Characterization of histone lysine ß-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 ß-hydroxybutyrate (BHB) causes a novel epigenetic modification, histone lysine ß-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. We searched for and identified that Kbhb is present in bovine tissues in vivo and confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. Maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation or to develop until the blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but faintly in oocytes. RNA-seq analysis in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The downregulated genes were mainly involved in glycolysis and ribosome assembly pathways, while the upregulated genes were involved in mitochondrial metabolism and oocyte development. The genes and pathways altered by BHB will provide entry points to carry out functional experiments aiming to mitigate metabolic disorders and improve fertility in cattle.

Resiliency of equid H19 imprint to somatic cell reprogramming by oocyte nuclear transfer and genetically induced pluripotency†.

Cell reprogramming by somatic cell nuclear transfer and in induced pluripotent stem cells is associated with epigenetic modifications that are often incompatible with embryonic development and differentiation. For instance, aberrant DNA methylation patterns of the differentially methylated region and biallelic expression of H19-/IGF2-imprinted gene locus have been associated with abnormal growth of fetuses and placenta in several mammalian species. However, cloned horses are born with normal sizes and with no apparent placental anomalies, suggesting that H19/IGF2 imprinting may be epigenetically stable after reprogramming in this species. In light of this, we aimed at characterizing the equid H19 gene to determine whether imprinting is altered in somatic cell nuclear transfer (SCNT)-derived conceptuses and induced pluripotent stem cell (iPSC) lines using the mule hybrid model. A CpG-rich region containing five CTCF binding sites was identified upstream of the equine H19 gene and analyzed by bisulfite sequencing. Coupled with parent-specific and global H19 transcript analysis, we found that the imprinted H19 remains monoallelic and that on average the methylation levels of both parental differentially methylated regions in embryonic and extra-embryonic SCNT tissues and iPSC lines remained unaltered after reprogramming. Together, these results show that, compared to other species, equid somatic cells are more resilient to epigenetic alterations to the H19-imprinted locus during SCNT and iPSC reprogramming.

The Infertility of Repeat-Breeder Cows During Summer Is Associated with Decreased Mitochondrial DNA and Increased Expression of Mitochondrial and Apoptotic Genes in Oocytes.

Oocyte quality is known to be a major cause of infertility in repeat-breeder (RB) and heat-stressed dairy cows. However, the mechanisms by which RB oocytes become less capable of supporting embryo development remain largely unknown. Thus, the aim of this study was to investigate whether the decreased oocyte competence of RB cows (RBs) during summer is associated with an altered gene expression profile and a decrease in mitochondrial DNA (mtDNA) copy number. Therefore, oocytes collected from heifers, non-RBs in peak lactation (PLs), and RBs were used to evaluate mtDNA amounts as well as the expression levels of genes associated with the mitochondria (MT-CO1, NRF1, POLG, POLG2, PPARGC1A, and TFAM), apoptosis (BAX, BCL2, and ITM2B), and oocyte maturation (BMP15, FGF8, FGF10, FGF16, FGF17, and GDF9). The oocytes retrieved from RBs during winter contained over eight times more mtDNA than those retrieved from RBs during summer. They also contained significantly less mtDNA than oocytes retrieved from heifers and PLs during summer. Moreover, the expression of mitochondria- (NRF1, POLG, POLG2, PPARGC1A, and TFAM) and apoptosis-related (BAX and ITM2B) genes, as well as of GDF9, in RB oocytes collected during summer was significantly greater than that in oocytes collected from heifers and PLs during the same season. In oocytes from heifers and PLs, the expression levels of these genes were lower in those collected during summer compared with winter, but this difference was not observed in oocytes collected from RBs. Altogether, these data provide evidence of altered gene expression and reduced mtDNA copy number in the oocytes collected from RBs during summer. This indicates a loss of fertility in RBs during summer, which might be caused by a possible mitochondrial dysfunction associated with a greater chance of oocytes to undergo apoptosis.

Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage.

The mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8-16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: 'RNA processing', 'translation' and 'mRNA metabolic process'. Genes that are important to the molecular functions of 'RNA binding' and 'translation factor activity, RNA binding' were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

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. We then describe our bovine SCNT protocol for delivering live cloned calves and addressing basic questions about nuclear reprogramming. Other research groups can benefit from our basic protocol and build up on it to improve SCNT in the future. Strategies to correct or mitigate epigenetic errors (e.g., correcting imprinting loci, overexpression of demethylases, chromatin-modifying drugs) can integrate the protocol described here.

Exogenous OCT4 and SOX2 Contribution to In Vitro Reprogramming in Cattle.

Mechanisms of cell reprogramming by pluripotency-related transcription factors or nuclear transfer seem to be mediated by similar pathways, and the study of the contribution of OCT4 and SOX2 in both processes may help elucidate the mechanisms responsible for pluripotency. Bovine fibroblasts expressing exogenous OCT4 or SOX2, or both, were analyzed regarding the expression of pluripotency factors and imprinted genes H19 and IGF2R, and used for in vitro reprogramming. The expression of the H19 gene was increased in the control sorted group, and putative iPSC-like cells were obtained when cells were not submitted to cell sorting. When sorted cells expressing OCT4, SOX2, or none (control) were used as donor cells for somatic cell nuclear transfer, fusion rates were 60.0% vs. 64.95% and 70.53% vs. 67.24% for SOX2 vs. control and OCT4 vs. control groups, respectively; cleavage rates were 66.66% vs. 81.68% and 86.47% vs. 85.18%, respectively; blastocyst rates were 33.05% vs. 44.15% and 52.06% vs. 44.78%, respectively. These results show that the production of embryos by NT resulted in similar rates of in vitro developmental competence compared to control cells regardless of different profiles of pluripotency-related gene expression presented by donor cells; however, induced reprogramming was compromised after cell sorting.

Placental scaffolds as a potential biological platform for embryonic stem cells differentiation into hepatic-like cells lineage: A pilot study.

Hepatic microenvironment plays an essential role in liver regeneration, providing the necessary conditions for cell proliferation, differentiation and tissue rearrangement. One of the key factors for hepatic tissue reconstruction is the extracellular matrix (ECM), which through collagenous and non-collagenous proteins provide a three-dimensional structure that confers support for cell adhesion and assists on their survival and maintenance. In this scenario, placental ECM may be eligible for hepatic tissue reconstruction, once these scaffolds hold the major components required for cell support. Therefore, this preliminary study aimed to access the possibility of mouse embryonic stem cells differentiation into hepatocyte-like cells on placental scaffolds in a three-dimensional dynamic system using a Rotary Cell Culture System. Following a four-phase differentiation protocol that simulates liver embryonic development events, the preliminary results showed that a significant quantity of cells adhered and interacted with the scaffold through outer and inner surfaces. Positive immunolabelling for alpha fetus protein and CK7 suggest presence of hepatoblast phenotype cells, and CK18 and Albumin positive immunolabelling suggest the presence of hepatocyte-like phenotype cells, demonstrating the presence of a heterogeneous population into the recellularized scaffolds. Periodic Acid Schiff-Diastase staining confirmed the presence of glycogen storage, indicating that differentiate cells acquired a hepatic-like phenotype. In conclusion, these preliminary results suggested that mouse placental scaffolds might be used as a biological platform for stem cells differentiation into hepatic-like cells and their establishment, which may be a promissing biomaterial for hepatic tissue reconstruction.

Recellularized rat testis scaffolds with embryoid bodies cells: a promising approach for tissue engineering.

Tissue engineering is gaining use to investigate the application of its techniques for infertility treatment. The use of pluripotent embryonic cells for in vitro production of viable spermatozoa in testicular scaffolds is a promising strategy that could solve male infertility. Due to cell-extracellular matrix (ECM) interactions, here we aim to investigate the differentiation of embryoid bodies (EBs) in cultured into decellularized rat testis scaffolds. Decellularized testis (P = 0.019) with a low concentration of gDNA (30.58 mg/ng tissue) was obtained by sodium dodecyl sulfate perfusion. The structural proteins (collagens type I and III) and the adhesive glycoproteins of ECM (laminin and fibronectin) were preserved according to histological and scanning electron microscopy (SEM) analyses. Then, decellularized rat testis were cultured for 7 days with EB, and EB mixed with retinoic acid (RA) in non-adherent plates. By SEM, we observe that embryonic stem cells adhered in the decellularized testis ECM. By immunofluorescence, we verified the positive expression of HSD17B3, GDNF, ACRV-1, and TRIM-36, indicating their differentiation using RA in vitro, reinforcing the possibility of EB in male germ cell differentiation. Finally, recellularized testis ECM may be a promising tool for future new approaches for testicular cell differentiation applied to assisted reproduction techniques and infertility treatment.Abbreviations: ACRV-1: Acrosomal vesicle protein 1; ATB: Penicillin-streptomycin; DAPI: 4,6-Diamidino-2-phenylindole; EB: Embryoid bodies; ECM: Extracellular matrix; ESCs: Pluripotent embryonic stem cells; GAGs: Glycosaminoglycans; gDNA: Genomic DNA; GDNF: Glial cell line-derived neurotrophic factor; H&E: Hematoxylin and eosin; HSD17B3: 17-beta-Hydroxysteroid dehydrogenase type 3; PBS: Phosphate-buffered saline; PGCLCs: Primordial germ-cell-like cells; RA: Retinoic acid; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SSCs: Spermatogonial stem cells; TRIM-36: Tripartite Motif Containing 36.

Ooplast-mediated developmental rescue of bovine oocytes exposed to ethidium bromide.

Ooplasm transfer has been used successfully to treat infertility in women with ooplasmic insufficiency and has culminated in the birth of healthy babies. To investigate whether mitochondrial dysfunction is a factor in ooplasmic insufficiency, bovine oocytes were exposed to ethidium bromide, an inhibitor of mitochondrial DNA replication and transcription, during in-vitro maturation (IVM). Exposure of immature oocytes to ethidium bromide for 24h during IVM hampered meiotic resumption and the migration of cortical granules. However, a briefer treatment with ethidium bromide during the last 4h of IVM led to partial arrest of preimplantation development without affecting oocyte maturation. Ooplasm transfer was then performed to rescue the oocytes with impaired development. In spite of this developmental hindrance, transfer of normal ooplasm into ethidium bromide-treated oocytes resulted in a complete rescue of embryonic development and the birth of heteroplasmic calves. Although this study unable to determine whether developmental rescue occurred exclusively through introduction of unaffected mitochondria into ethidium bromide-damaged oocytes, e.g. ethidium bromide may also affect other ooplasm components, these results clearly demonstrate that ooplasm transfer can completely rescue developmentally compromised oocytes, supporting the potential use of ooplasm transfer in therapeutic applications.

Lipid profile of extracellular vesicles and their relationship with bovine oocyte developmental competence: New players in intra follicular cell communication.

Cell communication within the ovarian follicle is crucial during folliculogenesis to assure an ideal environment for the oocyte to achieve full developmental competence. Intercellular communication is facilitated by the presence of follicular fluid, which mediates the transfer of signaling molecules. Recently, extracellular vesicles (exosomes and microvesicles) containing mRNAs, miRNAs and proteins were described in mammalian follicular fluid. Besides these molecules, extracellular vesicles (EVs) can mediate the transfer of lipids that can act as signal transducers activating second messengers and modulating intracellular pathways. Our goal was to determine the lipid profile of exosomes (small extracellular vesicles) and microvesicles (large extracellular vesicles) from bovine ovarian follicles containing oocytes with different developmental capabilities to verify potential relationships to competence. Using mass spectrometry, we examined the lipid content of EVs present in the follicular fluid of follicles enclosing oocytes that were either unable to cleave (NCLEAVE), arrested at cleavage stage (CLEAVE), or developed to the blastocyst stage (BLAST) after parthenogenetic activation. Although most of the 514 lipids identified in the follicular fluid EVs were common among all groups, 10 exosome-derived lipids and 15 microvesicle-derived lipids were present exclusively in the BLAST group, suggesting a potential relationship with developmental competence. Therefore, our data indicate that the EVs present in follicular fluid of antral follicles of similar morphology contain lipids that may be used as biomarkers associated with the developmental capability of the oocyte to develop to the blastocyst stage.

Pronounced segregation of donor mitochondria introduced by bovine ooplasmic transfer to the female germ-line.

Ooplasmic transfer (OT) has been used in basic mouse research for studying the segregation of mtDNA, as well as in human assisted reproduction for improving embryo development in cases of persistent developmental failure. Using cattle as a large-animal model, we demonstrate that the moderate amount of mitochondria introduced by OT is transmitted to the offspring's oocytes; e.g., modifies the germ line. The donor mtDNA was detectable in 25% and 65% of oocytes collected from two females. Its high variation in heteroplasmic oocytes, ranging from 1.1% to 33.5% and from 0.4% to 15.5%, can be explained by random genetic drift in the female germ line. Centrifugation-mediated enrichment of mitochondria in the pole zone of the recipient zygote's ooplasm and its substitution by donor ooplasm led to elevated proportions of donor mtDNA in reconstructed zygotes compared with zygotes produced by standard OT (23.6% +/- 9.6% versus 12.1% +/- 4.5%; P < 0.0001). We also characterized the proliferation of mitochondria from the OT parents-the recipient zygote (Bos primigenius taurus type) and the donor ooplasm (B. primigenius indicus type). Regression analysis performed for 57 tissue samples collected from the seven OT fetuses at different points during fetal development found a decreasing proportion of donor mtDNA (r(2) = 0.78). This indicates a preferred proliferation of recipient taurine mitochondria in the context of the nuclear genotype of the OT recipient expressing a B. primigenius indicus phenotype.

Decellularization of Extracellular Matrix from Equine Skeletal Muscle.

Equine represents an attractive animal model for musculoskeletal tissue diseases, exhibiting much similarity to the injuries that occur in humans. Cell therapy and tissue bioengineering have been widely used as a therapeutic alternative by regenerative medicine in musculoskeletal diseases. Thus, the aim of this study was to produce an acellular biomaterial of equine skeletal muscle and to evaluate its effectiveness in supporting the in vitro culture of equine induced pluripotency stem cells (iPSCs). Biceps femoris samples were frozen at -20°C for 4 days and incubated in 1% sodium dodecyl sulfate (SDS), 5 mM EDTA + 50 mM Tris and 1% Triton X-100; the effectiveness of the decellularization was evaluated by the absence of remnant nuclei (histological and 4',6-diamidino-2-phenylindole [DAPI] analysis), preservation of extracellular matrix (ECM) proteins (immunofluorescence and immunohistochemistry) and organization of ECM ultrastructure (scanning electron microscopy). Decellularized samples were recellularized with iPSCs at the concentration of 50,000 cells/cm2 and cultured in vitro for 9 days, and the presence of the cells in the biomaterial was evaluated by histological analysis and presence of nuclei. Decellularized biomaterial showed absence of remnant nuclei and muscle fibers, as well as the preservation of ECM architecture, vascular network and proteins, laminin, fibronectin, elastin, collagen III and IV. After cellularization, iPSC nuclei were present at 9 days after incubation, indicating the decellularized biomaterial-supported iPSC survival. It is concluded that the ECM biomaterial produced from the decellularized equine skeletal muscle has potential for iPSC adhesion, representing a promising biomaterial for regenerative medicine in the therapy of musculoskeletal diseases.

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. Either the donor cell (before reconstruction) or the early embryo was exposed to the probe to assess its effect on developmental rates and epigenetic marks. First, we showed that the treatment of bovine fibroblasts with UNC0638 did mitigate the levels of H3K9me2. Moreover, H3K9me2 levels were decreased in cloned embryos regardless of treating either donor cells or early embryos with UNC0638. Additional epigenetic marks such as H3K9me3, 5mC, and 5hmC were also affected by the UNC0638 treatment. Therefore, the use of UNC0638 did diminish the levels of H3K9me2 and H3K9me3 in SCNT-derived blastocysts, but this was unable to improve their preimplantation development. These results indicate that the specific reduction of H3K9me2 by inhibiting EHMT1/2 during nuclear reprogramming impacts the levels of H3K9me3, 5mC, and 5hmC in preimplantation bovine embryos.

A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome.

SummaryOocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

Metabolic gene expression and epigenetic effects of the ketone body ß-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 ß-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. Treatment with BOHB does not increase histone acetylation in cells but stimulates genes associated with ketolysis and master regulators of metabolism. We further demonstrated that maturing COCs with high levels of BOHB does not affect their maturation rate or histone acetylation but increases the expression of PPARA in cumulus cells. Treatment of somatic cell nuclear transfer zygotes with BOHB causes hyperacetylation, which is maintained until the blastocyst stage, causing enhanced FOXO3A expression and blastocyst production. Our data shed light on the epigenetic mechanisms caused by BOHB in bovine cells and embryos and provide a better understanding of the connection between nutrition and reproduction.

Morphological characteristics of mule conceptuses during early development.

Hybrids between species are often infertile and extremely rare among mammals. Mules, i.e. crossing between the horse and the donkey, on the other hand are very common in agricultural and leisure practices due to their enhanced post-natal physical characteristics that is believed to occur for outbreeding or hybrid vigor. Since no reports are availableon the effects of hybrid vigor during early development, this study focused on characterizing the intrauterine development of mule conceptuses during critical embryo-to-fetus transition period. Nine embryos and fetuses of early gestation, obtained after artificial insemination and transcervical flushing, were evaluated by means of gross anatomy and histology and compared to data available for the equine. We found that some events, such as C-shape turning, apearence of branchial archs, limb and tail buds, formation of primary and secondary brain vesicles, heart compartmentalization, and development of somites, occurred slightly earlier in the mule. Nonetheless, no major differences were observed in other developmental features, suggesting similarities between the mule and the horse development. In conclusion, these data suggest that the effect of hybrid vigor is present during intrauterine development in the mule, at least with regard to its maternal parent.

Author Correction: Metabolic gene expression and epigenetic effects of the ketone body ß-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.

The kinetics of donor cell mtDNA in embryonic and somatic donor cell-derived bovine embryos.

The mechanisms controlling the outcome of donor cell-derived mitochondrial DNA (mtDNA) in cloned animals remain largely unknown. This research was designed to investigate the kinetics of somatic and embryonic mtDNA in reconstructed bovine embryos during preimplantation development, as well as in cloned animals. The experiment involved two different procedures of embryo reconstruction and their evaluation at five distinct phases of embryo development to measure the proportion of donor cell mtDNA (Bos indicus), as well as the segregation of this mtDNA during cleavage. The ratio of donor cell (B. indicus) to host oocyte (B. taurus) mtDNA (heteroplasmy) from blastomere(NT-B) and fibroblast(NT-F) reconstructed embryos was estimated using an allele-specific PCR with fluorochrome-stained specific primers in each sampled blastomere, in whole blastocysts, and in the tissues of a fibroblast-derived newborn clone. NT-B zygotes and blastocysts show similar levels of heteroplasmy (11.0% and 14.0%, respectively), despite a significant decrease at the 9-16 cell stage (5.8%; p<0.05). Heteroplasmy levels in NT-F reconstructed zygotes, however, increased from an initial low level (4.7%), to 12.9% (p<0.05) at the 9-16 cell stage. The NT-F blastocysts contained low levels of heteroplasmy (2.2%) and no somatic-derived mtDNA was detected in the gametes or the tissues of the newborn calf cloned. These results suggest that, in contrast to the mtDNA of blastomeres, that of somatic cells either undergoes replication or escapes degradation during cleavage, although it is degraded later after the blastocyst stage or lost during somatic development, as revealed by the lack of donor cell mtDNA at birth.

Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains.

Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.

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. Herein we used valproic acid (VPA) in SCNT to test whether the treatment of nuclear donor cells with this HDACi improves pre- and post-implantation development of cloned cattle. We found that the treatment of fibroblasts with VPA increased histone acetylation without affecting DNA methylation. Moreover, the treatment with VPA resulted in increased expression of IGF2R and PPARGC1A, but not of POU5F1. However, when treated cells were used as nuclear donors no difference of histone acetylation was found after oocyte reconstruction compared to the use of untreated cells. Moreover, shortly after artificial activation the histone acetylation levels were decreased in the embryos produced with VPA-treated cells. With respect to developmental rates, the use of treated cells as donors resulted in no difference during pre- and post-implantation development. In total, five clones developed to term; three produced with untreated cells and two with VPA-treated cells. Among the calves from treated group, one stillborn calf was delivered at day 270 of gestation whereas the other one was delivered at term but died shortly after birth. Among the calves from the control group, one died seven days after birth whereas the other two are still alive and healthy. Altogether, these results show that in spite of the alterations in fibroblasts resulting from the treatment with VPA, their use as donor cells in SCNT did not improve pre- and post-implantation development of cloned cattle.