Beyond the centrosome: The mystery of microtubule organising centres across mammalian preimplantation embryos.

Mammalian preimplantation embryogenesis depends on the spatio-temporal dynamics of the microtubule cytoskeleton to enable exceptionally fast changes in cell number, function, architecture, and fate. Microtubule organising centres (MTOCs), which coordinate the remodelling of microtubules, are therefore of fundamental significance during the first days of a new life. Despite its indispensable role during early mammalian embryogenesis, the origin of microtubule growth remains poorly understood. In this review, we summarise the most recent discoveries on microtubule organisation and function during early human embryogenesis and compare these to innovative studies conducted in alternative mammalian models. We emphasise the differences and analogies of centriole inheritance and their role during the first cleavage. Furthermore, we highlight the significance of non-centrosomal MTOCs for embryo viability and discuss the potential of novel in vitro models and light-inducible approaches towards unravelling microtubule formation in research and assisted reproductive technologies.

Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins.

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. Traditional pharmacological disruption of the ubiquitous microtubule network using, for instance, nocodazole can have devastating consequences for any cell. Reversibly photoswitchable microtubule inhibitors have the potential to overcome the limitations by enabling drug effects to be implemented in a spatiotemporally-controlled manner. One such family of drugs is the azobenzene-based photostatins (PSTs). These compounds are inactive in dark conditions, and upon illumination with UV light, they bind to the colchicine-binding site of ß-tubulin and block microtubule polymerization and dynamic turnover. Here, the application of PSTs in the 3-dimensional (3D) live preimplantation mouse embryo is set out to disrupt the microtubule network on a subcellular level. This protocol provides instructions for the experimental setup, as well as light activation and deactivation parameters for PSTs using live-cell confocal microscopy. This ensures reproducibility and enables others to apply this procedure to their research questions. Innovative photoswitches like PSTs may evolve as powerful tools to advance the understanding of the dynamic intracellular microtubule network and to non-invasively manipulate the cytoskeleton in real-time. Furthermore, PSTs may prove useful in other 3D structures such as organoids, blastoids, or embryos of other species.

Isolation and in vitro Culture of Mouse Oocytes.

Females are endowed at birth with a fixed reserve of oocytes, which declines both in quantity and quality with advancing age. Understanding the molecular mechanisms regulating oocyte quality is crucial for improving the chances of pregnancy success in fertility clinics. In vitro culture systems enable researchers to analyse important molecular and genetic regulators of oocyte maturation and fertilisation. Here, we describe in detail a highly reproducible technique for the isolation and culture of fully grown mouse oocytes. We include the considerations and precautionary measures required for minimising the detrimental effects of in vitro culture conditions. This technique forms the starting point for a wide range of experimental approaches such as post-transcriptional gene silencing, immunocytochemistry, Western blotting, high-resolution 4D time-lapse imaging, and in vitro fertilization, which are instrumental in dissecting the molecular determinants of oocyte quality. Hence, this protocol serves as a useful, practical guide for any oocyte researcher beginning experiments aimed at investigating important oocyte molecular factors. Graphic abstract: A step-by-step protocol for the isolation and in vitro culture of oocytes from mice.

Nampt-mediated spindle sizing secures a post-anaphase increase in spindle speed required for extreme asymmetry.

Meiotic divisions in oocytes are extremely asymmetric and require pre- and post-anaphase-onset phases of spindle migration. The latter induces membrane protrusion that is moulded around the spindle thereby reducing cytoplasmic loss. Here, we find that depleting the NAD biosynthetic enzyme, nicotinamide phosphoribosyl-transferase (Nampt), in mouse oocytes results in markedly longer spindles and compromises asymmetry. By analysing spindle speed in live oocytes, we identify a striking and transient acceleration after anaphase-onset that is severely blunted following Nampt-depletion. Slow-moving midzones of elongated spindles induce cortical furrowing deep within the oocyte before protrusions can form, altogether resulting in larger oocyte fragments being cleaved off. Additionally, we find that Nampt-depletion lowers NAD and ATP levels and that reducing NAD using small molecule Nampt inhibitors also compromises asymmetry. These data show that rapid midzone displacement is critical for extreme asymmetry by delaying furrowing to enable protrusions to form and link metabolic status to asymmetric division.

Oocytes mount a noncanonical DNA damage response involving APC-Cdh1-mediated proteolysis.

In mitotic cells, DNA damage induces temporary G2 arrest via inhibitory Cdk1 phosphorylation. In contrast, fully grown G2-stage oocytes readily enter M phase immediately following chemical induction of DNA damage in vitro, indicating that the canonical immediate-response G2/M DNA damage response (DDR) may be deficient. Senataxin (Setx) is involved in RNA/DNA processing and maintaining genome integrity. Here we find that mouse oocytes deleted of Setx accumulate DNA damage when exposed to oxidative stress in vitro and during aging in vivo, after which, surprisingly, they undergo G2 arrest. Moreover, fully grown wild-type oocytes undergo G2 arrest after chemotherapy-induced in vitro damage if an overnight delay is imposed following damage induction. Unexpectedly, this slow-evolving DDR is not mediated by inhibitory Cdk1 phosphorylation but by APC-Cdh1-mediated proteolysis of the Cdk1 activator, cyclin B1, secondary to increased Cdc14B-dependent APC-Cdh1 activation and reduced Emi1-dependent inhibition. Thus, oocytes are unable to respond immediately to DNA damage, but instead mount a G2/M DDR that evolves slowly and involves a phosphorylation-independent proteolytic pathway.

The Presence of Immature GV- Stage Oocytes during IVF/ICSI Is a Marker of Poor Oocyte Quality: A Pilot Study.

Here we investigate whether the presence of germinal vesicle-stage oocytes (GV- oocytes) reflects poor oocyte developmental competence (or quality). This was a prospective, non-randomised, cohort pilot-study involving 60 patients undergoing in vitro fertilization/ intracytoplasmic sperm injection for whom complete pregnancy outcome data were available. Patients in whom GV- oocytes were retrieved (GV+) at transvaginal oocyte retrieval (TVOR) were compared with those from whom no GVs were retrieved (GV-). We found that GV+ (n = 29) and GV- (n = 31) patients were similarly aged (35.4 vs. 36.4 years; p = 0.446). GV+ patients had a mean of 2.41 ± 2.03 GVs and comparable yields of MII oocytes to GV- patients (11 ± 6.88 vs. 8.26 ± 4.84; p = 0.077). Compared with GV- patients, GV+ patients had markedly lower implantation rates (11.8% vs. 30.2%; p = 0.022) as well as oocyte utilisation rates for clinical pregnancy (2.3% vs. 6.8%; p = 0.018) and live-birth (1.9% vs. 5.7%; p = 0.029). DNA damage levels measured using γH2AX immunostaining were not different in oocytes from women <36 years versus those ≥36 years (p = 0.606). Thus, patients who have GV- stage oocytes at TVOR exhibit poor oocyte quality reflected in reduced per-oocyte pregnancy success rates and uniformly high levels of oocyte DNA damage.

Disruption of Spermatogenesis and Infertility in Ataxia with Oculomotor Apraxia Type 2 (AOA2).

Ataxia with oculomotor apraxia type 2 (AOA2) is a rare autosomal recessive cerebellar ataxia characterized by onset between 10 and 20 years of age and a range of neurological features that include progressive cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia in a majority of patients, and elevated serum alpha-fetoprotein (AFP). AOA2 is caused by mutation of the SETX gene which encodes senataxin, a DNA/RNA helicase involved in transcription regulation, RNA processing, and DNA maintenance. Disruption of senataxin in rodents led to defective spermatogenesis and sterility in males uncovering a key role for senataxin in male germ cell survival. Here, we report the first clinical and cellular evidence of impaired spermatogenesis in AOA2 patients. We assessed sperm production in three AOA2 patients and testicular pathology in one patient and compared the findings to those of Setx-knockout mice. Sperm production was impaired in all patients assessed (3/3, 100%). Analyses of testicular biopsies from an AOA2 patient recapitulate features of the histology seen in Setx-knockout mice, strongly suggesting an underlying mechanism centering on DNA-damage-mediated germ cell apoptosis. These findings support a role for senataxin in human reproductive function and highlight a novel clinical feature of AOA2 that extends the extra-neurological roles of senataxin. This raises an important reproductive counseling issue for clinicians, and fertility specialists should be aware of SETX mutations as a possible diagnosis in young male patients presenting with oligospermia or azoospermia since infertility may presage the later onset of neurological manifestations in some individuals.

Cdk1 inactivation induces post-anaphase-onset spindle migration and membrane protrusion required for extreme asymmetry in mouse oocytes.

Female meiotic divisions are extremely asymmetric, producing large oocytes and small polar bodies (PBs). In mouse oocytes, the spindle relocates to the cortex before anaphase of meiosis I (MI). It is presumed that by displacing the future midzone, pre-anaphase spindle repositioning alone ensures asymmetry. But how subsequent anaphase events might contribute to asymmetric PB extrusion (PBE) is unknown. Here, we find that inactivation of cyclin-dependent kinase 1 (Cdk1) induces anaphase and simultaneously triggers cytoplasmic formin-mediated F-actin polymerisation that propels the spindle into the cortex causing it to protrude while anaphase progresses. Significantly, if post-anaphase-onset spindle migration fails, protrusion and asymmetry are severely threatened even with intact pre-anaphase migration. Conversely, post-anaphase migration can completely compensate for failed pre-anaphase migration. These data identify a cell-cycle-triggered phase of spindle displacement occurring after anaphase-onset, which, by inducing protrusion, is necessary for extreme asymmetry in mouse oocytes and uncover a pathway for maximising unequal division.

Immunofluorescence Staining of K-Fibers in Mouse Oocytes Using Cold Fixation.

The kinetochore is a multiprotein complex that assembles on centromeric DNA and constitutes the main attachment interface between chromosomes and microtubules of the spindle apparatus. Kinetochores also provide the platform for integrating the surveillance mechanism known as the spindle assembly checkpoint (SAC) that regulates the timing of anaphase onset. Saturation of microtubule binding sites on kinetochores displaces SAC proteins leading to loss of SAC-mediated inhibition and the triggering of anaphase. Microtubule binding sites become saturated by bundles of microtubules attached in an end-on manner to kinetochores, termed kinetochore fibers or K-fibers. The appearance of K-fibers therefore signifies the completion of attachment between kinetochores and microtubules and the silencing of the SAC. Here we describe a method involving cold-fixation for immunostaining and imaging K-fibers during meiosis I in mouse oocytes.

Regulation of chromosome segregation in oocytes and the cellular basis for female meiotic errors.

BACKGROUND: Meiotic chromosome segregation in human oocytes is notoriously error-prone, especially with ageing. Such errors markedly reduce the reproductive chances of increasing numbers of women embarking on pregnancy later in life. However, understanding the basis for these errors is hampered by limited access to human oocytes. OBJECTIVE AND RATIONALE: Important new discoveries have arisen from molecular analyses of human female recombination and aneuploidy along with high-resolution analyses of human oocyte maturation and mouse models. Here, we review these findings to provide a contemporary picture of the key players choreographing chromosome segregation in mammalian oocytes and the cellular basis for errors. SEARCH METHODS: A search of PubMed was conducted using keywords including meiosis, oocytes, recombination, cohesion, cohesin complex, chromosome segregation, kinetochores, spindle, aneuploidy, meiotic cell cycle, spindle assembly checkpoint, anaphase-promoting complex, DNA damage, telomeres, mitochondria, female ageing and female fertility. We extracted papers focusing on mouse and human oocytes that best aligned with the themes of this review and that reported transformative and novel discoveries. OUTCOMES: Meiosis incorporates two sequential rounds of chromosome segregation executed by a spindle whose component microtubules bind chromosomes via kinetochores. Cohesion mediated by the cohesin complex holds chromosomes together and should be resolved at the appropriate time, in a specific step-wise manner and in conjunction with meiotically programmed kinetochore behaviour. In women, the stage is set for meiotic error even before birth when female-specific crossover maturation inefficiency leads to the formation of at-risk recombination patterns. In adult life, multiple co-conspiring factors interact with at-risk crossovers to increase the likelihood of mis-segregation. Available evidence support that these factors include, but are not limited to, cohesion deterioration, uncoordinated sister kinetochore behaviour, erroneous microtubule attachments, spindle instability and structural chromosomal defects that impact centromeres and telomeres. Data from mice indicate that cohesin and centromere-specific histones are long-lived proteins in oocytes. Since these proteins are pivotal for chromosome segregation, but lack any obvious renewal pathway, their deterioration with age provides an appealing explanation for at least some of the problems in older oocytes. WIDER IMPLICATIONS: Research in the mouse model has identified a number of candidate genes and pathways that are important for chromosome segregation in this species. However, many of these have not yet been investigated in human oocytes so it is uncertain at this stage to what extent they apply to women. The challenge for the future involves applying emerging knowledge of female meiotic molecular regulation towards improving clinical fertility management.

Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types.

SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types.