棕色田鼠睾丸和附睾雄激素受体表达的增龄变化

应用免疫组织化学方法研究了1、10、25、45及60日龄（成体）5个发育阶段的棕色田鼠（Lasiopodomys mandarinus）睾丸和附睾中雄激素受体（androgen receptor,AR）的表达。结果发现：①睾丸间质细胞中：1日龄有AR表达,至10日龄和25日龄AR表达减弱,45日龄AR表达最强,至成体AR表达又减弱（P＜0.05）;②肌样细胞中：从1日龄至成体均有AR表达,25日龄AR表达最弱,45日龄AR表达最强,至成体AR表达又减弱（P＜0.05）。③1日龄前精原细胞偶有AR表达,10日龄精原细胞没有AR表达;25日龄精子细胞有AR表达,45日龄精子细胞和部分精母细胞有AR表达,成体精原细胞和精母细胞及精子中有AR表达。④支持细胞中：性成熟前AR表达不明显,成体有AR表达。⑤从1日龄到成体,附睾中均有AR表达。这些结果表明,雄激素在棕色田鼠睾丸间质细胞、肌样细胞和生精细胞的表达随个体的发育阶段而变化;雄激素可促进青春期棕色田鼠间质细胞的功能与分化,肌样细胞在精子发生过程中有重要作用;同时,雄激素对附睾功能有调控作用。     

棕色田鼠睾丸和附睾胚后发育中的睾酮表达

应用免疫组织化学方法研究了产后1、10、25、45、60日龄(成体)5个发育阶段的棕色田鼠(Lasiopodomys mandarinus)睾丸和附睾组织内睾酮的免疫阳性反应.1日龄和10日龄,棕色田鼠睾丸生精小管内的前精原细胞胞质中有睾酮阳性表达.25日龄,有许多精子细胞产生,睾酮主要集中于精子细胞胞质表达.45日龄,精母细胞和精子中也有睾酮表达.成体精原细胞、精母细胞、精子细胞和精子中均有睾酮表达.1日龄至成体睾丸间质细胞和肌样细胞均有睾酮表达,25日龄时表达最强(P0.05).1日龄至成体附睾上皮细胞和连接组织有睾酮表达,成体附睾管内的大量精子有睾酮表达.这些结果说明,棕色田鼠从出生到性成熟过程中,在精子发生的各阶段,睾酮对生精细胞的分化增殖有直接的调控作用,这种调控作用随发育阶段不同具有可变性,同时,附睾的功能和精子的成熟也受到睾酮的调节.     

神经生长因子在不同周龄小鼠睾丸组织中的表达

目的研究神经生长因子在小鼠不同周龄睾丸组织中的定量和定位表达。方法分别剖取不同周龄雄性小鼠的睾丸组织,部分提取总RNA,real-time PCR相对定量分析神经生长因子mRNA的表达量;另外部分组织固定、包埋,进行SABC法免疫组化分析,以观察神经生长因子蛋白在各周睾丸组织中的定位。结果Real-timePCR定量分析表明:小鼠生后1周龄睾丸组织有神经生长因子mRNA的表达,生后3周龄表达量达峰值,5周之后随鼠龄的增加呈下降趋势,成年小鼠睾丸组织的神经生长因子mRNA表达维持在一定水平。免疫组化定位分析显示:睾丸组织的神经生长因子蛋白表达于小鼠出生后的各个时期内,1周龄睾丸组织免疫阳性反应主要位于支持细胞,精原细胞也有着色;3周龄睾丸组织的间质细胞、各级生精细胞、支持细胞、管周肌样细胞表达均呈现阳性;5周后的睾丸组织内神经生长因子呈低水平表达,主要表达于间质细胞和生精细胞内。结论神经生长因子mRNA的表达量随着小鼠睾丸的生长发育期存在着一定的规律性变化;神经生长因子蛋白的表达在小鼠睾丸生长发育的不同时期其主要表达部位不同。     

长吻鮠精巢发育的分期及精子的发生和形成

长吻鮠精巢的发育分为精原细胞增殖期、精母细胞生长期、精母细胞成熟期、精子细胞出现期,精子完全成熟期和精子退化吸收期。精巢的后1/3不产生也不贮存精子,精子的发生和形成经过精原细胞、精母细胞、精子细胞到精子的一系列过程。精原细胞有两种类型。精子无顶体,有中心粒帽,中片长,核凹窝和线粒体发达,鞭毛具侧鳍。     

长吻wei精巢发育的分期及精子的发生和形成

长吻wei精巢的发育分为精原细胞增殖期、精母细胞生长期、精母细胞成熟期、精子细胞出现期、精子完全成熟期和精子退化吸收期。精巢的后1/3不产生也不贮存精子,精子的发生和形成经过精原细胞、精母细胞、精子细胞到精子的一系列过程。精原细胞有两种类型。精子无顶体,有中心粒帽,中片长,核凹窝和线粒体发达,鞭毛具侧鳍。     

小鼠睾丸不同阶段生精上皮细胞的发育形态学研究

目的:研究小鼠生精上皮各类细胞发育过程中形体学特征以及异常发育的生精细胞形态方法:PAS染色正常成年小鼠睾丸,通过光学显微镜观察每种上皮细胞的形态学特征。结果:小鼠生精上皮分为Stage I-XII不同阶段,在精子细胞分化、分裂的同时呈现出动态变化,生精上皮中的精子细胞包括精原细胞、减数分裂间期细胞(L、Z、P、D)、次级精母细胞、圆形精子细胞、长形精子细胞。每种细胞都具有明显形态学特征。结论:根据生精上皮细胞的形态特征可以判断小鼠不同类型精子发育是否正常,对于利用小鼠动物模型来研究生殖系统功能的研究有重要意义。     

中华蟾蜍精子的发生和形成

中华蟾蜍精子的发生可以分为精原细胞期、初级精母细胞期、次级精母细胞期、精子细胞期和精子期5个时期,精子的形成过程包括核固缩、细胞质丢失、鞭毛形成等阶段.曲细精管中可以分出4种形态的精原细胞,其中有3种为增殖型精原细胞,另1种形态的精原细胞通过分裂形成1个精原干细胞和1个增殖型精原细胞,前者分裂以维持精原细胞的数量,后者发育分裂成精母细胞.Mallory三色法染色能清晰的区分精原干细胞和增殖型精原细胞.毕特氏器是异性生殖腺的残余,与精巢以被膜为界.性成熟个体的毕特氏器之卵母细胞圆形或椭圆形,多处于Ⅲ时相;滤泡细胞扁平或矮立方状;卵母细胞间富含微血管、间质细胞、嗜苦味酸细胞等.     

p38 MAPK在小鼠睾丸不同发育阶段的表达和定位

为探讨丝裂原活化蛋白激酶p38 MAPK在小鼠睾丸不同发育阶段的表达，应用蛋白质免疫印迹杂交技术和免疫组织化学SABC法检测1至7周龄小鼠睾丸p38 MAPK的表达、定位及发育变化，并通过图像分析技术对免疫组织化学结果进行统计学分析。免疫印迹杂交发现，p38 MAPK在2～7周龄小鼠睾丸中均有表达。免疫组织化学结果显示，在2周龄小鼠睾丸曲细精管上皮中即可观察到p38 MAPK免疫阳性反应，免疫反应阳性细胞为精原细胞；3、4、5周龄小鼠睾丸仅有个别曲细精管上皮可见p38 MAPK免疫阳性反应；6、7周龄小鼠睾丸中p38 MAPK表达较丰富，免疫反应阳性细胞为精原细胞和初级精母细胞，免疫阳性反应物均主要位于细胞核内。在7周龄小鼠睾丸中还可见到部分间质细胞的细胞质亦呈p38 MAPK阳性。这些结果提示，p38 MAPK可能对生精细胞的增殖分化具有调控作用。     

兔精子发生过程中cyclin B1基因表达和蛋白定位的发育阶段依赖性

利用原位杂交和免疫组化等方法,研究兔精子发生过程中生精细胞cyclin B1 mRNA的表达和蛋白定位特点,结果显示,兔生精上皮中Cyclin B1 mRNA的主要分布在初级精母细胞中,直至圆形精子细胞仍然存在,于精子细胞的变态过程中逐渐消失,在伸长的精子细胞和精子中未检测出cyclin B1 mRNA,Cyclin B1蛋白在进入分裂期的精原细胞和精母细胞中表达,在圆形精子细胞和伸长的精子细胞中呈现大量的cyclin B1蛋白,上述结果表明,在兔精子发生过程中,cyclin B1 mRNA表达和蛋白定位具有发育阶段依赖性的特征。     

中心体蛋白Cenexin在大鼠精子发生过程中的表达特征

中心体蛋白Cenexin是成熟中心粒的唯一标志分子。为阐明中心粒在大鼠精子发生过程中的成熟以及功能,我们首先通过RT-PCR技术从大鼠睾丸组织中扩增出了Cenexin cDNA片段,原核表达重组蛋白后,用其免疫小鼠制备了高滴度的抗Cenexin的多克隆抗体,然后利用免疫荧光染色、Western Blot和半定量RT-PCR方法,研究了大鼠精子发生过程中Cenexin蛋白和基因的表达特征。结果显示Cenexin mRNA水平在精原细胞和精母细胞中较高,随后表达水平下降,而蛋白质分子在精原细胞到精子细胞中都定位于细胞的一个中心粒上,表示有成熟中心粒的存在,在长形精子细胞中该蛋白位于鞭毛的基体部。附睾的绝大多数成熟精子中Cenexin免疫染色消失。中心体蛋白Cenexin在精子变态期的表达变化可能与精子鞭毛形成的起始有关。     

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.     

Immunohistochemical distribution of sulfhydryl oxidase in the human testis

Summary Sulfhydryl oxidase (SOx) is an enzyme that catalyzes the oxidation of sulfhydryl compounds. It is present in mitochondria of certain testicular cells at specific stages of functional activation. In the mature human testis moderate SOx immunoreactivity is found in Leydig cells, and lacking in Sertoli and in peritubular cells. The A_dark spermatogonia usually contain immuno-reactive mitochondria, while in A_pale spermatogonia immunoreactivity is mostly low. In stage V of spermatogenesis, A_pale spermatogonia were found containing immunoreactive material. Leptotene (stages IV and V) and zygotene (stage VI) primary spermatocytes display a moderate immunoreaction. It is strongest in pachytene spermatocytes of stages I–IV, decreases in stage V, and is low during diakinesis and in secondary spermatocytes. Late spermatids usually show a stronger immunoreactivity than early spermatids. At stage V of spermatogenesis the late spermatids contain only few immunoreactive particles. Spermatozoa are free of SOx-immunoreactive mitochondria. In residual bodies small amounts of SOx-immunoreactive particles are seen. Compared to rat and hamster testis, SOx immunoreactivity of the human testis is less clearly stage-dependent and it is not confined to certain germ cell stages. As deduced from the findings in patients with spermatogenic disorders, the SOx immunoreactivity of spermatogonia in human testis seems to be of diagnostic relevance.     

Testicular expression of small ubiquitin-related modifier-1 (SUMO-1) supports multiple roles in spermatogenesis: silencing of sex chromosomes in spermatocytes, spermatid microtubule nucleation, and nuclear reshaping

SUMO-1 is a member of a ubiquitin-related family of proteins that mediates important post-translational effects affecting diverse physiological functions. Whereas SUMO-1 is detected in the testis, little is known about its reproductive role in males. Herein, cell-specific SUMO-1 was localized in freshly isolated, purified male germ cells and somatic cells of mouse and rat testes using Western analysis, high-resolution single-cell bioimaging, and in situ confocal microscopy of seminiferous tubules. During germ cell development, SUMO-1 was observed at low but detectable levels in the cytoplasm of spermatogonia and early spermatocytes. SUMO-1 appeared on gonosomal chromatin during zygotene when chromosome homologues pair and sex chromatin condensation is initiated. Striking SUMO-1 increases in the sex body of early-to-mid-pachytene spermatocytes correlated with timing of additional sex chromosome condensation. Before the completion of the first meiotic division, SUMO-1 disappeared from the sex body when X and Y chromosomal activity resumed. Together, these data indicate that sumoylation may be involved in non-homologous chromosomal synapsis, meiotic sex chromosome inactivation, and XY body formation. During spermiogenesis, SUMO-1 localized in chromocenters of certain round spermatids and perinuclear ring and centrosomes of elongating spermatids, data implicating SUMO-1 in the process of microtubule nucleation and nuclear reshaping. STAT-4, one potential target of sumoylation, was located along the spermatid nuclei, adjacent but not co-localized with SUMO-1. Androgen receptor-positive Leydig, Sertoli, and some peritubular myoepithelial cells express SUMO-1, findings suggesting a role in modulating steroid action. Testicular SUMO-1 expression supports its specific functions in inactivation of sex chromosomes during meiosis, spermatid microtubule nucleation, nuclear reshaping, and gene expression.     

Rat synapsin 1 promoter mediated transgene expression in testicular cell types

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.     

Telomere length in male germ cells is inversely correlated with telomerase activity

Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.