Acentriolar spindle assembly in mammalian female meiosis and the consequences of its perturbations on human reproduction

The purpose of meiosis is to generate developmentally competent, haploid gametes with the correct number of chromosomes. For reasons not completely understood, female meiosis is more prone to chromosome segregation errors than meiosis in males, leading to an abnormal number of chromosomes, or aneuploidy, in gametes. Meiotic spindles are the cellular machinery essential for the proper segregation of chromosomes. One unique feature of spindle structures in female meiosis is spindles poles that lack centrioles. The process of building a meiotic spindle without centrioles is complex and requires precise coordination of different structural components, assembly factors, motor proteins, and signaling molecules at specific times and locations to regulate each step. In this review, we discuss the basics of spindle formation during oocyte meiotic maturation focusing on mouse and human studies. Finally, we review different factors that could alter the process of spindle formation and its stability. We conclude with a discussion of how different assisted reproductive technologies (ART) could affect spindles and the consequences these perturbations may have for subsequent embryo development.

Cryopreservation of Gametes and Embryos and Their Molecular Changes

The process of freezing cells or tissues and depositing them in liquid nitrogen at –196 °C is called cryopreservation. Sub-zero temperature is not a physiological condition for cells and water ice crystals represent the main problem since they induce cell death, principally in large cells like oocytes, which have a meiotic spindle that degenerates during this process. Significantly, cryopreservation represents an option for fertility preservation in patients who develop gonadal failure for any condition and those who want to freeze their germ cells for later use. The possibility of freezing sperm, oocytes, and embryos has been available for a long time, and in 1983 the first birth with thawed oocytes was achieved. From the mid-2000s forward, the use of egg vitrification through intracytoplasmic sperm injection has improved pregnancy rates. Births using assisted reproductive technologies (ART) have some adverse conditions and events. These risks could be associated with ART procedures or related to infertility. Cryopreservation generates changes in the epigenome of gametes and embryos, given that ART occurs when the epigenome is most vulnerable. Furthermore, cryoprotective agents induce alterations in the integrity of germ cells and embryos. Notably, cryopreservation extensively affects cell viability, generates proteomic profile changes, compromises crucial cellular functions, and alters sperm motility. This technique has been widely employed since the 1980s and there is a lack of knowledge about molecular changes. The emerging view is that molecular changes are associated with cryopreservation, affecting metabolism, cytoarchitecture, calcium homeostasis, epigenetic state, and cell survival, which compromise the fertilization in ART.

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

ABSTRACTWhile 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 mechanisms that promote acentrosomal spindle stability. To this end, we have optimized the auxin-inducible degron system in C. elegans to remove factors from pre-formed oocyte spindles within minutes and assess 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 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.

The Formation of Bivalents and the Control of Plant Meiotic Recombination

During the first meiotic division, the segregation of homologous chromosomes depends on the physical association of the recombined homologous DNA molecules. The physical tension due to the sites of crossing-overs (COs) is essential for the meiotic spindle to segregate the connected homologous chromosomes to the opposite poles of the cell. This equilibrated partition of homologous chromosomes allows the first meiotic reductional division. Thus, the segregation of homologous chromosomes is dependent on their recombination. In this review, we will detail the recent advances in the knowledge of the mechanisms of recombination and bivalent formation in plants. In plants, the absence of meiotic checkpoints allows observation of subsequent meiotic events in absence of meiotic recombination or defective meiotic chromosomal axis formation such as univalent formation instead of bivalents. Recent discoveries, mainly made in Arabidopsis, rice, and maize, have highlighted the link between the machinery of double-strand break (DSB) formation and elements of the chromosomal axis. We will also discuss the implications of what we know about the mechanisms regulating the number and spacing of COs (obligate CO, CO homeostasis, and interference) in model and crop plants.

Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. It is still poorly understood how efficient, centromere-dependent kinetochore assembly is accomplished from hundreds of individual protein building blocks in a cell cycle dependent manner. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-Phase and targets the protein for degradation. Binding of the Mtw1Mis12 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature-sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic in SCF mutants, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits facilitates efficient centromere-dependent kinetochore assembly. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.

P–245 Machine learning predicting oocyte’s fertilization and blastocyst potential based on morphological features

Study question Can machine learning (ML) predict oocyte’s fertilization and blastocyst development potential based on morphological features extracted from single static images? Summary answer AI accurately predicted 70.4% of fertilization and 60.4% of blastocyst development outcomes from a database of 1000 oocytes. What is known already Some morphological features of the oocyte have been associated with IVF-related outcomes, such as size, shape, and coloration of zona pellucida, polar body, perivitelline space, cytoplasm, and the meiotic spindle. Based on these characteristics, clinics might discard the low-quality oocytes according to a subjective assessment. AI-based algorithms could reduce the subjectivity and improve prediction on IVF outcomes such as successful fertilization and blastocyst development. Study design, size, duration Non-intervention study based on a cohort of 1000 oocytes’ micrographs collected between January 2019 and December 2020 from two IVF clinics. The inclusion criteria were known fertilization and blastocyst development outcome, and patient’s age between 25 and 45 years old undergoing IVF/ICSI treatment. Different features were considered for this study including metadata from oocyte’s (e.g. age, source), as well as manually extracted morphological features from the oocytes’ images (e.g. diameters, shape, granularity, presence/absence of spindle). Participants/materials, setting, methods We trained three machine-learning (ML) classifiers (i.e. Support Vector Machine, logistic regression, and neural networks) to predict successful fertilization and blastocyst development. For the training process we used a 10-fold cross validation approach to assess the model’s generalization capabilities. Besides we tested the statistical difference of each feature among groups (i.e. fertilized and no fertilized) using a two sided Student’s t-test for numerical and Z-test for categorial features (significance of p < 0.01). Main results and the role of chance Our database showed 68.2% of successful fertilization and 34.8% of blastocyst formation. To balance the training data (50% per training class), we aleatory selected 318 and 348 samples per branch of successful/unsuccessful fertilization and blastocyst formation, respectively. From all ML classifiers, the neural network obtained the best results with an accuracy of 0.70 (AUC of 0.74) for predicting fertilization; and an accuracy of 0.60 (AUC of 0.62), for predicting blastocyst formation. We found that spherical shape, presence of meiotic spindle, clear coloration, larger oocyte diameter, thicker zona pellucida, and smaller vacuoles are statistically associated with both successful outcomes. As expected, we also found a strong association between age groups and outcome. The younger group (<35 years) demonstrated to have a larger proportion of successful fertilization compared to the rest of the age groups (36–37, 38–39, 40–42, >42). For the blastocyst formation we observed a similar association. Limitations, reasons for caution It is relevant to note that all cycles were performed under a mini-IVF protocol. Oocytes extracted through conventional stimulation might show the same associations, but it would need further testing. Wider implications of the findings: The present study revealed that our system can predict fertilization success and blastocyst development potential based on metadata and morphometric features extracted from single digital micrographs of oocytes, offering a novel, adaptable and robust integration into clinical practice. Trial registration number CONBIOETICA–09-CEI–001–2017–0131

P–805 Artificial oocytes: from somatic cells to fertile pups

Study question We analyzed the efficacy of generating artificial oocytes using somatic cells (SCs) from two mouse strains (B6D2F1 and FVB) and followed their full pre-/post-implantation development. Summary answer While artificial oocytes generated from the new strain (FVB) had higher fertilization rates, those from the standard strain (B6D2F1) provided expanded blastocysts and fertile pups. What is known already B6D2F1 is a popular hybrid mouse strain for cloning and transgenic creation due to its geno-/pheno-typic uniformity and high oocyte yield and quality. Indeed, B6D2F1 oocytes have a distinct metaphase II (MII) spindle complex, making them an ideal candidate to generate ooplasts used in SC nuclear transfer (SCNT). However, because they lack genetic variance, they are less suitable for reciprocal SCNT studies. In contrast, FVB mice have single nucleotide polymorphisms and indels on each chromosome that can aid in tracing the pedigree of progeny. Study design, size, duration A total of 10 experiments were performed over the course of 3 months, using 30 stimulated mice. SCs were retrieved from cumulus oophorus harvested from FVB and B6D2F1 mice. SCs from both strains were injected into enucleated MII B6D2F1 oocytes. Unmanipulated B6D2F1 oocytes were piezo-ICSI inseminated, serving as controls. The occurrence of haploidization, fertilization, and full preimplantation development was compared. Some blastocysts were transferred into pseudo-pregnant CD–1 mice to obtain offspring. Participants/materials, setting, methods Oocyte enucleation was performed under Oosight™ visualization and cytochalasin B exposure. An FVB or B6D2F1 SC was transferred into the perivitelline space of the ooplast with Sendai virus to promote fusion. Haploidization was monitored by pseudo-meiotic spindle formation followed by extrusion of a pseudo-polar body after insemination. Conceptuses were cultured in a time-lapse imaging system, with piezo-ICSI controls. Expanded blastocysts were transferred into uterine horns of pseudo-pregnant mice. Offspring were mated to test their fertility. Main results and the role of chance FVB (n = 278) and B6D2F1 (n = 905) SCs at G0 phase, with a diameter <10 mm, were chosen for SCNT and transferred into enucleated B6D2F1 ooplasts. Enucleation of 1,212 oocytes yielded a survival rate of 97.6%. Both FVB and B6D2F1 SCNT resulted in similar survival rates of 100% and 98.5%, respectively. Successful haploidization, determined by the presence of a pseudo-meiotic spindle 2 hours after SCNT, was also comparable, with 59.9% of FVB and 63.7% of B6D2F1. Survival after piezo-ICSI was also comparable between FVB- and B6D2F1-reconstituted oocytes, with rates of 64.3% and 60.3%, respectively, albeit lower than the control (75.2%, P < 0.00001). FVB embryos fertilized at a rate of 88.7%, comparable to the control zygotes at 85.8%, while B6D2F1 conceptuses demonstrated a lower fertilization rate (70.8%, P < 0.00001). Blastulation of FVB- and B6D2F1-derived embryos was 15.1% and 24.0%, respectively, while the control was 80.7% (P < 0.00001). Whole-genome karyotyping of 9 B6D2F1-derived blastocysts confirmed 5 of the samples to be euploid. FVB blastocysts (N = 8) and B6D2F1 blastocysts (N = 81) were transferred into pseudo-pregnant mice, resulting in 3 fertile offspring only from the B6D2F1 conceptuses. Limitations, reasons for caution This is still a limited number of observations, and pups were delivered only from the B6D2F1 strain. The utilization of a strain with higher genetic variance may help facilitate offspring fingerprinting. Wider implications of the findings: This study demonstrates the ability to generate artificial genotyped conceptuses, yielding live offspring. The identification of a feasible donor cell, together with optimization of cell cycle stage and standardization of post-implantation development, will help promote this technique for human reproduction in couples with age-related infertility or poor ovarian reserve. Trial registration number N/A

P–800 Generation of artificial oocytes by two distinct mechanisms

Study question Are haploid genome replication and somatic cell haploidization feasible mechanisms for generating parentally genotyped oocytes? Summary answer Artificial oocytes can be generated by haploid genome replication and somatic cell haploidization. The latter is more efficient and capable of generating live offspring. What is known already A low number of mature oocytes is one of the major limitations to treating infertile women who have impaired ovarian reserve. Although it has been proposed that competent oocytes can be created by a phenomenon known as somatic cell haploidization (SCH), its clinical value has yet to be examined due to its poorly understood mechanism. On the other hand, spindle transfer has been clinically applied for mitochondrial replacement therapy. Therefore, we propose to utilize G2-phase haploid pseudo-blastomere (HpB), generated by parthenogenesis, as a nuclear donor to create oocyte replica. Study design, size, duration In the past 7 months, individual G0 phase cumulus cells (CCs) were transferred into 1,066 ooplasts for SCH. HpBs obtained from the activation of 80 oocytes were transferred into 464 ooplasts. Both cohorts were ICSI-inseminated and placed in the time lapse for embryo development. Another 379 unmanipulated oocytes were ICSI-inseminated, serving as control. Pre-implantation development was monitored and compared for both neogametogenesis techniques. Fully expanded blastocysts were transferred to obtain live pups. Participants/materials, setting, methods CCs were isolated from the cumulus oophorus of B6D2F1 mice. HpBs were obtained via oocyte activation, cultured to the 8-cell stage, and subsequently treated by nocodazole to synchronize at the G2-phase. In two experimental groups, CCs or HpBs were individually transferred into the perivitelline space of the ooplasts with inactivated Sendai virus. Reconstructed oocytes presenting with a pseudo-meiotic spindle were fertilized by piezo-actuated ICSI. Blastocysts were transferred into a pseudo-pregnant CD–1 surrogate to obtain pups. Main results and the role of chance A total of 1,769 oocytes underwent enucleation to generate ooplasts, with a survival rate of 97%. Survived ooplasts were allocated to SCH (n = 1,034) and HpB-SCNT (n = 458). To generate HpBs, 80 unmanipulated oocytes were activated; 58 of them progressed to the 8-cell stage and generated 464 HpB for SCNT. For SCH, CCs were selected based on morphology with a diameter <10 micron. Nuclear transfer of CCs and HpB yielded survival rates of 98.6% and 93.2%, respectively. Following SCH and HpB-SCNT, spindle development for SCH and HpB-SCNT was comparable at 63.5% for SCH and 66.7% for HpB-SCNT. The ICSI survival rates for SCH and HpB-SCNT were 58.9% and 64.9%, respectively, but lower than the control at 73.9% (P < 0.001). Fertilization rates for SCH and HpB-SCNT were also comparable at 61.3% and 64.3%, respectively, but lower than the control at 89.6% (P < 0.00001). Full pre-implantation development was achieved for both experimental groups. While the SCH group yielded a development rate of 24.6% (n = 94), the HpB-SCNT group yielded a lower rate at 12.4% (n = 23) (P < 0.001), both lower than the control (71.7%, P < 0.00001); however, the morphokinetics of the embryo development was retained. To date, only 3 live pups were obtained from SCH group. Limitations, reasons for caution While these techniques to manufacture oocytes are very new and highly experimental, our findings show a lower blastulation rate for oocytes generated by HpB. Both techniques require refinement and improvement of reliability and consistency before they can be considered a feasible technique for human reproduction. Wider implications of the findings: The study confirms the potential to create artificial oocytes capable of supporting full pre-implantation development and, in some cases, live pups. If further streamlining of both procedures demonstrates their safety, they may both represent a viable option to generate de novo gametes Trial registration number N/A