非特异性睾丸炎睾丸生精上皮的改变
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摘要
目的：本实验应用HE染色、免疫组织化学染色、原位杂交以及磁分离均相酶联免疫（磁酶免）睾酮、黄体生成素(luteinizing hormone, LH)定量测定法等技术方法，研究了注射脂多糖（lipopolysaccharide，LPS）、印度墨水诱导的非特异性睾丸炎时睾丸间质以及生精上皮的改变。主要观察非特异性睾丸炎时曲细精管生精上皮内增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、α-连接素（α-catenin）的变化，支持细胞(Sertoli细胞)表达雄激素结合蛋白（androgen binding protein，ABP）mRNA的变化以及睾丸内间质细胞（Leydig细胞）分泌睾酮量的改变、巨噬细胞诱导型一氧化氮合酶(iNOS)的表达，以期进一步了解睾丸在病理状态下生精功能的受损机制，探讨睾丸生精微环境维持和紊乱的影响因素，为临床诊治睾丸炎性不育提供参考。
方法：实验选用健康成年雄性Wistar大鼠，体重200克左右，由北京军科院实验动物中心提供，将实验动物随机分为3组：对照组（D）、实验组1（S1）、实验组2（S2）。D组大鼠尾静脉及腹腔内间隔注射生理盐水（NS），剂量为5ml/㎏，每天注射一次，共10次。S1组大鼠参考O’Bryan报道的方法加以改进，腹腔内注射LPS，剂量为1mg/kg，隔一天注射一次，共注射5次。S2组大鼠，参考肖长义报道的方法，尾静脉内注射20%的印度墨水，剂量为5ml/kg，每天注射一次，连续注射10次。每组均距第一次注射11天取材,常规乙
醚麻醉动物，迅速打开腹腔，从下腔静脉穿刺取血，静置凝血，高速离心2000rpm，20分钟，取上清以备检测血清睾酮及LH。取睾丸置Bourin’s固定液，常规石蜡包埋、切片，用于光镜观察睾丸病理改变以及免疫组织化学方法检测PCNA、α-catenin在睾丸生精上皮的表达情况，同时观察iNOS在睾丸间质的表达。原位杂交标本用含1/1000DEPC的Bourin’s固定液由睾丸动脉灌流固定睾丸，然后置Bourin’s液中固定2小时，常规脱水、浸蜡、包埋。用原位杂交方法检测ABP mRNA在支持细胞的表达。
结果
1 HE染色：正常对照组大鼠睾丸曲细精管分布均匀，生精上皮规则，自生精小管基底部至腔面，依次有精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子，各级生精细胞排列有序，管腔中可见大量精子。间质无水肿、渗出，可见少量间质细胞，核大而圆，偏位。两实验组均可见生精上皮脱落、变薄，生精细胞排列紊乱，生精上皮出现空泡。严重者曲细精管内生精细胞大量脱落，生精细胞也可以形成多核巨细胞，附在支持细胞之间或脱落入管腔。间质中血管增多，血管壁变厚，在血管周围和曲细精管界膜附近，可见炎性细胞浸润。
2 血清睾酮与LH检测结果
2.1 睾酮: D组血清睾酮量为3.576±1.124ng/ml；S1组由于LPS刺激睾酮的生物合成增加，其值为5.766±1.141ng/ml，与D组比较有明显差别（P﹤0.05）。S2组血清睾酮量无明显变化，其值为4.670±1.060ng/ml，与D组比较无明显差别（P﹥0.05）。
2.2 LH: D组血清LH含量为0.988±0.123mIu/ml；S1组血清中LH含量变化不明显，其值为1.136±0.251 mIu/ml，与D组相比无明显差别（P﹥0.05）；S2组血清LH含量明显减低，其值为0.690±0.134mIu/ml，与D组比较均有显著差别（P﹤0.05）
3 免疫组织化学染色
3.1 iNOS：各组睾丸内均存在iNOS的表达，其分布特点为：iNOS在睾丸间质内的血管平滑肌细胞以及血管内的巨噬细胞表达强阳性，免疫染色呈深棕黄色，而血管外巨噬细胞及其他细胞未着色。S1组着色部位主要位于血管平滑肌细胞，S2组主要位于血管内巨噬细胞。
3.2 PCNA：免疫阳性产物位于细胞核，通常呈颗粒状，小部分呈弥散状。精原细胞PCNA表达强阳性，免疫组化染色为深棕色，初级精母细胞、部分类肌细胞也有着色，精子细胞、精子未见着色。S1和S2组生精上皮内PCNA的表达量比对照组明显减少：对照组每个曲细精管的阳性细胞个数为80±6个，两个实验组每个曲细精管阳性细胞个数明显减少，分别为59±5个（S1）、56±3个（S2），与对照组相比均有明显差异（P﹤0.01）。另外，两个实验组仅精原细胞着色的曲细精管个数占总管数的百分率比对照组也显著升高（P﹤0.01），其值分别为：0.587±0.044（D）、0.673±0.054（S1）、0.732±0.045（S2）。
3.3 α- catenin：α- catenin主要表达于精子、精子细胞以及部分精母细胞，着色呈月牙状或线状，位于胞浆内靠近细胞核的一端或精子头部，精原细胞、支持细胞未着色。对照组阳性反应颗粒平均光密度为0.174±0.013；S1、S2组大鼠
睾丸内α- catenin表达量减少，阳性反应颗粒平均光密度分别为0.150±0.014、0.131±0.007，两实验组与对照组比较均有显著性差异（P﹤0.01）。其中，碳粒（S2组）对α- catenin在生精上皮中表达影响的程度要大（与S1组相比P﹤0.01）。
4 ABP mRNA原位杂交结果：ABP mRNA主要位于支持细胞核内，且多存在于核仁处，原位杂交染色为深棕黄色。D组每个曲细精管着色的细胞核约有16.80±1.98个；S1组睾丸曲细精管上皮表达ABP mRNA量增多，每个曲细精管的阳性细胞核数为21.59±2.68，且比D组有明显差异（P﹤0.05）；而S2组ABP mRNA表达也略有升高，每个曲细精管上皮内阳性细胞核数为
Objective: The present study has investigated the change of testicular spermatogenic epithelium and interstitial tissue with stimulated by a bacterial lipoplysaccharide (LPS) and Indian ink in vivo by using the methods of tissue hemotixylin and eosin (H＆E) staining, tissue immunohistochemistry staining, in situ hybrydization technique, magnetic enzyme immunoassay of serum testosterone and luteinizing hormone (LH).We observed mainly the change of proliferating cell nuclear antigen(PCNA)、α-catenin in the spermatogenic epithelium and the production of androgen binding protein (ABP) mRNA in Sertoli cell. Meanwhile, we also assayed the role of LPS and Indian ink on the production of testosterone by testicular Leydig cells、LH by pituitary and testicular macrophage expression of iNOS in LPS or Indian ink-treated rats. According to these, we expect to understand the testicular damaged mechanism in pathological conditions and discuss the effect factors of maitenance and disorder of testicular spermatogenic microenvironment in order to provide reliable reference for diagnosing and treating immune infertility.
Methods：Experiments were performed on healthy adult
male Wistar rats, weighting about 200-250g.The rats were divided into three groups: control group(D)、expriment group 1(S1)、expriment group 2(S2). The S1 rats were injected ip with saline containing 1mg LPS/㎏, once every two day, for ten days according to the improved way from O’Bryan report. The other experiment group (S2) rats were induced by intravascular injection of Indian Ink, once a day, for ten days. And the rats in the control group were treated with pyrogen-free saline instead of LPS or Indian Ink. After operation rats were anesthetized with ether, then abdomen were exposed via a midline incision and a sample of blood was collected via inferior vena cava puncture in order to examine serum LH and testosterone. Testes were removed and put in Bourin’s fixative, after the testes tissue was fixationed, embeded and sectioned as usual methods of light microscope and immunohistochemistry were involved in order to examine the expession of PCNA、α-catenin、iNOS in testes. The testicular sample used in in situ hybrydization technique was obtained after testicular artery douche with Bourin’s fixative containing 1/1000 DEPC.
Results: 1 HE staining: In normal control group testicular seminiferous tubules distributing were uniform, seminiferous epithelial was organized well. From the basement membrane to the lumen of the seminiferous tubule there were four or five concentric layers of cells including spermatogonium、primary spermatocyte、second spermatocyte、spermatid and spermatozoon. Testicular interstitial tissue was not damaged, it
contains Leydig cells which had a large, sphercal nucleus. Nevertheless, observed under light microscope, the testes of two experiment groups showed that in some seminiferous tubules the seminiferous epithelium became thin and the spermatogenic cells were selectively falling from seminiferous epithelium into the lumine of the tubule, at some region of tubules, the cells appeared to have reversed arranges. Meanwhile, in some othe tubules pathological changes were serious, the spermatogenic cells vanished completely from the seminiferous epithelium or vacuoles and coenocytes were visited in the seminiferous epithelium. In the interstitial tissue the number of cells increased obviously.
2 serum LH and testosterone: 1) serum testosterone: In normal control group serum testosterone concentration was 3.576±1.124ng/ml. After LPS treatment, steroidogenesis was promoted and serum testosterone level increased, it was 5.766±1.141ng/ml. Compared with that of D group it was significant (P<0.05). While Indian Ink had no effect on serum testosterone (P>.05), and it was 4.670±1.060ng/ml. 2) LH: After induced by intravascular injection of Indian Ink, serum LH declined and it was 0.690±0.134mIu/ml. While in control group it was 0.988±0.123mIu/ml (P<0.05). However, compared with that of the control group after determination
方法：实验选用健康成年雄性Wistar大鼠，体重200克左右，由北京军科院实验动物中心提供，将实验动物随机分为3组：对照组（D）、实验组1（S1）、实验组2（S2）。D组大鼠尾静脉及腹腔内间隔注射生理盐水（NS），剂量为5ml/㎏，每天注射一次，共10次。S1组大鼠参考O’Bryan报道的方法加以改进，腹腔内注射LPS，剂量为1mg/kg，隔一天注射一次，共注射5次。S2组大鼠，参考肖长义报道的方法，尾静脉内注射20%的印度墨水，剂量为5ml/kg，每天注射一次，连续注射10次。每组均距第一次注射11天取材,常规乙
醚麻醉动物，迅速打开腹腔，从下腔静脉穿刺取血，静置凝血，高速离心2000rpm，20分钟，取上清以备检测血清睾酮及LH。取睾丸置Bourin’s固定液，常规石蜡包埋、切片，用于光镜观察睾丸病理改变以及免疫组织化学方法检测PCNA、α-catenin在睾丸生精上皮的表达情况，同时观察iNOS在睾丸间质的表达。原位杂交标本用含1/1000DEPC的Bourin’s固定液由睾丸动脉灌流固定睾丸，然后置Bourin’s液中固定2小时，常规脱水、浸蜡、包埋。用原位杂交方法检测ABP mRNA在支持细胞的表达。
结果
1 HE染色：正常对照组大鼠睾丸曲细精管分布均匀，生精上皮规则，自生精小管基底部至腔面，依次有精原细胞、初级精母细胞、次级精母细胞、精子细胞和精子，各级生精细胞排列有序，管腔中可见大量精子。间质无水肿、渗出，可见少量间质细胞，核大而圆，偏位。两实验组均可见生精上皮脱落、变薄，生精细胞排列紊乱，生精上皮出现空泡。严重者曲细精管内生精细胞大量脱落，生精细胞也可以形成多核巨细胞，附在支持细胞之间或脱落入管腔。间质中血管增多，血管壁变厚，在血管周围和曲细精管界膜附近，可见炎性细胞浸润。
2 血清睾酮与LH检测结果
2.1 睾酮: D组血清睾酮量为3.576±1.124ng/ml；S1组由于LPS刺激睾酮的生物合成增加，其值为5.766±1.141ng/ml，与D组比较有明显差别（P﹤0.05）。S2组血清睾酮量无明显变化，其值为4.670±1.060ng/ml，与D组比较无明显差别（P﹥0.05）。
2.2 LH: D组血清LH含量为0.988±0.123mIu/ml；S1组血清中LH含量变化不明显，其值为1.136±0.251 mIu/ml，与D组相比无明显差别（P﹥0.05）；S2组血清LH含量明显减低，其值为0.690±0.134mIu/ml，与D组比较均有显著差别（P﹤0.05）
3 免疫组织化学染色
3.1 iNOS：各组睾丸内均存在iNOS的表达，其分布特点为：iNOS在睾丸间质内的血管平滑肌细胞以及血管内的巨噬细胞表达强阳性，免疫染色呈深棕黄色，而血管外巨噬细胞及其他细胞未着色。S1组着色部位主要位于血管平滑肌细胞，S2组主要位于血管内巨噬细胞。
3.2 PCNA：免疫阳性产物位于细胞核，通常呈颗粒状，小部分呈弥散状。精原细胞PCNA表达强阳性，免疫组化染色为深棕色，初级精母细胞、部分类肌细胞也有着色，精子细胞、精子未见着色。S1和S2组生精上皮内PCNA的表达量比对照组明显减少：对照组每个曲细精管的阳性细胞个数为80±6个，两个实验组每个曲细精管阳性细胞个数明显减少，分别为59±5个（S1）、56±3个（S2），与对照组相比均有明显差异（P﹤0.01）。另外，两个实验组仅精原细胞着色的曲细精管个数占总管数的百分率比对照组也显著升高（P﹤0.01），其值分别为：0.587±0.044（D）、0.673±0.054（S1）、0.732±0.045（S2）。
3.3 α- catenin：α- catenin主要表达于精子、精子细胞以及部分精母细胞，着色呈月牙状或线状，位于胞浆内靠近细胞核的一端或精子头部，精原细胞、支持细胞未着色。对照组阳性反应颗粒平均光密度为0.174±0.013；S1、S2组大鼠
睾丸内α- catenin表达量减少，阳性反应颗粒平均光密度分别为0.150±0.014、0.131±0.007，两实验组与对照组比较均有显著性差异（P﹤0.01）。其中，碳粒（S2组）对α- catenin在生精上皮中表达影响的程度要大（与S1组相比P﹤0.01）。
4 ABP mRNA原位杂交结果：ABP mRNA主要位于支持细胞核内，且多存在于核仁处，原位杂交染色为深棕黄色。D组每个曲细精管着色的细胞核约有16.80±1.98个；S1组睾丸曲细精管上皮表达ABP mRNA量增多，每个曲细精管的阳性细胞核数为21.59±2.68，且比D组有明显差异（P﹤0.05）；而S2组ABP mRNA表达也略有升高，每个曲细精管上皮内阳性细胞核数为
Objective: The present study has investigated the change of testicular spermatogenic epithelium and interstitial tissue with stimulated by a bacterial lipoplysaccharide (LPS) and Indian ink in vivo by using the methods of tissue hemotixylin and eosin (H＆E) staining, tissue immunohistochemistry staining, in situ hybrydization technique, magnetic enzyme immunoassay of serum testosterone and luteinizing hormone (LH).We observed mainly the change of proliferating cell nuclear antigen(PCNA)、α-catenin in the spermatogenic epithelium and the production of androgen binding protein (ABP) mRNA in Sertoli cell. Meanwhile, we also assayed the role of LPS and Indian ink on the production of testosterone by testicular Leydig cells、LH by pituitary and testicular macrophage expression of iNOS in LPS or Indian ink-treated rats. According to these, we expect to understand the testicular damaged mechanism in pathological conditions and discuss the effect factors of maitenance and disorder of testicular spermatogenic microenvironment in order to provide reliable reference for diagnosing and treating immune infertility.
Methods：Experiments were performed on healthy adult
male Wistar rats, weighting about 200-250g.The rats were divided into three groups: control group(D)、expriment group 1(S1)、expriment group 2(S2). The S1 rats were injected ip with saline containing 1mg LPS/㎏, once every two day, for ten days according to the improved way from O’Bryan report. The other experiment group (S2) rats were induced by intravascular injection of Indian Ink, once a day, for ten days. And the rats in the control group were treated with pyrogen-free saline instead of LPS or Indian Ink. After operation rats were anesthetized with ether, then abdomen were exposed via a midline incision and a sample of blood was collected via inferior vena cava puncture in order to examine serum LH and testosterone. Testes were removed and put in Bourin’s fixative, after the testes tissue was fixationed, embeded and sectioned as usual methods of light microscope and immunohistochemistry were involved in order to examine the expession of PCNA、α-catenin、iNOS in testes. The testicular sample used in in situ hybrydization technique was obtained after testicular artery douche with Bourin’s fixative containing 1/1000 DEPC.
Results: 1 HE staining: In normal control group testicular seminiferous tubules distributing were uniform, seminiferous epithelial was organized well. From the basement membrane to the lumen of the seminiferous tubule there were four or five concentric layers of cells including spermatogonium、primary spermatocyte、second spermatocyte、spermatid and spermatozoon. Testicular interstitial tissue was not damaged, it
contains Leydig cells which had a large, sphercal nucleus. Nevertheless, observed under light microscope, the testes of two experiment groups showed that in some seminiferous tubules the seminiferous epithelium became thin and the spermatogenic cells were selectively falling from seminiferous epithelium into the lumine of the tubule, at some region of tubules, the cells appeared to have reversed arranges. Meanwhile, in some othe tubules pathological changes were serious, the spermatogenic cells vanished completely from the seminiferous epithelium or vacuoles and coenocytes were visited in the seminiferous epithelium. In the interstitial tissue the number of cells increased obviously.
2 serum LH and testosterone: 1) serum testosterone: In normal control group serum testosterone concentration was 3.576±1.124ng/ml. After LPS treatment, steroidogenesis was promoted and serum testosterone level increased, it was 5.766±1.141ng/ml. Compared with that of D group it was significant (P<0.05). While Indian Ink had no effect on serum testosterone (P>.05), and it was 4.670±1.060ng/ml. 2) LH: After induced by intravascular injection of Indian Ink, serum LH declined and it was 0.690±0.134mIu/ml. While in control group it was 0.988±0.123mIu/ml (P<0.05). However, compared with that of the control group after determination
引文
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