大豆异黄酮对巴马香猪初情启动及卵泡发育的影响及其机制

发布时间：2018-04-20 06:34

本文选题：大豆异黄酮 + 下丘脑-垂体-卵巢轴　；参考：《湖南农业大学》2015年博士论文

【摘要】：大豆异黄酮(Soybean Isoflavones, SIF)是一种植物雌激素,广泛存在于豆科植物中。目前的观点认为,SIF会干扰动物的内分泌平衡,引发内分泌紊乱,导致繁殖力降低。本研究通过在无豆粕的日粮中添加不同浓度的SIF,探讨SIF对巴马香猪初情启动及卵泡发育的影响。试验采用单因子试验设计,从11窝胎龄相同、胎次相近的仔猪中,每窝选择5头35日龄的雌性巴马香猪,随机分配到5个处理组,分别给予0、250、500、1250 mg/kg的大豆异黄酮和0.66 mg/kg的戊酸雌二醇(相当于0.5 mg/kg的雌二醇),每个处理组11个重复,每个重复1头猪,进行动物饲养试验。试验期间,准确记录各组猪的初情日龄；在4月龄、发情第2天和第4天分别空腹采血,用于血清激素水平测定;在猪启动初情后的第4天,每组选择6头猪屠宰,采集下丘脑、垂体、卵巢等组织样品,用于荧光定量PCR、western blot、HE染色和免疫组化的检测；从剩下的猪中,每组选择3头猪于第二次发情时配种,饲喂至分娩,考察SIF对雌性巴马香猪繁殖性能的影响。此外,从附近屠宰场收集卵巢组织,分离原代颗粒细胞,探讨大豆苷元和染料木黄酮对原代颗粒细胞激素合成、GDF-9/BMP-15信号通路及凋亡的影响。试验结果如下：(1)巴马香猪日粮中添加不同浓度的SIF导致猪的初情期延迟,240日龄时猪的发情率降低,且1250 mg/kg的SIF显著增加了(P0.05)巴马香猪的初情日龄。日粮中添加SIF显著降低了(P0.05)4月龄巴马香猪血清中的GnRH和LH水平,增加了(P0.05)FSH水平。250mg/kg添加水平的SIF显著降低了巴马香猪下丘脑中ER β和卵巢中ER α的mRNA表达水平,500mg/kg的SIF显著降低了下丘脑中ERβ的表达水平,1250mg/kg的SIF显著降低了垂体和卵巢中ER α的表达水平,且各添加水平的SIF降低了(P0.05)香猪卵巢ER β的蛋白表达水平。1250 mg/kg的SIF增加了(P0.05)垂体FSHβ的表达水平,250 mg/kg的SIF显著降低了垂体GNRHR的表达。日粮中添加各水平的SIF极显著降低了(P0.01)巴马香猪下丘脑KISS1和卵巢StAR、 3β-HSD的mRNA表达水平和卵巢StAR的蛋白水平。250 mg/kg添加水平的SIF显著降低了(P0.05)卵巢CYP17A1和CYP19A1的表达水平。此外,日粮中添加250 mg/kg的SIF显著降低了(P0.05)巴马香猪卵巢组织FSHR mRNA的表达水平,各水平的SIF显著降低了(P0.05)卵巢中FSHR蛋白的表达水平。结果提示,SIF导致巴马香猪初情期的延迟,其机制是通过改变下丘脑、垂体、卵巢中雌激素受体的表达,引起下丘脑中KISS1基因、卵巢中StAR和3 β-HSD基因和蛋白的表达下调,从而引起猪血清中激素水平的改变,导致内分泌紊乱。(2)大豆异黄酮对巴马香猪的卵巢质量、卵巢指数及初情排卵数均没有显著影响(P0.05)。日粮中添加SIF减少了巴马香猪卵巢中各期卵泡的数量,其中,1250 mg/kgSIF显著减少了(P0.05)原始卵泡数量,500、1250 mg/kg大豆异黄酮显著减少了(P0.05)生长期卵泡数量。日粮中添加SIF显著降低了(P0.05)巴马香猪卵巢组织GDF-9、BMPR-1B mRNA的表达水平,250 mg/kg的SIF显著降低了(P0.05)卵巢BMP-15、BMPR2的mRNA的表达水平；各水平的SIF显著降低了(P0.05)巴马香猪卵巢中BMP-15蛋白的表达水平。日粮添加SIF显著降低了(P0.05)巴马香猪卵巢组织HAS2、PTGS2、Ptx3、Tnfaip6以及SMAD2的mRNA表达水平,250 mg/kg组显著降低了(P0.05) SMAD3和SMAD4的mRNA表达水平。日粮中添加不同水平的SIF减少了产仔母猪的头数,增加了返情母猪头数。结果表明：大豆异黄酮还可通过调节GDF-9/BMP-15信号通路中关键因子的表达,来干扰猪的卵泡发育,进而影响繁殖性能。(3)日粮中添加1250 mg/kg的SIF和0.66 mg/kg的EV减少了(P0.05)巴马香猪卵巢中闭锁卵泡数目。500、1250 mg/kg添加水平的SIF显著增加了(P0.05)巴马香猪卵巢组织Bc1-2的mRNA表达水平,250 mg/kg组降低了(P0.05)Bax的mRNA表达水平,且随着SIF含量的增加,Bcl-2的蛋白表达水平逐渐增加,Bax的蛋白表达水平逐渐降低,提示SIF可通过调节Bcl-2和Bax的表达水平减少卵泡闭锁。(4)在体外培养的颗粒细胞的培养基中加入100、500 μM的大豆苷元和1、10、100和500州的染料木黄酮,能显著抑制(P0.05)培养48 h时颗粒细胞的活力。大豆苷元和染料木黄酮均显著抑制了(P0.05)颗粒细胞ERβ的mRNA表达水平,染料木黄酮还显著抑制(P0.05)ER α的mRNA表达水平。0.1和1 μM的染料木黄酮则降低了(P0.05)培养基中孕酮的浓度。各添加水平的大豆苷元显著降低了(P0.05)颗粒细胞3β-HSD的mRNA表达水平,而染料木黄酮则显著降低了(P0.05)细胞StAR和3β-HSD的mRNA表达水平。同时,在培养基中加入0.1和10 gM的大豆苷元显著降低了(P0.05)猪颗粒细胞CYP11A1的表达水平,而1和10μM的大豆苷元则降低了(P0.05) CYP19A1的表达水平。说明大豆苷元和染料木黄酮均能通过下调类固醇激素合成通路中关键基因的表达来影响颗粒细胞雌二醇和孕酮的合成与分泌。在体外培养的猪颗粒细胞培养基中加入0.1μM的染料木黄酮能显著增加(P0.05)BMPR2的表达水平。大豆苷元和染料木黄酮均显著降低了(P0.05)猪颗粒细胞Ptx3和HAS2基因的表达水平,10 μM的大豆苷元降低了(P0.05) PTGS2的表达水平,1、10μM的大豆苷元显著降低了(P0.05) Tnfaip6的表达水平,且染料木黄酮均显著降低了(P0.05)颗粒细胞Tnfaip6的表达水平。说明给体外培养的猪颗粒细胞的培养基中加入不同浓度的大豆苷元和染料木黄酮,虽然不改变GDF-9/BMP-15受体的表达水平,但是能显著下调其下游调控因子的表达水平。在体外培养的猪颗粒细胞培养基中加入1、10 μM的大豆苷元减少了(P0.05)细胞的早期凋亡率,1 μM的大豆苷元还减少了(P0.05)细胞的晚期凋亡率和总凋亡率,0.1、1μM的大豆昔元减少了(P0.05)死亡细胞的百分率。不同浓度的染料木黄酮减少了(P0.05)猪颗粒细胞的早期凋亡率,但是增加了(P0.05)晚期凋亡率、总凋亡率和死亡细胞的百分率。添加0.1μM的大豆苷元显著增加了(P0.05)颗粒细胞Bcl-2/Bax的比例。在培养基中加入不同浓度的染料木黄酮,降低了颗粒细胞Bcl-2的表达水平,其中添加1 μM的水平达到显著性水平(P0.05)；添加10 μM的染料木黄酮则增加了Bax的表达水平(P0.05),说明低浓度的大豆苷元和染料木黄酮能抑制颗粒细胞的早期凋亡,而高浓度则能引起颗粒细胞凋亡甚至死亡。由以上试验结果得出,大豆异黄酮作用于巴马香猪,导致香猪初情期延迟,卵泡发育发生障碍,其机理主要为：1)大豆异黄酮能改变下丘脑、垂体、卵巢组织雌激素受体的表达水平,影响GnRH、LH、FSH及类固醇激素合成中关键因子的表达,从而影响激素的合成和分泌,引起香猪激素调节改变；2)大豆异黄酮能下调GDF-9/BMP-15信号通路中关键因子的表达,减少卵巢中各期卵泡的数量,从而影响卵泡的正常发育和香猪的繁殖性能；3)颗粒细胞的体外培养试验也表明,大豆苷元和染料木黄酮均能改变细胞雌激素受体、固醇激素合成关键基因、GDF-9下游因子的基因表达,从而影响固醇激素的合成和分泌。由此可知,大豆异黄酮作为饲料添加剂,或者后备母猪日粮配方中使用高水平的豆粕需要慎重考虑。
[Abstract]:Soybean Isoflavones (SIF) is a kind of phytoestrogens, which is widely found in leguminous plants. The present view is that SIF interferes with the endocrine balance of animals, causes endocrine disorders and leads to the decrease of fecundity. In this study, the initial condition of SIF to Bama Xiang pigs was discussed by adding different concentrations of SIF in the diet without soybean meal. The effect of dynamic and follicular development. A single factor test was used to select 5 female Bama pigs of 35 days of age at the same age of 11 nests and similar fetal times. 5 treatment groups were randomly assigned to 02505001250 mg/kg of soybean isoflavones and 0.66 mg/kg of valerate (equal to 0.5 mg/kg estradiol). Each treatment group had 11 repetitions, each repeated 1 pigs, and the animal feeding test was carried out. During the trial, the initial age of the pigs was recorded, and the blood was collected on the empty abdomen in 4 month old, second and fourth days, respectively, for the determination of serum hormone levels, and 6 pigs were slaughtered in each group for fourth days after the start of the initial situation, and the hypothalamus, pituitary and ovary were collected. Tissue samples were used for the detection of fluorescent quantitative PCR, Western blot, HE staining and immunohistochemical staining. From the remaining pigs, 3 pigs were selected for second estrus, feeding to childbirth, and the effects of SIF on the reproductive performance of female Bama Xiang Pigs. The effects of glycosides and genistein on the synthesis of hormone, GDF-9/BMP-15 signaling pathway and apoptosis in primary granulosa cells. The results were as follows: (1) the initial stage delay of pigs was delayed by adding different concentrations of SIF in the diet of Bama Xiang pig, and the oestrus rate of pigs decreased at the age of 240 days, and the 1250 mg/kg SIF significantly increased the initial day of (P0.05) Bama Xiang pig The addition of SIF in the diet significantly reduced the level of GnRH and LH in the serum of (P0.05) 4 month old Bama Xiang Pigs, and the SIF significantly reduced the mRNA expression of ER alpha in the hypothalamus of Bama Xiang Pigs, which was significantly reduced by SIF (P0.05) FSH level.250mg/kg addition level. F significantly reduced the expression level of ER alpha in the pituitary and ovary, and the levels of SIF decreased (P0.05) the protein expression level of ER beta in the ovary of the pigs increased (P0.05) the expression level of the pituitary FSH beta, and the 250 mg/kg SIF significantly reduced the expression of pituitary gland. The mRNA expression level of 3 beta -HSD in the hypothalamus of Bama Xiang pig and the mRNA expression level of 3 beta -HSD and the protein level of the ovarian StAR significantly decreased the level of the expression of CYP17A1 and CYP19A1 in the ovary (P0.05). In addition, the expression level of the ovarian tissue of Bama Xiang pig was significantly reduced by the addition of 250 mg/kg in the diet. The horizontal SIF significantly reduced the expression level of FSHR protein in the ovary of (P0.05). The results suggested that SIF led to the delay in the early stage of Bama Xiang pig. The mechanism was to change the expression of estrogen receptor in the hypothalamus, pituitary, and ovary, causing the KISS1 gene in the hypothalamus, the expression of StAR and 3 beta -HSD in the ovary, and thus causing swine blood. The changes in hormone levels in the Qing Dynasty led to endocrine disorders. (2) soybean isoflavones had no significant effects on the ovarian quality, ovarian index and initial oviposit number of Bama Xiang Pigs (P0.05). The addition of SIF to diet reduced the number of follicles in the ovary of Bama Xiang Pigs, of which 1250 mg/kgSIF significantly reduced the number of original follicles (P0.05), 5001250 M G/kg soybean isoflavones significantly reduced the number of follicles in the growth period of (P0.05). The addition of SIF in the diet significantly reduced the expression level of GDF-9 and BMPR-1B mRNA in the ovarian tissue of Bama Xiang Pigs (P0.05), and the SIF of 250 mg/kg decreased significantly (P0.05) ovary BMP-15, and the expression of BMPR2 was flat. BMP-15 protein expression level. Dietary supplementation of SIF significantly reduced the mRNA expression level of HAS2, PTGS2, Ptx3, Tnfaip6 and SMAD2 in the ovarian tissue of (P0.05) Bama Xiang pig. The 250 mg/kg group significantly reduced the level of SMAD3 and the expression level of SMAD2. The results showed that soybean isoflavones could also interfere with the development of porcine follicle by regulating the expression of key factors in the GDF-9/BMP-15 signal pathway, and then affect the reproductive performance. (3) adding 1250 mg/kg SIF and 0.66 mg/kg EV in the diet reduced the SIF display of the number of.5001250 mg/kg in the ovaries of (P0.05) Bama Xiang pig. The mRNA expression level of Bc1-2 in the ovarian tissue of Bama Xiang pig was increased (P0.05). The 250 mg/kg group reduced the mRNA expression level of (P0.05) Bax, and with the increase of SIF content, the expression level of Bcl-2 protein gradually increased and the protein expression level of Bax decreased gradually, suggesting that SIF could reduce the follicle atresia by regulating Bcl-2 and the expression level. (4) 100500 M daidzein and 1,10100 and genistein in 500 state were added to the culture medium of the cultured granulosa cells, which could significantly inhibit the activity of granulosa cells in (P0.05) culture of 48 h. Daidzein and genistein significantly inhibited the mRNA expression level of ER beta in (P0.05) granulosa cells, and genistein also significantly inhibited (P0.05) E The mRNA expression level of R alpha and genistein of 1 mu M decreased the concentration of progesterone in the (P0.05) medium. The levels of daidzein significantly reduced the mRNA expression level of 3 beta -HSD in (P0.05) granulosa cells, while genistein significantly reduced the mRNA expression level of StAR and 3 beta -HSD in (P0.05) cells. Daidzein into 0.1 and 10 gM significantly reduced the expression level of CYP11A1 in pig granulosa cells, while 1 and 10 M daidzein decreased the expression level of (P0.05) CYP19A1. It indicated that daidzein and genistein could affect the estradiol in granular cells by downregulating the expression of the key genes in the steroid hormone synthesis pathway. The synthesis and secretion of progesterone. The addition of 0.1 M genistein to the culture medium of porcine granulosa cells in vitro could significantly increase the expression level of (P0.05) BMPR2. Both daidzein and genistein significantly reduced the expression level of Ptx3 and HAS2 genes in (P0.05) pig granulosa cells, and 10 UG daidzein decreased the expression of (P0.05) PTGS2. Level, 1,10 mu M daidzein significantly reduced the expression level of (P0.05) Tnfaip6, and genistein significantly reduced the expression level of Tnfaip6 in (P0.05) granulosa cells. It indicated that the different concentrations of daidzein and genistein were added to the culture medium of pig granulosa cells in vitro, although the GDF-9/BMP-15 receptor was not changed. The expression level of the downstream regulatory factors was significantly reduced. The early apoptosis rate of (P0.05) cells was reduced by the addition of 1,10 mu M daidzein in the culture medium of porcine granulosa cells in vitro. The 1 u M daidzein also reduced the late apoptosis rate and total apoptosis rate of (P0.05) cells, and the soy celecoxib of 0.1,1 mu M decreased (P0.0). 5) the percentage of dead cells. The different concentrations of genistein reduced the early apoptosis rate of (P0.05) pig granulosa cells, but increased the rate of late apoptosis, the total apoptosis rate and the percentage of dead cells. The proportion of daidzein adding 0.1 mu M significantly increased the proportion of Bcl-2/Bax in the (P0.05) granulocyte. Different concentrations were added to the medium. Genistein reduced the expression level of Bcl-2 in granular cells, in which the level of adding 1 mu M reached a significant level (P0.05), and the expression level of Bax was increased by the addition of 10 M genistein (P0.05), indicating that the low concentration of daidzein and genistein could inhibit the early apoptosis of the granulosa cells, while the high concentration could cause a grain. The effects of soybean isoflavones on Bama Xiang pig, which resulted in the delayed initial stage and the development of follicle development, were mainly: 1) soybean isoflavones could change the expression level of estrogen receptor in the hypothalamus, pituitary and ovary, and affect the synthesis of GnRH, LH, FSH and steroid hormones. The expression of key factors, which affects the synthesis and secretion of hormone, causes the regulation changes of the corticosteroids; 2) soybean isoflavones can downregulate the expression of key factors in the GDF-9/BMP-15 signaling pathway, reduce the number of follicles in each stage of the ovary, and thus affect the normal development of follicles and the reproductive performance of the fragrant pigs; 3) in vitro culture test of granular cells It is also indicated that daidzein and Genistein can change the cell estrogen receptor, the key gene of steroid hormone synthesis, the gene expression of the downstream factor of GDF-9, thereby affecting the synthesis and secretion of steroid hormones. Thus, soy isoflavones as feed additives, or high level soybean meal in the reserve sow formula, need caution. Serious consideration.

【学位授予单位】：湖南农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S828

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