Oocyte Spindle Research Articles

Characteristics of oocytes that failed to fertilize after intracytoplasmic sperm injection.

We assessed the morphologies of meiotic spindles in oocytes that failed to fertilize following intracytoplasmic sperm injection (ICSI) and identified factors contributing to failed fertilization. A total of 225 unfertilized oocytes were collected after ICSI. Oocytes were fixed and stained for tubulin and chromosomes. Meiotic spindle morphologies, chromosome alignment, and sperm nuclear decondensation were assessed to identify contributing factors to fertilization failure. We identified relationships between several factors and both abnormal spindle morphologies and sperm nuclear decondensation in oocytes that failed to fertilize. Three causes for unfertilized oocytes after ICSI were identified: (I) the absence of a sperm nucleus in the ooplasm; (II) failed oocyte activation; and (III) defects in pronucleus formation or migration. The rate of disarranged polar spindles in oocytes collected from women older than 35 years (73.3%; 33/45 oocytes) was significantly higher than that of those collected from women 35 years and younger (50.4%; 68/135 oocytes; odds ratio [OR]: 2.71, 95% confidence interval [CI]: 1.29-5.69, p = 0.009). The proportion of unfertilized oocytes with abnormal spindles and chromosome misalignment was significantly higher in oocytes collected from women older than 35 years than those from women 35 years and younger (62.2% vs. 41.5%, p = 0.016). The proportion of partially decondensed chromatin in the abnormal sperm morphology group was significantly higher than in the normal sperm morphology group (66.7% versus 52.9%, OR: 1.78, 95% CI: 1.01-3.11, p = 0.044). The main contributor to the failure of oocytes to fertilize after ICSI is failed oocyte activation. The ICSI technique used, the maternal age, and sperm morphology are also contributing factors in fertilization failure after ICSI.

Unfertilized oocytes after intracytoplasmic sperm injection and women’s age

This study assessed the characteristics of meiotic spindles in unfertilized oocytes following intracytoplasmic sperm injection (ICSI) and their relationship with the women’s age. A total of 263 unfertilized oocytes were collected and were divided into 2 groups based on the age of the women. Meiotic spindles were stained and classified to identify causes of fertilization failure. Identified relationships between the spindle apparatus, causes of failed fertilization and the women’s age. The number of unfertilized oocytes and the rate of unfertilized oocytes per cycle were not significantly different between women aged 35 years and younger and those older than 35 years (3.6 ± 2.4 versus 2.6 ± 2.2 unfertilized oocytes, p=0.083; 22.0 ± 12.1% vs 21.8 ± 12.9% unfertilized oocytes per cycle, p=0.947). The rate of disarranged spindles in oocytes collected from women older than 35 years was significantly higher than that of women 35 years and younger (73.5 vs. 52.5%; OR = 2.50, CI = 1.23 – 5.07, p = 0.011). The main factor associated with the failure of oocytes to fertilize after ICSI was failed oocyte activation, and there was no statistically significant difference between the two age groups. The women’s age had a relationship with abnormal spindles.

HucMSC-EVs Facilitate In Vitro Development of Maternally Aged Preantral Follicles and Oocytes

Follicle developmental capacity and oocyte quality decline with advanced maternal age. Extracellular vesicles from human umbilical cord mesenchymal stem cells (HucMSC-EVs) act as a potential therapeutic product in the treatment of age-related ovarian dysfunction. In vitro culture (IVC) of preantral follicles is a useful method for understanding the mechanism of follicle development and is a promising means for improving female fertility. However, whether HucMSC-EVs have beneficial effects on aged follicle development during IVC has not yet been reported. Our research demonstrated that follicular development with single-addition withdrawal of HucMSC-EVs was better than that with continuous treatment with HucMSC-EVs. HucMSC-EVs facilitated the survival and growth of follicles, promoted the proliferation of granulosa cells (GCs), and improved the steroid hormone secretion of GCs during IVC of aged follicles. Both GCs and oocytes could uptake HucMSC-EVs. Moreover, we observed elevated cellular transcription in GCs and oocytes after treatment with HucMSC-EVs. The RNA sequencing (RNA-seq) results further validated that the differentially expressed genes are related to the promotion of GC proliferation, cell communication, and oocyte spindle organization. Additionally, the aged oocytes displayed a higher maturation rate, presented less aberrant spindle morphology, and expressed a higher level of the antioxidant protein Sirtuin 1 (SIRT1) after treatment with HucMSC-EVs. Our findings suggested that HucMSC-EVs can improve the growth and quality of aged follicles and oocytes in vitro through the regulation of gene transcription, which provides evidence for HucMSC-EVs as potential therapeutic reagents to restore female fertility with advanced age.Graphical

Nicotinamide mononucleotide restores oxidative stress-related apoptosis of oocyte exposed to benzyl butyl phthalate in mice.

Benzyl butyl phthalate (BBP) is a chemical softener and plasticizer commonly used in toys, food packaging, wallpaper, detergents and shampoos. The estrogenic actions of BBP have detrimental effects on humans and animals. In this study, the specific influence of BBP on mouse oocyte maturation was investigated using in vivo and in vitro models. The experiment first verified that BBP exposure significantly affected the rate of oocyte exclusion of the first polar body, although it did not affect germinal vesicle breakdown (GVBD) through in vitro oocyte culture system. Results of in vitro fertilization show that BBP exposure affects blastocyst rate. Subsequently, the results obtained by immunofluorescence staining technology showed that oocyte spindle organization, chromosomal arrangement and the distribution of cortical actin were disrupted by BBP exposure, and led to the failure of oocyte meiotic maturation and the subsequent early embryo development. Singe-cell transcriptome analysis found that BBP exposure altered the expression levels of 588 genes, most associated with mitochondria-related oxidative stress. Further analysis demonstrated that the detrimental effects of BBP involved the disruption of mitochondrial function and oxidative stress-induced early apoptosis. Nicotinamide mononucleotide (NMN) supplementation reduced the adverse effects of BBP. Collectively, these findings revealed a mechanism of BBP-induced toxicity on female reproduction and showed that NMN provides an effective treatment for BBP actions.

C. elegans XMAP215/ZYG-9 and TACC/TAC-1 act at multiple times during oocyte meiotic spindle assembly and promote both spindle pole coalescence and stability.

The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division. In C. elegans, XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibit multiple and indistinguishable oocyte spindle assembly defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect their requirements at high temporal resolution. Temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure, stabilizing pole foci as they grow and limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization that occurs upon nuclear envelope breakdown. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation. Third, we show that ZYG-9 and TAC-1 also are required for spindle assembly during meiosis II, independently of their meiosis I requirements. The metaphase pole stability requirement appears to be important for maintaining chromosome congression, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability.

Omega-3 fatty acids and L-carnitine prevent meiotic oocyte damage induced by follicular fluid from infertile women with endometriosis: an experimental study.

To assess whether follicular fluid (FF) from infertile women with endometriosis in advanced stages [moderate/severe (EIII/IV) without or with endometrioma (Endometrioma)] induce more oocyte damages than in early stages (minimal/mild: EI/II); and whether supplementation with L-carnitine (LC) and omega 3 (n3) can prevent these oocyte damages. Experimental study using bovine oocytes (obtained of ovaries from slaughterhouse), and human FF (samples were obtained during oocyte recovery for ICSI). Bovine oocytes were submitted to in vitro maturation (IVM) divided into 9 groups: no FF(No-FF), with 1% FF from infertile women without endometriosis (FFC), with EI/II, EIII/IV and Endometrioma, and with (or not) LC+n3 addition. After IVM, oocytes were fluorescently labelled and visualized by confocal microscopy to analyze chromosomes and spindle. FF from endometriosis decreased rate of normal MII (spindle assembly and chromosome alignment) compared to No-FF (87.2%) and FFC (87.2%). FFEIII/IV (80.7%) and FFEndometrioma (69.3%) decreased total MII rate compared to No-FF (91.9%) and FFC (89.2%), and FFEndometrioma had lower total MII rate compared to other groups. LC+n3 increased MII rate in the FFEIII/IV (80.7% vs. 90.8%) and the Endometrioma (69.3% vs. 86.4%), and it prevented damages in spindle and chromosomes in MII oocytes in the FFEI/II group (62.2% vs. 84.5%) and the FFEIII/IV group (70.2% vs. 84.1%). FF of endometriosis damaged the meiotic spindle of bovine MII oocytes. EIII/IV led to impaired nuclear maturation; FF from women with endometrioma had further negative impact in oocyte maturation. LC+n3 completely prevented the effects of FF from women with endometriosis on oocyte.

Phosphorylation of adducin-1 by TPX2 promotes interpolar microtubule homeostasis and precise chromosome segregation in mouse oocytes

BackgroundADD1 (adducin-1) and TPX2 (targeting protein for Xklp2) are centrosomal proteins and regulate mitotic spindle assembly. Mammalian oocytes that segregate homologous chromosomes in Meiosis I and sister chromatids in Meiosis II with a spindle lacking centrosomes are more prone to chromosome segregation errors than in mitosis. However, the regulatory mechanisms of oocyte spindle assembly and the functions of ADD1 and TPX2 in this process remain elusive.ResultWe found that the expression levels and localization of ADD1, S726 phosphorylated ADD1 (p-ADD1), and TPX2 proteins exhibited spindle assembly-dependent dynamic changes during mouse oocyte meiosis. Taxol treatment, which stabilizes the microtubule polymer and protects it from disassembly, made the signals of ADD1, p-ADD1, and TPX2 present in the microtubule organizing centers of small asters and spindles. Knockdown of approximately 60% of ADD1 protein levels destabilized interpolar microtubules in the meiotic spindle, resulting in aberrant chromosome alignment, reduced first polar body extrusion, and increased aneuploidy in metaphase II oocytes, but did not affect K-fiber homeostasis and the expression and localization of TPX2. Strikingly, TPX2 deficiency caused increased protein content of ADD1, but decreased expression and detachment of p-ADD1 from the spindle, thereby arresting mouse oocytes at the metaphase I stage with collapsed spindles.ConclusionPhosphorylation of ADD1 at S726 by TPX2 mediates acentriolar spindle assembly and precise chromosome segregation in mouse oocytes.

DELAYED INTRACYTOPLASMIC SPERM INJECTION(ICSI) EFFECT IN METHAPHASE II(MII) OOCYTES WITH NO SPINDLE VISUALIZATION

The nuclear and cytoplasmic maturation of the egg is essential for normal embryonic development, and when nuclear and cytoplasmic maturation is desynchronized, abnormal fertilization is increased, which is known to affect embryonic development. Synchronization of the nucleus and cytoplasm can be confirmed through the confirmation of the spindle and the polar body. In this study, the correlation between the synchronization of nuclear and cytoplasmic maturation, AMH, and age was investigated. In this study, ICSI results were compared with and without spindles in MII oocytes.

Investigation of the deterministic value of presence of visible spindle on fertilization and blastocytes utilization potential in frozen/thawed oocyte cycle

Background and Aim: To date, characteristics of the meiotic spindles in human oocytes studied by several authors and shown that they are associated with fertilization, embryo quality, pregnancy and live birth rates. Also, it has been shown that, meiotic spindle structures are very susceptible to cryodamage and can be disassembled and re-assembled depending on the changes in temperature. Thus, studying spindle characteristics of the vitrified-warmed oocytes are important and can provide information regarding the full recovery of the oocytes as well as association with fertilization, embryo development and pregnancy, which the data is limited in literature.

P-689 Mutated MDR-1 impairs chromosome alignment and reduces oocyte quality

Abstract Study question With this study we aim to assess the link between oocyte mitochondrial physiology and spindle formation with respect to Multidrug Resistance transporter 1 (MDR-1) function. Summary answer Our most recent data suggest that oocyte quality is compromised in young and old Mdr1 mutant mice. What is known already MDR-1 is a transmembrane ATP-dependent effluxer present in different organs that transport a variety of xenobiotics and byproducts from one side of the membrane to the other. Previous findings by Clark et al. 2019 demonstrated that this transporter is also present in the oocyte mitochondrial membrane and that its mutation led to abnormal mitochondrial homeostasis. Mitochondria are essential for normal oocyte physiology, quality and competence for fertilization and blastocyst development (Chappel, 2013; Dumollard et al., 2007). Therefore, any alteration of their bioenergetics and functions affects the quality of the egg, eventually compromising fertility. Study design, size, duration To carry out the experiments we used Mdr1mutant female mice, expressing an aberrant MDR-1 protein. We focused our analysis on ovaries and oocytes of young (2-3 months) and old (9-12 months) mutant mice, comparing the results with age-matched wild types. Participants/materials, setting, methods In these conditions, we performed ATP quantification and we utilized oocyte in vitro maturation (IVM), immunofluorescence analysis and Ca2+ stimulation analysis. Main results and the role of chance Clark et al. 2019 previously observed that young mutant animals ovulate more eggs compared to wild-types. However, oocytes were fragile and lysed easily and didn’t yield more pups (Clark et al 2019). Conversely, we observed that old Mdr1 mutant mice ovulated less eggs compared to age-matched controls (P < 0.01, n = 4), suggesting diminished ovarian reserve which is associated with poor oocyte quality. Our preliminary data concerning oocyte ATP concentration suggest that mutant germinal vesicle (GV) oocytes present lower ATP levels compared to wild types. Immunofluorescence analysis on metaphase I (MI) oocyte spindle revealed that mutant oocytes present a higher percentage of misaligned chromosomes compared to wild type oocytes (34% and 15%, respectively; P < 0.05, n = 3). Lastly, considering that mitochondria are fundamental for Ca2+ homeostasis, which in turn is necessary for the fertilization process, Ca2+ waves released from mutant vs wild-type metaphase II (MII) oocytes were analyzed using stimulation with strontium, ionomycin, thapsigargin and PLCZ. In all conditions, mutant MII oocytes had altered Ca2+ response (n=∼10 cells per genotype). Limitations, reasons for caution Although these findings demonstrate the importance of MDR-1 for proper maturation and subsequent oocyte development, further studies are necessary to elucidate the specific role and localization of this transporter in the oocyte’s mitochondrial membranes. Wider implications of the findings Our mouse model indicates the importance of mitochondrial homeostasis in infertility. Moreover, our study will provide enhanced understanding of the molecular mechanisms defining oocyte developmental competence, providing insights into human fertility studies. Trial registration number not applicable

P-126 Investigation of the deterministic value of presence of visible spindle on fertilization and blastocytes utilization potential in frozen/thawed oocyte cycle

Abstract Study question Does presence or absence of birefringent spindle after warming of the oocytes has deterministic value on fertilization and early embryonic development potentiaL? Summary answer The warmed oocytes with birefringent spindle seem to have a higher potential to fertilize and become good quality blastocytes. What is known already To date, characteristics of the meiotic spindles in human oocytes were studied by several authors by using a non-invasive method (PolScope) and shown that they are associated with fertilization, embryo quality, pregnancy, and live birth rates. Also, it has been known that meiotic spindle structures are very susceptible to cryodamage and can be disassembled and re-assembled depending on the change in temperature. Thus, studying spindle characteristics of the vitrified-warmed oocytes is important and can provide information about the full recovery of the oocytes, fertilization, embryo development, and pregnancy, which the data is limited in the literature. Study design, size, duration A retrospective study consisting 40 egg donation cycles by using vitrified-warmed oocytes between December 2017 and December 2019 at British Cyprus IVF Hospital. Participants/materials, setting, methods Spindle morphologies of vitrified-warmed oocytes were analyzed by PolScope (RI) combined with an image analysis system after approximately 1.5 hours post-warming. The data is divided into two according to the presence or absence of visible nucleus and correlation with embryonic development analyzed accordingly. To make a comparison in groups a new numerical scoring system for embryo quality on days 5 and 6 was created where embryos scored depending on the stage of expansion, Main results and the role of chance In total, 587 out of 648 vitrified/warmed oocytes survived (90.6%) and 447 of them were fertilized (75.0%). Among the survived oocytes, 503 (85.5%) oocytes were containing detectable spindle (Group I) while spindles were missing in 84 oocytes (Group II). The results showed that the fertilization rate (78.1% vs. 64.3%, p < 0.01), the proportion of good PN morphology (Z Score: Z1 and Z2 over other parameters: 50.4% vs 35.2%, p = 0.036), and viable embryo count (40.6% vs 15.1%, p < 0.01) which is defined as embryos that good enough to transfer or freezing, were higher in group I. Similarly, the degree of expansion on day 5/6 (p = 0.013, p = 0.015 respectively) and the quality of the cells in ICM and TE (p < 0.01) were found higher in group I. We further investigated the differences in embryo quality between groups by converting the embryo grades to numerical value starting from arrested (Score=0) to 6AA (Score=22) and found that embryo scores on day 5 (p = 0.01) and day 6 (p = 0.013) were higher in group I. Limitations, reasons for caution The main limitation of this study is the lack of data regarding the spindle status of the frozen oocytes before vitrification where some of the oocytes might not have visible spindle due to incomplete maturation rather than cryodamage. Wider implications of the findings The present data showed that the presence of birefringent meiotic spindles in warmed oocytes is a very important sign for a full recovery and early embryonic development potential, which can be useful for egg banks and IVF clinics. Trial registration number not applicable

P-198 An analysis of the size of micro pronucleus in 2.1 pronuclear zygotes by using time-lapse images

Abstract Study question Is it possible to determine the difference between 2.1 pronuclear (2.1PN) zygotes and tripronuclear (3PN) zygotes from time-lapse images? Summary answer A pronucleus of less than 15 μm in diameter can be considered the micro pronucleus (micro PN), and it is possible to classify 2.1PN zygotes. What is known already 2.1PN zygotes are defined as zygotes with two pronuclei and one smaller pronucleus. Capalbo et al. (2017) reported that most of the 2.1PN-derived blastocysts were diploid by preimplantation genetic testing for aneuploidy (PGT-A), including single-nucleotide polymorphisms (SNPs) analysis. Thus, the treatment with 2.1 PN zygotes should be performed with chromosome testing. In Japan, where PGT-A is not available in principle, 2.1PN zygotes are rarely used in the embryo transfer. On the other hand, the size of the micro pronucleus in 2.1PN zygotes has not been clearly defined, and it is difficult to determine differences between 2.1PN and 3PN zygotes. Study design, size, duration The study was performed retrospectively on 2463 cycles of in vitro fertilization (IVF) conducted at our clinic between August 2020 and December 2021. A total of 3073 embryos underwent conventional-IVF (c-IVF) or intracytoplasmic sperm injection (ICSI) and were cultured in the time-lapse incubator, of which 221 zygotes with three pronuclei were used in the study. Participants/materials, setting, methods The diameter of the three PNs at one hour before syngamy from time-lapse images; 2.1 PN and 3PN zygotes were classified in the report by Capalbo et al. (2017). The age of the patients and the method of insemination between the groups were compared, and the diameter of the micro PN was analysed. Moreover, logistic regression analysis was performed to investigate the predictor of 2.1PN zygotes from the morphological characteristics of oocytes at ICSI. Main results and the role of chance The mean age of each patient was 42.9 years for 2.1PN zygotes and 39.8 years for 3PN zygotes, significantly higher for 2.1PN zygotes (P =0.003). On the other hand, when comparing the stage of oocyte maturation at the time of oocyte retrieval, there was no significant difference (P =0.749). According to the insemination method, the incidence of 2.1PN zygotes was significantly higher in ICSI (including rescue-ICSI) compared to c-IVF: 32.9% [95%CI: 22.5-44.6%] vs 2.4% [95%CI: 0.1-12.9%] (P <0.001). In terms of ICSI-derived zygotes, the mean age was also significantly higher for 2.1PN zygotes compared to 3PN zygotes: 43.3 years vs. 40.9 years (P =0.03). The diameter of micro PNs calculated using the receiver operating characteristics (ROC) curve from the measurements of the diameter was less than 15 μm (AUC [95%CI]: AUC=0.988 [0.975-1.00]). Logistic regression analysis using age, position of the oocyte spindle at ICSI, cytoplasmic viscosity, and condition of the cell membrane as explanatory variables revealed a significant difference only in age (P =0.0154, odds ratio [95%CI]: 1.18 [1.03-1.35]) and no statistically significant oocyte morphological characteristics. Limitations, reasons for caution In this study, we have not investigated whether 2.1 PN zygotes become blastocysts. It will be necessary to further examine the criteria for 2.1PN along with chromosome testing to investigate the use of 2.1PN-derived blastocysts. Wider implications of the findings A pronucleus of less than 15 μm in diameter can be considered a micro PN. Compared to 3PN zygotes, 2.1PN zygotes were more frequently observed in older patients and in ICSI-derived zygotes. However, it is difficult to predict the incidence of 2.1PN zygotes from the oocytes’ morphological characteristics at ICSI. Trial registration number not applicable

Recovery of spindle morphology and mitochondrial function through extended culture after vitrification-warming of bovine oocytes

The present study aimed to determine the effects of vitrification on the meiotic spindle and mitochondrial function of bovine oocytes submitted to different times of post-warming culture. Partially denuded cumulus-oocyte complexes were vitrified at different maturation times (18-, 20-, and 24-h) using a two-step cryoprotectant addition protocol and submitted to 6-, 4-, or 0-h of post-warming extended culture in maturation medium. Microtubule configuration and chromosomal arrangement were analyzed after 0- and 6-h of extended culture, whereas mitochondrial membrane potential and ATP content were measured at 0-, 4-, and 6-h of post-warming recovery. Results of meiotic spindle integrity revealed that vitrified-warmed oocytes that underwent 6-h of culture had similar incidence of normal microtubule configuration and chromosomal arrangement as compared to fresh oocytes, but higher than oocytes in the vitrification control group (no culture). Mitochondrial membrane potential was not different in all the vitrification groups, but the oocytes that were cultured for 4-h after warming had similar levels compared to fresh oocytes. ATP concentration in all vitrification groups was lower than the control group. However, oocytes cultured for 6-h had the lowest rate of ATP depleted oocytes among the vitrification groups. The results of this study indicate that extended culture after warming promotes the recovery of the meiotic spindle and, to some extent, mitochondrial function of vitrified-warmed metaphase II bovine oocytes.

The oocyte spindle midzone pauses Cdk1 inactivation during fertilization to enable male pronuclear formation and embryo development.

Inactivation of cyclin-dependent kinase 1 (Cdk1), controlled by cyclin B1 proteolysis, orders events during mitotic exit. Here, we used a FRET biosensor to study Cdk1 activity while simultaneously monitoring anaphase II and pronuclear (PN) formation in live mouse eggs throughout fertilization. We find that Cdk1 inactivation occurs over two phases separated by a 3-h pause, the first induces anaphase II and the second induces PN formation. Although both phases require the inhibitory Cdk1 kinase Wee1B, only the first involves cyclin B1 proteolysis. Enforcing the 3-h pause is critical for providing the delay required for male PN formation and is mediated by spindle midzone-dependent sequestration of Wee1B between the first and second phases. Thus, unlike continuous Cdk1 inactivation driven by cyclin B1 proteolysis during mitotic exit, MII oocytes engineer a physiologically important pause during fertilization involving two different pathways to inactivate Cdk1, only the first of which requires proteolysis.

Mutation analysis of the TUBB8 gene in primary infertile women with oocyte maturation arrest

BackgroundOocyte maturation arrest at metaphase I leads to fertilization failure in humans. In early embryos, the tubulin beta 8 class VIII (TUBB8) encodes a β-tubulin isotype and aids in the assembling of the human oocyte spindle. Mutations in the TUBB8 potentially interfere with human oocyte maturation—a crucial prerequisite for fertilization and subsequent embryonic development. This study aims to investigate the novel mutations in TUBB8 and their prevalence.ResultsHundred fertile women (controls) and eleven infertile women with oocyte maturation arrest were chosen for the study. A total of five TUBB8 heterozygous/homozygous mutations were found in eleven infertile females (p.A313V, p.C239W, p.R251Q, p.P358L, and p.G96R). The Exome Aggregation Consortium (ExAC), SIFT, and PolyPhen-2 analyses revealed that p. A313V has unknown pathogenicity and p.C239W, p.R251Q, p.P358L, and p.G96R have possible pathogenicity. The wild-type (WT) and four mutant gene constructs were transfected to Hela cells. The Western blot analysis indicates that the TUBB8 expression of the p.C239W, p.R251Q, and p.G96R mutations was significantly decreased than that of WT. The immunofluorescence assay showed that the Hela cells transfected with either p.C239W, p.R251Q, or p.G96R mutations exhibited the disrupted microtubule structure, revealing a significant difference in the organization of the microtubule network compared to the WT.ConclusionsWe identified three novel variants and two reported variants out of 11 infertile women with oocyte metaphase I arrest. According to the present data, TUBB8 gene variants account for 31.96% of all participants (109/341) with oocyte maturation arrest.

Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification

The cryopreservation of mammalian oocytes and embryos has become an integral part of assisted reproduction in both humans and veterinary species. However, the methods used to cryopreserve bovine oocytes still have significant shortcomings. A wide variety of approaches has been used to try to improve and optimize methods of cryopreservation. However, these procedures employed are not always designed to specifically take account of the osmotic tolerance response of the cells according to the temperature and time of cryoprotectant (CPA) addition. When these properties are considered, optimal procedures for the addition of CPAs can be designed proactively. Based on in silico and in vitro osmotic observations, we propose shorter dehydration-based protocols at different temperatures (25°C vs. 38.5°C) towards defining an improved cryopreservation method. In vitro matured oocytes were exposed to equilibration solution (ES) at 25°C and 38.5°C and effects of optimized exposure times for each temperature were determined prior to vitrification/warming on oocyte spindle configuration, DNA fragmentation, and further embryo development. Upon exposure to standard ES (7.5% dimethyl sulfoxide + 7.5% ethylene glycol in TCM199 medium + 20% fetal bovine serum), original oocyte volume was recovered within 2 min 30 s at 38.5°C and 5 min 30 s at 25°C. In vitro matured oocytes were then exposed to the aforementioned cryoprotectants at both temperature/duration conditions and vitrified/warmed. While similar percentages of oocytes exhibiting a normally configured spindle and DNA fragmentation were observed in the fresh control group and oocytes vitrified at 38.5°C, significantly higher apoptosis rate and lower percentages of normal spindle configuration were observed in oocytes vitrified at 25°C when compared to control fresh oocytes. Similar cleavage rates and blastocyst yields were observed in the vitrified/38.5°C and fresh controls, while these rates were lower in vitrified/25°C. These results revealed that the limitation of the exposure time of the oocytes to the ES to the point of osmotic equilibrium volume recovery could be a more efficient approach to prepare them for vitrification. Therefore, exposure time to ES to 2 min 30 s at 38.5 °C appears to improve the quality of vitrified/warmed oocytes by protecting spindle integrity and reducing DNA fragmentation thus improving blastocyst rates and embryo quality.

Multiple motors cooperate to establish and maintain acentrosomal spindle bipolarity in C. elegans oocyte meiosis.

While centrosomes organize spindle poles during mitosis, oocyte meiosis can occur in their absence. Spindles in human oocytes frequently fail to maintain bipolarity and consequently undergo chromosome segregation errors, making it important to understand the mechanisms that promote acentrosomal spindle stability. To this end, we have optimized the auxin-inducible degron system in Caenorhabditis elegans to remove the factors from pre-formed oocyte spindles within minutes and assess the effects on spindle structure. This approach revealed that dynein is required to maintain the integrity of acentrosomal poles; removal of dynein from bipolar spindles caused pole splaying, and when coupled with a monopolar spindle induced by depletion of the kinesin-12 motor KLP-18, dynein depletion led to a complete dissolution of the monopole. Surprisingly, we went on to discover that following monopole disruption, individual chromosomes were able to reorganize local microtubules and re-establish a miniature bipolar spindle that mediated chromosome segregation. This revealed the existence of redundant microtubule sorting forces that are undetectable when KLP-18 and dynein are active. We found that the kinesin-5 family motor BMK-1 provides this force, uncovering the first evidence that kinesin-5 contributes to C. elegans meiotic spindle organization. Altogether, our studies have revealed how multiple motors are working synchronously to establish and maintain bipolarity in the absence of centrosomes.

Altered microfilament dynamics contribute to the formation of diploid metaphase spindles in porcine oocytes which fail to reach the metaphase-II stage during in vitro maturation.

Premature meiotic arrest during in vitro maturation (IVM) of porcine oocytes after germinal vesicle breakdown is associated with microfilament degradation. We aimed to clarify (1) if such arrest occurs at the metaphase-I (MI) stage or the oocyte progresses to a so-called diploid metaphase-II (MII) stage and (2) if microfilament degradation is the cause or result of the meiotic arrest. The number and morphology of chromosomes in oocytes showing premature meiotic arrest at 44 h IVM (38 monovalents) was similar to those cultured in the presence of the actin polymerization-inhibitor cytochalasin-B, but different from those of MI-stage (19 bivalents), and MII-stage oocytes (19 monovalents) at 33 and 44 h of IVM, respectively. Immunostaining revealed similar frequencies of microfilament degradation in prematurely arrested and cytochalasin-B-treated oocytes (58.7% and 57.2%, respectively), which were higher (P < 0.05) than those in MI- and MII-stage oocytes (10.6% and 6.8%, respectively). Induction of MI-arrest by nocodazole did not affect microfilament morphology. ATP and mRNA levels of microfilament-related genes in oocytes were similar among all groups. These results suggest that altered microfilament dynamics contribute to the formation of diploid metaphase spindles in oocytes, which fail to reach the MII stage. However, the cause of microfilament degeneration remains unclear.

CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes

Proper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V−/− oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V−/− oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V−/− oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

Two mutations in TUBB8 cause developmental arrest in human oocytes and early embryos

Research questionHow can the effect of genetic mutations that may cause primary female infertility be evaluated? DesignPatients and their family members underwent whole-exome sequencing and Sanger sequencing to detect the infertility-causing gene and inheritance pattern. To study the function of mutant proteins in vitro, vectors containing wild-type or mutant TUBB8 cDNA were constructed for transient expression in HeLa cells, and in-vitro transcribed mRNA were used for microinjection in germinal vesicle-stage mouse oocytes. Immunofluorescence staining was used to observe the microtubule structure in HeLa cells or meiotic spindle in mouse oocytes. ResultsA maternally inherited TUBB8 (Tubulin beta 8 class VIII) mutation (NM_177987.2: c. 959G>A: p. R320H) and a previously reported (NM_177987.2: c. 161C>T: p. A54V) recessive mutation from two infertile female patients were identified. The oocytes from the patient carrying p.A54V mutation failed fertilization, whereas oocytes with p.R320H mutation could be fertilized but showed heavy fragmentation during early development. In vitro, functional assays showed that p. A54V mutant disrupted the microtubule structure in HeLa cells (49.3% of transfected cells) and caused large polar body extrusion in mouse oocytes (27.5%), whereas the p.R320H mutant caused a higher abnormal rate (69.7%) in cultured cells and arrested mouse oocytes at meiosis I (38.7%). ConclusionTwo TUBB8 mutations (p.A54V and p.R320H) were identified and their pathogeny was confirmed by in-vitro functional assays.