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。     

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.     

受体性别及完整性对睾丸组织移植物发育的影响

目的以免疫缺陷小鼠为受体,探讨受体的性别及完整性对未成熟睾丸组织移植物生长发育的影响。方法将作为受体的免疫缺陷小鼠分为4组:雄性正常组、雄性去势组、雌性正常组和雌性去势组。移植供体组织均为新生1～2d的昆明小鼠睾丸,移植部位为受体背部皮下。移植7周后取材,计算移植物的回收率并称重,对各组移植物中生精小管结构和生精细胞的组成情况以及生精细胞的染色体进行观察分析。结果移植7周后,从4组受体中获得的移植物体积和重量与移植前相比均有明显增加。染色体观察显示,所有4组移植物中生精细胞染色体数量均为正常2倍体(40条)和单倍体(20条);HE染色结果显示,所有4组受体取出的移植物中均发现带有长形精子的生精小管,其中雄性正常组、雄性去势组、雌性正常组和雌性去势组分别有1、5、1和3只受体中观察到含有长形精子的生精小管。结论新生昆明小鼠睾丸组织分别移植到去势的、未去势的雄性和雌性免疫缺陷小鼠背部7周后,未成熟的生精细胞均可发育成为长形精子。     

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。     

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.     

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.     

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.     

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。     

Development of bovine fetal testis tissue after ectopic xenografting in mice

Testis tissue xenografting represents a versatile model to study testis biology, and to preserve fertility in immature animals. To evaluate whether bovine fetal testes can mature when grafted into mouse hosts, small fragments of testes from midgestation (125 to 145 days of gestation) bovine fetuses were grafted ectopically into immunodeficient castrated male mice. At grafting, donor tissue displayed the typical seminiferous cords composed of gonocytes and primitive Sertoli cells. At 5 or 10 months after grafting, weight of the seminal vesicles in recipient mice was indicative of production of bioactive testosterone by xenografts. Xenografts showed similar development regardless of donor age. At 5 months, tubule formation occurred but germ cell differentiation had not proceeded beyond the spermatogonia stage. At 10 months, an increase in tubule size was evident and pachytene spermatocytes were observed as the most advanced type of germ cells in the xenografts of 2 donors. The number of tubules with germ cells was reduced in xenografts compared to donor tissue, but at 10 months the number of germ cells per tubule was higher than in donors. Germ cell proliferation was similar in donor tissue and xenografts. However, Sertoli cells showed a higher proliferation rate in xenografts collected at 5 months than in donor fetal testes and xenografts collected at 10 months. Sertoli cells in xenografts showed a progressive but incomplete loss of expression of Müllerian inhibiting substance and weak androgen receptor expression, indicating an incomplete Sertoli cell maturation. In conclusion, fetal testis tissue developed partially, qualitatively similar to pubertal testes in situ.     

Oxytocin promotes spermiation and sperm transfer in the mouse

Spermatogenesis is a complex process during which developing germ cells move from the base of the seminiferous tubule towards the lumen where they are shed. Studies in the rat suggest that seminiferous tubule contraction, induced by exogenous oxytocin, promotes spermiation. This study examines the role of testicular oxytocin in development of the testes, spermatogenesis and spermiation in the mouse. Groups of wild-type (WT) mice, oxytocin knockout mice (OTKO) deficient in testicular oxytocin and mice containing an oxytocin transgene (bOT4.2) that over express testicular oxytocin were killed between days 5 and 45 post partum. The testes and epididymides were removed weighed and prepared either for histological and morphometric study by light microscopy, for sperm counts (epididymis), or extracted for determination of oxytocin content (testis - day 45 only). Testicular oxytocin concentrations were significantly greater (p < 0.05) in bOT4.2 mice than in WT or OTKO mice. No differences in testicular and epididymal weight, or in diameter and area of seminiferous tubules between the mice genotypes were found at any given time. Germ cell development was similar in all genotypes and was comparable with previous studies. The timing of spermiation between the groups was significantly different (p < 0.001) with bOT4.2 < WT < OTKO and the appearance of epididymal sperm was significantly different (p < 0.05) with bOT4.2 < WT < OTKO. There were significant correlations between the percentage of tubules containing residual bodies and epididymal sperm count (p < 0.05) and between the percentage of animals containing residual bodies and the percentage of animals containing epididymal sperm (p < 0.01). These data suggest that in the mouse oxytocin, whilst not involved in germ cell development, is important in the process of spermiation and sperm transfer in the mouse.     

Expression of extracellular matrix proteins and vimentin in testes of azoospermic man： an immunohistochemical and morphometric study

AIM: To investigate the changes in the extracellular matrix protein expression and the morphology of seminiferous tubules in the testis of 88 azoospermic men. METHODS: The patients were of the following categories: (1) 22 cases of Sertoli-cell-only syndrome, (2) 20 cases of spermatogenic arrest, and (3) 46 cases with hypospermatogenesis. Testicular sections were immunohistochemically stained for fibronectin, vimentin, laminin and collagen type IV. The seminiferous tubular diameter and the connective matrix zone (CMZ, the acellular zone between the basement membrane [BM] and the peritubular cells) thickness were measured. Seminiferous tubules were typed according to the thickness of the connective matrix in the lamina propria. The predominant tubule type and the Johnsen and Silber scores were determined. RESULTS: The mean tubular diameter were 119 +/- 27, 117 +/- 20, and 140 +/- 38 microm for Groups 1, 2, and 3, respectively. Both the laminin and the type IV collagen were localized to the epithelial BM and peritubular cells. In most of the tubules, BM and peritubular cells were separated by a homogenous acellular layer, the CMZ, in which laminin, type IV collagen, fibronectin and vimentin were not present. It is perceived that the worse the testicular histology, the higher the thickness of the CMZ. CONCLUSION: In testis with no or low sperm production, the diameter of the seminiferous tubules is decreased, the thickness of the seminiferous tubular wall is increased and a CMZ is formed between the peritubular cells and the BM. The thickness of CMZ is increasing with the advancement of testiclar deterioration. The most important morphologic predictive factor for spermiogenesis is the predominant     

A game of cat and mouse: xenografting of testis tissue from domestic kittens results in complete cat spermatogenesis in a mouse host

Loss of genetic diversity because of infertility or the premature death of valuable individuals is a significant problem in the conservation of rare and endangered felid species, as well as in the maintenance of lines of cats used to study inherited feline and human disease. Attempts to overcome loss of genetic diversity have focused on freezing sperm; however, sperm cannot be collected from immature males. Previously, we reported completion of spermatogenesis in testis tissue from newborn pigs and goats grafted ectopically into host mice. The objective of this study was to extend the technique of testis tissue xenografting to the domestic cat as a model for felid species. Testes from 1- to 5-week-old domestic shorthaired kittens (n = 9) were cut into small fragments (about 0.5-1 mm3 each), and up to 8 fragments were grafted under the back skin of each castrated immunodeficient host mouse (n = 16). Histologic examination of the testis xenografts was performed between 5 and 54 weeks posttransplantation. At the time of grafting, the seminiferous cords of the donor testis tissue contained only immature Sertoli cells and gonocytes. At 14 weeks after grafting, tubular expansion was evidently caused by the proliferation of Sertoli cells and tubular lumen formation. By 18 weeks after transplantation, the seminiferous epithelium contained spermatocytes, and by 20 weeks, round spermatids were the most advanced types of germ cells. By 36 weeks after transplantation, xenografts of cat testis tissue had completed spermatogenesis. These results demonstrate the potential of xenografting to achieve full spermatogenesis in testis tissue from kittens. Therefore, sperm production in a mouse host can provide an alternative for germ line preservation from immature felids where sperm cryopreservation is not an option.     

Human chorionic gonadotropin deteriorates the histology of rat testes

OBJECTIVES: It is not yet certain whether early hormonal treatment in cryptorchidism is safe for germ cells. We investigated the histologic effects of human chorionic gonadotropin (hCG) therapy on descended testes of rats. DESIGN AND SETTING: Thirty male Wistar albino rats were randomized into two groups. The rats of the hCG group (n=15) were administered 50 IU/kg/day hCG once daily via the subcutaneous route for 15 days. Fifteen rats received subcutaneous isotonic saline and acted as controls. At the first month, testicular tissue was obtained after scarification in both groups. The histological examination was performed to evaluate the seminiferous tubular diameter, germinal membrane thickness, and the percentage of the open seminiferous tubule lumen in each testis to compare the two groups. RESULTS: The percentage of the open seminiferous tubular lumen in testicular tissues of hCG-treated rats was higher than that of controls (p<0.05). The mean germinal membrane thickness in testicular tissues of the hCG group was statistically lower than that of the control group (p<0.05). There was no statistical difference between mean seminiferous tubular diameter in testicular tissues of hCG-treated rats and controls, as expected (p>0.05). Additionally, there were two interesting cases of Sertoli cell only appearance in the hCG group. CONCLUSIONS: We may assume that hCG impairs the seminiferous tubule histology in normal testes of rats. Thus, further experimental studies on dose dependency and the reversibility of these effects are warranted.