大鼠胚胎睾丸组织体外培养方法的建立

Establishment of a method for in vitro culture of fetal rat testis tissue

目的:建立大鼠胚胎睾丸组织体外培养方法。方法:取SPF级性成熟(体重200～250 g) SD雄性大鼠3只、雌性6只,根据阴道涂片判断雌鼠所属的发情时期,在其发情期及发情前期按照1∶2合笼,发现精子则记为怀孕0. 5 d(0. 5 dpc)。解剖显微镜下分离出15. 5 dpc胚胎睾丸,一式2份,1份直接进行HE染色,另1份置于软琼脂糖培养系统,37℃、5%CO_2进行培养,分为对照组和hC G组。培养当天为d0,倒置显微镜记录培养睾丸生长情况,每天更换并收集d1、d2、d3、d4培养液及d4的睾丸组织。检测培养液睾酮浓度,培养结束后睾丸固定脱水包埋进行组织学研究。结果:应用阴道涂片方法可高效获得15. 5 dpc大鼠,HE染色提示能准确分离出15. 5 dpc胚胎睾丸。倒置显微镜下可见培养睾丸发育良好,体积逐渐增大,生精小管逐渐增粗、迂曲、清晰。培养液中可检测到睾酮,且睾酮浓度逐渐增高,d3时达到峰值; hC G可刺激睾酮分泌。HE染色可见培养后睾丸组织结构完整,无中央坏死现象发生,组织内有典型的生精小管,可见生殖母细胞、支持细胞及间质细胞;透射电镜下可见生精小管管腔中的生殖母细胞、支持细胞以及管腔外的间质细胞,细胞内的线粒体、内质网等细胞器,未见明显肿胀。结论:大鼠胚胎睾丸组织可在软琼脂糖培养系统中生长良好、保持活性,并向培养液中分泌睾酮。

Objective：To establish a method for in vitro culture of the fetal rat testis tissue. Methods： 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. Results：Fetal rats were successfully obtained with the vaginal smear at 15.5 dpe, 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, gonoeytes, 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 mitoehondria and endoplasmic retieula in the cells. Conclusion： 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.