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Objective: To develop, assess, and refine an online educational tool, Plan for Clarity, to support financial and legal planning in dementia. Methods: A Delphi mixed-method study with three rounds of anonymous review by lay and professional stakeholders was designed to reach consensus about the content of the online tool and explore the socio-cultural and behavioral factors that could affect access and use. Results: Consensus showed that the online tool covered key information, knowledge, and communication skills for financial and legal planning. Study themes: 1) the online tool had to be easy to navigate with relevant, easily understood information; 2) people with early signs of memory difficulties would be able to use the tool; 3) a referral from a trusted source is a primary way to facilitate access and use of the tool; and 4) discussions about financial and legal issues should be held early, ahead of barriers that can block discussion. Conclusion: These data suggest this online tool is relevant and feasible for dementia care and support as well as aging more generally. Innovation: Plan for Clarity is innovative as an evidence and theory-driven online education tool to address financial and legal planning for dementia care, particularly among underserved older adults.
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Decreased oocyte quality is a major determinant of age-associated fertility decline. Similarly, individuals affected by early ovarian aging carry low-quality oocytes. Using an established bovine model of early ovarian aging, we investigated key features of 'quality' oocyte maturation, associated with the onset of egg aneuploidy and reproductive aging, such as histone modifications, mitochondria distribution and activity, reduced glutathione (GSH) content, and gap junction functionality. Bovine ovaries were classified according to the antral follicle count (AFC), and the retrieved oocytes were processed immediately or matured in vitro. We observed alterations in several cellular processes, suggesting a multifactorial etiology of the reduced oocyte quality. Furthermore, we performed a rescue experiment for one of the parameters considered. By adding cysteamine to the maturation medium, we experimentally increased the free radical scavenger ability of the 'low competence' oocytes and obtained a higher embryo development. Our findings show that adopting culture conditions that counteract the free radicals has a positive impact on the quality of 'compromised' oocytes. Specifically, cysteamine treatment seems to be a promising option for treating aging-related deficiencies in embryo development.
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Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence in compact and expanded cumulus-oocyte complexes (COCs) according to follicle size (<1, 1-2, >2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles <1 and 1-2cm in diameter on embryo development. Meiotic competence was not affected by prematuration, whereas COCs from follicles 1-2cm in diameter yielded embryos with a higher number of cells per blastocyst than oocytes that underwent direct IVM (P<0.01, unpaired Mann-Whitney test), suggesting improved developmental competence. Oocytes collected from follicles <1cm in diameter were not affected by prematuration. This study represents an extensive characterisation of the functional properties of immature horse oocytes and is the first report of the effects of cilostamide-based prematuration in horse oocyte IVM on embryo development.
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Cromatina/metabolismo , Junções Comunicantes/metabolismo , Cavalos , Técnicas de Maturação in Vitro de Oócitos , Oócitos/citologia , Folículo Ovariano/citologia , Animais , Comunicação Celular/fisiologia , Tamanho Celular , Células Cultivadas , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Feminino , Junções Comunicantes/efeitos dos fármacos , Cavalos/embriologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Meiose/efeitos dos fármacos , Meiose/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Quinolonas/farmacologia , Estações do Ano , Manejo de Espécimes/métodos , Manejo de Espécimes/veterináriaRESUMO
Total hip arthroplasty (THA) represents one of the commonest surgical procedures in the orthopedic field. Osteointegration of the implant with native bone is essential for an optimal result; thus, the quality of the patient's bone surrounding the implant (i.e., the bone stock) is crucial. However, in some cases, the bone stock is insufficient and needs to be improved with autologous grafts rich in multipotent cells (i.e., from the iliac crest, from the head of the femur, or from the subchondral bone harvested from the acetabulum) or allogenic frozen bone. It is not known if the harvesting site may influence the osteogenic potential of these cells. Thus, our aim was to characterize and compare multipotent cells collected from the bone marrow, acetabular subchondral bone, and trabecular bone on the femoral head with a focus on osteogenic differentiation. The cells from three sources had a fibroblast-like phenotype and expressed surface antigens CD73, CD90, and CD105 and are negative to CD11b, CD34, and CD45. Although all these cells could be induced to differentiate into osteoblasts, chondrocytes, and adipocytes, they displayed different differentiation potentials. In osteogenic differentiation condition, the cells from the acetabulum had the lowest accumulation of calcium deposit while the cells originated from the bone marrow and femur created a considerably increased amount of the deposit. These findings were confirmed by quantitative polymerase chain reaction (qPCR). In chondrogenic and adipogenic conditions, bone marrow cells possessed a predominant differential capacity compared with the others, illustrated by high collagen type II expression together with a cartilage-like lacuna structure and the presence of fat-specific markers, respectively. To our knowledge, this is the first study comparing and demonstrating that the progenitor cells obtained from diverse surgical sites in hip replacement procedure share common characteristics of MSC but differ about plasticity and may provide rational for clinical application in cell therapy and bone grafting. The project number L1033 is registered with ClinicalTrials.gov NCT03369457.
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BACKGROUND: The management of cartilage lesions is an open issue in clinical practice, and regenerative medicine represents a promising approach, including the use of autologous micrografts whose efficacy was already tested in different clinical settings. The aim of this study was to characterize in vitro the effect of autologous cartilage micrografts on chondrocyte viability and differentiation and perform an evaluation of their application in racehorses affected by joint diseases. MATERIALS AND METHODS: Matched human chondrocytes and micrografts were obtained from articular cartilage using Rigenera® procedure. Chondrocytes were cultured in the presence or absence of micrografts and chondrogenic medium to assess cell viability and cell differentiation. For the pre-clinical evaluation, three racehorses affected by joint diseases were treated with a suspension of autologous micrografts and PRP in arthroscopy interventions. Clinical and radiographic follow-ups were performed up to 4 months after the procedure. RESULTS: Autologous micrografts support the formation of chondrogenic micromasses thanks to their content of matrix and growth factors, such as transforming growth factor ß (TGFß) and insulin-like growth factor 1 (IGF-1). On the other hand, no significant differences were observed on the gene expression of type II collagen, aggrecan, and SOX9. Preliminary data in the treatment of racehorses are suggestive of a potential in vivo use of micrografts to treat cartilage lesions. CONCLUSION: The results reported in this study showed the role of articular micrografts in the promoting chondrocyte differentiation suggesting their potential use in the clinical practice to treat articular lesions.
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Cartilagem/transplante , Condrócitos/fisiologia , Doenças dos Cavalos/cirurgia , Artropatias/veterinária , Animais , Artroscopia , Autoenxertos , Diferenciação Celular , Sobrevivência Celular , Feminino , Cavalos , Humanos , Artropatias/cirurgia , Masculino , Plasma Rico em Plaquetas , Cultura Primária de CélulasRESUMO
BACKGROUND: Polycystic-ovary syndrome (PCOS) is a reproductive illness characterized by hyperandrogenism and anovulation. Using hyperandrogenized mice, it was demonstrated that the oral administration of incremental dose of follicle stimulating hormone (FSH) attenuated some of PCOS characteristics. This work aimed to study the effect of ultra-low doses of combined FSH and progesterone orally administered on PCOS murine model. Moreover, the effect of sequential kinetic activation of administered hormones was tested. METHODS: Thirty-two female mice were used as animal model (four groups of eight animals each). Mice were hyperandrogenized by injection of dehyidroepiandrosterone diluted in sesame oil. Control group received only oil. Simultaneously, each animal daily received per os an activated or a not-activated combination of FSH (0.44 pg) plus progesterone (0.44 pg) or saline solution as control. Serum testosterone, estradiol, progesterone and luteinizing hormone were analyzed as endocrine markers and a morphological study of antral follicle was conducted. Data were analyzed by one-way ANOVA, followed by multiple comparison test. The p<0.05 was considered significant. RESULTS: Dehyidroepiandrosterone treatment increased both estradiol and progesterone serum levels, besides testosterone, while reduced luteinizing hormone (p<0.05); histological examination revealed an increase of cystic follicles (p<0.05). Irrespective of activation, the combined FSH and progesterone treatments restored estradiol level (p>0.05 vs. control group) and reduced cystic signs in the follicles (p<0.05 vs. dehyidroepiandrosterone treatment). CONCLUSION: This study indicate that ultra-low doses of FSH and progesterone orally administrated can reduce the sternness of PCOS in the mouse model and open a route for the study of innovative approaches for PCOS treatment.
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An in-depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti-angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve-CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro-cartilaginous differentiation. Expression of VEGF and Ve-CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.
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Envelhecimento/metabolismo , Colágeno Tipo II/genética , Meniscos Tibiais/metabolismo , Neovascularização Fisiológica/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fatores Etários , Envelhecimento/genética , Animais , Animais Recém-Nascidos , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Endostatinas/genética , Endostatinas/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Articulações/citologia , Articulações/crescimento & desenvolvimento , Articulações/metabolismo , Meniscos Tibiais/irrigação sanguínea , Meniscos Tibiais/citologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Suínos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected. Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.
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Segregação de Cromossomos , Histonas/metabolismo , Cavalos/fisiologia , Oócitos/fisiologia , Fuso Acromático/ultraestrutura , Acetilação , Aneuploidia , Animais , Centrômero/ultraestrutura , Feminino , Expressão Gênica , Histona Acetiltransferases/biossíntese , Histona Acetiltransferases/genética , Histona Desacetilases/biossíntese , Histona Desacetilases/genética , Histonas/química , Técnicas de Maturação in Vitro de Oócitos , Metáfase , Oócitos/metabolismo , Óvulo , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Implantation failure and genetic developmental disabilities in mammals are caused by errors in chromosome segregation originating mainly in the oocyte during meiosis I. Some conditions, like maternal ageing or in vitro maturation (IVM), increase the incidence of oocyte aneuploidy. Here oocytes from adult mares were used to investigate oocyte maturation in a monovulatory species. Experiments were conducted to compare: (1) the incidence of aneuploidy, (2) the morphology of the spindle, (3) the acetylation of lysine 16 on histone H4 (H4K16) and (4) the relative amount of histone acetyltransferase 1 (HAT1), K(lysine) acetyltransferase 8 (KAT8, also known as MYST1), histone deacetylase 1 (HDAC1) and NAD-dependent protein deacetylase sirtuin 1 (SIRT1) mRNA in metaphase II stage oocytes that were in vitro matured or collected from peri-ovulatory follicles. The frequency of aneuploidy and anomalies in spindle morphology was increased following IVM, along with a decrease in H4K16 acetylation that was in agreement with our previous observations. However, differences in the amount of the transcripts investigated were not detected. These results suggest that the degradation of transcripts encoding for histone deacetylases and acetyltransferases is not involved in the changes of H4K16 acetylation observed following IVM, while translational or post-translational mechanisms might have a role. Our study also suggests that epigenetic instabilities introduced by IVM may affect the oocyte and embryo genetic stability.
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Segregação de Cromossomos/fisiologia , Histonas/metabolismo , Técnicas de Maturação in Vitro de Oócitos , Oócitos/metabolismo , Fuso Acromático/fisiologia , Acetilação , Animais , Feminino , Cavalos , Meiose/fisiologia , Oogênese/fisiologiaRESUMO
The degeneration of articular cartilage represents an ongoing challenge at the clinical and basic level. Tissue engineering and regenerative medicine using stem/progenitor cells have emerged as valid alternatives to classical reparative techniques. This review offers a brief introduction and overview of the field, highlighting a number of tissue sources for stem/progenitor cell populations. Emphasis is given to recent developments in both clinical and basic sciences. The relative strengths and weaknesses of each tissue type are discussed.
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STUDY QUESTION: Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). SUMMARY ANSWER: The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. WHAT IS KNOWN ALREADY: Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. STUDY DESIGN, SIZE, DURATION: Bovine cumulus-oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. PARTICIPANTS/MATERIALS, SETTING, METHOD: RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, quantitative RT-PCR results of a subset of five selected genes were consistent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as 'cell cycle' and 'cell death and survival', whereas genes included in cluster #5 were mostly related to 'gene expression'. Interestingly, 'lipid metabolism' was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was 'cell death and survival'. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apoptosis increased significantly in CC from oocytes with condensed chromatin, reaching a peak in CC_GV3 (P < 0.05). Finally, the tailored oocyte pre-maturation strategy, based on morphological features of the COC and the oocyte chromatin configuration, demonstrated that pre-IVM improved the developmental capability of oocytes at early stages of differentiation (GV1-enriched COC) but was detrimental for oocytes at more advanced stages of development (GV2 and GV3-enriched COC). LARGE SCALE DATA: The data are available through the GEO series accession number GSE79886. LIMITATIONS, REASONS FOR CAUTION: This study was conducted with bovine samples. Whether or not the results are applicable to human oocytes requests further elucidation. Embryo transfer experiments are required to determine whether the improvement in blastocyst rates in the tailored system leads to increased live birth rates. WIDER IMPLICATIONS OF THE FINDINGS: The identification of multiple non-invasive biomarkers predictive of oocyte quality can greatly strengthen the pre-IVM approach aimed to improve IVM outcomes. These results have potentially important implications in treating human infertility and in developing breeding schemes for domestic mammals. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by NSERC Strategic Network EmbryoGENE, Canada and in part by CIG-Marie Curie Actions-Reintegration Grants within the EU 7FP (n. 303640, 'Pro-Ovum'). The authors declare no potential conflict of interest.
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Células do Cúmulo/metabolismo , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Bovinos , Cromatina/metabolismo , Células do Cúmulo/fisiologia , Feminino , Técnicas de Maturação in Vitro de Oócitos , Meiose/genética , Meiose/fisiologia , Oócitos/fisiologia , Oogênese/genética , Oogênese/fisiologia , Folículo Ovariano/fisiologiaRESUMO
BACKGROUND: Polycystic Ovary Syndrome (PCOS) is a widespread reproductive disorder characterized by a disruption of follicular growth and anovulatory infertility. In women with PCOS, follicular growth and ovulation can be induced by subcutaneous injections of low doses of follicle stimulating hormone (FSH). The aim of this study was to determine the effect of oral administration of recombinant human FSH (rhFSH) on follicle development in a PCOS murine model. Moreover, since it is unlikely that intact rhFSH is present into the circulation after oral administration, the biological activity of a peptide fragment, derived from the predicted enzymatic cleavage sites with the FSH molecule, was investigated in vitro on cumulus-enclosed oocytes (COCs). METHODS: Female peripubertal mice were injected with dehydroepiandrosterone (DHEA) diluted in sesame oil for 20 consecutive days and orally treated with a saline solution of rhFSH. A control group received only sesame oil and saline solution. At the end of treatments, blood was analyzed for hormone concentrations and ovaries were processed for morphological analysis. The presumptive bioactive peptide was added during in vitro maturation of bovine COCs and the effects on cumulus expansion and on maturation rate were evaluated. RESULTS: DHEA treatment increased serum levels of testosterone, estradiol and progesterone as well as the percentage of cystic follicles. Orally administered rhFSH restored estradiol level and reduced the percentage of cystic follicles. Despite these results indicating a reduction of the severity of PCOS in the mouse model, the presumptive bioactive peptide did not mimic the effect of rhFSH and failed to induce bovine cumulus expansion and oocyte maturation in vitro. CONCLUSIONS: Although further studies are needed, the present data supports the concept that orally administrated FSH could attenuate some of the characteristic of PCOS in the mouse model.
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Hormônio Foliculoestimulante/farmacologia , Síndrome do Ovário Policístico/tratamento farmacológico , Adjuvantes Imunológicos/toxicidade , Administração Oral , Análise de Variância , Animais , Aromatase/metabolismo , Desidroepiandrosterona/toxicidade , Modelos Animais de Doenças , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/administração & dosagem , Hormônios Esteroides Gonadais/metabolismo , Hiperandrogenismo/induzido quimicamente , Hiperandrogenismo/tratamento farmacológico , Injeções Intradérmicas , Camundongos Endogâmicos BALB C , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Proteínas Recombinantes , Aumento de PesoRESUMO
The dramatic increase in the number of animals required for reproductive toxicity testing imposes the validation of alternative methods to reduce the use of laboratory animals. As we previously demonstrated for in vitro maturation test of bovine oocytes, the present study describes the transferability assessment and the inter-laboratory variability of an in vitro test able to identify chemical effects during the process of bovine oocyte fertilization. Eight chemicals with well-known toxic properties (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486) were tested in two well-trained laboratories. The statistical analysis demonstrated no differences in the EC50 values for each chemical in within (inter-runs) and in between-laboratory variability of the proposed test. We therefore conclude that the bovine in vitro fertilization test could advance toward the validation process as alternative in vitro method and become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.
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Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Acetoacetatos/toxicidade , Animais , Benzo(a)pireno/toxicidade , Bussulfano/toxicidade , Cloreto de Cádmio/toxicidade , Bovinos , Cicloeximida/toxicidade , Dietilestilbestrol/toxicidade , Cetoconazol/toxicidade , Laboratórios , Mifepristona/toxicidade , Oócitos , Reprodutibilidade dos TestesRESUMO
Oocyte in vitro maturation (IVM) has become a valuable technological tool for animal breeding and cloning and the treatment of human infertility because it does not require the administration of exogenous gonadotropin to obtain fertilizable oocytes. However, embryo development after IVM is lower compared to in vivo maturation, most likely because oocytes collected for IVM are heterogeneous with respect to their developmental competencies. Attempts to improve IVM outcome have relied upon either prematuration culture (PMC) or two-step maturation strategies in the hope of normalizing variations in developmental competence. Such culture systems invoke the pharmacological arrest of meiosis, in theory providing oocytes sufficient time to complete the acquisition of developmental competence after cumulus-enclosed oocytes isolation from the follicle. The present study was designed to test the efficiency of natriuretic peptide precursor C (NPPC) as a nonpharmacologic meiosis-arresting agent during IVM in a monoovulatory species. NPPC has been shown to maintain meiotic arrest in vivo and in vitro in mice and pigs; however, the use of this molecule for PMC has yet to have been explored. Toward this end, meiotic cell cycle reentry, gap-junction functionality, and chromatin configuration changes were investigated in bovine cumulus-enclosed oocytes cultured in the presence of NPPC. Moreover, oocyte developmental competence was investigated after IVM, in vitro fertilization, and embryo culture and compared to standard IVM-in vitro fertilization protocol without PMC. Our results suggest that NPPC can be used to delay meiotic resumption and increase the developmental competence of bovine oocytes when used in PMC protocols.
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Comunicação Celular , Células do Cúmulo/fisiologia , Junções Comunicantes/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Oocistos/citologia , Oogênese , Precursores de Proteínas/metabolismo , Matadouros , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Bovinos , Comunicação Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Células do Cúmulo/efeitos dos fármacos , Ectogênese/efeitos dos fármacos , Técnicas de Cultura Embrionária , Feminino , Fertilização in vitro , Junções Comunicantes/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Prófase Meiótica I/efeitos dos fármacos , Peptídeo Natriurético Tipo C/farmacologia , Oocistos/efeitos dos fármacos , Oocistos/metabolismo , Oogênese/efeitos dos fármacos , Inibidores da Fosfodiesterase 3/farmacologia , Precursores de Proteínas/farmacologia , Quinolonas/farmacologiaRESUMO
Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.
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Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Adenilato Quinase/metabolismo , Creatina Quinase/metabolismo , Mitocôndrias/metabolismo , Oócitos/metabolismo , Oogênese , Matadouros , Adenilato Quinase/antagonistas & inibidores , Adenilato Quinase/genética , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Blastocisto/ultraestrutura , Bovinos , Creatina Quinase/antagonistas & inibidores , Creatina Quinase/genética , Ectogênese/efeitos dos fármacos , Técnicas de Cultura Embrionária , Inibidores Enzimáticos/farmacologia , Feminino , Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Oogênese/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacosRESUMO
High-producing dairy cows are subfertile; however, the mechanisms responsible for the decreased fertility are unknown. The aim of the present study was to test the hypothesis that culled dairy cows (4-8 years old) characterised by 'Lo' ovaries (i.e. those with <10 mid-antral follicles) are affected by premature ovarian senescence. Cows in which both ovaries were 'Lo' ovaries represented 5% of the total population analysed, and exhibited reduced ovarian size (P<0.001) and increased perifollicular stroma (P<0.05) compared with age-matched controls (i.e. cows in which both ovaries had >10 mid-antral follicles; 'Hi' ovaries). The total number of follicles, including healthy and atretic primordial, primary, secondary and small antral follicles, was lower in Lo ovaries (P<0.01). Interestingly, the primordial follicle population in Lo ovaries was lower (P<0.05) than in the control. Finally, the follicular fluid of mid-antral follicles from Lo ovaries had reduced oestradiol and anti-Müllerian hormone levels (P<0.05), but increased progesterone concentrations (P<0.05). Together, these data account for the reduced fertility of cows with Lo ovaries and are in agreement with previous observations that oocytes isolated from Lo ovaries have reduced embryonic developmental competence. Cows with a specific Lo ovary condition may represent a suitable model to address the causes of low fertility in high-yielding dairy cows, as well as the condition of premature ovarian aging in single-ovulating species.
Assuntos
Doenças dos Bovinos/patologia , Senescência Celular , Indústria de Laticínios , Infertilidade Feminina/veterinária , Folículo Ovariano/patologia , Insuficiência Ovariana Primária/veterinária , Fatores Etários , Animais , Hormônio Antimülleriano/metabolismo , Bovinos , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/fisiopatologia , Estradiol/metabolismo , Feminino , Fertilidade , Líquido Folicular/metabolismo , Infertilidade Feminina/metabolismo , Infertilidade Feminina/patologia , Infertilidade Feminina/fisiopatologia , Folículo Ovariano/metabolismo , Folículo Ovariano/fisiopatologia , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/patologia , Insuficiência Ovariana Primária/fisiopatologia , Progesterona/metabolismo , Células Estromais/patologiaRESUMO
The LIVESTRONG Centers of Excellence were funded to increase the effectiveness of survivorship care in oncology practice. This study describes the ongoing process of adopting and implementing survivorship care using the framework of the diffusion of innovation theory of change. Primary data collection included telephone interviews with 39 members from the eight centers and site visits. Organizational characteristics, overall progress, and challenges for implementation were collected from proposals and annual reports. Creating an awareness of cancer survivorship care was a major accomplishment (relative advantage). Adoption depended on the fit within the cancer center (compatibility), and changed over time based on trial and error (trialability). Implementing survivorship care within the existing culture of oncology and breaking down resistance to change was a lengthy process (complexity). Survivorship care became sustainable as it became reimbursed, and more new patients were seen (observability). Innovators and early adopters were crucial to success. Diffusion of innovation theory can provide a strategy to evaluate adoption and implementation of cancer survivorship programs into clinical practice.
RESUMO
In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 µM cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P < 0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT.
Assuntos
Comunicação Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Técnicas de Transferência Nuclear/veterinária , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Quinolonas/farmacologia , Suínos/fisiologia , Animais , Células Cultivadas , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Relação Dose-Resposta a Droga , Feminino , Meiose/efeitos dos fármacos , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Partenogênese/fisiologiaRESUMO
PURPOSE: This study was aimed to test the hypothesis that gap junction mediated communications (GJC) are required to allow the progressive chromatin configuration remodeling (from GV1 to GV3) process to occur in fully grown oocytes in order to gain the final step of developmental competence acquisition, and that a premature disruption of GJC can alter this process. METHODS: Bovine cumulus-oocytes complexes collected from medium antral follicles were cultured for 2, 4, 6 and 8 h in the presence of 10(-4) IU/ml of r-hFSH and with 2 mM of the non-selective PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) to prevent meiotic resumption. GJC functionality and chromatin configuration were monitored during the culture period. After meiotic arrest, the developmental capability of oocytes was assessed after IVM and IVF. RESULTS: IBMX was effective in significantly sustaining GJC up to 6 h and maintaining meiotic arrest, when compared to control group. Moreover, the percentage of oocytes with less condensed chromatin (GV1) decreased within 4 h of culture, while the proportion of GV2 oocytes gradually increased up to 6 h. Interestingly, a decline in the proportion of GV2 oocytes and an increase in the proportion of GV3 oocytes were observed after 6 h of culture, when the major drop of GJC occurred. On the contrary, when GJC were uncoupled by adding 3 mM of 1-heptanol or through cumulus cells removal, chromatin condensation occurred rapidly throughout the culture period, more promptly in denuded oocytes. Moreover, the maintenance of GJC during meiotic arrest was accompanied by a significant increase of developmental competence compared to the control, as indicated by a higher percentage of hatched blastocysts and blastocyst cell number. CONCLUSIONS: Altogether, our data indicate that both paracrine and junctional mechanisms are involved in modulating large-scale chromatin structure during the final phase of oocyte differentiation.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/crescimento & desenvolvimento , Diester Fosfórico Hidrolases/administração & dosagem , Animais , Bovinos , Comunicação Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Feminino , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/genética , Técnicas de Maturação in Vitro de Oócitos , Meiose/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oogênese/efeitos dos fármacosRESUMO
Oocytes isolated from cows of reproductive age with reduced antral follicle counts (AFC) have a diminished capacity of embryonic development, which may be related to alterations in the mechanism that directs the proper segregation of chromosomes. Because we demonstrated that progesterone receptor membrane component 1 (PGRMC1) is involved in chromosome congression and metaphase II (MII) plate formation, the present study was designed to determine 1) if the decrease in oocyte developmental competence observed in dairy cows with a reduced AFC is due to a higher incidence of aneuploidy and 2) whether alterations in PGRMC1 contributes to the incidence of aneuploidy. Oocytes from ovaries with reduced AFC and age-matched controls were matured in vitro and the occurrence of aneuploidy determined as well as the mRNA level and localization of PGRMC1. Although oocytes from ovaries with reduced AFC were capable of undergoing meiosis in vitro, these oocytes showed a 3-fold increase in aneuploidy compared to oocytes isolated from control ovaries (P < 0.05). Although Pgrmc1 mRNA levels were not altered, PGRMC1 and aurora kinase B (AURKB) failed to localize to precise focal points on MII chromosomes of oocytes from ovaries with reduced AFC. Furthermore, when oocytes of control ovaries were cultured with an inhibitor of AURKB activity, their MII plate was disrupted and PGRMC1 was not properly localized to the chromosomes. These results suggest that alterations in PGRMC1 and/or AURKB localization account in part for the increased aneuploidy and low development competence of oocytes from ovaries with reduced AFC.