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Scientific Reports Mar 2020Three genes are known to be essential for gamete adhesion/fusion (Cd9, Izumo1 and Juno). Here, we confirmed that Spaca6 null males are infertile and showed that their...
Three genes are known to be essential for gamete adhesion/fusion (Cd9, Izumo1 and Juno). Here, we confirmed that Spaca6 null males are infertile and showed that their sperm accumulate in the perivitelline space but are unable to fuse with oocyte. Like IZUMO1, SPACA6 which is expressed by human sperm, is remained on the equatorial segment after acrosomal reaction and is involved in human fertilization since an anti-SPACA6 antibody inhibited it. Despite the similarity of the phenotypes caused by Spaca6 and Izumo1 knockouts, these are not redundant and the essential relocation of IZUMO1 is not affected by the lack of SPACA6. We propose a model in which IZUMO1 and SPACA6 would be part of a molecular complex necessary for gamete fusion and that their concomitant presence would be required for the recruitment of another essential molecular actor, such as a fusogen, for the fusion to take place.
Topics: Acrosome Reaction; Animals; COS Cells; Chlorocebus aethiops; Female; Fertilization in Vitro; Humans; Immunoglobulins; Infertility, Male; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Mice, Transgenic; Seminal Plasma Proteins; Sperm Head; Sperm Injections, Intracytoplasmic; Sperm-Ovum Interactions; Spermatozoa
PubMed: 32210282
DOI: 10.1038/s41598-020-62091-y -
BMC Cancer Oct 2013In vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive...
BACKGROUND
In vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive immune system until 14 days post-fertilization, human cancer cells can survive and metastasize when transplanted into zebrafish larvae. Despite isolated reports, there has been no systematic evaluation of the robustness of this system to date.
METHODS
Individual cell lines were stained with CM-Dil and injected into the perivitelline space of 2-day old zebrafish larvae. After 2-4 days fish were imaged using confocal microscopy and the number of metastatic cells was determined using Fiji software.
RESULTS
To determine whether zebrafish can faithfully report metastatic potential in human cancer cells, we injected a series of cells with different metastatic potential into the perivitelline space of 2 day old embryos. Using cells from breast, prostate, colon and pancreas we demonstrated that the degree of cell metastasis in fish is proportional to their invasion potential in vitro. Highly metastatic cells such as MDA231, DU145, SW620 and ASPC-1 are seen in the vasculature and throughout the body of the fish after only 24-48 hours. Importantly, cells that are not invasive in vitro such as T47D, LNCaP and HT29 do not metastasize in fish. Inactivation of JAK1/2 in fibrosarcoma cells leads to loss of invasion in vitro and metastasis in vivo, and in zebrafish these cells show limited spread throughout the zebrafish body compared with the highly metastatic parental cells. Further, knockdown of WASF3 in DU145 cells which leads to loss of invasion in vitro and metastasis in vivo also results in suppression of metastasis in zebrafish. In a cancer progression model involving normal MCF10A breast epithelial cells, the degree of invasion/metastasis in vitro and in mice is mirrored in zebrafish. Using a modified version of Fiji software, it is possible to quantify individual metastatic cells in the transparent larvae to correlate with invasion potential. We also demonstrate, using lung cancers, that the zebrafish model can evaluate the metastatic ability of cancer cells isolated from primary tumors.
CONCLUSIONS
The zebrafish model described here offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells. Using this model it is possible to critically evaluate whether genetic manipulation of signaling pathways affects metastasis and whether primary tumors contain metastatic cells.
Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Janus Kinases; Male; Mice; Neoplasm Grading; Neoplasm Metastasis; Neoplasms; Phenotype; Protein Kinases; Wiskott-Aldrich Syndrome Protein Family; Zebrafish
PubMed: 24089705
DOI: 10.1186/1471-2407-13-453 -
Animal Reproduction 2022The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research...
Relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos to their developmental competence : a preliminary assessment using time-lapse imaging.
The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of -derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm, respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of -produced ovine embryos albeit further confirmational studies are needed.
PubMed: 35432605
DOI: 10.1590/1984-3143-AR2022-0009 -
The Journal of Reproduction and... Oct 2007Although mouse oocytes progressively acquire meiotic competence during their growth in the ovaries, only half of full-grown oocytes can accomplish meiosis. Two types of...
Although mouse oocytes progressively acquire meiotic competence during their growth in the ovaries, only half of full-grown oocytes can accomplish meiosis. Two types of full-grown oocytes have been reported on the basis of their chromatin configuration, the surrounded-nucleolus (SN) type and the non-surrounded-nucleolus (NSN) type. Therefore, full-grown oocytes collected from the ovaries of adult animals comprise a heterogeneous population; some oocytes are meiotically incompetent (NSN-type), and some are competent (SN-type). In the present study, we found that full-grown oocytes could be classified into two groups using the criterion of formation of the perivitelline space (PVS) after culture with 3-isobutyl-1-methylxanthine (IBMX) for 1 h. In oocytes with a PVS, actin-filled processes within zona pellucidae originating from cumulus cells were reduced, while they were rich in oocytes without a PVS, suggesting that a reduction in these processes contributes to PVS formation. PVS formation was highly correlated with meiotic competence and SN-type configuration. The results of this study demonstrate that PVS formation is a useful criterion for easily distinguishing between SN- and NSN-type oocytes, without injury to the cells.
Topics: 1-Methyl-3-isobutylxanthine; Actins; Animals; Cell Nucleus; Cells, Cultured; Chromatin; Female; Meiosis; Mice; Oocytes; Oogenesis; Zona Pellucida
PubMed: 17587772
DOI: 10.1262/jrd.19064 -
Journal of Visualized Experiments : JoVE Apr 2017In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer...
In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.
Topics: Animals; Animals, Genetically Modified; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Humans; Neovascularization, Pathologic; Xenograft Model Antitumor Assays; Zebrafish
PubMed: 28518096
DOI: 10.3791/55459 -
Reports of Biochemistry & Molecular... Oct 2020Currently, the efficient production of chimeric mice and their survival are still challenging. Recent researches have indicated that preimplantation embryo culture media...
BACKGROUND
Currently, the efficient production of chimeric mice and their survival are still challenging. Recent researches have indicated that preimplantation embryo culture media and manipulation lead to abnormal methylation of histone in the promotor region and consequently alter their gene expression pattern. This investigation was designed to evaluate the relationship between the methylation state of histone H3 and expression in mice chimeric blastocysts.
METHODS
Mouse 129/Sv embryonic stem cells (mESCs) expressing the green fluorescent protein (mESCs-GFP) were injected into the perivitelline space of 2.5 days post-coitis (dpc) embryos (C57BL/6) using a micromanipulator. H3K4 and H3K9 methylation, and H19 and expression was measured by immunocytochemistry and q-PCR, respectively, in blastocysts.
RESULTS
Histone H3 trimethylation in H3K4 and H3K9 in chimeric blastocysts was significantly less and greater, respectively (p< 0.05), than in controls. expression was significantly less (p< 0.05), while expression was less, but not significantly so, in chimeric than in control blastocysts.
CONCLUSION
Our results showed, that the alteration ofH3K4me3 and H3K9me3 methylation, change expression in chimeric blastocysts.
PubMed: 33649730
DOI: 10.29252/rbmb.9.3.357 -
Reproduction & Fertility Jul 2022During fertilization, avian sperm preferentially penetrate into the perivitelline membrane that covers the germinal disk region where the female nucleus is present. This...
ABSTRACT
During fertilization, avian sperm preferentially penetrate into the perivitelline membrane that covers the germinal disk region where the female nucleus is present. This phenomenon has been observed not only in domestic birds but also in wild birds; however, the mechanisms controlling sperm preference are still unclear. In this study, we investigated the possible involvement of annexin family protein in sperm-egg interaction in Japanese quail. Microscopic examination of fertilized eggs indicated that quail sperm penetration only occurred in the germinal disk region, and sperm localized outside the germinal disk were trapped in the perivitelline membrane. Western blot analysis and immunofluorescence microscopy revealed the presence of annexin A1 and A6 in the oocyte membrane, while annexin A6 localized in the perivitelline space of the germinal disk region. Further, our sperm binding assay using recombinant annexin A6 demonstrated that ejaculated sperm specifically bound to annexin A6 expressed in mammalian cell lines. These results suggest that annexin A6, which is expressed on the surface of oocytes, may function in sperm-egg interaction in the germinal disk region and that this binding may ensure sperm retention on the surface of the egg plasma membrane until fertilization takes place in Japanese quail.
LAY SUMMARY
In bird species, fertilization takes place immediately after ovulation of the egg. Sperm preferentially penetrate a specific area of the egg coating that covers the 'germinal disk region' - this area contains the cell that needs to be fertilized by a sperm. However, since the bird egg is extremely large in size and sperm must reach the 'germinal disk region' to achieve fertilization, it is unclear how this happens. Annexin proteins support fertilization in mammals, and we found that annexin A6 protein exhibits a unique localization in the germinal disk region in the eggs of Japanese quail. To test this interaction, we incubated quail sperm with cells that produced annexin A6 and found that ejaculated sperm bound to the cells. These results suggest that annexin A6 may have a role in the sperm-egg interaction in the germinal disk region in Japanese quail.
Topics: Male; Female; Animals; Annexin A6; Coturnix; Semen; Sperm-Ovum Interactions; Fertilization; Quail; Mammals
PubMed: 35972319
DOI: 10.1530/RAF-21-0115 -
Advanced Biosystems Nov 2020This study shows for the first time the ability to rewarm cryopreserved zebrafish embryos that grow into adult fish capable of breeding normally. The protocol employs a...
This study shows for the first time the ability to rewarm cryopreserved zebrafish embryos that grow into adult fish capable of breeding normally. The protocol employs a single injection of cryoprotective agents (CPAs) and gold nanorods (GNRs) into the yolk and immersion in a precooling bath to dehydrate the perivitelline space. Then embryos are encapsulated within CPA and GNR droplets, plunged into liquid nitrogen, cryogenically stabilized, and rewarmed by a laser pulse. Postlaser nanowarming, embryos (n = 282) exhibit intact structure by 1 h (40%), continued development after 3 h (22%), movement after 24 h (11%), hatching after 48 h (9%), and swimming after Day 5 (3%). Finally, from fish that survives till Day 5, two larvae are grown to adulthood and spawned, yielding survival comparable to an unfrozen control. Future efforts will focus on improving the survival to adulthood and developing methods to cryopreserve large numbers of embryos for research, aquaculture, and biodiversity preservation.
Topics: Animals; Cryopreservation; Cryoprotective Agents; Embryo Culture Techniques; Embryo, Nonmammalian; Gold; Lasers; Metal Nanoparticles; Nanotechnology; Nanotubes; Vitrification; Zebrafish
PubMed: 32996298
DOI: 10.1002/adbi.202000138 -
Journal of Visualized Experiments : JoVE Nov 2018Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome...
Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.
Topics: Animals; Blastocyst; Embryonic Development; Female; Gene Transfer Techniques; Lasers; Lentivirus; Mice; Mice, Transgenic; Zygote
PubMed: 30451224
DOI: 10.3791/58327 -
BMC Pregnancy and Childbirth Sep 2023Previous studies looked into the connections between pregnancy and the Zona Pellucida (ZP) thickness and Zona Pellucida Thickness Variation (ZPTV), as well as the...
BACKGROUND
Previous studies looked into the connections between pregnancy and the Zona Pellucida (ZP) thickness and Zona Pellucida Thickness Variation (ZPTV), as well as the embryo's radius, circumference, perimeter and global symmetry. However, no research has linked embryo implantation and pregnancy to the percentage of ZP thinning, the reduction in ooplasm volume, and the increase in perivitelline space (PVS) volume. Our objective is to correlate the percentage of ZP thinning, the percentage of ooplasm volume shrinkage and the percentage of PVS increase to the implantation. These data will be used for embryo selection as well as it can be put into a software that will assist embryo selection.
MATERIALS AND METHODS
Retrospective study included 281 patients, all of them had 2 embryos transferred, 149 patients got pregnant with two gestation sacs and 132 patients did not get pregnant. All of the transferred embryos had the ZP thickness measured several times from time of ICSI till Embryo Transfer (ET), the ooplasm volume was calculated from time of ICSI till two Pronuclei (2PN) fading and the PVS was calculated from the ICSI time till the 2PN fading.
RESULTS
The first characteristic is the change in the average ZP thickness that decreased by 32.7% + 5.3% at 70 h for the implanted embryos (Group 1) versus 23.6% + 4.8% for non-implanted embryos (Group 2) p = 0.000. The second characteristic is the average reduction in the volume of the ooplasm which is 20.5% + 4.3% in Group 1 versus 15.1% + 5.2% in Group 2, p = 0.000. The third characteristic is the increase in the volume of the PVS which was 38.1% + 7.6% in Group 1 versus 31.6% + 9.7% in Group 2 p = 0.000.
CONCLUSION
The implanted embryos showed higher percent of ZP thinning, higher percent of ooplasm reduction and higher percent of PVS increase.
Topics: Pregnancy; Female; Humans; Retrospective Studies; Sperm Injections, Intracytoplasmic; Embryo Implantation; Embryo Transfer; Zona Pellucida
PubMed: 37770819
DOI: 10.1186/s12884-023-06025-2