Eight-cell Embryos Research Articles

Changes in DNA methylation and imprinting disorders in E9.5 mouse fetuses and placentas derived from vitrified eight-cell embryos.

Vitrification is increasingly used in assisted reproductive technology (ART) laboratories worldwide, and potential vitrification-induced risks require further exploration. The effect of vitrification on changes in DNA methylation and imprinting disorders was investigated in E9.5 mouse fetuses and placentas. Fetus and placental tissues were collected from the natural mating (nautural conception [NC]) group, in vitro culture (IVC) group and vitrified embryo transfer (VET) group. The fetal crown-rump length at E9.5 in both the IVC (0.210 ± 0.059 mm) and VET (0.205 ± 0.048 mm) groups was significantly reduced compared with the NC group (0.288 ± 0.083 mm). The global methylation levels of fetuses were decreased in the IVC group compared with the NC group and it was increased after vitrification compared with IVC (p < 0.05), similar to what was observed in the NC group (p > 0.05). The changes could be attributed to the disorders of DNA methyltransferases and ten-eleven translocations. In the IVC and VET fetuses, a majority of maternally expressed genes were upregulated, which repressed fetal growth. Furthermore, vitrification led to a change in the methylation level of KvDMR1, which resulted in the disturbance of gene imprinting. According to our results, vitrification could contribute to increased methylation compared with IVC and contributes to a gene imprinting disorder rather than recovery. Despite the routine use of embryo vitrification in clinical settings, the effect that this procedure may have on genomic imprinting deserves much greater attention.

Over Ten-Cell Good Embryo Transfers on Day Three have Equivalent Clinical Outcomes with Those of Eight-Cell Embryos in Female Patients Aged ≤35 Years: A Retrospective Cohort Study

Background/Aim: To compare the clinical outcomes between > 10- and 8-cell good quality embryos on day 3. Methods: A retrospective study of a cohort of 459 patients was included in the fresh embryo transfer (ET) cycle group from January 2009 to April 2016. In this group, 2 good quality embryos on day 3, were transferred on corresponding dates, in 75 patients (> 10-cell embryos), and in 384 patients (8-cell embryos). Seven hundred and forty four patients, with 1 blastocyst transferred derived from > 10-cell (n = 183) or 8-cell (n = 561) good quality embryos on day 3, were assigned in the frozen ET (FET) group. Results: In the ET group, the clinical pregnancy and live birth rates of the > 10-cell transfer patients were comparable with those of the 8-cell transfer group (62.67 vs. 69.27%, 60.00 vs. 59.90%, respectively); however, the miscarriage rate of > 10-cell transfers was significantly lower than that of 8-cell transfers (4.26 vs. 13.53%, p < 0.05). In the FET group, there were no statistically significant differences found in the clinical pregnancy, live birth, and miscarriage rates between one > 10-cell-derived and one 8-cell-derived blastocyst transfers (71.04 vs. 65.78%, 59.02 vs. 54.19%, and 16.92 vs. 17.62%, respectively). Conclusions: The results suggested that > 10- and 8-cell, good quality embryos on day 3, had comparable viability and clinical outcomes.

Intranuclear Actin Distribution at the Four- and Eight-Cell Stages of Mouse Embryo Development

Intranuclear distribution of actin was studied in mouse embryos in the middle and end of the four-cell stage of development, as well as in the blastomeres of uncompacted eight-cell embryos. Actin was visualized with direct fluorescence, indirect immunofluorescence and confocal microscopy. It was found that nuclei at these stages of development contained monomeric, as well as polymeric actin, differing from conventional filamentous. The pattern of actin distribution in the nuclei at the middle-stage four-cell embryos and uncompacted eight-cell embryos was similar. Comparison of this pattern with nuclear actin distribution in the two-cell embryos that we examined previously showed that at the early stages of embryo cleavage the intranuclear actin localization was not stage-specific and was stably reproduced in the nuclei of the embryos during transition from earlier to later stage of embryogenesis. Filamentous actin that had not been found in the nuclei of middle-stage four-cell embryos was detected by fluorescent phalloidin in the nuclei of the embryos at the end of a four-cell stage of development, shortly before the beginning of the third cleavage division. The appearance of nuclear filamentous actin is possibly related to the preparation of embryos to the following cleavage division.

Does blastomere removal alter the expression level of miR-Let7a and its target genes following mouse embryo biopsy?

Embryo manipulations may cause the misexpression of various genes, most of which play critical roles in the regulation of implantation. This study aimed to evaluate the effects of embryo biopsy on the expression of miR-Let-7a and its gene targets including ErbB4, Tgf-α, Itg-αv, Itg β3 on the implantation of mouse embryo. Embryos were produced by in vitro fertilization followed by blastomere biopsy at the eight-cell stage. The effects of blastomere removal on the expression of genes ErbB4, Tgf-α, Itg αv, Itg β3, and miR-Let-7a as well as the alteration of the blastocyst cell number were compared in both biopsied and non-biopsied groups. Finally, blastocyst attachment was assessed on culture dishes precoated with Fibronectin. The results revealed that there were no significant differences between the biopsied and non-biopsied embryos with reference to the blastocyst formation rates, the average inner cell mass, trophectoderm cell number, and percentage of attachment of blastocysts (P > 0.05). The expression of ErbB4, Itg-β3, Itg-αv, TGF-α transcripts, and miR-Let-7a in blastocysts biopsied embryos did not differ from the non-biopsied blastocysts (P > 0.05). The results demonstrated that the preimplantation embryo development and attachment of biopsied embryos in vitro is not adversely affected by one blastomere biopsy at the eight-cell stage embryo.

114 Insulin-Like Growth Factor (IGF)2 and IGF2 Receptor (IGF2R) Murine Blastocyst Transcriptional Response after High Gaseous Pressure Exposure at 8-Cell Stage

Insulin-like growth factor 2 (IGF2) is a pleiotropic hormone encoded by an imprinted gene expressed in the paternal allele of mammals, and acts in physiological responses including cell proliferation, differentiation, and development. It is mediated through the IGF1R signalling pathway, whereas the IGF2R, a maternally imprinted gene, acts on lysosomal IGF2 degradation. As imprinted genes, both Igf2 and Igf2r expressions are more susceptible to dysregulation by environmental factors. This study aimed to evaluate the effect of exposure of 8-cell-stage murine embryos to 16 MPa of high gaseous pressure (HGP) on the relative Igf2 and Igf2r mRNA abundance in resulting blastocysts following in vitro culture (IVC). Day-3 embryos were recovered from superovulated Mus musculus domesticus females. Eight-cell embryos were exposed to 16 MPa HGP for either 2 h (P1 group) or 4 h (P2 group), with a Control group not exposed to HGP. Immediately after recovery or HGP exposure, embryos were in vitro-cultured for 48 h in mKSOM medium supplemented with 0.4% BSA at 37.5°C, 5% CO2, 5% O2, 90% N2, and saturated humidity. Resulting blastocysts were collected in pools of 10 and stored at –80°C, pending analysis. Following total mRNA extraction, cDNA syntesis and RT-qPCR were performed according to manufacturers. Values were normalized to the internal control Ppia gene. Relative gene expression was calculated using the 2−ΔΔ Ct approach. Blastocyst rates after IVC were compared by the Chi-squared test (P &lt; 0.05), with relative Igf2 and Igf2r expression data and Igf:Igf2r ratio analysed by ANOVA, after log-transformation when needed, with pairwise comparisons done by the Tukey test (P &lt; 0.05). No differences in blastocyst rates after IVC were observed among groups (Control: 94.2%; P1: 95.4%; P2: 94.1%). However, the Igf2 mRNA relative abundances in blastocysts were 6.3- and 4.2-fold lower in P1 (P &lt; 0.01) and P2 (P = 0.07) than in the Control group, respectively. Likewise, the Igf2r relative transcription levels were 6.6- and 2.2-fold down-regulated in blastocysts from the P1 (P &lt; 0.001) and P2 (P &lt; 0.01) groups, respectively, when compared with controls. Although the relative expression for both genes followed a down-regulation pattern in blastocysts exposed to HGP at the 8-cell stage, the Igf2:Igf2r ratio was 1.9-fold lower in blastocysts in the P2 group (P &lt; 0.05) than Controls, which was similar to the P1 group, indicating a potential stress adaptation response for embryo growth and development after exposure to HGP in the P1 group. It is known that cells under certain conditions of stress may halter growth and development as a response to initiate cellular events to maintain viability. Results from this study appear to translate such response process to HGP in both experimental groups. However, as embryo development and the Igf2:Igf2r ratio in embryos were similar between the Control and the P1 group, exposure to 16 MPa HGP for 2 h at the 8-cell-stage embryo does not seem to affect cell signalling to growth and proliferation up to the blastocyst stage.

Survival and developmental competence of bovine embryos at different developmental stages and separated blastomeres after vitrification in different solutions.

Generating techniques to enhance the success of blastomere separation is important for bovine economy, because it increases the number of transferable embryos. This study aimed to identify the optimum cryoprotectants for the vitrification of bovine embryos and the separation of blastomeres at different stages. In experiment 1, expanded blastocysts were vitrified in two different vitrification solutions, either (1) ethylene glycol (EG)+propylene glycol (PG) or (2) EG. The survival rate of blastocysts in the EG+PG was higher than that of the EG. In experiment 2, intact two-cell and eight-cell stage embryos were vitrified in the same solutions used in experiment 1. The EG+PG produced more dead embryos than the EG (P<0.05). In the EG, the rate of blastocyst formation was similar for the vitrified two- and eight-cell embryos and the non-vitrified ywo-cell embryos. In experiment 3, separated blastomeres of two- and eight-cell embryos were vitrified in EG. There was no difference in the rate of blastocyst formation and total number of cells between the two vitrified groups. In summary, at the blastocyst stage, EG+PG was superior, based on both survival rates and cell numbers; however, at the 2-8 cell stage, the use of EG alone was better than the other groups.

MicroRNA expression in bovine preimplantation embryos.

We profiled 98 mature microRNAs (miRNAs) using a stem-loop reverse transcription polymerase chain reaction assay array based on human miRNAs. We demonstrated that one, but not two, base-pair changes in the miRNA recognition sequence at the 3' end only marginally affected copy number estimates. Absolute levels of miRNAs were measured in matured cattle oocytes, eight-cell embryos and normal and parthenogenetic blastocysts and Day-14 trophoblast. Most miRNA concentrations were below the expected functional threshold required for effective repression of moderately to highly abundant target RNA. In oocytes and peri-embryonic genome activation embryos, miRNA 320, a member of the Dgcr8/Drosha-independent class of miRNAs, was expressed at greater than 1000 copies per embryo. miRNAs were more abundant at the eight-cell than the oocyte stage. miRNA concentrations per cell increased from the eight-cell to the blastocyst stage. Both the number of miRNA species and their expression levels were reduced in trophoblast tissue at Day 14. The parthenogenetic samples were more related in their miRNA expression profiles to each other than to their wild-type (in vitro-produced cultured) counterparts. miRNAs 299 and 323, which have been shown to be maternally expressed in other species, were also more than 4-fold overexpressed in the cattle parthenogenetic samples.

A second successful pregnancy using oocytes from a woman with premature ovarian failure with embryos transferred to a gestational carrier

Purpose: To describe a second successful pregnancy in a woman with premature ovarian failure who was successfully able to induce ovulation by restoring down-regulated follicle stimulating hormone (FSH) receptors, by lowering elevated FSH by inhibiting FSH release from the pituitary, by using ethinyl estradiol with subsequent stockpiling of embryos following in vitro fertilization (IVF) for subsequent future transfer to a gestational carrier. Materials and Methods: A day 3 eight-cell embryo obtained from a fertilized oocyte 10.5 years before was transferred to a gestational carrier. Results: The gestational carrier conceived and she successfully carried to six months to date. Conclusions: This is the first case of two successful pregnancies from the same woman with premature ovarian failure. Also, this is the longest an embryo derived from a woman in ovarian failure has been frozen leading to a successful pregnancy.

Transfer of pathogenic and nonpathogenic cytosolic proteins between spinal cord motor neurons in vivo in chimeric mice

Recent studies have reported spread of pathogenic proteins in the mammalian nervous system, but whether nonpathogenic ones spread is unknown. We initially investigated whether spread of a mutant amyotrophic lateral sclerosis-associated cytosolic superoxide dismutase 1 (SOD1) protein between motor neurons could be detected in intact chimeric mice. Eight-cell embryos from G85R SOD1YFP and G85R SOD1CFP mice were aggregated, and spinal cords of adult chimeric progeny were examined for motor neurons with cytosolic double fluorescence. By 3 mo of age, we observed extensive double fluorescence, including in amyotrophic lateral sclerosis-affected cranial nerve motor nuclei but not in the relatively spared extraocular nuclei. Chimeras of nonpathogenic wtSOD1YFP and G85R SOD1CFP also exhibited double fluorescence. In a third chimera, mitochondrial mCherry did not transfer to G85R SOD1YFP motor neurons, suggesting that neither RNA nor organelles transfer, but mito-mCherry neurons received G85R SOD1YFP. In a chimera of ChAT promoter-EGFP and mito-mCherry, EGFP efficiently transferred to mito-mCherry+ cells. Thus, nonpathogenic cytosolic proteins appear capable of transfer. During study of both the SOD1FP and EGFP chimeras, we observed fluorescence also in small cells neighboring the motor neurons, identified as mature gray matter oligodendrocytes. Double fluorescence in the G85R SOD1FP chimera and observation of the temporal development of fluorescence first in motor neurons and then in these oligodendrocytes suggest that they may be mediators of transfer of cytosolic proteins between motor neurons.

ECAT1 is essential for human oocyte maturation and pre-implantation development of the resulting embryos.

ECAT1 is a subunit of the subcortical maternal complex that is required for cell cycle progression during pre-implantation embryonic development; however, its exact function remains to be elucidated. Here we investigated the expression of ECAT1 in human ovarian tissue, oocytes and pre-implantation embryos and assessed its function by using RNA interference (RNAi) in oocytes. ECAT1 mRNA was highly expressed in human oocytes and zygotes, as well as in two-cell, four-cell and eight-cell embryos, but declined significantly in morulae and blastocysts. ECAT1 was expressed in the cytoplasm of oocytes and pre-implantation embryos and was localized more specifically in the cortical region than in the inner cytoplasm. RNAi experiments demonstrated that down-regulation of ECAT1 expression not only impaired spindle assembly and reduced maturation and fertilization rates of human oocytes but also decreased the cleavage rate of the resulting zygotes. In conclusion, our study indicates that ECAT1 may play a role in meiotic progression by maintaining the accuracy of spindle assembly in human oocytes, thus promoting oocyte maturation and subsequent development of the embryo.

RhoA/ROCK pathway activity is essential for the correct localization of the germ plasm mRNAs in zebrafish embryos

Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos.

Embryo selection using time-lapse analysis (Early Embryo Viability Assessment) in conjunction with standard morphology: a prospective two-center pilot study.

Does prospective embryo selection using the results from the Eava Test (Early Embryo Viability Assessment) in combination with standard morphology increase the pregnancy rate of IVF and ICSI patients compared to embryo selection based on morphology only? Embryo selection using the Eeva Test plus standard morphology on Day 3 results in comparable pregnancy rates as conventional morphological embryo selection. Time-lapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. The Eeva Test records the development of each embryo with a cell-tracking system and predicts the likelihood (High, Medium or Low) that an embryo will form a blastocyst based on an automated analysis of early cell division timings. This trial was designed as a prospective, observational, two-center pilot study with a propensity matched control group. The analysis involved 280 of 302 enrolled patients who were included in the Eeva Test group in 2013 and 560 control patients who were treated in the years 2011-2013. The majority of transfers (98%) were single embryo transfers. Two academic hospitals (VUmc Amsterdam and UZ Gent) enrolled patients <41 years old, with <3 previous attempts and ≥5 normally fertilized eggs. Propensity matching was used to identify a propensity matched control group from a cohort of 1777 patients based on age, cycle number, oocyte number and number of fertilized oocytes. There was no difference in patient baseline characteristics between the two groups. The ongoing pregnancy rate (OPR) of patients enrolled in the Eeva Test group (34.3%; 96/280) did not differ significantly from the OPR in the propensity matched control group (34.6%, 194/560; P =0.92). However, significantly less top quality embryos (eight-cell embryos with ≤25% fragmentation) were transferred in the Eeva Test group compared to the propensity matched control group (70.4% vs. 82.3%; P<0.001). The transfer of Eeva High and Medium embryos resulted in a significantly higher OPR of 36.8% (89/242) compared to 18.4% (7/38) for Eeva Low embryos (P=0.02). This pilot study is limited by its nonrandomized design with a concurrent and historical control. Our pilot data did not reveal significant differences between time-lapse based and conventional embryo selection. Interestingly, the pregnancy rates were comparable in both groups even though the morphological quality of the transferred embryos was significantly lower in the Eeva Test group compared to the propensity matched control group. A sufficiently powered three-armed randomized controlled trial (RCT) with a solid design should be performed to generate decisive evidence in the future. Progyny Inc., formerly Auxogyn provided the Eeva scopes, software and technical support for this study. The funding sources did neither influence data collection, management, analysis and interpretation of the data, nor the preparation of the manuscript. ClinicalTrials.gov: NCT01671644.

Maternal obesity in mice not only affects fresh embryo quality but also aggravates injury due to vitrification.

The aims of the present study are to identify the mechanism(s) whereby obesity impairs fresh embryos and to clarify the effects of vitrification on lipid droplet content within embryos from maternally obese mice. The diet-induced obesity mouse model was established, and the zygotes were captured and cultured to day 3. The eight-cell embryos were selected and divided into fresh and vitrified groups. The blastocysts derived from fresh embryos were used as a control. The expression profiles of endoplasmic reticulum (ER) stress genes (Atf4, Grp78, and Hsp70) and other genes (MnSOD, p53, Gadd45g, caspase-3, IGF-II, ZO-1, and E-cadherin) on day-3 fresh and post-warming eight-cell embryos from obese and control groups were determined. For day-5 fresh blastocysts and blastocysts previously vitrified on day 3, the expression profiles for all of the above genes were also determined. For the fresh group, obesity significantly upregulated Hsp70, p53, IGF-II, and ZO-1 expression in embryos on day 3 and notably upregulated Atf4, MnSOD, Gadd45g, caspase-3, ZO-1, and E-cadherin expression in blastocysts on day 5. For vitrified ones, obesity significantly upregulated Atf4, MnSOD, and Gadd45g expression in embryos on day 3 and notably upregulated Hsp70 expression and downregulated MnSOD in day 5 blastocysts previously vitrified on day 3. Obesity impairs fresh embryos and aggravates embryonic vitrification injury at a molecular level.

A novel long intergenic noncoding RNA indispensable for the cleavage of mouse two-cell embryos.

Endogenous retroviruses (ERVs) are transcriptionally active in cleavage stage embryos, yet their functions are unknown. ERV sequences are present in the majority of long intergenic noncoding RNAs (lincRNAs) in mouse and humans, playing key roles in many cellular processes and diseases. Here, we identify LincGET as a nuclear lincRNA that is GLN-, MERVL-, and ERVK-associated and essential for mouse embryonic development beyond the two-cell stage. LincGET is expressed in late two- to four-cell mouse embryos. Its depletion leads to developmental arrest at the late G2 phase of the two-cell stage and to MAPK signaling pathway inhibition. LincGET forms an RNA-protein complex with hnRNP U, FUBP1, and ILF2, promoting the cis-regulatory activity of long terminal repeats (LTRs) in GLN, MERVL, and ERVK (GLKLTRs), and inhibiting RNA alternative splicing, partially by downregulating hnRNP U, FUBP1, and ILF2 protein levels. Hnrnpu or Ilf2 mRNA injection at the pronuclear stage also decreases the preimplantation developmental rate, and Fubp1 mRNA injection at the pronuclear stage causes a block at the two-cell stage. Thus, as the first functional ERV-associated lincRNA, LincGET provides clues for ERV functions in cleavage stage embryonic development.

CHD1 Regulates Deposition of Histone Variant H3.3 During Bovine Early Embryonic Development.

The CHD family of proteins is characterized by the presence of chromodomains and SNF2-related helicase/ATPase domains, which alter gene expression by modification of chromatin structure. Chd1-null embryos arrest at the peri-implantation stage in mice. However, the functional role of CHD1 during preimplantation development remains unclear, given maternal-derived CHD1 may mask the essential role of CHD1 during this stage in traditional knockout models. The objective of this study was to characterize CHD1 expression and elucidate its functional role in preimplantation development using the bovine model. CHD1 mRNA was elevated after meiotic maturation and remained increased through the 16-cell stage, followed by a sharp decrease at morula to blastocyst stage. Similarly, immunoblot analysis indicated CHD1 protein level is increased after maturation, maintained at high level after fertilization and declined sharply afterwards. CHD1 mRNA level was partially decreased in response to alpha-amanitin (RNA polymerase II inhibitor) treatment, suggesting that CHD1 mRNA in eight-cell embryos is of both maternal and zygotic origin. Results of siRNA-mediated silencing of CHD1 in bovine early embryos demonstrated that the percentages of embryos developing to the 8- to 16-cell and blastocyst stages were both significantly reduced. However, expression of NANOG (inner cell mass marker) and CDX2 (trophectoderm marker) were not affected in CHD1 knockdown blastocysts. In addition, we found that histone variant H3.3 immunostaining is altered in CHD1 knockdown embryos. Knockdown of H3.3 using siRNA resulted in a similar phenotype to CHD1-ablated embryos. Collectively, our results demonstrate that CHD1 is required for bovine early development, and suggest that CHD1 may regulate H3.3 deposition during this period.

Production of early monozygotic twin bovine embryos in vitro by the blastomere separation and coculture technique

The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres (groups of at least 30 at the eight-cell stage) were separated into eight segments (to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments (blastomeres) were cultured respectively in microwells on the bottom of the four-well dish (Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics (cocultured with nothing, intact embryos, bovine cumulus cells (bCCs), intact embryos & bCCs) were applied to the group of four segments (blastomeres). Finally, diameter and inner cell mass (ICM):trophectoderm (TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group (P<0.05). In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo & bCCs (P<0.05). Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo (P<0.05), more important quality indicator of the blastocyst diameter and ICM:TE cell ratio was similar between our experimental group and the control group (P>0.05). Thus, these results suggest that combined with intact embryos & bCCs coculture system, culturing four isolated segments (blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.

The role of diet and housing-temperature in the production of genetically modified mouse embryos and their developmental capacity after cryopreservation

Mutant mouse lines are unique models with an enormous scientific potential. Cryopreservation of preimplantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if sufficient specimens have been cryopreserved. Prerequisites to economically cryopreserve embryos are high yields of embryos prepared from donors and a high recovery rate after revitalization. Diets for laboratory animals are often produced from phytoestrogen-containing soy; the present study shows that feeding the donor animals with a phytoestrogen-poor diet is more efficient compared to a phytoestrogen-containing, soy-based diet. Additionally, a uterotrophic bioassay indicating the estrogenic role of compounds showed a significant increase of the relative uterus size of females fed with a phytoestrogen-rich diet. The role of the housing-temperature was investigated, too, showing that a housing-temperature of 24 °C results in the best embryo yields. The production of two-cell embryos is more economic than the production of eight-cell embryos. Investigating the recovery rate of frozen/thawed embryos, a very high recovery rate was determined when both, two- and eight-cell embryos were thawed. However, the capacity to develop to the next embryonic stage in vitro was dramatically reduced when two-cell embryos were compared to eight-cell embryos. After embryo transfer, the sex ratio became uneven and more males were delivered. This effect might be due to the procedures to which animals and embryos were subjected. These data show that many parameters can influence the production of animals when using (frozen/thawed) embryos. These parameters need continuous surveillance.

Histone methyltransferase Smyd3 regulates early embryonic lineage commitment in mice

SET and MYND domain-containing protein 3 (Smyd3) is a histone H3 lysine 4 (H3K4) di- and tri-methyltransferase that forms a transcriptional complex with RNA polymerase II and activates the transcription of oncogenes and cell cycle genes in human cancer cells. However, the study of Smyd3 in mammalian early embryonic development has not yet been addressed. In the present study, we investigated the expression pattern of Smyd3 in mouse preimplantation embryos and the effects of RNA interference (RNAi)-mediated Smyd3 repression on the development of mouse embryos. We showed that Smyd3 mRNA levels increased after the two-cell stage, peaked at the four-cell stage, and gradually decreased thereafter. Moreover, in two-cell to eight-cell embryos, SMYD3 staining was more intense in the nuclei than it was in the cytoplasm. In Smyd3-knockdown embryos, the percentage of inner cell mass (ICM)-derived colony formation and trophectoderm (TE)-derived cell attachment were significantly decreased, which resulted in a reduction in the number of viable offspring. Furthermore, the expression of Oct4 and Cdx2 during mid-preimplantation gene activation was significantly decreased in Smyd3-knockdown embryos. In addition, the transcription levels of ICM and epiblast markers, such as Oct4, Nanog, and Sox2, the transcription levels of primitive endoderm markers, such as Gata6, and the transcription levels of TE markers, such as Cdx2 and Eomes, were significantly decreased in Smyd3-knockdown blastocysts. These findings indicate that SMYD3 plays an important role in early embryonic lineage commitment and peri-implantation development through the activation of lineage-specific genes.

Sexually dimorphic gene expression emerges with embryonic genome activation and is dynamic throughout development.

BackgroundAs sex determines mammalian development, understanding the nature and developmental dynamics of the sexually dimorphic transcriptome is important. To explore this, we generated 76 genome-wide RNA-seq profiles from mouse eight-cell embryos, late gestation and adult livers, together with 4 ground-state pluripotent embryonic (ES) cell lines from which we generated both RNA-seq and multiple ChIP-seq profiles. We complemented this with previously published data to yield 5 snap-shots of pre-implantation development, late-gestation placenta and somatic tissue and multiple adult tissues for integrative analysis.ResultsWe define a high-confidence sex-dimorphic signature of 69 genes in eight-cell embryos. Sex-chromosome-linked components of this signature are largely conserved throughout pre-implantation development and in ES cells, whilst the autosomal component is more dynamic. Sex-biased gene expression is reflected by enrichment for activating and repressive histone modifications. The eight-cell signature is largely non-overlapping with that defined from fetal liver, neither was it correlated with adult liver or other tissues analysed. The number of sex-dimorphic genes increases throughout development. We identified many more dimorphic genes in adult compared to fetal liver. However, approximately two thirds of the dimorphic genes identified in fetal liver were also dimorphic in adult liver. Sex-biased expression differences unique to adult liver were enriched for growth hormone-responsiveness. Sexually dimorphic gene expression in pre-implantation development is driven by sex-chromosome based transcription, whilst later development is characterised by sex dimorphic autosomal transcription.ConclusionThis systematic study identifies three distinct phases of sex dimorphism throughout mouse development, and has significant implications for understanding the developmental origins of sex-specific phenotypes and disease in mammals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1506-4) contains supplementary material, which is available to authorized users.

Concise reviews: Assisted reproductive technologies to prevent transmission of mitochondrial DNA disease.

While the fertilized egg inherits its nuclear DNA from both parents, the mitochondrial DNA is strictly maternally inherited. Cells contain multiple copies of mtDNA, each of which encodes 37 genes, which are essential for energy production by oxidative phosphorylation. Mutations can be present in all, or only in some copies of mtDNA. If present above a certain threshold, pathogenic mtDNA mutations can cause a range of debilitating and fatal diseases. Here, we provide an update of currently available options and new techniques under development to reduce the risk of transmitting mtDNA disease from mother to child. Preimplantation genetic diagnosis (PGD), a commonly used technique to detect mutations in nuclear DNA, is currently being offered to determine the mutation load of embryos produced by women who carry mtDNA mutations. The available evidence indicates that cells removed from an eight-cell embryo are predictive of the mutation load in the entire embryo, indicating that PGD provides an effective risk reduction strategy for women who produce embryos with low mutation loads. For those who do not, research is now focused on meiotic nuclear transplantation techniques to uncouple the inheritance of nuclear and mtDNA. These approaches include transplantation of any one of the products or female meiosis (meiosis II spindle, or either of the polar bodies) between oocytes, or the transplantation of pronuclei between fertilized eggs. In all cases, the transferred genetic material arises from a normal meiosis and should therefore, not be confused with cloning. The scientific progress and associated regulatory issues are discussed. Stem Cells 2015;33:639–645