Computed tomography-based radiomics to predict early recurrence of hepatocellular carcinoma post-hepatectomy in patients background on cirrhosis.

BACKGROUND: The prognosis for hepatocellular carcinoma (HCC) in the presence of cirrhosis is unfavourable, primarily attributable to the high incidence of recurrence. AIM: To develop a machine learning model for predicting early recurrence (ER) of post-hepatectomy HCC in patients with cirrhosis and to stratify patients' overall survival (OS) based on the predicted risk of recurrence. METHODS: In this retrospective study, 214 HCC patients with cirrhosis who underwent curative hepatectomy were examined. Radiomics feature selection was conducted using the least absolute shrinkage and selection operator and recursive feature elimination methods. Clinical-radiologic features were selected through univariate and multivariate logistic regression analyses. Five machine learning methods were used for model comparison, aiming to identify the optimal model. The model's performance was evaluated using the receiver operating characteristic curve [area under the curve (AUC)], calibration, and decision curve analysis. Additionally, the Kaplan-Meier (K-M) curve was used to evaluate the stratification effect of the model on patient OS. RESULTS: Within this study, the most effective predictive performance for ER of post-hepatectomy HCC in the background of cirrhosis was demonstrated by a model that integrated radiomics features and clinical-radiologic features. In the training cohort, this model attained an AUC of 0.844, while in the validation cohort, it achieved a value of 0.790. The K-M curves illustrated that the combined model not only facilitated risk stratification but also exhibited significant discriminatory ability concerning patients' OS. CONCLUSION: The combined model, integrating both radiomics and clinical-radiologic characteristics, exhibited excellent performance in HCC with cirrhosis. The K-M curves assessing OS revealed statistically significant differences.

[Establishment of STO Cell Lines Expressing Green Fluorescent Protein and Mouse Leukemia Inhibitory Factor].

OBJECTIVE: To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer. METHODS: The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed. RESULTS: The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer. CONCLUSION: The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.

[Effect of Endomorphin-1 on Maturation and Expression of TLR4 in Peripheral Blood Dendritic Cells Induced by High Glucose].

OBJECTIVE: To investigate the effects of endomorphin-1 (EM-1) on the maturation phenotype, cytokine secretion, T cell proliferation and TLR4 expression in human peripheral blood dendritic cells (PBDCs) stimulated and induced by high glucose, and to explore the regulatory mechanism of EM-1 on DC immune function. METHODS: Peripheral blood mononuclear cells (PBMNCs) were induced into immature dendritic cells (imDCs). The high glucose was used as the stimulating factor, and the EM-1 was used as the interventional factor. Then, the experiments were divided into normal glucose group (NG group), high glucose group (HG group), high glucose plus EM-1 group (EM group) and high glucose plus EM-1 and naloxone group (Nal group), respectively. The PBDC's phenotype changes were detected by flow cytometry; ELISA was used to detect the changes of cytokines secreted by PBDCs co-cultured with autologous lymphocytes; CFSE was used to detect the proliferation of T lymphocytes. TLR4 expression on PBDC surface was detected by RT-PCR. RESULTS: Compared with HG group, the expression of PBDC surface molecules CD86, CCR7 and CD36 was up-regulated in EM group (P<0.01), while the change of CD83 expression was not statistically significant. However, IL-12 and IL-10 secreted by PBDCs and the proliferation index of T-lymphocytes stimulated by PBDCs were both decreased in EM group. Compared with EM group, the expression of CD86, CCR7 and CD36 was decreased in Nal group (P<0.01), while the expression of CD83 was almost unchanged (P>0.05). T-lymphocyte proliferation index was increased very significantly in Nal group (P<0.01). The gray ratio of TLR4 in HG group was higher than that in NG group, while the gray ratio in EM group's was very significantly lower than that in HG group's (P<0.01). These results indicate that the high glucose can promote the expression of PBDC TLR4, while the EM-1 inhibits the expression of TLR4. CONCLUSION: EM-1 up-regulates the expression of PBDC surface molecules CD86, CCR7 and CD36 stimulated and induced by high glucose, but inhibites the induction of PBDC to maturity by high glucose. And the secreted inflammatory cytokines IL-12 and IL-10 inhibites the proliferation of T lymphocytes derived from PBDCs, while naloxone inhibites the effect of EM-1. EM-1 inhibites the expression of TLR4 on PBDC surface induced by high glucose.

Inhibitory effects of the constituents of Hippophae rhamnoides on 3T3-L1 cell differentiation and nitric oxide production in RAW264.7 cells.

Three new flavonol glycosides, hippophaeosides A-C (1-3), together with 27 known constituents, were isolated from Hippophae rhamnoides L. leaves. Their structures were determined by spectroscopic analyses. Their inhibitory activities on 3T3-L1 preadipocyte differentiation and triglyceride accumulation in maturing adipocytes, and nitric oxide production in RAW264.7 cells were examined.

Triterpenoids from Hippophae rhamnoides L. and their nitric oxide production-inhibitory and DPPH radical-scavenging activities.

In our investigation on the chemical constituents of Hippophae rhamnoides L., the chloroform-soluble fraction of the 80% acetone extract of branch bark was observed to inhibit nitric oxide (NO) production in a lipopolysaccharide and recombinant mouse interferon-gamma-activated murine macrophage-like cell line, RAW 264.7 cells. Two new triterpenoids, 2-O-trans-p-coumaroyl maslinic acid (1) and 2-O-caffeoyl maslinic acid (2), and three known triterpenoids, oleanolic acid (3), 3-O-trans-p-coumaroyl oleanolic acid (4), and 3-O-caffeoyl oleanolic acid (5), and 6-methoxy-2H-1-benzopyran (6) and beta-sitosterol (7) were isolated from the branch bark extract. Their inhibitory activities on the production of NO in RAW 264.7 cells and radical-scavenging activities were examined.

[Micro machining of micro-cantilever probes for efficient deposition for biochips].

Biochip technology will bring a tremendous revolution to life science and medical research in 21 century. Microarray assays represent an essential technical advance in biomedical research. Recently, the demand for microarray assay technology has spring up. Therefore, low cost and flexible techniques are needed to meet specific requirements for increasingly integrated biochips. Also performance must be improved in terms of speed and sensitivity. To this end, promising approaches, mainly based on micro and nanotechnologies, have been developed. In this paper, the design and microfabrication of a novel type of micro-cantilever probe are introduced. These probes were fabricated using silicon dioxide by Micro-electromechanical System (MEMS) techniques, and they featured one micron split gap, microchannels and self-replenishing reservoirs. All fabricated micro-cantilever probe were tested on Nanoarrayer instrumentation. Cy3-streptavidin was loaded as biological sample and patterned on DSU gold surface. Results showed these probes were capable of generating high quality biological arrays with routine spot sizes of 2 - 3 microns and could deposit at least three thousand spots without reloading. The spot size could potentially achieve sub-micron when probe size was further shrunk down by the high-resolution lithography technique or more precise microfabrication technologies, such as E-beam lithography. To further improve sample loading efficiency, it is needed to modify the cantilever surface in order to better confine sample inside the microchannel and reservoir, which will be researched in the future.

Localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during the first meiosis and its functions as a spindle checkpoint protein.

The present study was designed to investigate the localization of mitotic arrest deficient 1 (MAD1) in mouse oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at various stages during the first meiosis were fixed and immunostained for MAD1, kinetochores, microtubules, and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes were treated with nocodazole or Taxol before examination. The anti-MAD1 antibody was injected into the oocytes at the germinal vesicle (GV) stage for examination of chromosome alignment and spindle formation. It was found that MAD1 was present in the oocytes from the GV to prometaphase I stages around the nuclei. When the oocytes reached the metaphase I (M-I) to metaphase II (M-II) stages, MAD1 was mainly localized at the spindle poles. However, MAD1 relocated to the vicinity of the chromosomes when spindles were disassembled by nocodazole or cooling, and the relocated MAD1 moved back to the spindle poles during spindle recovery. Taxol treatment did not affect the MAD1 localization. Although anti-MAD1 antibody injection did not affect nuclear maturation, significantly higher proportions of injected oocytes had misaligned chromosomes when the oocytes reached the M-I to M-II stages. The results of the present study indicate that MAD1 is present in mouse oocytes at all stages during the first meiosis and that it participates in spindle checkpoint during meiosis. However, MAD1 could not check misaligned chromosomes during spindle recovery after the spindles were destroyed by drug or cooling, which caused some chromosomes to scatter in the oocytes.

Reduced expression of MAD2, BCL2, and MAP kinase activity in pig oocytes after in vitro aging are associated with defects in sister chromatid segregation during meiosis II and embryo fragmentation after activation.

This study was conducted to examine expression of centromere protein B (CENPB), spindle checkpoint protein MAD2 (mitotic arrest deficient protein), and antiapoptotic protein BCL2; activities of MAPK (mitogen-activated protein kinase) and mitochondria distribution in pig oocytes during aging, and their relationship with sister chromatid separation during meiosis II and embryo fragmentation and apoptosis after activation. After immature oocytes were cultured for 40-72 h, CENPB, MAD2, tubulin, BCL2, and MAPK in the oocytes were examined by immunoblotting. Spindles, chromosomes, kinetochores, and mitochondria were examined by immunofluorescence staining and apoptosis was examined by TUNEL assay. It was found that tubulin and CENPB was not changed during 40-72 h of culture. However, the expression of MAD2 and BCL2 and the activity of MAPK were gradually reduced during oocyte aging. The percentages of oocytes with normal spindle, chromosomes, and kinetochores were also reduced as oocyte aged from 9.5% at 40 h to 17.3%, 34.6%, and 42.9% at 48, 60, and 72 h, respectively. Aggregated mitochondria were found in the aged oocytes as compared with the uniform distribution in young oocytes. After activation, the proportions of oocytes with abnormal anaphase II were significantly increased in aged oocytes. More (P<0.001) oocytes cultured for 60-72 h fragmented and showed apoptosis after activation as compared with the oocytes cultured for 40-48 h. This study indicates that aging reduces expression in spindle checkpoint protein and antiapoptosis protein and MAPK activity in pig oocytes. These events in turn cause abnormal sister chromatid segregation during meiosis II, embryo fragmentation, and apoptosis.

Isolation and culture of pluripotent cells from in vitro produced porcine embryos.

The present study was designed to examine whether in vitro produced porcine embryos can be used to establish an embryonic stem (ES) cell line. Porcine embryos were produced by in vitro maturation and in vitro fertilization. Embryos at the 4-cell to blastocyst stages were cultured in an ES medium containing 16% fetal bovine serum with mouse embryonic fibroblasts as a feeder layer. It was found that ES-like colonies were derived only from blastocysts. When these ES-like colonies were separated in 0.25% trypsin-0.02% EDTA solution and cultured again, ES-like colonies were further observed in the subsequent culture until the fourth passage. The cells from ES-like colonies showed positive alkaline phosphatase activity. Some cells from the colonies differentiated into several types of cells in vitro when they were cultured in the medium without feeder layers and leukemin inhibitory factor. Embryoid bodies were also formed when the cells were cultured in a suspension status. These results indicate that porcine ES-like cells can be derived from in vitro produced porcine blastocysts and these ES-like cells are pluripotent. The culture system used in the present study is useful to isolate and culture ES cells from in vitro produced porcine embryos.

Intra-oocyte localization of MAD2 and its relationship with kinetochores, microtubules, and chromosomes in rat oocytes during meiosis.

The present study was designed to investigate subcellular localization of MAD2 in rat oocytes during meiotic maturation and its relationship with kinetochores, chromosomes, and microtubules. Oocytes at germinal vesicle (GV), prometaphase I (ProM-I), metaphase I (M-I), anaphase I (A-I), telophase I (T-I), and metaphase II (M-II) were fixed and immunostained for MAD2, kinetochores, microtubules and chromosomes. The stained oocytes were examined by confocal microscopy. Some oocytes from GV to M-II stages were treated by a microtubule disassembly drug, nocodazole, or treated by a microtubule stabilizer, Taxol, before examination. Anti-MAD2 antibody was also injected into the oocytes at GV stage and the injected oocytes were cultured for 6 h for examination of chromosome alignment and spindle formation. It was found that MAD2 was at the kinetochores in the oocytes at GV and ProM-I stages. Once the oocytes reached M-I stage in which an intact spindle was formed and all chromosomes were aligned at the equator of the spindle, MAD2 disappeared. However, when oocytes from GV to M-II stages were treated by nocodazole, spindles were destroyed and MAD2 was observed in all treated oocytes. When nocodazole-treated oocytes at M-I and M-II stages were washed and cultured for spindle recovery, it was found that, once the relationship between microtubules and chromosomes was established, MAD2 disappeared in the oocytes even though some chromosomes were not aligned at the equator of the spindle. On the other hand, when oocytes were treated with Taxol, MAD2 localization was not changed and was the same as that in the control. However, immunoblotting of MAD2 indicated that MAD2 was present in the oocytes at all stages; nocodazole and Taxol treatment did not influence the quantity of MAD2 in the cytoplasm. Significantly higher proportions of anti-MAD2 antibody-injected oocytes proceeded to premature A-I stage and more oocytes had misaligned chromosomes in the spindles. The present study indicates that MAD2 is a spindle checkpoint protein in rat oocytes during meiosis. When the spindle was destroyed by nocodazole, MAD2 was reactivated in the oocytes to overlook the attachment between chromosomes and microtubules. However, in this case, MAD2 could not check unaligned chromosomes in the recovered spindles, suggesting that a normal chromosome alignment is maintained only in the oocytes without any microtubule damages during maturation.

Localization of CD9 in pig oocytes and its effects on sperm-egg interaction.

CD9 is a cell surface protein that participates in many cellular processes, such as cell adhesion. Fertilization involves sperm and oocyte interactions including sperm binding to oocytes and sperm-oocyte fusion. Thus CD9 may play an essential role during fertilization in mammals. The present study was conducted to examine whether CD9 is present in porcine gametes and whether it participates in the regulation of sperm-oocyte interactions. The presence of CD9 in ovarian tissues, oocytes and spermatozoa was examined by immunohistochemistry, immunofluorescence and immunoblotting. Sperm binding and penetration of oocytes treated with CD9 antibody were examined by in vitro fertilization. The results showed that CD9 was present on the plasma membrane of oocytes at different developmental stages. A 24 kDa protein was found in oocytes during in vitro maturation by immunoblotting and its quantity was significantly (P < 0.001) increased as oocytes underwent maturation and reached the highest level after the oocytes had been cultured for 44 h. No positive CD9 staining was found in the spermatozoa. Both sperm binding to ooplasma and sperm penetration into oocytes were significantly (P < 0.01) reduced in anti-CD9 antibody-treated oocytes (1.2 +/- 0.2 per oocyte and 16.6% respectively) as compared with oocytes in the controls (2.5 +/- 0.4 per oocyte and 70.3% respectively). These results indicated that CD9 is expressed in pig oocytes during early growth and meiotic maturation and that it participates in sperm-oocyte interactions during fertilization.

[Cytotoxicity of MICA-reactive V delta 1 gamma delta T cells towards epithelial tumor cells].

OBJECTIVE: To confirm whether human MHC class I chain-related A (MICA) induces the amplification of V delta 1 gamma delta tumor-infiltrating lymphocytes (TILs) in vitro and to identify the cytotoxicity of MICA-reactive V delta 1 gamma delta TILs towards epithelial tumor cells. METHODS: MICA protein was prokaryoticly expressed and purified by molecular cloning technology. The purified recombined MICA (rMICA) was used to induce V delta 1 gamma delta T cells from tumor tissues in vitro and the cytotoxicity of these V delta 1 gamma delta TILs were tested by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). RESULTS: The rMICA was expressed in prokaryocyte with pET30 as a vector. The immobilized rMICA protein could markedly induce the amplification of V delta 1 gamma delta T cells from tumor tissue in vitro. These V delta 1 gamma delta T cells showed strong cytolytic activities towards tumor cell lines expressing MICA. CONCLUSION: The MICA-reactive V delta 1 gamma delta T cell may be a candidate for adoptive cellular therapy of tumors.

Reduced polyspermic penetration in porcine oocytes inseminated in a new in vitro fertilization (IVF) system: straw IVF.

High incidence of polyspermy is still a major problem in the in vitro fertilization (IVF) of porcine oocytes matured in vitro. This study was designed to examine whether embryo cryopreservation straws can be used to conduct IVF in porcine oocytes. The efficiency of this system was further compared with traditional microdrop IVF. Immature oocytes were aspirated from antral follicles and matured in vitro. After maturation, oocytes were inseminated either in straws or in microdrops with frozen-thawed boar spermatozoa. For straw IVF, sperm concentration and the presence of air columns between insemination segment and oil column were examined. Sperm-oocyte binding and cortical granules (CGs) before and after sperm penetration were examined by confocal microscopy. When various sperm concentrations were used for IVF in the straws with air columns, it was found that 5 x 106 cells/ml of sperm concentration was the optimal concentration; a high penetration rate (94.0%) and normal fertilization (oocytes with both male and female pronuclei) rate (38.2%) were obtained. Increasing sperm concentration to 10 x 106 cells/ml increased polyspermic penetration (61.9%) without affecting sperm penetration (86.9%). Reducing sperm concentration to 1 x 106 cells/ml reduced polyspermic penetration (25.6%), but sperm penetration rate (69.9%) was also reduced. When IVF was conducted in the straws with or without air columns, and in the microdrops, it was found that sperm penetration in the straws with air columns (96.5%) was significantly (p < 0.05) higher than that in the straws without air columns (81.7%) and in the microdrop (72.9%). However, the incidence of polyspermic penetration in the straws with air columns (34.2%) and without air columns (36.6%) was significantly (p < 0.05) lower than that (52.4%) in the microdrops. The number of spermatozoa bound to the oocytes was increased gradually in the straws but not in the microdrops in which more spermatozoa bound to the oocytes soon after insemination. CG exocytosis was more complete and faster in the oocytes inseminated in the straws than in the microdrops. These findings indicate that IVF of porcine oocytes in the straws provides a better condition in which more oocytes are fertilized normally than that in the microdrop IVF.

Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes.

Meiotic spindle structure and chromosome alignment were examined after porcine oocytes were cooled at metaphase II (M II) stage. Cumulus-oocyte complexes (COCs) collected from medium size follicles were cultured in an oocyte maturation medium at 39 degrees C, 5% CO(2) in air for 44 hr. At the end of culture, oocytes were removed from cumulus cells and cooled to 24 or 4 degrees C for 5, 30, or 120 min in a solution with or without 1.5 M dimethyl sulfoxide (DMSO). After being cooled, oocytes were either fixed immediately for examination of the meiotic spindle and chromosome alignment or returned to maturation medium at 39 degrees C for 2 hr for examination of spindle recovery. Most oocytes (65-71%) cooled to 24 degrees C showed partially depolymerized spindles but 81-92% of oocytes cooled at 4 degrees C did not have a spindle after cooling for 120 min. Quicker disassembly of spindles in the oocytes was observed at 4 degrees C than at 24 degrees C. Cooling also induced chromosome abnormality, which was indicated by dispersed chromosomes in the cytoplasm. Limited spindle recovery was observed in the oocytes cooled to both 4 and 24 degrees C regardless of cooling time. The effect of cooling on the spindle organization and chromosome alignment was not influenced by the presence of DMSO. These results indicate that the meiotic spindles in porcine M II oocytes are very sensitive to a drop in the temperature. Both spindle and chromosomes were damaged during cooling, and such damage was not reversible by incubating the oocytes after they had been cooled.

Maturation of porcine oocytes after cooling at the germinal vesicle stage.

Maturation of porcine oocytes was examined after oocytes were cooled at the germinal vesicle stage. Cumulus-oocyte complexes (COCs) collected from medium-sized follicles were cooled at 24 degrees C or 4 degrees C for 5, 30 or 120 min in a solution with or without 1.5 M dimethylsulfoxide (DMSO). After rewarming, COCs were cultured in maturation medium at 39 degrees C, 5% CO2 in air for 44 h. Meiotic spindle organisation (by immunostaining and confocal microscopy), nuclear maturation (by orcein staining) and cytoplasmic maturation (by intracellular glutathione assay) of oocytes were examined after maturation. When COCs were cooled at 24 degrees C for various times in the medium without DMSO, a tendency to decreased spindle formation, nuclear maturation and cytoplasmic maturation was observed, but there was no statistical difference compared with controls. Addition of DMSO during cooling inhibited subsequent nuclear maturation and spindle formation. When COCs were cooled at 4 degrees C, both nuclear and cytoplasmic maturation as well as spindle formation were inhibited in most oocytes in a time-dependent manner. DMSO during cooling did not have any beneficial effect on subsequent oocyte maturation and spindle formation. These results suggest that porcine oocytes are very sensitive to a drop in the temperature before exposure to culture. Cooling oocytes before maturation inhibits their subsequent spindle organisation, nuclear and cytoplasmic maturation. Addition of DMSO to the cooling solution did not protect porcine oocytes from cooling-induced damage.

Synergetic effects of epidermal growth factor and estradiol on cytoplasmic maturation of porcine oocytes.

This study was conducted to examine the effect of epidermal growth factor (EGF) and 17beta-estradiol (E2) on nuclear and cytoplasmic (male pronuclear formation and early embryo development) maturation of porcine oocytes. Oocytes were aspirated from antral follicles and cultured in modified TCM-199 medium supplemented with 0.57 mM cysteine, 10 IU/ml eCG, 10 IU/ml hCG, with or without EGF and/or E2. In vitro fertilisation of matured oocytes was performed in a modified Tris-buffered medium (mTBM) with frozen-thawed ejaculated spermatozoa. Oocytes were transferred to NCSU-23 supplemented with 0.4% bovine serum albumin at 6 h after in vitro fertilisation. Significantly higher (p < 0.05) rates of nuclear maturation, pronuclear formation and cleavage (91.7%, 65.2% and 37.3%, respectively) were observed when oocytes were cultured in the medium containing both EGF (10 ng/ml) and E2 (1 microg/ml) than in the medium supplemented with either EGF or E2 or without both. Intracellular glutathione concentration in the oocytes cultured in the medium containing both E2 and EGF was also significantly higher (12.1 pmol per oocyte) than that of oocytes cultured in the medium with E2 or EGF alone or without both. These findings suggested that EGF and E2 have a synergestic effect on both nuclear and cytoplasmic maturation of porcine oocytes.

Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles.

Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 +/- 2.1 pmol/oocyte) and medium (13.69 +/- 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 +/- 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 +/- 5.18 nM and 52.25 +/- 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 +/- 68.6 ng/ml) than from medium (40.0 +/- 6.4 ng/ml) and small (41.2 +/- 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 +/- 45.9 ng/ml) and medium (267.5 +/- 38.6 ng/ml) follicles were significantly higher than that (174.7 +/- 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.