孔雀石綠對成年小鼠睪丸結(jié)構(gòu)與功能的影響
發(fā)布時間：2018-09-01 18:15
【摘要】： 目的 觀察孔雀石綠(malachite green，MG)急性和慢性染毒對雄性成年小鼠睪丸組織學(xué)結(jié)構(gòu)及超微結(jié)構(gòu)的影響，以了解MG對小鼠睪丸組織及細(xì)胞的損傷特點；研究MG對睪丸內(nèi)凋亡誘導(dǎo)蛋白Fas／Fas-L表達(dá)的影響，以明確MG對小鼠睪丸生精上皮的損傷特點；分析染毒后睪丸基因組隨機(jī)擴(kuò)增多態(tài)性DNA分子標(biāo)記擴(kuò)增圖譜的變化，以揭示MG對睪丸遺傳物質(zhì)的損傷。上述研究有助于深入地了解MG對雄性生殖系統(tǒng)的損傷及其特點，增加環(huán)境毒物與生殖健康相互關(guān)系的認(rèn)識，為雄(男)性不育癥的病因?qū)W提供參考資料。 材料與方法 1．實驗動物及分組 實驗動物為6-8周齡的雄性BALB／c清潔級小鼠，體重18-22g。用于急性實驗的小鼠共32只，隨機(jī)均分為4組，按照MG濃度設(shè)為：A組(0 mg／L)、B組(100 mg／L)、C組(400 mg／L)、D組(800 mg／L)；用于慢性實驗的小鼠共16只，隨機(jī)均分為4組后按照MG濃度設(shè)為A’組(0 mg／L)、B’組(100 mg／L)、C’組(300 mg／L)、D’組(600 mg／L)。急性實驗組連續(xù)灌胃給藥30d，慢性實驗組灌胃給藥持續(xù)90d，急、慢性實驗組中的MG均溶于生理鹽水，A和A’組則以等量生理鹽水代替MG。 2．檢測方法 各實驗組均檢測小鼠體重、睪丸及附睪重量；應(yīng)用HE染色及透射電鏡觀察睪丸組織結(jié)構(gòu)及超微結(jié)構(gòu)的變化；用免疫組化法檢測凋亡誘導(dǎo)蛋白Fas／Fas-L的表達(dá)部位及表達(dá)量的改變；用隨機(jī)擴(kuò)增多態(tài)性DNA分子標(biāo)記對睪丸基因組進(jìn)行PCR擴(kuò)增，通過比較擴(kuò)增圖譜的差異來鑒定睪丸基因組的損傷。 結(jié)果 1．小鼠體重、睪丸與附睪重量的變化 急、慢性染毒后，各實驗組小鼠體重均比對照組減輕，D和D’組的體重減輕具顯著性(P＜0.05)；各實驗組小鼠睪丸重量均比對照組有所降低，其中C’、D、D’組的睪丸重量降低具顯著性(P＜0.05，P＜0.01)；附睪重量僅在D’組明顯降低(P＜0.01)，在其它組無顯著變化(P＞0.05)。 2．小鼠睪丸組織學(xué)結(jié)構(gòu)的變化 急、慢性染毒均能造成睪丸的組織的病理學(xué)改變，急性染毒可使生精上皮細(xì)胞層數(shù)減少、生精細(xì)胞排列紊亂或脫落入管腔、精子生成減少等。慢性實驗組損傷比急性實驗組更加嚴(yán)重，隨劑量增加，急、慢性實驗組的病理變化均更為明顯。 3．小鼠睪丸超微結(jié)構(gòu)的改變 各實驗組小鼠的超微結(jié)構(gòu)均與對照組的有明顯差異，可見基膜皺褶，精原細(xì)胞和支持細(xì)胞胞漿空泡化，支持細(xì)胞與生精細(xì)胞間連接消失，線粒體腫脹、空泡化，精子形態(tài)異常等。慢性實驗組變化比急性實驗組更加明顯，而急、慢性實驗組的病理變化都隨劑量增大而加重。 4．小鼠睪丸內(nèi)Fas／Fas-L的表達(dá) Fas的陽性表達(dá)集中于支持細(xì)胞胞漿以及精子和其殘余胞漿，F(xiàn)as-L的陽性表達(dá)定位于支持細(xì)胞的胞漿及管腔中集合在一起的絮狀精子尾。Fas在除B組外的其它各組精子及支持細(xì)胞的表達(dá)均比對照組顯著增強(qiáng)(P＜0.05)；Fas-L在各實驗組支持細(xì)胞的表達(dá)均顯著強(qiáng)于對照組(P＜0.05)，而其在精子尾的表達(dá)則無明顯變化(P＞0.05)。各實驗組支持細(xì)胞內(nèi)Fas／Fas-L的表達(dá)水平均隨劑量增加而升高，急、慢性實驗組間無明顯差別。 5．小鼠睪丸基因組RAPD分子標(biāo)記的改變 各實驗組睪丸基因組DNA的RAPD分子標(biāo)記擴(kuò)增圖譜發(fā)生改變，包括擴(kuò)增條帶明亮的變化，擴(kuò)增條帶數(shù)目的改變和差異條帶的產(chǎn)生。各實驗組與對照組的差異條帶的比例隨染毒劑量增大而增大，急、慢性實驗組間無明顯差別。 結(jié)論 1．MG對小鼠的急、慢性染毒均能導(dǎo)致小鼠體重、睪丸重量的減輕，且減輕程度隨劑量增大而加大。提示MG能夠?qū)ΣG丸造成損傷，且損傷呈劑量效應(yīng)。 2．MG的急、慢性染毒均能造成睪丸的組織學(xué)結(jié)構(gòu)及超微結(jié)構(gòu)的病理損傷，慢性染毒組的病理損傷更嚴(yán)重，且急、慢性染毒后睪丸的損傷程度都隨劑量增加而加重。提示MG能夠?qū)ΣG丸內(nèi)多種細(xì)胞造成損傷，且損傷呈劑量效應(yīng)，，長期低劑量的MG染毒比短期高劑量的染毒更能導(dǎo)致睪丸損傷。 3．MG急、慢性染毒均可誘導(dǎo)睪丸內(nèi)的凋亡蛋白Fas／Fas-L在支持細(xì)胞的表達(dá)上調(diào)，且上調(diào)水平隨劑量增加而升高。提示MG可能通過Fas／Fas-L通路使生精細(xì)胞凋亡增加，從而對睪丸造成損傷，使精子生成減少，生育力降低。 4．MG急、慢性染毒均使睪丸基因組DNA的RAPD分子標(biāo)記擴(kuò)增圖譜改變，提示MG能對睪丸基因組DNA造成損傷，DNA的損傷可進(jìn)一步誘發(fā)睪丸癌，或者精子DNA的損傷致使受精后的胚胎異常。
[Abstract]:objective
To observe the effects of malachite green (MG) on histology and ultrastructure of testis in male adult mice, to understand the damage characteristics of MG on testis tissue and cells, to study the effect of MG on the expression of apoptosis-inducing protein Fas/Fas-L in testis, and to clarify the damage of MG on spermatogenic epithelium of testis in mice. The above research will help us to understand the damage of MG to male reproductive system and its characteristics, and increase the understanding of the relationship between environmental toxicity and reproductive health, so as to be male (male) infertility. Provide references for etiology.
Materials and methods
1. experimental animals and groups
Thirty-two male BALB/c clean-grade mice aged 6-8 weeks, weighing 18-22 g, were randomly divided into four groups according to MG concentration: group A (0 mg/L), group B (100 mg/L), group C (400 mg/L), and group D (800 mg/L); and 16 mice for chronic experiment were randomly divided into four groups and then set to A'according to MG concentration. Group A (100 mg / L), group B (100 mg / L), group C (300 mg / L) and group D (600 mg / L). The acute group was given continuous intragastric administration for 30 days, the chronic group for 90 days, the acute group and the chronic group were all dissolved in normal saline, and group A and A were given the same amount of normal saline instead of MG.
2. detection methods
The weight of mice, testis and epididymis were measured in each experimental group; the histological and ultrastructural changes of testis were observed by HE staining and transmission electron microscopy; the expression of apoptosis-inducing protein Fas/Fas-L was detected by immunohistochemistry; the genome of testis was amplified by random amplified polymorphic DNA molecular markers by PCR The difference in amplification map was used to identify testicular genome damage.
Result
1. body weight, testicular and epididymal weight in mice
After acute and chronic exposure, the weight of mice in each experimental group was lower than that in the control group, and the weight of mice in D and D'groups was significantly lower than that in the control group (P < 0.05). There was no significant change in other groups (P > 0.05).
2. histological changes of testis in mice
Acute and chronic exposure can cause pathological changes in testis. Acute exposure can reduce the number of spermatogenic epithelial cells, disorder of spermatogenic cells arrangement or fall into the lumen, and decrease spermatogenesis.
3. changes in the ultrastructure of testis in mice
The ultrastructure of the mice in each experimental group was obviously different from that in the control group. There were basement membrane folds, vacuolation of spermatogonia and Sertoli cells, disappearance of junction between Sertoli cells and spermatogenic cells, mitochondrial swelling, vacuolation, abnormal sperm morphology and so on. Pathological changes were aggravated with the increase of dose.
4. expression of Fas / Fas-L in testis of mice
The positive expression of Fas was concentrated in the cytoplasm of Sertoli cells, sperm and their residual cytoplasm. The positive expression of Fas-L was located in the cytoplasm of Sertoli cells and the flocculent sperm tail in lumen. The expression of Fas in sperm and Sertoli cells of all groups except group B was significantly higher than that of control group (P < 0.05). The expression of Fas/Fas-L in Sertoli cells was significantly higher than that in control group (P < 0.05), but there was no significant change in the expression of Fas/Fas-L in sperm tail (P > 0.05).
5. the change of RAPD molecular marker in mouse testis genome
The RAPD molecular marker amplification profiles of testicular genomic DNA in each experimental group changed, including the brightness of amplified bands, the number of amplified bands and the generation of differential bands.
conclusion
1. Both acute and chronic exposure to MG can result in weight loss and testicular weight loss in mice, and the degree of reduction increases with the increase of dose.
2. Both acute and chronic exposure to MG can cause histological and ultrastructural pathological damage of the testis. The pathological damage of the chronic exposure group is more serious, and the degree of the damage of the testis is aggravated with the increase of dosage. Exposure to poison is more likely to cause testicular injury than short-term high-dose exposure.
3. Both acute and chronic exposure to MG can induce the expression of apoptotic protein Fas/Fas-L in Sertoli cells to be up-regulated, and the up-regulated level increases with the increase of dosage.
4. Both acute and chronic exposure to MG can alter the RAPD molecular marker amplification profiles of testicular genomic DNA, suggesting that MG can cause damage to testicular genomic DNA, DNA damage can further induce testicular cancer, or sperm DNA damage can cause abnormal embryos after fertilization.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R698.2;R363
本文編號：2217969
[Abstract]:objective
To observe the effects of malachite green (MG) on histology and ultrastructure of testis in male adult mice, to understand the damage characteristics of MG on testis tissue and cells, to study the effect of MG on the expression of apoptosis-inducing protein Fas/Fas-L in testis, and to clarify the damage of MG on spermatogenic epithelium of testis in mice. The above research will help us to understand the damage of MG to male reproductive system and its characteristics, and increase the understanding of the relationship between environmental toxicity and reproductive health, so as to be male (male) infertility. Provide references for etiology.
Materials and methods
1. experimental animals and groups
Thirty-two male BALB/c clean-grade mice aged 6-8 weeks, weighing 18-22 g, were randomly divided into four groups according to MG concentration: group A (0 mg/L), group B (100 mg/L), group C (400 mg/L), and group D (800 mg/L); and 16 mice for chronic experiment were randomly divided into four groups and then set to A'according to MG concentration. Group A (100 mg / L), group B (100 mg / L), group C (300 mg / L) and group D (600 mg / L). The acute group was given continuous intragastric administration for 30 days, the chronic group for 90 days, the acute group and the chronic group were all dissolved in normal saline, and group A and A were given the same amount of normal saline instead of MG.
2. detection methods
The weight of mice, testis and epididymis were measured in each experimental group; the histological and ultrastructural changes of testis were observed by HE staining and transmission electron microscopy; the expression of apoptosis-inducing protein Fas/Fas-L was detected by immunohistochemistry; the genome of testis was amplified by random amplified polymorphic DNA molecular markers by PCR The difference in amplification map was used to identify testicular genome damage.
Result
1. body weight, testicular and epididymal weight in mice
After acute and chronic exposure, the weight of mice in each experimental group was lower than that in the control group, and the weight of mice in D and D'groups was significantly lower than that in the control group (P < 0.05). There was no significant change in other groups (P > 0.05).
2. histological changes of testis in mice
Acute and chronic exposure can cause pathological changes in testis. Acute exposure can reduce the number of spermatogenic epithelial cells, disorder of spermatogenic cells arrangement or fall into the lumen, and decrease spermatogenesis.
3. changes in the ultrastructure of testis in mice
The ultrastructure of the mice in each experimental group was obviously different from that in the control group. There were basement membrane folds, vacuolation of spermatogonia and Sertoli cells, disappearance of junction between Sertoli cells and spermatogenic cells, mitochondrial swelling, vacuolation, abnormal sperm morphology and so on. Pathological changes were aggravated with the increase of dose.
4. expression of Fas / Fas-L in testis of mice
The positive expression of Fas was concentrated in the cytoplasm of Sertoli cells, sperm and their residual cytoplasm. The positive expression of Fas-L was located in the cytoplasm of Sertoli cells and the flocculent sperm tail in lumen. The expression of Fas in sperm and Sertoli cells of all groups except group B was significantly higher than that of control group (P < 0.05). The expression of Fas/Fas-L in Sertoli cells was significantly higher than that in control group (P < 0.05), but there was no significant change in the expression of Fas/Fas-L in sperm tail (P > 0.05).
5. the change of RAPD molecular marker in mouse testis genome
The RAPD molecular marker amplification profiles of testicular genomic DNA in each experimental group changed, including the brightness of amplified bands, the number of amplified bands and the generation of differential bands.
conclusion
1. Both acute and chronic exposure to MG can result in weight loss and testicular weight loss in mice, and the degree of reduction increases with the increase of dose.
2. Both acute and chronic exposure to MG can cause histological and ultrastructural pathological damage of the testis. The pathological damage of the chronic exposure group is more serious, and the degree of the damage of the testis is aggravated with the increase of dosage. Exposure to poison is more likely to cause testicular injury than short-term high-dose exposure.
3. Both acute and chronic exposure to MG can induce the expression of apoptotic protein Fas/Fas-L in Sertoli cells to be up-regulated, and the up-regulated level increases with the increase of dosage.
4. Both acute and chronic exposure to MG can alter the RAPD molecular marker amplification profiles of testicular genomic DNA, suggesting that MG can cause damage to testicular genomic DNA, DNA damage can further induce testicular cancer, or sperm DNA damage can cause abnormal embryos after fertilization.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R698.2;R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 周立紅,陳學(xué)豪,徐長安;用微核技術(shù)研究孔雀石綠對魚的誘變作用[J];集美大學(xué)學(xué)報(自然科學(xué)版);1997年02期
2 邱緒建;林洪;王聯(lián)珠;江潔;王建華;;孔雀石綠及其代謝物無色孔雀石綠在鯽魚肌肉中的代謝規(guī)律[J];中國海洋大學(xué)學(xué)報(自然科學(xué)版);2006年05期
3 黃英,柯才煥,周時強(qiáng);幾種藥物對波部東風(fēng)螺早期發(fā)育的影響[J];廈門大學(xué)學(xué)報(自然科學(xué)版);2001年03期
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