Effect of in vitro growth on mouse oocyte competency, mitochondria and transcriptome

In vitro generation of fertile oocytes has been reported in several mammalian species. However, oocyte integrity is compromised by in vitro culture. Here, we aimed to understand the factors affecting oocyte competency by evaluating mitochondrial function and transcriptome as well as lipid metabolism in in vivo-derived oocytes and in vitro grown and matured (IVGM) oocytes under atmospheric (20%) and physiological (7%) O2 concentration. We used single-cell RNA-sequencing as well as Gene Ontology and KEGG analyses to identify the molecular pathways affecting the developmental competence of oocytes. Oocytes grown under 20% O2 conditions showed a significant decrease in mitochondrial membrane potential, upregulation of ceramide synthesis pathway-associated genes, and high ceramide accumulation compared with oocytes grown under 7% O2 conditions and in vivo-grown oocytes. This suggests that excess ceramide level causes mitochondrial dysfunction and poor developmental ability of the oocytes. Mitochondrial DNA copy number was lower in IVGM oocytes irrespective of O2 concentration in culture, although there was no common abnormality in the expression of genes related to mitochondrial biosynthesis. In contrast, some oocytes produced under 7% O2 conditions showed gene expression profiles similar to those of in vivo-grown oocytes. In these oocytes, the expression of transcription factors, including Nobox, was restored. Nobox expression correlated with the expression of genes essential for oocyte development. Thus, Nobox may contribute to the establishment of oocyte competency before and after the growth phase. The comprehensive analysis of IVGM oocytes presented here provides a platform for elucidating the mechanism underlying functional oocyte production in vivo.


Introduction
Gametes are the only cells capable of transmitting genetic and epigenetic information to their descendants after fertilization.Oocyte, the female gamete, is responsible for the transmission of organelles, such as mitochondria and endoplasmic reticula, and providing maternal factors necessary for embryonic development.At the initial stage of oogenesis, oocytes contain only a small amount of cytoplasm and mitochondria (Cao et al. 2007).These oocytes are functionally immature and lack meiotic and developmental competencies (Sorensen & Wassarman 1976, Eppig et al. 1994).Once oocytes enter the growth phase, transcription and translation of maternal factors and biosynthesis of organelles are drastically activated (Mtango et al. 2008).As a result, cytoplasmic volume increases by approximately 125-fold, and mitochondrial DNA (mtDNA) copy number increases by approximately 30-fold, as observed in mouse oocytes (Cao et al. 2007), leading to the acquisition of functional competency.However, the entire process of generating competent oocytes remains unknown.
Mitochondria perform multiple functions, including ATP production, Ca 2+ homeostasis, steroid hormone biosynthesis, and apoptosis (Green & Reed 1998).During oocyte growth, the mitochondrial matrix becomes highly dense, indicating the accumulation of factors essential for ATP production and mitochondrial biosynthesis in the fully grown oocytes (Sathananthan & Trounson 2000).As ATP produced in oocytes is utilized for meiosis and early embryonic development (Yu et al. 2010, Dalton et al. 2014), ATP content and mitochondrial membrane potential are indexes of oocyte competency.In fact, aging, obesity, and diabetes reportedly result in impaired female fertility and are associated with mitochondrial dysfunction, including reduced mitochondrial membrane potential, low ATP content, and abnormal cellular localization, in the oocytes (Wang et al. 2009, Wang & Moley 2010, Vural et al. 2015, Wu et al. 2015, Li et al. 2018).High negative potential of the inner mitochondrial membrane, which is created by H + pumps, is essential for the production of ATP and inhibition of cytochrome c release, that is, inhibition of apoptosis, and is an indicator of mitochondrial integrity.
Several studies have reported the production of fertile oocytes from immature ovarian follicles in mice, cows, and pigs.We and others have successfully produced functionally mature oocytes via in vitro growth (IVG) of mouse secondary follicles (Morohaku et al. 2016a,b, Mizumachi et al. 2018, Morohaku 2019).However, the competency of these IVG oocytes was markedly lower than that of in vivogrown oocytes, and only a few of them developed to term after in vitro maturation (IVM), in vitro fertilization (IVF), and embryo transfer (ET).More recently, we evaluated the IVG conditions and demonstrated a significant increase in the yield of fertile oocytes by the addition of high molecular weight polyvinylpyrrolidone (PVP) to the medium and reducing the O 2 concentration from 20 to 7% (Ota et al. 2021).These results were consistent with previous reports on optimal O 2 concentration for IVG of bovine and murine oocytes (Eppig & Wigglesworth 1995, Hirao et al. 2012).Indeed, physiological O 2 concentrations range from 10.5 to 1.4% in the reproductive tract (Fischer et al. 1992, de Castro et al. 2008, Redding et al. 2008).IVM oocytes are known to exhibit reduced developmental ability accompanied by decreased mitochondrial membrane potential and ATP content, increased reactive oxygen species (ROS), and abnormal mitochondrial localization (Nagai et al. 2006, Yuan et al. 2016, Hosseinzadeh Shirzeyli et al. 2020).In the somatic cell, O 2 concentration affects the efficiency of ATP production by mitochondria, regulation of gene expression, and cell fate (Pfander et al. 2003, Kenneth & Rocha 2008).Therefore, mitochondria are pivotal in influencing the developmental competence of IVG oocytes.However, whether O 2 concentration during IVG affects oocyte mitochondrial function and the mechanisms underlying reduced developmental competence of IVG oocytes remain to be elucidated.Identifying factors responsible for abnormalities in IVG oocytes will help understand the process of functional oocyte development in vivo.The present study aimed to determine the effect of O 2 concentration in culture on mitochondrial quality and quantity in IVG oocytes.Furthermore, we aimed to identify the molecular pathway impacting mitochondrial function and developmental competence of IVG oocytes through single-cell RNA-sequencing (scRNA-seq) and lipid analyses.

Animals
All animals were purchased from CLEA Japan Inc. B6D2F1 (C57BL/6N × DBA/2) mice were used for all experiments.The animals were maintained in accordance with the guidelines of the Science Council of Japan, and all experiments were approved by the Institutional Animal Care and Use Committee of the Tokyo University of Agriculture (approval number: 2020048).

Culture
All culture experiments for oocyte and embryo production were conducted as described previously (Morohaku et al. 2017) (Supplementary materials and methods, see section on supplementary materials given at the end of this article).

Oocyte isolation for analyses
Oocytes at the germinal vesicle (GV) stage were collected from explants at day 12 of IVG.As control, in vivo-grown GV oocytes were collected from Graafian follicles of adult mice 44-48 h after administration of 5 IU of equine chorionic gonadotropin (eCG; ASKA Pharmaceutical).If we needed to select a part of COCs to be subjected to two or more analyses, we numbered each COC and chose the number randomly without observation.Then, survived COCs were used for each analysis.To isolate IVG oocytes and in vivo-grown GV oocytes, all cumulus cells were removed by pipetting using fine pulledglass capillary in M2 medium containing 240 µM dibutyryl cyclic AMP (Sigma-Aldrich).
IVGM oocytes at the MII stage were isolated from expanded COCs 17 h after IVM.To obtain control oocytes, 5 IU of hCG was administered to adult mice 44-48 h after 5 IU eCG administration; after 14-16 h, these in vivo-derived MII oocytes were collected.To isolate in vivo-derived MII oocytes and IVGM oocytes, all cumulus cells were removed by pipetting in M2 medium with or without 300 mIU of hyaluronidase (Sigma-Aldrich).

Analysis of mitochondria
IVG/IVGM oocytes and in vivo-derived oocytes were simultaneously observed using the Nikon A1 confocal microscope (Nikon), and images were captured.Fluorescence intensity was measured with NIS-element software (Nikon).
To assess mitochondrial distribution, oocytes were observed after incubation with 100 nM MitoTracker DeepRed FM (Thermo Fisher) in M2 medium containing 1 µg/mL Hoechst 33342 (Dojindo) at 37°C for 1 h.The images which were acquired near the center of the oocytes in the Z-axis with GV in focus were subjected to the analysis.The circular regions of interest were drawn along the GV (perinuclear 30%) and oocyte (cortical 20%), and the oocyte was divided into two regions.Average intensities of perinuclear and cortical regions in each oocyte were measured.When the intensity ratio (perinuclear/ cortical) was within the mean ratio ± 1 s.d. of in vivo-grown oocytes, mitochondrial distribution was judged as a normal pattern.When the ratio was without the mean ratio ± 1 s.d. of in vivo-grown oocytes, mitochondrial distribution was judged as an abnormal pattern.
To evaluate mitochondrial membrane potential, oocytes were incubated with 1% (v/v) JC-1 (Cayman Chemical) in M2 medium at 37°C for 15 min.After washing in M2 medium, the oocytes were observed.Mitochondrial membrane potential was evaluated by Intensities of J-aggregate (red fluorescence)/ monomer (green fluorescence) in each oocyte.
ROS in oocytes were detected with CellROX Green (Thermo Fisher).Oocytes were observed after incubation with 5 µM CellROX Green in M2 medium at 37°C for 1 h.
Intercellular ATP content for each oocyte was measured with an Intracellular ATP assay kit ver.2 (Toyo Ink Group) using a SpectraMax i3 plate reader (Molecular Devices), following the manufacturer's instructions.All samples were analyzed at least in duplicate.
For quantitative analysis of mtDNA copy numbers, zona pellucida of oocytes were digested with 0.5% (w/v) pronase (Sigma-Aldrich), and polar bodies were removed by pipetting.Individual oocytes were lysed using QIAamp DNA Micro Kit (QIAGEN), and mtDNA from each oocyte was eluted in 40 µL of water.Six microliters of this elution were subjected to quantitative PCR (q-PCR) in a 20-µL reaction containing specific primers (forward: 5′-AACCTGGCACTGAGTCACCA-3′, reverse: 5′-GG GTCTG AGTGT ATATA TCATG AAGAG AAT-3 ′), customized Taqman probe (FAM-TCTGT AGCCC TTTTT GTCAC ATGAT C-TAM RA; Thermo Fisher), and TaqMan Universal Master Mix II (Thermo Fisher) (Cao et al. 2007).q-PCR was performed using QuantStudio 3 (Thermo Fisher) with the following conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 60 s.The external standard used was a plasmid (pGEM -T Easy Vector Systems; Promega) in which the PCR product of mtDNA was cloned.All samples were analyzed in duplicate.

scRNA-seq analysis of oocytes
Oocytes obtained via two independent culture experiments were subjected to scRNA-seq analysis.GV oocytes were treated with 0.5% (w/v) pronase in M2 medium to remove zona pellucida.Each oocyte was transferred into a microtube containing 7 µL PBS (−), and a cDNA library was generated using a QIAseq FX Single-Cell RNA Library kit (QIAGEN) following the manufacturer's instructions.The GeneRead Size Selection kit (QIAGEN) was used to clean up the synthesized library.Quality assessment was performed using an Agilent DNA1000 kit (Agilent).Thereafter, quantity assessment was performed using a KAPA Library Quantification kit (KAPA).Finally, all libraries were mixed and subjected to single-end 50 bp sequencing using NextSeq 500 (Illumina).

Measurement of ceramide content
GV oocytes (100-200) after removal of cumulus cells were transferred into a microtube containing 20 µL of water.Pooled oocytes were frozen in liquid nitrogen and stored at -80°C until further analysis.Sample preparation and liquid chromatographmass spectrometry (LC-MS) analysis was performed at the Environmental Technology Department at the Chemicals Evaluation and Research Institute (CERI; Tokyo, Japan).To measure ceramide, lipid was extracted from the pooled sample by the chloroform-methanol extraction method.Lipid extract was analyzed by LC-MS (Nexera XR, Shimadzu; QTRAP 5500, AB sciex).An L-column2 C8 (2.0 × 50 mm) with 3 µm particles was used with 0.3 mL/min flow rate and 3 µL injection volume.The detected peak area in each sample was corrected using the internal standard ceramide (d18:1/16:0; Merck).

Statistical analysis
Data were obtained from three or more independent culture experiments.Data are presented as mean ± s.d. for each group.Tukey-Kramer test was used for multiple comparisons among experimental groups to assess mitochondrial function and quantity.Chi-square test was used to assess the developmental ability of embryos produced from IVGM oocytes.P values less than 0.05 were considered to indicate statistical significance.

Mitochondrial distribution and membrane potential in oocytes produced by IVG and IVM
We found that mouse oocytes produced via IVG and IVM under 20% O 2 condition, hereafter referred to as 20%-IVG and 20%-IVGM oocytes (Fig. 1), respectively, were less capable of supporting development after fertilization than in vivo-derived oocytes.In contrast, oocytes produced by IVG and IVM under 7% O 2 condition, hereafter referred to as 7%-IVG and 7%-IVGM oocytes (Fig. 1), respectively, developed to blastocysts after fertilization at a higher frequency, although not high as the in vivo-derived oocytes (Supplementary Tables 1  and 2).We hypothesized that this poor competency of IVGM oocytes is due to mitochondrial damage.To test this hypothesis, we first analyzed the mitochondrial distribution and membrane potential in IVG/IVGM oocytes.In in vivo-grown oocytes, mitochondria were distributed throughout the cytoplasm and were highly localized around the GV.However, abnormal mitochondrial distribution was observed in 20%-IVG and 7%-IVG oocytes at high frequencies.Mitochondria aggregated extremely around GV or cortical region (Fig. 2A).The relative levels of J-aggregate/monomer at the GV stage were significantly lower in 20%-IVG oocytes (0.4 ± 0.49, n = 68, P < 0.05) compared to that in 7%-IVG oocytes (0.7 ± 0.80, n = 40) and in vivo-grown oocytes (1.0 ± 0.52, n = 46) (Fig. 2B).At the MII stage, relative levels of J-aggregate/monomer were also significantly lower in 20%-IVGM (0.2 ± 0.14, n = 52, P < 0.05) and 7%-IVGM (0.5 ± 0.37, n = 55, P < 0.05) oocytes than that in vivo-derived oocytes (1.0 ± 0.51, n = 44) (Fig. 2C).Thus, mitochondrial membrane potential was remarkably lower in IVG/IVGM oocytes than that in vivo-derived oocytes.However, reduction in mitochondrial membrane potential of IVG/IVGM oocytes was significantly relieved by culturing in 7% O 2 (P < 0.05).
To summarize, IVG/IVGM oocytes exhibited reduced mitochondrial membrane potential although the culture condition with 7% O 2 rescues this abnormality to some extent.Additionally, abnormal mitochondrial distribution and diminished mitochondrial quantity were common abnormality in 20%-IVG/IVGM and 7%-IVG/IVGM oocytes.

Comprehensive analysis of transcriptome of oocytes produced by IVG
To elucidate the cause of poor competency of IVGM oocytes and lower mitochondrial content and membrane potential in IVG/IVGM oocytes, we performed scRNAseq analysis in 20%-IVG, 7%-IVG, and in vivo-grown oocytes at the GV stage.A total of 24,335 genes were detected in 20%-IVG (n = 21), 7%-IVG (n = 24), and in vivo-grown (n = 23) oocytes.Hierarchical clustering analysis revealed differences among the three groups, and transcriptome profiles of 20%-IVG oocytes were distinct from those of 7%-IVG and in vivo-grown oocytes (Fig. 4A).Interestingly, three 7%-IVG oocytes (#44, #37, and #49) were included in the in vivo-grown oocyte cluster.PCA showed that the distance among 20%-IVG, 7%-IVG, and in vivo-grown oocytes was not widely separated but shifted to the left side on the PC1 axis (93.7%) in increasing order of oocyte competency (Fig. 4B).Two oocytes from each of 20%-IVG (#1 and #11) and 7%-IVG (#40 and #56) oocyte groups were plotted on the right side of the clusters.These results suggest that the differences shown by hierarchical clustering analysis and PCA reflect the differences in oocyte competency.Furthermore, GO analysis of the 1450 genes that contributed highly to the PC1 axis variation (more than 2 s.d.) showed enrichment of GO terms such as 'negative regulation of apoptotic process' (Supplementary Fig. 1).This suggests that the variance of the PC1 axis may be associated with the mitochondrial abnormalities in the IVG oocytes described above.
Then, we investigated mitochondria-associated genes (Supplementary Fig. 2), transcription factor A, mitochondria (Tfam), that are involved in mitochondria biogenesis was significantly downregulated in 20%-IVG oocytes (padj < 0.05) but not in 7%-IVG oocytes compared with in vivo-grown oocytes.The expression of nuclear respiratory factor 1 (Nrf1), a Tfam upstream regulator, did not significantly vary among the groups, while that of dynamin 1-like (Dnm1l, also known as Drp1) and mitofusin 1 (Mfn1), which are essential for mitochondrial fission and fusion, respectively (Hall  Novin et al. 2015), were significantly downregulated in 7%-IVG oocytes compared to in vivogrown oocytes (padj < 0.05).On the other hand, several mitochondria-associated genes coded by mtDNA that are involved in oxidative phosphorylation were upregulated in 20%-IVG oocytes (padj < 0.05) but not in 7%-IVG oocytes.The expression levels of well-known mitochondria-associated genes did not delineate the network.

Discussion
Maternal factors, including proteins, mRNA, and organelles, are directly responsible for successful  In the present study, we focused on the recovered developmental competency of IVGM oocytes by reducing O 2 concentration in the culture from atmospheric (20%) to physiological (7%) levels.We investigated the role of mitochondria in the recovery of oocyte competency and identified the pathway impacting mitochondrial function and oocyte competency by molecular analyses.First, we found that mitochondria in 20%-IVG and 7%-IVG oocytes were abnormally aggregated in perinuclear and cortical regions, respectively.The cause of this difference in distribution was unclear in this study.Several studies have shown that abnormal mitochondrial distribution in oocytes correlates with the low mitochondrial quality and low meiotic and developmental competencies (Stojkovic et al. 2001, Brevini et al. 2005, Nagai et al. 2006).However, IVGM oocytes reached the MII stage at a high frequency regardless of the O 2 environment.In contrast, the mitochondrial membrane potential of 20%-IVG/IVGM oocytes was significantly reduced compared with that in 7%-IVG/IVGM and in vivo-derived oocytes, suggesting that attenuation of mitochondrial abnormalities could be one of the causes responsible for recovered developmental competence of 7%-IVGM oocytes compared with those of 20%-IVGM oocytes.scRNA-seq analysis identified abnormalities in the expression of genes associated with apoptosis and sphingolipid signaling pathway, which includes ceramide metabolism, in 20%-IVG oocytes.Ceramide not only directly induces mitochondrial damage but plays a role as a lipid second messenger to mediate activation of apoptosis-associated genes and thereby indirectly induces mitochondrial damage (Gudz et al. 1997, Haimovitz-Friedman et al. 1997).In 20%-IVG oocytes, the expression of gene sets associated with ceramide synthesis was significantly upregulated.Cers6, which was markedly overexpressed in 20%-IVG oocytes, is required for the synthesis of C16-fatty acidcontaining ceramide.The most compelling evidence supporting abnormal ceramide metabolism was the significantly higher C16-fatty acid ceramide (d18:1/16:0) content in 20%-IVG oocytes than that in 7%-IVG and in vivo-grown oocytes.Itami et al. showed that adding ceramide or palmitate, a ceramide source, to the IVM medium detrimentally affects meiotic competency and mitochondrial function in porcine oocytes (Itami et al. 2018).Moreover, ceramide content was reportedly higher in oocytes collected from aged mice than that in oocytes from young mice (Perez et al. 2005).Taken together, we concluded that ceramide metabolism is affected by O 2 concentration during IVG, and excess ceramide accumulation in 20%-IVG oocytes leads to mitochondrial dysfunction and impaired developmental ability.Indeed, follicular O 2 concentration reportedly reduces with follicular growth in the ovaries of humans, cows, and pigs unlike in in vitro constitutive environment ( Fischer et al. 1992, de Castro et al. 2008).Thus, reduction in follicular O 2 concentration possibly inhibits excess ceramide production in oocytes, which in turn ensures mitochondrial integrity in in vivo-grown oocytes.Next, we found that mtDNA copy number was reduced in both IVGM oocytes.Additionally, the IVG oocytes contained higher ATP content than in vivo-grown oocytes at the GV stage although a significant reduction in mitochondrial membrane potential in IVG oocytes compared to that in in vivo-derived oocytes.A previous study showed that mitochondrial cristae change from irregular to transverse and mitochondrial matrix becomes more denser with progression from non-growing to fully grown stage (Sathananthan & Trounson 2000).This suggests that mitochondrial ATP production activity is acquired by the growth phase transition in oocytes.Genetic and biochemical analyses showed that glycolysis is not active in oocytes but the expression of genes associated with oxidative phosphorylation is increased after entering the growth phase (Shimamoto et al. 2019).In contrast, despite the oocyte-specific deletion of pyruvate dehydrogenase E1 alpha1 (Pdha1), growth and ATP accumulation were observed in GV oocytes (Johnson et al. 2007), indicating that ATP in oocytes is either completely supplied by follicle cells, as in the case of Pdha1 deletion, or partially supplemented by these cells during oocyte growth; nevertheless, ATP production in oocytes during growth remains to be elucidated.On the other hand, oocytes are known to produce ATP from pyruvate and consume ATP during meiotic maturation (Downs 1995, Yu et al. 2010, Dalton et al. 2014).In the present study, higher ATP content in IVG oocytes was found to be decreased to the same level as that in in vivoderived oocytes after IVM.This implies that the rate of ATP consumption was more than that of ATP production in IVG oocytes during IVM, which may be attributed to mitochondrial dysfunction.The effects of reduced mitochondrial membrane potential and quantity are evident after the meiotic resumption due to the reduction in excess ATP levels in IVG oocytes which is presumably associated with their subsequent developmental fate.The reason and mechanism for greater ATP accumulation in IVG oocytes than that in in vivo-grown oocytes remain unclear; however, our findings suggest that currently employed IVG culture conditions would cause metabolic alterations in oocytes and/or the surrounding follicle cells.Additionally, the causes of mitochondrial damage in IVG oocytes other than excess ceramide accumulation have not been identified.Recent studies have shown that STAT3 is localized in mitochondria and contributes to activating ATP production and inhibiting ROS production in somatic cells (Carbognin et al. 2016).Stat3 is also involved in transcription and chromatin dynamics in oocytes and early embryos.Considering the fact that STAT3, produced in the non-growing oocytes, persists even in conditionally Stat3-deleted fully grown oocytes by Gdf9-Cre (Haraguchi et al. 2020), understanding of the maternal STAT3 function is limited (Ou-Yang et al. 2021).However, Stat3 that was downregulated in both IVG oocytes and might be responsible for mitochondrial dysfunction in IVG oocytes.
Finally, we used a data mining strategy to identify candidate genes impacting oocyte competency.scRNAseq analysis showed that four IVG oocytes were classified into the same cluster as in vivo-grown oocytes.Among these, we focused on three 7%-IVG oocytes that were IVL oocytes, based on the hypothesis that these oocytes exhibit full competency similar to in vivo-grown oocytes.Then, we identified ten genes that were downregulated in IVG oocytes and found that the expression of genes annotated to 'transcription, DNA-templated' was recovered in IVL oocytes.Among these ten genes, only Nobox is known to be essential for oocyte development.In Nobox-deleted female mice, oogenesis ceased before oocyte growth with concomitant abolishment of Gdf9 expression (Rajkovic et al. 2004).NOBOX binds to the promoter of Gdf9 and Pou5f1 (Choi & Rajkovic 2006).Gdf9 is expressed in oocytes and is essential for follicle growth (Carabatsos et al. 1998).In the present study, expression levels of 87 genes including Gdf9 clearly correlated with that of Nobox.Kit and Nlrp5, whose expression levels were correlated with that of Nobox, are also essential maternal factors (Nishijima et al. 2014, Monk et al. 2017).Till date, Nobox function has been studied mainly before oocyte growth; however, Nobox may control the expression of gene networks throughout oogenesis and thereby regulate the acquisition of oocyte competency.
In conclusion, we showed that IVG oocytes exhibited mitochondrial dysfunction, and this may be partially attributed to excess ceramide accumulation resulting from abnormal metabolism.Our findings indicate that ceramide metabolism is regulated by O 2 concentration during oocyte growth.The expression levels of transcription factors were altered in IVG oocytes but recovered in IVL oocytes.Further studies on cell metabolism in IVG oocytes/follicles and functional analysis of Nobox may open new avenues for understanding the mechanisms responsible for acquiring oocyte competency.

Figure 3
Figure 3 Mitochondrial quantity, ATP content, and ROS accumulation in in vivo-derived and in vitro grown (IVG) and matured (IVGM) oocytes.(A and B) Quantification of mtDNA copy numbers per oocyte in in vivo-derived, 20%-IVG/IVGM, and 7%-IVG/IVGM oocytes at the GV (A) and the MII (B) stages.(C and D) Quantification of ATP content per oocyte in in vivo-derived, 20%-IVG/IVGM, and 7%-IVG/IVGM oocytes at the GV (C) and the MII (D) stages.(E and F) ROS accumulation in 20%-IVG/IVGM and 7%-IVG/IVGM oocytes in terms of relative green fluorescence intensity (CellROX Green) of in vivo-derived oocytes at the GV (E) and the MII (F) stages.Error bars indicate s.d.Different letters represent significant difference (Tukey-Kramer test, P < 0.05).

Figure 7
Figure 7 Identification of candidate genes responsible for close clustering of in vivogrown and in vitro grown (IVG) oocytes.(A) Venn diagram of 6343 DEGs (red and gray; padj < 0.05) between in vivo-grown oocytelike IVG (IVL) oocytes and in vivo-grown oocytes and 4121 DEGs (yellow and gray; padj < 0.05) between canonical 7%-IVG oocytes and IVL oocytes.The expression levels of 1623 genes (yellow) in IVL oocytes were restored to those in in vivo-grown oocytes.GO analysis of the 1623 DEGs showed enrichment of GO term 'regulation of transcription and DNA-templated'.Horizontal axis indicates −log 10 (q-value).(B) Gene expression levels in IVL (gray) and in vivo-grown (white) oocytes relative to that in canonical-7%-IVG oocytes (black).(C) Correlation between the expression of Nobox and other genes [log 2 (CPM+1)≥6].White and gray dots indicate a correlation score ≥ 0.7 and < 0.7, respectively.