| ObjectiveN-hexane is a widely used industrial soluent.The effects of N-hexane on the female reproductive endocrine disruption have aroused great attention in recent years. To study the effect and its mechanism on the ovarian granulose cell is of great significance for female reproductive occupational hazard prevention.MethodsThis experiment was designed to study the effect and mechanism of N-hexane on the three important aspects of the sex hormone synthesis of ovarian granulose cells.1.48 mature Wistar rats were weight and randomly assigned to four groups based on weight. N-hexane was given asp 4h/d, days/week for six weeks at dose of 0, 3.01,9.03,27.1g/m3. The rats were killed on the day of the estrus stage which was define by daily vaginal smears. The serum E2, P4 and E2, P4 of ovaries granule cells were mearsured by the ratio immunoassays. The expression of StAR, P450scc and P450arom were determinated with RT-PCR.2.48 hours after the pregnant mare serum gonadotropin(PMSG) was administrated subcutaneacsty at the dose of 100IU to Wistar rats. The ovaries granula cells were took out and incubated in Dulbecco modified Eagle medium (DMEM-F12) for the following study. Using orthogonal experiment design with orthogonal layout of L16(45), effects of 2,5-HD (0,20,40,60 mmol/L) and Gen (0,15,30,60 umol/L) on steroidogenesis in the ovaries granule cells were studied.Results1. In-vivo,①The period of estrus stage in rats treated with N-hexane was shorter than that of the control group, the period of estrus and metestrus in rats treated with 27.1 g/m3 was significantly shorted.②There were no significant differences on both E2 and P4 in the 4 group (P>0.05,respectively). ③As the concentration of N-hexane were increased, E2 levels were significantly declined in a dose-dependent manner in granule cells(P<0.05). P4 concentration secreted in vitro by the ovarian granulose cells of rats treated with 27.1 g/m3 were significantly decreased, and P4 concentration secreted in vitro by the ovarian granulose cells of rats treated with 3.01 g/m3 were significantly increased(P<0.05).④The expression of StAR mRNA and P450scc mRNA in 27.1 g/m3 were significantly lower than control,and the expression of StAR mRNA and P450scc mRNA in 3.01 g/m3 were significantly higher than control.⑤As the concentration of N-hexane were increased, the expression of P450arom mRNA were significantly declined in a dose-dependent manner (P<0.05).2.In-vitro,①the E2 and P4 level in the 20mmol/L were significantly higher than control (P<0.05). P4 concentration were significantly decreased in both the 40 and 60 mmol/L group (P<0.05).②As the concentration of Gen were increased, E2 and P4 levels were significantly declined in a dose-dependent manner in granule cells (P<0.05). Effect of 2,5-HD and Gen on P4 in granule cells were not significantly (P>0.05), and effect of 2,5-HD and Gen on E2 in granule cells were significantly (P<0.05).③The expression of StAR mRNA, P450scc mRNA and P450arom mRNA in the 20 mmol/L were significantly higher than control (P<0.05).④The expression of P450arom mRNA in both the 40 and 60 mmol/L were significantly lower than control (P<0.05).Conclusions1. N-hexane has obvious female reproductive endocrine disruption. There were the effects of N-hexane that may disturb the level of E2 and P4 of granule cells. And the period of estrous stages in rats decreased significantly.2. The change in the expression of StAR, P450scc and P450arom mRNA in the E2 and P4 synthesis of ovaries have been shown,and which is considered as important mechanism of endocrine disruption.3. The observed dose response suggests the presence of a hormesis effect (n-hexane inhalation for six weeks at dose of 3.01g/m3), but the result and the exact mechanism is still to be seen. However, the inhibition effect of the high dose of n-hexane on the progesterone synthesis can not be ignored.4. Binding of Soybean isoflavones results in inhibition of hormesis of lower dose 2,5-HD on the progesterone synthesis of granulose cells. |
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