Phagocytosis and clearance of apoptotic cells is mediated by MER

Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.     

基于Cre／Loxp系统Tyr03、Axl受体酪氨酸激酶转基因小鼠的建立

采用大肠杆菌β一半乳糖酶基因LacZ作为报告基因,建立Tyr03、Axl受体酪氨酸激酶含有loxp位点的转基因小鼠。与不同组织特异性Cre小鼠杂交,获得时空性表达Tyr03、Axl的转基因小鼠,为研究其在各个组织中的功能奠定基础。方法构建含有loxp位点的Tyr03、Axl受体酪氨酸激酶质粒,测序正确后,通过显微注射法将插入CAG启动子下游的Geo—STOP—msAxl及Geo—STOP—msTyr03基因的转基因载体注射入BDFI供体鼠受精卵,移植受精卵至ICR受体鼠,待产仔后,通过双引物PCR鉴定F。以及Fn代小鼠的基因型,RT—PCR,X—gal染色观察小鼠组织中LaeZ基因,运用WB（WesternBlot）检测转基因小鼠和野生型小鼠体内目的蛋白表达的差异。结果获得了含有目的基因的转基因阳性小鼠;通过RT—PCR对阳性小鼠证明LacZ基因的存在;对存在LacZ基因的小鼠进行X—gal染色,在Geo—STOP—msTyr03的脑、睾丸、胸腺中观察到蓝色沉淀,在Geo—STOP—msAxl的脑、心、肾中观察到蓝色沉淀,间接说明目的基因能表达的组织和器官;对双阳性的Geo．STOP．msTyr03、Geo．STOP—msAxl转基因小鼠和野生型小鼠,WB检验证实Tyr03、Axl基因的表达没有差异,说明在加入stop序列之后,Tyr03基因确实存在,而且表达受到抑制。结论成功建立Geo-STOP—msAxl及Geo—STOP—msTyr03酪氨酸受体转基因小鼠。     

类固醇激素受体在扬子鳄睾丸表达的免疫细胞化学研究

用免疫细胞化学方法对雄激素受体(AR)、雌激素受体(ER)和孕激素受体(PR) 在扬子鳄睾丸中进行定位研究.结果表明,三种类固醇激素受体在扬子鳄睾丸中都有分布:雄激素受体在睾丸的肌样细胞和支持细胞中呈阳性反应,雌激素受体在睾丸的间质细胞中呈 阳性反应,孕激素受体在睾丸的生精细胞中呈阳性反应.推测结果表明,雄激素受体在扬子 鳄精子发生过程有重要作用;雌激素受体可能在扬子鳄睾丸发育过程中影响睾丸间质细胞雄性激素的分泌,进而调节生精过程和精子发育成熟,孕激素受体可能通过转换成AR和ER来对精子发生进行调节.这些结果为证明性类固醇激素参与调节扬子鳄性腺生殖内分泌调控提供了重要的形态学新证据.     

绒山羊曲细精管生殖细胞的长期培养和精子发生过程的观察

哺乳动物精子发生过程是生殖生物学的重要研究课题之一.为了开展对山羊精子发生过程的研究,我们首次建立了绒山羊睾丸生殖细胞原代培养方法.原代生殖细胞和支持细胞(Sertolicells)由绒山羊睾丸曲细精管组织块产生,在体外共培养超过三个月.在共培养期间,观察到绒山羊的精原干细胞分化为精母细胞和精子细胞的形态变化过程.这一方法的建立为山羊精子发生过程的研究和应用打下了基础.实验结果显示,在没有添加任何生长因子的条件下,绒山羊睾丸生殖细胞长期增生分化,不断产生精子细胞.这一结果暗示了组织块和共生的支持细胞为生殖细胞的增生和分化提供了营养因子和调节因子.生成的游离精子细胞最后全部死亡,暗示这一共培养体系不能提供变态过程所需因子.     

Testis determination requires insulin receptor family function in mice

In mice, gonads are formed shortly before embryonic day 10.5 by the thickening of the mesonephros and consist of somatic cells and migratory primordial germ cells. The male sex-determining process is set in motion by the sex-determining region of the Y chromosome (Sry), which triggers differentiation of the Sertoli cell lineage. In turn, Sertoli cells function as organizing centres and direct differentiation of the testis. In the absence of Sry expression, neither XX nor XY gonads develop testes, and alterations in Sry expression are often associated with abnormal sexual differentiation. The molecular signalling mechanisms by which Sry specifies the male pathway and models the undifferentiated gonad are unknown. Here we show that the insulin receptor tyrosine kinase family, comprising Ir, Igf1r and Irr, is required for the appearance of male gonads and thus for male sexual differentiation. XY mice that are mutant for all three receptors develop ovaries and show a completely female phenotype. Reduced expression of both Sry and the early testis-specific marker Sox9 indicates that the insulin signalling pathway is required for male sex determination.     

Abnormal sexual development in transgenic mice chronically expressing müllerian inhibiting substance

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone, is a glycoprotein normally secreted by the Sertoli cells of the fetal and adult testis and by granulosa cells of the postnatal ovary. The production of MIS in the male fetus brings about the regression of the Müllerian ducts, the anlagen of the uterus, oviducts, and upper vagina. In addition, purified MIS induces the formation of seminiferous cord-like structures in fetal rat ovaries cultured in vitro, suggesting that MIS may influence testicular differentiation. We have produced transgenic mice chronically expressing human MIS under the control of the mouse metallothionein-1 promoter to investigate its role during sexual development. In females, chronic expression led to the inhibition of Müllerian duct differentiation, resulting in a blind vagina and no uterus or oviducts. At birth the ovaries had fewer germ cells than normal; during the next two weeks germ cells were lost and the somatic cells became organized into structures resembling seminiferous tubules. Apparently, these structures degenerate as they are undetectable in adult females. The majority of transgenic males developed normally. But in two lines with the highest levels of MIS expression, some males showed feminization of the external genitalia, impairment of Wolffian duct development, and undescended testes. These results suggest that MIS has several distinct roles in mammalian sexual development.     

Absence of growth by most receptor-expressing fetal thymocytes in the presence of interleukin-2

The growth of mature T cells is regulated by receptors for interleukin-2 (IL-2) and by IL-2 itself. Binding of antigen to T-cell antigen receptors induces the expression of IL-2 receptors, and binding of IL-2 to these receptors induces transferrin receptor expression and is sufficient to promote the growth of T cells for several days. However, nothing is known about the growth requirements of pre-T cells. We have therefore studied the dividing population of T-cell precursors which carry the Thy-1 surface antigen, but lack surface antigens Ly2 and L3T4; these cells are present in 14-day-old embryonic thymus. If the thymus is removed at this stage and placed in organ culture, all lymphocyte subpopulations normally present in thymuses of adult mice develop in vitro, that is, the nonfunctional Ly2+, L3T4+ population and the functional Ly2+, L3T4- and Ly2-, L3T4+ populations. We now report that, in contrast to their progeny, the early Ly2-, L3T4- cells express large amounts of IL-2 receptors, but most of them do not grow in IL-2-containing media outside the thymus. In contrast to dividing mature T cells, most fetal thymocytes express low amounts of transferrin receptors.     

鳜精巢的组织学和超微结构观察

对鳜精巢的组织学结构和超微结构进行观察．鳜精巢呈叶型结构,由精原细胞、初级精母细胞、次级精母细胞、精细胞、成熟精子等生殖细胞和支持细胞、间质细胞、边界细胞、类肌细胞、成纤维细胞、内皮细胞等非生殖细胞组成．鳜精子形成过程,精细胞大致经历了鞭毛发生,核质凝缩及核位置的改变,线粒体迁移等过程．鳜成熟精子无顶体结构,头短而圆,主要为核占据,核凹窝发达,尾细长,具侧鳍,尾部轴丝为“9 2”结构．     

Isolation of HTLV-transformed B-lymphocyte clone from a patient with HTLV-associated adult T-cell leukaemia

The human T-cell leukaemia/lymphoma virus (HTLV) is an exogenous retrovirus which has been associated with adult T-cell leukaemia/lymphoma (ATL). This malignancy of T lymphocytes is endemic to southern Japan, the West Indies, and to a lesser extent, the Middle East, Central Africa and the southeastern United States. ATL cells from patients of diverse geographical origins have been found to be infected with HTLV-1 (ref.6). HTLV is normally tropic for mature T lymphocytes, especially those expressing the helper-inducer surface antigen phenotype (OKT4 or Leu-3-positive), and the neoplastic T cells infected with HTLV generally express receptors for T-cell growth factor (detected by reactivity with anti-Tac antibody). However, we report here the isolation of a HTLV-infected B-lymphocyte clone from the peripheral blood of a patient with ATL. This clone is cytogenetically normal and is not infected with Epstein-Barr virus (EBV). Co-culture of cells from this clone with cord blood lymphocytes resulted in transmission of HTLV and the immortalization of either T or B lymphocytes. These results suggest that HTLV may be associated with a broader range of host cells than previously recognized.     

Ovulated oocytes in adult mice derive from non-circulating germ cells

Decades of research in reproductive biology have led to the generally accepted belief that in female mammals, all surviving germ cells enter meiosis at the end of fetal development and as a result, the postnatal ovary harbours a limited supply of oocytes that cannot be replenished or regenerated if lost to injury or disease. However, recent reports have challenged this view, suggesting instead that oocyte production is maintained through continual seeding of the ovary by circulating, bone-marrow-derived germ cells. To test directly the physiological relevance of circulating cells for female fertility, we established transplantation and parabiotic mouse models to assess the capacity of circulating bone marrow cells to generate ovulated oocytes, both in the steady state and after induced damage. Our studies showed no evidence that bone marrow cells, or any other normally circulating cells, contribute to the formation of mature, ovulated oocytes. Instead, cells that travelled to the ovary through the bloodstream exhibited properties characteristic of committed blood leukocytes.     

Characterization of murine cytolytic-helper hybrid T cell clones

L3T4, Lyt-2 and the T-cell receptor for antigen are cell-surface molecules involved in antigen specific T cell activation. We have constructed functional murine cytolytic-helper T-cell hybrid clones to study the link between expression of cell-surface molecules and specific cell function. Three of the clones express two antigen receptors and both Lyt-2 and L3T4, normally expressed on mutually exclusive subsets of mature T lymphocytes. The pattern of lymphokines produced by the hybrid cells in response to antigen was not controlled by the specific antigen receptor; both T-cell growth factor, produced only by the helper T-cell partner, and gamma-interferon, produced only by the cytolytic T-cell partner, were secreted when either antigen receptor was stimulated. However, cytolytic activity appeared to be restricted to the recognition of antigen by the T-cell receptor of the cytolytic partner. Thus cytolysis appears to be rightly linked to the antigen receptor of the cytolytic parent but lymphokine release is not tightly linked.     

Review: Actin-based dynamics during spermatogenesis and its significance

Actin can be found in all kinds of eukaryotic cells, maintaining their shapes and motilities, while its dynamics in sperm cells is understood less than their nonmuscle somatic cell counterparts. Spermatogenesis is a complicated process, resulting in the production of mature sperm from primordial germ cell. Significant structural and biochemical changes take place in the seminiferous epithelium of the adult testis during spermatogenesis. It was proved that all mammalian sperm contain actin, and that F-actin may play an important role during spermatogenesis, especially in nuclear shaping. Recently a new model for sperm head elongation based on the acrosome-acroplaxome-manchette complex has been proposed. In Drosophila, F-actin assembly is supposed to be very crucial during individualization. In this mini-review, we provide an overview of the structure, function, and regulation characteristics of actin cytoskeleton, and a summary of the current status of research of actin-based structure and movement is also provided, with emphasis on the role of actins in sperm head shaping during spermiogenesis and the cell junction dynamics in the testis. Research of the Sertoli ectoplasmic specialization is in the spotlight, which is a testis-specific actin-based junction very important for the movement of germ cells across the epithelium. Study of the molecular architecture and the regulating mechanism of the Sertoli ectoplasmic specialization has become an intriguing field. All this may lead to a new strategy for male infertility and, at the same time, a novel idea may result in devising much safer contraception with high efficiency. It is hoped that the advances listed in this review would give developmental and morphological researchers a favorable investigating outline and could help to enlarge the view of new strategies and models for actin dynamics during spermatogenesis.     

Generation of pluripotent stem cells from adult human testis

Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.     

Sperm from neonatal mammalian testes grafted in mice

Spermatogenesis is a productive and highly organized process that generates virtually unlimited numbers of sperm during adulthood. Continuous proliferation and differentiation of germ cells occur in a delicate balance with other testicular compartments, especially the supporting Sertoli cells. Many complex aspects of testis function in humans and large animals have remained elusive because of a lack of suitable in vitro or in vivo models. Germ cell transplantation has produced complete donor-derived spermatogenesis in rodents but not in other mammalian species. Production of sperm in grafted tissue from immature mammalian testes and across species has not yet been accomplished. Here we report the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs or goats into mouse hosts. This approach maintains structural integrity and provides the accessibility that is essential for studying and manipulating the function of testes and for preserving the male germ line. Our results indicate that this approach is applicable to diverse mammalian species.     

RT-PCR法检测大鼠组织中GLUT8基因表达特异性

采用半定量RT_PCR技术检测青春前期和成年大鼠的主要组织中GLUT8基因mRNA表达水平.实验结果显示,葡萄糖转运蛋白GLUT8基因在成年和青春前期大鼠的睾丸组织中都有高度表达,在心脏和肾脏组织中有少量表达,在肝、脾等组织中的表达是微量的;在成年大鼠睾丸组织中,Leydig细胞、睾丸生精细胞以及附睾的精子细胞中GLUT8基因都有大量的表达,其中以在Leydig细胞中的表达最丰富,而且GLUT8基因在睾丸生精细胞中的表达水平高于附睾的精子细胞.结果说明GLUT8蛋白主要分布在大鼠睾丸组织中,并且主要在Leydig细胞和精子细胞中参与葡萄糖转运和能量代谢与供应.     

Defective repair of alkylated DNA by human tumour and SV40-transformed human cell strains

We have identified a group of 8 (among 39) human tumour cell strains deficient in the ability to support the growth of adenovirus 5 preparations treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but able to support the growth of non-treated adenovirus normally. This deficient behaviour defines the Mer- phenotype. Strains having the Mer- phenotype were found to arise from tumours originating in four different organs. Relative to Mer+ strains, Mer- tumour strains showed greater sensitivity to MNNG-produced killing, greater MNNG-stimulated "DNA repair synthesis and a more rapid MNNG-produced decrease in semi-conservative DNA synthesis. Here we report that (1) Mer- strains are deficient in removing O6-methylguanine (O6-MeG) from their DNA after [Me-14C]MMNG treatment (Table 1); (2) Mer- tumour strains originate from tumours arising in patients having Mer+ normal fibroblasts (Fig. 1a, b); (3) SV40 transformation of (Mer+) human fibroblasts often converts them to Mer- strains (Fig. 1c, d); (4) MNNG produces more sister chromatid exchanges (SCEs) in Mer- than in Mer+ cell strains (Fig. 2).     

GDNF对睾丸功能的调节

在睾丸中,Sertoli’s细胞等细胞表达胶质细胞源神经营养因子（Glial cell line—derived neuotrophic factor,GDNF）,通过旁分泌或自分泌方式作用于生精细胞和Sertoli’s细胞等,调节精原干细胞（Spermatogonial stem cells,SSCs）的自我更新和分化,并促进Sertoli’s细胞的增殖以及血睾屏障的形成等。     

Both immature and mature T cells mobilize Ca2+ in response to antigen receptor crosslinking

T cells develop from prothymocytes which express no detectable antigen receptors to immature thymocytes with few receptors, eventually becoming mature thymocytes and peripheral T cells with 20,000-40,000 receptors per cell. Recent studies suggest that immature thymocytes are immunologically unresponsive. We have suggested that an early step in signal transduction following engagement of the T cell receptor might differ in immature and mature T cells. Here we examine anti-receptor antibody mediated induction of calcium mobilization in immature and mature T cells. Results indicate that antigen receptors on both immature and mature receptor-positive T cells transduce signals via calcium mobilization. Significant differences were observed, however, between these populations in the magnitude of influx of extracellular Ca2+ following binding of antireceptor antibody. Specifically immature cells show a much reduced Ca2+ influx response compared to mature cells which could result from a low Ca2+ channel frequency in the plasma membranes of immature T cells, or from less efficient activation of existing channels.