Biomedical subjects

X J Tao

Publications and source records attributed to X J Tao.

13 recordsLinked to original sources

PubMed2000

Expression of BCL-2, BAX and BAK in the trophoblast layer of the term human placenta: a unique model of apoptosis within a syncytium.

The regulation of apoptosis in the syncytiotrophoblast is of particular interest because this is the only true syncytial epithelium in human cell biology. Nuclei characteristic of apoptotic cells have been localized to this syncytium especially in association with fibrin-containing fibrinoid deposits. The factors responsible for regulating cell death-like features in the trophoblast syncytium are unknown. We tested the hypothesis that fibrin was required for trophoblast apoptosis. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP end-labelling) staining to detect DNA fragmentation typical of apoptosis was performed in term human placentae revealing labelled nuclei associated with fibrin-type fibrinoid, as well as labelled nuclei in discrete areas of syncytiotrophoblast without fibrin. We also hypothesized that members of the BCL-2 family of apoptosis-associated proteins contribute to the regulation of syncytiotrophoblast apoptosis. To identify members of this protein family that might regulate trophoblast apoptosis, we assessed expression of three important members of the bcl-2 gene family. We used immunohistochemistry with monoclonal antisera against human BCL-2 and polyclonal antisera against human BAX and BAK to study paraffin-embedded sections of human term placentae (n=5) from uncomplicated pregnancies. The anti-apoptotic BCL-2 protein was expressed throughout the syncytium of normal villi with much less staining in cytotrophoblast. Staining was also seen adjacent to fibrin deposits and in syncytium overlying fibrin deposits. Expression of the pro-apoptotic BAX protein was undetectable in the syncytiotrophoblast, was expressed in rare cytotrophoblast and was prominent in connective tissue and perivascular cells within the villous core. Localization of a second pro-apoptotic protein, BAK, revealed immunoreactivity in isolated areas of intact syncytium of normal villi. Additionally, fibrin deposits were associated with intense BAK staining in both syncytiotrophoblast and cytotrophoblast. From these data, we speculate that modulation of BAK expression is one factor regulating apoptosis in human trophoblast.

Adult↗

PubMed1999

The SCID mouse: an experimental model for endometriosis.

The purpose of this study was to validate the suitability of the severe combined immunodeficient (SCID) mouse as an experimental model for endometriosis, by defining the morphological and histological features of induced endometrial implants, and characterizing specific biochemical properties of these implants. Human secretory endometrial tissues were injected into the peritoneal cavity of SCID/SCID CB17 mature female mice. Successful peritoneal implantation was observed in 55 of 57 (96.5%) SCID mice and consisted of circumscribed elevated nodules. Haematoxylin-eosin staining of implanting lesions demonstrated the presence of endometrial glandular tissue in a mixed background of stromal and inflammatory cells. When progesterone was administered to mice, epithelial glands underwent well-defined secretory changes. Immunohistochemical analysis using polyclonal human pan-cytokeratin antibodies demonstrated selective positive staining in the glandular epithelium of the human implants with none in the surrounding stroma. In-situ hybridization analysis using complement component 3 cDNA radiolabelled riboprobes yielded significantly more intense signals in glands compared to stroma. As human endometrial implants in SCID mice were shown to retain specific histological, functional and biochemical properties, we conclude that the SCID mouse is an attractive animal model for the study of endometriosis.

Animals↗

PubMed1999

Fragmentation and death (a.k.a. apoptosis) of ovulated oocytes.

Accumulating evidence indicates that fragmentation of ovulated murine oocytes, resulting spontaneously or following exposure to lethal stimuli such as anticancer drugs during in-vitro culture, occurs with several hallmark features of apoptosis. However, recent work has failed to demonstrate a correlation between DNA cleavage, as assessed by DNA 3'-end-labelling, or of phosphatidylserine exposure on the outer leaflet of the plasma membrane, as measured by annexin V-staining, with fragmentation of ovulated mouse or human oocytes maintained in vitro. Consequently, these authors stated that it is 'premature to conclude that apoptosis occurs in ovulated oocytes or that such a mechanism is involved in the elimination or prevention of fertilization of oocytes with cytoplasmic or chromosomal defects'. Here, we have re-assessed DNA cleavage in normal and fragmented murine oocytes, have provided new evidence of an additional biochemical marker of apoptosis in fragmented oocytes (i.e. caspase activity), and have re-evaluated published reports regarding oocyte fragmentation, in an effort to clarify these discrepant findings. The results and discussions presented herein fully support previous conclusions reached by ourselves and others that fragmentation of ovulated oocytes is in fact an unequivocal example of apoptotic cell death.

Amino Acid Chloromethyl Ketones↗

PubMed1999

Requirement for phosphatidylinositol-3'-kinase in cytokine-mediated germ cell survival during fetal oogenesis in the mouse.

Apoptosis is responsible for primordial germ cell (PGC) attrition in the developing fetal ovary. In monolayer cultures of murine PGC, stem cell factor (SCF) and leukemia inhibitory factor (LIF) independently promote survival in vitro; however, the relevance of these data to fetal ovarian oogonium and oocyte survival, as well as the intracellular events involved in transducing the antiapoptotic actions of these cytokines in germ cells, remain to be elucidated. In this report, we investigated the effects of SCF and LIF, alone and in combination, on the survival of oogonia and oocytes, and elaborated on components of the signal transduction pathway used by these molecules, after validating a method of culturing fetal mouse ovaries. We further employed this system to also test the hypothesis that insulin-like growth factor-I (IGF-I), a classic antiapoptotic molecule, and transforming growth factor-beta (TGF-beta), a classic pro-apoptotic molecule, interact with the SCF/LIF pathway and function in a reciprocal fashion to precisely regulate germ cell numbers during fetal oogenesis. Freshly isolated embryonic day 13.5 ovaries contained nonapoptotic germ cells, as determined by histologic analysis of cellular morphology and in situ 3'-end-labeling of DNA integrity. In vitro culture of fetal ovaries without tropic support for 24, 48, and 72 h resulted in a time-dependent induction of germ cell apoptosis, such that most oogonia and oocytes present after 72 h were apoptotic. Morphometric analysis of serially sectioned ovaries indicated that the numbers of nonapoptotic germ cells remaining after 24, 48, and 72 h of culture were 78%, 38%, and 10%, respectively, of the number present before culture (P < 0.05 for all time points vs. 0 h). Inclusion of SCF (100 ng/ml) together with LIF (100 ng/ml) in the culture medium significantly attenuated germ cell apoptosis, with the SCF/LIF-treated ovaries retaining 5.5-fold more oogonia and oocytes after 72 h of culture as compared with control ovaries deprived of tropic support (P < 0.05). However, SCF or LIF, when added separately, had no (SCF) or little (LIF) inhibitory effect on germ cell apoptosis. Provision of 50 ng/ml IGF-I maintained survival of approximately two-thirds of the germ cells in cultured ovaries (P < 0.05), whereas a combination of all three growth factors (SCF, LIF, IGF-I) completely preserved the fetal ovary in culture to that resembling a freshly-isolated gonad. Cotreatment with 25 ng/ml TGF-beta partially reversed the survival actions of IGF-I or SCF/LIF, such that only one-third of the starting number of oogonia/oocytes remained after 72 h of culture (P < 0.05). Lastly, the antiapoptotic effects of SCF/LIF or IGF-I were almost entirely eliminated by cotreatment of fetal ovaries with either one of two inhibitors of phosphatidylinositol-3'-kinase (PI3K), LY294002 (5 microM) or wortmannin (50 nM), whereas cotreatment with an inhibitor of p70 S6 kinase (rapamycin, 25 ng/ml) was without effect. These data indicate that the combined actions of SCF, LIF, and IGF-I are required for maximal inhibition of apoptosis in germ cells of fetal mouse ovaries, and that the PI3K signaling pathway is an essential component of cytokine-mediated female germ cell survival. Moreover, TGF-beta can partially override the antiapoptotic actions of SCF/LIF or IGF-I in oogonia and oocytes, suggesting the existence of a complex signaling network that ultimately determines fetal ovarian germ cell fate.

Animals↗

PubMed1999

Localization, regulation and possible consequences of apoptotic protease-activating factor-1 (Apaf-1) expression in granulosa cells of the mouse ovary.

The recent characterization of apoptotic protease-activating factor-1 (Apaf-1) in vertebrates as a putative homolog of the Caenorhabditis elegans gene, ced-4, indicates that the third major arm of the C. elegans programmed cell death machinery has also been conserved through evolution. Although apoptosis is now known to be important for ovarian follicular atresia in vertebrates, nothing is known of the role of Apaf-1 in ovarian function. Herein we show by immunohistochemical analysis that Apaf-1 is abundant in granulosa cells of early antral follicles whereas in vivo gonadotropin priming completely suppresses Apaf-1 expression and granulosa cell apoptosis. Western blot analysis of fractionated protein extracts prepared from granulosa cells before and after in vitro culture without hormonal support to induce apoptosis indicated that mitochondrial cytochrome c release, a biochemical step required for the activation of Apaf-1, occurs in granulosa cells cultured in vitro. Moreover, Western blot analysis of procaspase-3 processing, a principal downstream event set in motion by activated Apaf-1, indicated that healthy granulosa cells possess almost exclusively the inactive (pro-) form of the enzyme whereas granulosa cells deprived of hormonal support rapidly process procaspase-3 to the active enzyme. Lastly, we show that serum-starved granulosa cells activate caspase-3-like enzymes both prior to and after nuclear pyknosis, as revealed by a single-cell fluorescent caspase activity assay. These data, combined with previous observations regarding the role of homologs of the two other C. elegans cell death regulatory genes, ced-9 (Bcl-2 family members) and ced-3 (caspases), in atresia fully support the hypothesis that granulosa cell apoptosis is precisely coordinated by all three major arms of a cell death program conserved through evolution.

Animals↗

PubMed1998

Elevated expression of the proapoptotic BCL-2 family member, BAK, in the human endometrium coincident with apoptosis during the secretory phase of the cycle.

OBJECTIVE: To characterize the distribution of BAK (BCL-2 homologous antagonist/killer) protein in the human endometrium relative to the occurrence of apoptosis. DESIGN: A prospective, controlled study. SETTING: An academic hospital. PATIENT(S): Premenopausal women with histologically normal endometrium who were undergoing hysterectomy and curettage. INTERVENTION(S): Endometrial tissues were collected. MAIN OUTCOME MEASURE(S): Detection of BAK protein by immunohistochemical and immunoblot analyses, and of apoptosis by in situ DNA end-labeling. RESULT(S): BAK protein was detected in secretory endometrium and was confined to the glandular epithelial cells of the functionalis layer. Immunoreactive BAK was absent from most of the cells of the proliferative endometrium. By immunoblot analysis, we confirmed the immunohistochemical data by demonstrating that a 30-kD protein corresponding to BAK was elevated in lysates prepared from secretory, as compared with proliferative, endometrium. The elevated levels of BAK coincided with the onset of apoptosis in endometrial glandular epithelial cells during the secretory phase of the menstrual cycle. CONCLUSION(S): BAK protein localizes to glandular epithelial cells on the verge of apoptosis in the human endometrium. Thus, BAK likely functions with other members of the BCL-2 family in the regulation of apoptosis in the human uterus.

Adult↗

PubMed1998

Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary.

Recent data support a role for apoptosis, under tight regulatory control by bcl-2, oxidative stress response, tumor suppressor, and CASP gene family members, in mediating granulosa cell demise during follicular atresia in the rodent and avian ovary. Herein we evaluated the occurrence of apoptosis in the human and baboon ovary relative to follicular health status, and analyzed expression of several cell death genes in these tissues. In situlocalization of DNA strand breaks in fixed human and baboon ovarian tissue sections indicated that apoptosis was essentially restricted to granulosa cells of atretic antral follicles. Biochemical analysis of DNA oligonucleosomes in individual follicles isolated from baboon ovaries during the ovulatory phase revealed the presence of apoptotic DNA fragments in subordinate but not dominant follicles, thus substantiating the in situ labeling studies. Messenger RNA transcripts encoded by the bax death susceptibility gene, the bcl-xlong survival gene, the bcl-xshort pro-apoptosis gene, the p53 tumor suppressor gene, and two members of the CASP gene family (CASP-2/Ich-1, CASP-3/CPP32), were detected by Northern blot analysis of total RNA prepared either from human ovaries or from Percoll-purified granulosa-lutein cells obtained from patients undergoing assisted reproductive technologies. Lastly, immunohistochemical localization of the BAX death-susceptibility protein in the human ovary revealed abundant expression in granulosa cells of early atretic follicles, whereas BAX protein was extremely low or non-detectable in healthy or grossly-atretic follicles. We conclude that apoptosis occurs during, and is probably responsible for, folicular atresia in the human and baboon ovary. Moreover, apoptosis in the human ovary is likely controlled by altered expression of the same cohort of cell death regulatory factors recently implicated as primary determinants of apoptosis induction or suppression in the rodent ovary.

Adult↗

PubMed1997

Induction of PreB cell apoptosis by 7,12-dimethylbenz[a]anthracene in long-term primary murine bone marrow cultures.

Numerous studies demonstrate that polycyclic aromatic hydrocarbons (PAH) suppress immunity by modifying the function of both B and T cells. Relatively few studies have assessed the effects of these common environmental chemicals on immature lymphocytes. In the present study, long-term primary bone marrow cultures were employed to investigate the effects of a prototypic PAH and aryl hydrocarbon receptor (AhR) agonist, 7,12-dimethylbenz[a]anthracene (DMBA), on immature B lymphocytes. In this system, immature preB cells are maintained in a supportive microenvironment provided by bone marrow stromal cells. Results presented here demonstrate that (1) exposure of primary bone marrow cultures to DMBA results in preB cell death by apoptosis; (2) notably low doses of DMBA (> or = 10(-8) M) induce preB cell apoptosis; (3) in long-term cultures, bone marrow stromal cells, but not preB cells, express AhR mRNA and protein as determined by in situ hybridization, RT-PCR, and immunoblotting; (4) freshly isolated unfractionated bone marrow cells, but not purified bone marrow B cells, express AhR protein as assessed by immunohistochemistry; (5) alpha-naphthoflavone, a competitive AhR inhibitor and cytochrome P450 antagonist, completely blocks DMBA-induced preB cell apoptosis in primary bone marrow cultures; and (6) DMBA or benzo[a]pyrene injection in vivo results in bone marrow cell apoptosis consistent with the death of hematopoietic cells clustered around stromal elements. The results implicate programmed cell death as a mechanism underlying DMBA-mediated immunosuppression and suggest that preB cell death is influenced by local interactions with AhR+ bone marrow stromal cells.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

Increased expression of complement component 3 in human ectopic endometrium compared with the matched eutopic endometrium.

OBJECTIVE: To compare the gene expression of complement component 3 (C3) in human eutopic and ectopic endometrium. DESIGN: A prospective, controlled study. SETTING: Academic hospital. PATIENT(S): Women with documented endometriosis. INTERVENTION(S): Eutopic and ectopic endometrial tissues were collected simultaneously at laparoscopy. MAIN OUTCOME MEASURE(S): Detection of C3 messenger RNA (mRNA) by in situ hybridization and C3 protein by immunohistochemistry and Western blot. RESULT(S): Expression of C3 mRNA increased in ectopic endometrium compared with that in the matched eutopic endometrium. The quantitative analysis of C3 mRNA by grain count (mean +/- SE) showed 175.60 +/- 40.02 and 39.97 +/- 8.17 grains per micron2 in ectopic and eutopic glands, respectively, and 67.65 +/- 29.82 and 15.02 +/- 5.80 grains per micron2 in ectopic and eutopic stroma, respectively. Expression of C3 mRNA in ectopic glands was significantly higher than that in eutopic glands. The pattern of immunoreactive staining of C3 protein was consistent with that of C3 mRNA. A higher level of C3 protein in ectopic endometrium than eutopic endometrium was detected by immunohistochemistry and Western blot. CONCLUSION(S): Expression of C3 mRNA and protein significantly increased in human ectopic endometrium compared with that in the matched eutopic endometrium.

Adult↗

PubMed1997

Endometrial alpha-2 macroglobulin; localization by in situ hybridization and effect on mouse embryo development in vitro.

alpha-2 macroglobulin (A2M) is a 718,000-kDA broad spectrum plasma protease inhibitor whose production by the human endometrium was recently reported. The multifunctional A2M receptor, also known as low-density lipoprotein receptor-related protein, was also recently immunolocalized to the endometrial stroma. The objective of this study was to further characterize the endometrial site of expression of A2M, and to study its effects on mouse embryo development in vitro, to gain some insight into the functional significance of its endometrial production. Formalin-fixed, paraffin-embedded human endometrium from hysterectomy and endometrial biopsy specimen was used for in situ hybridization analysis, with 35S-labeled riboprobes representing subcloned A2M complementary DNA (cDNA) fragments. Duplicate sections of human endometrium were hybridized with sense and antisense probe and coated with photographic emulsion. Resultant autoradiograms were analyzed qualitatively by light- and darkfield microscopy and quantitatively by a computerized analysis of the signal intensity. Immunohistochemistry and immunoblotting for endometrial tissues were performed using an affinity-purified polyclonal antibody to human A2M. The effect of A2M on mouse embryo development was studied by exposure of one cell mouse embryo in culture to physiological concentrations of biologically active and inactive A2M. Expression signals for A2M were more numerous and intense in the secretory endometrium, compared with proliferative endometrium. Endothelial cells lining the endometrial blood vessels seemed to be the main source of A2M expression. The A2M expression signals in secretory endothelium were 2- to 3-fold stronger than the proliferative endothelium, suggesting transcriptional activation of A2M expression in the secretory endothelium. Glandular expression was observed in secretory endometrium from two patients with endometriosis. Ectopic endometrial tissues also produced A2M. A2M at concentrations of 400-500 mumol/L significantly inhibited blastocyst development of mouse embryos in vitro. A2M is expressed predominantly by the endometrial endothelial cells and may be involved in endometrial physiology. Physiological concentrations of A2M inhibit mouse embryo development in vitro, suggesting that endometrial production of A2M may play a role in regulating preimplantation embryo development.

Adult↗

PubMed1997

Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax.

Glandular epithelial cells of the human endometrium initiate apoptosis in the secretory phase of the cycle. To better understand the regulation of apoptosis in this paradigm of endocrine-regulated cell turnover, we studied the expression of the cell death regulatory genes, bax, bcl-2, and bcl-x, in human proliferative and secretory endometria relative to the absence or presence of apoptosis. As assessed by immunohistochemistry, levels of BAX protein were modest in proliferative endometrium and increased dramatically in the secretory phase when apoptosis was most prevalent. Expression of BAX was predominantly localized to epithelial cells of the functionalis layer of the secretory endometrium. In contrast, BCL-2 immunoreactivity was maximal during the proliferative phase and decreased in the secretory phase. Moreover, BCL-2 was topographically concentrated in the basalis layer. Immunoreactive BCL-X protein was observed mostly in glandular epithelial cells of the human endometrium. Compared with proliferative endometrium, secretory endometrium showed stronger BCL-X staining, especially in the functionalis layer. By Western blotting we confirmed that both proliferative and secretory endometrium expressed the long or antiapoptosis form as well as the short or proapoptosis form of the BCL-X protein. Taken together, our results demonstrate a coordinated pattern of expression of bcl-2 gene family members in human endometrium during the menstrual cycle, with a shift toward greater levels of the proapoptosis protein, BAX, occurring in glandular epithelial cells during the secretory phase of the cycle. Therefore, we conclude that cyclic changes in endometrial growth and regression may be precisely regulated by shifts in the ratio or balance of BCL-2 and related proteins in glandular epithelial cells.

Apoptosis↗

PubMed1996

Localization of the expression of complement component 3 in the human endometrium by in situ hybridization.

C3 production by the human endometrium has been previously described. The objective of the current study was to localize the site of expression and regulation of the third component of complement, C3, in the endometrium. Eight secretory and eight proliferative archival endometrial samples from hysterectomy and endometrial biopsy specimens were used for in situ hybridization analysis. This analysis was performed with a radiolabeled riboprobe synthesized from a 736-bp template representing sequence 1944-2680 of the human C3 complementary DNA. Duplicate sections were hybridized with sense and antisense riboprobes. Resultant autoradiograms were analyzed qualitatively by light- and darkfield microscopy. In proliferative endometrium, minimal expression of C3 was observed and was limited to a few stromal patches and glands throughout the section. In the secretory samples, prominent C3 expression was observed in both the glands and stroma of the basalis layer. Endometrial lymphocytes did not express C3. Endometrial stromal and glandular cells express the C3 gene. Endometrial lymphocytes did not express C3, but other nondistinct lymphoid elements scattered in the stroma may be expressing C3. There was a visibly more intense expression of C3 in the basalis layer of the secretory endometrium than in proliferative endometrium. The spatial and temporal pattern of C3 expression may have implications in normal menstrual physiology and in the immunological response of the endometrium to the invading trophoblast during placentation.

Adult↗

PubMedDate not supplied

Immunohistochemical localization of alpha-2 macroglobulin receptor/low-density lipoprotein receptor-related protein, receptor-associated protein, and Gp330 in the human endometrium.

OBJECTIVE: We investigated the expression of alpha-2 macroglobulin receptor/low-density lipoprotein receptor-related protein (LRP), an analogous receptor Gp330, and the 39-kd receptor-associated protein (RAP) in the human endometrium. METHODS: Specific polyclonal and monoclonal antibodies against LRP, Gp330, and RAP were used in standard immunohistochemical analyses of normal secretory and proliferative archival endometrial tissue. RESULTS: There was prominent and uniform stromal staining for LRP in secretory and proliferative endometrium. In both phases, 10-25% of glands stained positive for Gp330, with no appreciable stromal staining for Gp330. Also in both phases, 15-30% of glands stained positive for RAP, with apical staining in proliferative glands and uniform staining in secretory glands. Stromal staining for RAP was patchy and appeared to be more intense in the secretory samples than in the proliferative ones. CONCLUSIONS: The human endometrium expresses LRP, RAP, and Gp330. These receptor proteins are known to be involved in endocytosis of multiple ligands. Some of these ligands, such as proteases, plasminogen activators, and cytokines, are produced by the endometrium and play a role in endometrial remodeling and receptivity to implantation. By virtue of their in vitro properties, it is conceivable that endometrial LRP, Gp330, and RAP are involved in endometrial physiology.

Antibodies↗