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Xenotransplantation of Human glioblastoma in Zebrafish larvae: imaging and proliferation assessment.Biology Open May 2019Glioblastoma (GBM) is the most prevalent type of primary brain tumor. Treatment options include maximal surgical resection and drug-radiotherapy combination. However,...
Glioblastoma (GBM) is the most prevalent type of primary brain tumor. Treatment options include maximal surgical resection and drug-radiotherapy combination. However, patient prognosis remains very poor, prompting the search for new models for drug discovery and testing, especially those that allow assessment of responses to treatment. Zebrafish xenograft models have an enormous potential to study tumor behavior, proliferation and cellular interactions. Here, an imaging and proliferation assessment method of human GBM xenograft in zebrafish larvae is introduced. Zebrafish larvae microinjected with fluorescently labeled human GBM cells were screened daily using a stereomicroscope and imaged by light sheet fluorescence microscopy (LSFM); volumetric modeling and composite reconstructions were done in single individuals. Larvae containing tumors were enzymatically dissociated, and proliferation of cancer cells was measured using dye dilution by flow cytometry. GBM micro-tumors formed mainly in the zebrafish yolk sac and perivitelline space following injection in the yolk sac, with an engraftment rate of 73%. Daily image analysis suggested cellular division, as micro-tumors progressively grew with differentiated fluorescence intensity signals. Using dye dilution assay by flow cytometry, at least three GBM cells' division cycles were identified. The combination of LSFM and flow cytometry allows assessment of proliferation and tumor growth of human GBM inside zebrafish, making it a useful model to identify effective anti-proliferative agents in a preclinical setting.
PubMed: 31085547
DOI: 10.1242/bio.043257 -
Developmental Biology May 2019Stereotyped left-right asymmetry both in external and internal organization is found in various animals. Left-right symmetry is broken by the neurula rotation in the...
Stereotyped left-right asymmetry both in external and internal organization is found in various animals. Left-right symmetry is broken by the neurula rotation in the ascidian, Halocynthia roretzi. Neurula embryos rotate along the anterior-posterior axis in a counterclockwise direction, and the rotation stops when the left side of the embryo is oriented downwards, resulting in contact of the left-side epidermis with the vitelline membrane at the bottom of perivitelline space. Then, such contact induces the expression of nodal and its downstream Pitx2 gene in the left-side epidermis. Vitelline membrane is required for the promotion of nodal expression. Here, we showed that a chemical signal from the vitelline membrane promotes nodal gene expression, but mechanical stimulus at the point of contact is unnecessary since the treatment of devitellinated neurulae with an extract of the vitelline membrane promoted nodal expression on both sides. The signal molecules are already present in the vitelline membranes of unfertilized eggs. These signal molecules are proteins but not sugars. Specific fractions in gel filtration chromatography had the nodal promoting activity. By mass spectrometry, we selected 48 candidate proteins. Proteins that contain both a zona pellucida (ZP) domain and epidermal growth factor (EGF) repeats were enriched in the candidates of the nodal inducing molecules. Six of the ZP proteins had multiple EGF repeats that are only found in ascidian ZP proteins. These were considered to be the most viable candidates of the nodal-inducing molecules. Signal molecules are anchored to the entire vitelline membrane, and contact sites of signal-receiving cells are spatially and mechanically controlled by the neurula rotation. In this context, ascidians are unusual with respect to mechanisms for specification of the left-right axis. By suppressing formation of epidermis monocilia, we also showed that epidermal cilia drive the neurula rotation but are dispensable for sensing the signal from the vitelline membrane.
Topics: Animals; Body Patterning; Cell Extracts; Cilia; Egg Proteins; Embryo, Nonmammalian; Epidermis; Gene Expression Regulation, Developmental; Glycosylation; Nodal Protein; Protein Domains; Quinazolinones; Rotation; Signal Transduction; Sugars; Urochordata; Vitelline Membrane
PubMed: 30710513
DOI: 10.1016/j.ydbio.2019.01.016 -
Reproduction, Fertility, and Development Mar 2020This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes... (Comparative Study)
Comparative Study
Morphometric, subcellular, in vitro fertilisation and embryonic developmental assessment of mouse oocytes produced by anti-inhibin serum or pregnant mare serum gonadotrophin superovulation.
This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes obtained from females superovulated with either anti-inhibin serum-human chorionic gonadotrophin (AIS-hCG) or pregnant mare serum gonadotrophin (PMSG)-hCG. The oocyte's quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs) and mitochondrial distribution were determined. Superovulation using AIS-hCG resulted in a higher numbers of oocyte/donor compared with PMSG-hCG (P=0.002). There was no difference in morphologically normal and abnormal oocytes between AIS-hCG and PMSG-hCG (P=0.425 and P=0.194, respectively). The morphometric measurements showed no difference in oocyte diameter between AIS-hCG and PMSG-hCG (P=0.289). However, the thickness of the ZP of oocytes from AIS-hCG females was decreased compared with PMSG-hCG (P<0.001). The PVS of oocytes from the AIS-hCG was larger than with PMSG-hCG (P<0.001). The microtubules of oocytes from both AIS-hCG and PMSG-hCG were normal, although there was an increased fluorescence intensity in the AIS-hCG oocytes (P<0.001). The F-actin and CGs distribution in oocytes from both AIS-hCG and PMSG-hCG were similar (P=0.330 and P=0.13, respectively). Although the oocytes from PMSG-hCG females had homogenously distributed mitochondria, AIS-hCG oocytes showed more peripheral distribution with no differences in fluorescence intensity (P=0.137). The blastocyst development rates after IVF with fresh sperm showed no difference between AIS-hCG and PMSG-hCG (P=0.235). These data suggested that AIS-hCG superovulation produces high numbers of morphologically normal oocytes that also possess normal subcellular structures, good morphological characteristics and had high invitro embryonic developmental potential.
Topics: Animals; Blastocyst; Chorionic Gonadotropin; Embryo Culture Techniques; Female; Fertility Agents, Female; Fertilization in Vitro; Gonadotropins, Equine; Immune Sera; Inhibins; Mice, Inbred C57BL; Mice, Inbred ICR; Oocyte Retrieval; Oocytes; Ovulation; Pregnancy; Superovulation
PubMed: 31972126
DOI: 10.1071/RD19131 -
Blood Jul 2016Patient-derived multiple myeloma (MM) cells are difficult to establish in culture or propagate in vivo in murine model. Here, we describe a zebrafish xenograft model...
Patient-derived multiple myeloma (MM) cells are difficult to establish in culture or propagate in vivo in murine model. Here, we describe a zebrafish xenograft model that permits rapid, reliable growth of human MM cells injected into the perivitelline space of albino zebrafish (Casper) embryos 48 hours postfertilization. MM1S and MM1R MM cell lines and primary CD138(+) MM cells were stained with CM-Dil red fluorescent dye and suspended in Matrigel prior to their injection. The cells grew at the site of injection and disseminated throughout the developing embryos and larvae. Tumor size was quantified by fluorescent microscopy, and cell fate was followed for 4 days. All of the cell line xenografts showed responses similar to those previously observed with in vitro assays. CD138(+) plasma cell xenografts derived from MM patients also grew and were inhibited by the same drugs patients had responded to clinically. Using this technique, we can assess drug sensitivity or resistance with a small number of MM cells in a short period. This raises the possibility that one might be able to assess drug sensitivity in real time with readily obtainable clinical samples.
Topics: Animals; Cell Line, Tumor; Heterografts; Humans; Multiple Myeloma; Neoplasm Transplantation; Neoplasms, Experimental; Zebrafish
PubMed: 27207793
DOI: 10.1182/blood-2016-03-704460 -
PloS One 2017Studies were conducted to explore the possibility of employing dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for the development of interspecies...
First cloned Bactrian camel (Camelus bactrianus) calf produced by interspecies somatic cell nuclear transfer: A step towards preserving the critically endangered wild Bactrian camels.
Studies were conducted to explore the possibility of employing dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for the development of interspecies somatic cell nuclear transfer (iSCNT) embryos using skin fibroblast cells of an adult Bactrian camel (Camelus bactrianus) and Llama (Llama glama) as donor nuclei. Also, the embryos reconstructed with Bactrian cells were transferred into the uterus of synchronized dromedary camel recipients to explore the possibility of using them as surrogate mothers. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated mature oocytes, collected from super-stimulated dromedary camels, and fused using an Eppendorf electroporator. After activation with 5μM ionomycin and 6-dimethylaminopurine, they were cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. In experiment 1, Day 7 blastocysts were stained with Hoechst to count their cell numbers, while in experiment 2, they were transferred to synchronized dromedary recipients. A lower number (P < 0.05) of blastocysts were obtained from reconstructs utilizing fibroblast cells from Llama when compared with those reconstructed with dromedary and Bactrian fibroblast cells. However, no difference was observed in their cell numbers. In experiment 2, a higher (P < 0.05) proportion of blastocysts were obtained from the cleaved embryos reconstructed with Bactrian fibroblast cells when compared to those reconstructed with dromedary cells. Twenty-six Day 7 blastocysts reconstructed with Bactrian cells were transferred to 23 synchronized dromedary recipients with 5 pregnancies established on Day 30, however, only one of the pregnancies developed to term and a healthy calf weighing 33 kgs was born after completing 392 days of gestation. Unfortunately, the calf died on day 7 due to acute septicemia. In conclusion, the present study reports, for the first time, birth of a cloned Bactrian calf by iSCNT using dromedary camel as a source for oocytes as well as a surrogate for carrying the pregnancy to term.
Topics: Animals; Animals, Newborn; Camelids, New World; Camelus; Cloning, Organism; Embryo Culture Techniques; Embryo Transfer; Endangered Species; Female; Nuclear Transfer Techniques; Ovulation Induction; Pregnancy
PubMed: 28545049
DOI: 10.1371/journal.pone.0177800 -
Electronic Physician Jan 2016Today, the use of electromagnetic waves in medical diagnostic devices, such as magnetic resonance imaging (MRI), has increased, and many of its biological effects have...
INTRODUCTION
Today, the use of electromagnetic waves in medical diagnostic devices, such as magnetic resonance imaging (MRI), has increased, and many of its biological effects have been reported. The aim of the present study was to assess the biological effects of 1.5 Tesla (T) magnetic resonance imaging (MRI) on fertility and reproductive parameters.
METHODS
Eighty adult male and female NMRI mice (NMRI: Naval Medical Research Institute) of age 6-8 weeks were studied and randomly divided into two study and control groups. After confirmation of pregnancy, the mice in the study group were exposed to the MRI (1.5 T) machine's waves over the next three weeks, once a week for 36 minutes. One day and thirty-five days after the last radiation, the mice were killed in order to do the in vitro fertilization (IVF) by neck beads' displacement and the impact on the evolution of embryos, and its quality was studied. Data were analyzed using SPSS version 20 and the significance level of less than 0.05 was considered.
RESULTS
Embryo morphometry showed that the total diameter and the cytoplasm diameter of the study group embryos suffered significant reduction compared to the control group, 1 day after the last irradiation (p < 0.05), but the diameter of the perivitelline space of this group's embryos had a significant increase (p < 0.05). The qualitative results during 35 days after irradiation showed that morphologically parameters of the embryos in the study group had no significant differences from the control group.
CONCLUSION
Exposure to MRI irradiation can transiently disturb the development of mouse embryos and fertility, but these effects are reversible 35 days after the last irradiation.
PubMed: 26955439
DOI: 10.19082/1701 -
Acta Endocrinologica (Bucharest,... 2019Polycystic ovarian syndrome (PCOS) occurs in 6-10% of all women in their reproductive age. In women with PCOS, controlled ovarian hyperstimulation (COH) often results in...
OBJECTIVES
Polycystic ovarian syndrome (PCOS) occurs in 6-10% of all women in their reproductive age. In women with PCOS, controlled ovarian hyperstimulation (COH) often results in an increased risk of ovarian hyperstimulation syndrome (OHSS). maturation (IVM) of human oocyte is an alternative technique for fertilization (IVF). The aim of this study was to compare the morphometric analysis and morphology of oocytes after maturation (IVM) between normal women and those suffering from polycystic ovary syndrome (PCOS).
MATERIAL AND METHODS
Thirty two women of 20 to 35 years of age that were undergoing controlled ovarian stimulation by the ICSI/IVF protocol were chosen for the study. The immature oocytes (n=108) were divided into two groups: the first oocyte group was comprised of 16 normal women (n=54); and the second group included 16 women with PCOS (n=54); then the oocytes were matured . After 24-48h of incubation, the oocyte maturation rate and morphometric and morphological characteristics were assessed using an inverted microscope, and then the images were compared.
RESULTS
There were significant differences in the maturity of oocytes between normal women and those with PCOS after IVM (P<0.05). Moreover, morphometric assessments revealed that there were no significant difference in the total diameter (μm) (zona thickness (ZPT) + perivitelline space width (PVS) + cytoplasm (CD) of oocytes between normal women and those with PCOS (156.3±6.8 and 137.7±9.9), respectively (P>0.05). Evaluation of morphological oocytes showed that morphological abnormalities, including ooplasmic vacuolization and granulation were higher in PCOS women compared to normal women (P<0.05).
CONCLUSION
The increased quality of oocytes after IVM reflected a positive impact of IVM oocytes on normal women as compared to women with PCOS.
PubMed: 32010346
DOI: 10.4183/aeb.2019.295 -
The Journal of Reproduction and... Apr 2016Contraction during the blastocyst stage is observed during embryonic development of various mammals, including humans, but the physiological role of this process is not...
Contraction during the blastocyst stage is observed during embryonic development of various mammals, including humans, but the physiological role of this process is not well understood. Using time-lapse monitoring (TLM), we studied the influence of vitrification and contractions on embryonic development in mice. Mouse embryos were cultured at the 2-cell stage. At the 8-cell stage, embryos were randomly divided into a fresh group (FG) and vitrified group (VG) and observed for up to 144 h. Strong contractions (i.e., contractions causing a decrease in volume of more than 20% and expansion of the perivitelline space) occurred significantly more often in unhatched embryos than hatching embryos in both groups. Regarding hatching embryos, contractions in the pre-hatching stage were significantly more frequent in the VG than the FG. Furthermore, mRNA expression levels of genes related to contractions were determined at three time points, the 8-cell stage, early blastocyst stage, and 20 h after blastocoel formation, with quantitative reverse transcription-polymerase chain reaction. There was no significant difference in Hspa1a expression between the FG and VG, but Hspa1a overexpression was observed just after thawing and tended to decrease gradually thereafter in some blastocysts. Furthermore, in the VG, Atp1a1 tended to show higher expression in the strong contraction group than in the weak contraction group. Overall, vitrification is an excellent method for cryopreservation but could increase contractions in the pre-hatching stage and may increase energy demands of the embryo. Observation of contraction by TLM may improve the evaluation of embryo quality.
Topics: Animals; Blastocyst; Chorionic Gonadotropin; Cryopreservation; Culture Media; Embryo Culture Techniques; Embryonic Development; Female; HSP70 Heat-Shock Proteins; Humans; Mice; Mice, Inbred C57BL; Pregnancy; Pregnancy, Animal; RNA, Messenger; Software; Time Factors; Vitrification
PubMed: 26806421
DOI: 10.1262/jrd.2015-150 -
Frontiers in Cell and Developmental... 2020Maternal regulatory factors endow the oocyte with developmental competence , which might be absent in current maturation (IVM) systems, thereby compromising oocyte...
Maternal regulatory factors endow the oocyte with developmental competence , which might be absent in current maturation (IVM) systems, thereby compromising oocyte quality. In the present study, by employing RNA sequencing data analysis, we expect to identify potential contributing factors to support porcine oocyte maturation through binding to their receptors on the oolemma. Here, C-X-C motif chemokine ligand 12 (CXCL12), vascular endothelial growth factor A (VEGFA), and Wingless-type MMTV integration site family member 5A (WNT5A), termed CVW, are selected and confirmed to be important maternal cytokines for porcine oocyte maturation. Combined supplementation of CVW promotes the nuclear maturation percentage from 57.2% in controls to 75.9%. More importantly, these maternal cytokines improve the developmental potential of matured oocytes by parthenogenesis, fertilization, and cloning, as their blastocyst formation efficiencies and total cell numbers are increased. CVW supplementation also enlarges perivitelline space and promotes cumulus expansion, which results in a more complete transzonal projection retraction on the zona pellucida, and a reduced incidence of polyspermy in fertilized oocytes. Meanwhile, inhibiting the CVW receptor-mediated signaling pathways severely impairs oocyte meiotic resumption and cumulus expansion during IVM. We further determine that maturation improvement by CVW is achieved through activating the MAPK pathway in advance and inhibiting the canonical WNT pathway at the end of the IVM period. These findings provide a new combination of three cytokines to promote the porcine IVM process, which also holds potential to be used in human assisted reproduction technologies as well as in other species.
PubMed: 32733887
DOI: 10.3389/fcell.2020.00578 -
Journal of Assisted Reproduction and... Mar 2024To identify whether follicular environment parameters are associated with mature oocyte quality, embryological and clinical outcomes.
PURPOSE
To identify whether follicular environment parameters are associated with mature oocyte quality, embryological and clinical outcomes.
METHODS
This retrospective study examined 303 mature oocytes from 51 infertile women undergoing ICSI cycles between May 2018 and June 2021. Exclusion criteria consisted of advanced maternal age (> 36 years old), premature ovarian failure, obesity in women, or use of frozen gametes. Luteal granulosa cells (LGCs) were analyzed for mitochondrial DNA/genomic (g) DNA ratio and vitality. The relationships between hormone levels in the follicular fluid and oocyte features were assessed. Quantitative morphometric measurements of mature oocytes were assessed, and the association of LGC parameters and oocyte features on live birth rate after single embryo transfer was examined.
RESULTS
Results indicated an inverse correlation between the mtDNA/gDNA ratio of LGCs and the size of polar body I (PBI). A 4.0% decrease in PBI size was observed with each one-unit increase in the ratio (p = 0.04). Furthermore, a 1% increase in LGC vitality was linked to a 1.3% decrease in fragmented PBI (p = 0.03), and a 1 ng/mL increase in progesterone levels was associated with a 0.1% rise in oocytes with small inclusions (p = 0.015). Associations were drawn among LGC characteristics, perivitelline space (PVS) debris, cytoplasmic inclusions, PBI integrity, and progesterone levels. Certain dysmorphisms in mature oocytes were associated with embryo morphokinetics; however, live birth rates were not associated with follicular parameters and oocyte quality characteristics.
CONCLUSION
Follicular markers may be associated with mature oocyte quality features.
Topics: Female; Humans; Adult; Progesterone; Infertility, Female; DNA, Mitochondrial; Retrospective Studies; Oocytes; Granulosa Cells; Fertilization in Vitro
PubMed: 38363455
DOI: 10.1007/s10815-024-03053-5