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N Koibuchi

Publications and source records attributed to N Koibuchi.

51 records · Page 3Linked to original sources

Expression of prolactin gene in human decidua during pregnancy studied by in situ hybridization histochemistry.

The prolactin (PRL) gene is known to be expressed not only in the anterior pituitary but also in the decidualized human endometrium. This study was designed to detect the site of synthesis of PRL during pregnancy by in situ hybridization histochemistry. Decidual and trophoblast tissues from early pregnancy were obtained from patients undergoing therapeutic abortion at 8-10 weeks of gestation. Term placentae were obtained from patients with uncomplicated deliveries at 38-40 weeks. Sections of these tissues were hybridized with 35S-labeled RNA probe complementary to human PRL mRNA. Specific hybridization signals were distributed over the decidual cells in early and term pregnancy. In the decidua capsularis of early pregnancy, labeled cells were concentrated close to the amniotic cavity, although decidual cells were distributed evenly. In the decidua parietalis, almost all decidual cells were labeled, but no specific labeling was seen in the endometrial glands or capillary endothelium. In the decidua basalis, greater signals were always detected over the decidual cells in early pregnancy than in term pregnancy, when sections, which were hybridized with the same probe and exposed simultaneously, were compared. No specific hybridization was detected in the trophoblast cells. These results not only confirm that PRL is specifically synthesized in the decidual cells but also indicate that there are regional and periodical differences in PRL gene expression in the decidual cells during pregnancy.

Blotting, Northern↗

Gonadotropin-releasing hormone (GnRH): expression during salmon migration.

In the seaward migrating chum salmon, immunocytochemical and in situ hybridization techniques revealed isolated GnRH neurons at the base of the nasal epithelium, along the nervus terminalis and as ganglia at the rostroventral (gROB) and caudalmost (GT) olfactory bulb. A novel GnRH ganglion was seen at the cribriform bone (gCB). GnRH immunoreactivity but not the hybridization signal was detected in the midbrain neurons. During the migratory period, there were trends towards an increase in GnRH mRNA in the gCB and the gROB and a significant surge in plasma thyroid hormones was also evident. Therefore, we hypothesise thyroid hormones might be crucial for the increased tendency of GnRH expression and the migratory behavior of chum salmon.

Animals↗

In situ hybridization detection of TSH beta subunit gene expression in the serum-free primary culture of the adult rat pituitary.

This study was designed to construct the primary culture system to detect the change in TSH beta subunit (TSH beta) gene expression in individual cells. Adult, male Wistar rats were sacrificed by transcardial perfusion of 0.25% trypsin solution under pentobarbital anesthesia (50 mg/kg body weight). Their anterior pituitaries were removed, dispersed and cultured for 1, 2, 3, or 6 days with or without 1 nM triiodothyronine (T3) under the serum-free condition. In some cultures, TRH was added to a final concentration of 1 microM on 6, 12 or 24 h before fixation. Then the culture media were removed to measure TSH concentration. Cells were fixed with paraformaldehyde and hybridized with 35S-labeled RNA probe complementary to TSH beta mRNA. Emulsion autoradiography was subsequently performed. T3 treatment markedly suppressed relative cellular levels of TSH beta mRNA on 2, 3 and 6 days after the onset of culture (day 2, 3 and 6) and suppressed TSH secretion on day 3 and 6. TRH treatment increased TSH beta mRNA on 12 and 24 h after the treatment on day 2 and 3 but did not increase TSH beta mRNA on day 6. TSH concentration in the culture medium was increased by TRH treatment on 6, 12 and 24 h after the treatment on day 2, on 12 h and 24 h on day 3, and 24 h on day 6. On day 2 and 3, although T3 treatment suppressed basal level of TSH beta mRNA, TRH-induced increase in TSH beta mRNA was not suppressed by T3 treatment. These results show that the thyroid hormone and TRH regulate TSH beta gene expression independently. Our culture system may provide a useful model to examine the action of individual substances on a specific subpopulation of the anterior pituitary cells.

Animals↗

[Expression of prolactin gene in human decidua].

UNLABELLED: This study was designed to detect the localization of prolactin gene expressing cells in the human utero-placental unit during pregnancy. METHODS: Human endometrium was obtained at hysterectomy from normally cycling women who underwent surgery for myoma uteri. Decidual and trophoblast tissues from early pregnancy were obtained by curettage from patients undergoing therapeutic abortion at 8-10 weeks of gestation. Term placenta was obtained from patients with uncomplicated deliveries at 38-40 weeks. Total RNAs of these tissues were extracted to perform Northern blot hybridization (NBH) with the radiolabelled human pituitary prolactin cDNA probe. Frozen sections of these tissues were cut and processed for in situ hybridization (ISH) with the radiolabelled RNA probe complementary for human prolactin mRNA. RESULTS: 1. By NBH, approximately a 1.3 kilobase (kb) size band was detected in the total RNA obtained from late secretory endometrium (day 26), decidua of early pregnancy and placenta of term pregnancy, and the decidua had the most significant signal. No hybridization signal was detected in the chorionic villi in early pregnancy. 2. By ISH, a significant hybridization signal was detected in the cytoplasm of the decidual cells in early and term pregnancy. No hybridization signal was detected in the endometrial grandsand trophoblast cells. CONCLUSION: These results indicate that prolactin is synthesized in the decidual cells in the human uterus during pregnancy.

Animals↗

Increase in c-erbA alpha 2 mRNA in the parvocellular region of the paraventricular nucleus of the hypothalamus following thyroidectomy in the adult male rat.

To examine thyroid hormone regulation of c-erbA alpha 2 mRNA expression in the parvo-cellular region of the paraventricular nucleus of the rat hypothalamus (pPVN), quantitative in situ hybridization was performed using 3H-labeled probe complementary to c-erbA alpha 2 mRNA. Thyroidectomy induced a significant increase in the number of silver grains overlying the cytoplasm in the pPVN relative to sham-operated controls. This effect was prevented by daily injection of thyroxine. These results indicate that hypothyroidism induced an increase in cellular c-erbA alpha 2 mRNA level in the pPVN.

Animals↗

Circadian rhythms of vasopressin release in primary cultures of rat suprachiasmatic nucleus.

We have developed a suprachiasmatic nucleus (SCN) cell culture system to study the cellular and molecular bases of the mammalian circadian pacemaker. The SCN regions were dissected from 4- to 6-day-old rat pups and dissociated cells were cultured in a defined medium. In all the cultures, the release of vasopressin showed clear circadian oscillation, which appeared within a few days in culture and lasted for more than a month. The peak of vasopressin release was observed at subjective day. These findings suggest that this culture system provides a valuable model for elucidating the mechanism of the circadian pacemakers.

Animals↗

Heterogeneous expression of ornithine decarboxylase gene in the proximal tubule of the mouse kidney following testosterone treatment.

The expression of the ornithine decarboxylase (ODC) gene in the mouse kidney following testosterone treatment was examined using in situ hybridization histochemistry. Testosterone (n = 5) or vehicle (n = 5) was subcutaneously injected (1 mg/animal) into male BALB/c mice (8 weeks in age) 14 h before sacrifice. Animals were sacrificed under ether anesthesia, their kidneys were removed and immediately frozen in liquid nitrogen. Frozen sections (10-microns-thick) were cut on a cryostat. Sections were hybridized with 35S-labeled sense or antisense RNA probe. The hybridization continued for 24 h at 50 degrees C and emulsion autoradiography was subsequently performed. A marked increase in ODC mRNA was exclusively detected in the proximal tubule of the renal cortex in the testosterone-treated animals. The hybridization signal was greater in the outer portion of the proximal tubule than in the inner portion. No significant hybridization signal was detected either in the distal tubule, renal corpuscle or peritubular tissues. These results indicate that testosterone induces the expression of the ODC gene in the proximal tubule of the renal cortex, leading to the increase in ODC activity in the same region.

Animals↗

Induction of ornithine decarboxylase immunoreactivity in the male mouse kidney following testosterone treatment: an axial heterogeneity in the proximal tubule.

The effect of testosterone on the activity of ornithine decarboxylase (ODC), its protein level and immunocytochemical distribution were examined in the mouse kidney. Male BALB C mice at 8 weeks of age were used throughout. Fourteen hours before death, they received a subcutaneous injection of testosterone (1 mg/animal) or solvent to measure renal ODC activity or to detect the distribution of ODC immunoreactivity in the kidney. Renal ODC activity and the content of the enzyme were markedly increased after testosterone treatment. Histologically, few cells that were obviously immunoreactive to ODC were observed in the control animals and in the testosterone-treated animals a marked increase in ODC immunoreactivity was observed only in the cortex. ODC immunoreactive cells were located diffusely in the proximal tubule. In the pars recta, cells were stained weakly and homogeneously, while in the pars convoluta, the luminal surface of the cells showed stronger immunoreactivity. Moreover, many granule-like particles that were strongly ODC immunoreactive were observed inside the lumen of the pars convoluta. These results show that testosterone treatment induces an increase in ODC content in certain cells located in the proximal tubule of the cortex.

Animals↗

Change in Fos-like immunoreactivity in the suprachiasmatic nucleus in the adult male rat.

The circadian change in the number of the Fos-like immunoreactive (IR) cells in the suprachiasmatic nucleus (SCN) was examined in the adult male rats, whose eyeballs were enucleated two months before sacrifice. Their circadian rhythms were determined by their locomotor activity. They were sacrificed in the middle of the active phase or inactive phase. Then brain sections were cut for immunocytochemistry for Fos. A marked increase in the number of the Fos-like IR cells was observed in the inactive phase in the SCN, whereas no such increase was observed in the supraoptic nucleus. These results indicate that, in the SCN, Fos expression was changed with endogenous circadian rhythm in the free-running rat.

Animals↗

Thyroidectomy induces Fos-like immunoreactivity within thyrotropin-releasing hormone-expressing neurons located in the paraventricular nucleus of the adult rat hypothalamus.

Effects of thyroidectomy on Fos-like immunoreactivity (IR) in the rat brain were examined using single and double-label immunocytochemical techniques. In particular, the possibility that Fos might be involved in thyroid hormone regulation of thyrotropin releasing hormone (TRH)-containing neurons located in the parvocellular region of the paraventricular nucleus of the hypothalamus (pPVN) was examined. Adult, male, Sprague-Dawley rats were used and all animals received either surgical removal of the thyroid gland or sham surgery. Two experiments were performed. In the first experiment, animals were killed 1, 3, or 6 days after surgery and numbers of Fos-like IR cells in the parvocellular (pPVN) and magnocellular (mPVN) regions of the PVN, the anterior hypothalamic nucleus (AH), the lateral hypothalamic nucleus (LH), and the pyriform cortex were determined. In the second experiment, animals received an intracerebroventricular injection of colchicine 5 days after surgery. The next day, animals were killed and numbers of Fos-like IR cells double-labeled for either TRH, corticotropin releasing factor (CRF), or methionine-enkephalin (met-Enk) were determined. Six days after thyroidectomy there was a significant increase in the number of Fos-like IR cells detected in the pPVN. No induction in the pPVN was observed 1 and 3 days after thyroidectomy, and no effects attributable specifically to thyroidectomy (as opposed to stress) on Fos expression in the mPVN, AH, LH, or pyriform cortex were observed. In addition, a rapid, stress-related, induction of Fos-like IR was detected in the mPVN, AH, and LH and was easily distinguished from Fos expression induced in the pPVN as a function of thyroidectomy. The time course for the effect of thyroidectomy on Fos expression in the pPVN paralleled increased plasma TSH concentration. A significant correlation between numbers of Fos-like IR cells in the pPVN and plasma TSH concentration following thyroidectomy was also observed, suggesting that plasma levels of TSH correlate directly with the number of activated TRH-containing neurons located in the pPVN. Double staining for Fos and TRH, CRF, or met-Enk revealed that thyroidectomy induced Fos-like IR specifically within TRH-, but not within CRF-, or met-Enk, expressing neurons in the pPVN. Taken together, the data suggest that Fos-like IR is induced within TRH-expressing neurons in the pPVN as a consequence of decreasing levels of circulating thyroid hormone (TH). Whether this reflects a direct effect of decreasing TH on Fos expression is not yet known; however, the data are consistent with the hypothesis that Fos is involved in TH-associated regulation of TRH production and release.

Animals↗

Localization by immunohistochemistry of renal ornithine decarboxylase in the mouse with and without testosterone treatment.

The immunohistochemical distribution of renal ornithine decarboxylase was studied in male mice both with and without testosterone treatment. Testosterone (1 mg per mouse) induced a marked increase in ornithine decarboxylase activity of the mouse kidney, whereas no significant immunohistochemical difference was observed either in immunoreactivity or its localization. In intact male as well as androgen-treated mice dense ornithine decarboxylase-immunoreactive cells were observed mainly in the cortex, especially many ornithine decarboxylase-immunoreactive cells were observed in the inner portion, while a much weaker immunoreactivity was observed in the medulla. The largest number of ornithine decarboxylase-immunoreactive cells seemed to be localized in the pars recta of the proximal tubule. The immunoreactivity was not detected in all the tubular cells but scattered among them. The renal corpuscles were not immunoreactive. In each ornithine decarboxylase-immunoreactive cell, the cytoplasm showed much denser immunoreactivity than the nucleus.

Animals↗

Suppression of human growth hormone (GH)-releasing hormone-induced GH secretion in pentobarbital-anesthetized rats after electrical stimulation of the midbrain central gray and several raphe nuclei.

To investigate the neural mechanism involved in suppression of GH secretion, we examined the effect of electrical stimulation of the midbrain central gray (CG) and several raphe nuclei on human (h) GRF-induced GH secretion in pentobarbital-anesthetized rats. A concentric bipolar stimulating electrode was implanted stereotaxically into each nucleus under pentobarbital anesthesia 1 week before stimulation. Blood samples were taken through a cannula placed in the right atrium via the right external jugular vein. Under pentobarbital anesthesia, 5 micrograms hGRF dissolved in 0.3 ml saline were systemically applied through this cannula. Ten minutes after the infusion, plasma GH was increased from the resting value of 28.5 +/- 7.1 ng/ml (mean +/- SE) to 686.1 +/- 62.0 ng/ml. Biphasic electrical stimulation was delivered for the first 10 min. When the CG was stimulated with a current of 500 microA, hGRF-induced GH secretion was markedly suppressed. However, even when the stimulation site was outside the CG, hGRF-induced GH secretion was suppressed. But with a smaller current (100 microA) the suppressive effect was observed only when the medial portion of the CG was stimulated. This suppression was abolished by prior lesioning of the hypothalamic periventricular nucleus (Pe), in which most of the somatostatin-immunoreactive fibers in the median eminence originate. The stimulation of the dorsal raphe with a current of 500 microA suppressed the GH increment at 10 min, but no suppression occurred with a current of 100 microA. This suppression was abolished by prior lesioning of the Pe. Electrical stimulation of the rest of the raphe nuclei had no effect on hGRF-induced GH secretion. These results suggest that electrical stimulation of the CG suppresses hGRF-induced GH secretion, and the most effective area is the ventromedial portion of the CG. These suppressive actions may be achieved by activation of the somatostatin neurons in the Pe.

Animals↗

Growth hormone release induced by electrical stimulation of the basolateral amygdala, observed in pentobarbital anesthetized rats.

The aim of this study was to clarify the neural pathway leading to the growth hormone (GH) release when the basolateral amygdala (ABL) was electrically stimulated. Concentric bipolar stimulating electrode was implanted in the unilateral ABL. Blood samples were taken from a cannula implanted into the right atrium via the right external jugular vein. Electrical stimulation of the ABL for 10 min caused a significant increase in plasma GH level from resting value 27.5 +/- 5.7 ng/ml (mean +/- S.E.M.) to 62.2 +/- 7.5 ng/ml at the termination of stimulation. This increase in GH level was markedly augmented to 152.0 +/- 23.0 ng/ml after lesion of the periventricular hypothalamic nucleus (Pe), where somatostatinergic neurons send their axons to the median eminence. Lesion of the stria terminalis (st) fully or partly abolished GH release induced by ABL stimulation. These results suggest that stimulation of the ABL accelerates GH secretion. The st is an essential pathway for this release, whereas the activity of Pe-neurons is rather inhibitory to this release.

Amygdala↗

Thyroid hormone action and brain development.

Thyroid hormone (TH) plays a crucial role in brain development. Developing rodent cerebellum might be an excellent model for studying the molecular mechanisms of TH action in the brain because perinatal hypothyroidism greatly affects its ontogeny. Although the TH-regulated genes that play crucial roles in cerebellar development have not yet been fully characterized, recent studies have provided novel insights into TH action in brain development.

Animals↗