polar bodies
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2022 ◽  
Vol 13 (1) ◽  
Zhaoyue Men ◽  
Meng Cao ◽  
Yuechan Gong ◽  
Lun Hua ◽  
Ruihao Zhang ◽  

Abstract Background Dietary fiber (DF) is often eschewed in swine diet due to its anti-nutritional effects, but DF is attracting growing attention for its reproductive benefits. The objective of this study was to investigate the effects of DF intake level on oocyte maturation and uterine development, to determine the optimal DF intake for gilts, and gain microbial and metabolomic insight into the underlying mechanisms involved. Methods Seventy-six Landrace × Yorkshire (LY) crossbred replacement gilts of similar age (92.6 ± 0.6 d; mean ± standard deviation [SD]) and body weight (BW, 33.8 ± 3.9 kg; mean ± SD) were randomly allocated to 4 dietary treatment groups (n = 19); a basal diet without extra DF intake (DF 1.0), and 3 dietary groups ingesting an extra 50% (DF 1.5), 75% (DF 1.75), and 100% (DF 2.0) dietary fiber mixture consisting of inulin and cellulose (1:4). Oocyte maturation and uterine development were assessed on 19 d of the 2nd oestrous cycle. Microbial diversity of faecal samples was analysed by high-throughput pyrosequencing (16S rRNA) and blood samples were subjected to untargeted metabolomics. Results The rates of oocytes showing first polar bodies after in vitro maturation for 44 h and uterine development increased linearly with increasing DF intake; DF 1.75 gilts had a 19.8% faster oocyte maturation rate and a 48.9 cm longer uterus than DF 1.0 gilts (P <  0.05). Among the top 10 microbiota components at the phylum level, 8 increased linearly with increasing DF level, and the relative abundance of 30 of 53 microbiota components at the genus level (> 0.1%) increased linearly or quadratically with increasing DF intake. Untargeted metabolic analysis revealed significant changes in serum metabolites that were closely associated with microbiota, including serotonin, a gut-derived signal that stimulates oocyte maturation. Conclusions The findings provide evidence of the benefits of increased DF intake by supplementing inulin and cellulose on oocyte maturation and uterine development in gilts, and new microbial and metabolomic insight into the mechanisms mediating the effects of DF on reproductive performance of replacement gilts.

Hieu Nguyen ◽  
Hongwen Wu ◽  
Anna Ung ◽  
Yukiko Yamazaki ◽  
Ben Fogelgren ◽  

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.

David Alonso-Gutiérrez ◽  
Silouanos Brazitikos

Abstract We prove several estimates for the volume, the mean width, and the value of the Wills functional of sections of convex bodies in John’s position, as well as for their polar bodies. These estimates extend some well-known results for convex bodies in John’s position to the case of lower-dimensional sections, which had mainly been studied for the cube and the regular simplex. Some estimates for centrally symmetric convex bodies in minimal surface area position are also obtained.

PLoS Biology ◽  
2021 ◽  
Vol 19 (9) ◽  
pp. e3001376
Benoit Dehapiot ◽  
Raphaël Clément ◽  
Bourdais Anne ◽  
Virginie Carrière ◽  
Huet Sébastien ◽  

Mammalian oocyte meiotic divisions are highly asymmetric and produce a large haploid gamete and 2 small polar bodies. This relies on the ability of the cell to break symmetry and position its spindle close to the cortex before anaphase occurs. In metaphase II–arrested mouse oocytes, the spindle is actively maintained close and parallel to the cortex, until fertilization triggers sister chromatid segregation and the rotation of the spindle. The latter must indeed reorient perpendicular to the cortex to enable cytokinesis ring closure at the base of the polar body. However, the mechanisms underlying symmetry breaking and spindle rotation have remained elusive. In this study, we show that spindle rotation results from 2 antagonistic forces. First, an inward contraction of the cytokinesis furrow dependent on RhoA signaling, and second, an outward attraction exerted on both sets of chromatids by a Ran/Cdc42-dependent polarization of the actomyosin cortex. By combining live segmentation and tracking with numerical modeling, we demonstrate that this configuration becomes unstable as the ingression progresses. This leads to spontaneous symmetry breaking, which implies that neither the rotation direction nor the set of chromatids that eventually gets discarded are biologically predetermined.

2021 ◽  
Vol 36 (Supplement_1) ◽  
E Molinari ◽  
M Yang ◽  
J Hu ◽  
L Zhang ◽  
D F Albertini ◽  

Abstract Study question What causes our patient’s repeated almost complete oocyte maturation arrest (OMA)? Summary answer Since we did not detect PATL2 and TUBB8 mutations, both known to cause OMA, this case was likely caused by mutations in HUS1 and ITGB3 What is known already OMA has been associated with loss-of-function in key genes, such as PATL2 and TUBB8. Such patients have, however, uniformly have been unable to conceive with IVF Study design, size, duration We here report the case of repeatedly presenting patient between 2009 until 2020 (age 30 at 1st and 41 at last visit). Participants/materials, setting, methods The couple underwent 7 IVF treatments under several ovarian stimulation protocols at different gonadotropin dosages and in different preparations to try to recruit mature eggs. She conceived in her 2nd IVF cycle in 2009 and delivered uneventfully in 2010. She then conceived spontaneously and delivered a healthy boy in 2014. The couple since then has been attempting another pregnancy. Remarkably, in all IVF cycles all eggs but one arrested at prophase. Main results and the role of chance The female demonstrates abnormally high ovarian reserve for age (AMH=5.9 ng/mL in 2019) (mean, 10.6 oocytes). In all cycles, all but one retrieved were immature. In vitro maturation rate for the GV oocytes was 28%. Resultant M2s, however, demonstrated morphological abnormalities, such as giant polar bodies. In vivo M2s, in contrast, were always morphologically unremarkable, and their fertilization rate was 85%. Embryo morphology deteriorated appreciatively with advancing age. Sanger sequencing for TUBB8 and PATL2 genes were unremarkable. Whole genome sequencing of her and her sister (who had no fertility problems) revealed mutations of genes belonging to the integrin family (ITGB3) and DNA repair checkpoint (HUS1), both of which could be determinants in the observed maturation arrest. Limitations, reasons for caution A functional study, coupled with imaging of the discarded material, will likely offer further information regarding the mechanisms leading to OMA in this female. Wider implications of the findings: This case report represents a new phenotype of female infertility, characterized by almost complete maturation arrest which, however, still offers opportunity for pregnancy. Further isolation of underlying mutation(s) may offer additional insights about checkpoints required for the transition of prophase to metaphase in human oocytes. Trial registration number NA

2021 ◽  
Vol 36 (Supplement_1) ◽  
C Racowsky

Abstract text Fertilization is a critical event in development in that it provides the connection between the gametes and the earliest stages of embryogenesis. Yet, despite the central importance of this process in contributing to embryo developmental fate, clinical embryologists have historically assessed fertilization merely by the number of pronuclei and, if two are present, perhaps, by the presence of two polar bodies. Even though over 20 years ago, time lapse imaging was applied for defining early events of fertilization (Payne et al., 1997), it is only with contemporary time-lapse imaging systems in the last few years that detailed evaluation of spatial and temporal events of fertilization have been described (Iwata & Yasuyuki, 2016; Cottichio et al., 2018). These careful analyses allow us to describe typical and atypical events of fertilization and how they are each associated with timing of the first cleavage division and subsequent embryo development. In this lecture, we will first describe the fundamental underpinnings of fertilization and highlight the normal events associated with this process. We will then discuss gross morphological abnormalities as visualized by light microscopy and highlight the unknowns associated with these events. Finally, we will focus on time-lapse imaging studies, which have revealed the remarkable spatial and temporal coordination of meiotic resumption, pronuclear dynamics, chromatin organization and cytoplasmic/cortical modifications that occur during fertilization and the implications of aberrations for the first cleavage division. At the conclusion of this presentation, attendees should be able to: Review the normal events associated with fertilization and the first cleavage division. 1 Describe gross morphological aberrations of these two fundamental processes. 2 Discuss temporal and spatial abnormalities in the coordinated sequence of events that underly these processes. 3 State the potential application of these abnormalities as predictors of abnormal embryo development. 4 Summarize the puzzling unknowns that underly these abnormalities.

2021 ◽  
Vol 36 (Supplement_1) ◽  
E Crugnola ◽  
A Gobbetti ◽  
N Fiandanese ◽  
G Filippini

Abstract Study question How to technically deal with the PGT-M set-up in case of de novo mutations in female or male affected patients with dominant disease due to de novo mutations. Summary answer PGT-M was performed for three couples carrying de novo mutations using direct and linkage analysis on sperm or polar bodies to define haplotypes and phase. What is known already Couples with a de novo mutation in a gene causing AD disease, such as FGFR3 (achondroplasia), NF1 (neurofibromatosis) and EXT2 (multiple exostosis) cannot undergo PGT-M via standard techniques like karyomapping, as the absence of affected relatives makes phasing impossible. However, linkage analysis combined with direct mutation analysis allows on haploid cells from the mutation carrier, such as sperm or polar bodies (PB), allows the correct association of a haplotype and the disease-causing mutation. Flanking informative STRs must be positioned at &lt; 1 Mb of the gene, in order to minimize the risk of recombination during meiosis. Study design, size, duration Couples underwent pre-test counselling with a geneticist and an IVF specialist. Pathogenic variants were identified and their absence from the couples’ parents confirmed. Four to six informative STRs were identified. For males we analysed 20–50 isolated sperm to define the haplotypes and the phase, before starting with the stimulation cycle; for females, we needed to wait after the oocyte pick-up and the biopsy of PBs. Point mutations are identified by SNaPshot, deletions by multiplexed STS. Participants/materials, setting, methods The 3 couples in the study presented in IVF centres, requesting PGT-M for either male or female AD disease. They had genetic testing reports from other laboratories. For FGFR3 and NF1, the described variants were confirmed. The patient with multiple exostosis came with a negative genetic result for EXT1 and EXT2 genes, but after diagnostic-quality NGS (Blueprint Genetics, Finland) we identified an EXT2 deletion. Diagnostic multiplex PCR was then performed on embryos or polar bodies. Main results and the role of chance The setup started with the confirmation of the mutations in the 3 couples and the confirmation of the de novo status. Four to six informative STRs were then identified for each couple. Multiplex PCR containing the STRs and the SNAPSHOT analysis for the point mutations was developed. To identify the phase and the disease-carrying haplotype in male carriers, we performed a multiplex PCR on 20–50 spermatozoa. In the female patient with NF1, the haplotype and the phase were determined on the polar bodies; the mutation was on her paternal allele, as predicted genetically. Prior to PGT, we evaluated the robustness of each multiplex on 20 to 50 single leukocytes of the couple. Each couple had at least one embryo not carrying the risk haplotype, suitable for transfer. The couples with NF1 and achondroplasia both delivered a healthy, unaffected baby. The pregnancy is ongoing in the couple with the EXT2 variant. PGT-M is now easily handled for standard situations, with semiautomated protocols that do not need extensive setups. De novo mutations however present a unique challenge, because of the impossibility in most cases of determining the phase of the disease-causing variant. We present a patient-centric approach with individualized protocols. Limitations, reasons for caution Allele drop-out could lead to misdiagnosis of the embryo. To avoid that, 6 flanking STRs (3 proximal and 3 distal) and genotyping of the variant should be performed. When possible, it is good practice to pre-define the different haplotypes with the parents of the patients. Wider implications of the findings: The increasing number of laboratories offering off-the-shelf testing with NGS panels and semi-automated PGT can fulfil demand for routine situations. However in more complex cases, diagnostic-quality NGS and individualized PGT-M programmes are needed. These cases also remind us that PGT-M requires extensive multidisciplinarity to maximize the chance of successful outcome. Trial registration number Not applicable

2021 ◽  
Tie-Gang Meng ◽  
Qian Zhou ◽  
Xue-Shan Ma ◽  
Xiao-Yu Liu ◽  
Qing-Ren Meng ◽  

Abstract This protocol presents ULI-NChIP-seq (ultra-low-input micrococcal nuclease-based native ChIP-seq) assay to generate high quality and complexity genome-wide histone mark profiles from rare oocytes andembryos populations. The procedure of ULI-NChIP-seq assay typically consists of five parts including Binding antibodies to magnatic beads, Chromatin shearing and nuclear membrane solubilization, Magnetic immunoprecipitation, Washes and DNA isolation. Sample preparation involves to remove the zona Pellucida of oocyte and polar body to avoid the genomic contamination of polar bodies.

2021 ◽  
Vol 33 (2) ◽  
pp. 148
O. Briski ◽  
A. Gambini ◽  
LD Ratner ◽  
DF Salamone

Pigs are considered an important experimental model for their biological similarities to humans, including their potential as organ donors in xenotransplantation. Unfortunately, in this species conventional invitro fertilization results in high polyspermic rates. ICSI avoids polyspermy and ICSI-mediated gene edition could be a powerful technique to produce genetically modified pigs. However, ICSI is not yet efficient in pigs. Moreover, the ATP-dependent chromatin remodeller, SMARCA4, translocates to the pronuclei soon after fertilization and its mislocalization or reduction leads to poor embryo development. The aim of this study was to assess whether assisted activation or the use of the piezo drill (PD) during ICSI improves pronuclear (PN) formation rates and to analyse SMARCA4 intensity levels in pronuclei. First, cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured invitro for 44h. Matured and denuded oocytes were subjected to (1) ICSI (n=47), (2) ICSI assisted by PD (ICSIp, n=21), (3) ICSI assisted by electrical activation (ICSIe, n=39), and (4) electrical activation as an haploid parthenogenetic control (HAP, n=21). Presumptive zygotes were fixed for 20min in 4% formaldehyde solution 18h after injection or activation and incubated with SMARCA4 antibody (1:100) and Alexa Fluor (1:1000) as a secondary antibody. Then, the zygotes were classified according to the presence of PN in 2 PN (2-PN), 1 PN with the presence of a semi-condensed or condensed sperm (1-PN), and semi-condensed or condensed sperm with no evidence of PN (no activation). Zygotes that exhibited a different pattern were included in the “other” category. A region of interest was drawn around each PN and the average pixel intensity of SMARCA4 was determined with ImageJ image processing software. Data were analysed by Fisher’s exact test and Kruskal–Wallis test using GraphPad software (GraphPad Inc.). Differences were considered significant at P&lt;0.05. We found no significant differences in 2-PN formation rates among groups after ICSI (ICSI n=16, 34.04%; ICSIe n=10, 25.64%; ICSIp n=6, 28.57%). As expected, the majority of the HAP zygotes exhibited 1 PN (n=14, 66.67%). In contrast, in most of the zygotes of all experimental groups, SMARCA4 was found to be localised in both PN, being absent in polar bodies, metaphase plate, or condensed sperm. Interestingly, out of the total 2-PN porcine ICSI zygotes of all experimental groups (n=25), 7 zygotes (28%) showed clear asymmetric intensity levels between PN. The rest of the ICSI zygotes (n=18, 72%) showed a similar SMARCA4 intensity level between PN. In conclusion, our results suggest that neither the use of piezo drill or electrical activation improves PN formation or SMARCA4 pattern. It remains to be determined whether the asymmetric levels of SMARCA4 between PN observed in some zygotes could be associated with a lower embryo developmental competence.

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