Mouse Blastocysts Research Articles

O-093 Endometrium-blastocyst cross talk: how far are we from understanding?

Abstract The early events characterising implantation of the human embryo represent a critical step in which proper activation of orchestrated molecular pathways regulate the success of pregnancy. Understanding this complex network is considered by many the last frontier of human reproduction. Although outstanding research advancements have provided clinicians tools to select the best quality embryo, identification of the optimal timing in which the endometrium is ready to accommodate it remains a challenge. Over the last decade several studies have identified many key players involved in the preparation of the endometrium to implantation and several diffusible signals guiding the embryo in its journey toward the maternal endometrial tissue have been uncovered (Massimiani et al. 2020). However, pieces of the puzzle are still missing. For convenience, the process of implantation is subdivided into several sequential processes, the completion of one being propaedeutic to the following. Once the blastocyst has arrived in close proximity to the uterine epithelium, the first event occurring is its escape from the zona pellucida. The molecular basis of blastocyst hatching remains poorly understood, and species-specific differences have been reported. Using the mouse model, we recently demonstrated that thyroid hormone up-regulates the expression of specific lytic enzymes (Isp1 and Isp2) known mediators of mouse embryo zona lysis. Interestingly, we observed that TH-mediated upregulation of Isp1 and Isp2 is strictly dependent on the presence of endometrial stromal cell feeder layer, clearly indicating that blastocyst–endometrium interaction is indispensable for TH-mediated increased expression of these proteases. So far, no molecular signals regulating hatching in humans have been identified, and only mechanical forces have been proposed to induce zona opening; however very recently the possibility that proteases may be involved also in humans has been suggested (Almagor et al., 2020), although further studies are needed to support this hypothesis. Once free from the zona, the embryo contacts the endometrial epithelium and starts its journey to further proceed in the consolidation of pregnancy. Hormones, pro- and anti-inflammatory mediators, growth factors and their receptors, adhesion molecules, and proteases and protease inhibitors have been reported in the regulation of the process of embryo apposition, adhesion, and invasion (Massimiani et al., 2020). Among the many molecular signals, the Notch pathway has been recognized key in embryo-endometrium cross talk (Cuman et al., 2014). Transient activation of the Notch1 receptor has been demonstrated in the process of decidualization (Afshar et al., 2012), a limiting step to implantation, and blastocyst-conditioned medium has been shown to induce the expression of Notch family members in decidual cells (Hess et al., 2007). Expression of notch receptors and ligands has been reported in the blastocyst trophectoderm as well (Cuman et al., 2014). We previously demonstrated that the secreted factor epidermal growth factor-like domain 7 (EGFL7) is a novel modulator of the Notch pathway, it is expressed by trophoblast cells of the mouse blastocyst (Fitch et al., 2004; Lacko et al., 2014) and it regulates human trophoblast migration and invasion ability by activating the Notch pathway (Massimiani et al., 2015). Data will be presented demonstrating that EGFL7 is expressed in the human endometrium and its expression is mainly localized in the glandular epithelium throughout the menstrual cycle. EGFL7 is significantly upregulated in whole endometrial tissue during the secretory phase, particularly in the stromal compartment; this observation is further supported by in vitro studies showing its upregulation in decidualized endometrial stromal cells, suggesting a role for EGFL7 in implantation. Further support is provided by the evidence that its levels are strongly downregulated in the endometrium of women with impaired fertility. We propose EGFL7 as a novel player involved in the embryo-endometrium cross talk.

Maternal RNA binding protein with multiple splicing 2 (RBPMS2) is involved in mouse blastocyst formation through the bone morphogenetic protein pathway

Research questionIs early embryo development in mice influenced by RNA binding protein with multiple splicing 2 (RBPMS2), a maternal factor that accumulates and is stored in the cytoplasm of mature oocytes? DesignThe expression patterns of RBPMS2 in mouse were analysed using quantitative real-time PCR (qRT PCR) and immunofluorescence staining. The effect of knockdown of RBPMS2 on embryo development was evaluated through a microinjection of specific morpholino or small interfering RNA. RNA sequencing was performed for mechanistic analysis. The interaction between RBPMS2 and the bone morphogenetic protein (BMP) pathway was studied using BMP inhibitor and activator. The effect on the localization of E-cadherin was determined by immunofluorescence staining. ResultsMaternal protein RBPMS2 is highly expressed in mouse oocytes, and knockdown of RBPMS2 inhibits embryo development from the morula to the blastocyst stage. Mechanistically, RNA sequencing showed that the differentially expressed genes were enriched in the transforming growth factor-β (TGF-β) signalling pathway. BMPs are members of the TGF-β superfamily of growth factors. It was found that the addition of BMP inhibitor to the culture medium led to a morula-stage arrest, similar to that seen in RBPMS2 knockdown embryos. This morula-stage arrest defect caused by RBPMS2 knockdown was partially rescued by BMP activator. Furthermore, the localization of E-cadherin to the membrane was impaired in response to a knockdown of RBPMS2 or inhibition of the BMP pathway. ConclusionThis study suggests that RBPMS2 activates the BMP pathway and thus influences the localization of E-cadherin, which is important for early mouse embryo development during blastocyst formation.

Characterization of epidermal growth factor-like domain 7 (EGFL7) expression in normal endometrium and in the endometrium of women with poor reproductive outcomes.

Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes? EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF). The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL. This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF. Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes. Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not. This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions. Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention. This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose. Not applicable.

Role of activated p21-activated kinase 2 in methylmercury-induced embryotoxic effects on mouse blastocysts.

Methylmercury (MeHg), a biotransformation product derived from mercury or inorganic mercury compounds in waterways, is a potent toxin that exerts hazardous effects on human health via environmental contamination. Previous studies have reported MeHg-induced impairment of nerve development in embryogenesis and placental development. However, the potential deleterious effects and regulatory mechanisms of action of MeHg on pre- and post-implantation embryo development are yet to be established. Experiments from the current study clearly demonstrate that MeHg exerts toxic effects on early embryonic development processes, including the zygote to blastocyst stage. Induction of apoptosis and decrease in embryo cell number were clearly detected in MeHg-treated blastocysts. Additionally, intracellular reactive oxygen species (ROS) generation and activation of caspase-3 and p21-activated protein kinase 2 (PAK2) were observed in MeHg-treated blastocysts. Importantly, prevention of ROS generation by pre-treatment with Trolox, a potent antioxidant, significantly attenuated MeHg-triggered caspase-3 and PAK2 activation as well as apoptosis. Notably, the downregulation of PAK2 via transfection of specifically targeted siRNA (siPAK2) led to marked attenuation of PAK2 activity and apoptosis and the deleterious effects of MeHg on embryonic development in blastocysts. Our findings strongly suggest that ROS serve as an important upstream regulator to trigger the activation of caspase-3, which further cleaves and activates PAK2 in MeHg-treated blastocysts. Activated PAK2 promotes apoptotic processes that, in turn, cause sequent impairment of embryonic and fetal development.

Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the developmentof aneuploid mouse embryos through a Trp53-dependent mechanism.

This study was designed to determine if DMO limits in vitro development of aneuploid-enriched mouse embryos by activating a Trp53-dependent mechanism. Mouse cleavage-stage embryos were treated with reversine to induce aneuploidy or vehicle to generate controls, and then cultured in media supplemented with DMO to reduce the pH of the culture media. Embryo morphology was assessed by phase microscopy. Cell number, mitotic figures, and apoptotic bodies were revealed by staining fixed embryos with DAPI. mRNA levels of Trp53, Oct-4, and Cdx2 were monitored by quantitative polymerase chain reactions (qPCRs). The effect of Trp53 on the expression of Oct-4 and Cdx2 was assessed by depleting Trp53 using Trp53 siRNA. Aneuploid-enriched late-stage blastocysts were morphologically indistinguishable from control blastocysts but had fewer cells and reduced mRNA levels of Oct-4 and Cdx2. Adding 1mM DMO to the culture media during the 8-cell to blastocyst transition reduced the formation of aneuploid-enriched late-stage blastocysts but not control blastocysts and further suppressed the levels of Oct-4 and Cdx2 mRNA. Trp53 RNA levels in aneuploid-enriched embryos that were exposed to DMO were > twofold higher than controls, and Trp53 siRNA levels reduced the levels of Trp53 and increased levels of Oct-4 and Cdx2 mRNA by > twofold. These studies suggest that the development of morphologically normal aneuploid-enriched mouse blastocysts can be inhibited by adding low amounts of DMO to the culture media, which results in elevated levels of Trp53 mRNA that suppresses Oct-4 and Cdx2 expression.

Estrogen signaling encourages blastocyst development and implantation potential.

Estrogen is well-known for preparing uterine receptivity. However, its roles in regulating embryo development and implantation are unclear. Our objective was to characterize estrogen receptor 1 (ESR1) in human and mouse embryos and determine the effect of estradiol (E2) supplementation on pre- and peri-implantation blastocyst development. Mouse embryos, 8-cell through hatched blastocyst stages, and human embryonic days 5-7 blastocysts were stained for ESR1 and imaged using confocal microscopy. We then treated 8-cell mouse embryos with 8nM E2 during in vitro culture (IVC) and examined embryo morphokinetics, blastocyst development, and cell allocation into the inner cell mass (ICM) and trophectoderm (TE). Finally, we disrupted ESR1, using ICI 182,780, and evaluated peri-implantation development. ESR1 exhibits nuclear localization in early blastocysts followed by aggregation, predominantly in the TE of hatching and hatched blastocysts, in human and mouse embryos. During IVC, most E2 was absorbed by the mineral oil, and no effect on embryo development was found. When IVC was performed without an oil overlay, embryos treated with E2 exhibited increased blastocyst development and ICM:TE ratio. Additionally, embryos treated with ICI 182,780 had significantly decreased trophoblast outgrowth during extended embryo culture. Similar ESR1 localization in mouse and human blastocysts suggests a conserved role in blastocyst development. These mechanisms may be underappreciated due to the use of mineral oil during conventional IVC. This work provides important context for how estrogenic toxicants may impact reproductive health and offers an avenue to further optimize human-assisted reproductive technology (ART) to treat infertility.

Evaluation of the post-implantation development of mouse embryos derived from round spermatid injection

Round spermatid injection (ROSI), one of the assistant reproductive technologies, was used to treat partial infertility patients suffering from non-obstructive azoospermia. However, the development efficiency and birth rate of ROSI embryos are extremely low, and it is urgent to investigate the underlying mechanisms of low efficiency to improve the clinical application of ROSI technology. Here, we analyzed and compared the genome stability of the mouse blastocyst and the post-implantation development between ROSI and ICSI embryos. We first sequenced the genome of blastocysts from mouse ROSI embryos that can correctly form male and female pronuclei (2 PN) and found that the genomes of 7 blastocysts were normal. Furthermore, the implantation rate of ROSI 2 PN embryos on embryonic day 7.5 is similar to that of ICSI embryos, and at this time, 37.50% (9/24) of deciduas have no normal gestational sac. The proportion of embryos that survived to embryonic day 11.5 in the ROSI 2 PN group, ROSI non-2 PN group, parthenogenesis group, and ICSI 2 PN group was 51.61%, 7.14%, 0.00%, and 55.00%, respectively. And two smaller fetuses were found in the ROSI 2 PN group, which is not found in the other three groups. In addition, the physiological indexes, including fetus and placenta weight, sex ratio, growth rate, and the natural breeding ability for the offspring obtained from mouse ROSI, were evaluated; ROSI mice exhibited no obvious defect or abnormality and implied that the progeny were safe. Our results provided new evidence to promote the clinical application of ROSI technology.

Polarity inversion reorganizes the stem cell compartment of the trophoblast lineage

The extra-embryonic tissues that form the placenta originate from a small population of trophectoderm cells with stem cell properties, positioned at the embryonic pole of the mouse blastocyst. During the implantation stages, the polar trophectoderm rapidly proliferates and transforms into extra-embryonic ectoderm. The current model of trophoblast morphogenesis suggests that tissue folding reshapes the trophoblast during the blastocyst to egg cylinder transition. Instead of through folding, here we found that the tissue scale architecture of the stem cell compartment of the trophoblast lineage is reorganized via inversion of the epithelial polarity axis. Our findings show the developmental significance of polarity inversion and provide a framework for the morphogenetic transitions in the peri-implantation trophoblast.

Direct Effects of Mifepristone on Mice Embryogenesis: An In Vitro Evaluation by Single-Embryo RNA Sequencing Analysis

The clinical use of mifepristone for medical abortions has been established in 1987 in France and since 2000 in the United States. Mifepristone has a limited medical period that lasts <9 weeks of gestation, and the incidence of mifepristone treatment failure increases with gestation time. Mifepristone functions as an antagonist for progesterone and glucocorticoid receptors. Studies have confirmed that mifepristone treatments can directly contribute to endometrium disability by interfering with the endometrial receptivity of the embryo, thus causing decidual endometrial degeneration. However, whether mifepristone efficacy directly affects embryo survival and growth is still an open question. Some women choose to continue their pregnancy after mifepristone treatment fails, and some women express regret and seek medically unapproved mifepristone antagonization with high doses of progesterone. These unapproved treatments raise the potential risk of embryonic fatality and developmental anomalies. Accordingly, in the present study, we collected mouse blastocysts ex vivo and treated implanted blastocysts with mifepristone for 24 h. The embryos were further cultured to day 8 in vitro to finish their growth in the early somite stage, and the embryos were then collected for RNA sequencing (control n = 3, mifepristone n = 3). When we performed a gene set enrichment analysis, our data indicated that mifepristone treatment considerably altered the cellular pathways of embryos in terms of viability, proliferation, and development. The data indicated that mifepristone was involved in hallmark gene sets of protein secretion, mTORC1, fatty acid metabolism, IL-2-STAT5 signaling, adipogenesis, peroxisome, glycolysis, E2F targets, and heme metabolism. The data further revealed that mifepristone interfered with normal embryonic development. In sum, our data suggest that continuing a pregnancy after mifepristone treatment fails is inappropriate and infeasible. The results of our study reveal a high risk of fetus fatality and developmental problems when pregnancies are continued after mifepristone treatment fails.

Supplementation of culture medium with quercetin improves mouse blastocyst quality and increases the expression of HIF-1α protein.

Regarding the low number of embryos that reach the blastocyst stage when cultured in vitro, this study aimed to evaluate the effects of quercetin on pre-implantation mouse (Mus musculus) embryos obtained using in vitro fertilization, especially during the passage from morula to blastocyst. Furthermore, we studied whether quercetin also affected the expression of hypoxia-inducible factor 1α (HIF-1α). The culture medium for the embryos was supplemented with quercetin, for long or short periods of time, and then the development potential, total cell number, apoptosis rates and expression of HIF-1α were studied to determine the effect of quercetin. Embryos failed to develop when cultured for long periods of time with quercetin, implying the possible toxic effects of this, alternatively antioxidant, compound. However, a short culture from morula to blastocyst significantly improved the development potential of in vitro produced embryos, increasing the final total cell number and reducing the apoptosis rate, observing similar results to those embryos cultured in low-oxygen concentrations or developed in utero. Furthermore, in embryos treated with quercetin for 2 or 4 h we found an increase in HIF-1α compared with untreated embryos. This work could imply a way to use quercetin in fertility clinics to improve the production of healthy blastocysts and, consequently, increase the success rates in assisted reproduction techniques.

DEVELOPMENT OF MURINE STEM CELLS WITH CONDITIONAL KNOCKOUT OF HUMANIZED SNCA GENE

α-synuclein is one of the key molecular links in the pathogenesis of Parkinson’s disease. The accumulated data indicate that pathogenic mutations in the Snca gene are associated with the development of neurodegenerative brain damage, indicating the relevance of studying the synuclein neurobiological role.The aim of the study was to create a genetically modified clone of mouse stem cells with a conditional knockout of humanized α-synuclein, which can be used for the reinjection into mouse blastocysts, as well as for basic and applied in vitro research in the field of pathophysiology and neuropharmacology.Materials and methods. To create mouse stem cells with a conditional knockout of the humanized Snca gene, a previously obtained clone with the first Snca exon flanked by LoxP sites, was used. The CRISPR/Cas9-mediated homologous recombination system with donor DNA oligonucleotides of the human sites of the corresponding gene sites was used to humanize the fourth and fifth exons. Cas9 nuclease, single guide RNA, and donor DNA were transfected into mouse cells.Results. An approach to obtaining clones of mouse genetically modified stem cells expressing pathological humanized α-synuclein, has been proposed and implemented. The resulting clones were plated on Petri dishes for propagation and a further genetic analysis. Clone 126-2F4 was found out carrying the necessary genetic modifications. The results obtained are fundamentally important not only for understanding the development of the pathological process in α-synucleinopathies, but which is more important, for the development of new therapeutic approaches that will stop the extension of the human α-synuclein aggregation pathology throughout the nervous system, and the validation of these approaches in preclinical trials.Conclusion. As a result of the study, a strategy for CRISPR/Cas9-assisted homologous recombination in the genome of mouse embryonic stem cells has been developed to create a fully humanized Snca gene encoding α-synuclein, and the clone genome of mouse embryonic stem cells has been edited using a CRISPR technology. The RNA and DNA oligonucleotides necessary for the creation of RNP complexes that carry out a directed homologous recombination in the Snca locus of the mouse genome have been synthesized. The developed cell clone can serve to create a line of genetically modified mice that serve as a test system for pathophysiological and neuropharmacological studies associated with synucleinopathies. Herewith, before the induction of the Cre-dependent recombination, this line is a representative model for studying a biological role of mutant Snca. At the same time, after a Cre-dependent knockout activation, it is possible to imitate the pharmacological inhibition of α-synuclein, which is of particular interest for applied research in neuropharmacology.

Optimized Method for Using Embryonic Microenvironment to Reprogram Cancer Stem Cells

Purpose: The embryonic microenvironment contains many properties that have not yet been fully explored. Our aim in this study is to report an optimized and efficient method that enables investigating the effects of the secretome of pluripotent embryonic stem cells on cancer stem cells.&#x0D; Methods: The study is performed with a chimeric model consisted of mouse blastocysts, human prostate cancer stem cells and non-cancer stem cells. Ovulation induced mice were used for blastocyst collection. DU145 prostate cancer cell line was separated into cancer stem cells and non-cancer stem cells according to cancer stem cells biomarker expressions by fluorescent activated cell sorting method. Human prostate cancer stem cells and non-cancer stem cells were microinjected into 4-day blastocyst culture in vitro by intracytoplasmic sperm injection method.&#x0D; Results: Chimeric models provide us great convenience in basic oncological studies. In this study, using a chimeric model, we were able to study the secretome of mouse embryonic stem cells and their effect on cancer stem cells. The method is efficient and yield promising result; and could be used to study the effects on other cells as well.&#x0D; Conclusion: The embryonic stem cell microenvironment is suggested to have a great regenerative capacity which is, nowadays, the center of attraction for cancer research studies. Ethical issues restrict the human embryo studies, however, mimicking the in vivo human microenvironment with 3D cell cultures or bioprinting are now possible. Finally, optimization of new methods including 3D cell cultures with human cell lines will be a great opportunity for better understanding the reprogramming notion.

The regulation and function of acetylated high-mobility group box 1 during implantation and decidualization.

High-mobility group box 1 (HMGB1) is a non-histone nuclear protein and can be extracellularly secreted to induce sterile inflammation. Although uterine deletion of HMGB1 causes implantation and decidualization defects, how secreted HMGB1 is involved in mouse early pregnancy is still unknown. Mouse models, mouse primary endometrial cells and human endometrial cell lines were used in this study. Both immunofluorescence and Western blot were performed to show the localization and relative level of HMGB1 and acetylated HMGB1, respectively. Relative mRNA levels were analyzed by real time RT-PCR. The secreted HMGB1 was detected in uterine lumen fluid in mouse periimplantation uterus. There is an obvious difference for secreted HMGB1 levels in uterine fluid between day 4 of pregnancy and day 4 of pseudopregnancy, suggesting the involvement of blastocysts during HMGB1 secretion. Trypsin is clearly detected in mouse blastocyst cavity and in the supernatant of cultured blastocysts. Trypsin significantly stimulates HB-EGF production through activating PAR2 and ADAM17. Uterine injection of PAR2 inhibitor into day 4 pregnant mice significantly reduces the number of implantation sites. HB-EGF released from luminal epithelium can induce mouse in vitro decidualization. The conditioned medium collected from trypsin-treated luminal epithelium is able to induce in vitro decidualization, which is suppressed by EGFR inhibitor. Intrauterine injection of glycyrrhizin (HMGB1 inhibitor) can significantly inhibit mouse embryo implantation. We also showed that exogenous HMGB1 released from human epithelial cells are able to induce human in vitro decidualization. Trypsin can induce decidualization of stromal cells via PAR2-HMGB1-ADAM17-HB-EGF from luminal epithelium.

Genome-wide assessment of DNA methylation alterations induced by superovulation, sexual immaturity and in vitro follicle growth in mouse blastocysts

BackgroundIn their attempt to fulfill the wish of having children, women who suffer from fertility issues often undergo assisted reproductive technologies such as ovarian stimulation, which has been associated with adverse health outcomes and imprinting disorders in children. However, given the crucial role of exogenous hormone stimulation in improving human infertility treatments, a more comprehensive analysis of the potential impacts on DNA methylation in embryos following ovarian stimulation is needed. Here, we provide genome-wide DNA methylation profiles of blastocysts generated after superovulation of prepubertal or adult mice, compared with blastocysts derived from non-stimulated adult mice. Additionally, we assessed the impact of the in vitro growth and maturation of oocytes on methylation in blastocysts.ResultsNeither hormone stimulation nor sexual maturity had an impact on the low global methylation levels characteristic of the blastocyst stage or was associated with extensive DNA methylation alterations. However, we found hormone- and age-associated changes at specific positions but dispersed throughout the genome. In particular, we detected anomalous methylation at a limited number of CpG islands. Additionally, superovulation in adult mice was associated with alterations at the Sgce and Zfp777 imprinted genes. On the other hand, in vitro culture of follicles from the early pre-antral stage was associated with globally reduced methylation and increased variability at imprinted loci in blastocysts.ConclusionsOur results indicate a minimal effect of ovarian stimulation of adult and prepubertal mice on the DNA methylation landscape attained at the blastocyst stage, but potentially greater impacts of in vitro growth and maturation of oocytes. These findings have potential significance for the improvement of assisted reproductive techniques, in particular for those related to treatments in prepubertal females, which could be crucial for improving human fertility preservation strategies.

Membrane lipid changes in mouse blastocysts induced by ovarian stimulation, IVF and oocyte vitrification

Research questionIs the membrane lipid profile of mice blastocysts affected by ovarian stimulation, IVF and oocyte vitrification? Could supplementation of vitrification media with L-carnitine and fatty acids prevent membrane phospholipid changes in blastocysts from vitrified oocytes? DesignExperimental study comparing the lipid profile of murine blastocysts produced from natural mating, superovulated cycles or after IVF submitted or not to vitrification. For in-vitro experiments, 562 oocytes from superovulated females were randomly divided into four groups: fresh oocytes fertilized in vitro and vitrified groups: Irvine Scientific (IRV); Tvitri-4 (T4) or T4 supplemented with L-carnitine and fatty acids (T4-LC/FA). Fresh or vitrified–warmed oocytes were inseminated and cultured for 96 h or 120 h. The lipid profile of nine of the best quality blastocysts from each experimental group was assessed by multiple reaction monitoring profiling method. Significantly different lipids or transitions between groups were found using univariate statistics (P < 0.05; fold change = 1.5) and multivariate statistical methods. ResultsA total of 125 lipids in blastocysts were profiled. Statistical analysis revealed several classes of phospholipids affected in the blastocysts by ovarian stimulation, IVF, oocyte vitrification, or all. L-carnitine and fatty acid supplements prevented, to a certain extent, changes in phospholipid and sphingolipid contents in the blastocysts. ConclusionOvarian stimulation alone, or in association with IVF, promoted changes in phospholipid profile and abundance of blastocysts. A short exposure time to the lipid-based solutions during oocyte vitrification was sufficient to induce changes in the lipid profile that were sustained until the blastocyst stage.

Decoding anterior-posterior axis emergence among mouse, monkey, and human embryos.

Anterior-posterior axis formation regulated by the distal visceral endoderm (DVE) and anterior visceral endoderm (AVE) is essential for peri-implantation embryogenesis. However, the principles of the origin and specialization of DVE and AVE remain elusive. Here, with single-cell transcriptome analysis and pseudotime prediction, we show that DVE and AVE independently originate from the specialized primary endoderm in mouse blastocysts. Along distinct developmental paths, these two lineages, respectively, undergo four representative states with stage-specific transcriptional patterns around implantation. Further comparative analysis shows that AVE, but not DVE, is detected in human and non-human primate embryos, defining differences in polarity formation across species. Moreover, stem cell-assembled human blastoids lack DVE or AVE precursors, implying that additional induction of stem cells with DVE/AVE potential could promote the current embryo-like models and their post-implantation growth. Our work provides insight into understanding of embryonic polarity formation and early mammalian development.

Upregulation of iNOS and phosphorylated eNOS in the implantation-induced blastocysts of mice.

This study aimed to examine expressions of iNOS and phosphorylated eNOS (p-eNOS) in implantation-induced blastocysts. We also examined the upstream of p-eNOS. To address the protein expressions in implantation-induced blastocysts, we performed immunohistochemical analysis using a delayed implantation mouse model. Immunostaining for iNOS, p-eNOS, and p-Akt was done. To address the relationship between p-eNOS and p-Akt, activated blastocysts were treated with an Akt inhibitor, MK-2206. iNOS expression was at low levels in dormant blastocysts, whereas the expression was significantly increased in the activated blastocysts. Double staining of p-eNOS and p-Akt in individual blastocysts showed colocalization of p-eNOS and p-Akt of the trophectoderm. p-eNOS and p-Akt expressions were at low levels in dormant blastocysts, whereas both of them were significantly increased in the activated blastocysts. Both dormant and activated blastocysts showed significant positive correlations between p-eNOS and p-Akt. MK-2206 treatment for activated blastocysts showed that blastocysts with lower p-Akt had significantly lower p-eNOS levels. iNOS and p-eNOS, Ca2+ independent NOS, are upregulated by E2 in the blastocysts during implantation activation. Furthermore, p-eNOS is upregulated in implantation-induced blastocysts downstream of p-Akt.

Association between uterine toxicity induced by chlorpyrifos and downregulation of heparin-binding epidermal growth factor and L-selectin genes.

Various factors are effective in reducing the fertility rate. This experiment aimed to investigate chlorpyrifos (CPF), an organophosphate, that could alter the structure of the uterus and the molecules involved in parental and fetal. CPF was injected intraperitoneally in thirty mice for five days in a week (six weeks). The animals were euthanized on the 5th day of gestation, then their blood and uterus were collected for biochemical and histopathological assays. Exposure to CPF resulted in a significant reduction in maternal weight gain and the number of litters. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were significantly increased in blood serum of the CPF group compared with the control. The number of uterus glands, endometrium thickness, and the uterine cavity were changed following CPF injection. Additional investigation indicated that the expressions of L-selectin, L-selectin ligand, and heparin-binding epidermal growth factor (HB-EGF) as initial adhesion of mice blastocysts and maternal endometrium biomarkers were downregulated in the CPF group. Nevertheless, any mortality and abnormal clinical symptoms were not observed in the treated mice. This study revealed a potential molecular mechanism of continuous CPF-induced toxicity in fetal-maternal attachment without clinical symptoms.

Early embryonic lethality of mice lacking POLD2.

As a highly conserved DNA polymerase (Pol), Pol δ plays crucial roles in chromosomal DNA synthesis and various DNA repair pathways. However, the function of POLD2, the second small subunit of DNA Pol δ (p50 subunit), has not been characterized in vivo during mammalian development. Here, we report for the first time, the essential role of subunit POLD2 during early murine embryogenesis. Although Pold2 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at gastrulation stages. Outgrowth assays reveal that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Notably, these phenotypes can be recapitulated by small interfering RNA (siRNA)-mediated knockdown, which also exhibit slowed cellular proliferation together with skewed primitive endoderm and epiblast allocation during the second cell lineage specification. In summary, our study demonstrates that POLD2 is essential for the earliest steps of mammalian development, and the retarded proliferation and embryogenesis may also alter the following cell lineage specifications in the mouse blastocyst embryos.

Melatonin protects against visible light-induced oxidative stress and promotes the implantation potential of mouse blastocyst in vitro

Improvement of embryo culture media using antioxidant agents could help to improve embryo quality against environmental factors such as visible light and could overcome implantation failures. The usefulness of the melatonin against the effect of light on the expression of the primary implantation receptors, ErbB1 and ErbB4 on pre-implantation mouse embryo was investigated. Two-cell mouse embryos were exposed to the 1600 LUX light for 30 min then randomly divided into 3 groups including: Melatonin-Treated; Luzindole Treated and Simple media as a Control group. After 72–96 The expanded blastocysts were examined for morphological quality of the embryos by Hoechst and propidium iodide staining and for the expression of ErbB1 and ErbB4 by Real-time PCR and immunocytochemistry. The expression of the Sirt3 gene was also assayed. Furthermore, intracellular reactive oxygen species (ROS) levels and the total antioxidant capacity (TAC) were examined by DCFH-DA fluorescence intensity and radical cation respectively.The number of cells in the inner cell mass (ICM) and outer cell mass (OCM) were elevated significantly in the Melatonin-treated group suggesting increased viability and proliferation. Furthermore, we found that melatonin significantly increased the expression levels of ErbB1, ErbB4, and Sirt3 genes, and the protein expression of ErbB1, ErbB4 correlated with intracellular ROS levels and TAC significantly increased after melatonin treatment. Together, these results demonstrate that melatonin could be helpful to improve preimplantation embryos through its effects in decreasing ROS levels and increasing expression of implantation-related genes.