92 results on '"Fidel Ovidio Castro"'
Search Results
2. Proteomic Analysis of Domestic Cat Blastocysts and Their Secretome Produced in an In Vitro Culture System without the Presence of the Zona Pellucida
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Daniel Veraguas-Dávila, Camila Zapata-Rojas, Constanza Aguilera, Darling Saéz-Ruiz, Fernando Saravia, Fidel Ovidio Castro, and Lleretny Rodriguez-Alvarez
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in vitro fertilization ,felid embryos ,zona pellucida ,embryo–maternal communication ,protein expression profile ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography–tandem mass spectrometry (LC–MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups (p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts.
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- 2024
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3. DNA Content in Embryonic Extracellular Vesicles Is Independent of the Apoptotic Rate in Bovine Embryos Produced In Vitro
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Diego Caamaño, Joel Cabezas, Constanza Aguilera, Ioanna Martinez, Yat Sen Wong, Daniela Sanhueza Sagredo, Belén Ibañez, Sebastián Rodriguez, Fidel Ovidio Castro, and Lleretny Rodriguez-Alvarez
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EVs ,DNA ,bovine ,embryo ,apoptosis ,Veterinary medicine ,SF600-1100 ,Zoology ,QL1-991 - Abstract
Pre-implantation embryos release extracellular vesicles containing different molecules, including DNA. The presence of embryonic DNA in E-EVs released into the culture medium during in vitro embryo production could be useful for genetic diagnosis. However, the vesicles containing DNA might be derived from embryos suffering from apoptosis, i.e., embryos of bad quality. This work intended to confirm that embryos release DNA that is useful for genotyping by evaluating the effect of embryonic apoptosis on DNA content in E-EVs. Bovine embryos were produced by parthenogenesis and in vitro fertilization (IVF). On Day 5, morulae were transferred to individual cultures in an EV-depleted SOF medium. On Day 7, embryos were used to evaluate cellular apoptosis, and each culture medium was collected to evaluate E-EV concentration, characterization, and DNA quantification. While no effect of the origin of the embryo on the apoptotic rate was found, arrested morulae had a higher apoptotic rate. E-EVs containing DNA were identified in all samples, and the concentration of those vesicles was not affected by the origin or quality of the embryos. However, the concentration of DNA was higher in EVs released by the arrested parthenogenetic embryos. There was a correlation between the concentration of E-EVs, the concentration of DNA-positive E-EVs, and the concentration of DNA. There was no negative effect of apoptotic rate on DNA-positive E-EVs and DNA concentration; however, embryos of the best quality with a low apoptotic rate still released EVs containing DNA. This study confirms that the presence of DNA in E-EVs is independent of embryo quality. Therefore, E-EVs could be used in liquid biopsy for noninvasive genetic diagnosis.
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- 2024
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4. Mare stromal endometrial cells differentially modulate inflammation depending on oestrus cycle status: an in vitro study
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Yat S. Wong, Ana C. Mançanares, Felipe I. Navarrete, Pamela M. Poblete, Lídice Méndez-Pérez, Graça M. L. Ferreira-Dias, Lleretny Rodriguez-Alvarez, and Fidel Ovidio Castro
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endometrosis ,endometrium stromal cells ,fibrosis-related genes ,pro-fibrotic miRNA ,anti-fibrotic miRNA ,extracellular vesicles ,Veterinary medicine ,SF600-1100 - Abstract
The modulation of inflammation is pivotal for uterine homeostasis. Here we evaluated the effect of the oestrus cycle on the expression of pro-inflammatory and anti-inflammatory markers in a cellular model of induced fibrosis. Mare endometrial stromal cells isolated from follicular or mid-luteal phase were primed with 10 ng/mL of TGFβ alone or in combination with either IL1β, IL6, or TNFα (10 ng/mL each) or all together for 24 h. Control cells were not primed. Messenger and miRNA expression were analyzed using real-time quantitative PCR (RT-qPCR). Cells in the follicular phase primed with pro-inflammatory cytokines showed higher expression of collagen-related genes (CTGF, COL1A1, COL3A1, and TIMP1) and mesenchymal marker (SLUG, VIM, CDH2, and CDH11) genes; p
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- 2023
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5. Extracellular Vesicles Secreted by Pre-Hatching Bovine Embryos Produced In Vitro and In Vivo Alter the Expression of IFNtau-Stimulated Genes in Bovine Endometrial Cells
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Constanza Aguilera, Alejandra Estela Velásquez, Miguel Angel Gutierrez-Reinoso, Yat Sen Wong, Barbara Melo-Baez, Joel Cabezas, Diego Caamaño, Felipe Navarrete, Daniela Rojas, Gonzalo Riadi, Fidel Ovidio Castro, and Llretny Rodriguez-Alvarez
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extracellular vesicles ,embryo-maternal communication ,interferon tau ,bovine embryos ,endometrial bovine cells ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.
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- 2023
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6. Advantages in Wound Healing Process in Female Mice Require Upregulation A2A-Mediated Angiogenesis under the Stimulation of 17β-Estradiol
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Felipe Troncoso, Kurt Herlitz, Jesenia Acurio, Claudio Aguayo, Katherine Guevara, Fidel Ovidio Castro, Alejandro S. Godoy, Sebastian San Martin, and Carlos Escudero
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adenosine ,A2A receptor ,angiogenesis ,wound healing ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Estrogenic steroids and adenosine A2A receptors promote the wound healing and angiogenesis processes. However, so far, it is unclear whether estrogen may regulate the expression and pro-angiogenic activity of A2A receptors. Using in vivo analyses, we showed that female wild type (WT) mice have a more rapid wound healing process than female or male A2A-deficient mice (A2AKO) mice. We also found that pulmonary endothelial cells (mPEC) isolated from female WT mice showed higher expression of A2A receptor than mPEC from male WT mice. mPEC from female WT mice were more sensitive to A2A-mediated pro-angiogenic response, suggesting an ER and A2A crosstalk, which was confirmed using cells isolated from A2AKO. In those female cells, 17β-estradiol potentiated A2A-mediated cell proliferation, an effect that was inhibited by selective antagonists of estrogen receptors (ER), ERα, and ERβ. Therefore, estrogen regulates the expression and/or pro-angiogenic activity of A2A adenosine receptors, likely involving activation of ERα and ERβ receptors. Sexual dimorphism in wound healing observed in the A2AKO mice process reinforces the functional crosstalk between ER and A2A receptors.
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- 2020
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7. Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells
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Ana Carolina Furlanetto Mançanares, Joel Cabezas, José Manríquez, Vanessa Cristina de Oliveira, Yat Sen Wong Alvaro, Daniela Rojas, Felipe Navarrete Aguirre, Lleretny Rodriguez-Alvarez, and Fidel Ovidio Castro
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CRISPR/cas9 ,prostaglandin E2 ,adipose mesenchymal stem cell ,equine ,Veterinary medicine ,SF600-1100 ,Zoology ,QL1-991 - Abstract
In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.
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- 2020
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8. Endometritis and In Vitro PGE2 Challenge Modify Properties of Cattle Endometrial Mesenchymal Stem Cells and Their Transcriptomic Profile
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Evelyn Lara, Alejandra Velásquez, Joel Cabezas, Nathaly Rivera, Paulina Pacha, Lleretny Rodríguez-Alvarez, Fernando Saravia, and Fidel Ovidio Castro
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Internal medicine ,RC31-1245 - Abstract
Mesenchymal stem cells (MSCs) were isolated and characterized from postpartum bovine endometrium of animals with subclinical (n=5) and clinical endometritis (n=3) and healthy puerperal females (n=5). Cells isolated displayed mean morphological features of MSCs and underwent osteogenic, chondrogenic, and adipogenic differentiation after induction (healthy and subclinical). Cells from cows with clinical endometritis did not undergo adipogenic differentiation. All cells expressed mRNAs for selected MSC markers. Endometrial MSCs were challenged in vitro with PGE2 at concentrations of 0, 1, 3, and 10 μM, and their global transcriptomic profile was studied. Overall, 1127 genes were differentially expressed between unchallenged cells and cells treated with PGE2 at all concentrations (763 up- and 364 downregulated, fold change > 2, and P
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- 2017
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9. Endometrial Stem Cells in Farm Animals: Potential Role in Uterine Physiology and Pathology
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Evelyn Lara, Nathaly Rivera, Joel Cabezas, Felipe Navarrete, Fernando Saravia, Lleretny Rodríguez-Alvarez, and Fidel Ovidio Castro
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mesenchymal stem cells ,endometrium ,livestock ,Technology ,Biology (General) ,QH301-705.5 - Abstract
The endometrium is an accessible source of mesenchymal stem cells. Most investigations of endometrial mesenchymal stem cells (eMSCs) have been conducted in humans. In animals, particularly in livestock, eMSC research is scarce. Such cells have been described in the bovine, ovine, caprine, porcine, and equine endometrium. Here we provide the state of the art of eMSCs in farm animals with a focus on the bovine species. In bovines, eMSCs have been identified during the phases of the estrous cycle, during which their functionality and the presence of eMSC-specific markers has been shown to change. Moreover, postpartum inflammation related to endometritis affects the presence and functionality of eMSCs, and prostaglandin E2 (PGE2) may be the mediator of such changes. We demonstrated that exposure to PGE2 in vitro modifies the transcriptomic profile of eMSCs, showing its potential role in the fate of stem cell activation, migration, and homing during pathological uterine inflammation in endometritis and in healthy puerperal endometrium. Farm animal research on eMSCs can be of great value in translational research for certain uterine pathologies and for immunomodulation of local responses to pathogens, hormones, and other substances. Further research is necessary in areas such as in vivo location of the niches and their immunomodulatory and anti-infective properties.
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- 2018
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10. Disruption of the Blood-Brain Barrier by Extracellular Vesicles From Preeclampsia Plasma and Hypoxic Placentae: Attenuation by Magnesium Sulfate
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Pablo Torres-Vergara, Felipe Troncoso, Jesenia Acurio, Lina Bergman, Carlos Escudero, Jose Leon, Manu Vatish, Juan José Jorge López, Fidel Ovidio Castro, and Anna-Karin Wikström
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medicine.medical_specialty ,Placenta ,chemistry.chemical_element ,Blood–brain barrier ,Extracellular vesicles ,Preeclampsia ,Extracellular Vesicles ,Magnesium Sulfate ,Mice ,Pre-Eclampsia ,Pregnancy ,Internal medicine ,Electric Impedance ,Internal Medicine ,medicine ,Animals ,Humans ,Hypoxia ,Magnesium ,Endothelial Cells ,medicine.disease ,Microvesicles ,Mice, Inbred C57BL ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Blood-Brain Barrier ,Female - Abstract
Preeclampsia, a pregnancy-related endothelial disorder, is associated with both cardiovascular and cerebrovascular complications. Preeclampsia requires the presence of a placenta as part of its pathophysiology, yet the role of this organ in the cerebrovascular complications remains unclear. Research has shown that circulating small extracellular vesicles (also known as exosomes) present in preeclampsia plasma can generate endothelial dysfunction, but it is unclear whether the impairment of function of brain endothelial cells at the blood-brain barrier is secondary to plasma-derived or placental-derived exosomes. In this study, we evaluated the effect of small extracellular vesicles isolated from plasma samples of women with preeclampsia (n=12) and women with normal pregnancy (n=11) as well as from human placental explants from normotensive pregnancies (n=6) subjected to hypoxia (1% oxygen) on the integrity of the blood-brain barrier, using both in vitro and animal models. Exposure of human-derived brain endothelial cell monolayers to plasma and plasma-derived small extracellular vesicles from preeclamptic pregnancies increased the permeability and reduced the transendothelial electrical resistance. A similar outcome was observed with hypoxic placental-derived small extracellular vesicles, which also increased the permeability to Evan’s blue in the brain of C57BL6 nonpregnant mice. Cotreatment with magnesium sulfate reversed the effects elicited by plasma, plasma-derived, and hypoxic placental-derived small extracellular vesicles in the employed models. Thus, circulating small extracellular vesicles in plasma from women with preeclampsia or from hypoxic placentae disrupt the blood-brain barrier, which can be prevented using magnesium sulfate. These findings provide new insights into the pathophysiology of cerebral complications associated with preeclampsia.
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- 2021
11. Domestic cat embryos generated without zona pellucida are capable of developing in vitro but exhibit abnormal gene expression and a decreased implantation rate
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Alejandro Gonzalez, Fernando Saravia, Fidel Ovidio Castro, Soledad Saez, Daniel Veraguas-Davila, Lleretny Rodriguez-Alvarez, Maria Francisca Cordero, and Darling Saez-Ruiz
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endocrine system ,medicine.medical_treatment ,Gene Expression ,Fertilization in Vitro ,Biology ,Morula ,Andrology ,Food Animals ,SOX2 ,Pregnancy ,medicine ,Animals ,Embryo Implantation ,Blastocyst ,Small Animals ,Zona pellucida ,Zona Pellucida ,reproductive and urinary physiology ,In vitro fertilisation ,urogenital system ,Equine ,Embryo culture ,Embryo ,Embryo transfer ,medicine.anatomical_structure ,embryonic structures ,Cats ,Somatic cell nuclear transfer ,Female ,Animal Science and Zoology - Abstract
The removal of the zona pellucida has been used to improve the in vitro development of domestic cat embryos generated by IVF and SCNT. However, the in vivo development of domestic cat embryos generated without the zona pellucida has not been evaluated. The objective of this study was to evaluate the effects of zona pellucida removal on the in vitro and in vivo development of domestic cat embryos generated by IVF. For this purpose, two experimental groups were created: 1) domestic cat embryos cultured in vitro (Zona-intact group, ZI) and 2) domestic cat embryos cultured in vitro without the zona pellucida (Zona-free group, ZF). Domestic cat embryos were generated by IVF and cultured in vitro for 8 days. In the ZF group, the zona pellucida was removed after IVF, and embryos were cultured using the well of the well system (WOW). Cleavage, morula and blastocyst rates were evaluated in both groups. The diameter and total cell number of blastocysts were assessed. Relative expression of pluripotency (OCT4, SOX2 and NANOG), differentiation (CDX2 and GATA6) and apoptotic markers (BAX and BCL2) was evaluated in blastocysts. Finally, to evaluate in vivo development, embryos at days 5, 6 and 7 of development were transferred into recipient domestic cats, and ultrasonography was performed to evaluate implantation. No differences were observed in the cleavage, morula or blastocyst rates between embryos from the ZI and ZF groups. The diameter (mean ± SD) of blastocysts from the ZF group was greater (253.4 ± 83.3 μm) than that from the ZI group (210.5 ± 78.5 μm). No differences were observed in the relative expression of OCT4, CDX2 or GATA6. However, the relative expression of SOX2 and NANOG was significantly reduced in ZF blastocysts compared to ZI blastocysts. Furthermore, the relative expression of BAX was higher in ZF blastocysts than in ZI blastocysts. Finally, four pregnancies were confirmed after the transfer of ZI embryos (n = 110). However, no pregnancies were observed after the transfer of ZF embryos at the morula or blastocyst stage (n = 56). In conclusion, domestic cat embryos cultured without the zona pellucida were able to develop in vitro until the blastocyst stage. However, the removal of the zona pellucida negatively affected the gene expression of pluripotency and apoptosis markers, and ZF embryos were unable to implant. This might indicate that the removal of the zona pellucida is detrimental for the implantation and in vivo development of domestic cat embryos.
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- 2021
12. Analysis of trophectoderm markers in domestic cat blastocysts cultured without zona pellucida
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Daniel Veraguas-Dávila, Darling Saéz-Ruíz, María Consuelo Álvarez, Fernando Saravia, Fidel Ovidio Castro, and Lleretny Rodríguez-Alvarez
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Blastocyst ,Caspase 3 ,Cats ,Animals ,Cell Biology ,Fertilization in Vitro ,Cadherins ,Zona Pellucida ,Developmental Biology - Abstract
SummaryDomestic cat embryos generated by in vitro fertilization (IVF) and cultured without the zona pellucida have a reduced implantation capacity after embryo transfer at the blastocyst stage. The objective of this study was to evaluate the expression of trophectoderm markers in domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were selected: (1) domestic cat embryos generated by IVF and cultured in vitro normally (zona intact group, ZI); and (2) domestic cat embryos generated by IVF and cultured in vitro without a zona pellucida (zona-free group, ZF). In the ZF group, the zona pellucida of the presumptive zygote was removed and these were cultured using the well of the well (WOW) system. In vitro culture was carried out for 7 days. The cleavage, morula and blastocyst rates were estimated. Finally, the relative expression levels of the trophectoderm markers TEAD4, YAP1, CDX2 and EOMES, the cell adhesion marker E-cadherin and the apoptosis marker CASP3 were evaluated by RT-qPCR in the blastocysts. The Wilcoxon test was used to evaluate differences (P < 0.05). No differences were observed in the cleavage, morula and blastocyst rates between the ZF and ZI groups. No differences were found in the expression of TEAD4, CDX2, E-cadherin and CASP3 between groups. The expression of YAP1 and EOMES was higher in ZF blastocysts than in ZI blastocysts. In conclusion, the in vitro culture without the zona pellucida generates an overexpression of YAP1 and EOMES in the domestic cat blastocysts. More studies are needed to confirm if this overexpression might affect in vivo development.
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- 2022
13. Efeito do ácido valpróico e dos fatores de crescimento na plasticidade dos fibroblastos dérmicos felinos / Effect of Valproic acid and growth factors on plasticity of feline dermal fibroblasts
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Daniela Michel Rojas Mansilla, Fidel Ovidio Castro, Lleretny Rodríguez Álvarez, Constanza Javiera Aguilera González, and Diana Maritza Echeverry Berrío
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Valproic Acid ,Chemistry ,medicine ,General Earth and Planetary Sciences ,Plasticity ,ácido valproico ,Molecular biology ,General Environmental Science ,medicine.drug - Abstract
As células-tronco mesenquimais (CTM) são utilizadas na terapia celular, isolando-se de diferentes tecidos, incluindo a pele. Os fibroblastos dérmicos têm mostrado características de potência semelhantes às CTMs como expressão de marcadores de superfície e diferenciação para outros tipos de linhagens de origem mesodérmica, principalmente sob a influência de moduladores epigenéticos e fatores de crescimento. A terapia celular em medicina veterinária, especificamente em gatos, representa um desafio devido à invasividade na obtenção de tecidos fonte de CTM, para os quais outras opções menos invasivas estão sendo buscadas para an obtenção dessas células. O objetivo deste estudo foi avaliar o efeito do ácido valpróico - VPA (modulador epigenético) e fatores de crescimento (PRP e h-PDGF-B) na expressão de marcadores de superfície, genes de pluripotência e capacidade de diferenciação mesodérmica de fibroblastos felinos. Fibroblastos de pele foram isolados de gatas e cultivados com VPA e fatores de crescimento por 12 dias. A expressão de Cd90, Cd44, E-Caderina, Snail, Nanog e Oct4 foi avaliada nos dias 5 e 12. O potencial de diferenciação adipogênico, condrogênico e osteogênico dos fibroblastos foi avaliado após 12 dias de tratamento. A expressão de Cd44 aumentou no dia 5 do tratamento com VPA + PRP (p = 0,01). A expressão de Oct4 e Nanog aumentou no dia 5 do tratamento com VPA + h-PDGF-B (p 0,05). Fibroblastos em tratamento com VPA e h-PDGF-B mostraram capacidade de se diferenciar para condrogênese e osteogênese. O protocolo de cultura de células para fibroblastos felinos com VPA e h-PDGF-B confere plasticidade aos fibroblastos felinos ao promover a expressão de Nanog e Oct4, bem como a diferenciação mesodérmica in vitro.
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- 2021
14. Evaluation of extracellular vesicles and gDNA from culture medium as a possible indicator of developmental competence in human embryos
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Carlos Henriquez, Barbara Melo-Baez, Fidel Ovidio Castro, C. Aguilera, Lleretny Rodriguez-Alvarez, Pedro Silva-Ibañez, D. Veraguas, and Alejandra Velásquez
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animal structures ,medicine.medical_treatment ,Nanoparticle tracking analysis ,Biology ,Intracytoplasmic sperm injection ,Embryo Culture Techniques ,Andrology ,Extracellular Vesicles ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,030304 developmental biology ,Comparative Genomic Hybridization ,0303 health sciences ,030219 obstetrics & reproductive medicine ,Embryo ,Cell Biology ,Embryo, Mammalian ,Embryo transfer ,In vitro ,Microvesicles ,genomic DNA ,Blastocyst ,embryonic structures ,Embryo quality ,Developmental Biology - Abstract
SummaryHuman embryos generated in vitro have a high incidence of chromosomal abnormalities that negatively affect pregnancy rate. Embryos generated in vitro secrete extracellular vesicles (EVs) into the culture medium that could be used potentially as indicators of embryo competence. This research aimed to evaluate the concentration and size of EVs and their gDNA content as an indicator of developmental competence in human embryos. Human embryos generated by intracytoplasmic sperm injection (ICSI) were classified morphologically as of either TOP, FAIR or POOR quality. Culture medium and developmentally arrested embryos (which were not able to be used for embryo transfer) were collected. Microvesicles, exosomes (MV/Exo) and apoptotic bodies (ABs) were isolated from culture medium. Nanoparticle tracking analysis (NTA) and array comparative genomic hybridization (aCGH) analysis were performed to evaluate EVs and their gDNA content. From NTA, the diameter (mean) of MVs/Exo from TOP quality embryos was higher (112.17 nm) compared with that of FAIR (108.02) and POOR quality embryos (102.78 nm) (P < 0.05). aCGH analysis indicated that MVs/Exo and ABs carried gDNA with the presence of 23 chromosome pairs. However, when arrested embryos were compared with their respective MVs/Exo and ABs, the latter had an increased rate of chromosomal abnormalities (24.9%) compared with embryos (8.7%) (P < 0.05). In conclusion, the size of EVs from culture medium might be an alternative for evaluating competence of human embryos, however more studies are needed to validate the use of gDNA from EVs as an indicator of embryo competence.
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- 2020
15. In vitro and in vivo development of domestic cat embryos generated by in vitro fertilization after eCG priming and oocyte in vitro maturation
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D. Veraguas, Diana Maritza Echeverry, Darling Saez-Ruiz, L. Rodriguez-Alvarez, C. Aguilera, Fidel Ovidio Castro, P. F. Gallegos, and S. Saez
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Ovariectomy ,medicine.medical_treatment ,Embryonic Development ,Fertilization in Vitro ,Biology ,Hysterectomy ,Chorionic Gonadotropin ,Andrology ,Human fertilization ,Food Animals ,Pregnancy ,medicine ,Animals ,Blastocyst ,Small Animals ,reproductive and urinary physiology ,In vitro fertilisation ,Equine ,Gene Expression Regulation, Developmental ,Embryo ,Embryo Transfer ,Embryo, Mammalian ,Oocyte ,Embryo transfer ,In Vitro Oocyte Maturation Techniques ,In vitro maturation ,Pregnancy rate ,medicine.anatomical_structure ,embryonic structures ,Cats ,Oocytes ,Female ,Animal Science and Zoology - Abstract
The objective of this study was to evaluate, in the domestic cat, the effect of ovarian stimulation with eCG prior to oocyte in vitro maturation (priming) on in vitro and in vivo development after in vitro fertilization (IVF). For this purpose, oocyte donors were either 1) treated with a single dose of 200 IU eCG four days before oocyte recovery (eCG group), or, 2) given no treatment before oocyte recovery (control group). Ovaries of both groups were collected by ovariohysterectomy (OVH) and cumulus-oocyte complexes (COCs) were recovered by slicing. Immature COCs from both groups were matured in vitro (IVM) for 26-28 h. IVF was done with refrigerated epididymal sperm. After 24 h co-incubation, presumptive zygotes were cultured in vitro for eight days. The rates of cleavage, morulae, blastocyst development and hatching were estimated. Some blastocysts were stained for total cell counting and others were used for gene expression analysis of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6). Additionally, to evaluate in vivo development, embryos from the eCG group were transferred at Day 5 and Days 7 or 8 of IVC to synchronized cat recipients. The results showed that, eCG priming increased significantly the rate of blastocyst development as compared to the control group (37.9 and 25.6%, respectively) (P 0.05). No differences were observed in total cell number of blastocysts and hatching blastocysts (mean ± SD) between the eCG and control groups (420.6 ± 193.6 and 347.0 ± 237.1, respectively) (P 0.05). In the gene expression analysis, blastocysts generated in the eCG group had higher expression of OCT4 than blastocysts from the control group (P 0.05). However, no significant differences were observed in the relative expression of SOX2, NANOG, CDX2 and GATA6 (P 0.05). Additionally, six embryo transfer (ET) procedures were done, three with Day 5 embryos and three with Day 7 or 8 embryos. Recipients from both ET groups delivered live kittens. The total pregnancy rate was 4/6 (67%), meanwhile the live birth rate was 2/6 (33%). In conclusion, eCG priming improved the rate of blastocyst development in vitro and increased relative expression of OCT4. These results demonstrate that eCG priming of oocytes donors before IVM improves oocyte competence, enhance in vitro embryo development and allows live births of healthy offspring after ET.
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- 2020
16. Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes
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J. Cabezas, E. Mellisho, D. Veraguas, Alejandra Velásquez, Barbara Melo-Baez, Fidel Ovidio Castro, Lleretny Rodriguez-Alvarez, and Diego Andrés Caamaño Escobar
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Homeobox protein NANOG ,culture medium ,03 medical and health sciences ,0302 clinical medicine ,SOX2 ,medicine ,bovine embryos ,Blastocyst ,EVs ,030219 obstetrics & reproductive medicine ,Zygote ,General Veterinary ,Chemistry ,Embryogenesis ,0402 animal and dairy science ,Embryo ,04 agricultural and veterinary sciences ,pluripotency genes ,040201 dairy & animal science ,Embryonic stem cell ,In vitro ,Cell biology ,medicine.anatomical_structure ,embryonic structures ,Animal Science and Zoology ,Original Article ,nanoparticles - Abstract
Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.
- Published
- 2021
17. The expression level of SOX2 at the blastocyst stage regulates the developmental capacity of bovine embryos up to day-13 of in vitro culture
- Author
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J. Cabezas, Alejandra Velásquez, L. Rodriguez-Alvarez, Fidel Ovidio Castro, J. Manríquez, and D. Veraguas
- Subjects
0301 basic medicine ,Homeobox protein NANOG ,030219 obstetrics & reproductive medicine ,Embryogenesis ,Embryo ,Cell Biology ,Biology ,Embryonic stem cell ,Andrology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,SOX2 ,embryonic structures ,Gene expression ,medicine ,Blastocyst ,Gene ,Developmental Biology - Abstract
SummaryQuality of in vitro-produced embryos is influenced by changes in gene expression in response to adverse conditions. Gene markers for predicting ‘good embryos’ do not exist at present. We propose that the expression of pluripotency markers OCT4–SOX2–NANOG in D9 (day 9) bovine demi-embryos correlated with development at D13 (day 13). Day 8 in vitro-produced blastocysts were split in two cloned halves, one half (D9) was subjected to analysis of pluripotency markers and the other was kept in culture until D13 of development. Embryo development was scored and correlated with its own status at D9 and assigned to one of two categories: G1, arrested/dead; or G2, development up to D13. SOX2 and NANOG expression levels were significantly higher in embryos from G1 and there was also negative correlation between SOX2 and embryo survival to D13 (G3; r = −0.37; P = 0.03). We observed a significant reduction in the expression of the three studied genes from D9 to D13. Furthermore, there was a correlation between the expression of pluripotency markers at D9 and embryo diameter and the expression of trophoblastic markers at D13 (TP1–EOMES–FGF4–CDX2–TKDP1). Finally, the quotient between the relative expression of SOX2 and OCT4 in the D9 blastocysts from G1 and G2 showed that embryos that were considered as competent (G2) had a quotient close to one, while the other group had a quotient of 2.3 due to a higher expression of SOX2. These results might indicate that overexpression of SOX2 at the blastocyst stage had a negative effect on the control of embryonic developmental potential.
- Published
- 2019
18. Effect of BMP15 and/or AMH during in vitro maturation of oocytes from involuntarily culled dairy cows
- Author
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Lleretny Rodriguez-Alvarez, Alejandra Velásquez, E. Mellisho, and Fidel Ovidio Castro
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Anti-Mullerian Hormone ,0301 basic medicine ,endocrine system ,Beef cattle ,Biology ,Andrology ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,medicine ,Animals ,Blastocyst ,reproductive and urinary physiology ,Dairy cattle ,Cumulus Cells ,030219 obstetrics & reproductive medicine ,Bone morphogenetic protein 15 ,Embryogenesis ,Cell Biology ,Oocyte ,Breed ,In Vitro Oocyte Maturation Techniques ,In vitro maturation ,030104 developmental biology ,medicine.anatomical_structure ,embryonic structures ,Oocytes ,Cattle ,Female ,Bone Morphogenetic Protein 15 ,Developmental Biology - Abstract
The high metabolic activity to which the dairy cattle are exposed to maintain milk production altered steroid metabolism that affects reproductive physiology and reduce oocyte competence. Our aims were (a) to characterize the competence of immature oocytes collected from dairy cattle based on the expression of genes in cumulus cells (CCs) and (b) to improve oocyte competence to support preimplantation embryo development by the supplementation of maturation medium with bone morphogenetic protein 15 (BMP15) and/or anti-mullerian hormone (AMH). Oocyte donors were identified at the moment of ovary collection and grouped by involuntarily culled dairy cows (Holstein breed) or beef cattle. The embryo development speed to blastocyst of the cull dairy cattle versus beef cattle (control group) was lower. Besides
- Published
- 2018
19. Distinctive Cellular Transcriptomic Signature and MicroRNA Cargo of Extracellular Vesicles of Horse Adipose and Endometrial Mesenchymal Stem Cells from the Same Donors
- Author
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Gonzalo Riadi, Fernando Saravia, Yat Sen Wong, Lleretny Rodriguez-Alvarez, Ana Carolina Furlanetto Mançanares, J. Cabezas, D. Rojas, J. Manríquez, Felipe Navarrete, and Fidel Ovidio Castro
- Subjects
Cell ,Biology ,Extracellular matrix ,Transcriptome ,Endometrium ,Extracellular Vesicles ,Gene expression ,microRNA ,medicine ,Animals ,Horses ,Cells, Cultured ,Mesenchymal stem cell ,Cell Differentiation ,Mesenchymal Stem Cells ,Cell Biology ,Transforming growth factor beta ,Cell biology ,MicroRNAs ,medicine.anatomical_structure ,MRNA Sequencing ,Adipose Tissue ,biology.protein ,Female ,Developmental Biology ,Biotechnology ,Signal Transduction - Abstract
Equine endometrial and adipose mesenchymal stem cells (eMSCs and aMSCs, respectively) were isolated from the same donors of thoroughbred mares. The cells displayed characteristic features of MSCs, including trilineage mesodermal and also neurogenic differentiation. We evaluated the influence of cellular origin on their transcriptome profile. Cellular RNA was isolated and sequenced and extracellular vesicles (EVs) were obtained from conditioned medium of cells cultured in medium depleted of EVs, and their microRNA (miRNA) cargo analyzed by sequencing. Differential expression of mRNAs and EV-miRNA was analyzed, as well as pathways and processes most represented in each cell origin. mRNA reads from all expressed genes clustered according to the cellular origin. A total of 125 up- and 51 downregulated genes were identified and 31 differentially expressed miRNAs. Based on mRNA sequencing, endometrial MSCs strongly upregulated genes involved in the Hippo, transforming growth factor beta, and pluripotency signaling pathways. Alongside with this, pathways involved in extracellular matrix reorganization were the most represented in the miRNA cargo of EVs secreted by eMSCs. The niche from which MSCs originated defined the transcriptomic signature of the cells, including the secretion of lineage-specific loaded EV to ensure proper communication and homeostasis. Identification and testing their biological functions can provide new tools for the therapeutic use of horse MSC.
- Published
- 2020
20. Advantages in Wound Healing Process in Female Mice Require Upregulation A2A-Mediated Angiogenesis under the Stimulation of 17β-Estradiol
- Author
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Carlos Escudero, Katherine Guevara, Claudio Aguayo, Kurt Herlitz, Alejandro Godoy, Felipe Troncoso, Jesenia Acurio, Fidel Ovidio Castro, and Sebastian San Martin
- Subjects
0301 basic medicine ,Male ,Adenosine ,Angiogenesis ,Estrogen receptor ,Stimulation ,wound healing ,Wounds, Penetrating ,lcsh:Chemistry ,A2A receptor ,angiogenesis ,Mice ,0302 clinical medicine ,Receptor ,lcsh:QH301-705.5 ,Lung ,Spectroscopy ,Mice, Knockout ,Estradiol ,Chemistry ,General Medicine ,Computer Science Applications ,Female ,Signal Transduction ,medicine.medical_specialty ,Receptor, Adenosine A2A ,medicine.drug_class ,Neovascularization, Physiologic ,Catalysis ,Article ,Inorganic Chemistry ,03 medical and health sciences ,Sex Factors ,Downregulation and upregulation ,Internal medicine ,Phenethylamines ,medicine ,Animals ,Estrogen Receptor beta ,Physical and Theoretical Chemistry ,Molecular Biology ,Cell Proliferation ,Organic Chemistry ,Estrogen Receptor alpha ,Endothelial Cells ,Receptor Cross-Talk ,Adenosine receptor ,030104 developmental biology ,Endocrinology ,Pyrimidines ,lcsh:Biology (General) ,lcsh:QD1-999 ,Gene Expression Regulation ,Estrogen ,Pyrazoles ,Wound healing ,030217 neurology & neurosurgery - Abstract
Estrogenic steroids and adenosine A2A receptors promote the wound healing and angiogenesis processes. However, so far, it is unclear whether estrogen may regulate the expression and pro-angiogenic activity of A2A receptors. Using in vivo analyses, we showed that female wild type (WT) mice have a more rapid wound healing process than female or male A2A-deficient mice (A2AKO) mice. We also found that pulmonary endothelial cells (mPEC) isolated from female WT mice showed higher expression of A2A receptor than mPEC from male WT mice. mPEC from female WT mice were more sensitive to A2A-mediated pro-angiogenic response, suggesting an ER and A2A crosstalk, which was confirmed using cells isolated from A2AKO. In those female cells, 17&beta, estradiol potentiated A2A-mediated cell proliferation, an effect that was inhibited by selective antagonists of estrogen receptors (ER), ER&alpha, and ER&beta, Therefore, estrogen regulates the expression and/or pro-angiogenic activity of A2A adenosine receptors, likely involving activation of ER&alpha, receptors. Sexual dimorphism in wound healing observed in the A2AKO mice process reinforces the functional crosstalk between ER and A2A receptors.
- Published
- 2020
21. Embryo aggregation allows the production of kodkod (Leopardus guigna) blastocysts after interspecific SCNT
- Author
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D. Veraguas, Lleretny Rodriguez-Alvarez, Darling Saez-Ruiz, C. Aguilera, Diana Maritza Echeverry, and Fidel Ovidio Castro
- Subjects
Homeobox protein NANOG ,Nuclear Transfer Techniques ,Somatic cell ,Cloning, Organism ,Embryonic Development ,Biology ,Cytoplast ,Leopardus guigna ,Andrology ,Food Animals ,SOX2 ,medicine ,Animals ,Blastocyst ,Chile ,Small Animals ,reproductive and urinary physiology ,Equine ,Embryo ,biology.organism_classification ,Embryo, Mammalian ,medicine.anatomical_structure ,embryonic structures ,Cats ,Somatic cell nuclear transfer ,Animal Science and Zoology - Abstract
The kodkod (Leopardus guigna) is a small felid endemic of Chile and is considered a vulnerable species. Domestic cat oocytes have been successfully used as recipient cytoplast to reprogram somatic cells from different felids by interspecific somatic cell nuclear transfer (iSCNT). The developmental competence of felid embryos generated by iSCNT can be improved by the aggregation method using a zona-free culture system. The objective of this research was to evaluate the developmental competence of kodkod embryos generated by iSCNT using domestic cat oocytes and the aggregation method. For this purpose, five experimental group were done: (1) cat embryos generated by IVF, (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by iSCNT (K1x) and (5) aggregated kodkod embryos generated by iSCNT (K2x). Cleavage, morulae and blastocyst rates were estimated. The blastocyst diameter was evaluated. The gene expression level of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6) was analyzed in blastocysts. Morulae rate was higher in the IVF group and when cloned embryos were cultured in aggregates (IVF: 68.2%, Ca2x: 58.0% and K2x: 62.4%) compared to individually cultured kodkod embryos (K1x: 37.0%) (P 0.05). Embryo aggregation increased blastocysts formation in the Ca2x group (30.9%) to a similar rate compared to the IVF group (44.5%) (P 0.05). No blastocysts were generated in the K1x group, whereas blastocysts formation was obtained in K2x group (5.9%). The diameter of blastocysts from the K2x group (172.8 μm) was significantly lower than blastocysts from the Ca2x group (P 0.05). The relative expression of OCT4 was lower in blastocysts from Ca1x than in blastocysts from IVF (P 0.05). Furthermore, CDX2 expression was lower in blastocysts from Ca2x than in blastocysts from Ca1x and IVF groups (P 0.05). In kodkod embryos, only one blastocyst from the K2x group expressed OCT4. No expression of SOX2, NANOG, CDX2 and GATA6 was detected in kodkod blastocysts. In conclusion, after iSCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. The aggregation method increases the morulae rate of kodkod cloned embryos and allows blastocysts formation. However, kodkod blastocysts have a poor morphological quality and a lacking expression of pluripotency and differentiation markers, probably caused by an incomplete nuclear reprogramming.
- Published
- 2020
22. Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells
- Author
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J. Cabezas, Lleretny Rodriguez-Alvarez, Yat Sen Wong Alvaro, J. Manríquez, Ana Carolina Furlanetto Mançanares, Felipe Navarrete Aguirre, D. Rojas, Vanessa Cristina de Oliveira, and Fidel Ovidio Castro
- Subjects
Sanger sequencing ,prostaglandin E2 ,lcsh:Veterinary medicine ,General Veterinary ,Prostaglandin E2 receptor ,Mesenchymal stem cell ,Adipose tissue ,Transfection ,Biology ,Molecular biology ,Article ,CRISPR/cas9 ,symbols.namesake ,lcsh:Zoology ,symbols ,lcsh:SF600-1100 ,Animal Science and Zoology ,lipids (amino acids, peptides, and proteins) ,lcsh:QL1-991 ,Receptor ,adipose mesenchymal stem cell ,Fetal bovine serum ,Homing (hematopoietic) ,equine - Abstract
Simple Summary Prostaglandin E2 plays a regulatory role in MSC self-renewal, and PGE2 receptor-mediated signalling is involved in cell proliferation and migration. The aim of this work was to establish a genetically modified equine cellular model in which receptor genes EP2 and EP4 were edited by the CRISPR/Cas9 system. We successfully targeted EP2 and EP4 receptors in horse adipose MSCs using the CRISPR/Cas9 system. This targeting did not affect the surface marker phenotype of the adipose MSCs generated. Gene edition significantly lowered the expression of each of the targeted receptors and affected the early migration ability of the edited MSCs. This opens the possibility of using these mutant cell lines as a model system to elucidate the role of EP2 and EP4 in MSCs and, particularly, as therapeutic tools in equine regenerative medicine. Abstract In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.
- Published
- 2020
23. Assessment of the anti-inflammatory and engraftment potential of horse endometrial and adipose mesenchymal stem cells in an in vivo model of post breeding induced endometritis
- Author
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Felipe Navarrete, Fernando Saravia, Pedro Pablo Silva, Lleretny Rodriguez-Alvarez, Ana Carolina Furlanetto Mançanares, J. Cabezas, D. Rojas, Fidel Ovidio Castro, Fernanda Rojas, and Gabriela Cisterna
- Subjects
medicine.medical_treatment ,Anti-Inflammatory Agents ,Adipose tissue ,Carboxyfluorescein diacetate succinimidyl ester ,Endometrium ,Andrology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Food Animals ,Biopsy ,medicine ,Animals ,Horses ,Small Animals ,Saline ,030219 obstetrics & reproductive medicine ,medicine.diagnostic_test ,Equine ,business.industry ,Mesenchymal stem cell ,0402 animal and dairy science ,Mesenchymal Stem Cells ,04 agricultural and veterinary sciences ,medicine.disease ,040201 dairy & animal science ,Sperm ,medicine.anatomical_structure ,chemistry ,Animal Science and Zoology ,Female ,Horse Diseases ,Endometritis ,business - Abstract
Horse mesenchymal stem cells (MSC) are potential anti-inflammatory tools for post-breeding induced endometritis (PBIE). In this research MSCs isolated from the endometrium or subcutaneous fat of the same donors were infused iu into mares with PBIE for assessment of their anti-inflammatory action and engraftment. PBIE was induced in nine gynecologically healthy mares by iu infusion of 500 million dead sperm in saline. Inflammatory markers were analyzed in uterine lavages and biopsies immediately before (phase I) and 3 h after infusion of sperm (phase II). Measurements: polymorph nuclear cells (PMN), proteins IL-6 and TNFα (ELISA in the lavages) and immunostaining in biopsies, transcripts of IL-1α, 6, 8, 10, TNFα and COX2 (qPCR of pelleted lavages). At 24 h after sperm deposition (phase III), mares were instilled iu with 20 ml of saline containing 2 × 107 adipose MSCs (n = 3, group 1) or endometrial MSCs (n = 3, group 2). Cells were labeled previously with carboxyfluorescein diacetate succinimidyl ester (CFDA SE). A third group (n = 3) received 20 mL of sterile saline alone. After 48 h another biopsy/lavage were done and the same parameters analyzed. For engraftment, additional biopsies were taken at days 10 and 30 of sperm infusion and analyzed by confocal microscopy. Dead sperm in saline markedly increased PMNs counts, IL-6 and TNFα expression in the ELISA (p
- Published
- 2020
24. Complimentary Diagnostic Tools for Endometrosis in Biopsies of Mares with Clinical Subfertility
- Author
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J. Cabezas, Rodriguez-Alvarez Lleretny, D. Rojas, Ghyslaine Ramírez, Fidel Ovidio Castro, and Fernando Saraiva
- Subjects
medicine.medical_specialty ,Pathology ,Endometrosis ,medicine.diagnostic_test ,biology ,Vimentin ,Histology ,General Medicine ,Immunofluorescence ,Staining ,Cytokeratin ,medicine ,biology.protein ,Histopathology ,Immunostaining - Abstract
Background: Endometrosis is a multifactorial disease and one of the main causes of infertility in mares, its etiology and pathogenesis are not completely understood. It is defined as peri glandular and/or stromal endometrial fibrosis with glandular alterations. Due to the few clinical symptoms, besides anamnesis and fertility data, endometrosis requires histological confirmation. The histo-morphology and immune histochemical characteristics of the endometrium vary among individuals according to the disease progression. The aim of this research was to combine histology with new immune and histochemical tools for a more precise detection of fibrotic changes of mares with endometrosis.Materials, Methods & Results: The endometrium of forty thoroughbred mares aged 5-18 years, that did not become pregnant during the last two breeding seasons in a Chilean commercial equine breeding center were biopsied. Samples were subjected to conventional histopathology with hematoxylin-eosin as well as to specific histological staining using specific techniques such as Alcian blue and Masson Fontana, aimed to ascertain what types of mucopolysaccharides were present in those samples. In order to have a deeper picture of the progression of the pathology, immune histochemical methods for the detection of vimentin, cytokeratin, progesterone receptor and lymphocyte marker CD3 were used. Finally in order to detect fibrillar collagen we used second harmonic generation (SHG) technique with detects fibrillar collagen without staining, due to intrinsic hyperpolarization ability of this type of collagen, which can be detected by atomic force microscopy. As a result of our research samples were categorized according to the scale of Keeney and Doig into categories I, IIa, IIb and III (45, 42, 7.5 and 5% respectively). These samples also were characterized by the methods listed earlier and a result we found specific staining in 15 samples coming from higher endometrial damage using Masson-Fontana, while acid staining indicative of acid mucopolysaccharides were detected in 97% of the samples. At immunostaining, we found cytokeratin and vimentin differentially expressed as a function of the degree of the lesions. Cytokeratin was detected in glands from healthy mares, but it was almost absent as the injury was increased. Vimentin in turn, was detected in 26 samples with different degree of intensity. In nine animals, vimentin expression was anomalous, and the marker was detected in fibrotic foci concentrically surrounding glands in biopsies of grade I and IIA only. Although progesterone receptor was detected, there was no correlation with endometrosis. Finally, the lymphocyte T specific CD3 marker was positive in 100% of cases analyzed in which moderate lymphocytic infiltration was found by hematoxylin-eosin; however no staining could be detected in mares with more advanced endometrosis. By combining immunofluorescence with the detection of second harmonic it was possible to detect in the same sample two proteins and collagen deposition at the same time, this had not been reported earlier for endometrosis and mares.Discussion: Our results suggest that the combination of the different methods mentioned is useful for validation and further characterization of the routine hematoxylin-eosin, and can be used as a complementary tool for the diagnosis. Of value, it was possible to combine immunofluorescence with SHG in a single sample in situ.
- Published
- 2020
25. Embryo splitting affects the transcriptome during elongation stage of in vitro–produced bovine blastocysts
- Author
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J.F. Cox, J. Manríquez, L. Rodriguez-Alvarez, Fidel Ovidio Castro, and Alejandra Velásquez
- Subjects
0301 basic medicine ,animal structures ,Microarray ,Embryonic Development ,Fertilization in Vitro ,Biology ,Transcriptome ,Andrology ,03 medical and health sciences ,Food Animals ,Pregnancy ,Gene expression ,medicine ,Animals ,Blastocyst ,Small Animals ,Gene ,Equine ,Gene Expression Regulation, Developmental ,Embryo ,Molecular biology ,In vitro ,Pregnancy rate ,030104 developmental biology ,medicine.anatomical_structure ,embryonic structures ,Cattle ,Female ,Animal Science and Zoology - Abstract
Embryo splitting has been used for the production of identical twins and to increase the pregnancy rate per available embryo. Split blastocysts can develop to term; however, little is known about the impact on gene expression of split embryos, especially at the whole transcriptome level. This work was aimed to evaluate the effect of blastocyst splitting on global gene expression profile at the elongation stage. For that, split and time-matched nonsplit (control group) bovine blastocysts were transferred to a bovine recipient and recovered at Day 17 of development. The number of collected embryos, their size, and global gene expression was compared between both groups. From 16 transferred split embryos, six (37.5%) were collected, whereas nine elongated were recovered from 17 nonsplit (52.9%). Neither the recovery rate nor the average length of the elongated embryos was significantly different between both groups. However more than 50% of embryos from the control group had a length surpassing 100 mm, whereas only 33% of the split embryos reached that size. Global gene expression was performed in individual elongated embryos from both groups using Two-Color Microarray-Based Gene Expression Analysis. From detected genes, 383 (1.31%) were differentially expressed between both groups, among them, 185 (0.63%) were downregulated and 198 (0.67%) genes were upregulated in split embryos. Bioinformatic analysis of differentially expressed genes revealed that embryo splitting affects transcriptomes of resulting elongated embryos, mainly downregulating genes involved in matrix remodelation, control of growth, detoxification, and transport of metabolites. These in turns might have a detrimental impact on the developmental potential of produced embryos.
- Published
- 2017
26. Extracellular vesicles secreted during blastulation show viability of bovine embryos
- Author
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J. Cabezas, Mario Briones, Lleretny Rodriguez-Alvarez, Alejandra Velásquez, E. Mellisho, and Fidel Ovidio Castro
- Subjects
Embryology ,Pregnancy Rate ,medicine.medical_treatment ,Population ,Embryonic Development ,Fertilization in Vitro ,Biology ,Andrology ,Embryo Culture Techniques ,Extracellular Vesicles ,Endocrinology ,Pregnancy ,medicine ,Animals ,Blastocyst ,education ,Cells, Cultured ,education.field_of_study ,In vitro fertilisation ,Embryogenesis ,Obstetrics and Gynecology ,Embryo ,Cell Biology ,Blastula ,Embryo Transfer ,Embryo, Mammalian ,Embryo transfer ,MicroRNAs ,medicine.anatomical_structure ,Reproductive Medicine ,embryonic structures ,RNA, Small Untranslated ,Cattle ,Female ,Embryo quality - Abstract
Extracellular vesicles (EVs) secreted by blastocysts may be clinically relevant, as indicator of embryo viability on in vitro fertilization. We tested if the characteristics of EVs secreted during blastulation are related to embryo viability. Morulae were individually cultured in SOF media depleted of EVs until day 7.5 post IVF. Viable embryos were determined by a system of extended in vitro culture of bovine embryos until day 11 (post-hatching development). Afterward, a retrospective classification of blastocyst and culture media was performed based on blastulation time (early blastulation (EB) or late blastulation (LB)) and post-hatching development at day 11 (viable (V) or non-viable embryo (NV)). A total of 254 blastocysts and their culture media were classified in four groups (V-EB, NV-EB, V-LB, NV-LB). Group V-EB had a larger blastocyst diameter (170.8 μm), higher proportion of good-quality blastocysts (77%) and larger mean size of population of EVs (122.9 nm), although the highest concentration of EVs (5.75 × 109 particles/mL) were in group NV-EB. Furthermore, small RNA sequencing detected two biotypes, miRNA (86–91%) and snoRNA (9–14%), with a total of 182 and 32 respectively. In differential expression analysis of miRNAs between V versus NV blastocysts, there were 12 miRNAs upregulated and 15 miRNAs downregulated. Binary logistic regression was used to construct a non-invasive novel model to select viable embryos, based on a combination of variables of blastocyst morphokinetics and EVs characteristics, the ROC-AUC was 0.853. We concluded that characteristics of EVs secreted during blastulation vary depending on embryo quality.
- Published
- 2019
27. Differentiation and multipotential characteristics of mesenchymal stem cells derived from adipose tissue of an endangered wild cat (Leopardus guigna)
- Author
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Lleretny Rodriguez-Alvarez, J. Cabezas, D. Veraguas, C. Aguilera, D. Rojas, Fidel Ovidio Castro, and Diana Maritza Echeverry
- Subjects
CATS ,General Veterinary ,biology ,Mesenchymal stem cell ,CD44 ,Adipose tissue ,biology.organism_classification ,Molecular biology ,wild cat ,Leopardus guigna ,stem cell ,Cell therapy ,guigna ,biology.protein ,CD90 ,cell therapy ,Induced pluripotent stem cell - Abstract
Adipose tissue derived mesenchymal stem cells (AMSCs) had been isolated and used for cell therapy in domestic cats. For wild cats, the isolation of AMSCs has only been reported in the black-footed cat (Felis nigripes). AMSCs obtained from wild cats may be useful to treat injuries of endangered cat species that remain in captivity or arrive at wildlife rehabilitation centers. Additionally, AMSCs might allow improvement of cloning techniques or assist in derivation of induced pluripotent stem cells. Endangered wild cats such as the guigna (Leopardus guigna), an endemic and endangered species from Chile and Argentina, might benefit greatly from the development of novel treatments or techniques that can be applied for its conservation. The objective of this study was to characterise putative AMSCs from guigna in terms of their main biological attributes, particularly, growth kinetics, differentiation ability and surface marker expression. Results obtained from this characterisation were compared with AMSCs isolated from domestic cats. AMSCs were isolated from peritoneal adipose tissue of female cats and subcutaneous tissue from a female guigna. Migration potential, colony-forming unit assay, mesodermal differentiation and surface marker expression (CD45, CD44, CD90, MHCI and MHCII) were evaluated. Domestic cat and guigna AMSCs displayed similar growth properties in culture. Both AMSC types showed mesodermal differentiation potential, in vitro homing potential and similar surface marker expression. These results indicate that AMSCs from subcutaneous tissue of guigna could have potential use as regenerative treatment for this species and might be considered for use in other biotechnological applications.
- Published
- 2019
28. MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence
- Author
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Fidel Ovidio Castro, C. Aguilera, Ana Carolina Furlanetto Mançanares, Gonzalo Riadi, J. Cabezas, Barbara Melo-Baez, Yat Sen Wong, and Lleretny Rodriguez-Alvarez
- Subjects
0301 basic medicine ,animal structures ,Embryonic Development ,embryo ,Biology ,Article ,Catalysis ,Embryo Culture Techniques ,lcsh:Chemistry ,Inorganic Chemistry ,Extracellular Vesicles ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,microRNA ,Gene expression ,medicine ,Animals ,Secretion ,Blastocyst ,Physical and Theoretical Chemistry ,lcsh:QH301-705.5 ,Molecular Biology ,Spectroscopy ,bovine ,Organic Chemistry ,biomarkers ,Embryo ,General Medicine ,Embryo, Mammalian ,microRNAs ,Computer Science Applications ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,lcsh:Biology (General) ,lcsh:QD1-999 ,IVF ,Culture Media, Conditioned ,030220 oncology & carcinogenesis ,embryonic structures ,Cattle ,Female ,Stem cell ,Signal transduction - Abstract
During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo&ndash, maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells, miRNAs participate in embryo&ndash, maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5&ndash, 5). Individual culture media from in vitro&ndash, produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8&ndash, 16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs, bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.
- Published
- 2020
29. Transient Expression of Functional Glucocerebrosidase for Treatment of Gaucher’s Disease in the Goat Mammary Gland
- Author
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Luciana Relly Bertolini, Jorge R. Toledo, Kaio César Simiano Tavares, Carlos Enrique Méndez Calderón, Felipe Ledur Ongaratto, Cicera R. Lazzarotto, Ana Christina de Oliveira Dias, Fidel Ovidio Castro, Antonio Pinto, I. S. Carneiro, Saul Gaudencio Neto, Marcelo Bertolini, Diógenes Santiago Santos, Jocelei Maria Chies, and L. H. Aguiar
- Subjects
0301 basic medicine ,Mammary gland ,Bioengineering ,Biology ,Glucosylceramides ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Biochemistry ,Adenoviridae ,law.invention ,03 medical and health sciences ,Mammary Glands, Animal ,In vivo ,law ,medicine ,Animals ,Humans ,Enzyme Replacement Therapy ,Molecular Biology ,Gaucher Disease ,Goats ,Enzyme replacement therapy ,medicine.disease ,Molecular biology ,Recombinant Proteins ,In vitro ,Milk ,030104 developmental biology ,medicine.anatomical_structure ,Gaucher's disease ,Recombinant DNA ,Glucosylceramidase ,Female ,Glucocerebrosidase ,Biotechnology - Abstract
Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.
- Published
- 2015
30. In vitro preconditioning of equine adipose mesenchymal stem cells with prostaglandin E2, substance P and their combination changes the cellular protein secretomics and improves their immunomodulatory competence without compromising stemness
- Author
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Yat Sen Wong, Fidel Ovidio Castro, Ana Carolina Furlanetto Mançanares, F Telleria, D. Rojas, Lleretny Rodriguez-Alvarez, J. Manríquez, and J. Cabezas
- Subjects
0303 health sciences ,MHC class II ,General Veterinary ,biology ,040301 veterinary sciences ,Immunology ,Mesenchymal stem cell ,FOXP3 ,04 agricultural and veterinary sciences ,Molecular biology ,0403 veterinary science ,03 medical and health sciences ,MHC class I ,biology.protein ,CD90 ,Tumor necrosis factor alpha ,IL-2 receptor ,Interleukin 8 ,030304 developmental biology - Abstract
Mesenchymal stem cells (MSC) are modern tools in regenerative therapies of humans and animals owed to their immunomodulatory properties, which are activated in a pro-inflammatory environment. Different preconditioning strategies had been devised to enhance the immunomodulatory properties of MSC. In this research, we evaluated the immunological attributes of equine adipose MSC (eAMSC) before and after preconditioning in vitro with prostaglandin E2 (PGE2), substance P (SP), their combination and IFNγ. PGE2/SP was the best combination to keep or enhance the mesodermal lineage differentiation of eAMSC. Alongside with this, preconditioning of eMSC with PGE2 and SP did not affect expression of stemness MSC surface phenotype: CD90+, CD44+, MHC class I+, MHC class II- and CD45-, assessed by cytometry. Both naive and preconditioned eAMSC expressed genes related with immune properties, such as MHC-I, PTGES, IL6, IL1A, TNFα and IL8 assessed by qPCR. Only TNFα was under expressed in treated cells, while the other markers were either overexpressed or not changed. In no cases MHC-II expression was detected. The antiproliferative effect of preconditioned eAMSC exposed to activated peripheral blood mononuclear cells (PBMC) showed that SP treatment significantly inhibited proliferation of LPS stimulated PBMC. When eAMSC were stimulated with Poly I:C, all the treatments significantly inhibited proliferation of stimulated PBMC (p < 0.05). Direct contact (coculture) between the preconditioned eAMSC and PBMC, induced a shift of significantly more (CD4/CD25/FOXP3)+ T-regulatory PBMC than naive eAMSC. In the experiments of this research, we investigated the secreted proteomic profile of naive and preconditioned eAMSC, 42 up-regulated and 40 down-regulated proteins were found in the proteomic assay. Our proteomic data revealed profound changes in the secretory pattern of MSC exposed to different treatments, compared to naive eAMSC as well as among treatments. In overall, compared to naive cells, the protein profile of preconditioned cells resembled the mesenchymal-epithelial transition (MET). Here we showed that the combined use of PGE2 and SP provoked in overall the highest expression of anti-inflammatory markers as well as lead to an increased acquisition of a T-regulatory phenotype in preconditioned eAMSC without affecting their "stemness".
- Published
- 2020
31. 210 Horse allogeneic mesenchymal stem cells perform homing and ameliorate endometrial inflammation after induced endometritis of mares
- Author
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Fernando Saravia, Fidel Ovidio Castro, D. Rojas, L. Rodriguez-Alvarez, Felipe Navarrete, G. Cisterna, and F. Rojas
- Subjects
Mesenchymal stem cell ,Uterus ,Inflammation ,Reproductive technology ,Biology ,Endometrium ,medicine.disease ,Andrology ,Endocrinology ,medicine.anatomical_structure ,Reproductive Medicine ,Cytology ,Genetics ,medicine ,Animal Science and Zoology ,Tumor necrosis factor alpha ,Endometritis ,medicine.symptom ,Molecular Biology ,Developmental Biology ,Biotechnology - Abstract
Post-mating induced endometritis (PMIE) is an acute inflammatory response of the endometrium to spermatozoa, linked to an incapability of some mares to drain out the fluids associated with inflammation. This is of pivotal importance for reproductive success in mares. Mesenchymal stem cells (MSCs) are potential candidates for anti-inflammatory uterine therapies. Here, we aimed to study inflammatory markers in the endometrium of healthy mares and of those with induced endometritis, before and after intrauterine inoculation of MSCs, and to characterise their homing potential invivo in an induced endometritis horse model. Nine mares during their ovulatory season were selected after gynaecologic examination (absence of free liquid in the uterus, no polymorphonuclear leucocytes (PMNs) at cytology, negative bacteriology, and grade I in Kenney's scale on uterine biopsies). Mares were infused in the uterine body with 2mL of 500×106 spermmL−1 previously killed by repeated frozen-thawing cycles. At 3h, uteri were flushed with 250mL of sterile saline and the inflammatory response was monitored in the lavages and biopsies. Parameters measured included cytology, protein expression of inflammatory markers (supernatant) after lavage centrifugation (800×g, 10min), ELISA, and immunostaining for interleukin (IL)-6 and tumor necrosis factor alpha (TNFα). The mares were divided into three groups (3 mares each). Then, 24h after dead sperm challenge, group 1 received intrauterine infusion of 2×107 adipose MSC in 0.9% sterile saline; group 2, received the same amount of endometrial MSCs in the same vehicle; and group 3 received only saline. The volume of infusion in the uterine body was 20mL for all groups. Cells (passage 4) were previously labelled with 10μM Vybrant CFDA SE Cell Tracer Kit (ThermoFisher Scientific). After 48h, the same lavages, biopsies, and measurements as described above were performed. Additional biopsies were taken at Days 10 and 30 after intrauterine infusions. Biopsies were split in two, one for confocal microscopy and the other for quantitative PCR. Endometritis was induced in all mares, as judged by cytology and expression of protein markers of inflammation. After 48h, reduction in IL-6 and TNFα was detected by immunostaining of biopsies and confirmed by ELISA in the lavages, as well as by PCR. Homing was detected in all mares infused with MSC and it persisted at Days 10 and 30 after infusion. No homing was found in the control mares. As a result of these experiments, we conclude that inoculation of MSCs significantly reduced inflammation independently of the origin of the cells (adipose or endometrial). Both types of cells were nested in the endometrium at low quantities, although the number of cells actually detected at fixed time points was not quantified. Overall, we can propose that, given the number of homed cells detected and the marked decrease in inflammatory markers after inoculation of cells, MSCs exert their anti-inflammatory function preferentially by a paracrine mechanism and not necessarily by nesting and proliferation, although both events occur. Funding for this study was provided by Fondecyt 1150757.
- Published
- 2020
32. 80 Evaluation of extracellular vesicles from culture medium of human embryos as a possible method of pre-implantation genetic diagnosis
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L. Rodriguez-Alvarez, D. Veraguas, C. Henriquez, Alejandra Velásquez, Fidel Ovidio Castro, and C. Aguilera
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0303 health sciences ,030219 obstetrics & reproductive medicine ,Embryo ,Embryo culture ,Reproductive technology ,Biology ,Cryopreservation ,Andrology ,Transgenesis ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Reproductive Medicine ,embryonic structures ,Genetics ,Animal Science and Zoology ,Molecular Biology ,Embryo quality ,Fertilisation ,030304 developmental biology ,Developmental Biology ,Biotechnology ,Comparative genomic hybridization - Abstract
Noninvasive methods are the clue to increase the efficiency of invitro-derived embryo selection without decreasing their competence. Embryos selection based on their morphology is the most used method but only 40% of selected embryos are able to implant and develop correctly. In humans, pre-implantation genetic diagnosis increases the efficiency of selection by excluding embryos with chromosomal abnormalities. However, pre-implantation genetic diagnosis needs embryonic cells, which might compromise embryo viability. On the other hand, embryos release extracellular vesicles (EVs: microvesicles and exosomes) to the culture medium that contain biological cargo-like proteins and mRNA lipids, and might contain genomic DNA (gDNA). For this study we evaluated the culture medium from embryos generated by intracytoplasmic sperm injection in a certified fertility clinic. Embryos were cultured in Global Total serum-free medium. The embryos were assessed at Day 3 of development and classified in three categories: top, fair, and poor quality. Corresponding medium was collected for isolation of EVs. The nature of EVs was confirmed by their size and concentration using nanoparticle tracking analysis (NTA), presence of surface markers (CD9, CD63, CD81, and CD40L), and morphology using transmission electron microscopy. A correlation analysis between NTA results (EV size and concentration) and embryo quality was performed. To evaluate chromosomal abnormalities of gDNA present in isolated EVs from embryo culture medium, microarray-based comparative genomic hybridization (aCGH) assay was performed. In a second experiment, aCGH analysis was performed and compared between arrested embryos and EVs isolated from corresponding culture medium. Isolated nanoparticles from embryo culture medium were positive to all markers CD9 (30.9%), CD63 (27.2%), CD81 (31.7%), CD40L (8.7%) and had a morphology accordingly to exosomes. The analysis of NTA data indicated that top-quality embryos had EVs with higher diameter (mean: 112.17nm, mode: 91.74nm) than embryos classified as fair (mean: 108.02nm, mode: 89.67nm) and poor quality (mean: 102.78nm, mode: 88.17 nm; P
- Published
- 2020
33. 208 Effect of growth factors and reprogramming molecules on induction to multipotency of dermal fibroblasts from colocolo (Leopardus colocolo)
- Author
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D. Rojas, Fidel Ovidio Castro, L. Rodriguez-Alvarez, Diana Maritza Echeverry, and C. Aguilera
- Subjects
Homeobox protein NANOG ,Growth factor ,medicine.medical_treatment ,Cellular differentiation ,Mesenchymal stem cell ,Reproductive technology ,Biology ,biology.organism_classification ,Cell biology ,Endocrinology ,Reproductive Medicine ,Genetics ,medicine ,Leopardus colocolo ,Animal Science and Zoology ,Molecular Biology ,Reprogramming ,Developmental Biology ,Biotechnology ,Transforming growth factor - Abstract
Reprogramming of terminally differentiated cells to higher plasticity levels can be achieved with small molecules. This can be of value for somatic cell nucleus transfer, deriving multi and pluripotent cells and conservation purposes. Recently, induced mesenchymal stem cells were derived from differentiated human and mouse cells by using small molecules and growth factors. The pampas cat or colocolo (Leopardus colocolo) is a South American felid categorrized as near threatened by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Major historical threats to the pampas cat include illegal hunting, habitat loss or transformation, and conflict retaliation for poultry predation. Here, we tested 5-azacytidine (an epigenetic modifier) and A8301 (a potent inhibitor of transforming growth factor-β type I receptor superfamily), linked to platelet-rich plasma (PRP) and platelet-derived growth factor (PDGF-B) to induce changes in the expression of pluripotency genes and differentiation capacity of colocolo fibroblasts towards other mesodermal lineages. For this, dermal fibroblasts were treated with (I) 5-azacytidine + PRP + A8301 + VitC, or (II) 5- azacytidine + VitC + A8301 + PDFG for 12 days. On Days 0, 5, and 12 of reprogramming, expression of OCT4, NANOG, E-cadherin and SNAIL was evaluated by reverse transcription-PCR, and tri-lineage differentiation was induced. For treatment I, no statistical difference was found in the expression of OCT4 and NANOG. Chondrogenic and osteogenic differentiation was observed. In treatment II, significant expression of OCT4 and NANOG (P
- Published
- 2020
34. 22 The zona pellucida is required for normal development of invitro-produced cat embryos
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Darling Saez-Ruiz, D. Veraguas, Fidel Ovidio Castro, C. Aguilera, L. Rodriguez-Alvarez, S. Saez, and Maria Francisca Cordero
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Zygote ,urogenital system ,Embryo culture ,Reproductive technology ,Biology ,Andrology ,Endocrinology ,medicine.anatomical_structure ,Human fertilization ,Reproductive Medicine ,embryonic structures ,Genetics ,medicine ,Animal Science and Zoology ,Blastocyst ,Zona pellucida ,Molecular Biology ,reproductive and urinary physiology ,Fertilisation ,Embryo quality ,Developmental Biology ,Biotechnology - Abstract
Limited information have been reported regarding to the role of the zona pellucida during embryo development in the domestic cat. In the present research we compared invitro and invivo development of cat embryos generated with versus without the zona pellucida. Embryos were produced by IVF and cultured up to blastocyst stage accordingly to experimental group: 1) with intact zona pellucida (ZI) and 2) without zona pellucida (ZF). Ovaries of domestic cats were collected by ovariohysterectomy and cumulus-oocyte complexes (COCs) were recovered by slicing. For IVM, COCs were cultured in supplemented medium-199 for 26-28h in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C. For IVF, 1.5-2.5×106 epididymal spermatozoa/mL were incubated with 20-30 COCs in supplemented Tyrode's albumin lactate pyruvate medium for 18h in a 5% CO2 atmosphere at 38.5°C. The zona pellucida of the presumed zygotes was removed by 2-4min of incubation in 2mgmL−1 pronase. In ZI and ZF groups, embryo culture was done in 4-well dishes; in the ZF group the well of the well system was used. The IVC was done in supplemented synthetic oviductal fluid medium, in a 5% CO2, 5% O2, and 90% N2 atmosphere at 38.5°C for 8 days. The frequencies of cleavage and development to the morula and blastocyst stages were determined. The relative expression of the pluripotency markers OCT4, SOX2, and NANOG was evaluated by RT-qPCR in the blastocysts using the standard curve method. The SDHA gene was used as internal control. Additionally, the invivo development was evaluated. For this, cat recipients were synchronized with 200IU equine chorionic gonadotrophin followed by 100IU human chorionic gonadotrophin 4 days later, and embryo transfers (ET) were made by mid-ventral laparotomy. The Wilcoxon nonparametric test was used to evaluate the developmental competence and the gene expression analysis. Nine and six replicates were performed in the ZI and ZF groups, respectively. No differences were observed between the ZI and ZF groups in the cleavage rate: 155/239 (64.9%) and 116/177 (65.5%), morulae rate: 115/155 (74.2%) and 68/116 (58.6%), and blastocysts rate: 51/155 (32.9%) and 36/116 (31.0), respectively (P>0.05). No differences were observed in the expression of OCT4 (P>0.05). However, the expression of SOX2 and NANOG was lower in blastocysts from the ZF group than in those from the ZI group (P
- Published
- 2020
35. 52 Blocking of embryonic development by nanoparticles derived from endometrial and oviductal cells isolated with an Amicon filter system
- Author
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J. Cabezas, D. Rojas, Fidel Ovidio Castro, Barbara Melo-Baez, L. Rodriguez-Alvarez, and M. Gutierrez
- Subjects
Embryo ,Embryo culture ,Reproductive technology ,Biology ,Endometrium ,Cryopreservation ,Andrology ,Endocrinology ,medicine.anatomical_structure ,Reproductive Medicine ,Genetics ,medicine ,Oviduct ,Animal Science and Zoology ,Blastocyst ,Molecular Biology ,Fertilisation ,Developmental Biology ,Biotechnology - Abstract
The success of development of in vitro embryo production needs to mimic culture conditions in the maternal environment. Recently, it has been seen that extracellular vesicles (EVs) secreted by oviducal or endometrial cells may improve development and quality of embryos produced in vitro. Extracellular vesicles are a mechanism of cellular communication; they carry molecules that are delivered into the target cells changing gene expression and function. Due to the size range and characteristics of EVs, they require specific methods for purification and characterisation. However, the possible contamination with other nanoparticles and their effect on embryo development have not been considered. Based on that, the goal of this work was to evaluate the effect on in vitro bovine embryo development, of the addition to culture medium EVs secreted by oviducal and endometrial cells and isolated by centrifugation and concentrates with Amicon filters. For this purpose, cells were isolated from bovine oviduct and endometrium collected in local abattoir and primary cultures of epithelial and stromal cells were derived. The primary cultures from both sources were exposed or not to progesterone (P4; 15 ng mL−1) for 4 days and then cultured for 24 h in EV depleted media. The supernatant was harvested and EVs were isolated by serial centrifugations and subsequently concentrated by a 100 kDa Amicon filter system. The isolated EVs were characterised by transmission electron microscopy, nanoparticle tracking analysis, and flow cytometry. Oocytes were obtained from ovaries collected in the abattoir. The cumulus-oocyte complexes were matured in vitro for 22 h and subsequently fertilised for 18 h. Presumptive zygotes were in vitro cultured in synthetic oviducal fluid with EVs (1000 ng mL−1 of total proteins) or not according to experimental group (1: EVs− (control); 2: EVs−OP4+; 3: EVs−OP4−; 4: EVs−EP4+ and 4: EVs−EP4−). Embryos were cultured for 7 days in 5% CO2, 5% O2, and 90% N2 (25 embryos/well in 4-well plates). At Day 7, embryo development was evaluated considering the blastocyst yield. Transmission electron microscopy showed typical structures and morphology of EVs and they were positive for CD9, CD63, and CD81 markers, and negative for CD40. According to nanoparticle tracking analysis, the mean size of EVs was 160 ± 62 nm and concentration of 3.29 × 1011 particles mL−1 for oviducal and endometrial cells, respectively. A significant reduction of blastocyst rate was observed when embryos were cultured with cell-derived EVs; control: 152/44 (28.9%) vs. treatments with EVs; OP4+: 74/3 (4.1%), OP4−:76/2 (2.6%), EP4+: 74/6 (8.1%), and EP4−: 73/2 (2.7%) (P ≤ 0.01). Our results indicate that the use of nanoparticles, including EVs, isolated from cells of oviduct or endometrium, has a blocking effect on embryonic development and compromises the performance of blastocysts on Day 7 when used at concentrations of 1000 ng mL−1 total protein, independent of the use or not of P4 and the source. These data provide insights regarding the use and protocols of acquiring exosomes for embryo supplementation. This research was supported by FONDECYT, Chile-1170310.
- Published
- 2020
36. 79 MicroRNAs of extracellular vesicles secreted by embryos as an early biomarker of competence
- Author
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C. Aguilera, J. Cabezas, L. Rodriguez-Alvarez, Fidel Ovidio Castro, Barbara Melo-Baez, and Yat Sen Wong
- Subjects
Embryo culture ,Embryo ,Reproductive technology ,Biology ,Embryonic stem cell ,Microvesicles ,Cell biology ,Endocrinology ,medicine.anatomical_structure ,Reproductive Medicine ,Gene expression ,Genetics ,medicine ,Animal Science and Zoology ,Blastocyst ,KEGG ,Molecular Biology ,Developmental Biology ,Biotechnology - Abstract
Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted by different cell types and participate in cellular communication by carrying molecules as microRNAs (miRNAs) that can interfere with gene expression of target cells. Extracellular vesicles have become relevant as a mechanism of embryo-maternal communication. The aim of this study was to evaluate miRNA content in EVs secreted after embryonic genome activation, by bovine embryos with different developmental potential. Bovine embryos were produced invitro and cultured in group until Day 3.5 in synthetic oviductal fluid (SOF) medium. Only 8-16-cell embryos were cultured individually in EVs-depleted SOF until Day 5. The SOF was EV depleted by ultrafiltration. Culture media (CM) were collected at Day 5 and embryos continued in culture until Day 7 with fresh SOF. Collected media were conserved individually and identified with the corresponding embryo. Then, CM were classified according to capacity of its embryo to reach blastocyst stage at Day 7: G1-CM (blocked embryos in 8-16 cell) and G2-CM (embryos that reach blastocyst stage). The EV isolation was carried out using the protocol described by Mellisho et al. (2017). Recovered EVs were evaluated by nanoparticle tracking analysis (NTA), Transmission electron microscopy and the presence of surface markers (CD9, CD63, CD81, and CD40L). After NTA, individual CM were pooled to organise 3 replicates of 10CM each, for G1 and G2. The whole miRNA isolation, library preparation, and sequencing was performed by Norgen Biotek facilities (Canada). The quality of libraries was analysed using the FastQC program platform followed by Trimmomatic to remove remnant adapters. For the miRNA library it accepted reads with value above 30 Phreads and 22 to 30bp length. The reads were mapped against the reference genome ARS-UCD1.2 using Bowtie2 software and miRDeep2 mapper, and the gene counts were calculated using HTSeq. Differential expression analysis was performed in EdgeR package. To expand this information, principal component analysis, Heatmap, and Volcano plot were plotted and pathway enrichment analysis was conducted. The NTA, transmission electron microscopy, and flow cytometry confirmed the presence of exosomes and microvesicles in isolated EVs. According to NTA, the mean size of EVs was 102.1-176.2nm and concentration of 8.4×107-8.6×108 particlesmL−1 in G1 and G2, respectively. We identified 96 miRNAs significantly expressed across the samples. Only eight miRNAs in EVs were differentially expressed between groups (G2 vs. G1). The bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were up-regulated (Log2 fold-change>1), whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were down-regulated (Log2 fold-change
- Published
- 2020
37. eCG stimulation in domestic cats increases the expression of gonadotrophin-induced genes improving oocyte competence during the non-breeding season
- Author
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P. F. Gallegos, Lleretny Rodriguez-Alvarez, Darling Saez-Ruiz, Fidel Ovidio Castro, Sandra R. Cuevas, and D. Veraguas
- Subjects
endocrine system ,Gonadotropins, Equine ,In Vitro Oocyte Maturation Techniques ,Parthenogenesis ,Embryonic Development ,Stimulation ,Biology ,Breeding ,Andrology ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,In vivo ,medicine ,Animals ,Blastocyst ,030219 obstetrics & reproductive medicine ,CATS ,Embryogenesis ,0402 animal and dairy science ,04 agricultural and veterinary sciences ,Oocyte ,040201 dairy & animal science ,In vitro maturation ,medicine.anatomical_structure ,Gene Expression Regulation ,Cats ,Oocytes ,Animal Science and Zoology ,Female ,Biotechnology - Abstract
The objective of this study was to evaluate the eCG stimulation on domestic cat oocyte competence during the non-breeding season. Four experimental groups were made: (a) untreated cycling cats (Breeding season group; BS), (b) untreated anestrous cats (Non-breeding group; NB), (c) anestrous cats treated with 200 IU of eCG (eCG group) and (d) anestrous cats treated with 200 IU of eCG and 100 IU of hCG four days later (hCG group). In the BS, NB and eCG groups, grade I and II immature cumulus-oocyte complexes (COCs) were subjected to in vitro maturation or used for the gene expression analysis of FSHR, LHCGR, EGFR, EGR1, ESR2, PTGS2, GDF9, BMP15 and GATM. The in vitro matured oocytes from the BS, NB and eCG groups and the in vivo matured oocytes from the hCG group were subjected to parthenogenetic activation. The grade I and II COCs from the eCG group had an increased expression of FSHR, LHCGR, EGFR, EGR1 and ESR2 and a higher maturation rate than the BS and NB groups (p 0.05). After parthenogenetic activation, the blastocyst rate from the hCG, eCG and BS groups was higher than the NB group (p 0.05). However, no significant improvement was observed in the blastocyst rate in the hCG group compared to the eCG group (p 0.05). In conclusion, the eCG treatment increases the expression of specific genes improving the oocyte competence during the cat non-breeding season, which is reflected in an enhanced in vitro maturation and in vitro embryo development after parthenogenetic activation.
- Published
- 2018
38. Applied Biotechnologies in the Conservation of Wild Felids: In Vitro Embryo Production and Cellular Regenerative Therapies
- Author
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LleretnyRodriguez-Alvarez, D. Veraguas, Diana Maritza Echeverry, and Fidel Ovidio Castro
- Subjects
0301 basic medicine ,0403 veterinary science ,03 medical and health sciences ,030104 developmental biology ,040301 veterinary sciences ,Embryo ,04 agricultural and veterinary sciences ,Biology ,GeneralLiterature_REFERENCE(e.g.,dictionaries,encyclopedias,glossaries) ,In vitro ,Cell biology - Published
- 2017
39. Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation
- Author
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Lleretny Rodriguez-Alvarez, J.F. Cox, Fidel Ovidio Castro, Mario Briones, D. Veraguas, Alejandra Velásquez, and E Lara
- Subjects
0301 basic medicine ,animal structures ,medicine.medical_treatment ,Fertilization in Vitro ,Biology ,Embryo Culture Techniques ,Andrology ,03 medical and health sciences ,0302 clinical medicine ,Pregnancy ,Gene expression ,medicine ,Animals ,Inner cell mass ,Blastocyst ,030219 obstetrics & reproductive medicine ,In vitro fertilisation ,Embryogenesis ,Gene Expression Regulation, Developmental ,Embryo culture ,Embryo ,Cell Biology ,Anatomy ,Embryo Transfer ,Embryo transfer ,030104 developmental biology ,medicine.anatomical_structure ,embryonic structures ,Cattle ,Female ,Developmental Biology - Abstract
SummaryEmbryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7–9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.
- Published
- 2014
40. Equine mesenchymal stem cells derived from endometrial or adipose tissue share significant biological properties, but have distinctive pattern of surface markers and migration
- Author
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G. Rivera, J. Cabezas, Fernando Saravia, Fidel Ovidio Castro, L. Rodriguez-Alvarez, Felipe Navarrete, D. Rojas, and R. Ortiz
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Stromal cell ,Population ,Adipose tissue ,Biology ,Endometrium ,03 medical and health sciences ,Food Animals ,Cell Movement ,medicine ,Animals ,CD90 ,Horses ,Small Animals ,education ,Cells, Cultured ,education.field_of_study ,Equine ,Mesenchymal stem cell ,CD44 ,Membrane Proteins ,Mesenchymal Stem Cells ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Adipose Tissue ,Gene Expression Regulation ,biology.protein ,Animal Science and Zoology ,Female ,Stem cell ,Biomarkers - Abstract
Adult stromal mesenchymal stem cells (MSCs) have been postulated as responsible for cell renewal in highly and continuously regenerative tissues such as the endometrium. MSCs have been identified in the endometrium of many species including humans, rodents, pets and some farm animals, but not in horses. The objective of this work was to isolate such cells from the endometrium of mares and to compare their main biological attributes with horse adipose-derived MSCs. Here we successfully isolated and characterized endometrial MSCs (eMSCs) from mares. Said cells showed fibroblast-like morphology, grew on plastic, had doubling population times of 46.4 ± 3.38 h, underwent tri-lineage (osteo, chondro and adipogenic) differentiation after appropriate inductions, migrated toward the attraction of fetal calf serum and displayed a pattern of surface markers commonly accepted for horse MSCs. All these are properties of MSCs. Some of these attributes were shared with equine adipose-derived MSCs, but the migration pattern of eMSC at 12 and 24 h after stimulation was reduced in comparison with adipose MSCs. Also, expression of CD44, CD90 and MHCI surface markers were dramatically down-regulated in eMSCs. In conclusion, equine-derived endometrial MSC share biological attributes with adipose MSC of this species, but displayed a different surface marker phenotype and an impaired migration ability. Conceivably, this phenotype is distinctive for MSC of this origin.
- Published
- 2017
41. Endometritis and
- Author
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Evelyn, Lara, Alejandra, Velásquez, Joel, Cabezas, Nathaly, Rivera, Paulina, Pacha, Lleretny, Rodríguez-Alvarez, Fernando, Saravia, and Fidel Ovidio, Castro
- Subjects
Research Article - Abstract
Mesenchymal stem cells (MSCs) were isolated and characterized from postpartum bovine endometrium of animals with subclinical (n = 5) and clinical endometritis (n = 3) and healthy puerperal females (n = 5). Cells isolated displayed mean morphological features of MSCs and underwent osteogenic, chondrogenic, and adipogenic differentiation after induction (healthy and subclinical). Cells from cows with clinical endometritis did not undergo adipogenic differentiation. All cells expressed mRNAs for selected MSC markers. Endometrial MSCs were challenged in vitro with PGE2 at concentrations of 0, 1, 3, and 10 μM, and their global transcriptomic profile was studied. Overall, 1127 genes were differentially expressed between unchallenged cells and cells treated with PGE2 at all concentrations (763 up- and 364 downregulated, fold change > 2, and P < 0.05). The pathways affected the most by the PGE2 challenge were immune response, angiogenesis, and cell proliferation. In conclusion, we demonstrated that healthy puerperal bovine endometrium contains MSCs and that endometritis modifies and limits some functional characteristics of these cells, such as their ability to proceed to adipogenic differentiation. Also, PGE2, an inflammatory mediator of endometritis, modifies the transcriptomic profile of endometrial MSCs. A similar situation may occur during inflammation associated with endometritis, therefore affecting the main properties of endometrial MSCs.
- Published
- 2017
42. Endometritis and In Vitro PGE2 Challenge Modify Properties of Cattle Endometrial Mesenchymal Stem Cells and Their Transcriptomic Profile
- Author
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Fernando Saravia, Lleretny Rodriguez-Alvarez, Paulina Pacha, Fidel Ovidio Castro, Nathaly Rivera, Alejandra Velásquez, E Lara, and J. Cabezas
- Subjects
0301 basic medicine ,medicine.medical_specialty ,lcsh:Internal medicine ,Article Subject ,Angiogenesis ,Inflammation ,Biology ,Endometrium ,Andrology ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Internal medicine ,medicine ,lcsh:RC31-1245 ,Molecular Biology ,030219 obstetrics & reproductive medicine ,Mesenchymal stem cell ,Cell Biology ,medicine.disease ,030104 developmental biology ,medicine.anatomical_structure ,Endocrinology ,Adipogenesis ,Endometritis ,medicine.symptom - Abstract
Mesenchymal stem cells (MSCs) were isolated and characterized from postpartum bovine endometrium of animals with subclinical (n=5) and clinical endometritis (n=3) and healthy puerperal females (n=5). Cells isolated displayed mean morphological features of MSCs and underwent osteogenic, chondrogenic, and adipogenic differentiation after induction (healthy and subclinical). Cells from cows with clinical endometritis did not undergo adipogenic differentiation. All cells expressed mRNAs for selected MSC markers. Endometrial MSCs were challenged in vitro with PGE2at concentrations of 0, 1, 3, and 10 μM, and their global transcriptomic profile was studied. Overall, 1127 genes were differentially expressed between unchallenged cells and cells treated with PGE2at all concentrations (763 up- and 364 downregulated, fold change > 2, andP<0.05). The pathways affected the most by the PGE2challenge were immune response, angiogenesis, and cell proliferation. In conclusion, we demonstrated that healthy puerperal bovine endometrium contains MSCs and that endometritis modifies and limits some functional characteristics of these cells, such as their ability to proceed to adipogenic differentiation. Also, PGE2, an inflammatory mediator of endometritis, modifies the transcriptomic profile of endometrial MSCs. A similar situation may occur during inflammation associated with endometritis, therefore affecting the main properties of endometrial MSCs.
- Published
- 2017
43. Characterization of mesenchymal stem cells in bovine endometrium during follicular phase of oestrous cycle
- Author
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E Lara, Nathaly Rivera, Fidel Ovidio Castro, L. Rodriguez-Alvarez, and D. Rojas
- Subjects
0301 basic medicine ,Homeobox protein NANOG ,Gene Expression ,Estrous Cycle ,Biology ,Stem cell marker ,Endometrium ,Andrology ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,SOX2 ,Follicular phase ,medicine ,Animals ,reproductive and urinary physiology ,Cells, Cultured ,030219 obstetrics & reproductive medicine ,urogenital system ,Mesenchymal stem cell ,Cell Differentiation ,Mesenchymal Stem Cells ,Embryonic stem cell ,030104 developmental biology ,medicine.anatomical_structure ,Follicular Phase ,Animal Science and Zoology ,Cattle ,Female ,Stem cell ,Biotechnology - Abstract
Contents Stem cells have been postulated as responsible for cell regeneration in highly and continuously regenerative tissues such as the endometrium. Few studies in cattle have identified and specified the presence of stem cells in the endometrium during the oestrous cycle. The aim of this study was to investigate the presence of mesenchymal stem cells (MSCs) in the bovine endometrium during the follicular phase (FP) of the oestrous cycle. Uterine tissue was collected in the time-frame comprising day 18 of the cycle and ovulation (day 0). We isolated, cultured and expanded four primary cell lines from endometrium and identified byRT-qPCR the expression of OCT4, SOX2 but not NANOG (undifferentiated/embryonic markers), CD44 (MSCs marker) and c-KIT (stem cell marker) genes; and the encoded Oct4, Sox2 and Cd44 proteins by Western blot or immunostaining of paraffin-embedded tissue in endometrium. We demonstrated that cells isolated from bovine endometrium displayed essentially the same gene expression pattern; however, at the protein level, Oct4 and Cd44 were not detected. Besides, they showed typical functional characteristics of MSCs such as fibroblast-like morphology, plastic adherence, high proliferative capacity, clone formation in vitro and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. We obtained for the first time an extensive characterization of undifferentiated cells populations contained in the bovine endometrium during the FP of the oestrous cycle.
- Published
- 2016
44. Cell cycle synchronization and analysis of apoptosis-related gene in skin fibroblasts from domestic cat (Felis silvestris catus) and kodkod (Leopardus guigna)
- Author
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D. Veraguas, L. Rodriguez-Alvarez, P. F. Gallegos, and Fidel Ovidio Castro
- Subjects
0301 basic medicine ,Felidae ,Nuclear Transfer Techniques ,Cell Survival ,Cloning, Organism ,Population ,Apoptosis ,Resting Phase, Cell Cycle ,Culture Media, Serum-Free ,Leopardus guigna ,Flow cytometry ,Andrology ,03 medical and health sciences ,Endocrinology ,medicine ,Animals ,Cell synchronization ,education ,education.field_of_study ,biology ,medicine.diagnostic_test ,Differential staining ,Contact Inhibition ,Felis ,Gene Expression Profiling ,Cell Cycle ,Contact inhibition ,Cell cycle ,Fibroblasts ,biology.organism_classification ,030104 developmental biology ,Animal Science and Zoology ,Biotechnology - Abstract
The kodkod population is in constant decrease and the somatic cell nuclear transfer (SCNT) might help to preserve the genetic pool of this species. The cell cycle synchronization of donor cells plays a crucial role in SCNT. The objective of this research was to evaluate two different methods for quiescence induction, serum starvation (SS) and contact inhibition (CI), both for 1, 3 and 5 days, on skin fibroblast from domestic cat and kodkod. Flow cytometry analysis revealed that in domestic cat, SS and CI, both at 3 and 5 days, increased the percentage of fibroblasts in G0/G1 compared to growing cells (GC) (p
- Published
- 2016
45. 185 Overexpression or CRISPr/Cas9-mediated inhibition of prostaglandin E2 receptors EP2 and EP4 in equine adipose mesenchymal stem cells: implications for cell migration and cellular therapies
- Author
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Ana Carolina Furlanetto Mançanares, J. Cabezas, Lleretny Rodríguez, F. Telleria, Fidel Ovidio Castro, and J. Manríquez
- Subjects
Prostaglandin E2 receptor ,Mesenchymal stem cell ,Adipose tissue ,Cell migration ,Transfection ,Biology ,Cell biology ,Paracrine signalling ,Endocrinology ,Immune system ,Reproductive Medicine ,Genetics ,lipids (amino acids, peptides, and proteins) ,Animal Science and Zoology ,Receptor ,Molecular Biology ,Developmental Biology ,Biotechnology - Abstract
Prostaglandin E2 (PGE2) acts through 4 cellular receptors: EP1, EP2, EP3, and EP4; only EP2 and EP4 are relevant for immunomodulation and migration in immune cells. Besides those, several cells express these receptors on their surface, including mesenchymal stem cells. Pharmacological inhibition of the EP2 receptor prevents migration of immune system cells to inflamed sites, where the concentration of PGE2 is high. Based on this, we hypothesised that overexpression of EP2 or EP4 receptors in equine mesenchymal stem cells (eMSC) will improve their migration to inflammatory sites and subsequent homing capability. Conversely, their suppression will lead to low or no migration, favouring the paracrine properties of MSC in the processes of tissue regeneration and reduction of inflammation. To test this, we manipulated the PGE2-EP2-EP4 axis and evaluated the effect of such modifications on transgenic cells in vitro. Equine MSC from adipose tissue were obtained from 5 animals. The coding sequences of both receptors were synthesised (GenScript, Hong Kong) based on the published horse genome (National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/) and cloned into pcDNA3.1 overexpression vectors (Addgene). The resulting constructs were lipofected into naïve adipose eMSC. For knockouts, we PCR-amplified and sequenced horse EP2 and EP4 receptors, and gRNAs were created based on the obtained sequences and ligated into LentiCRISPRV2 plasmid (Addgene, Cambridge, MA, USA). The lentiviral vector plus helping packaging plasmids were co-transfected into HEK293FT cells and the produced viral particles were harvested and transduced into adipose eMSC. After 48h of transfection (for overexpression) or transduction (for knockout, KO), cells were probed for the presence/absence of EP2 and EP4 receptors by immunohistochemistry and/or quantitative (q)PCR. Mitomycin-C-treated cells of both phenotypes and naïve, were subjected to migration in scratch assay, towards 3mM PGE2. Fetal calf serum (10 or 0%) was used as positive or negative control, respectively, in migration experiments. Receptors EP2 and EP4 were clearly overexpressed after transfection as determined by immunocytochemistry or qPCR assays (phenotype MSC-EP2+/EP4+), whereas in the cells that underwent KO, little or no expression of EP2 and EP4 was detected (phenotype MSC-EP2ko/EP4ko) compared with unmanipulated cells (naïve MSC-Ctr). In the migration experiments towards 3mM of PGE2, MSC-EP2+/EP4+ cells at 24h filled the scratch faster (P
- Published
- 2019
46. 17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer
- Author
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C. Aguilera, Darling Saez-Ruiz, L. Rodriguez-Alvarez, Diana Maritza Echeverry, D. Veraguas, and Fidel Ovidio Castro
- Subjects
Leopardus ,Embryo culture ,Embryo ,Reproductive technology ,Biology ,biology.organism_classification ,Andrology ,Transgenesis ,Endocrinology ,medicine.anatomical_structure ,Reproductive Medicine ,Genetics ,medicine ,Somatic cell nuclear transfer ,Animal Science and Zoology ,Blastocyst ,Molecular Biology ,Fertilisation ,Developmental Biology ,Biotechnology - Abstract
The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm−1 for 40 µs and then incubated for 5h in 10μg mL−1 of cycloheximide and 5μg mL−1 of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O2, 5% CO2, and 90% N2 atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P>0.05). However, the diameter of the blastocysts from the K2x group (172.8µm) tended to be lower than that of the blastocysts from the Ca2x group (P=0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P=0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P>0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.
- Published
- 2019
47. 128 Next-generation RNA sequencing of horse adipose and endometrial mesenchymal stem cells from the same donors unveils striking differences in their transcriptomic pattern
- Author
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J. Cabezas, Felipe Navarrete, Fernando Saravia, Y. Wang, L. Rodriguez-Alvarez, A. Navarro, E. Mellisho, and Fidel Ovidio Castro
- Subjects
Cell type ,Biological adhesion ,Biology ,Phenotype ,Cell biology ,Transcriptome ,Endocrinology ,Reproductive Medicine ,Genetics ,Animal Science and Zoology ,Stem cell ,Biological regulation ,Molecular Biology ,Gene ,Illumina dye sequencing ,Developmental Biology ,Biotechnology - Abstract
Earlier we successfully isolated and characterised endometrial (eMSC) and adipose (aMSC) mesenchymal stem cells from the same donors. Mesenchymal stem cells share biological traits but display different surface marker phenotype and migration ability. Here we extended our research to their mRNA signature using next-generation sequencing. The RNA from cells (3 biological replicates from each cell type and 3 technical replicates) at 90% confluence was extracted using a total RNA extraction kit and sent for mRNA-Seq (Norgen, Ontario, Canada; Illumina Sequencing Platform NextSEqn 500). Raw 76-bp single-end reads were aligned against the EquCab3 genome using RNA-STAR aligner. Counts were filtrated at a minimum of 5. Pairwise comparisons between the cell types were the input for gene ontology enrichment analysis. Only genes differentially expressed (DE) with 5 folds change (FC; P50×: 8 and 13; >20×, 10×, 5×, 2×
- Published
- 2019
48. 149 Bovine embryo selection can be improved by the characteristics of secreted extracellular vesicles
- Author
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Mario Briones, Fidel Ovidio Castro, L. Rodriguez-Alvarez, and E. Mellisho
- Subjects
Receiver operating characteristic ,Embryo culture ,Embryo ,Reproductive technology ,Biology ,Blastula ,Confidence interval ,Andrology ,Endocrinology ,medicine.anatomical_structure ,Reproductive Medicine ,embryonic structures ,Linear regression ,Genetics ,medicine ,Animal Science and Zoology ,Blastocyst ,Molecular Biology ,Developmental Biology ,Biotechnology - Abstract
Extracellular vesicles (EV) secreted by blastocysts might be relevant to predict competence of embryos produced in vitro. The aim of this study was to develop a model to select competent embryos that combines blastocyst morphokinetics data and morphological parameters of EV secreted during blastulation (Days 5-7.5). Embryos were cultured in groups up to Day 5; morulae were selected and individually cultured in SOFaa depleted of EV until Day 7.5 after IVF. Embryo competence was determined by in vitro post-hatching development up to Day 11. A retrospective classification of blastocyst and culture media was performed based on blastulation time [early (EB) or late (LB)] and competence at Day 11 [competent (C) or non-competent (NC)]. The EV were isolated from culture media of individual embryos, their properties determined by nanoparticle tracking analysis. The model was based on a binary logistic regression to describe the dichotomous-dependent variable of the blastocyst (C=1 and NC=0). A set of independent variables of blastocyst morphokinetics (blastulation time, blastocyst stage, blastocyst quality and blastocyst diameter at Day 7.5) and EV morphological parameters [mean size (ME), mode size (MO) and particle concentration (CO)] were analysed with multiple regression. The analysis generated the coefficients and their standard errors and significance level of an equation to calculate a probability, where values between 0.5 and 1 predict competent embryos. To verify the predictive power of the algorithm, the following indicators were used: the receiver operating characteristic with the determination of area under the curve, percentage correct predictions, and Omnibus tests. Statistical significance was determined at the P
- Published
- 2019
49. Effect of zona pellucida removal on early development of in vitro produced bovine embryos
- Author
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Alejandra Velásquez, Fidel Ovidio Castro, L. Rodriguez-Alvarez, and Manríquez
- Subjects
endocrine system ,General Veterinary ,urogenital system ,Embryogenesis ,Embryo culture ,Embryo ,Blastomere ,Parthenogenesis ,Biology ,Cleavage (embryo) ,Andrology ,medicine.anatomical_structure ,embryonic structures ,Immunology ,medicine ,Blastocyst ,Zona pellucida ,reproductive and urinary physiology - Abstract
SUMMARY During early embryo development the zona pellucida acts as a barrier against polyspermia and guarantees communication between blastomeres before and during compaction. However, the development of new technologies of embryo production such as “Handmade Cloning” demands removal of this membrane. The aim of this study was to determine the effect of zona pellucida removal on in vitro bovine embryo development. First, the consequences of zona pellucida removal was assessed by comparing the percentage of first cleavage, percentage of blastocysts and cell number among zona-included and zona-free embryos; either through the removal of the zona pellucida immediately after IVF or parthenogenesis. Embryo development was also evaluated when zona pellucida was removed before parthenogenesis. In a second set of experiments, the gene expression levels of BAX, BCL2, CASP3, CDH1, OCT4 and SOX2 were evaluated in zona-free and zona-included IVF-derived embryos. No significant differences were found in the percentage of first cleavage, percentage of blastocyst and cell number on IVF-embryos cultured with or without zona. Parthenogenetic embryos followed the same general pattern even when the zona pellucida was eliminated before activation; however there was a significant increase in the rate of first cleavage when the zona pellucida was removed after activation but this did not impact upon further development. Furthermore, no significant differences in gene expression level were found between zona-free and zona-included IVF-embryos for the studied genes. We concluded that the lack of zona pellucida did not affect the early development when an appropriate system is used for embryo culture to ensure blastomeric contact and normal compaction.
- Published
- 2013
50. FSH stimulation of anestrous cats improves oocyte quality and development of parthenogenetic embryos
- Author
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Fidel Ovidio Castro, P. F. Gallegos, Lleretny Rodriguez-Alvarez, D. Veraguas, and Alejandra Velásquez
- Subjects
0301 basic medicine ,endocrine system ,medicine.medical_specialty ,Parthenogenesis ,Stimulation ,Biology ,Real-Time Polymerase Chain Reaction ,Anestrus ,03 medical and health sciences ,Food Animals ,SOX2 ,Internal medicine ,Gene expression ,medicine ,Animals ,Blastocyst ,Small Animals ,reproductive and urinary physiology ,CATS ,Cumulus Cells ,Equine ,Hatching ,Ovary ,Gene Expression Regulation, Developmental ,Embryo ,Oocyte ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,embryonic structures ,Cats ,Oocytes ,RNA ,Animal Science and Zoology ,Female ,Follicle Stimulating Hormone ,Biomarkers - Abstract
In the domestic cat, the efficiency of in vitro embryo production systems is negatively affected during the nonbreeding season. The objective of this research was to evaluate the effect of FSH stimulation in anestrous cats, on quality of cumulus-oocyte complexes (COCs) and in vitro developmental competence after parthenogenetic activation. To accomplish this purpose, anestrous cats were grouped into: (1) FSH treated (serial doses of 5 mg of porcine FSH each, every 24 hours, for 4 days) and (2) untreated control. The COCs were classified morphologically and a proportion of grade I and II COCs was used for expression analysis of FSHR, LHCGR, EGFR, PTGS2, EGR1, GDF9, and GATM by RT-qPCR. In addition, another proportion of grade I and II COCs was matured in vitro and used for parthenogenetic activation. After 8 days in culture, blastocyst and hatching blastocyst rates were assessed, and the expression of OCT4, SOX2, NANOG, CDX2, and GATA6 was evaluated. The COCs in the FSH group had an enhanced quality, a higher expression of LHCGR and a lower expression of GATM than did COCs from the control group (P
- Published
- 2016
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