Mechanisms of Oocyte Maturation and Related Epigenetic Regulation

1Uncovering the signals involved in controlling the resumption of oocyte meiosis is a major issue in female reproductive biology. The meiosis initiation and resumption of oocytes is different from sperm in at least three aspects. Female germ cells enter and undergo the first meiotic progression during embryonic development, and arrest at the diplotene stage of prophase I before birth. And, some of the arrested oocytes within fully grown follicles will resume meiosis after puberty in response to luteinizing hormones (LHs) during each estrous (animal) or menstrual cycle (human). Last, the cell division of oocytes is known as asymmetric cytokinesis. Interestingly, whenever fully grown oocytes are released from follicles and cultured in appropriate medium in vitro, spontaneous resumption happens as well. Oocyte meiotic maturation is a complicated and vital process used to attain full competence required for the oocyte as well as early embryonic development. An oocyte arrested at meiotic prophase I contains a large nucleus covered by a nuclear envelope, which is known as the germinal vesicle (GV). With the arrival of LH surge, serial processes related to oocyte nuclear maturation, such as chromatin condensation and germinal vesicle breakdown (GVBD), occur in oocytes of fully grown follicles. After GVBD, oocytes enter the metaphase I (MI) stage. Later, after extrusion of the first polar body (PB1) containing a small portion of cytoplasm, an oocyte containing one set of chromosomes completes meiosis I. Very soon after that, the second meiosis starts and the oocyte (mature egg) arrests at metaphase II (MII) until fertilization. Actually, the oocyte accomplishes its meiosis progress only when fertilization happens.

2In humans and animals, multiple factors including epigenetic molecules and different signaling pathways have been identified and proven to be pivotal for meiotic maturation. They not only regulate oocytes maturation, but also coordinate with each other to ensure good oocyte quality. This article aims to review the events and development around the quality control of mammalian oocyte meiotic maturation in nuclear and cytoplasm aspects, of which, the underlying molecular mechanisms are discussed to provide detailed information for better understanding of meiosis.

3Before an oocyte is enclosed by ovarian granulosa cells to form primordial follicles, meiosis has been initiated and the cell has arrested at the diplotene stage of prophase I. When females are sexually mature, a small portion of primordial follicles will be activated and start to grow gradually. Previous studies have indicated that molecules such as cyclic adenosine monophosphate (cAMP) within growing oocytes and the natriuretic peptide precursor type C (NPPC)/natriuretic peptide receptor 2 (NPR2) system in granulosa cells play essential roles in maintaining oocyte meiotic arrest during the long developmental journey. Later, oocytes in fully grown follicles in response to gonadotropins stimulation possess the capability to resume meiosis and ovulate in vivo.

4In mammals, meiotic arrest is regulated by a high level of cAMP in the oocyte. When oocytes are isolated from the antral follicles, the cAMP levels within the oocytes decrease and meiosis resumes spontaneously. On the contrary, when they are cultured with the cAMP analog dibutyryl cAMP (dbcAMP) or cAMP phosphodiesterase (PDE) inhibitors such as isobutyl methyl xanthine (IBMX) and milrinone, the spontaneous meiotic maturation of mouse oocytes is prevented. Therefore, a constantly higher level of cAMP becomes the priority for oocytes to sustain meiosis at the GV stage.

5cAMP in oocytes plays a central role in the regulation of meiosis arrest. Oocytes possess all of the necessary proteins including adenylyl cyclase (AC), Gs protein, and G protein-coupled receptor 3 (GPR3) for producing cAMP themselves. AC is responsible for specifically catalyzes ATP to form cAMP, and Gs protein, which stimulates AC3 activity in oocytes. Mice oocytes lacking AC3 expression fail to maintain meiosis arrest. Similarly, blocking Gs function causes spontaneous resumption of meiosis in follicle-enclosed mouse oocytes. GPR3, which is located in the oocyte plasma membrane, is necessary to stimulate Gs activity and elevate the level of cAMP. This is approved by the fact that oocytes undergo spontaneous meiotic resumption at an early antral stage in GPR3 KO mice and the phenomenon can be reversed by injection of GPR3 mRNA into the oocyte. The studies in pig oocytes are consistent with those in mice. Although GPR3 is expressed in the human oocyte, it contributes nothing to premature ovarian failure, which is unlike the phenotype of GPR3 KO mice. While GPR and Gs are functional in generating intrinsic cAMP, PDE in mice oocytes is responsible for the degradation of cAMP. In a PDE3 knockout model, oocytes are permanently arrested at the GV stage and female mice are infertile. Specifically, inhibition of PDE3 elevates cAMP level and prevents oocyte spontaneous maturation simultaneously in cultured cumulus-oocyte-complexes (COCs) or denuded oocytes (DOs). Simultaneously knockout of GPR3 and PDE3A result in oocyte maturation.

6Meiosis inhibition is a process in which oocytes coordinate with granulosa cells to sustain a high level of cAMP. Cumulative data have proven that intrinsic cAMP produced by oocyte alone is not sufficient to maintain meiotic arrest. Instead, a sustained high level of cAMP in the oocyte depends on cGMP, which is produced in the surrounding granulosa cells, possibly by suppressing PDE3A activity. Generally, cGMP is produced from GTP by guanylyl cyclases in mural granulosa cells (MGCs) and cumulus granulosa cells (CGCs) and is transported to oocytes.

7cGMP production in CGCs relies on the coordination of MGCs-secreted NPPC conjugating with its receptor, guanylyl cyclase NPR2 which is found on the membrane of CGCs. NPPC and NPR2 are both highly expressed in follicular granulosa cells. NPPC inhibits the spontaneous GVBD in COCs, but not in DOs in vitro. Besides, NPR2 mutant mice are infertile due to premature resumption of meiosis because of the shortage of cGMP production in CGCs, which results in oocyte fragmentation and poor embryo development. Consistently, applying NPPC in cultured COCs contributes to preventing spontaneous oocyte maturation by increasing the cGMP levels in the CGCs. Together, these results suggest that cGMP produced in granulosa cells play a vital role in keeping the cAMP level high in the oocyte, and that maintaining oocyte meiotic arrest requires coordination between granulosa cells and an oocyte within a follicle.

8How is the NPPC/NPR2 signaling pathway regulated in granulosa cells? One of the important actions of follicle stimulation hormones (FSHs) on MGCs and CGCs of antral follicles is to sustain high levels of NPPC/NPR2 in humans, rodents, and pig. Pregnant mare serum gonadotrophin (PMSG) that possesses primarily FSH activity induces the expression of NPPC and NPR2 mRNA in the ovary. This is further approved by the fact that estrogen-promoted NPPC expression in granulosa cells can be enhanced by interaction with FSH. However, the oocytes within antral follicles did not show precocious resumption of meiosis after deletion of the estrogen receptor or Cyp19α1 (aromatase), possibly implying that there are other pathways mediating NPPC/NPR2 action. In line with this speculation, we have proved that the expression of the NPPC/NPR2 system in ovarian granulosa cells is up regulated by sex hormones, such as androgen and estrogen through respective hormone receptors (AR and ER) in physiological conditions, in polycystic ovary syndrome (PCOS) in mice ovaries, and in in vitro cultured granulosa cell lines. Therefore, NPPC/NPR2 as a specific pathway potentially helps to explain the mechanism of the ovulatory disruption in PCOS. In addition, proved that transforming growth factor β (TGF-β) could regulate the expression of NPPC in MGCs and oocyte maturation. In the presence of FSH, TGF-β further increased NPPC levels and inhibited the oocyte meiotic resumption of COCs. Interestingly, supplementary natriuretic peptide precursor type B (NPPB) and NPPC are effective at improving the developmental competence of oocytes recovered from small-sized antral follicles of porcine in vitro.

9Importantly, one of the important roles of LH surge is to downregulate the level of the NPPC/NPR2 system in MGCs and CGCs as well. The levels of NPPC/NPR2, as well as the activity of NPR2, are either completely decreased or inhibited in mouse and human ovaries after the activation of LH receptors, which occurs sufficiently earlier than GVBD. The underlined mechanism could be that LH significantly decreases AR and ER levels, and thus decreases NPPC/NPR2 levels and induces oocyte maturation. By suppressing the NPPC/NPR2 system, LH reduces cGMP level in CGCs as well as oocytes rapidly. Besides, the reduced cGMP level in oocytes releases PDE3A from the inhibitory state. As a result, cAMP is degraded and the maturation promoting factor (MPF) is activated, which induces the resumption of meiosis. However, it remains unclear how LH and FSH specifically regulate the expression of AR, ER, and TGF-β. The regulations of granulosa cells cooperate with oocytes to maintain oocyte meiotic arrest in mice, which are summarized in Figure 1.

10Fully grown oocytes in early antral and preovulatory follicles have the capability to resume meiosis before LH surge. According to the hypothalamus-pituitary-ovary axis feedback theory, an LH surge in response to a peak estrogen surge initiates oocytes meiosis in vivo through positive feedback regulation. LH surge produces rapid changes in MGCs via intracellular pathways and extracellular paracrine loops. In brief, LH binds to the LH receptor (LHR) located in the membrane of theca cells and the MGCs of a follicle. As a result, the activated LHR induces serial affairs in follicular granulosa cells and oocytes. It reduces the cAMP level within the oocyte through downregulating the NPPC/NPR2 system and shutting down gap junctions between the oocyte and CGCs. Also, it upregulates the activity of the epidermal-like growth factors (EGF) network in MGCs/CGCs. In the oocyte, reduced cAMP levels activates the activity of MPF, which in turn phosphorylates proteins including APC and initiates GVBD and chromosome segregation.

11The mechanism of how high levels of cAMP are necessary to prevent meiotic maturation in oocytes is more or less fully understood. It is clear that cAMP exerts its role by activating protein kinase A (PKA). PKA balances the activities of WEE1B/MYT1 kinase and CDC25 phosphatase, and thus regulates the activity of cyclin-dependent kinase 1 (CDK1). Briefly, the CDK1 and cyclin B complex, namely MPF, is essential for oocytes meiotic maturation. The ability of CDK1 to phosphorylate target proteins at specific serine and threonine residues depends on its activity and binding with the cyclin B. It has been found that activated CDK1 triggers CXXC-finger protein 1 (CXXC1, also known as CFP1) phosphorylation and degradation following meiotic resumption. The degradation of CFP1 ensures the absence of the SET domain containing 1 (SETD1)-CXXC1 complex from chromatin, thereby facilitating chromosome condensation during oocyte maturation. Besides, CFP1 coordinates histone H3 lysine-4 trimethylation and meiotic cell cycle progression in mouse oocytes. Therefore, one of the key points to initiate oocyte meiosis depends on when to activate CDK1. In arrested oocytes, a sustained high level of cAMP activates PKA, which in turn activates WEE1. WEE1 inactivates while CDC25 activates CDK1 through phosphorylates or dephosphorylates the Thr14 and Tyr15 residues of CDK1, respectively. Thus, the activity of MPF is indirectly controlled by the level of cAMP. Interestingly, epigenetic molecules, such as histone lysine demethylases KDM1A (also known as LSD1), are involved in regulating the expression of CDC25B to maintain meiotic arrest. Conditional deletion of LSD1 in growing oocytes results in precocious resumption of meiosis and spindle and chromosomal abnormalities.

12Synthesis and accumulation of cyclin B1 and its interaction with CDK1 have long been considered prerequisites for oocyte MPF activation as well. As part of the MPF, cyclin B1 must be constantly degraded by a multi-subunit ubiquitin E3 ligase named the anaphase promoting complex (APC) to maintain meiosis arrest. During this time, the role of cadherin 1 (CDH1) is important because it is an activator of the APC. Before GVBD happens, cyclin B1 translocation from the cytoplasm into the nucleus is required. Interestingly, cyclin B1-null oocytes resumed and finished meiosis I but are then arrested at the meiosis interphase when cyclin B2 is available, indicating that cyclin B2 compensates for the shortage of cyclin B1 in oocyte meiosis I.

13Gap junction provides a direct communication channel between cells which allow molecules smaller than 1,000 Da be transferred to the adjacent cells. In mice, as many as 20 connexins (Cxs) participate in forming the channels of the gap junction. Inside a follicle, cGMP produced in CGCs diffuses into oocytes through Cx43 and Cx37 GJs and thus elevates oocyte cGMP level. Importantly, the closure of GJs between MGCs and CGCs and between CGCs and oocytes are targets of LH signaling. For instance, LH inhibits Cx43 translation and breaks down GJs to prevent cAMP and cGMP diffusion into the oocyte, which results in PKA inactivation and triggers the initiation of oocyte maturation.

14Of all connexins, Cx43 and Cx37 are the most studied ones in the follicle and may possess equal importance to folliculogenesis. In mice, Cx43 is mainly expressed in gap junctions between GCs and is regulated by extracellular signal regulated kinase-1 and -2 (ERK1/ERK2) signals in response to LH surge in vivo. However, PKCε-mediated mitogen-activated protein kinase (MAPK)-dependent signals might contribute to Cx43 phosphorylation in CGCs during FSH-induced oocyte meiotic resumption in vitro. Ovaries lacking Cx43 do not proceed beyond the primary follicle stage. Also, Cx37, which is mainly expressed between the oocyte and CGCs, is essential to oocyte growth and survival, which in turn is necessary to maintain proper MGC function. In Cx37-knockout mice, folliculogenesis is arrested at the early antral stage and this disruption results in sterility because mutant oocytes grow slowly and cannot survive. To examine the roles that Cx37 and Cx43 play in oogenesis, a transgenic mouse model, in which Cx37 specifically replaced Cx43 in growing oocytes, was made. The generations of Cx43 transgene mice driven by zona pellucida 3 (ZP3) crossed with Cx37-null mice are fertile due to the restoration of oocyte–granulosa cell coupling, oocyte growth, and oocyte maturation. Thus, despite their different properties, Cx43 may be physiologically equivalent to Cx37 in coupling oocytes with granulosa cells. Both of them are indispensable in the regulation of oocyte maturation.

15EGF-related proteins are a set of proteins that respond to the LH signal and promote oocyte maturation. Different epidermal-like growth factors, such as amphiregulin (AREG), epiregulin (EREG), and beta-cellulin (BTC) are expressed in MGCs and CGCs in autocrine and paracrine manners through respective EGF receptors (EGFRs). The activation of EGFR is required for oocyte meiotic resumption and cumulus cell expansion. Studies using inhibitors and gene deletion mouse models have identified that EGFs mediate LH action through EGFR. For instance, the process of oocyte maturation, cumulus expansion, and ovulation stimulated by LH are either delayed in AREG or blocked in EGFR-deficient mice. Furthermore, in granulosa cell-specific EDFR deleted mice, oocytes cannot resume meiosis.

16How does EGF signaling regulate meiosis resumption response to LH surge? When the LH surge arrives, LH decreases NPPC/NPR2 expression levels, thereby blocking cGMP synthesis, and stimulates MGCs to secrete EGFs to activate EGFR signaling in cumulus cells, and activates phosphodiesterase 5 (PDE5). The activation of PDE5 suppresses the production of NPPC and closes the gap junction communication between granulosa cells, resulting in the decrease of cGMP levels and the reduction of oocyte cAMP levels. Then, cumulus expansion and oocyte maturation starts. Even though the expression of EGFR and the direct effects of EGF on oocytes has been reported, how LH regulates EGF in detailed molecular mechanisms remain unclear. Recently, found that LH surge-induced histone deacetylase 3 (HDAC3) downregulation in GCs is essential for oocyte maturation. HDAC3 in GCs is a negative regulator of EGF expression before the LH surge. HDAC3 in GCs is recruited by transcription factors, such as FOXO1, to the AREG promoter to suppress the expression of AREG. With the LH surge, the HDAC3 level decreases while histone H3K14 acetylation increases, which enables transcription factor SP1 binding to the AREG promoter to initiate AREG transcription. Moreover, granulosa cell-specific knockout of HDAC3 in vivo or inhibition of HDAC3 activity in vitro increases the proportion of the oocyte maturation independent of LH. Unfortunately, the mechanism of HDAC3 downregulation after the LH surge remains unclear.

17In addition, calcium signaling is involved in gonadotropin-induced oocyte maturation in many species. It was reported that EGFR signaling activates phospholipase Cγ, which may increase calcium levels. Moreover, the elevated calcium of cumulus granulosa cells inactivates NPR2, further decreasing the binding affinity of NPR2 for NPPC. As a result, cGMP levels and meiotic resumption decreases. The regulations of granulosa cells cooperate with oocytes to resume meiosis induced by LH surge in mice, which are summarized in Figure 2.

18As one the key factors that heavily determines the quality of an oocyte, the cytoplasmic maturation of oocytes is critically important, which includes the synthesis, activation, and degradation of maternal mRNA as well as orderly arrangement of organelles, thereby affecting fertilization and embryonic development.

19The expression and degradation of maternal mRNA is developmental stage dependent. Along with the growth of activated follicles, the transcription of genes necessary for oocyte growth and meiosis resumption accumulate and are stored in the cytoplasm. With the initiation of meiotic resumption, not only does the transcription in oocytes cease because of staining agglutination, but the maternal mRNAs stored in oocytes are degraded and gradually consumed. As to protein synthesis, although a large amount of maternal mRNA exists in fully grown oocytes at the GV stage, they are translationally dormant in mice until meiotic maturation. The freshly translated proteins after oocytes resume meiosis play important roles in meiotic spindle assembly, MII arrest maintenance, and mRNA clearance during maternal zygotic transition (MZT). Generally, novel mRNA synthesis is initiated in the late stage of fertilized eggs.

20How could the transcriptions of mRNA in growing oocytes remain stable before GVBD? Accumulative data show that there are stringent mRNA stabilizing mechanisms within GV-stage oocytes. For instance, cytoplasmic polyadenylation of the 3′-untranslated region (3′-UTR) is closely related to mRNA stability and mRNA translational activation, which plays an important role in oocyte maturation. Actually, the degree of polyadenylation of mRNA affects oocyte translation activation as well. Cytoplasmic polyadenylation is a key process that serves to unmask particular mRNAs and enables them to be translated. In its simplest form, masked mRNA refers to dormant transcripts in the oocyte that are to be translated during completion of meiotic divisions or in early embryos.

21The degradation of maternal mRNA is controlled strictly in oocytes undergoing meiotic resumption and in early embryos. In mouse oocytes, transcriptional destruction, especially the transcripts of oxidative phosphorylation, energy production, and protein synthesis during the transition from GV to MII, is selective rather than promiscuous. It is stated that the selective degradation of the transcription of maternal mRNAs is a prerequisite for the activation of the zygotic genome. Particularly, regulation of maternal mRNA translation and degradation mainly occurs in maturing oocytes rather than in fertilized eggs, but these mechanisms are essential for the oocyte and zygote to build up competence to accomplish MZT.

22The starting point of the MZT is oocyte activation from meiotic arrest rather than fertilization. About 90% of the maternal mRNA is degraded at the two-cell stage of early mice embryos. In the major pathway of mRNA degradation, shortening of the poly(A) tail, or deadenylation, is the first and rate-limiting step. Poly(A) tail shortening reduces the binding of poly(A) binding proteins (PABPs) and slows down translation. In this aspect, prepared RNA-binding proteins (RBPs) in fully grown oocytes are important for sustaining genome stability, stabilizing and/or degrading mRNAs, or even for protein synthesis and degradation. For instance, meiosis arrest female 1 (MARF1) is an essential regulator of important oogenic processes leading to female fertility and the development of healthy offspring by suppressing levels of specific transcripts. More information about oocyte-specific RBPs regulating genome stability and mRNA stability needs to be uncovered.

23The cytoplasmic maturation of oocytes also includes the maturation of various organelles, especially cortical granules, mitochondria, the endoplasmic reticulum (ER), and cytoskeleton. The time dependent distribution and structure of these organelles are indispensable for the respective functions.

24Cortical granules are membranous organelles derived from Golgi complexes, which are found in the cortex of unfertilized oocytes and play important roles during the fertilization process. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space, which result in the blocking of polyspermy by modifying the oocytes’ extracellular matrices, such as the zona pellucida in mammals. Besides, mitochondria are the key to ATP energy supply in oocytes. Impaired oocyte quality, including meiosis chromosome separation, maturation, and fertilization failure, correlates with both abnormal mitochondrial rearrangement and low ATP level. In addition, ER is responsible for the storage and release of free Ca2+ in the cytoplasm in oocytes, which is important for calcium reaction at fertilization. Since the cytoskeleton is mainly composed of microtubules and filaments and the recombination of spindles is strictly controlled by the microfilament network, anything that disrupts either microtubules or microfilament causes failure of chromosome movement and separation, in which case, the oocyte is arrested at the metaphase stage.

25Multiple posttranslational modifications exist in developing oocytes, including acetylation, phosphorylation, methylation, glycosylation, ubiquitination, and SUMOylation of various proteins, implying that epigenetic modification plays different but important roles during the oocyte maturation process under varying temporal and environmental conditions. As follows, the changes and regulation as well as functions of histone modifications during meiotic maturation of mammalian oocytes, with particular emphasis on histone acetylation and methylation are summarized.

26Lysine acetylation of histones is generally controlled by histone acetyl transferases (HATs) and histone deacetylases (HDACs). Acetylation of H3/4 leads to open chromatin configuration, enhances transcriptional activity, and thereby promotes transcription factor binding to DNA. Contrarily, deacetylation is associated with transcriptional inactivation. The key sites of histones for acetylation include at least four conserved lysines (K) in histone H4 (K5, K8, K12, and K16) and two conserved lysines (K) in H3 (K9 and K14). In general, all lysine residues are acetylated in fully grown GV oocytes, including H4K5ac, H4K8ac, H4K12ac, H4K16ac, H3K9ac, and H3K14ac, except for H4K8ac, which is deacetylated in condensed chromosomes and is maintained until the MII stage. In mammals, as many as 18 HDACs are identified and divided into four classes based on their homology with yeast proteins. In which, class I HDACs are nuclear-localized, including HDAC 1, 2, 3, and 8. Class II is divided into IIa (HDAC 4, 5, 7, and 9) and IIb (HDAC 6 and 10), both of which can shuttle between the nucleus and cytoplasm. Class III includes seven sirtuins (SIRT1-7), which are homologous with the yeast SIRT2 family proteins and require NAD+ as a cofactor to function. HDAC11 is the only member of class IV, which is homologous to both classes I and II. The respective actions of these proteins in oocytes are reviewed in the following.

27Class I HDACs are important in oocyte development and maturation. HDAC1 and HDAC2 share high amino acid homology and work together in almost all repressive transcriptional complexes. HDAC1 and HDAC2 are located in the nucleus throughout oocyte growth. HDAC1 in the nucleus decreases gradually during the growth of oocytes and co-localizes with chromosomes following meiosis resumption. In contrast, HDAC2 in the nucleus increases between 5 and 12 days post-partum, and is relatively stable during the growing period of mice oocytes. After GVBD, HDAC2 in an oocyte is uniformly dispersed throughout the cytoplasm. Germ-line deletion of either HDAC1 or HDAC2 will cause mouse embryo lethality. However, conditional knockout HDAC1 by ZP3-Cre has no obvious impact on fertility and oocyte maturation. Although, the deletion of HDAC2 by ZP3-Cre did result in reduced fertility, but the follicular development was normal. Further, deletion of both HDAC1 and HDAC2, however, results in infertility due to oocyte development arrest at the secondary follicle stage. The low level acetylation of H4K16 is essential for the function of centromeres. Interestingly, the deletion of maternal HDAC2 caused high level acetylation of H4K16 and resulted in disorder in chromosome segregation and kinetochore function during MII in oocytes. In summary, HDAC1/2 regulate oocyte growth with their compensatory function, and HDAC2 could be more critical than HDAC1 for oogenesis.

28HDAC3 is expressed in the nucleus of GV oocytes and disperses in the cytoplasm of oocytes after meiotic resumption. The signal of HDAC3 accumulates on the meiotic spindle region from pre-metaphase I to MII. Knockdown of HDAC3 in oocytes results in spindle/chromosome organization failure, with severely impaired kinetochore-microtubule attachments. In addition, overexpression of HDAC3 modulates the acetylation status of α-tubulin in mouse oocytes. HDAC3 also has functions in promoting meiotic apparatus assembly in aging mouse oocytes. Overexpression of HDAC3 in old oocytes not only partially prevents spindle/chromosome disorganization, but significantly lowers the incidence of aneuploidy. HDAC3 also plays important roles in GCs. Conditional knockout of HDAC3 in MGCs in vivo or inhibition of HDAC3 activity in vitro promotes the maturation of oocytes independent of LH. The above results indicate that HDAC3 in both granulosa cells and oocytes plays important regulatory roles in oocyte maturation.

29HDAC8 could be important for oocyte maturation according to its distribution in growing oocytes. It is widely distributed in the cytoplasm of mouse oocytes at the GV stage. After GVBD, it starts to accumulate around the chromosomes, and shows a spindle pole-like localization pattern in both MI and MII. Inhibition of HDAC8 in fully grown oocytes causes spindle defects and chromosome misalignment during oocyte meiotic maturation, accompanied by impaired kinetochore-microtubule attachments. Conditional deletion of HDAC8 by Vasa-Cre results in subfertile females, which is independent of chromosome segregation errors during meiosis. On the whole, HDAC8 is important for oocyte development and maturation, but the mechanisms of its action on oocytes needs further study.

30The function of class IIa HDACs in oocyte maturation has not been well studied. According to existing reports, the expression of HDAC4 is maintained at a high level in fully grown oocytes until the MII stage, and then dramatically decreased after fertilization, it may play specific roles during mouse oocyte maturation.

31In class IIb, HDAC6 has been studied extensively, while HDAC10 has hardly been reported. HDAC6 localizes in the cytoplasm of mouse GV oocytes. Overexpression of HDAC6 results in GV oocytes and pronuclear zygotes which results in altered nuclear structure and causes compaction of the chromatin. In addition, inhibition of HDAC6 in GV oocytes prevents PB1 extrusion later because of disrupted maturational progression and meiotic apparatus assembly. However, HDAC6 KO mice are viable and fertile and presented no major observable phenotype. Despite that, the TuA-treated group presented significant changes in the expression of HDAC subfamily genes such as HDAC6, 10, and 11 and sirtuin 2, 5, 6, and 7 by RNA-sequencing, which may indicate that TuA is a multifunctional inhibitor which targets both HDAC and sirtuin activity rather than being a HDAC6-specific inhibitor in mouse oocytes.

32Sirtuins are generally important for oocyte development. SIRT1, SIRT2, SIRT3, and SIRT6 are beneficial for improving the competence of oocytes grown or matured in vitro in humans and animals. SIRT4, SIRT5, and SIRT7 have seldom been studied so far. Activation of SIRT1 by resveratrol in vitro improves oocyte quality and embryo development in mice, pigs, and cows. SIRT1 relates to mitochondria biosynthesis and degradation in oocytes because resveratrol supplementation improves the mitochondrial function and the developmental capability of the oocytes. In contrast, specifically inhibition of SIRT1 results in increased ROS production and abnormal MII plates in mouse oocytes. Similarly, inhibition of SIRT2 during in vitro oocyte maturation or knockout of SIRT2 blocks the progression of oocyte development after GVBD. SIRT2 knockdown also affects spindle organization and chromosome alignment during meiosis. Besides, SIRT3 regulates the ROS level in oocytes. Overexpression of SIRT3 reduces the spindle defects and chromosome misalignment in oocytes. Last, SIRT6 is important in regulating meiotic progression as well. Depleted SIRT6 results in disruption of spindle morphology and chromosome alignment in oocytes.

33The expression of HDAC11 in oocytes decreases from the GV to MII stage. Inhibition of HDAC11 by JB3-22 significantly interrupted mouse oocyte meiosis progress, possibly because of abnormal spindle organization and misaligned chromosomes, impaired kinetochore-microtubule attachment, and spindle assembly checkpoint function.

34The function of HDACs during oocyte maturation are summarized in Table 1.

35Histone acetyl transferases, including MYST, GCNA5/PCAF, and p300/CREB-binding protein (CBP), regulate the acetylation of histones as well.

36MYST is an acronym of its four founding members, including human MOZ (monocytic leukemia zinc finger protein), yeast Ybf2 (renamed Sas3, for something about silencing 3), yeast Sas2, and mammalian TIP60 (HIV Tat-interacting 60 kDa protein). Importantly, K (lysine) acetyltransferase 8 (KAT8) is a highly conserved MYST family member who is specifically responsible for H4K16 acetylation and is important for mouse oocyte development, by regulating reactive oxygen species levels. The expression of KAT8 increases dramatically between 14 days and full-grown GV-stage oocytes, followed by a sharp decrease in GVBD-MI-stage oocytes. The protein is mainly located in the nucleus throughout the growth phase, but upon GVBD, the staining intensity decreases and the signal becomes uniformly dispersed throughout the oocyte. Oocyte KAT8 deletion results in female infertility with defects in follicle development and increased oocyte apoptosis.

37In bovine, the levels of MYST4 mRNA in both GV and MII oocytes are high. MYST4 protein accumulates in the nucleus of GV oocytes. It concentrates in the vicinity of the meiotic spindle rather than on chromosomes in the MI stage.

38Histone acetyltransferases p300 and the CBP subfamily are constitutively expressed in the GCs of growing and ovulatory follicles in a gonadotrophin-independent manner. ED-rich tail (CITED) protein CITED4 formed an endogenous protein complex with CBP and transcription factors CCAAT/enhancer binding protein C/EBP/b, which docked on the promoters of LH and ERK1/2 target genes. Both CITED4 expression and CBP acetyltransferase activity were indispensable for ovulation-related molecular and histological events. Moreover, the dynamic histone acetylation changes (histone H2B-Lys5 and H3-Lys9) in GCs were regulated by LH, CBP, and HDACs during ovulation. In addition to the above two subfamilies, the constant expression of HAT1 and GCN5 mRNA was also detected during bovine oocyte maturation.

39Taken together, HATs have essential roles in mouse follicle development and oocyte maturation, and the potential functions of HATs in oocytes maturation needs more exploration.

40Histone methylation correlates with chromatins activity. For instance, H3K4 methylation is associated with the activation of chromatins and occurs mainly in the promoter regions of active genes, while the methylations of either H3K9 or H3K27 relates to gene inactivation. In developing mouse oocytes, the level of H3K4me3 are elevated during the transition of chromatin configuration from the non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) type, the latter of which have better developmental competence after fertilization. H3K4me and H3K4me2 levels elevate, but H3K4me3 level decreases after GVBD and reaches its lowest point in anaphase I.

41Histone methylation modification is regulated by histone lysine methyl transferases (KMTs) and histone lysine demethylases (KDMs) via modifying lysine residues and the number of methyl groups. There are six known histone H3 methyltransferases, including SET domain containing 1A/B (SETD1A/B), lysine (K) methyltransferase 2A/B (KMT2A/B), and KMT3/4 in mammals.

42The SETD1/COMPASS histone methyltransferase complex is the primary enzyme that methylates histone H3K4 by recognizing its basic subunit, CXXC1. The expression of SETD1A and SETD2B persists from oocyte to blastocyst. SETD1A is first required at the epiblast stage, whereas SETD1B becomes essential after gastrulation. In GDF9-Cre-driven SETD1B deficient mice, the number of follicles decreases gradually with time, the ovulated MII oocytes exhibit meiotic spindle abnormalities. And the oocytes as well as zygotes display perturbed cytoplasmic organelles and aggregated lipid droplets. SETD1-CXXC1 conjugation regulates H3K4me3 in mice oocytes. A stabilized CXXC1 in fully grown GV oocytes resulted in decreased GVBD and PB1 emission rates as well as spindle assembly defects in mice.

43KMT2B activates gene expression by regulating H3K4me3. Loss of KMT2B in mouse oocytes induced by GDF9-Cre resulted in abnormal meiosis maturation, anovulation, oocyte death, and female sterility, in which H3K4 level decreased and gene expression was abnormal. KDMs consist of KDM1 and the KDM2-KDM7 subfamily, which contain a Jumonji C (JmjC) domain. KDM1A is expressed in the oocyte nucleus, which specifically catalyzes the demethylation of H3K4me1 and H3K4me2. KDM1A-null oocytes display defects in maintaining prophase I arrest and undergo precocious GVBD. Most KDM1A-null oocytes undergo apoptosis before the completion of meiotic maturation. KDM1B is highly expressed in growing oocytes and the level persists through later stages of oogenesis, but it is hardly detectible in oocytes of primordial and primary follicles in mice. The deletion of KDM1B in mice does not affect embryo development, animal survival, or oocyte growth. However, oocytes from KDM1B-deleted females show high levels of H3K4 methylation and fail-to-deposit DNA methylation marks at four out of seven imprinted genes. Early embryos derived from these oocytes show biallelic expression or suppression of the affected genes and died before mid-gestation.

44KDM4A and KDM4B are located in human oocytes, granulosa cells, theca cells, and luteal cells in reproductive-aged women. Deletion of KDM4A during oogenesis had no significant impact on ovulation since KDM4A–/– and wild-type females ovulated similar numbers of fertilizable oocytes. MII oocytes from KDM4A–/– females were euploid with no evidence of major chromosomal breakages or aneuploidies. KDM4A is the major demethylase functional in MII oocytes and is required to maintain the genomic stability of pre-implantation embryos.

45The KDM5 family consists of KDM5A to KDM5D. During knockout of KDM5A, the mice are viable and fertile, whereas knockout of KDM5B resulted in early embryonic lethality, suggesting that KDM5B is the major functional KDM family member in vivo.

46Protein phosphorylation occurs most often on serine, threonine, or tyrosine residues and competently regulates cell cycle stage-related affairs in a variety of different signal transduction pathways. For example, histone H3 phosphorylation at Ser10 and Ser28 affects chromatin condensation of either mitosis or meiosis. Aurora B phosphorylates histone H3 at Ser28 in mitotic cells and Ser10 and Ser28 in meiosis cell. ZM447439 (an inhibitor of the Aurora kinase family) treatment prevented Aurora B activity and significantly decreased the phosphorylation levels of both H3/Ser10 and H3/Ser28 in mouse oocytes, resulting in chromosome misalignment. In addition, protein phosphatase 1 (PP1) dephosphorylates H3 at Ser10 in budding yeast and nematodes. Inhibition of PP1/PP2a induces rapid chromosome condensation with hyperphosphorylated histone H3. Taken together, the balance of Aurora B kinase and PP1 activities regulate the meiotic phosphorylation of histone H3 in mammalian oocytes.

47The ubiquitination/deubiquitination system is important for the degradation of proteins, cell cycle progression, and transcriptional regulation, which is also important for oocyte maturation. Importantly, APC initiates the metaphase to anaphase transition by inducing the degradation of cyclin B and securin. Also, protein ubiquitin (Ub) E3 ligases trigger specific protein degradation and thus plays an important role in the process of both the meiotic and mitotic cell cycle.

48Interestingly, cullin ring-finger ubiquitin ligase 4 (CRL4) is one of E3 ligase members who exert multiple functions in the maintenance of oocyte survival and meiotic cell cycle progression. DCAF13, a CRL4 adaptor, stimulates the meiotic resumption-coupled activation of protein synthesis in oocytes. Deletion of DCAF13 in oocytes resulted in not only decreased CDK1 activity and impaired meiotic cell cycle progression as well as chromosome condensation defects, but also polyubiquitination and degradation of PTEN.

49In addition, protein-ubiquitination mediated CCNB1 and securin degradation is essential for the metaphase to anterograde transition during oocyte meiotic maturation.

50Ubiquitin C-terminal hydrolases (UCHs) are a deubiquitin enzyme that catalyzes the hydrolysis of peptides, isopeptides, or UB portions. UCHs present in oocytes in many species. UCHs have a complimentary distribution in porcine, bovine, and murine oocytes. UCHL1, one of the most abundant proteins in mammalian oocytes, accumulates in the oocyte cortex. UCHL3 is associated with oocyte spindle. Inhibiting UCH activity causes excessively large PB1, distorts the meiotic spindle, and disrupts other spindle attributes, such as chromosome alignment. In vitro, inhibition of UCHL3 reduces the expansion of cumulus cells.

51In follicular granulosa cells, the ubiquitin-proteasome system (UPS) was involved in regulating the deposition of the extracellular matrix of cumulus and steroidal formation during the expansion of cumulus cells, implying that this system may be pivotal for follicle development.

52In the female reproductive system, a large number of proteins, including FSH, LH, GDF9, BMP15, and AMH are glycosylated. Protein glycosylation is one of the most frequent post-translational modifications (PTMs), which affects many things, such as protein folding, distribution, stability, and activity. There are two main types of glycosylation in cells, N-linked and O-linked glycosylation. Defects in the process of protein glycosylation leads to many clinical diseases.

53Protein N-glycosylation in oocytes is crucial for female fertility. For example, DPAGT1 is an enzyme involved in the process of protein N-glycosylation. DPAGT1 missense mutation causes subfertility in females due to defective follicular development and less ovulation. Also, due to the decreased glycosylation of ZP proteins, the mutant oocytes have a thin and fragile ZP layer and have poor developmental ability after in vitro fertilization. Furthermore, the first meiotic division is accelerated in such mutant oocytes. Importantly, the phenotypes of conditional knockout of DPAGT1 in infertile mouse oocytes is consistent with those in humans.

54Protein O-glycosylation plays a small role in oocyte maturation. In vitro, O-glycosylation is elevated in bovine COCs exposed to glucosamine. Glucosamine treatment during in vitro maturation does not affect the meiotic maturation of cow, pig, or mouse oocytes, but blastocyst development was severely inhibited. This suggests that protein O-glycosylation does not affect oocyte maturation, but it affects the quality of oocytes.

55SUMOylation and de-SUMOylation modification refers to the reversible addition and removal of SUMO (small ubiquitin-related modifier) polypeptides on lysine residues. SUMO proteins, such as SUMO1, SUMO2, SUMO3, and UBE2I, are expressed in and are required for oocyte maturation in events like oocyte meiotic resumption and spindle formation.

56The localization of SUMO1, SUMO2, and SUMO3 in oocytes depends on the developmental stage of the oocytes. In immature oocytes, SUMO1 localizes to the nuclear membrane while SUMO2/3 are within the nucleoplasm. During oocyte meiosis, SUMO1 localizes to the spindle poles and around the chromosomes whereas SUMO2/3 locate near the centromeres. UBE2I primarily expresses in the nucleoplasm of mouse growing oocytes at least from postnatal day 13 to GV-stage fully grown oocytes. UBE2I is downregulated following meiotic resumption.

57The importance of SUMOylation on oocytes maturation could be highlighted by the following facts. After endogenous SUMO1 or UBC9 activities in oocytes were either inhibited or silenced, the percentage of GVBD and PB1 extrusion was significantly reduced, together with abnormal spindle organization, chromosome misalignment, segregation defects, and aneuploidy in matured oocytes. Similarly, inhibition of UBE2I for GV-stage mouse oocytes disrupts meiotic maturation and causes defects in spindle organization, while overexpression of UBE2I in meiotic incompetent oocytes stimulates gene transcription in vitro. Deletion of UBE21 by GDF9-Cre results in complex infertility phenotypes, including defects appearing at multiple critical oocyte transition points, such as unstable ovarian reserves, impaired communication with granulosa cells, and defective resumption of meiosis and meiotic progression.

58Other proteins that affect oocyte maturation are also involved in the process of SUMOylation. For instances, overexpression of the SUMO-specific isopeptidase, sentrin/SUMO-specific protease 2 (SENP2), leads to defects in MII spindle organization by changing the localization of SUMO-modified proteins in oocytes. Septin, a conserved GTP-binding protein that is modified by SUMO1, is also required for chromosome congression in mouse oocytes. And the spindle-assembly checkpoint protein Bub1-related kinase, or MAD3/Bub1b (BUBR1), may be SUMOylated by SUMO1 and is necessary for homologous chromosome alignment as well.

59Oocyte maturation is a complex process involving multiple steps and is regulated by many molecules and signaling pathways. In recent years, due to the rapid development and popularization of technologies like the genetic modification of animal models, molecular biology, and biochemistry, researchers have gained a better understanding of oocyte GV arrest and meiosis I resumption. The major cellular and molecular affairs, especially the epigenetic modification events related to oocyte maturation in response to hormone induction, and the major advances in this field, are highlighted in this review.

60Since the development of an oocyte depends not only on the oocyte itself, but on mutual communication and physical contact with follicular granulosa cells, it is important to focus more on epigenetic changes within oocytes, ovarian granulosa cells in response to hormones, and other extracellular molecules induction. Besides, applying microscopes with high resolution and a high-throughput analysis technique, such as mono-cellular based sequencing and omics techniques, should be emphasized to present clearer 3D or even time-dependent 4D representations of critical affairs that happen during oogenesis. Finding more specific oocyte-expressed proteins, such as RBPs and oocyte-derived paracrine molecules, may contribute to uncover the mysterious mechanisms of oocyte meiosis as well. Further, integration of analysis of sequencing data, comparing the data collected from different breeds, and verifying the function of each individual molecule in vitro and in vivo simultaneously based on multiple animal models are also plausible.

61MH and TZ collected the information and wrote the manuscript. CW and YY revised the manuscript. All authors read and approved the final manuscript.

62The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.