scholarly journals Deletion of Orc4 during oogenesis severely reduces polar body extrusion and blocks zygotic DNA replication

2022 ◽  
Author(s):  
Hieu Nguyen ◽  
Hongwen Wu ◽  
Anna Ung ◽  
Yukiko Yamazaki ◽  
Ben Fogelgren ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Origin Recognition Complex ◽  
Polar Body ◽  
Full Length ◽  
Primary Follicle ◽  
Cell Stage ◽  
Polar Bodies ◽  
Polar Body Extrusion

Abstract Origin Recognition Complex subunit 4 (ORC4) is a DNA binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted GV oocytes cultured in vitro arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. A reduced number of Orc4-depleted oocytes developed to the MII stage and after activation these oocytes arrested at the 2-cell stage, without undergoing DNA synthesis. This confirms that transcription of full length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint since cytokinesis progressed without DNA synthesis. Together the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis.

scholarly journals ALADIN is Required for the Production of Fertile Mouse Oocytes.

2016 ◽  
Author(s):  
Sara Carvalhal ◽  
Michelle Stevense ◽  
Katrin Koehler ◽  
Ronald Naumann ◽  
Angela Huebner ◽  
...  
Keyword(s):  
Polar Body ◽  
Spindle Assembly ◽  
Cell Periphery ◽  
Cell Stage ◽  
Spindle Positioning ◽  
Asymmetric Cell Divisions ◽  
Cell Divisions ◽  
Polar Bodies ◽  
Polar Body Extrusion

Asymmetric cell divisions depend upon the precise placement of the mitotic spindle. In mammalian oocytes, spindles assemble close to the cell center but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, non-developing polar bodies at anaphases. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently, we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygous deficient for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is impaired in a majority of oocytes due to problems in spindle orientation prior to the first meiotic anaphase. Those few oocytes that can mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.

scholarly journals ALADIN is required for the production of fertile mouse oocytes

2017 ◽  
Vol 28 (19) ◽  
pp. 2470-2478 ◽  
Author(s):  
Sara Carvalhal ◽  
Michelle Stevense ◽  
Katrin Koehler ◽  
Ronald Naumann ◽  
Angela Huebner ◽  
...  
Keyword(s):  
Polar Body ◽  
Spindle Assembly ◽  
Cell Periphery ◽  
Cell Stage ◽  
Spindle Positioning ◽  
Asymmetric Cell Divisions ◽  
Cell Divisions ◽  
Polar Bodies ◽  
Polar Body Extrusion

Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell’s center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.

scholarly journals In vitro aging of porcine oocytes

2011 ◽  
Vol 49 (No. 3) ◽  
pp. 93-98 ◽  
Author(s):  
I. Petrová ◽  
M. Sedmíková ◽  
E. Chmelíková ◽  
D. Švestková ◽  
R. Rajmon
Keyword(s):  
In Vitro Culture ◽  
Polar Body ◽  
Cell Stage ◽  
Subsequent Aging ◽  
Parthenogenetic Activation ◽  
In Vitro Aging ◽  
Polar Bodies ◽  
Porcine Oocyte ◽  
Parthenogenetic Embryos

Porcine oocytes matured in vitro develop in various ways if they are further cultivated. In our studies these oocytes were cultivated for 1 to 5 days (in vitro aging). During the 1st day of aging, most of them remained at the stage of metaphase II (98%). Then many oocytes underwent the spontaneous parthenogenetic activation. The portion of activated oocytes reached its peak after 2 or 3 days of aging in vitro (39 or 45%). The portion of fragmented oocytes peaked at the same time (28%). During subsequent aging in vitro (i.e. day 4 or 5 of aging), the portion of lysed oocytes significantly increased (30 or 37%). The highest portion of spontaneously activated parthenogenetic embryos at a pronuclear stage (35%) was observed during the 2nd day of aging in vitro. These pronuclear embryos had mainly one polar body with two pronuclei (47% of all pronuclear embryos) or two polar bodies with one pronucleus (38% of all pronuclear embryos). During the 3rd and 5th day of in vitro aging, there was a significant increase in the portion of parthenogenetic embryos cleaved to the 2-cell or 3-cell stage. When considering the prolonged in vitro culture of porcine oocyte, only the first day of aging should be taken into account, since beyond this time significant changes, i.e. parthenogenesis, fragmentation or lysis, occurred in oocytes under in vitro conditions.  

scholarly journals Intra-cytoplasmic sperm injection in a marsupial

Reproduction ◽  
2004 ◽  
Vol 128 (5) ◽  
pp. 595-605 ◽  
Author(s):  
Nadine M Richings ◽  
Geoffrey Shaw ◽  
Peter D Temple-Smith ◽  
Marilyn B Renfree
Keyword(s):  
Cell Adhesion ◽  
Polar Body ◽  
Tammar Wallaby ◽  
Mature Oocyte ◽  
Cell Stage ◽  
Intra Cytoplasmic Sperm Injection ◽  
Polar Body Extrusion ◽  
Cell Cell

Here we report the first use of intra-cytoplasmic sperm injection (ICSI) in a marsupial, the tammar wallaby (Macropus eugenii ), to achieve in vitro fertilization and cleavage. A single epididymal spermatozoon was injected into the cytoplasm of each mature oocyte collected from Graafian follicles or from the oviduct within hours of ovulation. The day after sperm injection, oocytes were assessed for the presence of pronuclei and polar body extrusion and in vitro development was monitored for up to 4 days. After ICSI, three of four (75%) follicular and four of eight (50%) tubal oocytes underwent cleavage. The cleavage pattern was similar to that previously reported for in vivo fertilized oocytes placed in culture, where development also halted at the 4- to 8-cell stage. One-third of injected oocytes completed the second cleavage division, but only a single embryo reached the 8-cell stage. The success of ICSI in the tammar wallaby provided an opportunity to examine the influence of the mucoid coat that is deposited around oocytes passing through the oviduct after fertilization. The presence of a mucoid coat in tubal oocytes did not prevent fertilization by ICSI and the oocytes cleaved in vitro to a similar stage as follicular oocytes lacking a mucoid coat. Cell–zona and cell–cell adhesion occurred in embryos from follicular oocytes, suggesting that the mucoid coat is not essential for these processes. However, blastomeres were more closely apposed in embryos from tubal oocytes and cell–cell adhesion was more pronounced, indicating that the mucoid coat may be involved in maintaining the integrity of the conceptus during cleavage.

Timing of the First Cleavage Division of Haploid Mouse Eggs, and the Duration of its Component Stages

1973 ◽  
Vol 13 (2) ◽  
pp. 553-566 ◽  
Author(s):  
M. H. KAUFMAN
Keyword(s):  
Polar Body ◽  
Time Lapse ◽  
Follicle Cells ◽  
Cleavage Division ◽  
Cell Stage ◽  
Cell Embryo ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Mouse Eggs

Mouse eggs were activated by treatment with hyaluronidase which removed the follicle cells, followed by culture in vitro, and examined at the first cleavage mitosis. Second polar body extrusion usually occurred and haploid parthenogenesis was initiated. Air-dried chromosome preparations were made between 11 and 15.5 h after activation. Out of the 308 eggs examined 74 had already progressed to the 2-cell stage; the remaining 234 at the 1-cell stage were examined in detail. All chromosome preparations of the first cleavage mitosis were classified into groups corresponding with the stages of prometaphase, metaphase (early or ‘pre-chromatid’, ‘chromatid’ and ‘late chromatid’) and anaphase. An indirect estimate was made of the duration of the first cleavage mitosis and of its component stages from the incidence of stages observed at different time intervals after activation. Similar eggs were also observed at 37 °C by time-lapse cine-photography and the interval between the disappearance of the pronucleus to the beginning of telophase of the first cleavage division was determined. The results of timing studies on the haploid eggs were compared with results obtained from similar observations on the first cleavage division of fertilized eggs which would of course normally be diploid. Artificially activated eggs with 2 pronuclei, resulting from second polar body suppression, were also examined, and serial chromosome preparations during mitosis showed that the 2 pronuclear chromosome groups unite on the first cleavage spindle and divide to give a hetero-zygous diploid 2-cell embryo.

Do embryonic polar bodies commit suicide?

Zygote ◽  
2012 ◽  
Vol 22 (1) ◽  
pp. 10-17 ◽  
Author(s):  
Dušan Fabian ◽  
Štefan Čikoš ◽  
Pavol Rehák ◽  
Juraj Koppel
Keyword(s):  
Polar Body ◽  
Point Of View ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Polar Bodies ◽  
First Polar Body ◽  
External Conditions ◽  
Living Organisms ◽  
Second Polar Body

SummaryThe extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis

1984 ◽  
Vol 4 (8) ◽  
pp. 1476-1482
Author(s):  
H Ariga
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Cell Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Sv40 Dna

The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixture with BLUR8 as a template was semiconservative and not primed by a putative RNA polymerase III transcript synthesized on the Alu family sequence in vitro. Pulse-chase experiments showed that the small-sized DNA produced in a short-term incubation was converted to full-length closed circular and open circular DNAs in alkaline sucrose gradients. DNA synthesis in extracts began in a region of the Alu family sequence and was inhibited 80% by the addition of anti-T serum. Furthermore, partially purified T antigen bound the Alu family sequence in BLUR8 by the DNA-binding immunoassay. These results suggest that SV40 T antigen recognizes the Alu family sequence, similar to the origin sequence of SV40 DNA, and initiates semiconservative DNA replication in vitro.

Expression of the HSP 70.1 gene, a landmark of early zygotic activity in the mouse embryo, is restricted to the first burst of transcription

Development ◽  
1995 ◽  
Vol 121 (1) ◽  
pp. 113-122 ◽  
Author(s):  
E. Christians ◽  
E. Campion ◽  
E.M. Thompson ◽  
J.P. Renard
Keyword(s):  
Dna Replication ◽  
Mouse Embryo ◽  
Culture Conditions ◽  
Hsp 70 ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Genome Activation ◽  
Alpha Amanitin ◽  
Zygotic Genome

Activation of the mouse embryonic genome at the 2-cell stage is characterized by the synthesis of several alpha-amanitin-sensitive polypeptides, some of which belong to the multigenic hsp 70 family. In the present work we show that a member of this family, the HSP 70.1 gene, is highly transcribed at the onset of zygotic genome activation. Transcription of this gene began as early as the 1-cell stage. Expression of the gene continued through the early 2-cell stage but was repressed before the completion of the second round of DNA replication. During this period we observed that the level of transcription was modulated by in vitro culture conditions. The coincidence of repression of HSP70.1 transcription with the second round of DNA replication was not found for other transcription-dependent polypeptides synthesized at the 2-cell stage.

scholarly journals Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability

Animals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 209 ◽  
Author(s):  
Ling Yang ◽  
Qingkai Wang ◽  
Maosheng Cui ◽  
Qianjun Li ◽  
Shuqin Mu ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Melatonin Receptors ◽  
Melatonin Treatment ◽  
In Vitro Development ◽  
First Polar Body ◽  
Mitochondrial Distribution ◽  
Polar Body Extrusion ◽  
Vitro Development

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus–oocyte complexes were cultured in TCM199 medium with non-treated (control), 10−5 M luzindole (melatonin receptor antagonist), 10−5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10−5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

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