Lysosomes Are Involved In Induction Of Steroidogenic Acute Regulatory Protein(StAR) Gene Expression And Progesterone Synthesis Through Low Density Lipoprotein In Cultured Bovine Granulosa Cells

In recent years, with the continuous development of dairy industry, the cow’s milk production level increased year by year. However it also brought a lot of problems, the postpartum fertility decline was the most prominent. A lot of high producing dairy cows became out of heat, insufficient heat performance or conception rate decrease. These leaded to offspring population decline of high yield cows,cow fetal spacing increase and cost increase and profits decline of the farms. Reproductive hormones had a very important role in regulating estrus cycle and pregnancy of the cow. And a lot of work had been performed on the regulating mechanism and application of reproductive hormones. But it still did not fundamentally solve the problem of cows postpartum fertility decline. The cows entered the peak milk production on 30 days after delivery, but it was not until 90 days postpartum cow began to enter the feeding peak. During this period, the energy of the cow was in a state of negative balance of energy. However, postpartum 60 to 90 days was the best period of insemination. A recent study reported that St AR gene expression was downregulated under the adverse metabolic environment caused by lactation or nutritional restriction in cattle, which directly resulted in the decrease of the level of progesterone synthesis in ovary. If the cows was inseminated at this time, it would lead to reduced conception rate. The study results suggest that certain nutrients may be involved in the regulation of the cow’s postpartum heat and conception. LDL, as a nutritive material or metabolic substrate, had been shown to have stimulatory effects on progesterone biosynthesis in cultured bovine luteal cells and swine granulosa cells. And St AR expression in mouse non-ovarian cells was upregulated by LDL. However, it was currently unknown whether LDL had a similar role for increasing St AR expression in bovine granulosa cells. The study of this problem would help to understand the roles of LDL in the process of granulosa cells differentiation after the follicle ovulation.The granulosa cells were cultured with or without LDL(20μg of protein/ml) for 24 h. And a series of comparisons were performed as below.The concentrations of progesterone in the medium were measured by progesterone RIA Kit. The results showed that treatment with LDL(20μg of protein/ml) stimulated more progesterone production(155.61±35 ng/105 cells) in granulosa cells than in untreated control cells(30.31±6.2 ng/105 cells; p<0.05). The genes expression levels were verified by quantitative PCR(q-PCR). Compared with the control, LDL treatment also induced approximately 2.29- and 2.33-fold increases in St AR and P450 scc m RNA levels, respectively(p<0.05). Western blot analyses showed that LDL treatment induced approximately 2.71-fold increases in St AR protein levels compared with the control(p<0.05). Oil red staining displayed that LDL treatment induced more lipid droplets(124.3±24.3/cell) than the control(40.6±12/cell; p<0.05). In addition, luteal cells from mid-luteal phase were cultured and stained with oil red. The results showed that luteal cells accumulated more lipid droplets(354.1±27.9/cells; p<0.05) than that in the LDL-treated granulosa cells and the control. In order to study the changes of lysosomes, we visualized lysosomes using two different approaches: acridine orange(AO) staining and immunostaining with anti-LAMP1 antibodies. As shown in the results, LDL induced a wider distribution of LAMP1 fluorescence in granulosa cells than in control cells, and the fluorescence intensity was significantly increased in the LDL-treated cells(0.93±0.02) compared with that in control cells(0.57±0.14, p<0.05). The AO staining showed that more lysosomes(58.2 ± 5.5/cell) appeared in the granulosa cells treated with LDL than in control cells(25.4±3.0/cell; p< 0.05). Also, luteal cells from mid-luteal phase were cultured and stained with AO staining and immunostaining with anti-LAMP1 antibodies. The results showed that luteal cells accumulated more fluorescence intensity and lysosomes(3.71±0.05 and 301.7±5.0/cell, respectively; p<0.05) than that in the LDL-treated granulosa cells and the control.To determine whether the synthesis of LDL-induced St AR and progesterone was mediated by lysosome activation, the granulosa cells were treated with control medium, LDL(20μg of protein/ml) or chloroquine(CQ, 50μmol/L) plus LDL(20μg of protein/ml). As shown in the results treatment with CQ strikingly abolished the stimulation of progesterone production by LDL(19.2±7.5ng/105 VS. 178.5±9.4 ng/105 cells, p<0.05), close to the baseline level observed in the control cells(27±4.7ng/105 cells, p>0.05). In addition, as shown in the results, CQ inhibited the LDL-induced St AR protein expression. In order to study the effect of the intracellular cholesterol homeostasis on the number of the lysosomes, the cultured granulosa cells were pretreated with cholesterol(60μmol/L) for 1 hour, and then the medium was changed by LDL(20μg of protein/ml) or cholesterol(60μmol/L) plus LDL(20μg of protein/ml). The LAMP1 fluorescence intensity(0.39±0.01) was significantly decreased by treated with CQ puls LDL than treated with LDL alone(0.93±0.02; p<0.05). In the same way, the number of lysosome stained by AO in LDL plus cholesterol treated cells(34.9±4.29/cell) was significantly lower than that in cells treated with LDL alone(75±8.25/cell; p<0.05).Thus, LDL regulated progesterone synthesis in granulosa cells by up-regulating the gene expression of St AR and P450 scc. LDL also induced more lysosomes in granulosa cells, and these lysosomes controled the regulation of LDL on St AR expression and progesterone synthesis. And the change of lysosome number was regulated by the cholesterol homeostasis of granulosa cells.