| Vitamin D3 is one fat soluble material,mainly stores up in the fatty tissue, 1,25- two hydroxy Vitamin D3 is its most main active form.Because Vitamin D3 has the important physiological action,in clinical it becomes an important assistant medication and in the people daily life it becomes an important supplement nutrient,but to health,specially to the underage child and the old age woman,vitD3 may reproduce the healthy question especially.This project takes the Vitamin D3 reproduction and internal secretion interference effect as the core, including the Vitamin D3 estrogenic effects and the Vitamin D3 female gonadal toxicity and its related mechanism,this systematic,comprehensive research focuses on Vitamin D3 reproduction and internal secretion interference effect and its related mechanism,then provides the scientific basis for the risk appraisal and preventing and controlling of the toxicities on Vitamin D3,and provides the instruction for the Vitamin D3 clinical practice and the reasonable supplement.Part 1 Study on the uterotrophic effect and its mechanism of VitD3Objectives:This experiment was designed to study the uterotrophic effect on young mice and its related mechanism of Vitamin D3Methods:66 mice,21 days of age,were randomly divided into 6 groups,exposure doses were respectively 0,0.00926,0.0556,0.33,2.0 mg / kg VitD3,positive control group for 30μg/kg estradiol.The mice were exposed to VitD3 once a day, for three consecutive days.Mice uterine weight,uterine organ coefficient, endometrial epithelial thickness,glands and PCNA,ER expression in endometrial lamina were observed.Results:1.The uterine wet weight and organ coefficient of estradiol group were significantly higher than those of the negative control group(p＜0.05),the uterine wet weight of all the vitamin D3 groups were not significantly different from that of the negative control group(p＞0.05),but it had an increasing trends;the uterus coefficient of O.0556mg/Kg,0.33mg/Kg and 2.0mg/Kg doses of VitD3 groups were significantly higher than the negative control group(p＜0.05);the uterine weight and organ coefficient of Estradiol group was significantly higher than those of all the vitamin D3 groups(p＜0.01);2.The endometrium epithelial thickness:the estradiol group was significantly higher than that of the control group and all the vitamin D3 dose groups;the number of endometrial glands:it increased with the increasing VitD3 doses,that in 0.33 mg / Kg,2.0 mg / Kg doses of VitD3 groups and the positive control group were higher than the control group,the positive control group was significantly higher than that of all the vitamin D3 dose group(p＜0.05 or p＜0.01);3.The PCNA positive percentage of endometrial:that of all the VitD3 groups and the estradiol group were significantly higher than that of the control group,and that of the estradiol group was higher than that of all the VitD3 groups;ER-positive rate:that of the control group was lower than that of 0.00926 mg / Kg,0.0556 mg / Kg,0.33 mg/Kg groups,and higher than that of 2.0 mg / Kg dose group;that of the estradiol group was significantly higher than those of the control and 0.0556 mg / Kg,0.33 mg / Kg,2.0 mg / Kg dose groups.PartⅡ:The study of female gonadal toxieities of VitD3Objectives:This experiment was designed to study on the effects of estrous cycle,serum hormone levels,uterine and ovarian pathology in female rats sub-chronicly exposure to VitD3,and to explore the female gonadal toxicities of VitD3.Methods:60 healthy female Wistar rats,weight 80±5g,were randomly divided into 5 groups,the doeses were respectively 0,0.0167,0.1,0.6,3.6 mg / kg,and the solvent was olive oil.The rats were exposed to VitD3 once a day and for 35 consecutive days.The exposure ended when the animals were in the estrus.The indicators such as weight,estrus stage,uterine wet weight and organ coefficient, ovarian wet weight and organ coefficient,the number of follicles,endometrial histology,serum follicle stimulating hormone(FSH),luteinizing hormone(LH), estradiol(E2) and progesterone(P4) levels were observed.Results:1.In the end of the experiment,the body weight of the rats exposed to the highest dose was significantly lower than other groups,and significantly lower than the control group(p＜0.01);2.The period of estrus stage in rats of 3.6mg/kg vitD3 was longer than that of the control group,and its metestrus period was significantly prolonged(p＜0.05);3.The wet weight of the uterus and the its coefficient in 3.6mg/kg group were significantly lower than the other groups(p＜0.01),and the ovarian wet weight was lighter than the control group,the difference has statistical significance(p＜0.01), but ovarian organ coefficient had no statistically significant difference compared with the control group(p＞0.05).4.Compared with the control group,the number of atretic follicles in all VitD3 doses group showed an upward trend,and the number of atretic follicles in 3.6mg/kg dose group was significantly higher than that of the control group(p＜0.05).5.The serum FSH level in 3.6mg/kg dose group was lower than the control group (p＜0.05),the level of LH in each group had no statistically significance compared with the control group,the levels of serum E2 in all vitD3 doses showed an upward trend,and the E2 level of 3.6mg/kg dose group was significantly higher than that of control group(p＜0.05);there were no statistical difference of the serum P4 levels in all doses group compared with the control group(p＞0.05),but there was a downward trend in serum P4 level.PartⅢStudy of the female gonadal toxicities mechanism(the hormone secretion of ovarian and ER expression in ovarian and uterine) of vitamin D3Objectives:This experiment was designed to study the effects on ovarian sex hormone secretion,the ER expression in both ovarian and uterine,and to explore part of the mechanisms of female gonadal toxicities exposured to VitD3Methods:1.Using orthogonal experimental design with orthogonal layout of L16(45),effects of VitD3(0,10-10,10-7,10-4mol/L )and estrous cycle(proestrous,estrous, postestrous,dioestrous) on steroidogenesis in the ovary were studied.2.Using orthogonal experimental design with orthogonal layout of L9(34),effects of VitD3(0,10-7,10-4mol/Land incubation time(4,8,24h) on steroidogenesis in granulosa cells were studied.3.50 healthy female wistar rats,weight 80±5g,were randomly divided into 5 groups,the doeses were respectively 0,0.0167,0.1,0.6,3.6 mg / kg,and the solvent was olive oil.The rats were exposed to VitD3 once a day and for 35 consecutive days.The exposure ended when the animals were in the estrus.Steroidogenesis in ovary in vitro,expression of ER in both ovary and uterus were observed.Results:1.In whole ovary culture,effects of VitD3 on estradiol secretion were significant (p＜0.05),and effects of VitD3 on progesterone secretion were not significant (p＞0.05).Effects of estrous cycle on estradiol and progesterone secretion in whole ovary culture were significant(p＜0.001 or p＜0.05);2.Effects of VitD3 on estradiol secretion in granulosa cells were significant (p＜0.01),and effects of VitD3 on progesterone secretion were not significant (p＞0.05).Effects of incubation time on estradiol secretion in granulosa cells were not significant(p＞0.05),and effects of incubation time on progesterone secretion were significant(p＜0.05);3.Estradiol and progesterone concentrations secreted in vitro by the ovaries of rats treated with 3.6mg/kg VitD3 were significantly decreased(p＜0.05);4.The expression of endometrial ER in 3.6mg/Kg group was significantly higher than that of the control group(p＜0.05);the expression of ER in ovarian cortex in 0.0167mg/kg,3.6mg/kg groups were lower than that of the control group,and that of 0.1mg / kg group was significantly higher than that of the control group(p＜0.01). PartⅣStudy of the apoptosis in rat ovary and its possible mechanism in female gonadal toxicities of vitamin D3Objectives:This experiment was designed to study on ovarian cell apoptosis induced by VitD3 s and its possible mechanism,and to explore the further mechanisms of female gonadal toxicities exposed to VitD3Methods:1.50 healthy female wistar rats,weight 80±5g,were randomly divided into 5 groups,the doeses were respectively 0,0.0167,0.1,0.6,3.6 mg/ kg,and the solvent was olive oil.The rats were exposed to VitD3 once a day and for 35 consecutive days.The exposure ended when the animals were in the estrus.The indicators such as the ultrastructure of ovarian tissue use of transmission electron microscopy, ovarian cell apoptosis by Tunel assay,the serum and ovarian tissue levels of calcium ions,ERα,ERβ,Caspase3,Caspase8 mRNA expression were observed.2.Using orthogonal experimental design with orthogonal layout of L9(34),effects of VitD3(0,10-7,10-4mol/L)and incubation time(4,8,24h) on apoptosis in granulosa cells were studied.The apoptosis of GC measured by FCMResults:1.The apoptosis in atretic follicles and luteal granulosa cell of 3.6 mg / kg dose group was significantly higher(p＜0.01),while the apoptosis in the growing follicles,mature follicles of all the VitD3 groups,the differences were not statistically significant.2.Apoptotic cells were visible in all groups;compared with the control group,an increased heterochromatin,chromatin margination,apoptotic bodies, mitochondrial edema,matrix-thinning,a decreased electron density,and even shaped vacuole were observed in 3.6mg/kg dose group;3.The calcium concentration of ovarian tissue in 3.6mg/kg group was higer than that of the control group(p＜0.05).4.The ERα,ERβmRNA expression and ERα/ ERβmRNA ratio in all vitD3 groups had no significant difference compared with the control group(p＞0.05).5.The expression of Caspase3mRNA of 0.0167 mg / kg dose group was significantly lower than that of the control group(p＜0.05),with the increasing VitD3 dose,Caspase3,Caspase8mRNA expression in each VitD3 group showed an increasing trend,and the Caspase3,Caspase8 mRNA expression of 3.6 mg / kg dose group were higher than the control group,but only Caspase8 mRNA expression was significantly higher than that of the control group(p＜0.01).6.The results showed that in vitro,10-7 and 10-4 mol / L of vitamin D3 increased the apoptosis rate of GC,and with the culture time extended,the apoptosis rate of GC increased.Conclusions:In this experimental condition,the following conclusions can be drawn:1.Vitamin D3 has the uterotrophic effect in young mice,showing the effect of uterine cell proliferation and the number of glands increasing,but its effects are weaker than that of estradiol;2.Vitamin D3(sub-chronic exposure) has the obvious female gonadal toxicities: showing the effects of the prolonged metestrus period,the increased atretic follicles number,the changed serum sex hormones.3.Ovarian is an important target organ of vitamin D3,and vitamin D3 can inhibit the sex hormones secretion of ovarian,and can affect the ER expression in both ovarian and uterine,which is the essential base of toxicological study and probably one of the mechanisms in its gonadal toxicities of uterotrophic effect vitamin D3;4.Vitamin D3 can induce apoptosis in ovarian cells:The main mechenisms were calcium homeostasis disorders;activated caspases family,but the detailed mechanisms need to be further explored. |
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