C1型尼曼–匹克病小鼠雄性不育的睾丸病理研究
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摘要
该文主要研究C1型尼曼–匹克病小鼠(Npc1~(–/–)小鼠)雄性不育的睾丸病理变化,并进一步探讨Npc1基因对雄性不育的影响。随机选取P60的Npc1~(–/–)和Npc1~(+/+)雄鼠各12只,观察隐睾发生率;然后,随机选取P20、P40和P60的Npc1~(–/–)和Npc1~(+/+)雄鼠睾丸组织,统计睾丸重量并计算睾丸指数, HE染色观察生殖细胞层数并测量生精小管直径, PAS糖原染色观察生殖细胞周期并统计精母细胞在总细胞数中的相对百分比,油红O染色观察间质细胞的脂质存储;最后,随机选择P60的Npc1~(–/–)和Npc1~(+/+)雄鼠睾丸,通过TUNEL染色观察生殖细胞凋亡,并通过Western blot检测凋亡蛋白Cleaved-caspase-3、Cleaved-caspase-9、p53、Bax和Bcl-2的表达水平。结果显示,与Npc1~(+/+)雄鼠相比, Npc1~(–/–)雄鼠均有隐睾发生,睾丸重量和睾丸指数均显著降低(P<0.05, P<0.001),生殖细胞层数不明显,生精小管管径显著减小(P<0.001),生精周期不规律且精母细胞数量显著减少(P<0.001),间质细胞的脂质存储显著减少(P<0.001);生殖细胞凋亡大幅增加(P<0.001), Cleaved-caspase-3、Cleaved-caspase-9、p53、Bax表达量显著升高(P<0.001),Bax/Bcl-2比率显著升高(P<0.001)。该文结果提示, Npc1基因突变导致隐睾、睾丸结构异常、间质细胞脂质储存减少及生殖细胞凋亡,因此, Npc1基因可能成为研究男性不育的新靶点。
In this paper, we mainly studied the testicular pathological changes of male infertility in Niemann-Pick disease type C1 mice(Npc1~(–/–)mice), and further explored the effect of Npc1 gene on male infertility.Twelve P60 Npc1~(–/–) and Npc1~(+/+) male mice were randomly selected to observe the incidence of cryptorchidism. The testicular tissues were collected from P20, P40 and P60 Npc1~(–/–) and Npc1~(+/+) male mice to calculate testicular weight and testicular index. HE staining was performed to observe the layers of germ cells and measure the diameter of seminiferous tubules. PAS staining was used to observe the cycle of germ cells and count relative percentage of spermatocyte in the total number of cells. Oil red O staining was performed to observe the lipid storage of leydig cells. Finally, testicular tissues of P60 Npc1~(–/–) and Npc1~(+/+) male mice were randomly selected to observe the apoptosis of germ cells by TUNEL staining, and the expression levels of apoptotic proteins Cleaved-caspase-3, Cleavedcaspase-9, p53, Bax and Bcl-2 were detected by Western blot. The results showed, compared with Npc1~(+/+) male mice, cryptorchidism occurred in all Npc1~(–/–) mice, testicular weight and index were decreased significantly(P<0.05, P<0.001), germ cell layers were not obvious, diameter of seminiferous tubules were also decreased significantly(P<0.001), spermatogenic cycle was irregular and the spermatogonial cells number were decreased significantly(P<0.001), and lipid storage in leydig cells were also decreased significantly(P<0.001). Apoptosis increased significantly(P<0.001), and the expression of Cleaved-caspase-3, Cleaved-caspase-9, p53 and Bax increased significantly(P<0.001) and the ratio of Bax/Bcl-2 increased significantly(P<0.001). The mutation of Npc1 gene leads to cryptorchidism, the decrease of lipid storage in leydig cells, and the apoptosis of germ cells. Therefore, Npc1 gene may become the new targets for male infertility.
In this paper, we mainly studied the testicular pathological changes of male infertility in Niemann-Pick disease type C1 mice(Npc1~(–/–)mice), and further explored the effect of Npc1 gene on male infertility.Twelve P60 Npc1~(–/–) and Npc1~(+/+) male mice were randomly selected to observe the incidence of cryptorchidism. The testicular tissues were collected from P20, P40 and P60 Npc1~(–/–) and Npc1~(+/+) male mice to calculate testicular weight and testicular index. HE staining was performed to observe the layers of germ cells and measure the diameter of seminiferous tubules. PAS staining was used to observe the cycle of germ cells and count relative percentage of spermatocyte in the total number of cells. Oil red O staining was performed to observe the lipid storage of leydig cells. Finally, testicular tissues of P60 Npc1~(–/–) and Npc1~(+/+) male mice were randomly selected to observe the apoptosis of germ cells by TUNEL staining, and the expression levels of apoptotic proteins Cleaved-caspase-3, Cleavedcaspase-9, p53, Bax and Bcl-2 were detected by Western blot. The results showed, compared with Npc1~(+/+) male mice, cryptorchidism occurred in all Npc1~(–/–) mice, testicular weight and index were decreased significantly(P<0.05, P<0.001), germ cell layers were not obvious, diameter of seminiferous tubules were also decreased significantly(P<0.001), spermatogenic cycle was irregular and the spermatogonial cells number were decreased significantly(P<0.001), and lipid storage in leydig cells were also decreased significantly(P<0.001). Apoptosis increased significantly(P<0.001), and the expression of Cleaved-caspase-3, Cleaved-caspase-9, p53 and Bax increased significantly(P<0.001) and the ratio of Bax/Bcl-2 increased significantly(P<0.001). The mutation of Npc1 gene leads to cryptorchidism, the decrease of lipid storage in leydig cells, and the apoptosis of germ cells. Therefore, Npc1 gene may become the new targets for male infertility.
引文
1 Inhorn MC,Patrizio P.Infertility around the globe:new thinking on gender,reproductive technologies and global movements in the 21st century.Hum Reprod Update 2015;21(4):411-26.
2 Kumar N,Singh AK.Trends of male factor infertility,an important cause of infertility:a review of literature.J Hum Reprod Sci2015;8(4):191-6.
3 Mazur DJ,Lipshultz LI.Infertility in the aging male.Curr Urol Rep 2018;19(7):54.
4 Kanatsu-Shinohara M.Spermatogonial stem cell self-renewal and development.Annu Rev Cell Dev Biol 2013;29:163-87.
5 Rego T,Farrand S,Goh AMY,Eratne D,Kelso W,Mangelsdorf S,et al.Psychiatric and cognitive symptoms associated with Niemann-Pick type C disease:neurobiology and management.CNS Drugs 2019;33(2):125-42.
6 Gévry NY,Murphy BD.The role and regulation of the NiemannPick C1 gene in adrenal steroidogenesis.Endocr Res 2002;28(8):403-12.
7 Kamiya S,Yamagami T,Umeda M,Sugiyama M,Daigo M.Lectin histochemistry of foamy cells in non-nervous tissues of feline sphingomyelinosis.J Comp Pathol 1991;105(2):241-5.
8 Roff CF,Strauss JF,Goldin E,Jaffe H,Patterson MC,Agritellis GC,et al.The murine Niemann-Pick type C lesion affects testosterone production.Endocrinology 1993;133(6):2913-23.
9 Fan J,Akabane H,Graham SN,Richardson LL,Zhu GZ.Sperm defects in mice lacking a functional Niemann-pick C1 protein.Mol Reprod Dev 2006;73(10):1284-91.
10 Gévry NY,Lopes FL,Ledoux S,Murphy BD.Aberrant intracellular cholesterol transport disrupts pituitary and ovarian function.Mol Endocrinol 2004;18(7):1778-86.
11 Donohue C,Marion S,Erickson RP.Expression of Npc1 in glial cells corrects sterility in Npc1-/-mice.J Appl Genet 2009;50(4):385-90.
12 Trussell JC,Lee PA.The relationship of cryptorchidism to fertility.Curr Urol Rep 2004;5(2):142-8.
13 Flesch FM,Gadella BM.Dynamics of the mammalian sperm plasma membrane in the process of fertilization.Biochim Biophys Acta 2000;1469(3):197-235.
14 Wang C,Ma Z,Scott MP,Huang X.The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis.Dev Biol 2011;351(1):146-55.
15白晨,张茨,杜贤进.小鼠隐睾模型研究进展.实用医学杂志(Bai Chen,Zhang Ci,Du Xianjin.Research progress of mouse cryptorchidism model.The Journal of Practical Medicine.)2011;27(7):1308-10.
16 Matzkin ME,Yamashita S,Ascoli M.The ERK1/2 pathway regulates testosterone synthesis by coordinately regulating the expression of steroidogenic genes in Leydig cells.Mol Cell Endocrinol2013;370(1/2):130-7.
17 Castillo ALF,Paredes RME,Vargas VC,Ruiz CH,Lasso CEB,Gómez Beltrán OD.Testicular cancer and cryptorchidism:myth or reality.Cir Pediatr 2013;26(2):98-101.
18 O'Donnell L.Mechanisms of spermiogenesis and spermiation and how they are disturbed.Spermatogenesis 2015;4(2):e979623.
19 Sasso-Cerri E,Giovanoni M,Hayashi H,Miraglia SM.Morphological alterations and(Jerome F.Strauss III,2002#3)intratubular lipid inclusions as indicative of spermatogenic damage in cimetidine-treated rats.Arch Androl 2001;46(1):5-13.
20 Strauss III JF,Liu P,Christenson LK,Watari H.Sterols and intracellular vesicular trafficking:lessons from the study of NPC1.Steroids 2002;67(12):947-51.
21 Stojkov NJ,Janjic MM,Bjelic MM,Mihajlovic AI,Kostic TS,Andric SA.Repeated immobilization stress disturbed steroidogenic machinery and stimulated the expression of cAMP signaling elements and adrenergic receptors in Leydig cells.Am J Physiol Endocrinol Metab 2012;302(10):E1239-51.
22 Koji T.Male germ cell death in mouse testes:possible involvement of Fas and Fas ligand.Med Electron Microsc 2001;34(4):213-22.
23 Tait SWG,Green DR.Mitochondrial regulation of cell death.Cold Spring Harb Perspect Biol 2013;5(9):a008706.
24 Wang XW,Harris CC.p53 tumor-suppressor gene:clues to molecular carcinogenesis.J Cell Physiol 1997;173(2):247-55.
25 Horn HF,Vousden KH.Coping with stress:multiple ways to activate p53.Oncogene 2007;26(9):1306-16.
26 Fouchécourt S,Livera G,Messiaen S,Fumel B,Parent AS,Marine JC,et al.Apoptosis of sertoli cells after conditional ablation of murine double minute 2(Mdm2)gene is p53-dependent and results in male sterility.Cell Death Differ 2016;23(3):521-30.
27 Cheng WL,Lin TY,Tseng YH,Chu FH,Chueh PJ,Kuo YH,et al.Inhibitory effect of human breast cancer cell proliferation via p21-mediated G1 cell cycle arrest by araliadiol isolated from Aralia cordata Thunb.Planta Med 2011;77(2):164-8.
28 Laptenko O,Prives C.Transcriptional regulation by p53:one protein,many possibilities.Cell Death Differ 2006;13(6):951-61.
2 Kumar N,Singh AK.Trends of male factor infertility,an important cause of infertility:a review of literature.J Hum Reprod Sci2015;8(4):191-6.
3 Mazur DJ,Lipshultz LI.Infertility in the aging male.Curr Urol Rep 2018;19(7):54.
4 Kanatsu-Shinohara M.Spermatogonial stem cell self-renewal and development.Annu Rev Cell Dev Biol 2013;29:163-87.
5 Rego T,Farrand S,Goh AMY,Eratne D,Kelso W,Mangelsdorf S,et al.Psychiatric and cognitive symptoms associated with Niemann-Pick type C disease:neurobiology and management.CNS Drugs 2019;33(2):125-42.
6 Gévry NY,Murphy BD.The role and regulation of the NiemannPick C1 gene in adrenal steroidogenesis.Endocr Res 2002;28(8):403-12.
7 Kamiya S,Yamagami T,Umeda M,Sugiyama M,Daigo M.Lectin histochemistry of foamy cells in non-nervous tissues of feline sphingomyelinosis.J Comp Pathol 1991;105(2):241-5.
8 Roff CF,Strauss JF,Goldin E,Jaffe H,Patterson MC,Agritellis GC,et al.The murine Niemann-Pick type C lesion affects testosterone production.Endocrinology 1993;133(6):2913-23.
9 Fan J,Akabane H,Graham SN,Richardson LL,Zhu GZ.Sperm defects in mice lacking a functional Niemann-pick C1 protein.Mol Reprod Dev 2006;73(10):1284-91.
10 Gévry NY,Lopes FL,Ledoux S,Murphy BD.Aberrant intracellular cholesterol transport disrupts pituitary and ovarian function.Mol Endocrinol 2004;18(7):1778-86.
11 Donohue C,Marion S,Erickson RP.Expression of Npc1 in glial cells corrects sterility in Npc1-/-mice.J Appl Genet 2009;50(4):385-90.
12 Trussell JC,Lee PA.The relationship of cryptorchidism to fertility.Curr Urol Rep 2004;5(2):142-8.
13 Flesch FM,Gadella BM.Dynamics of the mammalian sperm plasma membrane in the process of fertilization.Biochim Biophys Acta 2000;1469(3):197-235.
14 Wang C,Ma Z,Scott MP,Huang X.The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis.Dev Biol 2011;351(1):146-55.
15白晨,张茨,杜贤进.小鼠隐睾模型研究进展.实用医学杂志(Bai Chen,Zhang Ci,Du Xianjin.Research progress of mouse cryptorchidism model.The Journal of Practical Medicine.)2011;27(7):1308-10.
16 Matzkin ME,Yamashita S,Ascoli M.The ERK1/2 pathway regulates testosterone synthesis by coordinately regulating the expression of steroidogenic genes in Leydig cells.Mol Cell Endocrinol2013;370(1/2):130-7.
17 Castillo ALF,Paredes RME,Vargas VC,Ruiz CH,Lasso CEB,Gómez Beltrán OD.Testicular cancer and cryptorchidism:myth or reality.Cir Pediatr 2013;26(2):98-101.
18 O'Donnell L.Mechanisms of spermiogenesis and spermiation and how they are disturbed.Spermatogenesis 2015;4(2):e979623.
19 Sasso-Cerri E,Giovanoni M,Hayashi H,Miraglia SM.Morphological alterations and(Jerome F.Strauss III,2002#3)intratubular lipid inclusions as indicative of spermatogenic damage in cimetidine-treated rats.Arch Androl 2001;46(1):5-13.
20 Strauss III JF,Liu P,Christenson LK,Watari H.Sterols and intracellular vesicular trafficking:lessons from the study of NPC1.Steroids 2002;67(12):947-51.
21 Stojkov NJ,Janjic MM,Bjelic MM,Mihajlovic AI,Kostic TS,Andric SA.Repeated immobilization stress disturbed steroidogenic machinery and stimulated the expression of cAMP signaling elements and adrenergic receptors in Leydig cells.Am J Physiol Endocrinol Metab 2012;302(10):E1239-51.
22 Koji T.Male germ cell death in mouse testes:possible involvement of Fas and Fas ligand.Med Electron Microsc 2001;34(4):213-22.
23 Tait SWG,Green DR.Mitochondrial regulation of cell death.Cold Spring Harb Perspect Biol 2013;5(9):a008706.
24 Wang XW,Harris CC.p53 tumor-suppressor gene:clues to molecular carcinogenesis.J Cell Physiol 1997;173(2):247-55.
25 Horn HF,Vousden KH.Coping with stress:multiple ways to activate p53.Oncogene 2007;26(9):1306-16.
26 Fouchécourt S,Livera G,Messiaen S,Fumel B,Parent AS,Marine JC,et al.Apoptosis of sertoli cells after conditional ablation of murine double minute 2(Mdm2)gene is p53-dependent and results in male sterility.Cell Death Differ 2016;23(3):521-30.
27 Cheng WL,Lin TY,Tseng YH,Chu FH,Chueh PJ,Kuo YH,et al.Inhibitory effect of human breast cancer cell proliferation via p21-mediated G1 cell cycle arrest by araliadiol isolated from Aralia cordata Thunb.Planta Med 2011;77(2):164-8.
28 Laptenko O,Prives C.Transcriptional regulation by p53:one protein,many possibilities.Cell Death Differ 2006;13(6):951-61.