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Ultrastructure of the fertilized egg envelope from Melanotaenia praecox, Melanotaeniidae, Teleostei.Applied Microscopy Apr 2021We examined the morphology of fertilized egg and ultrastructures of fertilized egg envelopes of dwarf rainbowfish (Melanotaenia praecox) belong to Melanotaeniidae using...
We examined the morphology of fertilized egg and ultrastructures of fertilized egg envelopes of dwarf rainbowfish (Melanotaenia praecox) belong to Melanotaeniidae using light and electron microscopes. The fertilized eggs were spherical with adhesive filament, transparent, demersal, and had a narrow perivitelline space and small oil droplets. The size of fertilized egg was 1.02 ± 0.18 mm (n = 30), and there were two kinds of adhesive filament on the fertilized eggs. The long and thick (diameter 12.22 ± 0.52 μm, n = 20) adhesive filaments were only at the area of animal pole, and short and thin (diameter 1.99 ± 0.23 μm, n = 20) adhesive filaments were around the long filaments. A micropyle was conical shaped with adhesive filament and located near the animal pole of egg. The outer surface of fertilized egg was rough side. Also, the total thickness of the fertilized egg envelope was about 7.46 ± 0.41 μm (n = 20), the fertilized egg envelope consisted of two layers, an inner lamellae layer and an outer layer with high electron-density. And the inner layer was 8 layers. Collectively, these morphological characteristics and adhesive property of fertilized egg with adhesive filaments, and ultrastructures of micropyle, outer surface, and section of fertilized egg envelope are showed species specificity.
PubMed: 33797003
DOI: 10.1186/s42649-021-00052-z -
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 -
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 -
Frontiers in Cell and Developmental... 2021Human zona pellucida (ZP) matrix is composed of four glycoproteins designated as ZP glycoprotein -1 (ZP1), -2 (ZP2), -3 (ZP3), and -4 (ZP4). Mutations in the genes... (Review)
Review
Human zona pellucida (ZP) matrix is composed of four glycoproteins designated as ZP glycoprotein -1 (ZP1), -2 (ZP2), -3 (ZP3), and -4 (ZP4). Mutations in the genes encoding human ZP glycoproteins are one of the causative factors leading to abnormal ZP matrix and infertility in women. Relevance of the human ZP glycoproteins in 'sperm-oocyte' binding has been delineated by using either transgenic animal models expressing human zona proteins or purified native/recombinant human zona proteins. Studies based on the purified native/recombinant human zona proteins revealed that ZP1, ZP3, and ZP4 primarily bind to the capacitated acrosome-intact human spermatozoa whereas ZP2 binds to acrosome-reacted spermatozoa. On the contrary, human spermatozoa binds to the eggs obtained from transgenic mouse lines expressing human ZP2 but not to those expressing human ZP1, ZP3, and ZP4 suggesting that ZP2 has an important role in human 'sperm-oocyte' binding. Further studies using transgenic mouse lines showed that the N-terminus of human ZP2 mediate the taxon-specific human sperm-oocyte binding. Both glycans and protein-protein interactions have a role in human gamete interaction. Further studies have revealed that the purified native/recombinant human ZP1, ZP3, and ZP4 are competent to induce acrosome reaction. Human sperm binds to the mouse transgenic eggs expressing human ZP1-4 instead of mouse ZP1-3 proteins, penetrated the ZP matrix and accumulated in the perivitelline space, which were acrosome-reacted suggesting that human ZP2 in transgenic mouse model also induce acrosome reaction. In humans -linked glycosylation of zona proteins have been shown to play an important role in induction of the acrosome reaction. Hence in humans, based on studies using transgenic mouse model as well as purified native/recombinant zona proteins, it is likely that more than one zona protein is involved in the 'sperm-oocyte' binding and induction of the acrosome reaction.
PubMed: 33681199
DOI: 10.3389/fcell.2021.619868 -
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 -
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 -
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 -
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 -
Cancers May 2024Ovarian cancer (OC) is an umbrella term for cancerous malignancies affecting the ovaries, yet treatment options for all subtypes are predominantly derived from...
Ovarian cancer (OC) is an umbrella term for cancerous malignancies affecting the ovaries, yet treatment options for all subtypes are predominantly derived from high-grade serous ovarian cancer, the largest subgroup. The concept of "functional precision medicine" involves gaining personalized insights on therapy choice, based on direct exposure of patient tissues to drugs. This especially holds promise for rare subtypes like low-grade serous ovarian cancer (LGSOC). This study aims to establish an in vivo model for LGSOC using zebrafish embryos, comparing treatment responses previously observed in mouse PDX models, cell lines and 3D tumor models. To address this goal, a well-characterized patient-derived LGSOC cell line with the mutation c.35 G>T (p.(Gly12Val)) was used. Fluorescently labeled tumor cells were injected into the perivitelline space of 2 days' post-fertilization zebrafish embryos. At 1 day post-injection, xenografts were assessed for tumor size, followed by random allocation into treatment groups with trametinib, luminespib and trametinib + luminespib. Subsequently, xenografts were euthanized and analyzed for apoptosis and proliferation by confocal microscopy. Tumor cells formed compact tumor masses ( = 84) in vivo, with clear Ki67 staining, indicating proliferation. Zebrafish xenografts exhibited sensitivity to trametinib and luminespib, individually or combined, within a two-week period, establishing them as a rapid and complementary tool to existing in vitro and in vivo models for evaluating targeted therapies in LGSOC.
PubMed: 38791891
DOI: 10.3390/cancers16101812 -
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