CSIRO PUBLISHING
Reproduction, Fertility and Development, 2015, 27, 419–426 http://dx.doi.org/10.1071/RD13293
Aging alters histone H3 lysine 4 methylation in mouse germinal vesicle stage oocytes Gen-Bao Shao A,E, Jie Wang B, Liu-Ping Zhang A, Chao-Yang Wu B, Jie Jin A, Jian-Rong Sang C, Hong-Yan Lu D, Ai-Hua Gong A, Feng-Yi Du A and Wan-Xin Peng A A
Department of Biology, Jiangsu University, School of Medical Science and Laboratory Medicine, Zhenjiang 212013, P. R. China. B Department of Radiation Oncology, the Affiliated People’s Hospital of Jiangsu University, Zhenjiang 212002, P. R. China. C Department of Physiology, Jiangsu University, Zhenjiang 212013, P. R. China. D Department of Pediatrics, the Affiliated Hospital of Jiangsu University, Zhenjiang 212001, P. R. China. E Corresponding author. Email: [email protected]
Abstract. Decreasing oocyte competence with maternal aging is a major factor in mammalian infertility. One of the factors contributing to this infertility is changes to chromatin modifications, such as histone acetylation in old MII stage oocytes. Recent studies indicate that changes in histone acetylation at MII arise at the germinal vesicle (GV) stage. We hypothesised that histone methylation could also change in old GV oocytes. To test this hypothesis, we examined mono-, di- and trimethylation of histone H3 lysine 4 (H3K4 me1, me2 and me3, respectively) in young and older oocytes from 6–8- and 42–44-week-old mice, respectively. We found that H3K4 me2 and me3 decreased in older compared with young GV oocytes (100% vs 81% and 100% vs 87%, respectively; P , 0.05). H3K4 me2 later increased in older MII oocytes (21% vs 56%; P , 0.05). We also examined the expression of genes encoding the H3K4 demethylases lysine (K)-specific demethylase 1A (Kdm1a) and retinol binding protein 2 (Rbp2). Expression of Kdm1a increased at both the mRNA and protein levels in older GV oocytes, but decreased in older MII oocytes (P , 0.05), and was negatively correlated with H3K4 me2 levels. Conversely, expression of Rbp2 mRNA and protein decreased in older GV oocytes (P , 0.05), and this was not correlated with H3K4 me3 levels. Finally, we showed that inhibition of Kdm1a of older oocytes at the GV stage restored levels of H3K4 me2 at the MII stage to those seen in ‘young’ oocytes (41% vs 38%; P . 0.05). These results suggest that changes in expression of H3K4 me2 and Kdm1a in older GV oocytes may represent a molecular mechanism underlying human infertility caused by aging. Additional keyword: Kdm1a. Received 9 September 2013, accepted 22 November 2013, published online 3 January 2014
Introduction The recent tendency in many countries to postpone child bearing increases the risk of infertility. The main cause of agingrelated infertility in mammals is the poor quality of old oocytes. Several molecular and cellular defects have been reported in old oocytes, such as increased aneuploidy (Pan et al. 2008) and activity of maturation-promoting factor (MPF; Tarı´n et al. 2004), altered mitochondrial morphology (de Bruin et al. 2004) and differential gene expression (Hamatani et al. 2004; Pan et al. 2008). These defects are associated with abnormalities of chromatin modifications, such as histone acetylation and methylation (Kubicek and Jenuwein 2004; Akiyama et al. 2006; Liang et al. 2012). It has been shown that histone acetylation is altered by aging in MII stage oocytes (Huang et al. 2007). In Journal compilation Ó CSIRO 2015
particular, histone H4 acetylation is correlated with aginginduced defects of oocytes at the germinal vesicle (GV) stage (De La Fuente 2006; Manosalva and Gonzalez 2009; van den Berg et al. 2011). Conversely, methylation of histone H3 lysine 4 (H3K4) regulates lifespan and the expression of specific genes involved in longevity (Han and Brunet 2012). H3K4 methylation is generally associated with both gene activation and aging (Eissenberg and Shilatifard 2010; Manosalva and Gonzalez 2010). The di- and trimethylation of H3K4 (H3K4 me2 and me3, respectively) mark the transcriptional start sites of actively transcribed genes (Barski et al. 2007; Mikkelsen et al. 2007), whereas monomethylation of H3K4 (H3K4 me1) is correlated with enhancer sequences (Heintzman et al. 2007). Age-dependent changes in H3K4 me3 have been www.publish.csiro.au/journals/rfd
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observed in the human brain (Cheung et al. 2010; Kuzumaki et al. 2010). The genome-wide H3K4 me3 landscape in neurons reveals that neurons from infants exhibit a larger number of loci with H3K4 me3 than neurons from old adults (Cheung et al. 2010). H3K4 me2 shows higher levels in young MII oocytes than old MII oocytes (Manosalva and Gonzalez 2010). In addition, H3K4 me2 is upregulated during aging of rhesus macaque brain (Han et al. 2012). Several H3K4 demethylases have been implicated as affecting aging in various organisms. Knockdown of lysine (K)-specific demethylase 1A (Kdm1a, Lsd1), a H3K4 me1/2 demethylase, extends lifespan (McColl et al. 2008), whereas suppression of retinol binding protein 2 (Rbp2), a H3K4 me3 demethylase, shortens lifespan in worms (Greer et al. 2010). Deficiency of Rbp2 also shortens longevity in male Drosophila (Li et al. 2010). Moreover, Rbp2 can facilitate demethylation of H3K4 me3 and silencing of retinoblastoma target genes during senescence in primary human cells (Chicas et al. 2012). In the present study, we examined the presence of H3K4 methylation in older GV and MII oocytes. We then determined whether the presence of H3K4 methylation was correlated with the expression of Kdm1a and Rbp2. Finally, we evaluated whether methylation abnormalities due to aging could be corrected pharmacologically in cultured oocytes. Materials and methods Animals In the present study, Kunming (KM) white mice were used as oocyte donors. Young mice were 6–8 weeks of age, whereas older mice were 42–44 weeks old. We chose this age for the older mice because histone acetylation changes have been observed at this age (Akiyama et al. 2006). All mice were maintained under conditions of controlled temperature (25 28C) and lighting (lights from 0700 to 1900 hours) for at least 1 week before use. Animal care and experimental procedures were conducted in accordance with the Animal Research Committee Guidelines of Jiangsu University. Reagents and media Unless stated otherwise, all reagents and media used in the present study were from Sigma-Aldrich (St Louis, MO, USA). M2 (M7167) and M16 (M7292) media were prepared for the handling and culture of mouse oocytes, respectively. Oocyte collection To retrieve fully grown oocytes arrested at prophase I of meiosis, young and older mice were superovulated by injection of 10 IU pregnant mare serum gonadotrophin (PMSG; Ningbo Second Hormone Factory, Zhejiang, China). Ovaries were removed from the mice 48 h after PMSG injection and were transferred to M2 medium. The ovarian follicles were punctured with a 30-gauge needle to release the oocytes in the cumulus oophorus. The cumulus cells surrounding the oocytes were removed by gentle pipetting through a narrow glass pipette in M2 medium. Oocytes displaying GVs were collected. MII-arrested oocytes were retrieved by superovulation of young and older mice following injection of 10 IU PMSG,
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followed by 10 IU human chorionic gonadotropin (hCG; Ningbo Second Hormone Factory) 48 h later. Cumulus–oocyte complexes (COCs) were recovered from oviducal ampullae 14–16 h after hCG injection; cumulus cells were removed by brief incubation in 1 mg mL1 hyaluronidase. The oocytes were then washed with M2 medium until no cumulus cells were observed. IVM of GV oocytes The IVM procedure was performed as described previously (Manosalva and Gonzalez 2009), with few changes. Briefly, COCs were transferred to M16 medium supplemented with 3 mg mL1 bovine serum albumin and incubated in a humidified atmosphere of 5% CO2/95% air at 388C for 18 h. Tranylcypromine treatment Kdm1a demethylase activity during meiosis was inhibited by treating GV stage oocytes with 100 mM tranylcypromine (Biomol International, Plymouth Meeting, PA, USA). The oocytes treated with tranylcypromine for 1 h was washed with M16 medium and cultured for another 17 h. As a control, we used oocytes cultured in medium without tranylcypromine. A stock solution of 137 mM tranylcypromine was diluted with M16 medium immediately before use to the desired concentration. RNA extraction and quantitative real-time polymerase chain reaction RNA was extracted from 20 oocytes isolated from young or older mice using a PicoPure RNA Isolation Kit (Arcturus, Molecular Devices, Mountain View, CA, USA) according to the manufacturer’s instructions. The RNA was treated with DNase I and then reverse transcribed using a PrimeScript RT Reagent Kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. All gene transcripts were quantified by quantitative real-time polymerase chain reaction (PCR). The primer sequences used in the present study were as follows: Kdm1a (GenBank accession number NM_133872.2), 50 -GGTCTTATCAACTTCGG CATCT-30 (forward) and 50 -GCAACTCGTCCACCTACT CG-30 (reverse); Rbp2 (GenBank accession number NM_ 145997.2), 50 -GGTGTATCCGCAGAAATGG-30 (forward) and 50 -TAGGAAGGGAGGAGGTGGT-30 (reverse); and Gapdh (GenBank accession number GU214026.1), 50 -TGG CAAAGTGGAGATTGTTGCC-30 (forward) and 50 -AAGAT GGTGATGGGCTTCCCG-30 (reverse). All PCR were performed using a Bio-Rad CFX96 system with SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. To identify specific amplification of a single PCR product, the product was confirmed by 2% agarose gel electrophoresis. Negative controls, comprising of the PCR reaction mix without nucleic acid, were also run with each group of samples. The abundance of each single gene was determined relative to the abundance of the housekeeping gene Gapdh. Expression levels were quantified using the standard curve method as described previously (Shao et al. 2009). The experiments were repeated at least three times.
Aging alters H3K4 methylation in oocytes
Histology and immunohistochemistry For histological examination of tissue sections, ovaries were fixed in 4% paraformaldehyde overnight at 48C, embedded in paraffin and sectioned using a microtome (RM2145; Leica, Wetzlar, Germany). Immunohistochemical staining was performed on 5-mm sections. Briefly, tissues were deparaffinised with xylene and then rehydrated through three changes of alcohol. After washing in distilled water for 5 min, antigen retrieval was performed by transferring tissue sections to sodium citrate buffer, heating in the microwave at high temperature for 2 min, then low temperature for 28 min. The slides were then rinsed in phosphate-buffered saline (PBS) and endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 20 min at room temperature. The sections were subsequently washed in PBS three times for 5 min each time and then incubated overnight at 48C with either polyclonal rabbit anti-H3K4 me1 (1 : 300; Upstate Biotechnology, Lake Placid, NY, USA), anti-H3K4 me3 (1 : 500; Abcam, Cambridge, MA, USA), anti-Kdm1a (1 : 500; Cell Signaling Technology, Danvers, MA, USA), anti-Rbp2 (1 : 500; Cell Signaling Technology), monoclonal mouse anti-H3K4 me2 (1 : 300; Upstate) or control rabbit or mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) antibodies at the same concentration as the primary antibody. After washing with PBS containing 0.1% Tween-20 (PBST), the sections were incubated with secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (1 : 500; Jackson Immunoresearch) for 1 h at room temperature, followed by washing with PBST. Immune complexes were revealed by diaminobenzidine staining and sections were counterstained with haematoxylin. Slides were observed under a Zeiss Axio Observer microscope (Carl Zeiss, Oberkochen, Germany). Immunofluorescence Young and older oocytes were immunostained with antibodies against H3K4 me1, me2 and me3, Kdm1a and Rbp2, as described previously (Shao et al. 2008a). Briefly, oocytes were fixed with 3.7% paraformaldehyde for 30 min, permeabilised with 0.5% Triton X-100 for 15 min and then incubated with polyclonal rabbit anti-H3K4 me1 (1 : 500; Upstate), anti-H3K4 me3 (1 : 500; Abcam), anti-Kdm1a (1 : 400; Cell Signaling Technology), anti-Rbp2 (1 : 400; Cell Signaling Technology) or monoclonal mouse anti-H3K4 me2 (1 : 500; Upstate) antibodies for 1 h at room temperature. The antibodies that bound to the oocytes were probed with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit or anti-mouse IgG (1 : 300; Jackson Immunoresearch). Sections were counterstained with 40 ,60 -diamidino2-phenylindole (DAPI) to visualise the nuclei. Fluorescence was detected by using a Zeiss Axio Observer microscope equipped with epifluorescence (Carl Zeiss). Exposure times from fluorescent light were kept constant for the respective channel (FITC or DAPI). Quantitation of the fluorescence intensity was determined using the image analyser system SigmaScan-pro V5.01 (SPSS Inc., Chicago, IL, USA), as ratios of H3K4 me1, me2 or me3 to DAPI DNA signals. In each experiment, the average value of fluorescence intensity in young oocytes was set to 1 and the value in older oocytes was expressed relative to this value.
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Statistical analysis The frequency of positive immunostained oocytes was analysed by the Chi-squared test, whereas the intensity of fluorescence and gene expression were analysed by the independent samples t-test. Both tests were performed using SPSS 11.5 software (SPSS Inc.). Data are presented as the mean s.e.m. of three independent experiments. P , 0.05 was considered significant. Results Changes in H3K4 methylation in older GV and MII oocytes Signals for H3K4 me1, me2 and me3 in young and older GV and MII oocytes were examined by immunocytochemistry. All young GV oocytes exhibited H3K4 me1, me2 and me3 (Fig. 1a). Older GV oocytes always showed H3K4 me1 (100%; 118/118), but the percentage decreased for H3K4 me2 (81%; 87/ 107) and me3 (87%; 81/93; Fig. 1b; P , 0.05). In young MII oocytes, signals for H3K4 me2 and me3 were observed only in 21% (18/86) and 78% (62/80) of oocytes, respectively (Fig. 1b). In older MII oocytes, higher percentages were observed for H3K4 me2 (56%; 57/101; P , 0.05), but similar percentages were seen for H3K4 me3 (82%; 93/113; P . 0.05). In contrast, H3K4 me1 signals were not detected in either young (0%; 0/123) or older (0%; 0/110) MII oocytes (Fig. 1b). To specify the differences in methylation in GV and MII oocytes, we measured fluorescence intensities of methylation in young and older oocytes. We found that the fluorescence intensity decreased for H3K4 me2 (0.3-fold) in older GV oocytes (Fig. 1c; P , 0.05). In contrast, the fluorescence intensity increased significantly for both H3K4 me2 (1.4-fold) and me3 (1.6-fold) in older MII oocytes (Fig. 1c; P , 0.05). To confirm the differences in methylation in GV oocytes, we examined H3K4 me2 and me3 in young and older ovaries. We found that H3K4 me2 and me3 were present in both groups of ovaries, but that H3K4 me2 (74%; 17/23) and me3 (83%; 20/24) decreased in older compared with young ovaries (Fig. 1d ). Changes in H3K4 demethylases Kdm1a and Rbp2 in older GV and MII oocytes Because H3K4 methylation was previously related to the Kdm1a and Rbp2 genes (McColl et al. 2008; Greer et al. 2010; Li et al. 2010; Chicas et al. 2012), expression of Kdm1a and Rbp2 was evaluated in the present study using quantitative realtime PCR. There was a significant increase in Kdm1a mRNA levels in older GV oocytes, but a decrease in older MII oocytes (Fig. 2a; P , 0.05), which was negatively correlated with H3K4 me2 levels (Fig. 1b). Conversely, there were lower Rbp2 levels in older GV oocytes (P , 0.05). At the MII stage, there was no change in Rbp2 levels (Fig. 2a; P . 0.05). To examine the location of Kdm1a and Rbp2 proteins, we used immunofluorescence. The Kdm1a and Rbp2 proteins were predominantly localised in the nuclear region of GV and MII oocytes (Fig. 2b). In young oocytes, Kdm1a protein was observed in only 76% (22/29) of GV and 93% (25/27) of MII oocytes. In contrast, in older oocytes, Kdm1a protein was observed in oocytes at the GV (100%; 22/22) and MII (54%; 14/26) stages (Fig. 2c; P , 0.05). Conversely, Rbp2 protein was
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Fig. 1. Histone methylation at histone H3 lysine 4 (H3K4) in young and older oocytes and ovarian follicles. (a) There is a lack of H3K4 di- and trimethylation (me2 and me3, respectively) in older germinal vesicle (GV) oocytes, whereas H3K4 me2 is present in older MII oocytes. Histone methylation was observed by immunofluorescence using specific antibodies against H3K4 monomethylation (me1), me2 and me3 (green). DNA was stained with 40 ,60 -diamidino-2-phenylindole (DAPI; blue). Scale bar ¼ 30 mm. (b) There was a decrease in the percentage of older GV oocytes methylated at H3K4 me2 and me3. The percentage of older MII oocytes methylated at H3K4 me2 increased. *P , 0.05 compared with young oocytes (Chi-squared test). The number of oocytes examined for H3K4 me1, me2 and me3 was: 37, 36 and 36, respectively, in young GV oocytes; 41, 29 and 27, respectively, in young MII oocytes; 39, 36 and 31, respectively, in older GV oocytes; and 37, 34 and 38, respectively, in older MII oocytes. (c) The fluorescence intensity of H3K4 me2 decreased in older GV oocytes, whereas that of H3K4 me2 and me3 increased in older MII oocytes. *P , 0.05 compared with young oocytes (independent samples t-test). The number of oocytes examined for H3K4 me1, me2 and me3 was: 37, 36 and 36, respectively, in young GV oocytes; 41, 29 and 27, respectively, in young MII oocytes; 39, 36 and 31, respectively, in older GV oocytes; and 37, 34 and 38 in older MII oocytes, respectively. Data are the mean s.e.m. (d ) Histological analysis of ovarian follicles to show differential H3K4 methylation between young and older GV oocytes. Arrows indicate that the nucleolus. The number of older oocytes examined for H3K4 me2 and me3 was 23 and 24, respectively. Scale bar ¼ 100 mm.
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Fig. 2. Gene expression and protein immunostaining for lysine (K)-specific demethylase 1A (Kdm1a) and retinol binding protein 2 (Rbp2) in young and older oocytes. (a) Expression of Kdm1a, Rbp2 and Gapdh mRNA in young and older oocytes (germinal vesicle (GV) and MII stages) was detected by quantitative real-time polymerase chain reaction. Kdm1a mRNA expression increased in older GV oocytes and decreased in older MII oocytes. Rbp2 mRNA expression decreased in older GV oocytes. The mRNA levels in young GV oocytes were set to 1. Data are the mean s.e.m. *P , 0.05 compared with young oocytes (independent samples t-test). (b) Immunofluorescence showing Kdm1a protein immunostaining in young and older oocytes (green). Rbp2 immunofluorescence was present in young GV and MII oocytes (green) but not in older GV and MII oocytes. DNA was stained with 40 ,60 -diamidino-2-phenylindole (DAPI; blue). Scale bar ¼ 30 mm. (c) The percentage of older oocytes with Kdm1a protein increased at the GV stage, but decreased at the MII stage. Older oocytes lacked Rbp2 protein at the GV and MII stages. Data are the mean s.e.m. *P , 0.05 compared with young oocytes (Chi-squared test). The number of young GV and MII and older GV and MII oocytes examined for Kdm1a was 29, 27, 22 and 26, respectively. The number of young GV and MII and older GV and MII oocytes examined for Rbp2 was 25, 23, 23 and 24, respectively.
detected only in some young GV (48%, 12/25) and MII (13%, 3/23) oocytes (Fig. 2c). H3K4 methylation in IVM older oocytes To gain further insight into the function of H3K4 methylation, young and older oocytes were subjected to IVM in the presence
and absence of the demethylase inhibitor tranylcypromine. Immunofluorescence of H3K4 me2 after IVM revealed a higher percentage of positive older oocytes (74%; 23/31) than young oocytes (34%; 10/29; Fig. 3a; P , 0.05), in agreement with the in vivo data (Fig. 1b). Tranylcypromine treatment (18 h) further increased the percentage of young (100%; 27/27) and older
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Fig. 3. Histone methylation at histone H3 lysine 4 (H3K4) dimethylation (me2) in oocytes maturated in the absence and presence of tranylcypromine. Oocytes were maturated in vitro for 18 h (a), for 18 h in the presence of tranylcypromine (b) or for 1 h in the presence of tranylcypromine followed by 17 h without tranylcypromine (c). The percentage of older oocytes methylated at H3K4 me2 increased after 18 h maturation in the presence (b) and absence (a) of tranylcypromine maturation, but decreased after 1 h tranylcypromine followed by 17 h without tranylcypromine (c). Data are the mean s.e.m. *P , 0.05 compared with young oocytes (Chi-squared test). The number of young oocytes examined in (a), (b) and (c) was 29, 27 and 24, respectively; the number of older oocytes examined in (a), (b) and (c) was 31, 25 and 27, respectively.
(100%; 25/25) oocytes with H3K4 me2 (Fig. 3b). Remarkably, 1 h exposure to tranylcypromine followed by 17 h culture without tranylcypromine decreased H3K4 me2 levels in older oocytes (41%; 11/27) towards those seen in young oocytes (38%; 9/24; Fig. 3c). Transient tranylcypromine treatment (1 h) of young oocytes did not alter H3K4 me2 levels (38% vs 34%; Fig. 3c). Discussion Most studies into the effects of aging on oocytes have focused on the MII stage (Ottolenghi et al. 2004; Akiyama et al. 2006). These studies reported changes in chromatin modifications, such as DNA methylation and histone acetylation. Herein we report that aging alters H3K4 me2 and Kdm1a levels in GV oocytes. We demonstrated that H3K4 me2 decreased in older GV oocytes, but increased in older MII oocytes, which is negatively correlated with Kdm1a. Furthermore, inhibition of Kdm1a of older oocytes at the GV stage restored levels of H3K4 me2 at the MII stage to those seen in young oocytes. Previous studies reported that H3K4 me1, me2 and me3 are present in young GV oocytes (Sarmento et al. 2004; Kageyama et al. 2007). This was confirmed in the present study using a different mouse strain. Recently, Manosalva and Gonzalez (2010) reported that H3K4 me2 was also present in old GV oocytes. This is consistent with our finding that older oocytes show H3K4 me2/3, but the percentage decreases at the GV stage. Moreover, we observed that there was a higher percentage of older oocytes with H3K4 me2 at the MII stage. However, Manosalva and Gonzalez (2010) also found that young MII oocytes had H3K4 me2, but there was a lower percentage of older MII oocytes with H3K4 me2. The reason for this discrepancy in H3K4 me2 between young and older oocytes remains to be elucidated. Interestingly, we observed a decrease in H3K4 me2/3 in MII oocytes. H3K4 me2/3 is an activating histone methylation that decreases at the MII stage when gene expression is inactive (Eissenberg and Shilatifard 2010). Therefore, the decrease in H3K4 me2/3 may be related to the function of this methylation.
Transcript profiles of oocytes from old mice have been determined on a global scale using microarray analysis (Hamatani et al. 2004; Pan et al. 2008). Hamatani et al. (2004) compared the mRNA expression profiles of MII oocytes from young (5–6 weeks old) and older (42–45 weeks old) C57BL/6 mice. The authors found that approximately 5% (530/11000) of genes were affected by maternal aging. Among the disrupted gene transcripts, some of them, such as Dnmt1, Dnmt3a and Dmap1, were involved in epigenetic modification. Pan et al. (2008) used a different strain of mouse (B6D2F1) at an older age (66 weeks) in another microarray-based gene expression study, and detected many of the same changes, including those in Dnmt3a and Dmap1. In the present study, through real-time PCR and immunostaining analysis, we examined the expression profiles of histone demethylases during oocyte aging using oocytes from young (6–8 weeks) and older (42–44 weeks) KM female mice. We observed that Kdm1a mRNA and protein levels were higher in older GV oocytes, which was negatively correlated with H3K4 me2 levels. Inhibition of Kdm1a induced methylation of H3K4 and abnormalities in preimplantation development in a previous study (Shao et al. 2008b). These data suggest that Kdm1a may be responsible for abnormal demethylation of H3K4 in older oocytes. In contrast, Rbp2 levels were lower in older GV oocytes, like in other senescent cells (Chicas et al. 2012), which does not correlate with H3K4 me3. However, Kdm1a and Rbp2 were not detected in the aforementioned microarray studies in older oocytes (Hamatani et al. 2004; Pan et al. 2008). This is probably because of the different mouse strains and different culture media used. An IVM assay was performed to investigate the impact of extrinsic factors on H3K4 methylation. Our data indicated that the ovary was able to limit H3K4 me2 of older MII oocytes, because IVM of young oocytes increased this methylation. Oocyte-intrinsic factors also contributed to the control of H3K4 me2, because the increase of this methylation was higher in older compared with young oocytes. Interestingly, transient tranylcypromine treatment of older GV oocytes restored normal levels of H3K4 me2 at the MII stage, suggesting that oocyte maturation may require a precise pattern of H3K4 me2.
Aging alters H3K4 methylation in oocytes
A similar study reported that there was a correlation between histone methylation defects in older GV and MII oocytes, because there was a similar percentage of methylated oocytes (Manosalva and Gonzalez 2010). Therefore, changes in histone methylation observed in older MII oocytes may have originated from older GV oocytes. In summary, our data indicate that aging causes abnormalities in histone methylation and demethylase activity at the GV stage. Furthermore, brief tranylcypromine treatment alleviates some of the aging-induced GV abnormalities. Our results provide insights to inform future studies into the effects of reproductive aging on chromatin modifications. Acknowledgements The authors thank for Professor Qiong Lin for critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (81170573), the Open Research Fund Program of Jiangsu Province Key Laboratory for Photon Manufacturing Science and Technology (GZ200710), the Natural Science Foundation of the Jiangsu Higher Education Institutions (09KJB310002) and the Natural Science Foundation of Jiangsu Province (BK2011485).
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Reproduction, Fertility and Development, 2015, 27, 419–426 http://dx.doi.org/10.1071/RD13293
Aging alters histone H3 lysine 4 methylation in mouse germinal vesicle stage oocytes Gen-Bao Shao A,E, Jie Wang B, Liu-Ping Zhang A, Chao-Yang Wu B, Jie Jin A, Jian-Rong Sang C, Hong-Yan Lu D, Ai-Hua Gong A, Feng-Yi Du A and Wan-Xin Peng A A
Department of Biology, Jiangsu University, School of Medical Science and Laboratory Medicine, Zhenjiang 212013, P. R. China. B Department of Radiation Oncology, the Affiliated People’s Hospital of Jiangsu University, Zhenjiang 212002, P. R. China. C Department of Physiology, Jiangsu University, Zhenjiang 212013, P. R. China. D Department of Pediatrics, the Affiliated Hospital of Jiangsu University, Zhenjiang 212001, P. R. China. E Corresponding author. Email: [email protected]
Abstract. Decreasing oocyte competence with maternal aging is a major factor in mammalian infertility. One of the factors contributing to this infertility is changes to chromatin modifications, such as histone acetylation in old MII stage oocytes. Recent studies indicate that changes in histone acetylation at MII arise at the germinal vesicle (GV) stage. We hypothesised that histone methylation could also change in old GV oocytes. To test this hypothesis, we examined mono-, di- and trimethylation of histone H3 lysine 4 (H3K4 me1, me2 and me3, respectively) in young and older oocytes from 6–8- and 42–44-week-old mice, respectively. We found that H3K4 me2 and me3 decreased in older compared with young GV oocytes (100% vs 81% and 100% vs 87%, respectively; P , 0.05). H3K4 me2 later increased in older MII oocytes (21% vs 56%; P , 0.05). We also examined the expression of genes encoding the H3K4 demethylases lysine (K)-specific demethylase 1A (Kdm1a) and retinol binding protein 2 (Rbp2). Expression of Kdm1a increased at both the mRNA and protein levels in older GV oocytes, but decreased in older MII oocytes (P , 0.05), and was negatively correlated with H3K4 me2 levels. Conversely, expression of Rbp2 mRNA and protein decreased in older GV oocytes (P , 0.05), and this was not correlated with H3K4 me3 levels. Finally, we showed that inhibition of Kdm1a of older oocytes at the GV stage restored levels of H3K4 me2 at the MII stage to those seen in ‘young’ oocytes (41% vs 38%; P . 0.05). These results suggest that changes in expression of H3K4 me2 and Kdm1a in older GV oocytes may represent a molecular mechanism underlying human infertility caused by aging. Additional keyword: Kdm1a. Received 9 September 2013, accepted 22 November 2013, published online 3 January 2014
Introduction The recent tendency in many countries to postpone child bearing increases the risk of infertility. The main cause of agingrelated infertility in mammals is the poor quality of old oocytes. Several molecular and cellular defects have been reported in old oocytes, such as increased aneuploidy (Pan et al. 2008) and activity of maturation-promoting factor (MPF; Tarı´n et al. 2004), altered mitochondrial morphology (de Bruin et al. 2004) and differential gene expression (Hamatani et al. 2004; Pan et al. 2008). These defects are associated with abnormalities of chromatin modifications, such as histone acetylation and methylation (Kubicek and Jenuwein 2004; Akiyama et al. 2006; Liang et al. 2012). It has been shown that histone acetylation is altered by aging in MII stage oocytes (Huang et al. 2007). In Journal compilation Ó CSIRO 2015
particular, histone H4 acetylation is correlated with aginginduced defects of oocytes at the germinal vesicle (GV) stage (De La Fuente 2006; Manosalva and Gonzalez 2009; van den Berg et al. 2011). Conversely, methylation of histone H3 lysine 4 (H3K4) regulates lifespan and the expression of specific genes involved in longevity (Han and Brunet 2012). H3K4 methylation is generally associated with both gene activation and aging (Eissenberg and Shilatifard 2010; Manosalva and Gonzalez 2010). The di- and trimethylation of H3K4 (H3K4 me2 and me3, respectively) mark the transcriptional start sites of actively transcribed genes (Barski et al. 2007; Mikkelsen et al. 2007), whereas monomethylation of H3K4 (H3K4 me1) is correlated with enhancer sequences (Heintzman et al. 2007). Age-dependent changes in H3K4 me3 have been www.publish.csiro.au/journals/rfd
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observed in the human brain (Cheung et al. 2010; Kuzumaki et al. 2010). The genome-wide H3K4 me3 landscape in neurons reveals that neurons from infants exhibit a larger number of loci with H3K4 me3 than neurons from old adults (Cheung et al. 2010). H3K4 me2 shows higher levels in young MII oocytes than old MII oocytes (Manosalva and Gonzalez 2010). In addition, H3K4 me2 is upregulated during aging of rhesus macaque brain (Han et al. 2012). Several H3K4 demethylases have been implicated as affecting aging in various organisms. Knockdown of lysine (K)-specific demethylase 1A (Kdm1a, Lsd1), a H3K4 me1/2 demethylase, extends lifespan (McColl et al. 2008), whereas suppression of retinol binding protein 2 (Rbp2), a H3K4 me3 demethylase, shortens lifespan in worms (Greer et al. 2010). Deficiency of Rbp2 also shortens longevity in male Drosophila (Li et al. 2010). Moreover, Rbp2 can facilitate demethylation of H3K4 me3 and silencing of retinoblastoma target genes during senescence in primary human cells (Chicas et al. 2012). In the present study, we examined the presence of H3K4 methylation in older GV and MII oocytes. We then determined whether the presence of H3K4 methylation was correlated with the expression of Kdm1a and Rbp2. Finally, we evaluated whether methylation abnormalities due to aging could be corrected pharmacologically in cultured oocytes. Materials and methods Animals In the present study, Kunming (KM) white mice were used as oocyte donors. Young mice were 6–8 weeks of age, whereas older mice were 42–44 weeks old. We chose this age for the older mice because histone acetylation changes have been observed at this age (Akiyama et al. 2006). All mice were maintained under conditions of controlled temperature (25 28C) and lighting (lights from 0700 to 1900 hours) for at least 1 week before use. Animal care and experimental procedures were conducted in accordance with the Animal Research Committee Guidelines of Jiangsu University. Reagents and media Unless stated otherwise, all reagents and media used in the present study were from Sigma-Aldrich (St Louis, MO, USA). M2 (M7167) and M16 (M7292) media were prepared for the handling and culture of mouse oocytes, respectively. Oocyte collection To retrieve fully grown oocytes arrested at prophase I of meiosis, young and older mice were superovulated by injection of 10 IU pregnant mare serum gonadotrophin (PMSG; Ningbo Second Hormone Factory, Zhejiang, China). Ovaries were removed from the mice 48 h after PMSG injection and were transferred to M2 medium. The ovarian follicles were punctured with a 30-gauge needle to release the oocytes in the cumulus oophorus. The cumulus cells surrounding the oocytes were removed by gentle pipetting through a narrow glass pipette in M2 medium. Oocytes displaying GVs were collected. MII-arrested oocytes were retrieved by superovulation of young and older mice following injection of 10 IU PMSG,
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followed by 10 IU human chorionic gonadotropin (hCG; Ningbo Second Hormone Factory) 48 h later. Cumulus–oocyte complexes (COCs) were recovered from oviducal ampullae 14–16 h after hCG injection; cumulus cells were removed by brief incubation in 1 mg mL1 hyaluronidase. The oocytes were then washed with M2 medium until no cumulus cells were observed. IVM of GV oocytes The IVM procedure was performed as described previously (Manosalva and Gonzalez 2009), with few changes. Briefly, COCs were transferred to M16 medium supplemented with 3 mg mL1 bovine serum albumin and incubated in a humidified atmosphere of 5% CO2/95% air at 388C for 18 h. Tranylcypromine treatment Kdm1a demethylase activity during meiosis was inhibited by treating GV stage oocytes with 100 mM tranylcypromine (Biomol International, Plymouth Meeting, PA, USA). The oocytes treated with tranylcypromine for 1 h was washed with M16 medium and cultured for another 17 h. As a control, we used oocytes cultured in medium without tranylcypromine. A stock solution of 137 mM tranylcypromine was diluted with M16 medium immediately before use to the desired concentration. RNA extraction and quantitative real-time polymerase chain reaction RNA was extracted from 20 oocytes isolated from young or older mice using a PicoPure RNA Isolation Kit (Arcturus, Molecular Devices, Mountain View, CA, USA) according to the manufacturer’s instructions. The RNA was treated with DNase I and then reverse transcribed using a PrimeScript RT Reagent Kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. All gene transcripts were quantified by quantitative real-time polymerase chain reaction (PCR). The primer sequences used in the present study were as follows: Kdm1a (GenBank accession number NM_133872.2), 50 -GGTCTTATCAACTTCGG CATCT-30 (forward) and 50 -GCAACTCGTCCACCTACT CG-30 (reverse); Rbp2 (GenBank accession number NM_ 145997.2), 50 -GGTGTATCCGCAGAAATGG-30 (forward) and 50 -TAGGAAGGGAGGAGGTGGT-30 (reverse); and Gapdh (GenBank accession number GU214026.1), 50 -TGG CAAAGTGGAGATTGTTGCC-30 (forward) and 50 -AAGAT GGTGATGGGCTTCCCG-30 (reverse). All PCR were performed using a Bio-Rad CFX96 system with SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. To identify specific amplification of a single PCR product, the product was confirmed by 2% agarose gel electrophoresis. Negative controls, comprising of the PCR reaction mix without nucleic acid, were also run with each group of samples. The abundance of each single gene was determined relative to the abundance of the housekeeping gene Gapdh. Expression levels were quantified using the standard curve method as described previously (Shao et al. 2009). The experiments were repeated at least three times.
Aging alters H3K4 methylation in oocytes
Histology and immunohistochemistry For histological examination of tissue sections, ovaries were fixed in 4% paraformaldehyde overnight at 48C, embedded in paraffin and sectioned using a microtome (RM2145; Leica, Wetzlar, Germany). Immunohistochemical staining was performed on 5-mm sections. Briefly, tissues were deparaffinised with xylene and then rehydrated through three changes of alcohol. After washing in distilled water for 5 min, antigen retrieval was performed by transferring tissue sections to sodium citrate buffer, heating in the microwave at high temperature for 2 min, then low temperature for 28 min. The slides were then rinsed in phosphate-buffered saline (PBS) and endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 20 min at room temperature. The sections were subsequently washed in PBS three times for 5 min each time and then incubated overnight at 48C with either polyclonal rabbit anti-H3K4 me1 (1 : 300; Upstate Biotechnology, Lake Placid, NY, USA), anti-H3K4 me3 (1 : 500; Abcam, Cambridge, MA, USA), anti-Kdm1a (1 : 500; Cell Signaling Technology, Danvers, MA, USA), anti-Rbp2 (1 : 500; Cell Signaling Technology), monoclonal mouse anti-H3K4 me2 (1 : 300; Upstate) or control rabbit or mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) antibodies at the same concentration as the primary antibody. After washing with PBS containing 0.1% Tween-20 (PBST), the sections were incubated with secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (1 : 500; Jackson Immunoresearch) for 1 h at room temperature, followed by washing with PBST. Immune complexes were revealed by diaminobenzidine staining and sections were counterstained with haematoxylin. Slides were observed under a Zeiss Axio Observer microscope (Carl Zeiss, Oberkochen, Germany). Immunofluorescence Young and older oocytes were immunostained with antibodies against H3K4 me1, me2 and me3, Kdm1a and Rbp2, as described previously (Shao et al. 2008a). Briefly, oocytes were fixed with 3.7% paraformaldehyde for 30 min, permeabilised with 0.5% Triton X-100 for 15 min and then incubated with polyclonal rabbit anti-H3K4 me1 (1 : 500; Upstate), anti-H3K4 me3 (1 : 500; Abcam), anti-Kdm1a (1 : 400; Cell Signaling Technology), anti-Rbp2 (1 : 400; Cell Signaling Technology) or monoclonal mouse anti-H3K4 me2 (1 : 500; Upstate) antibodies for 1 h at room temperature. The antibodies that bound to the oocytes were probed with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit or anti-mouse IgG (1 : 300; Jackson Immunoresearch). Sections were counterstained with 40 ,60 -diamidino2-phenylindole (DAPI) to visualise the nuclei. Fluorescence was detected by using a Zeiss Axio Observer microscope equipped with epifluorescence (Carl Zeiss). Exposure times from fluorescent light were kept constant for the respective channel (FITC or DAPI). Quantitation of the fluorescence intensity was determined using the image analyser system SigmaScan-pro V5.01 (SPSS Inc., Chicago, IL, USA), as ratios of H3K4 me1, me2 or me3 to DAPI DNA signals. In each experiment, the average value of fluorescence intensity in young oocytes was set to 1 and the value in older oocytes was expressed relative to this value.
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Statistical analysis The frequency of positive immunostained oocytes was analysed by the Chi-squared test, whereas the intensity of fluorescence and gene expression were analysed by the independent samples t-test. Both tests were performed using SPSS 11.5 software (SPSS Inc.). Data are presented as the mean s.e.m. of three independent experiments. P , 0.05 was considered significant. Results Changes in H3K4 methylation in older GV and MII oocytes Signals for H3K4 me1, me2 and me3 in young and older GV and MII oocytes were examined by immunocytochemistry. All young GV oocytes exhibited H3K4 me1, me2 and me3 (Fig. 1a). Older GV oocytes always showed H3K4 me1 (100%; 118/118), but the percentage decreased for H3K4 me2 (81%; 87/ 107) and me3 (87%; 81/93; Fig. 1b; P , 0.05). In young MII oocytes, signals for H3K4 me2 and me3 were observed only in 21% (18/86) and 78% (62/80) of oocytes, respectively (Fig. 1b). In older MII oocytes, higher percentages were observed for H3K4 me2 (56%; 57/101; P , 0.05), but similar percentages were seen for H3K4 me3 (82%; 93/113; P . 0.05). In contrast, H3K4 me1 signals were not detected in either young (0%; 0/123) or older (0%; 0/110) MII oocytes (Fig. 1b). To specify the differences in methylation in GV and MII oocytes, we measured fluorescence intensities of methylation in young and older oocytes. We found that the fluorescence intensity decreased for H3K4 me2 (0.3-fold) in older GV oocytes (Fig. 1c; P , 0.05). In contrast, the fluorescence intensity increased significantly for both H3K4 me2 (1.4-fold) and me3 (1.6-fold) in older MII oocytes (Fig. 1c; P , 0.05). To confirm the differences in methylation in GV oocytes, we examined H3K4 me2 and me3 in young and older ovaries. We found that H3K4 me2 and me3 were present in both groups of ovaries, but that H3K4 me2 (74%; 17/23) and me3 (83%; 20/24) decreased in older compared with young ovaries (Fig. 1d ). Changes in H3K4 demethylases Kdm1a and Rbp2 in older GV and MII oocytes Because H3K4 methylation was previously related to the Kdm1a and Rbp2 genes (McColl et al. 2008; Greer et al. 2010; Li et al. 2010; Chicas et al. 2012), expression of Kdm1a and Rbp2 was evaluated in the present study using quantitative realtime PCR. There was a significant increase in Kdm1a mRNA levels in older GV oocytes, but a decrease in older MII oocytes (Fig. 2a; P , 0.05), which was negatively correlated with H3K4 me2 levels (Fig. 1b). Conversely, there were lower Rbp2 levels in older GV oocytes (P , 0.05). At the MII stage, there was no change in Rbp2 levels (Fig. 2a; P . 0.05). To examine the location of Kdm1a and Rbp2 proteins, we used immunofluorescence. The Kdm1a and Rbp2 proteins were predominantly localised in the nuclear region of GV and MII oocytes (Fig. 2b). In young oocytes, Kdm1a protein was observed in only 76% (22/29) of GV and 93% (25/27) of MII oocytes. In contrast, in older oocytes, Kdm1a protein was observed in oocytes at the GV (100%; 22/22) and MII (54%; 14/26) stages (Fig. 2c; P , 0.05). Conversely, Rbp2 protein was
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Fig. 1. Histone methylation at histone H3 lysine 4 (H3K4) in young and older oocytes and ovarian follicles. (a) There is a lack of H3K4 di- and trimethylation (me2 and me3, respectively) in older germinal vesicle (GV) oocytes, whereas H3K4 me2 is present in older MII oocytes. Histone methylation was observed by immunofluorescence using specific antibodies against H3K4 monomethylation (me1), me2 and me3 (green). DNA was stained with 40 ,60 -diamidino-2-phenylindole (DAPI; blue). Scale bar ¼ 30 mm. (b) There was a decrease in the percentage of older GV oocytes methylated at H3K4 me2 and me3. The percentage of older MII oocytes methylated at H3K4 me2 increased. *P , 0.05 compared with young oocytes (Chi-squared test). The number of oocytes examined for H3K4 me1, me2 and me3 was: 37, 36 and 36, respectively, in young GV oocytes; 41, 29 and 27, respectively, in young MII oocytes; 39, 36 and 31, respectively, in older GV oocytes; and 37, 34 and 38, respectively, in older MII oocytes. (c) The fluorescence intensity of H3K4 me2 decreased in older GV oocytes, whereas that of H3K4 me2 and me3 increased in older MII oocytes. *P , 0.05 compared with young oocytes (independent samples t-test). The number of oocytes examined for H3K4 me1, me2 and me3 was: 37, 36 and 36, respectively, in young GV oocytes; 41, 29 and 27, respectively, in young MII oocytes; 39, 36 and 31, respectively, in older GV oocytes; and 37, 34 and 38 in older MII oocytes, respectively. Data are the mean s.e.m. (d ) Histological analysis of ovarian follicles to show differential H3K4 methylation between young and older GV oocytes. Arrows indicate that the nucleolus. The number of older oocytes examined for H3K4 me2 and me3 was 23 and 24, respectively. Scale bar ¼ 100 mm.
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Fig. 2. Gene expression and protein immunostaining for lysine (K)-specific demethylase 1A (Kdm1a) and retinol binding protein 2 (Rbp2) in young and older oocytes. (a) Expression of Kdm1a, Rbp2 and Gapdh mRNA in young and older oocytes (germinal vesicle (GV) and MII stages) was detected by quantitative real-time polymerase chain reaction. Kdm1a mRNA expression increased in older GV oocytes and decreased in older MII oocytes. Rbp2 mRNA expression decreased in older GV oocytes. The mRNA levels in young GV oocytes were set to 1. Data are the mean s.e.m. *P , 0.05 compared with young oocytes (independent samples t-test). (b) Immunofluorescence showing Kdm1a protein immunostaining in young and older oocytes (green). Rbp2 immunofluorescence was present in young GV and MII oocytes (green) but not in older GV and MII oocytes. DNA was stained with 40 ,60 -diamidino-2-phenylindole (DAPI; blue). Scale bar ¼ 30 mm. (c) The percentage of older oocytes with Kdm1a protein increased at the GV stage, but decreased at the MII stage. Older oocytes lacked Rbp2 protein at the GV and MII stages. Data are the mean s.e.m. *P , 0.05 compared with young oocytes (Chi-squared test). The number of young GV and MII and older GV and MII oocytes examined for Kdm1a was 29, 27, 22 and 26, respectively. The number of young GV and MII and older GV and MII oocytes examined for Rbp2 was 25, 23, 23 and 24, respectively.
detected only in some young GV (48%, 12/25) and MII (13%, 3/23) oocytes (Fig. 2c). H3K4 methylation in IVM older oocytes To gain further insight into the function of H3K4 methylation, young and older oocytes were subjected to IVM in the presence
and absence of the demethylase inhibitor tranylcypromine. Immunofluorescence of H3K4 me2 after IVM revealed a higher percentage of positive older oocytes (74%; 23/31) than young oocytes (34%; 10/29; Fig. 3a; P , 0.05), in agreement with the in vivo data (Fig. 1b). Tranylcypromine treatment (18 h) further increased the percentage of young (100%; 27/27) and older
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Fig. 3. Histone methylation at histone H3 lysine 4 (H3K4) dimethylation (me2) in oocytes maturated in the absence and presence of tranylcypromine. Oocytes were maturated in vitro for 18 h (a), for 18 h in the presence of tranylcypromine (b) or for 1 h in the presence of tranylcypromine followed by 17 h without tranylcypromine (c). The percentage of older oocytes methylated at H3K4 me2 increased after 18 h maturation in the presence (b) and absence (a) of tranylcypromine maturation, but decreased after 1 h tranylcypromine followed by 17 h without tranylcypromine (c). Data are the mean s.e.m. *P , 0.05 compared with young oocytes (Chi-squared test). The number of young oocytes examined in (a), (b) and (c) was 29, 27 and 24, respectively; the number of older oocytes examined in (a), (b) and (c) was 31, 25 and 27, respectively.
(100%; 25/25) oocytes with H3K4 me2 (Fig. 3b). Remarkably, 1 h exposure to tranylcypromine followed by 17 h culture without tranylcypromine decreased H3K4 me2 levels in older oocytes (41%; 11/27) towards those seen in young oocytes (38%; 9/24; Fig. 3c). Transient tranylcypromine treatment (1 h) of young oocytes did not alter H3K4 me2 levels (38% vs 34%; Fig. 3c). Discussion Most studies into the effects of aging on oocytes have focused on the MII stage (Ottolenghi et al. 2004; Akiyama et al. 2006). These studies reported changes in chromatin modifications, such as DNA methylation and histone acetylation. Herein we report that aging alters H3K4 me2 and Kdm1a levels in GV oocytes. We demonstrated that H3K4 me2 decreased in older GV oocytes, but increased in older MII oocytes, which is negatively correlated with Kdm1a. Furthermore, inhibition of Kdm1a of older oocytes at the GV stage restored levels of H3K4 me2 at the MII stage to those seen in young oocytes. Previous studies reported that H3K4 me1, me2 and me3 are present in young GV oocytes (Sarmento et al. 2004; Kageyama et al. 2007). This was confirmed in the present study using a different mouse strain. Recently, Manosalva and Gonzalez (2010) reported that H3K4 me2 was also present in old GV oocytes. This is consistent with our finding that older oocytes show H3K4 me2/3, but the percentage decreases at the GV stage. Moreover, we observed that there was a higher percentage of older oocytes with H3K4 me2 at the MII stage. However, Manosalva and Gonzalez (2010) also found that young MII oocytes had H3K4 me2, but there was a lower percentage of older MII oocytes with H3K4 me2. The reason for this discrepancy in H3K4 me2 between young and older oocytes remains to be elucidated. Interestingly, we observed a decrease in H3K4 me2/3 in MII oocytes. H3K4 me2/3 is an activating histone methylation that decreases at the MII stage when gene expression is inactive (Eissenberg and Shilatifard 2010). Therefore, the decrease in H3K4 me2/3 may be related to the function of this methylation.
Transcript profiles of oocytes from old mice have been determined on a global scale using microarray analysis (Hamatani et al. 2004; Pan et al. 2008). Hamatani et al. (2004) compared the mRNA expression profiles of MII oocytes from young (5–6 weeks old) and older (42–45 weeks old) C57BL/6 mice. The authors found that approximately 5% (530/11000) of genes were affected by maternal aging. Among the disrupted gene transcripts, some of them, such as Dnmt1, Dnmt3a and Dmap1, were involved in epigenetic modification. Pan et al. (2008) used a different strain of mouse (B6D2F1) at an older age (66 weeks) in another microarray-based gene expression study, and detected many of the same changes, including those in Dnmt3a and Dmap1. In the present study, through real-time PCR and immunostaining analysis, we examined the expression profiles of histone demethylases during oocyte aging using oocytes from young (6–8 weeks) and older (42–44 weeks) KM female mice. We observed that Kdm1a mRNA and protein levels were higher in older GV oocytes, which was negatively correlated with H3K4 me2 levels. Inhibition of Kdm1a induced methylation of H3K4 and abnormalities in preimplantation development in a previous study (Shao et al. 2008b). These data suggest that Kdm1a may be responsible for abnormal demethylation of H3K4 in older oocytes. In contrast, Rbp2 levels were lower in older GV oocytes, like in other senescent cells (Chicas et al. 2012), which does not correlate with H3K4 me3. However, Kdm1a and Rbp2 were not detected in the aforementioned microarray studies in older oocytes (Hamatani et al. 2004; Pan et al. 2008). This is probably because of the different mouse strains and different culture media used. An IVM assay was performed to investigate the impact of extrinsic factors on H3K4 methylation. Our data indicated that the ovary was able to limit H3K4 me2 of older MII oocytes, because IVM of young oocytes increased this methylation. Oocyte-intrinsic factors also contributed to the control of H3K4 me2, because the increase of this methylation was higher in older compared with young oocytes. Interestingly, transient tranylcypromine treatment of older GV oocytes restored normal levels of H3K4 me2 at the MII stage, suggesting that oocyte maturation may require a precise pattern of H3K4 me2.
Aging alters H3K4 methylation in oocytes
A similar study reported that there was a correlation between histone methylation defects in older GV and MII oocytes, because there was a similar percentage of methylated oocytes (Manosalva and Gonzalez 2010). Therefore, changes in histone methylation observed in older MII oocytes may have originated from older GV oocytes. In summary, our data indicate that aging causes abnormalities in histone methylation and demethylase activity at the GV stage. Furthermore, brief tranylcypromine treatment alleviates some of the aging-induced GV abnormalities. Our results provide insights to inform future studies into the effects of reproductive aging on chromatin modifications. Acknowledgements The authors thank for Professor Qiong Lin for critical reading of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (81170573), the Open Research Fund Program of Jiangsu Province Key Laboratory for Photon Manufacturing Science and Technology (GZ200710), the Natural Science Foundation of the Jiangsu Higher Education Institutions (09KJB310002) and the Natural Science Foundation of Jiangsu Province (BK2011485).
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