Vitrification-induced activation of lysosomal cathepsin B perturbs spindle assembly checkpoint function in mouse oocytes

Abstract As the age of child-bearing increases and correlates with infertility, cryopreservation of female gametes is becoming common-place in ART. However, the developmental competence of vitrified oocytes has remained low. The underlying mechanisms responsible for reduced oocyte quality post-vitrification are largely unknown. Mouse cumulus–oocyte complexes were vitrified using a cryoloop technique and a mixture of dimethylsulphoxide, ethylene glycol and trehalose as cryoprotectants. Fresh and vitrified/thawed oocytes were compared for chromosome alignment, spindle morphology, kinetochore-microtubule attachments, spindle assembly checkpoint (SAC) and aneuploidy. Although the majority of vitrified oocytes extruded the first polar body (PB), they had a significant increase of chromosome misalignment, abnormal spindle formation and aneuploidy at metaphase II. In contrast to controls, vitrified oocytes extruded the first PB in the presence of nocodazole and etoposide, which should induce metaphase I arrest in a SAC-dependent manner. The fluorescence intensity of mitotic arrest deficient 2 (MAD2), an essential SAC protein, at kinetochores was reduced in vitrified oocytes, indicating that the SAC is weakened after vitrification/thawing. Furthermore, we found that vitrification-associated stress disrupted lysosomal function and stimulated cathepsin B activity, with a subsequent activation of caspase 3. MAD2 localization and SAC function in vitrified oocytes were restored upon treatment with a cathepsin B or a caspase 3 inhibitor. This study was conducted using mouse oocytes, therefore confirming these results in human oocytes is a prerequisite before applying these findings in IVF clinics. Here, we uncovered underlying molecular pathways that contribute to an understanding of how vitrification compromises oocyte quality. Regulating these pathways will be a step toward improving oocyte quality post vitrification and potentially increasing the efficiency of the vitrification program.

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Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

Reproduction Fertility and Development ◽

10.1071/rd12332 ◽

2014 ◽

Vol 26 (8) ◽

pp. 1084 ◽

Cited By ~ 2

Author(s):

Yu-Ting Shen ◽

Yue-Qiang Song ◽

Xiao-Qin He ◽

Fei Zhang ◽

Xin Huang ◽

...

Keyword(s):

Polar Body ◽

Meiotic Maturation ◽

Dependent Manner ◽

Germinal Vesicle Breakdown ◽

Mouse Oocytes ◽

First Polar Body ◽

Triphenyltin Chloride ◽

Dose Dependent

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

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Mitochondrial Ca2+ Overload Leads to Mitochondrial Oxidative Stress and Delayed Meiotic Resumption in Mouse Oocytes

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.580876 ◽

2020 ◽

Vol 8 ◽

Author(s):

Luyao Zhang ◽

Zichuan Wang ◽

Tengfei Lu ◽

Lin Meng ◽

Yan Luo ◽

...

Keyword(s):

Oxidative Stress ◽

Mitochondrial Function ◽

Maternal Obesity ◽

Polar Body ◽

Germinal Vesicle ◽

Oocyte Quality ◽

Obese Women ◽

Germinal Vesicle Breakdown ◽

Mouse Oocytes ◽

First Polar Body

Overweight or obese women seeking pregnancy is becoming increasingly common. Human maternal obesity gives rise to detrimental effects during reproduction. Emerging evidence has shown that these abnormities are likely attributed to oocyte quality. Oxidative stress induces poor oocyte conditions, but whether mitochondrial calcium homeostasis plays a key role in oocyte status remains unresolved. Here, we established a mitochondrial Ca2+ overload model in mouse oocytes. Knockdown gatekeepers of the mitochondrial Ca2+ uniporters Micu1 and Micu2 as well as the mitochondrial sodium calcium exchanger NCLX in oocytes both increased oocytes mitochondrial Ca2+ concentration. The overload of mitochondria Ca2+ in oocytes impaired mitochondrial function, leaded to oxidative stress, and changed protein kinase A (PKA) signaling associated gene expression as well as delayed meiotic resumption. Using this model, we aimed to determine the mechanism of delayed meiosis caused by mitochondrial Ca2+ overload, and whether oocyte-specific inhibition of mitochondrial Ca2+ influx could improve the reproductive abnormalities seen within obesity. Germinal vesicle breakdown stage (GVBD) and extrusion of first polar body (PB1) are two indicators of meiosis maturation. As expected, the percentage of oocytes that successfully progress to the germinal vesicle breakdown stage and extrude the first polar body during in vitro culture was increased significantly, and the expression of PKA signaling genes and mitochondrial function recovered after appropriate mitochondrial Ca2+ regulation. Additionally, some indicators of mitochondrial performance—such as adenosine triphosphate (ATP) and reactive oxygen species (ROS) levels and mitochondrial membrane potential—recovered to normal. These results suggest that the regulation of mitochondrial Ca2+ uptake in mouse oocytes has a significant role during oocyte maturation as well as PKA signaling and that proper mitochondrial Ca2+ reductions in obese oocytes can recover mitochondrial performance and improve obesity-associated oocyte quality.

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Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes

Reproduction ◽

10.1530/rep.1.00856 ◽

2005 ◽

Vol 130 (6) ◽

pp. 829-843 ◽

Cited By ~ 75

Author(s):

Hayden A Homer ◽

Alex McDougall ◽

Mark Levasseur ◽

Alison P Murdoch ◽

Mary Herbert

Keyword(s):

Spindle Assembly Checkpoint ◽

Polar Body ◽

Cyclin B ◽

Mutant Form ◽

Anaphase Promoting Complex ◽

Mouse Oocytes ◽

First Polar Body ◽

Polar Body Extrusion ◽

Meiosis I

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.

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Echinoderm Microtubule Associated Protein Like 1 Is Indispensable for Oocyte Spindle Assembly and Meiotic Progression in Mice

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.687522 ◽

2021 ◽

Vol 9 ◽

Author(s):

Hong Yin ◽

Teng Zhang ◽

Hao Wang ◽

Xin Hu ◽

Xuan Hou ◽

...

Keyword(s):

Spindle Assembly Checkpoint ◽

Polar Body ◽

Spindle Assembly ◽

Mass Spectrometry Analysis ◽

Spectrometry Analysis ◽

Meiotic Progression ◽

Spindle Poles ◽

First Polar Body ◽

Specific Localization ◽

Oocyte Meiotic Maturation

Completion of the first meiosis is an essential prerequisite for producing a functionally normal egg for fertilization and embryogenesis, but the precise mechanisms governing oocyte meiotic progression remains largely unclear. Here, we report that echinoderm microtubule associated protein (EMAP) like 1 (EML1), a member of the conserved EMAP family proteins, plays a crucial role in the control of oocyte meiotic progression in the mouse. Female mice carrying an ENU-induced nonsense mutation (c.1956T > A; p.Tyr652∗) of Eml1 are infertile, and the majority of their ovulated oocytes contain abnormal spindles and misaligned chromosomes. In accordance with the mutant oocyte phenotype, we find that EML1 is colocalized with spindle microtubules during the process of normal oocyte meiotic maturation, and knockdown (KD) of EML1 by specific morpholinos in the fully grown oocytes (FGOs) disrupts the integrity of spindles, and delays meiotic progression. Moreover, EML1-KD oocytes fail to progress to metaphase II (MII) stage after extrusion of the first polar body, but enter into interphase and form a pronucleus containing decondensed chromatins. Further analysis shows that EML1-KD impairs the recruitment of γ-tubulin and pericentrin to the spindle poles, as well as the attachment of kinetochores to microtubules and the proper inactivation of spindle assembly checkpoint at metaphase I (MI). The loss of EML1 also compromises the activation of maturation promoting factor around the time of oocyte resumption and completion of the first meiosis, which, when corrected by WEE1/2 inhibitor PD166285, efficiently rescues the phenotype of oocyte delay of meiotic resumption and inability of reaching MII. Through IP- mass spectrometry analysis, we identified that EML1 interacts with nuclear distribution gene C (NUDC), a critical mitotic regulator in somatic cells, and EML1-KD disrupts the specific localization of NUDC at oocyte spindles. Taken together, these data suggest that EML1 regulates acentrosomal spindle formation and the progression of meiosis to MII in mammalian oocytes, which is likely mediated by distinct mechanisms.

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156 EFFECTS OF MORPHOLOGY TYPE OF POLAR BODY ON PORCINE OOCYTE QUALITY AND DEVELOPMENTAL POTENTIAL AFTER IN VITRO FERTILIZATION

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab156 ◽

2014 ◽

Vol 26 (1) ◽

pp. 192

Author(s):

L. Cai ◽

E. Kim ◽

S. U. Hwang ◽

J. D. Yoon ◽

Y. Jeon ◽

...

Keyword(s):

Polar Body ◽

Oocyte Quality ◽

Developmental Competence ◽

Penicillin G ◽

Cleavage Rate ◽

Developmental Potential ◽

Vitro Fertilization ◽

First Polar Body

Evaluation of morphology of first polar body (1st PB) could be a method for the oocyte's quality and developmental competence. The developmental potential of oocyte with fragmented PB after in vitro maturation (IVM) is a controversial issue. The aim of this study is to investigate the effects of PB morphology type on oocyte quality and developmental competence after IVF. Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 2 h in physiological saline supplemented with 100 IU mL–1 penicillin G and 100 mg mL–1 streptomycin sulfate. The cumulus–oocyte complexes (COC) were aspirated using an 18-gauge needle attached to a 10-mL disposable syringe from superficial follicles 3 to 6 mm in diameter followed by IVM. After IVM, oocytes were classified into 3 types as follows, oocytes with normal PB (A type), oocytes with a little of fragmented PB (B type), and oocytes with separated 2 PBs (C type), respectively. As classification of PB types, we analysed the distribution ratio of each PB type after IVM, and then performed IVF for analysis of fertilization rate and developmental potential. The ratio of oocyte with A type (73%) was significantly (P < 0.05) higher than that of B type (24.5%) or C type (2.5%) after IVM. Only mature oocytes were selected from A and B type and were subjected to IVF because of a small number of oocytes with C type. In the IVF experiment, the efficiency of monospermy and fertilization were significantly higher in oocytes of A type (46.7%) than those of type B (20.0%). The cleavage rate of oocytes with A type (63.9%) was significantly (P < 0.05) higher than the oocytes with B type (43.8%). Embryonic developmental competence to the blastocyst stage after IVF was significantly (P < 0.05) higher in the A-type oocytes (26.3%) than in the B-type oocytes (16.9%). The levels of glutathione and reactive oxygen species were not affected by the morphological classification of the PB. In summary, these results suggest that polar body morphology could be a marker of oocyte quality after IVM. We are currently studying gene expression of each oocytes and blastocysts. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

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Iodoacetic acid disrupts mouse oocyte maturation by inducing oxidative stress and spindle abnormalities

10.1101/2020.09.02.279604 ◽

2020 ◽

Author(s):

Xiaofei Jiao ◽

Andressa Gonsioroski ◽

Jodi A Flaws ◽

Huanyu Qiao

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Oocyte Maturation ◽

Polar Body ◽

Spindle Assembly ◽

Iodoacetic Acid ◽

Mouse Oocyte ◽

Water Disinfection ◽

Dependent Manner ◽

Mouse Oocytes

AbstractDisinfection by-products (DBPs) are compounds produced during the water disinfection process. Iodoacetic acid (IAA) is one of the unregulated DBPs in drinking water, with potent cytotoxicity and genotoxicity in animals. However, whether IAA has toxic effects on oocyte maturation remains unclear. Here, we show that IAA exposure resulted in metaphase I (MI) arrest and polar-body-extrusion failure in mouse oocytes, indicating that IAA had adverse effects on mouse oocyte maturation in vitro. Particularly, IAA treatment caused abnormal spindle assembly and chromosome misalignment. Previous studies reported that IAA is a known inducer of oxidative stress in non-germline cells. Correspondingly, we found that IAA exposure increased the reactive oxygen species (ROS) levels in oocytes in a dose-dependent manner, indicating IAA exposure could induce oxidative stress in oocytes. Simultaneously, DNA damage was also elevated in the nuclei of these IAA-exposed mouse oocytes, evidenced by increased γ-H2AX focus number. In addition, the un-arrested oocytes entered metaphase II (MII) with severe defects in spindle morphologies and chromosome alignment after 14-hour IAA treatment. An antioxidant, N-acetyl-L-cysteine (NAC), reduced the elevated ROS level and restored the meiotic maturation in the IAA. exposed oocytes, which indicates that IAA-induced maturation failure in oocytes was mainly mediated by oxidative stress. Collectively, our results indicate that IAA exposure interfered with mouse oocyte maturation by elevating ROS levels, disrupting spindle assembly, inducing DNA damage, and causing MI arrest.

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The Aurora kinase inhibitor ZM447439 accelerates first meiosis in mouse oocytes by overriding the spindle assembly checkpoint

Reproduction ◽

10.1530/rep-10-0223 ◽

2010 ◽

Vol 140 (4) ◽

pp. 521-530 ◽

Cited By ~ 46

Author(s):

Simon I R Lane ◽

Heng-Yu Chang ◽

Phoebe C Jennings ◽

Keith T Jones

Keyword(s):

Spindle Assembly Checkpoint ◽

Kinase Inhibitor ◽

Polar Body ◽

Spindle Assembly ◽

Aurora Kinase ◽

Mitotic Cell ◽

Cyclin B1 ◽

Anaphase Promoting Complex ◽

Mouse Oocytes ◽

Polar Body Extrusion

Previous studies have established that when maturing mouse oocytes are continuously incubated with the Aurora inhibitor ZM447439, meiotic maturation is blocked. In this study, we observe that by altering the time of addition of the inhibitor, oocyte maturation can actually be accelerated by 1 h as measured by the timing of polar body extrusion. ZM447439 also had the ability to overcome a spindle assembly checkpoint (SAC) arrest caused by nocodazole and so rescue polar body extrusion. Consistent with the ability of the SAC to inhibit cyclin B1 degradation by blocking activation of the anaphase-promoting complex, we could also observe a rescue in cyclin B1 degradation when ZM447439 was added to nocodazole-treated oocytes. The acceleration of the first meiotic division by ZM447439, which has not been achieved previously, and its effects on the SAC are all consistent with the proposed mitotic role of Aurora B in activating the SAC. We hypothesize that Aurora kinase activity controls the SAC in meiosis I, despite differences to the mitotic cell cycle division in spindle architecture brought about by the meiotic mono-orientation of sister kinetochores.

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Evaluation of oocyte quality: morphological, cellular and molecular predictors

Reproduction Fertility and Development ◽

10.1071/rd06103 ◽

2007 ◽

Vol 19 (1) ◽

pp. 1 ◽

Cited By ~ 159

Author(s):

Qiang Wang ◽

Qing-Yuan Sun

Keyword(s):

Transforming Growth Factor ◽

Polar Body ◽

Morphological Characteristics ◽

Transforming Growth Factor Β ◽

Oocyte Quality ◽

Developmental Competence ◽

Meiotic Spindle ◽

Oocyte Competence ◽

Cumulus Oocyte Complex ◽

Glucose 6 Phosphate Dehydrogenase

Mounting evidence that oocyte quality profoundly affects fertilisation and subsequent embryo development drives the continued search for reliable predictors of oocyte developmental competence. In the present review, we provide an overall summary and analysis of potential criteria that can be used to evaluate oocyte quality. These criteria are specifically classified as morphological and cellular/molecular predictors. Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus–oocyte complex, polar body and/or meiotic spindle. Although the use of morphological characteristics as predictors of oocyte quality is controversial, such a grading system can provide valuable information for the preselection of oocytes with higher developmental competence and, therefore, may maximise embryo developmental outcome. Several intrinsic markers (such as mitochondrial status and glucose-6-phosphate dehydrogenase l activity) and extrinsic markers (such as apoptosis of follicular cells and levels of the transforming growth factor-β superfamily in follicular fluid or serum) have also been reported as useful indicators of oocyte competence and embryo quality. Compared with the morphological parameters, these cellular and molecular predictors of oocyte quality may prove to be more precise and objective, although further studies and refinement of techniques are needed.

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OLA1 is responsible for normal spindle assembly and SAC activation in mouse oocytes

10.7287/peerj.preprints.27837 ◽

2019 ◽

Author(s):

Di Xie ◽

Juan Zhang ◽

JinLi Ding ◽

Jing Yang ◽

Yan Zhang

Keyword(s):

Spindle Assembly Checkpoint ◽

Polar Body ◽

Germinal Vesicle ◽

Spindle Assembly ◽

Mouse Oocyte ◽

Immunofluorescent Staining ◽

Germinal Vesicle Breakdown ◽

Oocyte Meiosis ◽

Polar Body Extrusion ◽

Spread Analysis

Background. OLA1 is a member of the GTPase protein family, unlike other members, it can bind and hydrolyze ATP more efficiently than GTP. OLA1 participates in cell proliferation, oxidative response and tumorigenesis. However, whether OLA1 is also required for oocyte meiosis is still unknown. Methods. In this study, the localization, expression, and functions of OLA1 in the mouse oocyte meiosis were examined. Immunofluorescent and confocal microscopy were used to explore the location pattern of OLA1 in the mouse oocyte. Moreover, nocodazole treatment was used to confirm the spindle-like location of OLA1 during mouse meiosis. Western blot was used to explore the expression pattern of OLA1 in the mouse oocyte. Microinjection of siRNA was used to explore the OLA1 functions in the mouse oocyte meiosis. In addition, chromosome spreading was used to investigate the spindle assembly checkpoint (SAC) activity. Results. Immunoﬂuorescent staining showed that OLA1 evenly distributed in the cytoplasm at germinal vesicle (GV) stage. After meiosis resumption (GVBD), OLA1 co-localized with spindles, which was further identified by nocodazole treatment experiments. Knockdown of OLA1 impaired the germinal vesicle breakdown progression and finally resulted in a lower polar body extrusion rate. Immunofluorescence analysis indicated that knockdown of OLA1 led to abnormal spindle assembly, which was evidenced by multipolar spindles in OLA1-RNAi-oocytes. After 6 h post-GVBD in culture, an increased proportion of oocyte which has precociously entered into anaphase/telephase I (A/TI) was observed in OLA1-knockdown oocytes, suggesting that loss of OLA1 resulted in the premature segregation of homologous chromosomes. In addition, the chromosome spread analysis suggested that OLA1 knockdown induced premature anaphase onset was due to the precocious inactivation of SAC. Taken together, we concluded that OLA1 plays important role in GVBD, spindle assembly and SAC activation maintenance in oocyte meiosis.

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