scholarly journals Maternal Yes-Associated Protein Participates in Porcine Blastocyst Development via Modulation of Trophectoderm Epithelium Barrier Function

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1606 ◽  
Author(s):  
Zubing Cao ◽  
Tengteng Xu ◽  
Xu Tong ◽  
Yiqing Wang ◽  
Dandan Zhang ◽  
...  
Keyword(s):  
Tight Junction ◽  
Lineage Commitment ◽  
Hippo Signaling ◽  
Blastocyst Formation ◽  
Fluid Accumulation ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Downstream Effector ◽  
Morula Stage

The establishment of a functional trophectoderm (TE) epithelium is an essential prerequisite for blastocyst formation and placentation. Transcription coactivator yes-associated protein (YAP), a downstream effector of the hippo signaling pathway, is required for specification of both the TE and epiblast lineages in mice. However, the biological role of YAP in porcine blastocyst development is not known. Here, we report that maternally derived YAP protein is localized to both the cytoplasm and nuclei prior to the morula stage and is then predominantly localized to the TE nuclei in blastocysts. Functionally, maternal YAP knockdown severely impeded blastocyst formation and perturbed the allocation of the first two lineages. The treatment of embryos with verteporfin, a pharmacological inhibitor of YAP, faithfully recapitulated the phenotype observed in YAP deleted embryos. Mechanistically, we found that maternal YAP regulates multiple genes which are important for lineage commitment, tight junction assembly, and fluid accumulation. Consistent with the effects on tight junction gene expression, a permeability assay revealed that paracellular sealing was defective in the trophectoderm epithelium. Lastly, YAP knockdown in a single blastomere at the 2-cell stage revealed that the cellular progeny of the YAP+ blastomere were sufficient to sustain blastocyst formation via direct complementation of the defective trophectoderm epithelium. In summary, these findings demonstrate that maternal YAP facilitates porcine blastocyst development through transcriptional regulation of key genes that are essential for lineage commitment, tight junction assembly, and fluid accumulation.

scholarly journals K+ efflux through two-pore domain K+ channels is required for mouse embryonic development

Reproduction ◽  
2012 ◽  
Vol 143 (5) ◽  
pp. 625-636 ◽  
Author(s):  
Chang-Gi Hur ◽  
Eun-Jin Kim ◽  
Seong-Keun Cho ◽  
Young-Woo Cho ◽  
Sook-Young Yoon ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mammalian Cells ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Blastocyst Stage ◽  
Ruthenium Red ◽  
Channel Blockers ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Wide Range ◽  
Morula Stage

Numerous studies have suggested that K+ channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K+ channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K+ channel blockers to identify the functional role of K+ channels in mouse embryonic development. Voltage-dependent K+ channel blockers, such as tetraethylammonium and BaCl2, had no effect on embryonic development to the blastocyst stage, whereas K2P channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K2P channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ∼38% compared with scrambled siRNA injection (P<0.05). The blockade of K2P channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K2P channels could improve mouse embryonic development through the modulation of gating by activators.

Disruption of the cytokeratin filament network in the preimplantation mouse embryo

Development ◽  
1988 ◽  
Vol 104 (2) ◽  
pp. 219-234
Author(s):  
J.A. Emerson
Keyword(s):  
Mouse Embryo ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Trophectoderm Cells ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse Embryo ◽  
Preimplantation Mouse ◽  
Morula Stage ◽  
Cytokeratin Filaments

The distribution of the cytokeratin network in the intact preimplantation mouse embryo and the role of cytokeratin filaments in trophectoderm differentiation were investigated by means of whole-mount indirect immunofluorescence microscopy and microinjection of anti-cytokeratin antibody. Assembled cytokeratin filaments were detected in some blastomeres as early as the compacted 8-cell stage. The incidence and organization of cytokeratin filaments increased during the morula stage, although individual blastomeres varied in their content of assembled filaments. At the blastocyst stage, each trophectoderm cell contained an intricate network of cytokeratin filaments, and examination of sectioned blastocysts confirmed that extensive arrays of cytokeratin filaments were restricted to cells of the trophectoderm. Microinjection of anticytokeratin antibody into individual mural trophectoderm cells of expanded blastocysts resulted in a dramatic rearrangement of the cytokeratin network in these cells. Moreover, antibody injection into 2-cell embryos inhibited assembly of the cytokeratin network during the next two days of development. Despite this disruption of cytokeratin assembly, the injected embryos compacted and developed into blastocysts with normal morphology and nuclear numbers. These results suggest that formation of an elaborate cytokeratin network in preimplantation mouse embryos is unnecessary for the initial stages of trophectoderm differentiation resulting in blastocyst formation.

Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1 alpha+ isoform

Development ◽  
1997 ◽  
Vol 124 (10) ◽  
pp. 2027-2037 ◽  
Author(s):  
B. Sheth ◽  
I. Fesenko ◽  
J.E. Collins ◽  
B. Moran ◽  
A.E. Wild ◽  
...  
Keyword(s):  
Tight Junction ◽  
De Novo ◽  
Transmembrane Protein ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Cell Junctions ◽  
Cell Stage ◽  
Membrane Assembly ◽  
Morula Stage ◽  
Late Expression

The mouse preimplantation embryo has been used to investigate the de novo synthesis of tight junctions during trophectoderm epithelial differentiation. We have shown previously that individual components of the tight junction assemble in a temporal sequence, with membrane assembly of the cytoplasmic plaque protein ZO-1 occurring 12 hours before that of cingulin. Subsequently, two alternatively spliced isoforms of ZO-1 (alpha+ and alpha-), differing in the presence or absence of an 80 residue alpha domain were reported. Here, the temporal and spatial expression of these ZO-1 isoforms has been investigated at different stages of preimplantation development. ZO-1alpha- mRNA was present in oocytes and all preimplantation stages, whilst ZO-1alpha+ transcripts were first detected in embryos at the morula stage, close to the time of blastocoele formation. mRNAs for both isoforms were detected in trophectoderm and ICM cells. Immunoprecipitation of 35S-labelled embryos also showed synthesis of ZO-1alpha- throughout cleavage, whereas synthesis of ZO-1alpha+ was only apparent from the blastocyst stage. In addition, 33P-labelling showed both isoforms to be phosphorylated at the early blastocyst stage. The pattern and timing of membrane assembly of the two isoforms was also distinct. ZO-1alpha- was initially seen as punctate sites at the cell-cell contacts of compact 8-cell embryos. These sites then coalesced laterally along the membrane until they completely surrounded each cell with a zonular belt by the late morula stage. ZO-1alpha+ however, was first seen as perinuclear foci in late morulae before assembling at the tight junction. Membrane assembly of ZO-1alpha+ first occurred during the 32-cell stage and was zonular just prior to the early blastocyst stage. Immunostaining indicative of both isoforms was restricted to the trophectoderm lineage. Membrane assembly of ZO-1alpha+ and blastocoele formation were sensitive to brefeldin A, an inhibitor of intracellular trafficking beyond the Golgi complex. In addition, the tight junction transmembrane protein occludin co-localised with ZO-1alpha+ at the perinuclear sites in late morulae and at the newly assembled cell junctions. These results provide direct evidence from a native epithelium that ZO-1 isoforms perform distinct roles in tight junction assembly. Moreover, the late expression of ZO-1alpha+ and its apparent intracellular interaction with occludin may act as a final rate-limiting step in the synthesis of the tight junction, thereby regulating the time of junction sealing and blastocoele formation in the early embryo.

scholarly journals YAP activity is necessary and sufficient for basal progenitor abundance and proliferation in the developing neocortex

2018 ◽  
Author(s):  
Milos Kostic ◽  
Judith T.M.L. Paridaen ◽  
Katherine Long ◽  
Nereo Kalebic ◽  
Barbara Langen ◽  
...  
Keyword(s):  
Subventricular Zone ◽  
Potential Role ◽  
Activity Levels ◽  
Hippo Signaling ◽  
Hippo Signaling Pathway ◽  
Downstream Effector ◽  
Basal Progenitor ◽  
Developing Neocortex ◽  
Necessary And Sufficient

SummaryThe expansion of the neocortex during mammalian evolution has been linked to an enlargement of the subventricular zone during cortical development and an increase in the proliferation of the basal progenitors residing therein. Here, we explored a potential role of YAP, the major downstream effector of the Hippo signaling pathway, in proliferation of basal progenitors. We show that YAP expression and activity are high in ferret and human basal progenitors, which are known to exhibit high proliferative capacity, but low in mouse basal progenitors, which lack such capacity. To induce YAP activity in mouse basal progenitors, we expressed a constitutively active YAP (CA-YAP). This resulted in an increase in proliferation of basal progenitor. In addition, CA-YAP expressing mouse basal progenitors promoted the production of upper-layer neurons. To investigate if YAP is required for the proliferation of basal progenitors, we pharmacologically interfered with the function of YAP in the developing ferret and human neocortex. This resulted in a decrease of cycling basal progenitors. In concert, genetical interference with the function of YAP in ferret developing neocortex resulted in decreased abundance of basal progenitors. Together, our data indicate that YAP promotes the proliferation of basal progenitors and suggest that changes in YAP activity levels contributed to the evolutionary expansion of the neocortex.

scholarly journals ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1914 ◽  
Author(s):  
Jeongwoo Kwon ◽  
Nam-Hyung Kim ◽  
Inchul Choi
Keyword(s):  
Tight Junction ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Coiled Coil ◽  
Specific Inhibitor ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Genes Encoding ◽  
Rock Activity

The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P< 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins.

scholarly journals 272OVUM RECOVERY AND BLASTOCYST DEVELOPMENT FOLLOWING INTRACYTOPLASMIC SPERM INJECTION IN CHIMPANZEES

2004 ◽  
Vol 16 (2) ◽  
pp. 256
Author(s):  
I. Hayasaka ◽  
N. Yoshimoto ◽  
Y. Mori ◽  
K. Suzuki ◽  
R. Honda ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Early Embryogenesis ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Morula Stage ◽  
The One ◽  
Estradiol Levels

In the present study, we report on oocyte collection, intracytoplasmic sperm injection and early embryogenesis in chimpanzees. Eight adult female chimpanzees, 11–27 years of age, received a single s.c. injection of 3.75mg GnRH (Leuplin, Takeda Co. Ltd., Osaka, Japan) 1 to 3 days after the beginning of menstruation. Daily i.m. injections of hMG (Humegon, Nippon Organon K.K., Tokyo, Japan) were initiated the following day. The dose of hMG was altered from 75 to 300IU according to serum estradiol levels. When at least one follicle of 17mm or more in diameter was observed, 10000IU of hCG (Pregnyl, Nippon Organon K.K.) were administered by i.m injection. Oocytes were recovered by ultrasound-guided transvaginal follicular aspiration 30.5 to 35.5h after hCG injection. Mature oocytes were denuded of cumulus cells by treatment with 0.1% hyaluronidase, and injected with a frozen-thawed or fresh spermatozoan using a Piezo-driven micromanipulator. Zygotes were cultured in Quinn’s Advantage Fertilization Medium (Cooper Surgical, Inc., Trumbull, CT, USA) with 10 serum protein substitute (SPS) at 37°C in a 5% CO2 atmosphere until the pronucleus stage. The medium was replaced by Quinn’s Advantage Cleavage Medium with 10 SPS from the pronuclear to 8-cell stage, and Quinn’s Advantage Blastcyst Medium with 10 SPS, thereafter. Mild ovarian hyperstimulation syndrome (OHSS) occurred in one female chimpanzee with estradiol levels of 7520pgmL−1. No oocytes were collected from 2 chimpanzees in which large follicles were observed. Thirty-five mature oocytes, one immature oocyte and 6 degenerate/fragmented oocytes were retrieved from 6 chimpanzees, including the one with OHSS. Among 35 mature oocytes injected with spermatozoa, 26 oocytes (74%) produced two pronuclei;; 23 zygotes (66%) cleaved to the 2-cell stage, 22 (63%) to the 4-cell stage, 14 (40%) to the 8-cell stage, and 9 (26%) to the morula stage. Seven zygotes (20%) developed to the blastocyst stage by 120h. There were no differences in fertilization rate or early embryogenesis between frozen and fresh spermatozoa. Results indicate that techniques used for human-assisted reproduction may be applicable to the chimpanzee to help preserve this endangered species.

scholarly journals 114 PORCINE EMBRYO FRAGMENTATION, DEVELOPMENT AND APOPTOSIS: A CONFOCAL MICROSCOPY STUDY

2005 ◽  
Vol 17 (2) ◽  
pp. 207
Author(s):  
B. Mateusen ◽  
A. Van Soom ◽  
D. Maes ◽  
A. de Kruif
Keyword(s):  
Confocal Microscopy ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Developmental Arrest ◽  
Morula Stage ◽  
Embryonic Arrest

The relationship between embryonic fragmentation, embryonic arrest, and apoptosis has been the subject of some controversy (Hardy K 1999 Rev. Reprod. 4, 125–134). In order to investigate possible links, in vivo-produced, in vitro-cultured porcine embryos (n = 132) were scored for developmental stage and fragmentation at 7 days post insemination (dpi) and processed for propidium iodide and annexin V labelling. After fixation, embryos were processed for terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Using confocal microscopy, a cell was categorized apoptotic if (i) it had a fragmented or condensed nucleus, (ii) the cell membrane was annexin V-positive, and (iii) the nucleus was TUNEL labelled. An apoptotic cell ratio (ACR) was determined as the percentage of apoptotic cells per embryo. Differences in the % of fragmented and apoptotic embryos and correlations were analyzed using chi-square. Logistic regression was used to compare the average fragmentation % and the ACR. Sixty-one embryos (46%) arrested during the culture period, with 8 embryos arresting before or at the 4-cell stage. Significantly more arrested embryos were fragmented compared to embryos that were blastocysts at 7 dpi. Also, the average fragmentation percentage was significantly higher for arrested embryos compared to blastocysts. The correlation detected between developmental arrest and fragmentation was 0.60 (P < 0.05). None of the embryos without fragmentation had cells categorized as apoptotic, whereas 50 out of 55 embryos with fragmentation possessed apoptotic cells, which led to a correlation of 0.87 (P < 0.01) between fragmentation and apoptosis. The percentage of embryos with apoptotic cells was significantly higher for embryos arrested during the 5-cell to the morula stage compared to embryos that arrested before or at the 4-cell stage and embryos with blastocyst development at 7 dpi. The average ACR of embryos arrested during the 5-cell to the morula stage was significantly higher compared to the average ACR of blastocysts at 7 dpi. The correlation detected between the developmental arrest, during the 5-cell to the morula stage period and apoptosis was 0.57 (P < 0.01). Taken together, significant correlations between fragmentation, developmental arrest and apoptosis were detected. However, the association between embryonic arrest and apoptosis could be established only for embryos arrested after embryonic genome activation.

scholarly journals TAZ inhibits osteoclastogenesis by attenuating TAK1/NF-κB signaling

Bone Research ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Wanlei Yang ◽  
Xuanyuan Lu ◽  
Tan Zhang ◽  
Weiqi Han ◽  
Jianlei Li ◽  
...  
Keyword(s):  
Osteoclast Differentiation ◽  
Underlying Mechanism ◽  
Osteoclast Formation ◽  
Binding Motif ◽  
Hippo Signaling ◽  
Bone Homeostasis ◽  
Transcriptional Coactivator ◽  
Downstream Effector

AbstractOsteoporosis is an osteolytic disorder commonly associated with excessive osteoclast formation. Transcriptional coactivator with PDZ-binding motif (TAZ) is a key downstream effector of the Hippo signaling pathway; it was suggested to be involved in the regulation of bone homeostasis. However, the exact role of TAZ in osteoclasts has not yet been established. In this study, we demonstrated that global knockout and osteoclast-specific knockout of TAZ led to a low-bone mass phenotype due to elevated osteoclast formation, which was further evidenced by in vitro osteoclast formation assays. Moreover, the overexpression of TAZ inhibited RANKL-induced osteoclast formation, whereas silencing of TAZ reduced it. Mechanistically, TAZ bound to TGF-activated kinase 1 (TAK1) and reciprocally inhibited NF-κB signaling, suppressing osteoclast differentiation. Collectively, our findings highlight an essential role of TAZ in the regulation of osteoclastogenesis in osteoporosis and its underlying mechanism.

scholarly journals Clinical Relevance of Secreted Small Noncoding RNAs in an Embryo Implantation Potential Prediction at Morula and Blastocyst Development Stages

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1328
Author(s):  
Angelika V. Timofeeva ◽  
Ivan S. Fedorov ◽  
Maria A. Shamina ◽  
Vitaliy V. Chagovets ◽  
Nataliya P. Makarova ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Target Genes ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Specific Method ◽  
Hippo Signaling ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Morula Stage

Despite the improvements in biotechnological approaches and the selection of controlled ovarian hyperstimulation protocols, the resulting pregnancy rate from in vitro fertilization (IVF) protocols still does not exceed 30–40%. In this connection, there is an acute question of the development of a non-invasive, sensitive, and specific method for assessing the implantation potential of an embryo. A total of 110 subfertile couples were included in the study to undergo the IVF/ICSI program. Obtained embryos for transfer into the uterine cavity of patient cohort 1 (n = 60) and cohort 2 (n = 50) were excellent/good-quality blastocysts, and small noncoding RNA (sncRNA) content in the corresponding spent culture medium samples at the morula stage (n = 43) or at the blastocyst stage (n = 31) was analyzed by deep sequencing followed by qRT-PCR in real time. Two logistic regression models were developed to predict the implantation potential of the embryo with 100% sensitivity and 100% specificity: model 1 at the morula stage, using various combinations of hsa_piR_022258, hsa-let-7i-5p, hsa_piR_000765, hsa_piR_015249, hsa_piR_019122, and hsa_piR_008112, and model 2 at the blastocyst stage, using various combinations of hsa_piR_020497, hsa_piR_008113, hsa-miR-381-3p, hsa_piR_022258, and hsa-let-7a-5p. Protein products of sncRNA potential target genes participate in the selective turnover of proteins through the ubiquitination system and in the organization of the various cell cytoskeleton and nucleoskeleton structures, regulating the activity of the Hippo signaling pathway, which determines the fate specification of the blastomers.

Actin filament distribution in blocked and developing pig embryos

Zygote ◽  
2000 ◽  
Vol 8 (4) ◽  
pp. 353-358 ◽  
Author(s):  
Wei-Hua Wang ◽  
Lalantha R. Abeydeera ◽  
Randall S. Prather ◽  
Billy N. Day
Keyword(s):  
Embryo Development ◽  
Actin Filament ◽  
Actin Filaments ◽  
Culture Medium ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Cell Stage ◽  
The Relationship ◽  
Perinuclear Area

SummaryActin filaments play an important role in cell division. The present study was designed to examine the relationship between actin filament distribution and pig embryo development. When in vivo matured and fertilised pig oocytes were cultured in TCM 199 or NCSU 23, in various proportions, 45–65% of inseminated oocytes developed to the 2- to 4-cell stages but blastocyst development was observed only in NCSU 23 (34%) or NCSU 23 containing 10% TCM 199 (7%). Supplementation of NCSU 23 medium with 20% or more TCM 199 resulted in no blastocyst formation. Examination of actin filaments indicated that microfilaments were distributed in the cortex, at the junction of blastomeres and in the perinuclear area in the embryos cultured in NCSU 23, but perinuclear actin filaments were not observed in embryos cultured in TCM 199. When 2- to 4-cell stage embryos obtained from TCM 199 were transferred to NCSU 23 medium at 36 h after in vivo fertilisation, 57% of the cleaved embryos developed to blastocysts, which was no different from the proportion obtained after culture in NCSU 23 alone (56%). In addition, when 2- to 4-cell stage embryos obtained from TCM 199 were transferred to NCSU 23, most embryos showed perinuclear actin filaments within 6 h. The results indicate that the composition of the culture medium plays an important role in the polymerisation of actin filaments, which in turn influences embryo development. It is possible that pig embryo development was blocked by some components in TCM 199 which prevented actin filament polymerisation.

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