Immunohistochemical localization of gonadotropin-releasing hormone receptors (GnRHR) in the nervous system, Hatschek’s pit and gonads of amphioxus,Branchiostoma belcheri

Using gonadotropin-releasing hormone (GnRH) anti-idiotypic antibodies and APA immunohistochemical method, the immunoreactivity of GnRHR in the nervous system, Hatschek’s pit and gonads of amphioxus has been located. It is found for the first time that the immunoreactivity of GnRHR exists in the nerve cells and fibers in the amphioxus’s brain and nerve tube and the epithelial cells of Hatschek’s pit at the different stages of gonadal development. At the same time, it is also found that GnRHR also exists in the ovary and testis of different developed stages. These findings provide morphological new proof for the informative transfer and regulation between brain and Hatschek’s pit mediation by GnRHR, and for the understanding of the mechanism of action on the reproductive endocrine control axis among brain-Hatschek’s pit-gonads.     

Distribution of ghrelin-like immunoreactive cells in amphioxus, Branchiostoma belcheri – A study of immunohistochemistry

The distribution of ghrelin-like immunoreactive cells in amphioxus （Branchiostoma belcheri） was investigated by using immunohistochemical staining with rabbit antiserum against synthetical mammalian ghrelin. The results showed that ghrelin-like immunoreactive cells were distributed widely in the nervous system, Hatschek＇s pit, wheel organ, digestive tract and gonads （ovary and testis）. In nervous system, ghrelin-like immunoreactive neurons and their protrusions were distributed specifically on the dorsal side, ventral side and funnel part of brain vesicle, with a few dispersive immunoreactive nerve cells and their fibers in nerve tube. Ghrelin-like immunoreactivities were also detected in Hatschek＇s pit epithelial cells and wheel organ cells, with positive substance located along cell membrane. In digestive tract, ghrelin-like immunoreactive cells existed in hepatic diverticulum, anterior and posterior region of midgut, and could be classified into two types, closed- and opened-type endocrine cells. The number of positive cells was most in hepatic diverticulum, secondary in posterior region of midgut and least in anterior region of midgut. In gonads, ghrelin-like immunoreactive substance was detected in oogonia, oocytes and follicle cells in ovary at the small and large growth stages and in early spermatogenic cells and Sertoli cells in testis. The extensive distribution of ghrelin-like cells in amphioxus suggested that these kinds of cells are conservative in evolution and diversified in function. At the same time, we found for the first time that ghrelin-like immunoreactive cells existed in brain vesicle and Hatschek＇s pit, which provided new morphological evidence for the existence of an activation pathway between brain vesicle and Hatschek＇s pit for the regulation of growth hormone excretion.     

Distribution of growth hormone-like immunoreactive cells and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus, Branchiostoma belcheri

Using immunohistochemical method and double staining technique, the localization of growth hormone (GH) and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus has been investigated. The results showed that the growth hormone-like nerve cells and endocrine cells as well as three subtypes of somatostatin receptors exist in the nervous system and Hatschek's pit, and GH-like nerve cells and endocrine cells co-exist with three subtypes of somatostatin receptors in the brain vesicle and Hatschek's pit. It is suggested that a primitive control system of inhibitory growth hormone secretion in Hatschek's pit could have been developed in amphioxus, as in vertebrates. The present study provides new evidence for the endocrinology and the evolution of Hatschek's pit.     

Distribution of growth hormone-like immunoreactive cells and somatostatin receptors in the nervous system and Hatschek''s pit of amphioxus, Branchiostoma belcheri

Using immunohistochemical method and double staining technique, the localization of growth hormone (GH) and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus has been investigated. The results showed that the growth hormone-like nerve cells and endocrine cells as well as three subtypes of somatostatin receptors exist in the nervous system and Hatschek's pit, and GH-like nerve cells and endocrine cells co-exist with three subtypes of somatostatin receptors in the brain vesicle and Hatschek's pit. It is suggested that a primitive control system of inhibitory growth hormone secretion in Hatschek's pit could have been developed in amphioxus, as in vertebrates. The present study provides new evidence for the endocrinology and the evolution of Hatschek's pit.     

Neuroendocrine regulation of structure and function in the endostyle of amphioxus, Branchiostoma belcheri

Using histological and histochemical methods the structure of the endostyle in amphioxus at different gonadal developmental stages is studied, and the immunocytochemical localization of thyroglobulin (Tg) and thyrotropin (TSH) in the endostyle, Hatschek's pit and brain vesicle is investigated. The results not only confirm the previous report that the endostyle is composed of 6 zones and the cells of zone 5 are thyroid hormone synthesizing cells, but also find the thyroxine synthesis in and secretion from zone 3. In addition, the epithelial cells in Hatschek's pit and the neural cells in brain vesicle are immunopositive for TSH, and the immunoactivity is correlated with gonadal cycle. The present study may provide morphological proof for the hypothesis that the secretory activity of thyroid cells is regulated by TSH from Hatschek's pit and brain vesicle.     

Distribution of CRH-and ACTH-like immunoreactive cells in amphioxus,Branchiostoma belcheri

Immunocytochemical studies on the nervous system,Hatschek's pit,digestive tract and gonads tissues of an amphioxus(Branchiostoma belcheri)were performed using polyclonal antibodies against human corticotrophin-releasing hormone(CRH)and human adrenocorticotropin(ACTH).The results showed that many CRH-like immunoreactive neurons were distributed specifically on the dorsal side and ventral side of brain vesicle,while a few CRH-like neurons and their fibers in spinal cord.At the same time,the epithelial cells in the basic region of Hatschek's pit were shown immunopositive to CRH antibody.In gonads(ovary and testis),CRH-immunopositive substance was localized in the cytoplasm near oocyte nucleus and in early spermatogenic cells.ACTH-like immunoreactivities were observed specially in the neurons and their protrusions localized on the ventral side of the brain vesicle and in spinal cord,and also in epithelial cells of Hatschek's pit,enteric neurons of digestive tract,oocytes in ovary and in early spermatogenic cells as well.It was found for the first time that CRH-like neurons existed in the middle region of brain vesicle(corresponding to the hypothalamus of vertebrates)and ACTH-like immunopositive cells existed in Hatschek's pit,implying that a control mechanism between brain vesicle and Hatschek's pit maybe had been already built in amphioxus as that in vertebrates.The present study will provide new morphological evidence for the origin and evolution of ACTH.In addition,the immunoreactivities of CRH and ACTH in the digestive tract and gonads suggested other physiological function of CRH and ACTH in amphioxus.     

Distribution of α-MSH-like immunoreactive cells in the nervous system, Hatschek''''s pit and other tissues of amphioxus, Branchiostoma belcheri

Immunohistochemical localization of a-melanocyte-stimulating hormone (α-MSH) in the nervous system, Hatschek's pit and other tissues of amphioxus (Branchiostoma belcheri) was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of brain vesicle, the dorsal side and the surrounding of nerve tube, and in the epithelial cells of Hatschek's pit, the zone 1, 3, and 6 of endostyle and gut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek's pit in amphioxus does not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of the present study provided a new evidence for the endocrinology of Hatschek's pit and for the origin and evolution of vertebrate adenohypophysis.     

Distribution of growth hormone-like immunoreactive cells and somatostatin receptors in the nervous system and Hatschek''''s pit of amphioxus, Branchiostoma belcheri

Using immunohistochemical method and double staining technique, the localization of growth hormone (GH) and somatostatin receptors in the nervous system and Hatschek's pit of amphioxus has been investigated. The results showed that the growth hormone-like nerve cells and endocrine cells as well as three subtypes of somatostatin receptors exist in the nervous system and Hatschek s pit, and GH-like nerve cells and endocrine cells co-exist with three subtypes of somatostatin receptors in the brain vesicle and Hatschek s pit. It is suggested that a primitive control system of inhibitory growth hormone secretion in Hatschek s pit could have been developed in amphioxus, as in vertebrates. The present study provides new evidence for the endocrinology and the evolution of Hatschek's pit.     

Localization and expression of LHβmRNA in the brain and Hatschek's pit of amphioxus, Branchiostoma belcheri

To determine whether gonadotropin-like substance in the brain and Hatschek' s pit of amphioxus issynthesized by the tissue in situ or transported from other tissue, a histochemical study was carried out by in situ hy- bridization using digoxigenin (DIG)-labelled LHβRNAprobes. The results showed that LHβmRNA expressed in the nerve cells of brain and the epithelial cells of Hatschek' s pit, thus providing new evidence for the homology of pituitary of ver- tebrates with Hatschek' s pit and the functional evolution of gonadotropin.     

Evidence for coexistence of dopamine and CCK in meso-limbic neurones

Vanderhaeghen et al. reported the occurrence of gastrin-like immunoreactivity in the mammalian brain. Subsequent studies have revealed that this immunoreactivity corresponded mainly to the COOH-terminal octapeptide of cholecystokinin (CCK-8), which has a COOH-terminal pentapeptide identical to gastrin. Also, two peptides resembling the NH- and the COOH-terminal tetrapeptide fragments of CCK-8 are present in the central nervous system (CNS). Using COOH-terminal-specific antisera raised to gastrin and/or CCK, the distribution of CCK neurones has been described with immunohistochemical techniques. Although high numbers of cells and nerve terminals are found in cortical areas, the CCK systems are also present in most other parts of the brain and spinal cord. In the CNS, true gastrin molecules, gastrin-17 and gastrin-34 have been located only in the neurohypophysis, hypothalamus and occasionally in the medulla oblongata (unpublished results). We describe here the occurrence of peptides in meso-limbic dopamine neurones in the rat brain. Evidence has also been obtained that mesencephalic dopamine neurones in the human brain contain similar peptides.     

大鼠神经干细胞表达家蚕神经系统特异蛋白类似物

用免疫组织化学的方法,研究了大鼠胎脑中家蚕神经系统特异蛋白（Bombyrin）的分布以及与神经干细胞的关系．结果表明,家蚕Bornbyrin类似物主要分布在大鼠胎脑的脑室下区、纹状体等区域,特别是表达在大鼠的神经干细胞中,而分化的神经干细胞则没有Bombyrin类似物的免疫活性．western blot的分析表明,大鼠脑中存在77kDa和110kDa 2种Bornbyrin类似的蛋白．这些结果暗示,大鼠脑中Bombyrin类似蛋白可能与神经干细胞特性的维持有关.     

Position in the evolution of reproductive endocrine of amphioxus,Branchiostoma belcheri

This review summarizes the recent discoveries of many authors who found that in amphioxus Hatschek’s pit is capable of synthesizing vertebrate gonadotropin-like substance, and that the content of gonadotropin-releasing hormone in the amphioxus’ body shows a positive correlation with the reproductive cycle, and that the sex steroid hormone exists in gonads. Exogenous hormones could promote gonadal development, maturation and reproductive activity in amphioxus. A possible implication might be that the reproductive activity in amphioxus is regulated by reproductive hormones like vertebrate, indicating the existence of primitive reproductive endocrine regulatory axis, brain vesicle-Hatschek’s pit-gonads axis, as compared with regulatory axis of vertebrate. It will provide a new line for establishing the position of reproductive endocrine evolution in lancelet.     

Increase of corticotropin-releasing factor staining in rat paraventricular nucleus neurones by depletion of hypothalamic adrenaline

In response to stress, adrenocorticotropic hormone (ACTH) is released by corticotrophs in the anterior pituitary under the control of several central and peripheral factors including corticotropin-releasing factor (CRF), which was recently isolated from the brain and sequenced. Immunocytochemical studies have shown that most of the CRF-containing cell bodies that project to the median eminence are present in the hypothalamic paraventricular nucleus (PVN). A dense PNMT(phenylethanolamine-N-methyltransferase)-containing fibre network was also observed in the same region--PNMT is the final enzyme in the biosynthesis of adrenaline and has been demonstrated in the brain. In the present study we found an association of adrenergic nerve fibres and CRF neurones by immunohistochemistry using antisera to PNMT and CRF. To examine the functional significance of the adrenergic projection to the PVN, we blocked the synthesis of adrenaline using a specific inhibitor of PNMT. The depletion of adrenaline resulted in an increase in CRF immunoreactivity. The present results suggest that, as well as catecholamines which regulate ACTH release at the anterior pituitary level via a beta 2-adrenergic receptor mechanism, central catecholamines (mainly adrenaline) also affect ACTH release through their action on CRF cells. Peripheral catecholamines seem to have a direct stimulatory effect on the pituitary corticotroph cells, whereas the present findings suggest that central adrenaline-containing neurones have an inhibitory role in the physiological response to stress.     

钝化304不锈钢单晶在硫酸含氯介质中的孔蚀

采用交流阻抗技术,研究钝化304不锈钢单晶体在pH1.0,0.25mol/lNa_2SO_4含氯介质中的孔蚀行为。对孔蚀过程中的阻抗频谱特征进行了分析,提出孔蚀的发生和发展过程中都有复合物(MOHC1)形成。复合物进一步吸附更多的氯离子,导致溶解反应发生。并提出一个孔蚀的的反应机理模型,它与304不锈钢多晶体在硫酸含氯介质中的孔蚀机理没有本质的差别。     

Developmental study on the immunocytochemical localization of NOV protein-containing neurons in the rat brain

The localization and development of nephroblastoma overexpressed gene (nov) protein-immunoreactive neurons in the brain of E8-P300 rats have been studied using immunocytochemistry and image analysis. Results are as follows: No NOV protein-immunoreactive cells were detected in the rat brain during prenatal development. A few of positive cells were detected at the early postnatal stage. However, the number and the immunoreactivity of these cells increased gradually at later stages. NOV-immunoreactive cells were widely distributed in the rat brain during P30–P60. The number of immunoreactive cells and their intensity also peaked within this stage. The number and staining intensity of NOV-positive cells decreased gradually with age. The positive cells were mainly located in cingulate cortex, striatum, hippocampus, hypothalamus, cerebellum and brain stem. The present results indicate thatnov may play an important role in the development and differentiation of brain as well as maintaining the function of brain.     

金鱼视觉系统内中、高分子量神经丝免疫组织化学定位

RT97是一种特异性识别中、高分子量神经丝蛋白抗原簇的抗体.采用组织化学技术对金鱼(Carassiusauratus)视觉系统内RT97识别神经丝的表达分布进行了观察.结果表明,RT97在金鱼视网膜、视神经和视顶盖中均有阳性反应:①在视网膜内视纤维层、内网层、内核层的水平细胞、光感受器的外节有RT97阳性反应;②在视乳突部和视神经有少量生长纤维为RT97阳性;③在视顶盖中RT97阳性反应集中在顶盖的边缘层(SM),同时观察到有RT97阳性纤维从中央纵突区(TL)通过顶盖腹面延伸至顶盖边缘区.实验结果提示,RT97不仅可作为金鱼视觉系统视觉生长纤维的一种标记蛋白,同时在视顶盖由RT97识别的蛋白也可能是一种吸引视觉纤维生长的靶蛋白.     

Evidence for neurotensin as a non-adrenergic, non-cholinergic neurotransmitter in guinea pig ileum

Neurotensin is a 13-amino acid peptide that is widely distributed in central and peripheral tissues of various mammalian species. In peripheral tissues, the highest concentration of neurotensin-like immunoreactivity is found in the ileum, where it is present in endocrine-like cells and nerve fibres. The longitudinal smooth muscle layer of the guinea pig ileum, where neurotensin has both a direct relaxant and an indirect contractile action, has been used extensively as a biological assay system for neurotensin. We report here that the majority of specific 3H-neurotensin binding sites is present in the guinea pig ileum circular smooth muscle layer, which is known to be innervated by a large proportion of the ileal non-adrenergic inhibitory nerves. Neurotensin produces a dose-dependent, tetrodotoxin-resistant relaxation, whereas the relaxation produced by field stimulation of the inhibitory nerves is frequency-dependent and tetrodotoxin-sensitive. The calcium-dependent potassium channel blocker apamin inhibits both the neurotensin- and nerve stimulation-induced muscle relaxation. Incubation of the circular smooth muscle preparation with a neurotensin antiserum substantially inhibited the nerve stimulation-induced relaxation, indicating a direct relationship between the effects of neurotensin and of nerve stimulation.     

Peptidergic transmitters in synaptic boutons of sympathetic ganglia

In sympathetic ganglia of the bullfrog, a slow synaptic potential lasting for minutes--the late slow excitatory postsynaptic potential (e.p.s.p.)--was discovered. This slow response, unlike other previously known synaptic potentials in the autonomic nervous system, is not mediated by acetylcholine or monoamines. Similar non-cholinergic, non-adrenergic slow synaptic potentials have since been found in several other vertebrate autonomic ganglia. We found that the late slow e.p.s.p. is probably mediated by a peptide that is identical to, or closely resembles, mammalian luteinizing hormone releasing hormone (LHRH), because (1) when applied directly to sympathetic neurones, LHRH and its agonists elicit a slow depolarization, associated with similar changes in membrane conductance and excitability as those occurring during the late slow e.p.s.p. Furthermore, both peptide-induced and nerve-evoked responses are blocked by antagonists of LHRH; and (2) radioimmunoassays indicate that a chain of sympathetic ganglia contains 100-800 pg of a LHRH-like peptide. Its distribution among spinal nerves, the great reduction of this substance following denervation, and its release from ganglia following isotonic KCl treatment or nerve stimulation suggest that the LHRH-like material is contained in preganglionic nerve fibres. Here we report that immunohistochemical staining of sympathetic ganglia shows that LHRH-like immunoreactivity is indeed present in synaptic boutons. We also show that the two types of ganglion cells (B cells and C cells) receive strikingly different patterns of peptidergic innervation.     

Enkephalin-, VIP- and substance P-like immunoreactivity in the carotid body

The carotid body type I cell contains amines and has features, both morphological and cytochemical, which indicate that it may also produce a peptide. Many regulatory peptides are now known to be present in both central and peripheral tissues. In the periphery these neuropeptides occur in both classical endocrine (APUD) cells and the neurones of the autonomic nervous system. We have now investigated the possible presence of neuropeptides in the cat carotid body using both immunocytochemistry and radioimmunoassay. Met- and Leu-enkephalin-like material occurred in considerable quantities in carotid body extracts and enkephalin-like immunoreactivity was localised in type I cells. Both vasoactive intestinal polypeptide (VIP)- and substance P-like immunoreactivity was also present but was localised in nerve fibres distributed throughout the organ. These active neuropeptides are widely distributed in mammalian tissues, forming a diffuse regulatory system which now seems to include the carotid body.     

Cloning of a novel gene encoding human thioredoxin-like protein

mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed to screen the arrayed human fetal brain cDNA library. A1 .2-kb cDNA clone has been found. This cDNA consists of an 867 bp open reading frame, a 132 bp 5' untranslated sequence and a 209 bp 3' untranslated sequence with a typical polyadenylation signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like gene (hTRXL).