Effect Of DES And INSL3 On The Expressions Of LGR8 Mrna And Protein In Gubernacular Cells In Vitro

Objective:Recently, the effects of the environment estrogens (EEs) on the male reproductive system have become increasingly evident, most of which are correlated with testicular descent and dysplasia. The gubernaculum plays an essential role in the complex mechanism of testicular descent. Many researches indicated that the insulin-like factor 3 (INSL3) appears to play an important role in the testis descent, which involves development of the gubernaculums. INSL3 binds its specific receptor(LGR8), which was highly expressed in gubernaculum, to produce a crucial effect in the first transabdominal descent stage, but its mechanism also remains unclear. In our experiment, the expressions of LGR8 mRNA and protein in mouse gubernacular cells were measured to investigate their effects on LGR8 by administering DES and INSL3 of various doses.Materials and Methods:The gubernacula were removed from 3-day-old mice and cultured. The subcultured cells were randomly divided into 2 normal control groups and 9 experimental groups. The DES groups were administered 10μg/ml, 1μg/ml, 0.1μg/ml, 0.01μg/ml of diethylstilbestrol dissolved in DMSO respectively. And the INSL3 groups were administered 20ng/ml, 2ng/ml, 0.2ng/ml, 0.02ng/ml of insulin-like factor 3 dissolved in double distilled water respectively. The cell morphology was observed under an inverted microscope, and LGR8 was localized by immunofluorescence. After treated for 48 hours, total RNA was isolated by Trizol Reagent, the purity and integrity of RNA were identified by UV spectrophotometry and electrophoresis respectively. The expressions of LGR8 gene and protein in gubernacula cells was investigated by RT-PCR and Flow CytoMeter respectively.Results:1. The gubernacular cells were mainly fibroblasts-like, the high dose of DES can induce transformation of gubernacular cells evidently and lost the fibrous morphology, cytoplasmic shrinkage and irregular elliptic shape. 2. LGR8 was highly expressed in the cell membrane by immunofluorescence.3. Compared with the control group, the expression of LGR8 mRNA and protein in 10μg/ml DES group were upregulated (P <0.01), and reduced in 1μg/ml, 0.1μg/ml, 0.01μg/ml groups (P <0.01 or P <0.05). There was no significant difference in the DMSO group (P >0.05).4. The expression of LGR8 mRNA and protein were upregulated in 0.02ng/ml INSL3 group (P <0.01), and there were no significant differences in the 20ng/ml, 2ng/ml, 0.2ng/ml groups (P >0.05).Conclusion:1. The cultured mouse gubernacular cells were mainly fibroblasts-like, highly homogeneous morphology and viability. The high dose of DES can induce transformation and structural disorder of gubernacular cells evidently.2. LGR8 was highly expressed in the cell membrane by immunofluorescence.3. The high dose of DES can upregulate the expression of LGR8.4. Very low dose of INSL3 can increase the expression of LGR8.5. We demonstrated that high doses of DES and very low doses of INSL3 can upregulate the expression of LGR8 mRNA and protein in gubernacula cells, which might interfere with gubernacula development by LGR8 pathway.