Zhang Tong-Dian, Zhang Lian-Dong, Xu Li-Li, Li He-Cheng, Ma Yu-Bo, Wu Zhi-Zhong, Wang Zi-Ming
Department of Urology, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710004, China.
Department of Gynecological Endocrinology, Northwest Women and Children's Hospital, Xi'an, Shaanxi 710003, China.
Zhonghua Nan Ke Xue. 2018 Nov;24(11):967-973.
To establish a method for in vitro culture of the fetal rat testis tissue.
Nine sexually mature specific-pathogen-free rats, 3 males and 6 females, weighing 200－250 g, were used for this study. The estrus of the female rats was determined according to the results of the vaginal smear test. The female rats were mated with the male ones in proestrus and estrus at night in the ratio of 2∶1 and observed the following day for conception (0.5 day post-conception ［dpc］) based on the presence of sperm in the vaginal smear. At 15.5 dpc, the fetal testes were isolated under the anatomical microscope, some for HE staining and the rest divided into a control and an hCG group to be cultured in a soft agar culture system at 37 ℃ in a humidified atmosphere containing 5% CO2. From the first day of culture (d 0), the development of the testes was observed under the inverted microscope, the culture medium collected and replaced on d 1, d 2, d 3 and d 4, and the testis tissue obtained on d 4. The concentration of testosterone in the culture medium was determined and the testis tissues were fixed, dehydrated and embedded for histological examination.
Fetal rats were successfully obtained with the vaginal smear at 15.5 dpc, and the fetal testes effectively isolated, which were well developed, with gradual increase of their volume and enlargement of convoluted seminiferous tubules under the inverted microscope. Testosterone was observed in the culture medium, its concentration gradually increasing and reaching the peak on d 3, and its secretion stimulated by hCG. At 15.5 dpc. The fetal testes showed a histomorphological integrity, with typical seminiferous tubules, gonocytes, Sertoli cells and Leydig cells, but no central necrosis. Transmission electron microscopy revealed gonocytes and Sertoli cells within and Leydig cells between the seminiferous tubules, without obvious swelling of the mitochondria and endoplasmic reticula in the cells.
The fetal rat testis tissue cultured in the soft agar culture system can develop well, retain its normal activity, and excrete testosterone into the culture medium.
建立胎鼠睾丸组织体外培养方法。
选用9只体重200－250 g的性成熟无特定病原体大鼠，其中雄性3只，雌性6只。根据阴道涂片检查结果确定雌性大鼠的发情期。在夜间将处于发情前期和发情期的雌性大鼠与雄性大鼠按2∶1的比例交配，次日根据阴道涂片中精子的存在情况观察受孕情况（受孕后0.5天［dpc］）。在15.5 dpc时，在解剖显微镜下分离胎鼠睾丸，一部分用于苏木精-伊红（HE）染色，其余分为对照组和人绒毛膜促性腺激素（hCG）组，置于含5%二氧化碳的湿润环境中，在37℃的软琼脂培养系统中培养。从培养的第1天（d 0）开始，在倒置显微镜下观察睾丸的发育情况，在第1、2、3和4天收集并更换培养基，在第4天获取睾丸组织。测定培养基中睾酮的浓度，并将睾丸组织固定、脱水和包埋以进行组织学检查。
通过阴道涂片在15.5 dpc成功获得胎鼠，并有效分离出胎鼠睾丸，其发育良好，在倒置显微镜下体积逐渐增大，曲细精管扩张。在培养基中观察到睾酮，其浓度逐渐升高，在第3天达到峰值，且hCG刺激其分泌。在15.5 dpc时，胎鼠睾丸呈现组织形态学完整性，有典型的曲细精管、生殖细胞、支持细胞和间质细胞，无中央坏死。透射电子显微镜显示曲细精管内有生殖细胞和支持细胞，曲细精管之间有间质细胞，细胞内线粒体和内质网无明显肿胀。
在软琼脂培养系统中培养的胎鼠睾丸组织能良好发育，保持其正常活性，并向培养基中分泌睾酮。
