Soybean molasses increases subcutaneous fat deposition while reducing lipid oxidation in the meat of castrated lambs.

This study aimed to evaluate the effect of including soybean molasses (SM) on performance, blood parameters, carcass traits, meat quality, fatty acid, and muscle (longissimus thoracis) transcriptomic profiles of castrated lambs. Twenty Dorper × Santa Inês lambs (20.06 ±â€…0.76 kg body weight [BW]) were assigned to a randomized block design, stratified by BW, with the following treatments: CON: 0 g/kg of SM and SM20: 200 g/kg of SM on dry matter basis, allocated in individual pens. The diet consisted of 840 g/kg concentrate and 160 g/kg corn silage for 76 d, with the first 12 d as an adaptation period and the remaining 64 d on the finishing diet. The SM20 diet increased blood urea concentration (P = 0.03) while reduced glucose concentration (P = 0.04). Lambs fed SM showed higher subcutaneous fat deposition (P = 0.04) and higher subcutaneous adipocyte diameter (P < 0.01), in addition to reduced meat lipid oxidation (P < 0.01). SM reduced the quantity of branched-chain fatty acids in longissimus thoracis (P = 0.05) and increased the quantity of saturated fatty acids (P = 0.01). In the transcriptomic analysis, 294 genes were identified as differentially expressed, which belong to pathways such as oxidative phosphorylation, citric acid cycle, and monosaccharide metabolic process. In conclusion, diet with SM increased carcass fat deposition, reduced lipid oxidation, and changed the energy metabolism, supporting its use in ruminant nutrition.

This study investigated the effects of incorporating soybean molasses (SM) into the diet of castrated lambs on various aspects of their performance and meat quality. Twenty lambs were divided into two groups: one was fed a control diet without SM whereas the other was fed a similar diet but containing 20% of SM. The feeding trial lasted for 76 d. Results showed that the SM inclusion in the diet led to increased blood urea levels and decreased glucose concentrations. SM inclusion also resulted in lambs with higher levels of subcutaneous fat and larger adipocytes, while reducing meat lipid oxidation. Moreover, SM altered fatty acid composition in the meat, decreasing branched-chain fatty acids and increasing saturated fatty acids. In agreement with these findings, transcriptomic analysis revealed a significant change in the expression of genes related to energy metabolism in the muscle of lambs fed SM. In conclusion, incorporating SM in lamb's diet increased fat deposition, improved meat quality, and induced a transcriptomic change in the muscle energy metabolism, supporting its potential use in ruminant nutrition.

Mitochondrial Inheritance Following Nuclear Transfer: From Cloned Animals to Patients with Mitochondrial Disease.

Mitochondria are indispensable power plants of eukaryotic cells that also act as a major biochemical hub. As such, mitochondrial dysfunction, which can originate from mutations in the mitochondrial genome (mtDNA), may impair organism fitness and lead to severe diseases in humans. MtDNA is a multi-copy, highly polymorphic genome that is uniparentally transmitted through the maternal line. Several mechanisms act in the germline to counteract heteroplasmy (i.e., coexistence of two or more mtDNA variants) and prevent expansion of mtDNA mutations. However, reproductive biotechnologies such as cloning by nuclear transfer can disrupt mtDNA inheritance, resulting in new genetic combinations that may be unstable and have physiological consequences. Here, we review the current understanding of mitochondrial inheritance, with emphasis on its pattern in animals and human embryos generated by nuclear transfer.

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. As a result, micro-vibration led to lower incidence of apoptosis in blastocysts following vitrification-thawing. Further analyses revealed epigenetic and transcriptional changes in blastocysts derived from the micro-vibration treatment, with a total of 502 differentially expressed genes. Enrichment analyses linked differentially expressed genes to 'Oxidative phosphorylation', 'Cytokine-cytokine receptor interaction', and 'Signaling pathways regulating pluripotency of stem cells'. Yet, a meta-analysis indicated that the transcriptional changes induced by micro-vibration were not toward that of in vivo-derived embryos. In conclusion, micro-vibration increases the cryoresistance of bovine embryos, but caution should be taken given the unclear consequences of epigenetic and transcriptional abnormalities induced by the treatment.

Modulating mitochondrial DNA mutations: factors shaping heteroplasmy in the germ line and somatic cells.

Until recently it was thought that most humans only harbor one type of mitochondrial DNA (mtDNA), however, deep sequencing and single-cell analysis has shown the converse - that mixed populations of mtDNA (heteroplasmy) are the norm. This is important because heteroplasmy levels can change dramatically during transmission in the female germ line, leading to high levels causing severe mitochondrial diseases. There is also emerging evidence that low level mtDNA mutations contribute to common late onset diseases such as neurodegenerative disorders and cardiometabolic diseases because the inherited mutation levels can change within developing organs and non-dividing cells over time. Initial predictions suggested that the segregation of mtDNA heteroplasmy was largely stochastic, with an equal tendency for levels to increase or decrease. However, transgenic animal work and single-cell analysis have shown this not to be the case during germ-line transmission and in somatic tissues during life. Mutation levels in specific mtDNA regions can increase or decrease in different contexts and the underlying molecular mechanisms are starting to be unraveled. In this review we provide a synthesis of recent literature on the mechanisms of selection for and against mtDNA variants. We identify the most pertinent gaps in our understanding and suggest ways these could be addressed using state of the art techniques.

Autophagy deficiency abolishes liver mitochondrial DNA segregation.

Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific atg7 (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. prkn (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy.Abbreviations: Apob: apolipoprotein B; Atg1: autophagy-related 1; Atg7: autophagy related 7; Atp5a1: ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BL6: C57BL/6N mouse strain; BNIP3: BCL2/adenovirus E1B interacting protein 3; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mt-Atp8: mitochondrially encoded ATP synthase 8; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mt-Co3: mitochondrially encoded cytochrome c oxidase III; mt-Cytb: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MUL1: mitochondrial ubiquitin ligase activator of NFKB 1; nDNA: nuclear DNA; Ndufa9: NADH:ubiquinone oxireductase subunit A9; NDUFB8: NADH:ubiquinone oxireductase subunit B8; Nnt: nicotinamide nucleotide transhydrogenase; NZB: NZB/BINJ mouse strain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced putative kinase 1; Polg2: polymerase (DNA directed), gamma 2, accessory subunit; Ppara: peroxisome proliferator activated receptor alpha; Ppia: peptidylprolyl isomerase A; Prkn: parkin RBR E3 ubiquitin protein ligase; P10: post-natal day 10; P21: post-natal day 21; P100: post-natal day 100; qPCR: quantitative polymerase chain reaction; Rpl19: ribosomal protein L19; Rps18: ribosomal protein S18; SD: standard deviation; SEM: standard error of the mean; SDHB: succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1: sequestosome 1; Ssbp1: single-stranded DNA binding protein 1; TFAM: transcription factor A, mitochondrial; Tfb1m: transcription factor B1, mitochondrial; Tfb2m: transcription factor B2, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; UQCRC2: ubiquinol cytochrome c reductase core protein 2; WT: wild-type.

Mitochondrial DNA heteroplasmy is modulated during oocyte development propagating mutation transmission.

Heteroplasmic mitochondrial DNA (mtDNA) mutations are a common cause of inherited disease, but a few recurrent mutations account for the vast majority of new families. The reasons for this are not known. We studied heteroplasmic mice transmitting m.5024C>T corresponding to a human pathogenic mutation. Analyzing 1167 mother-pup pairs, we show that m.5024C>T is preferentially transmitted from low to higher levels but does not reach homoplasmy. Single-cell analysis of the developing mouse oocytes showed the preferential increase in mutant over wild-type mtDNA in the absence of cell division. A similar inheritance pattern is seen in human pedigrees transmitting several pathogenic mtDNA mutations. In m.5024C>T mice, this can be explained by the preferential propagation of mtDNA during oocyte maturation, counterbalanced by purifying selection against high heteroplasmy levels. This could explain how a disadvantageous mutation in a carrier increases to levels that cause disease but fails to fixate, causing multigenerational heteroplasmic mtDNA disorders.

Mice born to females with oocyte-specific deletion of mitofusin 2 have increased weight gain and impaired glucose homeostasis.

Offspring born to obese and diabetic mothers are prone to metabolic diseases, a phenotype that has been linked to mitochondrial dysfunction and endoplasmic reticulum (ER) stress in oocytes. In addition, metabolic diseases impact the architecture and function of mitochondria-ER contact sites (MERCs), changes which associate with mitofusin 2 (MFN2) repression in muscle, liver and hypothalamic neurons. MFN2 is a potent modulator of mitochondrial metabolism and insulin signaling, with a key role in mitochondrial dynamics and tethering with the ER. Here, we investigated whether offspring born to mice with MFN2-deficient oocytes are prone to obesity and diabetes. Deletion of Mfn2 in oocytes resulted in a profound transcriptomic change, with evidence of impaired mitochondrial and ER function. Moreover, offspring born to females with oocyte-specific deletion of Mfn2 presented increased weight gain and glucose intolerance. This abnormal phenotype was linked to decreased insulinemia and defective insulin signaling, but not mitochondrial and ER defects in offspring liver and skeletal muscle. In conclusion, this study suggests a link between disrupted mitochondrial/ER function in oocytes and increased risk of metabolic diseases in the progeny. Future studies should determine whether MERC architecture and function are altered in oocytes from obese females, which might contribute toward transgenerational transmission of metabolic diseases.

Evidence of Selection Against Damaged Mitochondria During Early Embryogenesis in the Mouse.

There is evidence of a purifying filter acting in the female germline to prevent the expansion of deleterious mutations in the mitochondrial DNA (mtDNA). Given our poor understanding of this filter, here we investigate the competence of the mouse embryo to eliminate dysfunctional mitochondria. Toward that, mitochondria were damaged by photoirradiation of NZB/BINJ zygotes loaded with chloromethyl-X-rosamine (CMXRos). The resultant cytoplasm was then injected into C57BL/6J zygotes to track the levels of NZB/BINJ mtDNA during the preimplantation development. About 30% of NZB/BINJ mtDNA was present after injection, regardless of using photoirradiated or non-photoirradiated cytoplasmic donors. Moreover, injection of photoirradiated-derived cytoplasm did not impact development into blastocysts. However, lower levels of NZB/BINJ mtDNA were present in blastocysts when comparing injection of photoirradiated (24.7% ± 1.43) versus non-photoirradiated (31.4% ± 1.43) cytoplasm. Given that total mtDNA content remained stable between stages (zygotes vs. blastocysts) and treatments (photoirradiated vs. non-photoirradiated), these results indicate that the photoirradiated-derived mtDNA was replaced by recipient mtDNA in blastocysts. Unexpectedly, treatment with rapamycin prevented the drop in NZB/BINJ mtDNA levels associated with injection of photoirradiated cytoplasm. Additionally, analysis of mitochondria-autophagosome colocalization provided no evidence that photoirradiated mitochondria were eliminated by autophagy. In conclusion, our findings give evidence that the mouse embryo is competent to mitigate the levels of damaged mitochondria, which might have implications to the transmission of mtDNA-encoded disease.

Mitofusin 1 is required for oocyte growth and communication with follicular somatic cells.

Mitochondrial function, largely regulated by the dynamics of this organelle, is inextricably linked to the oocyte health. In comparison with most somatic cells, mitochondria in oocytes are smaller and rounder in appearance, suggesting limited fusion. The functional implications of this distinct morphology, and how changes in the mitochondrial shape translate to mitochondrial function in oogenesis is little understood. We, therefore, asked whether the pro-fusion proteins mitofusins 1 (MFN1) and 2 (MFN2) are required for the oocyte development. Here we show that oocyte-specific deletion of Mfn1, but not Mfn2, prevents the oocyte growth and ovulation due to a block in folliculogenesis. We pinpoint the loss of oocyte growth and ovulation to impaired PI3K-Akt signaling and disrupted oocyte-somatic cell communication. In support, the double loss of Mfn1 and Mfn2 partially rescues the impaired PI3K-Akt signaling and defects in oocyte development secondary to the single loss of Mfn1. Together, this work demonstrates that the mitochondrial function influences the cellular signaling during the oocyte development, and highlights the importance of distinct, nonredundant roles of MFN1 and MFN2 in oogenesis.

Maternal transmission of mitochondrial diseases.

Given the major role of the mitochondrion in cellular homeostasis, dysfunctions of this organelle may lead to several common diseases in humans. Among these, maternal diseases linked to mitochondrial DNA (mtDNA) mutations are of special interest due to the unclear pattern of mitochondrial inheritance. Multiple copies of mtDNA are present in a cell, each encoding for 37 genes essential for mitochondrial function. In cases of mtDNA mutations, mitochondrial malfunctioning relies on mutation load, as mutant and wild-type molecules may co-exist within the cell. Since the mutation load associated with disease manifestation varies for different mutations and tissues, it is hard to predict the progeny phenotype based on mutation load in the progenitor. In addition, poorly understood mechanisms act in the female germline to prevent the accumulation of deleterious mtDNA in the following generations. In this review, we outline basic aspects of mitochondrial inheritance in mammals and how they may lead to maternally-inherited diseases. Furthermore, we discuss potential therapeutic strategies for these diseases, which may be used in the future to prevent their transmission.

Metabolic studies of a patient harbouring a novel S487L mutation in the catalytic subunit of AMPK.

AMP-activated protein kinase (AMPK) regulates many different metabolic pathways in eukaryote cells including mitochondria biogenesis and energy homeostasis. Here we identify a patient with hypotonia, weakness, delayed milestones and neurological impairment since birth harbouring a novel homozygous mutation in the AMPK catalytic α-subunit 1, encoded by the PRKAA1 gene. The homozygous mutation p.S487L in isoform 1 present in the patient is in a cryptic residue for AMPK activity. In the present study, we performed the characterization of mitochondrial respiratory properties of the patient, in comparison to healthy controls, through the culture of skin fibroblasts in order to understand some of the cellular consequences of the PRKAA1 mutation. In these assays, mitochondrial respiratory complex I showed lower activity, which was followed by a decrement in the mtDNA copy number, which is a probable consequence of the lower expression of PGC-1α and PRKAA1 itself as measured in our quantitative PCRs experiments. Confirming the effect of the patient mutation in respiration, transfection of patient fibroblasts with wild type PRKAA1 partially restore complex I level. The preliminary clinic evaluations of the patient suggested a metabolic defect related to the mitochondrial respiratory function, therefore treatment with CoQ10 supplementation dose started four years ago and a clear improvement in motor skills and strength has been achieved with this treatment.

Oocyte mitochondria: role on fertility and disease transmission.

Oocyte mitochondria are increased in number, smaller, and rounder in appearance than mitochondria in somatic cells. Moreover, mitochondrial numbers and activity are narrowly tied with oocyte quality because of the key role of mitochondria to oocyte maturation. During oocyte maturation, mitochondria display great mobility and cluster at specific sites to meet the high energy demand. Conversely, oocyte mitochondria are not required during early oogenesis as coupling with granulosa cells is sufficient to support gamete's needs. In part, this might be explained by the importance of protecting mitochondria from oxidative damage that result in mutations in mitochondrial DNA (mtDNA). Considering mitochondria are transmitted exclusively by the mother, oocytes with mtDNA mutations may lead to diseases in offspring. Thus, to counterbalance mutation expansion, the oocyte has developed specific mechanisms to filter out deleterious mtDNA molecules. Herein, we discuss the role of mitochondria on oocyte developmental potential and recent evidence supporting a purifying filter against deleterious mtDNA mutations in oocytes.

Correction: Is Placental Mitochondrial Function a Regulator that Matches Fetal and Placental Growth to Maternal Nutrient Intake in the Mouse?

[This corrects the article DOI: 10.1371/journal.pone.0130631.].

Is Placental Mitochondrial Function a Regulator that Matches Fetal and Placental Growth to Maternal Nutrient Intake in the Mouse?

BACKGROUND: Effective fetal growth requires adequate maternal nutrition coupled to active transport of nutrients across the placenta, which, in turn requires ATP. Epidemiological and experimental evidence has shown that impaired maternal nutrition in utero results in an adverse postnatal phenotype for the offspring. Placental mitochondrial function might link maternal food intake to fetal growth since impaired placental ATP production, in response to poor maternal nutrition, could be a pathway linking maternal food intake to reduced fetal growth. METHOD: We assessed the effects of maternal diet on placental water content, ATP levels and mitochondrial DNA (mtDNA) content in mice at embryonic (E) day 18 (E18). Females maintained on either low- (LPD) or normal- (NPD) protein diets were mated with NPD males. RESULTS: Fetal dry weight and placental efficiency (embryo/placental fresh weight) were positively correlated (r = 0.53, P = 0.0001). Individual placental dry weight was reduced by LPD (P = 0.003), as was the expression of amino acid transporter Slc38a2 and of growth factor Igf2. Placental water content, which is regulated by active transport of solutes, was increased by LPD (P = 0.0001). However, placental ATP content was also increased (P = 0.03). To investigate the possibility of an underlying mitochondrial stress response, we studied cultured human trophoblast cells (BeWos). High throughput imaging showed that amino acid starvation induces changes in mitochondrial morphology that suggest stress-induced mitochondrial hyperfusion. This is a defensive response, believed to increase mitochondrial efficiency, that could underlie the increase in ATP observed in placenta. CONCLUSIONS: These findings reinforce the pathophysiological links between maternal diet and conceptus mitochondria, potentially contributing to metabolic programming. The quiet embryo hypothesis proposes that pre-implantation embryo survival is best served by a relatively low level of metabolism. This may extend to post-implantation trophoblast responses to nutrition.

The interval between the emergence of pharmacologically synchronized ovarian follicular waves and ovum pickup does not significantly affect in vitro embryo production in Bos indicus, Bos taurus, and Bubalus bubalis.

The aim of the present study was to determine the optimal phase of the follicular wave to perform ovum pickup (OPU) for in vitro embryo production (IVEP) in various genetic groups. For this purpose, 27 heifers-nine Bos taurus (Holstein), nine Bos indicus (Nelore), and nine Bubalus bubalis (Mediterranean)-were maintained under the same nutritional, management, and environmental conditions. Heifers within each genetic group were submitted to six consecutive OPU trials with 14-day intersession intervals, at three different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5 after follicular wave emergence), in a 3 × 3 crossover design. When OPU was performed at different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5), no differences were found in the percent of oocytes recovered (70.5 ± 3.1%, 75.0 ± 3.1%, 76.0 ± 3.2%, respectively; P = 0.41) or blastocyst production rates (19.4 ± 2.9%, 16.6 ± 2.9%, 15.9 ± 2.6%, respectively; P = 0.36). Comparing genetic groups, B indicus showed a higher blastocyst rate (28.3(a) ± 2.8%; P < 0.01) than B taurus and B bubalis (14.1(b) ± 2.9% and 10.2(b) ± 2.0%, respectively). However, only B indicus heifers showed a variation in the number of visualized follicles and the total and viable oocytes along consecutive OPU sessions. In conclusion, different phases of the pharmacologically synchronized ovarian follicular wave did not affect OPU-IVEP in B indicus, B taurus, and B bubalis heifers. Additionally, B indicus heifers showed greater OPU-IVEP efficiency than did the other genetic groups, under the same management conditions.

Effect of nitric oxide on the cyclic guanosine monophosphate (cGMP) pathway during meiosis resumption in bovine oocytes.

Nitric oxide (NO) is a chemical messenger involved in the control of oocyte maturation. It stimulates guanylate cyclase to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases that may interfere with cAMP levels, a nucleotide also involved in meiosis resumption. The aim of this study was to determine the role played by NO on the cGMP/cAMP pathway during meiosis resumption in bovine oocytes. The effects of increasing NO generated by S-nitroso-N-acetylpenicillamine (SNAP; 10(-7)-10(-3) mol/L) and of other drugs that may affect the NO/cGMP pathway (proptoporfirin IX and 8-Br-cGMP) on meiosis resumption were investigated in bovine cumulus-oocyte complexes (COCs) matured for 9 hours in a semidefined medium (TCM199 + 3 mg/mL BSA). The COCs matured with 10(-7) mol/L SNAP associated or not with 100 µmol/L oxadiazole-one quinoxaline, a guanylate cyclase inhibitor, also had their cGMP and cAMP levels measured during the first hours of maturation (1, 3, and 6 hours). Quantitative polymerase chain reaction was performed by real-time polymerase chain reaction to determine the effects of NO on expression of genes encoding for enzymes of the NO/guanylate cyclase/cGMP and cAMP pathways during the first 9 hours of oocyte maturation. Increasing NO levels using 10(-7) mol/L SNAP resulted in lower rate of germinal vesicle breakdown (36% germinal vesicle breakdown; P < 0.05) at 9 hours IVM, whereas control group and the treatments with 10(-9) and 10(-8) mol/L SNAP showed about 70% germinal vesicle breakdown (P > 0.05). A temporary increase in cGMP levels was also observed with the same treatment (4.51 pmol/COC) at 1 hour IVM, which was superior to the control group (2.97 pmol/COC; P < 0.05) and was reversed by inhibiting guanylate cyclase activity with 100 µmol/L oxadiazole-one quinoxaline. Neither cAMP levels nor gene expression were affected by NO. These results suggest that NO acts via guanylate cyclase/cGMP and that even a temporary increase in cGMP levels leads to a delay in meiosis resumption, even when cAMP levels have declined. Nitric oxide does not act on oocyte maturation by affecting cAMP levels or the expression of genes related to the NO/guanylate cyclase/cGMP and cAMP pathways. Also, to our knowledge this is the first report to detect PKG1, PKG2, phosphodiesterase-5A, ADCY3, ADCY6, and ADCY9 transcripts in bovine oocytes.

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. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585±34,775 vs. 595,579±31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179±45,617 vs. 498,771±33,231) and blastocysts (816,627±40,235 vs. 765,332±51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

Transmission of mitochondrial DNA diseases and ways to prevent them.

Recent reports of strong selection of mitochondrial DNA (mtDNA) during transmission in animal models of mtDNA disease, and of nuclear transfer in both animal models and humans, have important scientific implications. These are directly applicable to the genetic management of mtDNA disease. The risk that a mitochondrial disorder will be transmitted is difficult to estimate due to heteroplasmy-the existence of normal and mutant mtDNA in the same individual, tissue, or cell. In addition, the mtDNA bottleneck during oogenesis frequently results in dramatic and unpredictable inter-generational fluctuations in the proportions of mutant and wild-type mtDNA. Pre-implantation genetic diagnosis (PGD) for mtDNA disease enables embryos produced by in vitro fertilization (IVF) to be screened for mtDNA mutations. Embryos determined to be at low risk (i.e., those having low mutant mtDNA load) can be preferentially transferred to the uterus with the aim of initiating unaffected pregnancies. New evidence that some types of deleterious mtDNA mutations are eliminated within a few generations suggests that women undergoing PGD have a reasonable chance of generating embryos with a lower mutant load than their own. While nuclear transfer may become an alternative approach in future, there might be more difficulties, ethical as well as technical. This Review outlines the implications of recent advances for genetic management of these potentially devastating disorders.

Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats.

This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca(2+) levels, and interleukin (IL)-1beta expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 microg ml(-1) single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca(2+), and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca(2+) levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

Embryo mitochondrial DNA depletion is reversed during early embryogenesis in cattle.

The extensive replication of mitochondria during oogenesis and the wide variability in mitochondrial DNA (mtDNA) copy numbers present in fully grown oocytes indicate that mtDNA amount may play an important role during early embryogenesis. Using bovine oocytes derived from follicles of different sizes to study the influence of mtDNA content on development, we showed that oocytes obtained from small follicles, known to be less competent in developing into blastocysts, contain less mtDNA than those originating from larger follicles. However, because of the high variability in copy number, a more accurate approach was examined in which parthenogenetic one-cell embryos were biopsied to measure their mtDNA content and then cultured to assess development capacity. Contrasting with previous findings, mtDNA copy number in biopsies was not different between competent and incompetent embryos, indicating that mtDNA content is not related to early developmental competence. To further examine the importance of mtDNA on development, one-cell embryos were partially depleted of their mtDNA (64% +/- 4.1% less) by centrifugation followed by the removal of the mitochondrial-enriched cytoplasmic fraction. Surprisingly, depleted embryos developed normally into blastocysts, which contained mtDNA copy numbers similar to nonmanipulated controls. Development in depleted embryos was accompanied by an increase in the expression of genes (TFAM and NRF1) controlling mtDNA replication and transcription, indicating an intrinsic ability to restore the content of mtDNA at the blastocyst stage. Therefore, we concluded that competent bovine embryos are able to regulate their mtDNA content at the blastocyst stage regardless of the copy numbers accumulated during oogenesis.