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1.
Anim Reprod Sci ; 208: 106125, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31405460

RESUMO

Somatic cell nuclear transfer (SCNT) is an important technique for producing cloned animals. It, however, is inefficient when there is use of SCNT for cloned animal production. Cytoplasm injection cloning technology (CICT) was developed to overcome the inefficiencies of SCNT use of this purpose. The use of CICT involves additional cytoplasm fusing with enucleated oocytes to restore the cytoplasmic volume, thus improving the in vitro developmental competence and quality of cloned embryos. In this study, there was application of CICT in cats to improve the in vitro developmental competence of cloned embryos, as well as the production of the offspring. The results of this study were that fusion rate of the cloned embryos with use of the CICT method was greater than that with SCNT (80.0 ± 4.8% compared with 67.8 ± 11.3%, respectively), and more blastocysts developed with use of CICT than SCNT (20.0 ± 2.0% compared with 13.5 ± 5.0%, respectively). The 62 cloned embryos that were produced with use of CICT were transferred into five estrous synchronized recipients, and 151 cloned embryos produced using SCNT were transferred to 13 estrous-synchronized recipients. After the embryo transfer, there was birth from surrogate mothers of one live-born kitten that resulted using SCNT compared with three live-born kittens using CICT. The number of CICT-cloned embryos born was greater than that of SCNT-cloned embryos (4.8 ± 2.3% compared with 0.7 ± 1.3%, P < 0.05). These results indicate that the CICT technique can be used to produce cloned kittens, including endangered feline species.


Assuntos
Gatos , Clonagem de Organismos/veterinária , Citoplasma , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Animais , Técnicas de Cultura Embrionária/veterinária , Transferência Embrionária/veterinária , Espécies em Perigo de Extinção , Feminino , Técnicas de Transferência Nuclear/veterinária , Gravidez
2.
J Anim Sci ; 97(9): 3786-3794, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31353395

RESUMO

Microchimerism is defined as the presence of a small population of cells or DNA in 1 organism originated from a genetically different organism. It is well established that this phenomenon occurs in humans and mice as cells are exchanged between mother and fetus during gestation. Currently, no information is available about the presence of maternal microchimerism in goats, and the only published study is limited to an evaluation of fetal and fetal-fetal microchimerism in blood samples following natural breeding. In order to determine whether bidirectional fetal-maternal cell or DNA trafficking occurs in goats, we assessed: 1) fetal microchimerism in surrogates that gave birth to somatic cell nuclear transfer (SCNT)-derived transgenic offspring (n = 4), 2) maternal microchimerism following natural breeding of SCNT-derived transgenic does with a nontransgenic buck (n = 4), and 3) fetal-fetal microchimerism in nontransgenic twins of transgenic offspring (n = 3). Neomycin-resistance gene (NEO) gene was selected as the marker to detect the presence of the αMHC-TGF-ß1-Neo transgene in kidney, liver, lung, lymph node, and spleen. We found no detectable maternal or fetal-fetal microchimerism in the investigated tissues of nontransgenic offspring. However, fetal microchimerism was detected in lymph node tissue of one of the surrogate dams carrying a SCNT pregnancy. These results indicate occurrence of cell trafficking from fetus to mother during SCNT pregnancies. The findings of this study have direct implications on the use and disposal of nontransgenic surrogates and nontransgenic offspring.


Assuntos
Quimerismo , Cabras/genética , Animais , Animais Geneticamente Modificados , DNA/genética , Feminino , Feto , Cabras/fisiologia , Técnicas de Transferência Nuclear/veterinária , Parto , Gravidez
3.
Theriogenology ; 135: 25-32, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31195358

RESUMO

Somatic cell nuclear transfer (SCNT), using transgenic donor cells, is a highly efficient method for producing transgenic embryos. We compared the developmental competence, quality and gene expression of transgenic embryos produced by Hand-made cloning from buffalo fetal fibroblasts (BFFs) containing human insulin gene, with non-transgenic embryos produced from BFFs (Controls). The expression vector (pAcISUBC), constructed by inserting human insulin gene between DNA fragments containing mammary gland-specific buffalo ß-lactoglobulin (buBLG) promoter and terminator buBLG 3'UTR regions into pAcGFP-N1 vector, was used for obtaining the 11 kb insert for transfection of BFFs by nucleofection. Presence of the transgene in embryos was confirmed by examining GFP expression by RT-PCR and immunofluorescence. The blastocyst rate was lower (P < 0.05) for transgenic embryos than for controls (35.7 ±â€¯1.8% vs 48.7 ±â€¯2.4%). The apoptotic index was higher (P < 0.05) for transgenic than for control blastocysts which, in turn, was higher (P < 0.05) than for IVF counterparts (6.9 ±â€¯0.9, 3.8 ±â€¯0.5 and 1.8 ±â€¯0.3, respectively). The total cell number was similar for transgenic and non-transgenic blastocysts (143.2 ±â€¯17.0 and 137.2 ±â€¯7.6, respectively). The expression level of pro-apoptotic genes BAX and BID but not that of CASP3 and CASP9, and cell cycle check point control-related gene P53 was higher (P < 0.05), and that of development- (IGF-1R and G6PD) and pluripotency-related gene NANOG was lower (P < 0.05) in transgenic than in control embryos. The expression level of epigenetic-related genes DNMT1, DNMT3a and HDAC1 and pluripotency-related gene OCT4 was similar in the two groups. The expression level of BAX, BID, CASP9, P53, DNMT1 and DNMT3a was higher (P < 0.05) and that of OCT4, NANOG IGF-1R and G6PD was lower (P < 0.05) in cloned transgenic than in IVF blastocysts whereas, that of CASP3 and HDAC1 was similar between the two groups. In conclusion, these results suggest that transgenic embryos produced by SCNT have lower developmental competence and quality, and altered gene expression compared to non-transgenic embryos.


Assuntos
Búfalos/embriologia , Búfalos/genética , Insulina/genética , Técnicas de Transferência Nuclear/veterinária , Animais , Clonagem de Organismos , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos/metabolismo , Fertilização In Vitro/veterinária , Regulação da Expressão Gênica , Humanos , Organismos Geneticamente Modificados
4.
Theriogenology ; 135: 85-93, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31203092

RESUMO

This document discusses recent developments in cloning of husbandry animals through somatic cell nuclear transfer, particularly with a view on improvements in their efficacy. Commercial developments in North and South America, Australia-New Zealand, and China are noted. The regulations and safety aspects surrounding the use of clones and their offspring for the purpose of food production are discussed. It is generally considered that foods from offspring of clones are no different than similar foods from conventional animals, yet besides safety, also ethical and animal welfare considerations come into play at the policy level. The related topic of detection and traceability of clones is discussed, which covers both molecular and documentary methods.


Assuntos
Clonagem de Organismos/veterinária , Técnicas de Transferência Nuclear/veterinária , Bem-Estar do Animal , Animais , Animais Domésticos , Animais Geneticamente Modificados , Clonagem de Organismos/métodos , Alimentos
5.
Nat Commun ; 10(1): 2852, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253764

RESUMO

Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA double-stranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. However, all reported cases only involved C-to-T substitution at a single targeted genomic site. Whether C-to-T substitution is effective in multiple sites/loci has not been verified in large animals. Here, by using pigs, an important animal for agriculture and biomedicine, as the subjective animal, we showed that CBEs could efficiently induce C-to-T conversions at multiple sites/loci with the combination of three genes, including DMD, TYR, and LMNA, or RAG1, RAG2, and IL2RG, simultaneously, at the embryonic and cellular levels. CBEs also could disrupt genes (pol gene of porcine endogenous retrovirus) with dozens of copies by introducing multiple premature stop codons. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Mutação Puntual , Suínos/genética , Desaminase APOBEC-1 , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR , DNA/genética , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Genoma , Técnicas de Transferência Nuclear/veterinária , RNA Guia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
Theriogenology ; 135: 164-168, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31216507

RESUMO

Though blastocyst production in vitro has been successful in several animal species, a culture system to produce viable and normal canine blastocysts in vitro remains to be established. In this study, we report the development of an in vitro culture system for canine preimplantation embryos produced via parthenogenetic activation (PA) and somatic cell nucleus transfer (SCNT). Our results show that the medium developed by us, named "Qingdao Agricultural University's (QAU)-4 medium", successfully breaks the developmental arrest observed at the eight-cell stage in canine embryos grown in other culture systems. The blastocysts produced in QAU-4 displayed normal blastocyst structures, including a clear inner cell mass and blastocyst cavity. We also found that blastocyst formation in PA embryos cultured in QAU-4 medium was quite high, though this was not so in the case of SCNT embryos. However, supplementation of QAU-4 medium with 100 nM of scriptaid caused a sharp increase in blastocyst formation in SCNT embryos. After culture, hatched blastocysts were also observed to successfully adhere to collagen-coated dishes, where further growth and differentiation occurred. To our knowledge, this is the first in vitro canine preimplantation embryo culture system that can successfully produce canine blastocysts.


Assuntos
Blastocisto/fisiologia , Cães/embriologia , Técnicas de Cultura Embrionária/veterinária , Técnicas de Transferência Nuclear/veterinária , Animais , Biomarcadores , Sobrevivência Celular , Células-Tronco Embrionárias/fisiologia , Feminino , Masculino , Partenogênese
7.
Zygote ; 27(3): 137-142, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31036094

RESUMO

SummaryChemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml-1 MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.


Assuntos
Blastocisto/efeitos dos fármacos , Núcleo Celular/metabolismo , Clonagem de Organismos/métodos , Mitomicina/farmacologia , Técnicas de Transferência Nuclear/normas , Oócitos/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/farmacologia , Blastocisto/citologia , Blastocisto/metabolismo , Bovinos , Clonagem de Organismos/veterinária , Transferência Embrionária , Desenvolvimento Embrionário , Feminino , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Oócitos/metabolismo , Partenogênese , Gravidez
8.
J Reprod Dev ; 65(3): 259-265, 2019 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-30905887

RESUMO

This study was carried out to examine the effects of manganese (Mn) on the developmental competence of porcine oocytes during in vitro maturation (IVM) after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). Upon treatment of porcine oocytes with different concentrations (0, 3, 6, and 12 ng/ml) of Mn during IVM, PA was performed to determine the optimum concentration. Following PA, the rate of blastocyst formation was higher significantly in treated porcine oocytes at 6 ng/ml of Mn than in other groups (P < 0.05). However, there was no substantial difference in the cleavage rate and total blastocyst cell numbers among all groups. SCNT was performed using the optimal concentration of Mn from PA, which showed an improved blastocyst formation rate in treated oocytes compared to that in control group (P < 0.05). However, the cleavage rate and total cell numbers per blastocyst were not different between the control and the Mn treated groups after SCNT. Additionally, oocyte nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels were assessed. There was no significant difference observed in nuclear maturation among all the groups. However, enhanced intracellular GSH levels while lower levels of ROS were seen in the Mn treated group compared to the control group (P < 0.05). Thus, these results indicate that Mn supplementation can improve the developmental competence of porcine PA and SCNT embryos by increasing GSH and decreasing ROS levels.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Manganês/farmacologia , Técnicas de Transferência Nuclear/veterinária , Oócitos/citologia , Animais , Antioxidantes/metabolismo , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Técnicas de Cultura Embrionária/veterinária , Feminino , Glutationa/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , Oogênese , Espécies Reativas de Oxigênio/metabolismo , Suínos
9.
J Dairy Sci ; 102(5): 4662-4673, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30879805

RESUMO

Accumulating studies have suggested that microRNA play a part in regulating multiple cellular processes, such as cell proliferation, apoptosis, the cell cycle, and embryo development. This study explored the effects of miR-101-2 on donor cell physiological status and the development of Holstein cow somatic cell nuclear transfer (SCNT) embryos in vitro. Holstein cow bovine fetal fibroblasts (BFF) overexpressing miR-101-2 were used as donor cells to perform SCNT; then, cleavage rate, blastocyst rate, inner cell mass-to-trophectoderm ratio, and the expression of some development- and apoptosis-related genes in different groups were analyzed. The miR-101-2 suppressed the expression of inhibitor of growth protein 3 (ING3) at mRNA and protein levels, expedited cell proliferation, and decreased apoptosis in BFF, suggesting that ING3, a target gene of miR-101-2, is a potential player in this process. Moreover, by utilizing donor cells overexpressing miR-101-2, the development of bovine SCNT embryos in vitro was significantly enhanced; the apoptotic rate in SCNT blastocysts was reduced, and the inner cell mass-to-trophectoderm ratio and SOX2, POU5F1, and BCL2L1 expression significantly increased, whereas BAX and ING3 expression decreased. Collectively, these findings suggest that miR-101-2 promotes BFF proliferation and vitality, reduces their apoptosis, and improves the early development of SCNT embryos.


Assuntos
Apoptose/genética , Bovinos/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica/genética , MicroRNAs/metabolismo , Animais , Blastocisto/metabolismo , Bovinos/crescimento & desenvolvimento , Linhagem Celular , Proliferação de Células/genética , Feminino , Fibroblastos/metabolismo , MicroRNAs/genética , Técnicas de Transferência Nuclear/veterinária , RNA Mensageiro/metabolismo
10.
Theriogenology ; 129: 121-129, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30844653

RESUMO

Atypical chemokine receptor (ACKR) 1, ACKR2, ACKR3, and ACKR4, chemokine decoy receptors that lack G-protein-mediated signaling pathways, internalize and degrade chemokines to control their availability and function. Chemokines play important roles in the endometrium during the estrous cycle and pregnancy, but the expression and regulation of ACKRs have not been determined in pigs. Therefore, we examined the expression of ACKRs in the endometrium throughout the estrous cycle and pregnancy and in conceptus tissues in pigs. ACKR1, ACKR2, ACKR3, and ACKR4 mRNA was expressed in the endometrium, with higher levels of ACKR3 on day 12 of the estrous cycle than in pregnancy and higher levels of ACKR4 on day 15 of pregnancy than in the estrous cycle. ACKR1, ACKR2, and ACKR3, but not ACKR4, mRNA was detected in conceptus and chorioallantoic tissues during pregnancy. ACKR2 and ACKR3 mRNA and ACKR4 protein were mainly localized to luminal epithelial cells and weakly to glandular epithelial cells in the endometrium. Increasing doses of progesterone increased the expression of ACKR2 and ACKR4 and decreased the expression of ACKR3 in endometrial tissues. On day 12 of pregnancy, the expression of ACKR4 mRNA was lower in the endometria of gilts with somatic cell nucleus transfer-derived conceptuses than in the endometria of gilts carrying conceptuses derived from natural mating. These results indicate that the expression of ACKRs is dynamically regulated at the maternal-conceptus interface, suggesting that ACKR proteins might play critical roles in regulating endometrial chemokines to support the establishment and maintenance of pregnancy in pigs.


Assuntos
Endométrio/metabolismo , Ciclo Estral/metabolismo , Prenhez/metabolismo , Receptores de Quimiocinas/metabolismo , Suínos/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Técnicas de Transferência Nuclear/veterinária , Gravidez , Receptores de Quimiocinas/genética , Suínos/genética
11.
Cell Reprogram ; 21(1): 51-60, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30735075

RESUMO

Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p < 0.05) in the CICT group than in the SCNT group (28.9 ± 0.8% vs. 20.2 ± 1.3%, respectively). Furthermore, the total cell number per day 8 blastocyst was significantly higher in the CICT group than in the SCNT group (176.2 ± 6.5 vs. 119.3 ± 7.7, p < 0.05). Moreover, CICT increased mitochondrial activity, as detected using MitoTracker® Green. The mRNA levels of DNA methyltransferase 1 and DNA methyltransferase 3a were significantly lower (p < 0.05) in the CICT group than in the SCNT group. The mRNA level of DNA methyltransferase 3b was lower in the CICT group than in the SCNT group; however, this difference was not significant (p > 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.


Assuntos
Blastocisto/citologia , Bovinos/embriologia , Clonagem de Organismos , DNA (Citosina-5-)-Metiltransferases/metabolismo , Oócitos/citologia , Animais , Citoplasma , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Desenvolvimento Embrionário , Feminino , Mitocôndrias/metabolismo , Técnicas de Transferência Nuclear/veterinária
12.
Cell Reprogram ; 21(1): 26-36, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30735078

RESUMO

The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days. The iPS-like cells demonstrated pluripotent characteristics such as self-renewal, high proliferation, expression of pluripotent markers, and aggregation ability. The embryo development through somatic cell nuclear transfer (SCNT), cleavage rate, and blastocyst formation rate did not show any significant differences. However, the total cell number of blastocysts was significantly increased with the established cell line. In conclusion, the iPS-like cell line, generated from porcine transcriptional factors using the PB transposon system, demonstrated pluripotency with the capacity for unlimited self-renewal, and could be used as donor cells to produce cloned embryos by SCNT. These cells will be suitable for gene modification and would contribute to the stability or safety of pig models in biomedical research.


Assuntos
Blastocisto/citologia , Técnicas de Cultura de Células , Clonagem de Organismos , Regulação da Expressão Gênica no Desenvolvimento , Suínos/embriologia , Animais , Animais Geneticamente Modificados , Blastocisto/fisiologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Células Cultivadas , Desenvolvimento Embrionário , Fibroblastos , Técnicas de Transferência Nuclear/veterinária , Células-Tronco Pluripotentes/citologia , Transfecção
13.
Reprod Fertil Dev ; 31(5): 855-866, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30641030

RESUMO

X (inactive)-specific transcript (Xist) is crucial in murine cloned embryo development, but its role in cloned goats remains unknown. Therefore, in this study we examined the expression and methylation status of Xist in somatic cell nuclear transfer (SCNT) embryos, as well as in ear, lung, and brain tissue of deceased cloned goats. First, the Xist sequence was amplified and a differentially methylated region was identified in oocytes and spermatozoa. Xist methylation levels were greater in SCNT- than intracytoplasmic sperm injection-generated female 8-cell embryos. In addition, compared with naturally bred controls, Xist methylation levels were significantly increased in the ear, lung, and brain tissue of 3-day-old female deceased cloned goats, but were unchanged in the ear tissue of female live cloned goats and in the lung and brain of male deceased cloned goats. Xist expression was significantly increased in the ear tissue of female live cloned goats, but decreased in the lung and brain of female deceased cloned goats. In conclusion, hypermethylation of Xist may have resulted from incomplete reprogramming and may be retained in 3-day-old female deceased cloned goats, subsequently leading to dysregulation of Xist.


Assuntos
Metilação de DNA , Técnicas de Transferência Nuclear/veterinária , Oócitos/metabolismo , RNA Longo não Codificante/metabolismo , Espermatozoides/metabolismo , Animais , Clonagem de Organismos , Feminino , Cabras , Masculino , RNA Longo não Codificante/genética
14.
Theriogenology ; 127: 80-87, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30677595

RESUMO

The objective of this study was to examine the effect of alanine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development in pigs. To this end, we investigated the nuclear maturation, intraoocyte glutathione (GSH) content of IVM oocytes, and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In addition, we analyzed the expression of genes associated with apoptosis and embryonic development in IVM oocytes, 4-cell stage embryos, and blastocysts produced via PA and SCNT. To determine the optimal concentration of alanine to promote the maturation and development of PA and SCNT embryos, various concentrations (0, 0.363, 1, 5, and 10 mM) of alanine were added to IVM medium during oocyte maturation. The proportion of metaphase II (MII) oocytes after IVM did not differ according to the concentration of alanine. However, significantly higher intraoocyte GSH content was observed in oocytes treated with 0.363 mM alanine compared with that in untreated oocytes. However, treatment of recipient oocytes with 5 or 10 mM alanine during IVM decreased the GSH content in mature oocytes compared to that in control oocytes. Oocytes matured in the presence of 0.363 mM alanine showed significantly increased rates of cleavage and blastocyst formation after PA and SCNT compared to untreated oocytes. PA and SCNT embryos from the 0.363 mM alanine-treated group of MII oocytes showed significantly higher transcript levels of POU5F1 and FGFR2, which are associated with oocyte quality and embryonic development, than the untreated group. Our results suggest that treatment of pig oocytes with 0.363 mM alanine during IVM improves embryonic developmental competence after PA and SCNT by increasing intraoocyte GSH content and increasing the mRNA expression of POU5F1 and FGFR2.


Assuntos
Alanina/farmacologia , Suplementos Nutricionais , Desenvolvimento Embrionário , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Transferência Nuclear/veterinária , Suínos , Animais , Apoptose , Feminino , Glutationa/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo
15.
Reprod Fertil Dev ; 31(5): 941-952, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30689958

RESUMO

Because of the growing importance of horses in leisure and several sports, somatic cell nuclear transfer (SCNT) is being used more frequently for cloning animals for performance and reproductive purposes. However, because of the need to perforate the zona pellucida during microsurgical reconstruction of the oocyte, it is possible that SCNT-derived embryos undergo premature hatching, resulting in embryo bisection and twinning. Therefore, because equine twin pregnancies often lead to abnormal embryo development and pregnancy failure, we performed a detailed comparative assessment of equine twin fetuses derived by SCNT with particular attention on the development of the central nervous system at 40 and 60 days gestation. The results of this study indicate that although cloned twin embryos show small differences in size, they do not exhibit apparent macro- or microscopic developmental discrepancies in the central nervous system, suggesting that the twining phenomenon resulting from SCNT does not affect fetal differentiation.


Assuntos
Sistema Nervoso Central/embriologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/fisiologia , Desenvolvimento Fetal/fisiologia , Técnicas de Transferência Nuclear/veterinária , Animais , Clonagem de Organismos , Transferência Embrionária/veterinária , Feminino , Cavalos , Gravidez
16.
Mol Biol Rep ; 46(2): 1737-1746, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30694456

RESUMO

Episomal plasmids based on a scaffold/matrix attachment region (S/MAR) are extrachromosomal DNA entities that replicate once per cell cycle and are stably maintained in cells or tissue. We generated minicircles, episomal plasmids devoid of bacterial sequences, and show that they are stably transmitted in clonal primary bovine fibroblasts without selection pressure over more than two months. Total DNA, plasmid extraction and fluorescence in situ hybridization (FISH) analyses suggest that the minicircles remained episomal and were not integrated into the genome. Minicircles survived extended periods in serum-starved cells, which indicates that ongoing transcription in non-proliferating cells is not necessary for the maintenance of S/MAR-episomes. To test whether minicircles endure the process of somatic cell nuclear transfer (SCNT), we used cell-cycle synchronized, serum-starved, minicircle-containing cells. Analysis of cells outgrown from SCNT-derived blastocysts shows that the minicircles are maintained through SCNT and early embryonic development, which raises the prospect of using cell lines with episomal minicircles for the generation of transgenic animals.


Assuntos
DNA Circular/fisiologia , Plasmídeos/genética , Plasmídeos/fisiologia , Animais , Animais Geneticamente Modificados/genética , Blastocisto , Bovinos , DNA Circular/genética , Vetores Genéticos/genética , Hibridização in Situ Fluorescente , Técnicas de Transferência Nuclear/veterinária
17.
Theriogenology ; 126: 8-16, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30508790

RESUMO

Application of cloning technology on a wide scale is severely limited by the very low live birth rate obtained with cloned embryos. Embryo quality is an important factor which affects the conception and live birth rate of cloned embryos. microRNA-21 (miR-21) has been implicated in the regulation of apoptosis and the expression level of several important genes which control apoptosis. We examined the effects of treatment of reconstructed buffalo embryos, produced by Hand-made cloning, with miR-21 mimic on developmental competence, quality and gene expression of cloned embryos. Expression level of miR-21, which increased from 2-cell to 8-cell stage and then decreased at the blastocyst stage, showed a similar pattern in cloned and IVF embryos. It was lower in cloned than in IVF embryos at 2-, 4- and 8-cell (P < 0.001) and blastocyst (P < 0.05) stages but not at morula stage. Treatment of reconstructed embryos with miR-21 mimic for 1 h after 1 h of electrofusion, increased (P < 0.05) the total cell number (251.3 ±â€¯10.7 vs 181.5 ±â€¯2.13). Blastocysts produced from miR-21-treated reconstructed embryos had lower (P < 0.05) apoptotic index than controls and IVF blastocysts (2.01 ±â€¯0.17, 5.46 ±â€¯0.26 and 4.19 ±â€¯0.15, respectively). The treatment also improved the inner cell mass:trophectoderm cell number ratio of blastocysts than in controls (0.21 ±â€¯0.01 vs 0.11 ±â€¯0.003) to values observed in IVF blastocysts (0.20 ±â€¯0.008). However, miR-21 mimic treatment did not affect the blastocyst rate, which was similar for treatment, control and negative control groups (36.58 ±â€¯3.64, 36.58 ±â€¯3.64 and 32.2 ±â€¯2.86%, respectively). miR-21 mimic treatment increased (P < 0.01) the expression level of apoptosis- (BCL2 and PTEN), pluripotency- (OCT4 and SOX2) and development-related genes (GLUT1, FGF4 and P53), but not that of CASPASE3 than in untreated controls in blastocysts. These results suggest that treatment of reconstructed embryos with miR-21 mimic improves blastocyst quality, reduces apoptosis and alters gene expression without improving the blastocyst rate.


Assuntos
Búfalos/embriologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , MicroRNAs/farmacologia , Animais , Apoptose/efeitos dos fármacos , Técnicas de Cultura Embrionária/métodos , Regulação da Expressão Gênica no Desenvolvimento , Marcação In Situ das Extremidades Cortadas , Técnicas de Transferência Nuclear/veterinária
18.
Reprod Domest Anim ; 54(2): 258-269, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30220080

RESUMO

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40 V/mm, 20 µs each) followed by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION) + 6-DMAP + CHX (12.07%) activation was higher than that of ION + 6-DMAP (8.6%) activation or ION + CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION + 6-DMAP + CHX and DC + 6-DMAP + CHX groups. The blastocyst rate of ION + 6-DMAP + CHX-activated oocytes in the basic rabbit culture medium (M-199) + 10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M-199 + 10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3-5 days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6-9 days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.


Assuntos
Blastocisto/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Transferência Nuclear/veterinária , Oócitos/efeitos dos fármacos , Partenogênese , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Blastocisto/fisiologia , Cicloeximida/farmacologia , Desenvolvimento Embrionário/fisiologia , Feminino , Ionomicina/farmacologia , Oócitos/fisiologia , Coelhos
19.
Reprod Domest Anim ; 54(2): 289-299, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30317681

RESUMO

The aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC-AF), adipose tissue (MSC-AT) and fibroblasts (FIB-control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3 and KAT2A genes was measured in embryos by qRT-PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC-AF were less hydroxymethylated for both animals. MSC-AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC-AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production. MSC-AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production.


Assuntos
Histona Acetiltransferases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Células-Tronco Mesenquimais/citologia , Acetilação , Animais , Bovinos , Clonagem de Organismos/métodos , Clonagem de Organismos/veterinária , Metilação de DNA , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Histona Acetiltransferases/genética , Histona Desacetilases/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Técnicas de Transferência Nuclear/veterinária
20.
Theriogenology ; 125: 259-267, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30476759

RESUMO

Artificial oocyte activation is an essential step in somatic cell nuclear transfer (SCNT) and can enhance viability of embryos as a form of assisted reproductive technology (ART) in clinics. Most artificial activation methods have been developed to increase cytosolic calcium (Ca2+) level in oocytes. Interestingly, recent studies have demonstrated that mammalian oocytes can be activated using N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn2+ chelator. Although effective, TPEN is also known to induce apoptosis and shows poor selectivity between free Zn2+ and protein-bound Zn2+. The aim of this study was to identify different Zn2+ chelators that can activate pig oocytes. Among five Zn2+ chelators examined, 1,10-phenanthroline (Phen), and tris(2-pyridylmethyl)amine (TPA) successfully activated pig oocytes. The level of available Zn2+ was reduced without any increase in Ca2+ in oocytes incubated with Phen or TPA, indicating that the oocyte activation occurred independently of Ca2+ signal. When various concentrations (100-500 µM) and incubation durations (10-120 min) of Phen and TPA were used to activate pig oocytes, 500 µM for 60 min and 100 µM for 60 min of Phen and TPA treatments, respectively, were found to be most effective in supporting embryo development. The frequency of blastocyst formation after the treatments was higher than 40% at day 7. When oocytes were incubated with TPEN, Phen, or TPA under their optimal treatment conditions, there was no significant difference in the frequencies of day 7 blastocyst formation among the three treatments. However, day 5 blastocyst formation was observed from the Phen- and TPA-treated oocytes, whereas no blastocyst was formed at day 5 in the TPEN-treated oocytes. The average total cell number in day 7 blastocysts was higher in the Phen treatment group than in the TPEN treatment (P < 0.05). These results suggest that Phen and TPA can be used as powerful agents to artificially activate oocytes and to increase the developmental potential of SCNT embryos or embryos going through clinical ART procedures.


Assuntos
Quelantes/farmacologia , Técnicas de Transferência Nuclear/veterinária , Oócitos/efeitos dos fármacos , Suínos , Zinco/metabolismo , Animais , Blastocisto/citologia , Feminino , Oócitos/fisiologia
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