Tannic Acid Affects The Physiological Mechanism Of Reproduction In Female Brandt’s Vole

The theory about the co-evolution between herbivores and plant suggests that plant secondary metabolites（PSMs）are the most important mediators of plant-herbivore interaction.Herbivores need to feed on plants to survive,but plants produce various PSMs to hinder this feeding.Then,animals adapt to or utilize PSMs in food by changing their physiologic reaction.Polyphenols are the most common PSMs found in plants and known as natural antioxidants.Tannin is the most widely studied phenolic substance,and tannic acid（TA）is the simplest hydrolyzed tannin.Many studies have found that tannins could enhance mammalian reproduction,but there were few studies to elucidate its mechanism.Reactive oxygen species（ROS）is rapier to animal ovary.It is involved in the regulation of oocyte growth,meiosis,ovulation and other physiological processes.The content of ovarian ROS in threshold level is necessary for animals to perform their reproductive function,but excessive ROS can induce reproductive dysfunction and disorders.Therefore,to study the antioxidative effect of TA on the morphology and function of animal gonads would be beneficial to reveal the mechanism for promoting animal reproduction by tannins.Brandt’s vole（Lasiopodomys brandtii）is small herbivorous mammal in Inner Mongolia grassland.It feeds on plants with tannins.In this study,the female Brandt’s voles were used as the objects.At first,they were given 0（control）,1.5 g·kg^-1 TA（low dose TA）and 3 g·kg^-1 TA（high dose TA）,respectively.And the influence of TA on the reproductive performance of Brandt’s voles were confirmed by observing the changes of ovarian morphology,ovarian index,follicle development,reproductive hormones at all levels of hypothalamic-pituitary-ovarian axis（HPO axis）,number of embryo and offspring.At the same time,the changes of antioxidant enzymes and ovarian autophagy related proteins were also determined to explore the possible mechanism of TA affecting the reproductive performance of Brandt’s vole.Secondly,adult female Brandt’s voles were treated with 3-nitropropionic acid（3-NPA）(12.5mg·kg^-1)to establish ovarian oxidative stress model,then,different doses TA were given them to observe the effects of TA on ovarian oxidative stress,and explore the antioxidative effects of TA on ovarian function.Thirdly,the pharmacokinetics of TA in Brandt’s voles were analyzed,and the metabolites of TA in plasma and ovary were determined.Fourthly,the granulosa cells of Brandt’s vole ovary were cultured in vitro and treated with H₂O₂ to establish granulosa cell oxidative stress model,and the effect of gallic acid,the main decomposition product of TA in voles,on these granulosa cells was observed,so that the antioxidative action of TA could be explored at the cellular level.Finally,all of the results above would supply the basic information for revealing the mechanism of tannins improving animal reproduction and provide new content for the theory of co-evolution between herbivores and plant.The results were as follows.1.Effects of tannic acid on reproduction of female Brandt’s volesBoth low and high doses of TA could promote the food intake of adult female Brandt’s voles,increase their ovarian index and the proportion of mature follicles.Low dose TA decreased the proportion of atretic follicles in adult female voles,while high dose TA increased the diameter of mature follicles in adult female voles.Compared with adult voles,TA had less the influence on adolescent voles because only their ovarian index was increased by high dose TA and the proportion of mature follicles increased by low dose TA.Low dose of TA increased the embryo number and litter number of vole,while high dose of TA only increased the embryo number.Low doses of TA increased the proportion of females in the offspring of voles,on the contrary,high doses TA increased the proportion of males in the offspring.2.Effects of tannic acid on the hormone levels of HPO axis in Brandt’s volesBoth low and high doses of TA could increase the level of GnRH in the hypothalamus of adult female Brandt’s voles.It also increased the serum levels of luteinizing hormone（LH）and follicle-stimulating hormone（FSH）,serum levels of estradiol（E2）,progesterone（P4）and anti-Muller hormone（AMH）in adolescent and adult female Brandt’s voles.3.Effects of tannic acid on antioxidant function and autophagy level of Brandt’s volesLow and high doses of TA not only increased the levels of superoxide dismutase（SOD）,catalase（CAT）and glutathione peroxidase（GSH-Px）in serum of adolescent and adult female Brandt’s voles,but also increased the levels of total antioxidant capacity（T-AOC）,CAT and SOD in ovary of adult Brandt’s voles.And both of them reduced MDA levels in serum and ovary in adolescent and adult Brandt’s voles.After treatment with TA,a greater number of autophagosomes were found in the ovaries of adult female Brandt’s voles under electron microscope.Low dose of TA increased the transcription levels of some autophagy related genes,such as Beclin1,LC3 and P62,in adult female Brandt’s voles,while high dose of TA only increased the transcription levels of LC3 and P62.Both of adolescent and adult female Brandt’s voles expressed higher levels of autophagy related proteins Beclinl and LC3 after treated with Low and high dose of TA.At the same time,adult Brandt’s voles also expressed more amount of P-AMPK and P-mTOR in the autophagy pathway.4.Effect of TA on oxidative damaged ovarian of Brandt’s volesThe ovarian oxidative stress model was established successfully after adult female Brandt’s voles were treated with 12.5mg·kg^-1 3-NPA for 18 days,and the ovary showed the oxidative damaged characteristic,that meant,the ovary had lower levels of T-AOC,CAT and SOD,and higher levels of MDA.The ovarian coefficient and the number of mature follicles decreased,but the number of atretic follicles increased.TA administration could increase T-AOC,CAT and SOD levels,reduce MDA levels,relieve oxidative stress and improve antioxidant capacity in ovary of model animals.TA treatment could decrease serum FSH and LH,and increase serum AMH,P4 and E2 levels in oxidative damaged animals,increased their ovarian index and the number of mature follicles,and decreased the number of atretic follicles;TA also increased the litter number of pregnant voles.The results of TUNEL showed that TA reduced the apoptosis of ovarian granulosa cells in model animals.Masson staining showed that TA decreased ovarian fibrosis and the levels of inflammatory cytokines,such as TNF-α,IL-6,IL-1β,PTEN and P-P65,and increased the levels of Nrf2.TA also alleviated the autophagy in the ovary of model animals by reducing their level of Beclinl and LC3 and increasing the levels of P-AKT and P-mTOR.5.Effects of gallic acid on oxidative stress in granulosa cellsLC/MS was used to detect metabolites of TA in plasma and ovary of Brandt’s voles.The results showed that the main metabolites of TA in plasma were gallic acid,4-O methylgallic acid,resorcinol and pyrogallol,and TA itself also appeared in plasma.The main metabolites in the ovaries of voles were gallic acid,4-O methyl gallic acid and resorcinol,but no tannic acid was detected.Ovarian granulosa cells were isolated,identified and cultured in vitro.Granulosa cell damage was induced by H₂O₂ and cell viability was detected by CCK8.The oxidative stress cell model was established successfully when cells treated with 200μM H₂O₂.The optimal concentration of gallic acid to protect cells from oxidative damage was 50 μM.DCFH-DA fluorescence probe showed that gallic acid could reduce ROS in granulosa cells damaged by oxidation.Annexin V/PI double staining（flow cytometry）and TUNEL assay showed that gallic acid reduced apoptosis caused by oxidative damage,and MDC and Lyso-tracker showed that gallic acid reduced autophagosome induced by oxidative stress.In addition,gallic acid increased the activity of T-AOC,CAT and SOD,and decreased the level of MDA in damaged cells.The expression of Beclinl and LC3 was decreased,while the expression of P62 protein was increased.Gallic acid also promoted the expression of P-AKT and P-mTOR,and decreased the expression of P-AMPK.All of these results strongly suggest that TA enhance the antioxidant capacity of Brandt’s voles,which in turn,affecting AMPK and AKT/mTOR signaling pathways to regulate ovarian autophagy and apoptosis,and regulate the hormone levels of HPO axis.Thus,it could improve vole’s anti-inflammatory ability,reduce its ovarian fibrosis and follicular atresia,increase follicular maturation and ovarian coefficient,and promote follicular development to reduce ovarian damage,and eventually,promote ovarian development and improve the reproductive ability of Brandt’s vole.