Reprint request to: Dr Toshifumi Takahashi, Department of Obstetrics and Gynecology, Yamagata University Faculty of Medicine, Yamagata 990-9585, Japan. Email: email@example.com
Oocyte quality is a key factor in determining embryo development; however, we have a poor understanding of what constitutes oocyte quality or the mechanisms governing it. Postovulatory aging of oocytes that have not been fertilized for a prolonged time after ovulation is known to significantly impair oocyte quality and subsequent embryo development after fertilization. Embryos derived from postovulatory-aged oocytes are prone to undergo apoptosis due to the decreased Bcl-2 expression. Postovulatory aging of oocytes changes the patterns of Ca2+ oscillations at fertilization as a result of impaired Ca2+ regulation in the endoplasmic reticulum. Moreover, postovulatory aging of oocytes impairs mitochondrial adenosine triphosphate production as a result of increasing oxidative stresses. Oxidative stresses also affect intracellular Ca2+ regulation and impair embryo development after fertilization. Collectively, the mechanism of postovulatory oocyte aging might be involved in reactive oxygen species-induced mitochondrial injury followed by abnormal intracellular Ca2+ regulation in the endoplasmic reticulum.
It is well known that decreased fertility with increasing female age has been determined on the basis of the data from demographic and epidemiological studies. Although the number of treatment cycles in assisted reproductive technology has steadily increased, there are no treatments available for infertility patients whose infertility arises from increasing maternal age; the oocyte donation program is an exception. Therefore, the age-associated decline in female fertility is largely attributable to the decrease in oocyte quality due to ovarian aging.[4, 5] The mechanism of oocyte aging might be a chronic process that damages the oocytes and/or ovarian follicle cells, such as the thecal and granulosa cells. However, the precise mechanism of oocyte aging due to ovarian aging remains unknown. There are several problems associated with the study of oocyte aging with respect to ovarian aging: it takes more than 1 year to obtain experimental animals that are old enough, and there are only a few available oocytes. Moreover, there is no appropriate animal model analogous to the one that is used in ovarian aging studies.
In contrast to ovarian aging, there is a concept of postovulatory oocyte aging. In mammals, ovulated oocytes are arrested at the metaphase stage of the second meiotic division until fertilization. The optimal period for oocyte fertilization is less than 10 h.[7, 8] Fertilization within this narrow window of developmental opportunity results in normal embryo development. Numerous investigators have reported that postovulatory aging of oocytes that are not fertilized for a prolonged time after ovulation significantly impairs oocyte quality and subsequent embryo development after fertilization.[9, 10] It is well known that postovulatory oocyte aging has a similar phenotype of reproductive failure as oocyte aging caused by ovarian aging. The aim of the present paper was to review the molecular mechanisms of time-dependent decreases in oocyte quality in postovulatory-aged oocytes.
Effects of Postovulatory Oocyte Aging on Female Reproduction
Postovulatory oocyte aging is classified into two types: in vivo- and in vitro-postovulatory oocyte aging. If fertilization does not occur during the optimal period after ovulation, an unfertilized oocyte that remains in the oviduct (in vivo-postovulatory oocyte aging) or in vitro culture (in vitro-postovulatory oocyte aging) goes through a time-dependent aging process. We and numerous other investigators have reported that in vivo- and in vitro-aged oocytes frequently show lower fertilization and blastocyst formation rates (Fig. 1a), polyspermy, digyny, chromosomal anomalies, and abnormal embryo development.[9, 11-13] These abnormalities in early embryo development result in decreased litter sizes in animals, lower pregnancy rates, and an increase in the number of spontaneous miscarriages in humans.[12, 14] In addition to abnormalities in early embryo development, postovulatory aging of oocytes is associated with retarded sensorimotor integration during pre-weaning development, high spontaneous motor activity, and high emotionality in adulthood in mice. A recent study has demonstrated that postovulatory aging affects epigenetic changes in mouse oocytes.
Cellular and Molecular Changes in Postovulatory-aged Oocytes
It is well established that in vivo- and in vitro-postovulatory aging of oocytes is associated with changes in various cellular and molecular pathways involved in intracellular signaling.[9, 10] In vivo- and in vitro-postovulatory aging of oocytes share many common properties.
Morphological and organelle changes
Postovulatory oocyte aging affects numerous morphological and organelle changes: changes in the structure of the oolemma, zona pellucida, cortical granules, mitochondria, cytoskeleton, meiotic spindle, and chromosome alignment.[9, 10] Thick and thin microfilament domains are lined along the oocyte cortex in fresh oocytes. Aged oocytes have a disrupted thick microfilament domain beneath the oolemma.[17, 18] Meiotic spindle assembly is an important cellular structure for accurate chromosomal distribution. There are many reports indicating that postovulatory aging of oocytes results in disruption and loss of meiotic spindle assembly in experimental animals and humans.[20, 21] In mouse studies, although the meiotic spindle is barrel-shaped and microtubules are clearly detected in fresh oocytes, microtubules gradually separate from the spindle in aged oocytes. Furthermore, in vitro-aged human oocytes show aberrant expression of γ-tubulin, which indicates disruption of the centrosome structure at the meiotic poles.[19, 22] These changes in aged oocytes lead to premature chromosomal separation, which is strongly associated with aneuploidy.[23, 24]
Biochemical and molecular changes
Postovulatory aging of oocytes leads to various biochemical and molecular changes in mammalian oocytes.[9, 10] The intracytoplasmic level of glutathione (GSH), which has a major role in protecting oocytes from damage by reactive oxygen species (ROS), decreases in aged oocytes. The level of lipid peroxidation, which is an indicator of the degree of oxidative stress, increases in in vivo-aged oocytes. Moreover, the amount of ROS increases in aged oocytes, with increasing in vitro culture time. The intracytoplasmic level of adenosine triphosphate (ATP) decreases in aged oocytes.[27, 28] The expression of Bcl-2, an anti-apoptotic protein, decreases in in vivo- and in vitro-aged oocytes.[13, 29, 30] Postovulatory aging of oocytes impairs intracellular Ca2+ regulation, which is one of the most important factors for early events after fertilization and subsequent embryo development.[31-34] Increased oxidative stress and abnormal intracellular Ca2+ regulation in aged oocytes are discussed later in the paper.
Mechanism of Poor Embryo Development in Postovulatory-aged Oocytes
As described above, morphological and biochemical changes in postovulatory-aged oocytes are translated into detrimental early and late phases of embryo development, such as lower fertilization rates, polyspermy, dingy, chromosomal anomalies, abnormal embryo development, and post-implantation mortality. Although the mechanism of poor embryo development by postovulatory aging of oocytes remains unknown, there are data that have contributed to the elucidation of the mechanism.
An apoptotic pathway is involved in poor embryo development in postovulatory-aged oocytes
Unfertilized aged oocytes undergo spontaneous cytoplasmic fragmentation. In addition, embryos derived from aged oocytes exhibit fragmentation after fertilization (Fig. 1a). Since these fragmented oocytes and embryos show terminal dUTP nick-end labeling (TUNEL)-positive staining (Fig. 1b),[13, 35, 36] these data suggest that apoptotic pathways are activated during the postovulatory aging period. Mammalian oocytes express several caspases as well as anti- and pro-apoptotic members of the BCL2 gene family.[37, 38] Pro-apoptotic molecules, such as Bax, induce the release of cytochrome c, which activates caspases, while anti-apoptotic molecules, such as Bcl2, prevent this release. In mouse and pig oocytes, the expression of Bcl-2 decreases, and the percentage of TUNEL-positive unfertilized oocytes increases with in vitro aging.[29, 30] In addition, we and Gordo et al. have reported that the expression of Bcl-2 decreases, whereas that of Bax remains unchanged in the oocytes aged in vitro.[13, 40] We have also confirmed that the expression of Bcl-2 decreases in in vivo-aged mouse oocytes (Takahashi and Kurachi, unpublished data). These results suggest that postovulatory aged oocytes are prone to undergo apoptosis due to decreased Bcl-2 expression.
Postovulatory oocyte aging affects Ca2+ homeostasis of the endoplasmic reticulum
In mammalian oocytes undergoing fertilization, sperm induces drastic changes in the intracellular Ca2+ concentration ([Ca2+]i), which consist of a single long-lasting rise in [Ca2+]i, followed by short repetitive changes in [Ca2+]i that last for several hours. These temporal changes in [Ca2+]i are termed ‘Ca2+ oscillations.’ The increase in [Ca2+]i plays important roles in fertilization, cortical granule exocytosis, resumption of meiosis, pronucleus formation, and subsequent embryo development.[42-45] In addition, the characteristics of Ca2+ oscillations, such as amplitude and frequency, affect early and post-implantation embryo development. We have demonstrated that in vivo- and in vitro-postovulatory aging alter the patterns of Ca2+ oscillations at fertilization in mouse oocytes.[13, 32] The frequency of Ca2+ oscillations at fertilization in aged oocytes is higher than that in freshly ovulated oocytes, while the amplitude of individual Ca2+ oscillations is lower in the former than in the latter (Fig. 2).[13, 32] Jones and Whittingham also reported that both the amplitude and rate of rise of individual Ca2+ oscillations at fertilization decrease in in vivo-aged mouse oocytes. We and other groups have reported that Ca2+ release from the inositol 1,4,5-triphosphate (InsP3)-sensitive Ca2+ stores decreases in in vivo-aged oocytes, unlike that observed in fresh oocytes.[45, 46] Furthermore, we have reported that the decrease in Ca2+ reuptake by both Ca2+-ATPases and Ca2+ stores in the endoplasmic reticulum (ER) in in vivo-aged oocytes is more than that in fresh oocytes.[32, 33] We have reported that Ca2+ stores in the ER in in vitro-aged oocytes also decrease. Taken together, these results indicate an impaired Ca2+ homeostasis in postovulatory aged oocytes.
Impaired Ca2+ homeostasis leads to poor embryo development
The abnormal intracellular Ca2+ signaling in aged oocytes leads to the apoptosis of oocytes. Gordo et al. reported that injection of Ca2+ oscillators, such as sperm cytosolic factor and adenophostin A, a potent agonist of the InsP3 receptor, into in vitro-aged oocytes causes increased fragmentation and caspase activation in oocytes. They also reported that the injection of Ca2+ oscillators into in vitro-aged oocytes induces abnormal Ca2+ oscillations with low amplitude and abrupt cessation. Moreover, we and another group have reported that decrease in the ER Ca2+ stores of fresh oocytes by thapsigargin, which is a specific inhibitor of smooth endoplasmic reticulum Ca2+-ATPases (SERCA), results in abnormal Ca2+ oscillations with low amplitude and high frequency at fertilization, unlike that observed in the case of vehicle-treated fresh oocytes.[13, 34, 49] Moreover, we have reported that thapsigargin treatment of fresh oocytes showed lower fertilization rate, lower blastocyst formation rate, and higher rate of fragmented embryo after in vitro fertilization compared to those observed in vehicle-treated fresh oocytes. These results suggest that abnormal Ca2+ handling in the postovulatory-aged oocytes might be related to poor embryo development after fertilization.
Does increased oxidative stress impair mitochondrial function and Ca2+ homeostasis in postovulatory-aged oocytes?
What mechanisms are involved in the impairment of Ca2+ homeostasis in postovulatory-aged oocytes? As mentioned above, we have reported that the activity of SERCA decreases in in vivo-aged mouse oocytes, and the ER Ca2+ stores decreases in both in vivo- and in vitro-aged mouse oocytes.[13, 33] The Ca2+ stores are maintained within the ER by the replenishment of Ca2+ from the cytosol through the activity of SERCA. The activity of SERCA is highly dependent on the availability of intracellular ATP. In fact, both in vivo- and in vitro-postovulatory-aged oocytes have decreased ATP content in unfertilized and fertilized mouse oocytes.[27, 28] Mitochondrial ATP production is a prerequisite for Ca2+ oscillations at fertilization and Ca2+ homeostasis in oocytes. Thus, impairment of fertilization-triggered mitochondrial ATP production is possibly linked to the impairment of Ca2+ homeostasis and abnormal patterns of Ca2+ oscillations at fertilization in aged oocytes.
A secondary question addresses the mechanisms responsible for impairment of mitochondrial function in aged oocytes. Mitochondria are organelles that produce ATP thorough oxidative phosphorylation to supply energy for various cell functions. Mitochondrial dysfunction has been linked to pathological conditions, including various reproductive failures. ROS, such as superoxide anion radical, hydrogen peroxide (H2O2), and hydroxyl radical, are produced endogenously by the proton electrochemical gradient during mitochondrial respiration. Because the mitochondria are a major source of ROS, they need continuous protection from free radical attack; the protection is by the ROS scavenger systems. Tarin et al. proposed a mechanism based on ‘the oxygen radical mitochondrial injury hypothesis of aging’ to explain the effects of postovulatory aging on the impairment of early and embryonic and fetal development. In this mechanism, the ROS harm the mitochondrial DNA, proteins, and lipids. We have reported that the magnitude of lipid membrane peroxidation in in vivo-aged oocytes is higher than that in fresh oocytes. We have also reported that the levels of ROS in in vitro-aged oocytes are higher than that in fresh oocytes (Fig. 3). Boerjan and de Boer reported that the amount of GSH, which is an ROS scavenger, decreases in in vivo-aged mouse oocytes. These results suggest that aged oocytes are prone to oxidative stresses resulting from decreased levels of ROS scavengers. Furthermore, we have reported that the exposure of fresh oocytes to 100-μM H2O2 resulted in abnormal patterns of Ca2+ oscillations with low amplitude and high frequency, which are similar to the patterns observed in postovulatory aged oocytes (Fig. 4). We have also reported that the H2O2 pretreatment of fresh oocytes results in poor embryo development after fertilization. In somatic cells, ROS are important mediators of intracellular signaling for numerous cell functions, including Ca2+ homeostasis through the modulation of SERCA and InsP3 receptor functions. These results suggest that the increase in ROS production in aged oocytes might directly affect Ca2+ homeostasis and/or impair mitochondrial function followed by ATP depletion.
Here, we have reviewed the molecular mechanism of poor embryo development in postovulatory aged oocytes (Fig. 5). ROS-induced mitochondrial injury followed by abnormal intracellular Ca2+ regulation of the ER may be involved in the mechanism of postovulatory aging of oocytes. According to this hypothesis, the antioxidant treatment in vivo and in vitro might prevent oocyte damage via postovulatory aging. However, it remains uncertain whether the mechanism of oocyte aging caused by ovarian aging is similar to that of postovulatory oocyte aging. Based on the evidence obtained from the study of postovulatory aging of oocytes, future research will be needed to address the decrease in oocyte quality due to ovarian aging.
This study was supported by Grants-in-aid for General Science Research no. 22591815 to Toshifumi Takahashi and 15790875, 17791102, and 20591905 to Hideki Igarashi.