Cyclin D1启动子在养精胶囊促进小鼠精原干细胞增殖中的作用※

目的 研究养精胶囊促进小鼠精原干细胞（SSCs）增殖的分子机制。方法 将不同浓度的养精胶囊提取物加入SSCs中培养48 h，CCK-8检测细胞的增殖活性，流式细胞仪检测细胞周期，荧光素酶报告基因检测Cyclin D1启动子的活性，qRT-PCR以及免疫荧光检测Cyclin D1的表达。之后进行阻断实验，在加入养精胶囊前预先加入siRNA-Cyclin D1，同样的方法检测细胞的增殖活性、细胞周期、Cyclin D1启动子的活性以及Cyclin D1的表达情况。结果 低、中、高浓度的养精胶囊可以促进SSCs的增殖，提高S期细胞的比例，增强Cyclin D1启动子的活性，促进Cyclin D1的表达。阻断Cyclin D1后，SSCs的增殖活性降低，S期细胞比例减少，Cyclin D1启动子的活性降低，Cyclin D1的表达减少。结论 养精胶囊通过增强Cyclin D1启动子的活性提高Cyclin D1的转录和翻译水平，进而促进SSCs增殖。     

Differentiation of murine male germ cells to spermatozoa in a soft agar culture system

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system (SACS), which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum (FCS). The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia (Vasa, Dazl, OCT-4, C-Kit, GFR-α-1, CD9 and α-6-integrin), meiotic cells (LDH, Crem-1 and Boule) and post-meiotic cells (Protamine-1, Acrosin and SP-10). Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.     

Development and Morphogenesis of Testis in Human Fetuses

Development and maturation of prenatal testis was studied on 104 human male fetuses. At 9 weeks, the testes were seen as elongated yellowish tissue superolateral to the developing urinary bladder and medial to mesonephros. The testis assumed ellipsoidal shape at 13 weeks. At about 16 weeks, the testes developed convexity on its anterior aspect. Epididymis and testis were at the same level until 20 weeks thereafter the upper testicular pole was encroached upon by the epididymis, with complete encroachment till its anterior aspect at 24 week. The sinus was also distinctly present between the two. The testis assumed miniature adult testis at term, but its size was approximately 1/15th of the adult. Cytoarchitecture of the testis at 9 weeks revealed radially disposed sex cords. At 13 weeks, tunica albuginea,tubular organization in the parenchyma and the Leydig cells were identifiable. At 17 weeks, tunica vasculosa and incomplete lobules were apparent. At 24 weeks, testis was marked by more but solid seminiferous tubules lined by 4–5 germ layers. Leydig cells were also identifiable till 24 weeks. Spermatogenic cells, both pale and dark type were distinguishable although pale spermatogonia were more numerous than dark cells. 28 weeks onwards tunica vaginalis and complete septa in the testicular parenchyma were evident. Moreover, the seminiferous tubules developed central vacuolation. By 30 weeks, seminiferous tubules became more complex as evident by their increase in number and coiled and tortuousity. At term, the fetal testis had not yet attained the cytoarchitecture of the adult testis suggesting that testicular maturation continues postnatally.     

Percoll离心联合多步差异黏附法分离、纯化人睾丸精原细胞

目的探讨分离、纯化人睾丸精原细胞的有效方法。方法用联合二步酶消化法获得人生殖细胞悬液,经Percoll不连续密度梯度离心,再用间隔不同时间(2、3、4h),不同次数(1、2次)差异黏附法纯化人精原细胞。结果未经纯化的精原细胞纯度为48.5%,间隔2、3、4h纯化1次所获得的精原细胞纯度分别为51.3%、59.0%、56.4%,纯度均高于未经纯化的纯度;间隔2、3、4h连续纯化2次所获精原细胞纯度分别为:57.7%、67.8%、57.8%,以间隔3h纯度最高;与间隔同样时间纯化1次精原细胞纯度相比,间隔2、3h纯化2次高于1次,间隔4h纯化2次与1次无明显差别。结论Percoll不连续密度梯度离心联合不同时间间隔差异黏附法可以有效地对人精原细胞进行分离和纯化;间隔2、3h,纯化2次高于1次;连续纯化2次时,间隔3h效果最佳。     

COMMENTS

Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor superfamily. It binds to and activates a receptor complex consisting of GFR-α1 and Ret receptor tyrosine kinase. In testis, GDNF is expressed by Sertoli cells. We have shown by transgenic loss- and gain-of-function mouse models that GDNF regulates the cell fate decision of undifferentiated spermatogonia. In the GDNF +/− mice, the spermatogonia differentiate in excess leading to the depletion of germ cells. In the mice overexpressing GDNF in testes, undifferentiated spermatogonia accumulate in the tubules, no sperm is produced, and the mice are infertile. After a year, the GDNF overexpressing mice frequently (89%) develop testicular tumours, and most of them are bilateral (56%). All these tumours show the same histological pattern. They are composed of round spermatogonial/gonocytic cells with only a scant cytoplasm. The tumours are locally invasive but do not metastasise. They express germ line markers, are positive for alkaline phosphatase, and aneuploid with a triploid peak. Thus, by several histological, molecular, and histochemical characteristics, the GDNF-induced tumours mimic classical seminomas in men, but the precursor lesions are apparently different in mouse and man.     

骨髓间充质干细胞的分离培养及在精原细胞培养条件下生物学特征的观察

目的体外分离、培养、纯化小鼠骨髓间充质干细胞(MSCs),并观察MSCs在精原细胞培养条件下的生物学特征,为体内诱导MSCs分化为精原细胞奠定基础。方法①分离5～6周龄的BALB/c小鼠胫骨、股骨,冲出骨髓,以Percoll密度梯度离心法和贴壁法相结合的方法分离、培养、纯化MSCs;②通过动态观察、苏木精伊红(HE)染色、透射电镜观察、免疫组化检测细胞表面标记等鉴定MSCs;③用ELISA法检测MSCs培养上清中白细胞介素6(IL6)、IL8、粒细胞集落刺激因子(G CSF)、干细胞因子(SCF)等细胞因子的相对含量,与相应对照组比较;④取第3代MSCs,分组进行诱导培养观察,对照组用基本培养液培养MSCs,实验组用条件培养液诱导培养MSCs。通过显微镜下动态观察、HE染色、免疫组化等方法观察诱导结果。结果①获得纯化的MSCs;②动态观察培养细胞具有不断增殖的干细胞特性,HE染色细胞呈梭形,透射电镜观察细胞较幼稚,免疫组化检测细胞表面标记CD44、CD90呈阳性,从而鉴定了MSCs;③MSCs培养上清中IL6、IL8、G CSF、SCF等的含量显著高于对照组(P<0.05);④实验组细胞形态发生变化,CD29、CD117和Oct4免疫组化染色阳性;对照组细胞形态不变,CD29、CD117和Oct4免疫组化染色阴性。结论①用Percoll密度梯度离心法和贴壁法相结合的方法可获得纯化的MSCs;②     

Protecting spermatogonia from apoptosis induced by doxorubicin using the luteinizing hormone-releasing hormone analog leuprorelin

PURPOSE: The present study was performed to investigate the protective effect of leuprorelin (LH-RH analog), on spermatogonia apoptosis induced by doxorubicin (DXR) in the Sprague-Dawley rat model. METHODS: Twenty-four adult male rats were divided into the following four groups: (i) control group; (ii) group given doxorubicin (intravenous injection, 8 mg/kg); (iii) group given leuprorelin (subcutaneous injection, 3 mg/kg); and (iv) group given both doxorubicin (intravenous injection, 8 mg/kg) and leuprorelin (subcutaneous injection, 3 mg/kg). Evaluation for quantification of apoptotic spermatogonia was made by the ratio of TUNEL-labeled spermatogonia versus 100 Sertoli cells in each seminiferous tubule. Two hundred seminiferous tubules of each rat were assessed. RESULTS: The ratio of apoptotic spermatogonia versus 100 Sertoli cells at stages II-IV of the groups given DXR (groups 2 and 4) were significantly higher than those of the other groups. However, the value at stages II-IV of the group given both DXR and leuprorelin (group 4) was significantly lower than that of the group given DXR (group 2). CONCLUSION: The significant prophylactic effect (P < 0.05) of LH-RH analog against doxorubicin-induced spermatogonial apoptosis was observed in a stage specific manner by microscopic evaluation with TUNEL.     

Mitotic and meiotic detection of radiation-induced translocations in mouse stem cell spermatogonia using fluorescencein situ hybridization

The efficiency of two methods of detection of translocations induced in mouse stem cell spermatogonia by X-ray doses of 2, 5 and 7 Gy was compared: classical multivalent analysis at diakinesis-metaphase I of meiosis and observation via fluorescencein situ hybridization analysis of mitotic or meiotic stages. Specific DNA libraries for chromosomes 1, 11 and 13 were used. The results obtained indicate that (a) chromosomes 1, 11 and 13 are more involved in multivalent formation than expected on the basis of DNA content and (b) if the mitotic FISH analysis data are corrected for the observed over-representation, the frequencies of induced translocations are similar to those recorded in the classical multivalent studies, suggesting equal scoring efficiencies in both systems.