Effects Of Diethylstilbestrol (DES) On Cultural Mouse Gubernacular Testis Cells

Objective:Recently, there is a positive correlation between the environmental factors and high incidence of congenital disorders of the urinary system, especially the environment estrogens(EEs). However, aside from male sexual differentiation and the development of testicular tissue, the mechanism of testicular descent remains controversial. The gubernaculums plays an essential role in the complex mechanism of testicular descent, but its mechanism also remains unclear. However, although a large amount of international research has taken place on the EEs disruption issue over the last decade, the precise cellular and molecular mechanisms underlying the action of estrogen on the testis and gubernaculums testis are still largely obscure. The cultured mouse gubernacular cells were treated with the synthetic estrogen diethylstilbestrol (DES) in this experiment. And then the morphology and functions of the cells were observed or measured. Not only the mechanism of how time-and dose-related effects of EEs on the gubernacular cells were explored, but also, more important, the idea of research and the method of precise cell and molecular biology on testis descent or even the congenital genital abnormalities.Methods: Gubernacula from 3-day-old mice were removed by operating magnifier and placed into phenol red-free Dulbecco's modified Eagle's medium (DMEM) supplemented with an enzyme solution containing typeⅠcollagenase ( 1mg/ml). The cultures were grown in 5% CO2 and 95% air in phenol red-free DMEM containing 5% charcoal dextran-treated FBS. After 3~5 days , primary cells were transferred into each wall of a 6-well culture plate. Cell viability was measured by trypan blue. Cell morphology was observed by HE stain. Subculturing cells were devided into six groups randomly, that is, normal group (not given any drug), control group (given DMSO,1μl/ml), and experimental groups 1~4: given 0.01μg/ml,0.10μg/ml,1.00μg/ml,10.00μg/ml DES respectively (DES dissolved in DMSO). After be treated 12h, 24h, 48h, the gubernacular cells were harvested to observe the morphology, proliferating and expression of PCNA, ER, F-actin, [Ca2+] I by Western Blot and cell immunity assay. Results: 1~2h after incubation, the primary cells had appeared to adhere to the wall. On days 3~5, a confluent monolayer of fibroblasts had formed, mainly were fibroblasts, and a few epithelioid. Fibroblasts were closely arranged, radial, knitted or circinate. 1~2 days after subculturing, the fibroblasts adhere the wall completely. The staining of HE of fibroblasts displayed the culture cells were highly homogeneous morphology. The cell viability was about 85-90%. DES can induce transformation of gubernacular cells evidently and the proliferation of experimental groups cells were inhibited by different dosage of DES and different measuring time in some degree, especially the high dose. The ER (ERα&ERβ) exited in the mouse gubernacular cells, both expressed in the cell nucleus and ERβalso expressed in the cytoplasm. The expression of [Ca2+] I and PCNA were obvious dosage-effect and time-effect correlations(P<0.05). Normal and Control cells exhibited short processes and a diffuse filamentous staining with FITC-Phalloidin around the cell periphery and nucelus. Cells exposed to DES exhibited the stress fibres distributed throughout the cytoplasm gradually instead of the F-actin around the cells, especially the high dose.Conclusion: The method of mouse gubernacular cell culture is established successfully. It can be applied to further investigation. DES is one of the classic EEs, the direct action in mouse gubernacular cells by DES can directly and accurately detect the dosage-effect and time-effect correlations of the expression of [Ca2+] I , PCNA and F-actin in gubernaculum testis. The ER proteins were abundant in the mouse gubernacular cells. It is proposed that EEs could lead to decline the contractive activity and inhibited the cell proliferation of gubernaculum, the effect androgen on gubernaculum testis was declined too. EEs may cause the anomogenesis and inhibition of descent of the mice gubernaculum testis or even testis.