Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.050
Filtrar
1.
Science ; 368(6487): 181-186, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32273467

RESUMO

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.


Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Microscopia Intravital/métodos , Crista Neural , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Divisão Celular , Movimento Celular , Quimera/embriologia , Quimera/fisiologia , Eletroporação , Feminino , Técnicas de Transferência de Genes , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica , Crista Neural/irrigação sanguínea , Crista Neural/citologia , Crista Neural/embriologia , Placenta/fisiologia , Gravidez , Retina/embriologia , Retina/fisiologia , Transmissão Sináptica , Útero
2.
J Dairy Sci ; 103(6): 5641-5646, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32307164

RESUMO

The aim of this study was to determine the association between estrous expression, measured using a breeding indicator and an automated activity monitor (AAM), and the success of embryo collection after superovulation. Holstein heifers (n = 51; 10.5 to 14.5 mo, and 325.0 ± 21.1 kg of body weight) were superovulated (n = 69 events) for the collection of embryos using a protocol based on sequential administration of FSH for follicle superstimulation and GnRH to induce ovulation. Artificial insemination (AI) was performed twice, once at the moment of GnRH administration and again 12 h later, using thawed, sexed semen. Ovaries were scanned via ultrasonography on the day of the first AI to count the total number of preovulatory follicles and 7 d later for the total number of corpora lutea present. Embryos were collected 7 d post-AI, counted, and assessed for viability. A breeding indicator (Estrotect, Rockway Inc., Spring Valley, WI) and a collar-mounted AAM (CowScout Activity Monitoring System, GEA, Dusseldorf, Germany) were used to measure standing mounts and an algorithmic estimate of estrous expression, respectively. A score for the breeding indicator was given as follows: score 1 = 100% of the indicator was intact; score 2 = 50% of the indicator was rubbed off; score 3 = greater than 50% of the indicator was rubbed off. Estrous expression detected by the AAM was quantified through the relative increase in physical activity and duration of time spent above a set threshold. Data were analyzed by ANOVA using the MIXED procedures of SAS (SAS Institute Inc., Cary, NC). The number of follicles present at AI was not affected by estrous expression. The mean (± SD) ovulatory response was 67.5 ± 26.3%. We found an effect of estrous expression as detected by the breeding indicator on the ovulatory response (42.1 ± 8.0, vs. 78.2 ± 9.0, vs. 74.0 ± 4.9%, for scores 1, 2, and 3, respectively) but not from the AAM. Heifers that had a score of 3 (versus those with scores of 1 and 2) on the breeding indicator had a greater number of embryos (4.1 ± 0.5, vs. 1.2 ± 1.0, vs. 1.8 ± 1.0 embryos), and a greater percentage of these embryos were viable (43.1 ± 0.05, vs. 35.5 ± 0.1, vs. 34.3 ± 0.1%). Similarly, heifers that showed a greater intensity of activity (as measured by the AAM) had a greater number of embryos collected (10.2 ± 1.2 vs. 6.0 ± 1.3 embryos), and a greater percentage of those embryos were viable (53.1 ± 5.0 vs. 23.4 ± 5.1%). Longer-duration estrus episodes were associated with a higher percentage of viable embryos (51.2 ± 5.2 vs. 25.3 ± 5.3%). In conclusion, stronger estrous intensity was associated with a greater number of total embryos collected and a greater percentage of viable embryos. These results suggest that monitoring the intensity of estrus could be used to predict superovulatory response as well as embryo quality in Holstein heifers.


Assuntos
Bovinos/embriologia , Embrião de Mamíferos/fisiologia , Estro/fisiologia , Superovulação , Animais , Corpo Lúteo/efeitos dos fármacos , Sincronização do Estro/métodos , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Inseminação Artificial/veterinária , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Progesterona/farmacologia , Coleta de Tecidos e Órgãos
3.
PLoS One ; 15(4): e0231108, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251418

RESUMO

Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated legal and ethical issues, and delaying childbearing years. While the outcome of oocyte cryopreservation has recently been improved, currently used vitrification method still suffer from increased biosafety risk and handling issues while slow freezing techniques yield overall low success. Understanding better the mechanism of cryopreservation-induced injuries may lead to development of more reliable and safe methods for oocyte cryopreservation. Using the mouse model, a microarray study was conducted on oocyte cryopreservation to identify cryoinjuries to transcriptionally active genome. To this end, metaphase II (MII) oocytes were subjected to standard slow freezing, and then analyzed at the four-cell stage after embryonic genome activation. Non-frozen four-cell embryos served as controls. Differentially expressed genes were identified and validated using RT-PCR. Embryos produced from the cryopreserved oocytes displayed 200 upregulated and 105 downregulated genes, associated with the regulation of mitochondrial function, protein ubiquitination and maintenance, cellular response to stress and oxidative states, fatty acid and lipid regulation/metabolism, and cell cycle maintenance. These findings reveal previously unrecognized effects of standard slow oocyte freezing on embryonic gene expression, which can be used to guide improvement of oocyte cryopreservation methods.


Assuntos
Criopreservação/normas , Embrião de Mamíferos/fisiologia , Congelamento/efeitos adversos , Oócitos/fisiologia , Transcriptoma/genética , Animais , Desenvolvimento Embrionário/genética , Feminino , Fertilização In Vitro/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Metáfase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mapas de Interação de Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real
4.
Am J Hum Genet ; 106(4): 525-534, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32220293

RESUMO

Despite next-generation sequencing, which now allows for the accurate detection of segmental aneuploidies from in vitro fertilization embryo biopsies, the origin and characteristics of these aneuploidies are still relatively unknown. Using a multifocal biopsy approach (four trophectoderms [TEs] and one inner cell mass [ICM] analyzed per blastocyst; n = 390), we determine the origin of the aneuploidy and the diagnostic predictive value of segmental aneuploidy detection in TE biopsies toward the ICM's chromosomal constitution. Contrary to the prevalent meiotic origin of whole-chromosome aneuploidies, we show that sub-chromosomal abnormalities in human blastocysts arise from mitotic errors in around 70% of cases. As a consequence, the positive-predictive value toward ICM configuration was significantly lower for segmental as compared to whole-chromosome aneuploidies (70.8% versus 97.18%, respectively). In order to enhance the clinical utility of reporting segmental findings in clinical TE biopsies, we have developed and clinically verified a risk stratification model based on a second TE biopsy confirmation and segmental length; this model can significantly improve the prediction of aneuploidy risk in the ICM in over 86% of clinical cases enrolled. In conclusion, we provide evidence of the predominant mitotic origin of segmental aneuploidies in preimplantation embryos and develop a risk stratification model that can help post-test genetic counseling and that facilitates the decision-making process on clinical utilization of these embryos.


Assuntos
Blastocisto/fisiologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/genética , Aneuploidia , Aberrações Cromossômicas , Cromossomos/genética , Hibridização Genômica Comparativa/métodos , Feminino , Fertilização In Vitro/métodos , Humanos , Incidência , Gravidez , Diagnóstico Pré-Implantação/métodos
5.
Proc Natl Acad Sci U S A ; 117(11): 5938-5942, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123078

RESUMO

Reproduction in mammals requires distinct cycles of ovulation, fertilization, pregnancy, and lactation often interspersed with periods of anoestrus when breeding does not occur. Macropodids, the largest extant species of marsupials, the kangaroos and wallabies, have a very different reproductive strategy to most eutherian mammals whereby young are born at a highly altricial stage of development with the majority of development occurring over a lengthy lactation period. Furthermore, the timings of ovulation and birth in some species occurs within a very short interval of each other (sometimes hours). Female swamp wallabies have an oestrous cycle shorter than their pregnancy length and were, therefore, speculated to mate and form a new embryo before birth thereby supporting two conceptuses at different stages of pregnancy. To confirm this, we used high-resolution ultrasound to monitor reproduction in swamp wallabies during pregnancy. Here, we show that females ovulate, mate, and form a new embryo prepartum while still carrying a full-term fetus in the contralateral uterus. This embryo enters embryonic diapause until the newborn leaves the pouch 9 mo later. Thus, combined with embryonic diapause, females are continuously pregnant and lactating at the same time throughout their reproductive life, a unique reproductive strategy that completely blurs the normal staged system of reproduction in mammals.


Assuntos
Macropodidae/fisiologia , Gravidez/fisiologia , Reprodução/fisiologia , Áreas Alagadas , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Ciclo Estral , Feminino , Lactação , Macropodidae/embriologia , Ovulação , Parto , Ultrassonografia , Vitória
7.
Animal ; 14(S1): s103-s112, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32024564

RESUMO

Assisted reproduction techniques (ARTs) provide access to early stage embryos whose analysis and assessment deliver valuable information. The handling of embryos, including the in vitro production of bovine embryos, is a rapidly evolving area which nonetheless exposes the embryos to unnatural conditions for a period of time. The Fallopian tube provides innumerable quantitative and qualitative factors, all of which guarantee the successful development of the embryo. It is well known that the Fallopian tube can be bypassed, using embryo transfer, resulting in successful implantation in the target recipient animal and the birth of calves. However, the question arises as to whether such circumvention has a negative impact on the embryo during this sensitive development period. First crosstalk between the embryo and its environment confirms mutual recognition activities and indicate bilateral effects. Nowadays, in vitro production of bovine embryos is a well-established technology. However, it is still evident that in vitro generated embryos are not qualitatively comparable to embryos obtained ex vivo. To counteract these differences, comparative studies between in vitro and ex vivo embryos are advantageous, as embryos grown in their physiological environment can provide a blueprint or gold standard against which to compare embryos produced in vitro. Attempts to harness the bovine oviduct were sometimes very invasive and did not result in wide acceptance and routine use. Long-term development and refinement of transvaginal endoscopy for accessing the bovine oviduct has meanwhile been routinely applied for research as well as in practice. Comparative studies combining in vitro development with development in the cattle oviduct revealed that the environmental conditions to which the embryo is exposed before activation of the embryonic genome can have detrimental and lasting effects on its further development. These effects are manifested as deviations in gene expression profiles and methylation signatures as well as frequency of whole chromosomal or segmental aberrations. Furthermore, it was shown that hormonal superstimulation (multiple ovulation and embryo transfer), varying progesterone concentrations as well as metabolic disorders caused by high milk production, markedly affected embryo development in the postpartum period. Assisted reproductive techniques that allow the production and handling of extra numbers of generated embryos promise to have a very high impact on scientific and practical application. Any influence on the early embryonic life, both in animals and in vitro, is accompanied by a sensitive change in embryonic activity and should be assessed in vivo on the basis of physiological conditions before being used for ART.


Assuntos
Bovinos/fisiologia , Desenvolvimento Embrionário/fisiologia , Meio Ambiente , Reprodução , Animais , Bovinos/embriologia , Implantação do Embrião , Transferência Embrionária/veterinária , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/fisiologia , Tubas Uterinas/embriologia , Tubas Uterinas/fisiologia , Feminino , Oviductos/embriologia , Oviductos/fisiologia , Gravidez , Progesterona/metabolismo
8.
Fertil Steril ; 113(3): 578-586.e1, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32044089

RESUMO

OBJECTIVE: To determine if a dynamic embryo culture system affects the reproductive potential of human embryos resulting from in vitro fertilization (IVF). DESIGN: Paired randomized controlled trial (RCT). SETTING: IVF center. PATIENT(S): IVF patients with normal ovarian reserve eligible for two-embryo transfer. INTERVENTION: IVF care was routine until fertilization was confirmed. Two-pronuclear embryos (2PNs) were then randomized: One-half of each patient's 2PNs were cultured in dynamic culture and one-half in static culture. Preimplantation genetic testing for embryonic aneuploidy was used to control for aneuploidy and allow for DNA fingerprinting. The best euploid blastocyst from each culture system was selected and patients underwent a frozen two-embryo transfer. If a singleton gestation resulted, DNA-fingerprinting was used to determine which of the two blastocysts implanted. The dynamic platform used was the NSSB-300 (Nepagene). MAIN OUTCOME MEASURE(S): The primary outcome was the proportion of usable blastocysts obtained. The secondary outcome was sustained implantation rate (SIR). RESULT(S): One hundred participants completed oocyte retrieval and blastocyst vitrification for frozen-thawed embryo transfer; 609 dynamic 2PNs and 615 static 2PNs were followed; and 304 blastocysts developed in dynamic culture and 333 blastocysts developed in static culture. In the paired analysis, the rate of usable blastulation was similar between dynamic and static culture (58.3% vs. 57.1%). In addition, there was no difference in the rate of aneuploidy (20.0% vs. 33.3%) or SIR (67.1% vs. 63.1%) between groups. CONCLUSION(S): In this paired RCT, dynamic culture did not improve usable blastulation rate or SIR. CLINICAL TRIAL REGISTRATION NUMBER: NCT02467725.


Assuntos
Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/fisiologia , Hidrodinâmica , Movimento (Física) , Adulto , Células Cultivadas , Implantação do Embrião/fisiologia , Transferência Embrionária , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilização In Vitro/métodos , Humanos , Gravidez , Taxa de Gravidez
9.
Dev Cell ; 52(3): 321-334.e6, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32049039

RESUMO

Epithelial fusion is a key process of morphogenesis by which tissue connectivity is established between adjacent epithelial sheets. A striking and poorly understood feature of this process is "zippering," whereby a fusion point moves directionally along an organ rudiment. Here, we uncover the molecular mechanism underlying zippering during mouse spinal neural tube closure. Fusion is initiated via local activation of integrin ß1 and focal anchorage of surface ectoderm cells to a shared point of fibronectin-rich basement membrane, where the neural folds first contact each other. Surface ectoderm cells undergo proximal junction shortening, establishing a transitory semi-rosette-like structure at the zippering point that promotes juxtaposition of cells across the midline enabling fusion propagation. Tissue-specific ablation of integrin ß1 abolishes the semi-rosette formation, preventing zippering and causing spina bifida. We propose integrin-mediated anchorage as an evolutionarily conserved mechanism of general relevance for zippering closure of epithelial gaps whose disturbance can produce clinically important birth defects.


Assuntos
Embrião de Mamíferos/fisiologia , Células Epiteliais/fisiologia , Adesões Focais , Integrina beta1/fisiologia , Crista Neural/embriologia , Tubo Neural/embriologia , Neurulação , Actomiosina/metabolismo , Animais , Fusão Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Crista Neural/metabolismo , Crista Neural/fisiologia , Tubo Neural/metabolismo , Tubo Neural/fisiologia
10.
Cell Mol Life Sci ; 77(16): 3177-3194, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32095869

RESUMO

The degradation of maternally provided molecules is a very important process during early embryogenesis. However, the vast majority of studies deals with mRNA degradation and protein degradation is only a very little explored process yet. The aim of this article was to summarize current knowledge about the protein degradation during embryogenesis of mammals. In addition to resuming of known data concerning mammalian embryogenesis, we tried to fill the gaps in knowledge by comparison with facts known about protein degradation in early embryos of non-mammalian species. Maternal protein degradation seems to be driven by very strict rules in terms of specificity and timing. The degradation of some maternal proteins is certainly necessary for the normal course of embryonic genome activation (EGA) and several concrete proteins that need to be degraded before major EGA have been already found. Nevertheless, the most important period seems to take place even before preimplantation development-during oocyte maturation. The defects arisen during this period seems to be later irreparable.


Assuntos
Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/fisiologia , Proteínas/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genoma/fisiologia , Humanos , Oócitos/metabolismo , Oócitos/fisiologia
11.
Cell Prolif ; 53(2): e12754, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31916359

RESUMO

The abnormalities of early post-implantation embryos can lead to early pregnancy loss and many other syndromes. However, it is hard to study embryos after implantation due to the limited accessibility. The success of embryo culture in vitro can avoid the challenges of embryonic development in vivo and provide a powerful research platform for research in developmental biology. The biophysical and chemical cues of the microenvironments impart significant spatiotemporal effects on embryonic development. Here, we summarize the main strategies which enable researchers to grow embryos outside of the body while overcoming the implantation barrier, highlight the roles of engineered microenvironments in regulating early embryonic development, and finally discuss the future challenges and new insights of early embryo culture.


Assuntos
Microambiente Celular/fisiologia , Implantação do Embrião/fisiologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Animais , Humanos
12.
PLoS One ; 15(1): e0226735, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31917811

RESUMO

The major milestones in mouse placental development are well described, but our understanding is limited to how the placenta can adapt to damage or changes in the environment. By using stereology and expression of cell cycle markers, we found that the placenta grows under normal conditions not just by hyperplasia of trophoblast cells but also through extensive polyploidy and cell hypertrophy. In response to feeding a low protein diet to mothers prior to and during pregnancy, to mimic chronic malnutrition, we found that this normal program was altered and that it was influenced by the sex of the conceptus. Male fetuses showed intrauterine growth restriction (IUGR) by embryonic day (E) 18.5, just before term, whereas female fetuses showed IUGR as early as E16.5. This difference was correlated with differences in the size of the labyrinth layer of the placenta, the site of nutrient and gas exchange. Functional changes were implied based on up-regulation of nutrient transporter genes. The junctional zone was also affected, with a reduction in both glycogen trophoblast and spongiotrophoblast cells. These changes were associated with increased expression of Phlda2 and reduced expression of Egfr. Polyploidy, which results from endoreduplication, is a normal feature of trophoblast giant cells (TGC) but also spongiotrophoblast cells. Ploidy was increased in sinusoidal-TGCs and spongiotrophoblast cells, but not parietal-TGCs, in low protein placentas. These results indicate that the placenta undergoes a range of changes in development and function in response to poor maternal diet, many of which we interpret are aimed at mitigating the impacts on fetal and maternal health.


Assuntos
Aclimatação , Dieta com Restrição de Proteínas/efeitos adversos , Embrião de Mamíferos/citologia , Retardo do Crescimento Fetal/etiologia , Privação de Alimentos , Placenta/citologia , Animais , Proliferação de Células , Embrião de Mamíferos/fisiologia , Feminino , Desenvolvimento Fetal , Retardo do Crescimento Fetal/patologia , Células Gigantes , Masculino , Fenômenos Fisiológicos da Nutrição Materna , Camundongos , Camundongos Endogâmicos C57BL , Placenta/fisiologia , Gravidez , Trofoblastos/citologia , Trofoblastos/fisiologia
13.
J Dairy Sci ; 103(3): 2773-2783, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31954558

RESUMO

This study aimed to evaluate the effects of plane of nutrition and advancing days of pregnancy (DP) on maternal body composition and fetal development. Differing planes of nutrition were established by 2 feeding regimens (FR): ad libitum (AL) or maintenance (MA). Sixty-two nonlactating multiparous Holstein × Gyr cows with average body weight of 480 ± 10.1 kg and an age of 5 ± 0.5 yr were used. Cows were divided into 3 groups: pregnant (n = 44), nonpregnant (n = 12), and baseline reference cows (n = 6). The 56 pregnant and nonpregnant cows were randomly allocated into 2 different FR: AL or MA. Cows fed at MA received 1.15% of their body weight on a dry matter (DM) basis, receiving corn silage and a concentrate-based diet at a ratio of 93:7 on a DM basis. Reference group cows were slaughtered at the beginning of the experimental period to estimate body composition and empty body weight. To evaluate the effects of DP, pregnant and nonpregnant animals were slaughtered at d 140, 200, 240, and 270 of gestation. Feeding regimen affected maternal tissue composition. Days of pregnancy affected fresh weight (FW), DM, and energy content, but no differences were observed for crude protein (CP) and ether extract (EE) because of DP. Feeding regimen affected mammary gland components (CP, EE, and energy content), but not fresh or dry weights. Days of pregnancy influenced almost all mammary gland components except energy content. Regarding the uterus, FR affected only fresh and dry weights; however, DP affected every uterus component measured. The only interaction between FR and DP in this study was observed for placental FW. Cows fed AL on d 270 presented the same placental FW as cows at MA and AL on d 200 and 240. Further, pregnant cows fed at MA on d 270 had greater placental FW than cows fed AL at this day. Days of pregnancy, but not FR, influenced the composition of fetal fluids in pregnant cows. Finally, cows fed at MA had greater FW for the fetus than cows fed AL; however, fetus composition changed over DP. The FW, DM, EE, and energy content increased until d 270, but CP decreased. In conclusion, the novelty of our data presents how changes due to FR and DP occur in maternal tissues and the conceptus.


Assuntos
Composição Corporal , Bovinos/fisiologia , Desenvolvimento Fetal , Silagem/análise , Animais , Peso Corporal , Bovinos/embriologia , Dieta/veterinária , Embrião de Mamíferos/fisiologia , Feminino , Lactação , Gravidez , Distribuição Aleatória , Útero/fisiologia , Zea mays
14.
Dev Cell ; 52(2): 139-140, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31991103

RESUMO

Embryonic diapause is the reversible arrest in development of mammalian embryos at the blastocyst stage. In this issue of Developmental Cell, Hussein et al. (2020) reveal that alternative splicing of Lkb1 is essential for diapause to persist and find the elevation of glycolytic and lipolytic pathways that were previously considered dormant.


Assuntos
Blastocisto/metabolismo , Implantação Tardia do Embrião/fisiologia , Embrião de Mamíferos/fisiologia , Proteínas Serina-Treonina Quinases/genética , Processamento Alternativo , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos
15.
FASEB J ; 34(1): 1637-1651, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914649

RESUMO

Studies on the effects of transcriptional memory on clone reprogramming in mammals are limited. In the present study, we observed higher levels of active histone H3 lysine 4 trimethylation (H3K4me3 and 5-hydroxymethylcytosine) and repressive (5-methylcytosine) epigenetic modifications in bovine early cloned embryos than in in vitro fertilized embryos. We hypothesized that aberrant epigenetic modification may result in transcriptional disorders in bovine somatic cell nuclear transfer (SCNT) embryos. RNA sequencing results confirmed that both abnormal transcriptional silencing and transcriptional activation are involved in bovine SCNT reprogramming. The cloned embryos exhibited excessive transcription in RNA processing- and translation-related genes as well as transcriptional defects in reproduction-related genes whose transcriptional profiles were similar to those in donor cells. These results demonstrated the existence of active and silent memory genes inherited from donor cells in early bovine SCNT embryos. Further, H3K4me3-specific demethylase 5B (KDM5B) mRNA was injected into the reconstructed embryos to reduce the increased H3K4me3 modification. KDM5B overexpression not only reduced the transcriptional level of active memory genes, but also promoted the expression of silent memory genes; in particular, it rescued the expression of multiple development-related genes. These results showed that transcriptional memory acts as a reprogramming barrier and KDM5B improves SCNT reprogramming via bidirectional regulation effects on transcriptional memory genes in bovines.


Assuntos
Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Transcrição Genética/genética , Animais , Bovinos , Reprogramação Celular/genética , Clonagem de Organismos/métodos , Epigênese Genética/genética , Fertilização In Vitro/métodos , Histonas/genética , Técnicas de Transferência Nuclear , Processamento de Proteína Pós-Traducional/genética
16.
Pol J Vet Sci ; 22(4): 711-716, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31867922

RESUMO

Feeder cells can promote cell proliferation and help overcome the developmental arrest of early embryos by producing growth factors. The objective of this study was to evaluate the effects of feeder cells on the development of all single porcine parthenogenetic embryos in vitro. Firstly, we showed that the cleavage and blastocyst formation rate of all single procine parthenogenetic embryos co-cultured with feeder cells increased in contrast to those cultured without feeder cells (p⟨0.05). However, no statistically significant differences were observed between the blastocyst formation rate in the embryos co-cultured with 3 different kinds feeder cells namely oviduct epithelial feeder cells, granulose feeder cells and porcine fetal fibroblast feeder cells (p>0.05). Secondly, highly significant differences were observed between the cleavage and blastocyst formation rate (p⟨0.05) when the embryos were co-cultured with oviduct epithelial feeder cells in different volume drops ranging from 3 to 20 µL and the cleavage rate were the highest when cultured in 5 µL drops. Thirdly, the tempospacial pattern of the development of single embryos co-cultured with oviduct epithelial feeder cells was consistent with that of traditional multi-embryo culture, indicating that the co-culturing does not affect the developmental competence of the porcine parthenogenetic embryos. Finally, highly significant differences were observed between the cleavage and blastocyst formation rate with and without zona pellucida in vitro (p⟨0.05). In this study, a new adaption of in vitro co-culture of single porcine parthenogenetic embryos using feeder cells has been successfully established and this will facilitate further investigations to discover the mechanistic mode of developmental arrest of porcine embryos.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos/fisiologia , Células Epiteliais/fisiologia , Células da Granulosa/fisiologia , Partenogênese , Suínos/embriologia , Animais , Técnicas de Cocultura , Tubas Uterinas/citologia , Feminino
17.
PLoS One ; 14(12): e0226419, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856190

RESUMO

Morphological assessment at defined developmental stages is the most important method to select viable embryos for transfer and cryopreservation. Timing of different developmental stages in embryo development has been shown to correlate with its potential to develop into a blastocyst. However, improvements in pregnancy rates by using time-lapse techniques have been difficult to validate scientifically. Therefore, there is a need for new methods, preferably non-invasive methods based on metabolomics, genomics and proteomics, to improve the evaluation of embryo quality even further. The aim of this study was to investigate if different levels of caspase-3 and histidine-rich glycoprotein (HRG), secreted by the embryo into the culture media, can be used as biomarkers of embryo quality. In this study, a total of 334 samples of culture media were collected from in vitro fertilization (IVF) treatments at three different clinics. Protein analysis of the culture media was performed using multiplex proximity extension protein analysis to detect levels of caspase-3 and HRG in the embryo secretome. Protein levels were compared in secretome samples from high- and low-quality blastocysts and embryos that became arrested during development. Correlation between protein levels and time to morula formation was also analyzed. Furthermore, protein levels in secretomes from day-2 cultured embryos were compared on the basis of whether or not pregnancy was achieved. The results showed that caspase-3 levels were lower in secretomes from high-quality vs. low-quality blastocysts and those that became arrested (p ≤ 0.05 for both). In addition, higher HRG levels correlated with a shorter time to morula formation (p ≤ 0.001). Caspase-3 levels were also lower in secretomes from day-2 cultured embryos resulting in a pregnancy vs. those that did not (p ≤ 0.05). Furthermore, it was shown that caspase-3 might be used as a marker for predicting potential success rate after transfer of day-2 cultured embryos, where a caspase-3 cutoff level of 0.02 gave a prediction probability of 68% (p = 0.038). In conclusion, in future prediction models, levels of caspase-3 and HRG might be used as potential markers of embryo quality, and secreted caspase-3 levels could to some extent predict the outcome after transfer of day-2 cultured embryos.


Assuntos
Blastocisto/enzimologia , Caspase 3/metabolismo , Embrião de Mamíferos/metabolismo , Proteínas/metabolismo , Adulto , Biomarcadores/metabolismo , Blastocisto/fisiologia , Criopreservação , Embrião de Mamíferos/fisiologia , Feminino , Fertilização In Vitro , Humanos , Masculino , Mórula/fisiologia , Gravidez , Resultado do Tratamento , Adulto Jovem
18.
Proc Natl Acad Sci U S A ; 116(51): 25677-25687, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31754036

RESUMO

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL's absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis-after organogenesis has begun-in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer.


Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas , Gônadas , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Feminino , Células Germinativas/metabolismo , Células Germinativas/fisiologia , Gônadas/citologia , Gônadas/fisiologia , Masculino , Camundongos , Neoplasias Ovarianas/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Suínos , Teratoma/genética , Neoplasias Testiculares/genética
19.
J Assist Reprod Genet ; 36(11): 2345-2355, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31696385

RESUMO

PURPOSE: To investigate whether the ability of human spermatozoa to decondense in vitro in the presence of heparin (Hep) and glutathione (GSH) is related to assisted reproduction (ART) success. METHODS: Cross-sectional pilot study involving male partners of 129 infertile couples undergoing ICSI with (45) or without (84) donor oocytes at two infertility clinics in CABA, Argentina, between October 2012 and December 2013. In vitro decondensation kinetics with Hep and GSH and DNA fragmentation (TUNEL) were determined on the same sample used for ICSI. The possible relationship of decondensation parameters (maximum decondensation and decondensation velocity) and TUNEL values with ART success was evaluated. RESULTS: Embryo quality correlated positively with decondensation velocity (D60/D30) (Spearman's correlation, p < 0.05). According to D60/D30 values, patients were classified as slow decondensers (SlowD) (n = 68) or fast decondensers (FastD) (n = 61). Embryo quality was better in FastD (unpaired t test, p < 0.05). FastD and SlowD were subdivided according to use of donor oocytes. Among SlowD, biochemical and clinical pregnancy rates per transfer were significantly higher in donor (n = 19) vs. in non-donor (n = 31) cycles (Fisher's exact test, p < 0.05). TUNEL values were not related to embryo quality, but no clinical pregnancies or live births were achieved in TUNEL+ SlowD (n = 7). CONCLUSION: Decondensation kinetics of human spermatozoa in vitro with Hep and GSH could be related to embryo quality and ART success.


Assuntos
Embrião de Mamíferos/fisiologia , Espermatozoides/fisiologia , Argentina , Estudos Transversais , Fragmentação do DNA , Feminino , Fertilização In Vitro/métodos , Humanos , Marcação In Situ das Extremidades Cortadas/métodos , Infertilidade/terapia , Nascimento Vivo , Masculino , Oócitos/fisiologia , Projetos Piloto , Gravidez , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas/métodos
20.
Reprod Biol Endocrinol ; 17(1): 84, 2019 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-31656205

RESUMO

BACKGROUND: In the absence of international guidelines indicating the usage of vitrification rather than slow-freezing, the study aim was to analyze a large cohort of slow-frozen/thawed embryos to produce a rationale supporting the standardization of IVF cryopreservation policy. METHODS: This retrospective analysis included 4779 cleavage stage embryos cryopreserved by slow-freezing/thawing from September 2009 to April 2017 at a single Center. Biological and clinical outcomes of three different commercial kits adopted sequentially, i.e. Vitrolife Cleave Kit® from Vitrolife (kit 1) vs. K-SICS-5000 Kit® and K-SITS-5000 Kit® from Cook Medical (kit 2) and Freeze/Thaw 1™ Kit® from Vitrolife (kit 3) were collected and compared in the light of cryoprotectants composition. RESULTS: Kit 3 compared to kit 1 and kit 2 showed significantly (P < 0.001) higher embryo survival (79.9% vs. 75.6 and 68.1%, respectively) and frozen embryo replacement (91.5% vs. 86.5 and 83.3%, respectively) rates, and significantly (P < 0.001) lower blastomere degeneration rate (41.5% vs. 43.6 and 52.4%, respectively). No significant difference for clinical outcomes was observed among kits. Only a slight positive trend was observed for kit 3 vs. kit 1 and kit 2 on delivery rate per thawing cycle (7.12% vs. 4.19 and 4.51%, respectively; P < 0.058) and live birth rate (3.07% vs. 2.59 and 1.93%, respectively, P < 0.069). Thawing solutions of kit 3 were similar to those of any warming protocol. CONCLUSIONS: A defined concentration of extracellular cryoprotectants in thawing/warming solutions had a beneficial effect on the embryo cryosurvival rate. Results could provide the rationale for the adoption of a single standardized warming protocol.


Assuntos
Blastômeros/fisiologia , Fase de Clivagem do Zigoto/fisiologia , Criopreservação/métodos , Crioprotetores/farmacologia , Embrião de Mamíferos/fisiologia , Vitrificação/efeitos dos fármacos , Blastômeros/citologia , Transferência Embrionária , Embrião de Mamíferos/citologia , Feminino , Humanos , Gravidez , Taxa de Gravidez , Estudos Retrospectivos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA