双酚A对小鼠附睾和性腺肥大细胞数量和分布的影响

将18只8周龄雄性昆明小鼠随机分为3组,适应饲养1周后分别腹腔注射50μL、每千克体重含0(对照组)、10(试验1组)和100 mg(试验2组)双酚A的乙醇,每天1次,连续2周。处理结束,取附睾和性腺制备组织切片,通过改良甲苯胺蓝染色法测定附睾和性腺组织肥大细胞数量与分布的变化。结果:在附睾头被膜,试验2组增加到45.37个/mm2,在附睾头间质,试验1、2组每1 mm2分别增加到36.74、38.16个,显著高于对照组(P<0.05);在附睾尾被膜,试验2组增加到50.24个/mm2,在附睾尾间质,100 mg/kg组每1 mm2增加到43.22个(P<0.01);在精囊腺被膜,试验2组增加到56.25个/mm2,在精囊腺间质,对照组肥大细胞数为43.35个,试验1、2组每1 mm2分别增加到54.43、58.65个(P<0.01);在前列腺被膜,试验组与对照组相比,均无明显变化;在前列腺间质,试验2组每1 mm2增加到66.15个(P<0.01)。结果表明,双酚A暴露影响小鼠附睾和性腺肥大细胞数量和分布,可能对附睾中生精细胞的进一步发育成熟也有影响。     

实验性乳腺炎小鼠乳腺肥大细胞的变化

应用改良甲苯胺蓝染色法和苏木精-伊红染色法对金黄色葡萄球菌诱发实验性乳腺炎小鼠乳腺肥大细胞的数量、分布、活性进行了研究。正常对照组小鼠乳腺组织中肥大细胞主要分布于乳腺小叶之间的结缔组织中,有些围绕血管或乳腺导管分布,腺上皮之间没有肥大细胞分布;阳性试验组小鼠乳腺肥大细胞主要分布在相邻腺泡周围或腺导管上皮周围,胞质内充满异染颗粒,脱颗粒状态显著,甚至表现有颗粒耗竭现象,腺泡中有一定程度的肥大细胞浸润。肥大细胞计数结果显示,阳性试验组母鼠乳腺内的肥大细胞数急剧增多,显著高于正常对照组(P<0.05),而脱颗粒肥大细胞数阳性试验组与正常对照组相比极显著增多(P<0.01)。结论:肥大细胞在小鼠乳腺炎的致病机制中发挥重要作用,参与了乳腺炎的发病过程。     

奶牛乳腺肥大细胞免疫形态学的增龄性变化

应用改良甲苯胺蓝染色法研究不同年龄奶牛乳腺肥大细胞分布特点的增龄性变化，并用Alcian bluc-safranin染色法对肥大细胞进行细胞化学分型研究。结果：青年奶牛乳腺中肥大细胞数量较少，中年奶牛乳腺中肥大细胞的数量增多，有趣的是，老年奶牛乳腺肥大的数量更多，AB-S染色显示，各年龄奶牛乳腺中只有黏膜型肥大细胞。结果表明：奶牛乳腺肥大细胞数量具有随年龄增长而增加的特点，但肥大细胞的类型没有发生改变。     

大鼠乳腺局部免疫的调节机制--泌乳期与静止期乳腺肥大细胞分布的比较

应用改良甲苯胺蓝染色法(MTB)观察了大鼠泌乳期和静止期乳腺肥大细胞的分布、形态、数量变化规律．并用阿尔新蓝一番红鉴别染色法(AB-S)进行了细胞化学分型研究。结果：AB-S染色显示，大鼠乳腺只存在黏膜型肥大细胞；无论是泌乳期还是静止期。乳腺肥大细胞大多分布于腺泡间和小叶问结缔组织中。细胞的形态各异，但细胞数量在静止期和泌乳期有显著差异。泌乳期的乳腺肥大细胞数量明显减少(P＜0．01)。乳腺肥大细胞的动态变化，可能与乳腺泌乳期腺泡上皮的生长和静止期腺泡问结缔组织细胞增生等结构变化有关。     

炔雌醚对小鼠附睾与副性腺肥大细胞数量和分布的影响

采用组织学和组织化学方法探讨炔雌醚暴露对小鼠附睾和副性腺肥大细胞数量及分布的影响。将15只8周龄雄性昆明小鼠随机分为3组,适应饲养1周后分别腹腔注射50μL含0、0.1、1mg/kg体重炔雌醚的橄榄油,每天1次,连续2周。处理结束,取附睾和副性腺并制备组织切片,通过甲苯胺蓝染色测定附睾和副性腺组织肥大细胞数量与分布的变化。结果显示,在附睾头被膜,0.1mg/kg和1mg/kg组分别增加到47.25个/mm2和59.38个/mm2(P0.01),在附睾头间质,0.1 mg/kg和1 mg/kg组分别增加到41.65个/mm2和49.45个/mm2(P0.01),显著高于对照组;在附睾尾被膜,0.1mg/kg和1mg/kg组分别增加到51.82个/mm2和57.47个/mm2(P0.01),在附睾尾间质,0.1mg/kg和1mg/kg组分别增加到44.43个/mm2和56.46个/mm2(P0.01);在精囊腺被膜,0.1 mg/kg和1mg/kg组分别增加到57.38个/mm2和62.35个/mm2(P0.01),在精囊腺间质,0.1mg/kg和1mg/kg组分别增加到48.27个/mm2和61.38个/mm2(P0.01);在前列腺被膜,0.1mg/kg和1mg/kg组分别增加到56.52个/mm2和64.75个/mm2(P0.01),在前列腺间质,0.1mg/kg和1mg/kg组分别增加到55.28个/mm2和82.47个/mm2(P0.01)。结果表明,炔雌醚暴露影响小鼠附睾和副性腺肥大细胞数量及分布,推测可能对附睾中生精细胞的进一步发育成熟有影响,其机制有待进一步研究。     

己烯雌酚对成年仓鼠附睾组织形态的影响

探讨环境内分泌干扰物已烯雌酚(DES)对成年动物附睾组织形态的影响,将DES皮下注射成年仓鼠(剂量为每天1mg/kg)连续1周后,用光镜和电镜观察附睾组织形态的变化,并用脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记法(TUNEL)检测附睾管上皮细胞和精子的凋亡情况。结果,DES处理后附睾头和附睾尾萎缩,附睾管变细。附睾管上皮细胞形态出现明显的改变,附睾上皮细胞和精子均出现大量凋亡,附睾管管腔内充满发育异常的生精细胞,少见长形精子。结果表明,DES能诱发成年动物附睾形态的改变,这可能是造成成年动物生精障碍的重要原因。     

不同外源性激素对雄性牛蛙睾丸中肥大细胞的影响

为了解外源性激素对雄性牛蛙生殖器官睾丸的作用,试验将复方甲地孕酮、苯甲酸雌二醇、丙酸睾酮注入雄牛蛙体内,饲养牛蛙7 d,用Bouin氏固定液固定,最后用改良的甲苯胺蓝染色法显示肥大细胞。结果表明:睾丸中肥大细胞主要分布在睾丸组织的被膜下、睾丸间质和输出小管内;睾丸肥大细胞呈圆形、椭圆形、梭形和不规则形,大小不一,可见肥大细胞的胞浆中充满紫红色颗粒;接受复方甲地孕酮、苯甲酸雌二醇、丙酸睾酮处理的雄性牛蛙,睾丸肥大细胞数量增多,与对照组相比差异极显著(P0.01),用丙酸睾酮处理的睾丸肥大细胞数量变化与对照组相比差异极显著(P0.01)。     

奶牛乳腺肥大细胞数量与胶原纤维面积的相关性研究

为了研究奶牛乳腺肥大细胞的数量与奶牛乳腺胶原纤维面积的相关性,试验对奶牛泌乳期和静止期乳腺进行采样,应用甲苯胺蓝组织化学染色法观察奶牛泌乳期和静止期乳腺肥大细胞数量的变化特点,应用苦味酸天狼猩红染色法观察奶牛泌乳期和静止期乳腺胶原纤维面积的变化特点。结果表明:静止期奶牛乳腺肥大细胞的数量明显比泌乳期多(P0.01),静止期奶牛乳腺胶原纤维的面积明显比泌乳期大(P0.01)。静止期奶牛乳腺肥大细胞的数量多,胶原纤维的面积也大;泌乳期的奶牛乳腺肥大细胞的数量少,胶原纤维的面积也小。说明奶牛乳腺肥大细胞的数量与胶原纤维的面积存在明显正相关性(r=0.677,P0.05)。     

高原型藏绵羊附睾细胞外基质的组织化学研究

旨在探索高原型藏绵羊附睾细胞外基质相关蛋白的分布特征。应用Masson’s、Gomori’s、PAS和ABPAS(pH 2.5)染色组织化学方法观察7只健康成年高原型藏绵羊附睾的组织结构特点,进而用免疫组织化学SP法观察层粘连蛋白(LN)、IV型胶原蛋白(Col IV)和硫酸乙酰肝素蛋白多糖(HSPG)的分布特征。高原型成年藏绵羊附睾各部分管腔为柱状纤毛上皮,附睾尾间质胶原纤维和网状纤维较附睾头及附睾体明显增多。PAS反应显示附睾尾阳性强于附睾头和附睾体,AB-PAS反应显示附睾头和附睾尾的阳性强于附睾体。免疫组织化学结果显示,LN在组织中表达最强,HSPG次之,Col IV表达较弱,HSPG和LN的阳性表达差异不显著(P0.05)。高原型成年藏绵羊附睾尾间质胶原纤维和网状纤维的分布较附睾头和附睾体多,附睾各部分中PAS的反应强弱与附睾上皮分泌功能的变化密切相关,附睾尾输精管的分泌功能增强;Col IV参与基膜的物质转运;HSPG与血-附睾屏障构成相关;LN与基膜的形成及其附睾中细胞外基质相关蛋白(ECM)的合成和分泌有关。     

奶牛发育各期乳腺肥大细胞的组织化学特性研究

本研究旨在对奶牛乳腺肥大细胞的组织化学特点进行研究。对奶牛发育各期乳腺进行取样,应用改良甲苯胺蓝染色法(MTB)和阿尔新蓝—番红鉴别染色法（AB-S）进行染色,观察肥大细胞的组织化学特点和数量变化特点。同时比较了甲苯胺蓝染色时两种固定液固定对乳腺肥大细胞着染的效果。奶牛不同发育时期乳腺肥大细胞只被阿尔新蓝染色,Carnoy氏液固定效果优于中性甲醛（NBF）,MTB的染色效果优于AB-S。奶牛乳腺肥大细胞的数量在泌乳各期和静止期有显著的变化。与静止期相比,泌乳期的乳腺肥大细胞数量明显减少(P＜0.05),分娩后60 d奶牛乳腺肥大细胞的数量最少。奶牛乳腺只存在黏膜型肥大细胞;用Carnoy氏液固定,用MTB法染色可以很好地显示肥大细胞;泌乳期乳腺肥大细胞数量与静止期相比,明显减少。     

不同繁殖季节成年牦牛附睾组织特征比较

为比较不同繁殖季节成年牦牛附睾组织结构特征变化,应用苏木精-曙红常规染色、Masson's和Gomori's特殊组织化学染色方法比较不同繁殖季节牦牛（6头繁殖期成年牦牛和9头繁殖间期成年牦牛）附睾的组织结构特点并用IPP图像分析软件进行定量分析。结果显示,与繁殖间期相比,繁殖期附睾尾间质胶原纤维和网状纤维较附睾头及附睾体明显增多;附睾头和附睾体柱状上皮厚度显著增加（P<0.05）,附睾头纤毛长度增加显著（P<0.05）;附睾头和附睾尾管腔内径及外径显著增高（P<0.05）;但是附睾尾管腔外径繁殖期与繁殖间期相比差异不显著（P>0.05）。本研究结果表明,成年牦牛附睾管外围网状纤维与胶原纤维分布一致,二者在附睾尾较为丰富,可能与其较强的收缩能力及精子运输有关;柱状纤毛上皮高度及纤毛长度、管腔内径与外径的变化与其所处的不同繁殖季节密切相关,附睾管腔的膨大和回缩亦可能是高原地区季节性繁殖的哺乳动物中一种普遍存在的现象。     

Histological Characteristics of Adult Yak Epididymis in Different Breeding Seasons

To compare the histological changes of the adult yak's epididymis in different breeding seasons,six adult yak testis in breeding season and nine adult yak testis in breeding interval were collected for structure investigation by HE staining,Masson's and Gomori's histochemistry methods,and IPP (Image-Pro Plus) statistics method was used to quantitative statistics.The results showed that comparing with the adult yak in the breeding interval,the epididymis ducts of adult yak in the breeding season were covered with the columnar ciliated epithelium.The collagen and reticular fiber in cauda epididymis were obviously more abundant than caput and corpus epididymitis.And the thickness of columnar epithelium cells in caput and corpus epididymitis,the length of the cilia in caput,and also the internal and external lumen diameter of caput and corpus epididymitis were all significantly increased in the breeding season (P<0.05),but the external lumen diameter of the cauda epididymis had no significant differences (P>0.05).In conclusion,the research showed that the distribution of collagen and reticular fiber in adult yak's epididymis interstitial were similar,and they were more rich in cauda epididymis,which might relate to the capacity and the sperm transport;The changes of the epithelial thickness,the length of cilia,the internal and external lumen diameter were close related to the different breeding seasons,and it might be a common phenomenon in plateau mammals that the enlargement and reduction of the epididymal duct in different breeding seasons.     

Immunolocalization of Aquaporins 1 and 9 in the Ram Efferent Ducts and Epididymis

Aquaporins (AQPs) are essential membrane protein channels for the transport of water across membranes. Fluid movement in the epididymis is important for modulation of the luminal environment, in which sperm mature and reside. This study was designed to understand the morphology and localization of AQPs in ram efferent ducts (ED) and epididymis. For this purpose, the epididymis of seven animals were removed for histologic and immunohistochemical analyses. AQP1 immunoreactivity was observed in the apex of the ED, and AQP9 was found adjacent to the nuclei of the epithelial cells of the ED. The epithelial lining of ram epididymis is pseudostratified columnar and presents principal, basal, apical and narrow cells. In the initial segment (IS), a moderate reaction for AQP1 was observed in the apical cytoplasm of epithelial cells. An intense reactivity for AQP1 was noted over the microvilli of principal cells and in spermatozoa in the caput. In the corpus and cauda, AQP1 was noted only over the endothelial cells of vascular channels located in intertubular spaces. A weak‐to‐moderate reaction for AQP9 was observed in the nuclei of epithelial cells in the IS, caput and corpus of the epididymis. In the cauda, an intense reaction to AQP9 was observed in the epithelial border. In the IS, caput and corpus, the reactivity for AQP9 differed from those observed in domestic animals. The cauda showed a pattern similar to that previously described. These results indicate that AQPs 1 and 9 have reversed locations and roles in rams, suggesting activity variations related with fluid and solute absorption throughout the epididymis.     

免疫细胞在牦牛附睾和输精管的分布规律

旨在研究免疫细胞在健康雄性牦牛附睾和输精管的分布。采用免疫组织化学和实时荧光定量(qRT-PCR)方法对幼龄(5~6月龄)及成年(3~4岁)牦牛附睾(头、体、尾)和输精管中CD68⁺巨噬细胞、CD3⁺ T淋巴细胞、CD79α⁺ B淋巴细胞、IgA⁺和IgG⁺浆细胞的分布特征及其表面标志分子的表达水平进行研究。结果显示：CD68⁺巨噬细胞、CD3⁺ T淋巴细胞、CD79α⁺ B淋巴细胞、IgA⁺和IgG⁺浆细胞主要分布在附睾管和输精管的上皮和间质;另外,CD68和CD3 mRNA和蛋白水平在各年龄组牦牛附睾头和附睾体显著高于附睾尾和输精管(P < 0.05),而CD79α、IgA和IgG mRNA和蛋白水平在附睾尾和输精管显著高于附睾头和附睾体(P < 0.05);此外,在成年牦牛附睾和输精管CD3、CD79α、IgA、IgG、CD68 mRNA和蛋白水平均显著高于幼龄牦牛(P < 0.05)。综上提示,牦牛附睾头可能主要是细胞免疫发生的位点,而附睾尾和输精管则主要进行体液免疫应答,此外,成年牦牛附睾和输精管的局部免疫可能更完善,以上数据为进一步研究高原牦牛局部生殖免疫和病理提供了形态学资料。     

ART3抑制剂3-MBA对小鼠睾丸生精功能的影响

旨在探究ADP-核糖基转移酶3（ADP-ribosyl transferase 3，ART3）调控精子发生机制，为改善精液品质，提高家畜繁殖性能提供理论依据。本试验设计了3个ART3抑制剂3-甲氧基苯甲酰胺（3-methoxybenzamide，3-MBA）浓度梯度（0.302、0.906和1.510 mg·mL^-1），分别对6～8周龄小鼠进行睾丸注射，并于注射后3 h、6 h、12 h、1 d、2 d、3 d和5 d采集小鼠睾丸和附睾组织，将睾丸组织制成石蜡切片进行HE染色，并对附睾尾精子进行动力学和形态学分析。结果发现，0.302 mg·mL^-13-MBA浓度组于注射后3 d小鼠睾丸曲细精管内空泡面积达到最大化，生精细胞减少，且排布散乱不规则，注射后5 d空泡面积减小，生精细胞有恢复趋势，而0.906和1.510 mg·mL^-1浓度组于注射后5 d空泡面积最大化，曲细精管出现空管现象；3个剂量组小鼠附睾尾精子密度、活力及前向运动精子均呈时间依赖性降低，畸形精子随时间推移逐渐增加，且精子尾部出现断裂、弯折和卷曲等异常形态。综上表明，ART3可能参与调控生精细胞增殖、分化和迁移及精子尾部形成等精子发生过程。     

Histochemical detection of glycoconjugates in the male reproductive system of the horse

Lectins are glycoproteins of plant and animal origin that have the ability to bind specific carbohydrate residues of cell glycoconjugates, particularly in terminal positions. In this study, the binding of lectins, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), Bandeiraea simplicifolia BS-1 (isolectin B4), Triticum vulgaris (WGA), Arachis hypogaea (PNA), and Ulex europaeus (UEA-I), was studied in the reproductive systems of male thoroughbred horses.DBA was detected in the stereocilia of the caput and corpus epididymis, and in the vas deferens. It was weakly detected in connective tissue of the corpus epididymis. Strong SBA staining was seen in epithelial cells in the testis, stereocilia of the corpus and cauda epididymis, and in the vas deferens. There were intense positive reactions for isolectin B4 in interstitial cells in all tissue and serosa of the vas deferens. PNA staining was seen only in stereocilia in the caput and corpus epididymis, and in the vas deferens. Strong WGA staining was seen throughout the testis, except in Sertoli cells, stereocilia, and connective tissue. UEA-I was detected in secondary spermatids, stereocilia, and epithelial cells of the cauda epididymis. These results show that degenerating cells in the testis, epididymal tubules, and vas deferens have differential affinities for lectins, and suggest that lectins play a role in the reproductive system of the horse. The heterogeneity of the lectin staining pattern in the reproductive tubules of adult horses suggests that the carbohydrate composition of each cell type is region specific.     

绵羊附睾褪黑素受体1的表达与定位

为研究褪黑素受体1（MT1）在不同年龄绵羊附睾中的表达模式，选用幼龄绵羊（2~3月龄）、青年绵羊（6~8月龄）和成年绵羊（2~3岁）的附睾，采用实时荧光定量PCR和免疫组化技术检测不同年龄绵羊附睾各部位MT1基因mRNA的表达量和MT1的分布情况。结果表明：幼龄绵羊附睾尾MT1基因的转录水平极显著高于附睾头和附睾体（P<0.01）；青年绵羊附睾体和附睾尾MT1基因的转录水平显著高于附睾头（P<0.05）；成年绵羊附睾尾MT1基因的转录水平显著高于附睾头和附睾体（P<0.05），附睾各部位MT1基因的转录水平随年龄增长呈明显降低趋势；定位结果显示，MT1在各年龄组绵羊附睾的各个部位均有分布，且主要分布在附睾上皮细胞中。综合上述结果，不同年龄绵羊附睾的不同部位均有MT1表达和分布，并随年龄增长各部位的表达量降低，相同年龄绵羊附睾尾MT1的表达水平较高。     

Morphological and acrosomal changes of canine spermatozoa during epididymal transit

Background

During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda.

Results

After the dilution required for the collection of epididymal content, sperm motility was significantly higher (P <0.0001) in the cauda compared to corpus and caput.Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda (P <0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P <0.0001) and cauda (P =0.006).Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P <0.0001) and in the cauda (P <0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P =0.0003) and in the cauda (P <0.05).Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P =0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes.

Conclusions

Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature spermatozoa which are then stored in the cauda epididymis. From this site, spermatozoa can be retrieved and used in assisted reproductive techniques as a valuable tool for propagating genetic traits of high value individuals that dies accidentally or undergoes orchiectomy for medical purposes. Further investigations should be also focused on the potential use of spermatozoa recovered from other epididymal regions.     

Transfer of MicroRNAs From Epididymal Epithelium to Equine Spermatozoa

All epididymal regions are lined with multiple epithelial cell types, each with different functions to provide the luminal environment for spermatozoal maturation. Epithelial cells also create apical blebs, which are released from the apical surface via apocrine secretion and disintegrate in the lumen, thereby releasing epididymosomes. Epididymosomes transport proteins to spermatozoa and contain microRNAs. We hypothesized that epididymosomes also transfer miRNA from epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine miRNA profiles of epididymal tissue from caput and cauda, epididymal spermatozoa from caput and cauda, and epididymosomes and from caput, proximal corpus, distal corpus, and cauda. Pathway analysis was performed using DIANA tools on the miRNA unique to caudal spermatozoa. We found 66 newly acquired miRNAs in spermatozoa located in the caudal epididymis. Predicted pathways targeted by these miRNAs suggest a role in cell motility and viability and factors in oocyte and embryo maturation and development. These findings suggest that miRNAs are transported to spermatozoa from epididymal epithelium via epididymosomes.