Understanding oocyte ageing.

Females are born with a finite and non-renewable reservoir of oocytes, which therefore decline both in number and quality with advancing age. A striking characteristic of oocyte quality is that "ageing" effects manifest whilst women are in their thirties and are therefore still chronologically and physically young. Furthermore, this decline is unrelenting and not modifiable to any great extent by lifestyle or diet. Since oocyte quality is rate-limiting for pregnancy success, as the proportion of good-quality oocytes progressively deteriorate, the chance of successful pregnancy during each 6-12-month period also decreases, becoming exponential after 37 years. Unlike oocyte quality, age-related attrition in the size of the ovarian reservoir is less impactful for natural fertility since only one mature oocyte is typically ovulated per menstrual cycle. In contrast, oocyte numbers are pivotal for in-vitro fertilization success, since larger numbers enable better-quality oocytes to be found and is important for buffering the inefficiencies of the IVF process. The ageing trajectory is accelerated in ~10% of women, so-called premature ovarian ageing, with ~1% of women at the extreme end of this spectrum with loss of ovarian function occurring before 40 years of age, termed premature ovarian insufficiency. The aim of this review was to analyze how ageing impacts the size and quality of the oocyte pool along with emerging interventions for combating low oocyte numbers and improving quality.

Two-step nuclear centring by competing microtubule- and actin-based mechanisms in 2-cell mouse embryos.

Microtubules typically promote nuclear centring during early embryonic divisions in centrosome-containing vertebrates. In acentrosomal mouse zygotes, microtubules also centre male and female pronuclei prior to the first mitosis, this time in concert with actin. How nuclear centring is brought about in subsequent acentrosomal embryonic divisions has not been studied. Here, using time-lapse imaging in mouse embryos, we find that although nuclei are delivered to the cell centre upon completion of the first mitotic anaphase, the majority do not remain stationary and instead travel all the way to the cortex in a microtubule-dependent manner. High cytoplasmic viscosity in 2-cell embryos is associated with non-diffusive mechanisms involving actin for subsequent nuclear centring when microtubules again exert a negative influence. Thus, following the first mitotic division, pro-centring actin-dependent mechanisms work against microtubule-dependent de-centring forces. Disrupting the equilibrium of this tug-of-war compromises nuclear centring and symmetry of the subsequent division potentially risking embryonic development. This circuitous centring process exposes an embryonic vulnerability imposed by microtubule-dependent de-centring forces.

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.

Senataxin: A New Guardian of the Female Germline Important for Delaying Ovarian Aging.

Early decline in ovarian function known as premature ovarian aging (POA) occurs in around 10% of women and is characterized by a markedly reduced ovarian reserve. Premature ovarian insufficiency (POI) affects ~1% of women and refers to the severe end of the POA spectrum in which, accelerated ovarian aging leads to menopause before 40 years of age. Ovarian reserve refers to the total number of follicle-enclosed oocytes within both ovaries. Oocyte DNA integrity is a critical determinant of ovarian reserve since damage to DNA of oocytes within primordial-stage follicles triggers follicular apoptosis leading to accelerated follicle depletion. Despite the high prevalence of POA, very little is known regarding its genetic causation. Another little-investigated aspect of oocyte DNA damage involves low-grade damage that escapes apoptosis at the primordial follicle stage and persists throughout oocyte growth and later follicle development. Senataxin (SETX) is an RNA/DNA helicase involved in repair of oxidative stress-induced DNA damage and is well-known for its roles in preventing neurodegenerative disease. Recent findings uncover an important role for SETX in protecting oocyte DNA integrity against aging-induced increases in oxidative stress. Significantly, this newly identified SETX-mediated regulation of oocyte DNA integrity is critical for preventing POA and early-onset female infertility by preventing premature depletion of the ovarian follicular pool and reducing the burden of low-grade DNA damage both in primordial and fully-grown oocytes.

Sirt1 sustains female fertility by slowing age-related decline in oocyte quality required for post-fertilization embryo development.

The NAD+ -dependent sirtuin deacetylase, Sirt1, regulates key transcription factors strongly implicated in ageing and lifespan. Due to potential confounding effects secondary to loss of Sirt1 function from the soma in existing whole-animal mutants, the in vivo role of Sirt1 in oocytes (oocyte-Sirt1) for female fertility remains unknown. We deleted Sirt1 specifically in growing oocytes and study how loss of oocyte-Sirt1 affects a comprehensive range of female reproductive parameters including ovarian follicular reservoir, oocyte maturation, oocyte mitochondrial abundance, oxidative stress, fertilization, embryo development and fertility during ageing. Surprisingly, eliminating this key sirtuin from growing oocytes has no effect in young females. During a 10-month-long breeding trial, however, we find that 50% of females lacking oocyte-Sirt1 become prematurely sterile between 9 and 11 months of age when 100% of wild-type females remain fertile. This is not due to an accelerated age-related decline in oocyte numbers in the absence of oocyte-Sirt1 but to reduced oocyte developmental competence or quality. Compromised oocyte quality does not impact in vivo oocyte maturation or fertilization but leads to increased oxidative stress in preimplantation embryos that inhibits cleavage divisions. Our data suggest that defects emerge in aged females lacking oocyte-Sirt1 due to concurrent age-related changes such as reduced NAD+ and sirtuin expression levels, which compromise compensatory mechanisms that can cover for Sirt1 loss in younger oocytes. In contrast to evidence that increasing Sirt1 activity delays ageing, our data provide some of the only in vivo evidence that loss of Sirt1 induces premature ageing.

Sirt3 is dispensable for oocyte quality and female fertility in lean and obese mice.

Mammalian oocytes rely heavily on mitochondrial oxidative phosphorylation (OXPHOS) for generating ATP. However, mitochondria are also the primary source of damaging reactive oxygen species (ROS). Mitochondrial de-regulation, therefore, underpins poor oocyte quality associated with conditions such as obesity and aging. The mitochondrial sirtuin, Sirt3, is critical for mitochondrial respiration and redox regulation. Interestingly, however, Sirt3 knockout (Sirt3-/- ) mice do not exhibit systemic compromise under basal conditions, only doing so under stressed conditions such as high-fat diet (HFD)-induced obesity. Mouse oocytes depleted of Sirt3 exhibit increased ROS in vitro, but it is unknown whether Sirt3 is necessary for female fertility in vivo. Here, we test this for the first time by investigating ovarian follicular reserve, oocyte maturation (including detailed spindle assembly and chromosome segregation), and female fertility in Sirt3-/- females. We find that under basal conditions, young Sirt3-/- females exhibit no defects in any parameters. Surprisingly, all parameters also remain intact following HFD-induced obesity. Despite markedly increased ROS levels in HFD Sirt3-/- oocytes, ATP levels nevertheless remain normal. Our data support that ATP is sustained in vivo through increased mitochondrial mass possibly secondary to compensatory upregulation of another sirtuin, Sirt1, which has overlapping functions with Sirt3.

NAD+ Repletion Rescues Female Fertility during Reproductive Aging.

Reproductive aging in female mammals is an irreversible process associated with declining oocyte quality, which is the rate-limiting factor to fertility. Here, we show that this loss of oocyte quality with age accompanies declining levels of the prominent metabolic cofactor nicotinamide adenine dinucleotide (NAD+). Treatment with the NAD+ metabolic precursor nicotinamide mononucleotide (NMN) rejuvenates oocyte quality in aged animals, leading to restoration in fertility, and this can be recapitulated by transgenic overexpression of the NAD+-dependent deacylase SIRT2, though deletion of this enzyme does not impair oocyte quality. These benefits of NMN extend to the developing embryo, where supplementation reverses the adverse effect of maternal age on developmental milestones. These findings suggest that late-life restoration of NAD+ levels represents an opportunity to rescue female reproductive function in mammals.

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.

First meiotic anaphase requires Cep55-dependent inhibitory cyclin-dependent kinase 1 phosphorylation.

During mitosis, anaphase is triggered by anaphase-promoting complex (APC)-mediated destruction of securin and cyclin B1, which leads to inactivation of cyclin-dependent kinase 1 (Cdk1). By regulating APC activity, the mitotic spindle assembly checkpoint (SAC) therefore has robust control over anaphase timing to prevent chromosome mis-segregation. Mammalian oocytes are prone to aneuploidy, the reasons for which remain obscure. In mitosis, Cep55 is required post-anaphase for the final steps of cytokinesis. We found that Cep55-depleted mouse oocytes progress normally through early meiosis I, but that anaphase I fails as a result of persistent Cdk1 activity. Unexpectedly, Cdk1 inactivation was compromised following Cep55 depletion, despite on-time SAC silencing and intact APC-mediated proteolysis. We found that impaired Cdk1 inactivation was caused by inadequate inhibitory Cdk1 phosphorylation consequent upon failure to suppress Cdc25 phosphatase, identifying a proteolysis-independent step necessary for anaphase I. Thus, the SAC in oocytes does not exert exclusive control over anaphase I initiation, providing new insight into vulnerability to error.

Immunofluorescence staining of spindles, chromosomes, and kinetochores in human oocytes.

Understanding how human oocytes execute chromosome segregation is of paramount importance as errors in this process account for the overwhelming majority of human aneuploidies and increase exponentially with advancing female age. The spindle is the cellular apparatus responsible for separating chromosomes at anaphase. For accurate chromosome segregation, spindle microtubules must establish appropriately configured attachments to chromosomes via kinetochores. With regard to understanding the mechanistic basis for human aneuploidies therefore, it will be important to explore the molecular underpinnings of spindle structure and the interaction of its microtubules with chromosomes in human oocytes. Here we describe a technique for simultaneously immunolabelling chromosomes, spindle microtubules and kinetochores in human oocytes.

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

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

RNA interference in meiosis I human oocytes: towards an understanding of human aneuploidy.

Although female meiosis I errors account for the majority of human aneuploidy, their molecular basis is largely unknown. By elucidating gene function, gene knockdown using RNA interference (RNAi) could shed light on this enigmatic process. In practice, however, the extreme paucity of immature human oocytes makes the evaluation of gene-targeting tools difficult. Here, we undertake RNAi in human oocytes and describe an approach employing mouse oocytes which could overcome the problem of limited biological material. We designed a short interfering RNA (siRNA) designated si539 to target the human mitotic arrest deficient 2 (hMad2) spindle checkpoint component. In human oocytes microinjected with si539, the hMad2 signal detected by Western blotting was 85-92% less intense than in oocytes injected with control siRNA indicating efficient silencing. Further examination of si539's targeting efficiency was undertaken using a green fluorescent protein (GFP)-tagged hMad2 mRNA construct in mouse oocytes. Consistent with Western blot analysis, si539 reduced hMad2-GFP expression in mouse oocytes by approximately 94% and relieved the meiosis I arrest otherwise induced by hMad2-GFP in mouse oocytes. By facilitating the investigation of candidate genes involved in regulating human female meiosis I, this approach can bring us closer to understanding the origins of aneuploidies such as Down's syndrome.

Restaging the spindle assembly checkpoint in female mammalian meiosis I.

In mammalian somatic cells, the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation by delaying cell-cycle progression in the face of even a single misaligned chromosome. In contrast, the role of the SAC in unperturbed mammalian oocytes is less well defined as progression through meiosis I is unaltered in mouse oocytes in the presence of one or a few misaligned chromosomes. Furthermore, attempts to disable the function of the SAC protein, Mad2, in mouse oocytes have produced conflicting results. To gain further insight into SAC function during female mammalian meiosis I, we recently utilised a morpholino-based antisense approach to deplete the majority of Mad2 in mouse oocytes. Our results define a clear role for Mad2 in ensuring the proper timing of meiosis I events and ultimately, in ensuring the fidelity of homologue disjunction. We discuss the implications of these results for the regulation of meiosis I in mammalian oocytes and for the genesis of human aneuploidy.

Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes.

In mitosis, the spindle checkpoint protein Mad2 averts aneuploidy by delaying anaphase onset until chromosomes align. Here we show that depletion of Mad2 in meiosis I mouse oocytes induced an increased incidence of aneuploidy. Proteolysis of cyclin B and securin commenced earlier in Mad2-depleted oocytes, resulting in a shortened duration of meiosis I. Furthermore, overexpression of Mad2 inhibited homolog disjunction. We conclude that Mad2 delays the onset of cyclin B and securin degradation and averts aneuploidy during meiosis I in mammalian oocytes. The data suggest a link between trisomies such as Down syndrome and defective oocyte spindle checkpoint function.