RESUMEN
CONTEXT: In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. AIM: This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus-oocyte complexes (COC) to improve oocyte metabolism. METHODS: COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. KEY RESULTS: Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. CONCLUSIONS: The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. IMPLICATIONS: The coculture system with PLC could be an alternative for the conventional maturation medium in pigs.
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Células Lúteas , Zona Pelúcida , Femenino , Animales , Porcinos , Zona Pelúcida/metabolismo , Técnicas de Cocultivo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/metabolismo , Fertilización In Vitro/veterinaria , Lípidos/análisisRESUMEN
The aim of this study was to evaluate the effect of different progesterone (P4) concentrations during the follicular growth on the intensity of estrous expression, ovarian response to the superovulatory treatment, and embryo production and quality in superovulated heifers. A total of 63 Holstein heifers were randomly assigned into 2 experimental groups: Low P4 (n = 31) and High P4 (n = 32). Animals received a pre-synchronization protocol followed by a protocol of superovulation that included the allocated P4 treatment. Activity was monitored continuously by an automated activity monitor, and estrus characteristics (maximum intensity and duration) were recorded. Embryo collection was performed 7 d post artificial insemination (AI). Embryos were counted and graded from good or excellent (1) to degenerated (4). The outcomes of interest were: number and diameter of follicles at the time of AI, ovulation success (confirmed 7 d post-AI), time to estrus event, maximum intensity and duration of estrus, number and quality of embryos. Data were analyzed according to the type of outcome variable using logistic, linear, or Poisson regression models. A total of 105 embryos (High P4: n = 42; Low P4: n = 63) were graded for quality. Different P4 levels did not affect the maximum intensity (High P4 = 497.8 ± 23.9%; Low P4 = 542.2 ± 23.5%) or the duration (High P4 = 13.5 ± 1.5 h; Low P4 = 14.3 ± 1.4 h) of estrus. Heifers in the High P4 treatment had greater number of follicles at time of AI (High P4 = 16.6 ± 1.6 follicles; Low P4 = 13.9 ± 1.2 follicles), but with smaller diameter (High P4 = 11.3 ± 0.1 mm; Low P4 = 12.0 ± 0.1 mm) compared with Low P4. High P4 heifers tended to have better embryo quality compared with Low P4 heifers (odds ratio = 1.98; 95% CI = 0.90-4.35). High P4 heifers had less embryos than Low P4 heifers, but this was modified by the CIDR (intravaginal implant of P4) removal to estrus interval (interval 0-21 h: mean ratio = 1.15, 95% CI = 0.42-1.87; interval 22-46 h: mean ratio = 0.58, 95% CI = 0.27-0.96). Although estrous expression was not associated with embryo quality, as the duration and the maximum intensity of estrous expression increased, the number of embryos recovered 7 d post-AI increased (duration: mean ratio = 1.04; 95% CI = 1.03-1.05; maximum intensity: mean ratio = 1.50; 95% CI = 1.42-1.58). In conclusion, P4 during the follicular growth, and intensity of estrus, are playing a role in regulating the quality and the number of embryos produced by superovulated heifers. This study was supported by contributions from Resilient Dairy Genome Project and the Natural Sciences and Engineering Research Council.
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Progesterona , Superovulación , Femenino , Bovinos , Animales , Progesterona/farmacología , Sincronización del Estro/métodos , Estro/fisiología , Ovario , Inseminación Artificial/veterinaria , Inseminación Artificial/métodos , Dinoprost/farmacología , Hormona Liberadora de Gonadotropina/farmacologíaRESUMEN
CONTEXT: With two northern white rhinos (NWR) remaining, the continued existence of this species relies on studying their relative, the southern white rhino (SWR). AIMS: (1) Characterise gene expression in granulosa cells (GC) from SWR cumulus oocyte complexes (COCs) prior to (Pre-) and after (Post-) in vitro maturation (IVM), comparing culture media and oocytes from donors treated with or without gonadotropin stimulation prior to ovum recovery; and (2) evaluate COC glucose consumption in spent media. METHODS: COCs were retrieved from four SWRs. Granulosa cells were collected before and after IVM in SDZ or IZW medium. Total RNA was evaluated by qPCR. KEY RESULTS: Oocyte maturation was greater in SDZ than IZW media. Expression of genes associated with follicle development increased in Pre-IVM GC. Six genes were differentially expressed in Post-IVM GC from stimulated compared to unstimulated donors. COCs from stimulated animals consumed more glucose. Fifty seven percent of oocytes in SDZ medium consumed all available glucose. CONCLUSIONS: Gene expression changed upon in vitro maturation and gonadotropin stimulation. Higher glucose availability might be needed during IVM. IMPLICATIONS: This is the first study examining GC gene expression and COC metabolic requirements in rhinoceros, which are critical aspects to optimise IVM of rhinoceros oocytes.
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Células del Cúmulo , Técnicas de Maduración In Vitro de los Oocitos , Animales , Células del Cúmulo/metabolismo , Femenino , Expresión Génica , Glucosa/metabolismo , Gonadotropinas , Células de la Granulosa/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/metabolismo , Perisodáctilos/genéticaRESUMEN
Oocyte maturation in culture is still the weakest part of in vitro fertilization (IVF) and coculture with somatic cells may be an alternative to improve suboptimal culture conditions, especially in the pig in which maturation takes more than 44 h. In the present study, we investigated the effect of a coculture system of porcine luteal cells (PLC) during in vitro maturation (IVM) on embryo development and gene expression. Cumulus-oocyte complexes were matured in vitro in TCM-199 with human menopausal gonadotrophin (control) and in coculture with PLC. IVF was performed with frozen-thawed boar semen in Tris-buffered medium. Presumptive zygotes were cultured in PZM for 7 days. The coculture with PLC significantly increased blastocysts rates. Gene expression changes were measured with a porcine embryo-specific microarray and confirmed by RT-qPCR. The global transcription pattern of embryos developing after PLC coculture exhibited overall downregulation of gene expression. Following global gene expression pattern analysis, genes associated with lipid metabolism, mitochondrial function, endoplasmic reticulum stress, and apoptosis were found downregulated, and genes associated with cell cycle and proliferation were found upregulated in the PLC coculture. Canonical pathway analysis by Ingenuity Pathway revealed that differential expression transcripts were associated with the sirtuin signaling pathway, oxidative phosphorylation pathway, cytokines and ephrin receptor signaling. To conclude, the coculture system of PLC during IVM has a lasting effect on the embryo until the blastocyst stage, modifying gene expression, with a positive effect on embryo development. Our model could be an alternative to replace the conventional maturation medium with gonadotrophins with higher rates of embryo development, a key issue in porcine in vitro embryo production.
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Células Lúteas , Animales , Blastocisto , Técnicas de Cocultivo/veterinaria , Desarrollo Embrionario , Femenino , Fertilización In Vitro/veterinaria , Expresión Génica , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Masculino , Oocitos , PorcinosRESUMEN
Increasing evidence indicates that maternal malnutrition leads to decreased female fertility and dysregulated metabolic homeostasis in offspring. High levels of non-esterified fatty acids (NEFAs) in follicular fluid were reported to be involved in these maternal nutritional effects, but the mechanisms remain unclear. This study explored the mechanisms of action of abnormal NEFA levels during porcine oocyte in vitro maturation (IVM) on early embryo development (blastocysts) using phenotypic, transcriptomic, and epigenetic analysis. The oocytes were treated during IVM with, in addition to the 1% (v/v) porcine follicular fluid in the control group, a combination of 468 µmol/L palmitic acid, 194 µmol/L stearic acid, and 534 µmol/L oleic acid supplemented to North Carolina State University-23 (NCSU-23) maturation medium to achieve a high level of NEFAs. After IVM, oocytes were in vitro fertilized and then cultured in regular conditions for blastocysts. Expanded blastocysts were collected to complete transcriptomic and epigenetic analysis. Macroscopically, high level of NEFAs impaired embryo development by reducing the blastocyst rate. Analysis of the transcriptome revealed that pathways related to inflammation, apoptosis, metabolism, and oxidative stress were the most affected. Moreover, DNA methylation data demonstrated differentially methylated regions in genes related to cellular metabolism and inflammation processes. Therefore, our conclusion is that high level of NEFAs during IVM might affect porcine early embryo development by diminishing blastocyst rate and altering gene expression, especially at the metabolism and cell status levels, which could further decrease the embryo quality.
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Ácidos Grasos no Esterificados , Transcriptoma , Animales , Blastocisto , Desarrollo Embrionario , Epigenoma , Ácidos Grasos no Esterificados/metabolismo , Ácidos Grasos no Esterificados/farmacología , Femenino , Humanos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/metabolismo , PorcinosRESUMEN
Cows at the beginning of lactation often do not meet their energy needs by feeding and therefore mobilize body fat, which produces ketone bodies, including ß-hydroxybutyrate (BHB). They are nevertheless usually inseminated around 60 d postpartum, when they are still in this characteristic period of energy deficit. The aim of this study was to observe the effects of negative energy balance on embryo quality and to identify ways to improve the fertility of dairy cows. Holstein cows (n = 18) grouped as high or low BHB based on blood measurement at day 45 postpartum were estrus-synchronized and treated with follicle-stimulating hormone to obtain multiple follicle development, induced to ovulate and inseminated with sexed semen around day 60 postpartum. Of the 290 embryos collected over 16 mo, 159 were of quality I to IV. Based on microarray analysis of gene expression, exposure to an energy deficit metabolic environment (high BHB) during early development appeared to modify signaling by the mTOR and sirtuins pathways in the embryo, implying mitochondrial dysfunction and inhibition of transcription, leading to slower cell division, thus programming the embryo to be more energy efficient. Altered methylation markers suggested that such coping mechanisms might persist into adulthood.
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Ácido 3-Hidroxibutírico/farmacología , Bovinos/embriología , Metabolismo Energético/fisiología , Periodo Posparto , Ácido 3-Hidroxibutírico/metabolismo , Animales , Técnicas de Cultivo de Embriones , Ácidos Grasos no Esterificados , Femenino , Regulación del Desarrollo de la Expresión Génica , Lactancia , Embarazo , Análisis por Matrices de Proteínas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Cumulus cells have an important role to play in the final preparation of the oocyte before ovulation. During the final phase of follicular differentiation, FSH levels are low and LH maintains follicular growth; however, it is not known if at that time LH has an influence on cumulus cells inside the follicle. In humans, LH is often inhibited to avoid a premature ovulatory LH surge. This procedure provides a tool to investigate the role of LH in follicular development. In this study, we investigated the impact of suppressing LH using the GnRH antagonist cetrorelix during an ovarian coasting stimulation protocol on the transcriptome of bovine cumulus cells (CC). Oocytes were collected twice from 6 dairy cows. For the first collection, the cows received FSH twice daily for 3 d, followed by FSH withdrawal for 68 h as a control protocol. For the second collection, the same stimulation protocol was used, but the cows also received, starting on day 2 of FSH stimulation, a GnRH antagonist once a day until recovery of the cumulus-oocyte complexes (COC). Half of the COC were subjected to in vitro maturation, fertilization, and culture to assess blastocyst rates. The other half of the COC underwent microarray analysis (n = 3 cows, 2 treatments, 6 oocyte collections) and qRT-PCR (n = 6 cows: 3 microarray cows +3 other cows, 2 treatments, 12 oocyte collections). The differential expression of specific genes was confirmed by RT-qPCR: decrease of ATP6AP2, SC4MOL, and OSTC and increase of PTGDS in the LH-inhibited condition. The global transcriptomic analysis of cumulus cells demonstrated that the inhibition of LH secretion may decrease survival and growth of the follicle. Moreover, the results suggested that LH may be important to cumulus for the maintenance of cellular mechanisms such as global RNA expression, protein and nucleic acid metabolism, and energy production. These results support the hypothesis that LH support is important during the final part of follicle maturation through its influence on the cumulus cells.
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Bovinos , Células del Cúmulo/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/análogos & derivados , Fase Luteínica/fisiología , Hormona Luteinizante/antagonistas & inhibidores , Animales , Células Cultivadas , Células del Cúmulo/metabolismo , Femenino , Hormona Folículo Estimulante/farmacología , Hormona Liberadora de Gonadotropina/farmacología , Antagonistas de Hormonas/farmacología , Inducción de la Ovulación , SuperovulaciónRESUMEN
BACKGROUND: Prolongation of superstimulatory treatment appears to be associated with a greater superovulatory response and with greater oocyte maturation in cattle. A genome-wide bovine oligo-microarray was used to compare the gene expression of granulosa cells collected from ovarian follicles after differing durations of the growing phase induced by exogenous FSH treatment. Cows were given a conventional (4-day) or long (7-day) superstimulatory treatment (25 mg FSH im at 12-h intervals; n = 6 per group), followed by prostaglandin treatment with last FSH and LH treatment 24 h later. Granulosa cells were harvested 24 h after LH treatment. RESULTS: The expression of 416 genes was down-regulated and 615 genes was up-regulated in the long FSH group compared to the conventional FSH group. Quantification by RT-PCR of 7 genes (NTS, PTGS2, PTX3, RGS2, INHBA, CCND2 and LRP8) supported the microarrays data. Multigene bioinformatic analysis indicates that markers of fertility and follicle maturity were up-regulated in the long FSH group. CONCLUSION: Using the large gene expression dataset generated by the genomic analysis and our previous associated with the growth phase and gene expression changes post LH, we can conclude that a prolonged FSH-induced growing phase is associated with transcriptomic characteristics of greater follicular maturity and may therefore be more appropriate for optimizing the superovulatory response and developmental competence of oocytes in cattle.
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Bovinos/genética , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/metabolismo , Transcriptoma/efectos de los fármacos , Animales , Bovinos/metabolismo , Femenino , Hormona Folículo Estimulante/administración & dosificación , Líquido Folicular/química , Perfilación de la Expresión Génica , Células de la Granulosa/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , SuperovulaciónRESUMEN
During growth, the oocyte accumulates mRNAs that will be required in the later stages of oogenesis and early embryogenesis until the activation of the embryonic genome. Each of these developmental stages is controlled by multiple regulatory mechanisms that ensure proper protein production. Thus mRNAs are stabilized, stored, recruited, polyadenylated, translated and/or degraded over a period of several days. As a consequence, understanding the biological significance of changes in the abundance of transcripts during oocyte growth and differentiation is rather complex. Nevertheless the availability of transcriptomic platforms applicable to scarce samples such as oocytes has generated large amounts of data that depict the transcriptome of oocytes under different conditions. Despite several technical constrains related to protein determination in oocytes that still limit the possibility to verify certain hypothesis, it is now possible to use mRNA levels to start building plausible scenarios. To start deciphering the changes in the level of specific mRNAs involved in chromatin remodelling, we have performed a meta-analysis of existing microarray datasets from germinal vesicle (GV) stage bovine oocytes during the final stages of oocyte differentiation. We then analysed the expression profiles of histone and histone-remodelling enzyme mRNAs and correlated these with the major histone modifications known to occur at the same period, based on data available in the literature. We believe that this approach could reveal the function of specific enzymes in the oocyte. In turn, this information will be useful in future studies, which final ambitious goal is to decipher the 'oocyte-specific histone code'.
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Ensamble y Desensamble de Cromatina , Histonas/genética , Oocitos/enzimología , Oocitos/metabolismo , Animales , Bovinos , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Femenino , Oogénesis/genéticaRESUMEN
Medically assisted reproductive technologies, such as in vitro embryo production, are increasingly being used to palliate infertility. Eggs are produced following a hormonal regimen that stimulates the ovaries to produce a large number of oocytes. Collected oocytes are then fertilized in vitro and allowed to develop in vitro until they are either frozen or transferred to mothers. There are controversial reports on the adverse impacts of these technologies on early embryos and their potential long-term effects. Using newly developed technological platforms that enable global gene expression and global DNA methylation profiling, we evaluated gene perturbations caused by such artificial procedures. We know that cells in the early embryo produce all cells in the body and are able to respond to their in vitro environment. However, it is not known whether gene perturbations are part of a normal response to the environment or are due to distress and will have long-term impacts. While the mouse is an established genetic model used for quality control of culture media in clinics, the bovine is a large mono-ovulating mammal with similar embryonic kinetics as humans during the studied developmental window. These model systems are critical to understand the effects of assisted reproduction without the confounding impact of infertility and without the limitations imposed by the scarcity of donated human samples and ethical issues. The data presented in this review come mostly from our own experimentation, publications, and collaborations. Together they demonstrate that the in vitro environment has a significant impact on embryos at the transcriptomic level and at the DNA methylation level.
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Técnicas de Cultivo de Embriones/métodos , Epigénesis Genética/fisiología , Fertilización In Vitro/métodos , Modelos Animales , Animales , Bovinos , Técnicas de Cultivo de Embriones/tendencias , Fertilización In Vitro/tendencias , Humanos , Inducción de la Ovulación/métodos , Inducción de la Ovulación/tendenciasRESUMEN
BACKGROUND: Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts. RESULTS: Bovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological ("BASAL") concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological ("HIGH COMBI") concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P < 0.05). HIGH COMBI-exposed oocytes and embryos resulted in blastocysts with altered DNA methylation and transcriptomic fingerprints, compared to BASAL-exposed counterparts. Differences in gene expression and methylation were more pronounced after exposure during culture compared to maturation suggesting that zygotes are more susceptible to adverse environments. Main gene networks affected were related to lipid and carbohydrate metabolism, cell death, immune response and metabolic disorders. CONCLUSIONS: Overall, high variation in methylation between blastocysts made it difficult to draw conclusions concerning methylation of individual genes, although a clear overview of affected pathways was obtained. This may offer clues regarding the high rate of embryonic loss and metabolic diseases during later life observed in offspring from mothers displaying lipolytic disorders.
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Blastocisto/metabolismo , Embrión de Mamíferos/metabolismo , Epigénesis Genética/efectos de los fármacos , Ácidos Grasos no Esterificados/toxicidad , Oocitos/metabolismo , Transcriptoma/efectos de los fármacos , Animales , Bovinos , ADN/química , ADN/aislamiento & purificación , ADN/metabolismo , Metilación de ADN/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Histonas/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oocitos/efectos de los fármacos , Análisis de Secuencia de ADN , Proteínas Nucleares snRNP/genéticaRESUMEN
STUDY HYPOTHESIS: We hypothesized that a better discrimination between follicles containing oocytes with high developmental competence and those containing oocytes with low competence, based on a combination of a follicle's size and transcriptomic signature, will provide a reliable method to predict embryonic outcome of IVF. STUDY FINDING: This study provides new insights on the impact of follicular size on oocyte quality as measured by embryonic development and demonstrates that medium follicles yield a better percentage of transferable embryos. WHAT IS KNOWN ALREADY: Although it is generally accepted that large ovarian follicles contain better eggs, other studies report that a better follicular size subdivision and a better characterization are needed. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Individual follicles (n = 136), from a total of 33 women undergoing IVF, were aspirated and categorized on the basis of their follicular liquid volume (small, medium or large) and the embryonic outcome of the enclosed oocyte: poor or good development. Comprehensive gene expression analysis between cells from the different sized follicles was performed using microarrays and quantitative RT-PCR to find molecular markers associated with follicular maturity and oocyte developmental competence. MAIN RESULTS AND THE ROLE OF CHANCE: The analysis of embryonic outcome in relation to follicular size indicates that the medium-sized follicles category yielded more transferable embryos (35%) compared with the largest follicles (30%) (NS). Gene expression analysis revealed expression markers with significant (P < 0.05) discrimination between the poor development groups for all three follicle sizes, and good development medium-size follicles, including up-regulation of thrombomodulin, transforming growth factor, beta receptor II and chondrolecti, and those associated with hyaluronan synthesis, coagulation and hepatocyte growth factor signalling. LIMITATIONS, REASONS FOR CAUTION: These analyses were performed in a single cohort of patients coming from a single clinic and the biomarkers generated will require validation in different geographical and biological contexts to ensure their global applicability. WIDER IMPLICATIONS OF THE FINDINGS: Medium-size follicles seem to be the optimal size for a positive embryonic outcome and are associated with competence markers that may help in understanding the ideal differentiation status during late folliculogenesis. LARGE SCALE DATA: The data discussed in this publication have been deposited in The National Center for Biotechnology Information Gene Expression Omnibus database and are accessible through GEO Series accession number GSE52851. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council of Canada (NSERC) to M.A.S. There are no competing interests to declare.
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Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Femenino , Líquido Folicular/citología , Líquido Folicular/metabolismo , Humanos , Oocitos/citología , Oocitos/metabolismo , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: This study describes the generation and analysis of the transcriptional profile of bovine inner cell mass (ICM) and trophectoderm (TE), obtained from in vivo developed embryos by using a bovine-embryo specific array (EmbryoGENE) containing 37,238 probes. RESULTS: A total of 4,689 probes were differentially expressed between ICM and TE, among these, 2,380 and 2,309 probes were upregulated in ICM and TE tissues, respectively (P ≤ 0.01, FC ≥ 2.0, FDR: 2.0). Ontological classification of the genes predominantly expressed in ICM emerged a range of functional categories with a preponderance of genes involved in basal and developmental signaling pathways including P53, TGFß, IL8, mTOR, integrin, ILK, and ELF2 signalings. Cross-referencing of microarray data with two available in vitro studies indicated a marked reduction in ICM vs. TE transcriptional difference following in vitro culture of bovine embryos. Moreover, a great majority of genes that were found to be misregulated following in vitro culture of bovine embryos were known genes involved in epigenetic regulation of pluripotency and cell differentiation including DNMT1, GADD45, CARM1, ELF5 HDAC8, CCNB1, KDM6A, PRDM9, CDX2, ARID3A, IL6, GADD45A, FGFR2, PPP2R2B, and SMARCA2. Cross-species referencing of microarray data revealed substantial divergence between bovine and mouse and human in signaling pathways involved in early lineage specification. CONCLUSIONS: The transcriptional changes occur during ICM and TE lineages specification in bovine is greater than previously understood. Therefore, this array data establishes a standard to evaluate the in vitro imprint on the transcriptome and to hypothesize the cross-species differences that allow in vitro acquisition of pluripotent ICM in human and mice but hinder that process in bovine.
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Masa Celular Interna del Blastocisto/citología , Ectodermo/citología , Regulación del Desarrollo de la Expresión Génica/genética , Transcriptoma/genética , Trofoblastos/citología , Animales , Bovinos , Diferenciación Celular/genética , Linaje de la Célula , Ectodermo/embriología , Embrión de Mamíferos/embriología , Desarrollo Embrionario/genética , Femenino , Perfilación de la Expresión Génica , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa , Transcripción Genética/genética , Activación Transcripcional/genéticaRESUMEN
The fertility of dairy cows is challenged during early lactation, and better nutritional strategies need to be developed to address this issue. Combined supplementation of folic acid and vitamin B12 improve energy metabolism in the dairy cow during early lactation. Therefore, the present study was undertaken to explore the effects of this supplement on gene expression in granulosa cells from the dominant follicle during the postpartum period. Multiparous Holstein cows received weekly intramuscular injection of 320 mg of folic acid and 10 mg of vitamin B12 (treated group) beginning 24 (standard deviation=4) d before calving until 56 d after calving, whereas the control group received saline. The urea plasma concentration was significantly decreased during the precalving period, and the concentration of both folate and vitamin B12 were increased in treated animals. Milk production and dry matter intake were not significantly different between the 2 groups. Plasma concentrations of folates and vitamin B12 were increased in treated animals. Daily dry matter intake was not significantly different between the 2 groups before [13.5 kg; standard error (SE)=0.5] and after (23.6 kg; SE=0.9) calving. Average energy-corrected milk tended to be greater in vitamin-treated cows, 39.7 (SE=1.4) and 38.1 (SE=1.3) kg/d for treated and control cows, respectively. After calving, average plasma concentration of ß-hydroxybutyrate tended to be lower in cows injected with the vitamin supplement, 0.47 (SE=0.04) versus 0.55 (SE=0.03) for treated and control cows, respectively. The ovarian follicle ≥12 mm in diameter was collected by ovum pick-up after estrus synchronization. Recovered follicular fluid volumes were greater in the vitamin-treated group. A microarray platform was used to investigate the effect of treatment on gene expression of granulosa cells. Lower expression of genes involved in the cell cycle and higher expression of genes associated with granulosa cell differentiation before ovulation were observed. Selected candidate genes were analyzed by reverse transcription quantitative PCR. Although the effects of intramuscular injections of folic acid and vitamin B12 on lactational performance and metabolic status of animals were limited, ingenuity pathway analysis of gene expression in granulosa cells suggests a stimulation of cell differentiation in vitamin-treated cows, which may be the result of an increase in LH secretion.
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Bovinos/metabolismo , Ácido Fólico/administración & dosificación , Expresión Génica/efectos de los fármacos , Células de la Granulosa/metabolismo , Periodo Posparto/metabolismo , Vitamina B 12/administración & dosificación , Ácido 3-Hidroxibutírico/sangre , Animales , Suplementos Dietéticos , Metabolismo Energético , Femenino , Inyecciones Intramusculares/veterinaria , Lactancia/fisiología , Leche/químicaRESUMEN
Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.
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Células del Cúmulo/fisiología , AMP Cíclico/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/métodos , 1-Metil-3-Isobutilxantina/farmacología , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Colforsina/farmacología , Células del Cúmulo/citología , Células del Cúmulo/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dipiridamol/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Understanding the mechanisms regulating oocyte developmental competence is essential to enhance the clinical efficiency of assisted reproduction. FSH orchestrates the acquisition of oocyte competence, both in vivo and in vitro. Multiple pathways are implicated in FSH signalling; however, their precise coordination remains unresolved. A robust system to investigate FSH signalling is oocyte in vitro maturation (IVM) and we have previously demonstrated better bovine embryo development after FSH addition for the first 6 h during IVM. Using this model, we investigated FSH signalling in cumulus through transcriptomic and pharmacological tools. We demonstrate modulation of cumulus transcriptome by FSH mainly through protein kinase A (PKA) and epidermal growth factor (EGF) pathways. Differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism and oocyte competence. FSH required rouse-sarcoma oncogene (SRC) for EGF receptor transactivation. PKA and EGF pathway crosstalk was investigated using extracellular signal-regulated kinases (ERK1/2) phosphorylation as the functional end-point. FSH enhanced ERK1/2 activation by the EGF pathway with a simultaneous diminution through PKA. More specifically, FSH increased dual specific phosphatase (DUSP1) transcripts via PKA although DUSP1 protein did not change since EGF was required to prevent degradation. Our findings implicate FSH in PKA and EGF pathway activation, which interact to maintain appropriate levels of ERK1/2 phosphorylation and eventually cumulus expansion, metabolism and steroidogenesis. Moreover, considering the implication of the EGF pathway in GDF9 and BMP15 actions, our findings suggest that FSH may have a role in modulation of the cumulus response to oocyte-secreted factors. This information has implications for improvement of IVM and hence oocyte developmental competence.
Asunto(s)
Células del Cúmulo/efectos de los fármacos , Fármacos para la Fertilidad/farmacología , Hormona Folículo Estimulante/farmacología , Oocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Bovinos , Biología Computacional , Células del Cúmulo/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfatasa 1 de Especificidad Dual/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Técnicas de Maduración In Vitro de los Oocitos , Oocitos/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Transducción de Señal/genética , Factores de Tiempo , TranscriptomaRESUMEN
Major remodelling of the chromatin enclosed within the germinal vesicle occurs towards the end of oocyte growth in mammals, but the mechanisms involved in this process are not completely understood. In bovine, four distinct stages of chromatin compaction-ranging from a diffused state (GV0) to a fully compacted configuration (GV3)-are linked to the gradual acquisition of developmental potential. To better understand the molecular events and to identify mRNA modulations occurring in the oocyte during the GV0-to-GV3 transition, transcriptomic analysis was performed with the EmbryoGENE microarray platform. The mRNA abundance of several genes decreased as chromatin compaction increased, which correlates with progressive transcriptional silencing that is characteristic of the end of oocyte growth. On the other hand, the abundance of some transcripts increased during the same period, particularly several histone gene transcripts from the H2A, H2B, H3, H4, and linker H1 family. In silico analysis predicted RNA-protein interactions between specific histone transcripts and the bovine stem-loop binding protein 2 (SLBP2), which helps regulate the translation of histone mRNA during oogenesis. These results suggest that some histone-encoding transcripts are actively stored, possibly to sustain the needs of the embryo before genome activation. This dataset offers a unique opportunity to survey which histone mRNAs are needed to complete chromatin compaction during oocyte maturation and which are stockpiled for the first three cell cycles following fertilization.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Histonas/biosíntesis , Oocitos/metabolismo , Oogénesis/fisiología , ARN Mensajero/biosíntesis , Animales , Bovinos , Femenino , Análisis de Secuencia por Matrices de Oligonucleótidos , Oocitos/citologíaRESUMEN
Maternal metabolic disorders linked to lipolysis are major risk factors for reproductive failure. A notable feature of such disorders is increased non-esterified fatty acid (NEFA) concentrations in the blood, which are reflected in the ovarian follicular fluid. Elevated NEFA concentrations impact on the maturing oocyte and even alter subsequent embryo physiology. The aetiological mechanisms have not been fully elucidated. Therefore, in the present study, bovine in vitro maturing cumulus-oocyte complexes were exposed (24 h) to three different maturation treatments containing: (1) physiological (72 µM) NEFA concentrations (=control); (2) elevated (75 µM) stearic acid (SA) concentrations (=HIGH SA); and (3) elevated (425 µM) NEFA concentrations (=HIGH COMBI). Zygotes were fertilised and cultured following standard procedures. Transcriptomic analyses in resulting Day 7.5 blastocysts revealed that the major pathways affected are related to lipid and carbohydrate metabolism in HIGH COMBI embryos and to lipid metabolism and cell death in HIGH SA embryos. Furthermore, lower glutathione content and a reduced number of lipid droplets per cell were observed in HIGH SA-exposed oocytes and resulting morulae, respectively, compared with their HIGH COMBI-exposed counterparts. Vitrified embryos originating from HIGH SA-exposed oocytes tended to exhibit lower survival rates compared with controls. These data suggest possible mechanisms explaining why females across species suffering lipolytic disorders experience difficulties in conceiving.
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Blastocisto/metabolismo , Ácidos Grasos no Esterificados/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/efectos de los fármacos , Fenotipo , Análisis de Varianza , Animales , Blastocisto/efectos de los fármacos , Bovinos , Cartilla de ADN/genética , Ácidos Grasos no Esterificados/sangre , Femenino , Perfilación de la Expresión Génica/veterinaria , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Ácidos EsteáricosRESUMEN
Studying cumulus cell (CC) transcriptome is of great interest as it could provide a noninvasive method to assess oocyte quality. In cattle, the search for quality markers has not been done with cumulus-oocyte complexes (COCs) cultured individually from maturation to blastocyst stage. Here, differences between high- and low-potential COCs were examined by transcriptomic analysis of CC biopsies obtained from COCs of 2 to 6 mm follicles (n = 249; eight replicates) before individual in vitro maturation, fertilization, and culture until Day 8 after fertilization. Each COC was individually tracked and categorized based on his fate: embryo at blastocyst stage (CC-Blast) or embryo arrested at 2- to 8-cell stage (CC-2-8-cells). Average blastocyst rates were 27.7% for individual culture and 31.2% for group control (not significantly different). For transcriptomic analysis, five cumulus biopsies per replicate were pooled for each fate. Three CC replicates underwent transcriptomic analysis using RNA microarray assay. Some clear differences in gene expression between the CC-Blast and the CC-2-8-cell groups were identified. Considering a 1.5-fold change (P < 0.05), 68 genes were differentially expressed between the CC-Blast and CC-2-8-cells. Quantitative reverse transcription-polymerase chain reaction validations were performed for 12 selected genes: six upregulated genes for each COC fate. Higher expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) (lipid metabolism), Chloride intracellular channel 3 (CLIC3), Keratin 8 (KRT8), and Lumican (LUM) (molecular transport) was observed in CC-2-8-cells (P < 0.05). The CC-Blast fate analysis revealed a significantly higher expression of Glycine amidinotransferase (L-arginine:glycine amidinotransferase) (GATM) (posttranslational modification, amino acid metabolism, and free radical scavenging). This newly identified set of genes could provide new markers to distinguish COCs associated with good quality embryos from COCs with limited developmental potential.
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Bovinos/embriología , Células del Cúmulo/química , Células del Cúmulo/fisiología , Fertilización In Vitro/veterinaria , Perfilación de la Expresión Génica/veterinaria , Oocitos/fisiología , Animales , Apoptosis , Blastocisto/fisiología , Recuento de Células , Técnicas de Cultivo de Embriones , Femenino , Etiquetado Corte-Fin in Situ , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Análisis por Micromatrices , ARN/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinariaRESUMEN
The review presents an overview of studies that examined the effects of follicular aging and maternal aging in the bovine model. The first of three main sections is a discussion of the developmental competence of oocytes from (1) the ovulatory follicle of 2-wave and 3-wave estrous cycles, (2) dominant follicles that develop under high or low LH pulse frequency, and (3) natural versus FSH-stimulated ovulatory follicles. The second section highlights the effects of maternal aging. Maternal aging in cattle is associated with (1) elevated circulating FSH concentrations, (2) reduced response to superstimulatory treatment, and (3) markedly decreased early embryonic development in cows >12 year of age. The third and final section on superstimulation protocols addresses the effects of the duration of FSH stimulation and withdrawal (i.e., FSH "starvation" or "coasting") on oocyte competence. Ovarian superstimulation for 4 days altered the expression of genes related to angiogenesis, and activated oxidative stress-response genes. Extending the duration of FSH stimulation from 4 to 7 days resulted in a greater and more synchronous ovulatory response and optimal oocyte maturation. The highest rates of blastocyst development in vitro were obtained when FSH support was discontinued for 44 to 68h and granulosa cell SMAD7 mRNA was predictive of this period. Longer periods of FSH starvation resulted in a loss of oocyte competence or ovulatory capability. By extending the bovine model to the transcriptome level, new approaches and treatments may be devised to resolve subfertility in women and animals.