High-resolution ribosome profiling reveals translational selectivity for transcripts in bovine preimplantation embryo development.

High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome and the polysome- and non-polysome-bound RNA profiles of bovine oocytes (germinal vesicle and metaphase II stages) and early embryos at the two-cell, eight-cell, morula and blastocyst stages, and revealed four modes of translational selectivity: (1) selective translation of non-abundant mRNAs; (2) active, but modest translation of a selection of highly expressed mRNAs; (3) translationally suppressed abundant to moderately abundant mRNAs; and (4) mRNAs associated specifically with monosomes. A strong translational selection of low-abundance transcripts involved in metabolic pathways and lysosomes was found throughout bovine embryonic development. Notably, genes involved in mitochondrial function were prioritized for translation. We found that translation largely reflected transcription in oocytes and two-cell embryos, but observed a marked shift in the translational control in eight-cell embryos that was associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become more synchronized in morulae and blastocysts. Taken together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development.

Epigenetic reprogramming in the transition from pluripotency to totipotency.

Mammalian development commences with the zygote, which can differentiate into both embryonic and extraembryonic tissues, a capability known as totipotency. Only the zygote and embryos around zygotic genome activation (ZGA) (two-cell embryo stage in mice and eight-cell embryo in humans) are totipotent cells. Epigenetic modifications undergo extremely extensive changes during the acquisition of totipotency and subsequent development of differentiation. However, the underlying molecular mechanisms remain elusive. Recently, the discovery of mouse two-cell embryo-like cells, human eight-cell embryo-like cells, extended pluripotent stem cells and totipotent-like stem cells with extra-embryonic developmental potential has greatly expanded our understanding of totipotency. Experiments with these in vitro models have led to insights into epigenetic changes in the reprogramming of pluri-to-totipotency, which have informed the exploration of preimplantation development. In this review, we highlight the recent findings in understanding the mechanisms of epigenetic remodeling during totipotency capture, including RNA splicing, DNA methylation, chromatin configuration, histone modifications, and nuclear organization.

Maternal RNF114-mediated target substrate degradation regulates zygotic genome activation in mouse embryos.

Zygotic genomic activation (ZGA) is a landmark event in the maternal-to-zygotic transition (MZT), and the regulation of ZGA by maternal factors remains to be elucidated. In this study, the depletion of maternal ring finger protein 114 (RNF114), a ubiquitin E3 ligase, led to developmental arrest of two-cell mouse embryos. Using immunofluorescence and transcriptome analysis, RNF114 was proven to play a crucial role in major ZGA. To study the underlying mechanism, we performed protein profiling in mature oocytes and found a potential substrate for RNF114, chromobox 5 (CBX5), ubiquitylation and degradation of which was regulated by RNF114. The overexpression of CBX5 prevented embryonic development and impeded major ZGA. Furthermore, TAB1 was abnormally accumulated in mutant two-cell embryos, which was consistent with the result of in vitro knockdown of Rnf114. Knockdown of Cbx5 or Tab1 in maternal RNF114-depleted embryos partially rescued developmental arrest and the defect of major ZGA. In summary, our study reveals that maternal RNF114 plays a precise role in degrading some important substrates during the MZT, the misregulation of which may impede the appropriate activation of major ZGA in mouse embryos.

Outcomes of BRCA pre-implantation genetic testing according to the parental mutation origin: a cohort study.

BACKGROUND: The process of gamete formation and early embryonic development involves rapid DNA replication, chromosome segregation and cell division. These processes may be affected by mutations in the BRCA1/2 genes. The aim of this study was to evaluate BRCA mutation inheritance and its effect on early embryonic development according to the parental origin of the mutation. The study question was approached by analyzing in vitro fertilization cycles (IVF) that included pre-implantation testing (PGT-M) for a BRCA gene mutation. METHODS: This retrospective cohort study compared cycles of pre-implantation genetic testing for mutations (PGT-M) between male and female patients diagnosed with BRCA 1/2 mutations (cases), to a control group of two other mutations with dominant inheritance (myotonic dystrophy (MD) and polycystic kidney disease (PKD)). Results were compared according to mutation type and through a generalized linear model analysis. RESULTS: The cohort included 88 PGT-M cycles (47 BRCA and 41 non-BRCA) among 50 patients. Maternal and paternal ages at oocyte retrieval were comparable between groups. When tested per cycle, FSH dose, maximum estradiol level, oocytes retrieved, number of zygotes, and number of embryos available for biopsy and affected embryos, were not significantly different among mutation types. All together 444 embryos were biopsied: the rate of affected embryos was comparable between groups. Among BRCA patients, the proportion of affected embryos was similar between maternal and paternal mutation origin (p = 0.24). In a generalized linear model analysis, the relative oocyte yield in maternal BRCA patients was significantly lower (0.7, as related to the non BRCA group)(p < 0.001). Zygote formation and blastulation were not affected by the BRCA gene among paternal cases (P = 0.176 and P = 0.293 respectively), nor by paternal versus maternal BRCA carriage (P = 0.904 and P = 0.149, respectively). CONCLUSIONS: BRCA PGT-M cycles performed similarly compared to non-BRCA cycles. Inheritance rate and cycle parameters were not affected by the parental origin of the mutation.

Maternal factor Trim75 contributes to zygotic genome activation program in mouse early embryos.

BACKGROUND: Zygotic genome activation (ZGA) is an important event in the early embryo development, and human embryo developmental arrest has been highly correlated with ZGA failure in clinical studies. Although a few studies have linked maternal factors to mammalian ZGA, more studies are needed to fully elucidate the maternal factors that are involved in ZGA. METHODS AND RESULTS: In this study, we utilized published single-cell RNA sequencing data from a Dux-mediated mouse embryonic stem cell to induce a 2-cell-like transition state and selected potential drivers for the transition according to an RNA velocity analysis. CONCLUSIONS: An overlap of potential candidate markers of 2-cell-like-cells identified in this research with markers generated by various data sets suggests that Trim75 is a potential driver of minor ZGA and may recruit EP300 and establish H3K27ac in the gene body of minor ZGA genes, thereby contributing to mammalian preimplantation embryo development.

Lineage segregation in human pre-implantation embryos is specified by YAP1 and TEAD1.

STUDY QUESTION: Which processes and transcription factors specify the first and second lineage segregation events during human preimplantation development? SUMMARY ANSWER: Differentiation into trophectoderm (TE) cells can be initiated independently of polarity; moreover, TEAD1 and YAP1 co-localize in (precursor) TE and primitive endoderm (PrE) cells, suggesting a role in both the first and the second lineage segregation events. WHAT IS KNOWN ALREADY: We know that polarity, YAP1/GATA3 signalling and phospholipase C signalling play a key role in TE initiation in compacted human embryos, however, little is known about the TEAD family of transcription factors that become activated by YAP1 and, especially, whether they play a role during epiblast (EPI) and PrE formation. In mouse embryos, polarized outer cells show nuclear TEAD4/YAP1 activity that upregulates Cdx2 and Gata3 expression while inner cells exclude YAP1 which upregulates Sox2 expression. The second lineage segregation event in mouse embryos is orchestrated by FGF4/FGFR2 signalling which could not be confirmed in human embryos; TEAD1/YAP1 signalling also plays a role during the establishment of mouse EPI cells. STUDY DESIGN, SIZE, DURATION: Based on morphology, we set up a development timeline of 188 human preimplantation embryos between Day 4 and 6 post-fertilization (dpf). The compaction process was divided into three subgroups: embryos at the start (C0), during (C1), and at the end (C2) of, compaction. Inner cells were identified as cells that were entirely separated from the perivitelline space and enclosed by cellular contacts on all sides. The blastulation process was divided into six subgroups, starting with early blastocysts with sickle-cell shaped outer cells (B0) and further on, blastocysts with a cavity (B1). Full blastocysts (B2) showed a visible ICM and outer cells referred to as TE. Further expanded blastocysts (B3) had accumulated fluid and started to expand due to TE cell proliferation and zona pellucida (ZP) thinning. The blastocysts then significantly expanded further (B4) and started to hatch out of the ZP (B5) until they were fully hatched (B6). PARTICIPANTS/MATERIALS, SETTING, METHODS: After informed consent and the expiration of the 5-year cryopreservation duration, 188 vitrified high quality eight-cell stage human embryos (3 dpf) were warmed and cultured until the required stages were reached. We also cultured 14 embryos that were created for research until the four- and eight-cell stage. The embryos were scored according to their developmental stage (C0-B6) displaying morphological key differences, rather than defining them according to their chronological age. They were fixed and immunostained for different combinations of cytoskeleton (F-actin), polarization (p-ERM), TE (GATA3), EPI (NANOG), PrE (GATA4 and SOX17), and members of the Hippo signalling pathway (YAP1, TEAD1 and TEAD4). We choose these markers based on previous observations in mouse embryos and single cell RNA-sequencing data of human embryos. After confocal imaging (LSM800, Zeiss), we analysed cell numbers within each lineage, different co-localization patterns and nuclear enrichment. MAIN RESULTS AND THE ROLE OF CHANCE: We found that in human preimplantation embryos compaction is a heterogeneous process that takes place between the eight-cell to the 16-cell stages. Inner and outer cells are established at the end of the compaction process (C2) when the embryos contain up to six inner cells. Full apical p-ERM polarity is present in all outer cells of compacted C2 embryos. Co-localization of p-ERM and F-actin increases steadily from 42.2% to 100% of the outer cells, between C2 and B1 stages, while p-ERM polarizes before F-actin (P < 0.00001). Next, we sought to determine which factors specify the first lineage segregation event. We found that 19.5% of the nuclei stain positive for YAP1 at the start of compaction (C0) which increases to 56.1% during compaction (C1). At the C2 stage, 84.6% of polarized outer cells display high levels of nuclear YAP1 while it is absent in 75% of non-polarized inner cells. In general, throughout the B0-B3 blastocyst stages, polarized outer/TE cells are mainly positive for YAP1 and non-polarized inner/ICM cells are negative for YAP1. From the C1 stage onwards, before polarity is established, the TE marker GATA3 is detectable in YAP1 positive cells (11.6%), indicating that differentiation into TE cells can be initiated independently of polarity. Co-localization of YAP1 and GATA3 increases steadily in outer/TE cells (21.8% in C2 up to 97.3% in B3). Transcription factor TEAD4 is ubiquitously present throughout preimplantation development from the compacted stage onwards (C2-B6). TEAD1 displays a distinct pattern that coincides with YAP1/GATA3 co-localization in the outer cells. Most outer/TE cells throughout the B0-B3 blastocyst stages are positive for TEAD1 and YAP1. However, TEAD1 proteins are also detected in most nuclei of the inner/ICM cells of the blastocysts from cavitation onwards, but at visibly lower levels as compared to that in TE cells. In the ICM of B3 blastocysts, we found one main population of cells with NANOG+/SOX17-/GATA4- nuclei (89.1%), but exceptionally we found NANOG+/SOX17+/GATA4+ cells (0.8%). In seven out of nine B3 blastocysts, nuclear NANOG was found in all the ICM cells, supporting the previously reported hypothesis that PrE cells arise from EPI cells. Finally, to determine which factors specify the second lineage segregation event, we co-stained for TEAD1, YAP1, and GATA4. We identified two main ICM cell populations in B4-6 blastocysts: the EPI (negative for the three markers, 46.5%) and the PrE (positive for the three markers, 28.1%) cells. We conclude that TEAD1 and YAP1 co-localise in (precursor) TE and PrE cells, indicating that TEAD1/YAP1 signalling plays a role in the first and the second lineage segregation events. LIMITATIONS, REASONS FOR CAUTION: In this descriptive study, we did not perform functional studies to investigate the role of TEAD1/YAP1 signalling during the first and second lineage segregation events. WIDER IMPLICATIONS OF THE FINDINGS: Our detailed roadmap on polarization, compaction, position and lineage segregation events during human preimplantation development paves the way for further functional studies. Understanding the gene regulatory networks and signalling pathways involved in early embryogenesis could ultimately provide insights into why embryonic development is sometimes impaired and facilitate the establishment of guidelines for good practice in the IVF lab. STUDY FUNDING/COMPETING INTERESTS: This work was financially supported by Wetenschappelijk Fonds Willy Gepts (WFWG) of the University Hospital UZ Brussel (WFWG142) and the Fonds Wetenschappelijk Onderzoek-Vlaanderen (FWO, G034514N). M.R. is doctoral fellow at the FWO. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Similar fertilization rates and preimplantation embryo development among testosterone-treated transgender men and cisgender women.

RESEARCH QUESTION: What are the effects of testosterone treatment on oocyte fertilization and preimplantation embryo development among transgender men who have undergone fertility preservation? DESIGN: A retrospective study was undertaken in a university-affiliated tertiary hospital between April 2016 and November 2021. Embryos were divided into three groups by source: 210 embryos from 7 testosterone-exposed transgender men, 135 from 10 cisgender women who cryopreserved embryos, and 276 from 24 cisgender women who underwent fertility treatment. Statistical analyses compared assisted reproductive technology outcomes between the group of transgender men and both groups of cisgender women. Morphokinetic and morphological parameters were compared between the embryos derived from these three groups. RESULTS: The transgender men (30.2 ± 3.5 years of age) were significantly younger than the cisgender women who cryopreserved embryos (35.1 ± 1.8 years; P = 0.005) and the cisgender women who underwent fertility treatment (33.8 ± 3.2 years; P = 0.017). After adjusting for participant age, the fertilization rate was comparable between the transgender men and both groups of cisgender women (P = 0.391 and 0.659). There were no significant differences between the transgender men and the cisgender women who preserved fertility in terms of number of cryopreserved embryos (7.2 ± 5.1 and 3.5 ± 2.6; P = 0.473) or the distribution of embryo age at cryopreservation (P = 0.576). All morphokinetic parameters evaluated by time-lapse imaging, as well as the morphological characteristics, were comparable for the embryos in all three groups. CONCLUSIONS: Testosterone exposure among transgender men has no adverse impact upon fertilization rates or preimplantation embryo development and quality.

Anatomical and cellular heterogeneity in the mouse oviduct-its potential roles in reproduction and preimplantation development†.

The oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive organ that provides an environment for internal fertilization and preimplantation development. However, our knowledge of its regional and cellular heterogeneity is still limited. Here, we examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines. We found that there are consistent coiling patterns and turning points in the coiled mouse oviduct that serve as reliable landmarks for luminal morphological regionalities. We also found previously unrecognized anatomical structures in the isthmus and uterotubal junction, which likely play roles in reproduction. Furthermore, we demarcated the ampulla-isthmus junction as a distinct region. Taken together, the oviduct mucosal epithelium has highly diverse structures with distinct epithelial cell populations, reflecting its complex functions in reproduction.

Time-lapse imaging of human embryos fertilized with testicular sperm reveals an impact on the first embryonic cell cycle.

Testicular sperm is increasingly used during in vitro fertilization treatment. Testicular sperm has the ability to fertilize the oocyte after intracytoplasmic sperm injection (ICSI), but they have not undergone maturation during epididymal transport. Testicular sperm differs from ejaculated sperm in terms of chromatin maturity, incidence of DNA damage, and RNA content. It is not fully understood what the biological impact is of using testicular sperm, on fertilization, preimplantation embryo development, and postimplantation development. Our goal was to investigate differences in human preimplantation embryo development after ICSI using testicular sperm (TESE-ICSI) and ejaculated sperm. We used time-lapse embryo culture to study these possible differences. Embryos (n = 639) originating from 208 couples undergoing TESE-ICSI treatment were studied and compared to embryos (n = 866) originating from 243 couples undergoing ICSI treatment with ejaculated sperm. Using statistical analysis with linear mixed models, we observed that pronuclei appeared 0.55 h earlier in TESE-ICSI embryos, after which the pronuclear stage lasted 0.55 h longer. Also, significantly more TESE-ICSI embryos showed direct unequal cleavage from the 1-cell stage to the 3-cell stage. TESE-ICSI embryos proceeded faster through the cleavage divisions to the 5- and the 6-cell stage, but this effect disappeared when we adjusted our model for maternal factors. In conclusion, sperm origin affects embryo development during the first embryonic cell cycle, but not developmental kinetics to the 8-cell stage. Our results provide insight into the biological differences between testicular and ejaculated sperm and their impact during human fertilization.

Effects of Cigarette Smoking on Preimplantation Embryo Development.

In this chapter, we first gave a brief introduction to the detriments of cigarette smoking, with an emphasis on its adverse effects on female reproductive health. Then, we outlined recent advances about the impacts of cigarette smoke on preimplantation embryo development. Additionally, toxicities of cadmium and benzo(a)pyrene (BaP) at this specific developmental window were also discussed, to illustrate the potential mechanisms involved in cigarette smoke-associated embryotoxicity. Finally, we provide an overview of the issues to be solved in the future research. Further studies about the molecular mechanism of cigarette smoking-associated female infertility may provide vital insights into developing new interventions for the women smokers and thus improving their reproductive outcomes.

Advanced glycation end products present in the obese uterine environment compromise preimplantation embryo development.

RESEARCH QUESTION: Proinflammatory advanced glycation end products (AGE), highly elevated within the uterine cavity of obese women, compromise endometrial function. Do AGE also impact preimplantation embryo development and function? DESIGN: Mouse embryos were cultured in AGE equimolar to uterine fluid concentrations in lean (1-2 µmol/l) or obese (4-8 µmol/l) women. Differential nuclear staining identified cell allocation to inner cell mass (ICM) and trophectoderm (TE) (day 4 and 5 of culture). Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay (day 5). Day 4 embryos were placed on bovine serum albumin/fibronectin-coated plates and embryo outgrowth assessed 93 h later as a marker of implantation potential. AGE effects on cell lineage allocation were reassessed following pharmacological interventions: either 12.5 nmol/l AGE receptor (RAGE) antagonist; 0.1 nmol/l metformin; or combination of 10 µmol/l acetyl-l-carnitine, 10 µmol/l N-acetyl-l-cysteine, and 5 µmol/l alpha-lipoic acid. RESULTS: 8 µmol/l AGE reduced: hatching rates (day 5, P < 0.01); total cell number (days 4, 5, P < 0.01); TE cell number (day 5, P < 0.01), and embryo outgrowth (P < 0.01). RAGE antagonism improved day 5 TE cell number. CONCLUSIONS: AGE equimolar with the obese uterine environment detrimentally impact preimplantation embryo development. In natural cycles, prolonged exposure to AGE may developmentally compromise embryos, whereas following assisted reproductive technology cycles, placement of a high-quality embryo into an adverse 'high AGE' environment may impede implantation success. The modest impact of short-term RAGE antagonism on improving embryo outcomes indicates preconception AGE reduction via pharmacological or dietary intervention may improve reproductive outcomes for overweight/obese women.

Ameliorative effects of quercetin on the preimplantation embryos development in diabetic pregnant mice.

AIM: Maternal diabetes adversely retards the development of preimplantation embryos. Quercetin is a flavonoid belonging to phytoestrogens family and may be useful in treatment of reproductive disorders. The aim of this study was investigation of the ameliorative effects of quercetin administration on preimplantation embryo development in diabetic pregnancy. METHODS: Diabetic and healthy female mice were treated with 30 mg/kg/day quercetin 4 weeks before conception. Blastocysts were recovered at the 4th day of pregnancy for protein and mRNA expression changes. Plasma sex-steroid levels were also analyzed. RESULTS: Quercetin significantly decreased blood glucose levels in diabetic mice. Embryos retrieved from diabetic mice exhibited a considerable delay in morphological development. In diabetic mice with quercetin treatment, morphological distribution was shifted considerably to the well-developed stages. Serum estradiol level reduced in diabetic mice but, treatment with quercetin significantly increased serum estradiol level. While IGF1R, integrin αvß3, and Cox2 mRNA expression in the blastocyst of diabetic mice decreased significantly, quercetin treatment caused increasing expression levels of these genes. Expression of the Caspase3 gene increased dramatically in the collected blastocysts from diabetic mice and reduced following quercetin treatment. Besides, the inactive ß-catenin protein level in the blastocysts of diabetic mice was higher than that in normal mice, while treatment with quercetin decreased the level of inactive ß-catenin protein in the blastocyst of diabetic mice. CONCLUSION: Quercetin protects preimplantation embryos from destructive effects of diabetes. The amelioration of sex hormones disturbance in early pregnancy may help to treat reproductive disorders in diabetic women. Quercetin can be considered as a novel solution to the improvement of reproductive disorders in the diabetic females.

Dynamics of alternative polyadenylation in human preimplantation embryos.

Alternative polyadenylation (APA) affects the length of the 3' untranslated region (3'-UTR) and the regulation of microRNAs. Previous studies have shown that cancer cells tend to have shorter 3'-UTRs than normal cells. A plausible explanation for this is that it enables cancer cells to escape the regulation of microRNAs. Here, we extend this concept to an opposing context: changes in 3'-UTR length in the development of the human preimplantation embryo. Unlike cancer cells, during early development 3'-UTRs tended to become longer, and gene expression was negatively correlated with 3'-UTR length. Moreover, our functional enrichment results showed that length changes are part of the development mechanism. We also investigated the analogy of 3'-UTR length variation with respect to lncRNAs and found that, similarly, lncRNA length tended to increase during embryo development.

Low-dose irradiation of mouse embryos increases Smad-p21 pathway activity and preserves pluripotency.

PURPOSE: To study the outcomes of mouse preimplantation embryos irradiated with low doses of X-rays (≤ 1 Gy) and investigate apoptosis and pluripotency of the irradiated embryos. METHODS: Mouse embryos at the 2-cell stage were collected for in vitro culture. After reaching the 8-cell stage, embryos were irradiated with various low doses of X-rays (0-1 Gy). Blastocysts with a normal appearance were transferred into a pseudopregnant uterus. The developmental rate to blastocysts and the survival rate following embryo transfer were examined. Expression levels of p21, Smad2, Foxo1, Cdx2, Oct4, and Nanog genes were measured by RT-PCR. Apoptotic cells in mouse blastocysts were examined immunofluorescently by staining for cleaved caspase-3. RESULTS: More than 90% of non-irradiated and low-dose X-ray-irradiated preimplantation embryos developed to morphologically normal blastocysts that could be implanted and survive in the uterus. However, embryos irradiated with X-rays had more apoptotic cells in a dose-dependent manner. Expression of p21, Smad2, and Foxo1 genes in X-ray-irradiated embryos was increased significantly, while expression of Cdx2, Oct4, and Nanog genes was maintained in comparison with non-irradiated embryos. CONCLUSIONS: Although irradiated embryos contained apoptotic cells, the low doses of irradiation did not disturb development of 8-cell stage embryos to blastocysts or their survival in utero. The underlying mechanisms might involve anti-apoptotic systems, including the Smad-p21 pathway, and preservation of pluripotency.

Roles and regulations of Hippo signaling during preimplantation mouse development.

During preimplantation development, mouse embryos form two types of cells, the trophoectoderm (TE) and inner cell mass (ICM), by the early blastocyst stage. This process does not require maternal factors localized in the zygotes, and embryos self-organize at the blastocyst stage through intercellular communications. In terms of the mechanisms of cell fate specification, three historical models have been proposed: the positional model, and the original and newer versions of the polarity model. Recent studies have revealed that the intercellular Hippo signaling pathway plays a central role in the specification of the first cell fates. Hippo signaling is active in the inner cells but inactive in the outer cells. The Hippo-active inner and Hippo-inactive outer cells take the fates of the ICM and the TE, respectively. At the 32-cell stage, E-cadherin-mediated cell-cell adhesion and cell polarization by the Par-aPKC system activates and inactivates the Hippo pathway, respectively. Both mechanisms involve regulation of angiomotin, and cooperation of these mechanisms establishes cell position-dependent activation of Hippo signaling. At the 16-cell stage, however, asymmetric cell division produces the initial differences in Hippo signaling. At this stage, cell polarity is controlled by both Par-aPKC-dependent and -independent mechanisms. All three historical models are explained by the different regulations and roles of Hippo signaling. Based on these findings, I would like to propose the model by which the differences in Hippo signaling among blastomeres is first produced by asymmetric cell division and then enhanced and stabilized by cell position-dependent mechanisms until their fates are fixed.

The interferon α-responsive gene, Ifrg15, plays vital roles during mouse early embryonic development.

The interferon alpha-responsive gene (Ifrg15) mRNA is highly expressed in various stages during preimplantation mammalian embryo development. Unfortunately, few studies have investigated the effect of Ifrg15 in this process. In mammals, the fusion of male and female pronuclei generates a diploid zygote, and is an important step for subsequent cleavage and blastocyst formation. Here, by using RNA interference, rescue experiments, immunofluorescence staining and live cell observations, we found that preimplantation embryo development was arrested at the 1-cell stage after knocking down Ifrg15 expression. This induced DNA damage and prevented the cleavage of embryos. Furthermore, the effect of Ifrg15 deficiency in arresting preimplantation embryo development produced by specific short interfering RNA microinjection was concentration-dependent. Using transcriptome expression profiles, gene ontogeny functional annotation and enrichment analysis, we gained 197 enriched pathways based on 1445 differentially expressed genes (DEGs). Of these, 12 pathways and about one third of the DEGs were involved in DNA damage, DNA repair, cell cycle, and developmental processes. Thus, the IFRG15 protein might be an important molecule for maintaining genomic integrity and stability through upregulating or downregulating a cascade of genes to permit normal preimplantation embryo development.

CDK2 Is Required for the DNA Damage Response During Porcine Early Embryonic Development.

Cyclin-dependent kinase (CDK) 2 inhibition plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine embryos. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or meiosis II arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage, as demonstrated by the formation of distinct gammaH2AX foci in nuclei of Day-3 and Day-5 embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activation of the ataxia telangiectasia mutated (ATM)-P53-P21 pathway. However, the mRNA expression of genes involved in nonhomologous end joining or homologous recombination pathways for double-strand break repair were reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in Day-7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression.

Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells.

The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well-understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte-to-embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4-cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells.

Effects of High-Butterfat Diet on Embryo Implantation in Female Rats Exposed to Bisphenol A.

Bisphenol A (BPA) is an endocrine disruptor associated with poor pregnancy outcomes in human and rodents. The effects of butterfat diets on embryo implantation and whether it modifies BPA's actions are currently unknown. We aimed to determine the effects of butterfat diet on embryo implantation success in female rats exposed to an environmentally relevant dose of BPA. Female Sprague-Dawley rats were exposed to dietary butterfat (10% or 39% kcal/kg body weight [BW]) in the presence or absence of BPA (250 µg/kg BW) or ethinylestradiol (0.1 µg/kg BW) shortly before and during pregnancy to assess embryo implantation potentials by preimplantation development and transport, in vitro blastulation, outgrowth, and implantation. On gestational day (GD) 4.5, rats treated with BPA alone had higher serum total BPA level (2.3-3.7 ng/ml). They had more late-stage preimplantation embryos, whereas those receiving high butterfat (HBF) diet had the most advanced-stage embryos; dams cotreated with HBF and BPA had the most number of advanced embryos. BPA markedly delayed embryo transport to the uterus, but neither amount of butterfat had modifying effects. An in vitro implantation assay showed HBF doubled the outgrowth area, with BPA having no effect. In vivo, BPA reduced the number of implanted embryos on GD8, and cotreatment with HBF eliminated this adverse effect. HBF diet overall resulted in more and larger GD8 embryos. This study reveals the implantation disruptive effects of maternal exposure to an environmentally relevant dose of BPA and identifies HBF diet as a modifier of BPA in promoting early embryonic health.

Osteopontin is expressed in the oviduct and promotes fertilization and preimplantation embryo development of mouse.

Osteopontin (OPN) is a multifunctional phosphoprotein that is detected in various tissues, including male and female reproductive tracts. In this study, we evaluated OPN expression in mouse oviducts during the estrus cycle, and at days 1-5 of pregnancy and pseudopregnancy by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The mice oocytes, sperm and embryos were treated with different concentrations of anti-OPN antibody in vitro to detect the function of OPN in fertilization and preimplantation embryo development. OPN mRNA and protein expression in mouse oviducts were cyclic dependent throughout the estrous cycle, which was highest at estrous and lowest at diestrous. Such a phenomenon was consistent with the change in estrogen level in mice. The expression levels of OPN in mice oviduct of normal pregnancy and pseudopregnancy were significantly different, which indicated that OPN expression in mouse oviducts was depend on estrogen and preimplantation embryo. Furthermore, anti-OPN antibody treatment could reduce the rates of fertilization, cleavage and blastocyst formation in vitro in a dose-dependent way. Overall, our results indicated that the expression of OPN in mouse oviducts during the estrous cycle and early pregnancy is likely regulated by estrogen and the embryo, and OPN may play a vital role in oocyte fertilization and preimplantation embryo development.