Next Generation Sequencing Detects Premeiotic Errors in Human Oocytes

Autosomal aneuploidy is the leading cause of embryonic and foetal death in humans. This arises mainly from errors in meiosis I or II of oogenesis. A largely ignored source of error stems from germinal mosaicism, which leads to premeiotic aneuploidy. Molecular cytogenetic studies employing metaphase fluorescence in situ hybridization and comparative genomic hybridisation suggest that premeiotic aneuploidy may affect 10–20% of oocytes overall. Such studies have been criticised on technical grounds. We report here an independent study carried out on unmanipulated oocytes that have been analysed using next generation sequencing (NGS). This study confirms that the incidence of premeiotic aneuploidy in an unselected series of oocytes exceeds 10%. A total of 140 oocytes donated by 42 women gave conclusive results; of these, 124 (88.5%) were euploid. Sixteen out of 140 (11.4%) provided evidence of premeiotic aneuploidy. Of the 140, 112 oocytes were immature (germinal vesicle or metaphase I), of which 10 were aneuploid (8.93%); the remaining 28 were intact metaphase II - first polar body complexes, and six of these were aneuploid (21.4%). Of the 16 aneuploid cells, half contained simple errors (one or two abnormal chromosomes) and half contained complex errors. We conclude that germinal mosaicism leading to premeiotic aneuploidy is a consistent finding affecting at least 10% of unselected oocytes from women undergoing egg collection for a variety of reasons. The importance of premeiotic aneuploidy lies in the fact that, for individual oocytes, it greatly increases the risk of an aneuploid mature oocyte irrespective of maternal age. As such, this may account for some cases of aneuploid conceptions in very young women.

Polycystic ovary syndrome phenotype does not have impact on oocyte morphology

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.

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

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.

How Cold Shock Affects Ploidy Level and Early Ontogenetic Development of the Sterlet, A. ruthenus L.

The objective of the present research was to study the effect of cold shock (3 °C and 6 °C) on fertilized eggs of the sterlet, Acipenser ruthenus L. Cold shock was applied for various durations (30, 60 and 90 min) and the ploidy levels, survival, and genotypes of the treated embryos/larvae were recorded. Analysis of ploidy levels confirmed the presence of diploid, triploid, and mosaic (1n/2n, 2n/3n, and 1n/2n/3n) genotypes in experimental groups, while it was strictly diploid in control groups. Microsatellite genotyping confirmed both the incidence of polyspermy and retention of the 2nd polar body in experimental groups. However, patterns of inheritance in all diploid offspring in experimental and control groups revealed classical Mendelian disomic inheritance. Interestingly, the observed mosaic sterlets had normal morphology and were alive. However, some larvae had abnormal morphology which may be due to haploid syndrome. In all treatment groups (treatments: 3 °C–30 min; 3 °C–60 min; 3 °C–90 min; 6 °C–60 min), where the percentage of polyploid/mosaic larvae were high, the mortality was also high. Whereas, in the control groups (where there were only diploid (2n) larvae), the mortality was relatively low.

Pterostilbene Alleviates Chlorpyrifos-Induced Damage During Porcine Oocyte Maturation

Chlorpyrifos (CPF), a widely used organophosphate pesticide, is reported to severely impair mammalian reproductive system. Pterostilbene (PTS), an effective free radical scavenger, is considered as beneficial for mammalian reproduction. However, the toxicity of CPF on oocyte maturation and whether PTS can eliminate the detrimental effect of CPF on oocytes remain unclear. Here, porcine oocytes were applied to investigate the potential effect and possible mechanism of CPF and PTS during oocyte maturation. This work demonstrated that CPF significantly delayed the meiotic progression and decreased the polar body extrusion by disturbing spindle assembly and chromosome alignment and causing DNA damage in oocytes (p < 0.05). And, CPF significantly impaired oocyte cytoplasmic maturation by inducing the high level of reactive oxygen species and decreasing glutathione content (p < 0.05). Moreover, CPF significantly triggered embryo apoptosis and reduced the blastocyst rate and cell number following parthenogenetic activation (p < 0.05). Whereas CPF-exposed oocytes were treated with PTS, these defects caused by CPF were obviously rescued, and oocyte maturation and subsequent embryonic development were also significantly ameliorated (p < 0.05). In conclusion, these results revealed that CPF exerted the toxic effect on porcine oocytes, while PTS effectively alleviated CPF-induced damage on oocytes. This work provides a potential strategy to protect oocyte maturation in mammalian species.

In vitro maturation of goat oocytes selected using Brilliant cresyl blue staining

The present study was conducted to assess the developmental competence of goat oocytes selected using Brilliant cresyl blue (BCB) staining. Goat ovaries were collected from the slaughtered animals with unknown reproductive history. The oocytes retrieved by aspiration technique were selected based on morphology and subjected to BCB staining. Brilliant cresyl blue staining is based on the activity of glucose-6-phospahte dehydrogenase (G6PDH) enzyme synthesised by the oocytes. The cytoplasm remains blue in oocytes that have finished the growth phase (BCB+) while the growing oocytes remain colourless (BCB-). The stained and unstained oocytes were subjected to in vitro maturation separately to assess cumulus cell expansion index and polar body extrusion. A total of 206 culture grade oocytes were subjected to study, out of which, 76.75 ± 2.38 per cent of oocytes showed positive to BCB staining and 23.21 ± 2.38 per cent were negatively stained. Significantly higher maturation rate was observed in BCB+(92.89 ± 2.37%) oocytes than BCB-(29.72 ± 2.46%). The present study concluded that BCB staining can be used for selecting goat oocytes with good cytoplasmic maturation for further in vitro embryo production

Glycine Ameliorates Endoplasmic Reticulum Stress Induced by Thapsigargin in Porcine Oocytes

The endoplasmic reticulum (ER) is a multifunctional organelle in the cytoplasm that plays important roles in female mammalian reproduction. The endoplasmic reticulum and mitochondria interact to maintain the normal function of cells by maintaining intracellular calcium homeostasis. As proven by previous research, glycine (Gly) can regulate the intracellular free calcium concentration ([Ca2+]i) and enhance mitochondrial function to improve oocyte maturation in vitro. The effect of Gly on ER function during oocyte in vitro maturation (IVM) is not clear. In this study, we induced an ER stress model with thapsigargin (TG) to explore whether Gly can reverse the ER stress induced by TG treatment and whether it is associated with calcium regulation. The results showed that the addition of Gly could improve the decrease in the average cumulus diameter, the first polar body excretion rate caused by TG-induced ER stress, the cleavage rate and the blastocyst rate. Gly supplementation could reduce the ER stress induced by TG by significantly improving the ER levels and significantly downregulating the expression of genes related to ER stress (Xbp1, ATF4, and ATF6). Moreover, Gly also significantly alleviated the increase in reactive oxygen species (ROS) levels and the decrease in mitochondrial membrane potential (ΔΨ m) to improve mitochondrial function in porcine oocytes exposed to TG. Furthermore, Gly reduced the [Ca2+]i and mitochondrial Ca2+ ([Ca2+]m) levels and restored the ER Ca2+ ([Ca2+]ER) levels in TG-exposed porcine oocytes. Moreover, we found that the increase in [Ca2+]i may be caused by changes in the distribution and expression of inositol 1,4,5-triphosphate receptor (IP3R1) and voltage-dependent anion channel 1 (VDAC1), while Gly can restore the distribution and expression of IP3R1 and VDAC1 to normal levels. Apoptosis-related indexes (Caspase 3 activity and Annexin-V) and gene expression Bax, Cyto C, and Caspase 3) were significantly increased in the TG group, but they could be restored by adding Gly. Our results suggest that Gly can ameliorate ER stress and apoptosis in TG-exposed porcine oocytes and can further enhance the developmental potential of porcine oocytes in vitro.

Cofilin regulates actin network homeostasis and microvilli length in mouse oocytes

How multiple actin networks coexist in a common cytoplasm, while competing for a shared pool of monomers, is still an ongoing question. This is exemplified by meiotic maturation in the mouse oocyte, which relies on the dynamic remodeling of distinct cortical and cytoplasmic F-actin networks. Here we show that the conserved actin-depolymerizing factor cofilin is activated in a switch-like manner at meiosis resumption from prophase arrest. Interfering with cofilin activation during maturation resulted in widespread microvilli elongation, while cytoplasmic F-actin was depleted, leading to defects in spindle migration and polar body extrusion. In contrast, cofilin inactivation in metaphase II-arrested oocytes resulted in a shutdown of F-actin dynamics, along with a dramatic overgrowth of the polarized actin cap. However, inhibition of the Arp2/3 complex to promote actin cap disassembly elicited ectopic microvilli outgrowth in the polarized cortex. These data establish cofilin as a key player in actin network homeostasis in oocytes, and reveal that microvilli can act as a sink for monomers upon disassembly of a competing network.

Grape Seed Proanthocyanidin Ameliorates FB1-Induced Meiotic Defects in Porcine Oocytes

Fumonisin B1 (FB1), as the most prevalent and toxic fumonisin, poses a health threat to humans and animals. The cytotoxicity of FB1 is closely related to oxidative stress and apoptosis. The purpose of this study is to explore whether Grape seed proanthocyanidin (GSP), a natural antioxidant, could alleviate the meiotic maturation defects of oocytes caused by FB1 exposure. Porcine cumulus oocyte complexes (COCs) were treated with 30 μM FB1 alone or cotreated with 100, 200 and 300 μM GSP during in vitro maturation for 44 h. The results show that 200 μM GSP cotreatment observably ameliorated the toxic effects of FB1 exposure, showing to be promoting first polar body extrusion and improving the subsequent cleavage rate and blastocyst development rate. Moreover, 200 μM GSP cotreatment restored cell cycle progression, reduced the proportion of aberrant spindles, improved actin distribution and protected mitochondrial function in FB1-exposed oocytes. Furthermore, reactive oxygen species (ROS) generation was significantly decreased and the mRNA levels of CAT, SOD2 and GSH-PX were obviously increased in the 200 μM GSP cotreatment group. Notably, the incidence of early apoptosis and autophagy level were also significantly decreased after GSP cotreatment and the mRNA expression levels of BAX, CASPASE3, LC3 and ATG5 were markedly decreased, whereas BCL2 and mTOR were observably increased in the oocytes after GSP cotreatment. Together, these results indicate that GSP could exert significant preventive effects on FB1-induced oocyte defects by ameliorating oxidative stress through repairing mitochondrial dysfunction.