细辛对D-半乳糖所致衰老小鼠睾丸的形态学研究

本文研究了细辛对D－半乳糖所致衰老小鼠的抗衰老作用，应用光镜电镜技术测定小鼠睾丸的重量、生精小管直径、生精上皮细胞数、随龄变化的间质细胞数，同时观察了细辛对上述指标的影响。结果 1．随增龄、睾丸重量减轻，生精小管直径缩小。衰老时租精细胞缺如，仅有支持细胞或散在分布，间质细胞随龄递减。2．细辛可以使小鼠的曲细精管增粗，生精过程活跃，生精细胞增多，间质细胞增多。结论 细辛有一定抗衰老作用。     

猕猴输精管内注射高分子聚合物HFMC后对睾丸组织结构的影响

本实验选用具有生育力成年雄性猕猴７只，在直视下行双侧ＨＦＭＣ输精管内注射，每侧剂量分别为３０ｍｇ１只，６０ｍｇ和１００ｍｇ各３只；于注射后２．５年和３．５年分别处死动物，取睾丸组织进行光镜和电镜观察．结果发现：猕猴注射ＨＦＭＣ２．５年后，睾丸光镜大部分曲细精管生精上皮结构完整，排列整齐。仅见局部少数管腔生精上皮层数减少，上皮细胞轻度水样变性等病理改变。电镜下曲细精管内除支持细胞内脂褐素增多，轻度基底膜增厚和精母细胞内质网扩张外，各级生精细胞，支持细胞及细胞间连接复合体等超微结构未见明显异常。注射ＨＦＭＣ３．５年后猕猴的光镜、电镜结果与注射后２．５年结果相似，但局部改变较２．５年组轻。上述结果表明：猕猴输精管内注射一定剂量ＨＦＭＣ节育不会引起睾丸组织的严重病理改变。但是，由于注射ＨＦＭＣ后，ＨＦＭＣ释放Ｈ＋及其对输精管的暂时阻塞，改变了精子生存的内环境，使睾丸出现局部轻度病理改变，随着ＨＦＭＣ逐渐溶解排出，睾丸功能相继恢复正常，配对产仔。为ＨＦＭＣ应用提供了安全性依据。     

精原干细胞移植后的体内迁移、增殖和分化

背景:精原干细胞移植对男性不育的治疗具有潜在的临床应用价值,但移植后干细胞体内迁移、增殖、分化的过程目前尚不完全清楚。目的:观测精原干细胞移植后的体内迁移、增殖和分化过程。方法:以出生后6～10d的雄性C57BL/6小鼠为供体,通过复合酶消化、差速贴壁结合非连续性Percoll密度梯度离心的方法获取精原干细胞;以出生后6周的雄性C57BL/6小鼠为受体,腹腔注射白消安,破坏其内源性生精功能。实验组采用曲细精管微注射法将供体精原干细胞移植入受体睾丸内,对移植后细胞进行PKH26-GL荧光追踪分析,观察其体内迁移过程,以Western Blot和RFQ-PCR法检测睾丸组织α6-Integrin,c-kit,SCF蛋白及mRNA的变化。以未接受化疗和细胞移植的正常同系生小鼠作为阳性对照组,以单侧睾丸曲细精管微注射移植细胞递质作为阴性对照组。结果与结论:PKH26-GL荧光追踪移植细胞,移植后1周部分精原干细胞已向曲细精管基底膜迁移,移植后1个月精原干细胞已从曲细精管管腔迁移至曲细精管基底膜,并分裂增殖,移植后3个月曲细精管管腔内可见大量精子细胞形成。移植后1,2,3个月,各组α6-Integrin,c-kit蛋白表达均呈增加趋势(P0.01),阴性对照组、实验组SCF蛋白表达有增加趋势(P0.05);各组α6-Integrin,c-kit,SCF mRNA的表达均有增加趋势(P0.05)。提示大剂量化疗后,生精上皮内仍存在一定数量的Sertoli细胞,这种精子发生的微环境并未完全破坏,外源性精原干细胞移植后能在受体Sertoli细胞所提供的微环境中增殖和分化。     

淋巴管阻断对大鼠睾丸生精上皮的影响

用外科手术的方法将正常成年ＳＤ大鼠双侧睾丸淋巴管阻断后，观察术后不同时间睾丸生精上皮的组织学变化。术后２天出现生精上皮的形态改变，生精细胞排列松散，部分精子细胞脱落，聚集在管腔部位。术后６天多数曲细精管的精子细胞全部脱落。假手术对照组，睾丸生精上皮具有正常的形态结构。     

海洛因对小鼠初级精母细胞影响的形态定量分析

将昆明小鼠随机分为低(0.025)、中(0.05)、高(0.1)剂量组及生理盐水对照组,以2号海洛因经腹腔注射染毒,20天后,取睾丸组织制成石蜡切片.HE染色,光镜下观察生精小管上皮细胞的形态变化,并应用图像分析系统对初级精母细胞的形态参数进行了分析;结果表明,(1)光镜观察 对照组及低剂量组小鼠睾丸组织未见明显的变化;中剂量组小鼠睾丸曲细精管上皮有不同程度的组织改变;高剂量组小鼠生精管壁细胞明显地出现上皮层次减少,精子细胞和精子减少,并发现曲细精管内有脱落的生精细胞;(2)形态定量分析显示 低剂量组及中剂量组各形态参数(中剂量组圆形度除外)较对照组无显著差异;高剂量组初级精母细胞核面积、核灰度与对照组相比无显著差异,细胞面积、细胞周长及浆面积较对照组明显增大(P<0.05).而核浆比例、圆形度较对照组有减小(P<0.05).结果提示 海洛因可明显改变小鼠生殖细胞的形态结构.     

超声波对小白鼠生精上皮的影响

用功率为５Ｗ／ｃｍ２、频率为１．１０ＭＨｚ的超声波照射小白鼠睾丸５分钟（实验组１）、１０分钟（实验组Ⅱ），分别于处理后２４小时，４８小时及７天时间切取睾丸组织，制作石蜡切片，在光镜下观察生精上皮的组织学变化并与对照组进行比较。结果显示：（１）小白鼠睾丸经超声波照射后，曲细精管萎缩，管径变小；生精上皮变薄，精子发生时相消失；生精细胞减少，没有精子形成。（２）超声波照射１０分钟对生精上皮的损伤比照射５分钟更为严重。（３）超声波照射后２４小时，生精上皮即受到破坏，精子细胞减少；照射后４８小时，上皮受损伤的程度增大；照射后７天时间，曲细精管的组织学结构开始恢复，但仍无精子形成。（４）超声波对生精上皮的影响主要限于精母细胞、精子细胞和精子，而精原细胞与支持细胞没有明显变化。上述结果表明，超声波能够抑制小白鼠的精子发生，该抑制作用可能可逆。     

牦牛犊牛睾丸结构特征及细胞外基质相关蛋白的分布

目的观察9头健康牦牛犊牛睾丸结构特征及细胞外基质相关蛋白的分布特点。方法采用组织化学染色和透射电镜技术,观察睾丸显微及超微结构特点,免疫组织化学SP法及IPP图像分析技术,研究层黏连蛋白(LN)、Ⅳ型胶原(ColⅣ)和硫酸乙酰肝素糖蛋白(HSPG)的分布特征。结果睾丸生精上皮由Sertoli细胞和生殖母细胞构成1~2层,未形成空腔。电镜下,Sertoli细胞异染色质丰富,相邻Sertoli细胞胞膜下分布有典型外质特化与肌动蛋白丝形成的胞膜下纤维束;生殖母细胞较大,胞质丰富。免疫组织化学显示,LN、ColⅣ和HSPG在生精小管基膜和肌样细胞表达较弱,LN在生殖母细胞内的表达量显著低于ColⅣ和HSPG(P0.05),而在Sertoli细胞及Leydig细胞均无表达;ColⅣ和HSPG在Sertoli细胞均为强阳性表达;HSPG在Leydig细胞的表达量显著高于ColⅣ(P0.05)。结论牦牛犊牛生精上皮以幼稚型Sertoli细胞为主,Leydig细胞处于胚胎型向成熟型过渡时期;细胞外基质(ECM)相关Ⅰ及Ⅳ型Col、LN及HSPG的分布适应于其在高原低氧环境中的发育。     

大鼠睾丸Sertoli细胞内微管分布的研究

本研究应用免疫荧光和透射电镜方法,对大鼠睾丸Setroli细胞内微管的分布进行了观察。结果表明,Serto|i细胞内微管主要分布于核上方胞质内,沿细胞长轴排列,并随Sertoli细胞主干及分支伸展于各生精细胞之间,直至上皮顶部。在生精上皮周期中,伴随上皮构筑的改变及Sertoli细胞的变形,微管分布呈现规律性变化。Ⅰ～Ⅴ期长形精子细胞周围微管密集,沿细胞长轴顺行。Ⅶ期时,富含微管Sertoli细胞突起包绕于精子细胞头部周围,并将其送入和悬吊子曲管腔内,部分微管伸展方向与精子细胞头部镰状外形一致。精子释放后(Ⅷ～Ⅸ期),Sertoli细胞突起及其微管回缩;在开始伸长变形的精子细胞周围微管再次聚集。本研究结果提示,Sertoli细胞内微管分布及其规律性变化与生精上皮构筑,Sertoli细胞外形变化及精子细胞的运动和释放密切相关。     

42 大鼠出生前后睾丸中两种中间丝蛋白的免疫组化观察

<正>本文旨在探讨细胞角蛋白和波蛋白在大鼠发育中睾丸内的表达,特别是在支持细胞内的表达.阴栓龄17～20天的Wistar胎鼠和生后1、5、10、15、20和40天的幼鼠购自中国医学科学院实验动物中心,生后1天的仔鼠及胎鼠取右侧睾丸于4%多聚甲醛中浸泡固定.生后5天以上的幼鼠先经心脏灌流固定,再取睾丸浸泡固定.石蜡包埋后组织块作5μm厚切片.PAP法或ABC法显示细胞角蛋白,Biotin-Streptoavidin法显示波蛋白.兔抗牛角蛋白多克隆抗体和小鼠抗猪波蛋白单克隆抗体均为DAKO公司产品.结果如下:(1)17天龄胎鼠睾丸可见两种中间纵蛋白的表达.角蛋白主要见于表面上皮,纵隔及其附近的睾丸索(testicular cords)内的上皮细胞以及其它睾丸索的支持细胞.在纵隔以外的睾丸索,角蛋白的反应弱,只见于支持细胞的核下区.波蛋白分布于白膜、问质及血管内皮.在睾丸索内则分布在支持细胞的核下区.(2)随年龄增长,睾丸索内支持细胞的角蛋白表达减弱.纵隔及其附近的睾丸索除外,它们发展成为直细精管和睾丸网.生后15天以后,睾丸索(曲细精管)支持细胞的角蛋白用PAP法已不能显示.(3)用ABC法,在出生后15天以上的曲细精管内确可见到少数角蛋白阳性细胞,大多数支持细胞的波蛋白表达增强.本结果提示,支持细胞可能有两种类型.随着发育,大多数支持细?     

磁水对家兔睾丸生精功能的影响

用磁水饲养家兔，经3和6个月的观察，实验组动物 精液量、精子数、精子存活率，附睾尾中精子数以及每100mg附睾尾组织的精子烽，曲细精管直径及曲细精管生精细胞数都比对照组显著增加；精子畸形率比对照组明显减少，光镜及扫描电镜观察显示实验组精母细胞、精子细胞、精子数量都比对照组显著增加，表明饮用磁水能促进生精功能。     

Immunohistochemical staining of IL-1 alpha and IL-1 receptor antagonist but not IL-1 beta in cultures of sertoli cells

The interleukin-1 (IL-1) system has been suggested to be involved in the cell cell cross talk within the testis. To identify a testicular cell source of IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (IL-1ra), immature mouse Sertoli cells were isolated, purified, cultured and examined for the cellular compartment localization of these cytokines by immunohistochemical staining. Our results show that both Germ cells and Sertoli cells in unpurified Sertoli cell cultures (before hypotonic shock) and purified culture of Sertoli cells (after hypotonic shock) were stained for IL-1 alpha. The levels of this cytokine were increased in Sertoli cells when the purified cultures were stimulated with lipopolysaccharide (LPS) (5 microg/mL). However, we could not identify a positive staining for IL-1 beta when Sertoli cell cultures were stained for this cytokine, even after stimulation with various concentrations of LPS (0.1-10 microg/mL). On the other hand, immunohistochemical staining of isolated Sertoli cells without treatment with hypotonic shock (cultures containing Sertoli cells and Germ cells) for IL-1ra showed constitutive positive staining of both cell types (Sertoli cells and Germ cells). Our results, using immunohistochemical staining, may indicate the different expression of IL-1 alpha, IL-1 beta and IL-1ra in Sertoli cells. These results may suggest the involvement of IL-1 system in the autocrine and paracrine regulation of testicular cell functions.     

Testis morphology in patients with idiopathic hypogonadotropic hypogonadism

BACKGROUND: Adult patients with idiopathic hypogonadotropic hypogonadism (IHH) typically present with absent puberty and therefore have prepubertal testes. IHH is recognized as one of the few curable causes of male infertility and is often effectively treated with either gonadotropins or pulsatile GnRH therapy. The objective of this study was to determine the structure of the testis prior to initiation of treatment. METHODS AND RESULTS: Eight adult IHH patients with prepubertal testes (<4 ml), with no previous gonadotropin therapy and with no history of cryptorchidism underwent open bilateral testicular biopsy prior to the initiation of hormonal treatment. The testes of all patients showed seminiferous cords separated by interstitium composed of blood vessels, connective tissue cells and collagen fibres but typical adult Leydig cells were absent. The cords contained only Sertoli cells and early type A spermatogonia. The spermatogonia mostly resided in the centre of the cords and were often large, typical of gonocytes. Sertoli cells appeared immature with ovoid nuclei devoid of infoldings and cytoplasm that lacked polarity. Tight junctional complexes commonly found connecting adult Sertoli cells were lacking. CONCLUSIONS: These results demonstrate that the immature testes from patients with the severe form of IHH possess early spermatogonia that could possibly reinitiate spermatogenesis with appropriate hormone stimulation. Therefore, the immature testis of this IHH subset resembles those of prepubertal boys and may provide important biologic and genetic insights into testicular development.     

The morphogenesis of cyclohexylamine-induced testicular atrophy in the rat: in vivo and in vitro studies

Male Wistar strain rats were fed a diet providing an intake of 0 or 400 mg cyclohexylamine (CHA)/kg body weight/day for 1, 3, 7, 9, or 13 weeks. At the end of the appropriate feeding period the rats were perfused-fixed with Karnovsky's fixative. The weights of the fixed testes were recorded and the testes, epididymides, and spermatic cord were sampled and processed into methacrylate resin. Histopathological examination of the testes showed changes after 3 weeks of CHA administration. The most frequent and consistent lesion consisted of a focal, basal vacuolation of the Sertoli cell cytoplasm associated with the local loss of spermatocytes and spermatogonia. After a 7-week administration, the Sertoli cell vacuolation was extensive, while the germ cell population showed mild to moderate degeneration and depletion. After longer periods of treatment the lesion was more severe and affected a greater number of tubules leading to general disruption of the germinal epithelium. Cocultures of Sertoli and germ cells were prepared from the testes of Wistar strain rats and exposed to (CHA) or its metabolite 4-aminocyclohexanol (4ACH) at concentrations ranging from 0.1 to 10 mM for periods of 24-72 hr. The cultures were fixed, stained, and examined by light microscopy. Cultures exposed to CHA or 4ACH showed morphological changes comparable with those seen in vivo. Sertoli cell vacuolation was the earliest change with progressive germ cell degeneration and exfoliation from the Sertoli cell monolayer. At equimolar concentrations, CHA produced more marked changes than 4ACH. These results suggest that CHA itself acts directly on the testis and that its primary cellular target is the Sertoli cell.     

The effect of dimensionality on growth and differentiation of neural progenitors from different regions of fetal rat brain in vitro: 3-dimensional spheroid versus 2-dimensional monolayer culture

Three-dimensional (3-D) spheroids are widely used for culturing cells. However, 2-dimensional (2-D) monolayer cultures have also been adopted for culture and used in a broad range of cell biology studies. To address the effect of dimensionality on the growth and differentiation of neuroprogenitor cells in 3-D spheroids and 2-D monolayer cultures, cells were isolated from cerebral cortex, cerebella and brainstem of fetal rat brain then cultured in serum-free DMEM/F12 medium or DMEM with 10% FBS. The growth and differentiation of neuroprogenitor cells from three brain regions in spheroids was compared with that in monolayer cultures, and the differentiation components of neuroprogenitor cells were compared with in vivo brain sections. Neuroprogenitor cells in spheroids proliferate actively over 10 days in culture as showed by Ki67 incorporation and increase in spheroid diameter. More neuroprogenitor cells underwent neuronal differentiation in spheroids than in monolayer cultures. In comparison with fixed rat brain sections, the neuron to astrocyte ratio, as shown by neurofilament to glial fibrillary acidic protein immunoreactivity, in spheroids is similar to that found in adult rat tissue sections. Our results suggest that the spheroid culture system mimics the in vivo cytoarchitecture to a greater extent and more closely reflects the cellular composition in adult brain tissue. This supports the notion that the intercellular niche in spheroids is more favorable for the survival and differentiation of neuronal precursors, while the cues in monolayer cultures may favor glial cell survival. It is therefore concluded that dimensionality plays a significant role in determining cellular behavior in vitro.     

人胎儿睾丸发生过程中表皮生长因子与细胞增殖核抗原的表达

目的　观察表皮生长因子 (EGF)与细胞增殖核抗原 (PCNA)在人胎儿睾丸发生过程中的表达特征 ,探讨两者在睾丸发生中的作用。　方法　用SP免疫细胞化学技术检测了人胎儿 12～ 32周睾丸间质细胞、支持细胞、生殖细胞EGF、PCNA阳性细胞表达率。　结果　EGF在胎儿 12周龄睾丸组织未见表达 ,16～ 32周龄睾丸间质细胞呈阳性表达 ,16～ 2 0周为表达高峰 ,随着胎龄的增长呈逐渐下降趋势 (P <0 0 1) ;PCNA在胎儿睾丸间质细胞、支持细胞和生殖细胞均有不同程度表达 ,16～ 2 0周为表达高峰 ,随着胎龄的增长呈逐渐下降趋势 (P <0 0 1)。　结论　EGF与PCNA在人胎儿睾丸发育过程中有不同程度的表达 ,表明EGF与PCNA在睾丸发育过程中起着一定的作用     

Primary epithelial cell cultures derived from canine prostate: Isolation,culture, and characterization

Epithelial-cell-enriched primary cultures were established from canine prostate. Minced tissue was dissociated with 750 units/ml of collagenase in F12K tissue culture medium containing 1% fetal bovine serum. This treatment resulted in the gradual removal of stromal elements from the base of the epithelial cells. After 30 minutes of digestion, aggregates of epithelial cells free of stroma were dislodged from the minced pieces of prostate. These aggregates were washed and plated at high density in F12K plus 10% fetal bovine serum. After 12–16 hours in vitro the unattached cellular aggregates were removed from the culture dishes, washed, and reinoculated into new culture vessels containing fresh medium. After 48 hours in vitro, the aggregates had attached to the culture vessels and spread out to yield discrete patches of epithelial cells. By 120 hours in vitro the patches of cells had grown and coalesced to form a confluent monolayer of epithelial cells. Ultrastructural examination of these cultures indicated that adjacent cells were joined by desmosomes and tight junctions and had tonofilaments and microvilli, giving the cells an epithelial appearance. The cells contained rough endoplasmic reticulum, Golgi apparatus, and secretory granules similar to those of the epithelial cells in the intact organ. In addition, intracellular “blebs” containing acid phosphatase were observed in the monolayers and were found to increase in size and number with time in vitro. Differentiated function of the cultures was demonstrated by the presence of ornithine decarboxylase and acid phosphatase and the ability of the cultures to metabolize testosterone to primarily 5α-reduced metabolites.     

大鼠Sertoli细胞与精原细胞的分离及共培养

目的 分离纯化14 d大鼠Sertoli细胞与精原细胞,用Sertoli细胞作为饲养层对大鼠精原细胞进行体外培养研究.方法 酶消化法制备大鼠睾丸组织单细胞悬液,采用牛血清白蛋白(BSA)不连续密度梯度沉降法分离Sertoli细胞和精原细胞.结果 经分离的Sertoli细胞与精原细胞的纯度分别达到92.73%和78.36%.Sertoli细胞与精原细胞共培养,可见精原细胞发生分裂增殖等行为.结论 在添加EGF、bFGF和GDNF等细胞因子的10%胎牛血清DMEM/F12培养基中,精原细胞能够存活一定时间;而Sertoli细胞表现为旺盛的生长状态,并可促进精原细胞的分裂与增殖.     

Characterization and distribution of hyaluronan and the proteoglycans decorin, biglycan and perlecan in the developing embryonic mouse gonad

The morphogenesis of tissues and organs requires dynamic changes in cells and in extracellular matrix components. It is known that various extracellular matrix molecules are of fundamental importance for gonad differentiation and growth. In the adult testis, the extracellular matrix represents an important component of the interstitium, participating in the transport of biologically active substances needed for the communication between different cellular components, as well as for the regulation of spermatogenesis and hormone production. The present study was designed in order to identify the proteoglycans biglycan, decorin and perlecan, as well as the glycosaminoglycan hyaluronan, during testis development in mouse embryos. Our data profile the chronology of testis differentiation, as well as the distribution of these extracellular matrix components during testis development in mice. We show that these extracellular matrix molecules are present early in the development of the gonads, suggesting that they play a role in gonad development. In addition, we found no decorin in the testicular cords. Furthermore, of the proteoglycans analysed, only biglycan was seen surrounding immature Sertoli cells and Leydig cell precursors in the testicular cords. This indicates that specific sets of extracellular matrix molecules are required in the various compartments of the developing gonad.     

Structural response of adult rat sertoli cells to peritubular fibroblasts in vitro

Sertoli cells were harvested from sexually mature rats and maintained in vitro for up to ten days (Sertoli cell-enriched cultures) or co-cultured with rat peritubular fibroblasts. Cultures were examined by differential light microscopy and by scanning or transmission electron microscopy. The presence of peritubular fibroblasts in co-culture greatly enhanced plating efficiency and viability of adult Sertoli cells. Sertoli cell aggregates preferentially adhered to peritubular cells, and by ten days had flattened and spread across these cells. Sertoli cells retained their characteristic ultrastructural features. Early in co-culture a collagen-like extracellular material was seen bridging Sertoli and peritubular cells, and its appearance was coincident with the presence of swollen rough endoplasmic reticulum in peritubular cells. Interperitubular cell spaces became engorged with a fibrillar material morphologically similar to the basal lamina of the seminiferous tubule wall. In the absence of peritubular cells, in culture medium “conditioned” by prior incubation with peritubular cells but not containing them, or when cultured with other fibroblastic cells, plating efficiency was low; Sertoli cells never flattened and cell ultrastructure progressively degenerated. The results indicate that peritubular cells support adult Sertoh cells in culture, possibly via extracellular material derived from peritubular cells, and suggest that Sertoli cells have an inductive effect on peritubular cell secretory activity.     

Morphological development of testes in ostrich (Struthio camelus) embryo

Although the histological structure of ostrich testis has been studied, very little information is currently available on the embryonic development of this organ. The aim of this study was to determine the sequence of the histological changes in diverse components of the testis in ostrich embryo from embryonic day (E) 20 to E42. The main findings were categorized into four histological features, i.e., development of sex cords, interstitial tissue and rete ducts, and the appearance of defective septa. While the lumen of sex cords, tunica albuginea, capsular rete ducts and Leydig cell precursors appeared at E26, the filum-shaped defective septa were visible at E36. The emersion of the lumen in the primary sex cords and formation of capsular rete ducts in the ostrich embryo is considerably different from that in other birds. However, tunica albuginea and Leydig cell precursors appeared in a similar pattern to those of other birds. An interesting observation was that the primordial germ cell (PGC)-like cells were completely distinct, while the capsular rete ducts were formed by trapping of some Sertoli cell aggregations in the tunica albuginea. This suggests that similar to the primary sex cords, the capsular rete ducts may originate from the Sertoli cell aggregations which had corralled some PGCs. Stereological estimations in the ostrich embryo testis showed the major proportion of testis is occupied by the seminiferous tubules, which is unlike the fowl embryo testis.