老龄哈多利系博美犬睾丸组织结构分析

为比较哈多利系博美犬老龄与青年阶段睾丸的组织结构差异及相关蛋白分布特征,探索博美犬不同年龄阶段睾丸组织结构变化及其对生殖功能的影响,本试验应用特殊染色、免疫组织化学法结合免疫荧光观察比较青年与老龄博美犬睾丸组织化学特点,并用IPP图像分析软件进行定量分析。结果显示,与青年博美犬相比,老龄博美犬生精上皮层数与厚度降低,Leydig细胞数及Sertoli细胞数显著增加(P0.05),生精小管基底膜及血管管壁层胶原纤维与网状纤维含量明显增加。免疫组织化学结果显示,老龄博美犬睾丸细胞外基质(exreacellular matrix,ECM)相关蛋白Ⅳ型胶原(collage,ColⅣ)、硫酸乙酰肝素蛋白多糖(heparan sulfate proteoglycan,HSPG)含量极显著低于青年博美犬(P0.01),层黏连蛋白(laminin,LN)含量显著低于青年博美犬(P0.05)。免疫荧光结果显示,ColⅣ、HSPG和LN主要在Leydig细胞及Sertoli细胞强表达。因此,老龄博美犬胶原纤维与网状纤维含量增加,Leydig细胞数及Sertoli细胞数增多可能与其生精功能的维持相关,其生精功能的下降与ColⅣ和HSPG的变化密切相关。     

老龄牦牛睾丸细胞外基质相关蛋白的分布特征

探索细胞外基质相关蛋白在老龄牦牛睾丸的分布特征。应用组织化学方法比较、观察9头健康老龄牦牛和10头青年牦牛睾丸组织结构特点及层黏连蛋白(LN)、Ⅳ型胶原(ColⅣ)和硫酸乙酰肝素糖蛋白(HSPG)的分布特征。结果显示:光镜下,老龄牦牛生精上皮部分或完全退化,生精小管固有膜及间质胶原纤维及网状纤维较青年牦牛丰富;老龄睾丸间质血管及生精小管固有膜中AB-PAS阳性反应较青年牦牛增强。数据统计表明,老龄牦牛Sertoli细胞及Leydig细胞数均明显减少,生精小管横截面积以及平均间质组织面积极显著大于青年牦牛(P0.01)。免疫组织化学显示,LN在老龄牦牛睾丸Sertoli细胞和肌样细胞表达与青年牦牛相近,LN在生精细胞表达降低,而在Leydig细胞几乎无表达;ColⅣ在不同年龄牦牛睾丸组织表达位置及强弱相似,但其平均吸光度检测结果无统计学差异;老龄牦牛睾丸组织HSPG主要在Leydig细胞表达降低,平均吸光度检测统计极显著低于青年牦牛(P0.01);同一年龄段LN、ColⅣ和HSPG表达无明显差异。高原环境中,老龄牦牛生精上皮退化伴随着间质成分增加、间质面积增大、Leydig细胞及Sertoli细胞数量减少等形态学变化;睾丸组织ColⅣ分泌增加且胶原纤维合成增强,LN和HSPG的显著降低可能影响Leydig细胞合成分泌能力。     

犬隐睾及睾丸肿瘤中PGP9.5和NPY的分布比较

试验旨在分析肽能神经递质蛋白基因产物9.5（protein gene product 9.5,PGP9.5）和神经肽Y（neuropeptide Y,NPY）在犬隐睾及睾丸肿瘤中的分布和表达,并与同年龄正常睾丸组织进行比较,为认识犬睾丸肿瘤恶变临床诊断提供参考。应用HE染色、Masson三色染色、Gomori银浸染、甲苯胺蓝染色观察各组织中网状纤维、胶原纤维及肥大细胞等组织特征,采用免疫组织化学SP法及免疫荧光法结合IPP统计分析PGP9.5和NPY在组织中的表达及定位。结果显示,正常犬睾丸生精上皮由4~7层生精细胞及Sertoli细胞构成,间质组织胶原纤维和网状纤维分布稀疏。隐睾生精小管基底膜胶原纤维厚度增加,Sertoli细胞核浓缩位于生精小管基底,间质网状纤维增多。睾丸肿瘤组织结构不清晰,胶原纤维和网状纤维无规则分布,肥大细胞较正常组及隐睾组显著增多。免疫荧光定位表明,PGP9.5在正常睾丸Leydig细胞中呈中等阳性表达,生精细胞中无明显表达;隐睾Leydig细胞及生精细胞中呈强阳性表达;睾丸肿瘤中偶有表达。NPY在正常睾丸Leydig细胞中偶见阳性表达,生精细胞中无表达;隐睾Leydig细胞及生精上皮中无表达,间质小血管管壁呈高密度强阳性表达;睾丸肿瘤组织中无明显表达。免疫组化统计表明,睾丸肿瘤组织中PGP9.5和NPY较正常组极显著降低（P<0.01）,隐睾组PGP9.5和NPY表达显著或极显著增加（P<0.05;P<0.01）。因此,犬隐睾时PGP9.5及NPY的表达增高,提示犬隐睾时已有发展为肿瘤的趋势,且与肿瘤恶变程度相关。     

FGF22及其受体在庆阳黑山羊正常睾丸与隐睾中的表达比较

试验旨在研究成纤维生长因子22（fibroblast growth factor 22,FGF22）及其受体2（fibroblast growth factor receptor 2,FGFR2）、硫酸乙酰肝素糖蛋白（heparan sulfate proteoglycans,HSPG）在庆阳黑山羊正常睾丸与隐睾中的分布与表达,探究其在山羊睾丸发育和隐睾形成中的作用。采用HE和特殊染色观察其组织学结构特征,进而以免疫组织化学及免疫荧光法结合形态计量学统计研究FGF22、FGFR2和HSPG在山羊正常睾丸及隐睾中的定位。结果表明,山羊隐睾较正常睾丸生精小管缩窄,腔内各级生精细胞排列紊乱,间质的胶原纤维和网状纤维增多,糖原类物质阳性反应较弱,FGF22在隐睾组织的Leydig、Sertoli细胞、管周肌样细胞及血管内皮细胞整体表达密度相较于正常睾丸显著减弱（P<0.05）。HSPG在正常睾丸表达显著强于隐睾（P<0.05）,间质组织变化尤其明显。FGFR2在隐睾组表达显著增高（P<0.05）,且以Sertoli细胞强阳性表达为主。庆阳黑山羊隐睾较正常睾丸发育异常,间质组织有纤维化趋势,糖原类物质含量减少;FGF22及HSPG表达降低应与隐睾局部环境温度变化密切相关;FGFR2在隐睾组表达增高提示其在发生隐睾时可能通过Sertoli细胞进行适应性调节。     

性激素受体在子午岭黑山羊隐睾及正常睾丸中的分布比较

旨在研究性激素受体[雄激素受体（androgen receptor,AR）、雌激素受体（estrogen receptor,ER）]在子午岭黑山羊正常睾丸与隐睾组织中的分布与隐睾症的关系,应用免疫组织化学及免疫荧光技术方法结合形态计量学统计软件,比较了正常睾丸与隐睾的组织化学特点。特殊染色结果显示：与正常组相比,隐睾组间质组织疏松,管腔面积明显减小,糖原含量明显减少,胶原纤维及网状纤维含量增多。免疫组化及免疫荧光结果显示：1） AR在正常睾丸组Leydig细胞呈高密度强阳性表达,在各级生精细胞呈中等强度阳性表达,隐睾组中Leydig细胞及各级生精细胞表达明显减弱,且在精原细胞偶见表达;2） ER在正常睾丸Leydig细胞呈高密度强阳性表达,管周肌样细胞呈中等强度阳性表达,Sertoli细胞偶见表达,各级生精细胞无表达;3） ER在隐睾Leydig细胞、初级精母细胞、精原细胞和Sertoli细胞中均呈中等强度阳性表达;4）统计结果显示,隐睾组AR的平均光密度较正常组显著降低（P<0.05）,而ER的平均光密度则显著高于正常组（P<0.01）,且隐睾组AR与ER表达量比值基本接近1：1。子午岭黑山羊隐睾组织胶原纤维和网状纤维分布较正常睾丸多,生精小管基膜主要成分以中性糖蛋白为主,酸性糖蛋白含量明显降低影响精子的正常形成;隐睾组织精原细胞及Sertoli细胞ER与AR表达失常尤为明显,可为哺乳动物隐睾的相关研究提供一定参考。     

老龄牦牛睾丸组织结构研究

探讨高原老龄牦牛睾丸组织结构特点与其生殖机能的关系。采集9~13岁健康老龄牦牛睾丸组织,应用组织化学染色和透射电镜技术,观察其细胞化学特点及结构特征。结果显示,老龄牦牛睾丸体积及重量左侧略大于右侧,光镜下睾丸被膜、间质组织胶原纤维及网状纤维丰富,小叶不明显,Leydig细胞散在于结缔组织之间,生精小管不同程度退化,生精上皮层数为2~5层,细胞间隙变大,部分脱落,Sertoli细胞低矮,细胞质较少。电镜观察Sertoli细胞核外形不规则,内质网呈疏松的泡状,次级溶酶体增加,体积增大。相邻Sertoli细胞间没有观察到并行的内质网层和细丝束这一血-睾屏障所特有外质特化区形态结构,部分生精小管固有膜胶原纤维增生,上皮皱缩;Leydig细胞内脂滴异常丰富,毛细血管内皮细胞间存在紧密连接。间质组织偶有肥大细胞,间质血管及生精小管固有膜中PAS及AB-PAS阳性反应明显。老龄牦牛Sertoli细胞细胞器结构异常,血-睾屏障中Sertoli细胞间的连接处缺陷,且Sertoli细胞与基膜连接的改变可能影响了Sertoli细胞和生精细胞之间的相互作用,进而抑制了生精细胞的功能。     

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。     

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。     

高原牦牛睾丸组织VEGF及其受体的增龄性分布特点

探索VEGF及其受体(VEGFR2)在不同年龄牦牛睾丸的分布特点。应用免疫组织化学SP法检测VEGF及其受体(VEGFR2)分布特征并通过IPP图像分析软件进行定量统计。免疫组织化学显示,不同年龄段牦牛睾丸中,VEGF表达于各级生精细胞以及Sertoli细胞和Leydig细胞,强表达于长形精子,小血管内皮细胞见强阳性表达,肌样细胞未见表达;其受体VEGFR2表达于各级生精细胞及Sertoli细胞,且Leydig细胞内强表达,小血管弱表达;牦牛幼龄期至性成熟期,睾丸中VEGF表达量呈明显上升趋势(P0.05),性成熟期达到最高水平,进入老龄期后表达量明显下降(P0.05);其受体VEGFR2表达量性成熟前期最高,进入老龄期下降不明显。因此,牦牛睾丸中VEGF及其受体随年龄增加存在差异性表达,幼龄期随增加明显,性成熟期趋于稳定,老龄后下降显著,提示其可能参与调节牦牛生后睾丸发育的各个阶段,且与高原牦牛睾丸血管发育,生精及其低氧适应具有一定的关系。     

性成熟期辽宁绒山羊与子午岭黑山羊睾丸发育比较

本研究旨在探究性成熟期辽宁绒山羊与子午岭黑山羊睾丸发育是否存在差异,并对两品种繁殖性能进行比较。选取性成熟期健康的辽宁绒山羊和子午岭黑山羊各5只,采集睾丸组织,通过大体解剖和苏木精-伊红（HE）染色石蜡切片,比较两品种山羊睾丸组织发育及形态学差异;ELISA检测雄激素浓度;实时荧光定量PCR （real time quantitative PCR,RT-qPCR）、蛋白免疫印迹（Western blot）检测两品种山羊睾丸组织中死盒多肽4（DEAD box polypeptide 4,DDX4）和类无精症缺失基因（deleted in azoospermia-like gene,DAZL）的表达情况。结果显示,辽宁绒山羊睾丸总重和睾丸长周径极显著高于子午岭黑山羊（P<0.01）,而睾丸短周径、睾丸脏体比和睾丸胴体比均差异不显著（P>0.05）;辽宁绒山羊生精上皮厚度显著高于子午岭黑山羊（P<0.05）,而两者精细管面积、直径和单位面积内精细管数量均无显著差异（P>0.05）;两品种山羊睾丸中雄激素分泌无显著差异（P>0.05）;辽宁绒山羊DDX4 mRNA及蛋白表达量均显著高于子午岭黑山羊（P<0.01或P<0.05）,DAZL mRNA表达量极显著高于子午岭黑山羊（P<0.01）,而蛋白表达量差异不显著（P>0.05）。以上结果表明,性成熟期辽宁绒山羊性腺发育程度与子午岭黑山羊一致,但生精上皮较子午岭黑山羊厚,生殖标记基因表达量存在差异,推测可能会影响两品种的生精能力。     

Comparison of the Distribution of PGP9.5 and NPY in Cryptorchidism and Testicular Tumors in Dogs

The purpose of this study was to analyze the distribution and expression of peptidergic neurotransmitters protein gene product 9.5 (PGP9.5) and neuropeptide Y (NPY) in cryptorchidism and testicular tumors of dogs,compare them with normal testicular tissues of the same age,and provide reference for clinical diagnosis of malignant transformation in testicular tumors of dogs.HE staing,Masson trichrome staining,Gomori silver staining and toluidine blue staining were used to observe the tissue characteristics of reticular fibers,collagen fibers and mast cells.Immunohistochemical SP method and immunofluorescence combined with IPP were used to analyze the expression and localization of PGP9.5 and NPY in tissues.The results showed that the seminiferous epithelium of normal dog testis was composed of 4-7 layers of spermatogenic cells and Sertoli cells,and the distribution of collagen fibers and reticular fibers in interstitial tissue was sparse.The thickness of collagen fibers in the basement membrane of cryptorchidism seminiferous tubules increased,the nucleus of Sertoli concentrated at the base of seminiferous tubules,and the interstitial reticular fibers increased.The tissue structure of testicular tumor was unclear,collagen fibers and reticular fibers were irregularly distributed,and mast cells increased significantly compared with normal and cryptorchid groups.The immunofluorescence results showed that PGP9.5 was moderately positive in Leydig cells of normal testis,no significant expression in spermatogenic cells,strongly positive in Leydig cells and spermatogenic cells of cryptorchidism,and occasional expression in testicular tumors.NPY was occasionally expressed in normal testicular Leydig cells,but not in spermatogenic cells,strong positive expression in Leydig cells and seminiferous epithelium of cryptorchidism,high density and strong positive expression in interstitial vessels,and no obvious expression in testicular tumors.Immunohistochemical statistics showed that the expression of PGP9.5 and NPY in testicular tumor tissue were extremely significantly lower than that in normal group (P<0.01),while the expression of PGP9.5 and NPY in cryptorchidism group were significantly or extremely significantly increased (P<0.05;P<0.01).Therefore,the expression of PGP9.5 and NPY in cryptorchidism of dogs was increased suggesting that the cryptorchidism of dogs had a tendency to develop into a tumor,and was related to the degree of malignant transformation of tumor.     

母猪妊娠期不同能量水平对后代公猪生长性能、睾丸发育与免疫的影响

试验旨在研究母猪妊娠期能量水平对后代睾丸发育与免疫的影响及其作用机理。选取30头体重、背膘相近的7~9胎长白×约克夏（LY）经产母猪,按照体重和胎次随机分为2组,每组15个重复,每个重复1头母猪,从妊娠当天分别饲喂正常能量（CON）和低能量饲粮（LE,12.55 MJ/kg）,直到分娩,所有母猪哺乳期均饲喂同一饲粮。分娩后,分别从CON和LE组中挑选体重为平均体重±0.05 kg的后代公猪各15头,断奶后所有公猪按阶段饲喂相同饲粮,于仔猪120日龄时结束试验。记录公猪每个月的体重并计算阶段平均日增重和平均日采食量;采集28和120 d的血液进行血清生化指标和免疫相关细胞因子检测,采集28和120 d的睾丸进行睾丸细胞计数和睾丸免疫相关基因检测。结果表明,与对照组相比,LE组0~59 d平均日增重极显著降低（P<0.01）,28~89 d平均日采食量和0 d睾丸重显著降低（P<0.05）,28 d睾丸指数显著增加（P<0.05）;LE组0和120 d睾丸组织内间质细胞数目、120 d生殖细胞数目、28和120 d支持细胞数目均显著降低（P<0.05）;LE组血清中28 d甘油三酯（TG）和120 d睾酮（T）含量均极显著升高（P<0.01）、雌二醇（E₂）含量极显著降低（P<0.01）,TG、T和E₂含量均存在时间和能量的交互作用（P<0.01）;LE组血液中胆固醇（TC）、高密度脂蛋白胆固醇（HDL-C）的含量120 d时极显著降低（P<0.01）,低密度脂蛋白胆固醇（LDL-C）、TC、HDL-C三者均无时间和能量的交互作用（P>0.05）。随着公猪日龄增加,血液中白细胞介素-1α（IL-1α）、肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、免疫球蛋白（IgG）浓度均极显著增加（P<0.01）,其中TNF-α和IL-1β有能量和时间的交互作用。与对照组相比,LE组28 d时TNF-α含量显著降低（P<0.05）,120 d时IL-1β水平显著升高（P<0.05）。LE组28 d紧密连接蛋白1（ZO1）及0和28 d闭合蛋白（Occludin）基因相对表达量均显著降低（P<0.05）,28和120 d时连接黏附分子1（JAM1）基因相对表达量显著增加（P<0.05）。LE组0 d时CCL4基因和28 d时IL-1α基因相对表达量均显著降低（P<0.05）,0和120 d时趋化因子2（CCL2）基因及28 d时细胞间黏附分子1（ICAM1）和酪氨酸激酶2（JAK2）基因相对表达量均显著增加（P<0.05）。与对照组相比,28 d时Toll样受体1（TLR1）基因、0 d时肿瘤坏死因子受体超家族成员Ⅰ型（TNFRSF1A）基因相对表达量均显著降低（P<0.05）,0 d时B细胞κ轻肽基因增强子核因子抑制因子（NFKBIA）、IL-1β和干扰素（IFNG）基因相对表达量均显著增加（P<0.05）。综上所述,母猪妊娠期摄入12.55 MJ/kg能量水平饲粮可降低后代公猪日增重、平均日采食量和睾丸重;降低后代公猪睾丸内生殖细胞数量及免疫相关细胞因子和睾丸免疫相关基因的表达,从而对后代成年后的免疫能力和繁殖性能产生深远影响。     

Localization of Insulin‐Like Growth Factor‐I (IGF‐I) and IGF‐I Receptor (IGF‐IR) in Equine Testes

The insulin‐like growth factor‐I (IGF‐I) is a key regulator of reproductive functions. IGF‐I actions are primarily mediated by IGF‐IR. The main objective of this research was to evaluate the presence of IGF‐I and IGF‐I Receptor (IGF‐IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF‐I and IGF‐IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF‐I and IGF‐IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti‐human polyclonal antibodies against IGF‐I and IGF‐IR were used as primary antibodies for immunohistochemistry and Western blot. At the pre‐pubertal and pubertal stages, IGF‐I immunolabelling was present in spermatogonia and Leydig cells. At post‐pubertal, adult and aged stages, immunolabelling of IGF‐I was observed in spermatogenic cells (spermatogonia, spermatocyte, spermatid, and spermatozoa) and Leydig cells. Immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at the pre‐pubertal stage. The immunolabelling becomes stronger as the age of animals advance through the post‐pubertal stage. Strong immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at post‐puberty, adult and aged stallions; and faint labelling was seen in spermatocytes at these ages. Immunolabelling of IGF‐I and IGF‐IR was not observed in Sertoli cells. In conclusion, IGF‐I is localized in equine spermatogenic and Leydig cells, and IGF‐IR is present in spermatogonia, spermatocytes and Leydig cells, suggesting that the IGF‐I may be involved in equine spermatogenesis and Leydig cell function as a paracrine/autocrine factor.     

Activation of Akt/protein kinase B and extracellular signal-regulated kinase in rats with acute experimental testicular torsion

The Akt/protein kinase B (PKB) and extracellular signal-regulated kinase (ERK) pathways are involved in cell survival. This study examined the temporal profiles and localization of Akt/PKB and ERK1/2 activation in rat testis after ischemia/reperfusion (I/R). Testicular tissue was collected from normal control rats and rats exposed to reperfusion for 6, 24, and 48 hr after ischemic injury; the tissues were analyzed via Western blotting and immunohistochemistry. Western blot analysis showed that the levels of phosphorylated Akt/PKB (pAkt/PKB) and ERK1/2 (pERK1/2) increased significantly during the first 6-24 hr of reperfusion after ischemia. However, both of these activated proteins were decreased slightly at 48 hr after reperfusion. Immunohistochemically, low levels of pAkt/PKB expression were observed in Sertoli cells from the normal control. After I/R, pAkt/PKB expression increased mainly in the adluminal portion of the Sertoli cells, as well as in spermatogenic cells. In addition, pERK1/2 expression was observed in Sertoli and Leydig cells in the normal control. After I/R, pERK1/2 expression increased in some surviving spermatogenic cells (mainly spermatocytes), as well as in the adluminal portion of Sertoli cells. These results suggest that both Akt/PKB and ERK1/2 are involved in the survival of testicular cells during the early phase of testicular I/R. These pathways may represent important targets for increasing cell survival in testicular injury, including testicular torsion.     

不同饲喂水平对绵羊睾丸发育、睾酮合成相关基因及雄激素受体(AR)表达的影响

旨在研究不同饲喂水平对绵羊睾丸发育、睾酮（T）合成相关基因与雄激素受体（androgen receptor, AR）表达的影响。本研究采用单因素完全随机试验设计,将18只健康、体重相近（（35±0.5） kg）的杜泊羊（♂）×晋中绵羊（♀）杂F1代4月龄公羔随机分为3组,每组6只,分别按照自由采食（AL组）、自由采食量的65%（AL65组）和自由采食量的40%（AL40组）3个水平进行饲喂。当AL组任意1只羔羊体重达到50 kg时全部屠宰,测定睾丸的周径与长度后采集睾丸组织,通过苏木精-伊红（hematoxylin-eosin,H-E）染色观察曲精细管生精上皮结构;酶联免疫（enzyme linked immunosorbent assay,ELISA）法测定睾酮（testosterone,T）的水平;用定量PCR（quantitative real-time PCR,qRT-PCR）检测T合成相关基因和AR mRNA的表达情况;通过免疫组化和Western blotting的方法对绵羊睾丸组织中的AR进行定位与定量分析。结果表明,AL40组羔羊睾丸周径和长度显著低于AL组（P<0.05）,但与AL65组差异不显著（P>0.05）;AL40组曲精细管生精上皮厚度与AL65组差异不显著（P>0.05）,但均显著低于AL组（P<0.05）。随着饲喂水平的提高,生精细胞和间质细胞密度显著增加（P<0.05）,T水平和STAR、3β-HSD基因以及AR mRNA和蛋白表达量均显著提高（P<0.05）;睾丸间质细胞、初级精母细胞、精子细胞、管壁肌样细胞层和间质血管平滑肌细胞中均观察到AR阳性产物。综上所述,绵羊的睾丸发育、T含量、T合成相关基因和AR的表达均受到日粮营养水平的调控。日粮营养水平可能通过改变T水平和生精细胞对T的敏感性来调控精子发生过程,从而影响其性成熟和繁殖性能。     

INSL-3 protein expression in normal and cryptorchid testes of Ziwuling black goats

Ziwuling black goats are typically found in loess plateaus regions and the Ziwuling Nature Reserve. Cryptorchidism is a common disease in this inbred goat, and its pathogenesis has been linked with the expression of insulin-like factor 3 (INSL-3). Therefore, this study aimed to investigate anatomical alterations caused by cryptorchism and the expression and distribution of INSL-3 in normal and cryptorchid testicular tissues. The testicular tissues of 6-month-old Ziwuling black goats were collected for microscopic analyses using histochemical, immunohistochemical, immunofluorescence and biometrical methods, as well as Western blotting to compare the expression and distribution of INSL-3. A lower expression of INSL-3 was observed in cryptorchid compared with normal testicular tissues (p < .01). Cryptorchidism caused a significant reduction in layers of spermatogenic epithelium and tubule areas in Ziwuling black goat (p < .01). The interstitial to seminiferous tubule area ratio was larger in cryptorchid than in normal group. Periodic Acid-Schiff (PAS) staining revealed pronounced positive bands in the interstitial tissue, while positive Alcian blue (AB) staining was not clear, and AB-PAS staining revealed a positive red band in the basement membrane of cryptorchid group. Immunofluorescence revealed a strong signal of INSL-3 expression in Sertoli and peritubular myoid cells, and moderate signal in Leydig and spermatogenic cells in the normal group. However, in cryptorchid testicular tissues, the signal of INSL-3 expression was strong in primary spermatocytes, occasional in Sertoli cells, limited in Leydig cells and absent in peritubular myoid cells. Furthermore, immunohistochemistry showed that INSL-3 expression was higher in normal testes compared with cryptorchid testicular tissues (p < .05), especially in primary spermatocytes and Sertoli cells. Collectively, our results indicate that cryptorchidism is closely related to the disorder of acid glycoprotein metabolism and the reduction in release of INSL-3 from Leydig cells. Moreover, Sertoli and peritubular myoid cells are crucial for INSL signalling and could underpin further research on the mechanism of cryptorchidism in animal.