INTRODUCTION
Female fertility decreases with age, and one of the main manifestations of this is the increase in aneuploidy due to abnormal spindle assembly and chromosome segregation during oocyte maturation [1,2]. The process of oocyte senescence is accompanied by dynamic changes in histone acetylation, in which insufficient histone deacetylation leads to increased aneuploidy rates in aged female mice [3,4]. In old germinal vesicle (GV) oocytes, acetylation of histone H4K12 and H4K16 decreased [4]. The acetylation levels of histone H3K14, H4K8 and H4K12 in mouse oocytes gradually increased during aging in vivo and in vitro after ovulation [3]. In contrast, inhibition of histone deacetylases (HDACs) leads to abnormal spindle assembly during oocyte maturation [5,6]. These results suggest that histone acetylation plays an important role during oocyte maturation.
Acetylation and deacetylation of histones are catalyzed by histone acetyltransferases (HATs) and HDACs, respectively. Different histone acetylation patterns are recognition codes that recruit different transcription factors upon transcriptional activation [7]. There is a dynamic balance between the activities of HAT and HDAC. Upon resumption of meiosis, oocytes undergo a reduction in histone acetylation associated with maturation [8,9]. The gene expression pattern of differentiated oocytes is reprogrammed during meiosis to allow the next generation of allotropic zygotes to initiate a new program [8]. Inhibition of HDAC activity (HDAC11, HDAC3, etc.) during oocyte maturation leads to histone hyperacetylation, which in turn alters meiotic progression [5,6,10]. Although HDACs affect gene expression during oocyte maturation by controlling histone acetylation, it is not clear how HDACs are regulated during oocyte maturation.
Nucleosome remodeling and histone deacetylation (NuRD) complex is a 1 MDa multi-subunit protein complex which comprises many different subunits, including Hdac1/2, Mta1/2, Rbbp4/7, Chd3/4, and Gatad2a/2b [11,12]. Among them Gatad2b, also called p66b, recruits Mbd2, another component of the NuRD complex, to DNA and histones, affecting histone deacetylation and repression of transcription [13]. An opposing regulation of Gatad2b by Mkk6 phosphorylation elevates histone acetylation levels and enhances pluripotency gene expression [14]. Recently, Gatad2b was reported to be sumoylated to enhance the formation of NuRD complexes [15,16]. And Gatad2b can directly interact with histone acetylation sites H3k9ac, H3K27ac and H4K16ac [14].
Rbbp7 is one of the components of the NuRD complex, a dormant maternal mRNA that is recruited for translation during oocyte maturation to regulate histone deacetylation. During oocyte meiotic maturation, RBBP7 loss of function leads to abnormalities in chromosomal passenger complex (CPC) localization and function [17]. RBBP4 is a ubiquitously expressed nuclear protein that belongs to the WD-40 family [18]. In mouse oocytes, RBBP4 can regulate bipolar spindle assembly by promoting Aurora kinase (AURK) C function [19]. Deletion of RBBP4 and RBBP7 function both result in oocyte histones H3K4, H4K8, H4K12, and H4K16 exhibit acetylation hyperacetylated, increasing the incidence of abnormal spindles, chromosome misalignment, and aneuploidy at metaphase II (MII) [17,19], and this deacetylation is required for bipolar spindle assembly via AURKC [19].
In this article, we demonstrated that Gatad2b is a novel regulator of histone deacetylation during meiosis maturation of mouse oocytes. Depletion of Gatad2b leads to abnormal spindle formation during metaphase I (MI) meiosis, followed by first polar body extrusion defects, chromosomal misalignment, and increased aneuploidy of MII. Intriguingly, we examined that the localization and activity of the microtubule organizing center (MTOC) complex was impaired within Gatad2b-depleted oocytes. Our contributions reveal new insights into the regulation of Gatad2b function and its role, especially associated with histone deacetylation, in spindle assembly.
MATERIALS AND METHODS
Animals
All animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee of Nanjing Drum Tower Hospital (2025AE01013). ICR mice were purchased from Beijing Vital River Laboratory Animal Technology. Mice were fed regular chow and housed in a controlled room under a 12:12-hour light-dark cycle at 22°C.
Oocyte collection and culture
Fully grown oocytes at GV-stage were achieved from the ovaries of 3 weeks old females. The female mice were superovulated with 5 IU pregnant mare serum gonadotropin, PMSG (Prospec) by intraperitoneal injection 46–48 hours ago. Female mice were used for oocyte retrieval 46–48 hours after intraperitoneal injection of 5 IU of PMSG (Sansheng). Cumulus-oocyte complexes were collected and cumulus cells were removed by mouth pipetting repeatedly. Oocytes was collected in MEM-alpha containing 3 mg/mL of bovine serum albumin (BSA, Sigma-Aldrich) and 5 μmol/L Milrinone (Calbiochem) to prevent meiotic resumption. Oocytes medium for in vitro maturation was MEM (Gibco, USA) supplemented with 3 mg/mL BSA, 75 μg/mL penicillin G (Sigma-Aldrich), 50 μg/mL streptomycin sulfate (Sigma-Aldrich), 25 μg/mL pyruvate (Sigma-Aldrich) and 38 μg/mL EDTA (Sigma-Aldrich). The oocytes were obtained under mineral oil at 37°C in a 5% O2, 5%CO2 and 90%N2 incubator.
Antibodies
Rabbit polyclonal antibodies were purchased from proteintech: anti-GATAD2B (Cat#:25679-1-AP, 1:200) and anti-γ-TUBB (Cat#:15176-1-AP, 1:400). Rabbit polyclonal antibodies were purchased from abcam: anti-H4K16ac (Cat#: ab109463, 1:400). Mouse monoclonal FITC conjugated anti α-tubulin (Cat#: F2168; 1:500), was purchased from Sigma-Aldrich. Human antibody, anti-centromere CREST (Cat#: 15-234; 1:500), was purchased from Antibodies Incorporated. 555-Rhodamine (TRITC) donkey anti-human IgG (Cat#:709-025-149; 1:750) was purchased from Jackson Immuno-Research Laboratory. Mouse anti-β-actin (Cat#:AF0003; 1:1,000) was purchased from Beyotime. Rhodamine-Phalloidin (Cat#: YP0063-50T; 1:1,000) was purchased from US EVERBRIGHT. Alexa Fluor Plus 555 donkey anti-rabbit IgG (Cat#: A31572; 1:1,000) and Alexa Fluor Plus 488 donkey anti-mouse IgG (Cat#: A-21202; 1:1,000) were from Thermo Fisher Scientific. Anti-acetylcysteine (Cat#: PTM-101, 1:200) was purchased from PTM BIO. The specificity of the antibodies was confirmed by Western blotting, showing a distinct band at the expected molecular weight.
Microinjection
The mouse oocytes were microinjected with approximately 10 pl of siRNA (25 μM) to knock down Gatad2b using a FemtoJet microinjector (Eppendorf). A combination of three different siRNAs targeting Gatad2b (5′-GCAGCCAAUAGCG AGUUUATT-3′, 5′-GGGACAACAAGGCUUAUCUTT-3′, 5′-CCCGAUCCAUGCUUUCAAATT-3′) was purchased, diluted with RNase-free water and stored at −80°C. The control oocytes were injected with control siRNAs (GenePharma). After injection, the oocytes were cultured in the medium containing 5 μM milrinone for 24 h to maintain GV-arrest. The oocytes were then cultured in fresh medium under mineral oil at 37°C in a 5% O2, 5%CO2 and 90%N2 incubator.
Immunofluorescence
Mouse oocytes were fixed in 4% paraformaldehyde (PFA) at room temperature for 30 minutes and then blocked with PBS containing 5% BSA and 0.5% Triton X-100 at room temperature for 1 hour. After blocking, the oocytes were incubated with primary antibodies at 4°C overnight. Secondary antibodies were incubated at room temperature for 1 hour. The details of antibodies were provided above. The nuclei were stained with DAPI (Hoechst 33342) for 10 minutes. The oocytes were mounted on glass slides and examined under a laser scanning confocal microscope (LSM 780, Carl Zeiss). Fluorescence intensity was quantitatively analyzed by Leica software. To analyze the length of spindle poles, the measurement tools that come with Leica Software was used. All fluorescent images were taken at the same scale. One side of the spindle pole was selected using the line segment tool to the other side and the software will provide the length information. The length information was collected for analysis.
Western blotting
A total of eighty mouse oocytes were lysed in 50 mM Tris-HCl (pH 6.8) containing 1% SDS, 1% β-mercaptoethanol, 20% glycerol, and denatured at 95°C for 5 minutes. Denatured proteins separated by 7.5% SDS-PAGE and transferred to PVDF membrane. Membranes were blocked by incubation in PBS with 5% low-fat dry milk and 0.1% Tween-20 (PBST) at room temperature for 1 hour. Membranes were then incubated with the primary antibodies, rabbit anti-GATAD2B (1:2,000, Proteintech) and mouse anti–β-actin (1:1,000, Beyotime), at 4°C overnight. Subsequently, membranes were incubated with secondary antibodies labeled with horseradish peroxidase in blocking buffer for 1 hour after washing 3 times in PBST. Finally, the signal was detected by ECL Plus Western Blotting Detection System (GE Healthcare) after washing in PBST for three times. ImageJ software was used to analyze the gray value of the obtained image, and the relative content of the target protein was calculated by the gray ratio between the target protein and the beta-actin. Finally, SPSS software was used for quantitative statistical analysis.
Chromosome spreading
Chromosome spread was conducted as described previously [20,21]. Zona pellucida was removed with exposure to Tyrode’s buffer (pH 2.5) (T1788, Sigma-Aldrich) at 37°C for 30 seconds. The oocytes, recovering in fresh medium for ten minutes, were fixed in 1% PFA with 0.15% Triton X-100 on a glass slide. The oocytes, after air drying, were incubated with the human anti-centromere antibodies (1:500, Antibodies Incorporated) at 4°C overnight, followed by incubation with secondary antibodies, 555-Rhodamine (TRITC) donkey anti-human IgG, at room temperature for 2 h. The chromosomes were then stained with Hochest33342. The slides were examined with laser scanning confocal microscope (LSM 780, Carl Zeiss).
Quantitative reverse transcription polymerase chain reaction analyses
Fifty oocytes for average at the indicated development with certain treatment were collected. To characterize the varied expression of different genes, total RNA was extracted, purified, reverse transcribed and amplified with Single Cell Sequence Specific Amplification Kit (P621, Vazyme) following the manufacturer’s protocol. Then, 2X Realtime polymerase chain reaction (PCR) Mix (Mei5Biotech, MF013) and Applied Biosystems Stepone Plus Real-Time PCR System (Thermo Fisher Scientific) were used to quantify the cDNA. Gene expression was normalized to GAPDH.
Statistical analysis
Data, unless otherwise indicated, was presented as mean± standard error of the mean (SEM). Student’s t-test was used to evaluate differences between 2 groups. Multiple comparisons between more than 2 groups were analyzed by one-way ANOVA followed by Tukey’s honest significant difference (HSD) test using Prism 5.0. The differences of p≤0.05 were considered to be significant. Data are expressed as mean± SEM from at least three independent experiments.
RESULTS
Expression and subcellular distribution of GATAD2B during mouse oocyte meiosis
By re-analyzing single-cell transcriptome sequencing data during human, marmoset, and mouse pre-implantation embryonic development, we found that Gatad2b is expressed in oocytes of three species. Gatad2b was expressed at abundant levels during the oocyte period, especially in mouse oocyte, while mRNA expression levels gradually decreased after fertilization (Figure 1A) [22]. To further characterize the dynamics of Gatad2b during oocyte meiotic maturation, we reanalyzed the mRNA level (blue line) and ribosome profiling (low-input Ribo-seq) (red line) in public database and it revealed an increase in both mRNA level and translation efficiency of Gatad2b from GV to MII period (Figure 1B) [23]. This result was further confirmed by western blot (Figure 1C). Subsequently, we investigated subcellular localization of GATAD2B during meiotic maturation. In GV-oocytes, GATAD2B does not exhibit specific localization. During the resumption of meiosis in oocytes, GATAD2B shows a certain degree of co-localization with α-TUBULIN at the Pro-MI and MI stages. In the TI and MII stages, GATAD2B continues to localize on the spindle (Figure 2). The specific localization of GATAD2B during oocyte maturation suggests a potential relationship between GATAD2B and spindle formation, and its involvement in regulating the process of oocyte meiotic division.
Knockdown of GATAD2B disrupts maturational progression of mouse oocytes
In order to explore the role of GATAD2B in oocyte maturation, we reduced GATAD2B expression by siRNA interference in GV stage (Figure 3A). After microinjection, the GV oocytes were cultured in medium supplemented with milrinone for 20 h to facilitate GATAD2B mRNA degradation. The mRNA and protein expression level of GATAD2B in GATAD2B knocked-down (GATAD2B-KD) oocytes were significantly reduced (Figures 3B–3F). The germinal vesicle breakdown (GVBD) rate of GATAD2B-KD oocytes was significantly lower than that of the control group one hour after meiosis recovery. Moreover, two hours after meiosis recovery, the GVBD rate of GATAD2B-KD oocytes was still lower than 60% (Figures 4A, 4B). In addition, after eight hours of maturation in vitro, the extrusion of first polar body by GATAD2B-KD oocytes was delayed, compared with that by control oocytes. Even after 14 hours of maturation, the rate of Pb1 extrusion in GATAD2B-KD oocytes was notably lower than that in control group (Figures 4C, 4D). In order to observe oocyte maturation process in real time, we further confirmed the result above by live cell imaging. Indeed, it took nearly 14 hours for certain GATAD2B-KD oocytes to discharge Pb1 in vitro (Figure 4E). These results indicated that GATAD2B knockdown disrupted the oocyte meiosis maturation process.
GATAD2B knockdown perturbs chromosome alignment and increases the incidence of aneuploidy in oocyte meiosis
The meiosis process of oocytes was disrupted after GATAD2B knockdown, which prompted us to explore whether there were disorders in spindle assembly and chromosome arrangement. After fourteen hours in vitro, it was found that GATAD2B-KD oocytes, with α-Tubulin-labeled microtubules and DAPI-labeled chromosomes, had a lower normal MII-oocytes ratio (Figures 5A, 5B). The abnormal GATAD2B-KD MII-oocytes are mainly manifested as incomplete separation from the first polar body (a), disordered chromosomes (b) and spindle with abnormal shape and size (c). In the GATAD2B-KD oocytes, we looked closely at the details of spindle assembly abnormalities and found a significant narrowing of the width of the spindle poles. Spindle poles were shortened by nearly 40% in GATAD2B-KD oocytes (Figures 5C, 5D).
The GATAD2B-KD oocytes showed intense chromosome misalignment. Accordingly, the incidence of aneuploidy may increase eventually. To demonstrate this hypothesis, karyotype analysis of MII oocytes was performed by chromosome spreading with labeled-kinetochore. As expected, the rate of aneuploidy increased significantly in GATAD2B-KD oocytes (Figure 6). Taken together, these results suggest that GATAD2B deletion disrupts spindle assembly and chromosome alignment and leads to an increasing incidence of aneuploidy.
GATAD2B knockdown results in abnormal distribution of microtubule organizing center in oocyte
GATAD2B-KD oocytes exhibited maturation defect when cultured to MII stage. However, it remains unclear whether GATAD2B-KD affects microtubule and, accordingly, chromosome arrangement. When resuming meiosis, oocytes first form MTOC. Subsequently, microtubules, centered on the MTOC, extend rapidly. The MTOCs will be distributed at both ends of the spindle, leading the movement of spindle. With GATAD2B knockdown, disorganized enrichment of spindle pole was significantly observed, which suggested the presence of abnormalities in the MTOCs. In order to confirm the formation and function of MTOCs, one core constituent protein, γ-tubublin, of the MTOCs were stained. Notable is, γ-tubublin, ahorseshoe-like-distributed protein in normal MI oocytes, showed abnormal distribution in GATAD2B-KD oocytes, leading to abnormal microtubule attachment (Figures 7A, 7C). Therefore, it is reasonable that during the MI period, the morphology of the spindle labeled by α-tubulin has been abnormal in GATAD2B-KD oocytes. Similarly, chromosome arrangement is already disordered in GATAD2B-KD MI oocytes (Figure 7B). Specifically, after 9 hours of oocyte maturation, only about 20% of the GATAD2B-KD oocytes entered the typical MI stage and in more than 40% of GATAD2B-KD oocytes, the chromosomes were not neatly arranged. This suggests that GATAD2B-KD affects chromosome arrangement and oocyte maturation as early as MI stage.
Knockdown of GATAD2B perturbs histone deacetylation
To figure out the role of GATAD2B in oocyte maturation, fully grown oocytes with intact nucleus were injected with a mixture of siRNA oligonucleotide, which is necessary to effectively deplete GATAD2B’s RNA stores. This approach significantly suppressed the translation of GATAD2B protein associated with maturation (Figure 3). Gatad2b is a key component of the NuRD complex, and we sought to investigate whether Gatad2b knockdown would affect the expression of other components of the NuRD complex. Multiple components of the NuRD complex show different patterns of change during oocyte maturation and preimplantation embryonic development (Figures 8A, 8B). We detected the mRNA expression of main components, such as Rbbp4, Rbbp7, Chd4, and Hdac2, of NuRD complex. These NuRD complex components did not decrease significantly with knockdown of Gatad2b during GV and MI. However, the mRNA expression of Rbbp7, Chd4, and Hdac2 was significantly increased in Gatad2b-KD oocytes during the MII period (Figure 8C).
Gatad2b is a key component of the NuRD complex, and Gatad2b knockdown may affect oocyte protein acetylation. The mRNA expressions of Multiple deacetylation-related factors, Sirt1 and Hdac3, also decreased to varying degrees. Thereinto, deacetylase Sirt1 decreased by 5 times nearly (Figure 9A). Next, to evaluate the role of GATAD2B in regulating protein deacetylation in oocytes, we examined GATAD2B-KD oocyte lysine acetylation by immunocytochemistry. Lysine acetylation in MII oocytes appears to be pervasive in the cytoplasm, but there is an abnormal accumulation of lysine acetylation in the cytoplasm of knockout oocytes (Figure 9B). Acetylation not only exists in cytoplasmic proteins, but also in intranuclear proteins. Deacetylation of H4K16 was significantly inhibited in GATAD2B-KD oocytes. Quantitative analysis showed that the acetylation level of H4K16 was significantly increased by nearly 2 times (Figures 9C, 9D). In addition, we reconfirmed by Western Blot that the total lysine acetylation level in GATAD2B-KD oocytes significantly increased compared with the control (Figures 9E, 9F). These results suggest that GATAD2B may regulate the process of protein deacetylation during oocyte maturation.
DISCUSSION
Here, we show for the first time that Gatad2b can regulate lysine deacetylation of non-nuclear proteins and histone deacetylation of nuclear proteins H4K16 (Figure 9). In addition, Gatad2b expression was most abundant during the MII period of meiotic maturation (Figure 1). The specific localization of Gatad2b at each stage of meiotic maturation in oocytes was also described in detail (Figure 2). GATAD2B-KD oocytes exhibit delayed oocyte meiotic resumption, abnormal chromosome arrangement, abnormal spindle assembly, and increased aneuploidy (Figures 4, 5, 7). This phenotype is consistent with the deletion of RBBP4, RBBP7, and HDAC2 in oocytes [17,19,24]. RBBP7-mediated histone acetylation is required for localization and function of the CPC [17]. RBBP4 regulates bipolar spindle assembly by affecting AURKC function [19]. Unlike these two, we found that Gatad2b loss of function resulted in significant abnormalities in MTOC assembly (Figure 6).
In GATAD2B-KD oocytes, spindle poles were significantly narrowed (Figure 5). Spindle poles are enriched MTOC in oocytes, similar to mitotic centrosomes with attached microtubules. Numerous protein profiling databases in recent years suggest that in addition to chromosome-associated proteins such as histones, numerous components of centrosomes are also acetylated [25–28]. The acetylation of centrosomal proteins is widely distributed in eukaryotic cells. In mitosis, the centrosome protein PLK2 is acetylated and undergoes deacetylation by SIRT1. Acetylation protects PLK2 from ubiquitination, and SIRT1-mediated deacetylation promotes ubiquitin-dependent degradation of PLK2. SIRT1 controls centriole duplication by temporally modulating centrosomal PLK2 levels [29]. Centrosomal proteins such as pericentry and γ-tubulin, undergo acetylation and deacetylation during oocyte meiosis [30]. In oocyte meiosis, RBBP4 is one of the major components of the NuRD deacetylation complex. In RBBP4-depleted oocytes, MTOC migration processes into two spindle poles requires deacetylation of γ-tubulin because the number of γ-tubulin foci increased more [19]. Similarly, in GATAD2B-KD oocytes,γ-tubulin, which is major component of MTOC, do not form the typical horseshoe-like distribution but are diffused in the oocyte cytoplasm (Figures 6, 10).
During oocyte maturation, histone deacetylation is essential for accurate chromosome segregation that is required to generate healthy, developmentally competent oocytes [24]. Gatad2b, which is one of the NuRD complex components, can recruit Mbd2, another component of the NuRD complex, to DNA and histones and affect histone deacetylation and repression of transcription [13,31]. Gatad2b can also be sumoylated to enhance the formation of the NuRD complex [15,16]. In mouse ESC, Co-Immunoprecipitation showed that Gatad2b interacted with H3k9ac, H3K27ac and H4K16ac [14]. Insufficient histone deacetylation of H4K12 during meiosis in mouse oocytes is associated with chromosome misalignment and aneuploidy [32]. Inhibition of HDAC activity by trichostatin A (TSA) treatment resulted in severe chromosome misalignment, chromosome lag, abnormal cytoplasmic segregation, and abnormal chromosome segregation during mouse oocyte maturation [33]. In RBBP4 or RBBP7-depleted oocytes, histones H3K4, H4K8, H4K12, and H4K16 were hyperacetylated [17,19]. A similar result was found for a significant increase in histone H4K16ac in GATAD2B-KD oocytes. In addition to nuclear histone H4K16ac proteins, a large number of non-nuclear proteins were also hyperacetylated in GATAD2B-depleted oocytes (Figure 9) [7]. These results provide evidence that GATAD2B as a novel regulator of histone deacetylation during oocyte maturation.
The Oocyte-to-Embryo transition (OTE) process is initiated with the resumption of oocyte meiosis. This process involves the initiation of degradation and translational activation of numerous unstable maternal mRNAs stored in the cytoplasm [34,35]. Although translation and degradation of maternal mRNA occur primarily in mature oocytes, these mechanisms are essential for oocytes and zygotes to establish their ability to complete Maternal-to-zygotic transition (MZT) [36–39]. Interestingly, we observed that Gatad2b expression was highest during MII and was enriched in the first polar body (Figure 1). Further, Gatad2b maintained high expression in zygotes (Figure 8). This suggests that Gatad2b may have an important role in the OTE process. Gatad2b expression trend from oocytes to preimplantation embryos is different from other components of the deacetylation complex NuRD, and it can be speculated that Gatad2b has a unique function during early embryonic development. Therefore, the function of Gatad2b in the OTE process needs to be further explored.
CONCLUSION
This study describes Gatad2b expression and localization during oocyte maturation. The absence of GATAD2B will lead to the abnormal distribution of the MTOC, the abnormal spindle assembly, chromosome misalignment, and the increase of aneuploidy. These results implicate GATAD2B as a novel regulator of histone deacetylation during oocyte maturation and provide evidence that such deacetylation is required for proper spindle assembly (Figure 10).
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