Cytoplasm Of Zygotes Research Articles

Creation of true interspecies hybrids: Rescue of hybrid class with hybrid cytoplasm affecting growth and metabolism.

Interspecies hybrids have nuclear contributions from two species but oocyte cytoplasm from only one. Species discordance may lead to altered nuclear reprogramming of the foreign paternal genome. We reasoned that initial reprogramming in same species cytoplasm plus creation of hybrids with zygote cytoplasm from both species, which we describe here, might enhance nuclear reprogramming and promote hybrid development. We report in Mus species that (i) mammalian nuclear/cytoplasmic hybrids can be created, (ii) they allow development and viability of a previously missing and uncharacterized hybrid class, (iii) different oocyte cytoplasm environments lead to different phenotypes of same nuclear hybrid genotype, and (iv) the novel hybrids exhibit sex ratio distortion with the absence of female progeny and represent a mammalian exception to Haldane's rule. Our results emphasize that interspecies hybrid phenotypes are not only the result of nuclear gene epistatic interactions but also cytonuclear interactions and that the latter have major impacts on fetal and placental growth and development.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

“Usage a CRISPR/Cas9-based for obtaining knockouts of economically significant cattle genes”

An experimental work dealing with the gene modification using the Cas9 RNA-based editing system was performed. Point site-specific breakpoints in gDNA were introduced at the zygote stage by microinjection of spCas9 mRNA protein and guide RNAs into the zygote cytoplasm. Oocytes that extruded the first and second polar bodies were used for the injection. 2 series of microinjections of gene editing designs for early bovine embryos were made. The degeneration ranged from 10% to 56% in different groups. A total of 100 injections were performed. Cleavage was started by 78% of the surviving oocytes; 5 embryos reached the blastocyst stage, which was 16% of the number of dividing embryos. All the resulting embryos were analyzed to evaluate the efficiency of editing. gDNA was isolated from all embryos that had reached the blastocyst stage. Using Sanger sequencing of genes of interest in pre-implantation bovine embryos and biopsies from them, it was shown that in 5 out of 17 embryos resulting from microinjections of guide RNA against the BLG gene and SpCas9 mRNA, and in 2 out of 9 embryos after microinjections of guide RNA against CD209 gene and SpCas9 mRNA, the required genome modifications were found. This is indicative of the high efficiency of this delivery method of the editing system.

Activated macrophages of CD 163 gene edited pigs generated by direct cytoplasmic microinjection with CRISPR gRNA/Cas9 mRNA are resistant to PRRS virus assault

Porcine reproductive and respiratory syndrome virus (PRRSV) infects placental and lung macrophages, causing a global epidemic with economic loss. Attempts to develop an effective vaccine to control the disease have not been effective. Currently, developing PRRSV disease-resistant pigs via a gene editing (GE) strategy to mutate the PRRSV receptor or to delete the binding domain on the macrophage appears promising. In this study, we used the strategy of Edinburg University to construct two guide RNAs (gRNAs) located on the proximal front and post sites of exon 7. Directive microinjection of two gRNAs and Cas9 mRNA into the cytoplasm of pronuclear zygotes efficiently generated four piglets confirmed as CD163 knockout (KO) and/or CD163 exon 7 deleted (CD163ΔE7). In four GE piglets, three pigs carried two chromosome CD163 KO or ΔE7. Peripheral blood mononuclear cells (PBMCs) from three GE and wild-type (WT) pigs were activated into macrophages for in vitro transfection. The results showed that the activated macrophages from all GE pigs were significantly more viable than those from WT pig. Current results suggest that we have successfully generated PRRSV-resistant pigs, although in vivo challenge is needed to validate that the pigs are PRRSV resistant.

The effect of CRISPR constructs microinjection on the expression of developmental genes in Rag1 knocked-out mice embryo.

Despite all the advances in the production of transgenic mice, the production efficiency of these animal models is still low. Given that the expression of developmental genes has a critical role in growth and development of embryo, we determined the expression pattern of pluripotency, trophectoderm and imprinting genes in the Rag1 (recombination‐activating gene 1) knocked‐out blastocysts resulting from microinjection of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9) constructs into the zygote cytoplasm of C57bl6 mice. Following microinjection, the embryos were cultured and the gene expression of developed blastocysts and natural blastocysts (Sham and control groups) were evaluated using real‐time PCR. The agarose gel to confirm the deletion in the Rag1 gene in Rag1 knocked‐out blastocyst. Our results showed that the expression of trophectoderm genes (‐TEAD‐4 and Cdx2), pluripotency genes (Nanog and Oct‐4) and imprinting gene (H19) in the Rag1 knocked‐out group was significantly lower compared with the embryos obtained from Natural fertilization. According to these findings, manipulation, embryo culture and microinjection of CRISPR constructs into the zygote cytoplasm of mice led to reduced expression of imprinting, pluripotency and trophectoderm genes. Therefore, the Rag1 knocked‐out embryos produced by the CRISPR/Cas9 system are of low quality, which reduces the chances of live birth in these animals and may cause various abnormalities in fetuses.

Automated and precise recognition of human zygote cytoplasm: A robust image-segmentation system based on a convolutional neural network

In this study, we evaluated the precision and reproducibility of convolutional neural network (CNN) measurements of zygote cytoplasm segmentation relative to experienced embryologists. We developed an automated system for segmenting human zygote cytoplasm using images and a CNN trained on a set of 550 zygote images labeled by embryologists. Further, we compared the consistency in cytoplasmic area measurements using images segmented by the CNN and the trained human eye (embryologists), as well as the precision and reproducibility of the measurements and the effect of several common image attributes. Moreover, the use of CNN results for 8377 zygote images for regularity classification revealed inter-observer agreement between two embryologists (98 ± 1.02 %) and between embryologists and the CNN regarding the cytoplasmic area (97.75 ± 1.45 %), with intra-observer agreement for the CNN system at 100 %. Furthermore, neither luminance nor image noise significantly affected CNN measurement precision; however, highly irregular zygote shapes reduced precision to 95 % for both. For classification, automatic segmentation of images of 8377 zygotes (area measurement: 9741.34 ± 7951.83 μm2; shapes: regular, 65.37 %; slightly irregular, 15.10 %; moderately irregular, 9.91 %; and highly irregular, 9.62 %) revealed good performance by the CNN in classifying both regularly and irregularly shaped zygotes (area under curve: 0.874 ± 0.043). These results demonstrated the efficacy of the CNN system for precisely measuring the zygote cytoplasmic area, classifying the shape regularity, and assisting with embryo assessment.

Optimizing injection time of GFP plasmid into sheep zygote.

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6-8hpi, n: 120; 8-10hpi, n: 122; 10-12hpi, n: 110 and 12-14hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p< 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8-10hpi compared with other injected groups (4 % versus 0 %, p <0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8-10hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.

Ratio of the zygote cytoplasm to the paternal genome affects the reprogramming and developmental efficiency of androgenetic embryos.

Uniparental embryos have uniparental genomes and are very useful models for studying the specific gene expression of parents or for exploring the biological significance of genomic imprinting in mammals. However, the early developmental efficiency of androgenetic embryos is significantly lower than that of parthenogenetic embryos. In addition, oocytes are able to reprogram sperm nuclei after fertilization to guarantee embryonic development by maternally derived reprogramming factors, which accumulate during oogenesis. However, the importance of maternal material in the efficiency of reprogramming the pronucleus of androgenetic embryos is not known. In this study, androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS). Compared with DS embryos, PT embryos that were derived from two zygotes contained more maternal material, like 10-11 translocation methylcytosine deoxygenase 3 (Tet3) and histone variant 3.3 (H3.3). Our experiments confirmed the better developmental potential of PT embryos, which had higher blastocyst rates, a stronger expression of pluripotent genes, a lower expression of apoptotic genes, and superior blastocyst quality. Our findings indicate that the aggregation of more maternal materials in the paternal pronucleus facilitate the reprogramming of the paternal genome, improving embryonic development in PT androgenesis.

Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes.

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T (C>T) or A→G (A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing (GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MII transition. Injection of cytosine base editor 3 (BE3) mRNA and X-linked Dmd-specific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MII oocytes developed into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4% ±2.1%. There 40%-78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five (5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine (9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.

Anephrogenic phenotype induced by SALL1 gene knockout in pigs

To combat organ shortage in transplantation medicine, a novel strategy has been proposed to generate human organs from exogenous pluripotent stem cells utilizing the developmental mechanisms of pig embryos/foetuses. Genetically modified pigs missing specific organs are key elements in this strategy. In this study, we demonstrate the feasibility of using a genome-editing approach to generate anephrogenic foetuses in a genetically engineered pig model. SALL1 knockout (KO) was successfully induced by injecting genome-editing molecules into the cytoplasm of pig zygotes, which generated the anephrogenic phenotype. Extinguished SALL1 expression and marked dysgenesis of nephron structures were observed in the rudimentary kidney tissue of SALL1-KO foetuses. Biallelic KO mutations of the target gene induced nephrogenic defects; however, biallelic mutations involving small in-frame deletions did not induce the anephrogenic phenotype. Through production of F1 progeny from mutant founder pigs, we identified mutations that could reliably induce the anephrogenic phenotype and hence established a line of fertile SALL1-mutant pigs. Our study lays important technical groundwork for the realization of human kidney regeneration through the use of an empty developmental niche in pig foetuses.

Optimization Strategy for Generating Gene-edited Tibet Minipigs by Synchronized Oestrus and Cytoplasmic Microinjection.

The Tibet minipig is a rare highland pig breed worldwide and has many applications in biomedical and agricultural research. However, Tibet minipigs are not like domesticated pigs in that their ovulation number is low, which is unfavourable for the collection of zygotes. Partly for this reason, few studies have reported the successful generation of genetically modified Tibet minipigs by zygote injection. To address this issue, we described an efficient way to generate gene-edited Tibet minipigs, the major elements of which include the utilization of synchronized oestrus instead of superovulation to obtain zygotes, optimization of the preparation strategy, and co-injection of clustered regularly interspaced short palindromic repeat sequences associated protein 9 (Cas9) mRNA and single-guide RNAs (sgRNAs) into the cytoplasm of zygotes. We successfully obtained allelic TYR gene knockout (TYR-/-) Tibet minipigs with a typical albino phenotype (i.e., red-coloured eyes with light pink-tinted irises and no pigmentation in the skin and hair) as well as TYR-/-IL2RG-/- and TYR-/-RAG1-/- Tibet minipigs with typical phenotypes of albinism and immunodeficiency, which was characterized by thymic atrophy and abnormal immunocyte proportions. The overall gene editing efficiency was 75% for the TYR single gene knockout, while for TYR-IL2RG and TYR-RAG1 dual gene editing, the values were 25% and 75%, respectively. No detectable off-target mutations were observed. By intercrossing F0 generation minipigs, targeted genetic mutations can also be transmitted to gene-edited minipigs' offspring through germ line transmission. This study is a valuable exploration for the efficient generation of gene-edited Tibet minipigs with medical research value in the future.

Establishment of a technological system via zygote injection of CRISPR/Cas9 system for the generation of gene modified ziwuling black goats

Zygote injection of CRISPR/Cas9 system is an efficient method for generation of gene modified animals. Efficiency of zygote collection, embryo survival rate after micro-injection and pregnancy rate of surrogate are the key influence factors for the generation of gene modified animals using this strategy. Here, we investigated the influence of CIDR treatment at different time during the estrus cycle to the superovulation, and analyzed the influence of the estrus terminal time between embryo donor and embryo receptor to the pregnancy rate in Ziwuling black goats. We designed three sgRNAs targeting myostatin ( MSTN ) and also optimized the micro-injected parameter of the CRISPR/Cas9 system. The results showed that embryo donor goats effectively increase the corpus luteum amount (15.25±3.69) and zygote amount (13.875±4.015) when they were treated with CIDR at the fourth day of estrus cycle. The mixture including Cas9 mRNA (60 ng/μL) and sgRNAs (60 ng/μL) targeting MSTN was injected into zygote cytoplasm using a micromanipulator at the optimized injection parameters (Pi, Ti and Pc was 300, 0.5 and 60, respectively). Embryo cleavage rate was 76.79 % after 24 h of embryo injection. Pregnancy rate of embryo receptors whose terminal estrus time was delayed 12–16 h compared with that of embryo donor was the highest (38.46%). A total of 19 living lambs were obtained, and indels were detected in all the lambs using TA cloning sequencing. Overall, we established the technological system based on the zygote injection of CRISPR/Cas9 in Ziwuling black goats. The established technological system is the basic for generation of gene modified goats.

57 Disruption of TET1 Leads to Abnormal Expression of Pluripotency-Related Genes in Porcine Embryos

DNA methylation is one of the principal epigenetic modifications playing an essential role in regulating gene expression. The TET family (1-3) is implicated in initiating the demethylation process by converting 5-methylcytosine (5mC) to 5-hydroxymethyl cytosine (5hmC) during embryogenesis. Previous studies in mice suggest that TET1 is required for pluripotency and maintenance of embryonic stem cells by managing their epigenetic marks, specifically DNA methylation. This raises the possibility that TET1 is capable of establishing distinct epigenetic marks during embryo development, thus regulating pluripotency-related genes. However, this has not been demonstrated in any species. Previously we have demonstrated that the level of TET1 (mRNA and protein) was high in porcine blastocysts. In this study, we generated TET1 knockout embryos and analysed expression patterns of pluripotency-related genes in blastocysts to study the role of TET1 in maintaining pluripotency during porcine embryo development. The CRISPR/Cas9 system was applied to disrupt the TET1 gene during embryogenesis. Three single-guide RNAs (sgRNAs) were designed based on our previous cloning of the TET1 gene. In vitro-synthesised Cas9 mRNA (20 ng µL−1) and sgRNAs (10 ng µL−1 each) were injected into the cytoplasm of zygotes after IVF. A total of 605 zygotes were used for microinjection and subsequently 54 blastocysts were formed. As a control, 89 IVF blastocysts were developed from 240 embryos. Nine to ten blastocysts per group were collected on Day 7 to analyse gene expression patterns of TET family and pluripotency-related genes using quantitative RT-PCR. Three biological and 3 experimental replications were used. Differences in the gene expression were evaluated by ANOVA. As expected, there was a 2-fold decrease in the transcript level of TET1 in TET1-knockout blastocysts compared with that in control IVF blastocysts (P &lt; 0.05). Interestingly, an increase in TET3 mRNA (P &lt; 0.01) and numeric increase of TET2 mRNA was observed in TET1-knockout blastocysts. We could also detect an elevated level of pluripotency-related genes in TET1-knockout blastocysts; the expression of NANOG, ESRRB, ZFP42, and TCL1A was up-regulated. However, there was no significant change in the expression level of other pluripotency-related genes (POU5F1, SOX2, KLF2, PRDM14, and DPPA3) in TET1 knockout blastocysts. In this study, we found that TET1 is involved in regulating expression of pluripotency-related genes: NANOG, ESRRB, ZFP42, and TCL1A. The loss of functional TET1 resulted in elevated expression of these genes. The reason for this is still under investigation, although TET3, known to have a positive correlation with the level of NANOG, could be involved. A 3-fold increase in TET3 mRNA response to the TET1-knockout may suggest a compensatory mechanism between TET1 and TET3 during porcine embryogenesis. To further understand these actions, we intend to analyse DNA methylation (5mc and 5hmc) levels on the promoter region of the genes. In addition, embryos lacking functional TET1 and TET3 will be generated to explore a potential compensatory effect of TET3 under the absence of TET1.

Cytoplasmic cleavage of DPPA3 is required for intracellular trafficking and cleavage-stage development in mice

Degradation of maternal proteins by the ubiquitin-proteasome system (UPS) accompanies the maternal-to-zygotic transition. DPPA3/Stella/PGC7, encoded by a maternal effect gene, is present in the nucleus and cytoplasm of zygotes and has been associated with protecting the female pronucleus from TET3-mediated demethylation. We now report that cytoplasmic DPPA3 is partially cleaved by the ubiquitin-proteasome system and an N-terminus fragment remains in the cytoplasm where it associates with early and re-cycling endosomes. If DPPA3 is absent or if cleavage is prevented, multiple vesicles coalesce/aggregate and markers of lysosomes are decreased. Fertilized eggs develop poorly into blastocysts, which results in significantly decreased fecundity of Dppa3R60A transgenic mice. This phenocopies aspects of Lamp1/2 knockdowns and Dppa3KO embryos can be partially rescued in vitro by DPPA31–60 and to a lesser extent by LAMP1/2. Thus, the N-terminus of DPPA3 has a significant role in cytoplasmic vesicular trafficking in addition to its previously reported nuclear function.

Effect of progesterone and its synthetic analogs on reproduction and embryonic development of a freshwater invertebrate model

The presence of a mixture of progestogens at ng/L concentration levels in surface waters is a worldwide problem. Only a few studies explore the effect of progestogen treatment in a mixture as opposed to individual chemicals to shed light on how non-target species respond to these contaminants. In the present study, we used an invertebrate model species, Lymnaea stagnalis, exposed to a mixture of four progestogens (progesterone, levonorgestrel, drospirenone, and gestodene) in 10ng/L concentration for 3 weeks. Data at both physiological and cellular/molecular level were analyzed using the ELISA technique, stereomicroscopy combined with time lapse software, and capillary microsampling combined with mass spectrometry.The treatment of adult Lymnaeas caused reduced egg production, and low quality egg mass on the first week, compared to the control. Starting from the second week, the egg production, and the quality of egg mass were similar in both groups. At the end of the third week, the egg production and the vitellogenin-like protein content of the hepatopancreas were significantly elevated in the treated group. At the cellular level, accelerated cell proliferation was observed during early embryogenesis in the treated group. The investigation of metabolomic changes resulted significantly elevated hexose utilization in the single-cell zygote cytoplasm, and elevated adenylate energy charge in the egg albumen. These changes suggested that treated snails provided more hexose in the eggs in order to improve offspring viability. Our study contributes to the knowledge of physiological effect of equi-concentration progestogen mixture at environmentally relevant dose on non-target aquatic species.

Manipulations with early mouse embryos for generation of genetically modified animals

Recently, genome-editing technologies have become more efficient and accessible. The discovery of nucleases for directional genome editing (CRISPR/Cas9, TALEN, ZFNs) significantly accelerated and simplified the production of mice with targeted gene editing in the genome. Until last time, the CRISPR/Cas9 system noticeably simplified the preparation of knockout or transgenic mice. CRISPR/Cas9 technology was successfully applied for gene knockout and knock-in, generation of large deletions or directed insertions in targeted genome regions in embryonic stem cells (ESCs).When injected into blastocysts, such modified ESCs are able to generate chimeras producing gametes with an identical genotype with ESC. Thus, it can identify animals with modified genomes. More recently, CRISPR/Cas9 technology was successfully applied to mouse zygotes and the birth of genetic modified mice was observed, i. e., the time required for generating genome-modified animals decreased significantly. The CRISPR/Cas9 system allows making gene knockout, large deletions or directed insertions into the target region of the genome by cytoplasm or pronuclear microinjection into zygotes. In addition, this is faster and simpler than similar work with mouse ESCs. Meanwhile, methods of manipulation with early embryos and their transplantation to surrogate mothers may be somewhat tricky. Therefore, it is important to use modern technologies for directional genome editing and perfect mastery in the embryological technics. In this article, we describe the protocols of microinjection into the pronucleus or cytoplasm of zygotes and injection of embryonic stem cells into the blastocyst cavity. We also describe embryological methods, such as superovulation, preparation of early stage embryos, surgical operation, production of foster mice. In addition, we describe the assembly and necessary components for the isoflurane anesthetic apparatus and isoflurane anesthesia.

91 DISRUPTION OF TET1 DURING PORCINE EMBRYOGENESIS USING CRISPR/Cas9 SYSTEM

Ten-eleven translocation (TET) enzymes catalyse oxidation of 5-methylcytosine to 5-hydroxymethyl cytosine. This TET-mediated conversion of 5-methylcytosine to 5-hydroxymethyl cytosine is implicated in initiating the DNA demethylation process, observed post-fertilization. Three members (TET1–3) of the TET family are differentially expressed during embryo development and appear to have different roles. Previous studies in mice suggest that TET1 is a key regulator in maintaining pluripotency in embryonic stem cells by managing epigenetic marks such as DNA methylation. This would imply that TET1 should be a regulator of epigenetic marks during embryo development, although this has not been demonstrated. Previously, we have cloned porcine TET1 from blastocysts (GenBank accession number KC137683) and demonstrated that the level of TET1 (mRNA and protein) was high in blastocysts. The protein level was greater in the inner cell mass compared with the trophectoderm. In this study, we generated TET1 knockout porcine embryos using CRISPR/Cas9 system to study the role of TET1 in controlling epigenetic marks during porcine embryo development. First, 2 sgRNA, immediately downstream of the presumable translation initiation site, were designed and synthesised; location of the sgRNA were nucleotide position at 2 to 21 bp and 23 to 42 bp, respectively (KC137683). Then, sgRNA (10 ng μL−1 each) and Cas9 mRNA (20 ng μL−1) were injected into the cytoplasm of IVF zygotes, and Day 7 blastocysts were genotyped. All embryos carried mutations on both alleles of TET1 (10/10), one homozygous and 9 biallelic mutations. However, immunocytochemistry analysis of other CRISPR/Cas9 injected embryos revealed that TET1 was not removed (10/10), indicating that the sgRNA may have not introduced a premature stop codon 3′ to the presumable translation initiation site. Therefore, 2 new sgRNA were designed to generate a premature stop codon at the 5′ side of a key functional domain, the 2-oxoglutarate-Fe(II)-dependent oxygenase domain (4690 to 5160 bp); the locations of the 2 sgRNA were 4450 to 4469 bp and 4501 to 4520 bp, respectively. Similarly, all of the embryos carried mutations in TET1 (7/7), 2 homozygous and 5 biallelic mutations. In addition, TET1 proteins were not detected in 11 of 16 blastocysts, confirmed by immunocytochemistry. In this study, we successfully generated embryos lacking TET1 by introducing designed CRISPR/Cas9 system during embryogenesis. Presence of TET1 from the first injection experiment suggests that the presumable translation initiation site is not accurate. Discrepancy between genotyping and immunocytochemistry results from the second injection experiment indicates that embryos possessing TET1 protein probably have mutations in triplets, thus no premature stop codon was synthesised. Further studies will focus on identifying the role of TET1 in maintaining pluripotency and epigenetic modification during pre-implantation stage using these embryos.

Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs.

The CRISPR/Cas9 genome editing tool has increased the efficiency of creating genetically modified pigs for use as biomedical or agricultural models. The objectives were to determine if DNA editing resulted in a delay in development to the blastocyst stage or in a skewing of the sex ratio. Six DNA templates (gBlocks) that were designed to express guide RNAs that target the transmembrane protease, serine S1, member 2 (TMPRSS2) gene were in vitro transcribed. Pairs of CRISPR guide RNAs that flanked the start codon and polyadenylated Cas9 were co-injected into the cytoplasm of zygotes and cultured in vitro to the blastocyst stage. Blastocysts were collected as they formed on days 5, 6 or 7. PCR was performed to determine genotype and sex of each embryo. Separately, embryos were surgically transferred into recipient gilts on day 4 of estrus. The rate of blastocyst development was not significantly different between CRISPR injection embryos or the non-injected controls at day 5, 6 or 7 (p = 0.36, 0.09, 0.63, respectively). Injection of three CRISPR sets of guides resulted in a detectable INDEL in 92–100 % of the embryos analyzed. There was not a difference in the number of edits or sex ratio of male to female embryos when compared between days 5, 6 and 7 to the controls (p > 0.22, >0.85). There were 12 resulting piglets and all 12 had biallelic edits of TMRPSS2. Zygote injection with CRISPR/Cas9 continues to be a highly efficient tool to genetically modify pig embryos.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-016-9989-6) contains supplementary material, which is available to authorized users.

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes.

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

Observations on Fertilization and a Novel Cytological Mechanism for Preventing Polyspermy in the FernOsmunda japonica

Premise of research. In advanced ferns that possess an egg envelope and a fertilization pore, a sac that persistently blocks the fertilization pore is considered a barrier for preventing polyspermy. However, for those ferns with no egg envelope and fertilization pore, the cytological mechanism for preventing polyspermy is still unclear. In this investigation, the main cytological processes during fertilization and zygote formation of the primitive fern Osmunda japonica are observed, and a novel cytological mechanism for preventing polyspermy is described.Methodology. The fertilization process was observed under stereomicroscope, and the fertilization time was recorded. The selected plants were fixed with glutaraldehyde and osmic acid and embedded in pure Spurr’s resin. Specimens were first thick-sectioned and then thin-sectioned for LM and TEM observations.Pivotal results. The observations showed that only one spermatozoid penetrates into the egg at the center in the upper surface of the mature egg. The egg immediately shrinks as the spermatozoid enters into the cytoplasm of the egg. The volume of the fertilized egg decreases remarkably, and simultaneously, a large vesicle forms above the fertilized egg. This vesicle can block other spermatozoids approaching the egg. During nuclear fusion, the male cytoskeleton defines a spatial framework, within which the male chromatin progressively fuses with the egg nucleus. The male organelles, including the microtubular ribbon, multilayered structure, flagella, and male mitochondria, are finally digested in the zygotic cytoplasm. A functional zygote is then formed at the venter of the archegonium.Conclusions. In the primitive fern O. japonica, the large vesicle, formed during fertilization above the egg, can block other spermatozoids approaching the egg, which is considered to be a novel cytological mechanism for preventing polyspermy.