The DNA damage response in mammalian oocytes

Abstract

DNA damage is one of the most common insults that challenge all cells. To cope, an elaborate molecular and cellular response has evolved to sense, respond to and correct the damage. This allows the maintenance of DNA fidelity essential for normal cell viability and the prevention of genomic instability that can lead to tumor formation. In the context of oocytes, the impact of DNA damage is not one of tumor formation but of the maintenance of fertility. Mammalian oocytes are particularly vulnerable to DNA damage because physiologically they may lie dormant in the ovary for many years (>40 in humans) until they receive the stimulus to grow and acquire the competence to become fertilized. The implication of this is that in some organisms, such as humans, oocytes face the danger of cumulative genetic damage for decades. Thus, the ability to detect and repair DNA damage is essential to maintain the supply of oocytes necessary for reproduction. Therefore, failure to confront DNA damage in oocytes could cause serious anomalies in the embryo that may be propagated in the form of mutations to the next generation allowing the appearance of hereditary disease. Despite the potential impact of DNA damage on reproductive capacity and genetic fidelity of embryos, the mechanisms available to the oocyte for monitoring and repairing such insults have remained largely unexplored until recently. Here, we review the different aspects of the response to DNA damage in mammalian oocytes. Specifically, we address the oocyte DNA damage response from embryonic life to adulthood and throughout oocyte development.

Keywords: DNA damage checkpoint; DNA damage response; apoptosis; meiosis; meiotic recombination; oocytes; p63; prophase arrest.

FIGURE 1

Schematic representation of the major steps of the G1 DNA damage checkpoint. Double strand breaks (DSB) and single strand breaks (SSB) cause the activation of the master DNA damage checkpoint kinases ATM and ATR, respectively. ATM phosphorylates and activates the downstream effector checkpoint kinases Chk1 and Chk2, while ATR activates Chk1. ATM/ATR and the checkpoint kinases activate the transcription factor p53. p53 drives the transcription of the cyclin-dependent kinase (CDK) inhibitor p21. p21 binds and inhibits CDKs responsible for progression into S-phase, such as Cyclin E-CDK2. As a result, the cell cycle arrests at G1. When the DNA damage cannot be repaired, p53 drives the cell to apoptosis through the transcription of pro-apoptotic genes, such as PAX, PUMA, and NOXA. It must be noted that this figure is an oversimplified representation of the pathways enabled in response to DNA damage at G1 phase. activating phosphate.

FIGURE 2

Schematic representation of the major steps of the G2 DNA damage checkpoint. At S/G2 phase, DSBs and SSBs activate ATM and ATR, respectively. As a consequence, checkpoint kinases Chk1 and Chk2 become activated. Chk1 and Chk2 can directly phosphorylate and activate Wee1 or Myt1 kinases. Wee1/Myt1 impose inhibitory phosphorylations on the M-phase kinase Cyclin B-CDK1 in order to block M-phase entry. At the same time, the checkpoint kinases directly phosphorylate and inhibit Cdc25 phosphatases. Unlike the inhibitory phosphorylations of Cdc25B and Cdc25C, checkpoint kinase-dependent phosphorylation of Cdc25A allows its recognition by the SCF/βTrCP ligase. Subsequent ubiquitination of Cdc25A renders the phosphatase a substrate for the proteasome leading to its degradation. As a result of their inhibition, the Cdc25 phosphatases cannot remove the inhibitory phosphate from CDK1. Consequently, the cell cycle arrests at G2 due to inhibition of CDK1 activation. The activating CDK1 phosphorylation is introduced by CDK-activating kinases but is masked by the Wee1/Myt1 inhibitory phosphorylations. activating phosphate. inhibiting phosphate.

FIGURE 3

Regulation of prophase arrest and resumption of meiosis. (A) Regulation of prophase arrest. During the arrested state, a signaling pathway that is established in response to the interactions of the oocyte with its neighboring granulosa cells leads to the accumulation of cAMP in the oocyte. cAMP causes the phosphorylation of protein kinase A (PKA). Similarly to Chk1 and Chk2 during the G2 DNA damage checkpoint, PKA in prophase-arrested oocytes phosphorylates and activates Wee1 kinase and specifically the Wee1 isoform Wee1B. Furthermore, PKA phosphorylates and inhibits Cdc25B phosphatase. As a result, Cyclin B-CDK1 remains inactive and the oocyte arrested in meiotic prophase. (B) Resumption of meiosis. During the estrus cycle, the surge of the luteinizing hormone (LH) drives a signaling cascade that results in the decline of cAMP levels in the oocyte. This leads to PKA inactivation, ending the PKA-dependent phosphorylation of Wee1B and Cdc25B. Just as in M-phase entry in somatic cells, Wee1B becomes inactive and Cdc25B is activated in order to remove the inhibitory phosphates from CDK1. In response to Cyclin B-CDK1 activation, the oocyte enters the first meiotic M-phase. activating phosphate. inhibiting phosphate.

FIGURE 4

Mammalian oocyte DNA damage checkpoints in relation to follicular and oocyte development. In most mammals, only a few hundred oocytes reach the competence to become fertilized. At the beginning of oogenesis, mitotically dividing oogonia proliferate to form a population of a few million. Most become destroyed through apoptosis while all the remaining enter meiosis before birth. These oocytes become surrounded by a single layer of epithelial cells forming primordial follicles. Following birth, ovarian follicles from this primordial pool mature spontaneously into primary and secondary follicles. During this stage of follicular maturation the oocyte grows in size and becomes surrounded by more layers of proliferating follicular cells which are in turn surrounded by layers of theca cells. However, these pre-antral follicles never reach full maturity and soon become atretic and deteriorate. At puberty and following the rise in the levels of the follicle stimulating hormone (FSH), during every estrus cycle, a small number of follicles mature beyond the pre-antral stage forming an antrum (antral follicle). From these follicles only a few reach the pre-ovulatory stage (Rodrigues et al., 2008). In the mouse, sustained unrepaired recombination-induced DSBs trigger oocyte apoptosis following the pachytene stage of meiotic prophase during embryonic life. These oocytes rarely survive to form primordial follicles around the time of birth. Genotoxic stress activates TAp63-dependent apoptosis at the diplotene stage of prophase. TAp63-induced apoptosis affects oocytes from the primordial stage of follicle development up to the pre-antral stage (primary and secondary follicles). Apoptosis does not appear in later stages of oocyte development. From the large pre-antral to the pre-ovulatory follicular stage, the oocyte remains in prophase arrest which may allow any inflicted DNA damage to be repaired. The fully grown oocyte which possesses the competence to resume meiosis and enter the first meiotic M-phase (MI) cannot establish cell cycle arrest checkpoints in response to DNA damage. It is possible that such checkpoints may be activated during meiotic M-phase. E, embryonic day; P, postnatal day; MII, meiotic M-phase II.