduced. Discussion Exposing diazinon during pregnancy can reduce brain development and would be neurotoxic to the developing brain and can lead to behavioral changes in the offspring.

Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences

What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model? POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation. Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage. The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos. A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing. Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts. One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes. Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests. N/A.

Transient prenatal ruxolitinib treatment suppresses astrogenesis during development and improves learning and memory in adult mice

Ruxolitinib is the first janus kinase 1 (JAK1) and JAK2 inhibitor that was approved by the United States Food and Drug Administration (FDA) agency for the treatment of myeloproliferative neoplasms. The drug targets the JAK/STAT signalling pathway, which is critical in regulating the gliogenesis process during nervous system development. In the study, we assessed the effect of non-maternal toxic dosages of ruxolitinib (0–30 mg/kg/day between E7.5-E20.5) on the brain of the developing mouse embryos. While the pregnant mice did not show any apparent adverse effects, the Gfap protein marker for glial cells and S100β mRNA marker for astrocytes were reduced in the postnatal day (P) 1.5 pups' brains. Gfap expression and Gfap+ cells were also suppressed in the differentiating neurospheres culture treated with ruxolitinib. Compared to the control group, adult mice treated with ruxolitinib prenatally showed no changes in motor coordination, locomotor function, and recognition memory. However, increased explorative behaviour within an open field and improved spatial learning and long-term memory retention were observed in the treated group. We demonstrated transplacental effects of ruxolitinib on astrogenesis, suggesting the potential use of ruxolitinib to revert pathological conditions caused by gliogenic-shift in early brain development such as Down and Noonan syndromes.

Renin-angiotensin system is involved in embryonic emergence of hematopoietic stem/progenitor cells.

Angiotensin-converting enzyme (ACE), a key element of the renin-angiotensin system (RAS), has recently been identified as a new marker of both adult and embryonic human hematopoietic stem/progenitor cells (HSPCs). However, whether a full renin-angiotensin pathway is locally present during the hematopoietic emergence is still an open question. In the present study, we show that this enzyme is expressed by hematopoietic progenitors in the developing mouse embryo. Furthermore, ACE and the other elements of RAS-namely angiotensinogen, renin, and angiotensin II type 1 (AT1) and type 2 (AT2) receptors-are expressed in the paraaortic splanchnopleura (P-Sp) and in its derivative, the aorta-gonad-mesonephros region, both in human and mouse embryos. Their localization is compatible with the existence of a local autocrine and/or paracrine RAS in these hemogenic sites. in vitro perturbation of the RAS by administration of a specific AT1 receptor antagonist inhibits almost totally the generation of blood CD45-positive cells from dissected P-Sp, implying that angiotensin II signaling is necessary for the emergence of hematopoietic cells. Conversely, addition of exogenous angiotensin II peptide stimulates hematopoiesis in culture, with an increase in the number of immature c-Kit+ CD41+ CD31+ CD45+ hematopoietic progenitors, compared to the control. These results highlight a novel role of local-RAS during embryogenesis, suggesting that angiotensin II, via activation of AT1 receptor, promotes the emergence of undifferentiated hematopoietic progenitors.

Balanced Notch-Wnt signaling interplay is required for mouse embryo and fetal development

This study investigated the role of Notch and Wnt cell signaling interplay in the mouse early embryo, and its effects on fetal development. Developmental kinetics was evaluated in embryos in vitro cultured from the 8-16-cell to the hatched blastocyst stage in the presence of signaling inhibitors of Notch (DAPT) and/or Wnt (DKK1). An embryo subset was evaluated for differential cell count and gene transcription of Notch (receptors Notch1-4, ligands Dll1, Dll4, Jagged1-2, effectors Hes1-2) and Wnt (Wnt3a, Lrp6, Gsk3β, C-myc, Tcf4, β-catenin) components, E-cadherin and pluripotency and differentiation markers (Sox2, Oct4, Klf4, Cdx2), whereas a second subset was evaluated for implantation ability and development to term following transfer into recipients. Notch and Wnt blockades had significant opposing effects on developmental kinetics - Notch blockade retarded while Wnt blockade fastened development. This evidences that Notch and Wnt regulate the pace of embryo kinetics by respectively speeding and braking development. Blockades significantly changed the transcription profile of Sox2, Oct4, Klf4 and Cdx2, and Notch and double blockades significantly changed embryonic cell numbers and cell ratio. The double blockade induced more severe phenotypes than those expected from the cumulative effects of single blockades. Implantation ability was unaffected, but Notch and double blockades significantly decreased fetal development to term. Compared to control embryos, Notch blockade and Wnt blockade embryos originated, respectively, significantly lighter and heavier fetuses. In conclusion, Notch and Wnt signaling interplay in the regulation of the pace of early embryo kinetics, and their actions at this stage have significant carry-over effects on later fetal development to term.

Effect of retinoic acid signaling on Ripply3 expression and pharyngeal arch morphogenesis in mouse embryos.

Pharyngeal arches (PA) are sequentially generated in an anterior-to-posterior order. Ripply3 is essential for posterior PA development in mouse embryos and its expression is sequentially activated in ectoderm and endoderm prior to formation of each PA. Since the PA phenotype of Ripply3 knockout (KO) mice is similar to that of retinoic acid (RA) signal-deficient embryos, we investigated the relationship between RA signaling and Ripply3 in mouse embryos. In BMS493 (pan-RAR antagonist) treated embryos, which are defective in third and fourth PA development, Ripply3 expression is decreased in the region posterior to PA2 at E9.0. This expression remains and its distribution is expanded posteriorly at E9.5. Conversely, high dose RA exposure does not apparently change its expression at E9.0 and 9.5. Knockout of retinaldehyde dehydrogenase 2 (Raldh2), which causes more severe PA defect, attenuates sequential Ripply3 expression at PA1 and reduces its expression level. EGFP reporter expression driven by a 6kb Ripply3 promoter fragment recapitulates the endogenous Ripply3 mRNA expression during PA development in wild-type, but its distribution is expanded posteriorly in BMS493-treated and Raldh2 KO embryos. Spatio-temporal regulation of Ripply3 expression by RA signaling is indispensable for the posterior PA development in mouse.

Ovarian superstimulatory response and embryo development using a new recombinant glycoprotein with eCG-like activity in mice

Pharmacological ovarian control required for the implementation of artificial insemination and embryo-related technologies usually includes the use of eCG, naturally produced in pregnant mares. In this study, we report the superovulatory response and embryo development in mice obtained with a new glycoprotein with eCG-like activity (reCG) produced by recombinant DNA technology. A total of 150 females from three different mouse strains (C57BL/6J, BALB/cJ and B6D2F1/J) were subjected to a superstimulatory protocol consisting of 5 IU of natural eCG (n = 50), 5 IU of reCG (n = 50), or received a placebo (no-eCG, n = 50) by intraperitoneal route, followed by 5 IU of human chorionic gonadotropin 48 h later. Overall, no significant differences were observed in the total number of ova/zygotes (33.6 ± 2.4 vs 28.7 ± 2.6; P = NS) and viable ova/zygotes (31.5 ± 2.4 vs 25.8 ± 2.5; P = NS) collected per female among eCG and reCG treated females, respectively, which were greater (P < 0.05) than those obtained in no-eCG treated females (6.9 ± 0.7 and 5.9 ± 0.7, respectively). Zygotes derived from the three experimental groups (n = 2914) were subjected to in vitro culture until hatching 4.5 days post coitum (dpc). Regardless of the mouse strain, no differences were observed among eCG and reCG treated females for overall cleavage rate 1.5 dpc (58.5% vs 60.5%), development rate 3.5 dpc (47.2% vs 48.9%) and hatching rate 4.5 dpc (49.5% vs 54.5) (P = NS). Control females from no-eCG treated group showed lower cleavage and development rates (36.4% and 29.7%, respectively; P < 0.05). In conclusion, this study reports for the first time comparable superovulatory response and embryo development between recombinant and natural eCG treatment, which has important implications for reproductive technologies in several species.

Ctdp1 deficiency leads to early embryonic lethality in mice and defects in cell cycle progression in MEFs

ABSTRACTRNA polymerase II subunit A Carboxy-Terminal Domain Phosphatase 1 (CTDP1), a member of the haloacid dehalogenase superfamily phosphatases, has a defined role in transcriptional regulation, but emerging evidence suggests an expanded functional repertoire in the cell cycle and DNA damage response. In humans, a splice site mutation in CTDP1 gives rise to the rare Congenital Cataracts Facial Dysmorphism and Neuropathy syndrome, and recent evidence from our lab indicates CTDP1 is required for breast cancer growth and proliferation. To explore the physiological function of CTDP1 in a mammalian system, we generated a conditional Ctdp1 knockout mouse model by insertion of loxP sites upstream of exon 3 and downstream of exon 4. Biallelic deletion of Ctdp1 results in lethality before embryonic day 7.5, with morphological features indicating embryo cell death and resorption. However, Ctdp1+/− mice are haplosufficient for phenotypic traits including body weight, hematological parameters, exploratory and locomotive functions. To investigate the potential mechanisms of the embryonic death caused by biallelic Ctdp1 knockout, mouse embryonic fibroblasts (MEFs) were established from Ctdp1+/+ and Ctdp1flox/flox mice. Lentivirus delivered Cre-mediated biallelic deletion of Ctdp1 in MEFs results in cell death preceded by impaired proliferation characterized by an increase in G1- and G2-phase populations and a reduction in the S-phase population. These cell cycle alterations caused by deletion of Ctdp1 are associated with an increase in p27 protein expression and a decrease in phosphorylated RB, phosphorylated Histone H3, and Cyclin B expression. Together, these results reveal that Ctdp1 plays an essential role in early mouse embryo development and cell growth and survival in part by regulating the cell cycle.

Cell Specific Expression of Vascular Endothelial Growth Factor Receptor-2 (Flk-1/KDR) in Developing Mice Embryo and Supporting Maternal Uterine Tissue during Early Gestation (D4-D7).

BackgroundVascular endothelial growth factor (VEGF) and the corresponding receptors play key role in vascu- logenesis and angiogenesis processes. VEGF is one of the prime candidates in regulating embryo implantation by increasing vascular permeability. VEGF receptor-2, also called Flk-1/KDR, is one of the prime receptor which is actively involved in the execution of various functions of VEGF. However, precise role of this receptor during early gestation period is yet to be addressed. In the present study, expression of Flk-1/KDR during peri-implantation mice uterus as well as fetal-maternal tissues from day 4-day 7 (D4-D7) of gestation was investigated.Materials and MethodsIn this experimental study, localization of Flk-1/KDR was investigated by immunohisto- chemistry and immunofluorescence techniques, in paraffin embedded tissue sections. Flk-1/KDR protein and mRNA expressions were investigated by western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Effects of ovarian steroids on expression of Flk-1/KDR were also assessed by estrogen and progesterone antagonist treatment.ResultsUterine tissue on D4 showed strong expression of Flk-1/KDR in luminal and uterine glandular epithelium. On D5 and D6, differential expression of Flk-1/KDR was evidenced in certain cell types of the embryo, maternal tissues and fetal-maternal interface with varied intensity. Flk-1/KDR was specifically expressed in the ectoplacental cone (EPC) and various cells of the embryo on D7. Flk-1/KDR expression was not evidenced in the estradiol-17β (E2) and progesterone (P4) antagonist treated uterus. Western blotting result revealed presence of Flk-1/KDR protein in the all gestation days, except antagonist treated uterus. qRT-PCR analysis showed significant increase of Flk-1/KDR mRNA transcript on D6 and D7.ConclusionSpatial-temporal expression of Flk-1/KDR during peri-implntation period in mice uterus especially in the feto-maternal interface was observed. This spatio-temporal specificity as well as increased expression of Flk-1/KDR could be one of the determinants for establishment of fetal-maternal cross talk during the critical period of develop- ment.

Inducible Stem-Cell-Derived Embryos Capture Mouse Morphogenetic Events InVitro.

SummaryThe development of mouse embryos can be partially recapitulated by combining embryonic stem cells (ESCs), trophoblast stem cells (TS), and extra-embryonic endoderm (XEN) stem cells to generate embryo-like structures called ETX embryos. Although ETX embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ESCs transiently expressing transcription factor Gata4, which drives the extra-embryonic endoderm fate, and combined them with ESCs and TS cells to generate induced ETX embryos (iETX embryos). We show that iETX embryos establish a robust anterior signaling center that migrates unilaterally to break embryo symmetry. Furthermore, iETX embryos gastrulate generating embryonic and extra-embryonic mesoderm and definitive endoderm. Our findings reveal that replacement of XEN cells with ESCs transiently expressing Gata4 endows iETX embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system.

Single-cell transcriptome analysis reveals that maternal obesity affects DNA repair, histone methylation, and autophagy level in mouse embryos.

Obesity causes many reproductive dysfunctions such as reduced conception, infertility, and early pregnancy loss, and this is largely due to the negative effects of obesity on oocyte and embryo quality. In the present study, we employed single-cell RNA transcriptome sequencing to investigate the potential causes for the maternal obesity effects on mouse embryos. Our results showed that the 4-cell and morula/blastocyst rates were all significantly decreased during embryo development in obese mice. Genome-wide analysis indicated that obesity altered the expression of more than 1100 genes in 2-cell embryos, including the genes which were related to the p53 signaling pathway and apoptosis. Further analysis showed that the expression of 47 genes related to DNA damage was changed, and apositive γH2A signal and the altered expression of Rad51 and Tex15 were observed in the obese embryos. Obesity also affected histone methylation, shownbythe decrease of the H3K4-me2 level. Besides this, we observed the occurrence of autophagy and apoptosis in the embryos of obese mice. There were 42 genes that were related to autophagy/apoptosis that showed aberrant expression, and the positive LC3 signal and the decrease of Clec16a, Rraga, and Atg10 level were also observed. In summary, our study suggested that obesity affected early embryonic development by inducing DNA damage, aberrant histone methylation, and autophagy levels in mice.

Perspective: One-Cell and Cleavage-Stage Mouse Embryos Thrive in Hyperosmotic Oviductal Fluid Through Expression of a Glycine Neurotransmitter Transporter and a Glycine-Gated Chloride Channel: Clinical and Transgenerational Implications.

The osmolality of mouse oviductal fluid ranges from about 300 mOsmol/kg in the ampulla 0–3 h post coitus (h p.c.) to more than 350 mOsmol/kg in the isthmus 34–36 h p.c. Thus, it has been surprising to find that development of one-cell and cleavage-stage mouse embryos arrests in vitro in media exceeding 300 mOsmol/kg, and they develop best in unphysiological, hypotonic media. The glycine concentration in oviductal fluid can, however, rescue development in hypertonic media, so physiological conditions in vivo and in vitro likely work together to foster embryo well-being. Glycine acts on one-cell and cleavage-stage mouse embryos through the glycine-gated chloride channel, GLRA4, and uptake via the glycine neurotransmitter transporter, GLYT1. Since these processes lead to further signaling in neurons, the presence and function of such signaling in preimplantation embryos also should be investigated. The more we know about the interactions of physiological processes and conditions in vivo, the better we would be able to reproduce them in vitro. Such improvements in assisted reproductive technology (ART) could improve patient outcomes for IVF and potentially help prevent unwanted developmental abnormalities in early embryos, which might include undesirable epigenetic DNA and histone modifications. These epigenetic modifications may lead to transgenerational adult disorders such as metabolic syndrome and related conditions.

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice.

The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus-oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

Ability of Ureaplasma parvum to invade mouse sperm, fertilize eggs through infected sperm, and impair mouse sperm function and embryo development

To examine the effect of Ureaplasma parvum (U. parvum) infection on mouse sperm motility, structure, and fertilizing ability and on embryo development. Invitro model of the effects of U. parvum serovar 3 infection on mouse sperm. Basic research laboratory. None. Mice. Mouse sperm motility was examined using the swim-up method, and their motility parameters were analyzed using the sperm motility analysis system. Localization and invasion of U. parvum were observed with fluorescence, confocal, and scanning electron microscopy. After invitro fertilization with U. parvum-infected sperm, the quality of the fertilized egg and embryo development were assessed. U. parvum was attached and internalized into mouse sperms and localized mainly at the sperm head and midpiece. U. parvum-infected mouse sperms exhibited decreased motility in a dose- and duration-dependent manner. Electron micrographs revealed that U. parvum infection induced the aggregation and morphological destruction of mouse sperm. Infected mouse sperm transported U. parvum into the fertilized egg with reduced fertilization rates, and infected embryo development was impaired. U. parvum infection caused deterioration of the mouse sperm quality and its functions, which affected the fertilization rate and embryo development.

Importance of endothelial Hey1 expression for thoracic great vessel development and its distal enhancer for Notch-dependent endothelial transcription

Thoracic great vessels such as the aorta and subclavian arteries are formed through dynamic remodeling of embryonic pharyngeal arch arteries (PAAs). Previous work has shown that loss of a basic helix-loop-helix transcription factor Hey1 in mice causes abnormal fourth PAA development and lethal great vessel anomalies resembling congenital malformations in humans. However, how Hey1 mediates vascular formation remains unclear. In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre–mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells and in the pharyngeal epithelium and somites, respectively. The novel endothelial enhancer was conserved across species and was specific to large-caliber arteries. Its transcriptional activity was regulated by Notch signaling in vitro and in vivo, but not by ALK1 signaling and other transcription factors implicated in endothelial cell specificity. The distal endothelial enhancer was not essential for basal Hey1 expression in mouse embryos but may likely serve for Notch-dependent transcriptional control in endothelial cells together with the proximal regulatory region. These findings help in understanding the significance and regulation of endothelial Hey1 as a mediator of multiple signaling pathways in embryonic vascular formation.

Reduced growth capacity of preimplantation mouse embryos in chronic unpredictable stress model.

Psychological stress can affect female reproduction by deteriorating oocyte quality, but the molecular mechanism is unclear. In this study, we used the chronic unpredictable stress model to study the effect of psychological stress on mouse oocyte competence during preimplantation stage, and RNA sequencing in single oocytes to analyze differential gene expression at the transcription level. Stress changed the serum levels of glucocorticoids and reduced oocyte developmental potential, depending on the strength of the stress. Strong stress (two stressors per day) reduced the fertilization rate and induced significant apoptosis in blastocysts. Moderate stress (one stressor per day) reduced the cleavage rate and blastocyst formation rate. Weak stress (one stressor every 2 days) did not have any significant negative effect on the fertilization, cleavage, and blastocyst formation. Hatching rate was not affected by stress, but stress retarded the development of the expanded blastocysts and inhibited the embryo development at early stages. Transcriptome analysis revealed that stress disturbed the expression of cell cycle regulators and apoptotic genes. The hub genes identified through protein-protein interaction analysis include Msln, Ceacam12, Psg16, Psg17, and Psg23, which are all carcinoembryonic or related genes involved in cell adhesion, proliferation, and migration. Thus, stress was inhibitory on fertilization and early embryo development in mice.