DNA double strand breaks but not interstrand crosslinks prevent progress through meiosis in fully grown mouse oocytes

There is some interest in how mammalian oocytes respond to different types of DNA damage because of the increasing expectation of fertility preservation in women undergoing chemotherapy. Double strand breaks (DSBs) induced by ionizing radiation and agents such as neocarzinostatin (NCS), and interstrand crosslinks (ICLs) induced by alkylating agents such as mitomycin C (MMC), are toxic DNA lesions that need to be repaired for cell survival. Here we examined the effects of NCS and MMC treatment on oocytes collected from antral follicles in mice, because potentially such oocytes are readily collected from ovaries and do not need to be in vitro grown to achieve meiotic competency. We found that oocytes were sensitive to NCS, such that this ionizing radiation mimetic blocked meiosis I and caused fragmented DNA. In contrast, MMC had no impact on the completion of either meiosis I or II, even at extremely high doses. However, oocytes treated with MMC did show ?-H2AX foci and following their in vitro maturation and parthenogenetic activation the development of the subsequent embryos was severely compromised. Addition of MMC to 1-cell embryos caused a similarly poor level of development, demonstrating oocytes have eventual sensitivity to this ICL-inducing agent but this does not occur during their meiotic division. In oocytes, the association of Fanconi Anemia protein, FANCD2, with sites of ICL lesions was not apparent until entry into the embryonic cell cycle. In conclusion, meiotic maturation of oocytes is sensitive to DSBs but not ICLs. The ability of oocytes to tolerate severe ICL damage and yet complete meiosis, means that this type of DNA lesion goes unrepaired in oocytes but impacts on subsequent embryo quality

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 surfacebound. 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 surfacebound 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

Reduced chromosome cohesion measured by interkinetochore distance is associated with aneuploidy even in oocytes from young mice

It is becoming clear that reduced chromosome cohesion is an important factor in the rise of maternal age-related aneuploidy. This reduction in cohesion has been observed both in human and mouse oocytes, and it can be measured directly by an increase with respect to maternal age in interkinetochore (iKT) distance between a sister chromatid pair. We have observed variations in iKT distance even in oocytes from young mice and wondered if such differences may predispose those oocytes displaying the greatest iKT distances to be becoming aneuploid. Therefore, we used two methods, one pharmacological (Aurora kinase inhibitor) and one genetic (Fzr1 knockout), to raise aneuploidy rates in oocytes from young mice (age, 1-3 mo) and to examine if those oocytes that were aneuploid had greater iKT distances. We observed that for both Aurora kinase inhibition and Fzr1 knockout, iKT distances were significantly greater in those oocytes that became aneuploid compared to those that remained euploid. Based on these results, we propose that individual oocytes undergo loss in chromosomal cohesion at different rates and that the greater this loss, the greater the risk for becoming aneuploid.Supported by an NHMRC project grant (569202) to K.T.J., S.M., and E.A.M. J.E.H. is supported by an Australian Research Council DECRA Fellowship. I.G.-H. and S.M. are supported by grants BFU2007-67464, BFU2008-01808, Consolider CSD2007-00015, and Junta de Castilla y León Grupo de Excelencia GR 265.Peer Reviewe

Repetitive sperm-induced Ca²⁺ transients in mouse oocytes are cell cycle dependent

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca²⁺ transients triggered by the sperm at fertilization. These Ca²⁺ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca²⁺ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca²⁺ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca²⁺ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca²⁺ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4–5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca²⁺ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca²⁺ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca²⁺ transients in oocytes is modulated in a cell cycle-dependent manner

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

Comparable impaired embryo development following ICL induction at either GV or 1-embryo stage.

<p>(A) Schematic showing the experimental design. MMC was either added after milrinone washout or following parthenogenetic activation with Sr²⁺. Blastocyst rates were assessed at 120 hours. (B) Blastocyst rates from those oocytes treated as in (A) with the MMC concentration stated. In parentheses, number of eggs examined from 3 pooled replicates; different letters denote significantly difference (p&lt;0.05; Fisher's exact test).</p

Induction of γH2AX foci in GV oocytes following MMC addition.

<p>(A) γH2AX immunostaining shown in GV oocytes treated with or without 20 µg/ml MMC. Scale bar, 10 μm. (B) γH2AX foci (mean ± standard deviation) counted per oocyte from images in (A). In parentheses, total number of oocytes examined, pooled from 2 independent experiments. *p&lt;0.0001 (t-test).</p

FANCD2 association with spindle microtubules and poles but not nuclear foci in oocytes.

<p>(A) FANCD2 immunostaining of the nuclei in GV oocytes with or without previous MMC addition at the concentrations stated. No FANCD2 foci were observed. (B, C) FANCD2 immunostaining of either meiotic spindles in meiosis I (B) or at metaphase II arrest (C). FANCD2 was present on spindle microtubules and poles, and its localisation was unaffected by MMC addition. Images are representative of at least 10 oocytes from at least two replicates. Scale bar, 10 μm.</p

Neocarzinostatin, an ionizing radiation mimetic, fragments DNA and blocks meiosis in oocytes.

<p>(A) First polar body (PB1) extrusion rates following maturation of oocytes treated with NCS. (B) Chromatin in oocytes from (A); (i) depicting a typical metaphase II egg that was not treated with NCS, (ii, iii) meiotic arrest caused by NCS with varying levels of fragmentation. White dotted line represents egg and polar body outlines. (C) Percentage of oocytes treated with or without NCS that display fragmented DNA. (D) Nuclear staining of γH2AX in GV oocytes with or without NCS treatment; asterisks is at center of nucleolus. (E) Percentage of γH2AX positive oocytes treated with or without NCS. (A, C, E) Pooled data from 2 replicates. In parentheses, total number of oocytes examined. (A) Different letters denote significant difference, p&lt;0.05 (Fisher's exact test). (B, D) Scale bar, 10 µm.</p