Immunohistochemical study of the responsiveness of LH cells of fetal rats to synthetic LHRH in vitro

Summary Fetal rat pituitaries on days 17–19 of gestation were maintained in serum-free Medium 199 for 24 h in the presence of 1, 10 and 100 ng/ml of synthetic LHRH. Immunohistochemical examination of such stimulated tissue reveals a complete depletion of immunoreactive material in most of the LH cells, irrespective of the LHRH concentrations tested, though some cells remain weakly immunopositive in the pituitaries of later developmental stages. Once discharge has occurred, there is little reaccumulation of secretory material in LH cells during prolonged incubation for 48 h in LHRH-free medium containing 10% calf serum. The LHRH treatment causes no immunohistochemical change in TSH cells.It is concluded that in fetal rats recently differentiated LH cells can release the secretory product if they are stimulated by hypothalamic LHRH.     

In vivo and in vitro studies on the appearance of LHRH neurons in the hypothalamus of perinatal rats

Summary Ontogenetic development of LHRH-containing neurons was studied by fluorescence and enzyme immunohistochemistry in rats. In in vitro studies, the tissues of the septal-chiasmatic and mediobasal hypothalamic areas of fetal rats on day 16.5 or 18.5 of gestation were trypsinized separately for dissociation of the neural cells, and cultured for several days. Immunopositive reaction against LHRH was first detected in nerve cells derived from both areas of the hypothalamus of the fetuses on days 16.5 and 18.5 of gestation, after 8 and 6 days culture, respectively. The cells were small, and seemed to be bipolar in morphology indicating an axon and arborized dendrites. Immunopositive material occurred in the cell soma as well as in the cellular processes. In in vivo studies, immunopositive material, possibly deposited in nerve fibers, appeared first in OVLT and simultaneously in the external layer of the median eminence of fetuses on day 20.5 of gestation. The immunoreactive fibers increased in number in both parts with development, especially after birth in the median eminence. No immunopositive material was detected within any neural cell bodies nor in the cytoplasm of any ependymal cells.This work was financed by the Ministry of Education, Japan. No. 257008. We would like to thank Dr. Katsuhiko Saito (Department of Surgery, Tokushima University) for his kind advice on the preparation of the antibody used for the immunofluorescence study.     

Immunocytochemical and immuno-electron-microscopical study of growth hormone cells in male and female rats of various ages

Summary Growth hormone (GH) secretory cells were identified by immunogold cytochemistry, and were classified on the basis of the size of secretory granules. Type I cells contained large secretory granules (250\2-350 nm in diameter). Type II cells contained the large secretory granules and small secretory granules (100\2-150 nm in diameter). Type III cells contained the small secretory granules. The percentages of each GH cell type changed with aging in male and female rats of the Wistar/Tw strain. Type I cells predominated throughout development; the proportion of type I cell was highest at 6 months of age, and decreased thereafter. The proportion of type II and type III cells decreased from 1 month to 6 months of age, but then increased at 12 and 18 months of age. The pituitary content of GH was highest at 6 months of age, and decreased thereafter. Estrogen and androgen, which are known to affect GH secretion, caused changes in the proportion of each GH cell type. The results suggest that when GH secretion is more active the proportion of type I GH cell increased, and when GH secretion is less active the proportion of type II and type III cells increased. The type III GH cell may therefore be an immature type of GH cell, and the type I cell the mature type of GH cell. Type II cells may be intermediate between type I and III cells.     

参乌胶囊及其有效成分二苯乙烯苷对老年大鼠海马区神经生长因子及其受体表达的影响

目的观察本室自行研制的中药新复方参乌胶囊（SW）及其有效成分何首乌四羟基二苯乙烯苷（TSG）对老年大鼠海马区神经生长因子（NGF）及其受体表达的影响。方法老年sD大鼠从21月龄始,分别灌胃给予SW低、高剂量（0．8s／kg、1．6g／kg）和TSG低、高剂量（0．03s／kg、0．06g／kg）至24月龄。以6月龄大鼠为青年对照,未给药的24月龄大鼠为老年对照。应用实时定量PCR方法检测大鼠海马区NGF及其受体TrkA的mRNA表达水平,免疫组织化学染色和Westernblot方法检测海马区NGF及TrkA表达水平。结果从基因和蛋白质水平观察到,老年大鼠海马区NGF及其受体TrkA的mRNA表达和蛋白表达明显降低。老年大鼠灌胃给予SW能够上调其海马区NGFmRNA和TrkAmRNA水平;SW和TSG对NGF蛋白表达有明显的增高作用,高剂量SW和TSG对TrkA表达的上调作用显著。结论SW和TSG能够增高老年大鼠海马区NGF及其受体水平,提示它们可作用在神经元信号转导通路的起始端,有利于提高神经元的存活和功能。     

Electron microscopic study of immunoreactive LHRH perikarya with special reference to neuronal regulation

Summary In early postnatal rats, immunoreactive LHRH perikarya in the preoptic area were studied by light and electron microscopy. Synaptic junctions were found between the immunoreactive perikaryon or its process, and the immunonegative nerve fibers. The significance of these synapses is discussed in relation to possible mechanisms by which the activities of LHRH neurons are regulated.     

Two types of luliberin-immunoreactive perikarya in the preoptic area of the rat

Summary At the light microscopic level, following immunostaining with a single antiserum against luliberin (LRF), two types of hormone-producing perikarya in the preoptic area are demonstrated. The two cell types differ in their morphological features: a bipolar, smooth-contoured cell type can be differentiated from an irregularly contoured unipolar type. Intermediate forms between both cell types occurring in the same area are not observed. Electron microscopically, both cell types contain labeled granules of similar size and immunoreactivity. It is dicussed whether the uneven surface of the one cell type is due to areas of synaptic contacts, and whether both cell types are integrated in different neuronal and functional circuits. Moreover, at the ultrastructural level, from the irregularly contoured LRF-producing perikarya a further positively stained cell type, probably a glial cell, can be differentiated. The specific labeling of the latter is caused by its content of immunoreactive lysosomal bodies. Differentiation between the labeled glial cells and the irregularly contoured LRF-producing perikarya is not possible at the light microscopic level.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and by the Stiftung Volkswagenwerk     

Immunohistochemical study on fetal raphe samples transplanted into the leptomeningeal tissue of 5,6-dihydroxytryptamine-treated adult rats

Summary Pieces of fetal midbrain raphe containing serotonergic and dopaminergic neurons were transplanted into the leptomeningeal tissue (see Fig. 3) of adult host rats that had previously been denervated by treatment with 5,6-dihydroxytryptamine. One, 2 and 5 months after transplantation, the rate of neuronal survival in the grafted tissue and the extent of axonal outgrowth into the host brain were studied by use of serotonin and tyrosine hydroxylase (TH) immunohistochemistry. The survival rate of the grafts in the 1-month group was approximately 70%. Neurons containing either serotonin or catecholamine were demonstrated by means of immunocytochemical procedures in the grafts. Two and 5 months after transplantation, serotonin-immunoreactive nerve fibers were densely distributed throughout the graft tissue, while TH-immunoreactive fiber elements were restricted to an area near the somata of TH-positive neurons. Numerous serotonin-immunoreactive fibers derived from the transplant were found in the leptomeningeal tissue surrounding the graft, on the wall of neighboring blood vessels, and also in the adjacent parenchyma of the host brain. Outgrowing TH-immunoreactive nerve fibers were not observed in the host brain, although such elements occurred in the leptomeningeal tissue and the wall of the larger blood vessels. These results suggest that the serotonergic and catecholaminergic (dopaminergic) neurons located in transplants of the raphe nuclei show different patterns when reinnervating the host tissue.     

Catecholamine-synthesizing enzymes in paraganglia of aged Fischer-344 rats

Summary The catecholamine-synthesizing enzymes, tyrosine hydroxylase, dopamine--hydroxylase and phenylethanolamine-N-methyltransferase were examined by immunohistochemistry in hypertrophied paraganglia of aged male Fischer-344 rats. All paraganglionic cells reacted with antibodies against tyrosine hydroxylase. Dopamine -hydroxylase was identified in most paraganglionic cells, indicating that they synthesized norepinephrine. A variable number of paraganglia were positive for phenylethanolamine-N-methyltransferase, which suggested that they synthesized epinephrine. The formaldehyde-induced fluorescence method demonstrated greenish-yellow fluorescence or yellowish-brown fluorescence. The intensity of the fluorescence was in the same range as in adrenal medullary cells. The observations indicate that paraganglia are capable of synthesizing epinephrine.Dr. J.M. Stolk is the recipient of RSDA K2-00018 from the National Institute of Health.     

Immunocytochemical localization of the gonadotropin-releasing hormone-associated peptide portion of the LHRH precursor in the hypothalamus and extrahypothalamic regions of the rat central nervous system

Summary The gonadotropin-releasing hormone-associated peptide (GAP) of the LHRH precursor and the decapeptide LHRH were localized in the rat brain by immunocytochemistry in 12 to 18-day-old animals, by use of thick Vibratome sections and nickel intensification of the diaminobenzidinereaction product. Our results indicate that the GAP portion of the LHRH precursor is present in the same population of neurons that contain LHRH in the rat brain. An important difference observed was that the GAP antiserum, in contrast to LHRH antisera, stained several perikarya in the medial basal hypothalamus. GAP-immunoreactive perikarya were observed in the following regions: the olfactory bulb and tubercle, diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and several regions of the hippocampus. In addition to the preoptico-terminal and the septopreoptico-infundibular pathways, we also observed GAPimmunopositive processes in several major tracts and areas of the brain, including the amygdala, stria terminalis, stria medullaris thalami, fasciculus retroflexus, stria longitudinalis medialis, periventricular plexus, periaqueductal gray of the mesencephalon and extra-cerebral regions, such as the nervus terminalis and its associated ganglion. These results confirm the specificity of previous immunocytochemical results obtained with antisera to LHRH. The presence of GAP immunoreactivity in nerve terminals of the rat brain indicates that GAP or a GAP-like peptide is located in the proper site to serve as a hypophysiotropic substance and/or as a neurotransmitter or neuromodulator.Supported by AKA No. 419427, OTKA No. 104, OKKFT 2.1.5.1 and NSF No. INT-8602688     

Pattern of follicular development during the estrous cycle of aged rats

Summary The pattern of follicular development during the estrous cycles of aged rats was examined and compared with that of mature rats. In both, preovulatory follicles are derived from a select group of small pre-Graafian follicles which begin to develop at estrus and reach the preovulatory size by the morning of proestrus, but the rate of growth, as judged by an increase in the percentage of granulosa cells incorporating ³H-thymidine, is accelerated in the follicles of aged rats. A second mechanism, which accounts for preovulatory follicles in aged rats, involves the rescue from atresia of pre-Graafian and preovulatory follicles. The existence of this mechanism is supported by the observation that at metestrus in aged rats virtually all follicles, regardless of their state of atresia, possess a high percentage of granulosa cells incorporating ³H-thymidine, indicating that the follicles are growing rapidly. However, some of these rapidly growing follicles show signs of atresia such as pyknotic nuclei within their granulosa cell layers. Since follicles in the initial stage of atresia contain defective oocytes (Peluso et al. 1979b), their rescue and development into preovulatory follicles would result in the ovulation of defective oocytes, a fact which accounts in part of the lower fertility in these older animals.     

Lymphocytes apoptosis: young versus aged and humans versus rats

This paper deals with a comparative study of lymphocyte apoptosis in young versus aged and humans versus rats. Apoptotic rate achieved by the use of different apoptogenic inducers, acting at different cellular levels, and cell surface modifications were analyzed. The results showed that aged human lymphocytes and freshly isolated rat lymphocytes were more prone to undergo apoptosis. Therefore, the same apoptotic signal is recognized by human and rat lymphocytes, but the extent of the answer is related to the species, to the intensity of the apoptotic stimulus and to the metabolic/developmental condition of the cells. Surface modifications (lipids and glycans), typical of apoptosis, were observed. Our data showed that cell surface changes are species and age dependent. They are early events, progressively achieved in the course of the apoptotic process involving lateral membrane movements of molecules.     

Correlative ontogenetic development of catecholamine- and LHRH-containing nerve endings in the median eminence of the rat

Summary The ontogenetic development of catecholamine (CA)-and LHRH-containing nerve endings in the median eminence of the rat was investigated by combining fluorescence histochemistry and immunohistochemistry in the same tissue section. LHRH-terminals appeared earlier than CA-terminals and were already detectable in the lateral part of the external layer of the central ME on the first day after birth. CA-nerve endings were first seen in a corresponding region of the ME on the seventh postnatal day. At this stage both types of terminals showed the earliest manifestation of a correlative pattern of their distribution. Subsequently the development of both types of nerve endings proceeded rapidly, and at 14 days their distribution pattern corresponded to that in adult animals. The authors conclude that at this stage the CA-neurons play a constant and significant role in the release of LHRH into the portal capillaries. The correlation between both types of nerve endings and the ontogenetic development of the capillary plexuses of the hypophysial portal system is discussed.This work was supported in part by a grant (No. 248093, 321426) from the Ministry of Education, Science and Culture, Japan     

Immunohistochemical study on the development of CRF-containing neurons in the hypothalamus of the rat

Summary Appearance of immunoreactive corticotropin-releasing factor (CRF)-containing neurons was studied in developing hypothalamus of the rat by use of antisera against rat- and ovine CRF. These neurons were first recognized in the lateral and paraventricular nuclei on days 15.5 and 16.5 of gestation, respectively, when antiserum against rat CRF was employed. Antiserum against ovine CRF revealed the cells two days later exclusively in the latter nucleus. In both nuclei, the neurons increased in number with development. The neurons in the paraventricular nucleus appeared to project their immunoreactive processes to the median eminence via the periventricular and lateral pathways. In the median eminence, the immunoreaction with antiserum to rat CRF was first recognized in its anterior portion in the form of dots on day 16.5 of gestation but as beaded fibers in the external layer on day 17.5; these structures increased in amount with development in rostro-caudal direction. Although antiserum to ovine CRF was less potent in immunostainability than antiserum to rat CRF, it also revealed the beaded fibers in the median eminence on day 17.5 of gestation. Since evidence is available that the paraventricular nucleus is involved in corticotropin release, it is concluded that, in rats, the hypothalamic regulatory mechanism controlling the release of corticotropin initially appears on days 16.5–17.5 of gestation.     

Mitochondrial alterations and apoptosis in smooth muscle from aged rats

We studied changes in mitochondrial morphology and function in the smooth muscle of rat colon. Under confocal microscopy, tissues loaded with potentiometric dye displayed rapid and spontaneous depolarization. Cyclosporin A (CsA), inhibitor of the permeability transition pore (PTP), caused an increase in mitochondrial membrane potential (ΔΨ_m) in tissues from adult young animals. In aged rats these changes were not observed. This suggests that physiological activation of PTP in aged rats is reduced. Electron microscopy showed alterations of the mitochondrial ultrastructure in tissues from aged rats involving a decreased definition of the cristae and fragmentation of the mitochondrial membranes. We also detected an increase in apoptotic cells in the smooth muscle from aged animals. Our results show that the aging process changes PTP activity, the ability to maintain ΔΨ_m and mitochondrial morphology. It is suggested that these can be associated with mitochondrial damage and cell death.     

Immunohistochemical mapping of galanin-like neurons in the rat central nervous system

Using an antiserum generated in rabbits against synthetic galanin (GA) and the indirect immunofluorescence method, the distribution of GA-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system (CNS) and a detailed stereotaxic atlas of GA-like neurons was prepared. GA-like immunoreactivity was widely distributed in the rat CNS. Appreciable numbers of GA-positive cell bodies were observed in the rostral cingulate and medial prefrontal cortex, the nucleus interstitialis striae terminalis, the caudate, medial preoptic, preoptic periventricular, and preoptic suprachiasmatic nuclei, the medial forebrain bundle, the supraoptic, the hypothalamic periventricular, the paraventricular, the arcuate, dorsomedial, perifornical, thalamic periventricular, anterior dorsal and lateral thalamic nuclei, medial and central amygdaloid nuclei, dorsal and ventral premamillary nuclei, at the base of the hypothalamus, in the central gray matter, the hippocampus, the dorsal and caudoventral raphe nuclei, the interpeduncular nucleus, the locus coeruleus, ventral parabrachial, solitarii and commissuralis nuclei, in the A1, C1 and A4 catechaolamine areas, the posterior area postrema and the trigeminal and dorsal root ganglia. Fibers were generally seen where cell bodies were observed. Very dense fiber bundles were noted in the septohypothalamic tract, the preoptic area, in the hypothalamus, the habenula and the thalamic periventricular nucleus, in the ventral hippocampus, parts of the reticular formation, in the locus coeruleus, the dorsal parabrachial area, the nucleus and tract of the spinal trigeminal area and the substantia gelatinosa, the superficial layers of the spinal cord and the posterior lobe of the pituitary. The localization of the GA-like immunoreactivity in the locus coeruleus suggests a partial coexistence with catecholaminergic neurons as well as a possible involvement of the GA-like peptide in a neuroregulatory role.     

Immunohistochemical study on adenohypophysial primordia in organ culture

Summary Rathke's pouches isolated from rat fetuses on day 12 were maintained in organ culture for 9 days and investigated immunohistochemically to test whether or not the hypothalamus is involved in the cytodifferentiation of the adenohypophysis. The unlabeled antibody enzyme method demonstrated that the cultured tissue contains different types of glandular cells, i.e., adrenocorticotropin (ACTH)-, growth hormone (GH)-, luteinizing hormone (LH)-, thyrotropin (TSH)-, and prolactin-producing cells. Indirect evidence was also obtained to indicate the presence of melanocyte stimulating hormone (MSH)-cells. These findings suggest that adenohypophysial primordial cells of rats start to synthesize their respective hormones without stimuli from neurosecretory substances of the brain which are known to be essential for the maintenance of the secretory activity of the adult gland.We wish to express our thanks to Dr. A. Kawaoi for providing anti-porcine ^1–39ACTH, to Dr. S.S. Spicer for the supply of anti-porcine ^17–39ACTH and to Dr. P. Petrusz for the gift of antisera against bovine GH, bovine TSH, HCG and rat prolactin. We should also like to thank Mr. Y. Okamura for technical help and Mr. I. Shimada for preparation of the photographs.     

A comparative in vitro study on LHRH responsiveness of LH cells of the pars tuberalis and pars distalis

Summary The aim of the present study was to test whether the luteinizing-hormone (LH) cells in the pars tuberalis (PT) of the rat and mouse respond to LH-releasing hormone (LHRH) as do those of the pars distalis. A part of the basal hypothalamus containing the pituitary stalk, median eminence and the pars tuberalis (H-PT), was dissected out and incubated in vitro.The LH-secreting capacity of the PT was investigated after removal of the pituitary body (i.e., partes distalis, intermedia and nervosa). First, some rat and mouse H-PT tissues were treated with synthetic LHRH (100ng/ml), while others were incubated without LHRH. After 24 h of incubation, variable amounts of LH release were detected in the medium. This LH discharge, however, was not LHRH-dependent but proportional to the number of PT LH cells that were immunohistochemically detected in each incubated tissue. Since there was marked individual variation in the number of LH cells in the PT, the LH levels in the incubation medium were next compared before and after LHRH treatment using the same H-PT of the rat. An effect of LHRH could not clearly be shown in this experiment.Finally, the cytological response of the PT to LHRH was investigated by incubating both the H-PT and pituitary body connected to the intact pituitary stalk. Immunohistochemical examination of LHRH-treated tissues after 24 h revealed that, in females of both rats and mice, hormone depletion occurred in LH cells of the pars distalis but not in those of the PT. These results indicate that although LH cells in the PT can release LH in vitro, their mode of hormone synthesis and/or discharge differs from that of LH cells in the pars distalis. Since there was a marked individual variation and small LH-secreting capacity by the PT tissue, it seems unlikely, at least in rats and mice, that LH of PT origin plays an important role in the normal physiological state.