Mitosis-specific phosphorylation of vimentin by protein kinase C coupled with reorganization of intracellular membranes.

Using two types of anti-phosphopeptide antibodies which specifically recognize vimentin phosphorylated by protein kinase C (PKC) at two distinct PKC sites, we found that PKC acted as a mitotic vimentin kinase. Temporal change of vimentin phosphorylation by PKC differed form changes by cdc2 kinase. The mitosis-specific vimentin phosphorylation by PKC was dramatically enhanced by treatment with a PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), while no phosphorylation of vimentin by PKC was observed in interphase cells treated with TPA. By contrast, the disruption of subcellular compartmentalization of interphase cells led to vimentin phosphorylation by PKC. Cytoplasmic and nuclear membranes are fragmented and dispersed in the cytoplasm and some bind to vimentin during mitosis. Thus, targeting of activated PKC, coupled with the reorganization of intracellular membranes which contain phospholipids essential for activation, leads to the mitosis-specific phosphorylation of vimentin. We propose that during mitosis, PKC may phosphorylate an additional subset of proteins not phosphorylated in interphase.

Optical switching and image storage by means of azobenzene liquid-crystal films.

Liquid crystals are promising materials for optical switching and image storage because of their high resolution and sensitivity. Azobenzene liquid crystals (LCs) have been developed, in which azobenzene moieties play roles as both mesogens and photosensitive chromophores. Azobenzene LC films showed a nematic phase in trans isomers and no LC phase in cis isomers. Trans-cis photoisomerization of azobenzene with a laser pulse resulted in a nematic-to-isotropic phase transition with a rapid optical response of 200 microseconds.

A physiological role of epidermal growth factor in male reproductive function.

Epidermal growth factor (EGF) stimulates the proliferation of various mammalian cells in culture, but its physiological role is not well defined. In mature male mice, large amounts of EGF are produced in the submandibular gland; it is present in the circulation at approximately 5 nanograms of EGF per milliliter of plasma. Sialoadenectomy (removal of the submandibular glands) decreased the amount of circulating EGF to an undetectable level but did not affect the circulating levels of testosterone or follicle-stimulating hormone. The number of mature sperm in the epididymis decreased by as much as 55 percent; the number of spermatids in the testis decreased by 40 to 50 percent; and the number of spermatocytes increased by about 20 percent. Administration of EGF to sialoadenectomized mice restored both the sperm content of the epididymis and the number of spermatids in the testis to normal. Thus, EGF may play a role in male reproductive function by stimulating the meiotic phase of spermatogenesis.

Epidermal growth factor-like, corneal wound healing substance in mouse tears.

We have identified the presence of a putative corneal wound healing substance in mouse tears, which has a molecular size and immunological properties similar to those of epidermal growth factor (EGF). The substance was capable of binding to EGF receptors in mouse parenchymal cells and this binding was inhibited by anti-EGF serum. The concentration of the EGF-like substance in the tears of male and female mice was estimated to be 79.3 +/- 7.0 (SD) ng/ml and 76.5 +/- 8.1 (SD) ng/ml, respectively, by EGF radioimmunoassay. Removal of the submandibular glands, which produce large amounts of EGF, reduced plasma EGF to an undetectable level and also decreased the concentration of the EGF-like substance in tears to 27.3 +/- 3.9 (SD) ng/ml in male mice and 25.8 +/- 3.7 (SD) ng/ml in female mice. Approximately 50% of sialoadenectomized (submandibular glands removed) male mice with deep corneal wounds developed severe ocular lesions or loss of sight whereas none of normal male mice with similar wounds did. Topical application of EGF to deeply wounded eyes of sialoadenectomized mice eliminated the various complications and restored the healing rate and incidence of recovery to virtually normal levels.

Importance of epidermal growth factor in implantation and growth of mouse mammary tumor in female nude mice.

In female nude mice, epidermal growth factor (EGF) was present at a mean concentration of 42.8 +/- 16.9 (SD) ng/mg wet tissue in the submandibular gland and 0.28 +/- 0.13 ng/ml in the plasma. Sialoadenectomy (removal of the submandibular glands) decreased circulating EGF to undetectable levels (less than 0.1 ng/ml). The possible role of EGF in transplantation and growth of mouse mammary tumors in nude mice was examined by sialoadenectomy, anti-EGF treatment, and EGF replacement therapy. The success rate of transplantation of a spontaneous mouse mammary tumor into nude mice was 55% in normal females and 17% in sialoadenectomized animals. Anti-EGF treatment of sialoadenectomized mice completely abolished the implantation of the tumor. Daily administration of EGF at a dose of 5 micrograms per mouse to both normal and sialoadenectomized animals enhanced the success rate of tumor implantation to more than 80%. Sialoadenectomy and/or anti-EGF treatment of tumor-bearing nude mice reduced the growth of implanted mammary tumors. These results suggest that EGF is important for implantation and growth of spontaneous mouse mammary tumor in female nude mice.

Caspase-2 deficiency prevents programmed germ cell death resulting from cytokine insufficiency but not meiotic defects caused by loss of ataxia telangiectasia-mutated (Atm) gene function.

It is well established that programmed cell death claims up to two-thirds of the oocytes produced during gametogenesis in the developing fetal ovaries. However, the mechanisms underlying prenatal germ cell loss in females remain poorly understood. Herein we report that caspase-11 null female mice are born with a reduced number of oocyte-containing primordial follicles. This phenotype is likely due to failed cytokine processing known to occur in caspase-11 mutants since neonatal female mice lacking both interleukin (IL)-1alpha and IL-1beta also exhibit a reduced endowment of primordial follicles. In addition, germ cell death in wild-type fetal ovaries cultured ex vivo is suppressed by either cytokine, likely via ligand activation of type 1 IL-1 receptors expressed in fetal germ cells. Normal oocyte endowment can be restored in caspase-11 null female mice by simultaneous inactivation of the gene encoding the cell death executioner enzyme, caspase-2. However, caspase-2 deficiency cannot overcome gametogenic failure resulting from meiotic recombination defects in ataxia telangiectasia-mutated (Atm) null female mice. Thus, genetically distinct mechanisms exist for developmental deletion of oocytes via programmed cell death, one of which probably functions as a meiotic quality-control checkpoint that cannot be overridden.

Fas/APO-1/CD95 system as a mediator of granulosa cell apoptosis in ovarian follicle atresia.

Current studies have shown that atresia of ovarian follicles is induced through apoptosis in granulosa cells. Several articles have been devoted to study of the molecular mechanisms responsible for APO-1/CD95 (Fas) is a cell surface protein that can mediate apoptosis in lymphoid cells, and Fas ligand was recently identified in a cytotoxic T cell line. To clarify the involvement of the Fas-Fas ligand system in granulosa cell apoptosis, we investigated the expression of Fas and Fas ligand at an individual cell level. For this purpose, we raised specific polyclonal antibodies against Fas and Fas ligand. Western blotting confirmed that our anti-Fas antibodies (anti-P2 and anti-P4) detect a specific band with a mol wt of 45 kDa in the lysate of ovaries from immature PMSG-treated rats or adult cyclic rats. In immature PMSG-treated rats, immunohistochemical analysis with these antibodies revealed specific staining of granulosa cells in secondary and tertiary follicles at an early stage of atresia, but not in healthy follicles. Fas messenger RNA was also found in granulosa cells of early atretic follicles using in situ hybridization. On the other hand, the anti-Fas ligand antibody (anti-P5) detected a specific 31-kDa band on a Western blot of the oocytes lysate, and the staining with the serum was localized to oocytes in most of developing follicles. Colocalization of Fas and Fas ligand in certain follicles intimately correlated with granulosa cell apoptosis, which was revealed by terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling staining of DNA strand breaks. Finally, we found that interferon-gamma increased Fas expression on granulosa cells in vitro. Coculturing interferon-gamma-pretreated granulosa cells with zona-free oocytes induced granulosa cell apoptosis, which was confirmed by Hoechst 33342 dye staining and terminal deoxynucleotidyl transferase-mediated deoxy-UTP-biotin nick end labeling, and the killing effect of oocytes was abolished by the addition of anti-P2, which was expected to interrupt the interaction between Fas and Fas ligand. These results demonstrate that activation between Fas and Fas ligand. These results demonstrate that activation of the Fas-Fas ligand system is capable of initiating apoptosis in the ovary, as are a number of other stimuli, outside the immune system.

Maternal epidermal growth factor deficiency causes fetal hypoglycemia and intrauterine growth retardation in mice: possible involvement of placental glucose transporter GLUT3 expression.

We investigated the physiological role of epidermal growth factor (EGF) in fetal growth in mice in which midgestational sialoadenectomy induced maternal EGF deficiency. Sialoadenectomy decreased the fetal weight significantly, indicating that maternal EGF deficiency caused intrauterine growth retardation. The weight of the fetal liver in the sialoadenectomized mice was reduced in proportion to the decrease in body weight (82.7+/-10.2 vs. 70.9+/-10.9 mg), whereas the brain weight was not reduced. Sialoadenectomy significantly decreased the glucose concentration in fetal plasma (86.0+/-13.0 vs. 63.0+/-11.8 mg/dl) without affecting the maternal plasma level of glucose. Transplacental transfer of 3H-2-deoxyglucose was significantly decreased by sialoadenectomy (5.17+/-1.25 vs. 2.94+/-1.02%), but transfer of 14C-aminoisobutyric acid was not affected. Northern blot analysis and in situ hybridization of glucose transporter isoform GLUT1 and GLUT3 messenger RNAs (mRNAs) in placenta revealed that sialoadenectomy significantly reduced the expression of GLUT3 mRNA without affecting GLUT1 mRNA levels. Administration of anti-EGF antiserum enhanced the effects of EGF deficiency, which were almost completely corrected by EGF supplementation. These results indicate that EGF plays an important role in fetal growth by regulating the transplacental supply of glucose via GLUT3 expression in the placenta.

Evidence for the presence of angiogenin in human follicular fluid and the up-regulation of its production by human chorionic gonadotropin and hypoxia.

Angiogenesis is an essential event during the development of the ovarian follicle and ensuing formation of the corpus luteum. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human ovary. Follicular fluid (FF) and granulosa cells (GCs) were collected from women undergoing in vitro fertilization and embryo transfer. The presence of angiogenin in FF and GCs was demonstrated by Western blot analysis. The production of angiogenin by cultured GCs was stimulated with the addition of human CG or cAMP or under the hypoxic milieu. Concentrations of angiogenin in FF from an individual follicle were positively correlated with those of progesterone, but not estradiol and testosterone. Given the presence of angiogenin in FF and up-regulation of its production by human CG and hypoxia, it seems logical to assume that angiogenin may play a role as a local angiogenic factor in the human ovary.

Demonstration of angiogenin in human endometrium and its enhanced expression in endometrial tissues in the secretory phase and the decidua.

Angiogenesis is thought to be crucial for normal physiology of the endometrium, where dynamic vascular remodeling occurs during the menstrual cycle and pregnancy. We investigated the presence of angiogenin, a potent inducer of angiogenesis, and the regulatory mechanisms of its production in the human endometrium. Western blot analysis demonstrated that angiogenin protein expression increased by 3- to 4-fold in the endometrium in the mid and late secretory phases and in early gestation relative to that during the proliferative phase. Quantitative mRNA analysis showed the similar tendency in the expression of angiogenin mRNA in the endometrium, with the highest levels observed in the mid and late secretory phases and early gestation. An immunohistochemical study showed that angiogenin was expressed in both stromal cells and epithelial cells, with indistinguishable intensity between these cells regardless of phases of the menstrual cycle. In support of the Western blot analysis, the intensity of staining appeared to be highest in the mid to late secretory phases relative to other phases. Consistent with these in vivo results, decidualized cultured stromal cells, after treatment with progesterone or progesterone plus E2, exhibited the capacity to secrete significantly increased amounts of angiogenin compared with untreated or E2 alone-treated control group. Both the treatment with (Bu)2cAMP and hypoxic conditions stimulated angiogenin secretion by stromal cells. For isolated epithelial cells, hypoxia stimulated angiogenin secretion, whereas (Bu)2cAMP had no appreciable effect. In summary, we demonstrated the presence of angiogenin in human endometrium and its possible local regulatory factors, such as progesterone, cAMP, and hypoxia. These findings along with its enhanced expression in the endometrium in the secretory phase and in decidual tissues raise the possibility that angiogenin may play a role in establishing pregnancy.

Differential expression of secreted frizzled-related protein 4 in decidual cells during pregnancy.

During pregnancy, the uterus shows marked morphological and physiological changes under the regulation of ovarian steroid. To elucidate the molecular cues of these changes, we tried to identify the transcripts differentially expressed in the pregnant rat uterus by using the suppression subtractive hybridization method. Seven independent clones were isolated and one of the up-regulated genes was secreted frizzled-related protein 4 (sFRP4). sFRP4 contains a Wnt-binding domain and belongs to the secreted frizzled protein family whose members are assumed to function as modulators of the Wnt signal. The expression level of sFRP4 mRNA reached a peak in the pregnant uterus on day 12, when uterine decidualization was almost complete in the rat. In situ hybridization histochemistry revealed that sFRP4 transcripts were observed in the decidual cells. In addition, proliferating cell nuclear antigen (PCNA)-positive cells were shown to be overlapped in decidua, suggesting that sFRP4 mRNA expression was accompanied by the late phase of decidual cell proliferation. Moreover, sFRP4 and estrogen receptor-alpha transcripts were co-localized. Furthermore, we analyzed the regulation of sFRP4 by estrogen using 17 beta-estradiol-treated ovariectomized rats. sFRP4 mRNA was detected in the uterus at 48 h after estrogen treatment, especially in endometrial stroma where PCNA-positive cells were also observed. The results in this study led us to the notion that sFRP4 mRNA may be up-regulated after estrogen treatment in the late phase of uterine cell proliferation.

Differential immunolocalization of estrogen receptor alpha and beta in rat ovary and uterus.

In order to investigate the localization of estrogen receptor (ER) alpha and ERbeta in the reproductive organs in the rat, polyclonal antibodies were raised to each specific amino acid sequence. The Western blot with anti-ERalpha antibody showed a 66 kDa band in rat ovary and uterus, while that with anti-ERbeta antibody detected a 55 kDa band in rat ovary, uterus and prostate. The ligand-independent nuclear localization of the two receptors was verified by immunocytochemistry. By immunohistochemistry, the nuclei of glandular and luminal epithelial cells in the uterus were stained with anti-ERalpha antibody, whereas only the nuclei of glandular epithelium cells were stained with anti-ERbeta antibody. In rat ovary, positive signals were shown with anti-ERbeta antibody in the nuclei of granulosacells. No specific immunostaining was observed with anti-ERalpha antibody. Although ERbeta was immunostained at the proestrous, metestrous and diestrous stages, the immunoreactivity of ERbeta was hardly detected at the estrous stage in rat ovary. Thus, we show differential expression of ERalpha and ERbeta in rat uterus and ovary at the protein level, which may provide a clue for understanding the roles of the two receptors in reproductive organs.

Effects of cyclopiazonic acid on rhythmic contractions in uterine smooth muscle bundles of the rat.

1. We studied the effects of cyclopiazonic acid (CPA) on rhythmic contractions and on Ca2+ uptake by the intracellular stores in longitudinal muscle strips of the rat uterus at 30 degrees C. 2. Oxytocin (1 microM) in Ca(2+)-free solution induced a transient rise in the intracellular Ca2+ concentration ([Ca2+]i) and contraction after Ca2+ loading of the stores in high-K(+)- and Ca(2+)-containing solution. CPA inhibited oxytocin-induced Ca2+ release and contraction, the half and full inhibitory concentrations of CPA being 0.3 and 10 microM, respectively. In contrast, addition of CPA after Ca2+ loading exerted no significant inhibitory effects. 3. Oxytocin (10 nM) applied in Ca(2+)-containing solution induced rhythmic increases in both force and [Ca2+]i. CPA (10 microM) had no effect on oxytocin-induced rhythmic contractions. 4. At a high concentration (300 microM), CPA inhibited the rhythmic contractions induced by 10 nM oxytocin; the frequency and the peak height were decreased, and in many bundles contractions were completely abolished. These inhibitory effects were reversed after CPA washout. 5. CPA (300 microM) inhibited the rate of rise of [Ca2+]i due to depolarization induced by high-K(+)-containing solution. 6. These results suggest that low concentrations of CPA inhibit the loading of Ca2+ into intracellular stores in intact tissue strips, and that the Ca2+ stores are not directly involved in the uterine rhythmic contractions. It is also suggested that a high concentration of CPA inhibits the mechanism that is responsible for the generation of rhythmic contractions as well as voltage-dependent Ca2+ channels.

The uterine growth-promoting action of epidermal growth factor and its function in the fertility of mice.

Epidermal growth factor (EGF) levels in the submandibular glands and plasma are increased in pregnant and aged female mice. The possible role of EGF in fertility was studied in virgin and pregnant mice ranging in age from 10 to 90 weeks of age, employing sialoadenectomy, administration of EGF antibody and EGF replacement. The uterine weight in pregnant, 10-week-old, sialoadenectomized mice was significantly less than in normal mice and the administration of EGF antibody to these mice further decreased uterine weight, resulting in an increased rate of abortion. Replacement EGF treatment in the sialoadenectomized mice prevented these changes. Uterine weight was about 70 mg at 10 weeks of age, and significantly increased from 30 to 80 weeks when it reached a plateau level of 275 mg. These changes closely followed the increase in the concentration of EGF in the submandibular glands and plasma and coincided with the decline in fertility. In contrast, uterine weight in the sialoadenectomized mice decreased immediately after the operation and remained at about 50-60 mg throughout the experimental period. Pregnancy, as judged by implantation, was achieved in the sialoadenectomized mice at later ages than in the controls. These findings suggest that elevated EGF levels may have a dual function in the control of fertility via uterine growth, depending on the age of mice.

Effect of sialoadenectomy, treatment with epidermal growth factor (EGF) antiserum and replacement of EGF on the epidermis in mice.

Epidermal growth factor (EGF) was present at a mean concentration of 266 +/- 18 (S.E.M.) pmol/mg wet tissue in the submandibular gland of 3-month-old male mice; it was also present in plasma at a concentration of 364 +/- 149 pmol/l. Sialoadenectomy (removal of the submandibular glands) decreased the plasma EGF content to undetectable levels (less than 16.5 pmol/l), lowered the concentration of EGF in the skin from 1.22 +/- 0.11 to 0.47 +/- 0.08 fmol/mg wet tissue and reduced the thickness of the epidermis from 28.9 +/- 2.7 to 11.0 +/- 0.8 micron in 3 weeks (P less than 0.001). Epidermal growth factor antiserum given to sialoadenectomized mice further decreased the thickness of the epidermis to 8.3 +/- 0.6 micron. No appreciable change was observed in the dermis and subcutaneous tissue. In sialoadenectomized mice, replacement of EGF prevented the decrease in thickness of the epidermis in a dose-dependent manner when started immediately after the operation. Treatment with EGF also effectively restored the normal morphology of the epidermis when its thickness had declined to its lowest level. These results suggest that EGF plays a physiological role in the maintenance of the epidermis.

Quantitative analysis of the spreading of the mouse trophoblast in vitro: a model for early invasion.

The outgrowth of the mouse blastocyst in culture represents an in vitro model of trophoblastic invasion. In the present study we analysed trophoblast spreading by time lapse video microscopy. Trophoblast spreading consists of (1) the migration and (2) the giant cell transformation of trophoblast cells, (3) the proliferation of ectoplacental cone (EPC) cells and (4) the subsequent transformation of EPC cells into the secondary giant cells. During migration, ruffling of the trophoblast cell membrane is followed by the formation of lamellipodia. The mean surface areas of the spreading trophoblast, measured in more than 100 cultured blastocysts, increased linearly from 48 to 96 h of culture, while the linear migratory speed at the periphery of the outgrowth declined as the time of culture advanced. The EPC cells increased in size approximately eightfold during the giant cell transformation. The apparent nuclear:cytoplasmic ratios, i.e., ratios between the size of nucleus and that of the cytoplasm, measured as the surface areas on the photomicrographs, of EPC cells increased between 40-46 h of culture, but a sharp decline in the ratio occurred between 50 and 51 h of culture, reflecting either the sudden and tremendous increase in the cellular volume and/or spreading of the cytoplasm. The rates of trophoblast spreading varied considerably among the blastocysts of different genetic constitution examined (ICR, C57BL/6, C3H/He and (B6 x C3)F1. It was fastest in blastocysts obtained from matings of males and females of (B6 x C3)F1, and slowest in the C57BL/6 embryos. The differences in the rate of outgrowth observed may not simply be ascribed to difference in the developmental speed of the early embryos, because the rate of outgrowth reached a plateau at about 96-120 h and no "catch-up' was observed by leaving the blastocysts in culture longer. Our results strongly suggest the possible presence of genetic regulatory mechanisms underlying trophoblast outgrowth; further analysis of the phenomenon may provide clues to understand the molecular mechanisms of trophoblastic invasion during the early phase of implantation, hopefully leading to improved success rates of in vitro fertilization-embryo transfer.

No evidence of PEG1/MEST gene mutations in Silver-Russell syndrome patients.

Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.

Expression and imprinting status of human PEG8/IGF2AS, a paternally expressed antisense transcript from the IGF2 locus, in Wilms' tumors.

A large imprinted gene cluster in human chromosome 11p15.5 has been implicated in Beckwith-Wiedemann syndrome and Wilms' tumor. We have identified a paternally expressed imprinted gene, PEG8/IGF2AS, in this locus. It is transcribed in the opposite direction to the IGF2 transcripts and some genomic regions are shared with the IGF2 gene, as in the case of the mouse imprinted Igf2as gene reported previously by T. Moore et al. As to the relationship between these genomic regions, the human and mouse genes are very similar but there is no homology in their middle parts. Interestingly, PEG8/IGF2AS and IGF2 were found to be overexpressed in Wilms' tumor samples, at levels over ten and a hundred times higher than that in normal kidney tissues neighboring the tumors, respectively. These findings indicate that PEG8/IGF2AS is a good marker of Wilms' tumor and also suggest the possibility of PEG8/IGF2AS being one of the candidate Wilms' tumor genes.