Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells

The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte.     

Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes

The synthesis and intracellular distribution of actin were studied in isolated dictyate and metaphase II mouse oocytes by (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of newly synthetized oocyte protein and (2) cytochemical F-actin labeling by fluorescent phalloidin. Unpermeabilized, fully grown oocytes bound phalloidin intensely at the level of the zona pellucida (ZP), such ZP-associated actin representing a significant portion of total actin found in these cells. In contrast, phalloidin binding to ZP was very low in growing oocytes and was undetectable in ovulated, metaphase II eggs. When ZP-associated actin of fully grown oocytes was removed by prolongedly exposing oocytes to α-chymotrypsin, the amount of newly synthesized actin displayed by cumulus-enclosed oocytes was reduced to a level comparable to that shown by oocytes isolated from granulosa cells. We demonstrate that ZP-associated actin belongs to granulosa cell processes that remain within the ZP as a consequence of oocyte isolation procedures. We conclude that actin synthesis of mouse oocytes is not regulated by granulosa cells.     

Trypanosoma cruzi: strain selection by different schedules of mouse passage of an initially mixed infection

From an initial double infection in mice, established by simultaneous and equivalent inocula of bloodstream forms of strains Y and F of Trypanosoma cruzi, two lines were derived by subinoculations: one (W) passaged every week, the other (M) every month. Through biological and biochemical methods only the Y strain was identified at the end of the 10th and 16th passages of line W and only the F strain at the 2nd and 4th passages of line M. The results illustrate strain selection through laboratory manipulation of initially mixed populations of T. cruzi.     

Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells

The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.     

Synthesis of LDH-1 during mamalian oogenesis and early development.

Nuclear multiplication stage embryos were punctured in either the anterior, midlateral, or posterior regions. Both embryos and adults were examined for defects, and the defects were correlated with whether there had been any leakage of cytoplasmic material from the egg at the time of puncturing. Embryonic defects were found, correlated to the site of damage, in all three regions. A number of embryos was followed through development and it was found that 15.1% of the embryos which leaked cytoplasm hatched into larvae, compared to 82.3% of those which did not leak any cytoplasm. Morphological defects arising as a result of lateral puncture only were observed in adults. Many sterile adults were obtained from eggs in which the posterior region had been punctured. The results show that nuclear multiplication embryos are well able to tolerate the disturbance of the cortical cytoplasm created by puncture, but only rarely are they able to compensate for the actual loss of material by regulation. The results were similar to those observed after puncturing Drosophila embryos at the cellular blastoderm stage.     

VEGF,HIF-1α Expression and MVD as an Angiogenic Network in Familial Breast Cancer

Angiogenesis, which plays an important role in tumor growth and progression of breast cancer, is regulated by a balance between pro- and anti-angiogenic factors. Expression of vascular endothelial growth factor (VEGF) is up-regulated during hypoxia by hypoxia-inducible factor-1α (HIF-1α). It is known that there is an interaction between HIF-1α and BRCA1 carrier cancers, but little has been reported about angiogenesis in BRCA1-2 carrier and BRCAX breast cancers. In this study, we investigated the expression of VEGF and HIF-1α and microvessel density (MVD) in 26 BRCA1-2 carriers and 58 BRCAX compared to 77 sporadic breast cancers, by immunohistochemistry. VEGF expression in BRCA1-2 carriers was higher than in BRCAX cancer tissues (p = 0.0001). Furthermore, VEGF expression was higher in both BRCA1-2 carriers and BRCAX than the sporadic group (p<0.0001). VEGF immunoreactivity was correlated with poor tumor grade (p = 0.0074), hormone receptors negativity (p = 0.0206, p = 0.0002 respectively), and MIB-1-labeling index (p = 0.0044) in familial cancers (BRCA1-2 and BRCAX). The percentage of nuclear HIF-1α expression was higher in the BRCA1-2 carriers than in BRCAX cancers (p<0.05), and in all familial than in sporadic tumor tissues (p = 0.0045). A higher MVD was observed in BRCA1-2 carrier than in BRCAX and sporadic cancer tissues (p = 0.002, p = 0.0001 respectively), and in all familial tumors than in sporadic tumors (p = 0.01). MVD was positively related to HIF-1α expression in BRCA1-2 carriers (r = 0.521, p = 0.006), and, in particular, we observed a highly significant correlation in the familial group (r = 0.421, p<0.0001). Our findings suggest that angiogenesis plays a crucial role in BRCA1-2 carrier breast cancers. Prospective studies in larger BRCA1-2 carrier series are needed to improve the best therapeutic strategies for this subgroup of breast cancer patients.     

Nuclear NHERF1 expression as a prognostic marker in breast cancer

Our purpose was to investigate whether Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) expression could be linked to prognosis in invasive breast carcinomas. NHERF1, an ezrin-radixin-moesin (ERM) binding phosphoprotein 50, is involved in the linkage of integral membrane proteins to the cytoskeleton. It is therefore believed to have an important role in cell signaling associated with changes in cell cytoarchitecture. NHERF1 expression is observed in various types of cancer and is related to tumor aggressiveness. To date the most extensive analyses of the influence of NHERF1 in cancer development have been performed on breast cancer. However, the underlying mechanism and its prognostic significance are still undefined. NHERF1 expression was studied by immunohistochemistry (IHC) in a cohort of 222 breast carcinoma patients. Association of cytoplasmic and nuclear NHERF1 expression with survival was analyzed. Disease-free survival (DFS) and overall survival (OS) were determined based on the Kaplan–Meier method. Cytoplasmic NHERF1 expression was associated with negative progesterone receptor (PgR) (P=0.017) and positive HER2 expression (P=0.023). NHERF1 also showed a nuclear localization and this correlated with small tumor size (P=0.026) and positive estrogen receptor (ER) expression (P=0.010). Multivariate analysis identified large tumor size (P=0.011) and nuclear NHERF1 expression (P=0.049) to be independent prognostic variables for DFS. Moreover, the nuclear NHERF1(−)/ER(−) immunophenotype (27%) was statistically associated with large tumor size (P=0.0276), high histological grade (P=0.0411), PgR-negative tumors (P<0.0001) and high proliferative activity (P=0.0027). These patients had worse DFS compared with patients with nuclear NHERF1(+)/ER(+) tumors (75.4% versus 92.6% P=0.010). These results show that the loss of nuclear NHERF1 expression is associated with reduced survival, and the link between nuclear NHERF1 and ER expression may serve as a prognostic marker for the routine clinical management of breast cancer patients.