| Soybean isoflavones (SIF) is a kind of phytoestrogen, they are abundant in soybean meal. It is received that SIF can interrupt the endocrine balance, result in endocrine disorders and subfertility or infertility of mammals. This study was to investigate the effects of SIF on onset of puberty and follicular development of female Bama miniature pigs by adding different levels of SIF into soybean meal free fomula. A total of fifty five (55),35-days old pigs with average weight 2.80±1.0 kg were obtained from a local pig herd in Bama County and used for this experiment. Pigs used in this study were selected from a total of eleven (11) farrowing mothers amidst the herd. Five healthy female pigs were selected from each mother and randomly assigned into five treatment groups. Thus, there were a total of eleven pigs assigned to each treatment. Four experimental diets were subsequently formulated by supplementing the basal diet with 0,250,500 and 1250 mg/kg SIF (80% purity), respectively. An additional fifth treatment designated as positive control was formulated to include 0.66 mg/kg estradiol valerate (EV:Equivalent to 0.5 mg/kg estradiol). In feeding period, estrus age of pigs were recorded; at 4 months of age, the second day and the forth day of estrus, blood samples were collected for the measurement of hormones; at the forth day of estrus,6 pigs from each group were selected and slaughtered, and hypothalamus, pituitary and ovary samples were collected for real-time PCR, western blot and histology analysis; 3 pigs of the remaining pigs in each group were mated at the second estrus to test the fertility parameters. In addition, ovaries were collected from slaughter house and primary granulosa cells were isolated to test the effects of daidzein (DAI) and genistein (GEN) on steroid hormone synthesis GDF-9/BMP-15 signaling and apoptosis of granulosa cells. The results were as follows:(1) Different levels of SIF supplemented to the diet of Bama miniature pigs resulted in the decrease of estrus rate at 240 d of age,1250 mg/kg of SIF increased the age of puberty. The GnRH and LH concentration of Bama miniature pigs were decreased (P<0.05) but FSH concentration was increased (P<0.05) when supplemented SIF to the diet of the pigs.250 mg/kg of SIF supplemented in the dietary decreased (P<0.05) the ER 3 in hypothalamus and ER a in ovary of Bama miniature pigs,500 mg/kg of SIF decreased (P<0.05) the expression of ER? in hypothalamus,1250 mg/kg SIF decreased (P<0.05) the ER? expression in pituitary and ovary of the pigs. All levels of SIF supplemented in the diet of the pigs decreased (P<0.05) the protein expression of ER?.1250 mg/kg SIF increased (P<0.05) the FSH-? gene expression, and 250 mg/kg soybean isoflavones supplemented in the diet decreased (P<0.05) the GNRHR expression in the pituitary of the pigs. The KISS1 gene expression in the hypothalumus and StAR,3?-HSD gene expression in the ovary was significantly decreased (P<0.01) by supplementing SIF to the diet of the pigs; 250 and 1250 mg/kg SIF supplemented in the diet decreased (P<0.05) CYP17A1 expression,250 mg/kg SIF decreased (P<0.05) the CYP19A1 expression.250 mg/kg SIF supplemented in the diet of the pigs decreased (P<0.05) the FSHR mRNA expression in the ovary of Bama miniature pigs, and all levels of soybean isoflavones decreased (P<0.05) the FSHR protein expression. These results indicate that soybean isoflavones resulted in the puberty delay of Bama miniature pigs, the mechanism is to modulate the mRNA and protein expression of estrogen receptor in hypothalamus, pituitary gland and ovary, and induced KISS1 expression in hypothalamus, StAR and 3?-HSD expression in the ovary, and induced the change of hormone level and endocrinology disorders.(2) Soybean isoflavones supplemented into the diet of Bama miniature pigs did not change (P>0.05) the weight, index of the ovaries and the number of ovulated eggs. Soybean isoflavones supplemented to the diet decreased (P<0.05) the number of follicles at different stages,1250 mg/kg of SIF decreased the primidial follicle number, and 500,1250 mg/kg of SIF decreased (P<0.05) the number of developing follicles. SIF supplemented in the diet of Bama miniature pigs decreased (P<0.05) the mRNA expression of GDF-9, BMPR-1B, and 250 mg/kg of SIF decreased (P<0.05) the mRNA expression of BMP-15, BMPR2 in the ovary of the pigs; all levels of SIF supplemented in the diet of the pigs decreased (P<0.05) the protein level of BMP-15,1250 mg/kg SIF decreased (P<0.05) the protein level of GDF-9. Soybean isoflavones supplemented in the diet of Bama miniature pigs decreased (P<0.05) the mRNA expression of HAS2, PTGS2, Ptx3, Tnfaip6 and Smad2 of the ovary,250 mg/kg of SIF decreased (P<0.05) the Smad3 and Smad4 expression. Different levels of SIF supplemented in the diet decreased (P<0.05) the number of farrowed sows, increased the number of and average litter weight, but with no difference. These results suggested that SIF interupted follicular development and reproductive parameters by regulating expression of key factors in GDF-9/BMP-15 signaling.(3) Dietary supplemented with 1250 mg/kg of SIF decreased the number of Atretie follicles in the ovary of Bama miniature pigs.500 and 1250 mg/kg of SIF supplemented in the diet increased (P<0.05) the mRNA expression of Bcl-2, while 250 mg/kg of SIF decreased (P<0.05) the mRNA expression of Bax in the ovary of Bama miniature pigs.500, 1250 mg/kg SIF have the tendency to increase the protein expression of Bcl-2, and decrease the expression of Bax in the ovary. These results indicates that SIF supplemented in the diet of Bama miniature pigs decreased the atretie follicles by regulationg the expression of apoptosis proteins in the ovary.(4) 100,500 ?M of DAI and 1,10,100 and 500 ?M of GEN supplemented into the medium of granulosa cells cultured in vitro inhibited (P<0.05) cell viability significantly. DAI and GEN decreased (P<0.05) the mRNA expression of ER ?, and GEN decreased (P<0.05) the mRNA expression of ER? in the granulosa cells.0.1 and 1 ?M of GEN supplemented in the medium decreased (P<0.05) P4 concentration. All levels of DAI decreased (P<0.05) 3?-HSD mRNA expression, while GEN decreased (P<0.05) StAR and 3?-HSD mRNA expression. Meanwhile,0.1 and 10 ?M of DAI supplemented in the medum significantly decreased (P<0.05) CYP11A1 expression in granulosa cells,1 and 10 ?M GEN decreased (P<0.05) CYP19A1 and 10?M decreased (P<0.05) 17?-HSD expression in granulosa cells. These indicates that daidzein and genistein can down-regulation of key genes expression of steroidogenesis to affect the E2 and P4 synthesis and secretion.Different levels of DAI supplemented into the medium of granulosa cells did not affect (P>0.05) gene expression of BMPR-1B and BMPR2, but 0.1 ?M of GEN increased (P<0.05) BMPR2 expression. Different levels of DAI and GEN decreased (P<0.05) Ptx3 and HAS2 expression in the granulosa cells, and 10 ?M DAI decreased (P<0.05) PTGS2 expression,1 and 10 ?M of DAI decreased (P<0.05) Tnfaip6 expression in the granulosa cells. These indicates that different levels of daidzein and genistein supplemented in to the medium of granulosa cells cultured in vitro did not change GDF-9/BMP-15 receptors expression, but down-regulated their down stream factors.1 and 10 ?M DAI supplemented into the medium of granulosa cells decreased (P<0.05) early apoptosis rate, and 1 ?M DAI decreased (P<0.05) late and total apoptosis rate. However, GEN supplemented in the medium of granulosa cells decreased (P<0.05) early apoptosis rate but increased (P<0.05) late and total apoptosis rate, and neiosis rate.0.1 ?M of DAI increased (P<0.05) Bcl-2/Bax ratio in granulosa cells. Different levels of GEN decreased Bcl-2 expression in granulosa cells, and 1 ?M of GEN significantly decreased (P<0.05) Bcl-2 expression; 10 ?M of GEN increased (P<0.05) Bax expression in granulosa cells. These indicate that low levels of daidzein and genistein could inhibit apoptosis, but high levels of daidzein and genistein induced apoptosis and neiosis of granulosa cells.In conclusion, soybean isoflavones supplemented into the diet of female Bama miniature pigs induced puberty delay and follicular develop disorders, the mechanism are:1) SIF changed the expression of estrogen receptor in hypothalamus, pituitary gland and ovary, and affected GnRH, LH, FSH and key factors's expression of steroidogenesis synthesis, and to affect the synthesis and scretion of hormones; 2) SIF supplemented into the diet down-regulated the key factors'expression in GDF-9/BMP-15 signaling, decreased follicle numbers in the ovary, and affected follicular development and reproductive parameters; 3) The in vitro study of granulosa cell suggested that daidzein and genistein affect the expression of estrogen receptors, key factors in steroidogenesis and GDF-9 down-stream genes, subsequently affect the synthesis and secretion of hormones. These suggested that soybean isoflavones as a feed additive, or high level of soybean meal used in gilts should carefully consideration. |
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