Light and electron microscope localization of binding sites of antibodies against ovine luteinizing hormone and its two subunits in rat adenohypophysis using peroxidase-labeled antibody technique

The binding sites of antisera generated in the guinea pig against ovine luteinizing hormone (oLH) and its two subunits (oLHα and oLHβ) have been localized in rat anterior pituitaries taken from normal or castrated males and from ovariectomized females with the peroxidase-labeled antibody method, using light and electron microscopy. With the light microscope, the cells positive with antiserum to ovine luteinizing hormone (A-oLH) were violet after the Alcian blue-periodic acid-Schiff (AB-PAS) staining; they were also positive for A-oLHα and for A-oLHβ and, from castrated males, they displayed an increased affinity for A-oLHβ. Another cell type which was blue after the AB-PAS method reacted with the A-oLHα only; these cells, presumably thyrotropic cells, were retracted after castration and, besides their affinity for A-oLHα, acquired an affinity for A-oLHβ. As seen through the electron microscope, two cell types were positive for A-oLH, A-oLHβ, and A-oLHα and may be identified as luteinizing hormone-secreting cells. Type A cells were characterized by two classes of rounded, secretory granules. Type B cells were smaller and contained only small secretory granules. 1 mo after the rats were castrated the type A cells were hypertrophied and vacuolized. In both cases the secretory granules were the main sites of the antigenicity with the three antisera. A positive reaction was also found in the cytoplasm, particularly in hypertrophied cells from ovariectomized females and with A-oLHβ. The cisternae of the rough endoplasmic reticulum were usually negative, except in highly degranulated cells from ovariectomized females and with A-oLHβ.     

An immunohistochemical study of somatostatin in the ovine, porcine and rodent pineal gland

Using anti-somatostatin in an immunoperoxidase technique at the light microscope level, somatostatin-like immunoreactivity was detected in ovine, porcine and rodent pineal glands. Positively stained cell bodies of various sizes and cellular processes were found throughout the glands. The chemical identity of this immunoreactive material and its physiologic significance remain to be determined.     

四种细胞因子在中华蟾蜍脑中的分布

采用免疫组织化学SABC法,研究白介素-1α、干扰素-γ、神经生长因子-β和肿瘤坏死因子-α在成体中华蟾蜍脑中的表达和分布特点。结果发现,白介素-1α阳性细胞数量很多,分布于脑的各个区域。白介素-1α多在细胞的胞体中,而原始海马锥体细胞,中脑的背前侧被盖核和腹后侧被盖核中的细胞可见阳性的突起。干扰素-γ阳性细胞数量较多,分布在端脑的原始海马和隔区,丘脑腹外侧核,下丘脑的视前区、视交叉上核和腹侧漏斗核,中脑被盖的背前侧被盖核、腹前侧被盖核、背后侧被盖核和腹后侧被盖核中,小脑的Purkinje细胞层和延髓的网状核,其中原始海马,背前侧被盖核和背后侧被盖核,视交叉上核,Purkinje细胞层和网状核中的细胞中可见阳性突起。神经生长因子-β阳性细胞数量较少,主要存在于下丘脑的视前区和视交叉上核,中脑被盖的腹前侧被盖核,小脑的Purkinje细胞层和延髓的网状核中,其中视前区、Purkinje细胞层和网状核中细胞可见阳性突起。肿瘤坏死因子-α阳性细胞数量最少,分布范围仅限于中脑被盖背前侧区和延髓的网状核及中缝核,但细胞具有阳性突起。因此,白介素-1α和干扰素-γ在成体动物脑中分布较为广泛,可能是神经细胞生命活动所必需的;而神经生长因子-β和肿瘤坏死因子-α在成体动物脑中分布范围狭窄,其作用可能仅限于脑中的某些特殊区域。     

An immunohistochemical study of somatostatin in the ovine,porcine and rodent pineal gland

Summary Using anti-somatostatin in an immunoperoxidase technique at the light microscope level, somatostatin-like immunoreactivity was detected in ovine, porcine and rodent pineal glands. Positively stained cell bodies of various sizes and cellular processes were found throughout the glands. The chemical identity of this immunoreactive material and its physiologic significance remain to be determined.     

Ontogenesis of the myenteric plexus in the cat gastro-intestinal sphincters. I. Development of the neuronal perikarya and dendrites

The pre- and postnatal development of the myenteric nerve perikarya and processes in gastro-intestinal sphincters was studied by means of light and electron microscopes. In the early fetal period, when migrate neuroblats were still seen, the myenteric ganglia were not formed. A peculiarity of each period of development was the presence of relative proportions of immature, transitional and mature nerve cells. With the progress of development the number of the immature neurons decreased, although single undifferentiated neurons were observed in adult cats. Multi-, bi- and pseudounipolar cells were distinguished in the late fetal period. On the electron microscope different myenteric neuronal types were differentiated in this period too. At birth and during the first postnatal weeks the impregnation showed an intensive dendritic branching and the Dogiel nerve types were well distinguished. To that corresponded a great variety in the fine structure of the dendrites. The nerve perikarya displayed a larger size and a richer content of organelles. Special attention was directed to the differences in the impregnation and fine structural features of the myenteric perikarya and dendrites the sphincters studied during the development.     

氨溴索与肝素联合应用对急性肺损伤兔TNF-α和IL-1β的影响

目的：探讨联合应用氨溴索与小剂量肝素对急性肺损伤（ALI）时氧化应激,TNF-α和IL-1β变化的干预及其机制。方法：健康日本大耳白兔24只,随机分成3组（n=8）：①生理盐水对照组（NC）,②油酸损伤组（OA）,③氨溴索＋小剂量肝素治疗组（AH）。各组分别在给药前和给药后6 h采血及测定动脉血氧分压（PaO2）、肿瘤坏死因子-α（TNF-α）、白介素-1β（IL-1β）的含量,实验结束后肉眼观察肺病理改变,测定支气管肺泡灌洗液（BALF）以及肺组织匀浆中TNF-α、IL-1β、超氧化物歧化酶（SOD）、丙二醛（MDA）、黄嘌呤氧化酶（XO）、谷胱甘肽过氧化物酶（GSH-Px）的含量,检测肺组织原位凋亡细胞变化、肺组织湿干比（W/D）,光镜观察肺组织病理改变,电镜观察肺组织超微结构变化。结果：①光镜,电镜观察结果以及W/D提示氨溴索＋小剂量联合治疗减轻了ALI造成的肺组织形态学改变。②OA组中显著降低的PaO2均在AH组明显升高（P〈0.01）。③抗氧化指标GSH-Px和SOD活力检测,发现AH组比OA组有不同程度升高（P〈0.01或P〈0.05）,而氧化性指标XO活力和MDA含量则较OA组显著降低（P〈0.01）。④除给药前IL-1β外,在OA组中IL-1β、TNF-α含量均显著高于NC组（P均〈0.01）,但在AH组中有显著的降低（P〈0.01）。⑤AH组凋亡指数（AI）比OA组显著降低（P〈0.01）。结论：在OA致ALI时,TNF-α和IL-1β明显升高,参与了ALI的发生与发展。联合应用氨溴索与小剂量肝素可减轻氧化应激反应,抑制促炎细胞因子TNF-α和IL-1β释放,发挥对ALI的治疗作用。     

Sexual dimorphism in the regulation of meiotic process in the rabbit

Meiosis, mitosis, and apoptosis during fetal and postnatal periods were investigated in order to explore mechanisms of sexual dimorphism in initiation of germ cell meiosis. Gonads were obtained from Japanese white rabbits from 23 to 51 days postcoitum (dpc). Gonadal thin sections were stained with hematoxylin and eosin. Germ cell alkaline phosphatase and apoptosis were detected with histochemical and immunohistochemical methods, respectively. In the ovary, meiotic germ cells were initially recognized at 29 dpc and arrested after enclosure within follicles. Similarly, meiotic germ cells were recognized outside seminiferous tubules at 29 dpc, but no meiotic figures were identified in intratubular spaces. Apoptotic germ cells were not recognized in the intratubular spaces before 35 dpc, and no apoptotic figures were recognized in the ovary during the period studied. In conclusion, the initiation of meiosis in testicular interstitial tissue at the time comparable to that in the ovary indicates that germ cells of both sexes have the ability to enter meiosis during the same stage of fetal development; and it appears most likely that delayed initiation of meiosis in the intratubular space is attributable to meiosis-inhibiting substance(s) present in seminiferous tubules.     

雌激素受体α和β免疫反应在雄性和雌性棕色田鼠脑区分布的比较

单配制和多配制动物社会行为有差异,这些差异可能与雌激素受体类型有关（ERs）。虽然多配制大鼠和小鼠中枢神经雌激素受体α（ERα）和β（ERβ）免疫反应在大脑的分布已有报道,单配制雄性草原田鼠中枢神经ERα的分布也有报道,但单配制田鼠ERα和（或）ERβ在雌性和雄性分布差异未见报道。本研究对雄性和雌性棕色田鼠前脑区域ERα和ERβ免疫反应（IR）细胞数量进行比较。研究结果表明：（1）免疫反应主要分布在细胞核中。 （2）ERα-IR和ERβ-IR细胞广泛分布于整个雌性和雄性前脑区域,在许多脑区表达有重叠。然而,不同受体在雌雄不同脑核中的分布数量是不同的。（3）ERα 和ERβ的分布存在性别差异。例如,雌性ERα在视前核中部（MPN）,终纹床和（BNST）和杏仁内侧核（MeA）比雄性多,相反雄性ERβ在MPN和BNST比雌性多。这些研究结果可能为我们理解如何通过ERα和ERβ调节动物的社会行为,及雌性和雄性社会行为的差异提供一个重要的神经解剖学基础。     

Immunohistochemical identification of cells in the pars distalis of the pituitary of the lizard Anolis carolinensis

Immunocharacteristics of the pars distalis cells of the pituitary of the male lizard A. carolinensis are determined by employing the immunoperoxidase technique with antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, 5 different cell types with characteristic anatomical distribution are recognized. ACTH cells are found in the rostral half of the pars distalis, and PRL cells in the rostral two thirds of the pars distalis. GH and TSH cells are located in the caudal half of the pars distalis. GTH cells are distributed throughout the gland. When consecutive sections are stained with antiserum to ovine FSH or its beta-subunit and to ovine LH, the same cells show immunoreactivity to all the three antisera. None of the GTH cells show positive immunoreactivity to ovine beta-LH antiserum. The results suggest the existence of one gonadotropic cell type in the pituitary of this lizard.     

Distinct systemic distribution of salusin-α and salusin-β in the rat

Salusin-α and salusin-β are multifunctional bioactive peptides that were initially predicted using in silico analyses. These peptides should be concomitantly biosynthesized from prosalusin in humans. However, little information is available yet on the biosynthesis and mode of presence of salusin-α and salusin-β in non-human species. In the present study, we examined whether salusin-α and salusin-β are conserved in the rat and whether salusin-α and salusin-β show distinct systemic distributions. Immunohistochemical analysis of rat tissues using a specific anti-rat salusin-α antibody detected immunoreactivity extensively in neuronal cells and fibers, and abundantly in the epithelial tissues throughout the organs. This distribution contrasts sharply with that of salusin-β, which is mainly localized to the neuroendocrine and hematopoietic systems. Western blot analysis of rat spleen extracts showed the presence of cleaved fragments corresponding to putative rat salusin-α. Reverse-phase and gel filtration high performance liquid chromatography analyses coupled with radioimmunoassay detection of rat urine extracts revealed a major immunoreactive component that co-eluted with synthetic putative rat salusin-β. These data support the processing of rat prosalusin into salusin-α and salusin-β despite absent dibasic amino acids between the two.     

Ontogenesis of Oxidative Reaction of 17β-hydroxysteroid Dehydrogenase and 11β-hydroxysteroid Dehydrogenase in Rat Leydig Cells, a Histochemical Study

The enzyme 17β-hydroxysteroid dehydrogenase is required for the synthesis and 11β-hydroxysteroid dehydrogenase for the regulation of androgens in rat Leydig cells. This histochemical study describes ontogenetic changes in distribution and intensity of these enzymes in Leydig cells from postnatal day (pnd) 1–90. Using NAD or NADP as the cofactor, 17β-hydroxysteroid dehydrogenase (substrate: 5-androstene-3β, 17β-diol) peaks were observed on pnd 16 for fetal Leydig cells and on pnd 19 and 37 for adult Leydig cells. Between pnd 13 and 25 the fetal cells showed a higher intensity for the 17β-enzyme than the adult cells; more fetal Leydig cells were stained with NADP, whereas more adult cells were positive with NAD on pnd 13 and 16. A nearly identical distribution of 11β-hydroxysteroid dehydrogenase (substrate: corticosterone) was observed with NAD or NADP as the cofactor; the reaction was present from pnd 31 onwards, first in a few adult Leydig cells and later in almost all these cells homogeneously. The ontogenetic curves of the two enzymes show an inverse relationship. To conclude: (1) Generally, a stronger reaction for 17β-hydroxysteroid dehydrogenase is shown with NAD as cofactor than with NADP; using NADP, fetal Leydig cells show a stronger staining than adult Leydig cells. (2) The data possibly support the notion of a new isoform of 11β -hydroxysteroid dehydrogenase in addition to types 1 and 2.     

Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.     

Light and electron microscopic immunocytochemistry of the same nerves from whole mount preparations

A technique for performing correlated light and electron microscopic immunocytochemical studies on whole mount preparations has been developed using myenteric plexus from guinea pig small intestine as a model. With this method a structure containing a particular antigen can first be located by light microscopy and then examined with the electron microscope. Pieces of intestine pinned on balsa were incubated in oxygenated Krebs solution at 37 degrees C for 90-120 min and then fixed for 1 hr at room temperature in 4% formaldehyde, 0.05% glutaraldehyde, and 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4. The tissue was washed vigorously in several changes of 50% ethanol until the picric acid had been removed, stored overnight in phosphate buffer, and then exposed to 0.1% sodium cyanoborohydride in buffer for 30 min. Vasoactive intestinal peptide (VIP) was localized in separated layers containing myenteric plexus and longitudinal muscle using the peroxidase-antiperoxidase technique with imidazole intensification of the diaminobenzidine reaction product. At the light microscope level, tissue stained by this technique showed VIP-immunoreactive nerve cell bodies and processes throughout the thickness of the myenteric ganglia in numbers approximately equivalent to those seen in whole mounts processed by an established technique for the light microscopic demonstration of VIP, which does not involve exposure of tissue to glutaraldehyde. VIP-immunoreactive structures that were first identified at the light microscope level were subsequently examined at the electron microscope level. VIP-immunoreactive axon profiles were found to form synapses on both immunoreactive and nonimmunoreactive myenteric neurons. The fine structural appearance of the different cell types present in whole mount preparations prepared by this method was similar to that seen in conventionally fixed tissue, except that free and bound ribosomes were absent from the tissue processed for immunocytochemistry. The method described here is reliable and no more difficult than presently available methods for preembedding electron microscopic immunocytochemistry on sections. Its main advantage is that immunoreactive structures for ultrastructural study can be selected from the entire population of chemically identified nerves within a whole mount rather than from a smaller sample present within a section. This technique is applicable to other tissues that can be stained immunohistochemically in whole mounts. The fixation and penetration enhancement procedures can also be adapted for immunocytochemical studies on vibratome or frozen sections.     

The expression of platelet endothelial cell adhesion molecule-1 in mouse primordial germ cells during their migration and early gonadal formation

The platelet endothelial cell adhesion molecule-1 (PECAM-1), or CD31, a member of the immunoglobulin superfamily, is located on the plasma membrane of endothelial and hematopoietic cells and involved in vascular development and inflammation. In this study, by use of immunohistochemistry at light and electron microscopic levels in combination with enzyme histochemistry for alkaline phosphatase, we demonstrated that PECAM-1/CD31 is expressed in the mouse primordial germ cell (PGC). Up to 8 days postcoitum (dpc), PGCs with alkaline phosphatase activity showed no PECAM-1/CD31 immunoreactivity. At 9 dpc, PECAM-1/CD31 immunoreactivity was first detected with low intensity in some PGCs located in the hindgut. Between 10 and 11 dpc, intense immunoreactivity was shown on the entire surface of PGCs migrating along the dorsal wall. After arrival and settlement of PGCs in the genital ridges around 11.5 dpc, the intense immunoreactivity was maintained on the entire surface of PGCs. By electron microscopy, the immunoreactivity was localized exclusively on the plasma membrane of PGCs, being as strong at the portions adjacent to neighboring PGCs as those adjacent to somatic cells. As the male and female gonads began to differentiate, PECAM-1/CD31 immunoreactivity remained strong in germ cells until 13 dpc, after which it gradually decreased in intensity and disappeared by 16 dpc. These results suggested that cell-to-cell interaction through PECAM-1/CD31 plays roles in the development of PGCs during their migration on the dorsal wall and homing in the gonads.     

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in human and cat gastric glands.

In the present work we have studied the occurrence of pituitary adenylate cyclase activating polypeptide (PACAP) in human and cat stomach mucosa using immunohistochemistry. As seen under a light microscope, there were many large rounded and ovoid cells that were PACAP immunopositive, mainly in the neck of the gastric glands of both species. The immunopositive material was predominant in the perinuclear area. The PACAP immunolabeling was specific because the preincubation of the antiserum with PACAP abolished the immunostaining. In human samples under electron microscope, the PACAP immunoreactive cells have shown the characteristics of parietal cells. In faintly stained cells, the localization of DAB reaction product was associated with the surface of the intracellular canaliculi. Cell labeling could not be observed besides parietal cells.