OBJECTIVE: To investigate new signal transduction cascade through integrin, FAK and ERK in the suppressed cell proliferation by GnRH-I and -II. METHOD: Human endometrial cancer cells (HEC1A) were cultured under the following condition: DMEM/F12 (10% FBS). GnRH-I and -II were treated time (0, 5, 10, 15, 20, 30 min; 100 nM) and dose (10 nM or 100 nM; 20 min) dependent manner according to experimental purposes. Cell proliferation was measured using [3H] thymidine incorporation assay. Immunoblotting was utilized to detect proteins. RESULTS: GnRH-I and -II inhibited proliferation of HEC1A cells and induced expression of integrin beta3. Phosphorylation of FAK and ERK were induced by GnRH-I and -II. CONCLUSION: GnRH inhibited cell proliferation via the expression of integrin and FAK, ERK phosphorylation.
Cell Proliferation* ; Endometrial Neoplasms* ; Female ; Gonadotropin-Releasing Hormone* ; Humans ; Immunoblotting ; Integrin beta3 ; Phosphorylation ; Signal Transduction ; Thymidine

OBJECTIVE: To investigate the role of ERK and PPAR gamma on the TGF-beta1 induced human endometrial stromal cell decidualization in vitro. METHOD: Endometrial stromal cells are cultured under the following condition: DMEM/F12 (10% FBS, 1 nM E2 and 100 nM P4). TGF-beta1 (5 ng/ml), Rosiglitazone (50 nM), and PD98059 (20 microgram) were added according to experimental purposes. Trypan-Blue and hematocytometer were utilized to count cell number. Enzyme-linked immunosorbent assay (ELISA) and western blotting were utilized to detect proteins. RESULT: TGF-beta1 inhibited proliferation of cultured human endometrial stromal cells and induced expression of PGE2 and prolactin. This effect was mediated by Smad and ERK activation. Administration of rosiglitazone, PPAR gamma agonist, prevented TGF-beta1 effect on cell proliferation. Furthermore, Rosiglitazone inhibited TGF-beta1 induced activation of ERK, consequently reduced PGE2 and prolactin production. CONCLUSION: TGF-beta1 induced decidualization of endometrial stromal cell through Smad and ERK phosphorylation. PPAR gamma acts as a negative regulator of human endometrial cell decidualization in vitro.
Blotting, Western ; Cell Count ; Cell Proliferation ; Dinoprostone ; Enzyme-Linked Immunosorbent Assay ; Humans* ; Peroxisome Proliferator-Activated Receptors* ; Phosphorylation ; PPAR gamma ; Prolactin ; Stromal Cells* ; Transforming Growth Factor beta1*

OBJECTIVE: To determine the effects of hCG on extracellular ATP induced apoptosis in cultured human luteinized granulosa cells (hLGCs) METHODS: The addition of various concentrations of ATP (0, 0.1, 0.25, 0.5, 0.75 mM) and 5 IU hCG to luteinized granulosa cells obtained during in vitro fertilization ovum pickup procedures. After culture for 24 hours, purinoceptor activity and functional changes in mitochondria were measured by patch clamp, flow cytometry, and confocal microscopy. RESULTS: Calcium imaging with fura-2 revealed that ATP elevated [Ca2+]i by mobilizing intracellularly stored Ca2+. A patch clamp study showed that ATP exerted its effect by initially binding to the P2Y type purinoceptor, as evidenced by the ATP-evoked outward Ca2+-activated K+ current. Probing mitochondria with JC-1, a mitochondrial transmembrane potential-sensitive dye, revealed that ATP induced mitochondrial depolarization in a concentration-dependent manner. A quantitative flow cytometric analysis with Annexin V showed that apoptotic cells were increased in number in proportion to the concentration of ATP, having 18.57% of apoptotic cell populations in the presence of 0.75 mM ATP compared to 7.88% in the control. Moreover, treatments with human chorionic gonadotropin (hCG) at 5 IU reversed both the ATP-induced mitochondrial depolarization and apoptosis (18.57 vs 6.32%). CONCLUSION: Taken together, these results indicate, first, extracellular ATP recognized by the P2Y type purinoceptor on h-LGCs increases the intracellular Ca2+. Second, the increased intracellular Ca2+ triggers the apoptotic cascade by acting at least, in part, on mitochondria. Third, hCG reverses the ATP-induced apoptosis, raising a possible clinical implication of hCG in the treatment of degeneration of granulosa cells such as follicular atresia.
Adenosine Triphosphate* ; Annexin A5 ; Apoptosis* ; Calcium ; Chorionic Gonadotropin ; Female ; Fertilization in Vitro ; Flow Cytometry ; Follicular Atresia ; Fura-2 ; Granulosa Cells* ; Humans* ; Lutein* ; Microscopy, Confocal ; Mitochondria ; Ovum ; Receptors, Purinergic

Stem cells provide an important means for regenerative medicine due to the capacity to generate multiple types of differentiated cells and at the same time to maintain self-renewal. To identify the therapeutic effect of the transplantation of neural stem cells, differentiation and migration capacity of the neural stem cells that were isolated from E14 rat embryo and maintained in culture were examined after transplantation to the striatum of the quinolinic acid (QA)-induced Huntington's disease rat model. in vitro co-culture of the neural stem cells with the mixture of primary neurons and astrocytes promoted the maturation and the synapse formation of neuronal progenies of neural stem cells. Following the implantation, the neural stem cells survived, differentiated, and migrated in the damaged striatum region, exhibiting immunoreactivities against nestin, Tuj-1, GFAP, GAD(67) and synapsin 1 to a varying degree. These data provide clear evidence supporting that the neural stem cells isolated from the rat embryo and maintained in the primary culture have a multiple capacity to differentiate into neurons or glial cells both in vitro and in vivo.
Animals ; Astrocytes ; Brain ; Coculture Techniques ; Embryonic Structures ; Huntington Disease ; Intermediate Filament Proteins ; Nerve Tissue Proteins ; Neural Stem Cells ; Neuroglia ; Neurons ; Quinolinic Acid ; Rats ; Regenerative Medicine ; Synapses ; Transplants

OBJECTIVES: To investigate the role of TGF (Transforming growth factor-beta) involved in the paracrinic communication during decidualization between UEC (uterine epithelial cells) and USC (uterine stromal cells), we have employed a co-culture system composed of human endometrial epithelial and stromal cells in defined hormonal conditions. DESiGN: in the co-culture, endometrial epithelial cells cultured in the matrigel-coated cell culture insert are seeded on top of the endometrial stromal cells cultured within a collagen gel. The co-culture was maintained for 48 hours under the following hormonal conditions: progesterone dominant condition (100 nM P4 and 1 nM E2) or estrogen-dominant condition (100 nM E2 and 1 nM P4). 10 ng/ ml HGF and/or 10 ng/ml TGF-beta1 are added. METHODS: RT-PCR is utilized to detect mRNAs quantitatively. Enzyme-linked immunosorbent assay (ELiSA) and immunohistochemical staining are utilized to detect proteins in the tissue. RESULTS: Prolactin mRNA is expressed in the co-cultured stromal cells under the progesterone dominant condition. TGF-beta1 and its receptors are expressed in both the co-cultured epithelial and stromal cells irrespective of the steroid present, which is in contrast with no or negligible expression of TGF-beta1 or its receptor in cells separately cultured. Both estrogen and progesterone significantly elevate the concentration of hepatocyte growth factor (HGF) in the conditioned medium of the co-culture with the value of 4,325 pg/ml in E2-dominant and 2,000 pg/ml in P4-dominant condition compare to 150 pg/ ml in no hormone. in separately cultured stromal cells, administration of HGF induces the expression of TGF receptor 1 in both hormonal conditions, but induction of TGF receptor 2 is only manifest in the P4-dominant condition. Administration of TGF-beta and HGF directly induce the decidualization marker prolactin mRNA in separately cultured stromal cells. CONCLUSION: it is likely that steroid hormones induces prolactin mRNA indirectly by promoting the cell to cell communication between the stromal and the epithelial cells. TGF-beta and HGF are two possible paracrine mediators in the human endometrial decidualization.
Cell Communication ; Cell Culture Techniques ; Coculture Techniques ; Collagen ; Culture Media, Conditioned ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; Estrogens ; Hepatocyte Growth Factor ; Humans* ; Progesterone ; Prolactin ; RNA, Messenger ; Stromal Cells ; Transforming Growth Factor beta ; Transforming Growth Factor beta1

OBJECTIVE: Aberrant expression of the cell surface proteoglycan, syndecan-1, is found in many malignancies. The current study describes the immunohistochemical study of syndecan-1 expression in normal, hyperplastic, and malignant endometrial tissues for evaluation of application as a parameter of cancer progression in patients with endometrial hyperplasia. METHODS: Immunohistochemical staining of syndecan-1 was performed in 101 formalin fixed, paraffin embedded sections of normal, hyperplastic, and malignant endometrial tissues. We analyzed specimens from patients with normal endometrium (NE, N=10) as controls, and those of simple hyperplasia (SH, N=20), complex hyperplasia without atypia (CH, N=20), atypical hyperplasia (AH, N=20), and endometrial cancer (EC, N=31). RESULTS: The mean rank of expression scores based on the frequency of syndecan-1 staining were 31.6, 20.5, 52.9, 72.1, and 62.1 for NE, SH, CH, AH and EC, respectively (p<0.001). Syndecan-1 expression was significantly greater in CH (p<0.001) or AH (p<0.001) than in SH, and significantly greater in AH compared to CH (p=0.028). Syndecan-1 is more frequently expressed in CH (p=0.042), AH (p<0.001), or EC (p=0.002) than in NE. Syndecan-1 expression did not differ significantly between NE and SH (p=0.248). CONCLUSION: Syndecan-1 expression appears to be useful as a predictive indicator in endometrial hyperplasia.
Endometrial Hyperplasia ; Endometrial Neoplasms ; Endometrium ; Female ; Formaldehyde ; Humans ; Hyperplasia ; Paraffin ; Proteoglycans ; Syndecan-1

OBJECTIVE: To determine the effects of extracellular ATP on mitochondrial function and apoptosis during human luteinized granulosa cell cultures. METHODS: The addition of various concentrations of ATP (0, 0.1, 0.25, 0.5, 0.75 mM) to luteinized granulosa cells obtained during In vitro fertilization ovum pickup procedures. After culture for 24 hours, purinoceptor activity and functional changes in mitochondria were measured by patch clamp, flow cytometry, and confocal microscopy. RESULTS: Measurement by patch clamp of the granulosa cell membrane potential after ATP addition to cultured granulosa cells showed that both the inward and outward currents were expressed. After treatment of the granulosa cells with JC-1, measurement of the mitochondrial activity by confocal microscopy showed that the with increasing concentrations of ATP the relative ratio of undamaged mitochondria (red/green ratio) tended to decrease (P=0.027). After double staining of the cultured granulosa cells with Annexin V and Propidium Iodide, quantitative flow cytometry analysis showed that apoptosis increased with increasing concentrations of ATP (7.88%, 8.44%, 11.40%, 13.52%, 18.57%). CONCLUSION: The results of this study shows that apoptosis of granulosa cells increases with increasing extracellular ATP concentrations in cultured human luteinized granulosa cells. This is observed to be a consequence of cell membrane purinoceptor activity and functional changes in the mitochondria. It is therefore thought that remodelling processes of the ovarian tissue is regulated by neuroendocrine factors of the extracellular ATP.
Adenosine Triphosphate ; Annexin A5 ; Apoptosis* ; Cell Membrane ; Female ; Fertilization in Vitro ; Flow Cytometry ; Granulosa Cells* ; Humans* ; Lutein* ; Membrane Potentials ; Microscopy, Confocal ; Mitochondria* ; Ovum ; Propidium ; Receptors, Purinergic