Expression of p68 RNA helicase is closely related to the early stage of adipocyte differentiation of mouse 3T3-L1 cells.

We have previously identified the genes expressed early in the differentiation of mouse 3T3-L1 cells into adipocytes. Since these genes were isolated as small fragments, many were unknown. In this study, we have cloned two full-length cDNAs and identified them as p68 RNA helicase and mc3s5/mtCLIC. The expression of these genes was rapidly induced, and specific to the adipocyte differentiation. When the expression of p68 RNA helicase was inhibited using an inducible antisense system, the differentiation into adipocytes was partially blocked, and the expression levels of some marker genes decreased. These findings strongly indicate that the expression of the above two genes was closely related to the adipocyte differentiation, and p68 RNA helicase in particular is crucial to the differentiation.

Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens.

Within developing ovaries of many insects, some developing follicles or oocytes usually degenerate (follicular atresia or oosorption), while the others may continue to grow to maturity, thus maintaining the balance between the number of eggs and reproductive circumstances such as available nutrients. To help clarify the phenomenon of follicular atresia during ovarian development, we examined cysteine proteinases stored in mosquito Culex pipiens pallens ovaries. First, analysis using synthesized substrates showed that cathepsin B- and L-like proteinases gradually accumulated in the developing ovaries after a blood meal, which required more than 10 min of preincubation under acidic conditions to reach their maximum activities. However, homogenates of degenerating follicles 3 days after feeding showed proteolytic activities without acid treatment, suggesting that the proteinases had already been activated, while the extract of normally developing follicles collected from the same ovaries required more than 10 min of acid preincubation to reach the optimum activities, suggesting that the enzymes remained as inactive forms. Chemical and immunohistochemical analyses showed that more proteinases are located in the cytoplasm, rather than being associated with yolk granules. Ovarian proteinases, which are believed to become activated at the onset of embryogenesis, should also be activated during oogenesis, presumably to enhance oosorption.

RGS2 promotes adipocyte differentiation in the presence of ligand for peroxisome proliferator-activated receptor gamma.

The events at the earliest stage of adipocyte differentiation are yet to be fully elucidated. Previously, we cloned the genes that are induced at the beginning of the differentiation of mouse 3T3-L1 preadipocyte cells. We found that the gene expression of regulators of G protein signaling-2 (RGS2) rapidly increased after the addition of inducers and decreased at 3-12 h. The expression pattern of RGS2 mRNAs differed among growth-arrested and proliferating 3T3-L1 cells and NIH-3T3 cells, indicating a specificity for adipogenesis. Here we report that the ectopic expression of RGS2 using a retroviral system in mouse NIH-3T3 cells promotes adipogenesis only in the presence of BRL49653, which is a ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma). These results strongly suggest that RGS2 play a crucial role in the program of adipocyte differentiation and may contribute to the function of PPARgamma.

Occurrence of a N-terminal proteolytic fragment of neurocan, not a C-terminal half, in a perineuronal net in the adult rat cerebrum.

Neurocan is a nervous tissue-unique chondroitin sulfate proteoglycan (CSPG) whose expression and proteolytic cleavage are developmentally regulated. In the adult rat brain, neurocan is completely cleaved into some proteoglycan fragments including the C-terminal half known as neurocan-C and a N-terminal fragment with a 130 kDa core glycoprotein (neurocan-130). We describe here the differential distribution of these two neurocan-derived CSPGs in the adult rat cerebrum and the occurrence of neurocan-130 as a new member of a perineuronal net-constituting molecule. At the light microscopic level, neurocan-130 exhibited pericellular localization around a subset of neurons in addition to diffuse distribution in the neuropil. In contrast, neurocan-C was distributed only diffusely in the neuropil. Double staining with anti-neurocan-130 and anti-synaptophysin antibodies suggested that neurocan-130 was localized in the vicinity of the synapses, but not at the synapses. Immunoelectron microscopy showed that neurocan-130 was mainly localized in the cytoplasm of glial cell processes, the so-called glial perineuronal net, encompassing the cell bodies of certain neurons. The presence of neurocan-130 in a limited number of glial cells may reflect some functional heterogeneity of the glia.

Cell surface-associated extracellular distribution of a neural proteoglycan, 6B4 proteoglycan/phosphacan, in the olfactory epithelium, olfactory nerve, and cells migrating along the olfactory nerve in chick embryos.

The immunocytochemical and immuno-electron microscopic distribution of a neural proteoglycan (PG) was investigated with a monoclonal antibody, MAb 6B4, in the olfactory epithelium, the olfactory nerve, and the cells originating the epithelium and migrating along the olfactory nerve toward the forebrain in chick embryos. The PG recognized by MAb 6B4, that is 6B4 PG, in the brain of early postnatal rats, is identical to phosphacan. In chick embryos, immunoreactivity to 6B4 PG appeared on embryonic day (ED) 3-3.5 in a thin layer beneath the olfactory epithelium. It disappeared immediately, then becoming apparent in the bundles of the olfactory nerve. The immunoreactivity in the nerve bundles gradually increased during ED 5-11. On the other hand, cell surface-associated extracellular localization of the immunoreactivity was seen in the olfactory epithelium on ED 6 and afterwards. Immunofluorescent double-labeling of 6B4 PG and gonadotropin-releasing hormone (GnRH) revealed that the cell bodies of both GnRH-containing cells and other cells migrating along the olfactory nerve were surrounded by a rim immunoreactive to the PG. Under an electron microscope, the surfaces of the cell bodies and of the neurites in the nerve bundles were surrounded by deposits immunoreactive to 6B4 PG. These results indicate that 6B4 PG in chick embryos is one type of cell surface-associated extracellular matrix molecule, and that 6B4 PG covered the surfaces of migrating cells and of elongating olfactory nerve. The cell surface-associated extracellular localization of 6B4 PG found in the nasal region, taken together with the binding properties of this PG with cell adhesion molecules shown in rat brains, suggested that 6B4 PG played a role in guiding the migration of cells along the olfactory nerve in chick embryos.

Glycosaminoglycans in the olfactory epithelium and nerve of chick embryos: an immunocytochemical study.

Immunoreactivity specific to keratan sulfate (KS), heparan sulfate (HS), and chondroitin sulfate (CS) in the nasal region of chick embryos on embryonic day (ED) 3.5-7, was investigated with six antibodies and three glycosidases. Immunoreactivity specific to HS, KS, and CS, and their localized distribution was seen in the olfactory epithelium, olfactory nerve, and cells located along the bundles of the olfactory nerve on ED 3.5-7. The immunoreactivity to HS was seen in the cells and their neurites located in the olfactory nerve bundles and in cells in the ventral forebrain on ED 3.5-5. The cells, i.e., gonadotropin-releasing hormone (GnRH)-or somatostatin-containing cells, are reported to originate in the olfactory epithelium, migrate along the olfactory nerve, and then invade the forebrain in chick embryos. These findings taken together, indicated that a subset of cells migrating along the nerve to the forebrain contained HS. Limited immunoreactivity to KS in the olfactory epithelium and in the underlying mesenchyme, but not in the olfactory nerve, ruled out the involvement of this glycosaminoglycan (GAG) in the migration of cells along the olfactory nerve. The immunoreactivity to CS and to its derivatives suggested the presence of CS in proteoglycan form in the olfactory structures and migrating cells.

Androgen enhances neuronal degeneration in the developing preoptic area: apoptosis in the anteroventral periventricular nucleus (AVPvN-POA).

Perinatal treatment of female rats with androgen decreases the nuclear volume of the anteroventral periventricular nucleus of the preoptic area (AVPvN-POA). In order to examine the effect of androgen on neurogenesis, bromodeoxyuridine (BrdU) was given once on Day 15 of gestation (= E15) to pregnant rats that also received testosterone propionate (TP) injections. When examined at E17, the number of BrdU-labeled neurons in the AVPvN-POA was not significantly different among control female, male, and androgenized female fetuses, suggesting that androgen does not interfere with neurogenesis. At E21, a significant reduction of BrdU-labeled AVPvN neurons was observed in males and androgenized females. These findings support the hypothesis that elimination of a population by cell death is enhanced in males and androgenized females. Similar selective elimination of the AVPvN neurons occurred in the female following neonatal TP treatment. In order to investigate the nature of androgen-induced cell death in the AVPvN-POA, specific labeling of nuclear DNA fragmentation was performed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method. The number of TUNEL-positive cells was significantly greater in neonatally androgenized females, compared to that in control females. Since DNA fragmentation is considered the most characteristic feature of apoptosis, and TUNEL method is based on direct, specific labeling of DNA fragmentation in nuclei in situ, the neuronal death in the AVPvN-POA is apoptotic, and perinatal androgen may induce the selective apoptotic cell death in the AVPvN-POA.

Formation of neurons in the sexually dimorphic anteroventral periventricular nucleus of the preoptic area of the rat: effects of prenatal treatment with testosterone propionate.

An examination was made of neurogenesis in the anteroventral periventricular nucleus (AVPv) of the preoptic area of the rat using bromodeoxyuridine (BrdU), a thymidine analog, and a BrdU-specific antibody. Cells in the AVPv of adult rats were labeled with the antibody when BrdU was injected into pregnant rats once during day 13 to 18 of gestation, but not during day 10 to 12 nor 19 to 20 of gestation nor on postnatal day 1, indicating the neurogenesis of the AVPv occurs during a limited period from day 13 to 18 of gestation. Next, to examine the effects of androgen on neurogenesis, BrdU was injected once on day 15 into pregnant rats that also received injections of testosterone propionate (TP). The number of BrdU-labeled cells in the AVPv was similar in control female and male fetuses and female fetuses from pregnant rats that received daily injections of TP during days 14 to 16, when fetuses were examined on day 17 of gestation. These results suggest that the neurogenesis that was recognized by labeling with BrdU was not affected by the treatment with TP. On day 21 of gestation, BrdU-labeled cells in the AVPv of control male fetuses and female fetuses that received TP during days 14 to 18 were fewer in number than those in female fetuses of the control group, whereas treatments with TP during days 14 to 16 and during days 17 to 18 did not cause any significant decrease in number of BrdU-labeled cells. These findings can support the hypothesis that elimination of a population of cells, for example, by cell death as described previously, is enhanced in male fetuses and in female fetuses treated with TP repetitively.

Sex differences in the anteroventral periventricular nucleus of the preoptic area and in the related effects of androgen in prenatal rats.

The density of cells in the anteroventral periventricular nucleus (AVPv) of the preoptic area of female rats was greater than that of males on day 21 of gestation. It appears that this difference between the sexes is caused by the action of androgen since the density of cells in the AVPv of female fetuses fell to the density in males when the mother received injections of testosterone propionate (TP) on days 14-18 of gestation. Pycnotic cells were more frequently found in oil-treated control males and TP-treated female fetuses than in control female fetuses. This result suggests that the prenatal injections of TP enhanced the rate of degeneration of the cells in the AVPv.

Persistence of reorganized synaptic connectivity in the amygdala of kindled rats.

Kindling stimulation was applied to the basolateral amygdala of adult rats, and the density of dendritic synapses was examined under the electron microscope in the medial amygdaloid nucleus (MAN) contralateral to the site of stimulation, and an unfolding correction of biasedness was made. When generalized motor seizures had been induced for 5 days consecutively, the kindling was considered to be complete. The number of dendritic synapses, but not the number of somatic synapses, was markedly decreased in the MAN of the kindled rats. Reductions in numbers were marked in the case of both dendritic shaft and spine synapses. The reductions in numbers of shaft and spine synapses were similarly evident in the MAN of kindled rats 100 days after stimulation was discontinued. The numbers of dendritic synapses were similarly decreased in the rats that received additional bouts of stimulation subsequent to the completion of kindling. Thus, once the kindling was completed, the newly acquired synaptic connectivity was preserved in the MAN. These findings indicate that the remodeling of synaptic connectivity was a morphological correlate of the kindling in the MAN.

Induction by nerve growth factor (NGF) of NGF receptor-like immunoreactivity in the preoptic area and hypothalamus of neonatal rats.

Nerve growth factor (NGF) was injected in the brain of neonatal rats. In eleven of thirty-one rats that received 1 microgram of mouse NGF (2.5S form) on postnatal day 3, NGF receptor-like immunoreactivity was found in the preoptic area (POA) and hypothalamus until 12 days after birth. However, no immunoreactivity was seen in the region of rats in the same group examined on day 20 or 45 and of uninjected control rats on days 1-45. These results indicate that NGF induces the transient appearance of the receptor for NGF in the POA and hypothalamus of neonatal rats.

Age- and sex-related differences in the nerve growth factor distribution in the rat brain.

Levels of the nerve growth factor (NGF) have been measured in various brain regions of young and aged male and female rats of Wistar strain by means of a highly sensitive two-site enzyme immunoassay system for beta-NGF. Among the ten regions examined, the amount of NGF per wet weight of tissue was found to be highest in the hippocampus, irrespective of the sex and age. The NGF concentration in the hippocampus of female rats at 3 months of age was comparable to that of same aged males. Further, there was no significant difference in the NGF levels of the hippocampus between young and age males. However, the NGF level was significantly lower in aged females as compared to that in 3- or 4-month-old females, and hence the marked male-female difference was found in the NGF levels in aged Wistar rats.

Loss of dendritic synapses in the medial amygdala associated with kindling.

Kindling stimulation was given in the basolateral amygdala (BLA), the septal area or the corpus callosum in the right hemisphere of adult rats. The density of dendritic synapses was electron microscopically studied in the medial amygdaloid nucleus (MAN) ipsi- and contralateral to the stimulation side. The number of dendritic synapses was markedly decreased in both sides of the MAN of 3 groups of kindled rats. Such reduction occurred in both dendritic shaft and spine synapses. The most remarkable decrease was obtained in the BLA kindling. These results suggest that a decrease of synapses may provide a morphological basis for kindling.

Effect of nerve growth factor (NGF) on survival of superior cervical ganglion (SCG) transplanted into the third ventricle in rats.

Effect of short-term exposure to nerve growth factor (NGF) on neuron survival of superior cervical ganglion (SCG) transplanted into the third ventricle in rats and that on neurite outgrowth of SCG neurons in a tissue culture system were examined. Because SCG taken from 3 week-old rats exhibited a high survival rate in transplantation and they were highly sensitive to NGF in culture, they were used in the short-term NGF treatment experiment. SCG from 3 week-old rats were preincubated with NGF for 30 min at room temperature, and then they were cultured for 2 days or grafted for 14 days. Short-term exposure to NGF at the concentration of 10 micrograms/ml enhanced the survival of transplanted SCG neurons. The same concentration of NGF enhanced neurite outgrowth of SCG neurons in culture. These results suggest that short-term NGF pretreatment could increase the efficiency of neuronal transplantation.

Prevalence of human alveolar echinococcosis in Hokkaido as evaluated by western blotting.

The Western blotting (WB) method was used to evaluate the prevalence of human alveolar echinococcosis in Hokkaido, Japan. One hundred and sixteen WB-positive serum specimens were found in 1987 and 1988, and were grouped into two serologically different types from their WB-immunostaining patterns: "complete type", that showed multiple bands with various molecular weights ranging from 29 to 205 kilodaltons (kDa), and "incomplete type", that revealed a few bands with low molecular weights of 30-35 kDa and/or with molecular weights higher than 90 kDa. Therefore, we found that the WB method is applicable for the sero-epidemiological studies of human alveolar echinococcosis in Hokkaido.

Ultrastructural characterization of prolactin-like immunoreactivity in rat medial basal hypothalamus.

Prolactin-like immunoreactivity has been reported in the medial basal hypothalamus at the light microscopic level, in hypophysectomized rats. Here, with preembedding immunocytochemistry at the electron microscopic level, we have observed prolactin-immunoreactive neurons and synapses in the hypothalamus. Reaction product was discovered in medial basal hypothalamic neurons, which had typical large nucleoli and received axosomatic synapses. In the cytoplasm, reaction product was distinctly granular. Immunoreactive neurons were usually surrounded by nonreactive cells. Reaction product was also seen in dendrites, some of which had spines. Some axons in the hypothalamus contained reaction product, usually surrounded by nonreactive axons, and immunopositive synapses were detected both in the hypothalamus and in the midbrain. In a small number of cases immunoreactive axons could be seen synapsing on immunoreactive dendrites.

Modification of nerve growth factor and sex steroid in development of hypothalamic neurons.

The medial basal hypothalamic (MBH) tissues were taken from rat fetuses at day 17-18 of gestation and transplanted into the third ventricle of adult female rats. 2.5 S mouse nerve growth factor (NGF) was infused into the third ventricle of some hosts for 2 weeks using a mini-osmotic pump. The other hosts were implanted silastic capsules containing estradiol. Three weeks after the operation, the transplants were immunocytochemically stained with anti-tyrosine hydroxylase (TH) antibody. A number of immunoreactive neurons was obtained in all of NGF-treated, estrogen-exposed and control transplants. Immunoreactive axons distributed densely in NGF-treated transplants. However, the distribution of immunoreactive axons was less dense in estrogen-exposed and control MBH transplants. These results suggest that NGF infused into the host ventricle stimulates the growth and/or regeneration of axons of hypothalamic neurons. In contrast, action of estrogen on transplanted hypothalamic tissues was not evident in the present study.

Intrinsic synapses in the ventromedial nucleus of the hypothalamus: an ultrastructural study.

To test the ideas that neurons intrinsic to a hypothalamic region might participate in steroid hormone effects and that intrinsic synapses might have unusual morphological features, we deafferented the ventromedial nucleus of the hypothalamus (VMH) in ovariectomized female rats with a Halasz knife. Some rats were given estrogen; others were endocrine controls. The main result of this study was that a large number of synapses in VMH survived the circumscribing cut. In fact, they comprised between one-half and two-thirds the number counted in unoperated controls. These were apparently from neuron cell bodies intrinsic to the medial basal hypothalamic island. Their morphology was not clearly different from that expected from unoperated controls or from other brain regions. A significant estrogen effect, an increase in the number of axodendritic synapses per unit area as seen in animals without surgical transections, was not present in the deafferented groups. This suggests that the hormone effect is due to synapses on VMH neurons from cell groups outside the medial basal hypothalamic island. If functional effects of estrogen mediated through VMH do not rely on intrinsic neurons, they must use estrogen-concentrating neurons that project to other brain regions.

Medial preoptic islands in the rat brain: electron microscopic evidence for intrinsic synapses.

Neurons intrinsic to the preoptic area might participate in the control of neuroendocrine or behavioral events. To determine their existence and features, we deafferented the preoptic area of female rats, using completely circumscribing cuts with a Halasz knife. Despite obvious signs of degeneration of synapses originating from nerve cell bodies outside the preoptic island, some synapses survived complete deafferentation. We saw synaptic contacts not only on the neuronal cell body, but also on the dendritic shaft and spine. There were no peculiar morphological features, as might suggest unique physiologic functions of these intrinsic synapses. The prominence of intrinsic synapses in the preoptic area suggests that, in addition to hormone effects on preoptic neurons, and long ascending afferents, intrinsic synapses might play significant roles in neuroendocrine controls.