Replacing sperm with genotyped haploid androgenetic blastomeres to generate cattle with predetermined paternal genomes†.

Unlabelled: Although meiosis plays an essential role for the survival of species in natural selection, the genetic diversity resulting from sexual reproduction impedes human-driven strategies to transmit the most suitable genomes for genetic improvement, forcing breeders to select diploid genomes generated after fertilization, that is, after the encounter of sperm and oocytes carrying unknown genomes. To determine whether genomic assessment could be used before fertilization, some androgenetic haploid morula-stage bovine embryos derived from individual sperm were biopsied for genomic evaluation and others used to reconstruct "semi-cloned" (SC) diploid zygotes by the intracytoplasmic injection into parthenogenetically activated oocytes, and the resulting embryos were transferred to surrogate females to obtain gestations. Compared to controls, in vitro development to the blastocyst stage was lower and fewer surrogates became pregnant from the transfer of SC embryos.

Haploid embryos and embryonic stem cells to produce offspring with predetermined parental genomes in cattle.

Selection strategies are performed post-fertilization when the random combination of paternal and maternal genomes has already occurred. It would be greatly advantageous to eliminate meiotic uncertainty by selecting genetically superior gametes before fertilization. To achieve this goal, haploid embryonic cells and embryonic stem cell lineages could be derived, genotyped, and used to substitute gametes.

Biosensor capability of the endometrium is mediated in part, by altered miRNA cargo from conceptus-derived extracellular vesicles.

We tested the hypothesis that the biosensor capability of the endometrium is mediated in part, by the effect of different cargo contained in the extracellular vesicles secreted by the conceptus during the peri-implantation period of pregnancy. We transferred Bos taurus taurus embryos of different origin, in vivo (high developmental potential (IV)), in vitro (intermediate developmental potential (IVF)), or cloned (low developmental potential (NT)), into Bos taurus indicus recipients. Extracellular vesicles (EVs) recovered from Day 16 conceptus-conditioned medium were characterized and their microRNA (miRNA) cargo sequenced alongside RNA sequencing of their respective endometria.

Human proinsulin production in the milk of transgenic cattle.

Background: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk.

Methods And Results: Pseudo-lentivirus containing the bovine β-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced.

Haploid androgenetic development of bovine embryos reveals imbalanced WNT signaling and impaired cell fate differentiation†.

Haploid embryos have contributed significantly to our understanding of the role of parental genomes in development and can be applied to important biotechnology for human and animal species. However, development to the blastocyst stage is severely hindered in bovine haploid androgenetic embryos (hAE). To further our understanding of such developmental arrest, we performed a comprehensive comparison of the transcriptomic profile of morula-stage embryos, which were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of transcripts associated with differentiation in haploid and biparental embryos.

Cattle Cloning by Somatic Cell Nuclear Transfer.

Cloning by somatic cell Nuclear Transfer (SCNT) is a powerful technology capable of reprograming terminally differentiated cells to totipotency for generating whole animals or pluripotent stem cells for use in cell therapy, drug screening, and other biotechnological applications. However, the broad usage of SCNT remains limited due to its high cost and low efficiency in obtaining live and healthy offspring. In this chapter, we first briefly discuss the epigenetic constraints responsible for the low efficiency of SCNT and current attempts to overcome them.

Micro-vibration results in vitro-derived bovine blastocysts with greater cryotolerance, epigenetic abnormalities, and a massive transcriptional change.

Much effort has been employed to improve the quality of embryos obtained by in vitro production (IVP) given the relevance of this technology to current livestock systems. In this context, dynamic IVP systems have proved beneficial to the embryo once they mimic fluid flows and mechanical forces resulting from the movement of ciliated cells and muscle contraction in the reproductive tract. In the present study, we sought to confirm these initial findings as well as assess potential molecular consequences to the embryo by applying micro-vibration (45 Hz for 5 s once per 60 min) during both oocyte maturation and embryo culture in cattle.

Characterization of histone lysine β-hydroxybutyrylation in bovine tissues, cells, and cumulus-oocyte complexes.

Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through histone posttranslational modifications. Ketone body β-hydroxybutyrate (BHB) causes a novel epigenetic modification, histone lysine β-hydroxybutyrylation (Kbhb), which is associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation, and the effects of this increase on cellular epigenomes are unknown.

Catalytic inhibition of H3K9me2 writers disturbs epigenetic marks during bovine nuclear reprogramming.

Orchestrated events, including extensive changes in epigenetic marks, allow a somatic nucleus to become totipotent after transfer into an oocyte, a process termed nuclear reprogramming. Recently, several strategies have been applied in order to improve reprogramming efficiency, mainly focused on removing repressive epigenetic marks such as histone methylation from the somatic nucleus. Herein we used the specific and non-toxic chemical probe UNC0638 to inhibit the catalytic activity of the histone methyltransferases EHMT1 and EHMT2.

Author Correction: Metabolic gene expression and epigenetic effects of the ketone body β-hydroxybutyrate on H3K9ac in bovine cells, oocytes and embryos.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Metabolic gene expression and epigenetic effects of the ketone body β-hydroxybutyrate on H3K9ac in bovine cells, oocytes and embryos.

The rapid decline in fertility that has been occurring to high-producing dairy cows in the past 50 years seems to be associated with metabolic disturbances such as ketosis, supporting the need for research to improve our understanding of the relations among the diet, metabolism and embryonic development. Recently, the ketone body β-hydroxybutyrate (BOHB) was demonstrated to be a potent inhibitor of histone deacetylases (HDACs). Herein, we performed a series of experiments aiming to investigate the epigenetic effects of BOHB on histone acetylation in somatic cells, cumulus-oocyte complexes (COCs) and somatic cell nuclear transfer (SCNT) embryos.

Epigenetic remodeling in preimplantation embryos: cows are not big mice.

Epigenetic mechanisms allow the establishment and maintenance of multiple cellular phenotypes from a single genomic code. At the initiation of development, the oocyte and spermatozoa provide their fully differentiated chromatin that soon after fertilization undergo extensive remodeling, resulting in a totipotent state that can then drive cellular differentiation towards all cell types. These remodeling involves different epigenetic modifications, including DNA methylation, post-translational modifications of histones, non-coding RNAs, and large-scale chromatin conformation changes.

Effect of POU5F1 Expression Level in Clonal Subpopulations of Bovine Fibroblasts Used as Nuclear Donors for Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) success is partially hindered by the low epigenetic reprogramming efficiency of the donor cell. Previous studies suggest cellular heterogeneity among donor nuclei in regard to reprogramming potential, which precludes comparison among different strategies to increase cloning success. In this context, we evaluated the effect of using clonal cell populations (CPs) of bovine adult fibroblasts established by single-cell plating in SCNT.

Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development.

Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques.

Cat amniotic membrane multipotent cells are nontumorigenic and are safe for use in cell transplantation.

Amnion-derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages. AMSCs can be acquired through noninvasive methods, and therefore are exempt from the typical ethical issues surrounding stem cell use. The objective of this study was to isolate and characterize AMSCs from a cat amniotic membrane for future application in regenerative medicine.

Development to term of cloned cattle derived from donor cells treated with valproic acid.

Cloning of mammals by somatic cell nuclear transfer (SCNT) is still plagued by low efficiency. The epigenetic modifications established during cellular differentiation are a major factor determining this low efficiency as they act as epigenetic barriers restricting reprogramming of somatic nuclei. In this regard, most factors that promote chromatin decondensation, including histone deacetylase inhibitors (HDACis), have been found to increase nuclear reprogramming efficiency, making their use common to improve SCNT rates.

MT3 melatonin binding site, MT1 and MT2 melatonin receptors are present in oocyte, but only MT1 is present in bovine blastocyst produced in vitro.

Background: Melatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP). The aim of our study was to investigate the presence of melatonin membrane receptors (MT1and MT2) and MT3, which is the melatonin binding site of NQO2 enzyme, in both oocytes and hatched blastocysts to consider an additional subcellular mechanism responsible for the effects of melatonin on IVP.

Methods: The presence of the high affinity melatonin receptors was investigated through an autoradiographic binding assay, using the non-permeable ligand [125I]-iodomelatonin (17 pM) in embryos.

Embryo production by parthenogenetic activation and fertilization of in vitro matured oocytes from Cebus apella.

The efficiency of in vitro fertilization (IVF) depends on the viability of spermatozoa. For capuchin monkeys (Cebus apella), in vitro capacitation of spermatozoa is challenging because of their unique seminal coagulum. Motile spermatozoa can be obtained after liquefaction of the semen coagulum in coconut water-based solution.

Viable calves produced by somatic cell nuclear transfer using meiotic-blocked oocytes.

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced.