Hiroyoshi Konishi

Localization of Erythropoietin and Erythropoietin- Receptor in Postimplantation Mouse Embryos

We used RT‐PCR (reverse transcription‐polymerase chain reaction) and immunocytochemical methods to demonstrate the presence of erythropoietin (Ep) and its receptor (EpR) in postimplantation mouse embryos from the egg‐cylinder to the unturned stage. Expression of mRNA for EpR was detected in total RNA from embryos and decidua in all these stages, but Ep mRNA was confined to embryos in the primitive streak stage and beyond and was not detected in the decidua. Staining of Ep and EpR was seen in all tissues, embryo proper and extra‐embryonic. Moreover, regions of marked staining of Ep and EpR were detected in the extra‐embryonic endoderm, embryonic ectoderm, neural folds and yolk sac, chronologically. No conspicuous differences were present in the staining patterns between Ep and EpR until primitive streak stage; however, after this stage, Ep was predominantly present in the nucleus and EpR on the surface of almost all cells; in the visceral yolk sac endoderm EpR was also detected in adsorption vacuoles and lipid droplets. These studies suggest that Ep first of exogenous and then endogenous origin and EpR of endogenous origin are involved not only in embryogenesis but also in neurogenesis and hematopoiesis in early postimplantation mouse embryos.

Induction of avascular yolk sac due to reduction of basic fibroblast growth factor by retinoic acid in mice

Vasculogenesis depends on autocrine secretion of basic fibroblast growth factor (bFGF) from capillary endothelial cells. Retinoic acid (RA) induced avascular yolk sac (AVY) of mouse embryos of dams given 60 mg/kg of RA orally on Day 8 of gestation and sacrificed 3 days later. We studied the localization and transcriptional expression of bFGF and FGF-receptor (flg), heparin-binding growth factor (HBGF) activity, localization of lysosomal enzymes and alpha 1-antitrypsin (AAT), and electron microscopy of the normal mouse visceral yolk sac (VYS) and AVY. bFGF, which is normally present in the endoderm of the VYS of 8-day-old embryos and in all components of the VYS by Day 11 of gestation, was reduced in the AVY. However, in the presence of bFGF in vitro capillary nets were restored in the AVY. The mRNA for bFGF was not detectable in either VYS or AVY, while flg mRNA was detected equally in both organs in Northern blotting. The characteristic distribution pattern of lysosomal enzymes, acid phosphatase, lysozyme, and cathepsin D, and AAT was altered in the AVY. The level of acid phosphatase and AAT was reduced to 10% in the AVY. Electron microscopy revealed a partial or total loss of lysosomal membranes where the contents of lysosomes fused with adjacent lysosomes and the external organelles. These results suggest that vitelline blood vessels are not developed by endogenous autocrine bFGF but by exogenous transcellular bFGF from absorptive endodermal cells. Retinoic acid does not affect the angiogenic capacity of the VYS mesenchyme but destroys lysosomes, which release hydrolytic enzymes, leading to degradation of AAT in the endodermal cells and then digestion of endocytosed bFGF.

Effects of saikosaponin-d on aminonucleoside nephrosis in rats

The effects of saikosaponin-d extracted from the roots of Bupleurum falcatum L. on aminonucleoside nephrosis were studied in rats. Urine protein excretion in rats receiving aminonucleoside alone was significantly elevated on the 2nd day after the last injection of aminonucleoside and reached a peak on the 11th day. Urinary protein on the 11th day was reduced by 48 and 46%, respectively in animals treated with saikosaponin-d from the 2nd and 8th day after the last injection of aminonucleoside. Electron microscopically, the degree of abnormality, e.g. loss or fusion of foot processes, in the glomerular epitherial cells was significantly lower in the rats treated with saikosaponin-d after aminonucleoside injection than in the rats treated with aminonucleoside alone. It is concluded that saikosaponin-d prevents the development of proteinuria induced by aminonucleoside in the rat.

Accelerated differentiation in seminiferous tubules of fetal mice prenatally exposed to ethinyl estradiol

SummaryEthinyl estradiol (EE) in olive oil (0.02, 0.2, or 2.0 mg/kg) administered to pregnant mice on days 11 to 17 of pregnancy induced abnormal differentiation of gonocytes and fetal Sertoli cells in male fetuses on day 18 of gestation. Light and electron microscopic examination of the testes showed fewer darkly stained prospermatogonia and more lightly stained prospermatogonia in the experimental than in the control fetuses. Widespread degeneration and lysis of gonocytes were seen only in the experimental mice. No spermatogonia type A could be detected. In spite of comparable mitotic rates in the Sertoli cells of the experimental and control mice and more dark Sertoli cells with well developed smooth endoplasmic reticulum (SER) in the experimental mice, one of the functions of fetal Sertoli cells was suppressed: there were fewer dark slender Sertoli cells with long processes extending to the centers of tubules and more contact areas with gonocytes, phenomena which may play a role in the migration of gonocytes towards the periphery of the tubules. More Sertoli cells were detected in the undescended than the descended testes exposed to the highest dose of EE. These morphological findings indicate that prenatal exposure to EE induces acceleration of prespermatogenesis and disturbances in the initiation of spermatogenesis and in the mechanical function of Sertoli cells.

Erythropoietin in mouse avascular yolk sacs is increased by retinoic acid

Erythropoiesis begins first in the visceral yolk sac (VYS) of the embryo; however, the involvement of erythropoietin (EPO) in yolk‐sac erythropoiesis has not been studied adequately. This study reports the expression of EPO in normal and hypoxic VYSs and alterations in yolk sac components induced by retinoic acid (RA) in mice. Gravid mice (plug day = day 0 of gestation) were given one oral dose of 60 mg/kg of RA in olive oil on days 6, 6.5, 7, 7.5, or 8 of gestation and were sacrificed 2.5, 3, or 3.5 days later. Control mice received olive oil without RA. None of the dams developed anemia, but more than 80% of the embryos of the dams that received RA on day 6, 6.5, or 7 of gestation had avascular yolk sacs (AVYs) and anemia. In these AVYs, the adenosine triphosphate (ATF) level was as low as 18–59% of that in the control VYSs. Reverse transcription‐polymerase chain reaction and Southern analysis of products demonstrated that mRNA for EPO receptor (EPR) was expressed in both VYSs and AVYs on days 9–11 of gestation, and EPO mRNA was present in VYSs and AVYs on days 9 and 10 of gestation and in vehicle‐exposed VYSs on day 11 of gestation. Furthermore, enzyme immunoassay of EPO indicated that AVYs contained more EPO protein than control VYSs. Light microscopy revealed that, in AVYs, in addition to the defective hemopoietic cells the endodermal layer was exclusively altered: The presence of focal proliferated regions and the separation from the mesenchyme led to a single layer from which some immature cells seemed to be migrating. Immunolocalization of EPO showed its presence in all components of VYSs with a characteristic distribution pattern: In the endodermal layer, cells with positive EPO staining decreased as gestation advanced, and erythroid precursor cells showed positive staining. In AVYs, the proliferated endodermal cells had EPO in abundance; in the separated regions, the distinction between positive and negative EPO staining became clearer than that in the control VYSs, and the immature cells in the lumens also had EPO. EPR was seen on the cell surface of the corresponding cells that reacted to EPO. These findings suggest that VYSs not only produce EPO temporarily but also respond to the oxygen content in situ. EPO and EPR appear to be synthesized in the endodermal cells of the VYSs that are likely to respond to the circumstances induced by RA.

Effects of saikosaponin‐d on enhanced CCl4‐hepatotoxicity by phenobarbitone

The effects of saikosaponin‐d extracted from the roots of Bupleurum falcatum L. on increased toxicity of CCl4 and increased activities of microsomal enzymes induced by phenobarbitone have been examined. Saikosaponin‐d showed protection against the CCl4‐hepatotoxicity enhanced by phenobarbitone. It also inhibited increases in the content of cytochrome P450 and NADPH‐cytochrome c reductase activity, which are induced by the phenobarbitone treatment, but the spectral characteristics of P450 were not altered. The rate of microsomal lipid peroxidation by NADPH and CCl4 was significantly lowered in‐vitro in rats pretreated with phenobarbitone and saikosaponin‐d compared with those pretreated with phenobarbitone alone.

Long-term effects on male reproductive organs of prenatal exposure to ethinyl estradiol.

The effects of oral contraceptives on human offspring have not been established. This study shows the long-term effects of prenatal exposure to ethinyl estradiol, a common estrogenic component of oral contraceptives, on the testes and epididymides of aged mice. Five (83%) of the six aged male mice examined showed atrophy of seminiferous tubules, four (67%) had Leydigs cell hyperplasia, and one (17%) had precancerous changes in the epididymis. These phenomena may be results of decreases in testosterone content and its drastic conversion into estradiol-17 beta in the fetal testes at critical periods induced by ethinyl estradiol.

Death-resistant and nonresistant malignant human cell lines under anoxia in vitro

BackgroundErythropoietin supports the survival of erythroblasts. We previously demonstrated that 24 malignant human cell lines expressed erythropoietin and its receptor and that erythropoietin secretion was enhanced under anoxia. In this study, we examined the viability of 22 of these cell lines excluding two leukemia cell lines under anoxia.MethodsTwenty-two cancer cell lines of various origins were cultured under anoxia or normoxia for 4 days, and their viability was examined at 1-day intervals. The levels of lactate and ATP were measured. The expressions of hypoxia-inducible transcription factor 1α (HIF-1α) and Bcl-2 family proteins were examined by western blotting analysis. The cellular and mitochondrial features were examined by microscopy.ResultsEleven of the 22 cancer cell lines examined showed 80% to 100% cell viability after 4 days under anoxia; 2 cell lines showed similar viability for 3 days, 3 cell lines showed similar viability for 2 days, and 6 cell lines showed similar viability for 1 day or less. These 11 death-resistant cell lines, which secrete various amounts of erythropoietin under anoxia, produced significantly more lactate during 2 days under anoxia than under normoxia, with ATP levels about 60% of those before anoxia. ATP returned to the normal level when normoxia was restored after 4 days of anoxia. However, the nonresistant cell lines responded to anoxia by yielding significantly more lactate without a reduction of the ATP level. The expression patterns of Bcl-2 family proteins revealed that apoptosis-inhibiting signals predominated over proapoptotic signals in the death-resistant cells under anoxia.ConclusionThe majority of the cancer cell lines examined survived under anoxia in vitro, through the Pasteur effect, in a dormant state without direct support of erythropoietin.

Vascular endothelial growth factor in edematous mouse embryos induced by retinoic acid in utero

ABSTRACT  Vascular endothelial growth factor (VEGF) is induced by hypoxic environment and contributes to vascular formation in both developing embryos and adults. Exogenous retinoic acid (RA) induces avascular yolk sacs with anemic stunted embryos of day 9 and 10 of gestation when RA is given to pregnant mice on day 6, 6.5 or 7 of pregnancy (Yasuda et al., 1996). We undertook the present studies to find out whether VEGF is activated and plays any role in those RA‐exposed embryos. Embryos were obtained from dams given 60 mg/kg of RA on day 6 or 7 of pregnancy and sacrificed three days later. Most RA‐exposed embryos showed edematous swelling without prominent vascular nets, but had beating heart tubes on day 9 and day 10 of gestation. Microscopic examination of developing tissue components showed various degrees of degeneration, and distension of the dorsal aorta when the body cavity was dosed. Northern blot analysis revealed expression of VEGF mRNA in the RA‐exposed and control embryos. The highest expression of VEGF mRNA was seen in the embryos of day 10 exposed to RA on day 7, and these embryos had a significantly lower ATP content than did the controls (p < 0.01). Immunoreactive VEGF was detectable in both experimental and control embryos; in the former it was especially visible in the distended neuroepithelium, endothelium and membranes. These VEGF‐immunoreactive regions also expressed another permeability factor, bradykinin. These findings suggest that VEGF upregulated by hypoxic conditions in edematous embryos induced by RA exposure in utero acts as hyperpermeability.