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。     

高原地区小尾寒羊附睾细胞外基质相关蛋白分布特征研究

旨在研究高原地区小尾寒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AB-PAS(pH=2.5)组织化学染色方法观察5只健康成年高原地区小尾寒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(ColⅣ)和硫酸乙酰肝素蛋白多糖(HSPG)的分布特征。高原地区小尾寒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反应显示附睾头和附睾尾阳性强于附睾体,AB-PAS反应显示附睾头、体、尾阳性差异较小。免疫组织化学显示,ColⅣ在附睾头和附睾体组织中表达最强,HSPG次之,LN表达较弱,HSPG和LN的阳性表达差异不显著(P0.05);而HSPG在附睾尾组织中表达最强,LN次之,ColⅣ表达较弱,HSPG和LN的阳性表达差异不显著(P0.05)。高原地区小尾寒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,附睾头和附睾尾输精管的分泌功能增强。附睾头上皮基细胞ColⅣ、HSPG和LN分泌增加,且ColⅣ、HSPG和LN与血管的调节作用密切相关。     

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。     

性成熟前不同日龄伊拉兔睾丸和附睾形态学与组织学特征

【目的】探究不同日龄伊拉兔睾丸和附睾组织在性成熟前的变化规律,为其初情期及性成熟的判定提供组织学依据。【方法】将45只伊拉兔随机分为9组,每组5只,从30日龄开始每隔15日采集1组试验兔的睾丸及其附睾,运用形态学与组织解剖学方法对其生长发育规律进行研究分析。【结果】随着日龄的增加,睾丸指数、睾丸重、睾丸长径、睾丸短径及厚径等指标逐步增加,且150日龄时各指标均显著高于其他组（P＜0.05）;30、45、60日龄睾丸内生精小管排列稀疏,75、90、105日龄时睾丸间质成分逐渐增多,管腔逐步形成,120、135、150日龄时间质细胞进一步增生并成群分布,90日龄起出现圆形和长形未成型精子,120日龄的睾丸生精小管、附睾管腔中出现少量成型精子,150日龄时有大量精子密集分布于睾丸管腔内,且在120、135、150日龄时生精小管面积、上皮细胞厚度、支持细胞数均显著大于其他组（P＜0.05）;30日龄以上各组间质细胞个数在各组间差异不显著（P＞0.05）,但均显著高于30日龄;120、135、150日龄时附睾头、体、尾的直径均极显著高于其他组（P＜0.05）,而附睾头的柱状上皮厚度和纤毛长度则从105日龄起就显著高于30、45、60、75和90日龄组（P＜0.05）。【结论】伊拉兔睾丸和附睾随日龄增加同步发育,在120日龄时达到初情期,在150日龄时睾丸和附睾已发育成熟,基本达到性成熟。     

还原型谷胱甘肽对氟中毒小鼠附睾的影响

试验旨在研究还原型谷胱甘肽（reduced glutathione,GSH）对氟中毒小鼠附睾的影响。将50只3周龄ICR雄性小鼠随机分为5组,分别为对照组和4组染氟组。对照组饮用蒸馏水,染氟组分别供给含100 mg/L NaF的蒸馏水,自由饮水30 d。之后在染氟组中选择3组每日分别灌胃200、400和800 mg/（kg·只）GSH,同时各组小鼠再自由饮含氟水30 d。试验结束后,摘取小鼠附睾进行活性氧（ROS）、丙二醛（MDA）、GSH含量及谷胱甘肽过氧化物酶（GPx）、谷胱甘肽硫转移酶（GSTs）和谷胱甘肽还原酶（GR）活性检测,同时制作石蜡切片,HE染色后进行组织形态结构分析。结果显示,与对照组相比,100 mg/L NaF组精子密度、精子活力、GSH含量、GPx和GR活性均显著下降（P<0.05）,而GSH添加组较100 mg/L NaF组均有所升高;100 mg/L NaF组精子畸形率、ROS和MDA含量较对照组均显著升高（P<0.05）,而GSH添加组较100 mg/L NaF组均降低,其中800 mg/kg GSH组效果明显。与对照组相比,100 mg/L NaF组附睾头、附睾体及附睾尾管腔厚度升高,管腔内精子数量下降;GSH添加组附睾头、附睾体及附睾尾的管腔厚度与管腔内精子数量都有所恢复,其中800 mg/kg GSH组效果较好。综上所述,添加GSH后氟中毒小鼠谷胱甘肽抗氧化系统酶活性有所升高,附睾组织形态结构一定程度上有所恢复,对附睾有一定的改善作用。     

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。     

牦牛发情周期中卵巢卵泡发育状况的组织学观察

运用组织学技术对36头处于发情周期的健康成年母牦牛卵巢卵泡的结构与发育状况进行了观察。结果表明，牦牛发情周期中卵巢卵泡发育的组织结构与其他牛基本相似。每对卵巢中原始卵泡数和生长卵泡数在发情前期、发情期、发情后期和发情间期之间差异不显著（P〉0．05）；发情前期的囊状卵泡数与发情期、发情后期和发情间期之间差异不显著（P〉0．05），发情期和发情后期均与发情间期差异显著（P〈0．05）。闭锁生长卵泡数在4个时期之间差异不显著（P〉0．05）；发情期的闭锁囊状卵泡数与其他3期之间差异极显著（P〈0．01），发情前期和发情间期均与发情后期差异显著（P〈0．05）。各级卵泡的闭锁形式各有特点。在生长卵泡、囊状卵泡及相应闭锁卵泡的卵泡膜中均见到了胶原纤维和网状纤维。     

不同繁殖节律母牦牛冷暖季血浆褪黑激素RP-HPLC测定

为研究不同季节、不同繁殖节律和繁殖活动期与静止期成年母牦牛血浆MLT分泌水平的差异,应用反相高效液相色谱法(RP-HPLC)检测成年母牦牛血浆 MLT 浓度.供试母牦牛63头,分为A、B、C 3组,每组21头.A组:1年1胎,繁殖活动期;B组:2年1胎,全奶,繁殖静止期;C组:2年1胎,半奶,繁殖活动期.在暖季的7月21-23日和冷季的11月21-23日,自然光制:12.00L:12.00D和14.65L:9.35D,日落前2 h开始采血,持续50min,颈静脉采血每头20 mL,肝素抗凝,离心取血浆.1年1胎母牦牛血浆MLT浓度((68.90±5.82) pg/mL)高于2年1胎全奶个体((44.38±2.61)pg/mL) (P< 0.01)和2年1胎半奶个体((57.51±3.82)pg/mL)(P<0.05);母牦牛血浆 MLT 浓度暖季高于冷季(P<0.05),表明其具有季节波动,并提示除光照外牦牛 MLT 分泌还与气温等环境因子有关;母牦牛血浆 MLT 浓度繁殖活动期高于繁殖静止期(P<0.05).母牦牛MLT分泌存在季节波动,母牦牛季节性繁殖和繁殖节律受到MLT水平的影响.     

山羊Lcn5的表达特点及其在繁殖器官中定位

为探明附睾视黄酸结合蛋白5(Lcn 5)mRNA在公山羊体组织及不同月龄附睾中的表达特性,及其在睾丸、附睾和精子中的定位。采用实时荧光定量PCR方法检测18种组织和不同月龄附睾Lcn5mRNA表达规律;利用蛋白印迹技术对成年公羊睾丸和附睾Lcn 5蛋白表达进行定量分析;利用免疫组化技术对5月龄公羊睾丸和附睾Lcn 5进行定位,利用免疫荧光技术对精子Lcn 5进行定位。Lcn5mRNA在不同组织中均有表达,其中在性腺中的表达量最高,附睾头附睾尾附睾体睾丸;在5月龄前附睾的Lcn5mRNA表达量随月龄逐渐升高,5月龄时达到最高,随后又逐渐降低。Western blotting结果显示,Lcn 5蛋白表达量:附睾头附睾尾附睾体睾丸,支持了Lcn5mRNA定量的结果。免疫组化结果显示,5月龄山羊在附睾管壁的柱状细胞、纤毛细胞检测到较强Lcn 5蛋白表达的阳性信号,附睾尾管腔检测到较强的阳性信号。在睾丸各级生精细胞中也检测到了微弱阳性信号。Lcn 5蛋白包裹在精子头部顶体帽上。Lcn5在山羊附睾中高度表达,其表达具有时空特异性,推测其在精子成熟和维持正常生理功能方面发挥重要作用,其生物学功能需要进一步深入研究。     

氟化钠对附睾形态结构及紧密连接相关基因表达的影响

为了研究氟对小鼠附睾结构形态及紧密连接相关基因的影响,将60只健康雄性小鼠随机分成4组,对照组饮用蒸馏水,3个氟处理组分别饮用含氟化钠（NaF） 25、50和100 mg/L的蒸馏水。染氟60 d,取小鼠股骨,采用氟离子选择电极法测其氟含量;取右侧附睾制作石蜡切片,HE染色后进行组织形态结构观察;左侧附睾提取RNA,采用实时荧光定量PCR技术（qRT-PCR）检测紧密连接相关基因的表达。结果显示,与对照组相比,氟处理组小鼠股骨氟含量随摄氟剂量的增加呈剂量依赖性,其中50、100 mg/L剂量组差异极显著（P<0.01）;氟处理组附睾尾管腔厚度极显著增厚（P<0.01）;紧密连接相关基因Claudin-2 mRNA表达量极显著下降（P<0.01）,100 mg/L剂量组ZO-1 mRNA表达量显著下降（P<0.05）。摄入过量NaF影响附睾形态及其紧密连接相关基因的表达,从而可能对血-附睾屏障造成损伤。     

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.     

A light and scanning electron microscopic study of the epididymis active state of the endemic Mexican rodent Peromyscus winkelmanni (Carleton) (Rodentia: Muridae)

The macroscopic and microscopic anatomy of the epididymis of the sexually mature Peromyscus winkelmanni (carleton) was examined using light and scanning electron microscopy. The epididymis was divided into three regions: caput, corpus and cauda. The epididymal duct was lined with columnar and cubic epithelium with stereocilia and covered by a muscular connective tissue sheath. Capillaries appear to penetrate directly into the epithelium from the underlying connective tissue in the initial segment. The epididymal epithelium presents four cell types: principal, basal, apical and clear cells. Based on morphological differences (height of epithelial cells, length of the stereocilia, luminal area, larger diameter and spermatic index), the epididymis of P. winkelmanni, presents seven zones. The stereocilia of the epididymal ducts of zones I, II, IV and V are thick and tall, while in zone III they are thin and short. The stereocilia in zone VI are thin, while in zone VII they are short but thick. The secretory products observed in the lumen of the epididymal ducts have vesicular, granular and fibrous form in the seven zones. This study contributes to an understanding of the morphofunctional features of the epididymis in sperm maturation in a species that shows seasonal reproductive activity.     

达乌尔黄鼠精子形成和细胞色素芳香化酶免疫位置的季节变化

用组织学和免疫组织化学方法调查达乌尔黄鼠精子形成季节性变化和细胞色素芳香化酶(P450 arom)在精巢和附睾中的免疫位置。黄鼠繁殖期与非繁殖期精巢大小、重量、生精小管直径存在显著差异;黄鼠繁殖期精巢中存在从精原细胞到有尾精子各期生殖细胞,非繁殖期精巢中只存在精原细胞和初级精母细胞。另外,繁殖期黄鼠附睾管中存在大量有尾精子,而非繁殖期附睾中未见精子存在。繁殖期P450 arom在黄鼠精巢的间质细胞、支持细胞、精子细胞和附睾头部输出小管上皮细胞都有发现,而在非繁殖期没有发现它的活力。这些结果表明达乌尔黄鼠精子形成、成熟是伴随着精巢复发和退行呈现显著季节性变化,雌激素在精子形成和成熟过程中起着重要的生理性作用。     

牦牛GPX5基因CDS区的克隆和表达研究

GPX5作为谷胱甘肽过氧化物酶的成员之一,表达于牦牛附睾之中,其主要作用为抗氧化应激。本实验旨在通过分子技术对牦牛GPX5基因进行克隆,分析其生物信息学的特征和附睾组织的表达特点。结果显示:牦牛GPX5基因CDS区有630 bp、209个氨基酸被编码,所编码蛋白带正电,具有亲水性且不稳定,无跨膜域却存在信号肽;GPX5基因在附睾头的表达高于附睾的颈和尾(P<0.01)。牦牛附睾中的GPX5能有效抵抗氧化应激以保护精子。     

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.     

Histology of lamb epididymal development

To describe the distribution of the histological regions and their morphometry during epididymal development, 10 Corriedale lambs were castrated monthly from 90 to 180 days of age (n = 24), and their testes and epididymides were weighed. All animals were weighed monthly. Epididymides were divided into caput, corpus and cauda, and cut sagitally so that sections included all the length of the organ. The diameter of the epididymal duct, the smooth muscle depth and the epididymal epithelium height were measured. The quantitative histology of the ovine epididymal development was described. Epididymal development advanced from caput to cauda. The distribution of the histological regions varied according to epididymal weight. Transient histological regions were found during epididymal development. The present results indicate a new way of epididymal development in sheep, which courses from caput to cauda with transient histological regions appearing, varying in location and disappearing during ovine epididymis development.     

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.