Antonin Bukovsky

Origin of germ cells and formation of new primary follicles in adult human ovaries

Recent reports indicate that functional mouse oocytes and sperm can be derived in vitro from somatic cell lines. We hypothesize that in adult human ovaries, mesenchymal cells in the tunica albuginea (TA) are bipotent progenitors with a commitment for both primitive granulosa and germ cells. We investigated ovaries of twelve adult women (mean age 32.8 ± 4.1 SD, range 27–38 years) by single, double, and triple color immunohistochemistry. We show that cytokeratin (CK)+ mesenchymal cells in ovarian TA differentiate into surface epithelium (SE) cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex are overgrown by TA, forming solid epithelial cords, which fragment into small (20 micron) epithelial nests descending into the lower ovarian cortex, before assembling with zona pellucida (ZP)+ oocytes. Germ cells can originate from SE cells which cover the TA. Small (10 micron) germ-like cells showing PS1 meiotically expressed oocyte carbohydrate protein are derived from SE cells via asymmetric division. They show nuclear MAPK immunoexpression, subsequently divide symmetrically, and enter adjacent cortical vessels. During vascular transport, the putative germ cells increase to oocyte size, and are picked-up by epithelial nests associated with the vessels. During follicle formation, extensions of granulosa cells enter the oocyte cytoplasm, forming a single paranuclear CK+ Balbiani body supplying all the mitochondria of the oocyte. In the ovarian medulla, occasional vessels show an accumulation of ZP+ oocytes (25–30 microns) or their remnants, suggesting that some oocytes degenerate. In contrast to males, adult human female gonads do not preserve germline type stem cells. This study expands our previous observations on the formation of germ cells in adult human ovaries. Differentiation of primitive granulosa and germ cells from the bipotent mesenchymal cell precursors of TA in adult human ovaries represents a most sophisticated adaptive mechanism created during the evolution of female reproduction. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. An essential mission of such follicular turnover might be elimination of spontaneous or environmentally induced genetic alterations of oocytes in resting primary follicles.

Oogenesis in cultures derived from adult human ovaries

Ten years ago, we reported that in adult human females the ovarian surface epithelium (OSE) is a source of germ cells. Recently, we also demonstrated that new primary follicles are formed by assembly of oocytes with nests of primitive granulosa cells in the ovarian cortex. The components of the new primary follicles, primitive granulosa and germ cells, differentiated sequentially from the OSE, which arises from cytokeratin positive mesenchymal progenitor cells residing in the ovarian tunica albuginea. In the present study, we investigated the possibility that the oocytes and granulosa cells may differentiate in cultures derived from adult human ovaries. Cells were scrapped from the surface of ovaries and cultured for 5 to 6 days, in the presence or absence of estrogenic stimuli [phenol red (PhR)]. The OSE cells cultured in the medium without PhR differentiated into small (15 micron) cells of granulosa phenotype, and epithelial, neural, and mesenchymal type cells. In contrast, OSE cells cultured in the presence of PhR differentiated directly into large (180 micron) cells of the oocyte phenotype. Such cells exhibited germinal vesicle breakdown, expulsion of the polar body, and surface expression of zona pellucida proteins, i.e. characteristics of secondary oocytes. These in vitro studies confirm our in vivo observations that in adult human ovaries, the OSE is a bipotent source of oocytes and granulosa cells. Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition, other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ totipotent embryonic stem cells developing from fertilized oocytes.

Multifaceted Regulation of Cell Cycle Progression by Estrogen: Regulation of Cdk Inhibitors and Cdc25A Independent of Cyclin D1-Cdk4 Function

ABSTRACT Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G1/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16INK4a to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16INK4a inhibited G1/S transition induced in MCF-7 cells by 17-β-estradiol (E2) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G1 and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21Cip1 and p27Kip1 was decreased, however, in both control and p16INK4a-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E2 in control and p16INK4a-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16INK4a. Inhibition of Cdc25A activity in p16INK4a-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E2-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisenseCDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16INK4a-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21Cip1 and p27Kip1.

Regression of endometrial explants in a rat model of endometriosis treated with the immune modulators loxoribine and levamisole.

OBJECTIVE To investigate the effects of the immune modulators levamisole and loxoribine in a rat model of endometriosis. DESIGN Prospective, placebo-controlled study. SETTING Hospital-based research facility. ANIMAL(S) Nineteen rats with experimentally induced endometriosis. INTERVENTION(S) Rats were treated with three weekly intraperitoneal injections of levamisole (2 mg per rat; n = 6), loxoribine (1 mg per rat; n = 6), or saline (control; n = 7) and killed 8 weeks after treatment. MAIN OUTCOME MEASURE(S) Histologic and immunohistochemical analysis of endometriotic explants. RESULT(S) The loxoribine-treated group showed marked regression of both epithelial and stromal components. Epithelial regression was noted in the control group, but the epithelium was strikingly preserved in the levamisole group. There were significantly greater numbers of dendritic cells in the explants of animals treated with loxoribine and levamisole. The number of natural killer cells was significantly reduced in loxoribine-treated explants. CONCLUSION(S) Loxoribine, a potent immunomodulatory drug, appeared to cause regression in both stromal and epithelium components in a rat model of endometriosis. Further, specific cell-mediated immune responses in this model of endometriosis were elucidated.

Signal transduction through the ras/Erk pathway is essential for the mycoestrogen zearalenone-induced cell-cycle progression in MCF-7 cells

Zearalenone is a naturally occurring estrogenic contaminant of moldy feeds and is present in high concentrations in dairy products and cereals. Zearalenone was postulated to contribute to the overall estrogen load of women, but the mechanisms of its action are not known. We demonstrated that zearalenone could stimulate the growth of estrogen receptor–positive human breast carcinoma cell line MCF‐7. In addition, zearalenone functioned as an antiapoptotic agent by increasing the survival of MCF‐7 cell cultures undergoing apoptosis caused by serum withdrawal. Treatment of these cells with 100 nM zearalenone induced cell‐cycle transit after increases in the expression of c‐myc mRNA and cyclins D1, A, and B1 and downregulation of p27Kip‐1. G1/G2‐phase kinase activity and phosphorylation of the retinoblastoma gene product was also evident. Flow cytometric analysis demonstrated entry of cells into the S and G2/M phases of the cell cycle, and phosphorylation of histone H3 occurred 36 h after zearalenone treatment. Ectopic expression of a dominant‐negative p21ras completely abolished the zearalenone‐induced DNA synthesis in these cells, and the specific inhibitor PD98059 for mitogen/extracellular‐regulated protein kinase kinase arrested S‐phase entry induced by zearalenone. These data suggest that the mitogen‐activated protein kinase signaling cascade is required for zearalenones effects on cell‐cycle progression in MCF‐7 cells. Given the presence of this mycotoxin in cereals, milk, and meat, the possibility that zearalenone is a potential promoter of breast cancer tumorigenesis should be investigated further. Mol. Carcinog. 30:88–98, 2001.

HARNESS and fault tolerant MPI

Abstract Initial versions of MPI were designed to work efficiently on multi-processors which had very little job control and thus static process models. Subsequently forcing them to support a dynamic process model would have affected their performance. As current HPC systems increase in size with greater potential levels of individual node failure, the need arises for new fault tolerant systems to be developed. Here we present a new implementation of MPI called fault tolerant MPI (FT-MPI) that allows the semantics and associated modes of failures to be explicitly controlled by an application via a modified MPI API. Given is an overview of the FT-MPI semantics, design, example applications, debugging tools and some performance issues. Also discussed is the experimental HARNESS core (G_HCORE) implementation that FT-MPI is built to operate upon.

Expression and localization of estrogen receptor-alpha protein in normal and abnormal term placentae and stimulation of trophoblast differentiation by estradiol

Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression.

Oogenesis in adult mammals, including humans: a review.

The origin of oocytes and primary follicles in ovaries of adult mammalian females has been a matter of dispute for over 100 yr. The prevailing belief that all oocytes in adult mammalian females must persist from the fetal period of life seems to be a uniquely retrogressive reproductive mechanism requiring humans to preserve their gametes from the fetal period for several decades. The utilization of modern techniques during last 10 yr clearly demonstrates that mammalian primordial germ cells originate from somatic cell precursors. This indicates that if somatic cells are precursors of germ cells, then somatic mutations can be passed on to progeny. Mitotically active germline stem cells have been described earlier in ovaries of adult prosimian primates and recently have been reported to also be present in the ovaries of adult mice. We have earlier shown that in adult human females, mesenchymal cells in the ovarian tunica albuginea undergo a mesenchymal-epithelial transition into ovarian surface epithelium cells, which differentiate sequentially into primitive granulosa and germ cells. Recently, we have reported that these structures assemble in the deeper ovarian cortex and form new follicles to replace earlier primary follicles undergoing atresia (follicular renewal). Our current observations also indicate that follicular renewal exists in rat ovaries, and human oocytes can differentiate from ovarian surface epithelium in fetal ovaries in vivo and from adult ovaries in vitro. These reports challenge the established dogma regarding the fetal origin of eggs and primary follicles in adult mammalian ovaries. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. Yet, the follicular renewal may cease at a certain age, and this may predetermine the onset of the natural menopause or premature ovarian failure. A lack of follicular renewal in aging ovaries may cause an accumulation of spontaneously arising or environmentally induced genetic alterations of oocytes, and that may be why aging females have a much higher chance of having oocytes with more mutations in persisting primary follicles.

Placental expression of estrogen receptor beta and its hormone binding variant – comparison with estrogen receptor alpha and a role for estrogen receptors in asymmetric division and differentiation of estrogen-dependent cells

During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single ~52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize ~64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a ~64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of ~64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.

Estrogen, Progesterone, and Androgen Receptor Expression in Levator Ani Muscle and Fascia

Previous studies have reported controversial data on estrogen receptor (ER) expression in levator ani muscle. We investigated ER expression in levator ani muscle and fascia and compared it with the expression of progesterone receptor (PR) and androgen receptor (AR). The study included 55 women undergoing surgery for gynecological (asymptomatic, n = 10) or urogynecological conditions (symptomatic, n = 45). The asymptomatic and 21 of the symptomatic women received no hormone replacement therapy (HRT). The remaining 24 symptomatic women received some form of HRT. Biopsies were taken from the levator ani muscle and the overlying fascia, and quantitative measurements of immunohistochemical staining by image analysis were made. None of the levator ani muscle samples showed any evidence of nuclear ER expression in striated muscle fibers, but some cells in the muscular stroma did express ER. However, PR and AR expression was found in both muscle and stromal cells. Levator ani fascia showed nuclear ER, PR, and AR expression to varying degrees. There was a significant increase (p < 0.03) in ER expression in levator ani fascia of symptomatic patients without HRT when compared with asymptomatic age-matched women. The ER expression was significantly lower (p < 0.001) in postmenopausal symptomatic women receiving long-term estrogen replacement compared with age-matched women without HRT. Our data indicate that ER expression is significantly higher in symptomatic women compared with age-matched asymptomatic females. However, long-term estrogenization causes significant decrease of ER expression.