Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice

Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.     

Warm-coding deficits and aberrant inflammatory pain in mice lacking P2X3 receptors

ATP activates damage-sensing neurons (nociceptors) and can evoke a sensation of pain. The ATP receptor P2X3 is selectively expressed by nociceptors and is one of seven ATP-gated, cation-selective ion channels. Here we demonstrate that ablation of the P2X3 gene results in the loss of rapidly desensitizing ATP-gated cation currents in dorsal root ganglion neurons, and that the responses of nodose ganglion neurons to ATP show altered kinetics and pharmacology resulting from the loss of expression of P2X(2/3) heteromultimers. Null mutants have normal sensorimotor function. Behavioural responses to noxious mechanical and thermal stimuli are also normal, although formalin-induced pain behaviour is reduced. In contrast, deletion of the P2X3 receptor causes enhanced thermal hyperalgesia in chronic inflammation. Notably, although dorsal-horn neuronal responses to mechanical and noxious heat application are normal, P2X3-null mice are unable to code the intensity of non-noxious 'warming' stimuli.     

P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury

Pain after nerve damage is an expression of pathological operation of the nervous system, one hallmark of which is tactile allodynia-pain hypersensitivity evoked by innocuous stimuli. Effective therapy for this pain is lacking, and the underlying mechanisms are poorly understood. Here we report that pharmacological blockade of spinal P2X4 receptors (P2X4Rs), a subtype of ionotropic ATP receptor, reversed tactile allodynia caused by peripheral nerve injury without affecting acute pain behaviours in naive animals. After nerve injury, P2X4R expression increased strikingly in the ipsilateral spinal cord, and P2X4Rs were induced in hyperactive microglia but not in neurons or astrocytes. Intraspinal administration of P2X4R antisense oligodeoxynucleotide decreased the induction of P2X4Rs and suppressed tactile allodynia after nerve injury. Conversely, intraspinal administration of microglia in which P2X4Rs had been induced and stimulated, produced tactile allodynia in naive rats. Taken together, our results demonstrate that activation of P2X4Rs in hyperactive microglia is necessary for tactile allodynia after nerve injury and is sufficient to produce tactile allodynia in normal animals. Thus, blocking P2X4Rs in microglia might be a new therapeutic strategy for pain induced by nerve injury.     

粗糙沼虾雄性生殖系统的组织学研究

粗糙沼虾雄性生殖系统由精巢和输精管组成，精巢内分隔成许多生精小管。生发区位于生精小管的一侧，生殖细胞的发育是非同步的。根据其外形和结构特征，整个输精管可分成四个部分：近端输精管、中部输精管、远端输精管及未端输精管。     

锯齿米虾雄性生殖系统的组织学研究

锯齿米虾雄性生殖系统由精巢和输精管组成。精巢内分隔成许多生精小管,生发区位于生精小管的一侧。生殖细胞的发育是非同步的。根据外形和结构特征,整个输精管可划分成四个部分：近端输精管、中部输精管、远端输精管和末端输精管。     

供精个体和异种动物精浆对小鼠精子介导转染外源基因效率影响的研究

目的探讨供精个体和异种动物精浆对小鼠精子转染外源DNA效率的影响,以探索精子作为外源基因的载体建立转基因小鼠的影响因素。方法从性成熟小鼠的输精管及部分附睾管中取出精子,用DIG免疫组化方法检测和比较精予转染效率,因素为供精个体和异种动物精浆。结果不同供精个体间转染效率存在一定差异,但有的个体问差异极显著（P〈0．01）,而有的个体间差异不显著：共孵育体系中异种动物精浆可显著降低转染效率（实验组与对照组分别为（8．6±1．7）％VS（51．3±5.3）％,P〈0．01）。结论小鼠供精个体和异种动物精浆可影响小鼠精予转染外源DNA的效率。     

MSG is involved in sperm gelatinolytic activity in the prawn, Macrobrachium rosenbergii

Proteases in male reproductive tract are considered to play key roles in fertilization processes.In contrast to mammals,there are limited data concerning crustacean sperm proteases.We previously identified a novel sperm gelatinase(MSG) from Macrobrachium rosenbergii that was inhibited by a male reproduction-related Kazal-type protease inhibitor(MRPINK) specifically.In the present study,MSG was found to be distributed on the vas deferens and terminal ampullae by Western blot.Immunohistochemical analyses revealed that MSG was expressed in secretory epithelial cells and sperm distributed mainly at the base zone.RNA interference(RNAi) mediated knock-down of MSG resulted in a marked loss of sperm gelatinolytic activity.Taken together,the results show that MSG is closely linked to and probably involved in the fertilization process.     

An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.     

Molecular mechanism of ATP binding and ion channel activation in P2X receptors

P2X receptors are trimeric ATP-activated ion channels permeable to Na+, K+ and Ca2+. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body β-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.     

Two functionally distinct alpha2-adrenergic receptors regulate sympathetic neurotransmission

The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors in the heart, blood vessels and kidney. Alpha2-adrenergic receptors are known to have a critical role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system; however, the individual roles of the three highly homologous alpha2-adrenergic-receptor subtypes (alpha2A, alpha2B, alpha2C) in this process are not known. We have now studied neurotransmitter release in mice in which the genes encoding the three alpha2-adrenergic-receptor subtypes were disrupted. Here we show that both the alpha2A- and alpha2C-subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. Alpha2A-adrenergic receptors inhibit transmitter release at high stimulation frequencies, whereas the alpha2C-subtype modulates neurotransmission at lower levels of nerve activity. Both low- and high-frequency regulation seem to be physiologically important, as mice lacking both alpha2A- and alpha2C-receptor subtypes have elevated plasma noradrenaline concentrations and develop cardiac hypertrophy with decreased left ventricular contractility by four months of age.     

IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice

Studies in invertebrates have led to the identification of a number of genes that regulate lifespan, some of which encode components of the insulin or insulin-like signalling pathways. Examples include the related tyrosine kinase receptors InR (Drosophila melanogaster) and DAF-2 (Caenorhabditis elegans) that are homologues of the mammalian insulin-like growth factor type 1 receptor (IGF-1R). To investigate whether IGF-1R also controls longevity in mammals, we inactivated the IGF-1R gene in mice (Igf1r). Here, using heterozygous knockout mice because null mutants are not viable, we report that Igf1r(+/-) mice live on average 26% longer than their wild-type littermates (P < 0.02). Female Igf1r(+/-) mice live 33% longer than wild-type females (P < 0.001), whereas the equivalent male mice show an increase in lifespan of 16%, which is not statistically significant. Long-lived Igf1r(+/-) mice do not develop dwarfism, their energy metabolism is normal, and their nutrient uptake, physical activity, fertility and reproduction are unaffected. The Igf1r(+/-) mice display greater resistance to oxidative stress, a known determinant of ageing. These results indicate that the IGF-1 receptor may be a central regulator of mammalian lifespan.     

ATP mediates fast synaptic transmission in mammalian neurons.

In addition to its diverse functions inside cells, ATP can act at several types of cell-surface receptor. One of these (P2X-purinoceptor) is believed to be a ligand-gated cation channel. The presence of P2X receptors on autonomic, sensory and central neurons suggests that ATP might be released to act as a fast excitatory synaptic transmitter. Here we record excitatory synaptic potentials and currents from cultured coeliac ganglion neurons which are mimicked by ATP, blocked by the P2-purinoceptor antagonist suramin, desensitized by alpha,beta-methylene-ATP and unaffected by antagonists acting at nicotine, 5-hydroxytryptamine, N-methyl-D-aspartate (NMDA), non-NMDA glutamate, gamma-aminobutyric acid (GABA), noradrenaline or adenosine receptors. We conclude that ATP is the neurotransmitter at this neuroneuronal synapse.     

秀丽白虾繁殖生物学研究Ⅰ.雄性生殖系统的结构与发育

利用组织学方法对秀丽白虾（ Ｅｘｏｐａｌａｅｍ ｏｎ ｍ ｏｄｅｓｔｕｓ） 雄性生殖系统的结构及发育进行研究,结果表明：秀丽白虾雄性生殖系统由精巢、输精管和雄性生殖孔组成．精巢由许多生精小管组成,生发区位于小管一侧,不同区段的生精小管内精子发生不同步．精巢的发育有季节性：１２ 月～次年２月为精原细胞增殖期,３月～４ 月为精母细胞形成期,５月～８月为精子形成期,９月～１１月精巢退化,越年虾死亡．输精管可分为前、中、后、末四部分,各部管壁上皮构成有所不同,繁殖季节后,上皮细胞分泌活动明显减弱。     

Evidence for two populations of excitatory receptors for noradrenaline on arteriolar smooth muscle

We have recorded the responses of arteriolar smooth muscle cells to iontophoretically applied noradrenaline. Records of both muscle movement and muscle membrane potential were made. We found that two distinct types of response could be detected, depending on the position of the noradrenaline micropipette. One type of response consisted of a localised constriction near the noradrenaline source: this effect could be abolished by the alpha-antagonist phentolamine and was not associated with a change in arteriolar membrane potential. The other type of response was a depolarisation similar to the excitatory junction potentials (e.j.ps) produced by sumpathetic nerve stimulation. These observations suggest that there are two populations of receptors for noradrenaline on arterioles, and could explain the paradoxical failure of alpha-antagonists to block neuromuscular transmission at some sutonomic end organs such as the vas deferens, arteries and arterioles.     

中华绒螯蟹精荚形成的超微结构研究

中华绒螯蟹的精荚形成始于前输精细管处,呈椭球形,由精子、精荚基质、精荚壁组成,其精荚壁由包被精子的外周精荚基质浓缩而成,故与内部精荚基质之间无界限,精荚表面光滑,无上皮细胞分泌物沉积。精子均匀分布于精荚基质中,基质为较低电子密度物质,呈网状,其间具有一些小的空泡。输精管上皮细胞分泌物分为四种：高电子密度物质,较低电子密度物质,泡状精液,颗粒状精液。     

在单细胞水平建立原位PCR基因鉴别技术平台——以精子基因型鉴别为例

目的精子发生是一个包括有丝分裂和减数分裂的精细调控过程,这一过程是从精原干细胞开始在雄性睾丸的曲细精管中完成的,由哺乳动物雌雄性比例接近1∶1这一现象可以推知:受精时带X基因的精子和带Y基因的精子比率应该是1∶1,但事实上精子发生的过程中,分化出来的X、Y精子的比例是否也为1∶1并不清楚,因为精子发生过程中并不是所有细胞都能最终形成精子,然而要研究雌雄出生比率就需要先研究精子本身是否严格地按照1∶1形成,因此本实验通过建立快速可靠原位PCR技术平台为进一步研究精子发生的调控过程提供支持。步骤用HSL基因优化荧光原位PCR实验方案,包括蛋白酶K作用时间和浓度,原位PCR前先对精子进行解聚,再用SRY基因对精子进行原位PCR鉴定。结果在荧光显微镜下可清楚地看到精子头部的荧光信号,头部有绿色荧光信号的精子为带有SRY基因的Y染色体精子(Y精子),实验检测到昆明小鼠精子共493个,有信号的共273个,X∶Y=55.37%,经卡方检验,X精子与Y精子的实验结果趋近于1∶1。结论可利用荧光原位PCR技术在单细胞水平快速鉴定精子"性别",理论上可用于任何细胞的任何基因的鉴定。     

Mutation in the porcine SERPINA7 gene and its association with boar fertility

The porcine SERPINA7 gene is considered as a positional candidate gene responsible for testis size for its location on X chromosome and its biologically critical role in the development of testis. A nonsynonymous polymorphism (His226Asn or C678A) in the ligand-binding domain of SERPINA7 has been identified, which alters SERPINA7’s affinity to thyroxine and is closely associated with testis size. In this study, a primer mutagenesis strategy was developed to genotype this polymorphism in Chinese indigenous pigs and some western commercial pigs. The C allele existed in all tested Chinese indigenous and wild pigs, while the A allele is specific for western commercial breeds, indicating the occurrence of the mutation is of western origin. The correlation of this polymorphism with different boar fertility traits was assessed using a White Duroc × Erhualian intercross which included 110 F₂ mature boars. The results showed that the C678A polymorphism was closely associated with testis weight and epididymis weight (P＜0.0001 and P=0.0016, respectively) with significant heavier testis weight and epididymis weight in boars carrying the A allele than boars with the C allele. A significant correlation was also observed between this polymorphism and total sperm in the ejaculate (P＜0.01) as well as semen volume (P＜0.05). No statistically significant association of the C678A polymorphism with sperm concentration and sperm motility was found.     

ATP is a mediator of chemosensory transduction in the central nervous system

Extracellular signalling by the purine nucleotide ATP has long been associated with sensory function. In the periphery, ATP mediates nociception, mechanosensitivity, thermal sensitivity and O2 chemosensitivity. These processes share a common mechanism that involves the release of ATP to excite afferent fibres via activation of ionotropic P2X and/or metabotropic P2Y receptors. Chemosensors located in the brainstem are crucial for the maintenance of physiological levels of blood gases through the regulation of breathing. Here we show that an increase in pCO2 in the arterial blood triggers the immediate release of ATP from three chemosensitive regions located on the ventral surface of the medulla oblongata. Blockade of ATP receptors at these sites diminishes the chemosensory control of breathing, suggesting that ATP release constitutes a key step in central chemosensory transduction. These new data suggest that ATP, a phylogenetically ancient, unique and simple molecule, has been widely used in the evolution of afferent systems to mediate distinct forms of sensory transduction not only in the periphery but also within the central nervous system.     

曼氏无针乌贼雄性生殖系统的组织学研究

曼氏无针乌贼雄性生殖系统由精巢和生殖导管组成,生殖导管根据各段的形态、结构、功能分为输精管、贮精囊、前列腺、精荚囊和阴茎。精巢由众多生精小管组成,生精小管内分布着不同发育阶段的生殖细胞,成熟精子悬浮在管腔中央,有规则地朝着一个方向排列,精子形态为稍弯的子弹头型或长柱状纺锤形。输精管细长,兼具运输和贮存精子的作用。贮精囊体积相对缩小,具发达的分泌细胞,为精子运输提供营养和润滑作用。     

先天性双侧输精管缺如患者的睾丸生殖病理学及睾丸精子观察

对先天性双侧输精管缺如不育患者,采用睾丸活检组织观察睾丸组织生殖病理学（n=18）及其睾丸精子（n＝8）特征。结果显示这类患者宰丸曲细精管界膜呈现不同程度的病理改变。大部分标本的曲细精管扩张,生精上皮变薄、剥落。精子发生障碍以轻、中度为主。间质无明显病变。睾丸组织切片（n＝18）见曲细精管腔内有精子,睾丸活检组织（n＝8）可分离出精子,睾丸精子可呈微弱运动,精于畸形率高。