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1.
Biol Reprod ; 109(6): 821-838, 2023 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-37788061

RESUMO

Haploid embryos have contributed significantly to our understanding of the role of parental genomes in development and can be applied to important biotechnology for human and animal species. However, development to the blastocyst stage is severely hindered in bovine haploid androgenetic embryos (hAE). To further our understanding of such developmental arrest, we performed a comprehensive comparison of the transcriptomic profile of morula-stage embryos, which were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of transcripts associated with differentiation in haploid and biparental embryos. Among numerous disturbances, results showed that pluripotency pathways, especially the wingless-related integration site (WNT) signaling, were particularly unbalanced in hAE. Moreover, transcript levels of KLF4, NANOG, POU5F1, SOX2, CDX2, CTNNBL1, AXIN2, and GSK3B were noticeably altered in hAE, suggesting disturbance of pluripotency and canonical WNT pathways. To evaluate the role of WNT on hAE competence, we exposed early Day-5 morula stage embryos to the GSK3B inhibitor CHIR99021. Although no alterations were observed in pluripotency and WNT-related transcripts, exposure to CHIR99021 improved their ability to reach the blastocysts stage, confirming the importance of the WNT pathway in the developmental outcome of bovine hAE.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Via de Sinalização Wnt , Humanos , Animais , Bovinos , Via de Sinalização Wnt/genética , Haploidia , Diferenciação Celular/genética , Blastocisto/metabolismo , Desenvolvimento Embrionário/genética
2.
Zygote ; 30(4): 440-463, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35652626

RESUMO

Over the last decades, extracellular vesicles (EVs) have been found to be implicated in a complex universal mechanism of communication between different cell types. EVs are nanostructures of lipid nature that have an exosomal or ectosomal biogenesis, responsible for the intercellular transport of proteins, lipids, carbohydrates, nucleic acids, ions, among other molecules. The content of EVs can vary due to various factors such as hormonal stimuli, non-physiological conditions, metabolic state, etc. Once EVs reach their target cell, they can modulate processes such as gene expression, metabolism, response to external factors, and can even be associated with the delivery of molecules involved in epigenetic inheritance processes in germ cells. In mammalian reproduction, EVs have been shown to play an important role, either in vivo or in vitro, modulating a variety of processes in sperm, oocytes and embryos, and in their respective environments. Moreover, EVs represent a biodegradable, harmless and specific vehicle, which makes them attractive allies to consider when improving assisted reproductive technologies (ARTs). Therefore, the present review aims to describe the content of the main EVs involved in mammalian reproduction and how they can vary due to different factors, as well as to detail how EVs modulate, directly or indirectly, different molecular processes in gametes and embryos. In addition, we will highlight the mechanisms that remain to be elucidated. We will also propose new perspectives according to the characteristics of each particular EV to improve the different ARTs.


Assuntos
Vesículas Extracelulares , Sêmen , Animais , Vesículas Extracelulares/metabolismo , Masculino , Mamíferos , Oócitos/fisiologia , Reprodução , Espermatozoides
3.
Mol Biol Rep ; 47(12): 9959-9965, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33226564

RESUMO

In-vitro fertilization is a routine livestock-breeding technique widely used around the world. Several studies have reported the interaction of bovine viral-diarrhea virus (BVDV) with gametes and in-vitro-produced (IVP) bovine embryos. Since, gene expression in BVDV-infected IVP bovine embryos is scarcely addressed. The aim of this work was to evaluate the differential expression of genes involved in immune and inflammatory response. Groups of 20-25 embryos on Day 6 (morula stage) were exposed (infected) or not (control) to an NCP-BVDV strain in SOF medium. After 24 h, embryos that reached expanded blastocyst stage were washed. Total RNA of each embryo group was extracted to determine the transcription levels of 9 specific transcripts related with antiviral and inflammatory response by SYBR Green real time quantitative (RT-qPCR). Culture media and an aliquot of the last embryos wash on Day 7 were analyzed by titration and virus isolation, respectively. A conventional PCR confirmed BVDV presence in IVP embryos. A significantly higher expression of interferon-α was observed in blastocysts exposed to NCP-BVDV compared to the controls (p < 0.05). In this study, the upregulation of INFα and TLR7 genes involved in inflammatory and immune response in BVDV-infected IVP bovine embryos is a new finding in this field. This differential expression suggest that embryonic cells could function in a manner like immune cells by recognizing and responding early to interaction with viral pathogens. These results provide new insights into the action of BVDV on the complex molecular pathways controlling bovine early embryonic development.


Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Bovinos , Vírus da Diarreia Viral Bovina/imunologia , Desenvolvimento Embrionário/imunologia , Expressão Gênica/imunologia , Interferon-alfa , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/embriologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos/embriologia , Bovinos/imunologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Embrião de Mamíferos/imunologia , Embrião de Mamíferos/virologia , Feminino , Fertilização in vitro , Interferon-alfa/imunologia , Receptor 7 Toll-Like/imunologia
4.
Biol Res ; 46(4): 452-62, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24510147

RESUMO

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01) in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.


Assuntos
Blastocisto/fisiologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Transferência Nuclear/veterinária , Animais , Bovinos , Técnicas de Cultura Embrionária/métodos , Feminino
5.
Biol Res ; 37(3): 449-60, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15515969

RESUMO

The feasibility of ablating differentiated adipocytes and the mechanism of cell ablation with a suitable prodrug activating system is described. The system is based on the use of E. coli nitroreductase (NTR) enzyme that activates certain nitro compounds, such as the antitumor drug CB1954, into cytotoxic DNA interstrand cross-linking agents. Differentiated preadipocyte cells (3T3L1) transfected with an aP2 driven nitroreductase construct were efficiently killed after incubation with medium containing the prodrug CB1954, while untransfected cells were not affected. It was demonstrated that the mechanism of cell ablation is apoptosis and that the system has a bystander effect mediated by a toxic metabolite of the prodrug. The described system should provide a good alternative approach for gene therapy studies and a new inducible approach to manipulating the number of cells in tissues of transgenic animals and the ability to study the recovery of the tissue from cell damage or loss.


Assuntos
Adipócitos/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose , Aziridinas/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Escherichia coli/genética , Genes Transgênicos Suicidas/efeitos dos fármacos , Nitrorredutases/genética , Células 3T3-L1 , Animais , Apoptose/genética , Técnicas de Cultura de Células , Diferenciação Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Escherichia coli/efeitos dos fármacos , Genes Transgênicos Suicidas/genética , Camundongos , Nitrorredutases/efeitos dos fármacos , Fatores de Tempo , Transfecção/métodos
6.
Biol. Res ; 37(3): 449-460, 2004. ilus, graf
Artigo em Inglês | LILACS | ID: lil-394438

RESUMO

The feasibility of ablating differentiated adipocytes and the mechanism of cell ablation with a suitable prodrug activating system is described. The system is based on the use of E. coli nitroreductase (NTR) enzyme that activates certain nitro compounds, such as the antitumor drug CB1954, into cytotoxic DNA interstrand cross-linking agents. Differentiated preadipocyte cells (3T3L1) transfected with an aP2 driven nitroreductase construct were efficiently killed after incubation with medium containing the prodrug CB1954, while untransfected cells were not affected. It was demonstrated that the mechanism of cell ablation is apoptosis and that the system has a bystander effect mediated by a toxic metabolite of the prodrug. The described system should provide a good alternative approach for gene therapy studies and a new inducible approach to manipulating the number of cells in tissues of transgenic animals and the ability to study the recovery of the tissue from cell damage or loss.


Assuntos
Animais , Camundongos , Adipócitos/efeitos dos fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Aziridinas/farmacologia , Proteínas de Escherichia coli/genética , Genes Transgênicos Suicidas/efeitos dos fármacos , Técnicas de Cultura de Células , Diferenciação Celular , Estudos de Viabilidade , Fatores de Tempo , Transfecção/métodos
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