| Soy isoflavones (SIF) are a kind of phytochemicals and phytoestrogens whichare found in soy and other legumes. Epidemiological evidence and experimentalstudies have repeatedly linked the consumption of soy isoflavones or soy products toa variety of beneficial health effects, such as the prevention of cancer andcardiovascular disease, as well as osteoporosis in menopausal and postmenopausalwomen. Children have low estrogen levels and the development of hypothalamic-pituitary-ovarian axis is immature, which makes them more sensitive to exogenousestrogenic compounds. Therefore, it is reasonable to assume that soy isoflavones maypose a greater hazard to the developing reproductive system in children, and publicconcern about this issue has recently increased. Previous studies had shown that soyisoflavones may adversely affect estrogen-sensitive target tissues, including the uterus,oviduct, vagina and mammary gland. However, whether the ovary, especially theovarian follicle, which as an endogenous estrogen-producing tissue, is affected by soyisoflavones remains unclear and is seldom been intensively studied. Based on theseobservations, the purpose of this study was to determine the influence of theadministration of soy isoflavones from weaning to sexual maturity on ovarian follicledevelopment by using the animal model of Wistar rats. The studies of our researchinclude the effects of soy isoflavones on ovarian development in general; themetabolomic variations in follicular fluid; apoptosis of ovarian cells and theregulation function of Fas and Bcl-2/Bax signal pathway. The results would haveguiding instuction for the prevention and control of soy isoflavones on female gonadalreproductive development and for the formulation of soy foods and soy isoflavonesformula in safe intake of immature female. Objective:To investigate the general effect of soy isoflavones on ovarian development byestablishing animal models of soybean isoflavones exposure from weaning to sexualmaturity, then to investigate the metabolomic variations in follicular fluid usingHPLC-MS and uncover useful toxic biomarkers. Finally to explore the effects of soyisoflavones on apoptosis of ovarian granulosa cells and the regulation function of Fasand Bcl-2/Bax signaling pathway. the results would have guiding instruction for theprevention and control of soy isoflavones on female gonadal reproductivedevelopment and for the formulation of soy foods and soy isoflavones formula in safeintake of immature female.Methods:1. Eighty female21day old Wistar rats weighing45±5g were randomly dividedinto four groups of twenty rats each. Rats of each group were treated daily byintragastric administration of SIF (0,50,100or200mg/kg body weight, respectively)until they reached sexual maturity. Weight gain was measured every day. Age ofvaginal opening and estrus cycle were observed before killing. After the treatmentperiod, the animals were killed by decapitation at the oestrus of estrous cycle, andblood, ovarian samples were harvested to determine serum estradiol (E2) andprogesterone (P4) levels and enumerate ovarian follicles in each stage.2. Follicular fluid was collected from the antral follicles of the ovary using amicroinjector under an inverted microscope. Follicular fluid collected from the leftovaries of three rats from the same group was pooled, up to a total volume of15μl.Each group consists of6samples.The follicular fluids were then prepared successivepassing through a series of centrifugation, methanol extraction, HPLC-MS detection,chromatography and mass spectrometry data dimension reduction and denoising.Principal component analysis (PCA) and partial least-squares-discriminant analysis(PLS-DA) was used to recognize pattern. Variable importance in projection (VIP) anddatabases were employed to identify differences in metabolites and reveal useful toxic biomarkers. Finally, Standards of metabolic interest were used to confirm thestructures.3. Two ovarian tissues were randomly selected to observe cell apoptosis by usingtransmission electron microscopy. Ovarian sections in part one were also used todetect cell apoptosis by TUNEL method. Six ovarian tissues were selected to detectmRNA level of Caspase-3,8,9, Fas, Bax and Bcl-2by real time PCR. The rest6ovarian tissues were selected to determine protein expression level of Caspase-3, Fas,Bax and Bcl-2by Western-blot method.Results:1. The general effect of soy isoflavones on ovarian development (1)There was nosignificant difference in body, ovary and uterus weight among any of the groups (P>0.05).(2)Compared with the control group, age of vaginal opening was accelerated inhigh-dose groups (P<0.05). There was no significant change in estrus cycle of SIFgroups (P>0.05).(3)SIF treatment led to significant reductions in serum E2levels inhigh-dose SIF group as compared to the control group (P <0.05). However, there wereno significant differences (P>0.05) in the levels of serum P4among any of thegroups.(4)In the control and low-dose SIF group, a large number of primordial andprimary follicles were found. However, in the middle and high dose SIF groups, thenumbers of atretic follicles and corpora lutea were higher than those of the controlgroup (P <0.05)2. Metabolomic changes in follicular fluid induced by soy isoflavonesadministered to rats from weaning until sexual maturity:(1) Total ion intensitychromatogram: Visual comparison of these chromatograms indicated that themetabolic profiles of the groups treated with soy isoflavones differ somewhat fromthat of control group.(2)PCA analysis: Through data dimension reduction anddenoising, approximately1300peaks (defined by a pair of m/z values and RT) wereidentified. the control and low, middle and high dose SIF rats are appreciablyseparated in the pseudo3D-PCA score plot.(3)PLS-DA analysis: The score plots of the PLS-DA between the control group and the groups treated with soy isoflavonesare clearly separated.(4)Based on the VIP value, database retrieval and standardverification, The24most significantly altered metabolites were identified, includingprimary sex hormones, amino acids, fatty acids and metabolites involved in energymetabolism.3. The effects of soy isoflavones on apoptosis of ovarian cells and the regulationfunction of Fas and Bcl-2/Bax signaling pathway:(1) Granulosa cell apoptosisincreased in SIF groups from Transmission electron microscope. Lipid accumulationwas also found in SIF groups.(2) By using TUNEL assay, the results showed thatgranulosa cell apoptosis rate of the antral follicle in the high-dose group wasobviously higher than that of the control group (P<0.05), while granulosa cellapoptosis rate of the atretic follicle was also higher than that of the control group(P<0.05). Granulosa cell apoptosis rate of corpora lutea in middle and high SIFgroups was significantly higher than that of control group (P<0.05).(3)mRNA levelsof caspase-3,8,9, Fas and Bax in middle and high-dose groups were up-regulated(P<0.05). mRNA level of Bcl-2in low-dose SIF group was higher than that of controlgroup (P<0.05).(4)Consistent with the results of real time PCR, protein expression ofCaspase-3, Fas, Bax gene was significantly up-regulated (P<0.05), but Bcl-2proteinexpression in high-dose group was significance lower than that of control group,which was inconsistent with the results of real time PCR.Conclusions:1. Continuous exposure of soy isoflavones from weaning to sexual maturity canaffect ovarian development, resulting in atretic follicle and corpus luteum increasing,which indicate that the ovary is one of the main target tissues of soy isoflavones.2. Soy isoflavones can induce metabolic alterations in follicular fluid, whichinclude primary sex hormones, amino acids, fatty acids and metabolites involved inenergy metabolism. These findings indicate that soy isoflavones affect ovarian follicledevelopment by inducing metabolomic variations in follicular fluid. 3. Soy isoflavones can induce apoptosis of granulosa cells and lipids accumulationin ovary. the possible mechanism may relate to the activation of Bcl-2/Bax and Fassystem, and then activating the Caspase family signaling pathway. |
