Global deletion of Trp53 reverts ovarian tumor phenotype of the germ cell-deficient white spotting variant (Wv) mice.

White spotting variant (Wv) mice are spontaneous mutants attributed to a point mutation in the c-Kit gene, which reduces the tyrosine kinase activity to around 1% and affects the development of melanocytes, mast cells, and germ cells. Homozygous mutant mice are sterile but can live nearly a normal life span. The female Wv mice have a greatly reduced ovarian germ cell and follicle reserve at birth, and the remaining follicles are largely depleted soon after the females reach reproductive stage at around 7 weeks of age. Consequently, ovarian epithelial tumors develop in 100% of Wv females by 3 to 4 months of age. These tumors, called tubular adenomas, are benign but can become invasive in older Wv mice. We tested if additional genetic mutation(s) could convert the benign ovarian epithelial tumors to malignant tumors by crossing the Wv mutant into the Trp53 knockout background. Surprisingly, we found that global deletion of Trp53 suppressed the development of ovarian tubular adenomas in Wv mice. The ovaries of Wv/Wv; Trp53 (-/-) mice were covered by a single layer of surface epithelium and lacked excessive epithelial proliferation. Rather, the ovaries contained a small number of follicles. The presence of ovarian follicles and granulosa cells, as indicated by Pgc7 and inhibin-alpha expression, correlated with the absence of epithelial lesions. A reduction of Pten gene dosage, as in Wv/Wv; Pten (+/-) mice, produced a similar, though less dramatic, phenotype. We conclude that deletion of Trp53 prolongs the survival of ovarian follicles in Wv mice and consequently prevents the proliferation of ovarian epithelial cells and development of ovarian tubular adenomas. The results suggest that various cell types within the ovary communicate and mutually modulate, and an intact tissue environment is required to ensure homeostasis of ovarian surface epithelial cells. Especially, the current finding emphasizes the importance of ovarian follicles in suppressing the hyperplastic growth of ovarian epithelial cells, dominating over the loss of p53.

Cyclooxygenase-1 inhibition prolongs postnatal ovarian follicle lifespan in mice.

Menopause is the permanent cessation of menstruation that results from depletion of ovarian germ cells and follicles. Although most animals experience reproductive senescence, the mechanisms differ from that in women, who may live more than one-third of their lives after menopause and consequently face the risk of a number of menopause-associated health problems. Understanding factors that influence ovarian aging may provide strategies to delay or alleviate physiological alterations that take place in postmenopausal women. The germ cell-deficient Wv mice recapitulate follicle loss, prolong postreproductive lifespan, and model many physiological changes that take place in postmenopausal women. Here, using genetic and pharmacological approaches, we found that inhibition of cyclooxygenase-1 but not cyclooxygenase-2 in Wv mice delays germ cell depletion and preserves ovarian follicles. Cyclooxygenase-1 inhibition slows down follicle maturation at the conversion of primary to secondary follicles and prolongs postnatal ovarian follicle lifespan. The current study suggests that inhibition of cyclooxygenase-1 may be able to delay ovarian aging and modulate menopausal timing.

White spotting variant mouse as an experimental model for ovarian aging and menopausal biology.

OBJECTIVE: Menopause is a unique phenomenon in modern women, as most mammalian species possess a reproductive period comparable with their life span. Menopause is caused by the depletion of germ cell-containing ovarian follicles and in laboratory studies is usually modeled in animals in which the ovarian function is removed through ovariectomy or chemical poisoning of the germ cells. Our objective was to explore and characterize the white spotting variant (Wv) mice that have reduced ovarian germ cell abundance, a result of a point mutation in the c-kit gene that decreases kinase activity, as a genetic model for use in menopause studies. METHODS: Physiological and morphological features associated with menopause were determined in female Wv/Wv mice compared with age-matched wildtype controls. Immunohistochemistry was used to evaluate the presence and number of follicles in paraffin-embedded ovaries. Bone density and body composition were evaluated using the PIXImus x-ray densitometer, and lipids, calcium, and hormone levels were determined in serum using antigen-specific enzyme immunoassays. Heart and body weight were measured, and cardiac function was evaluated using transthoracic echocardiography. RESULTS: The ovaries of the Wv/Wv females have a greatly reduced number of normal germ cells at birth compared with wildtype mice. The remaining follicles are depleted by around 2 months, and the ovaries develop benign epithelial lesions that resemble morphological changes that occur during ovarian aging, whereas a normal mouse ovary has numerous follicles at all stages of development and retains some follicles even in advanced age. Wv mice have elevated plasma gonadotropins and reduced estrogen and progesterone levels, a significant reduction in bone mass density, and elevated serum cholesterol and lipoprotein levels. Moreover, the Wv female mice have enlarged hearts and reduced cardiac function. CONCLUSIONS: The reduction of c-kit activity in Wv mice leads to a substantially diminished follicular endowment in newborn mice and premature depletion of follicles in young mice, although mutant females have a normal life span after cessation of ovarian function. The Wv female mice exhibit consistent physiological changes that resemble common features of postmenopausal women. These alterations include follicle depletion, morphological aging of the ovary, altered serum levels of cholesterol, gonadotropins and steroid hormones, decreased bone density, and reduced cardiac function. These changes were not observed in male mice, either age-matched male Wv/Wv or wildtype mice, and are improbably caused by global loss of c-kit function. The Wv mouse may be a genetic, intact-ovary model that mimics closely the phenotypes of human menopause to be used for further studies to understand the mechanisms of menopausal biology.

Loss of A-type lamin expression compromises nuclear envelope integrity in breast cancer.

Through advances in technology, the genetic basis of cancer has been investigated at the genomic level, and many fundamental questions have begun to be addressed. Among several key unresolved questions in cancer biology, the molecular basis for the link between nuclear deformation and malignancy has not been determined. Another hallmark of human cancer is aneuploidy; however, the causes and consequences of aneuploidy are unanswered and are hotly contested topics. We found that nuclear lamina proteins lamin A/C are absent in a significant fraction (38%) of human breast cancer tissues. Even in lamin A/C-positive breast cancer, lamin A/C expression is heterogeneous or aberrant (such as non-nuclear distribution) in the population of tumor cells, as determined by immunohistology and immunofluorescence microscopy. In most breast cancer cell lines, a significant fraction of the lamin A/C-negative population was observed. To determine the consequences of the loss of lamin A/C, we suppressed their expression by shRNA in non-cancerous primary breast epithelial cells. Down-regulation of lamin A/C in breast epithelial cells led to morphological deformation, resembling that of cancer cells, as observed by immunofluorescence microscopy. The lamin A/C-suppressed breast epithelial cells developed aneuploidy as determined by both flow cytometry and fluorescence in situ hybridization. We conclude that the loss of nuclear envelope structural proteins lamin A/C in breast cancer underlies the two hallmarks of cancer aberrations in nuclear morphology and aneuploidy.

Ectopic expression of GATA6 bypasses requirement for Grb2 in primitive endoderm formation.

Gene knockouts in mice have showed that Grb2 and GATA6 are essential for the formation of primitive endoderm in blastocysts. Here, we confirmed that implanted Grb2-null blastocysts lack primitive or extraembryonic endoderm cells either at E4.5 or E5.5 stages. We analyzed the relationship between Grb2 and GATA6 in the differentiation of embryonic stem (ES) cells to primitive endoderm in embryoid body models. Upon transfection with GATA6 expression vector, Grb2-null ES cells underwent endoderm differentiation as indicated by the expression of the extraembryonic endoderm markers Dab2 and GATA4. Transfection of GATA4 expression vector also had the same differentiation potency. When GATA6- or GATA4-transfected Grb2-null ES cells were allowed to aggregate, fragments of an endoderm layer formed on the surface of the spheroids. The results suggest that GATA6 is downstream of Grb2 in the inductive signaling pathway and the expression of GATA6 is sufficient to compensate for the defects caused by Grb2 deficiency in the development of the primitive and extraembryonic endoderm.

Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells.

Background. Mouse embryonic stem (ES) cells can be differentiated in vitro by aggregation and/or retinoic acid (RA) treatment. The principal differentiation lineage in vitro is extraembryonic primitive endoderm. Dab2, Laminin, GATA4, GATA5, and GATA6 are expressed in embryonic primitive endoderm and play critical roles in its lineage commitment. Results. We found that in the absence of GATA4 or GATA5, RA-induced primitive endoderm differentiation of ES cells was reduced. GATA4 (-/-) ES cells express higher level of GATA5, GATA6, and hepatocyte nuclear factor 4 alpha marker of visceral endoderm lineage. GATA5 (-/-) ES cells express higher level of alpha fetoprotein marker of early liver development. GATA6 (-/-) ES cells express higher level of GATA5 as well as mesoderm and cardiomyocyte markers which are collagen III alpha-1 and tropomyosin1 alpha. Thus, deletion of GATA6 precluded endoderm differentiation but promoted mesoderm lineages. Conclusions. GATA4, GATA5, and GATA6 each convey a unique gene expression pattern and influences ES cell differentiation. We showed that ES cells can be directed to avoid differentiating into primitive endoderm and to adopt unique lineages in vitro by modulating GATA factors. The finding offers a potential approach to produce desirable cell types from ES cells, useful for regenerative cell therapy.

Nuclear entry of activated MAPK is restricted in primary ovarian and mammary epithelial cells.

BACKGROUND: The MAPK/ERK1/2 serine kinases are primary mediators of the Ras mitogenic signaling pathway. Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription. PRINCIPAL FINDINGS: Here, however, we observe that in primary cultures of breast and ovarian epithelial cells, phosphorylation and activation of ERK1/2 are disassociated from nuclear translocalization and transcription of downstream targets, such as c-Fos, suggesting that nuclear translocation is limited in primary cells. Accordingly, in import assays in vitro, primary cells showed a lower import activity for ERK1/2 than cancer cells, in which activated MAPK readily translocated into the nucleus and activated c-Fos expression. Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells. Additionally, reduction in expression of nucleoporin 153 by siRNA targeting reduced ERK1/2 nuclear activity in cancer cells. CONCLUSION: ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors.

A reduction of cyclooxygenase 2 gene dosage counters the ovarian morphological aging and tumor phenotype in Wv mice.

Menopausal ovaries undergo morphological changes, known as ovarian aging, which are implicated in the high incidence of ovarian cancer occurring during the perimenopausal and immediate postmenopausal periods. The germ cell-deficient Wv mice recapitulate these postmenopausal alterations in ovarian morphology and develop tubular adenomas. We demonstrate that a reduction of cyclooxygenase 2 gene dosage rescued the ovarian aging phenotype of the Wv mice, whereas homozygous deletion was accompanied by a compensatory increase in ovarian cyclooxygenase 1 expression and prostaglandin E(2) synthesis. Cyclooxygenase inhibitors also reduced the tumor phenotype in a preliminary study. These findings suggest that increased cyclooxygenase activity contributes to the preneoplastic morphological changes of the ovarian surface epithelium, which can be reversed by a reduction of gene dosage achieved by either genetic or pharmacological approaches.

Perception of differentiation cues by GATA factors in primitive endoderm lineage determination of mouse embryonic stem cells.

The formation of the primitive endoderm covering the inner cell mass of early mouse embryos can be simulated in vitro by the differentiation of mouse embryonic stem (ES) cells in culture following either aggregation of suspended cells or stimulation of cell monolayers with retinoic acid. The developmentally regulated transcription factors GATA-4 and GATA-6 have determining role in mouse extraembryonic endoderm development. We analyzed the in vitro differentiation of mouse embryonic stem cells deficient of GATA factors and conclude that GATA-4 is required for ES cells to perceive a cell positioning (cell aggregation) signal and GATA-6 is required to sense morphogenic (retinoic acid) signal. The collaboration between GATA-6 and GATA-4, or GATA-6 and GATA-5 which can substitute for GATA-4, is involved in the perception of differentiation cues by embryonic stem cells in their determination of endoderm lineage. This study indicates that the lineage differentiation of ES cells can be manipulated by the expression of GATA factors.

Regulation of Ras-MAPK pathway mitogenic activity by restricting nuclear entry of activated MAPK in endoderm differentiation of embryonic carcinoma and stem cells.

In response to retinoic acid, embryonic stem and carcinoma cells undergo differentiation to embryonic primitive endoderm cells, accompanied by a reduction in cell proliferation. Differentiation does not reduce the activation of cellular MAPK/Erk, but does uncouple mitogen-activated protein kinase (MAPK) activation from phosphorylation/activation of Elk-1 and results in inhibition of c-Fos expression, whereas phosphorylation of the cytoplasmic substrate p90RSK remains unaltered. Cell fractionation and confocal immunofluorescence microscopy demonstrated that activated MAPK is restricted to the cytoplasmic compartment after differentiation. An intact actin and microtubule cytoskeleton appears to be required for the restriction of MAPK nuclear entry induced by retinoic acid treatment because the cytoskeletal disrupting agents nocodazole, colchicine, and cytochalasin D are able to revert the suppression of c-Fos expression. Thus, suppression of cell proliferation after retinoic acid-induced endoderm differentiation of embryonic stem and carcinoma cells is achieved by restricting nuclear entry of activated MAPK, and an intact cytoskeleton is required for the restraint.

Ras/MAPK pathway confers basement membrane dependence upon endoderm differentiation of embryonic carcinoma cells.

The formation of extraembryonic endoderm is one of the earliest steps in the differentiation of pluripotent cells of the inner cell mass during the early stages of embryonic development. The primitive endoderm cells and the derived parietal and visceral endoderm cells gain the capacity to produce collagen IV and laminin. The deposition of these components results in the formation of basement membrane and epithelium of the endoderm, with polarized cells covering the inner surface of the blastocoels. We used retinoic acid-induced endoderm differentiation of stem cell-like F9 embryonic carcinoma cells to study the role of the Ras pathway and its regulation in the formation of the visceral endoderm. Upon endoderm differentiation of F9 cells induced by retinoic acid, c-Fos expression, the downstream target of the Ras pathway, is suppressed by uncoupling Elk-1 phosphorylation/activation to MAPK activity. However, attachment to matrix gel greatly enhances the activation of MAPK in endoderm cells but not in undifferentiated F9 cells. Enhanced MAPK activation as a result of contact with basement membrane is able to compensate for reduced Elk-1 phosphorylation and c-Fos expression. We conclude that endoderm differentiation renders the activation of the Ras pathway basement membrane dependent, contributing to the epithelial organization of the visceral endoderm.