Effects Of Diethylstilbestrol (DES) On Insulin-like 3 Hormone Receptor LGR8 In Mouse Gubernaculum Testis

Objective:Recently, there is a positive correlation between the environment estrogens (EEs) and high incidence of congenital disorders of the urogenital system, which have much connection with male sexual differentiation and the development of testicular tissue. The gubernaculum plays an essential role in testicular descent, but the mechanism remains unclear. Many researches indicated that the Insulin-like factor 3 (INSL3) appears to play an important role in the transabdominal phase of testicular descent, which involves development of the gubernacula. Other studies have demonstrated that in utero exposure to diethylstilbestrol (DES), a synthetic estrogen, can lead to cryptorchidism both in human and in animal models. Furthermore, the effect of DES on INSL3 mRNA expression was determined. The present study was undertaken to investigate whether prenatal DES-exposure might interfere with LGR8, the special receptor of INSL3. The LGR8 protein and mRNA was detected by immunohistochemistry and western blot. To explore the effects and mechanism of different doses EEs on gubernaculum development.Methods: To obtain timed pregnancies, female mice were placed in individual cages with male mice and the morning a vaginal plug was found was designated embryonic day 0 (E0) of pregnancy. Pregnant mice were injected subcutaneously on days E9 through E17, with diethylstilbestrol (DES: 0.1μg/kg, 1.0μg/kg, 10.0μg/kg, 100μg/kg body weight) in dimethyl- sulfoxide or with dimethyl sulfoxide alone (control). Pregnant mice were sacrificed on E18 by cervical dislocation and fetuses were quickly removed. The postnatal male offsprings were sacrificed on postnatal day 20 (PND20) by cervical dislocation. E18 fetuses and PND20 male offsprings were examined under a dissection microscope, and established the position of the testes. E18 fetuses and gubernaculums from PND20 male offsprings were used for histological analysis. These were fixed in 10% formalin and embedded in paraffin. Serial sections of 4μm were cut and stained with hematoxylin-eosin. The slices were stained by immunohistochemistry with the LGR8 antibody (sc-22017) subsequently. The expression intensity was quantitated by image analysis in terms of their optical density (OD), and statistically analyzed thereafter. Gubernaculums from E18 fetuses PND20 male offsprings were isolated, frozen and stored in liquid nitrogen until RNA and protein isolation. Total RNA and protein were isolated by TRIzol Reagent, then separately detected by the method of RT-PCR and western blot. Results: Male fetuses at E18 were examined under a dissection microscope to study the position of the testis. At E18, the testis of control fetuses were located next to the bladder, at the bottom of the abdomen. In DES-treated male fetuses at the same fetal age, the testis were always positioned higher in the abdomen when compared with the controls, ranging from firm attachment to the posterior pole of the kidneys to a location well above the bladder. Histological analysis at E18 showed the gubernaculum bulb is well-development, with an inner mesenchymal core and muscular outlayer in the control male fetus, in contrast, it appears to be undifferentiated in the DES-treated male fetus. While gubernaculum from PND20 mice in the DES-treated seems thinner and shorter than in the control and there are any cells in mesenchymal core. Positive immunostaining is present in mesenchymal core and muscular outlayer. Immunoreactivity is localized at the peripheral cell membrance of muscle cells. All groups have positive staining but the DES-treated male fetus staining weaker than the control group, the more DES the weaker staining (P<0.05). LGR8 messenger RNA can be detected in both E18 and PND20 gubernaculum by RT-PCR. And the expression of LGR8 mRNA increased in high dose group of DES ( 10.0μg.kg^-1.d^-1, 100.0μg.kg^-1.d^-1) (P<0.05). We were not able to detect LGR8-specific bands by Western blot in tissue extracts using the LGR8 antibody (sc-22017).Conclusion: Prenatal DES-exposure resulted in cryptorchidism by altering the expression of LGR8 to interfer with the development of gubernaculums. DES-treated male fetus showed different expression between LGR8 mRNA and protein suggested that the DES maybe interfere with LGR8 after its transcription. The effect of DES to LGR8 protein maybe retrieved at some degree.