Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle

The alterations in morphology and function of the ovarian follicle as it matures, ovulates, and becomes a corpus luteum are dramatic. A variety of steroid and polypeptide hormones influence these processes, and the ovary in turn produces specific hormonal signals for endocrine regulation. One such signal is inhibin, a heterodimeric protein that suppresses the secretion of follicle-stimulating hormone from pituitary gonadotrophs. Rat inhibin complementary DNA probes have been used to examine the levels and distribution of inhibin alpha-and beta A-subunit messenger RNAs in the ovaries of cycling animals. Striking, dynamic changes have been found in inhibin messenger RNA accumulation during the developmental maturation of the ovarian follicle.     

性腺肽类激素在动物生殖调控中的作用

阐述了性腺肽类激素抑制素、活化素及卵泡抑素对动物生殖机能的影响及其分泌的调节。抑制素和卵泡抑素均可抑制垂体FSH的活性，从而影响动物的生殖功能。抑制素受内分泌、旁分泌及自分泌作用；卵泡抑素以自分泌方式调节颗粒细胞功能，促进卵泡的黄体化；活化素以自分泌方式调节颗粒细胞的分化，阻止排卵卵泡的黄体化，并有诱导产生和增加FSH受体的功能。     

Activin-binding protein from rat ovary is follistatin

Activin, a member of the transforming growth factor beta protein family, was originally isolated from gonadal fluids and stimulates the release of pituitary follicle-stimulating hormone (FSH). Activin has numerous functions in both normal and neoplastic cells. Various cells synthesize activin and have a specific binding site for this peptide. However, the molecular basis for its actions is unknown. A binding protein for activin was purified from rat ovary and was identical to follistatin, a specific inhibitor of FSH release. It is likely that the binding protein participates in the diverse regulatory actions of activin.     

Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice

Class IA phosphoinositide 3-kinases (PI3Ks) are a family of p85/p110 heterodimeric lipid kinases that generate second messenger signals downstream of tyrosine kinases, thereby controlling cell metabolism, growth, proliferation, differentiation, motility, and survival. Mammals express three class IA catalytic subunits: p110alpha, p110beta, and p110delta. It is unclear to what extent these p110 isoforms have overlapping or distinct biological roles. Mice expressing a catalytically inactive form of p110delta (p110delta(D910A)) were generated by gene targeting. Antigen receptor signaling in B and T cells was impaired and immune responses in vivo were attenuated in p110delta mutant mice. They also developed inflammatory bowel disease. These results reveal a selective role for p110delta in immunity.     

Induction of interleukin 2 messenger RNA inhibited by cyclosporin A

Cyclosporin A blocked production of the lymphokine interleukin 2 by activated T lymphocytes. In a human and a murine cell line this inhibition reflected an absence of interleukin 2 messenger RNA. Under conditions in which these cells are normally stimulated to secrete high levels of interleukin 2, they failed to do so in the presence of cyclosporin A. In both cell lines this failure was accompanied by an absence of interleukin 2 messenger accumulation.     

Underexpression of beta cell high Km glucose transporters in noninsulin-dependent diabetes

The role of defective glucose transport in the pathogenesis of noninsulin-dependent diabetes (NIDDM) was examined in Zucker diabetic fatty rats, a model of NIDDM. As in human NIDDM, insulin secretion was unresponsive to 20 mM glucose. Uptake of 3-O-methylglucose by islet cells was less than 19% of controls. The beta cell glucose transporter (GLUT-2) immunoreactivity and amount of GLUT-2 messenger RNA were profoundly reduced. Whenever fewer than 60% of beta cells were GLUT-2-positive, the response to glucose was absent and hyperglycemia exceeded 11 mM plasma glucose. We conclude that in NIDDM underexpression of GLUT-2 messenger RNA lowers high Km glucose transport in beta cells, and thereby impairs glucose-stimulated insulin secretion and prevents correction of hyperglycemia.     

大鼠垂体抑制素免疫组织化学定位

[目的]研究垂体本身能否分泌抑制素,探讨抑制素作用于垂体的方式。[方法]采用免疫组织化学定位技术对大鼠垂体组织进行免疫组化学分析。[结果]试验组与对照组大鼠的垂体组织上存在明显的抑制素阳性反应区,而阴性对照试验组未见阳性反应区。[结论]垂体本身能分泌抑制素,抑制素可能以旁分泌的方式作用于垂体,同时抑制素可能以旁分泌的方式与其受体结合。     

A novel method to improve sow reproductive performance: Combination of pre-weaning immunization against inhibin and post-insemination hCG treatment

The feasibility of a novel method to improve sow reproductive performance by combining inhibin immunization and hCG treatment was tested using in vivo and in vitro experiments. In the in vivo experiment, 106 sows were administered an inhibin immunogen on day 7 prior to weaning, and 56 non-treated sows served as the controls. Sows exhibiting oestrous behaviour on day 5 after weaning were artificially inseminated. On day 5 post-insemination, a subset of 50 inhibin-immunized sows received an injection of 1 000 IU human chorionic gonadotropin(hCG). Our results showed that pre-weaning immunization against inhibin marginally improved(P=0.068) total litter size and significantly increased(P=0.044) the live litter size. The overall differences in farrowing rates and live litter size tended toward significance(P=0.10) in the three groups, and the differences in total litter size were not significant(P=0.18). In the in vitro experiment, activin and hCG dose-dependently suppressed(P0.001) and stimulated(P0.001) progesterone(P4) secretion in cultured pig granulosa cells(GCs), respectively, and the suppression effect of activin was reversed(P0.001) by hCG. Activin suppressed P4 secretion mainly by downregulating(P0.001) the expression of StAR, Cyp11 a1, and 3β-HSDII, whereas hCG prevented(P0.001) the suppression effects. These results indicate that the combination of pre-weaning immunization against inhibin and postinsemination hCG treatment provides a novel method for improving sow reproductive performance.     

Characterization of T cell receptor gamma chain expression in a subset of murine thymocytes

While much information exists about the structure and function of the clonally distributed T cell receptor (TCR) alpha beta heterodimer, little is known about the gamma protein, the product of a third rearranging TCR gene. An antiserum to a carboxyl-terminal peptide common to several of the murine gamma chain constant regions and a monoclonal antibody to the murine T3 complex were used to identify products of this TCR gene family in a subpopulation of Lyt2-, L3T4- thymocytes. This subpopulation does not express TCR alpha or full-length TCR beta messenger RNA. The gamma chain is a 35-kilodalton (kD) protein that is disulfide-bonded to a 45-kD partner and is associated with the T3 complex. Analysis of the glycosylation pattern of this thymic gamma chain revealed that the major variable region gamma (V gamma) gene transcribed in activated peripheral T cells is absent from this subpopulation. The cells that bear this second T cell receptor may therefore represent a distinct lineage differentiating within the thymus.     

胃泌素RT-PCR方法的建立及同源性分析

从猪胃窦和十二指肠提取总RNA,采用美国Am b ion RNA公司生产的Q uan tum RNA class ic 18S为内标,建立了半定量RT-PCR方法,并对胃窦和十二指肠胃泌素mRNA进行了定量检测、测序和比较分析。结果表明,扩增的猪胃窦和十二指肠胃泌素核苷酸序列与猪前胃泌素(propregastrin)核苷酸的同源性达到99%。将其与已发表的人、牛、大鼠和小鼠等物种相应序列的比较结果表明,无论是核苷酸水平,还是氨基酸水平,都显示出了较高的同源性(80%~99%)。另外,建立的RT-PCR方法灵敏度高,操作快捷方便,可用于定量检测少量的组织样品中低丰度表达的胃泌素mRNA。     

Inhibin-mediated feedback control of follicle-stimulating hormone secretion in the female rat

The secretion of follicle-stimulating hormone (FSH) by the anterior pituitary gland is regulated by the interaction of hypothalamic and gonadal hormones. Recently, proteins termed inhibins that selectively suppress FSH secretion have been purified and characterized from the gonadal fluids of several species. Antibodies to a synthetic peptide encompassing the amino terminal 25 residues of the recently characterized porcine inhibin were used to develop a specific radioimmunoassay (RIA) for inhibin and to neutralize endogenous inhibin during the estrous cycle of the rat. The administration of 20 international units of pregnant mare serum gonadotropin (PMSG) stimulated the secretion of inhibin in intact immature female rats, whereas ovariectomy caused an abrupt decrease in plasma inhibin concentrations that were not prevented by the injection of PMSG. The infusion of a polyclonal antiserum to inhibin, from 12 noon on proestrus to 1 a.m. on the morning of estrus, as well as its acute intravenous injection during diestrus I or II, caused an increase in plasma FSH (but not luteinizing hormone) concentrations. These results support the hypothesis of a feedback loop between the release of ovarian inhibin and FSH in the female rat.     

Amyloid beta protein precursor is possibly a heparan sulfate proteoglycan core protein

The amyloid beta protein peptide is a major constituent of amyloid plaque cores in Alzheimer's disease and is apparently derived from a higher molecular weight precursor. It is now shown that the core protein of a heparan sulfate proteoglycan secreted from a nerve cell line (PC12) has an amino acid sequence and a size very similar to those of the amyloid beta protein precursor and that these molecules are antigenically related. This amyloid beta protein precursor-related protein is not found in the conditioned medium of a variant cell line (F3 PC12) that does not secrete heparan sulfate proteoglycan. The synaptic localization and metabolism of this class of proteoglycans are consistent with its potential involvement in central nervous system dysfunction.     

Localization of amyloid beta protein messenger RNA in brains from patients with Alzheimer's disease

The distribution of cells containing messenger RNA that encodes amyloid beta protein was determined in hippocampi and in various cortical regions from cynomolgus monkeys, normal humans, and patients with Alzheimer's disease by in situ hybridization. Both 35S-labeled RNA antisense and sense probes to amyloid beta protein messenger RNA were used to ensure specific hybridization. Messenger RNA for amyloid beta protein was expressed in a subset of neurons in the prefrontal cortex from monkeys, normal humans, and patients with Alzheimer's disease. This messenger RNA was also present in the neurons of all the hippocampal fields from monkeys, normal humans and, although to a lesser extent in cornu ammonis 1, patients with Alzheimer's disease. The distribution of amyloid beta protein messenger RNA was similar to that of the neurofibrillary tangles of Alzheimer's disease in some regions, but the messenger RNA was also expressed in other neurons that are not usually involved in the pathology of Alzheimer's disease.     

一株分泌抗猪γ-干扰素单克隆抗体的杂交瘤细胞的鉴定

[目的]应用杂交瘤技术获得稳定分泌抗猪γ-干扰素单克隆抗体的杂交瘤细胞株。[方法]先将原核表达产物His—PoIFN一γ免疫BALB／c小鼠4次,然后进行细胞融合,再以纯化的GST—PoIFN一1作为包被抗原,用间接ELISA筛选阳性杂交瘤无性系,最终获得能稳定分泌抗猪IFN-γ单克隆抗体的杂交瘤细胞株,对其进行问接ELISA和Westernblot检测。[结果]试验获得l株能稳定分泌抗猪IFN一1单克隆抗体的杂交瘤细胞株,被命名为B3株,经间接ELISA和Westernblot检测,该株分泌的抗体能与融合蛋白His—PoIFN一γ和GST—PoIFN一γ发生特异性反应,而与其他表达鸡γ-干扰素、His和GST蛋白等的重组质粒均不发生反应。B3株分泌的抗体小鼠腹水的ELISA效价达到1：160000,抗体亚型为IgG1型,且轻链为κ链。[结论]该研究为建立猪γ-干扰素检测方法,研究机体的免疫状态及免疫机制提供了技术基础。     

Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism

The aims of the study were to determine the distribution of inhibin and its α subunit in some rat tissues by an immunohistochemical method of streptavidin-biotin complex （SABC） and to assess the transport mechanism of inhibin by investigating the localization of inhibin and α subunits in the central nervous system （mainly in hypothalamus and pituitary） of ovariectomized rats. Investigations on the extragonadal tissues of ovariectomized rats showed positive expression of inhibin and its α subunit in heart, kidney, spleen, pancreatic gland cells, but no positive reaction sites were seen in lung, liver, submaxillary gland, and adrenal gland. After injection with inhibin α subunit fragment or inhibin extract, positive reaction sites were observed in hypothalamus and pituitary of ovariectomized rats by SABC. Inhibin and its subunit was present in a wide variety of nonreproductive organs and tissues, and its expression was tissue specific, which indicated that inhibin might play a role in regulating tissue function through autocrine/paracrine mechanisms. Inhibin dimer and α subunit could be transported through the BBB by the method of “separation and reconstruction”.     

Bovine x mouse hybridomas that secrete bovine immunoglobulin G1

The interspecific fusion of normal bovine lymphocytes with a nonsecreting mouse hybridoma produced stable cell lines secreting bovine immunoglobulins. One of these lines has continued to secrete immunoglobulin G1 (5 to 10 micrograms per milliliter) for over 16 months. The bovine x mouse hybrid cells can be expected to provide bovine monoclonal immunoglobulins for sequencing studies and for use as serological standards as well as to provide messenger RNA for cloning bovine immunoglobulin genes.     

T cell CD3-zeta eta heterodimer expression and coupling to phosphoinositide hydrolysis

The T cell antigen receptor consists of an antigen-binding heterodimer that is noncovalently associated with at least five CD3 subunits (gamma, delta, epsilon, zeta, and eta). The CD3-zeta chains are either disulfide-linked homodimers (CD3-zeta 2) or disulfide-linked heterodimers with eta (CD3-zeta eta). Variants of a murine antigen-specific T cell hybridoma that express normal amounts of CD3-zeta 2 but decreased amounts of CD3-zeta eta were isolated. When activated, the parental cell line increased both phosphatidylinositol hydrolysis and serine-specific protein kinase activity to a much greater extent than the variants. In contrast, the activation of a tyrosine-specific kinase after stimulation with a cross-linking antibody to CD3 was similar among these cells. There was a positive linear relation between the expression of CD3-zeta eta and phosphoinositide hydrolysis stimulated by the TCR, suggesting a differential coupling of the T cell alpha beta heterodimer to signal transduction mechanisms due to alpha beta association with either CD3-zeta 2 or CD3-zeta eta.     

Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor

A new type of agonist-binding subunit of rat neuronal nicotinic acetylcholine receptors (nAChRs) was identified. Rat genomic DNA and complementary DNA encoding this subunit (alpha 2) were cloned and analyzed. Complementary DNA expression studies in Xenopus oocytes revealed that the injection of messenger RNAs (mRNAs) for alpha 2 and beta 2 (a neuronal nAChR subunit) led to the generation of a functional nAChR. In contrast to the other known neuronal nAChRs, the receptor produced by the injection of alpha 2 and beta 2 mRNAs was resistant to the alpha-neurotoxin Bgt3.1. In situ hybridization histochemistry showed that alpha 2 mRNA was expressed in a small number of regions, in contrast to the wide distribution of the other known agonist-binding subunits (alpha 3 and alpha 4) mRNAs. These results demonstrate that the alpha 2 subunit differs from other known agonist-binding alpha-subunits of nAChRs in its distribution in the brain and in its pharmacology.     

抑制素主动免疫对母牛卵泡发育的影响

本试验以猪精液抑制素粗提物主动免疫母牛,研究了对母牛卵泡发育和内分泌的影响。免疫,母牛的卵泡发育率为１４５．５％,排卵率为１１８．２％,分别比对照组提高４５．５％和１８．２％。通过放射免疫法对免疫牛血液中ＦＳＨ,ＬＨ,Ｅ２水平测定发现,发情时免疫组母牛ＦＳＨ水平高于对照组,免疫组中多卵发育牛ＦＳＨ水平高于单卵牛,免疫组与对照组中ＬＨ水平相差不大,多卵牛发情时Ｅ２水平较高。