| A large amount of n-hexane is widely used in industry as a solvent or a component of mixed solvents, used in the production of adhesives, paint, cleaning products. n-Hexane is organic solvent that has been described as altering different reproductive functions in mammals. However, few data have been published concerning the effects this organic solvent has on oocyte maturation and granulose cells apoptotic. The aim of this study was to determine whether n-hexane could affect maturation of mouse oocytes and granulose cells apoptotic and it's mechanisms.Part 0ne: Effect of n-hexane on maturation of mouse oocytesObjectives: The aim of this study was to determine whether n-hexane and 2, 5-HD could affect maturation of mouse oocytes.Methods 1: Forty female 21 days old ICR mice and 56 days old ICR mouse were used in these experiments. n-Hexane was given to four groups of mice for a one-week period (8 hours of exposure per day 7days/week) at doses of 0, 5.7, 22.5 and 90.9 ml/m3 respectively. GVBD and formation of the first polar body were observed under a Leica light microscope. The mitochondrial membrane potentials were visualized using Rhodamine123. The early apoptosis was visualized using JC-1.Methods 2: After exposure to n-hexane, female mice were super ovulated by intraperitoneal injections of pregnant mare's gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) 48 h apart. And then females in the morning were mated with normal male mice (ICR) overnight (two females per one male). The presence of spermatozoa in the vaginal smear on the next morning was indicative of copulation and was considered as day-zero of pregnancy. Fertilization eggs were collected at 24 and 48 hours post-pregnant for the determination of number of embryo by light microscope.Methods 3: Forty immature female 21 days old ICR mice and 56 days old ICR mouse were used in these experiments. Oocytes were exposed to 0, 20, 40, 60 mmol/L of 2, 5-HD, GVBD and formation of the first polar body were observed under a Leica light microscope. The mitochondrial membrane potentials were visualized using Rhodamine123. The early apoptosis was visualized using JC-1.Results:1. GVBD occurred, but experiment groups (5.7 and 90.9 ml/m3 n-hexane) abnormally with the different efficiency and significant delay (or not shown) when compared to the control experiment (P<0.01). But there are no significantly after exoposure to 2,5-HD.2. The presence of n-hexane prevents the formation of the first polar body This event occurred both in 0 hours and after 24 hours culture, 90.9 ml/m3 n-hexane-treated eggs expelled polar bodies that were significantly lower than those observed in control eggs (P<0.01). 60mmol/L 2, 5-HD-treated eggs expelled polar bodies that were significantly lower than those observed in control eggs (P<0.01).3. After 24 hours culture, death ratio induced by 22.5 and 90.9 ml/m3 n-hexane were higher than the control group (P<0.01 or P<0.05). 60mmol/L 2, 5-HD-treated eggs expelled death ratio that were significantly lower than those observed in control eggs (P<0.01).4. After fertilization, the eggs in the mouse were significantly got less by n-hexane.5. Furthermore, The mitochondrial potential in oocyte cells after 5.7,22.5 and 90.9-ml/m3 treatments, which were significantly lower than that of the control group (P<0.01) Also, after exposure to 2,5-HD in 60mmol/L, mitochondrial membrane potentials were also altered in mouse oocytes, and oocytes in the mouse became apoptosis.Part Two: Effect of n-hexane on apoptosis of mouse granulose cells and its mechanism Objectives: The aim of this study was to determine whether n-hexane could affect apoptosis of mouse granulose cells.Methods1: N-hexane was given to four groups of mice (doses were 0, 5.7, 22.5 and 90.9ml/m3 respectively) for a period of one week (8 hours of exposure per day). After treatment, the indicators such as the ultra structure of ovarian tissue use of transmission electron microscopy, granulose cell by TUNEL method in order to detect apoptotic cells. Methods 2: n-Hexane was given to four groups of mice (doses were 0, 3.0, 15.1 and 75.8 ml/m3 respectively) for a period of five weeks (35 days, 4 hours of exposure per day, 7days/week). After treatment, the indicators such as the ultra structure of ovarian tissue use of transmission electron microscopy, granulose cell by TUNEL method in order to detect apoptotic cells, and ovarian tissue levels of Caspase3, Caspase8 and Caspase8 mRNA expression were observed by RT-PCR. Ovarian tissue levels of Fas, Bax and Bel-2 protein expression were observed by Westen-Blot.Results:1. 7days study: apoptotic cells were invisible in all groups; compared with the control group.2. Apoptotic cells were visible in all groups; compared with the control group, an increased heterochromatin, chromatin margination, apoptotic bodies, mitochondrial edema, matrix thinning, and a decreased electron density.3. We observed that the apoptotic rate of the mature follicle and corpus Luteum/albicans increased significantly in the 15.1ml/m3,75.8ml/m3 groups compared to the control.4. Caspase3, Caspase8 and Caspase9 mRNA expression significantly highter than that of the control group (p<0.01)5. Bax protein expression significantly highter than that of the control group (p<0.05).Part Three: A new principle of analysis of DNA methylation on follicle cellsObjectives: The aim to build a method to study DNA methylation on follicle cells in rat.Methods: Ten mature female 84 days old ICR rats were used in these experiments. Ovary slice were made. Immunohistochemistry for 5-methylcytosine were performed to study the 5-methylcytosin methylation on follicle cells. HELMET was perfoemed to study the CCGG sites of DNA methylation on follicle cells. HELMET was perfoemed to study the GATCG sites of DNA methylation on follicle cells.Results:1. The levels of methylation of CCGG sites were higer in mature follicle.2. The levels of methylation of GATCG sites were higer in mature follicle.Conclusions: In this experimental condition, the following conclusions can be drawn:1. N-hexane maybe affects the maturation of oocytes and thenΔΨm alter and cells apoptosis maybe one of the most important mechanisms.2. 2, 5-HD maybe affect the maturation of oocytes and thenΔΨm alter and cells apoptosis maybe one of the most important mechanisms.3. N-hexane can induce apoptosis in granulose cells: The main mechanisms were activated caspases family; activated Bcl-2/Bax family, but the detailed mechanisms need to be further explored.4. HELMET can used to study the follical DNA methylation.5. Muture follical were high DNA methylation.
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