Abstract 202: Engineering Artificial Sinus Node by Reprogrammed Cardiomyocytes

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Hu Yufeng ◽  
Shih-Ann Chen
Keyword(s):  
Sinus Node ◽  
Neonatal Rat ◽  
Immunofluorescent Staining ◽  
Three Dimension ◽  
Over Expression ◽  
Pilot Model ◽  
Engineered Tissues ◽  
Beating Rate ◽  
Engineered Tissue ◽  
Supportive Cells

The lack of clinically relevant sinoatrial node (SAN) disease model makes the pathophysiological investigation and therapeutic development stagnant. We hypothesize that engineering SAN by TBX18 somatic-reprogrammed cardiomyocytes on the three-dimension (3D) scaffold could create an in vitro SAN model, sharing similar features with a native SAN. Methods: In addition to neonatal rat ventricular cardiomyocytes (NRVMs) alone, we chose cardiosphere-derived cells (CDCs), or fibroblasts as supportive cells with different mixing ratios to construct engineered SAN. Hydrogel scaffolds including matrigels or platelet gels were used and compared. The engineered tissue was reprogrammed by TBX18 over-expression. Results: The over-expression of TBX18 increased HCN4 and CX45 transcriptions in cardiomyocytes. A stable spontaneous beating rate could be created in TBX18-reprogrammed engineered tissue, made of NRVMs and fibroblasts with matrigel scaffold (beating rate, TBX18 vs. control: 105.0 ± 10.7 bpm vs. 35.5±7.1 bpm, n=12, P<0.001). Although spontaneous beating could be observed in reprogrammed engineered tissues by NRVM alone, NRVM with CDCs, or NRVMs with CDCs and fibroblasts, the beating rates were not stable and slower. The beating rate in engineered tissue did not differ between scaffolds of matrigel and platelet gel. However, inter-experimental variation is higher in platelet gels, compared to matrigels. By immunofluorescent staining, an unique spatial distribution of NRVMs and fibroblasts was identified. NRVMs formed the central core of engineered tissues, encapsulated by fibroblasts, which was similar to a native SAN. The application of a sympathomimetic drug (epinephrine) doubled the beating rate of reprogrammed engineered tissue (P=0.02, n=6-8). Conclusions: A pilot model of engineered SAN was established by TBX18-reprogrammed cardiomyocytes. The supportive cells such as fibroblasts played an important role in tissue engineering of SAN.

scholarly journals Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells

2014 ◽  
Vol 143 (5) ◽  
pp. 577-604 ◽  
Author(s):  
Michael D. Stern ◽  
Larissa A. Maltseva ◽  
Magdalena Juhaszova ◽  
Steven J. Sollott ◽  
Edward G. Lakatta ◽  
...  
Keyword(s):  
Hierarchical Clustering ◽  
Calcium Release ◽  
Sinoatrial Node ◽  
Sinus Node ◽  
Ryanodine Receptors ◽  
Membrane Currents ◽  
Immunofluorescent Staining ◽  
Adrenergic Stimulation ◽  
Beating Rate ◽  
Sinoatrial Node Cells

The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This “calcium clock” takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by &gt;1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic–mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating rate in response to β-adrenergic stimulation. The model indicates that the hierarchical clustering of surface RyRs in SANCs may be a crucial adaptive mechanism. Pathological desynchronization of the clocks may explain sinus node dysfunction in heart failure and RyR mutations.

scholarly journals Qiliqiangxin Attenuates Phenylephrine-Induced Cardiac Hypertrophy through Downregulation of MiR-199a-5p

2016 ◽  
Vol 38 (5) ◽  
pp. 1743-1751 ◽  
Author(s):  
Haifeng Zhang ◽  
Shanshan Li ◽  
Qiulian Zhou ◽  
Qi Sun ◽  
Shutong Shen ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cardiac Hypertrophy ◽  
Natriuretic Peptide ◽  
Neonatal Rat ◽  
Immunofluorescent Staining ◽  
Mouse Heart ◽  
Mrna Levels ◽  
Elevated Protein ◽  
Ischemic Stress

Background/Aims: Qiliqiangxin (QL), a traditional Chinese medicine, has long been used to treat chronic heart failure. Previous studies demonstrated that QL could prevent cardiac remodeling and hypertrophy in response to hypertensive or ischemic stress. However, little is known about whether QL could modulate cardiac hypertrophy in vitro, and (if so) whether it is through modulation of specific hypertrophy-related microRNA. Methods: The primary neonatal rat ventricular cardiomyocytes were isolated, cultured, and treated with phenylephrine (PE, 50 µmol/L, 48 h) to induce hypertrophy in vitro, in the presence or absence of pretreatment with QL (0.5 µg/ml, 48 h). The cell surface area was determined by immunofluorescent staining for α-actinin. The mRNA levels of hypertrophic markers including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (MYH7) were assayed by qRT-PCRs. The protein synthesis of cardiomyocytes was determined by the protein/DNA ratio. The miR-199a-5p expression level was quantified in PE-treated cardiomyocytes and heart samples from acute myocardial infarction (AMI) mouse model. MiR-199a-5p overexpression was used to determine its role in the anti-hypertrophic effect of QL on cardiomyocytes. Results: PE induced obvious enlargement of cell surface in cardiomyocytes, paralleling with increased ANP, BNP, and MYH7 mRNA levels and elevated protein/DNA ratio. All these changes were reversed by the treatment with QL. Meanwhile, miR-199a-5p was increased in AMI mouse heart tissues. Of note, the increase of miR-199a-5p in PE-treated cardiomyocytes was reversed by the treatment with QL. Moreover, overexpression of miR-199a-5p abolished the anti-hypertrophic effect of QL on cardiomyocytes. Conclusion: QL prevents PE-induced cardiac hypertrophy. MiR-199a-5p is increased in cardiac hypertrophy, while reduced by treatment with QL. miR-199a-5p suppression is essential for the anti-hypertrophic effect of QL on cardiomyocytes.

Abstract 14951: Maresin 1 Induces Cardiomyocyte Hypertrophy Through RORα/IGF-1/PI3K/Akt Pathway

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Tri Wahyuni ◽  
Arisa Kobayashi ◽  
Shota Tanaka ◽  
Yoshiaki Miyake ◽  
Ayaha Yamamoto ◽  
...  
Keyword(s):  
Surface Area ◽  
Neonatal Rat ◽  
Immunofluorescent Staining ◽  
Akt Pathway ◽  
Cardiomyocyte Hypertrophy ◽  
Lipid Mediator ◽  
Myocardial Inflammation ◽  
Rat Cardiomyocytes ◽  
Phosphorylated Akt ◽  
Maresin 1

Myocardial inflammation is a critical event for the onset and progression of the heart failure. Maresin 1 (MaR1) was originally identified as a macrophage lipid mediator that exhibits anti-inflammatory and pro-resolving activities. Though it is widely accepted that macrophages positively and negatively regulate myocardial inflammation through cytokines and growth factors, the biological functions of lipid mediators, such as MaR1, in cardiomyocytes remain to be addressed. This study explored the functional roles of MaR1 in cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) were stimulated with MaR1 for 48 hours. Immunofluorescent staining with anti-sarcomeric α-actinin antibody revealed that MaR1 (50 nM) induced a significant increase in cardiomyocyte surface area (1760.34±66.86μm 2 vs. 960.83±29.46μm 2 ). Quantitative RT-PCR analyses revealed that the treatment with MaR1 upregulated the expression of IGF-1 mRNA (2.9±0.6 folds), accompanied by the enhanced level of total and phosphorylated Akt. Interestingly, MaR1 did not influence the expression of BNP and skeletal actin significantly, suggesting that MaR1 induced physiological hypertrophy. Since MaR1 is a ligand of RORα, we examined the effects of RORα blockade (SR3335) and found that this compound inhibited the increase of cardiomyocyte surface area by abrogating MaR1-mediated activation of IGF-1/PI3K/Akt pathway. Importantly, treatment with wortmannin or NVP-AEW541, inhibitors for PI3K or IGF-1 receptor, respectively, suppressed MaR1-induced cardiomyocyte hypertrophy, indicating that IGF-1/PI3K/Akt pathway is essential for MaR1-induced hypertrophy. In conclusion, MaR1 is a novel lipid mediator that induces physiological cardiomyocyte hypertrophy by activating RORα/IGF-1/PI3K/Akt pathway. Thus, MaR1 could coordinate the resolving process and tissue recovery in myocardial inflammation.

Carbonic anhydrase II promotes cardiomyocyte hypertrophy

2012 ◽  
Vol 90 (12) ◽  
pp. 1599-1610 ◽  
Author(s):  
Brittany F. Brown ◽  
Anita Quon ◽  
Jason R.B. Dyck ◽  
Joseph R. Casey
Keyword(s):  
Carbonic Anhydrase ◽  
Ventricular Myocytes ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Carbonic Anhydrase Ii ◽  
Cardiomyocyte Hypertrophy ◽  
Wild Type ◽  
Over Expression ◽  
Pathological Cardiac Hypertrophy ◽  
Neonatal Rat Ventricular Myocytes

Pathological cardiac hypertrophy, the maladaptive remodelling of the myocardium, often progresses to heart failure. The sodium–proton exchanger (NHE1) and chloride–bicarbonate exchanger (AE3) have been implicated as important in the hypertrophic cascade. Carbonic anhydrase II (CAII) provides substrates for these transporters (protons and bicarbonate, respectively). CAII physically interacts with NHE1 and AE3, enhancing their respective ion transport activities by increasing the concentration of substrate at their transport sites. Earlier studies found that a broad-spectrum carbonic anhydrase inhibitor prevented cardiomyocyte hypertrophy (CH), suggesting that carbonic anhydrase is important in the development of hypertrophy. Here we investigated whether cytosolic CAII was the CA isoform involved in hypertrophy. Neonatal rat ventricular myocytes (NRVMs) were transduced with recombinant adenoviral constructs to over-express wild-type or catalytically inactive CAII (CAII-V143Y). Over-expression of wild-type CAII in NRVMs did not affect CH development. In contrast, CAII-V143Y over-expression suppressed the response to hypertrophic stimuli, suggesting that CAII-V143Y behaves in a dominant negative fashion over endogenous CAII to suppress hypertrophy. We also examined CAII-deficient (Car2) mice, whose hearts exhibit physiological hypertrophy without any decrease in cardiac function. Moreover, cardiomyocytes from Car2 mice do not respond to prohypertrophic stimulation. Together, these findings support a role of CAII in promoting CH.

scholarly journals Modulation of cardiac myocyte beating rate and hypertrophy by cardiac fibroblasts isolated from neonatal rat ventricle.

1993 ◽  
Vol 57 (9) ◽  
pp. 912-920 ◽  
Author(s):  
HIROYUKI ORITA ◽  
MANABU FUKASAWA ◽  
SHIGEKI HIROOKA ◽  
HIDEAKI UCHINO ◽  
KANA FUKUI ◽  
...  
Keyword(s):  
Cardiac Myocyte ◽  
Cardiac Fibroblasts ◽  
Neonatal Rat ◽  
Beating Rate ◽  
Rat Ventricle

Cytotoxic effects of cardioplegic solutions and cytoprotective effects of insulin on immature cardiac myocytes during hypothermic preservation

1995 ◽  
Vol 5 (3) ◽  
pp. 243-250
Author(s):  
Hiroyuki Orita ◽  
Manabu Fukasawa ◽  
Hideaki Uchino ◽  
Tetsuro Uchida ◽  
Satoshi Shiono ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Neonatal Rat ◽  
Basic Solution ◽  
Cytotoxic Effects ◽  
Beneficial Effects ◽  
Ringer’S Solution ◽  
Beating Rate ◽  
Lactated Ringer's Solution ◽  
Immature Myocardium ◽  
Cardioplegic Solutions

AbstractThe purpose of this study was to evaluate the functional and biochemical effects of cardioplegic solutions on immature cardiac myocytes incubated under hypothermic conditions. In addition, the effects of insulin as an additive were evaluated in each solution. Cardiac myocytes were isolated from neonatal rat ventricles and cultured for four days; 12.5 x 105myocytes/flask were then incubated at 4 °C for three, six and 12 hours in three types of cardioplegic solutions—glucose-potassium solution (glucose: 50 gm/l, NaHCO3: 20 mEq, KCl: 20 mEq), lactated Ringer's solution (KCl: 20 mEq) and St. Thomas' Hospital solution. After each hypothermic incubation, enzymes were measured in the incubation solutions. The myocytes were then cultured for an additional 24 hours at 37 °C to evaluate the recovery of the myocyte beating rate. In the Ringer's group, the recovery ratio of the myocyte beating rate was complete at three hours, then decreased to 48.8 percent of control (beating rate prior to hypothermic incubation) at 12 hours. The glucose-potassium and St. Thomas' groups had significantly lower recovery ratios than the Ringer's group, beginning at three hours (63.4, 72.9, 95.6 percent, respectively). Release of enzymes (CPK and LDH) in the Ringer's group gradually increased and at 12 hours was 29.0 mIU/flask and 260.0 mIU/flask, respectively. The St. Thomas' group, in contrast, had significantly increased values for CPK at 12 hours to 116.0 mIU/flask, and the greatest increases of both enzymes were observed in the glucose-potassium group at 12 hours (CPK: 115.5, LDH: 1163.9). By addition of 20 IU/l insulin, marked improvements were observed in the Ringer's and glucose-potassium groups both functionally and biochemically. Thus, the lactated Ringer's solution had the least cytotoxic effects that might be suitable for a basic solution of various cardioplegic solutions during the neonatal period, and insulin may have beneficial effects on immature myocardium under hypothermic conditions.

Effects of K-channel blockers, calcium, and verapamil suggest different pacemaker mechanisms in cultured neonatal rat and embryonic chick ventricle cells

1989 ◽  
Vol 67 (7) ◽  
pp. 795-800 ◽  
Author(s):  
Otto F. Schanne ◽  
L. Boutin ◽  
J. Derosiers
Keyword(s):  
Cardiac Cells ◽  
Neonatal Rat ◽  
K Channel ◽  
Chronotropic Effect ◽  
Inward Current ◽  
Diastolic Depolarization ◽  
K Current ◽  
Beating Rate ◽  
Rat Ventricle ◽  
Diastolic Potential

We compared the determinants of spontaneous activity in explanted neonatal (2-day-old) rat ventricle cells and in reaggregates derived from 15-day-old chick embryos. We studied the beating rate with an optical recording method and the underlying electrical activity with glass microelectrodes using the K current blockers cesium (Cs) and tetraethylammonium, varied Ca concentrations, and the Ca antagonist verapamil. In the rat (i) Cs increased the beating rate that was mediated by an increase in the slope of the diastolic potential, (ii) Ca increased the beating rate dramatically at low and medium concentrations to decrease it again at 8 mM Cao.2This increase in the beating rate was mediated by an increase of the slope of the diastolic depolarization. (iii) The beating rate decreased with verapamil at concentrations between 0.5 and 2.0 μM. The effects of Cs and Ca suggest that an increase in net inward current (block of IK1) underlies the positive chronotropic effect of Cs and that the pacemaker mechanism is determined by a Ca inward current or an IT1 type current modulated by variations of Cai. In the chick reaggregates (i) Cs and tetraethylammonium decreased the beating rate that was mainly brought about by a decrease in the slope of diastolic depolarization. (ii) Ca increased the beating rate but to a lesser degree than in the rat and there was no decrease of the beating rate at higher concentrations. (iii) The increase in the beating rate was not mediated by an increase in the slope of the diastolic potential but mainly by a depolarization of the maximum diastolic potential. (iv) Verapamil inhibited electrogenesis before any change in the diastolic potential was evident. The negative chronotropic effect of Cs and tetraethylammonium is compatible with the notion that a voltage- and time-dependent K current was inhibited and that this current determines the pacemaker. Moreover, the Ca component of the pacemaker mechanism in explanted rat ventricle cells resembles either that of the sinoatrial node or represents triggered activity.Key words: pacemaker mechanism, cultured cardiac cells, K-channel blocker, calcium, verapamil.

scholarly journals COX5A over-expression protects cortical neurons from hypoxic ischemic injury in neonatal rats associated with TPI up-regulation

2020 ◽  
Author(s):  
Ya Jiang ◽  
Xue Bai ◽  
Ting-Ting Li ◽  
Mohammed AL Hawwas ◽  
Yuan Jin ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Molecular Mechanisms ◽  
Cerebral Injury ◽  
Neurological Dysfunction ◽  
Neurological Injury ◽  
Immunofluorescent Staining ◽  
Neonatal Rats ◽  
Over Expression ◽  
Qrt Pcr

Abstract Background: Neonatal hypoxic-ischemic encephalopathy (HIE) represents as a major cause of neonatal morbidity and mortality. However, the underlying molecular mechanisms in brain damage are still not fully elucidated. This study was conducted to determine the specific potential molecular mechanism in the hypoxic-ischemic induced cerebral injury. Methods: Here, hypoxic-ischemic (HI) animal models were established and primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) to mimic HIE model in-vivo and in - vitro . The HI-induced neurological injury was evaluated by Zea-longa scores, Triphenyte-trazoliumchloride (TTC) staining the Terminal Deoxynucleotidyl Transferased Utp Nick End Labeling (TUNEL) and immunofluorescent staining. Then the expression of Cytochrome c oxidase subunit 5a (COX5A) was determined by immunohistochemistry, western blotting (WB) and quantitative real time Polymerase Chain Reaction (qRT-PCR) techniques. Moreover, HSV-mediated COX5A over-expression virus was transducted into OGD neurons to explore the role of COX5A in - vitro , and the underlying mechanism was predicted by GeneMANIA, then verified by WB and qRT-PCR. Results: HI induced a severe neurological dysfunction, brain infarction, and cell apoptosis as well as obvious neuron loss in neonatal rats, in corresponding to the decrease on the expression of COX5A in both sides of the brain . What’s more, COX5A over-expression significantly promoted the neuronal survival, reduced the apoptosis rate, and markedly increased the neurites length after OGD. Moreover, Triosephosephate isomerase (TPI) was predicted as physical interactions with COX5A, and COX5A over-expression largely increased the expressional level of TPI. Conclusions: The present findings suggest that COX5A plays an important role in promoting neurological recovery after HI, and this process is related to TPI up-regulation.

Abstract 1060: Regulation of Protease Activated Receptor-1 signaling by G-protein coupled receptor kinase 3

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Liam M Casey ◽  
Jason Greenman ◽  
Frederick Aguilar ◽  
Olga Dunaevsky ◽  
Burns C Blaxall
Keyword(s):  
Heart Failure ◽  
G Protein ◽  
Ischemic Injury ◽  
Neonatal Rat ◽  
Cell Surface Receptor ◽  
Immunofluorescent Staining ◽  
Nuclear Accumulation ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) play crucial roles in normal heart function and dysregulated GPCR signaling contributes to heart failure (HF). Protease-activated receptors (PARs) are one class of GPCR expressed in the heart. Emerging evidence demonstrates that excessive PAR-1 signaling induces cardiac dysfunction. An important component of PAR signaling is ERK1/2, which is phosphorylated in response to PAR stimulation and can influence myocyte hypertrophy and survival. Cytoplasmic pERK1/2 accumulation depends in part on scaffolding complexes that assemble with internalized GPCRs. Activation of an internalization-defective PAR mutant leads to enhanced nuclear pERK1/2 accumulation upon stimulation. The nuclear/cytoplasmic distribution of pERK1/2 may be a key factor in determining the cellular effects of PAR stimulation as elevated nuclear pERK in cardiomyocytes is suggested to promote survival and physiological hypertrophy. Phosphorylation of PARs by G-protein coupled receptor kinase 3 (GRK3) is thought to promote receptor internalization, potentially influencing overall pERK1/2 accumulation and subcellular distribution. We have used a dominant negative form of GRK3 lacking the kinase domain (GRK3ct) to test the hypothesis that GRK3 influences PAR1 internalization and ERK1/2 phosphorylation. By measuring cell surface receptor levels we demonstrate that GRK3ct interferes with PAR1 internalization. Immunofluorescent staining and cellular fractionation techniques further show that GRK3ct enhances nuclear accumulation of pERK1/2 in COS-7 cells and adult mouse cardiomyocytes. Furthermore we find that GRK3ct overexpression in neonatal rat cardiomyocytes increases PAR1-activation induced physiologic hypertrophy. In summary these results may explain recent unpublished reports that mice overexpressing GRK3ct in the heart are protected against ischemic injury, a heart failure model that involves pathologic PAR signaling. Thus we conclude that following ischemic injury, reducing PAR1 internalization via interfering with endogenous GRK3 activity or promoting nuclear pERK accumulation might improve cardiac recovery.

Evidence that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovaryThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (4) ◽  
pp. 621-628 ◽  
Author(s):  
Xu-Xia Sui ◽  
Li-Li Luo ◽  
Jin-Jie Xu ◽  
Yu-Cai Fu
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Caspase 3 ◽  
Neonatal Rat ◽  
Immunofluorescent Staining ◽  
Oocyte Nucleus ◽  
Caspase 8 ◽  
Double Labeling ◽  
Cycle Arrest ◽  
Old Rats

Previous studies have proposed that the forkhead transcription factor FOXO3a is involved in cell cycle arrest and apoptosis and that it may also repress follicular development by inducing cell cycle arrest in ovaries. We have recently demonstrated that FOXO3a induces oocyte apoptosis of neonatal rat ovaries under in vitro conditions. In the present study, we evaluated the role of FOXO3a in oocyte apoptosis under in vivo conditions. Ovaries from rats were obtained from newborns on postnatal day (PD) 1, 2, 3, and 4. TUNEL assay results showed that oocyte apoptosis occurred mainly on PD 1 and 2. Immunohistochemical staining of FOXO3a, Bim, Fas ligand (FasL), p27KIP1, caspase-8, and caspase-3 showed that they were all expressed mainly in naked oocytes on PD 1 and 2. The percentage of positive FOXO3a staining of oocytes reached peak levels in the ovaries of 2-day-old rats, which was consistent with the rate of the apoptotic profiles determined by TUNEL. The percentage between TUNEL-positive and FOXO3a-positive oocytes in the nucleus showed no statistical differences within the 4-day-old rat ovaries. Furthermore, the positive oocyte percentage of the target factors of FOXO3a (Bim, p27KIP1, and FasL) and pro-apoptotic proteins (caspase-3 and caspase-8) also reached peak levels in the ovaries of 2-day-old rats, which was similar to the rate of FOXO3a-positive oocytes. These results suggest that FOXO3a in the oocyte nucleus is involved in oocyte apoptosis; that is, FOXO3a-positive oocytes may be the apoptotic cells. To verify this, rat oocytes were subjected to TUNEL and immunofluorescent double-labeling assays. We found that TUNEL-positive cells were also FOXO3a-, Bim-, or FasL-positive. To identify the downstream target of FOXO3a, double immunofluorescent staining with antibodies to Bim and FasL was performed. We found that FOXO3a-positive cells were also Bim- and FasL-positive. We conclude that the overexpression of FOXO3a in the oocyte nucleus of neonatal rat ovaries may play an important role in the apoptosis of naked oocytes, and that Bim, FasL, and p27KIP1 are the key downstream factors of FOXO3a.

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