冷冻保存未成熟睾丸组织移植后生精细胞的生长发育木

目的采用睾丸组织移植模型,探讨通过简单易行的冷冻保存程序保存未成熟睾丸组织,并使冷冻后的组织恢复生精过程的可能性,为该动物模型应用在保存雄性生殖细胞、帮助癌症患者保存生殖能力以及保存濒危物种等方面提供进一步的实验依据。方法用DMEM培养基加入二甲基亚砜配制冷冻保存液,将新生1～2d昆明小鼠睾丸组织按分步冷冻的方法进行冷冻,最终放入-80℃超低温冰箱中保存起来。2周后取出冻存的睾丸组织,移植到雄性去势免疫缺陷小鼠背部皮下,分别于移植后4周、5周和8周取材,制作HE染色切片,观察移植物中生精小管结构和生精细胞的组成,并与新鲜移植的睾丸组织进行对比分析。结果采用实验中的冷冻程序保存的新生小鼠睾丸组织,在冷冻保存一段时间后再移植,其表现与新鲜睾丸组织移植相同,其中末成熟的生精细胞可以在受体中继续生长发育,并完成整个生精过程,发育成为精子。结论本实验中所采用的睾丸组织冷冻保存方法具有不需特殊设备、简便易行、适用范围广泛等优点,适用于多种场合中生精细胞的冷冻保存。     

受体激素水平对未成熟睾丸移植物中生精细胞的生长发育影响

目的 采用已建立的未成熟睾丸组织异体异位移植模型,研究受体小鼠激素水平变化对未成熟睾丸组织移植物生长发育的影响.方法 以免疫缺陷小鼠为受体,将新生1～2d龄昆明小鼠睾丸移植到受体小鼠背部皮下.在移植后不同阶段取出移植物,进行组织形态学观察,分析移植物中生精细胞的发育情况,同时采用放射免疫分析法检测受体动物血清中睾酮(T)、促卵泡刺激素(FSH)和促黄体生成素(LH)水平,并以同龄正常昆明小鼠作对照.结果 受体小鼠血清中LH水平没有明显波动,而T和FSH浓度在移植的前8周呈逐渐上升趋势,从9周以后开始下降,且显著低于正常受体内睾酮和FSH浓度,与组织学中所观察到的生精上皮受损程度的增加是同步的.结论 以上结果提示,应注意获取移植物中精子的最佳时间.对于小鼠来说,从新生小鼠睾丸移植物中获取精子的最佳时间是术后5-8周.     

小鼠睾丸消化细胞异体移植重构生精小管的研究

目的:采用免疫缺陷小鼠作为受体,通过对小鼠睾丸消化细胞异位移植后不同时期移植物的研究,观察生精小管重构、生精细胞归巢及精子发生情况。方法:取新生ICR小鼠的睾丸消化成单细胞悬液,将其与Matrigel基质胶混匀后移植于雄性裸鼠背部皮下,术后裸鼠行去势。移植后分别于4、6、8、10周处死5只裸鼠,计算移植成功率,取移植物测量直径,并进行HE染色和免疫组化检测,观察生精小管的重构、生精细胞归巢及精子发生情况。结果:20只受体鼠接受睾丸消化细胞移植后全部存活。睾丸消化细胞移植后10周内可见明显隆起的包块,包块直径由第4周的(3.91±0.71)mm增加到(6.69±0.50)mm,移植物表面有血管生成。对移植物石蜡切片进行HE染色可见生精小管样结构,部分生精小管管腔内可见由精原细胞发育至精子细胞的各级生殖细胞,未见明显精子产生。对8周移植物进行免疫组化观察,可见生殖细胞标志物Mvh、支持细胞标志物Gata4和间质细胞标志物P450Scc表达。结论:新生小鼠睾丸消化细胞移植于裸鼠背部皮下后可重构生精小管,为研究睾丸组织工程及睾丸发育和精子发生过程中睾丸各组成细胞之间的相互作用提供了理想的研究模型。     

新生小鼠睾丸组织和作为异种移植物的人类胎儿睾丸组织在免疫缺陷小鼠中的发育情况(英文)

目的:采用同种和异种睾丸组织移植的方法,研究新生小鼠睾丸组织及人类未成熟睾丸组织异种移植物在免疫缺陷小鼠体内发育不同时期生精细胞的组成和基因表达情况中生精细胞的发育情况。方法:以免疫缺陷小鼠为受体,新生小鼠睾丸组织和人类未成熟睾丸组织为供体,分别进行同种和异种移植。通过对移植物的组织形态学观察和分子生物学检测,对各个时期同种移植物中的生精细胞组成及其特异性基因的表达情况进行评估并与末受损小鼠的情况相比较;对人睾丸组织异种移植物的存活及其生精细胞在异体异位的发育情况进行探讨。结果:新生小鼠睾丸组织在成年雄性去势免疫缺陷小鼠体内的发育状况在移植开始的一个阶段与在体睾丸组织的发育情况基本相同,各级生精细胞的出现及其基因表达均与在体睾丸组织中相类似,而移植7-8星期后生精小管发生退化现象。人未成熟睾丸组织在受体中存活并且进一步生长;组织学观察还发现,生精细胞的发育速度与在体相比具有加速的倾向。结论:新生小鼠睾丸组织同种移植物的发育与在体情况基本相同,而人类未成熟睾丸组织异种移植物的发育与正常生理状态相比较呈现出加速的倾向。     

小鼠原始生精细胞裸鼠皮下移植动物模型的制作

目的制作睾丸组织异体异位移植动物模型,并观察原始生精细胞在异体异位继续生长、发育情况。方法将鼠龄为1～2d昆明小鼠睾丸组织移植至去势裸鼠背部(n=29),于移植后30～110d取出移植物,观察移植物的生长情况、表观形状以及各级生精细胞、生精上皮发育的期相情况。结果术后29只受鼠全部存活,在其背部均观察到移植物的生长,移植物直径由移植前的(0.73±0.05)mm增加到(6.75±0.73)mm,湿重由移植前(5.67±0.72)mg增加到(113.12±78.23)mg,受鼠皮肤血管已广泛深入到移植物内;光镜下可见移植物中生精小管结构清晰,移植物与受鼠皮肤之间建立了紧密连接,移植物中含有各级生精细胞及精子,在过碘酸Schiff(PAS)染色的移植物标本可见整个精子发生周期(从StageⅠ到StageⅫ)各阶段的生精细胞。结论新生小鼠睾丸组织移植到去势裸鼠背部能够继续生长、发育,可作为研究生精细胞增殖发育规律和精子发生调控机制的动物模型。     

小鼠睾丸移植物中3种生精阶段特异性基因的表达

目的 采用逆转录-聚合酶链反应，探讨新生小鼠睾丸组织移植到裸鼠体内后，3种阶段特异性基因，缺失型无精子基因（Daz1）、磷酸甘油激酶2（Pgk2）和鱼精蛋白-2（Prm2）在不同发育阶段移植物中的表达情况。方法 将180只出生1-2d昆明小鼠睾丸移植到60只7-12周去势雄性免疫缺陷小鼠背部；在移植后不同时间段（分为3d、1周-8周和12周10个组）取出移植物，对在发育不同阶段表达的3种基因出现的时间及表达情况进行分析测定，并与相应各年龄段正常小鼠睾丸中的基因表达相比较。同时进行组织形态学观察，对比各种基因出现的时间与小鼠睾丸发育周期的吻合性。结果 在10个时间段取出的移植物中，所测定的3种基因表达趋势与在正常昆明小鼠睾丸中所见基本相同，并与小鼠发育周期基本吻合。结论 新生小鼠睾丸组织移植到免疫缺陷小鼠体内后，生精细胞的发育在形态学和几种受试基因出现的时间上与在正常小鼠中表现均相似。从而对该睾丸组织移植模型用于生精细胞异体异位生长发育研究及基因调控机制研究的可行性提供了进一步的理论依据。     

小鼠睾丸组织移植物二次移植后的生长发育研究

目的 通过将第一次移植后的移植物进行再移植,探讨二次移植的睾丸组织生长发育情况,为今后采用睾丸组织裸鼠移植模型探讨性成熟期较长动物(包括人类)睾丸组织的体外成熟提供实验依据.方法 将1～2 d龄昆明小鼠睾丸组织植入去势裸鼠背部,于移植后2周取出移植物,并将其立即移植到另一只去势裸鼠背部,并在二次移植4周后取出移植物,制作HE染色切片,在光镜下观察二次移植后睾丸组织的生长发育情况;同时采用检测鱼精蛋白-2 mRNA的方法对二次移植物的生长发育程度进行鉴定.结果 移植2周后的小鼠睾丸组织再次移植后,仍可观察到移植物中的血管组织再生以及生精小管的继续发育;二次植入的44块组织经4周生长发育后收集到22个二次移植物,回收率为50%;在仅含有精原细胞和精母细胞的移植物二次移植后收集到的移植物中,观察到了圆形精子细胞甚至长形精子细胞,并在所有的二次移植物中检测到鱼精蛋白-2 mRNA的表达.结论 新生小鼠睾丸组织移植物转移到另一只受体中后可继续存活并生长发育.     

异体异位发育小鼠睾丸组织中凋亡相关基因和DNA损伤修复基因表达的研究

目的 探讨异体异位移植睾丸中生精细胞退化的可能机制.方法 以新生1～2 d小鼠睾丸组织为供体,免疫缺陷的雄性去势裸鼠为受体进行组织移植,分别在移植后不同时间(3 d和1～8周共9个时间段)收集移植物组织,提取移植物总RNA,采用半定量RT-PCR检测不同发育阶段移植物中凋亡相关基因fas、bc1-2、bax和caspase3以及DNA损伤修复基因xrcc1、ogg1、ercc1、brca1和brca2的表达,并与相应年龄段正常小鼠睾丸组织中的基因表达进行比较分析.结果 与正常发育的小鼠睾丸组织相比,小鼠睾丸移植物中生精细胞退化增加,凋亡相关基因bc1-2表达降低,fas、bax和caspase3表达增加:DNA损伤修复基因xrcc1、ogg1、ercc1、brca1和brca2的表达量均有不同程度的降低.结论 新生小鼠睾丸组织移植到免疫缺陷裸鼠体内后,生精细胞退化增加,这可能与促凋亡基因表达增加和抑制凋亡基因表达降低,以及DNA损伤修复基因表达降低有关.     

异体异位发育小鼠睾丸组织中精子功能相关基因和蛋白的表达研究

目的:采用已建立的未成熟睾丸组织移植模型,研究异体异位生长发育的小鼠睾丸组织中精子功能相关基因Asrgl1,Gpx4,Asp,Prdx4和Spa17及其相关蛋白GPX-4和PRX-Ⅳ的表达,以迸一步评估该移植模型在生殖医学中应用的可行性.方法:以1～2 d小鼠睾丸组织为供体,7～12周雄性免疫缺陷小鼠为受体进行组织移植;移植8周后收取移植物组织,采用荧光定量PCR法检测移植物中Asrgl1,Opx4,Asp,Prdx4和Spa17的表达情况,并与正常小鼠睾丸组织进行对比;同时采用Western blot检测GPX-4和PRX-Ⅳ 2种蛋白的表达情况.结果:基因检测显示,5种受试基因在移植物中的表达与8周正常小鼠睾丸组织相比均有下调;蛋白分析提示,GPX-4和PRX-Ⅳ 2种蛋白在8周正常小鼠睾丸组织中表达而在小鼠睾丸组织移植物中表达缺陷.结论:几种主要在精子运动、精子顶体形成及精子的受精功能中发挥作用的基因和蛋白在移植物中表达下调及缺失提示,异体异位睾丸组织移植在临床应用的可行性还需进一步评估     

睾丸移植物中支持细胞相关基因和蛋白表达的研究

目的 研究新生小鼠睾丸组织异体异位移植后,几种在睾丸支持细胞中起重要作用的基因和蛋白表达情况,为异体异位睾丸组织移植模型用于科研及临床的可行性提供进一步实验数据.方法 将162只1～2d昆明小鼠的睾丸移植到54只7～12周去势雄性免疫缺陷小鼠背部;在移植后9个时间段(3d和1～8周)取出移植物;选取4种在睾丸支持细胞中表达或高表达的基因abp、amh、vim和clu,采用聚合酶链反应,对发育不同阶段移植物中4种基因的表达情况进行分析,并与正常小鼠相应各年龄段睾丸中的基因表达相比较;同时采用免疫组织化学方法对支持细胞的GATA-4蛋白在移植物组织中的表达量及分布情况进行分析.结果 在9个时间段取出的移植物中,所测定4种基因的表达趋势与在正常小鼠睾丸中所见基本相同;免疫组化结果显示,4周和8周移植物支持细胞中GATA-4蛋白呈高表达,与在正常小鼠睾丸组织支持细胞中的表达基本一致.结论 新生小鼠睾丸组织异体异位移植到免疫缺陷小鼠体内后,支持细胞的发育在组织形态学以及几种受试基因的表达趋势和蛋白的表达情况与在正常小鼠中的表现基本相同.     

睾丸组织异体异位移植研究进展

自从2002年《自然》杂志发表了第1篇用同种或异种睾丸组织移植进行的生精细胞体外发育研究以来,该技术被广泛应用于不同种类动物(包括人类)睾丸组织的体外成熟的探讨。这种采用免疫缺陷动物作为受体,同种或异种睾丸组织为供体的技术与其他生精细胞体外成熟方法相比具有环境条件较易控制、重复性较好等优点。迄今为止,在用小鼠、仓鼠、猫、兔、猪、山羊、牛和恒河猴等动物睾丸组织进行的移植研究中,未成熟生精细胞在作为受体的裸鼠体内均发育到精子阶段,并通过卵细胞胞质内单精子注射(ICSI)技术,用发育成的小鼠、兔精子分别产生了下一代。本文总结了供体组织的种类和年龄,受体的性别、完整性、免疫状况和移植部位以及移植时间对移植物发育的影响,概述了近几年来采用该技术所进行的睾丸组织移植研究发展现状,并对该技术的应用以及存在的问题进行了讨论。     

胎儿睾丸组织异种移植后生精细胞发育初探

目的 :以人胎儿睾丸组织为供体 ,免疫缺陷小鼠为受体 ,研究人类睾丸生精细胞异种移植后的继续发育情况。　方法 :将 2 6周胎儿的睾丸组织植入去势裸鼠背部 ,于移植后 1 35d取出移植物 ,进行组织形态学观察 ,分析原始生精细胞在异种异位的发育情况。　结果 :移植 1 35d后取出的移植物显示 ,其生长幅度已由移植前直径约 1mm和湿重约 5mg ,分别增加到移植后大于 3mm和 2 0mg。组织形态学观察发现 ,移植前的睾丸主要是由直径为(6 0± 1 5 ) μm的精曲小管索构成 ,其中包含的细胞主要是原始Sertoli细胞和少量原始生精细胞 ,细胞排列呈弥散无规则状态 ;而移植后 1 35d的精曲小管索已发育成具有管腔的精曲小管 ,出现由Sertoli细胞和生精细胞组成的完整生精上皮 ,直径增大到 (80± 2 5 ) μm。原本呈不规则分布的原始Sertoli细胞和生精细胞大部分已迁移到基膜处 ,其中有少数生精细胞发育成为精原细胞。　结论 :人胎儿睾丸组织移植到去势裸鼠背部后 ,可以继续存活并进一步生长发育。     

The length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts

Xenografting of immature mammalian testis tissue into mice can accelerate sperm production. To determine whether this shortened time to sperm production is because of reduced length of the spermatogenic cycle, we applied bromodeoxyuridine (BrdU) incorporation to analyze the spermatogenic cycle in porcine and ovine testis xenografts. Small testis fragments from newborn pigs and sheep were ectopically grafted into mice. Once complete spermatogenesis was present in grafted tissue, mice were injected with BrdU and grafts were recovered at different time points thereafter. In porcine grafts, the most advanced germ cells labeled 1 hour, 9 days, 12.3 days, and 18 days after BrdU injection were stage 1 preleptotene/leptotene primary spermatocytes, stage 1 pachytene primary spermatocytes, stage 5 newly-formed round spermatids, and late stage 2 elongating spermatids, respectively. In ovine grafts, the most advanced labeled germ cells at 1 hour, 11 days, and 22 days post-BrdU injection were stage 2 preleptotene/leptotene primary spermatocytes, late stage 1 pachytene primary spermatocytes, and stage 2 elongating spermatids, respectively. These results indicate that each spermatogenic cycle in porcine and ovine xenografts lasts approximately 9 and 11 days, respectively, which is similar to their durations in situ. Therefore, the length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts. This is consistent with earlier reports showing that the cycle length is inherent to the germ cell genotype. The shortened time to sperm production in xenografts therefore appears attributable to accelerated maturation of the testicular somatic compartments. Our results suggest that testis xenografts provide a useful model to study the timing of testicular maturation and spermatogenesis in different mammalian species.     

Cisplatin-induced germ cell apoptosis in mouse testes

The purpose of this study was to investigate whether exposure of male mice to cisplatin induces apoptosis in male germ cells and the possible role of apoptosis in cisplatin-induced testicular damage. Forty-eight male BALB/c mice were divided into cisplatin and control groups. The mice from the cisplatin group received a single intraperitoneal injection of cisplatin of either 1, 5, or 10 mg/kg. The control group received a single intraperitoneal injection of saline alone. The testes were removed on days 1, 3, and 7 after cisplatin administration, respectively. Following histological examination, apoptotic indices (AIs) were measured within seminiferous tubules of the mouse testes by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. A low incidence of spontaneous apoptosis was observed in controls, particularly in spermatogonia and spermatocytes of the mouse testes. After cisplatin administration, both increased Als and decreased spermatozoa and spermatids were found in the seminiferous tubules of the mouse testes. Cisplatin-induced apoptosis was found in spermatogonia, spermatocytes, and spermatids of the mouse testes. In comparison to the control values, AIs increased 2.6- to 6.8-fold in cisplatin-treated mouse testes. AIs reached the highest level on day 1 following 1 mg/ kg, on day 3 following 5 mg/kg, and on day 7 following treatment of 10 mg/kg cisplatin. The study showed that cisplatin-induced germ cell apoptosis in the mouse testes was related to both the dose response and the time course of response. It is suggested that cisplatin-induced germ cell apoptosis may result in decreased spermatogenesis, and the higher dose of cisplatin may delay the occurrence of apoptosis in the mouse testes.     

Bovine sertoli cells colonize and form tubules in murine hosts following transplantation and grafting procedures

The contribution of somatic cells to nonrodent male germ cell transplantation success has not been well established due to lack of cell type-specific markers to distinguish donor cells from host cells. In the present study, we first screened antibodies and a lectin to identify markers suitable for unequivocal distinction between germ cells and Sertoli cells in bovine testes compared with mouse testes. Anti-vimentin and the Dolichos biflorus agglutinin (DBA) lectin detected only bovine Sertoli cells and spermatogonia, respectively; anti-NONO and anti-GCNA1 detected only mouse Sertoli and germ cells, respectively. The outcome of transplanting bovine testis cells into nude mouse testes was then studied using these markers. Our results clearly showed that immature bovine Sertoli cells survive and colonize mouse testes at 2.5 months after transplantation and that tubular structures composed of donor Sertoli cells formed adjacent to murine tubules within the host mouse testis. Bovine germ cell colonization and survival in mouse testes after transplantation were confirmed, but this was restricted to areas of bovine Sertoli cell colonization. In addition, ectopic grafts of intact bovine testis tissue and cell aggregates from hanging drop cultures were placed under the back skin and testis capsule of nude mice. Bovine Sertoli cells in ectopic grafts and aggregates were able to form tubular structures, and some bovine germ cells were observed around 2 months after implantation. This study therefore identifies a practical strategy to assess the outcome of testicular cell transplantation using different antibodies and a lectin to distinguish bovine cells from mouse cells. It identifies an approach that can readily be adapted to study other nonrodent species.     

Inhibition of growth and differentiation of fetal hamster gonads grafted into the adult testis

A cytomorphological analysis of the effect of adult testes on growth and differentiation of grafted fetal testis or ovaries was performed in hamsters. Fetal gonads, taken at 14 days post-coital age, were grafted for 30 days either under the renal capsule or testicular capsule of scrotal or cryptorchid testes of adult hamsters (weight 115 +/- 23 g). Renal grafts were also performed in males castrated 30 days prior to receiving the fetal gonads. Growth and differentiation of the fetal gonads (testis or ovary) was totally inhibited by the scrotal testis. When the cryptorchid testis was the recipient of fetal gonads, inhibition was correlated inversely with the degree of spermatogenic damage elicited in the cryptorchid testis. No inhibition was observed in fetal gonads grafted under the kidney capsule, nor in castrated, normal or cryptorchid animals. As normal growth and differentiation of both testis and ovary occurred when grafted under the kidney capsule, the inhibitory effect of adult gonads seems to be unrelated to plasma testosterone levels in the host, as levels were undetectable in castrated hamsters and reduced drastically in cryptorchid animals. At the same time, the testicular-inhibiting substance in normal animals did not act at a distance, since its effect was restricted to fetal gonads grafted under the testicular capsule. This inhibitory substance may correspond to the spermatogonial chalone, known to be produced by differentiating spermatogenic cells (mainly spermatocytes and round spermatids in the rat and mouse); these chalones prevent spermatogonial proliferation and, consequently, the critical number of spermatogonia needed to enter meiosis is not attained. It is doubtful if the same substance has the ability to differentiate the fetal ovary or if this effect can be ascribed to a more complex situation involving other testicular peptides of paracrine action and/or locally high levels of androgens.