Expression And Regulation Of Scavenger Receptor Class B Type 1 In The Rat Ovary And Uterus

Scavenger receptor class B type 1（SR-B1）preferentially mediates the selective uptake of high density lipoprotein-cholesterol ester and the delivery of cholesterol for steroidogenesis.Although multiple analyses have investigated the function of SR-B1 in the liver,adrenal and ovary,study about its expression in rat ovary and uterus during the estrous cycle is lacking.In the present study,immunohistochemistry（IHC）,real-time PCR and western blot were used to investigate SR-B1 expression in the rat ovary and uterus during the estrous cycle.Meanwhile,we also analyzed its expression in the immature rat ovary and uterus after different hormones treatment,which could provide experimental evidence for the potential role of SR-B1 in ovarian follicular development,luteal function and uterine function.In addition,we studied the special rat ovarian oxidative stress model induced by sodium arsenite,then detected ovarian steroidogenic genes SR-B1,StAR,P450scc and 3β-HSD expression in the ovary.Our results could contribute to further understanding the function and regulation of SR-B1 and other steroidogenic genes involved in the steroid hormone production,and to explaining the mechanism of sodium arsenite interfering ovarian normal reproductive physiology.The main results are as follows:1 Expression patterns of SR-B1 in rat ovary and uterus during the estrous cycleTo investigate the localization and expression of SR-B1 in rat ovary and uterus during the estrous cycle,we detected estrous cycle phases of rats using vaginal smears.The levels of SR-B1 mRNA in rat ovary measured by real-time PCR were continuously increased from proestrus to metoestrus,and reached the highest level during metoestrus,then decreased significantly during diestrus（P<0.05）,and SR-B1 protein expression was consistent with SR-B1 mRNA expression pattern.In contrast,SR-B1 mRNA and protein expression in rat uterus were decreased from proestrus to diestrus（P<0.05）.Immunohistochemistry result revealed that SR-B1 protein was mainly localized in oocytes,theca internal cells（T-I）of follicles,interstitial cells（IC）as well as steroidogenic luteal cells（CL）in the ovary during the estrous cycle.The staining in CL was very strong in all stages,but no staining was seen in the granulosa cells（GC）at any stage.In all cell types,expression was observed throughout the cytoplasm and plasma membrane but appeared absent from the nucleus.In the uterus,SR-B1 protein was mainly localized in endometrial luminal epithelial cells（LEC）and glandular epithelial cells（GEC）.In addition,strong staining was found in the circular muscle（CM）cells while weak staining in stromal cells（SC）during the whole estrous cycle.Taken together,SR-B1 expression in the ovary and uterus across the estrus cycle demonstrate that SR-B1 may be involved in luteal function,follicular development as well as uterine function.2 The effects of exogenous sex steroid hormones on SR-B1 expression in rat ovary and uterusTo investigate whether ovarian and uterine SR-B1 expression were affected by sex steroid hormones,immature rats were treated with 17 β-estradiol（E₂）,progesterone（P₄）,or their antagonists from postnatal days 24 to 26.The results showed that steroid hormones could increase ovarian and uterine weights.Both mRNA and protein levels of SR-B1 in ovary and uterus were significantly increased by E2 compared to the control（P<0.05）.While single P4 treatment did not affect SR-B1 expression（P>0.05）,E2 and P4 co-treatment increased SR-B1 expression in both the ovary and uterus compared to the P4 group and vehicle（P<0.05）.Besides,pre-treatment with the ICI 182780（ER antagonist）completely reversed the E2-induced increase in SR-B1 mRNA and protein expression,while RU486（PR antagonist）had no significant inhibition on SR-B1 expression.These results suggest E2 increased SR-B1 expression in rat ovary and uterus,and ER was involved in E2-mediated regulation of SR-B 1 expression.3 Establishment of rat ovarian oxidative stress model and expression of steroidogenic genesSteroidogenic genes,such as SR-B1 and StAR,play an important role in the synthesis of steroid hormones in the body.To further examine SR-B1 and other steroidogenic genes expression under rat ovarian oxidative stress,sodium arsenite was applied to establish the rat ovarian oxidative stress model.Twenty adult rats were randomly divided into the normal control group and treatment group,each rat was intraperitoneally injected with saline solution or sodium arsenite every other day,respectively.HE was used to detect the ovary morphology,RIA was used to detect serum estradiol（E2）and progesterone（P4）levels and biochemical assay was used to detect the contents of reactive oxygen species（ROS）,malondialdehyde（MDA）,superoxide dismutase（SOD）and glutathione peroxidase（GSH-PX）in the ovary homogenate.Then,we detected the expression levels of ovarian SR-B1,StAR,P450scc and 3β-HSD genes by real-time PCR.The results showed that compared with the control group,ovarian organizational structure was damaged and atretic follicules were found in the model group;E₂ and P₄ levels were significantly reduced;the contents of ROS and MDA significantly increased,but SOD and GSH-PX activities were significantly reduced;and SR-B1,StAR,P450scc and 3β-HSD mRNA levels were significantly lower（P<0.05）.Our present study confirmed that these steroidogenic genes expressions under rat ovarian oxidative stress were interfered and damaged,which provided a theoretical basis for the explanation of the mechanism of sodium arsenite interfereing mammalian ovarian normal reproductive physiology.