RESUMO
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.
Assuntos
Técnicas de Transferência Nuclear , Células-Tronco Pluripotentes , Bovinos , Animais , Técnicas de Transferência Nuclear/veterinária , Clonagem de Organismos/métodos , Biotecnologia , Clonagem MolecularRESUMO
We evaluated the effect of the antral follicle count (AFC) on ovarian follicular dynamics, pregnancy rates, progesterone concentrations, and transcriptional patterns of genes in Nelore cattle (Bos taurus indicus) after a timed artificial insemination (TAI) programme. Cows were separated based on the AFC, and those with a high AFC showed a larger (P < 0.0001) ovarian diameter and area than those with a very low AFC. Females with a very low AFC exhibited a larger (P < 0.01) diameter of the dominant follicle at TAI (13.6 ± 0.3 vs. 12.2 ± 0.4 mm) and a tendency (P = 0.06) to have different serum progesterone concentrations (2.9 ± 0.3 vs. 2.1 ± 0.3 ng/mL; on day 18, considering day 0 as the beginning of the synchronization protocol) than those with a high AFC. The pregnancy rate was higher (P ≤ 0.05) in animals with a very low (57.9%) and low (53.1%) AFC than in those with a high AFC (45.2%). The expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis and cellular stress response was assessed on day 5. In females with a low AFC, 8 and 21 genes in oocytes and cumulus cells, respectively, were upregulated (P < 0.05), while 3 and 6 genes in oocytes and cumulus cells, respectively, were downregulated. The results described here will help elucidate the differences in ovarian physiology and the reproductive success of Bos indicus females with a low or high AFC.
Assuntos
Folículo Ovariano/fisiologia , Taxa de Gravidez , Progesterona/sangue , Transcriptoma , Animais , Bovinos , Células do Cúmulo/citologia , Feminino , Inseminação Artificial/veterinária , Oócitos/citologia , Folículo Ovariano/citologia , Ovário/citologia , Ovário/fisiologia , GravidezRESUMO
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.
Assuntos
Reprogramação Celular/genética , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Histona Metiltransferases/genética , Animais , Blastocisto , Bovinos , Diferenciação Celular , Clonagem de Organismos/métodos , Transferência Embrionária/métodos , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Técnicas de Transferência Nuclear , Oócitos/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional/genética , Quinazolinas/farmacologiaRESUMO
The objective of the present study was to evaluate efficiency of in vitro fertilization (IVF) in Nelore, Brangus, and Girolando oocyte donors. Ovum pickup (OPU) from the donors was conducted every 15 days to assess oocyte recovery, IVF, and post-transfer pregnancy percentage. For Nelore, the mean numbers of total and viable oocytes recovered (23.5 ± 1.1 and 14.0 ± 1.0, respectively) were higher (p < 0.05) than those for Brangus (12.7 ± 1.9 and 6.6 ± 1.0, respectively) and Girolando (12.5 ± 1.4 and 6.8 ± 0.7, respectively); Brangus and Girolando did not differ from each other (p > 0.05). The percentage of blastocyst production differed (p < 0.05) between Nelore (48.4 ± 2.4%), Brangus (40.3 ± 3.6%), and Girolando (38.9 ± 2.6%), but those in Brangus and Girolando did not differ (p > 0.05). The percentage of blastocysts (transferred) that resulted in pregnancy did not differ (p > 0.05) between Nelore (45.5 ± 3.8%), Brangus (41.7 ± 4.1%), and Girolando (40.7 ± 3.7%). Of the breeds studied, Nelore donors are more efficient for IVF, but conditions of this study.
Assuntos
Fertilização in vitro/veterinária , Doação de Oócitos/veterinária , Animais , Blastocisto , Cruzamento , Bovinos , Feminino , Fertilização in vitro/estatística & dados numéricos , Doação de Oócitos/estatística & dados numéricos , Oócitos , Gravidez , Taxa de GravidezRESUMO
We evaluated the competence of bovine oocytes via morphological selection associated with follicular diameter and by brilliant cresyl blue (BCB) staining, in order to improve oocyte selection and the efficiency of in vitro embryo production. Follicles with small (2<4 mm), large (48 mm) or mixed (28 mm) diameters were aspirated from slaughterhouse ovaries. Next, the morphologically categorized oocytes were stained and evaluated with 26 μM of BCB (90 min), followed by an assessment of in vitro maturation (MII) and blastocyst production. Oocytes from the large (61.1 ± 3.9%) and mixed (52.9 ± 4.6%) follicular groups showed comparable (p>0,05) BCB+ rates, but both differed (p<0.05) as compared to the small-diameter group (41.0 ± 4.5%). Between the BCB+ and BCB oocytes, there was no difference (p>0,05) in MII rate in the mixed (respectively 86.7 ± 3.4 and 75.8 ± 2.2), large (respectively 89.4 ± 2.6 and 79.7 ± 2.3) and small (respectively 76.7 ± 2.9 and 67.2 ± 2.5) diameter groups. Comparing BCB (+ and -) oocytes, we observed (p>0,05) no change in the percentage of embryos in the large group (respectively 67.3 ± 3.3 and 53.5 ± 5.8) and in the small group (respectively 43.8 ± 5.3 and 33.9 ± 2.2), but there was a difference (p<0.05) in the mixed group (respectively 57.8 ± 3.9 and 39.6 ± 7.5). Furthermore, when we compared between follicular groups, the BCB+/large-diameter group (61.1 ± 3.9%) had a significant higher percentage of embryos (p<0.05) than the small-diameter BCB+ (43.8 ± 5.3) groups. To conclude, selection procedures that use follicle size and/or BCB staining may be used as an additional tool to improve the in vitro production of embryos.(AU)
Avaliou-se a competência de oócitos bovinos através da seleção morfológica associada ao diâmetro folicular e pela coloração azul cresil brilhante (BCB), a fim de melhorar a seleção de oócitos e a eficiência da produção in vitro de embriões. Folículos com diâmetro pequeno (2<4 mm), grande (4-8 mm) ou misto (2-8 mm) foram aspirados de ovários de matadouro. Em seguida, os oócitos classificados morfologicamente foram corados e avaliados com 26 μM de BCB (90 min), seguido de uma avaliação da maturação in vitro (MII) e produção de blastocisto. Os oócitos dos grupos folículo grande (61,1 ± 3,9%) e misto (52,9 ± 4,6%) apresentaram taxas de BCB+ comparáveis (p>0,05), mas ambos diferiram (p<0,05) em relação ao grupo folículo pequeno (41,0 ± 4,5%). Entre os oócitos BCB+ e BCB, não houve diferença (p>0,05) na taxa de MII nos grupos mistos (respectivamente 86,7 ± 3,4 e 75,8 ± 2,2), grande (respectivamente 89,4 ± 2,6 e 79,7 ± 2,3) e pequeno (respectivamente 76,7 ± 2,9 e 67,2 ± 2,5). Comparando os oócitos BCB (+ e -), não observamos alteração (p>0,05) na porcentagem de embriões no grupo grande (respectivamente 67,3 ± 3,3 e 53,5 ± 5,8) e no grupo pequeno (respectivamente 43,8 ± 5,3 e 33,9 ± 2,2), mas há houve diferença (p<0,05) no grupo misto (respectivamente 57,8 ± 3,9 e 39,6 ± 7,5). Além disso, quando comparamos os grupos foliculares, o grupo BCB+/grande (61,1 ± 3,9%) apresentou uma porcentagem significativamente maior de embriões (p<0,05) do que o grupo BCB+/pequeno (43,8 ± 5,3). Para concluir, os procedimentos de seleção que usam tamanho do folículo e/ou coloração com BCB podem ser usados como uma ferramenta adicional para melhorar a produção in vitro de embriões.(AU)
Assuntos
Animais , Feminino , Bovinos , Pesquisas com Embriões , Desenvolvimento Embrionário , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/crescimento & desenvolvimento , Blastocisto , Embrião de Mamíferos/embriologia , Técnicas In Vitro/veterinária , Técnicas In Vitro/métodosRESUMO
We evaluated the competence of bovine oocytes via morphological selection associated with follicular diameter and by brilliant cresyl blue (BCB) staining, in order to improve oocyte selection and the efficiency of in vitro embryo production. Follicles with small (20,05) BCB+ rates, but both differed (p0,05) in MII rate in the mixed (respectively 86.7 ± 3.4 and 75.8 ± 2.2), large (respectively 89.4 ± 2.6 and 79.7 ± 2.3) and small (respectively 76.7 ± 2.9 and 67.2 ± 2.5) diameter groups. Comparing BCB (+ and -) oocytes, we observed (p>0,05) no change in the percentage of embryos in the large group (respectively 67.3 ± 3.3 and 53.5 ± 5.8) and in the small group (respectively 43.8 ± 5.3 and 33.9 ± 2.2), but there was a difference (p<0.05) in the mixed group (respectively 57.8 ± 3.9 and 39.6 ± 7.5). Furthermore, when we compared between follicular groups, the BCB+/large-diameter group (61.1 ± 3.9%) had a significant higher percentage of embryos (p<0.05) than the small-diameter BCB+ (43.8 ± 5.3) groups. To conclude, selection procedures that use follicle size and/or BCB staining may be used as an additional tool to improve the in vitro production of embryos.
Avaliou-se a competência de oócitos bovinos através da seleção morfológica associada ao diâmetro folicular e pela coloração azul cresil brilhante (BCB), a fim de melhorar a seleção de oócitos e a eficiência da produção in vitro de embriões. Folículos com diâmetro pequeno (20,05), mas ambos diferiram (p0,05) na taxa de MII nos grupos mistos (respectivamente 86,7 ± 3,4 e 75,8 ± 2,2), grande (respectivamente 89,4 ± 2,6 e 79,7 ± 2,3) e pequeno (respectivamente 76,7 ± 2,9 e 67,2 ± 2,5). Comparando os oócitos BCB (+ e -), não observamos alteração (p>0,05) na porcentagem de embriões no grupo grande (respectivamente 67,3 ± 3,3 e 53,5 ± 5,8) e no grupo pequeno (respectivamente 43,8 ± 5,3 e 33,9 ± 2,2), mas há houve diferença (p<0,05) no grupo misto (respectivamente 57,8 ± 3,9 e 39,6 ± 7,5). Além disso, quando comparamos os grupos foliculares, o grupo BCB+/grande (61,1 ± 3,9%) apresentou uma porcentagem significativamente maior de embriões (p<0,05) do que o grupo BCB+/pequeno (43,8 ± 5,3). Para concluir, os procedimentos de seleção que usam tamanho do folículo e/ou coloração com BCB podem ser usados como uma ferramenta adicional para melhorar a produção in vitro de embriões.
Assuntos
Feminino , Animais , Bovinos , Blastocisto , Desenvolvimento Embrionário , Embrião de Mamíferos/embriologia , Folículo Ovariano/crescimento & desenvolvimento , Oócitos/crescimento & desenvolvimento , Pesquisas com Embriões , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas In Vitro/métodos , Técnicas In Vitro/veterináriaRESUMO
Semen fertilizing potential is dependent upon the morphological, functional and molecular attributes of sperm. Sperm microRNAs (miRNAs) were recently shown to hold promise regarding their association with different fertility phenotypes. However, their role in fertility regulation remains to be determined. We postulated that sperm miRNAs might regulate early embryonic development. From this perspective, sperm quality and 380 sperm miRNAs were investigated in frozen-thawed semen from high (HF; 54.3 ± 1.0% pregnancy rate) and low (LF; 41.5 ± 2.3%) fertility bulls. Out of nine miRNAs that showed different levels in sperm cells, miR-216b was present at lower levels in HF sperm cells and zygotes. Among miR-216b target genes (K-RAS, BECN1 and JUN), K-RAS, related to cell proliferation, revealed a higher level in HF two-cell embryos. First cleavage rate, blastocyst cell number and division number were also higher in HF. In addition, by using a model based on polyspermy embryos, we demonstrated an increase in miR-216b levels in zygotes associated with sperm cell entry. Our results shed light on a possible mechanism of paternal contribution involving sperm-borne miR-216b that modulates levels of miR-216b in zygotes and K-RAS in two-cell embryos. This modulation might regulate early development by interfering with the first cleavage and blastocyst quality.
Assuntos
Blastômeros/metabolismo , Desenvolvimento Embrionário/fisiologia , Genes ras , Espermatozoides/química , Zigoto/metabolismo , Animais , Bovinos , Divisão Celular , Desenvolvimento Embrionário/genética , Fertilidade , Fertilização , Masculino , Proteínas Proto-Oncogênicas p21(ras)/análise , Análise do Sêmen , Espermatozoides/fisiologiaRESUMO
The present study evaluated Brangus cows treated with single doses of follicle stimulating hormone (FSH) subjected to follicular aspiration after 24 h to assess oocyte recovery, in vitro fertilization and pregnancy rate. Follicles exceeding 3 millimeters in diameter were aspirated, 200 mg of FSH was administered 2 days later, and a new ovum pickup was performed 24 h afterward. These methods were performed 3 times every 3 days. In control, follicular aspirations occurred at intervals of 1-week without FSH administration o. The aspirated oocytes were evaluated, submitted to in vitro fertilization and the embryos were transferred to the recipients. The average recovery of oocytes was higher (p 0.05) in control cows (12.4±1.8) than in treated cows (9.4±1.3). There was no difference (p>0.05) in the mean percentage of viable oocytes (52.0±3.9 and 62.7±4.7%) or the mean percentage of embryos (41.4±4.8 and 41.5±4.2%) among control and treated cows, respectively. The mean percentage of pregnancy did not differ (p>0.05) for control cows (43.8±2.7%), and treated cows (40.9±6.8%). In conclusion, FSH treatment did not improve oocyte recovery, in vitro fertilization, and pregnancy percentage. However, there is possibility of several consecutive ovum pickup every t3 days, concentrating the in vitro fertilization and the pregnancy percentage.
O presente estudo avaliou vacas Brangus tratadas com doses únicas de hormônio folículo estimulante (FSH) submetidas a aspiração folicular após vinte e quatro horas, para avaliação da recuperação oocitária, fertilização in vitro e taxa de prenhez. Folículos superiores a três milímetros de diâmetro foram aspirados, 200 mg de FSH foram administrados dois dias depois e uma nova aspiração folicular foi realizada 24 horas após. Esses métodos foram efetivados três vezes a cada três dias. No controle, as aspirações foliculares ocorreram em intervalos de uma semana sem administração de FSH. Os oócitos aspirados foram avaliados, submetidos à fertilização in vitro e os embriões foram transferidos em receptoras. A recuperação média dos oócitos foi superior (p 0,05) nas vacas controle (12,4±1,8) do que nas vacas tratadas (9,4±1,3). Não houve diferença (p>0,05) na porcentagem média de oócitos viáveis (52,0±3,9 e 62,7±4,7%) ou na porcentagem média de embriões (41,4±4,8 e 41,5±4,2%) entre vacas controle e vacas tratadas, respectivamente. A porcentagem média de prenhez não diferiu (p>0,05) para as vacas controle (43,8±2,7%) e as tratadas (40,9±6,8%). Em conclusão, o tratamento com FSH não melhorou a recuperação de oócitos, a fertilização in vitro e o percentual de prenhez. No entanto, existe a possibilidade de várias aspirações foliculares consecutivas a cada três
RESUMO
The present study evaluated Brangus cows treated with single doses of follicle stimulating hormone (FSH) subjected to follicular aspiration after 24 h to assess oocyte recovery, in vitro fertilization and pregnancy rate. Follicles exceeding 3 millimeters in diameter were aspirated, 200 mg of FSH was administered 2 days later, and a new ovum pickup was performed 24 h afterward. These methods were performed 3 times every 3 days. In control, follicular aspirations occurred at intervals of 1-week without FSH administration o. The aspirated oocytes were evaluated, submitted to in vitro fertilization and the embryos were transferred to the recipients. The average recovery of oocytes was higher (p<0.05) in control cows (12.4±1.8) than in treated cows (9.4±1.3). There was no difference (p>0.05) in the mean percentage of viable oocytes (52.0±3.9 and 62.7±4.7%) or the mean percentage of embryos (41.4±4.8 and 41.5±4.2%) among control and treated cows, respectively. The mean percentage of pregnancy did not differ (p>0.05) for control cows (43.8±2.7%), and treated cows (40.9±6.8%). In conclusion, FSH treatment did not improve oocyte recovery, in vitro fertilization, and pregnancy percentage. However, there is possibility of several consecutive ovum pickup every t3 days, concentrating the in vitro fertilization and the pregnancy percentage.
O presente estudo avaliou vacas Brangus tratadas com doses únicas de hormônio folículo estimulante (FSH) submetidas a aspiração folicular após vinte e quatro horas, para avaliação da recuperação oocitária, fertilização in vitro e taxa de prenhez. Folículos superiores a três milímetros de diâmetro foram aspirados, 200 mg de FSH foram administrados dois dias depois e uma nova aspiração folicular foi realizada 24 horas após. Esses métodos foram efetivados três vezes a cada três dias. No controle, as aspirações foliculares ocorreram em intervalos de uma semana sem administração de FSH. Os oócitos aspirados foram avaliados, submetidos à fertilização in vitro e os embriões foram transferidos em receptoras. A recuperação média dos oócitos foi superior (p<0,05) nas vacas controle (12,4±1,8) do que nas vacas tratadas (9,4±1,3). Não houve diferença (p>0,05) na porcentagem média de oócitos viáveis (52,0±3,9 e 62,7±4,7%) ou na porcentagem média de embriões (41,4±4,8 e 41,5±4,2%) entre vacas controle e vacas tratadas, respectivamente. A porcentagem média de prenhez não diferiu (p>0,05) para as vacas controle (43,8±2,7%) e as tratadas (40,9±6,8%). Em conclusão, o tratamento com FSH não melhorou a recuperação de oócitos, a fertilização in vitro e o percentual de prenhez. No entanto, existe a possibilidade de várias aspirações foliculares consecutivas a cada três
RESUMO
The present study evaluated Brangus cows treated with single doses of follicle stimulating hormone (FSH) subjected to follicular aspiration after 24 h to assess oocyte recovery, in vitro fertilization and pregnancy rate. Follicles exceeding 3 millimeters in diameter were aspirated, 200 mg of FSH was administered 2 days later, and a new ovum pickup was performed 24 h afterward. These methods were performed 3 times every 3 days. In control, follicular aspirations occurred at intervals of 1-week without FSH administration o. The aspirated oocytes were evaluated, submitted to in vitro fertilization and the embryos were transferred to the recipients. The average recovery of oocytes was higher (p<0.05) in control cows (12.4±1.8) than in treated cows (9.4±1.3). There was no difference (p>0.05) in the mean percentage of viable oocytes (52.0±3.9 and 62.7±4.7%) or the mean percentage of embryos (41.4±4.8 and 41.5±4.2%) among control and treated cows, respectively. The mean percentage of pregnancy did not differ (p>0.05) for control cows (43.8±2.7%), and treated cows (40.9±6.8%). In conclusion, FSH treatment did not improve oocyte recovery, in vitro fertilization, and pregnancy percentage. However, there is possibility of several consecutive ovum pickup every t3 days, concentrating the in vitro fertilization and the pregnancy percentage.
O presente estudo avaliou vacas Brangus tratadas com doses únicas de hormônio folículo estimulante (FSH) submetidas a aspiração folicular após vinte e quatro horas, para avaliação da recuperação oocitária, fertilização in vitro e taxa de prenhez. Folículos superiores a três milímetros de diâmetro foram aspirados, 200 mg de FSH foram administrados dois dias depois e uma nova aspiração folicular foi realizada 24 horas após. Esses métodos foram efetivados três vezes a cada três dias. No controle, as aspirações foliculares ocorreram em intervalos de uma semana sem administração de FSH. Os oócitos aspirados foram avaliados, submetidos à fertilização in vitro e os embriões foram transferidos em receptoras. A recuperação média dos oócitos foi superior (p<0,05) nas vacas controle (12,4±1,8) do que nas vacas tratadas (9,4±1,3). Não houve diferença (p>0,05) na porcentagem média de oócitos viáveis (52,0±3,9 e 62,7±4,7%) ou na porcentagem média de embriões (41,4±4,8 e 41,5±4,2%) entre vacas controle e vacas tratadas, respectivamente. A porcentagem média de prenhez não diferiu (p>0,05) para as vacas controle (43,8±2,7%) e as tratadas (40,9±6,8%). Em conclusão, o tratamento com FSH não melhorou a recuperação de oócitos, a fertilização in vitro e o percentual de prenhez. No entanto, existe a possibilidade de várias aspirações foliculares consecutivas a cada três
RESUMO
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RESUMO
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.
Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Fertilidade/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Oócitos/metabolismo , Ácido 3-Hidroxibutírico/biossíntese , Ácido 3-Hidroxibutírico/genética , Acetilação , Animais , Blastocisto/citologia , Bovinos , Linhagem Celular , Células do Cúmulo/metabolismo , Feminino , Proteína Forkhead Box O3/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Técnicas de Transferência Nuclear , Estresse Oxidativo/efeitos dos fármacos , PPAR alfa/biossíntese , GravidezRESUMO
Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return." In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 µM of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx+) and Caspase-9-positive (Casp-9+) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx+ (4.6% in control cells; p<0.01) and 24.9% were Casp-9+ (2.4% in control cells; p<0.01). Fusion and cleavage were not affected by the use apoptotic cells (p>0.05). Also, the use of Anx+ cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p>0.05). However, blastocyst formation was affected by the use of Casp-9+ cells (12.3%; p<0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return" for apoptosis may be located around activation of Caspase-9.
Assuntos
Apoptose , Bovinos , Reprogramação Celular/fisiologia , Fibroblastos/fisiologia , Técnicas de Transferência Nuclear , Animais , Apoptose/fisiologia , Bovinos/embriologia , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Células Cultivadas , Clonagem de Organismos/métodos , Clonagem de Organismos/veterinária , Desenvolvimento Embrionário/genética , Feminino , Fibroblastos/citologia , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Técnicas de Transferência Nuclear/veterinária , Cultura Primária de CélulasRESUMO
Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.