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

ABSTRACT

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 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. CONCLUSIONS: 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.