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1.
Reprod Domest Anim ; 59(6): e14627, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38837827

RESUMO

The efficiency of bovine in vitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of in vitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve in vitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.


Assuntos
Blastocisto , Blastômeros , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Animais , Bovinos/embriologia , Feminino , Técnicas de Cultura Embrionária/veterinária , Blastômeros/citologia , Fertilização in vitro/veterinária , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Gravidez
2.
Cell ; 187(11): 2838-2854.e17, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38744282

RESUMO

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.


Assuntos
Blastômeros , Linhagem da Célula , Embrião de Mamíferos , Feminino , Humanos , Blastômeros/citologia , Blastômeros/metabolismo , Divisão Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Masculino , Animais , Camundongos
3.
Eur J Obstet Gynecol Reprod Biol ; 297: 209-213, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38688135

RESUMO

The present study investigated whether day 3 blastomere number has an effect on the clinical outcomes during single vitrified-warmed blastocyst transfer cycles. A total of 3294 vitrified-warmed single day 5 blastocyst transferred cycles were analyzed in this retrospective study from January 2018 to December 2021. The cycles were divided into ≥ 7 and < 7 blastomere groups depending on the day 3 embryo blastomere number. The clinical outcomes were compared between the two groups, moreover multivariate logistic regression analysis was conducted to investigate the correlation between the number of day 3 blastomeres and clinical outcomes. The chi-square test demonstrated that the rates of clinical pregnancy and live birth were significantly higher in the ≥ 7 blastomere group compared to the < 7 blastomere group with respect to single high-quality blastocyst transfer cycles. Conversely, these rates were similar in the two groups with respect to single low-quality blastocyst transfer cycles. These results were confirmed by multivariate logistic regression analysis. However, the miscarriage rate was higher in the < 7 blastomere group than in ≥ 7 group during low-quality blastocyst transfer cycles. These results suggested that day 3 blastomere number should be considered during single vitrified-warmed blastocyst transfer cycles. Thus, blastocsyts derived from ≥ 7 blastomere embryos are preferred when choosing the same quality blastocysts.


Assuntos
Blastômeros , Transferência Embrionária , Taxa de Gravidez , Vitrificação , Humanos , Feminino , Estudos Retrospectivos , Gravidez , Blastômeros/citologia , Transferência Embrionária/métodos , Adulto , Criopreservação
4.
Reprod Biomed Online ; 48(5): 103763, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38452604

RESUMO

RESEARCH QUESTION: Embryo blastomeres and the zona pellucida are occasionally damaged during vitrification; is this a result of crack-induced mechanical damage in the glass state, caused by external bending of the device? DESIGN: A stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilized eggs were used to assess embryo damage under two external bending conditions: forward bending and backward bending, with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Damage was assessed by looking at blastomere lysis rates, and overall rates of damaged and surviving embryos. RESULTS: A series of parallel cracks were identified in the cryoprotectant used for external bending, which led to damage to the embryo blastomeres. Compared with forward bending and control, the embryos were found to be more easily damaged by backward bending, indicated by significantly higher blastomere lysis and embryo damage rates, and lower embryo survival rate of backward bending than forward bending (P < 0.001). The degree of embryo damage also increased as the degree of external forces increased. Embryo position correlated with degree of embryo damage. CONCLUSIONS: Cryoprotectant crack-induced damage was identified as the cause of embryo damage. Mechanical damage to the glass state occurs because of improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip.


Assuntos
Blastômeros , Criopreservação , Crioprotetores , Vitrificação , Humanos , Crioprotetores/farmacologia , Feminino , Embrião de Mamíferos/efeitos dos fármacos
5.
Artif Intell Med ; 149: 102773, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38462274

RESUMO

The selection of embryos is a key for the success of in vitro fertilization (IVF). However, automatic quality assessment on human IVF embryos with optical microscope images is still challenging. In this study, we developed a clinical consensus-compliant deep learning approach, named Esava (Embryo Segmentation and Viability Assessment), to quantitatively evaluate the development of IVF embryos using optical microscope images. In total 551 optical microscope images of human IVF embryos of day-2 to day-3 were collected, preprocessed, and annotated. Using the Faster R-CNN model as baseline, our Esava model was constructed, refined, trained, and validated for precise and robust blastomere detection. A novel algorithm Crowd-NMS was proposed and employed in Esava to enhance the object detection and to precisely quantify the embryonic cells and their size uniformity. Additionally, an innovative GrabCut-based unsupervised module was integrated for the segmentation of blastomeres and embryos. Independently tested on 94 embryo images for blastomere detection, Esava obtained the high rates of 0.9940, 0.9121, and 0.9531 for precision, recall, and mAP respectively, and gained significant advances compared with previous computational methods. Intraclass correlation coefficients indicated the consistency between Esava and three experienced embryologists. Another test on 51 extra images demonstrated that Esava surpassed other tools significantly, achieving the highest average precision 0.9025. Moreover, it also accurately identified the borders of blastomeres with mIoU over 0.88 on the independent testing dataset. Esava is compliant with the Istanbul clinical consensus and compatible to senior embryologists. Taken together, Esava improves the accuracy and efficiency of embryonic development assessment with optical microscope images.


Assuntos
Aprendizado Profundo , Gravidez , Feminino , Humanos , Consenso , Fertilização in vitro/métodos , Desenvolvimento Embrionário , Blastômeros
6.
Cell Rep ; 43(3): 113840, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38386558

RESUMO

Recent studies have elucidated Nr5a2's role in activating zygotic genes during early mouse embryonic development. Subsequent research, however, reveals that Nr5a2 is not critical for zygotic genome activation but is vital for the gene program between the 4- and 8-cell stages. A significant gap exists in experimental evidence regarding its function during the first lineage differentiation's pivotal period. In this study, we observed that approximately 20% of embryos developed to the blastocyst stage following Nr5a2 ablation. However, these blastocysts lacked inner cell mass (ICM), highlighting Nr5a2's importance in first lineage differentiation. Mechanistically, using RNA sequencing and CUT&Tag, we found that Nr5a2 transcriptionally regulates ICM-specific genes, such as Oct4, to establish the pluripotent network. Interference with or overexpression of Nr5a2 in single blastomeres of 2-cell embryos can alter the fate of daughter cells. Our results indicate that Nr5a2 works as a doorkeeper to ensure ICM formation in mouse blastocyst.


Assuntos
Blastocisto , Desenvolvimento Embrionário , Gravidez , Feminino , Animais , Camundongos , Desenvolvimento Embrionário/genética , Diferenciação Celular/genética , Blastômeros , Zigoto , Regulação da Expressão Gênica no Desenvolvimento , Receptores Citoplasmáticos e Nucleares/genética
7.
Methods Mol Biol ; 2740: 125-140, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38393473

RESUMO

The geometry of reductive divisions that mark the development of early embryos instructs cell fates, sizes, and positions, by mechanisms that remain unclear. In that context, new methods to mechanically manipulate these divisions are starting to emerge in different model systems. These are key to develop future innovative approaches and understand developmental mechanisms controlled by cleavage geometry. In particular, how cell cycle pace is regulated in rapidly reducing blastomeres and how fate diversity can arise from blastomere size and position within embryos are fundamental questions that remain at the heart of ongoing research. In this chapter, we provide a detailed protocol to assemble and use magnetic tweezers in the sea urchin model and generate spatially controlled asymmetric and oriented divisions during early embryonic development.


Assuntos
Fase de Clivagem do Zigoto , Desenvolvimento Embrionário , Animais , Diferenciação Celular , Divisão Celular , Blastômeros , Fenômenos Magnéticos , Ouriços-do-Mar
8.
Dev Dyn ; 253(3): 333-350, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37698352

RESUMO

BACKGROUND: Some marine invertebrate organisms are considered not to develop tumors due to unknown mechanisms. To gain an initial insight into how tumor-related genes may be expressed and function during marine invertebrate development, we here leverage sea urchin embryos as a model system and characterize the expressions of Myc and p53/p63/p73 which are reported to function synergistically in mammalian models as an oncogene and tumor suppressor, respectively. RESULTS: During sea urchin embryogenesis, a combo gene of p53/p63/p73 is found to be maternally loaded and decrease after fertilization both in transcript and protein, while Myc transcript and protein are zygotically expressed. p53/p63/p73 and Myc proteins are observed in the cytoplasm and nucleus of every blastomere, respectively, throughout embryogenesis. Both p53/p63/p73 and Myc overexpression results in compromised development with increased DNA damage after the blastula stage. p53/p63/p73 increases the expression of parp1, a DNA repair/cell death marker gene, and suppresses endomesoderm gene expressions. In contrast, Myc does not alter the expression of specification genes or oncogenes yet induces disorganized morphology. CONCLUSIONS: p53/p63/p73 appears to be important for controlling cell differentiation, while Myc induces disorganized morphology yet not through conventional oncogene regulations or apoptotic pathways during embryogenesis of the sea urchin.


Assuntos
Blastocisto , Proteína Supressora de Tumor p53 , Animais , Proteína Supressora de Tumor p53/genética , Blastômeros , Desenvolvimento Embrionário/genética , Ouriços-do-Mar/genética , Mamíferos
9.
Anim Sci J ; 94(1): e13907, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38102887

RESUMO

In mammals, around fertilization, the nucleolus of embryos transforms into the nucleolus precursor bodies (NPBs), which continue to mature until the blastocyst stage, leading to distinct morphological changes. In our study, we observed two types of nucleolar morphology in mouse in vitro fertilized embryos at the four-cell stage, which we refer to single nucleolus (SN) and multiple nucleoli (MN). To visualize nucleolar morphology, four-cell embryos were immunostained with anti-NOPP140 antibody. These embryos were categorized into five types based on the number of blastomeres carrying SN: SN4/MN0, SN3/MN1, SN2/MN2, SN1/MN3, and SN0/MN4, with percentages of 13, 27, 21, 23 and 9, respectively. Next, using a light microscope, we divided the four-cell in vitro fertilized embryos without fixation into two groups: those with at least two blastomeres displaying SN (SN embryos) and those without (MN embryos). Notably, significantly more SN embryos developed into blastocysts and offspring at 18.5 dpc compared with MN embryos. Furthermore, SN embryos displayed a higher NANOG-positive cell number at the blastocyst stage, significantly lower body and placental weights, resulting in a higher fetal/placental ratio. These findings suggest a close association between nucleolar state at the four-cell stage and subsequent developmental potential.


Assuntos
Nucléolo Celular , Placenta , Feminino , Gravidez , Animais , Camundongos , Blastocisto , Blastômeros , Fertilização in vitro/veterinária , Mamíferos
10.
Nat Commun ; 14(1): 7918, 2023 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-38097571

RESUMO

The combination of genome editing and primordial germ cell (PGC) transplantation has enormous significance in the study of developmental biology and genetic breeding, despite its low efficiency due to limited number of donor PGCs. Here, we employ a combination of germplasm factors to convert blastoderm cells into induced PGCs (iPGCs) in zebrafish and obtain functional gametes either through iPGC transplantation or via the single blastomere overexpression of germplasm factors. Zebrafish-derived germplasm factors convert blastula cells of Gobiocypris rarus into iPGCs, and Gobiocypris rarus spermatozoa can be produced by iPGC-transplanted zebrafish. Moreover, the combination of genome knock-in and iPGC transplantation perfectly resolves the contradiction between high knock-in efficiency and early lethality during embryonic stages and greatly improves the efficiency of genome knock-in. Together, we present an efficient method for generating PGCs in a teleost, a technique that will have a strong impact in basic research and aquaculture.


Assuntos
Blastômeros , Peixe-Zebra , Masculino , Animais , Peixe-Zebra/genética , Blástula , Células Germinativas
11.
Curr Biol ; 33(17): 3711-3721.e5, 2023 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-37607549

RESUMO

Some species undergo programmed DNA elimination (PDE), whereby portions of the genome are systematically destroyed in somatic cells. PDE has emerged independently in several phyla, but its function is unknown. Although the mechanisms are partially solved in ciliates, PDE remains mysterious in metazoans because the study species were not yet amenable to functional approaches. We fortuitously discovered massive PDE in the free-living nematode genus Mesorhabditis, from the same family as C. elegans. As such, these species offer many experimental advantages to start elucidating the PDE mechanisms in an animal. Here, we used cytology to describe the dynamics of chromosome fragmentation and destruction in early embryos. Elimination occurs once in development, at the third embryonic cell division in the somatic blastomeres. Chromosomes are first fragmented during S phase. Next, some of the fragments fail to align on the mitotic spindle and remain outside the re-assembled nuclei after mitosis. These fragments are gradually lost after a few cell cycles. The retained fragments form new mini chromosomes, which are properly segregated in the subsequent cell divisions. With genomic approaches, we found that Mesorhabditis mainly eliminate repeated regions and also about a hundred genes. Importantly, none of the eliminated protein-coding genes are shared between closely related Mesorhabditis species. Our results strongly suggest PDE has not been selected for regulating genes with important biological functions in Mesorhabditis but rather mainly to irreversibly remove repeated sequences in the soma. We propose that PDE may target genes, provided their elimination in the soma is invisible to selection.


Assuntos
Caenorhabditis elegans , Rhabditoidea , Animais , Caenorhabditis elegans/genética , Mitose , Blastômeros , DNA
12.
Sci Rep ; 13(1): 13050, 2023 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-37567923

RESUMO

Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.


Assuntos
Blastômeros , Embrião de Mamíferos , Animais , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Blastômeros/metabolismo , Citosol/metabolismo , Interferência de RNA , Embrião de Mamíferos/metabolismo
13.
Theriogenology ; 210: 42-52, 2023 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-37473595

RESUMO

One of the most typical abnormal cleavage patterns during early embryonic development is uneven division, but the first uneven division of pig zygote is common. Uneven division results in different daughter cell sizes and an uneven distribution of organelles such as lipid droplet, mitochondria, but the developmental capacity of daughter cells and proteomic changes of daughter cells are still unclear. Therefore, the developmental ability and proteomic quantification were investigated on blastomeres from even division (ED) or uneven division (UD) embryos at 2-cell stage in the present study. Firstly, the developmental ability was affected by the blastomeric size, when compared with medium blastomeres (MBs), the large blastomeres (LBs) with the higher cleavage rate but the small blastomeres (SBs) with the lower rate was observed. Subsequently, proteomic analysis was performed on blastomeres of LBs, MBs and SBs, a total of 109 DEPs were detected, which were involved in protein metabolism and processing, energy metabolism and ribosome. In particular, DEPs in LBs vs. SBs were focused on RNA binding and actin cytoskeletal tissue. Two protein-dense networks associated with RNA binding and cytoskeleton were revealed by further protein-protein interaction (PPI) analysis of DEPs in LBs vs. SBs, that DDX1 related to RNA binding and ACTB related to cytoskeleton were confirmed in UD embryos. Therefore, a briefly information of DEPs in blastomeres of 2-cell stage pig embryos was described in the present study, and it further confirmed that the formation of uneven division of the first cell cycle of pig embryos might be controlled by the cytoskeleton; the developmental capacity of daughter cells might be affected by the energy metabolism, RNA binding and ribosome, and further account for the developmental potential of the whole embryo.


Assuntos
Desenvolvimento Embrionário , Proteômica , Gravidez , Feminino , Animais , Suínos , Blastômeros/metabolismo , Embrião de Mamíferos , RNA/metabolismo
14.
Development ; 150(14)2023 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-37435786

RESUMO

The preimplantation mammalian (including mouse and human) embryo holds remarkable regulatory abilities, which have found their application, for example, in the preimplantation genetic diagnosis of human embryos. Another manifestation of this developmental plasticity is the possibility of obtaining chimaeras by combining either two embryos or embryos and pluripotent stem cells, which enables the verification of the cell pluripotency and generation of genetically modified animals used to elucidate gene function. Using mouse chimaeric embryos (constructed by injection of embryonic stem cells into the eight-cell embryos) as a tool, we aimed to explore the mechanisms underlying the regulatory nature of the preimplantation mouse embryo. We comprehensively demonstrated the functioning of a multi-level regulatory mechanism involving FGF4/MAPK signalling as a leading player in the communication between both components of the chimaera. This pathway, coupled with apoptosis, the cleavage division pattern and cell cycle duration controlling the size of the embryonic stem cell component and giving it a competitive advantage over host embryo blastomeres, provides a cellular and molecular basis for regulative development, ensuring the generation of the embryo characterised by proper cellular composition.


Assuntos
Blastocisto , Embrião de Mamíferos , Animais , Humanos , Camundongos , Apoptose/genética , Blastocisto/metabolismo , Blastômeros , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias , Mamíferos
15.
Dev Growth Differ ; 65(7): 384-394, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37392142

RESUMO

Spiralians, one of the major clades of bilaterians, share a unique development known as spiralian development, characterized by the formation of tiers of cells called quartets, which exhibit different developmental potentials along the animal-vegetal axis. Recently, spiralian-specific TALE-type homeobox genes (SPILE) have been identified, some of which show zygotic and staggered expression patterns along the animal-vegetal axis and function in quartet specification in mollusks. However, it is unclear which maternal molecular components control the zygotic expression of these transcription factors. In this study, we focused on SPILE-E, a maternal transcription factor, and investigated its expression and function in mollusks. We found that the maternal and ubiquitous expression of SPILE-E in the cleavage stages is conserved in molluskan species, including limpets, mussels, and chitons. We knocked down SPILE-E in limpets and revealed that the expression of transcription factors specifically expressed in the first quartet (1q2 ; foxj1b) and second quartet (2q; SPILE-B) was abolished, whereas the macromere-quartet marker (SPILE-C) was ectopically expressed in 1q2 in SPILE-E morphants. Moreover, we showed that the expression of SPILE-A, which upregulates SPILE-B but represses SPILE-C expression, decreased in SPILE-E morphants. Consistent with changes in the expression pattern of the above transcription factors, SPILE-E-morphant larvae exhibited patchy or complete loss of expression of marker genes of ciliated cells and shell fields, possibly reflecting incomplete specification of 1q2 and 2q. Our results provide a molecular framework for quartet specification and highlight the importance of maternal lineage-specific transcription factors in the development and evolution of spiralians.


Assuntos
Blastômeros , Genes Homeobox , Animais , Genes Homeobox/genética , Moluscos/genética , Fatores de Transcrição/genética
16.
Nat Commun ; 14(1): 1219, 2023 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-36882397

RESUMO

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.


Assuntos
Blastocisto , Edição de Genes , Humanos , Blastômeros , Embrião de Mamíferos , Alelos
17.
Reprod Fertil ; 4(2)2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36943184

RESUMO

Abstract: The transition of maternal to zygotic gene expression regulation is critical for human preimplantation embryo development. In recent years, single-cell RNA sequencing (scRNA-seq) had been applied to detect the factors that regulate human oocyte maturation and early embryo development. Here, the evaluation of transcriptomes in single blastomere from the embryo collected from patients by scRNA-seq was performed. There were 20 blastomeres biopsied from 8-cell embryos of seven patients who received more than two ART cycles due to low embryo competence. Meanwhile, ten cells were collected from 8-cell embryos of four patients who received ART treatment due to male or tubal factors. The blastomeres were then evaluated using the previously established scRNA-seq method to determine the associations between their gene expression and developmental competence. The total number of genes detected in 8-cell embryos that failed to form blastocyst including maternal and zygotic mRNAs was reduced. There were 324 differently expressed genes detected among the 8-cell embryos including 65 genes that were significantly suppressed in the 8-cell embryos that failed to form blastocyst. Further analysis found these 8-cell embryos arrested at the cleavage stage due to the dysfunction of the cell cycle, DNA transcription activity, histone methylation, and cell division-related genes such as SMCO-1, ZNF271P,ZNF679, ASF1b, BEX3, DPPA2, and ORC4. The alterations of gene expression detected in human 8-cell embryos are tightly associated with its developmental competence and could be used as targets to enhance embryo development or parameters to predict the embryo's development outcomes. Lay summary: Many females are suffering infertility due to the failure of embryonic development at early stages due to unknown causes. At the very beginning of human embryo development, the embryos start to express its own genes, which should be achieved at 8-cell stage. In current research, we isolated one cell from 8-cell embryos and detected the gene expression at single-cell level. Then the remaining cells of these embryos were cultured to form blastocyst. Meanwhile, the data was analyzed according to the outcomes of embryo development. We detected 324 differently expressed genes between the 8-cell embryos that succeeded and failed to form blastocyst. Our research showed the association between the gene expression and the developmental competence of 8-cell embryos. The findings could be used to predict the embryo quality and potential therapy target to improve the efficiency of assisted reproductive techniques.


Assuntos
Blastocisto , Desenvolvimento Embrionário , Gravidez , Feminino , Humanos , Masculino , Animais , Desenvolvimento Embrionário/genética , Blastocisto/metabolismo , Blastômeros/fisiologia , Embrião de Mamíferos , Análise de Sequência de RNA/veterinária , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo
18.
Genetics ; 224(1)2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-36988082

RESUMO

Whereas both sperm and egg contribute nuclear genetic material to the zygote in metazoan organisms, the inheritance of other cellular constituents is unequal between the 2 gametes. Thus, 2 copies of the centriole are contributed solely by the sperm to the zygote in most species. Centrioles can have a stereotyped distribution in some asymmetric divisions, but whether sperm-contributed centrioles are distributed in a stereotyped manner in the resulting embryo is not known. Here, we address this question in Caenorhabditis elegans using marked mating experiments, whereby the presence of the 2 sperm-contributed centrioles is monitored in the embryo using the stable centriolar component SAS-4::GFP, as well as GFP::SAS-7. Our analysis demonstrates that the distribution of sperm-contributed centrioles is stochastic in 4-cell stage embryos. Moreover, using sperm from zyg-1 mutant males that harbor a single centriole, we show that the older sperm-contributed centriole is likewise distributed stochastically in the resulting embryo. Overall, we conclude that, in contrast to the situation during some asymmetric cell divisions, centrioles contributed by the male germ line are distributed stochastically in embryos of C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans , Centríolos , Masculino , Animais , Centríolos/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Blastômeros/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismo , Proteínas Quinases/genética
19.
Genes (Basel) ; 14(3)2023 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-36980841

RESUMO

Recent advances in preimplantation embryo diagnostics enable a wide range of applications using single cell biopsy and molecular-based selection techniques without compromising embryo production. This study was conducted to develop a single cell embryo biopsy technique and gene expression analysis method with a very low input volume to ensure normal embryo development and to see if there are differences in gene expression profiles between day-5 biopsied bovine embryos that developed into blastocysts and embryos arrested at morula stage. Out of the 65 biopsied morulae, 32 developed to blastocysts (49.2%). Out of the 13,580 successfully annotated genes, 1204 showed a difference in mRNA expression level. Out of these, 155 genes were expressed in embryos developing to blastocysts. The pathway enrichment analysis revealed significant enrichment in "organelle biogenesis and maintenance", "mRNA splicing" and "mitochondrial translation" pathways. These findings suggest principal differences in gene expression patterns and functional networks of embryos able to reach the blastocyst stage compared to embryos arrested in development. Our preliminary data suggest that single blastomere biopsy and selected gene expression profiles at morula stage could offer additional possibilities for early preimplantation embryo selection before transfer.


Assuntos
Blastômeros , Diagnóstico Pré-Implantação , Gravidez , Feminino , Animais , Bovinos , RNA-Seq , Diagnóstico Pré-Implantação/métodos , Fertilização in vitro/métodos , Desenvolvimento Embrionário/genética , RNA Mensageiro
20.
Curr Biol ; 33(4): R143-R146, 2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36854271

RESUMO

Cutting up food into small pieces is well known to improve digestion. New work now shows that this concept also applies in the cellular world, by demonstrating that phagolysosome vesiculation promotes cell corpse degradation in Caenorhabditis elegans blastomeres.


Assuntos
Fagocitose , Fagossomos , Animais , Blastômeros , Cadáver , Caenorhabditis elegans
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