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1.
Arq. bras. med. vet. zootec. (Online) ; 73(4): 799-811, Jul.-Aug. 2021. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1285263

ABSTRACT

This study aimed to evaluate the ultrastructural morphometry of bovine embryos produced in vitro grown at different concentrations of antioxidants. After in vitro maturation and fertilization, the presumptive zygotes were assigned into five treatments. T1) without the addition of any antioxidants (negative control); T2) addition of 50µM/mL cysteamine; and T3, T4 and T5) adding 2.5µg/mL, 5.0µg/mL or 10.0µg/mL of the antioxidants derived from the oily extract from Lippia origanoides, respectively. On D7 of culture, the embryos in the blastocyst stage were fixed and prepared for electron transmission microscopy. These were evaluated for the proportion of cytoplasm-to-nucleus, cytoplasm-to-mitochondria, cytoplasm-to-vacuoles, cytoplasm-to-autophagic vacuoles and cytoplasm-to-lipid droplets. Blastocysts cultured in media containing oily extract of Lippia origanoides presented morphological characteristics such as high cell:mitochondria ratio and low cell:vacuoles and cell:autophagic vacuole ratio, possibly been morphological indicators of embryonic quality. Inner cell mass (ICM) from blastocysts cultured in media without any antioxidants had the highest cell:vacuole ratio. Similar results were found in the trophectoderm (TE) cells of blastocysts from treatment 2. Embryo culture media supplemented with antioxidants derived from Lippia origanoides oil produced embryos with a higher cytoplasmic proportion of organelles, such as mitochondria. Also, treatments without any antioxidants or with the addition of cysteamine presented cytoplasmic vacuolization, a characteristic related to production of poor-quality embryos.(AU)


Este estudo teve como objetivo avaliar a morfometria ultraestrutural de embriões bovinos produzidos in vitro e cultivados em diferentes concentrações de antioxidantes. Após a maturação e a fertilização in vitro, os possíveis zigotos foram divididos em cinco tratamentos: T1) sem adição de antioxidantes (controle negativo); T2) adição de 50µM/mL de cisteamina; e T3, T4 e T5) adição de 2,5µg/mL, 5,0µg/mL ou 10,0µg/mL dos antioxidantes derivados do extrato oleoso de Lippia origanoides, respectivamente. No D7 de cultivo, os embriões em estágio de blastocisto foram fixados e preparados para microscopia eletrônica de transmissão. Estes foram avaliados para a proporção entre citoplasma e núcleo, citoplasma e mitocôndria, citoplasma e vacúolos, citoplasma e vacúolos autofágicos e citoplasma e gotículas lipídicas. Blastocistos cultivados em meio contendo extrato oleoso de Lippia origanoides apresentaram características morfológicas como alta relação célula:mitocôndria e baixa relação célula:vacúolos e célula:vacúolo autofágico, possíveis indicadores morfológicos de qualidade embrionária. A massa celular interna (MCI) de blastocistos cultivados em meio sem quaisquer antioxidantes teve a maior razão célula:vacúolo. Resultados semelhantes foram encontrados nas células do trofectoderma (TE) de blastocistos do tratamento 2. Portanto, o meio de cultivo embrionário suplementado com antioxidantes derivados do óleo de Lippia origanoides produziu embriões com maior proporção citoplasmática de organelas, como mitocôndrias. Além disso, tratamentos sem antioxidantes ou com adição de cisteamina apresentaram vacuolização citoplasmática, característica relacionada à produção de embriões de baixa qualidade.(AU)


Subject(s)
Blastocyst , Cysteamine , Lippia , Embryo, Mammalian/ultrastructure , In Vitro Techniques/veterinary , Antioxidants
2.
Arq. bras. med. vet. zootec. (Online) ; 73(1): 256-260, Jan.-Feb. 2021. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1153048

ABSTRACT

As vantagens dos animais transgênicos têm sido demonstradas em diferentes aplicações, entretanto muitas metodologias usadas para gerar animais geneticamente modificados (GM) apresentam baixas taxas de eficiência. O objetivo deste estudo foi avaliar a entrega dos vetores lentivirais (VLs) em zigotos durante a fertilização in vitro (FIV), para gerar embriões GM, com o gene da proteína verde fluorescente (GFP) ou do fator IX de coagulação humana (FIX). Vetores lentivirais com os genes GFP (pLGW-GFP-LV) ou FIX (pLWE2-FIX-LV) foram utilizados na FIV ou na cultura de embriões in vitro (CIV). A coincubação de pLWE2-FIX-LV com espermatozoides e complexos oócitos-células do cumulus (COCs) durante a FIV diminuiu (P<0,05) as taxas de clivagem e de blastocistos, enquanto com pLGW-GFP-LV diminuiu (P<0,05) a taxa de blastocisto quando se comparou ao controle sem VL. A coincubação de pLWE2-FIX-LV e pLGW-GFP-LV com presumíveis zigotos durante a CIV não afetou (P>0,05) o desenvolvimento embrionário. A expressão da proteína GFP não foi detectada em embriões após a coincubação de FIV ou CIV, embora as células do cumulus expressassem a proteína até o dia oito de cultivo in vitro. Reações em cadeia da polimerase (PCR) não detectaram os genes GFP ou FIX em embriões, mas ambos foram detectados em células do cumulus. Assim, a coincubação de VL com espermatozoide bovino e COCs não é eficaz para produzir embriões geneticamente modificados por meio de FIV.(AU)


Subject(s)
Animals , Cattle , Zygote , Animals, Genetically Modified/genetics , Transgenes , Embryo, Mammalian , Genetic Vectors/analysis , Fertilization in Vitro/veterinary , Gene Transfer Techniques/veterinary
3.
Rev. bras. ginecol. obstet ; 43(1): 28-34, Jan. 2021. tab, graf
Article in English | LILACS | ID: biblio-1156078

ABSTRACT

Abstract Objective To determine embryo quality (mean graduated embryo score [GES]) in infertile patients with endometriosis undergoing in vitro fertilization with embryo transfer (IVF-ET) compared with infertile patients without endometriosis. Methods A case-control study was performed comparing 706 embryos (162 patients) divided into 2 groups: 472 embryos derived from patients without endometriosis (n= 109, infertile patients with tubal infertility) and 234 embryos from patients in the study group (n= 53, infertile patients with peritoneal endometriosis). All patients were subjected to IVF using an oestradiol-antagonist-recombinant follicle-stimulating hormone (FSH) protocol for ovarian stimulation. Themean GESwas performed to evaluate all embryos at 3 points in time: 16 to 18 hours, 25 to 27 hours, and 64 to 67 hours. Embryo evaluation was performed according to the following parameters: fragmentation, nucleolar alignment, polar body apposition, blastomere number/morphology, and symmetry. The primary outcomemeasure was the mean GES score.We also compared fertilization, implantation, and pregnancy rates. Results Although the number of embryos transferred was greater in patients with endometriosis than in the control group (2.38 ± 0.66 versus 2.15 ± 0.54; p= 0.001), the meanGESwas similar inbothgroups (71 ± 19.8 versus 71.9 ± 23.5; p= 0.881). Likewise, the fertilization ratewas similar in all groups, being 61% in patients with endometriosis and 59% in the control group (p= 0.511). No significant differences were observed in the implantation (21% versus 22%; [p= 0.989]) and pregnancy rates (26.4% versus 28.4%; p= 0.989). Conclusion Embryo quality measured by the mean GES was not influenced by peritoneal endometriosis. Likewise, the evaluated reproductive outcomes were similar between infertile patients with and without endometriosis.


Resumo Objetivo Determinar a qualidade do embrião (média de escore embrionário graduado [EEG]) em pacientes inférteis com endometriose submetidas à fertilização in vitro com transferência de embrião (FIV-TE) em comparação com pacientes inférteis sem endometriose. Métodos Realizamos um estudo de caso-controle comparando 706 embriões (162 pacientes) divididos em dois grupos: 472 embriões derivados de pacientes sem endometriose (n = 109, pacientes inférteis com infertilidade tubária) e 234 embriões de pacientes do grupo de estudo (n= 53, inférteis pacientes com endometriose peritoneal). Todos os pacientes foram submetidos à fertilização in vitro usando um protocolo follicle-stimulating hormone (FSH) recombinante de estradiol-antagonista para estimulação ovariana. A média do EEGfoi realizada para avaliar todos osembriõesemtrêsmomentos: de 16 a 18 horas, 25 a 27 horas e 64 a 67 horas.A avaliaçãoembrionária foi realizada de acordo comos seguintes parâmetros: fragmentação, alinhamento nucleolar, aposição do corpo polar, número de blastômeros/morfologia e simetria. A medida de desfecho primário foi o escore médios embrionário (EEG). Também avaliamos como desfechos secundários as taxas de fertilização, implantação e gravidez. Resultados Embora o número de embriões transferidos tenha sido maior em pacientes com endometriose do que no grupo controle (2,38 ± 0,66 versus 2,15 ± 0,54; p = 0,001), o EEGmédio foi semelhante nos dois grupos (71 ± 19,8 versus 71,9 ± 23,5; p = 0,881). Da mesma forma, a taxa de fertilização foi semelhante em todos os grupos, sendo 61% nos pacientes com endometriose e 59% no grupo controle (p = 0,511). Não foram observadas diferenças significativas nas taxas de implantação (21% versus 22%; [p = 0,989]) e nas taxas de gravidez (26,4% versus 28,4%; p = 0,989). Conclusão A qualidade embrionária medida pelo EEGmédio não foi influenciada pela endometriose peritoneal. Da mesma forma, os resultados reprodutivos avaliados foram semelhantes entre pacientes inférteis com e sem endometriose.


Subject(s)
Humans , Female , Pregnancy , Adult , Embryo, Mammalian , Embryo Transfer , Endometriosis , Infertility, Female , Ovulation Induction , Pregnancy Outcome , Fertilization in Vitro , Case-Control Studies , Prospective Studies
4.
Protein & Cell ; (12): 7-28, 2021.
Article in English | WPRIM | ID: wpr-880895

ABSTRACT

Mammalian fertilization begins with the fusion of two specialized gametes, followed by major epigenetic remodeling leading to the formation of a totipotent embryo. During the development of the pre-implantation embryo, precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality, but the underlying molecular mechanisms remain elusive. For the past few years, unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development, taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies. The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals, including DNA methylation, histone modifications, chromatin accessibility and 3D chromatin organization.


Subject(s)
Animals , Chromatin Assembly and Disassembly , DNA Methylation , DNA Transposable Elements , Embryo, Mammalian , Embryonic Development/genetics , Epigenesis, Genetic , Epigenome , Female , Fertilization/physiology , Gene Expression Regulation, Developmental , Histone Code , Histones/metabolism , Male , Mice , Oocytes/metabolism , Spermatozoa/metabolism
5.
Arq. bras. med. vet. zootec. (Online) ; 72(1): 33-39, Jan.-Feb. 2020. graf
Article in Portuguese | LILACS, VETINDEX | ID: biblio-1088915

ABSTRACT

A biópsia embrionária associada à genotipagem permite a obtenção de informações genômicas antes mesmo da transferência dos embriões. Neste estudo, foram avaliadas amostras biopsiadas de blastocistos bovinos transferidos para receptoras (n=47), sob a hipótese de que a raça (Gir ou Girolando), o estádio embrionário (blastocisto ou blastocisto expandido) e a competência para estabelecimento de prenhez (positiva ou negativa) afetariam a quantidade e a qualidade do DNA da amostra obtida. O DNA foi extraído, amplificado, quantificado por eletroferograma e genotipado. O parâmetro call rate (CR) foi adotado para mensurar a qualidade da genotipagem. Obteve-se concentração de DNA de 86,07±171,66ng/µL e CR 0,73±0,17. O CR não variou em função da quantidade de DNA nas amostras. As variáveis raça e estádio embrionário não influenciaram a concentração de DNA, nem o CR. Houve efeito da prenhez sobre o CR (P=0,0187), mas, como houve maior CR nas amostras provenientes do grupo prenhez negativa, não foi possível associar esse parâmetro à qualidade embrionária. Concluiu-se que a raça e a qualidade embrionária não afetam os parâmetros aqui estudados em amostras embrionárias, ou seja, embriões com maiores chances de implantação não refletem alta qualidade nas amostras de biópsia genotipadas.(AU)


Embryo biopsy associated with genotyping allows genomic information before embryo transfer. In this study, blastocyst biopsy samples from embryos transferred to recipients (n= 47) were evaluated, under the hypothesis that breed (Gyr or Girolando), embryonic stage (blastocyst or expanded blastocyst) and competence to establish pregnancy (positive or negative) would affect the quantity and DNA quality of samples. DNA was extracted, amplified, quantified by electropherogram and genotyped. The parameter call rate (CR) was used to measure the quality of genotyping. DNA concentration of 86.07±171.66ng/µl, and CR 0.73±0.17 was obtained. CR did not vary according to the amount of DNA in the samples. The variables breed and embryonic stage had no influence on DNA concentration or CR. There was pregnancy effect on the CR (P= 0.0187), but since there was a higher CR in the samples from the negative pregnancy group, it was not possible to associate this parameter with the embryonic quality. We conclude that the breed and embryo quality do not affect the evaluated parameters in embryonic samples. Embryos with higher chances of implantation do not reflect high quality in embryo biopsy genotyped samples.(AU)


Subject(s)
Animals , Cattle , Selection, Genetic , Biopsy/veterinary , Sequence Analysis, DNA/veterinary , Embryo, Mammalian , Genotyping Techniques/veterinary , In Vitro Techniques/veterinary
6.
Braz. arch. biol. technol ; 63: e20200234, 2020. tab, graf
Article in English | LILACS | ID: biblio-1132219

ABSTRACT

Abstract Curcumin (CUR) shows potential use for treating cancer. However, CUR has low solubility and reduced bioavailability, which limit its clinical effect. Therefore, the development of nanocarriers can overcome these problems and can ensure the desired pharmacological effect. In addition, it is mandatory to prove the quality, the efficacy, and the safety for a novel nanomedicine to be approved. In that sense, this paper aimed (a) to prepare CUR-loaded polyethylene glycol-poly(ε-caprolactone) nanocapsules; (b) to validate an analytical method by high performance liquid chromatography (HPLC) for quantifying CUR in these nanoformulations; (c) to evaluate the physicochemical stability of these formulations; and to investigate their cytotoxicity on NIH-3T3 mouse fibroblast cells. The HPLC method was specific to CUR in the loaded nanocapsules, linear (r = 0.9994) in a range of 10.0 to 90.0 µg.mL-1 with limits of detection and quantification of 0.160 and 0.480 µg.mL-1, respectively. Precision was demonstrated by a relative standard deviation lower than 5%. Suitable accuracy (102.37 ± 0.92%) was obtained. Values of pH, particle size, polydispersity index, and zeta potential presented no statistical difference (p > 0.05) for CUR-loaded nanoparticles. No cytotoxicity was observed against NIH-3T3 mouse embryo fibroblast cell line using both the tetrazolium salt and sulforhodamine B assays. In conclusion, a simple and inexpensive HPLC method was validated for the CUR quantification in the suspensions of nanocapsules. The obtained polymeric nanocapsules containing CUR showed suitable results for all the performed assays and can be further investigated as a feasible novel approach for cancer treatment.


Subject(s)
Animals , Mice , Curcumin/pharmacology , Embryonic Stem Cells/drug effects , Fibroblasts/drug effects , Chromatography, High Pressure Liquid , Toxicity Tests , Nanotechnology , NIH 3T3 Cells , Embryo, Mammalian/cytology , Nanocapsules
8.
Rev. bras. ginecol. obstet ; 41(9): 525-530, Sept. 2019. tab, graf
Article in English | LILACS | ID: biblio-1042339

ABSTRACT

Abstract Objective The etiology of embryonic demise is multifactorial, with chromosomal abnormalities being the most common (40%). The purpose of the present study is to evaluate the correlation between a serum biomarker, progesterone, and an ultrasonographic parameter, the distance between yolk sac and embryo (DYSE) in assessing the prognosis of pregnancy outcome in the 1st trimester. Methods The present study is a prospective case-control analysis that includes 2 groups of patients: 81 patients with first-trimester normal evolutive pregnancy and 89 patients with embryonic demise, all of the patients having between 6 and 11 weeks of amenorrhea. Endovaginal ultrasonographic exploration was performed to evaluate the distance between the lower pole of the embryo and the yolk sac. From each subject enrolled in the study, 20ml of blood was collected for progesterone serum level measurement. Results Regarding the DYSE in the case group, lower values were observed compared with the control group, the difference being statistically significant. In the statistical analysis of serum progesterone values, statistically significant differences were observed between the 2 groups (p<0.05). Conclusion The DYSE has a high positive predictive value in identifying pregnancies with potentially reserved outcome, with the present study demonstrating that a DYSE<3mm causes an unfavorable evolution of the pregnancy. Low serum levels of progesterone are associated with an increased rate of nonviable embryos. The correlation between these two parameters increases the effectiveness of screening methods in prenatal monitoring and improves the diagnostic methods for the firsttrimester pregnancies whose outcome potential can be reserved.


Subject(s)
Humans , Female , Pregnancy , Pregnancy Trimester, First/physiology , Pregnancy Trimester, First/blood , Progesterone/blood , Pregnancy Outcome/epidemiology , Ultrasonography, Prenatal , Prognosis , Yolk Sac/diagnostic imaging , Case-Control Studies , Embryo, Mammalian/diagnostic imaging
9.
Int. j. morphol ; 36(3): 1087-1094, Sept. 2018. graf
Article in English | LILACS | ID: biblio-954235

ABSTRACT

Catha edulis Forsk leaves (Khat) is a flowering plant. A high proportion of the adult population in the Arabian Peninsula and the Horn of Africa chews it for its mild stimulant effect. The aim of the current study was to investigate the embryotoxic and teratogenic effects of the Khat extract using 60 female pregnant rats. These were divided to a Khat extract-treated group and a control group. Methanolic extract of Khat was orally given to the treated group 4 days before mating and up to day 16 of pregnancy with a dose of 100 mg/kg. Our results showed that significant number of embryos of the Khat-treated mothers were malformed and different in size and shape compared to embryos from the mothers of the control group. At day 8 of pregnancy, malformed embryos had ill developed primitive layers. By day 10 of pregnancy, neural tube and the somite were not formed compared to the control embryos. At later stages of pregnancy, embryos of the Khat-treated mothers appeared severely abnormal with opened neural groove and visceral pouches. Disrupted normal neural tube development, undifferentiated brain vesicles, incomplete closure of the brain flexures were also observed in these embryos. Highly significant increase in the number of the resorbed embryos of the Khat-treated mothers were observed (P < 0.01). The resorbed embryos appeared as a cellular collection in their placenta with some of their decidua had no visible embryonic tissues. In conclusions, Khat induced embryotoxic effects as well as severely affected the early normal embryonic development in rat.


Catha edulis (Khat) es una planta floreciente. Una alta proporción de la población adulta en la Península Arábiga y el Cuerno de África la mastica por su efecto estimulante. El objetivo del presente estudio fue investigar los efectos embriotóxicos y teratogénicos del extracto de Khat utilizando 60 ratas hembras preñadas. Estas se dividieron en un grupo tratado con extracto de Khat y un grupo control. El extracto metanólico de Khat se administró por vía oral al grupo tratado 4 días antes del apareamiento y hasta el día 16 de preñez con una dosis de 100 mg / kg. Los resultados mostraron que una cantidad significativa de embriones de las madres tratadas con Khat tenían malformaciones y eran diferentes en tamaño y forma en comparación con los embriones de las madres del grupo control. En el día 8 de preñez, los embriones malformados tenían capas primitivas mal desarrolladas. Para el día 10 de preñez, el tubo neural y el somito no se formaron en comparación con los embriones del grupo control. En etapas posteriores de la preñez, los embriones de las madres tratadas con Khat parecían severamente anormales con surcos neurales abiertos y bolsas viscerales. También se observaron alteraciones en el desarrollo normal del tubo neural, vesículas cerebrales indiferenciadas y el cierre incompleto de las flexiones cerebrales en estos embriones. Se observó un aumento altamente significativo en el número de embriones reabsorbidos de las madres tratadas con Khat (P <0,01). Los embriones reabsorbidos aparecieron como una colección celular en su placenta con algunas de sus deciduas sin tejidos embrionarios visibles. Khat indujo efectos embriotóxicos y afectó severamente el desarrollo embrionario normal temprano en la rata.


Subject(s)
Animals , Female , Pregnancy , Rats , Plant Extracts/toxicity , Catha/chemistry , Embryo, Mammalian/drug effects , Teratogens , Rats, Sprague-Dawley
10.
Pesqui. vet. bras ; 38(7): 1259-1263, July 2018. graf
Article in English | LILACS, VETINDEX | ID: biblio-976452

ABSTRACT

This study aimed to characterize the embryotoxic, teratogenic and abortifacient effect of Poincianella pyramidalis in goats. Twenty pregnant goats with 18 days of gestation were divided into five groups of four animals each. After collection, the leaves of P. pyramidalis were dried in the shade and crushed. The daily feed provided to the goats was equivalent to 3% of their body weight, being 1% concentrated feed and 2% roughage. In Group 1 (control), the provided roughage was Cynodon dactylon (Tifton) hay; in Groups 2, 3 and 4, 10%, 20% and 80% of the C. dactylon roughage was replaced by dry and ground P. pyramidalis, respectively. In Group 5, all the roughage was replaced by green P. pyramidalis ad libitum, collected daily. Ultrasonographic examination was performed twice a week throughout the pregnancy. Goats in Groups 1, 2 and 3, delivered normal kids. Two goats in Group 4 aborted at 127 and 90 days of gestation. In group 5, three goats showed embryonic death at 25, 30 and 31 days of gestation and the other goat aborted at 39 days of pregnancy. Malformations were not observed. It is suggested that P. pyramidalis, which is very common in the semiarid region of northeastern Brazil, should be considered as an important cause of reproductive losses in this area. Due to its high palatability, it is important to avoid the ingestion of P. pyramidalis by pregnant and mating goats.(AU)


O objetivo deste trabalho foi estudar o potencial embriotóxico, abortivo e teratogênico da Poincianella pyramidalis em caprinos. Para tanto foram utilizadas 20 cabras prenhes com 18 dias de gestação, divididas em cinco grupos de quatro animais. Depois da coleta, as folhas de P. pyramidalis era secas a sombra e trituradas. A alimentação diária fornecida aos caprinos foi proporcional a 3% do seu peso vivo, sendo 1% de alimento concentrado e 2% de volumoso. No Grupo 1 (controle), o volumoso fornecido foi apenas feno de Cynodon dactylon (Tifton). Já nos Grupos 2, 3 e 4, 10%, 20% e 80% do volumoso foi substituído por folhas secas e trituradas de P. pyramidalis, respectivamente. No Grupo 5, todo o volumoso foi constituído por P. pyramidalis verde ad libitum, coletadas diariamente. Para o acompanhamento das gestações, exames ultrassonográficos foram realizados duas vezes por semana, durante toda a gestação. As cabras dos Grupos 1, 2 e 3 pariram cabritos normais. Duas cabras no Grupo 4 abortaram, sendo uma com 127 dias de gestação e outra com 90 dias. No grupo 5, três cabras apresentaram morte embrionária no 25º, 30º e 31º dia de gestação e uma cabra abortou no 39º dia de gestação. No presente estudo não foi observada nenhuma malformação. Com esses resultados e considerando a ampla difusão de P. pyramidalis na região semiárida do nordeste Brasileiro sugere-se que esta planta é uma importante causa de perdas reprodutivas na região. Devido a sua alta palatabilidade, recomenda-se evitar a permanência de cabras prenhes em áreas onde ocorre P. pyramidalis.(AU)


Subject(s)
Animals , Abortion, Induced/mortality , Caesalpinia/toxicity , Embryo, Mammalian , Plants, Toxic/embryology
11.
Int. j. morphol ; 36(2): 677-686, jun. 2018. tab, graf
Article in English | LILACS | ID: biblio-954171

ABSTRACT

The aim of this study was to know the embryonic and fetal development of the female rabbit genital system (Oryctolagus cuniculus), describing its main phases and the moment of sexual differentiation. Eleven pregnant New Zealand female rabbits were used in different gestational phases. The day of coitus was determined as day 0. For each stage a minimum of two animals was considered. The samples were obtained every two days from the ninth day post-coitus (dpc) until the 28th dpc. The gestational period was divided in two: animals with undifferentiated sex (group 1) and animals with differentiated sex (group 2). The ages of embryos and fetuses were estimated through the crown-rump method. Subsequently, embryos and fetuses were dissected, fixed and processed to be embedded in paraffin (Histosec). The histological analysis was performed on sections stained with hematoxylin and eosin. Immunohistochemical analysis to determine sexual differentiation was performed on samples from the 16th, 18th and 28th dpc. Desert Hedgehog (Dhh) and Indian Hedgehog (Ihh) primary antibodies, respectively, were used to identify cells of the male and female germinal epithelium. The immunohistochemical results showed that at the 16th dpc, female sexual differentiation was evident, since positive expression of the Ihh protein was observed. Sexual differentiation was obtained through histological analysis on the 18th dpc and through anatomical observation of the external genitalia on the 24th dpc. Knowing the characteristics of the embryonic and fetal development of the female rabbit genital system as well as the moment of sexual differentiation make it possible to establish bases for future research that address the physiology and pathology of these organs. Thus, any alteration in the chain of events of sexual determination and differentiation must search for an explanation from the knowledge of the possible normal mechanisms affected.


El objetivo de esta investigación fue conocer el desarrollo embrionario y fetal del sistema genital femenino de conejo (Oryctolagus cuniculus), describiendo sus principales fases y el momento de la diferenciación sexual. Se utilizaron 11 conejos hembras gestantes neozelandesas, en diferentes fases gestacionales. El día del coito se determinó como día 0. Para cada etapa fue considerado un mínimos de dos animales. Las muestras fueron obtenidas cada dos días, a partir del noveno día post-coito (dpc) hasta el 28 dpc. El periodo gestacional fue dividido en dos: animales con sexo indiferenciado (grupo 1) y, animales con sexo diferenciado (grupo 2). Las edades de los embriones y los fetos fueron estimadas a través del método de crown-rump. Posteriormente, embriones y fetos fueron disecados, fijados y procesados para su inclusión en parafina (Histosec). El análisis histológico se realizó en secciones teñidas con Hematoxilina y Eosina. El análisis inmunohistoquímico para determinar la diferenciación sexual fue realizado en muestras de 16, 18 y 28 dpc. Para identificar células del epitelio germinativo masculino y feminino se utilizaron los anticuerpos primarios Desert Hedgehog (Dhh) e Indian Hedgehog (Ihh), respectivamente. Los resultados inmunohistoquímicos mostraron que a los 16 dpc se evidenció diferenciación sexual femenina, ya que se observó expresión positiva de la proteína Ihh. La diferenciación sexual, a través del análisis histológico fue obtenida a los 18 dpc y a través de la observación anatómica de los genitales externos a los 24 dpc. Conocer las características del desarrollo embrionario y fetal del sistema genital femenino de conejo, así como, el momento de la diferenciación sexual, permiten sentar bases para futuras investigaciones que aborden la fisiología y patología de estos órganos. Así, cualquier alteración en la cadena de eventos de la determinación y diferenciación sexual deberá buscar una explicación a partir del conocimiento de los posibles mecanismos normales afectados.


Subject(s)
Animals , Male , Female , Pregnancy , Rabbits/embryology , Sex Differentiation/physiology , Embryo, Mammalian/anatomy & histology , Embryonic and Fetal Development/physiology , Immunohistochemistry
12.
Int. j. morphol ; 36(2): 693-698, jun. 2018. graf
Article in Spanish | LILACS | ID: biblio-954173

ABSTRACT

Sonic hedgehog (Shh) es un morfógeno esencial para el desarrollo de diversas estructuras, tales como notocorda, placa del piso del tubo neural, miembros, entre otros. Se buscó determinar la inmunolocalización de Shh en embriones y fetos de ratón. Para ello, se eutanasiaron 10 ratones gestantes (Mus musculus) BALB/c, un grupo de 5 animales a los 12,5 días post-coito (dpc), y otro grupo a los 17,5 dpc. Los embriones y fetos obtenidos fueron fijados en formalina al 10 % tamponada en PBS e incluidos en paraplast. Se realizaron cortes transversales seriados. Se utilizó anticuerpo policlonal Shh (Santa Cruz Biotechnology, H-160, conejo), dilución 1:100. Se identificó y describió la inmunolocalización de las muestras marcadas positivamente. La expresión de Shh en los embriones de 12,5 dpc fue inmunopositiva en notocorda, placa del piso del tubo neural, precartílago de radio y ulna, y prácticamente todos los epitelios: bronquial, intestinal, vejiga y uretra. En la etapa fetal, a los 17,5 dpc la inmunopositividad desaparece en el cartílago a excepción de zonas de osificación, disminuye en la epidermis pero aparece en folículos pilosos. La mucosa intestinal se ha diferenciado en segmentos, mostrando una inmunotinción mayor a nivel de las vellosidades intestinales. Shh actúa en distintos estadios del periodo gestacional, siendo clave en la diferenciación de distintas estructuras. En etapas embrionaria, es vital en la formación del sistema nervioso, organogénesis y formación de miembros, por lo que su expresión se encuentra en estas zonas. Sin embargo, en la etapa fetal la expresión cambia a estructuras de mayor especialización como folículo piloso y vellosidades intestinales.


Sonic hedgehog (Shh) is an essential morphogen for the development of various structures, such as notochord, neural tube floor plate, limbs, among others. We sought to determine the immunolocalization of Shh in embryos and mouse fetuses. To do this, 10 pregnant mice (Mus musculus) BALB /c were euthanized, a group of 5 animals at 12.5 days postcoitus (dpc), and another group at 17.5 dpc. Embryos and fetuses obtained were fixed in 10 % formalin buffered in PBS and embedded in paraplast. Serial cross sections were made. Polyclonal antibody Shh (Santa Cruz Biotechnology, H-160, rabbit), dilution 1:100 was used. The immunolocalization of the positively labeled samples was identified and described. Shh expression in 12.5 dpc embryos was immunopositive in notochord, neural tube floor plate, radius precartilage and ulna, and practically all epithelia: bronchial, intestinal, bladder and urethra. In the fetal stage, at 17.5 dpc the immunopositivity disappears in the cartilage except for areas of ossification, decreases in the epidermis but appears in hair follicles. The intestinal mucosa has differentiated into segments, showing greater immunostaining at the level of the intestinal villi. Shh acts in different stages of the gestational period, being key in the differentiation of different structures. In embryonic stages, it is vital in the formation of the nervous system, organogenesis and formation of limbs, so its expression is found in these areas. However, in the fetal stage the expression changes to more specialized structures such as hair follicles and intestinal villi.


Subject(s)
Animals , Female , Mice , Organogenesis/physiology , Hedgehog Proteins/metabolism , Embryonic and Fetal Development , Immunohistochemistry , Embryo, Mammalian , Mice, Inbred BALB C
13.
Neuroscience Bulletin ; (6): 527-533, 2018.
Article in English | WPRIM | ID: wpr-777035

ABSTRACT

Oligodendrocytes (OLs) are myelinating glial cells that form myelin sheaths around axons to ensure rapid and focal conduction of action potentials. Here, we found that an axonal outgrowth regulatory molecule, AATYK (apoptosis-associated tyrosine kinase), was up-regulated with OL differentiation and remyelination. We therefore studied its role in OL differentiation. The results showed that AATYK knockdown inhibited OL differentiation and the expression of myelin genes in vitro. Moreover, AATYK-deficiency maintained the proliferation status of OLs but did not affect their survival. Thus, AATYK is essential for the differentiation of OLs.


Subject(s)
Animals , Animals, Newborn , Apoptosis Regulatory Proteins , Genetics , Metabolism , Cell Differentiation , Physiology , Cell Proliferation , Genetics , Cells, Cultured , Cuprizone , Toxicity , Demyelinating Diseases , Metabolism , Pathology , Embryo, Mammalian , Gene Expression Regulation, Developmental , Genetics , Ki-67 Antigen , Metabolism , Mice , Mice, Inbred C57BL , Myelin Basic Protein , Metabolism , Myelin Proteolipid Protein , Metabolism , Myelin Sheath , Metabolism , Oligodendroglia , Metabolism , Protein-Tyrosine Kinases , Genetics , Metabolism , RNA, Small Interfering , Genetics , Metabolism , Rats , Rats, Sprague-Dawley
14.
Arq. bras. med. vet. zootec. (Online) ; 69(6): 1385-1392, nov.-dez. 2017. ilus, tab
Article in Portuguese | LILACS, VETINDEX | ID: biblio-909701

ABSTRACT

Avaliou-se o efeito da adição do ácido linoleico conjugado (CLA) ao meio de cultivo in vitro na viabilidade pós-vitrificação de embriões F1 Holandês x Zebu. Foram utilizados três meios de cultivo: controle (n=340 oócitos): meio SOF e soro fetal bovino (SFB), sem o CLA; SFB+CLA (n=359 oócitos): meio SOF, SFB e CLA; CLA (n=339 oócitos): meio SOF e CLA, sem o SFB. Todos os blastocistos produzidos foram submetidos à vitrificação, pelo método de Open Pulled Straw. Quinze blastocistos de cada tratamento foram fixados para quantificação lipídica por coloração com Sudan Black B. Para avaliar a viabilidade embrionária, foi observada a capacidade de reexpansão e eclosão pós-aquecimento dos embriões (controle=27; SFB+CLA=30; CLA=17). Foram realizadas transferências em um ou dois embriões por receptora para avaliação da sobrevivência in vivo: T1 [receptoras que receberam um blastocisto (n=17 embriões, sendo controle=5, SFB+CLA=6 e CLA=6)]; T2 [receptoras que receberam dois blastocistos, (n= 54 embriões, sendo controle=18, SFB+CLA=14 e CLA=22)]. Não houve diferença nas taxas de clivagem (62,1%; 74,0%; 74,0% para controle; SFB+CLA; CLA, respectivamente), produção de blastocistos em relação aos clivados (59,7%; 47,7%; 38,3% para controle; SFB+CLA; CLA, respectivamente) e produção de blastocistos em relação ao total de oócitos (37,1%; 35,4%; 28,3% para controle; SFB+CLA; CLA, respectivamente) (P>0,05). Houve diminuição de gotículas lipídicas nos embriões cultivados em meio suplementado com CLA em relação aos embriões cultivados na presença do SFB e na ausência do CLA (P<0,05). A taxa de reexpansão foi maior no grupo controle (70,4%) em relação ao CLA (47,1%) e menor no grupo SFB+CLA (43,3%) (P<0,05). O CLA foi eficaz em reduzir a deposição de lipídeos intracitoplasmáticos nas células embrionárias, porém não houve diferença de viabilidade após a desvitrificação dos embriões.(AU)


The effect of adding conjugated linoleic acid (CLA) to the culture media on the viability after cryopreservation of F1 Holstein X Zebu embryos was evaluated. Three different culture media were tested: control (n = 340 oocytes): SOF medium and fetal bovine serum (FBS) without the CLA; FBS + CLA (n = 359 oocytes): SOF, FBS and CLA; CLA (n = 339 oocytes): SOF and CLA without the FBS. The produced blastocysts were subjected to vitrification, by the Open Pulled Straw method. Fifteen blastocysts per treatment were fixed for lipid quantification by staining with Sudan Black B. Embryo re-expansion and hatching capability were used to assess viability (control = 27; FBS + CLA = 30; CLA = 17). Transfers of one or two embryos to recipients were performed to evaluate in vivo survival: T1 [recipients that received one blastocyst (n=17 embryos, Control=5, FBS+CLA=6 and CLA=6)]; T2 [recipients that received two blastocysts (n =54 embryos, Control=18, FBS+CLA=14 and CLA=22)]. There was no difference in cleavage rate (62.1%; 74%; 74% for Control; FBS + CLA, CLA, respectively), blastocyst production in relation to the cleaved structures (59.7%; 47.7%; 38 3% for Control; FBS + CLA, CLA, respectively) and blastocyst production relative to the total oocytes (37.1%, 35.4%, 28.3% for Control; FBS + CLA, CLA, respectively) between treatments (P> 0.05). A reduction of lipid droplets was observed in embryos cultured in medium supplemented with CLA compared to embryos cultured in the FCS in the absence and presence of CLA (P <0.05). The reexpansion rate was higher in the Control group (70.4%) compared to the CLA (47.1%) and lowest for FBS+CLA (43.3%) (P<0.05). The hatching rates were similar among treatments, 42.1%; 23.1%; 25% for control; SFB + CLA; CLA respectively (P>0.05). Only one pregnancy was observed in early and confirmatory diagnosis, as the result of a Control group embryo transfer. Although embryos cultured with CLA have shown smaller intracytoplasmic lipid content, no difference was observed in viability following vitrification between treatments.(AU)


Subject(s)
Animals , Cattle , Cryopreservation/veterinary , Linoleic Acids, Conjugated/therapeutic use , Embryo, Mammalian , Vitrification , In Vitro Techniques/veterinary
15.
An. acad. bras. ciênc ; 89(1,supl): 635-647, May. 2017. tab, graf
Article in English | LILACS | ID: biblio-886672

ABSTRACT

ABSTRACT The development of DBA/2J mouse strain embryos is nearly 12 h - or 6 somite pairs - delayed as compared to the outbred NMRI mouse embryos of the same age on gestation days (GD) 8-12. To evaluate inter-strain differences in susceptibility to teratogens, dams were treated with methylnitrosourea (MNU, 5 mg/kg body weight i.p.) on defined gestation days (NMRI: GD 9, 91/2 or 10; DBA/2J: GD 10 or 101/2). Skeletal anomalies produced by MNU on both mouse strains varied with the GD of treatment. The pattern of anomalies produced by MNU on a given GD markedly differed between the two mouse strains, yet they were similar -with a few exceptions- when exposures at equivalent embryonic stages are compared. Findings from this study indicated that strain-dependent differences in the developmental stage of mouse embryos of the same gestational age occur, a possibility that has been often neglected when inter-strain differences in susceptibility to developmental toxicants are interpreted.


Subject(s)
Animals , Female , Pregnancy , Rats , Skeleton/abnormalities , Teratogens/toxicity , Somites/abnormalities , Embryonic Development/drug effects , Embryo, Mammalian/abnormalities , Methylnitrosourea/toxicity , Skeleton/drug effects , Skeleton/embryology , Somites/drug effects , Somites/embryology , Embryo, Mammalian/drug effects , Mice, Inbred DBA
16.
Pesqui. vet. bras ; 37(4): 395-400, Apr. 2017. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-895428

ABSTRACT

The aim of the present study was to evaluate the intracytoplasmic lipid content, development and cryotolerance of in vitro-produced bovine embryos treated with different concentrations of forskolin before vitrification. Embryos were produced from abattoir-derived ovaries and allocated into four groups. In the treatment groups, forskolin was added to the in vitro culture medium on Day 6 and incubated for 24 hours in one of the following concentrations: 2.5µM (Forsk 2.5 group), 5.0µM (Forsk 5.0 group) or 10.0µM (Forsk 10.0 group). Embryos from the control group were cultured without forskolin. On Day 7 of culture, the expanded blastocysts were stained with the lipophilic dye Sudan Black B for determination of the intracytoplasmic lipid content or were cryopreserved via the Vitri-Ingá® procedure. Although there were no significant differences (P>0.05) in the blastocyst rates between the Control group (44.9%) and the other treatments, the embryo production was lower (P<0.05) in Forsk 10.0 group (38.8%) compared to Forsk 2.5 (50.5%) and Forsk 5.0 (54.7%) groups. The intracytoplasmic lipid content (expressed in arbitrary units of pixels) in blastocysts from the Control group (1.00±0.03) was similar (P>0.05) to that found in Forsk 2.5 (0.92±0.03) and Forsk 10.0 groups (1.06±0.03) groups; however the lipid accumulation in blastocysts from Forsk 5.0 group (0.82±0.04) was lower than in the Control group (P<0.05). Based on these results, Forsk 5.0 treatment was tested for cryotolerance and it was observed that the blastocoel re-expansion rate evaluated 24 hours after warming was greater (P<0.05) in Forsk 5.0 group (72.2%) compared to the Control group (46.2%). In conclusion, pre-treatment with forskolin at a concentration of 5.0 µM for 24 hours before vitrification is effective in reducing the intracytoplasmic lipid content and, consequently, improves cryotolerance of IVP bovine embryos.(AU)


Os embriões foram produzidos a partir de ovários obtidos em abatedouro e foram alocados em quatro grupos experimentais. Nos grupos tratados, o forskolin foi adicionado ao meio de cultivo in vitro no dia 6 do cultivo e os embriões foram incubados durante 24 horas com uma das seguintes concentrações: 2,5µM (grupo Forsk 2,5), 5,0µM (grupo Forsk 5,0) ou 10,0µM (grupo Forsk 10,0). Os embriões do grupo controle foram cultivados na ausência de forskolin. No dia 7 do cultivo, os blastocistos expandidos foram corados com o corante lipofílico Sudan Black B para a determinação do teor de lípidos intracitoplasmáticos ou foram criopreservados através do protocolo Vitri-Ingá®. Não foi observada diferença significativa (P>0,05) na taxa de produção de blastocistos entre o grupo Controle (44,9%) e os demais tratamentos, todavia observou-se menor produção de embriões (P<0,05) no grupo Forsk 10,0 (38,8%) em comparação com os grupos Forsk 2,5 (50,5%) e Forsk 5,0 (54,7%). A quantidade de lipídeos intracitoplasmáticos do grupo Controle (1,00±0,03) foi semelhante (P>0,05) a dos grupos Forsk 2,5 (0,92±0,03) e Forsk 10,0 (1,06±0,03); no entanto, o acúmulo de lípidos nos blastocistos do grupo Forsk 5.0 (0,82 ± 0,04) foi menor do que no grupo controle (P<0,05). A partir destes resultados, o grupo Forsk 5,0 foi testado quanto à criotolerância e foi observado que a taxa de re-expansão da blastocele 24 horas após o aquecimento foi maior (P<0,05) no grupo Forsk 5,0 (72,2%) quando comparado ao grupo Controle (46,2%). Em conclusão, o pré-tratamento com forskolin na concentração de 5,0 µM durante 24 horas antes da vitrificação foi eficiente para promover a redução da quantidade de lipídeos intracitoplasmáticos e, consequentemente, melhorou a criotolerância de embriões bovinos produzidos in vitro.(AU)


Subject(s)
Animals , Cattle , Colforsin , Embryo, Mammalian/physiology , Vitrification , Acclimatization/physiology , Lipids/analysis
17.
Braz. J. Vet. Res. Anim. Sci. (Online) ; 54(2): 168-177, 2017. ilus
Article in English | LILACS, VETINDEX | ID: biblio-875125

ABSTRACT

The interest in embryology, the science of the development of a zygote into a completely developed foetus, has increased greatly in recent years due to a number of studies involving embryonic and induced pluripotent stem cells. In addition, the development of techniques such as cloning has aided to understand the critical events that occur during embryonic development. In this study, we describe the morphology of two sheep embryos and one foetus using macroscopic and microscopic techniques. We investigated sheep without defined breed on days 24, 32, and 50 of gestation (estimated by crown-rump length [CR]). Macroscopically, we observed the development of E1 (24 days), with visible optic vesicle, but without retinal pigmentation and the forelimbs bud in development. In the E2 (32 days), we noticed the presence of optic retinal pigmentation and forelimbs more developed in comparison with E1. As expected, F1 revealed an eyeball already covered and the forelimbs developed. Meanwhile, microscopic analysis revealed somite, ventricle, atrium, and oral cavity in development in E1. However, in F1 we were able to identify more complex structures, such as ossification in the spine, ventricle, atrium, intraventricular septum, pericardial sac, and oral cavity with tongue. This work brings more precise and detailed data on the morphological characteristics of the major organ systems (nervous, circulatory, respiratory, digestive, and urinary) at each embryonic and foetal stage analysed.(AU)


O interesse em Embriologia, a ciência do desenvolvimento de um zigoto em um feto completamente desenvolvido, tem aumentado consideravelmente nos últimos anos devido a uma série de estudos envolvendo células-tronco pluripotentes embrionárias e induzidas. Além disso, o desenvolvimento de técnicas como a clonagem tem ajudado a compreender os eventos críticos que ocorrem durante o desenvolvimento embrionário. Neste estudo, descrevemos a morfologia de dois embriões de ovinos e um feto utilizando técnicas macroscópicas e microscópicas. Obtivemos ovelhas sem raça definida com 24, 32 e 50 dias de gestação (estimado pelo método de Crown-Rump, CR). Os conceptos foram mensurados, pesados e caracterizados a olho nu. Macroscopicamente, observamos o desenvolvimento dos embriões E1 (24 dias), apresentando globo ocular sem pigmentação de retina e broto do membro torácico e pélvico. Já o E2 (32 dias), apresentava globo ocular com pigmentação na retina e os membros torácicos e pélvicos mais desenvolvidos. O F1 apresentou olhos cobertos com uma membrana e membros torácicos e pélvicos mais desenvolvidos. Enquanto isso, microscopicamente observamos no E1 somitos, ventrículo, átrio e cavidade oral ainda em desenvolvimento. Porém, no F1 já era possível observar ossificação da coluna espinhal, coração com estruturas mais complexas, como ventrículo, átrio, septo interventricular e saco pericárdio. Além disso, na cavidade oral observamos a formação da língua. Este trabalho fornece informações precisas e detalhadas sobre as características morfológicas dos principais órgãos dos sistemas (nervoso, circulatório, respiratório, digestivo e urinário) em cada fase embrionária e fetal analisadas.(AU)


Subject(s)
Animals , Embryo, Mammalian/anatomy & histology , Embryonic Development , Fetal Development , Fetus/anatomy & histology , Sheep/embryology
18.
Braz. J. Vet. Res. Anim. Sci. (Online) ; 54(3): 209-214, 2017. tab, ilus, graf
Article in English | LILACS, VETINDEX | ID: biblio-879381

ABSTRACT

A new and effective protocol to culture bovine embryos without coculture and with individualized culture media has been established, which would allow the study of a single embryo's metabolism. For this purpose, bovine embryos were produced in vitro by standard protocols in three different types of media: KSOM, SOFaa, and KSOM followed by SOFaa at day 2. Presumptive zygotes were divided into six groups: control, cultured in groups (C-KSOM, C-SOFaa, and C-KS), and individual well system (W-KSOM, W-SOFaa, and W-KS). Cleavage and blastocyst rates were assessed on days 2 and 7 respectively. Relative quantification of transcripts related to important metabolic processes (GLUT1, GLUT3, GSK3, SOD1, HSPD1, G6PD) were assessed in C-KS and W-KS blastocysts. Results show that cleavage was significantly higher only in W-KSOM when compared to C-KSOM, while blastocyst rates differ only between C-SOF and W-SOF. All the other comparisons did not present statistical difference. Moreover, gene expression analysis revealed that blastocysts cultured in groups and in the individual well system present similar transcription patterns. Thus, the obtained conclusion was that the individual well system performed could be used as an effective alternative protocol for individual culture of bovine embryos, since the rates are similar to routine group culture.(AU)


Estabeleceu-se um protocolo novo e eficaz de cultivo individual de embriões bovinos sem o uso de cocultivo e sem compartilhamento de meio visando à análise do metabolismo individual do embrião. Para isso, embriões foram produzidos in vitro por protocolos convencionais em três diferentes tipos de meio: KSOM, SOFaa e KSOM seguido por SOFaa no dia 2. Os zigotos presumíveis foram divididos em seis grupos: controles (cultivo em grupo ­ C-KSOM, C-SOFaa e C-KS) e sistema de poços individuais (W-KSOM, W-SOFaa e W-KS). As taxas de clivagem foram avaliadas nos dias 2 e 7, respectivamente. Além disso, a quantificação relativa de transcritos relacionados a importantes processos metabólicos (GLUT1, GLUT3, GSK3, SOD1, HSPD1 e G6PD) foi avaliada nos blastocistos dos grupos C-KS e W-KS. Os resultados mostram que as taxas de clivagem foram maiores apenas no grupo W-KSOM quando comparado ao grupo C-KSOM, enquanto a taxa de blastocistos diferiu apenas entre os grupos C e W-SOF. Além disso, a análise da expressão gênica mostrou que blastocistos cultivados em grupo ou em sistema de poços individuais são semelhantes quanto à expressão gênica. Assim, a conclusão obtida foi que o sistema individual proposto pode ser utilizado como um protocolo alternativo eficiente para o cultivo individual de embriões de bovino, uma vez que suas características permanecem semelhantes àquelas do sistema convencional de produção de embriões.(AU)


Subject(s)
Animals , Cattle , Embryo Culture Techniques , Embryo, Mammalian
19.
Protein & Cell ; (12): 601-611, 2017.
Article in English | WPRIM | ID: wpr-756965

ABSTRACT

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.


Subject(s)
APOBEC-1 Deaminase , Genetics , Metabolism , Animals , Bacterial Proteins , Genetics , Metabolism , Base Sequence , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Cytidine , Genetics , Metabolism , Embryo Transfer , Embryo, Mammalian , Endonucleases , Genetics , Metabolism , Gene Editing , Methods , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Mice , Mice, Inbred C57BL , Microinjections , Plasmids , Chemistry , Metabolism , Point Mutation , RNA, Guide , Genetics , Metabolism , Thymidine , Genetics , Metabolism , Zygote , Metabolism , Transplantation
20.
Protein & Cell ; (12): 811-822, 2017.
Article in English | WPRIM | ID: wpr-756922

ABSTRACT

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.


Subject(s)
APOBEC-1 Deaminase , Genetics , Metabolism , Base Sequence , Blastomeres , Cell Biology , Metabolism , CRISPR-Cas Systems , Embryo, Mammalian , Metabolism , Pathology , Female , Fibroblasts , Metabolism , Pathology , Gene Editing , Methods , Gene Expression , HEK293 Cells , Heterozygote , Homozygote , Humans , Point Mutation , Primary Cell Culture , Promoter Regions, Genetic , Sequence Analysis, DNA , beta-Globins , Genetics , Metabolism , beta-Thalassemia , Genetics , Metabolism , Pathology , Therapeutics
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