scholarly journals Role of PB1 Midbody Remnant Creating Tethered Polar Bodies during Meiosis II

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1394
Author(s):  
Alex McDougall ◽  
Celine Hebras ◽  
Gerard Pruliere ◽  
David Burgess ◽  
Vlad Costache ◽  
...  
Keyword(s):  
Polar Body ◽  
Dynamic Imaging ◽  
Meiotic Spindle ◽  
Left Behind ◽  
Meiotic Chromosomes ◽  
Polar Bodies ◽  
The Future ◽  
Previous Round ◽  
Small Structure ◽  
Fertilized Egg

Polar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. Using ascidians, we observed that PB1 becomes tethered to the fertilized egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small structure around 1 µm in diameter protruded from the cortical outpocket that will form the future PB2, which we define as the “polar corps”. As emission of PB2 progressed, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 outpocket was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in fertilized eggs that had PB1 removed suggested that the midbody remnant remained within the fertilized egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 since our data are correlative in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in several species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates.

scholarly journals Role of midbody remnant in meiosis II creating tethered polar bodies

2017 ◽  
Author(s):  
Alex McDougall ◽  
Celine Hebras ◽  
Gerard Pruliere ◽  
David Burgess ◽  
Vlad Costache ◽  
...  
Keyword(s):  
Polar Body ◽  
Dynamic Imaging ◽  
Meiotic Spindle ◽  
Left Behind ◽  
Meiotic Chromosomes ◽  
Polar Bodies ◽  
The Future ◽  
Previous Round ◽  
Ascidian Embryos

AbstractPolar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. We observed that PB1 becomes tethered to the egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small corps around 1μm in diameter protruded from the center of the cortical outpocket that will form the future PB2, which we call the “polar corps”. During emission of PB2, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with LifeAct::mCherry/GFP or TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in eggs that had PB1 removed showed that the midbody remnant remained within the egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 in other species as in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in a number of species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates.

scholarly journals A casein kinase 1 prevents expulsion of the oocyte meiotic spindle into a polar body by regulating cortical contractility

2017 ◽  
Vol 28 (18) ◽  
pp. 2410-2419 ◽  
Author(s):  
Jonathan R. Flynn ◽  
Francis J. McNally
Keyword(s):  
Polar Body ◽  
Spindle Pole ◽  
Casein Kinase ◽  
Meiotic Spindle ◽  
Contractile Ring ◽  
Direct Inhibition ◽  
Casein Kinase 1 ◽  
Asymmetric Cell Divisions ◽  
Polar Bodies ◽  
Cortical Contractility

During female meiosis, haploid eggs are generated from diploid oocytes. This reduction in chromosome number occurs through two highly asymmetric cell divisions, resulting in one large egg and two small polar bodies. Unlike mitosis, where an actomyosin contractile ring forms between the sets of segregating chromosomes, the meiotic contractile ring forms on the cortex adjacent to one spindle pole, then ingresses down the length of the spindle to position itself at the exact midpoint between the two sets of segregating chromosomes. Depletion of casein kinase 1 gamma (CSNK-1) in Caenorhabditis elegans led to the formation of large polar bodies that contain all maternal DNA, because the contractile ring ingressed past the spindle midpoint. Depletion of CSNK-1 also resulted in the formation of deep membrane invaginations during meiosis, suggesting an effect on cortical myosin. Both myosin and anillin assemble into dynamic rho-dependent cortical patches that rapidly disassemble in wild-type embryos. CSNK-1 was required for disassembly of both myosin patches and anillin patches. Disassembly of anillin patches was myosin independent, suggesting that CSNK-1 prevents expulsion of the entire meiotic spindle into a polar body by negatively regulating the rho pathway rather than through direct inhibition of myosin.

scholarly journals Cytoplasmic Microtubule Organizing Centers Regulate Meiotic Spindle Positioning in Mouse Oocyte

2020 ◽  
Author(s):  
Daniela Londono Vasquez ◽  
Katherine Rodriguez-Lukey ◽  
Susanta K. Behura ◽  
Ahmed Z. Balboula
Keyword(s):  
Polar Body ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Cytoplasmic Microtubule ◽  
Spindle Positioning ◽  
Nuclear Envelope Breakdown ◽  
Oocyte Meiosis ◽  
Polar Bodies ◽  
Polar Body Extrusion

ABSTRACTDuring oocyte meiosis, migration of the spindle and its positioning must be tightly regulated to ensure elimination of the polar bodies and provide developmentally competent euploid eggs. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known whether microtubules (MTs) participate in regulating these processes. Here, we characterize a pool of MTOCs in the oocyte that does not contribute to spindle assembly but instead remains free in the cytoplasm during metaphase I (metaphase cytoplasmic MTOCs; mcMTOCs). In contrast to spindle pole MTOCs, which primarily originate from the perinuclear region in prophase I, the mcMTOCs are found near the cortex of the oocyte. At nuclear envelope breakdown, they exhibit robust nucleation of MTs, which diminishes during polar body extrusion before returning robustly during metaphase II. The asymmetric positioning of the mcMTOCs provides the spindle with a MT-based anchor line to the cortex opposite the site of polar body extrusion. Depletion of mcMTOCs, by laser ablation, or manipulating their numbers, through autophagy inhibition, revealed that the mcMTOCs are required to regulate the timely migration and positioning of the spindle in meiosis. We discuss how forces exerted by F-actin in mediating movement of the spindle to the oocyte cortex are balanced by MT-mediated forces from the mcMTOCs to ensure spindle positioning and timely spindle migration.

scholarly journals C. elegans CLASP/CLS-2 negatively regulates membrane ingression throughout the oocyte cortex and is required for polar body extrusion

2020 ◽  
Author(s):  
Aleesa J. Schlientz ◽  
Bruce Bowerman
Keyword(s):  
Polar Body ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Contractile Apparatus ◽  
Contractile Ring ◽  
C Elegans ◽  
Cell Divisions ◽  
Polar Bodies ◽  
Polar Body Extrusion ◽  
The Relationship

AbstractThe requirements for oocyte meiotic cytokinesis during polar body extrusion are not well understood. In particular, the relationship between the oocyte meiotic spindle and polar body contractile ring dynamics remains largely unknown. We have used live cell imaging and spindle assembly defective mutants lacking the function of CLASP/CLS-2, kinesin-12/KLP-18, or katanin/MEI-1 to investigate the relationship between meiotic spindle structure and polar body extrusion in C. elegans oocytes. We show that spindle bipolarity and chromosome segregation are not required for polar body contractile ring formation and chromosome extrusion in klp-18 mutants, but oocytes with severe spindle assembly defects due to loss of CLS-2 or MEI-1 have penetrant and distinct polar body extrusion defects: CLS-2 is required early for contractile ring assembly or stability, while MEI-1 is required later for contractile ring constriction. We also show that CLS-2 negatively regulates membrane ingression throughout the oocyte cortex during meiosis I, and we explore the relationship between global cortical dynamics and oocyte meiotic cytokinesis.Author SummaryThe precursor cells that produce gametes—sperm and eggs in animals—have two copies of each chromosome, one from each parent. These precursors undergo specialized cell divisions that leave each gamete with only one copy of each chromosome; defects that produce incorrect chromosome number cause severe developmental abnormalities. In oocytes, these cell divisions are highly asymmetric, with extra chromosomes discarded into small membrane bound polar bodies, leaving one chromosome set within the much larger oocyte. How oocytes assemble the contractile apparatus that pinches off polar bodies remains poorly understood. To better understand this process, we have used the nematode Caenorhabditis elegans to investigate the relationship between the bipolar structure that separates oocyte chromosomes, called the spindle, and assembly of the contractile apparatus that pinches off polar bodies. We used a comparative approach, examining this relationship in three spindle assembly defective mutants. Bipolar spindle assembly and chromosome separation were not required for polar body extrusion, as it occurred normally in mutants lacking a protein called KLP-18. However, mutants lacking the protein CLS-2 failed to assemble the contractile apparatus, while mutants lacking the protein MEI-1 assembled a contractile apparatus that failed to fully constrict. We also found that CLS-2 down-regulates membrane ingression throughout the oocyte surface, and we explored the relationship between oocyte membrane dynamics and polar body extrusion.

scholarly journals Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes

2013 ◽  
Vol 24 (24) ◽  
pp. 3832-3841 ◽  
Author(s):  
Zhen-Bo Wang ◽  
Zong-Zhe Jiang ◽  
Qing-Hua Zhang ◽  
Meng-Wen Hu ◽  
Lin Huang ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Homologous Chromosome ◽  
Polar Body ◽  
Conditional Knockout ◽  
Meiotic Spindle ◽  
Spindle Positioning ◽  
Mouse Oocytes ◽  
Polar Bodies ◽  
And Migration ◽  
Specific Deletion

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.

Establishment of embryonic axes in larvae of the starfish, Asterina pectinifera

Development ◽  
1983 ◽  
Vol 75 (1) ◽  
pp. 87-100
Author(s):  
Tetsuya Kominami
Keyword(s):  
Cleavage Plane ◽  
Anterior Part ◽  
Polar Body ◽  
Cleavage Pattern ◽  
Early Cleavage ◽  
Embryonic Axes ◽  
Cell Stage ◽  
Polar Bodies ◽  
Asterina Pectinifera ◽  
Fertilized Egg

In order to clarify the relationships between the first cleavage plane and the embryonic axes, early cleavage pattern of the fertilized eggs of the starfish, Asterina pectinifera was reexamined . It was ascertained that the polar bodies were formed at the site to which the germinal vesicle had closely located before the initiation of the meiotic division, and that the first cleavage plane passed near this site of polar body formation. While some of the early embryos of this starfish were observed to show various cleavage patterns during early cleavage stage, more than 70% of the embryos developed according to, so to say, the ‘typical’ cleavage pattern. Next, horseradish peroxidase (HRP) was injected into one of the blastomeres of the 2-cellor 8-cell-stage embryos. The embryos were allowed to develop up to either the early gastrula or the early bipinnaria stage and stained to detect the descendants of the blastomere injected with HRP. In early gastrulae still retaining radial symmetry, the activity of HRP injected at the 2-cell stage was found only in one side of the embryo partitioned by one of the symmetrical planes. When one of the four blastomeres lying nearer to the polar bodies at the 8-cell stage was marked with HRP, its descendants constituted one quarter of the anterior part of the gastrula, and descendants of a blastomere opposite the polar bodies were found in the posterior region of the embryo. It was concluded that the animal-vegetal (AV) axis was preexisting in the fertilized egg and that the first cleavage plane contained this primary axis. In early bipinnariae with their dorsoventral (DV) axes already established, the region of activity of the HRP injected at the 2-cell stage was still demarcated by a plane which passed through the AV axis, but the plane of the boundary had no fixed relation to the DV axis. The results indicate that the first cleavage plane does not necessarily correspond to the median plane of the starfish larva, unlike the case in sea-urchin eggs (Hörstadius & Wolsky, 1936). In other words, the DV axis of the starfish embryo is not predetermined in the fertilized egg, and might be established in the course of development through cell-to-cell interactions, while the AV axis is established mainly according to the pre-existing egg polarity.

scholarly journals PAR-1 and the microtubule-associated proteins CLASP2 and dynactin-p50 have specific localisation on mouse meiotic and first mitotic spindles

Reproduction ◽  
2005 ◽  
Vol 130 (3) ◽  
pp. 311-320 ◽  
Author(s):  
Catherine A Moore ◽  
Magdalena Zernicka-Goetz
Keyword(s):  
Polar Body ◽  
Model Systems ◽  
Regulatory Proteins ◽  
Meiotic Spindle ◽  
Mitotic Spindles ◽  
Microtubule Associated Proteins ◽  
Mouse Oocytes ◽  
Polar Bodies ◽  
Associated Proteins ◽  
Second Polar Body

The site of second meiotic division, marked by the second polar body, is an important reference point in the early mouse embryo. To study its formation, we look at the highly asymmetric meiotic divisions. For extrusion of the small polar bodies during meiosis, the spindles must be located cortically. The positioning of meiotic spindles is known to involve the actin cytoskeleton, but whether microtubules are also involved is not clear. In this study we investigated the patterns of localisation of microtubule regulatory proteins in mouse oocytes. PAR-1 is a member of the PAR (partitioning-defective) family with known roles in regulation of microtubule stability and spindle positioning in other model systems. Here we show its specific localisation on mouse meiotic and first mitotic spindles. In addition, the microtubule-associated proteins CLASP2 (a CLIP associating protein) and dynactin-p50 are found on kinetochores and a subset of microtubule-organising centres. Thus we show specific localisation of microtubule regulatory proteins in mouse oocytes, which could indicate roles in meiotic spindle organisation.

P–805 Artificial oocytes: from somatic cells to fertile pups

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
O Kocur ◽  
A Trout ◽  
P Xie ◽  
A Petrini ◽  
Z Rosenwaks ◽  
...  
Keyword(s):  
Genetic Variance ◽  
Imaging System ◽  
Polar Body ◽  
Somatic Cells ◽  
Human Reproduction ◽  
Meiotic Spindle ◽  
Mouse Strains ◽  
Age Related ◽  
Pregnant Mice ◽  
Post Implantation

Abstract Study question We analyzed the efficacy of generating artificial oocytes using somatic cells (SCs) from two mouse strains (B6D2F1 and FVB) and followed their full pre-/post-implantation development. Summary answer While artificial oocytes generated from the new strain (FVB) had higher fertilization rates, those from the standard strain (B6D2F1) provided expanded blastocysts and fertile pups. What is known already B6D2F1 is a popular hybrid mouse strain for cloning and transgenic creation due to its geno-/pheno-typic uniformity and high oocyte yield and quality. Indeed, B6D2F1 oocytes have a distinct metaphase II (MII) spindle complex, making them an ideal candidate to generate ooplasts used in SC nuclear transfer (SCNT). However, because they lack genetic variance, they are less suitable for reciprocal SCNT studies. In contrast, FVB mice have single nucleotide polymorphisms and indels on each chromosome that can aid in tracing the pedigree of progeny. Study design, size, duration A total of 10 experiments were performed over the course of 3 months, using 30 stimulated mice. SCs were retrieved from cumulus oophorus harvested from FVB and B6D2F1 mice. SCs from both strains were injected into enucleated MII B6D2F1 oocytes. Unmanipulated B6D2F1 oocytes were piezo-ICSI inseminated, serving as controls. The occurrence of haploidization, fertilization, and full preimplantation development was compared. Some blastocysts were transferred into pseudo-pregnant CD–1 mice to obtain offspring. Participants/materials, setting, methods Oocyte enucleation was performed under Oosight™ visualization and cytochalasin B exposure. An FVB or B6D2F1 SC was transferred into the perivitelline space of the ooplast with Sendai virus to promote fusion. Haploidization was monitored by pseudo-meiotic spindle formation followed by extrusion of a pseudo-polar body after insemination. Conceptuses were cultured in a time-lapse imaging system, with piezo-ICSI controls. Expanded blastocysts were transferred into uterine horns of pseudo-pregnant mice. Offspring were mated to test their fertility. Main results and the role of chance FVB (n = 278) and B6D2F1 (n = 905) SCs at G0 phase, with a diameter <10 mm, were chosen for SCNT and transferred into enucleated B6D2F1 ooplasts. Enucleation of 1,212 oocytes yielded a survival rate of 97.6%. Both FVB and B6D2F1 SCNT resulted in similar survival rates of 100% and 98.5%, respectively. Successful haploidization, determined by the presence of a pseudo-meiotic spindle 2 hours after SCNT, was also comparable, with 59.9% of FVB and 63.7% of B6D2F1. Survival after piezo-ICSI was also comparable between FVB- and B6D2F1-reconstituted oocytes, with rates of 64.3% and 60.3%, respectively, albeit lower than the control (75.2%, P < 0.00001). FVB embryos fertilized at a rate of 88.7%, comparable to the control zygotes at 85.8%, while B6D2F1 conceptuses demonstrated a lower fertilization rate (70.8%, P < 0.00001). Blastulation of FVB- and B6D2F1-derived embryos was 15.1% and 24.0%, respectively, while the control was 80.7% (P < 0.00001). Whole-genome karyotyping of 9 B6D2F1-derived blastocysts confirmed 5 of the samples to be euploid. FVB blastocysts (N = 8) and B6D2F1 blastocysts (N = 81) were transferred into pseudo-pregnant mice, resulting in 3 fertile offspring only from the B6D2F1 conceptuses. Limitations, reasons for caution This is still a limited number of observations, and pups were delivered only from the B6D2F1 strain. The utilization of a strain with higher genetic variance may help facilitate offspring fingerprinting. Wider implications of the findings: This study demonstrates the ability to generate artificial genotyped conceptuses, yielding live offspring. The identification of a feasible donor cell, together with optimization of cell cycle stage and standardization of post-implantation development, will help promote this technique for human reproduction in couples with age-related infertility or poor ovarian reserve. Trial registration number N/A

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