Oocyte Spindle Research Articles

Long-range repulsion between chromosomes in mammalian oocyte spindles.

During eukaryotic cell division, a microtubule-based structure called the spindle exerts forces on chromosomes. The best-studied spindle forces, including those responsible for the separation of sister chromatids, are directed parallel to the spindle's long axis. By contrast, little is known about forces perpendicular to the spindle axis, which determine the metaphase plate configuration and thus the location of chromosomes in the subsequent nucleus. Using live-cell microscopy, we find that metaphase chromosomes are spatially anti-correlated in mouse oocyte spindles, evidence of previously unknown long-range forces acting perpendicular to the spindle axis. We explain this observation by showing that the spindle's microtubule network behaves as a nematic liquid crystal and that deformation of the nematic field around embedded chromosomes causes long-range repulsion between them.

The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.

Distinct checkpoint and homolog biorientation pathways regulate meiosis I in Drosophila oocytes.

Mitosis and meiosis have two mechanisms for regulating the accuracy of chromosome segregation: error correction and the spindle assembly checkpoint (SAC). We have investigated the function of several checkpoint proteins in meiosis I of Drosophila oocytes. Evidence of a SAC response by several of these proteins is found upon depolymerization of microtubules by colchicine. However, unattached kinetochores or errors in biorientation of homologous chromosomes does not induce a SAC response. Furthermore, the metaphase I arrest does not depend on SAC genes, suggesting the APC is inhibited even if the SAC is silenced. Two SAC proteins, ROD of the ROD-ZW10-Zwilch (RZZ) complex and MPS1, are also required for the biorientation of homologous chromosomes during meiosis I, suggesting an error correction function. Both proteins aid in preventing or correcting erroneous attachments and depend on SPC105R for localization to the kinetochore. We have defined a region of SPC105R, amino acids 123-473, that is required for ROD localization and biorientation of homologous chromosomes at meiosis I. Surprisingly, ROD removal, or "streaming", is independent of the dynein adaptor Spindly and is not linked to the stabilization of end-on attachments. Instead, meiotic RZZ streaming appears to depend on cell cycle stage and may be regulated independently of kinetochore attachment or biorientation status. We also show that dynein adaptor Spindly is also required for biorientation at meiosis I, and surprisingly, the direction of RZZ streaming.

P-185 Shortening the time from ovulation trigger to insemination is effective in reducing multiple pronuclei formation in anti-centromere antibody-positive patients

Abstract Study question Does shortening the time from ovulation trigger to insemination reduce multiple pronuclei formation (MPF) and improve the outcome after fertilization? Summary answer Shortening the time from ovulation trigger to insemination increased the normal fertilization rate and increased the number of cycles in which good embryos were obtained. What is known already When anti-centromere antibody (ACA)-positive patients undergo IVF, low oocyte maturation rates and MPF may be observed, which tend to occur when ACA antibody titers are high. In this case, the patient tends to present with poor embryonic development. There is currently no effective treatment for ACA-positive patients. It has been reported that MPF is caused by chromosome dispersion due to significant abnormal spindle deformation. In such cases, it has been reported that the oocyte spindle is often not visualized by polarized light microscopy. The oocyte is affected by ACA during the meiosis, but the mechanism is not well understood. Study design, size, duration A retrospective study was conducted on 764 mature oocytes from 174 cycles in 45 patients who underwent oocyte retrieval and inseminated by intra-cytoplasmic sperm injection (ICSI) with consent from 2009 to 2022. Patients were limited to those who tested positive for ACA by blood antinuclear antibody testing and whose oocytes occurred MPF. Participants/materials, setting, methods Oocyte retrieval was performed 36 hours after ovulation trigger. After ICSI, embryos were cultured until day 3 or blastocyst. The group was divided into two groups: the time reduced from ovulation trigger to ICSI (S group, n = 95) and the normal protocol (N group, n = 79). The normal fertilization rate, MPF rate, the rate of good embryo on day 3, the percentage of IVF cycles with good embryo, and clinical pregnancy rate per transfer were compared. Main results and the role of chance The mean time from maturation trigger to insemination was shorter in the S group than in the N group (38.2h vs. 41.8h, P < 0.01). The normal fertilization rate was significantly increased in the S group compared to the N group (49.9 vs. 40.7, P < 0.01) and the MPF rate was significantly decreased (39.1 vs. 28.3, P < 0.01). The rate of transferable embryo on day 3 tended to increase in the S group compared to the N group (70.6 vs. 61.9, P = 0.09). In addition, the percentage of IVF cycles with good embryo was significantly increased in the S group compared to the N group (57.4 vs. 28.4, P < 0.01). There was no difference in pregnancy rate per embryo transfer. There was no difference in the age of the wife at the time of oocyte retrieval. Limitations, reasons for caution The data were conducted from a single facility. and the limited number of cases, resulting in a small sample size. The process of visualizing the oocyte spindle was not performed in this study because it is time consuming and increases the time from ovulation trigger to insemination. Wider implications of the findings Shortening the time from maturation trigger to insemination was decreased MPF, suggesting that the oocyte spindle is affected by ACA in a time-dependent manner. It is recommended that ICSI be performed as soon as possible after oocyte retrieval. Trial registration number not applicable

P-580 Benzophenone interrupted maturation of oocytes in vitro by promoting mitochondrial activity and inflammatory response in mice

Abstract Study question As an endocrine disrupting chemical, does benzophenone affect the competency of oocytes? Summary answer Benzophenone interfered with the in vitro maturation of oocytes and increased the number of mitochondria in oocytes by inducing of inflammatory response. What is known already Benzophenone (BP) is the organic compound used in ultraviolet (UV) filters due to its ability to absorb UV radiations and the applications of BP have a wide range in commercial/industrial products such as sunscreens, shampoos, lipsticks, food packaging, and plastics. However, BP is suspected to be an endocrine disrupting chemical (EDC) that can cause adverse effects on the endocrine system by mimicking or blocking the response of hormones in the body. Study design, size, duration Intraperitoneal injection of pregnant mare serum gonadotropin was performed at 7-week-old female ICR mice for acquire the number of available cumulus-oocyte-complexes (COCs). After 46 hours post injection, COCs were harvested from antrum follicles in the ovary and cultured in in-vitro maturation medium for 16 hours. After that, oocytes were retrieved. Participants/materials, setting, methods During in vitro maturation, BP was treated at COCs and Metaphase II (MII) oocytes were separated from retrieved oocytes. Intracellular tubulin of MII oocytes was stained for spindle alignment and their mitochondria were quantified by Mito-tracker and quantitative PCR (qPCR). Also, the expression levels of inflammation-related genes were confirmed by reverse transcription-qPCR. Main results and the role of chance In the present study, MII rates (MII/total oocyte) of each group were 77.50% of control, 79.17% of 1 μM BP, 68.10% of 10 μM BP, and 59.35% of 100 μM BP. Compared with the control group, the MII rate were significantly reduced at 100 μM of BP-treated group. We evaluated the morphology of meiotic spindle in MII oocytes with well-organized tubulin fibers and tightly aligned chromosomes. Abnormal morphology of spindle and chromosomal misalignment were found in the oocytes from BP-treated group. Because it has been known that having the optimal number of functional mitochondria is important for the maturation of oocytes, mitochondrial DNA (mtDNA) copy number and the distribution of mitochondria were confirmed in retrieved MII oocytes. There was a significant 4.2-fold increase in the number of mtDNA copies in 100 μM BP treatment group compared to the control group. Also, the number of activated mitochondria was raised when BP was treated, which was assessed using Mitotracker. Additionally, levels of inflammation-related genes (IL-1β, IL-6) were elevated in BP treatment groups. Limitations, reasons for caution Oocytes used in this study were collected from mice, not human. And since in vitro fertilization did not perform by using the MII oocytes, we couldn’t confirm whether BP-treated MII oocytes were capable of fertilization. Wider implications of the findings As industries evolve, novel chemicals, including EDCs, are being developed, used, and exposed to humans. Therefore, we need to understand the effects of these chemicals and assess their reproductive toxicity. Collectively, we suggest that BP causes an inflammatory response which activate mitochondria and has detrimental effects on the oocyte’s maturation. Trial registration number not applicable

Spermatocytes have the capacity to segregate chromosomes despite centriole duplication failure

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.

An optofluidic system for the concentration gradient screening of oocyte protection drugs

Oocytes protective drug screening is essential for the treatment of reproductive diseases. However, few studies construct the oocyte in vitro drug screening microfluidic systems because of their enormous size, scarcity, and sensitivity to the culture environment. Here, we present an optofluidic system for oocyte drug screening and state analysis. The system consists of two parts: an open-top drug screening microfluidic chip and an optical Fourier filter analysis part. The open-top microfluidic chip anchors single oocyte with hydrogel and allows nutrient and gas environment updating which is essential for oocyte culturing. The optical filter analysis part is used to accurately analyse the status of oocytes. Based on this system, we found that fluorene-9-bisphenol (BHPF) damaged the oocyte spindle in a dose-dependent manner, a high dose of melatonin (10−3 M) effectively reduces the percentage of abnormally arranged chromosomes of oocytes exposed to 40 μM BHPF. This optofluidic system shows great promise for the culture of oocytes and demonstrates the robust ability for convenient multi-concentration oocytes drug screening. This technology may benefit further biomedicine and reproductive toxicology applications in the lab on a chip community.

Effect of Moderate Static Magnetic Fields on Mice Oocyte Vitrification: Calcium-Related Genes Expression.

The ability to cryopreserve oocytes without ultrastructural injury has been a concern in the development and use of methods to preserve female reproduction. The stability of the cell membrane must be preserved to reduce the damage caused by ice crystals during vitrification. One approach that has been explored is the use of static magnetic fields (SMFs), which are believed to influence cell membrane stability. In this study, the in vitro effects of SMF that range between 20-80 mT on the vitrification of mice germinal vesicle (GV) oocytes were studied. The viability and mitochondrial (Mt) membrane potential of both vitrified and nonvitrified oocytes were assessed using Trypan blue and JC1 staining. The high in vitro maturation (IVM) rate and high Mt membrane potential in metaphase II (MII) oocytes were taken into account to determine the optimal magnetic field intensity, that is, 20 mT. None of the SMF conditions resulted in intact spindles in MII oocytes. The study also explored the expression of store-operated calcium entry (Stim1, Orai1, and Ip3r) and meiosis resumption (Ccnb, Cdk) genes in GV and MII oocytes of both vitrified and control groups. The results show that the expressions of Orai1 and Ccnb genes in Vit-MII-SMF oocytes were considerably increased. However, no significant difference in Stim1 expression was observed between the groups. The Vit-MII-SMF group exhibited a significantly higher Ccnb expression compared to other groups. In vitro fertilization (IVF) was performed to evaluate the 2 pronuclear (2PN) rates. The findings demonstrated that using 20 mT SMF improved 2PN rates compared to the nonvitrified groups. This study provides a deeper understanding of the effects of moderate SMF and vitrification on the expression of calcium channel genes in GV and MII oocytes. The results suggest that applying a 20 mT SMF can help prevent cryoinjury and enhance the characteristics of vitrified-warmed oocytes.

Male meiotic spindle poles are stabilized by TACC3 and cKAP5/chTOG differently from female meiotic or somatic mitotic spindles in mice.

Transforming acidic acid coiled-coil protein 3 (TACC3) and cytoskeleton associated protein 5 (cKAP5; or colonic hepatic tumor overexpressed gene, chTOG) are vital for spindle assembly and stabilization initiated through TACC3 Aurora-A kinase interaction. Here, TACC3 and cKAP5/chTOG localization with monospecific antibodies is investigated in eGFP-centrin-2- expressing mouse meiotic spermatocytes. Both proteins bind spermatocyte spindle poles but neither kinetochore nor interpolar microtubules, unlike in mitotic mouse fibroblasts or female meiotic oocyte spindles. Spermatocytes do not display a liquid-like spindle domain (LISD), although fusing them into maturing oocytes generates LISD-like TACC3 condensates around sperm chromatin but sparse microtubule assembly. Microtubule inhibitors do not reduce TACC3 and cKAP5/chTOG spindle pole binding. MLN 8237 Aurora-A kinase inhibitor removes TACC3, not cKAP5/chTOG, disrupting spindle organization, chromosome alignment, and impacting spindle pole γ-tubulin intensity. The LISD disruptor 1,6-hexanediol abolished TACC3 in spermatocytes, impacting spindle bipolarity and chromosome organization. Cold microtubule disassembly and rescue experiments in the presence of 1,6-hexanediol reinforce the concept that spermatocyte TACC3 spindle pole presence is not required for spindle pole microtubule assembly. Collectively, meiotic spermatocytes without a LISD localize TACC3 and cKAP5/chTOG exclusively at spindle poles to support meiotic spindle pole stabilization during male meiosis, different from either female meiosis or mitosis.

RNF20 Regulates Oocyte Meiotic Spindle Assembly by Recruiting TPM3 to Centromeres and Spindle Poles.

Previously a ring finger protein 20 (RNF20) is found to be essential for meiotic recombination and mediates H2B ubiquitination during spermatogenesis. However, its role in meiotic division is still unknown. Here, it is shown that RNF20 is localized at both centromeres and spindle poles, and it is required for oocyte acentrosomal spindle organization and female fertility. RNF20-depleted oocytes exhibit severely abnormal spindle and chromosome misalignment caused by defective bipolar organization. Notably, it is found that the function of RNF20 in spindle assembly is not dependent on its E3 ligase activity. Instead, RNF20 regulates spindle assembly by recruiting tropomyosin3 (TPM3) to both centromeres and spindle poles with its coiled-coil motif. The RNF20-TPM3 interaction is essential for acentrosomal meiotic spindle assembly. Together, the studies uncover a novel function for RNF20 in mediating TPM3 recruitment to both centromeres and spindle poles during oocyte spindle assembly.

Predicting Infertility: How Genetic Variants in Oocyte Spindle Genes Affect Egg Quality.

Successful reproduction relies on the union of a single chromosomally normal egg and sperm. Chromosomally normal eggs develop from precursor cells, called oocytes, that have undergone accurate chromosome segregation. The process of chromosome segregation is governed by the oocyte spindle, a unique cytoskeletal machine that splits chromatin content of the meiotically dividing oocyte. The oocyte spindle develops and functions in an idiosyncratic process, which is vulnerable to genetic variation in spindle-associated proteins. Human genetic variants in several spindle-associated proteins are associated with poor clinical fertility outcomes, suggesting that heritable etiologies for oocyte dysfunction leading to infertility exist and that the spindle is a crux for female fertility. This chapter examines the mammalian oocyte spindle through the lens of human genetic variation, covering the genes TUBB8, TACC3, CEP120, AURKA, AURKC, AURKB, BUB1B, and CDC20. Specifically, it explores how patient-identified variants perturb spindle development and function, and it links these molecular changes in the oocyte to their cognate clinical consequences, such as oocyte maturation arrest, elevated egg aneuploidy, primary ovarian insufficiency, and recurrent pregnancy loss. This discussion demonstrates that small genetic errors in oocyte meiosis can result in remarkably far-ranging embryonic consequences, and thus reveals the importance of the oocyte's fine machinery in sustaining life.

CRMP5 participates in oocyte meiosis by regulating spastin to correct microtubule-kinetochore misconnection.

Our previous studies have suggested that spastin, which aggregates on spindle microtubules in oocytes, may promote the assembly of mouse oocyte spindles by cutting microtubules. This action may be related to CRMP5, as knocking down CRMP5 results in reduced spindle microtubule density and maturation defects in oocytes. In this study, we found that, after knocking down CRMP5 in oocytes, spastin distribution shifted from the spindle to the spindle poles and errors in microtubule-kinetochore attachment appeared in oocyte spindles. However, CRMP5 did not interact with the other two microtubule-severing proteins, katanin-like-1 (KATNAL1) and fidgetin-like-1 (FIGNL1), which aggregate at the spindle poles. We speculate that, in oocytes, due to the reduction of spastin distribution on chromosomes after knocking down CRMP5, microtubule-kinetochore errors cannot be corrected through severing, resulting in meiotic division abnormalities and maturation defects in oocytes. This finding provides new insights into the regulatory mechanisms of spastin in oocytes and important opportunities for the study of meiotic division mechanisms.

#267 : The Spindle Localization ICSI (SL-ICSI) Improves the Fertilization Rate Decreased by Aging

Background and Aims: The position of meiotic spindle in metaphase II oocyte can be different from the underneath of the first polar body. In order to prevent the meiotic spindle damage during ICSI, confirming the position of the spindle is effective. In this study, we analyzed the meiotic spindle deviation and outcomes of ICSI with the patient age. Method: From 2017 to 2022, 5620 oocytes with the first polar body underwent SL-ICSI were analyzed. SL-ICSI was performed 4-5 hours after oocyte retrieval. Furthermore, the meiotic spindle deviation from the polar body was classified according to the angle; Group A: 0-5∘, Group B: 6-45∘, Group C: 46-90∘, Group D: 90∘<. The spindle invisible oocytes were regarded as group E. The average age, the fertilization rate and good blastocyst rate in each group were analyzed. Statistical analysis was performed by one-way ANOVA and Tukey’s post-hoc test. Results: The proportions of spindle-visible oocyteswere 98.7%; 41.2%, 36.1%, 14.6%, 6.8%, and 1.3% respectively, in group A to E. The mean agesin each group were 39.0, 39.6, 40.1, 40.6, and 40.7, respectively. In addition, the correlation between the age and the location of spindle was revealed (A-BCDE: [Formula: see text] < 0.001). The fertilization rates were 84.8%, 84.9%, 84.7%, 83.5%, 50.7%, respectively, and significantly decreased in group E ([Formula: see text] < 0.001). Abnormal fertilization rates were 4.3%, 5.1%, 6.4%, 7.3%, 20.5%, respectively, and significantly increased in group E ([Formula: see text] < 0.001). Good blastocyst rates were 49.8%, 52.4%, 47.6%, 50.5%, 3.3%, respectively, and significantly decreased in group E ([Formula: see text] < 0.001). Conclusion: The introduction of SL-ICSI can be effective for elderly patients. The meiotic spindle of oocytes is highly likely to deviate from the first polar body by aging. Furthermore, if the spindle-invisible oocyte is observed, ICSI should be carefully carried out.

Microtubules oppose cortical actomyosin-driven membrane ingression during C. elegans meiosis I polar body extrusion.

During C. elegans oocyte meiosis I cytokinesis and polar body extrusion, cortical actomyosin is locally remodeled to assemble a contractile ring that forms within and remains part of a much larger and actively contractile cortical actomyosin network. This network both mediates contractile ring dynamics and generates shallow ingressions throughout the oocyte cortex during polar body extrusion. Based on our analysis of requirements for CLS-2, a member of the CLASP family of proteins that stabilize microtubules, we recently proposed that a balance of actomyosin-mediated tension and microtubule-mediated stiffness limits membrane ingression throughout the oocyte during meiosis I polar body extrusion. Here, using live cell imaging and fluorescent protein fusions, we show that CLS-2 is part of a group of kinetochore proteins, including the scaffold KNL-1 and the kinase BUB-1, that also co-localize during meiosis I to structures called linear elements, which are present within the assembling oocyte spindle and also are distributed throughout the oocyte in proximity to, but appearing to underlie, the actomyosin cortex. We further show that KNL-1 and BUB-1, like CLS-2, promote the proper organization of sub-cortical microtubules and also limit membrane ingression throughout the oocyte. Moreover, nocodazole or taxol treatment to destabilize or stabilize oocyte microtubules leads to, respectively, excess or decreased membrane ingression throughout the oocyte. Furthermore, taxol treatment, and genetic backgrounds that elevate the levels of cortically associated microtubules, both suppress excess membrane ingression in cls-2 mutant oocytes. We propose that linear elements influence the organization of sub-cortical microtubules to generate a stiffness that limits cortical actomyosin-driven membrane ingression throughout the oocyte during meiosis I polar body extrusion. We discuss the possibility that this regulation of sub-cortical microtubule dynamics facilitates actomyosin contractile ring dynamics during C. elegans oocyte meiosis I cell division.

Stable expression and multi-site location of Odf2 in mouse oocytes, sperm and early embryos

In mammals, centriole is degenerated during early oogenesis, but it is still not known about the expression and function of centriolar structural components in oocyte meiosis. Here we found that Odf2 (outer dense fiber of sperm tails 2), a key centriolar appendage protein, was stably expressed in mouse oocytes during meiotic progression. Distinct from its single location at centrosomes in somatic mitosis, Odf2 was multiply located at microtubule organizing centers (MTOCs), chromosome centromeres and vesicles in oocyte meiosis. In addition, the vesicle-associated Odf2 disappeared in oocytes treated with the vesicle inhibitor Brefeldin A. Odf2 was mainly co-localized with the mitochondrial sheath in the sperm tail and presented as double spots, similar to γ-tubulin, in the sperm neck region. After fertilization, Odf2 remained on vesicles in embryos from 1-cell to 4-cell stage but was only detected on centrosomes at blastocyst stage. Taken together, Odf2 is expressed precisely in mouse oocytes even in the absence of intact centriole structure, and may regulate oocyte spindle assembly and positioning, additionally, the sperm motility and early embryo development.

Follicular cells protect Xenopus oocyte from abnormal maturation via integrin signaling downregulation and O-GlcNAcylation control

Xenopus oocytes are encompassed by a layer of follicular cells that contribute to oocyte growth and meiosis in relation to oocyte maturation. However, the effects of the interaction between follicular cells and the oocyte surface on meiotic processes are unclear. Here, we investigated Xenopus follicular cell function using oocyte signaling and heterologous-expressing capabilities. We found that oocytes deprotected from their surrounding layer of follicular cells and expressing the epidermal growth factor (EGF) receptor (EGFR) and the Grb7 adaptor undergo accelerated prophase I to metaphase II meiosis progression upon stimulation by EGF. This unusual maturation unravels atypical spindle formation but is rescued by inhibiting integrin β1 or Grb7 binding to the EGFR. In addition, we determined that oocytes surrounded by their follicular cells expressing EGFR-Grb7 exhibit normal meiotic resumption. These oocytes are protected from abnormal meiotic spindle formation through the recruitment of O-GlcNAcylated Grb7, and OGT (O-GlcNAc transferase), the enzyme responsible for O-GlcNAcylation processes, in the integrin β1-EGFR complex. Folliculated oocytes can be forced to adopt an abnormal phenotype and exclusive Grb7 Y338 and Y188 phosphorylation instead of O-GlcNAcylation under integrin activation. Furthermore, an O-GlcNAcylation increase (by inhibition of O-GlcNAcase), the glycosidase that removes O-GlcNAc moieties, or decrease (by inhibition of OGT) amplifies oocyte spindle defects when follicular cells are absent highlighting a control of the meiotic spindle by the OGT-O-GlcNAcase duo. In summary, our study provides further insight into the role of the follicular cell layer in oocyte meiosis progression.

P-613 Deletion of the CLPP gene in oocytes exacerbates chemotherapy-induced ovarian damage

Abstract Study question We attempted to investigate the role of the Clpp in chemotherapy-induced ovarian damage and the decline of oocyte function Summary answer We clarified that the deletion of Clpp gene in oocytes will aggravate the ovarian damage and the decline of oocyte function caused by chemotherapeutic drugs. What is known already Chemotherapy is extensively used to treat cancers and is often associated with ovarian damage which leads to premature ovarian insufficiency and infertility. Mitochondria have the critical role of producing energy and maintaining normal cellular activities for oocyte maturation and folliculogenesis. Study design, size, duration This study used a mouse model with oocyte-specific deletion of mitochondrial stress response gene Caseinolytic peptidase P (Clpp) to investigate mitochondrial homeostasis in oocytes from mice receiving a chemotherapeutic drug cyclophosphamide (CTX). Participants/materials, setting, methods First, analyze the reproductive phenotype and fertility test of mice, observe the ovarian development, the proportion of follicles at all levels, the spindle of oocytes and the aneuploidy rate of chromosomes at the MII stage, and using multiple mitochondrial function indicator kit to detect mitochondrial function. Then, we modeled 8-week-old mice with a single injection of 100ul 75uM cyclophosphamide. After 14 days, check the above indicators again. the impact of cyclophosphamide modeling will be analyzed. Main results and the role of chance We found the specific deletion of Clpp in oocytes reduced fecundity of the mice at advanced age. The Clpp deletion led to meiotic defects and impaired mitochondrial distribution and function in the MII oocytes. CTX induction further affected oocyte competence in Clpp cKO mice and lead to an exhaustive disruption of blastocytes formation due to the severe effects on mitochondrial distributions and functions. Limitations, reasons for caution We only clarified that the deletion of Clpp gene in oocytes will through affecting the mitochondrial function aggravate the ovarian damage and the decline of oocyte function caused by chemotherapeutic drugs.But the underlying mechanism has not been able to explore further. Wider implications of the findings This is first study specifically on the impact of oocyte specific deletion of Clpp on oocyte competence and early embryo development. At the same time, the role of Clpp gene in the ovary damaged by chemotherapy drugs was further clarified. Trial registration number not applicable

P-423 Effect of pergafast 201 on in vitro meiotic maturation of pig oocytes

Abstract Study question Does pergafast 201 (PF201) have harmful effects on selected markers of in vitro maturation of pig oocytes? Summary answer PF201 affects the course of in vitro oocyte maturation, reaching the metaphase II stage, formation of the meiotic spindle and methylation of selected epigenetic markers. What is known already PF201 is a chemical used as a color developer and currently serves as a replacement for bisphenol A and S in related paper and other products. Bisphenol A and S are already proven endocrine disruptors that can released into the environment and from there enter the human body, where they disrupt physiological hormonal functions. In the case of PF201, toxicity to aquatic organisms, persistence in the environment and affinity to some proteins associated with non-infectious diseases have already been confirmed. Therefore, further testing of the biological effect of PF201 is necessary to assess the risks to human health and reproduction. Study design, size, duration Cumulus-oocytes complexes (COCs) from the ovaries of slaughtered prepubertal sows were used. In vitro maturation dynamics of PF201-treated COCs were compared with control COCs (n ≥ 30). The meiotic spindle was assessed under a confocal microscope after fluorescence labeling of meiotic chromosomes and spindle microtubules. Dimethylation of histone H3 at lysine K4 (H3K4me2) and trimethylation at lysine K9 (H3K4me3) were imaged under a fluorescence microscope after fluorescent labeling, the intensity was subsequently evaluated in the ImageJ software. Participants/materials, setting, methods COCs were treated with PF201 in at least five independent experiments using the following concentrations: 300 pM, 30 nM and 3 µM, dissolved in DMSO to a final concentration of 0.05%. A vehicle control was used when COCs were cultured in medium with the same concentration of DMSO. COCs were cultured for 48 hours to metaphase II in 5.0% CO2 at 39 °C. Main results and the role of chance PF201 exposure in vitro affected meiotic maturation of pig oocytes, their ability to reach metaphase II, meiotic spindle formation, and epigenetic histone modifications. Treatment with 30 nM concentration of PF201 in vitro caused a significant accumulation of oocytes in metaphase I stage (35% vs. 7% in controls, p < 0.05), consistent with a non-linear effect. Moreover, according to our results, PF201 also disrupts the formation of the meiotic spindle in pig oocytes and probably affects the organization of the meiotic spindle at multiple levels. After 300 pM (30%) and 30 nM (25% vs. 10% in controls, p < 0.05) in vitro treatment of PF201, a significant increase in abnormalities of the meiotic spindle, most often irregular arrangement of the spindle, reduction of the meiotic figure, faulty attachment of chromosomes to tubulin and elongated poles of the meiotic spindle located far apart. A significant increase in the relative fluorescence intensity of epigenetic markers was also observed after treatment of oocytes with PF201. For histone H3K4me2, a significant increase in relative fluorescence intensity was observed after treatment with 300 pM and 3 µM concentrations, while for histone H3K9me3, a significant increase in relative intensity was observed after treatment with 30 nM and 3 µM concentrations. Limitations, reasons for caution As work with human oocytes encounters a number of ethical and legal regulations, a porcine model was chosen, which is physiologically close to the human oocyte. However, it would be great benefit if the effects of PF201 were evaluated in human discarded oocytes that can be obtained from IVF laboratories. Wider implications of the findings It is increasingly difficult for the human population to avoid exposure to environmentally toxic substances, and this fact could explain the decline in human fertility in recent decades. The finding that PF201 negatively affects meiotic maturation suggests, that it may reduce fertilization rates, thus requiring a re-evaluation of its use. Trial registration number MZE-RO0718

P-233 Novel, non-invasive imaging techniques in quality assessment of mammalian eggs

Abstract Study question Can Optical Coherence Microscopy (OCM) imaging and Cytoplasmic Movement Velocity (CMV) analysis be useful in quality assessment of mature mammalian oocytes? Summary answer OCM allows for assessment of oocytes’ metaphase spindles, and CMV – the actomyosin cytoskeleton functionality, therefore they are potentially useful in oocyte selection for IVF. What is known already The success rate of IVF has significantly improved over the recent years, yet its efficiency plummets with the increasing age of female patients. Therefore, novel approaches to oocyte/embryo quality assessment are in high demand. Among the most promising new methods are OCM, a label-free, 3D tool for subcellular structure visualization, such as the meiotic spindle in mature oocytes or nuclear structure in immature oocytes, and the analysis of CMV, a time-lapse imaging-based method for studying properties of the actomyosin cytoskeleton, crucial for a plethora of cellular process. Both these methods provide unique insights into oocyte competence. Study design, size, duration MII spindle dimensions were measured in OCM-imaged freshly ovulated oocytes collected from young mice (FO-MII, n = 108) and correlated with the outcome of preimplantation development achieved after the oocytes’ activation. Spindles of FO-MII (n = 45) were also compared to spindles of oocytes collected from mice in advanced maternal age (AMA-MII, n = 44). CMV was compared in time-lapse imaged FO-MII (n = 24), postovulatory aged oocytes collected from young mice (PA-MII, n = 16), and AMA-MII (n = 19). Participants/materials, setting, methods Oocytes were collected from hormonally primed mice and imaged using a customized OCM protocol. They were then activated parthenogenetically and cultured for 5 days, followed by immunofluorescence staining to assess the total number of cells and number of cells in the first embryonic cell lineages. FO-MII, PA-MII, and AMA-MII were time-lapse imaged (6 frames per minute; 15 min). The images were subjected to particle image velocimetry analysis and the mean CMV was calculated. Main results and the role of chance Oocytes that achieved the blastocyst stage had smaller spindle volume (p < 0.001) and width (p < 0.05) as compared to those failing to do so. Univariate logistic regression analysis indicated that a 1 μm rise in the spindle width decreases the odds of blastocyst formation by 28% and the number of cells on average by 8, whereas a 100 μm3 rise in the volume – by almost 10% and 2.7 cells respectively. Moreover, the spindle volume is also related to the number of trophectodermal cells, and the spindle length - to the number of primitive endoderm cells in the blastocysts. Noteworthily, young oocytes’ spindles had significantly smaller volume (p < 0.0001), as well as were narrower (p < 0.0001) compared to their counterparts from aged females. A multivariate linear regression model combining spindle length with three morphokinetic parameters (t3, cc2b, tSB) explained 60% of the variability in the total number of cells in 5-day-old embryos (R2 = 0.604) while models based solely on a single morphokinetic parameters explained only up to approx. 54% (R2 = 0.537, for tSB). On the other hand, CMV analysis revealed that freshly ovulated eggs display faster cytoplasmic movements than those postovulatory-aged (p < 0.0001). Reduced cytoplasmic speed was also observed in oocytes collected from aged mice (p < 0.0001). Limitations, reasons for caution All studies presented here were performed on a mouse model, so the obtained data needs to be verified in the target species for validation of these methods as novel tools in oocyte selection protocols. Wider implications of the findings Our results suggest that OCM-based assessment of the MII spindle and short, 15-minute CMV imaging allow for the selection of higher quality oocytes and could potentially find application in assisted reproductive technologies, as they are non-invasive and may enhance the universally acclaimed oocyte/embryo quality assessment protocols. Trial registration number not applicable

P-248 The meiotic spindle is preserved during freezing but post-thawing rehydration causes its destabilization in human eggs

Abstract Study question Is the human egg spindle integrity impaired by vitrification and thawing procedures? Summary answer Cryoprotective-caused dehydration stabilizes the MII spindle, but the re-entry of water during thawing induces depolymerization of microtubules, thus promoting instability of the division apparatus. What is known already Freezing sperm and preimplantation embryos have become routine IVF procedures with excellent clinical results, whereas cryopreservation of oocytes remains problematic. The major factor underlying the human oocytés notorious propensity to cryoinjury is the temperature sensitivity of the meiotic spindle, the fidelity of which is critical for faithful post-fertilization development. Most studies focus on evaluating vitrified-thawed oocytes' post-fertilization outcome, but behavior of the meiotic spindle during the freezing process has received only a little attention. Study design, size, duration The experimental study involved a total of 165 human oocytes donated for research. The presence and morphology of the meiotic spindle were examined by polarized light microscopy (PLM) and fluorescent confocal imaging to map out the MII spindle dynamics during the vitrification-thawing procedure. Participants/materials, setting, methods A total of 114 women gave informed consent for their surplus immature oocytes to be used in this project. Oocytes that completed maturation in vitro were subjected to spindle imaging (Oosight) and vitrified using a Kitazato/Cryotop open system. The PLM-positive/negative oocytes were fixed at 6 steps of the vitrification-thawing protocol and 2 hours after thawing (15 oocytes in each subgroup). Microtubules and DNA were fluorescently labeled to inspect chromosome-spindle configuration. Main results and the role of chance Time-course experiments showed that PLM-detectable bipolar spindle remained intact in all but one oocyte (44/45) fixed during pre-vitrification equilibration. Notably, the PLM signal became more prominent after adding a cryoprotectant that displaced intracellular water. In contrast, the MII spindle signal progressively disappeared during thawing. Specific tubulin labeling revealed that the microtubule mass was still present in most (41/45) oocytes from the vitrified-thawed sample group. However, the division apparatus tended to lose its bipolarity and disintegrate (8/45) during the washing steps. This trend was accented in the group of oocytes that lacked PLM-detectable spindles before freezing. Here, only a minority of cells (11/45) undergoing rehydration displayed characteristic chromosome-spindle configuration. The difference in eggś capability to ensure spindle bipolarity was apparent even 2 hours after thawing when 13/15 of initially PLM-positive but only 7/15 PLM-negative oocytes exhibited normal-shaped spindles. Based on these data, we hypothesize that the transient disappearance of the PLM spindle signal during warming may be attributed to the restoration of microtubule dynamics and spindle destabilization. The speed and efficiency of spindle reconstruction seem to depend on the overall egǵs fitness. Limitations, reasons for caution This research study involved a limited number of hormonally-primed oocytes that extruded a polar body in vitro shortly after retrieval. The obtained imaging data represent snapshots of the process. A technically challenging live imaging study is needed to complement the description of the oocyte spindle dynamics during post-vitrification recovery. Wider implications of the findings Our results demonstrate that meiotic spindles in frozen-thawed eggs are not products of de novo spindle assembly but are built on preexisting microtubule mass. Suboptimal handling and reducing the interval between thawing and ICSI may enhance unavoidable spindle instability and thus compromise the developmental potential of vitrified eggs. Trial registration number not-applicable