Shigeto Kanda

Detection of manganese superoxide dismutase mRNA in the theca interna cells of rat ovary during the ovulatory process by in situ hybridization

To determine the possible involvement of reactive oxygen species in ovulation, dynamic aspects of superoxide dismutase (SOD) isozyme were studied in the ovaries of rats by in situ hybridization histochemistry. Previously, mRNA levels of ovarian manganese superoxide dismutase (Mn-SOD) were reported markedly to increase whilst enzymic activity of Mn-SOD decreased during the ovulatory process after treating immature rats with 10 and 5 Units, respectively, of pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Levels of Cu/Zn-SOD activity and Cu/Zn-SOD mRNA were reported to remain unchanged throughout ovulation. This increase in the Mn-SOD mRNA level was shown in the present study by in situ hybridization to be localized to the theca interna cells throughout the PMSG/HCG-induced ovulatory process. The observations suggest that the turnover rate of Mn-SOD but not Cu/Zn-SOD increases specifically in the mitochondria of these cells. SOD has been postulated to play important roles in steroidogenesis. The relationship is discussed between mitochondrial functions in steroid-secreting cells and superoxide radicals and related metabolite(s).

Does TUNEL staining during peri- and post-natal development of the mouse inner ear indicate apoptosis ?

Terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) is being used more frequently to investigate programmed cell death (PCD). We have applied this method in order to examine how PCD is involved in the development of the mouse inner ear. In a series of studies, we identified a population of TUNEL-positive cells in the perinatal mouse ear that could not be regarded as apoptosis based upon morphological features of the nuclei. Theoretically, TUNEL detects DNA fragmentation, which can also occur in necrosis. Other authors regard TUNEL-positive cells in the sensory epithelia of the rat equilibrium organs between gestational day (GD) 19 and 7 days after birth (DAB) as apoptosis. We determined whether or not cells in the inner ear of perinatal and post-natal mice were TUNEL-positive due to apoptosis. We stained the inner ears of BALB/c mice aged GD17.5-4 weeks by the TUNEL method and analysed morphology by light microscopy and transmission electron microscopy (TEM). TUNEL-positive cells were distinct in the saccule from DAB3, and in the cochlea from DAB8. The number of TUNEL-positive cells in the hair cells of the saccule and in the cochlea increased with age, and seemed to reach a plateau just before 2 weeks of age. However, morphological analyses did not reveal findings characteristic of apoptosis. We conclude that these TUNEL-positive cells were labelled not because of apoptosis, but due to necrosis or post-mortem autolysis. We surmise that TUNEL staining can identify vulnerable cells of the inner ear that consume high levels of oxygen and easily undergo autolysis.

Expression of manganese superoxide dismutase mRNA in reproductive organs during the ovulatory process and the estrous cycle of the rat

Expression of managanese superoxide dismutase (Mn-SOD) mRNA during the pregnant mare serum gonadotrophin (PMSG)/human chorionic gonadotrophin (HCG)-induced ovulatory process, and during the estrous cycle was examined in rat female reproductive organs. Mn-SOD mRNA levels in theca interna cells markedly increased in PMSG-primed rats and high levels of the transcripts were maintained after HCG injection. The PMSG-enhanced expression of Mn-SOD mRNA in follicular epithelial cells increased concomitantly with luteinization of these cells. The levels of Mn-SOD mRNA remained high and became equivalent in both granulosa and theca lutein cells 24 h after HCG injection. Neither luteinization nor the expression of Mn-SOD mRNA was observed in the epithelial cells of unovulated follicles. Luteal bodies had formed 3 days after HCG injection, and the same level of Mn-SOD mRNA expression continued in lutein cells, but not in stromal cells. During the estrous cycle, Mn-SOD mRNA was localized to theca interna cells on proestrus, to the epithelial cells of luteinizing follicles on estrus, and to newly formed luteal bodies on diestrus. The epithelial cells in the oviduct did not express Mn-SOD mRNA throughout the ovulatory process or the estrous cycle. Expression of Mn-SOD mRNA in the luminal epithelial cells of the uterus increased after PMSG injection, reaching a maximum after 24 h, and became relatively negative 3 days after HCG injection when corpora lutea had formed in the ovary. During the estrous cycle, uterine epithelial cells and leukocytes showed marked increases in Mn-SOD mRNA expression on estrus and on proestrus, respectively. Expression in the vaginal epithelium became apparent 3 days after HCG injection and continued for at least 12 days after HCG injection. The expression was localized to the superficial layer of the epithelium. During the estrous cycle, expression occurs in the basal layer on proestrus and estrus, transferring to the superficial layer on diestrus day 1, and expression stops on diestrus day 2. The relationship between the expression of Mn-SOD mRNA and hormone-induced metabolic changes, including steroidogenesis, is discussed.

Histochemical and immunohistochemical changes in rat hepatocytes after halothane exposure

AbstractPurpose. Histochemical and immunohistochemical changes were observed in hepatocytes to study the developing and recovery processes of halothane-induced hepatic injury from 0 to 7 days after halothane exposure. Methods. A total of 330 7-week-old male Sprague-Dawley rats, with or without phenobarbital preteatment, were exposed to halothane in 100%, 21%, 10% oxygen or oxygen alone for 2 h. Results. In the phenobarbital group, degenerated hepatocytes were observed immediately after exposure to 10% oxygen, both with and without halothane: glycogen and ribosomal ribonucleic acid (rRNA) disappeared immediately and 6 h after exposure, respectively, and necrosis developed in zones 3 to 2 at 6 h after exposure. From 12 h to day 1, the necrosis was more marked and more widely observed in the cells with halothane than those without halothane. However, all tissues returned to normal by day 7. Conclusion. Disappearance of glycogen at 0 h and rRNA at 6 h after exposure to halothane under 10% oxygen is considered to be one of the factors inducing necrosis around the central vein. Recovery of the hepatolobular structure was attributed to the rearrangement of the remaining hepatocytes in the portal vein area.