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Improvement in vitro maturation culture conditions has been achieved by mimicking in vivo culture environments such as the follicular fluid. Acetic acid is an energy substrate that is abundantly present in the follicular fluid but has not been considered in vitro maturation. This study examined the effects of acetic acid on oocyte quality during nuclear maturation. Cumulus cells and oocyte complexes were collected from the porcine antral follicles of gilt ovaries and matured with 0, 0.1 or 1 mmol/L of acetic acid. After 44 h of in vitro maturation, the energy status, mitochondrial quality and function and embryonic developmental rate following parthenogenetic activation were determined. RNA-sequencing and protein expression analyses were conducted to predict the effects of acetic acid. Supplementation of the in vitro maturation medium with acetic acid (1 mmol/L) improved embryonic development. Oocytes matured with acetic acid had low adenosine triphosphate and lipid contents, mitochondrial membrane potential and reactive oxygen species levels. RNA-sequencing revealed differential expression of genes associated with the adenosine monophosphate-activated protein kinase signalling pathway. Immunostaining revealed that acetic acid increased the levels of phospho-adenosine monophosphate-activated protein kinase, phospho-acetyl-coenzyme A carboxylase, and sirtuin 1 and decreased those of fatty acid synthase and acetyl-coenzyme A synthetase 1. In summary, the use of acetic acid during oocyte maturation improved oocyte developmental ability and metabolism by altering mitochondrial activity and lipid metabolism. • Supplementation of acetic acid during in vitro maturation increases embryonic development in pigs. In brief: Despite its abundance in follicular fluid, acetic acid has not been considered in oocyte maturation. The present study demonstrated that acetic acid treatment during oocyte maturation improves embryonic development and induces metabolic changes. [ABSTRACT FROM AUTHOR]

Mitochondria in oocytes play important roles in many processes, including early embryo development. Promotion of mitochondrial degradation and biogenesis through Sirtuin 1 (SIRT1) activation enhances mitochondrial function and oocyte quality. Previous studies that used somatic cells have shown that short-term heat stress (SHS) induces SIRT1-regulated mitochondrial biogenesis. In this study, we examined whether SHS can induce mitochondrial degradation and biogenesis in porcine oocytes. We collected cumulus cell-oocyte complexes (COCs) from prepubertal gilt ovaries acquired from a slaughterhouse. COCs were treated at 41.5 °C (vehicle: 38.5 °C) for the first one hour of in vitro maturation, and the mitochondrial kinetics, oocyte function, and developmental competence of oocytes were examined. SHS increased the expression level of heat shock protein 72, which induced the high expression of SIRT1 and the phosphorylation of AMP-activated protein kinase. SHS did not alter the mitochondrial DNA copy number in oocytes, but induced mitochondrial degradation and biogenesis, which enhanced the mitochondrial membrane potential and ATP content in oocytes, and improved the ability of the oocytes to develop into blastocysts. [ABSTRACT FROM AUTHOR]

Maternal obesity, a major risk factor for adverse pregnancy complications, results in inflammatory cytokine release in the placenta. Levels of free fatty acids are elevated in the plasma of obese human. These fatty acids include obesity-related palmitic acids, which is a major saturated fatty acid, that promotes inflammatory responses. Increasing evidence indicates that nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasomes mediate inflammatory responses induced by endogenous danger signals. We hypothesized that inflammatory responses associated with gestational obesity cause inflammation. To test this hypothesis, we investigated the effect of palmitic acid on the activation of NLRP3 inflammasomes and inflammatory responses in a human Sw.71 trophoblast cell line. Palmitic acid stimulated caspase-1 activation and markedly increased interleukin (IL)-1β secretion in Sw.71 cells. Treatment with a caspase-1 inhibitor diminished palmitic acid-induced IL-1β release. In addition, NLRP3 and caspase-1 genome editing using a CRISPR/Cas9 system in Sw.71 cells suppressed IL-1β secretion, which was stimulated by palmitic acid. Moreover, palmitic acid stimulated caspase-3 activation and inflammatory cytokine secretion (e.g., IL-6 and IL-8). Palmitic acid-induced cytokine secretion were dependent on caspase-3 activation. In addition, palmitic acid-induced IL-1β, IL-6, and IL-8 secretion was depended on reactive oxygen species (ROS) generation. In conclusion, palmitic acid caused activation of NLRP3 inflammasomes and inflammatory responses, inducing IL-1β, IL-6, and IL-8 secretion, which is associated with ROS generation, in human Sw.71 placental cells. We suggest that obesity-related palmitic acid induces placental inflammation, resulting in association with pregnancy complications. [ABSTRACT FROM AUTHOR]

• pDCs in early-onset preeclampsia was significantly lower than in normal pregnancy. • pDCs in early-onset preeclampsia was significantly lower than in non-pregnancy. • pDCs in late-onset preeclampsia was not different from those in normal pregnancy. • pDCs in late-onset preeclampsia was significantly lower than in non-pregnancy. • Th17 cells in early-onset preeclampsia was higher than in normal pregnancy. The role of plasmacytoid dendritic cells (pDCs), and myeloid dendritic cells (mDCs) in women with preeclampsia has not been elucidated. We compared the frequency of peripheral pDCs, mDCs, NK cells, and T helper 17 (Th17) cells among non-pregnant/pregnant women, and women with early-/late-onset preeclampsia. We examined pDCs and mDCs using Anti-Human Lineage Cocktail 3 (CD3, CD14, CD19, and CD20), HLA-DR, CD11c, and CD123. We detected NK cells using Lineage cocktail, CD8, CD16, and CD56. We determined Th17 cells using CD3, CD4, CD8, CXCR3, and CCR6. We recruited 13 non-pregnant women, 50 normal pregnant women, 13 women with early-onset preeclampsia (onset at <34 gestational weeks), and 10 women with late-onset preeclampsia. The fraction of pDCs in women with early-onset preeclampsia was significantly lower than in non-pregnant women and normal pregnant women at 19−29 gestational weeks (4.1 % vs. 41.2 % and 19.0 %, respectively [ p = 0.0005, and p = 0.025]), however, the fraction of pDCs in late-onset preeclampsia was not significantly different from normal pregnant women at 37 gestational weeks (11.1 % vs. 29.1 %, respectively [ p = 0.149]), although it was significantly lower than in non-pregnant women (11.1 % vs. 41.2 %, respectively [ p = 0.044]). The fraction of Th17 cells in women with early-onset preeclampsia was significantly higher than in normal pregnant women at 19−29 gestational weeks (p = 0.022). In conclusion, the level of circulating pDCs was lower in early-onset preeclampsia than in non-pregnant and pregnant women, suggesting the role of pDCs in the pathogenesis of early-onset preeclampsia. [ABSTRACT FROM AUTHOR]

The first 7 days post-insemination are critical for establishment of pregnancy. The pre-ovulatory luteinizing hormone (LH) surge induces ovulation through disruption of the follicle structure that elucidates pro-inflammatory (Th1) responses. Various types of immune cells are recruited into the corpus luteum (CL) to regulate luteal angiogenesis and progesterone (P4) secretion into the circulation to establish pregnancy. The active sperm-uterine crosstalk also induces Th1 responses, mainly via Toll-like receptor (TLR) 2/4 signaling pathway in vitro. The endometrial glands serve as sensors for sperm signals, which trigger Th1 responses. Conversely, the sperm-oviduct binding generates anti-inflammatory (Th2) responses to support sperm survival until fertilization. It is well-established that embryo-maternal crosstalk starts after the embryo hatches out from the zona pellucida (ZP). However most recently, it was shown that the 16-cell stage bovine embryo starts to secrete interferon-tau (IFNT) that induces Th2 immune responses in the oviduct. Once developing embryos descend into the uterine horn, they induce Th2 responses with interferon-stimulated genes (ISGs) expression in the uterine epithelium and local immune cells mainly via IFNT release. Likewise, multiple embryos in the uterus of superovulated donor cows on D7 post-insemination induce Th2 immune responses with ISGs expressions in circulating immune cells. These findings strongly suggest that the maternal immune system reacts to the embryo during the first 7 days post-insemination to induce fetal tolerance. It became evident that the innate immunity of the developing CL, oviduct, and uterus works together to provide optimal conditions for fertilization and early embryonic development during the first 7 days post-insemination. [ABSTRACT FROM AUTHOR]

The present study investigated the effects of low ethanol exposure on bovine oocytes. Cumulus-oocyte complexes (COCs) were aspirated for the antral follicles of slaughterhouse-derived ovaries. These COCs were incubated in maturation medium containing 0, 0.1, and 0.2% ethanol for 21 h and subjected to fertilization and in vitro development, and then the rates of nuclear maturation, mitochondrial DNA copy number (Mt-cn) and protein (TOMM40), ATP content and lipid content in oocyte, fertilization, and blastulation were examined. Furthermore, COCs were incubated with 0 or 0.1% ethanol and then mitochondrial membrane potential (MMP) and the glucose consumption of COCs was determined. In addition, gene expression in oocytes was examined by RNA sequencing. Ethanol (0.1 and 0.2%) increased Mt-cn and Mt-protein levels whereas 0.2% ethanol increased the blastulation rate and ATP content in oocytes and decreased lipid content in oocytes. Ethanol (0.1%) increased MMP in oocytes and decreased glucose consumption of COCs. Eight stage embryos derived from 0.1% ethanol treated oocytes had higher levels of trimethyl-H3K9 compared with that of nontreated counterpart. RNA sequencing revealed that differentially expressed genes were associated with glycolysis/gluconeogenesis, carbon metabolism, sphingolipid metabolism, amino acid metabolism, and fatty acid degradation pathways. In conclusion, even 0.1% concentrations of ethanol during in vitro maturation considerably affects oocyte metabolism and histone configuration of embryos. • Even low concentration of ethanol affects metabolism and developmental ability of oocytes. • RNA sequencing revealed genes affected by low ethanol exposure. • Mitochondria are major target of ethanol exposure. [ABSTRACT FROM AUTHOR]

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m -chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP. [ABSTRACT FROM AUTHOR]

[Display omitted] • Mitochondrial DNA (mtDNA) copy number and telomere length (TL) correlates in blastocysts. • Paternal aging affects the mtDNA and TL in blastocysts. • Paternal aging affects the mtDNA in issues of offspring. Mitochondrial DNA (mtDNA) copy number and telomere length (TL) in blastocysts derived from the same male mice at young (10–19-week-old) and aged (40–49-week-old) time points and mtDNA and TL in the hearts of offspring derived from young and aged male mice were examined. Paternal aging correlated with reduced mtDNA and TL in blastocysts. mtDNA and TL were significantly correlated, which was also observed in bovine blastocysts. Moreover, mtDNA in the heart of offspring was reduced in male mice with paternal aging. In conclusion, paternal aging affects embryonic mtDNA and TL, potentially impacting their offspring. [ABSTRACT FROM AUTHOR]

Oocyte nuclear maturation depends on sufficient energy supply through oxidative phosphorylation and β-oxidation. AMP-activated protein kinase (AMPK) is an energy sensor controlling the oocyte energy metabolism. The main aim of this study was to examine the effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a potent activator of AMPK, on the ATP content and mitochondrial DNA copy number (Mt-number) of bovine oocytes and on their developmental ability. Oocytes were collected from slaughterhouse-derived bovine ovaries. When these oocytes were cultured in a maturation medium containing 0-, 50-, 250-, and 500-μM AICAR, higher AICAR concentrations reduced the rate of meiotic maturation and the ATP content in oocytes, whereas lower AICAR increased the ATP content in oocytes without affecting the maturation rate. Supplementation of the maturation medium with a low concentration of AICAR (50 and 250 μM) increased phospho-AMPK expression level, as determined by immunostaining. In addition, AICAR treatment increased the ATP content in oocytes, which remained elevated for as long as 2 days after fertilization. On culturing the oocytes with AICAR (250 μM), the fertilization outcome, rate of blastulation, and total cell number of the blastocysts significantly improved. When the proteosomal mitochondrial degradation was inhibited by supplementing the maturation medium with MG132, the Mt-number, as determined by real-time polymerase chain reaction, significantly increased. However, the treatment of oocytes with AICAR did not affect the Mt-number in the presence or absence of MG132. From these data, we conclude that low concentrations of AICAR improved the embryonic developmental ability, presumably via the upregulation of the ATP content in oocytes, but the increase in the ATP content was not due to the upregulation of mitochondrial biogeneration. [ABSTRACT FROM AUTHOR]

Granulosa cells (GCs) contribute to oocyte development. The present study addressed the effect of cryopreservation on the ability of GCs to support oocyte development. GCs were collected from antral follicles. Oocyte granulosa cell complexes (OGCs) derived from early antral follicles were cultured with additional fresh-GCs or frozen-thawed-GCs for 14 days, and the developmental ability and characteristics of the oocytes grown in vitro were examined. Furthermore, fresh- or frozen-thawed-GCs were cultured for two days, and the effects of cryopreservation on the characteristics of GCs were examined. The developmental ability of blastocysts and the acetylation levels of H4K12 in oocytes grown in vitro did not significantly differ among the three culture conditions: OGCs cultured with additional fresh-GCs, frozen-thawed-GCs, or without additional GCs. Although both fresh- and frozen-thawed-GCs exhibited increased ATP content compared with that in oocytes developed without additional GCs, only fresh-GCs showed significantly increased lipid content in oocytes grown in vitro. ATP content, reactive oxygen content, mitochondrial membrane potential, and mitochondrial DNA copy number were greater in cultured frozen-thawed-GCs compared with fresh-GCs. In contrast, lipid content of cultured frozen-thawed-GCs was lower than that of fresh-GCs. Both fresh- and frozen-GCs support oocyte growth, but cryopreservation changes the properties of GCs in a manner that affects the energy status of oocytes grown in vitro. • Addition of granulosa cells to culture systems support growth of oocyte derived from early antral follicles. • Cryopreservation reduces the ability of granulosa cells to support oocytes growth. • Cryopreservation changes the mitochondrial activities in granulosa cells. [ABSTRACT FROM AUTHOR]

The present study addresses molecular backgrounds underlying low oxygen induced metabolic changes and 1.2-fold change in bovine granulosa cell (GCs) proliferation. RNA-seq revealed that low oxygen (5%) upregulated genes associated with HIF-1 and glycolysis and downregulated genes associated with mitochondrial respiration than that in high oxygen level (21%). Low oxygen level induced high glycolytic activity and low mitochondrial function and biogenesis. Low oxygen level enhanced GC proliferation with high expression levels of HIF-1, VEGF, AKT, mTOR, and S6RP, whereas addition of anti-VEGF antibody decreased cellular proliferation with low phosphorylated AKT and mTOR expression levels. Low oxygen level reduced SIRT1, whereas activation of SIRT1 by resveratrol increased mitochondrial replication and decreased cellular proliferation with reduction of phosphorylated mTOR. These results suggest that low oxygen level stimulates the HIF1-VEGF-AKT-mTOR pathway and up-regulates glycolysis, which contributes to GC proliferation, and downregulation of SIRT1 contributes to hypoxia-associated reduction of mitochondria and cellular proliferation. [ABSTRACT FROM AUTHOR]

The developmental origins of health and disease (DOHaD) hypothesis is that future lifestyle diseases in offspring are associated with intrauterine origins in the mother; stress during pregnancy is a risk factor for these diseases in offspring. This study aimed to clarify association of maternal stress with placental dysfunction and offspring development in mice. We applied water stress for 24 h during late pregnancy to explore the metabolic response of offspring to a normal diet (ND) and high-fat diet (HFD). Placental functions were altered by maternal stress, reducing the birth weight of the offspring. In the later life of offspring fed with ND, maternal stress impaired systemic glucose tolerance and altered adipokine secretion in adipose tissue and/or liver. The female offspring of stress-induced dams were light in body weight with lower adipose tissue and smaller adipocytes in both the ND and HFD groups. Abnormal situations, such as dysregulation of plasma glucose levels and fatty liver despite and lower increases in body weight, were observed in the female offspring of stress-induced dams, especially in the HFD-treated group. These findings suggest that long-lasting abnormal conditions and responses to metabolic challenges in maternal stress-induced offspring are linked to placental dysregulation and fetal programming. • Severe maternal stress during pregnancy induces placental dysfunction. • Severe maternal stress alters post-growth adipose and liver function in the offspring. • The effect of severe maternal stress on offspring differs between male and female offspring. [ABSTRACT FROM AUTHOR]

An appropriate immune response by seminal plasma (SP) is essential for a successful pregnancy and is regarded as a sterile inflammatory response. In contrast, the overactivation of NLRP3 inflammasome is involved in pregnancy complications. We investigated whether NLRP3 inflammasome regulates SP-induced immune response for establishment of pregnancy. The pregnancy continuation rate of wild-type (WT) female mice mated with NLRP3-knockout (KO) male mice was lower, and immune response in their uterus were higher than mated with WT male mice. SP of WT or NLRP3-KO mice was injected into the uterus of mice. The implantation numbers were found to decrease in NLRP3-KO-SP group compared with the WT-SP group. Also, cytokine array analysis showed that most cytokine levels of NLRP3-KO-SP were lower than those of WT-SP. In murine macrophages, treatment with WT-SP increased mRNA expression of inflammatory cytokines and protein expression of macrophage differentiation markers, but not in treatment with NLRP3-KO-SP, suggesting that regulatory capacities of SP differed between WT and NLRP3-KO mice. Moreover, NLRP3 protein was detected in WT-SP, but not in NLRP3-KO-SP. In murine macrophages, treatment with NLRP3 protein increased protein expression of M2 type marker. These results suggest that NLRP3 plays an important role in regulating postcoitus immune response for establishment of pregnancy. [ABSTRACT FROM AUTHOR]

This study identified microRNAs (miRNAs) in bovine oviductal fluids (OFs) and examined the effect of miR-17-5p in OFs on embryonic development to the blastocyst stage. Small RNA-seq of extracellular vesicles of OFs revealed 242 miRNAs. Additionally, analyzing expressions of randomly selected OF-miRNAs with RT-qPCR in the culture medium of oviductal epithelial cells indicated that the abundance of miRNAs in OFs increased during the luteal phase. miR-17-5p mimic-treated eight-cell-stage zona pellucida-free embryos showed improved embryonic development to the blastocyst stage. The effect of the miR-17-5p mimic was confirmed using a dual-luciferase assay and immunostaining. In addition, RNA-seq of the miR-17-5p mimic- or control-treated embryos revealed differentially expressed genes (DEGs), suggesting possible pathways that overlapped with the in silico -predicted pathways for miR-17-5p targeting genes. Furthermore, ingenuity pathway analysis of DEG predicted miR-17 to be a significant upstream regulator. Our results suggest that miR-17-5p in OFs regulates embryonic development in bovines. • miR-17-5p presents in OFs of ovulated oviduct at high frequency. • OECs secrete miRNA including miR-17-5p into the culture medium. • RNA-Seq followed by IPA supports the effect of miR-17-5p transfection. • miR-17-5p improves embryonic development to the blastocyst stage. [ABSTRACT FROM AUTHOR]

• The ketone body BHB ameliorates inflammatory cytokine production in human placenta. • BHB suppresses activation of the NLRP3 inflammasome in placenta, resulting in inhibition of IL-1β secretion. • BHB reduces absorption induced by LPS in pregnant mice. The immune system contributes to the regulation of pregnancy, and the disruption of well-controlled immune functions leads to pregnancy complications. Recently, the nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome mechanisms [(a protein complex of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1)] have been reported to play roles in controlling placental inflammation involved in pregnancy pathologies. The ketone body β-hydroxybutyrate (BHB) can suppress NLRP3 inflammasome activation and improve various inflammatory diseases. Therefore, we hypothesized that BHB could suppress activation of the NLRP3 inflammasome in the placenta, resulting in the improvement of pregnancy complications. In human placental tissue culture, treatment with BHB suppressed the secretion levels of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and IL-8, but did not affect the mRNA expression levels of NLRP3 inflammasome-associated factors. Treatment with BHB reduced IL-1β secretion and the amount of mature IL-1β protein induced by lipopolysaccharide (LPS) stimulation in the placenta. In human trophoblast cells, BHB reduced ASC and activated-caspase-1 expression, resulting in the inhibition of IL-1β secretion. To investigate the effect of BHB during pregnancy, we used an animal model of LPS (100 μg/kg intraperitoneally [i.p.] on gestational day 14)-induced pregnancy complications. Administration of BHB (100 mg/kg i.p.) clearly suppressed the absorption rate and IL-1β production in the placenta induced by LPS in pregnant mice. Moreover, LPS-induced pregnancy abnormalities were improved in NLRP3-deficient mice. These findings suggest that BHB play a role in reducing placental inflammation and pregnancy complications via inhibition of NLRP3 inflammasome activation. [ABSTRACT FROM AUTHOR]

Abstract: Zearalenone (Zen) and its metabolites are estrogenic and may be important factors involved in reproductive disorders in domestic animals. We aimed to (1) simultaneously detect Zen and its metabolites in bovine follicular fluids (FFs) by liquid chromatography–tandem mass spectrometry and (2) examine the in vitro effects of Zen on bovine oocytes. Zen and its metabolites were detected in 6 of 32 normal follicles and 7 of 20 cystic follicles. Bovine oocytes were cultured in a maturation media containing various Zen concentrations (0 [control], 1, 10, 100, and 1000μg/L), fertilized, and cultured further. Maturation rates decreased dose-dependently. Further, maturation of 62 (50%) of 124 oocytes examined in the 1000-μg/L group was arrested in metaphase I, without affecting the fertilization rate. Blastocyst-formation rates did not significantly differ among the groups. Zen and its metabolites were detectable in bovine FFs. High Zen concentration may adversely affect meiotic competence but not the fertilization and development rates. [Copyright &y& Elsevier]

Lipid content, ATP content, and histone acetylation are thought to reflect the energy state of cells. In addition, the energy state closely associates with growth and developmental ability of oocytes. Oocyte growth is accompanied by active proliferation of the surrounding granulosa cells (GCs), and GCs play a key role in the provision of energy substrates to the oocytes. In the present study, we first examined the relationship among the average number of GCs per follicle, the average number of cumulus cells (CCs) per oocyte, and the average lipid content in oocytes that developed in vivo within individual donor gilts. Second, we validated the relationship between the number of cells surrounding oocytes and the energy states of oocytes by using an IVC system of oocyte granulosa cell complexes (OGCs) derived from early antral follicles. We collected cumulus cells and oocyte complexes (COCs) from antral follicles (3–5 mm in diameter) and found that average lipid content in oocytes significantly correlated with the average number of both GCs/follicle and CCs/oocyte (P < 0.05). In the next series of experiments, we collected OGCs from early antral follicles (0.5–0.7 mm in diameter), and cultured them for 14 days, and then determined the cell number of OGCs, as well as the lipid content, ATP content, and acetylation level of H4K12 in oocytes grown in vitro . In addition, glucose consumption by OGCs was calculated from the sample media collected at Days 13 and 14. The lipid content of oocytes grown in vitro , significantly correlated with the number of cells surrounding the oocytes (P < 0.01) and with the level of glucose consumption by OGCs (P < 0.01). In addition, both ATP content and H4K12 acetylation levels of oocytes grown in vitro significantly correlated with the number of cells surrounding the oocytes (P < 0.05) and glucose consumption by OGCs (P < 0.05). In conclusion, the lipid content of oocytes depends on the number of cells surrounding the oocytes, and glucose uptake by OGCs is crucial for lipid content and ATP content, and H4K12 acetylation in oocytes. [ABSTRACT FROM AUTHOR]