Fas-Fas ligand system as a possible mediator of spermatogenic cell apoptosis in human maturation-arrested testes

To elucidate the mechanism of maturation arrest, known as one of the male infertility, we addressed whether germ cell apoptosis occurs during maturation arrest, and if so, whether Fas and Fas ligand expressions are involved in the apoptosis. By electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), typical apoptotic features were frequently found around the spermatocytic stage in maturation arrest, compared to that in normal testes. When paraffin-embedded sections reacted with anti-Fas antiserum, staining for Fas was found in the plasma membranes of spermatocytes in the maturation-arrested testes, while no positive spermatogenic cells were seen in the normal testes. On the other hand, positive immunostaining for Fas ligand was restricted to Sertoli cells in the maturation-arrested testes as well as in the normal testes, although the intensity of staining for Fas ligand in normal testicular Sertoli cells was much weaker than that of maturation-arrested ones. Thus, these findings demonstrate that "maturation arrest" is characterized by frequent apoptosis of spermatocytes, and that Fas and Fas ligand staining are associated with a high frequency of apoptosis.     

Intracellular mechanisms of gonadotropin-stimulated gene expression in granulosa cells.

Previous studies have shown that the gonadotropins follicle-stimulating hormone and luteinizing hormone stimulate proopiomelanocortin (POMC) promoter activity and mRNA levels in ovarian granulosa cells. The objective of these studies was to determine the role of cAMP-dependent protein kinases (pKA) in gonadotropin-stimulated gene expression. Primary cultures of rat granulosa cells were transfected with a gene construct consisting of the POMC promoter (-150 to +63; designated pOMC-CAT) fused to the chloramphenicol acetyltransferase (CAT) reporter gene either alone or cotransfected with an expression plasmid (designated mutant RI), which overexpresses a mutant form of the murine RI subunit incapable of binding cAMP and serving as an irreversible inhibitor of the catalytic subunit of pKA. Follicle-stimulating hormone or isoproterenol caused a significant stimulation of pOMC-CAT activity in transfected cells. Cotransfection of pOMC-CAT with mutant RI caused a significant inhibition of basal pOMC-CAT activity and abolished the gonadotropin stimulation. As a control, transfection of the SV-40 viral enhancer-promoter fused to CAT (pSV2-CAT) was unresponsive to follicle-stimulating hormone stimulation and cotransfection with mutant RI had no significant effect on pSV2-CAT activity. These studies suggest that gonadotropin regulation of the POMC promoter is mediated by pKA and that promoter activity is stringently controlled by pKA.     

Subcellular trafficking of antisense oligonucleotides and down-regulation of bcl-2 gene expression in human melanoma cells using a fusogenic liposome delivery system

Antisense oligonucleotides (ODN) targeted to specific genes have shown considerable potential as therapeutic agents. The polyanionic charges carried by these molecules, however, present a barrier to efficient cellular uptake and consequently their biological effects on gene regulation are compromised. To overcome this obstacle, a rationally designed carrier system is desirable for antisense delivery. This carrier should assist antisense ODN penetrate the cell membrane and, once inside the cell, then release the ODN and make them available for target binding. We have developed a carrier formulation employing programmable fusogenic vesicles (PFV) as the antisense delivery mediator. This study investigates the intracellular fate of PFV–ODN and bioavailability of antisense ODN to cells. The subcellular distribution of PFV and ODN was examined by monitoring the trafficking of FITC-labeled ODN and rhodamine/phosphatidylethanolamine (Rh-PE)-labeled PFV using confocal microscopy. Fluorescently tagged ODN were first co-localized with the liposomal carrier in the cytoplasm, presumably in endosome/lysosome compartments, shortly after incubation of PFV–ODN with HEK 293 and 518A2 cells. Between 24 and 48 h incubation, however, separation of FITC–ODN from the carrier and subsequent accumulation in the nucleus was observed. In contrast, the Rh-PE label was localized to the cell cytoplasm. The enhanced cellular uptake achieved using the PFV carrier, compared to incubation of free ODN with cells, and subsequent release of ODN from the carrier resulted in significant down-regulation of mRNA expression. Specifically, G3139, an antisense construct targeting the apoptotic antagonist gene bcl-2, was examined in the human melanoma cell line 518A2. Upon exposure to PFV-encapsulated G3139, cells displayed a time-dependent reduction in bcl-2 message levels. The bcl-2 mRNA level was reduced by 50% after 24 h treatment and by ~80% after 72 h when compared to cells treated with free G3139, empty PFV or PFV–G3622, a control ODN sequence. Our results establish that ODN can be released from PFV after intracellular uptake and can then migrate to the nucleus and selectively down-regulate target mRNA.     

Formation of estrogen glucuronides by human granulosa-luteal cells isolated from human menopausal gonadotropin-stimulated cycles for in vitro fertilization

The formation of steroid glucuronides by human granulosa cells isolated from human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG)-stimulated cycles for in vitro fertilization was studied. From granulosa cells in suspension, 5 x 10(-7) M androstenedione was converted into estradiol (2.50 +/- 0.21 ng/ml), estrone (1.84 +/- 0.16 ng/ml), estradiol glucuronide (0.38 +/- 0.07 ng/ml), as well as estrone glucuronide (0.24 +/- 0.04 ng/ml). When 5 x 10(-7) M estradiol was incubated, estrone (15.5 +/- 0.9 ng/ml) and estradiol glucuronide (0.12 +/- 0.05 ng/ml) were detected in medium. Using the same preparation of granulosa cells, we have observed that androsterone could uniquely be transformed into androstane-3 alpha, 17 beta-diol (1.42 +/- 0.56 ng/ml), and only low amounts of steroid glucuronides could be detected. Since the formation of steroid glucuronides was extremely small when granulosa cells in suspension were used, we subsequently studied granulosa cells in culture. When 5 x 10(-7) M estradiol was added, estrone (7.8 +/- 1.3 ng/ml) and estradiol glucuronide (0.68 +/- 0.08 ng/ml) were formed. The addition of follicle-stimulating hormone did not cause a further increase in estrone or estradiol glucuronide levels. As observed with granulosa cells in suspension, incubation with androsterone led to the formation of androstane-3 alpha, 17 beta-diol (24.2 +/- 0.07 ng/ml). Our data demonstrated the presence of glucuronyltransferase in human granulosa cells obtained from preovulatory follicles of hMG/hCG-treated women. In addition, since the conversion of androsterone into C-19 steroid glucoronide was relatively small, the present finding also indicates that the glucoronyltransferase enzymatic activity in granulosa-luteal cells preferentially conjugated estrogens.     

Contributions of Fas-Fas ligand interactions to the pathogenesis of mouse hepatitis virus in the central nervous system

The pathogenesis of the neurotropic strain of mouse hepatitis virus in Fas-deficient mice suggested that Fas-mediated cytotoxicity may be required during viral clearance after the loss of perforin-mediated cytotoxicity. The absence of both Fas- and perforin-mediated cytolysis resulted in an uncontrolled infection, suggesting a redundancy of cytolytic pathways to control virus replication.     

Abnormal gene expression profiles in human ovaries from polycystic ovary syndrome patients

Polycystic ovary syndrome (PCOS) represents the most common cause of anovulatory infertility and affects 5-10% of women of reproductive age. The etiology of PCOS is still unknown. The current study is the first to describe consistent differences in gene expression profiles in human ovaries comparing PCOS patients vs. healthy normoovulatory individuals. The microarray analysis of PCOS vs. normal ovaries identifies dysregulated expression of genes encoding components of several biological pathways or systems such as Wnt signaling, extracellular matrix components, and immunological factors. Resulting data may provide novel clues for ovarian dysfunction in PCOS. Intriguingly, the gene expression profiles of ovaries from (long-term) androgen-treated female-to-male transsexuals (TSX) show considerable overlap with PCOS. This observation provides supportive evidence that androgens play a key role in the pathogenesis of PCOS. Presented data may contribute to a better understanding of dysregulated pathways in PCOS, which might ultimately reveal novel leads for therapeutic intervention.     

Regulation of FAS ligand expression by chemokine ligand 2 in human endometrial cells

Human endometrium is a dynamic tissue under the influence of numerous hormones, growth factors, and cytokines interacting to maintain a balance of cellular growth, differentiation, and apoptosis. We have previously demonstrated that several factors including interleukin-8, extracellular matrix, and steroid hormones modulate FASLG, one of the apoptotic molecules, in human endometrium. Chemokine ligand 2 (CCL2), a monocyte chemoattractant and activating factor, is a cytokine involved in endometrial function. CCL2 is elevated in the peritoneal fluid of women with endometriosis. We hypothesize that increased levels of CCL2 in the endometriotic environment may upregulate FASLG expression in human endometrial stromal cells and induce a local immunotolerance in endometriosis. To test our hypothesis, we studied the in vitro regulation of FASLG expression and apoptosis by CCL2 in endometrial stromal cells. Western blot analysis revealed that CCL2 upregulated FASLG protein expression in cultured endometrial stromal cells. Based on semiquantitative RT-PCR analysis, CCL2 did not alter either FAS or FASLG mRNA expression in endometrial stromal cells. Immunocytochemistry results from the same cells treated with CCL2 demonstrated upregulation of FASLG protein expression. CCL2 did not change rate of apoptosis in endometrial stromal cells as evaluated by TUNEL assay. However, an increased apoptotic rate was detected in Jurkat (T lymphocytes) cells cocultured with endometrial stromal cells previously treated with CCL2. We speculate that increased FASLG expression by CCL2 may induce apoptosis of T lymphocytes and thus produce an immunotolerant environment for the development of ectopic implants.     

Presence of angiotensin-converting enzyme in follicular fluids of porcine ovaries and its possible involvement in the intrafollicular breakdown of bradykinin

The follicular fluid of porcine ovaries contains a metalloenzyme capable of hydrolyzing the synthetic substrate, benzyloxycarbonyl-Val-Lys-Met-MCA. This enzyme was purified by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose, CM-cellulose, Zn(2+)-chelating Cellulofine, and Diol-300 gel-filtration columns. The molecular weight of the purified enzyme was estimated to be 170,000 by SDS-PAGE and 400,000 by gel-filtration analysis, suggesting that the native enzyme is a dimer of the 170-kDa subunit polypeptide. The enzyme activity was drastically enhanced by the presence of chloride ion, and strongly inhibited by captopril and bradykinin potentiator B. A 9-residue peptide containing a processing site of human amyloid precursor protein was degraded by its dipeptidyl carboxypeptidase activity. Furthermore, the purified protein was recognized by specific antibody raised against human angiotensin-converting enzyme. The enzyme rapidly degraded bradykinin in vitro. These results indicate that benzyloxycarbonyl-Val-Lys-Met-MCA-hydrolyzing enzyme is a porcine angiotensin-converting enzyme, and that the enzyme may play a role in bradykinin turnover within the follicles of porcine ovaries.     

Reinforcing apoptosis-resistance of COS and myeloma cells by transfecting with bcl-2 gene

COS, myeloma and HeLa cells, which are commonly used for protein production by cell culture, were transfected with human bcl-2 gene encoded on the shuttle vector BCMGS. Expression of human bcl-2 improved survival of cells remarkably, mildly, or negligibly for COS, myeloma, and HeLa, respectively. Four clones were obtained from the human bcl-2 expressing cell population of COS cells. They expressed human bcl-2 almost at the same level. The viable cell numbers were 6, 2.5, 2.5, and 0.8 times as many for the clones #8, #5, #6, and #7, respectively, as for the control COS cells, when they were cultured at low (0.2%) serum concentration for 9 days. The bcl-2 overexpressing COS cells showed morphology different from that of the control COS cells in serum limited condition. When transfected with mouse lambda protein gene carried by an SV40-derived vector, clone #8 of the bcl-2 transfected COS cells continued the transient expression of lambda protein longer than the control COS cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Amplification of specific gene products from human serum

The polymerase chain reaction (PCR) is an in vitro method for the primer-directed enzymatic amplification of specific DNA sequences. Ordinarily, sources with obvious DNA content such as cells, viruses, and plasmids serve as the origin of templates for the PCR. Here we report a simple and efficient method to obtain cellular DNA from serum suitable for use in PCR reactions or gene analysis. This procedure should facilitate the detection of disease and provide a basis for the examination of mutations in genes before the onset as well as during the progression of various diseases.     

bcl-2-specific siRNAs restore gemcitabine sensitivity in human pancreatic cancer cells

Gemcitabine has been shown to ameliorate disease related symptoms and to prolong overall survival in pancreatic cancer.Yet, resistance to Gemcitabine is commonly observed in this tumour entity and has been linked to increased expression of anti-apoptotic bcl-2. We therefore investigated if and to what extend silencing of bcl-2 by specific siRNAs (siBCL2) might enhance Gemcitabine effects in human pancreatic carcinoma cells. siBCL2 was transfected into the pancreatic cancer cell line YAP C alone and 72 hrs before co-incubation with different concentrations of Gemcitabine. Total protein and RNA were extracted for Western-blot analysis and quantitative polymerase chain reaction. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siBCL2 alone, Gemcitabine and control siRNA or Gemcitabine and siBCL2 for 21 days. Combination of both methods lead to a synergistic induction of apoptosis at otherwise ineffective concentrations of Gemcitabine. Tumour growth suppression was also potentiated by the combined treatment with siBCL2 and Gemcitabine in vivo and lead to increased TUNEL positivity. In contrast, non-transformed human foreskin fibroblasts showed only minor responses to this treatment. Our results demonstrate that siRNA-mediated silencing of anti-apoptotic bcl-2 enhances chemotherapy sensitivity in human pancreatic cancer cells in vitro and might lead to improved therapy responses in advanced stages of this disease.     

Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles.

Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100-200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (相似文献   

The endothelin system in human and monkey ovaries: in situ gene expression of the different components

The endothelin system is composed of three endothelin isoforms (ET-1, ET-2, and ET-3), the endothelin receptors ETA and ETB, and the endothelin-converting enzyme (ECE). Besides having a major vasoactive role, endothelins have roles in different cell types at a local level. We investigated the presence of the different components of the endothelin system in primate ovaries. Human ovaries and gonadotropin-stimulated monkey ovaries were studied using immunohistochemistry for endothelin, and in situ hybridization with probes for ET-1, ET-2, ET-3, ETA and ETB receptors, and ECE. ET-1 and ETA receptors were detected in endothelial cells and vascular smooth muscle cells, respectively, in stromal vessels adjacent to follicles and corpora lutea. ETB receptors and ET-1 were found in the endothelial cells of capillaries of corpora lutea. ECE was present in internal theca cells of secondary, de Graaf, atretic follicles, and in luteinized granulosa cells of the corpora lutea. The endothelin system components are present in or around the follicles of human and monkey ovaries. Although the components are not expressed in the same cell types, they are synthesized, mainly in follicles, by cells that are in close proximity. Thus, the endothelin system could act in a paracrine manner. ECE expression in steroid-producing cells changes its compartmentalization during follicle maturation.     

Fas-Fas ligand interactions are essential for the binding to and killing of activated macrophages by gamma delta T cells

Gammadelta T cells have a direct role in resolving the host immune response to infection by eliminating populations of activated macrophages. Macrophage reactivity resides within the Vgamma1/Vdelta6.3 subset of gammadelta T cells, which have the ability to kill activated macrophages following infection with Listeria monocytogenes (Lm). However, it is not known how gammadelta T cell macrophage cytocidal activity is regulated, or what effector mechanisms gammadelta T cells use to kill activated macrophages. Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta-/- mice were incubated with splenocytes from wild-type and Fas ligand (FasL)-deficient mice (gld), the ability of Vgamma1 T cells to bind macrophages was shown to be dependent upon Fas-FasL interactions. Combinations of anti-TCR and FasL Abs completely abolished binding to and killing of activated macrophages by Vgamma1 T cells. In addition, confocal microscopy showed that Fas and the TCR colocalized on Vgamma1 T cells at points of contact with macrophages. Collectively, these studies identify an accessory or coreceptor-like function for Fas-FasL that is essential for the interaction of Vgamma1 T cells with activated macrophages and their elimination during the resolution stage of pathogen-induced immune responses.     

Tolerance induction by veto CTLs in the TCR transgenic 2C mouse model. II. Deletion of effector cells by Fas-Fas ligand apoptosis

The direct assay of veto CTLs in the 2C mouse model enables monitoring, by FACS, the fate of the TCR transgenic effector CD8(+) T cells, the transgene of which can be stained with clonotypic Ab 1B2. After the addition of veto cells, CD8(+)1B2(+) effector cells increasingly express annexin V, and maximal apoptosis is attained 72 h after initiation of MLR. This veto activity can be partially blocked by anti-CD8 Abs directed against the allele expressed by the veto CTLs, but not by the effector cells. When effector CD8(+) T cells were from 2C mice, which lack Fas expression ((2CX lpr)F(2)), deletion of effector cells was not exhibited by veto cells. The protein levels of the apoptosis inhibitors FLIP and Bcl2 in purified CD8(+)1B2(+) effector cells at different time points after MLR showed an initial up-regulation of these inhibitors, with marked reduction of FLIP, but not of Bcl2, by 48 h after initiation of culture. Taken together, these results are in accordance with a Fas-FasL-based mechanism in which prolonged binding between the effector cell and the veto cell might be required to allow FLIP to be down-regulated. Such prolonged interaction might be afforded through the interaction of CD8 molecules on the veto cell with the alpha3 domain of H2 class 1 on the effector cell.     

益坤宁对围绝经期大鼠卵巢bcl-2和bax表达的影响

目的:研究中药益坤宁(Yikunning,YKN)对围绝经期大鼠卵巢bcl-2和bax基因表达的影响,探讨益坤宁治疗围绝经期综合征的作用机制。方法:选用自然衰老的围绝经期雌性大鼠,随机分为3组(n=10):围绝经期对照组、利维爱(Livial)对照组和益坤宁组,另选10只青年雌性大鼠作为青年对照组。连续灌胃处理4周后,采用逆转录-聚合酶链反应(RT-PCR)检测大鼠卵巢中凋亡相关基因bcl-2和baxmRNA表达,采用蛋白印迹(Westernblot)检测大鼠卵巢中Bcl-2和Bax蛋白表达。结果:益坤宁组大鼠卵巢中Bcl-2、BaxmRNA及其蛋白表达高于围绝经期对照组,差异有统计学意义(P0.01);益坤宁组大鼠卵巢中Bcl-2/BaxmRNA比值和蛋白比值高于围绝经期对照组,差异有统计学意义(P0.05或P0.01)。结论:中药益坤宁通过上调围绝经期大鼠卵巢中凋亡相关基因bcl-2和bax的表达,上调Bcl-2/BaxmRNA比值和蛋白比值,从而延缓卵巢衰老,这可能是其治疗围绝经期综合征的分子机制之一。     

Enhancement of survivability of mammalian cells by overexpression of the apoptosis-suppressor gene bcl-2

Cell lines derived from the hemopoetic lineages are widely used as hosts for the production of biologicals. These cell lines have been demonstrated to undergo high levels of the active death program commonly referred to as apoptosis. The effects of overexpression of the apoptosis suppressor gene bcl-2 on the properties of a Burkitt lymphoma were compared with the control cell line (transfected with a negative control plasmid) under a variety of conditions relevant to cell culture production technology. In stationary batch cultures, there was a clear reduction in both the rate of total cell death and the level of apoptosis during the decline phase of the bcl-2 transfected cell cultures as compared with that of the control cell cultures. Nutrient analysis revealed that the onset of death during the control cell cultures occurred following complete exhaustion of glutamine. However, the bcl-2 transfected cell cultures continued to grow even though glutamine had been exhausted, and a significant decline in viability only occurred when glucose had also been completely exhausted.When cells were cultured in suspension without prior adaptation, the bcl-2 transfected cells grew significantly better, suggesting that the bcl-2 gene protected the cells from apoptosis triggered by either the lack of substrate or the hydrodynamic environment. Fluorescence microscopy revealed that death of the control cells was almost entirely by apoptosis, whereas death was almost exclusively by necrosis in the delayed decline phase of the transfected cell cultures. In both instances, death occurred before total exhaustion of glucose and glutamine.The induction of apoptosis following growth arrest is a major impediment to the development of culture strategies that optimize specific productivity by reducing the growth rate. Results presented here suggest that suppression of apoptosis by bcl-2 under the condition of excess thymidine allows the maintenance of cells in a growth-arrested state for much longer than would otherwise be possible.When cells were transferred to a range of commercial serum-free media, cell growth was, in all cases, much better for the bcl-2 transfected cell line. Moreover, when cells were cultivated in glutamine-free medium, the control cells exhibited a decrease in viable cell number within the first 24 h whereas, for the bcl-2 transfected cell cultures, viable cell number did not exhibit any clear decrease until after 75 h. Clearly, these results indicate that the metabolic engineering approach can be used to alter advantageously the survival and proliferative capacity of cells in cell culture environments. (c) 1996 John Wiley & Sons, Inc.