Early Blastocyst Research Articles

The transition from local to global patterns governs the differentiation of mouse blastocysts.

During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterized by the expression of the transcription factors NANOG and GATA6, respectively. Here, we investigate the spatio-temporal distribution of NANOG and GATA6 expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single cell-based neighbourhood analyses. We define the cell neighbourhood by local features, which include the expression levels of both fate markers expressed in each cell and its neighbours, and the number of neighbouring cells. We further include the position of a cell relative to the centre of the ICM as a global positional feature. Our analyses reveal a local three-dimensional pattern that is already present in early blastocysts: 1) Cells expressing the highest NANOG levels are surrounded by approximately nine neighbours, while 2) cells expressing GATA6 cluster according to their GATA6 levels. This local pattern evolves into a global pattern in the ICM that starts to emerge in mid blastocysts. We show that FGF/MAPK signalling is involved in the three-dimensional distribution of the cells and, using a mutant background, we further show that the GATA6 neighbourhood is regulated by NANOG. Our quantitative study suggests that the three-dimensional cell neighbourhood plays a role in Epi and PrE precursor specification. Our results highlight the importance of analysing the three-dimensional cell neighbourhood while investigating cell fate decisions during early mouse embryonic development.

Development of Mice Embryo (Mus musculus L.) after Closed Pulled Straw Vitrification in CZB Medium

The objective of our present experiment was to investigate the effects of closed pulled straw (CPS) vitrification on the viability and development of mouse embryo. The experiment was arranged according to completely randomized design (CRD) consisting of 4 treatments, namely not vitrification (NV) is control, CPS 1 (mCZB Hepes + 20% Bovine Serum Albumin (BSA) +0.5 M sucrose + 10% EG + 10% DMSO), CPS 2 (mCZB Hepes + 20% BSA + 0.5 M sucrose + 15% EG + 15% DMSO), and CPS 3 (mCZB Hepes + 20% BSA +0.5 M sucrose + 20% EG + 20% DMSO) with 6 replications. The viability of embryos (%) was determined after 24 - 72 h of the culture period, while we also observed the percentage of embryos reaching the blastocyst stage (early blastocyst, expanded, hatching, and hatched). As a result, the treatments did not give a significant difference in the viability of embryos (P<0,05) but showed significant effects on embryo development (P<0,05). Furthermore, this present work conclusively found that CPS vitrification in CZB medium with cryoprotectants ethylene glycol (EG) and dimethyl sulfoxide (DMSO) noticeably influenced the development of mice embryo to reach the blastocyst stage, but showed no remarkable difference in the viability of embryo after culture for 24 – 72 h.

Estágio de desenvolvimento no envase afeta a viabilidade de embriões bovinos produzidos in vitro

O objetivo deste estudo foi avaliar a viabilidade de embriões produzidos in vitro (IVPE) bovinos em diferentes estágios de desenvolvimento, mantidos a 36ºC em diferentes tempos de transporte. No sétimo dia de cultura in vitro, os embriões grau I e II nos estágios iniciais do blastocisto, blastocisto e blastocisto expandido foram selecionados, envasados e distribuídos aleatoriamente por três tempos de transporte em uma incubadora portátil a 36°C: 6, 9 e 12 horas. Após o tempo de tratamento, os embriões foram re-cultivados em meio de cultivo por 72 horas e avaliados quanto à qualidade e desenvolvimento. Os dados de taxa de eclosão e degeneração foram analisados pelo teste do qui-quadrado. Os embriões envasados no estágio de blastocisto expandido apresentaram maiores taxas de eclosão e menores taxas de degeneração. Os embriões envasados no estágio de blastocisto expandido mostraram maior viabilidade em comparação com os do blastocisto inicial, independentemente do tempo de transporte, e os blastocistos transportados por 6 e 9 horas.

Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks.

Simple SummaryThis study was conducted to demonstrate the potential hazards of cross-infection and cross-contamination of embryos during storage in liquid nitrogen biobanks. For the harmless and successful cryopreservation of embryos, the vitrification method must be chosen meticulously to guarantee not only a high post-thaw survival of embryos, but also to reduce the risk of disease transmission when those embryos are in storage for long periods.In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the effects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae–early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures.

A stepwise approach to move from a cleavage-stage to a blastocyst-stage transfer policy for all patients in the IVF clinic

A stepwise approach to move from a cleavage-stage to a blastocyst-stage transfer policy for all patients in the IVF clinic

Impact of the corpus luteum on survival of the developing embryo and early pregnancy in mares

It has been more than a hundred years that studies aiming to elucidate the processes involved in cyclicity and pregnancy pointed out the requirement of ovaries and corpora lutea for embryo survival and pregnancy establishment. For horses, luteal progesterone is essential for pregnancy only during the first trimester. This progestational support is complex among domestic animals as ovarian luteal function is further enhanced by the LH-action role of equine chorionic gonadotropin (eCG) starting ∼ on Day 35 of pregnancy. Increased eCG secretion leads to the formation of supplementary corpora lutea resulting from follicles that luteinize (accessory corpora lutea) or ovulate (secondary corpora lutea), thus increasing concentrations of blood progesterone. Physiological details of progesterone-driven embryo-maternal interactions continue to be elucidated. In recent years, researchers studying the transcriptomes and secretomes of uterine tubes, endometrium and early embryo provided insight into the composition of molecular and cellular events that enable embryo survival and remodeling of the endometrium before a functional placenta is formed. Aluteal pregnancy models have also shown that while fertilization and early embryo development until the early blastocyst stage can occur under a progesterone-deprived environment, dysregulation of important pregnancy-related genes occur; embryo development is compromised unless progestin supplementation is provided once the embryo arrives into the uterus. As the body of knowledge on embryo-maternal interactions in the horse continues to grow, a fact remains true: luteal support is essential for embryo survival mainly at the uterine stage, driving directly or indirectly gene expression that promotes adequate embryo-maternal physiological interactions until a full competent placenta is formed, resulting in optimal chances of delivering a live foal at term.

Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos

Embryo cryopreservation is an important tool to preserve endangered species. As a cryoprotectant for mouse oocytes, antifreeze protein from Anatolica polita (ApAFP914) has demonstrated utility. In the present study, the effects of controlled slow freezing and vitrification methods on the survival rate of sheep oocytes fertilized in vitro after freezing-thawing were compared. Different ApAFP914 concentrations were added to the vitrification liquid for exploring the effect of antifreeze protein on the warmed embryos. The results showed that the survival and hatching rates of in vitro derived embryos were significantly higher than that of the slow freezing method. Furthermore, among the cryopreserved embryos at different developmental stages, the survival and hatching rates of the expanded blastocyst were significantly higher than those of the blastocysts, early blastocysts and morula. The survival and the hatching rates of the fast-growing embryos were both significantly higher than that of the slow-growing embryos. Additionally, treatment of ApAFP914 (5–30 μg/mL) did not increase the freezing efficiency of the 6–6.5 d embryos. However, addition of 10 μg/mL of ApAFP914 significantly increased the hatching rate of slow-growing embryos. In conclusion, our study suggests that the vitrification is better than the slow freezing method for the conservation of in vitro sheep embryos, and supplementation of ApAFP914 (10 μg/mL) significantly increased the hatching rate of slow-growing embryos after cryopreservation.

Efficient one-step direct transfer to recipients of thawed bovine embryos cultured in vitro and frozen in chemically defined medium

Direct transfer (DT) of cryopreserved embryos to recipients facilitates on-farm application. We analyzed a new freezing/thawing (F/T) procedure for in vitro produced (IVP) embryos, integrating: 1) an ethylene-glycol based system; 2) a culture step without protein; and 3) a synthetic protein substitute (CRYO3) in cryopreservation medium. IVP embryos from abattoir ovaries were cultured in groups in BSA-containing synthetic oviduct fluid with or without 0.1% fetal calf serum (FCS) until Day-6. Morulae and early blastocysts were subsequently cultured without protein from Day-6 onwards. Day 7 and Day 8 expanded blastocysts (EXB) were subjected to F/T or vitrification/warming (V/W). Thawed and warmed EXB were cultured in vitro, and development rates, cell counts and dead cells were analyzed in surviving embryos. V/W improved survival over F/T (live and hatching rates at 2 h, 24 h and 48 h) (P < 0.0001), and FCS before Day 6 did not affect in vitro survival. After F/T, embryos had lower cell counts in the ICM, TE and total cells than after V/W. Day-7 embryos after F/T showed % apoptotic, % pycnotic and % total dead cells higher (p < 0.05) than their Day-8 counterparts, probably because F/T reduced the numbers of ICM cells within Day-8 embryos. Thereafter, Day-7 blastocysts were transferred to heifers in an experimental herd. There were no differences in birth rates with frozen (-FCS [n = 40]: 45%; +FCS [n = 14]: 28%), vitrified (-FCS [n = 47]: 53%; +FCS [n = 11]: 36%) and fresh (-FCS [n = 30]: 47%; +FCS [n = 17]: 53%) embryos. However, frozen embryos produced with FCS showed 5/9 miscarriages after Day-40. Calves born from frozen (n = 22), vitrified (n = 29) and fresh (n = 22) transfers did not differ in birth weight, gestation length and daily gain weight (P > 0.10). Subsequently, transfer of frozen embryos (n = 29) derived from oocytes collected from live, hormonally stimulated cows in experimental herd, led to pregnancy rates of 57% (heifers) and 40% (dry cows). with EXB on Day-62 Finally, embryos produced with BSA were transferred to cows in an on-field trial (frozen [n = 80]; fresh [n = 58]), with no differences in pregnancy rates (days 30–40). Pregnancy and birth rates could not be predicted from in vitro approaches. The new F/T system yields pregnancy and birth rates comparable to vitrified and fresh embryos without birth overweight. The absence of products of animal origin, defined chemical composition, and direct transfer entail sanitary, manufacturing and application advantages.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P 0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P 0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P 0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

Adaptation within embryonic and neonatal heart environment reveals alternative fates for adult c-kit+ cardiac interstitial cells.

Cardiac interstitial cells (CICs) perform essential roles in myocardial biology through preservation of homeostasis as well as response to injury or stress. Studies of murine CIC biology reveal remarkable plasticity in terms of transcriptional reprogramming and ploidy state with important implications for function. Despite over a decade of characterization and in vivo utilization of adult c‐Kit+ CIC (cCIC), adaptability and functional responses upon delivery to adult mammalian hearts remain poorly understood. Limitations of characterizing cCIC biology following in vitro expansion and adoptive transfer into the adult heart were circumvented by delivery of the donated cells into early cardiogenic environments of embryonic, fetal, and early postnatal developing hearts. These three developmental stages were permissive for retention and persistence, enabling phenotypic evaluation of in vitro expanded cCICs after delivery as well as tissue response following introduction to the host environment. Embryonic blastocyst environment prompted cCIC integration into trophectoderm as well as persistence in amniochorionic membrane. Delivery to fetal myocardium yielded cCIC perivascular localization with fibroblast‐like phenotype, similar to cCICs introduced to postnatal P3 heart with persistent cell cycle activity for up to 4 weeks. Fibroblast‐like phenotype of exogenously transferred cCICs in fetal and postnatal cardiogenic environments is consistent with inability to contribute directly toward cardiogenesis and lack of functional integration with host myocardium. In contrast, cCICs incorporation into extra‐embryonic membranes is consistent with fate of polyploid cells in blastocysts. These findings provide insight into cCIC biology, their inherent predisposition toward fibroblast fates in cardiogenic environments, and remarkable participation in extra‐embryonic tissue formation.

Metabolic compounds within the porcine uterine environment are unique to the type of conceptus present during the early stages of blastocyst elongation.

The objective of this study was to identify metabolites within the porcine uterine milieu during the early stages of blastocyst elongation. At Days 9, 10, or 11 of gestation, reproductive tracts of White cross‐bred gilts (n = 38) were collected immediately following harvest and flushed with Roswell Park Memorial Institute‐1640 medium. Conceptus morphologies were assessed from each pregnancy and corresponding uterine flushings were assigned to one of five treatment groups based on these morphologies: (a) uniform spherical (n = 8); (b) heterogeneous spherical and ovoid (n = 8); (c) uniform ovoid (n = 8); (d) heterogeneous ovoid and tubular (n = 8); and (e) uniform tubular (n = 6). Uterine flushings from these pregnancies were submitted for nontargeted profiling by gas chromatography–mass spectrometry (GC–MS) and ultra performance liquid chromatography (UPLC)–MS techniques. Unsupervised multivariate principal component analysis (PCA) was performed using pcaMethods and univariate analysis of variance was performed in R with false discovery rate (FDR) adjustment. PCA analysis of the GC–MS and UPLC–MS data identified 153 and 104 metabolites, respectively. After FDR adjustment of the GC–MS and UPLC–MS data, 38 and 59 metabolites, respectively, differed (p < .05) in uterine flushings from pregnancies across the five conceptus stages. Some metabolites were greater (p < .05) in abundance for uterine flushings containing earlier stage conceptuses (i.e., spherical), such as uric acid, tryptophan, and tyrosine. In contrast, some metabolites were greater (p < .05) in abundance for uterine flushings containing later stage conceptuses (i.e., tubular), such as creatinine, serine, and urea. These data illustrate several putative metabolites that change within the uterine milieu during early porcine blastocyst elongation.

56 Mammalian pre-implantation embryos at the single cell level: The bovine as a model for early human embryonic development

Single-cell transcriptomics and gene editing on human pre-implantation embryos have proved that mechanisms previously identified and well characterised in mouse pre-implantation development may not hold true in the human embryo. However, ethical and legal concerns limit the availability of surplus human embryos for research, resulting in the need for novel animal models. Bovine embryos share morphological and temporal resemblance with human early development; still, key molecular and cellular developmental mechanisms need to be further explored. In the present study, we performed a comparative single-cell RNA sequencing analysis across multiple pre-implantational developmental stages of invitro-derived bovine, human (Petropoulos et al. 2016 Cell 165, 1012-1026; https://doi.org/10.1016/j.cell.2016.03.023), and mouse embryos (Cheng et al. 2019 Cell Rep. 26, 2593-2607, https://doi.org/10.1016/j.celrep.2019.02.031; Posfai et al. 2017 eLife 6, e22906, https://doi.org/10.7554/eLife.22906; Chen et al. 2016 Genome Res. 26, 1342-1354). In total 497 blastomeres corresponding to embryonic days (E) E4-E8 were prepared using Smart sEqn 2, Illumina HiSEqn 2500 sequencing with 50-bp single-end reads. Embryonic stage was defined by cell numbers and morphology as multicellular stage (Mu, ~E4), morulae (M; E5), early blastocysts (EB, ~6), mid-expanded blastocysts, (MB, ~E7), and late expanded/hatched blastocysts (LB, ~E8). We found top differentially expressed genes elucidating how lineage specification and pluripotency is controlled in the early bovine embryo. Our transcriptomic data showed that the first lineage segregation in the pre-implantation bovine embryo occurs after cavitation at E6 in the early blastocyst, suggesting a similarity with the early human blastocyst. In addition, E7 showed distinctive and more mature inner cell mass (ICM) and trophectoderm (TE) profiles. Different from the mouse, where the first lineage segregation of TE-ICM is suggested to occur at the morula stage due to CDX2-OCT4 antagonism, we found that the expression of OCT4 protein cattle expanded blastocysts is not restricted to the ICM, similar to what has been reported to occur in the human embryo. Interestingly, we also found that the second lineage segregation, which segregates the epiblast from the primitive endoderm within the ICM, starts in bovine at E8 expanded hatched late blastocysts, suggesting a potential difference with humans, where TE-ICM and epiblast-primitive endoderm has previously been reported to occur almost concurrently. We are now complementing our findings with genome editing in bovine embryos by generating knockout embryos of different target genes part of an evolutionarily conserved signaling pathway to study their effects in the early pre-implantation embryo. We aim to elucidate how early lineage specification and pluripotency establishment occurs while offering theoretical support for efficient derivation and culture of bovine embryonic stem cells.

11 Invivo-derived embryo pregnancy rates at Maddox Dairy from 2008 to 2018

11 Invivo-derived embryo pregnancy rates at Maddox Dairy from 2008 to 2018

66 Lipid profiling of bovine blastocysts produced invitro with and without a stimulator of cyclic guanosine monophosphate synthesis by multiple reaction monitoring profiling

Cryopreservation of oocytes and embryos is an essential technique for invitro-produced cattle worldwide. One of the great difficulties of cryopreservation of oocytes and blastocysts is the accumulation of lipids in the cytoplasm when produced invitro. The lipid metabolism of oocytes and embryos is classically regulated by the cAMP pathway. Furthermore, previous studies have suggested that the cyclic guanosine monophosphate (cGMP) pathway may also be involved in modulating lipid metabolism through protein kinase G activation. The objective of this study was to investigate the lipid profile of bovine blastocysts produced invitro when stimulated by specific stimulator of cGMP synthesis (NPPB). Pools of oocytes were matured invitro for 24h in tissue culture medium 199, with 15% bovine serum, 0.5µgmL−1 FSH, 5µgmL−1 LH, 0.8mM L-glutamine, and 50µgmL−1 gentamicin at 38.5°C and 10−6 M NPPB. The control group was matured without NPPB. After 22h, the oocytes were fertilised invitro with frozen sperm. The IVM oocytes were fertilised and cultured according standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). After the 7 days (Day 7), the blastocysts (from early blastocyst to expanded blastocyst) were collected, washed in methanol:water (vol/vol) 1:3, and frozen at −80°C. The lipid extraction of the samples was performed based on the standard protocol (Bligh and Dyer 1959 Can. J. Biochem. Physiol. 37, 911-917) but adapted for small samples. The samples were diluted and analysed in the Agilent 6410 QQQ (Agilent Technologies) mass spectrometer and analysed according to the multiple reaction monitoring method described by de Lima et al. (2018 J. Mass Spectrom. 53, 1247-1252). Data for 3 replicates/group were normalized and then submitted to t-test statistical analysis and principal component analysis, by Metaboanalyst 4.0, with a significance level of 5%. The rates of cleavage and blastocysts were not affected when we used the mGC stimulator presenting a 61% rate of cleavage for both groups, and 24.4% and 25% of blastocyst rate for control and NPPB, respectively (P&amp;lt;0.05). The results, regarding 164 lipids analysed, showed that the lipid profile was not affected when we used NPPB, maintaining the same profile of lipid classes. When we observe the quantitative values, we see a nonsignificant decrease in the lipid classes sphingomyelin, phosphatidylcholine, and triacylglycerol. The values for each class for control and NPPB, respectively, were 0.70 and 0.64 ng/blastocyst for sphingomyelin, 6.45 and 6.07 ng/blastocyst for phosphatidylcholine, and 11.82 and 10.51 ng/blastocyst for triacylglycerol (P&amp;lt;0.05). For the other classes of phospholipids (PE, PG, and PI), we observed a small increase when treated with NPPB, also not significant. We conclude that although we do not have significant differences between the control and the treatment, each class of lipid can respond differently when stimulated with cGMP synthesis.

82 Stage-specific expression of lineage marker genes and pluripotency marker distribution in porcine pre-implantation embryos

Lineage specification in pre-implantation embryos has been revealed, and it was expedited recently by single cell studies. However, data on expression marker genes and proteins in porcine embryos were still missing. We aimed to investigate the expression and distribution of marker genes and proteins, respectively, in IVF and parthenogenetically activated (PA) embryos. For this, cumulus-free oocytes were co-incubated with sperm in modified Tris-buffered medium (mTBM) for 5h and PA was performed using an electric pulse in activation medium. Following this, the embryos were incubated in porcine zygote medium 3 (PZM3). We first tested gene expression level of lineage candidates (internal control; β-actin). In IVF embryos (30, 25, 20, 15, 10, and 5 embryos pooled on Day 2, 3, 4, 5, 6, and 7; replicated 3 times), trophectoderm (TE)-specific genes (Dab2, Gata3) showed peaks on Day 4-5. Within the 2 genes, Dab2 had an earlier peak than Gata3. Inner cell mass (ICM) marker candidates (Nanog, Sox2, and Hnf4a) had diverse patterns. The Nanog and Sox2 genes had peak expression on Day 3. The Nanog expression dropped gradually, but Sox2 dropped suddenly on Day 4. Otherwise, Hnf4a expressed little in Day 3 and expression was sustained from Day 4 to 7. Primitive endoderm markers showed the highest expression on Day 4. We also checked expression level of ICM markers (Sox2, Oct4, and Nanog) in PA embryos (20, 20, 20, 10, and 5 embryos were pooled in 2, 4, 6-8 cells, morula, early, and late blastocyst stages; replicated 3 times). Expression of markers was similar (the highest in the 6-8-cell stage; at least 7.3-, 4.5-, and 3.7-fold compared with the other stages in Sox2, Oct4, and Nanog). We used analysis of variance and Tukey's test for statistical analysis. Following this, we conducted immunocytochemistry with both IVF and PA embryos (20 in each condition). Primary antibodies were treated overnight at 4°C and appropriate secondary antibodies were treated 1h at room temperature. In the case of IVF, well-known ICM markers (SOX2, OCT4, NANOG, and SOX17) showed restricted distribution in nuclei of ICM cells. However, DAB2 was distributed in the cytoplasm of TE cells. In PA embryos, SOX2 and NANOG distributions were similar to IVF. The OCT4 in ICM cells from morula to early blastocyst was restricted, but not in Day 7 embryos. In conclusion, marker genes showed diverse expression pattern in IVF, but all ICM-specific genes had a similar pattern in PA. Also, ICM marker proteins were restricted in nuclei of ICM cells only except Day 7 PA. Our results provide eye-opening information on marker contribution to lineage specification of porcine embryos. This work was supported by the National Research Foundation in Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03032256, NRF-2019R1C1C1004514).

Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig.

The inner cell mass (ICM) in blastocyst is the origin of all somatic and germ cells in mammals and pluripotent stem cells (PSCs) in vitro. As the conserved principles between pig and human, here we performed comprehensive single-cell RNA-seq for porcine early embryos from oocyte to early blastocyst (EB). We show the specification of the ICM and trophectoderm in morula and the molecular signature of the precursors. We demonstrate the existence of naïve pluripotency signature in morula and ICM of EB, and the specific pluripotent genes and the activity of signalling pathways highlight the characteristics of the naïve pluripotency. We observe the absence of dosage compensation with respect to X-chromosome (XC) in morula, and incomplete dosage compensation in the EB. However, the dynamics of dosage compensation may be independent of the expression of XIST induced XC inactivation. Our study describes molecular landmarks of embryogenesis in pig that will provide a better strategy for derivation of porcine PSCs and improve research in regenerative medicine.

MLL1 Inhibition and Vitamin D Signaling Cooperate to Facilitate the Expanded Pluripotency State

SUMMARYDynamic establishment of histone modifications in early development coincides with programed cell fate restriction and loss of totipotency beyond the early blastocyst stage. Causal function of histone-modifying enzymes in this process remains to be defined. Here we show that inhibiting histone methyl-transferase MLL1 reprograms naive embryonic stem cells (ESCs) to expanded pluripotent stem cells (EPSCs), with differentiation potential toward both embryonic and extraembryonic lineages in vitro and in vivo. MLL1 inhibition or deletion upregulates gene signatures of early blastomere development. The function of MLL1 in restricting induction of EPSCs is mediated partly by Gc, which regulates cellular response to vitamin D signaling. Combined treatment of MLL1 inhibitor and 1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3) cooperatively enhanced functionality of EPSCs, triggering an extended 2C-like state in vitro and robust totipotent-like property in vivo. Our study sheds light on interplay between epigenetics and vitamin D pathway in cell fate determination.

Interleukin-6 requires JAK to stimulate inner cell mass expansion in bovine embryos.

Supplementing interleukin-6 (IL6) to in vitro-produced bovine embryos increases inner cell mass (ICM) cell numbers in blastocysts. A series of studies were completed to further dissect this effect. Treatment with IL6 increased ICM cell numbers in early, regular and expanded blastocysts but had no effect on morulae total cell number. Treatment with IL6 for 30 min induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and nuclear translocation in all blastomeres in early morulae and specifically within the ICM in blastocysts. Also, IL6 supplementation increased SOCS3 mRNA abundance, a STAT3-responsive gene, in blastocysts. Chemical inhibition of Janus kinase (JAK) activity from day 5 to day 8 prevented STAT3 activation and the IL6-induced ICM cell number increase. Global transcriptome analysis of blastocysts found that transcripts for IL6 and its receptor subunits (IL6R and IL6ST) were the most abundantly expressed IL6 family ligand and receptors. These results indicate that IL6 increases ICM cell numbers as the ICM lineage emerges at the early blastocyst stage through a STAT3-dependent mechanism. Also, IL6 appears to be the primary IL6 cytokine family member utilized by bovine blastocysts to control ICM cell numbers.

The role of glyphosate in infertility: the mechanistic link

In light of the recent Roundup lawsuit, glyphosate has been widely accepted as a significant environmental toxin that may affect humans in various ways including cancer and infertility. Exposure to even low doses of glyphosate-based herbicides during pregnancy has been found to impair fertility, cause intrauterine growth restriciton and induce fetal malformations. In this study we sought to determine the underlying mechanism by which glyphosate negatively impacts oocyte quality, fertilization rates as well as embryo development. Experimental case-control study of mouse oocytes and pronuclear embryos, exposed in vitro to increasing glyphosate concentrations and followed through day 5 of development. We utilized multiple assays including reactive oxygen species (ROS) generation and zinc depletion to examine the possible underlying detrimental mechanisms. Metaphase II mouse oocytes (n=200) were retrieved from 8-10 week female mice and a subset (n=100) were fertilized using IVF. The oocytes as well as the fertilized mouse embryos were then exposed to increasing concentrations of glyphosate (0-200 μM) for 2h - 4h as per protocol. The oocytes were divided into four groups that were treated as follows: Group A: ROS detection assay, Group B: Zinquin ethyl ester assay, Group C: fixed, stained and scored based on the spindle structure (microtubule morphology -MT and chromosomal alignment - CH) as indicators of the oocyte's capacity to sustain exposure. All groups were compared to Group 4: untreated controls. Exposed embryos were incubated for up to 120 hours post fertilization and evaluated for full and hatching blast rate conversion. They were photographed and graded daily based on their appearance and development using published embryo grading protocols. A subset of the treated embryos (n = 10 for each concentration) were treated, in a similar fashion as the oocytes, in order to evaluate for ROS overproduction and zinc depletion. Confocal microscopy was used to assess the embryos. Statistical analysis was performed using t-test, ANOVA and chi-square. A p-value < 0.05 was considered statistically significant. Oocytes treated with increasing glyphosate concentrations > 50 μΜ were found to have poor scores for MT and CH and that difference was statistically significant as compared to controls (p< 0.05). Embryos followed to 96 hours post fertilization (early blastocyst) and 120 hours (full and hatching blastocyst) after glyphosate exposure (0-200 μM) were assessed and those exposed to glyphosate concentrations > 100 μM showed significantly increased arrest rates and poor morphology scores compared to controls. ROS overproduction as well as zinc depletion was evident in embryos treated with high glyphosate concentrations. These observations were statistically significant compared to untreated controls (p<0.05). This work suggests the possible underlying mechanisms by which glyphosate negatively affects reproductive health in the mouse model. Possible fertility implications in humans will require further research.

Embryos frozen within a short time of reaching the expanded blastocysts from the early blastocysts have high viability: time-lapse investigation of 5177 blastocysts

Embryos frozen within a short time of reaching the expanded blastocysts from the early blastocysts have high viability: time-lapse investigation of 5177 blastocysts