Efficiency of the zinc chelator 1,10-phenanthroline for assisted oocyte activation following ICSI in pigs.

Context In vitro embryo production in pigs is an important tool for advancing biomedical research. Intracytoplasmic sperm injection (ICSI) circumvents the polyspermy problems associated with conventional IVF in porcine. However, the suboptimal efficiency for ICSI in pigs requires new strategies to increase blastocyst formation rates.

Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation.

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and β4GalNT2.

Practical Approaches for Knock-Out Gene Editing in Pigs.

Pigs are an important resource for meat production and serve as a model for human diseases. Due to their physiological and anatomical similarities to humans, these animals can recapitulate symptoms of human diseases, becoming an effective model for biomedical research. Although, in the past pig have not been widely used partially because of the difficulty in genetic modification; nowadays, with the new revolutionary technology of programmable nucleases, and fundamentally of the CRISPR-Cas9 systems, it is possible for the first time to precisely modify the porcine genome as never before.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos.

CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos.

CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system.

A eukaryotic nicotinate-inducible gene cluster: convergent evolution in fungi and bacteria.

Nicotinate degradation has hitherto been elucidated only in bacteria. In the ascomycete , six loci, /AN9178 encoding the molybdenum cofactor-containing nicotinate hydroxylase, AN11197 encoding a Cys2/His2 zinc finger regulator HxnR, together with AN11196/, AN11188/, AN11189/ and AN9177/, are clustered and stringently co-induced by a nicotinate derivative and subject to nitrogen metabolite repression mediated by the GATA factor AreA. These genes are strictly co-regulated by HxnR.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos.

Bovine parthenogenotes produced by inhibition of first or second polar bodies emission.

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission.

Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos.

The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos.

Effects of bone morphogenic protein 4 (BMP4) and its inhibitor, Noggin, on in vitro maturation and culture of bovine preimplantation embryos.

Background: BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine.

Methods: For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin.

Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI.

Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams.

Activation with ionomycin followed by dehydroleucodine and cytochalasin B for the production of parthenogenetic and cloned bovine embryos.

In this work, Dehydroleucodine (DhL) was evaluated as a chemical activator of bovine oocytes and somatic cell nuclear transfer (SCNT) reconstituted embryos. Oocytes were activated with 5 microM Ionomycin (Io) and exposed for 3 h to 1 or 5 microM DhL alone (Io-Dhl1 or Io-DhL5) or combined with Cytochalasin B (Io-DhL1/CB; Io-DhL5/CB). Control groups were Io (Io), Io followed by 1.

High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation.

In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated.

Nucleosome positioning and histone H3 acetylation are independent processes in the Aspergillus nidulans prnD-prnB bidirectional promoter.

In Aspergillus nidulans, proline can be used as a carbon and nitrogen source, and its metabolism requires the integration of three signals, including proline induction and nitrogen and carbon metabolite derepression. We have previously shown that the bidirectional promoter in the prnD-prnB intergenic region undergoes drastic chromatin rearrangements such that proline induction leads to the loss of positioned nucleosomes, whereas simultaneous carbon and nitrogen metabolite repression results in the partial repositioning of these nucleosomes. In the proline cluster, the inhibition of deacetylases by trichostatin A leads to partial derepression and is associated with a lack of nucleosome positioning.

The tightly regulated promoter of the xanA gene of Aspergillus nidulans is included in a helitron.

In Aspergillus nidulans the xanA gene codes for a xanthine alpha-ketoglutarate-dependent dioxygenase, an enzyme only present in the fungal kingdom. The 5' region of this gene, including its putative promoter and the first 54 codons of the open reading frame, together with the first intron is duplicated in the genome. This duplication corresponds to a helitron, a eukaryotic element proposed to transpose replicatively by the rolling circle mechanism.

Convergent evolution of hydroxylation mechanisms in the fungal kingdom: molybdenum cofactor-independent hydroxylation of xanthine via alpha-ketoglutarate-dependent dioxygenases.

The xanthine oxidases and dehydrogenases are among the most conserved enzymes in all living kingdoms. They contain the molybdopterin cofactor Moco. We show here that in the fungi, in addition to xanthine dehydrogenase, a completely different enzyme is able to catalyse the oxidation of xanthine to uric acid.

A gene of the opsin family in the carotenoid gene cluster of Fusarium fujikuroi.

Opsins are membrane photoreceptors closely related to the heat-shock proteins of the HSP30 family. Their functions include light-driven ion pumping in archaea and light detection in algae and animals, using the apocarotenoid retinal as a light-absorbing prosthetic group. We describe a gene of Fusarium fujikuroi, carO, coding for a polypeptide resembling opsins and HSP30-like proteins and contiguous to the genes of the carotenoid pathway, carRA and carB.