Both diet and gene mutation induced obesity affect oocyte quality in mice

Obesity was shown to cause reproductive dysfunctions such as reduced conception, infertility and early pregnancy loss. However, the possible effects of obesity on oocyte quality are still not fully understood. In this study we investigated the effects of both diet and gene mutation induced obesity on impairments in mouse oocyte polarization, oxidative stress, and epigenetic modifications. Our results showed that high-fat diet induced obesity (HFD) and gene mutation induced obesity (ob/ob) could both impair oocyte meiotic maturation, disrupt spindle morphology, and reduce oocyte polarity. Oocytes from obese mice underwent oxidative stress, as shown by high DHE and ROS levels. Abnormal mitochondrial distributions and structures were observed in oocytes from obese groups of mice and early apoptosis signals were detected, which suggesting that oxidative stress had impaired mitochondrial function and resulted in oocyte apoptosis. Our results also showed that 5 mC levels and H3K9 and H3K27 methylation levels were altered in oocytes from obese mice, which indicated that DNA methylation and histone methylation had been affected. Our results showed that both HFD and ob/ob induced obesity affected oocyte maturation and that oxidative stress-induced early apoptosis and altered epigenetic modifications may be the reasons for reduced oocyte quality in obese mice.

showed that abnormal mitochondrial structures and functions in oocytes were associated with poor fertilization rates and abnormal embryo development in mice 18 . Furthermore, it was shown that cells that were suffering from oxidative stress resulted in their apoptosis, which may have resulted from impaired mitochondrial function and the activation of apoptotic factors in mouse kidneys 19 .
Epigenetic phenomena are heritable changes in gene expression that do not involve alterations in nucleotide sequences. These modifications include chromatin methylation and post-translational modifications of histones or other chromatin-associated proteins. DNA methylation is a key epigenetic modification that is essential for normal embryonic development 20 , and epigenetic information linked to environmental factors participates in the early stages of development 21 . A previous study showed that DNA methylation was altered in the oocytes of HFD induced obesity mice and in the oocytes and livers of their offspring 22 . Moreover, changes in histone modifications are key components of an epigenetic network that controls energy homeostasis 23 . For example, H3K9 and H3K27 play important roles in regulating gene expression in mitotic cells 24 . H3K9 methylation is also an epigenetic marker of parental genome origin during early preimplantation embryo development 25 .
In this study, we investigated maturation capability, spindle morphology, changes in oocyte polarization, oxidative stress levels, mitochondrial distributions and morphologies, and any epigenetic modifications in oocytes from both HFD and ob/ob mice. We compared the effects of both diet-induced and gene mutation-induced obesity on oocyte quality, and investigated the mechanisms possibly involved in these oocytes.
Obesity disrupts meiotic spindle morphology and reduces oocyte polarity. We next examined spindle morphology and the localization of actin filaments to assess any oocyte quality changes. The majority of MI oocytes in the control group had well-aligned chromosomes and normal spindle morphologies. However, a higher proportion of oocytes had misaligned chromosomes and disrupted spindle morphologies in the obesity groups. The abnormal rates of spindle morphology were: 7.07 ± 1.91% (n = 115) for controls vs. 15.91 ± 3.62% (n = 155) for HFD oocytes and 30.77 ± 2.51% (n = 127) for ob/ob oocytes (P < 0.05; Fig. 2A).
We next examined actin cap formation and the distributions of cortical granules (CGs) at MІ stage. For metaphase I (MI) oocytes from HFD and ob/ob mice, actin fluorescence intensity at the plasma membrane and in the cytoplasm was significantly lower (0.65 ± 0.04 for membrane actin and 0.31 ± 0.03 for cytoplasmic actin in HFD oocytes vs. 0.74 ± 0.14 for membrane actin and 0.62 ± 0.06 for cytoplasmic actin in ob/ob oocytes) than that in control MI oocytes (1.00 ± 0.00 for membrane actin and 1.00 ± 0.00 for cytoplasmic actin) (P < 0.05; Fig. 2B). We also examined CGs, which is an indicator of oocyte polarity. In control oocytes, CGs were clearly located under the plasma membrane and absent in areas close to chromosomes, whereas CGs were uniformly localized at the plasma membrane and CG signals were weaker in oocytes from mice with obesity. The formation of cortical granules-free domain (CGFD) was analyzed as a further feature of oocyte polarity. The proportions of CGFD were decreased in HFD oocyte (9.33 ± 1.76%, n = 65) and in ob/ob oocyte (8.75 ± 1.25%, n = 49), while it was 66.67 ± 4.41% (n = 76) in control oocyte (P < 0.05; Fig. 2C).
Obesity induces oxidative stress in mouse oocytes. Oxidative stress is a cellular response to noxious external stimuli. To determine whether obesity induced oxidative stress in diet and gene mutation induced mice with obesity, we first investigated dihydroethidium (DHE) and reactive oxygen species (ROS) levels in GV oocytes. This showed that DHE and ROS levels were both increased in GV oocytes from the two obesity groups. We then assessed the fluorescence intensity ratios of oxidative stress signals in oocytes from the HFD and ob/ ob groups. The fluorescence intensity ratios of DHE in GV oocytes from obesity groups (1.55 ± 0.17 for HFD mice and 2.70 ± 0.28 for ob/ob mice) were significantly higher than those of controls (P < 0.05; Fig. 3A). And the fluorescence intensity ratios of ROS in GV oocytes from both obesity mice (34.38 ± 7.56 for HFD mice and 26.53 ± 5.66 for ob/ob mice) were significantly increased compared to controls (P < 0.05; Fig. 3B).
Next, we examined mRNA expression levels of oxidative stress related genes. This showed that the mRNA levels of the genes for anti-oxidative enzymes, including GSH-Px, SOD, and CAT, were significantly increased in HFD and ob/ob oocytes as compared to those in controls (Fig. 3C). The relative mRNA levels as compared to those in controls were: 4.42 ± 0.32 for GSH-Px, 3.11 ± 0.35 for SOD, and 4.21 ± 0.47 for CAT in HFD oocytes; and 1.86 ± 0.50 for GSH-Px, 2.91 ± 0.35 for SOD, and 1.63 ± 0.23 for CAT in ob/ob oocytes (Fig. 3C).
Next, we used transmission electron microscopy to examine for any changes in mitochondrial morphology. This showed that the oocyte mitochondria from mice on the high fat diet had broken membranes, fewer cristae, increased swelling, and more vacuoles (Fig. 4B), which was consistent with previous results 11 .
These results indicated that HFD and ob/ob obesity had profound effects as early as the oocyte meiosis stage and might interfere with oocyte metabolism due to induced oxidative stress and impaired mitochondria.
We also examined for early apoptosis signals in oocytes. This showed that Annexin V/PI fluorescence signals were increased in oocytes from HFD (14.38 ± 2.94) and ob/ob (13.19 ± 3.43) mice with obesity as compared to those in controls (1.00 ± 0.00) (P < 0.05; Fig. 4C). Furthermore, we examined Bak and Bcl-2 mRNA expressions in oocytes, which results confirmed the results of Annexin V/PI staining. The fold-changes in Bak and Bcl-2 mRNA expression of ob/ob group and the Bcl-2 mRNA expression of HFD group were increased (1.31 ± 0.23, p > 0.1 for Bak and 3.71 ± 0.80, p < 0.05 for Bcl-2 of HFD group; 2.07 ± 0.57, p < 0.05 for Bak and 2.85 ± 0.39, p < 0.05 for Bcl-2 of ob/ob group) compared to the controls (Fig. 4D).
Obesity affects epigenetic modification levels in mouse oocytes. Two typical epigenetic modifications are well known to significantly influence DNA function: DNA methylation and histone modifications. Then we attempted to examine the epigenetic modifications in the NSN (no Hoechst-positive rim surrounding the nucleolus) GV oocytes, since NSN stage oocytes had a higher gene transcription activity which could reflect the early methylation levels 27 . We assessed the levels of 5 mC in GV oocytes, which showed that 5 mC expression levels both in HFD (0.61 ± 0.06) and ob/ob (0.36 ± 0.01) oocytes were reduced as compared to controls (1.00 ± 0.00) (P < 0.05; Fig. 5A). We also found that the H3K27-me2 fluorescence intensity ratios were reduced in ob/ob (0.49 ± 0.12, p < 0.05) oocytes as compared to controls (1.00 ± 0.00; Fig. 5B) but not in HFD oocytes (0.74 ± 0.09, p > 0.1); whereas the H3K9-me2 fluorescence intensity ratios were increased in ob/ob (1.62 ± 0.10, p < 0.05) oocytes as compared to controls (1.00 ± 0.00; Fig. 5C) but not in HFD oocytes (1.09 ± 0.17, p > 0.1).

Discussion
The burdens of obesity and the associated health problems have become global issues. Our results suggest that oocytes from both HFD and ob/ob mice with obesity had low rates of maturation, disrupted spindle morphologies, and reduced oocyte polarization. We also found that oocytes from HFD and ob/ob mice with obesity were suffering from oxidative stress, had alterations in mitochondria distributions, and experienced early apoptosis. Additionally, these oocytes had changes in DNA cytosine methylation and the methylation patterns of H3K9-me2 and H3K27-me2.
A previous study demonstrated that oocytes from HFD induced mice with obesity were smaller and had delayed maturation as compared to those from control mice 10 . In our study, we found that the numbers of oocytes from both HFD and ob/ob induced mice with obesity were reduced as compared to those from normal mice, and the maturated proportions and polar body extrusion percentages were reduced as compared to those oocytes from control mice. Abnormal spindles can impair oocyte meiosis and suppress polar body extrusion 28 . Other studies showed that the abnormal spindle morphologies of oocytes from HFD induced mice with obesity and diabetic model mice were increased 11,18 . We also found high percentages of abnormal spindle morphologies in the oocytes from both HFD and ob/ob induced mice with obesity. Moreover, during the late metaphase I stage, cortical granules (CGs) were lost in the region overlying the chromosomes and microfilaments were enriched under the membrane near the spindle to form an actin cap, which are features of oocyte polarity formation 29 . Our results showed that these two features were lost in most oocytes from mice with obesity, which further confirmed our hypothesis that HFD and ob/ob induced obesity both affected oocyte quality. Previous work showed that the fertility of mice with obesity was reduced which was due to the early embryonic loss in high fat diet induced obesity mice 11 . Our results, together with previous reports indicated that the infertility in mice with obesity might be due to the low quality of oocytes, which was reflected by the disrupted subcellular structures like cytoskeleton, mitochondria or chromosomes.
To determine a possible mechanism for how obesity affected oocyte quality, we investigated oxidative stress, apoptosis, and epigenetic modifications. HFD induced maternal obesity has been shown to be associated with increased ROS generation in different tissues 30,31 . Obesity also resulted in oxidative DNA damage and related genes' expression changes in breast tissue 32 . In this study, we showed that oocytes were suffering from oxidative stress and that anti-oxidative gene mRNA expression levels were increased in the oocytes from both HFD and ob/ob induced mice with obesity. Impairments in mitochondria were shown to be related to oxidative stress and apoptosis 5 , and excessive nutrient intake was associated with poor reproductive outcomes in women with obesity and mice and their early embryo mitochondrial metabolism exhibited alterations 33,34 .
Other previous work indicated that this abnormal distribution pattern of mitochondria was related to apoptosis 7 , and HFD induced mice with obesity had fewer follicles and more apoptotic granulosa cells in their follicles as compared to normal mice 8 . In this study, we found that the oocytes from both HFD mice with obesity and ob/ob mice with obesity had higher proportions of the clustered distribution of mitochondria, early apoptosis signals, and higher levels of apoptosis related genes' mRNA expression as compared to the oocytes from control mice. This indicated that similar to other cell types, oocytes from mice with obesity were also suffering from oxidative stress and early apoptosis. This may be one reason for the low quality of the oocytes from mice with obesity. 5 mC genomic levels are related to the metabolism and proliferation of cells 35,36 . In a study of oocytes and embryos, it was confirmed that the maternal DNA methylation pattern was maintained until the 16-cell stage 37 . Other reports showed that H3K27 was related to lipid metabolism 38 , and that H3K9 modulation in chromatin might be a new target for treating obesity and metabolic syndrome 39 . Studies of type 2 diabetes mellitus (T2DM) and HFD induced obesity demonstrated that DNA methylation was changed to promote genes' expression related to mitochondrial biogenesis and function 4 . In our study, we found that the histone modification H3K9-me2 was enhanced, while the DNA methylation of 5 mC and histone modification H3K27-me2 were reduced in oocytes from H3K27-me2 genomic contents. The H3K27-me2 fluorescence intensity ratio was reduced in oocytes from both HFD and ob/ob induced mice with obesity as compared to that in controls. (C) H3K9-me2 genomic contents. The H3K9-me2 fluorescence intensity ratio was increased in oocytes from both HFD and ob/ob induced mice with obesity as compared to that in controls.
Scientific RepoRts | 6:18858 | DOI: 10.1038/srep18858 both HFD and ob/ob mice with obesity. These results indicated that epigenetic modifications were altered in both HFD and ob/ob oocytes, which may have been another reason for the low quality of oocytes from mice with obesity.
In conclusion, we found that both HFD and ob/ob induced obesity in mice resulted in low oocyte quality, and that oxidative stress, early apoptosis, and epigenetic modification alterations might have been reasons for this phenomenon.
Scientific RepoRts | 6:18858 | DOI: 10.1038/srep18858 oocytes were incubated at room temperature for 1 h with a mouse anti-5 mC antibody (1:1000). After three washes in PB1, they were labelled with an Alexa Fluor 594 goat anti-mouse antibody (1:1000) at room temperature for 40 min. Then they were co-stained with Hoechst 33342 (10 μ g/mL in PBS) for 10 min, then samples were analyzed by confocal microscopy.
For single staining of H3K27-me2 or H3K9-me2, oocytes were fixed in 4% paraformaldehyde in PBS at room temperature for 30 min. They were then transferred to membrane permeabilization solution (0.5% Triton X-100) for 20 min. After washing with PBS containing 1% BSA (PB1), oocytes were incubated with rabbit anti-H3K27me2 and H3K9me2 antibodies (1:200) overnight at 4 °C and then stained with Alexa Fluor 488 goat anti-rabbit antibody (1:500) at room temperature for 40 min. Then they were co-stained with Hoechst 33342 (10 μ g/mL in PBS) for 10 min, then samples were analyzed by confocal microscopy.
Oocytes were then mounted on glass slides and examined with a Zeiss LSM 700 Meta scanning confocal microscope (Carl Zeiss, Inc.; Jena, Germany). At least 30 oocytes were examined for each experimental group. And the fluorescence intensity was analyzed by Image J.
Transmission Electron Microscopy. Ovary tissue fragments were cut into 1 mm 3 cubes, pre-fixed with 1% osmium tetroxide at RT for 1 h, fixed with 2.5% glutaraldehyde at 4 °C overnight, dehydrated using a graded series of ethanol and 100% acetone, and then embedded in epoxy resin. Ultrathin sections were cut, mounted on grids, and stained with uranyl acetate. These sections were observed with an H-7650 transmission electron microscope (Hitachi High-Technologies Corporation) and scanned with a Model S-3000N Scanning Electron Microscope. We get 10 sections per ovary, and analyzed the morphology of mitochondria in the sections.