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JBRA Assisted Reproduction Aug 2023To evaluate the origin and ultrastructure of the coarse granules in the perivitelline space (PVS) of oocytes of a group of couples attending assisted reproduction...
OBJECTIVE
To evaluate the origin and ultrastructure of the coarse granules in the perivitelline space (PVS) of oocytes of a group of couples attending assisted reproduction treatment.
METHODS
The ultrastructure of five oocytes with coarse granulues in the PVS obtained from three patients were evaluated by transmission electron microscopy (TEM). The influence of the ovulation induction regimen on the formation of granules in the PVS of the oocytes of 214 couples and the developmental capacity of these oocytes presenting granules in the PVS was analyzed retrospectively.
RESULTS
In TEM analysis, the microvilli structure was irregular, short, and loosely scattered through the oolemma in the oocytes presenting coarse granules in the PVS. Furthermore, dense lipid droplets were identified within the PVS and the surrounding cumulus cells. In retrospective analysis, the number of oocytes with coarse granules in the PVS was positively correlated with the duration of antagonist administration (r=0.23, p=0.013). Regardless of the type of granule, the presence of coarse or moderately coarse granules in the PVS was positively correlated with low-quality embryos on D3 (r=0.29, p=0.005) and the total number of arrested embryos up to D3 (r=0.33, p<0.001). Furthermore, the presence of coarse granules in the PVS severely exacerbated miscarriage rates.
CONCLUSIONS
Our findings suggest that the presence of especially coarse granules in the PVS is correlated with the reduction of further embryonic developmental capacity in post-implantation stages of embryonic development, indicating a negative impact from aggressive ovulation induction protocols on developing oocytes.
PubMed: 37579275
DOI: 10.5935/1518-0557.20230032 -
Cell Death and Differentiation Aug 2022Soon after fertilization, the block mechanisms are developed in the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding,...
Soon after fertilization, the block mechanisms are developed in the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, penetration, and fusion. However, the molecular basis and underlying mechanism for the post-fertilization block to sperm penetration through ZP has not yet been determined. Here, we find that transglutaminase 2 (Tgm2), an enzyme that catalyzes proteins by the formation of an isopeptide bond within or between polypeptide chains, crosslinks zona pellucida glycoprotein 3 (ZP3) to result in the ZP hardening after fertilization and thus prevents polyspermy. Tgm2 abundantly accumulates in the subcortical region of the oocytes and vanishes upon fertilization. Both inhibition of Tgm2 activity in oocytes by the specific inhibitor in vitro and genetic ablation of Tgm2 in vivo cause the presence of additional sperm in the perivitelline space of fertilized eggs, consequently leading to the polyploid embryos. Biochemically, recombinant Tgm2 binds to and crosslinks ZP3 proteins in vitro, and incubation of oocytes with recombinant Tgm2 protein inhibits the polyspermy. Altogether, our data identify Tgm2 as a participant of zona block to the post-fertilization sperm penetration via hardening ZP surrounding fertilized eggs, extending our current understanding about the molecular basis of block to polyspermy.
Topics: Animals; Female; Male; Mice; Oocytes; Protein Glutamine gamma Glutamyltransferase 2; Proteins; Semen; Sperm-Ovum Interactions; Spermatozoa; Zona Pellucida; Zona Pellucida Glycoproteins
PubMed: 35017645
DOI: 10.1038/s41418-022-00933-0 -
PloS One 2023Generating non-human primate models of human diseases is important for the development of therapeutic strategies especially for neurodegenerative diseases. The common...
Generating non-human primate models of human diseases is important for the development of therapeutic strategies especially for neurodegenerative diseases. The common marmoset has attracted attention as a new experimental animal model, and many transgenic marmosets have been produced using lentiviral vector-mediated transgenesis. However, lentiviral vectors have a size limitation of up to 8 kb in length for transgene applications. Therefore, the present study aimed to optimize a piggyBac transposon-mediated gene transfer method in which transgenes longer than 8 kb were injected into the perivitelline space of marmoset embryos, followed by electroporation. We constructed a long piggyBac vector carrying the gene responsible for Alzheimer's disease. The optimal weight ratio of the piggyBac transgene vector to the piggyBac transposase mRNA was examined using mouse embryos. Transgene integration into the genome was confirmed in 70.7% of embryonic stem cells established from embryos injected with 1000 ng of transgene and transposase mRNA. Under these conditions, long transgenes were introduced into marmoset embryos. All embryos survived after transgene introduction treatment, and transgenes were detected in 70% of marmoset embryos. The transposon-mediated gene transfer method developed in this study can be applied to the genetic modification of non-human primates, as well as large animals.
Topics: Animals; Mice; Callithrix; Genetic Vectors; Gene Transfer Techniques; Transgenes; Callitrichinae; Transposases; RNA, Messenger; DNA Transposable Elements
PubMed: 37294815
DOI: 10.1371/journal.pone.0287065 -
Frontiers in Cell and Developmental... 2020The presence of cellular fragments in the perivitelline space is a commonly used parameter to determine quality before transfer of produced (IVP) embryos. However, this...
The presence of cellular fragments in the perivitelline space is a commonly used parameter to determine quality before transfer of produced (IVP) embryos. However, this parameter is difficult to assess after blastocyst expansion. In this study, we used mechanical hatching to confirm the presence of cellular fragments in the perivitelline space of bovine IVP blastocysts. We further looked for associations between possible apoptosis within extruded cells/ cellular fragments and the quality of bovine blastocysts using quantitative RT-PCR and immunofluorescence. Surprisingly, more than 42% of expanded blastocysts had cellular fragments in the perivitelline space; however, more than 37% of extruded cells were TUNEL negative. We observed no significant difference in embryo quality between expanded blastocysts with and without cellular fragments in the perivitelline space. Overall, our data suggest that embryos extrude abnormal cells to maintain their developmental potential. The presence of fragmented cells is not an indicator of embryo quality.
PubMed: 33469540
DOI: 10.3389/fcell.2020.616801 -
International Journal of Molecular... Jan 2022Embryo fragmentation represents a phenomenon generally characterized by the presence of membrane-bound extracellular cytoplasm into the perivitelline space. Recent... (Review)
Review
Embryo fragmentation represents a phenomenon generally characterized by the presence of membrane-bound extracellular cytoplasm into the perivitelline space. Recent evidence supports the cellular and molecular heterogeneity of embryo fragments. In this narrative review, we described the different embryo fragment-like cellular structures in their morphology, molecular content, and supposed function and have reported the proposed theories on their origin over the years. We identified articles related to characterization of embryo fragmentation with a specific literature search string. The occurrence of embryo fragmentation has been related to various mechanisms, of which the most studied are apoptotic cell death, membrane compartmentalization of altered DNA, cytoskeletal disorders, and vesicle formation. These phenomena are thought to result in the extrusion of entire blastomeres, release of apoptotic bodies and other vesicles, and micronuclei formation. Different patterns of fragmentation may have different etiologies and effects on embryo competence. Removal of fragments from the embryo before embryo transfer with the aim to improve implantation potential should be reconsidered on the basis of the present observations.
Topics: Apoptosis; Blastomeres; Cell Division; Cell-Derived Microparticles; Embryo Implantation; Embryo Transfer; Embryo, Mammalian; Embryonic Development; Humans; Micronucleus, Germline
PubMed: 35163271
DOI: 10.3390/ijms23031349 -
Open Biology Aug 2021Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation...
Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well-understood. For many insects, egg activation can be triggered independently of fertilization. In , egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte. Here we use physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in mature oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. We show that the swelling process is regulated by the conserved osmoregulatory channels, aquaporins and DEGenerin/Epithelial Na channels. Furthermore, through pharmacological and genetic disruption, we reveal a concentration-dependent requirement of transient receptor potential M channels to transport calcium, most probably from the perivitelline space, across the plasma membrane into the mature oocyte. Our data establish osmotic pressure as a mechanism that initiates egg activation in and are consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and show remarkable similarities to the mechanism of egg activation in some plants.
Topics: Animals; Calcium; Calcium Signaling; Drosophila Proteins; Drosophila melanogaster; Embryonic Development; Female; Fertilization; Oocytes; Osmolar Concentration
PubMed: 34343463
DOI: 10.1098/rsob.210067 -
Biomolecules Sep 2020In the teleost egg, the embryo is immersed in an extraembryonic fluid that fills the space between the embryo and the chorion and partially isolates it from the external...
In the teleost egg, the embryo is immersed in an extraembryonic fluid that fills the space between the embryo and the chorion and partially isolates it from the external environment, called the perivitelline fluid (PVF). The exact composition of the PVF remains unknown in vertebrate animals. The PVF allows the embryo to avoid dehydration, to maintain a safe osmotic balance and provides mechanical protection; however, its potential defensive properties against bacterial pathogens has not been reported. In this work, we determined the global proteomic profile of PVF in zebrafish eggs and embryos, and the maternal or zygotic origin of the identified proteins was studied. In silico analysis of PVF protein composition revealed an enrichment of protein classes associated with non-specific humoral innate immunity. We found lectins, protease inhibitors, transferrin, and glucosidases present from early embryogenesis until hatching. Finally, in vitro and in vivo experiments done with this fluid demonstrated that the PVF possessed a strong agglutinating capacity on bacterial cells and protected the embryos when challenged with the pathogenic bacteria Our results suggest that the PVF is a primitive inherited immune extraembryonic system that protects the embryos from external biological threats prior to hatching.
Topics: Agglutination; Animals; Computer Simulation; Edwardsiella tarda; Embryo, Nonmammalian; Immunity, Innate; Maternal Inheritance; Proteomics; Zebrafish
PubMed: 32899417
DOI: 10.3390/biom10091274 -
Journal of the Turkish German... 2014In assisted reproductive technology (ART), high embryo quality is closely related to high-quality oocytes. Cytoplasmic maturation and extracytoplasmic maturation are the...
OBJECTIVE
In assisted reproductive technology (ART), high embryo quality is closely related to high-quality oocytes. Cytoplasmic maturation and extracytoplasmic maturation are the most important components in determining oocyte quality. One of the most important components of extracytoplasmic maturation is perivitelline abnormalities. The aim of this study is to determine the effect of perivitelline abnormalities on the development of high-quality embryos.
MATERIAL AND METHODS
The study material consisted of 217 of 1154 oocytes from 98 intracytoplasmic sperm injection (ICSI) cycles undertaken due to male factor infertility. Only cycles with long gonadotropin-releasing hormone analogs combined with recombinant Follicle-stimulating hormone (rec-FSH) were included in study. We compared 105 metaphase-II oocytes that had dominantly perivitelline space abnormalities (large perivitelline space with or without granules) with 112 normal metaphase-II oocytes, based on the embryo grade determined by Alpha Scientists in Reproductive Medicine and the European Society of Human Reproduction and Embryology (ESHRE) Special Interest Group of Embryology. Normal metaphase-II oocytes were characterized by a round, clear zona pellucida; a small perivitelline space containing a single unfragmented first polar body; and a pale, moderately granular cytoplasm with no inclusions.
RESULTS
The development rates of Grade I, II, and III embryos were 68.5%, 23.8%, and 7.7%, respectively, in the 105 oocytes with perivitelline abnormalities. The development rates of Grade I, II, and III embryos were 82.1%, 17.9%, and 0%, respectively, in the 112 morphologically normal oocytes. When compared with normal oocytes, Grade I (68.5% vs. 82.1%, p value; 0.019) and Grade III (7.7% vs. 0%, p value; 0.003) embryo development rates were significantly lower in oocytes that had perivitelline abnormalities.
CONCLUSION
It is important to analyze oocyte quality using multiple parameters, including the perivitelline space. Perivitelline space abnormalities might negatively affect embryo development in male factor-infertile couples that are stimulated with rec-FSH. Therefore, when choosing embryos for transfer, we must take into consideration the historical oocyte data.
PubMed: 25317044
DOI: 10.5152/jtgga.2014.13091 -
Reproductive Biology and Endocrinology... Jan 2022The primary objective of the present study of women participating in an ICSI program was to determine whether the morphologic quality of oocytes was related to the...
PURPOSE
The primary objective of the present study of women participating in an ICSI program was to determine whether the morphologic quality of oocytes was related to the polycystic ovary syndrome (PCOS) phenotype.
METHODS
We performed a retrospective cohort study in the IVF unit at the Lille University Medical Center (Lille, France) between 2006 and 2015. Oocyte morphology (fragmented first polar body, abnormal zona pellucida, large perivitelline space, material in perivitelline space, abnormal shape of oocyte, granular cytoplasm and intracytoplasmic vacuoles) was evaluated in PCOS women and according to different subgroup (depending on the presence or absence of the cardinal features polycystic ovarian morphology (PCOM), hyperandrogenism (HA), and oligo-anovulation (OA)).
RESULTS
A total of 1496 metaphase II oocytes (n = 602 for phenotype A combining PCOM + HA + OA, n = 462 oocytes for phenotype C: PCOM + HA, and n = 432 for phenotype D: PCOM + OA) were assessed. The phenotypes A, C and D did not differ significantly with regard to the proportion of normal oocytes (adjusted percentages (95%CI): 35.2% (31.5 to 39.1%), 25.8% (21.9 to 29.9%) and 34.0% (29.7 to 38.6%), respectively: adjusted p = 0.13). Likewise, there were no significant intergroup differences in oocyte morphology. The ICSI outcome was not significantly associated with the PCOS phenotype.
CONCLUSION
The present study is the first to show that the PCOS phenotype (notably the presence vs. absence of OA and/or HA) is not significantly associated with the morphological quality of oocytes.
Topics: Adolescent; Adult; Case-Control Studies; Cell Shape; Cohort Studies; Female; France; Humans; Infertility, Female; Male; Oocytes; Phenotype; Polycystic Ovary Syndrome; Pregnancy; Pregnancy Outcome; Retrospective Studies; Sperm Injections, Intracytoplasmic; Young Adult
PubMed: 34986863
DOI: 10.1186/s12958-021-00874-2 -
Life (Basel, Switzerland) Sep 2023Invasive and noninvasive features are normally applied to select developmentally competent oocytes and embryos that can increase the take-home baby rates in assisted... (Review)
Review
Invasive and noninvasive features are normally applied to select developmentally competent oocytes and embryos that can increase the take-home baby rates in assisted reproductive technology. The noninvasive approach mainly applied to determine oocyte and embryo competence has been, since the early days of IVF, the morphological evaluation of the mature cumulus-oocyte complex at the time of pickup, first polar body, zona pellucida thickness, perivitelline space and cytoplasm appearance. Morphological evaluation of oocyte quality is one of the options used to predict successful fertilization, early embryo development, uterine implantation and the capacity of an embryo to generate a healthy pregnancy to term. Thus, this paper aims to provide an analytical revision of the current literature relating to the correlation between ovarian stimulation procedures and oocyte/embryo quality. In detail, several aspects of oocyte quality such as morphological features, oocyte competence and its surrounding environment will be discussed. In addition, the main noninvasive features as well as novel approaches to biomechanical parameters of oocytes that might be correlated with the competence of embryos to produce a healthy pregnancy and live birth will be illustrated.
PubMed: 37895371
DOI: 10.3390/life13101989