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Effects of Diethylstilbestrol (DES) on Insulin-like 3 Hormone Receptor LGR8 in Mouse Gubernaculum Testis

Author: YangXinBin
Tutor: JiangXueWu
School: Shantou University
Course: Surgery
Keywords: Diethylstilbestrol Environmental estrogens Gubernaculum testis INSL3/LGR8
CLC: R697.22
Type: Master's thesis
Year: 2011
Downloads: 24
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Abstract

Objective: In recent years, many studies have shown that environmental estrogens (EEs) can cause male reproductive system differentiation and developmental abnormalities, and most of the testis, down imperfecta, and gubernaculum testis and testicular development, down close. During fetal development, the testicles descend into the scrotum from the peak of the abdominal cavity is divided into two phases, the downward phase of abdominal and groin scrotum phase. Transabdominal testicular descent phase controlled mainly by INSL3. Knockout INSL3 or its receptor LGR8 gene will cause the mice cryptorchidism. Whether EEs LGR8 receptors by affecting INSL3 INSL3-LGR8 interference signal transduction unclear. In this study, classical EEs diethylstilbestrol (DES) during pregnancy direct effect on mice, rats and rats by observing the fetal gubernaculum testis morphology changes and detect LGR8 protein and mRNA expression, to explore the exogenous estrogen ( EEs) affect testicular development lead with possible ways. Materials and Methods: SPF grade 8 to 10 week old female mice 120 randomly divided into six groups (n = 20): normal control group, the experimental group 1, the experimental group 2, the experimental group 3, the experimental group 4. Experimental groups 1-4 were given DES 0.1,1.0,10.0, and 100.0μg.kg ^{-1 . D ^{-1 . DES in dimethylsulfoxide (DMSO) and saline as the solvent. The control group only received DMSO and saline; normal group does not give any drugs. Male and female mice were mated routine, with the advent of vaginal plug as pregnancy signs. Found that vaginal plug day as day 0 of gestation, denoted by E0 (Embryonic day). In E9 ~ E17 8:00 ~ 9:00 subcutaneous injection, each used the same amount of DES were dissolved in DMSO and saline to ensure that each kilogram of body weight per mouse per DMSO and saline given the same . Pregnant rats were sacrificed by cervical E18, fetuses quickly removed, cut by 10% whichever is the lower abdomen fixed in formalin for 24 hours, then give washed, graded alcohol dehydration, xylene, embedded in paraffin. Pups group until their natural childbirth, birth day 1 mice denoted PND0 (Postnatal day). Male mice were sacrificed at PND20, anatomical get gubernaculum testis, I fixed, paraffin-embedded. Right-embedded tissue blocks for 4μm thick coronal serial sections and special routine HE staining and immunohistochemical studies gubernaculum testis observed changes in the structure, to detect the gubernaculum testis tissue of insulin-like factor 3 receptor (LGR8) expression. Application 3 times anatomic surgical loupe and microsurgical instruments to obtain fetal rat testis E18 gubernaculum, gross anatomy get PND20 testes of a belt. Extracted using TRIzol gubernaculum Organization RNA, agarose gel electrophoresis and UV spectrophotometry identification RNA integrity and purity. RNA samples DNase treatment, and reverse transcription first strand synthesis of cDNA. OK identified by PCR with primers exist LGR8 testis expression. PCR products were sequenced, and take a certain amount of agarose gel electrophoresis analysis system using the amplified product gel bands were scanned and the image input gel electrophoresis gel image analysis software (BandScan 5.0) for expression analysis. Total RNA was extracted TRIzol extraction while total protein, Western Blot to detect the protein expression of testicular gubernaculum LGR8. Results: 1. Anatomical visible fetal rat testes experimental group position in the abdominal cavity was significantly high compared with the normal group, the experimental group rats incidence of cryptorchidism. Light microscopy shows normal fetal rat control group lead with a well-developed testicles, in the middle of mesenchymal tissue and peripheral clear boundaries between myogenic cells; experimental group, especially the high-dose group showed testicular gubernaculum dysplasia, mesenchymal tissue and muscle-derived cells no obvious boundaries between organizational structure disorder. Pups gubernaculum testis visible fibrous muscle-derived cells, mesenchymal cells no obvious structure. Normal group and the experimental group had no significantly different. 2 Immunohistochemical staining LGR8 positive cells showed brown or tan, expressed in testis lead with the cell membrane, the normal group, control group and experimental group were expressed, the same developmental stage of the normal group and the control group a strong positive expression, weak positive expression of the experimental group, and with increasing doses of DES expression decreased. Fetal rat (E18) in each experimental group compared with the normal group was statistically significant (P lt; 0.05), rats (PND20) DES1μg, 10μg, 100μg, compared with the control group was statistically significant (P lt; 0.05) . The same dose group rats (PND20) than fetuses (E18) LGR8 expression intensity (P lt; 0.05). 3. The normal group, control group and experimental group gubernaculum testis is present in both LGR8 mRNA expression. The same developmental stage normal group and control group, no statistically significant difference, DES high dose (10μg, 100μg) group and the normal group and the control group compared to LGR8 mRNA expression increased (P lt; 0.05). The same dose of fetal rat LGR8 mRNA was no difference compared with rats (P gt; 0.05). PCR products were sequenced experimental group showed no mutations. 4. Western Blot not detected LGR8-specific expression. Conclusions: 1. DES exposure during pregnancy may lead to offspring with morphological abnormalities lead testis, organizational structural disorder, and dose are closely related. 2. LGR8 expressed in different developmental stages gubernaculum testis tissue, subcellular localization in the cell membrane. Offspring exposed to DES during pregnancy can lead testis tissue LGR8 with reduced levels of protein expression, the same dose of DES on fetal rat LGR8 protein inhibition is more obvious. With the growth of offspring mice, LGR8 a certain increase in protein expression. 3. DES can upregulate gubernaculum testis tissue LGR8 mRNA expression, but did not find mutations. 4. DES on testicular tissue LGR8 cited protein and mRNA with different effects, suggesting that DES for LGR8 protein expression may occur in post-transcriptional. DES may affect protein expression by interfering with LGR8 gubernaculum testis development, the role of this interference can be obtained with the growth and development of a certain degree of correction.}}

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