人胎睾丸发生过程中VEGF的表达研究

目的:研究血管内皮生长因子(VEGF)在人胚胎睾丸组织发生过程中的表达特征,探讨其在睾丸发生中的作用。方法:采用HE染色观察睾丸发育,SP免疫组织化学法检测VEGF在不同胎龄睾丸组织中的表达变化。结果:人胚胎睾丸发育正常;VEGF在胎儿睾丸间质细胞、生殖细胞中均有不同程度表达,间质细胞以12～24周为表达高峰,生殖细胞以16～24周为表达高峰,24周后随着胎龄的增长均呈下降趋势(P<0.01)。结论:VEGF在人胎睾丸发生、发育过程中有不同程度的表达,表明其在睾丸发生过程中起着一定的作用。     

胎儿睾丸发生过程中转化生长因子β1与Smad4的表达

目的:研究转化生长因子β1(TGF-β1)与Smad4在人胚胎睾丸组织发生过程中的表达特征,探讨其在睾丸发生中的作用.方法:采用H-E染色和SP免疫组织化学检测TGF-β1与Smad4在不同胎龄睾丸组织中的表达变化.结果:TGF-β1与Smad4在胎儿睾丸间质细胞、生殖细胞中均有不同程度表达,TGF-β1在胎儿睾丸中以12～20周为表达高峰,Smad4在胎儿睾丸以12～24周为表达高峰,24周后随着胎龄的增长均呈下降趋势.结论:TGF-β1与Smad4在人胎睾丸发生、发育过程中有不同程度的表达,表明其在睾丸发生过程中起着一定的作用.     

胎儿睾丸组织发育过程中c-fos的表达

目的:研究原癌基因c-fos在胎儿睾丸组织发育过程中的表达变化。方法:H-E染色、免疫组化和图像分析。结果:c-fos在胎儿睾丸组织间质细胞(Leydig cell,LC)、生精细胞、管周肌样细胞均呈阳性反应。支持细胞未见阳性表达。c-fos在12周胎儿睾丸组织中已有阳性表达,16周可明显区分阳性间质细胞与生精细胞,24周达到高峰,此后逐渐减弱,以后间质细胞几乎不可见。结论:c-fos在胎儿睾丸的组织发育中有不同的表达,表明c-fos在睾丸发育过程中可能起着重要的作用。     

人胎儿晶状体上皮细胞增殖、凋亡及相关因子的研究

本实验应用TUNEL和免疫组化法，选用人正常胎儿晶状体，分为9，10，12，16，20，24，28，32周，每组6例，观察了晶状体发育过程中，晶状体上皮细胞凋亡率以及PCNA、EGF、TGF-β1的变化，以期为基础医学和临床医学对晶状体研究提供理论依据和形态学资料。PCNA、EGF、TGF-β1在人胎儿不同胎龄晶状体上皮细胞均有表达，     

咖啡因对胎鼠及乳鼠睾丸生殖细胞数量和PCNA表达的影响

为了深入了解咖啡因(Caffeine,Caf.)对胎儿、新生儿生殖细胞数量及其PCNA(增殖细胞核抗原)表达的影响,本实验采用低(0.3mM)、中(0.6mM)、高(1.2mM)浓度Caf.体外培养SD孕18天胎鼠、0天及4天乳鼠睾丸组织块,培养时间分别为1周、2周、3周,观察Caf.对睾丸内生殖细胞数量及其表达PCNA的影响.结果如下:(1)18天胎鼠睾丸培养组织内生殖细胞数量受Caf.影响最小,0天乳鼠次之,4天乳鼠受影响较大.(2)低浓度的Caf.对生殖细胞数量影响较少;中等浓度的Caf.在培养三周后,生殖细胞的数量才减少;而高浓度的Caf.在培养二周后,生殖细胞数量已有下降,三周更明显.(3)Caf.对生殖细胞PCNA表达阳性率无明显影响,但PCNA含量在大多数实验组都有降低.由此可知高浓度Caf.长时间培养后使生殖细胞数量减少,降低了生殖细胞PCNA的含量.     

人胎睾丸的组织发生

用7～38周人胎35例,取睾丸固定于 Carnoy 液,石蜡包埋,以 HE、PAS 反应及甲绿哌喏宁法染色。7周初的性腺尚未分化,可见散在的原始生殖细胞。13周睾丸特征已明显,白膜厚,睾丸素清楚,索间有密集的嗜酸性间质细胞。14.5周部分睾丸索出现小腔,而成管状。睾丸索包含大而着色淡的原始生殖细胞及色深的原始支持细胞。胎早期原始生殖细胞含有丰富的糖原颗粒。睾丸门、白膜及表面上皮内均见散在或成群的生殖细胞。睾丸间质细胞分幼稚型、成熟型及退化型。13～15周以幼稚型为主;16～18周以成熟型为主,成熟型的胞质内含有 RNA 颗粒;20周后退化型增多,成熟型减少。38周时,睾丸间质细胞单个或成行存在,数量大为减少。     

中脑红核突触素的表达与突触发育的关系

目的:为探讨突触素在不同周龄阶段胎儿中脑红核的表达及其与红核突触发育的关系.方法:收集因故终止妊娠的胎儿32例,胎龄为16～39周,按周龄大小分为5组.利用免疫组织化学方法观察突触素在不同周龄阶段胎儿红核的表达情况,应用计算机图像分析技术测量突触素在不同周龄阶段胎儿红核表达的平均光密度.同时中脑取材,常规透射电镜技术处...     

突触素在中脑动眼神经核发育中的表达及意义

目的:观察突触素(synaptophysin,SYN)在不同周龄阶段胎儿中脑动眼神经核的表达情况,探讨SYN表达与动眼神经核发育之间的关系。方法:收集因故终止妊娠的胎儿32例,胎龄为16~39周,按周龄大小分为5组。利用免疫组织化学方法观察SYN在不同周龄阶段胎儿中脑动眼神经核的表达情况,通过图像分析技术测量SYN在动眼神经核表达的平均光密度值。结果:16~39周各组动眼神经核区域均可见SYN免疫反应产物表达,SYN表达量在16~24周增加迅速,25~29周增加减慢,30~39周又迅速增加。SYN表达量随周龄的增大而增强,各组间差异性明显(P<0.05)。SYN免疫反应产物于16~24周间在神经元胞体和周围突起部位均有表达,24~39周主要分布在神经元突起周围,神经元胞体表达不明显。SYN免疫反应产物呈棕黄色的点状或颗粒状,随着周龄的增大反应产物的量和颜色均增加。结论:SYN在动眼神经核表达的情况可以反映胎儿中脑神经元的发育程度,SYN的表达与动眼神经核的发育是一致的。SYN在动眼神经核发育过程中表达变化可能与中脑发育的规律有关。     

Oct-4和人端粒酶逆转录酶在不同胎龄人胚原始生殖细胞的表达变化

目的 检测不同胎龄人胚原始生殖细胞中Oct-4、人端粒酶逆转录酶(hTERT)的表达变化.方法 取人胚胎生殖嵴,通过RT-PCR检测5～13周龄人胚生殖嵴中Oct-4、hTERT的表达变化;同时,取人胚胎生殖嵴,经石蜡切片,HE染色、免疫组织化学染色等技术,观察不同胎龄人胚原始生殖细胞的形态,检测Oct-4、hTERT的表达情况.结果 胎龄6～7周时,生殖嵴中可见少量原始生殖细胞,且表达Oct-4及hTERT;胎龄8～12周时,生殖嵴中原始生殖细胞数量增多,Oct-4及hTERT表达增强(P<0.05);胎龄13周以后,生殖嵴中Oct-4阳性的原始生殖细胞数量逐渐减少,Oct-4表达减弱,但hTERT仍然高表达.结论 不同胎龄人胚原始生殖细胞中Oct-4的表达呈动态变化,胎龄8～12周时表达较强,但hTERT的表达量始终维持在较高水平,没有明显变化.     

大鼠睾丸支持细胞发育的形态学研究

对７０例不同胎龄及生后不同发育阶段Ｗｉｓｔａｒ大鼠睾丸支持细胞的发育进行了光镜、电镜研究。结果：１．依形态学特点，将支持细胞的发育过程初步分为４期：分化前期（胚胎１１～１５ｄ）、分化期（胚胎１５ｄ～生后１至２周）、成熟前期（生后２周～青春期前）、成熟期（青春期后）。２．胚胎及新生时，在支持细胞间见到两侧胞膜紧密相贴及不典型的桥粒连接等；自生后４周，见到紧密连接。３．自胚胎１７ｄ至成熟以后，在支持细胞与其周围的生殖细胞间见到大量类似桥粒和类似中间连接等不典型的细胞连接。４．胚胎１９ｄ及新生期，睾丸索内除支持细胞及生殖细胞外，尚可见一种特殊的细胞。     

小白鼠睾丸发育分化的组织学及组织化学观察

本文报道用组织学和组织化学方法观察小鼠睾丸胚胎天时睾丸即已有SDH活性,生后以间质细胞和精母细胞反应最强。4.AlP反应在胚胎时以生殖母细胞较强,生后以界膜反应较强。5.支持细胞、间质细胞AcP活性较强。6.生殖细胞和支持细胞5′-Nase呈阳性,间质反应极微。7.胚胎时,睾丸各种细胞ATPase反应均为阴性。生后界膜、支持细胞和生殖细胞渐出现活性且不断增强。8.胚胎14天睾丸内NSE即有微弱的反应,生后主要以间质细胞反应强烈。至生后的发育。结果表明:1.核糖核酸以代谢旺盛的生殖母细胞最为丰富,支持细胞也较多,精子细胞较少。2.胚胎14天时睾丸各种细胞均有丰富的糖原颗粒,16天起仅分布于间质,性索内糖原阴性。3.胚胎14     

Human fetal testis: source of estrogen and target of estrogen action

BACKGROUND: Estrogens are involved in masculine fertility and spermatogenesis. However, little is known about estrogen involvement in human testicular organogenesis. Therefore the aim of this study was to investigate the cellular sources and targets of estrogens and their variations in the human testis during fetal development. Expression profiles of aromatase (CYP19) and estrogen receptors (ER) alpha and beta were analysed in human fetal testes at various gestational stages by immunohistochemistry and quantitative RT-PCR. METHODS: Fifty-four archival paraffin-embedded and four frozen fetal testes were studied by immunohistochemistry and real-time PCR. Tissue quality was confirmed by histology and expression of specific functional markers: androgenic enzymes for Leydig cells, anti-Müllerian hormone for Sertoli cells and Steel factor receptor for germ cells. RESULTS: We demonstrate that the human fetal testes express aromatase and ERbeta simultaneously in Sertoli, Leydig and germ cells but are devoid of ERalpha. Quantification of positive cells indicates a window of protein expression, especially between 13 and 22-24 weeks. Quantitative RT-PCR confirmed that the human fetal testis expresses CYP19 and ERbeta but not ERalpha mRNA. CONCLUSIONS: Our findings suggest that locally produced estrogens influence human testicular development through autocrine and paracrine mechanisms, most notably during the period of maximal testicular susceptibility to endocrine disruptors.     

Origin and evolution of somatic cell testicular tumours in transgenic mice

Transgenic mice bearing a construct in which the expression of the SV40 oncogene is directed by the AMH promoter (AT mice) develop testicular tumours in adult life. We aimed to study early steps of tumour development and characterize tumours at different ages by histological, morphometric, and immunohistochemical techniques. One‐ to 3‐month‐old AT mice depicted multifocal Leydig cell hyperplasia. The testicular volume occupied by interstitial tissue was significantly higher in 3‐month‐old AT mice in comparison with littermate controls. Between 5 1/2 and 7 months, microscopic interstitial tumours developed that progressively evolved to form large confluent areas of high mitotic index in 7‐ to 14‐month‐old AT mice. Tumour cells had the characteristics and histoarchitecture of Leydig cells, or formed solid cord‐like structures reminiscent of those seen in Sertoli cell tumours. Hyperplastic areas and tumours diffusely expressed 3β‐hydroxysteroid dehydrogenase (3β‐HSD) in Leydig cell areas. AMH expression was negative in Leydig cell conglomerates and tumours and variable in cord‐like tumours. The SV40 T antigen and markers of cell proliferation (PCNA) were intensely positive in hyperplastic cells and tumours. Control mice of similar ages showed neither hyperplasia nor tumours, and SV40 T expression was always negative. In conclusion, transgenic mice develop large testicular tumours that are preceded by interstitial hyperplasia and microtumours. The histological and immunohistochemical phenotype of tumours (Leydig and Sertoli cell differentiation, positive 3β‐HSD, and variable AMH) suggests a mixed differentiation of somatic cells of the specialized gonadal stroma. The finding that an oncogene directed by a promoter specifically active in fetal Sertoli cells has given rise to testicular tumours of mixed differentiation is compatible with a common origin of Leydig and Sertoli cells from the specific stroma of the gonadal ridge, as supported by double labelling experiments in fetal mice showing co‐localization of the transgene with Sertoli and Leydig cell markers. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Accelerated germ cell apoptosis in sex chromosome aneuploid fetal human gonads

The purpose of the study was to examine the occurrence of programmed cell death (apoptosis) in normal and chromosomally aneuploid testis and ovaries during the second trimester of human development. Such information may be useful in understanding normal and abnormal germ cell development and disorders associated with infertility in adult life. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) analysis in human fetal ovaries (n = 16) and testis (n = 14) between 9 and 23 weeks of development, in ovaries of four Turner's syndrome fetuses (45X) and in the gonad of an XO/XY fetus. In normal fetal testis, a small proportion of germ cells, Sertoli cells and Leydig cells undergo apoptosis. In normal fetal ovaries, some developing oocytes and granulosa cells were detected as TUNEL positive. Semiquantitative analysis of fetal ovaries revealed that approximately 3-7% of oocytes were apoptotic. In abnormal fetal testis (XO/XY genotype). TUNEL analysis revealed that only germ cells not enclosed in seminiferous tubules undergo apoptosis. TUNEL analysis of the Turner's syndrome (45X) ovaries studied at 15 and 20 weeks of development revealed massive apoptosis of the oocytes. Nearly 50-70% of the oocytes were TUNEL positive in these ovaries. These results suggest that germ cell apoptosis is a common event occurring during development of human gonads. Chromosomal defects by some means accelerates apoptosis that probably leads to gonadal dysgenesis later in life.     

Developmental regulation of connexin 43 expression in fetal mouse testicular cells

Multiple connexins have been identified in testicular cells. Several lines of evidences indicate that, among them, connexin 43 (Cx43) may be unique for control of gonad development and spermatogenesis. To date, however, it is not known whether Cx43 is expressed in the fetal testis and what possible types of cellular interactions mediated by this connexin are critical to male fertility. In the present work, expression of Cx43 was investigated at various developmental ages in cryosections from mouse testis by using specific antibodies against Cx43. In serial or double-labeled sections, Cx43 localization was compared with immunocytochemical distribution of steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3betaHSD), Mullerian inhibitory hormone (MIH), and germinal nuclear cell antigen (GCNA1), which are specific markers, respectively, of interstitial Leydig, Sertoli, and germinal cells. Sections were analyzed by fluorescence microscopy. We found that Cx43 immunofluorescence (IF) was uniformly distributed in the undifferentiated gonad at 11.5 days post coitus (dpc) and in cells of the mesonephric tubules. In the undifferentiated gonad, Cx43 was localized between primordial germ cells and somatic cells. At 12.5 dpc, when the gonad has undergone sexual differentiation, in the interstitium Cx43 was localized in Leydig cells and in the seminiferous cord it was localized between adjacent Sertoli cells. In Leydig and Sertoli cells, Cx43 labeling increased at 14.5, 16.5, and 18.5 dpc. From day 12.5 up to 18.5 dpc, Cx43 was also localized in cell borders between germinal and Sertoli cells. In conclusion, this study demonstrates that from the earliest stages of gonadal development, Cx43 is expressed in the principal cell types that participate in the control of male fertility. It also shows that Cx43 expression in Leydig and Sertoli cells increase during fetal life. Finally, it provides evidence that, throughout embryonic life, Cx43 forms gap junctions between Sertoli and germinal cells.     

Human 'testicular dysgenesis syndrome': a possible model using in-utero exposure of the rat to dibutyl phthalate

BACKGROUND: The disorders comprising human 'testicular dysgenesis syndrome' (TDS) may be increasing in incidence. TDS originates in fetal life but the mechanisms are not known, and discerning them requires an animal model. METHODS AND RESULTS: The study investigated whether male rats exposed in utero to dibutyl phthalate [DBP; 500 mg/kg on gestational days (GD) 13-21] would provide a suitable model for human TDS. DBP induced a high rate (>60%) of cryptorchidism (mainly unilateral), hypospadias, infertility and testis abnormalities, similar to those in human TDS. Cell-specific immunohistochemistry and confocal microscopy were used to track development of Sertoli [anti-Müllerian hormone (AMH), Wilm's tumour (WT-1) protein, p27(kip)], Leydig [3beta-hydroxysteroid dehydrogenase (3beta-HSD)], germ (DAZL protein) and peritubular myoid (smooth muscle actin) cells from fetal life to adulthood. In scrotal and cryptorchid testes of DBP-exposed males, areas of focal dysgenesis were found that contained Sertoli and Leydig cells, and gonocytes and partially formed testicular cords; these dysgenetic areas were associated with Leydig cell hyperplasia at all ages. Suppression ( approximately 90%) of testicular testosterone levels on GD 19 in DBP-exposed males, coincident with delayed peritubular myoid cell differentiation, may have contributed to the dysgenesis. Double immunohistochemistry using WT-1 (expressed in all Sertoli cells) and p27(kip) (expressed only in mature Sertoli cells) revealed immature Sertoli cells in dysgenetic areas. DBP-exposed animals also exhibited Sertoli cell-only (SCO) tubules, sporadically in scrotal and predominantly in cryptorchid, testes, or foci of SCO within normal tubules in scrotal testes. In all SCO areas the Sertoli cells were immature. Intratubular Leydig cells were evident in DBP-exposed animals and, where these occurred, Sertoli cells were immature and spermatogenesis was absent. Abnormal Sertoli cell-gonocyte interaction was evident at GD 19 in DBP-exposed rats coincident with appearance of multinucleated gonocytes, although these disappeared by postnatal day 10 during widespread loss of germ cells. CONCLUSIONS: Abnormal development of Sertoli cells, leading to abnormalities in other cell types, is our hypothesized explanation for the abnormal changes in DBP-exposed animals. As the testicular and other changes in DBP-exposed rats have all been reported in human TDS, DBP exposure in utero may provide a useful model for defining the cellular pathways in TDS.     

胎儿巩膜碱性成纤维细胞生长因子和转化生长因子β表达的研究

目的 观察不同胎龄胎儿眼球巩膜碱性成纤维细胞生长因子(bFGF)和转化生长因子β(TGF-β)的表达情况,了解bFGF和TGF-β在正常眼球发育过程中的作用及变化规律.方法 收集12～20周米非司酮配伍米索前列醇或利凡诺羊膜腔注射引产的胎儿20例,其40只眼,立即摘除眼球,取4～5mm范围巩膜,用实时定量PCR方法检测巩膜bFGF和TGF-β的表达情况.结果 各胎龄阶段巩膜bFGF均有表达,胎龄12～14周时表达最高.以后逐渐下降并保持稳定水平,各胎龄阶段巩膜均有TGF-β表达,胎龄12周时表达较低,以后逐渐升高并保持稳定水平.结论 各胎龄阶段胎儿巩膜均有bFGF和TGF-β的表达,bFGF和TGF-β的表达水平随着胎龄的变化而变化.     

Different Localization of ATP Sensitive K+ Channel Subunits in Rat Testis

ATP sensitive K⁺ (K_ATP) channels are important linkage of cell membrane excitability to its cellular bioenergetic state. These channels are composed of pore‐forming subunits and regulatory subunits. The present study focused on the cellular expressions and localizations of these subunits in rat testis. RT‐PCR analysis showed that rat testis contained five K_ATP channel subunits, Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B. Immunoblot assay showed that proteins of Kir6.1, Kir6.2, SUR2A and SUR2B were expressed in rat testis. Immunohistochemistry revealed these K_ATP channel subunits were positive in different localizations of spermatogenic cells, Sertoli cells and Leydig cells, which implies these subunits playing important roles in spermatogenesis. Co‐localization of Kir6.2 with SUR2B was determined in acrosome or head cap of spermatids by double immunofluorescence analysis by indicating K_ATP channel might be formed by Kir6.2 and SUR2B in acrosome of spermatids. Different localizations of the K_ATP channel subunits in the cell membrane and membranous organelles of spermatogenic cells and Sertoli cells indicated the complex and multiple functions of K_ATP channels in rat testis. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Leydig cells of the human testis possess astrocyte and oligodendrocyte marker molecules

It has been established, that Leydig cells of the human testis possess neuroendocrine properties and are therefore a member of the diffuse neuroendocrine (paraneuron) system. In the present study, we examined whether Leydig cells of adult (51-86 year of age) and developing (between the 15th and 36th week of gestation) human testes are immunopositive for glial cell-specific antigens such as glial fibrillary acidic protein (GFAP), galactocerebroside (GalC), cyclic 2',3'-nucleotide-3'-phosphodiesterase (CNPase), A2B5-antigen (A2B5) and O4-antigen (O4). With the use of Western blots and dot blot analyses, respectively, GFAP, CNPase, GalC, A2B5 and O4 were found in whole testes and Leydig cell protein extracts of adult men. Corresponding immunohistochemical studies revealed presence of these antigens in the cytoplasm of Leydig cells both of adult testes and testes during prenatal development. Some differences in staining intensity of single antigens were observed probably depending on the functional and/or developmental stage of the single cells. In addition, GFAP-, GalC- and CNPase-immunopositivity was found in numerous Sertoli cells of the seminiferous tubules. Moreover, some connective tissue cells (compartmentalizing cells or Co-cells) of the intertubular space showed immunopositivity for CNPase, A2B5 and GalC. The results obtained show that Leydig cells of the human testis, in addition to their endocrine, neuronal and neuroendocrine features, possess qualities of both astrocytes and oligodendrocytes and thus show qualities of multipotential cells. Leydig cells probably differentiate to a phenotype that is characteristic for cells in the developing nervous system. Furthermore, the established immunohistochemical similarities are consistent with the assumption that foetal and postnatal Leydig cells are of common origin.