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The aim of this study was to investigate the growth and development of animals produced from demi-embryos and compare them with whole embryos from fetus to adult life. To achieve this, calves produced from fresh demi-embryos and whole embryos were individually transferred and monitored from 60 days of pregnancy until slaughter at 550 days. Ultrasound scans were conducted on fetuses at 60 and 90 days to evaluate the biparietal, abdominal, umbilical cord, orbital, and aorta diameters. Subsequently, morphological traits of newborn calves were measured at 0, 7, and 21 days (N = 18). Live weight was recorded at birth, weaning, and every 30 days thereafter until slaughter at 550 days. The growth curve of each group was modeled using logistic regression, and the factors of the respective functions were compared. As early as 60 days of pregnancy, ultrasound evaluations revealed no morphometric differences between fetuses produced from demi-embryos and those from whole embryos. This lack of differentiation persisted in the morphometric evaluations of newborns up to 21 days of age, as well as in live weight and the growth curve from birth to slaughter. Moreover, there were no significant differences between the groups in terms of rib eye area and fat thickness evolution. Consequently, individuals from demi-embryos exhibited no discernible disparities to those whole embryos in growth and development from 60 days of gestation, through birth, and into adulthood.
Assuntos
Animais Recém-Nascidos , Animais , Bovinos/embriologia , Feminino , Gravidez , Desenvolvimento Fetal/fisiologia , Transferência Embrionária/veterinária , Ultrassonografia Pré-Natal/veterinária , Fertilização in vitro/veterinária , Desenvolvimento Embrionário/fisiologiaRESUMO
Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and ß4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and ß4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Suínos/genética , Humanos , Masculino , Animais Geneticamente Modificados , Edição de Genes/veterinária , Transplante Heterólogo/veterinária , Injeções de Esperma Intracitoplásmicas/veterinária , Sêmen , Fertilização in vitro/veterináriaRESUMO
Horse cloning by somatic cell nuclear transfer (SCNT) is an attractive scientific and commercial endeavor. Moreover, SCNT allows generating genetically identical animals from elite, aged, castrated, or deceased equine donors. Several variations in the horse SCNT method have been described, which may be useful for specific applications. This chapter describes a detailed protocol for horse cloning, thus including SCNT protocols using zona pellucida (ZP)-enclosed or ZP-free oocytes for enucleation. These SCNT protocols are under routine use for commercial equine cloning.
Assuntos
Técnicas de Transferência Nuclear , Zona Pelúcida , Cavalos , Animais , Técnicas de Transferência Nuclear/veterinária , Oócitos , Clonagem de Organismos/métodosRESUMO
After sperm-oocyte fusion, intracytoplasmic rises of calcium (Ca) induce the release of zinc (Zn) out of the oocyte (Zn sparks). Both phenomena are known to play an essential role in the oocyte activation process. Our work aimed to explore different protocols for activating bovine and porcine oocytes using the novel zinc chelator 1,10-phenanthroline (PHEN) and to compare developmental rates and quality to bovine IVF and parthenogenetic ionomycin-induced embryos in both species. Different incubation conditions for the zinc chelator were tested, including its combination with ionomycin. Embryo quality was assessed by immunofluorescence of SOX2, SOX17, OCT4, and CDX2 and total cell number at the blastocyst stage. Even though blastocyst development was achieved using a zinc chelator in bovine, bypassing calcium oscillations, developmental rates, and blastocyst quality were compromised compared to embryos generated with sperm-induced or ionomycin calcium rise. On the contrary, zinc chelation is sufficient to trigger oocyte activation in porcine. Additionally, we determined the optimal exposure to PHEN for this species. Zinc chelation and artificial induction of calcium rise combined did not improve developmental competence. Our results contribute to understanding the role of zinc during oocyte activation and preimplantation embryo development across different mammalian species.
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The CRISPR-on system is a programmable, simple, and versatile gene activator that has proven to be efficient in cultured cells from several species and in bovine embryos. This technology allows for the precise and specific activation of single endogenous gene expression and also multiplexed gene expression in a simple fashion. Therefore, CRISPR-on has unique advantages over other activator systems and a wide adaptability for studies in basic and applied science, such as cell reprogramming and cell fate differentiation for regenerative medicine.In this chapter, we describe the materials and methods of the CRISPR-on system for activation of the endogenous SMARCA4 expression in bovine embryos.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Animais , Bovinos , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/genéticaRESUMO
Somatic cell nuclear transfer (SCNT) is a method with unique ability to reprogram the epigenome of a fully differentiated cell. However, its efficiency remains extremely low. In this work, we assessed and combined two simple strategies to improve the SCNT efficiency in the bovine. These are the use of less-differentiated donor cells to facilitate nuclear reprogramming and the embryo aggregation (EA) strategy that is thought to compensate for aberrant epigenome reprogramming. We carefully assessed the optimal time of EA by using in vitro-fertilized (IVF) embryos and evaluated whether the use of adipose-derived mesenchymal stem cells (ASCs) as donor for SCNT together with EA improves the blastocyst rates and quality. Based on our results, we determined that the EA improves the preimplantation embryo development per well of IVF and SCNT embryos. We also demonstrated that day 0 (D0) is the optimal aggregation time that leads to a single blastocyst with uniform distribution of the original blastomeres. This was confirmed in bovine IVF embryos and then, the optimal condition was translated to SCNT embryos. Notably, the relative expression of the trophectoderm (TE) marker KRT18 was significantly different between aggregated and nonaggregated ASC-derived embryos. In the bovine, no effect of the donor cell is observed on the developmental rate, or the embryo quality. Therefore, no synergistic effect of the use of both strategies is observed. Our results suggest that EA at D0 is a simple and accessible strategy that improves the blastocyst rate per well in bovine SCNT and IVF embryos and influence the expression of a TE-related marker. The aggregation of two ASC-derived embryos seems to positively affect the embryo quality, which may improve the postimplantation development.
Assuntos
Blastocisto/citologia , Clonagem de Organismos/veterinária , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Células-Tronco Mesenquimais/citologia , Animais , Bovinos , Embrião de Mamíferos/química , Feminino , Fertilização in vitro , GravidezRESUMO
Assisted reproductive technologies in canine species are limited due to the low efficiency of in vitro maturation (IVM). Unlike other mammals, bitches ovulate oocytes in the germinal vesicle stage and complete metaphase II (MII) after 48-72 h in the oviductal environment and become fertilizable. For this reason, we compared two different IVM media, synthetic oviductal fluid (SOF) supplemented with 8% bovine serum albumin (BSA) or a mixture of 8% BSA-2.5% fetal bovine serum (FBS) and TCM-199 with 10% FBS. Additionally, we evaluated the effect of supplementation with insulin-transferrin-selenium (ITS) and low O2 tension in oocyte maturation, reactive oxygen species (ROS) levels, membrane integrity, and embryo development following parthenogenetic activation (PA). After 72 h of culture, SOF + BSA, SOF + BSA + FBS, and TCM-199 + FBS show 5, 7, and 4% of MII, respectively, without a statistical difference. However, SOF + BSA produced significantly higher degeneration rates compared to SOF + BSA + FBS (44 and 23%, respectively). Remarkably, supplementation with 1 µl/ml of ITS under high O2 tension demonstrated a beneficial effect by improving maturation rates up to 20% compared to the other groups. Low O2 tension increased maturation rates to 36.5%, although there were no statistical differences compared to high O2 tension in the presence of ITS. Lower ROS levels and higher integrity of the cytoplasmic membrane were found in the presence of ITS despite no differences in maturation rates under low O2 tension groups. Additionally, after PA, 1% development until the eight-cell stage was obtained after activation of in vitro-matured oocytes in the presence of ITS. Taken together, these results indicate that SOF supplemented with 8% BSA and 2.5% FBS is suitable for IVM of canine oocytes and ITS supplementation was beneficial for both high and low O2 tension. Furthermore, the addition of ITS in the cultured system lowers ROS levels and increases membrane integrity in domestic dog oocytes after IVM.
RESUMO
Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.
Assuntos
Blastômeros/fisiologia , Fusão Celular , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Fertilização in vitro/veterinária , Tetraploidia , Animais , Blastômeros/citologia , Gatos , Transferência Embrionária , Embrião de Mamíferos/metabolismo , Feminino , Masculino , Panthera , GravidezRESUMO
Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be independent of the genetic background of the injected sperm. No differences were found in blastocyst rate of ICSI hybrid (zebra spermatozoon into horse egg) embryos relative to the homospecific horse control group. Interestingly, zebra cloned blastocyst rate was significantly higher at day 8. Moreover, most ICSI and cloned horse and zebra blastocysts showed a similar expression pattern of SOX2 and nuclear YAP1 with the majority of the nuclei positive for YAP1, and most SOX2+ nuclei negative for YAP1. Here we demonstrated that horse oocytes support zebra preimplantation development of both, ICSI and cloned embryos, without compromising development to blastocyst, blastocyst cell number neither the expression of SOX2 and YAP1. Our results support the use of domestic horse oocytes as a model to study in vitro zebra embryos on behalf of preservation of valuable genetic.
Assuntos
Desenvolvimento Embrionário , Equidae/embriologia , Equidae/genética , Cavalos/fisiologia , Oócitos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Núcleo Celular/fisiologia , Clonagem de Organismos/veterinária , Citoplasma/fisiologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Espécies em Perigo de Extinção , Equidae/metabolismo , Feminino , Perfilação da Expressão Gênica , Cavalos/genética , Técnicas In Vitro , Masculino , Técnicas de Transferência Nuclear/veterinária , Fatores de Transcrição SOXB1/genética , Injeções de Esperma Intracitoplásmicas/veterinária , Sus scrofaRESUMO
The successful use of assisted reproduction techniques (ART) depends in part on the sperm physiological status. Several sperm selection procedures have been applied to improve quality of sperm population when using the ART. There has previously been development of a Sperm Selection Assay (SSA) for humans which is based on the attraction of capacitated sperm by chemotaxis towards progesterone (P), resulting in an enriched sperm population with an optimal physiological status similar to capacitated spermatozoa, with these cells having very little DNA fragmentation and optimal concentrations of reactive oxygen species (ROS). In the present study, the aim was to adapt the SSA for frozen-thawed stallion semen samples and evaluate the functional status of those sperm selected using the SSA procedure, and to determine whether this enriched sperm population has a greater capacity to bind to the zona pellucida of cattle oocytes. There were experimental conditions developed to conduct the SSA with stallion sperm. Using these conditions, the indexes of induced acrosome reaction, protein tyrosine phosphorylation, mitochondrial membrane potential, mitochondrial and cytoplasmic reactive oxygen species, and number of sperm bound to the zona pellucida of cattle were greater when the sperm population was selected using the SSA. Consistently, the DNA fragmentation and phospholipase C zeta indexes were less for the selected sperm. In conclusion, stallion sperm selected using chemotaxis utilizing the SSA provides a sperm population of greater quality, which when used may improve the outcomes with use of the ART.
Assuntos
Criopreservação/veterinária , Cavalos/fisiologia , Preservação do Sêmen/veterinária , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologia , Adaptação Fisiológica , Animais , Quimiotaxia , Congelamento , Masculino , Reprodutibilidade dos TestesRESUMO
CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.
Assuntos
DNA Helicases/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Proteínas Nucleares/metabolismo , Fator de Transcrição AP-2/metabolismo , Fatores de Transcrição/metabolismo , Animais , Bovinos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Helicases/genética , Fertilização in vitro/veterinária , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/veterinária , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Fator de Transcrição AP-2/genética , Fatores de Transcrição/genéticaRESUMO
The aim of this study was to assess the presence and distribution of apoptosis in porcine cumulus-oocyte complexes (COCs) and its relations with COC morphology and developmental competence. The COCs were obtained from slaughterhouse ovaries, classified into A1 (top category), A2, B1, B2, C, and D based on their morphology. A1, A2, and B1 were matured and fertilized in vitro, and blastocyst rate was compared among them. Before and after in vitro maturation (IVM), annexin-V staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to assess early and late apoptosis, respectively. There was a significant increase in both annexin-V (+) oocytes and TUNEL (+) cumulus cells as morphology further deteriorated. There were no statistical differences regarding annexin-V (+) oocytes within immature and post-IVM COCs, but TUNEL (+) oocytes were only observed in post-IVM COCs. Early and late apoptosis was detected in cumulus cells of all categories of immature and post-IVM COCs. However, the difference was only significant for annexin-V (+). There were no significant differences in embryo development. Therefore, apoptosis increases as the morphological features of the immature COCs decrease. In conclusion, the selection of COCs from Categories A1, A2, and B1 may be used as a selection criterion for in vitro development.
Assuntos
Apoptose/fisiologia , Células do Cúmulo/citologia , Desenvolvimento Embrionário/fisiologia , Fertilização in vitro/métodos , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/citologia , Animais , Blastocisto/citologia , Técnicas de Cultura Embrionária , Feminino , Marcação In Situ das Extremidades Cortadas , Masculino , Sêmen , SuínosRESUMO
Pigs are an important resource for meat production and serve as a model for human diseases. Due to their physiological and anatomical similarities to humans, these animals can recapitulate symptoms of human diseases, becoming an effective model for biomedical research. Although, in the past pig have not been widely used partially because of the difficulty in genetic modification; nowadays, with the new revolutionary technology of programmable nucleases, and fundamentally of the CRISPR-Cas9 systems, it is possible for the first time to precisely modify the porcine genome as never before. To this purpose, it is necessary to introduce the system into early stage zygotes or to edit cells followed by somatic cell nuclear transfer. In this review, several strategies for pig knock-out gene editing, using the CRISPR-Cas9 system, will be summarized, as well as genotyping methods and different delivery techniques to introduce these tools into the embryos. Finally, the best approaches to produce homogeneous, biallelic edited animals will be discussed.
RESUMO
Assisted reproduction techniques (ARTs) have become widespread in the equine breeding industry. In particular, the combination of oocyte recovery from live mares followed by IVM and intracytoplasmic sperm injection (ICSI) has increased markedly among the ARTs used with valuable or low-fertility animals. There is currently no consensus among research groups regarding the optimal oocyte maturation period to produce high-quality embryos. In this study, we report the maturation dynamics of equine oocytes at different time points, from 20 to 40h (Experiment 1). In addition, in Experiment 2, equine ICSI blastocysts were produced from oocytes that exhibited early (up to 24h) or late (28-30h) extrusion of the first polar body (PB). Blastocyst rates and diameter were recorded and embryo quality was assessed by analysing the number of apoptotic cells and Yes-associated protein 1 (YAP1) expression. By 20h of IVM, 42% of oocytes were mature, and the remaining oocytes matured within the next 17h of IVM. Although no differences were found in cell apoptosis or the number of YAP1-positive cells between groups exhibiting early and late PB extrusion, embryos from the early group (Group I) exhibited an improved total cell number and blastocyst rate compared to embryos from the late group (Group II) (18.60% vs 10.17% respectively).
Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário/fisiologia , Cavalos , Corpos Polares/fisiologia , Injeções de Esperma Intracitoplásmicas , Animais , Blastocisto/citologia , Blastocisto/ultraestrutura , Células Cultivadas , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos , Feminino , Cavalos/embriologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Recuperação de Oócitos/métodos , Recuperação de Oócitos/veterinária , Oogênese/fisiologia , Injeções de Esperma Intracitoplásmicas/métodos , Injeções de Esperma Intracitoplásmicas/veterinária , Fatores de TempoRESUMO
In bovine, correct oocyte artificial activation is a key step in ICSI and other reproductive biotechnologies, and still needs to be improved. The current study was designed to compare the activating efficiency of ionomycin (Io) followed by: a 4â¯h time window and ethanol (4h-Et), roscovitine (Rosc), dehydroleucodine (DhL), cycloheximide (CHX) or PD0325901 (PD), each as a single treatment, and then combine them in novel protocols. Parthenogenetic haploid activation was evaluated in terms of pronuclear (PN) formation, second polar body (2PB) extrusion, ploidy of day 2 embryos and in vitro development. Combined treatments with Io-4h-Et-Rosc and Io-Rosc/CHX increased PN formation (92.2% and 96%, respectively) compared with Io-Rosc, Io-CHX or Io-4h-Et, which were equally efficient at inducing PN formation (82-84%) and 2PB extrusion (62.1-70.5%). Oocyte activation with Io-DhL and Io-Rosc/DhL resulted in higher 2PB extrusion rates (90% and 95.9%, respectively) but lower PN formation (49.4-58.8%) and cleavage rates (36-57.9%), as occurred with Io-CHX/DhL (76.4% and 70.4%, respectively). For the first time, results show that Io followed by the MAPK inhibitor PD induces PN formation and 2PB extrusion, but PD combined with Rosc or CHX resulted in low rates of haploid day 2 embryos. In conclusion, DhL strongly induces 2PB extrusion but leads to poor PN formation and embryo development. PD induces bovine oocyte activation but results in low rates of haploid embryos. In contrast, the improved PN formation rates after treatment with combined Io-4h-Et-Rosc and Io-Rosc/CHX suggest they should be further evaluated in ART, aiming to increase success rates in bovine.
Assuntos
Benzamidas/administração & dosagem , Difenilamina/análogos & derivados , Lactonas/administração & dosagem , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Técnicas de Reprodução Assistida , Sesquiterpenos/administração & dosagem , Animais , Bovinos , Difenilamina/administração & dosagem , Etanol , Feminino , Ionomicina , Fator Promotor de Maturação/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , RoscovitinaRESUMO
BACKGROUND: The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. RESULTS: We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. CONCLUSIONS: This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 µM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.
Assuntos
Blastocisto/citologia , Embrião de Mamíferos/embriologia , Camadas Germinativas/embriologia , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição SOXF/metabolismo , Animais , Benzamidas/farmacologia , Bovinos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Camadas Germinativas/citologia , Fator Inibidor de Leucemia/biossíntese , Proteína Homeobox Nanog/genética , Proteína Quinase C/biossíntese , Fatores de Transcrição SOXF/genética , Transdução de Sinais/fisiologia , Proteína Wnt1/biossínteseRESUMO
The ICSI-sperm mediated gene transfer (ICSI-SMGT) has been used to produce transgenic mice with high efficiency; however, the efficiency of this technique in farm animals is still less than desirable. Pretreatment of sperm with membrane destabilizing agents can improve the efficiency of ICSI in cattle. The objective of the present study was to evaluate streptolysin-O (SLO) as a novel treatment to permeabilize the bovine sperm membrane and assess its effect on efficiency of generating transgenic embryos by ICSI-SMGT. First, there was evaluation of the plasma membrane integrity (SYBR/PI), acrosome membrane integrity (PNA/FITC), DNA damage (TUNEL) and binding capacity of exogenous DNA (Nick Translation) in bull sperm treated with SLO. Subsequently, there was assessment of embryonic development and the efficiency in generating transgenic embryos with enhanced expression of the gene for green fluorescent protein (EGFP). Results indicate that SLO efficiently permeabilizes the plasma and acrosome membranes of bull spermatozoa and increases binding of exogenous DNA mostly to the post-acrosomal region and tail without greatly affecting the integrity of the DNA. Furthermore, treatment of bull spermatozoa with SLO prior to the injection of oocytes by ICSI-SMGT significantly increased the rate of embryo expression of the EGFP gene. Future experiments are still needed to determine the effect of this treatment on the development and transgene expression in fetuses and animals produced by ICSI-SMGT.
Assuntos
Bovinos/embriologia , Técnicas de Transferência de Genes/veterinária , Proteínas de Fluorescência Verde/metabolismo , Injeções de Esperma Intracitoplásmicas/veterinária , Espermatozoides/fisiologia , Animais , Feminino , Masculino , Gravidez , Espermatozoides/efeitos dos fármacos , Estreptolisinas/farmacologiaRESUMO
Intracytoplasmic sperm injection (ICSI) has become a useful technique for clinical applications in the horse-breeding industry. However, both ICSI blastocyst and offspring production continues to be limited for most farm and wild species. This article reviews technical differences of ICSI performance among species, possible biological and methodological reasons for the variable efficiency and potential strategies to improve the outcomes. One of the major applications of ICSI in animal production is the reproduction of high-value specimens. Unfortunately, some domestic species like the bovine show low rates of pronuclei formation after sperm injection, which led to the development of various artificial activation protocols and sperm pre-treatments that are discussed in this article. The impact of ICSI technique on equine breeding programs is considered in detail, since in contrast to other species, its use for elite horse reproduction has increased in recent years. ICSI has also been used to produce genetically modified animals; however, despite numerous attempts in several domestic species, only transgenic pigs have been consistently produced. Finally, the ICSI is a promising tool for genetic rescue of endangered and wild species. In conclusion, while ICSI has become a consistent ART for some species, it needs further development for others. The low results obtained for some domestic species, the high training needed and the equipment required have limited this technique to the production of elite specimens or for research purposes.
Assuntos
Fertilização in vitro/veterinária , Injeções de Esperma Intracitoplásmicas , Animais , Animais Domésticos , MamíferosRESUMO
Vários sistemas para a produção de proteínas recombinantes têm sido utilizados para a otimização daprodução das mesmas. A glândula mamária é considerada um sistema muito interessante para a produção destasproteínas devido ao seu alto nível de expressão e à sua capacidade para realizar modificações pós-traducionais.Caprinos podem produzir elevadas quantidades de leite durante um longo período de lactação e, portanto,tornam-se importantes candidatos como biorreatores para a expressão de proteínas recombinantes no leite. Noentanto, caprinos transgênicos não são de fácil obtenção devido à baixa eficiência dos métodos e à expressãoimprevisível da proteína recombinante. O incremento na eficiência dos métodos na transgênese em caprinos éum grande desafio a superar. Esta revisão tem por objetivo apresentar o estado atual dos métodos tradicionais,bem como apresentar técnicas emergentes para obtenção eficiente de caprinos transgênicos.
Aiming the optimization for recombinant protein production, various systems have been used. Themammary gland is considered to be a very interesting system for the production of these proteins due to its highlevel of expression and its ability to perform post-translational modifications. Goats can produce largequantities of milk over a long lactation period, and therefore this species is an important candidate forrecombinant protein expression in milk. However, it is not easy to generate transgenic goats due to the lowefficiency of methods and unpredictable expression of recombinant protein. An increase in efficiency fortransgenic methodologies for goats is a big challenge to overcome. This review aims to present the state of art ofthe traditional methods, as well as the emerging technologies for obtaining transgenic goats efficiently.
Assuntos
Feminino , Animais , Lactente , Glândulas Mamárias Animais , Proteínas Recombinantes/análise , Ruminantes/genética , Ruminantes/metabolismo , BiotecnologiaRESUMO
Vários sistemas para a produção de proteínas recombinantes têm sido utilizados para a otimização daprodução das mesmas. A glândula mamária é considerada um sistema muito interessante para a produção destasproteínas devido ao seu alto nível de expressão e à sua capacidade para realizar modificações pós-traducionais.Caprinos podem produzir elevadas quantidades de leite durante um longo período de lactação e, portanto,tornam-se importantes candidatos como biorreatores para a expressão de proteínas recombinantes no leite. Noentanto, caprinos transgênicos não são de fácil obtenção devido à baixa eficiência dos métodos e à expressãoimprevisível da proteína recombinante. O incremento na eficiência dos métodos na transgênese em caprinos éum grande desafio a superar. Esta revisão tem por objetivo apresentar o estado atual dos métodos tradicionais,bem como apresentar técnicas emergentes para obtenção eficiente de caprinos transgênicos.(AU)
Aiming the optimization for recombinant protein production, various systems have been used. Themammary gland is considered to be a very interesting system for the production of these proteins due to its highlevel of expression and its ability to perform post-translational modifications. Goats can produce largequantities of milk over a long lactation period, and therefore this species is an important candidate forrecombinant protein expression in milk. However, it is not easy to generate transgenic goats due to the lowefficiency of methods and unpredictable expression of recombinant protein. An increase in efficiency fortransgenic methodologies for goats is a big challenge to overcome. This review aims to present the state of art ofthe traditional methods, as well as the emerging technologies for obtaining transgenic goats efficiently.(AU)