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Biology of Reproduction Sep 2012In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the...
In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the mouse zygotes and two-cell embryos. In somatic cells ORC2 remains bound to DNA replication origins throughout the cell cycle, while MCM7 is one of the last proteins to bind to the licensing complex. We found that MCM7 but not ORC2 was bound to DNA in metaphase II oocytes and remained associated with the DNA until S-phase. Shortly after fertilization, ORC2 was detectable at the metaphase II spindle poles and then between the separating chromosomes. Neither protein was present in the sperm cell at fertilization. As the sperm head decondensed, MCM7 was bound to DNA, but no ORC2 was seen. By 4 h after fertilization, both pronuclei contained DNA bound ORC2 and MCM7. As expected, during S-phase of the first zygotic cell cycle, MCM7 was released from the DNA, but ORC2 remained bound. During zygotic mitosis, ORC2 again localized first to the spindle poles, then to the area between the separating chromosomes. ORC2 then formed a ring around the developing two-cell nuclei before entering the nucleus. Only soluble MCM7 was present in the G2 pronuclei, but by zygotic metaphase it was bound to DNA, again apparently before ORC2. In G1 of the two-cell stage, both nuclei had salt-resistant ORC2 and MCM7. These data suggest that licensing follows a unique pattern in the early zygote that differs from what has been described for other mammalian cells that have been studied.
Topics: Animals; Cell Cycle Proteins; Chromatin; DNA Replication; DNA Replication Timing; DNA-Binding Proteins; Embryo, Mammalian; Female; Fertilization; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Minichromosome Maintenance Complex Component 7; Models, Biological; Nuclear Proteins; Origin Recognition Complex; Tissue Distribution; Zygote
PubMed: 22674395
DOI: 10.1095/biolreprod.112.101774 -
Human Reproduction (Oxford, England) Jan 2015Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal...
STUDY QUESTION
Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage?
SUMMARY ANSWER
The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods.
WHAT IS KNOWN ALREADY
The use of more 'steps' of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time.
STUDY DESIGN, SIZE, DURATION
Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem-Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification.
MAIN RESULTS AND THE ROLE OF CHANCE
The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less osmotic stress resulting in better morphology, higher cell quality and improved developmental competence. This microfluidic procedure resulted in murine zygotes with a significantly smoother cell surface (P < 0.001), more spherical cellular morphology (P < 0.001), increased cytoplasmic lipid retention in vitrified and warmed bovine oocytes (P < 0.01), decreased membrane perforations and cytoplasmic leakage in CPA-exposed murine zygotes (P < 0.05) and improved developmental competence of vitrified and warmed murine zygotes (P < 0.05) than CPA exposure using the current clinically used manual pipetting method.
LIMITATIONS, REASONS FOR CAUTION
It is necessary to design the microfluidic device to be more user-friendly for widespread use.
WIDER IMPLICATIONS OF THE FINDINGS
The theory and approach of eliminating osmotic stress by decreasing shrinkage rate is complementary to the prevalent osmotic stress theory in cryobiology which focuses on a minimum cell volume at which the cells shrink. The auto-microfluidic protocol described here has immediate applications for improving animal and human oocyte, zygote and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sublethal damage should be broadly useful for cryobiology.
STUDY FUNDING/COMPETING INTERESTS
This project was funded by the National Institutes of Health and the University of Michigan Reproductive Sciences Program. The authors declare no conflicts of interest.
Topics: Animals; Cattle; Cryopreservation; Mice; Microfluidics; Oocytes; Osmotic Pressure; Vitrification; Zygote
PubMed: 25355589
DOI: 10.1093/humrep/deu284 -
Animal Reproduction Science Jan 2013The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl...
The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26μM of BCB for 90min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB-: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB-: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB-). After fertilization, the zygotes produced from OBCB+ and OBCB- oocytes were further stained with BCB for 10min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB-, OBCB-/ZBCB++, OBCB-/ZBCB+ and OBCB-/ZBCB-). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P<0.05) than ZBCB treatment (ZBCB-; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB-: 28.8%) was less (P<0.01) than single application of BCB test (ZBCB-: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB- zygotes (36.6%) were greater (P<0.05) than that from C oocytes (25.7%), OBCB- oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P>0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB- zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB-: 48.0%) was greater (P<0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB-: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test.
Topics: Animals; Blastocyst; Cattle; Female; Fertilization in Vitro; Oocyte Retrieval; Oocytes; Oxazines; Pregnancy; Zygote
PubMed: 23228698
DOI: 10.1016/j.anireprosci.2012.11.002 -
Journal of Cell Science Aug 1994The isolation of the cortex of the sea urchin blastomere by detergent lysis was explored with the aim of analyzing components important in the structure and function of...
The isolation of the cortex of the sea urchin blastomere by detergent lysis was explored with the aim of analyzing components important in the structure and function of the cortical cytoskeleton, and their relationship to such phenomena as contraction. Buffered EGTA medium supplemented with isotonic glycerol and with magnesium, at a level close to the reported internal cellular concentration, yields stable cytoskeletal cortices that retain their spherical shape. Cortices prepared this way contain actin, myosin, fascin and spectrin, components normally associated with the cortical cytoskeleton in a similar distribution to that in intact zygotes. They retain the organized cortical filamentous structure, including the actin-fascin bundles that form cores of microvilli. ATP and NaCl caused changes in cortical shape, described as either contraction or expansion, respectively. Spectrin, but not myosin, was partially extracted by NaCl, resulting in expansion of the cortex that suggests a role for spectrin in maintenance of cortical structure. ATP (but not ADP nor ATP gamma S), which caused the partial removal of myosin and spectrin, led to the contraction of the cortex, consistent with a role for myosin in cortical tension. In cortices isolated from dividing eggs, the zygotes retained their cleavage furrows and ATP induced continuation of furrow progression. This preparation appears to be a useful in vitro model for cytokinesis.
Topics: Actins; Adenine Nucleotides; Animals; Blastomeres; Cell Division; Cell Size; Contractile Proteins; Cytoskeleton; Fertilization; Sea Urchins; Sodium Chloride; Spectrin; Zygote
PubMed: 7983183
DOI: 10.1242/jcs.107.8.2239 -
Fertility and Sterility Jun 2008To evaluate the impact of the first division morphology on embryo development and IVF-embryo transfer outcome.
OBJECTIVE
To evaluate the impact of the first division morphology on embryo development and IVF-embryo transfer outcome.
DESIGN
Prospective study.
SETTING
Teaching hospital, France.
PATIENT(S)
All zygotes from 201 couples were checked for early cleavage. We defined as "even," early cleaved (EC) zygotes with 2 cells of even size; as "uneven," EC zygotes with 2 cells of uneven size; and as "fragmented," EC zygotes with more than 20% fragmentation rate. Day 2 embryo quality was assessed as "top" embryo or "non-top," with the evaluation of multinucleated blastomeres.
INTERVENTION(S)
None.
MAIN OUTCOME MEASURE(S)
Day 2 embryo quality, pregnancy and implantation rates.
RESULT(S)
Among EC zygotes, 59.1% were even, 13.0% were uneven, and 27.9% were fragmented. Even EC yielded more "top" embryos and less multinucleated blastomere embryos than uneven EC (77.0% vs. 46.3%) and fragmented EC (77.0% vs. 13.9%). The 125 double embryo transfers that comprised at least one embryo derived from even EC zygote led to higher pregnancy rate (PR) (64.0% vs. 43.4%) and implantation rate (42.0% vs. 27.6%) compared to the 76 double embryo transfers with embryos derived from breakdown or 2PN zygotes.
CONCLUSION(S)
The morphology of the early cleaved zygote is involved in embryo development. Evaluation of this morphology is an effective and valuable method of assessing the embryo quality.
Topics: Adult; Cell Division; Culture Media; Embryo Transfer; Embryonic Development; Female; Fertilization; Fertilization in Vitro; Humans; Ovulation Induction; Pregnancy; Pregnancy Outcome; Pregnancy Rate; Prospective Studies; Zygote
PubMed: 18068162
DOI: 10.1016/j.fertnstert.2007.04.047 -
Reproduction (Cambridge, England) Nov 2020Aneuploidy is the most frequent single cause leading into the termination of early development in human and animal reproduction. Although the mouse is frequently used as...
Aneuploidy is the most frequent single cause leading into the termination of early development in human and animal reproduction. Although the mouse is frequently used as a model organism for studying the aneuploidy, we have only incomplete information about the frequency of numerical chromosomal aberrations throughout development, usually limited to a particular stage or assumed from the occurrence of micronuclei. In our study, we systematically scored aneuploidy in in vivo mouse embryos, from zygotes up to 16-cell stage, using kinetochore counting assay. We show here that the frequency of aneuploidy per blastomere remains relatively similar from zygotes until 8-cell embryos and then increases in 16-cell embryos. Due to the accumulation of blastomeres, aneuploidy per embryo increases gradually during this developmental period. Our data also revealed that the aneuploidy from zygotes and 2-cell embryos does not propagate further into later developmental stages, suggesting that embryos suffering from aneuploidy are eliminated at this stage. Experiments with reconstituted live embryos revealed, that hyperploid blastomeres survive early development, although they exhibit slower cell cycle progression and suffer frequently from DNA fragmentation and cell cycle arrest.
Topics: Aneuploidy; Animals; Blastomeres; Cell Cycle; Embryo, Mammalian; Embryonic Development; Female; Fertilization in Vitro; Mice; Pregnancy; Zygote
PubMed: 33065541
DOI: 10.1530/REP-20-0086 -
Experimental Cell Research Jun 1984Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this...
Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this phenomenon is poorly understood, an examination of the cytology of the C. reinhardi plastid DNA was made in gametes, newly formed zygotes, maturing zygotes, and at zygote germination. The single plastid per cell of Chlamydomonas contains a small number of DNA aggregates ('nucleoids') which can be seen after staining with DNA-binding fluorochromes. In zygotes formed by pre-stained gametes, the fluorescing nucleoids disappear from the plastid of mating type minus (male) gamete plastids but not from the plastid of mating type plus (female) gamete plastids about 1 h after zygote formation. Subsequently, nucleoids aggregate slowly to a final average of two or three in the single plastid of the mature zygote. Quantitative microspectrofluorimetry indicates that gametes of both mating types have equal amounts of plastid DNA, and that zoospores arising from zygotes have 3.5 X as much as gametes. Assuming degradation of male plastid DNA, there must be a very major synthesis of plastid DNA between zygote formation and zoospore release when zygotes produce the typical 8-16 zoospores. That synthesis appears to occur at germination, where there is a massive increase in plastid DNA and nucleoid number beginning just prior to meiosis. The results support the theory that uniparental inheritance results from degradation of plastid DNA entering the zygote via the male gamete and suggest further studies, using mutants and altered conditions, which might explain how male plastid DNA sometimes survives.
Topics: Cell Fusion; Chlamydomonas; Chloroplasts; DNA; Extrachromosomal Inheritance; Female; Spectrometry, Fluorescence; Zygote
PubMed: 6539223
DOI: 10.1016/0014-4827(84)90655-4 -
Reproductive Biomedicine Online Apr 2012Some patients in IVF programmes repeatedly display an abnormal embryonic development characterized as soon as day 2 post fertilization by a high rate (>60%) of highly... (Clinical Trial)
Clinical Trial
Some patients in IVF programmes repeatedly display an abnormal embryonic development characterized as soon as day 2 post fertilization by a high rate (>60%) of highly fragmented embryos (⩾40% of cytoplasmic fragments) leading to recurrent IVF failures. This study postulated that, for various maternal reasons, some embryos were unable to withstand the in-vitro environment and an early pronucleate-stage transfer was proposed to these couples. Fifty-three patients with recurrent IVF failures (a mean of 2.8±1.0 previous attempts) characterized by low embryonic quality (a mean of 62.7% of the embryos with extended fragmentation) were included this transfer protocol. As in previous cycles, the mean number of oocytes retrieved and the fertilization rate were normal. The mean number of zygotes per transfer was 2.24. Fourteen clinical pregnancies were obtained, representing a pregnancy rate and a delivery rate per oocyte retrieval of 26.4% and 18.9%, respectively. Recurrent heavy and early embryo fragmentation in vitro characterizes around 3% of IVF couples and leads to lack of transfer or implantation failure. These data on fresh zygote transfers are encouraging and may provide a valid alternative solution for some of these patients. Some patients in IVF programmes repeatedly display an abnormal embryonic development characterized as soon as day 2 post fertilization by a high rate of highly fragmented embryos, leading to recurrent IVF failures. We hypothesized that, for various reasons, some embryos were unable to withstand the in-vitro environment and an early pronucleate stage transfer was proposed to these couples. Fifty-three patients with recurrent IVF failures characterized by low embryonic quality were included in this transfer protocol. As in previous cycles, the mean number of oocytes retrieved and the fertilization rate were normal. The mean number of zygotes per transfer was 2.24. Fourteen clinical pregnancies were obtained, representing a pregnancy rate and a delivery rate per oocyte retrieval of 26.4% and 18.9%, respectively. Recurrent early and heavy embryo fragmentation in vitro characterizes around 3% of IVF couples and leads to lack of transfer or implantation failure. Our data on fresh zygote transfers are encouraging and may provide a valid alternative solution for these patients.
Topics: Adult; Birth Rate; Blastocyst; Cell Shape; Embryo Transfer; Female; Fertilization in Vitro; Humans; Infant, Newborn; Infertility; Male; Phenotype; Pregnancy; Quality Control; Recurrence; Salvage Therapy; Sperm Injections, Intracytoplasmic; Zygote
PubMed: 22377150
DOI: 10.1016/j.rbmo.2012.01.004 -
PloS One 2013Covalent modification of cytosine nucleotides within the genome encode essential epigenetic information, with methylation (5meC) and hydroxymethylation (5hmC) having...
Covalent modification of cytosine nucleotides within the genome encode essential epigenetic information, with methylation (5meC) and hydroxymethylation (5hmC) having received most attention. It has been proposed that the formation of 5hmC is an intermediate in the active demethylation of 5meC. Some reports show that global loss of 5meC in the newly fertilised embryo is accompanied by increased 5hmC, but others have failed to confirm this finding. These analyses have relied on immuno-localization of these modifications. In this study we have established the conditions required for equilibrium binding of antibodies to 5meC and 5hmC in zygotes. Simultaneous detection of these antigens required denaturation of chromatin by acid treatment followed by antigen retrieval by tryptic digestion. Equilibrium binding then required incubation at 4°C for greater than 6 h. These are more demanding conditions than generally reported and resulted in the consistent detection of 5meC and 5hmC in both male and female pronuclei throughout zygotic maturation. No dynamic reciprocal change in the level of 5meC relative to 5hmC was observed. Both 5meC and 5hmC accumulated within the peri-nucleolar regions and this was more pronounced in the male pronucleus. Staining of 5meC was relatively more intense within the cortical and 5hmC in the central regions of pronuclei. The results are not consistent with a role for 5hmC in global demethylation in the zygote. The persistence of both modifications throughout zygotic maturation, and their differing patterns of localization and solvent exposure infer each modification provides its own epigenetic information to the early embryo.
Topics: 5-Methylcytosine; Animals; Cell Nucleus; Cytosine; Dose-Response Relationship, Drug; Epigenesis, Genetic; Female; Male; Mice; Solvents; Temperature; Zygote
PubMed: 23691085
DOI: 10.1371/journal.pone.0063689 -
Nature Reviews. Molecular Cell Biology Feb 2023
Topics: Nuclear Pore; Zygote; Genome; Gene Expression Regulation, Developmental
PubMed: 36609652
DOI: 10.1038/s41580-022-00575-7