Ablation effect of busulfan on pig endogenous spermatogonial stem cells and transplantation of exogenous spermatogonial stem cells
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摘要:目的
研究猪睾丸组织注射白消安消融猪内源精原干细胞(Spermatogonial stem cell，SSC)的效果，以及猪SSC同种异体移植后对内源性SSC消融受体生殖能力恢复的影响。
方法采用3 mg/kg剂量的白消安对9头6周龄大白公猪进行睾丸注射，另外3头注射2 mL 二甲基亚砜作为对照。3周后，对试验组公猪以相同剂量进行第2次睾丸注射。第2次注射3周后，采集试验组和对照组公猪睾丸进行相关检测，评估内源性SSC消融情况。第2次白消安注射1个月后，以两步酶消化法处理5~7日龄大白仔猪睾丸分离得到睾丸单细胞悬液，并用明胶差速贴壁法进行纯化，纯化后以免疫荧光和流式细胞术分析SSC的纯度。异体移植SSC 4个月后，用微卫星标记检测受体公猪精液以及睾丸组织中供体来源SSC的存在。
结果以3 mg/kg剂量的白消安处理大白公猪2次后，睾丸组织苏木精−伊红染色以及免疫组织化学染色结果显示，试验组睾丸曲细精管中各级生精细胞消融但其支持细胞结构完好，可以支持外源性SSC的定植及发育。免疫荧光以及流式细胞术结果表明，分离得到的睾丸单细胞悬液经纯化后UCHL-1阳性细胞占比由差速贴壁前的16.3%提高到了50.8%。苏木精−伊红染色以及免疫组织化学染色结果显示，猪SSC移植4个月后，移植组睾丸组织生精细胞恢复，在受体睾丸曲细精管基底膜上可检测到UCHL-1阳性SSC。受体睾丸组织的微卫星标记分析显示了供体SSC的存在，表明移植进入受体睾丸中的供体SSC可以在受体睾丸中定植存活超过4个月；精液微卫星标记未检测到供体来源的精子。
结论以3 mg/kg剂量的白消安注射公猪睾丸能有效消融内源性SSC，可以用来制备SSC移植的受体猪。两步酶消化及明胶差速贴壁法可成功分离纯化猪SSC。猪SSC经同种异体移植后可以在受体睾丸中定植存活超过4个月。
Abstract:ObjectiveTo study the effect of intratubular injection of busulfan on ablation of pig endogenous spermatogonial stem cells (SSCs) and allotransplantation of SSCs on the recovery of the reproductive ability of the recipient after ablation of endogenous SSCs.
MethodNine 6-week-old Large-white boars were injected through seminiferous tubules with busulfan at a dosage of 3 mg/kg, and other three pigs were injected with 2 mL DMSO as controls. After three weeks, each boar in the test group was given a second injection at the same dosage. At three weeks after the second injection, testes from test and control groups were collected to evaluate the endogenous SSC ablation conditions. At one month after the second busulfan injection, testicular single-cell suspension was isolated from the testes of 5−7-day-old Large-white piglets by two-step enzyme digestion method and purified by gelatin differential adherence method. After purification, the purity of SSCs was analyzed by immunofluorescence and flow cytometry. At four months after allotransplantation of SSCs, microsatellite marker analysis was used to detect the presence of donor-derived SSCs in recipient boar semen and testicular tissue.
ResultAfter treating the Large-white boar twice with busulfan at a dosage of 3 mg/kg, the results of HE staining and immunohistochemistry staining of testis tissue showed that germ cells at all levels in the seminiferous tubules of the testes in test group were ablated but the structure of sertoli cell was intact, which could support the colonization and development of transplanted exogenous SSC. The results of immunofluorescence and flow cytometry showed that the rate of UCHL-1 positive cells in the isolated testicular single-cell suspension increased from 16.3% before differential adhesion to 50.8% after purification. At four months after allotransplantation of porcine SSC, HE staining and immunohistochemical staining of testicular tissues in the transplanted group showed recovery of germ cell layers compared with the group injected with busulfan but not transplanted with donor cells. UCHL-1 positive SSCs were detected on the basement membrane of seminiferous tubules in recipient testes. Microsatellite marker analysis of recipient testis tissue showed the presence of donor SSCs, suggesting that donor SSCs transplanted into recipient testes could colonize and survive in recipient testes for more than four months. However, microsatellite marker analysis of semen did not detect donor-derived sperms.
ConclusionIntratubular injection of busulfan into boar testes at a dosage of 3 mg/kg can effectively ablate endogenous SSCs and can be used to prepare recipient pigs for SSC transplantation. Two-step enzyme digestion and gelatin differential adhesion method can be used to successfully isolate and purify porcine SSCs. Porcine SSCs can colonize in recipient testes and survive for more than four months after allotransplantation.
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Keywords:
- pig /
- spermatogonial stem cell /
- busulfan /
- cell transplantation /
- spermatogenesis
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图 1 白消安注射后睾丸形态学分析揭示内源性精原干细胞(SSC)消融
Figure 1. Morphological analysis of testis after busulfan injection showed endogenous spermatogonial stem cell(SSC) ablation
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图 2 白消安处理6周后血常规分析
a、c中的“*”和“**”分别表示差异达到0.05和0.01的显著水平(t检验)
Figure 2. Blood routine analysis at six weeks after busulfan treatment
“*” and “**” in figure a and c indicate significant difference at the levels of 0.05 and 0.01, respectively(t test)
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图 4 免疫荧光染色鉴定纯化后精原干细胞(SSC)纯度
Figure 4. Immunofluorescence staining to identify purity of spermatogonial stem cells (SSCs) after purification
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图 5 流式细胞术分析纯化前后睾丸单细胞悬液中精原干细胞(SSC)的比例
对照组未经UCHL-1抗体染色，试验组经由UCHL-1抗体染色；各图中，横坐标FL1-A表示绿色荧光通道(Alexa Flour 488)；0、0、16.3%和50.8%表示各处理细胞群中绿色荧光标记的阳性细胞占比
Figure 5. Flow cytometry analysis of the proportion of spermatogonial stem cells (SSCs) in testicular single cell suspension before and after purification
The control group was not stained with UCHL-1 antibody, and the test group was stained; In each graph, the horizontal axis FL1-A indicates the green fluorescence channel (Alexa Flour 488); 0, 0, 16.3% and 50.8% indicate the proportion of positive cells with green fluorescent labels in cell population
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图 6 移植后受体睾丸精原干细胞(SSC)的恢复
c中红色箭头指向未成熟精子细胞，d中红色箭头指向位于曲细精管基底膜上的SSC
Figure 6. Recovery of spermatogonial stem cells (SSCs) in testis of recipients after transplantation
Red arrow in figure c points at immature sperm cells, and red arrow in figure d points at SSCs located on the basement membrane of seminiferous tubules
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图 7 精原干细胞(SSC)移植后受体睾丸组织微卫星标记分析
图中红色箭头指示在S0155位点出现供体基因型信号；S0355、Sw857位点结果不能区分供体和受体基因型
Figure 7. Microsatellite marker analysis of testes of recipients with spermatogonial stem cell (SSC) transplantation
The red arrows in the figure indicate that the donor genotype signal appears at S0155；The microsatellite results of S0355 and Sw857 loci cannot distinguish the genotype of donor and recipient
表 1 微卫星多态性分析引物序列
Table 1 Primer sequences for polymorphic microsatellites analysis
位点名称
Locus name所在染色体
Chr.引物序列1)(5′→3′)
Primer sequence退火温度/ ℃
Annealing temperature等位基因长度范围/bp
Allele rangeS0155 1 D-TGTTCTCTGTTTCTCCTCTGTTTG
AAAGTGGAAAGAGTCAATGGCTAT55 142~162 S0355 15 D-TCTGGCTCCTACACTCCTTCTTGATG
TTGGGTGGGTGCTGAAAAATAGGA55 244~271 Sw857 14 D-TGAGAGGTCAGTTACAGAAGACC
GATCCTCCTCCAAATCCCAT55 141~159 1)字母“D”表示带荧光标记的引物
1) Letter“D” indicates dye labelled primer
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[1] KERR J, LOVELAND K, OBRYAN M, et al. Cytology of the testis and intrinsic control mechanisms[M]//NEILL J D. Knobil and Neill’s Physiology of Reproduction. Amsterdam: Elsevier, 2006: 827-947.
[2] MULDER C L, ZHENG Y, JAN S Z, et al. Spermatogonial stem cell autotransplantation and germline genomic editing: A future cure for spermatogenic failure and prevention of transmission of genomic diseases[J]. Human Reproduction Update, 2016, 22(5): 561-573. doi: 10.1093/humupd/dmw017
[3] BRINSTER R L, ZIMMERMANN J W. Spermatogenesis following male germ-cell transplantation[J]. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(24): 11298-11302. doi: 10.1073/pnas.91.24.11298
[4] BRINSTER R L, AVARBOCK M R. Germline transmission of donor haplotype following spermatogonial transplantation[J]. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(24): 11303-11307. doi: 10.1073/pnas.91.24.11303
[5] HONARAMOOZ A, MEGEE S O, DOBRINSKI I. Germ cell transplantation in pigs[J]. Biology of Reproduction, 2002, 66(1): 21-28. doi: 10.1095/biolreprod66.1.21
[6] MIKKOLA M, SIRONEN A, KOPP C, et al. Transplantation of normal boar testicular cells resulted in complete focal spermatogenesis in a boar affected by the immotile short-tail sperm defect[J]. Reproduction in Domestic Animals, 2006, 41(2): 124-128. doi: 10.1111/j.1439-0531.2006.00651.x
[7] ZENG W X, TANG L, BONDAREVA A, et al. Viral transduction of male germline stem cells results in transgene transmission after germ cell transplantation in pigs[J]. Biology of Reproduction, 2013, 88(1): 27.
[8] KIM B G, KIM Y H, LEE Y A, et al. Production of transgenic spermatozoa by lentiviral transduction and transplantation of porcine spermatogonial stem cells[J]. Tissue Engineering and Regenerative Medicine, 2014, 11(6): 458-466. doi: 10.1007/s13770-014-0078-8
[9] IZADYAR F, DEN OUDEN K, STOUT T A E, et al. Autologous and homologous transplantation of bovine spermatogonial stem cells[J]. Reproduction, 2003, 126(6): 765-774. doi: 10.1530/rep.0.1260765
[10] HERRID M, VIGNARAJAN S, DAVEY R, et al. Successful transplantation of bovine testicular cells to heterologous recipients[J]. Reproduction, 2006, 132(4): 617-624. doi: 10.1530/rep.1.01125
[11] STOCKWELL S, HERRID M, DAVEY R, et al. Microsatellite detection of donor-derived sperm DNA following germ cell transplantation in cattle[J]. Reproduction, Fertility and Development, 2009, 21(3): 462-468. doi: 10.1071/RD08130
[12] HONARAMOOZ A, BEHBOODI E, MEGEE S O, et al. Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats[J]. Biology of Reproduction, 2003, 69(4): 1260-1264. doi: 10.1095/biolreprod.103.018788
[13] HONARAMOOZ A, BEHBOODI E, BLASH S, et al. Germ cell transplantation in goats[J]. Molecular Reproduction and Development, 2003, 64(4): 422-428. doi: 10.1002/mrd.10205
[14] KAUL G, KAUR J, RAFEEQI T A. Ultrasound guided transplantation of enriched and cryopreserved spermatogonial cell suspension in goats[J]. Reproduction in Domestic Animals, 2010, 45(6): e249-e254. doi: 10.1111/j.1439-0531.2009.01549.x
[15] RODRIGUEZ-SOSA J R, DOBSON H, HAHNEL A. Isolation and transplantation of spermatogonia in sheep[J]. Theriogenology, 2006, 66(9): 2091-2103. doi: 10.1016/j.theriogenology.2006.03.039
[16] HERRID M, DAVEY R, STOCKWELL S, et al. A shorter interval between irradiation of recipient testis and germ cell transplantation is detrimental to recovery of fertility in rams[J]. International Journal of Andrology, 2011, 34(5 Pt 1): 501-512.
[17] HERRID M, OLEJNIK J, JACKSON M, et al. Irradiation enhances the efficiency of testicular germ cell transplantation in sheep[J]. Biology of Reproduction, 2009, 81(5): 898-905. doi: 10.1095/biolreprod.109.078279
[18] STOCKWELL S, HILL J R, DAVEY R, et al. Transplanted germ cells persist long-term in irradiated ram testes[J]. Animal Reproduction Science, 2013, 142(3/4): 137-140. doi: 10.1016/j.anireprosci.2013.09.012
[19] SCHLATT S, FOPPIANI L, ROLF C, et al. Germ cell transplantation into X-irradiated monkey testes[J]. Human Reproduction, 2002, 17(1): 55-62. doi: 10.1093/humrep/17.1.55
[20] SCHLATT S, ROSIEPEN G, WEINBAUER G F, et al. Germ cell transfer into rat, bovine, monkey and human testes[J]. Human Reproduction, 1999, 14(1): 144-150. doi: 10.1093/humrep/14.1.144
[21] JAHNUKAINEN K, EHMCKE J, QUADER M A, et al. Testicular recovery after irradiation differs in prepubertal and pubertal non-human primates, and can be enhanced by autologous germ cell transplantation[J]. Human Reproduction, 2011, 26(8): 1945-1954. doi: 10.1093/humrep/der160
[22] HERMANN B P, SUKHWANI M, WINKLER F, et al. Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm[J]. Cell Stem Cell, 2012, 11(5): 715-726. doi: 10.1016/j.stem.2012.07.017
[23] GOOSSENS E, TOURNAYE H. Functional sperm produced after spermatogonial stem cell transplantation into rhesus[J]. Asian Journal of Andrology, 2013, 15(2): 216-217. doi: 10.1038/aja.2012.155
[24] SHETTY G, MITCHELL J M, MEYER J M, et al. Restoration of functional sperm production in irradiated pubertal rhesus monkeys by spermatogonial stem cell transplantation[J]. Andrology, 2020, 8(5): 1428-1441. doi: 10.1111/andr.12807
[25] KIM Y, TURNER D, NELSON J, et al. Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog[J]. Reproduction, 2008, 136(6): 823-831. doi: 10.1530/REP-08-0226
[26] HARKEY M A, ASANO A, ZOULAS M E, et al. Isolation, genetic manipulation, and transplantation of canine spermatogonial stem cells: progress toward transgenesis through the male germ-line[J]. Reproduction, 2013, 146(1): 75-90. doi: 10.1530/REP-13-0086
[27] LI C H, YAN L Z, BAN W Z, et al. Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring[J]. Cell Research, 2017, 27(2): 241-252. doi: 10.1038/cr.2016.156
[28] HERRID M, NAGY P, JUHASZ J, et al. Donor sperm production in heterologous recipients by testis germ cell transplantation in the dromedary camel[J]. Reproduction, Fertility and Development, 2019, 31(3): 538-546. doi: 10.1071/RD18191
[29] GUL M, HILDORF S, DONG L, et al. Review of injection techniques for spermatogonial stem cell transplantation[J]. Human Reproduction Update, 2020, 26(3): 368-391. doi: 10.1093/humupd/dmaa003
[30] GONZÁLEZ R, DOBRINSKI I. Beyond the mouse monopoly: Studying the male germ line in domestic animal models[J]. ILAR Journal, 2015, 56(1): 83-98. doi: 10.1093/ilar/ilv004
[31] KUBOTA H, BRINSTER R L. Spermatogonial stem cells[J]. Biology of Reproduction, 2018, 99(1): 52-74. doi: 10.1093/biolre/ioy077
[32] SAVVULIDI F, PTACEK M, SAVVULIDI VARGOVA K, et al. Manipulation of spermatogonial stem cells in livestock species[J]. Journal of Animal Science and Biotechnology, 2019, 10: 46. doi: 10.1186/s40104-019-0355-4
[33] OGAWA T, ARÉCHAGA J M, AVARBOCK M R, et al. Transplantation of testis germinal cells into mouse seminiferous tubules[J]. International Journal of Developmental Biology, 1997, 41(1): 111-122.
[34] GIASSETTI M I, CICCARELLI M, OATLEY J M. Spermatogonial stem cell transplantation: Insights and outlook for domestic animals[J]. Annual Review of Animal Biosciences, 2019, 7: 385-401. doi: 10.1146/annurev-animal-020518-115239
[35] OATLEY J M. Recent advances for spermatogonial stem cell transplantation in livestock[J]. Reproduction, Fertility and Development, 2017, 30(1): 44-49.
[36] IWAMOTO T, HIRAKU Y, OIKAWA S, et al. DNA intrastrand cross-link at the 5'-GA-3' sequence formed by busulfan and its role in the cytotoxic effect[J]. Cancer Science, 2004, 95(5): 454-458. doi: 10.1111/j.1349-7006.2004.tb03231.x
[37] LIN Z H, BAO J J, KONG Q F, et al. Effective production of recipient male pigs for spermatogonial stem cell transplantation by intratesticular injection with busulfan[J]. Theriogenology, 2017, 89: 365-373. doi: 10.1016/j.theriogenology.2016.10.021
[38] HONARAMOOZ A, BEHBOODI E, HAUSLER C L, et al. Depletion of endogenous germ cells in male pigs and goats in preparation for germ cell transplantation[J]. Journal of Andrology, 2005, 26(6): 698-705. doi: 10.2164/jandrol.05032
[39] FAO/ISAG. Secondary guidelines for development of national farm animal genetic resources management plans measurement of domestic animal diversity (MoDAD): Recommended microsatellite markers[EB/OL]. Joint ISAG/FAO Standing Committee. 2004, (2004-09-09)[2020-05-01]. http://wwwuser.gwdg.de/~uatz/FAO/cattel.htm (5 of 5)09/09/2004 16: 53: 05.
[40] YANG H Q, WU Z F. Genome editing of pigs for agriculture and biomedicine[J]. Frontiers in Genetics, 2018, 9: 360. doi: 10.3389/fgene.2018.00360
[41] WANG X Y, CHEN T F, ZHANG Y N, et al. Isolation and culture of pig spermatogonial stem cells and their in vitro differentiation into neuron-like cells and adipocytes[J]. International Journal of Molecular Sciences, 2015, 16(11): 26333-26346. doi: 10.3390/ijms161125958
[42] ZHENG Y, TIAN X E, ZHANG Y Q, et al. In vitro propagation of male germline stem cells from piglets[J]. Journal of Assisted Reproduction and Genetics, 2013, 30(7): 945-952. doi: 10.1007/s10815-013-0031-0
[43] KIM Y H, KIM B J, KIM B G, et al. Stage-specific embryonic antigen-1 expression by undifferentiated spermatogonia in the prepubertal boar testis[J]. Journal of Animal Science, 2013, 91(7): 3143-3154. doi: 10.2527/jas.2012-6139
[44] ZHANG P F, QIN Y W, ZHENG Y, et al. Phospholipase D family member 6 is a surface marker for enrichment of undifferentiated spermatogonia in prepubertal boars[J]. Stem Cells and Development, 2018, 27(1): 55-64. doi: 10.1089/scd.2017.0140
[45] QUESENBERRY P J, STEWART F M, ZHONG S J, et al. Lymphohematopoietic stem cell engraftment[J]. Annals of the New York Academy of Sciences, 1999, 872: 40-47. doi: 10.1111/j.1749-6632.1999.tb08451.x
