Loss of centromeric RNA activates the spindle assembly checkpoint in mammalian female meiosis I.

The repetitive sequences of DNA centromeric regions form the structural basis for kinetochore assembly. Recently they were found to be transcriptionally active in mitosis, with their RNAs providing noncoding functions. Here we explore the role, in mouse oocytes, of transcripts generated from within the minor satellite repeats. Depletion of minor satellite transcripts delayed progression through meiosis I by activation of the spindle assembly checkpoint. Arrested oocytes had poorly congressed chromosomes, and centromeres were frequently split by microtubules. Thus, we have demonstrated that the centromeric RNA plays a specific role in female meiosis I compared with mitosis and is required for maintaining the structural integrity of centromeres. This may contribute to the high aneuploidy rates observed in female meiosis.

Chromosome structural anomalies due to aberrant spindle forces exerted at gene editing sites in meiosis.

Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules significantly diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.

Spindle tubulin and MTOC asymmetries may explain meiotic drive in oocytes.

In the first meiotic division (MI) of oocytes, the cortically positioned spindle causes bivalent segregation in which only the centre-facing homologue pairs are retained. 'Selfish' chromosomes are known to exist, which bias their spindle orientation and hence retention in the egg, a process known as 'meiotic drive'. Here we report on this phenomenon in oocytes from F1 hybrid mice, where parental strain differences in centromere size allows distinction of the two homologue pairs of a bivalent. Bivalents with centromere and kinetochore asymmetry show meiotic drive by rotating during prometaphase, in a process dependent on aurora kinase activity. Cortically positioned homologue pairs appear to be under greater stretch than their centre-facing partners. Additionally the cortex spindle-half contain a greater density of tubulin and microtubule organising centres. A model is presented in which meiotic drive is explained by the impact of microtubule force asymmetry on chromosomes with different sized centromeres and kinetochores.

Regulation of the meiotic divisions of mammalian oocytes and eggs.

Initiated by luteinizing hormone and finalized by the fertilizing sperm, the mammalian oocyte completes its two meiotic divisions. The first division occurs in the mature Graafian follicle during the hours preceding ovulation and culminates in an extreme asymmetric cell division and the segregation of the two pairs of homologous chromosomes. The newly created mature egg rearrests at metaphase of the second meiotic division prior to ovulation and only completes meiosis following a Ca2+ signal initiated by the sperm at gamete fusion. Here, we review the cellular events that govern the passage of the oocyte through meiosis I with a focus on the role of the spindle assembly checkpoint in regulating its timing. In meiosis II, we examine how the egg achieves its arrest and how the fertilization Ca2+ signal allows the initiation of embryo development.

Chromosome biorientation and APC activity remain uncoupled in oocytes with reduced volume.

The spindle assembly checkpoint (SAC) prevents chromosome missegregation by coupling anaphase onset with correct chromosome attachment and tension to microtubules. It does this by generating a diffusible signal from free kinetochores into the cytoplasm, inhibiting the anaphase-promoting complex (APC). The volume in which this signal remains effective is unknown. This raises the possibility that cell volume may be the reason the SAC is weak, and chromosome segregation error-prone, in mammalian oocytes. Here, by a process of serial bisection, we analyzed the influence of oocyte volume on the ability of the SAC to inhibit bivalent segregation in meiosis I. We were able to generate oocytes with cytoplasmic volumes reduced by 86% and observed changes in APC activity consistent with increased SAC control. However, bivalent biorientation remained uncoupled from APC activity, leading to error-prone chromosome segregation. We conclude that volume is one factor contributing to SAC weakness in oocytes. However, additional factors likely uncouple chromosome biorientation with APC activity.

DNA damage induces a kinetochore-based ATM/ATR-independent SAC arrest unique to the first meiotic division in mouse oocytes.

Mouse oocytes carrying DNA damage arrest in meiosis I, thereby preventing creation of embryos with deleterious mutations. The arrest is dependent on activation of the spindle assembly checkpoint, which results in anaphase-promoting complex (APC) inhibition. However, little is understood about how this checkpoint is engaged following DNA damage. Here, we find that within minutes of DNA damage checkpoint proteins are assembled at the kinetochore, not at damage sites along chromosome arms, such that the APC is fully inhibited within 30 min. Despite this robust response, there is no measurable loss in k-fibres, or tension across the bivalent. Through pharmacological inhibition we observed that the response is dependent on Mps1 kinase, aurora kinase and Haspin. Using oocyte-specific knockouts we find the response does not require the DNA damage response kinases ATM or ATR. Furthermore, checkpoint activation does not occur in response to DNA damage in fully mature eggs during meiosis II, despite the divisions being separated by just a few hours. Therefore, mouse oocytes have a unique ability to sense DNA damage rapidly by activating the checkpoint at their kinetochores.

Imaging Chromosome Separation in Mouse Oocytes by Responsive 3D Confocal Timelapse Microscopy.

Accurate chromosome segregation is necessary so that genetic material is equally shared among daughter cells. However, maturing mammalian oocytes are particularly prone to chromosome segregation errors, making them a valuable tool for identifying the causes of mis-segregation. Factors such as aging, cohesion loss, DNA damage, and the roles of a plethora of kinetochore and cell cycle-related proteins are involved. To study chromosome segregation in oocytes in a live setting is an imaging challenge that requires advanced techniques. Here we describe a method for examining chromosomes in live oocytes in detail as they undergo maturation. Our method is based on tracking the "center of brightness" of fluorescently labeled chromosomes. Here we describe how to set up our software and run experiments on a Leica TCS SP8 confocal microscope, but the method would be transferable to other microscopes with computer-aided microscopy.

The sensitivity of the DNA damage checkpoint prevents oocyte maturation in endometriosis.

Mouse oocytes respond to DNA damage by arresting in meiosis I through activity of the Spindle Assembly Checkpoint (SAC) and DNA Damage Response (DDR) pathways. It is currently not known if DNA damage is the primary trigger for arrest, or if the pathway is sensitive to levels of DNA damage experienced physiologically. Here, using follicular fluid from patients with the disease endometriosis, which affects 10% of women and is associated with reduced fertility, we find raised levels of Reactive Oxygen Species (ROS), which generate DNA damage and turn on the DDR-SAC pathway. Only follicular fluid from patients with endometriosis, and not controls, produced ROS and damaged DNA in the oocyte. This activated ATM kinase, leading to SAC mediated metaphase I arrest. Completion of meiosis I could be restored by ROS scavengers, showing this is the primary trigger for arrest and offering a novel clinical therapeutic treatment. This study establishes a clinical relevance to the DDR induced SAC in oocytes. It helps explain how oocytes respond to a highly prevalent human disease and the reduced fertility associated with endometriosis.

FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells.

Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4, and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers, and furthermore were not immortal but died after a few months. Our results suggest that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes and that adult de novo production is a rare or insignificant event.

DNA damage responses in mammalian oocytes.

DNA damage acquired during meiosis can lead to infertility and miscarriage. Hence, it should be important for an oocyte to be able to detect and respond to such events in order to make a healthy egg. Here, the strategies taken by oocytes during their stages of growth to respond to DNA damaging events are reviewed. In particular, recent evidence of a novel pathway in fully grown oocytes helps prevent the formation of mature eggs with DNA damage. It has been found that fully grown germinal vesicle stage oocytes that have been DNA damaged do not arrest at this point in meiosis, but instead undergo meiotic resumption and stall during the first meiotic division. The Spindle Assembly Checkpoint, which is a well-known mitotic pathway employed by somatic cells to monitor chromosome attachment to spindle microtubules, appears to be utilised by oocytes also to respond to DNA damage. As such maturing oocytes are arrested at metaphase I due to an active Spindle Assembly Checkpoint. This is surprising given this checkpoint has been previously studied in oocytes and considered to be weak and ineffectual because of its poor ability to be activated in response to microtubule attachment errors. Therefore, the involvement of the Spindle Assembly Checkpoint in DNA damage responses of mature oocytes during meiosis I uncovers a novel second function for this ubiquitous cellular checkpoint.

DNA damage induces a meiotic arrest in mouse oocytes mediated by the spindle assembly checkpoint.

Extensive damage to maternal DNA during meiosis causes infertility, birth defects and abortions. However, it is unknown if fully grown oocytes have a mechanism to prevent the creation of DNA-damaged embryos. Here we show that DNA damage activates a pathway involving the spindle assembly checkpoint (SAC) in response to chemically induced double strand breaks, UVB and ionizing radiation. DNA damage can occur either before or after nuclear envelope breakdown, and provides an effective block to anaphase-promoting complex activity, and consequently the formation of mature eggs. This contrasts with somatic cells, where DNA damage fails to affect mitotic progression. However, it uncovers a second function for the meiotic SAC, which in the context of detecting microtubule-kinetochore errors has hitherto been labelled as weak or ineffectual in mammalian oocytes. We propose that its essential role in the detection of DNA damage sheds new light on its biological purpose in mammalian female meiosis.

Reduced ability to recover from spindle disruption and loss of kinetochore spindle assembly checkpoint proteins in oocytes from aged mice.

Currently, maternal aging in women, based on mouse models, is thought to raise oocyte aneuploidy rates, because chromosome cohesion deteriorates during prophase arrest, and Sgo2, a protector of centromeric cohesion, is lost. Here we show that the most common mouse strain, C57Bl6/J, is resistant to maternal aging, showing little increase in aneuploidy or Sgo2 loss. Instead it demonstrates significant kinetochore-associated loss in the spindle assembly checkpoint protein Mad2 and phosphorylated Aurora C, which is involved in microtubule-kinetochore error correction. Their loss affects the fidelity of bivalent segregation but only when spindle organization is impaired during oocyte maturation. These findings have an impact clinically regarding the handling of human oocytes ex vivo during assisted reproductive techniques and suggest there is a genetic basis to aneuploidy susceptibility.

Non-canonical function of spindle assembly checkpoint proteins after APC activation reduces aneuploidy in mouse oocytes.

The spindle assembly checkpoint (SAC) prevents aneuploidy by coupling anaphase onset, through anaphase-promoting complex (APC) activation, with chromosome attachment to spindle microtubules. Here, we examine APC activity in oocytes, noted for their susceptibility to chromosome mis-segregation during the first meiotic division (MI). We find that MI oocytes only contain sub-maximal APC activity, measured through cyclin B1-GFP degradation, because inhibition of SAC proteins when the APC is normally fully active increases cyclin B1 degradation twofold and reduces the length of this division by 2 h. In addition, inhibiting the SAC component Mps1 only when the APC is already active increases aneuploidy rates in the resulting egg by up to 30%. We therefore establish that the activities of SAC proteins and the APC co-exist in oocytes, and such concurrence has a vital role in reducing aneuploidy rates by extending MI, probably by allowing time for numerous erroneous microtubule attachments to be corrected.

The APC/C activator FZR1 is essential for meiotic prophase I in mice.

Fizzy-related 1 (FZR1) is an activator of the Anaphase promoting complex/cyclosome (APC/C) and an important regulator of the mitotic cell division cycle. Using a germ-cell-specific conditional knockout model we examined its role in entry into meiosis and early meiotic events in both sexes. Loss of APC/C(FZR1) activity in the male germline led to both a mitotic and a meiotic testicular defect resulting in infertility due to the absence of mature spermatozoa. Spermatogonia in the prepubertal testes of such mice had abnormal proliferation and delayed entry into meiosis. Although early recombination events were initiated, male germ cells failed to progress beyond zygotene and underwent apoptosis. Loss of APC/C(FZR1) activity was associated with raised cyclin B1 levels, suggesting that CDK1 may trigger apoptosis. By contrast, female FZR1Δ mice were subfertile, with premature onset of ovarian failure by 5 months of age. Germ cell loss occurred embryonically in the ovary, around the time of the zygotene-pachytene transition, similar to that observed in males. In addition, the transition of primordial follicles into the growing follicle pool in the neonatal ovary was abnormal, such that the primordial follicles were prematurely depleted. We conclude that APC/C(FZR1) is an essential regulator of spermatogonial proliferation and early meiotic prophase I in both male and female germ cells and is therefore important in establishing the reproductive health of adult male and female mammals.

Premature dyad separation in meiosis II is the major segregation error with maternal age in mouse oocytes.

As women get older their oocytes become susceptible to chromosome mis-segregation. This generates aneuploid embryos, leading to increased infertility and birth defects. Here we examined the provenance of aneuploidy by tracking chromosomes and their kinetochores in oocytes from young and aged mice. Changes consistent with chromosome cohesion deterioration were found with age, including increased interkinetochore distance and loss of the centromeric protector of cohesion SGO2 in metaphase II arrested (metII) eggs, as well as a rise in the number of weakly attached bivalents in meiosis I (MI) and lagging chromosomes at anaphase I. However, there were no MI errors in congression or biorientation. Instead, premature separation of dyads in meiosis II was the major segregation defect in aged eggs and these were associated with very low levels of SGO2. These data show that although considerable cohesion loss occurs during MI, its consequences are observed during meiosis II, when centromeric cohesion is needed to maintain dyad integrity.

Molecular causes of aneuploidy in mammalian eggs.

Mammalian oocytes are particularly error prone in segregating their chromosomes during their two meiotic divisions. This results in the creation of an embryo that has inherited the wrong number of chromosomes: it is aneuploid. The incidence of aneuploidy rises significantly with maternal age and so there is much interest in understanding this association and the underlying causes of aneuploidy. The spindle assembly checkpoint, a surveillance mechanism that operates in all cells to prevent chromosome mis-segregation, and the cohesive ties that hold those chromosomes together, have thus both been the subject of intensive investigation in oocytes. It is possible that a lowered sensitivity of the spindle assembly checkpoint to certain types of chromosome attachment error may endow oocytes with an innate susceptibility to aneuploidy, which is made worse by an age-related loss in the factors that hold the chromosomes together.

Loss of GGN leads to pre-implantation embryonic lethality and compromised male meiotic DNA double strand break repair in the mouse.

The integrity of male germ cell genome is critical for the correct progression of spermatogenesis, successful fertilization, and proper development of the offspring. Several DNA repair pathways exist in male germ cells. However, unlike somatic cells, key components of such pathways remain largely unidentified. Gametogenetin (GGN) is a testis-enriched protein that has been shown to bind to the DNA repair protein FANCL via yeast-two-hybrid assays. This finding and its testis-enriched expression pattern raise the possibility that GGN plays a role in DNA repair during spermatogenesis. Herein we demonstrated that the largest isoform GGN1 interacted with components of DNA repair machinery in the mouse testis. In addition to FANCL, GGN1 interacted with the critical component of the Fanconi Anemia (FA) pathway FANCD2 and a downstream component of the BRCA pathway, BRCC36. To define the physiological function of GGN, we generated a Ggn null mouse line. A complete loss of GGN resulted in embryonic lethality at the very earliest period of pre-implantation development, with no viable blastocysts observed. This finding was consistent with the observation that Ggn mRNA was also expressed in lower levels in the oocyte and pre-implantation embryos. Moreover, pachytene spermatocytes of the Ggn heterozygous knockout mice showed an increased incidence of unrepaired DNA double strand breaks (DSBs). Together, our results suggest that GGN plays a role in male meiotic DSB repair and is absolutely required for the survival of pre-implantation embryos.

Melatonin prevents postovulatory oocyte aging in the mouse and extends the window for optimal fertilization in vitro.

The quality of metaphase II oocytes deteriorates rapidly following ovulation as the result of an aging process associated with impaired fertilizing potential, disrupted developmental competence, and increased likelihood of embryonic resorption. Because oxidative stress accelerates the onset of apoptosis in oocytes and influences their capacity for fertilization, this study aimed to characterize the significance of such stress in the postovulatory aging of mouse oocytes in vitro. We investigated the ability of the potent antioxidant melatonin to arrest the aging process when used to supplement oocyte culture medium. This study demonstrated that oxidative stress may occur in oocytes after as little as 8 h in culture and coincides with the appearance of early apoptotic markers such as phosphatidylserine externalization, followed 16 h later by caspase activation (P < 0.05) and morphological evidence of oocyte senescence. Importantly, supplementation of oocyte culture medium with 1 mM melatonin was able to significantly relieve the time-dependent appearance of oxidative stress in oocytes (P < 0.05) and, as a result, significantly delay the onset of apoptosis (P < 0.05). Furthermore, melatonin supplementation extended the optimal window for fertilization of oocytes aged for 8 and 16 h in vitro (P < 0.05) and significantly improved the quality of the resulting embryos (P < 0.01). We conclude that melatonin may be a useful tool in a clinical setting to prevent the time-dependent deterioration of oocyte quality following prolonged culture in vitro.

The control of meiotic maturation in mammalian oocytes.

Mammalian oocytes spend the majority of their lives in a dormant state, residing in primordial follicles. This arrest, most analogous to the G2 stage of the mitotic cell cycle division, is only broken in the hours preceding ovulation, when a hormonal rise induces meiotic resumption and entry into the first meiotic division. At a molecular level, this event is triggered by CDK1 activity, and here, we examine how CDK1 is suppressed during meiotic arrest and raised for oocyte maturation. We focus on signaling: intercellular signaling between the oocyte and the somatic cells of the follicle, and spatial signaling involving the anaphase-promoting complex (APC) within the oocyte. Meiotic arrest is achieved through APC(FZR1)-mediated cyclin B1 degradation. Once meiotic resumption resumes, CDK1 levels rise, but its activity eventually needs to be suppressed for completion of the first meiotic division. This is achieved by APC(CDC20), whose activity is critically regulated by the spindle assembly checkpoint, and which induces both a loss in CDK1 activity as well as the cohesive ties holding chromosomes together.