| It is essential for the ensurement of male reproduction and ethnic continuity to guarantee the development of testis and spermatogenesis.But the decline in fertility and even infertility of males is normal in current livestock.The source of mammalian spermatogenesis is SSCs.The proliferation and differentiation of SSCs was the key to spermatogenesis.Therefore,exploring the testicular development and spermatogenesis process plays an important role in the maintenance of male fertility and the protection of germplasm resources.In spring and winter of China,the lack of feed resources and the environment with high temperature and high humidity in summer are responsible for feed mildew,which affects the healthy growth of male animals.Both nutrient deficiencies and mycotoxins can cause stress in the animal’s body and destroy the body’s metabolic balance.Metabolic balance is essential for maintaining the normal physiological state of cells and the health of humans and mammals.The body can clear the damaged organelles and metabolic waste through cell autophagy to adapt to the unfavorable environment,and this process is a highly conserved cell self-protection pathway.UNC-51-like kinase 1(ULK1)is an essential protein for autophagy and inducing autophagosome formation.Studies have shown that 5’AMP-activated protein kinase(AMPK)is a highly conserved serine/threonine(Ser/Thr)protein kinase in eukaryotic cells and is a major sensor for regulating energy homeostasis and metabolic stress.It is involved in basic physiological processes such as cell growth,proliferation,autophagy and apoptosis.In the absence of energy,AMPK inhibits the Mammalian target of rapamycin complex 1(mTORC1)to activate the ULK1 complex to induce autophagy,and AMPK also directly phosphorylates ULK1 to promote autophagy.However,it was rarely reported about AMPK-ULK1-mediated autophagy pathway involved in the regulation of testis development and the proliferation and differentiation of SSCs.Therefore,AMPK-ULK1 signaling pathway was the key point in study to study the changes in autophagy levels in rams under energy deficit and oxidative stress,revealing the AMPK-ULK1 autophagy signaling pathway in ram development and goat SSCs.The study provides a reference for enriching and perfecting the molecular regulation theory of reproductive physiology of male animals.1.Effect of energy stress on testicular development in lambs24 male lambs with similar body conditions(3 month,22.02±0.14 kg)were randomly divided into three groups:C group(100%energy,control group,n=8),15%ER group(15%energy restriction,n=8)and 30%ER group(30%energy restriction,n=8).After 60 days,12 lambs(4 lambs for each group)were euthanized,then weighing testicular weight,collect blood and testicular tissue.Unslaught 16 lambs were continued to re-fed with 100%energy diet to compensate for energy restriction.Then renamed C group(100%energy,control group,n=4),15%ER-C(energy compensation after 15%energy restriction group,n=4)and 30%ER-C(30%energy restriction after feeding,n=4).After 90 days,weighing testicular weight,the blood and testicular tissues were collected(n=4).Plasma testosterone levels,histomorphology,autophagy and apoptosis-related genes and protein expression levels were detected by ELLSA,HE staining,qRT-PCR and Western-blot.The results showed that with the decreasing of energy level,the testicular weight decreased significantly(P<0.05),the testicular organ index decreased(P>0.05),the number and type of spermatogenic cells decreased significantly;the ER group Beclinl and LC3 mRNA and protein levels increased significantly,and the LC3Ⅱ/Ⅰ ratio increased significantly(P<0.05).Compared with the C group,the mRNA and protein levels of BAX/Bcl-2 in the ER group were significantly increased(P<0.05).However,after energy compensation,the testicular weight,testicular organ index and testosterone level were recovered to common condition(P>0.05);Beclinl protein expression level and LC3Ⅱ/I ratio were down-regulated in ER-C group(P<0.05),BAX/Bcl-2 mRNA and protein levels were also significantly decreased(P<0.05).In summary,insufficient energy induced increasing the level of autophagy and apoptosis in pre-pubertal male lambs,inhibiting the testis development of lambs.In addition,the testicular indicators of lambs in ER group were returned to normal levels,and the possible mechanisms for this phnomenon needed to be further studied.2.Effect of AMPK-ULK1 autophagy pathway on energy stress influencing testicular development in lambsThe distribution of p-AMPK(Thrl72)in testis tissue was determined by immunohistochemistry(IHC).Expression of genes and proteins related to the upstream and downstream of AMPK signaling pathway in the testis tissues of ER and EC groups was detected by qRT-PCR and Western-blot.The results showed that p-AMPK(Thr172)was mainly localized in the nucleus of spermatogenic cells;compared with the control group,the expression levels of LKB1,AMPK and ULK1 mRNA in the ER group were significantly increased(P<0.05),AMPK,p-AMPK and the expression of ULK1 protein was increased(P<0.05);the expression of LKB1 mRNA,p-AMPK and ULK1 protein was significantly decreased after energy compensation(P<0.05),while the expression of AMPK mRNA was significantly increased in 30%ER-C group(P<0.05).),AMPK protein expression was significantly elevated in all ER-C groups(P<0.05).In summary,insufficient energy activates LKB1,which in turn activates the AMPK signaling pathway,up-regulates the expression of ULK1,induces autophagy,and inhibits the development of testis tissue in lambs.After supplementation,the expression of LKB1 and p-AMPK protein in the upstream gene of AMPK in the treatment group decreased,indicating that energy compensation may down-regulate the level of autophagy and apoptosis through the AMPK-ULK1 pathway,thereby promoting the normalization of the lambs testis in the ER group.3.Effects of AMPK-ULK1 autophagy pathway on oxidative stress affecting SSCs in goatsAfter treatment of goat SSCs in vitro with HT-2 toxin in different concentration for 12 h,the appropriate concentration was selected for subsequent experiments.After treatment of SSCs with HT-2 toxin for 12 h,we detected the intracellular ROS levels,antioxidant enzymes GSH-px,SOD and CAT activities and oxidation product MDA content.The expresstion levels of cell proliferation,differentiation,autophagy and apoptosis related mRNA or protein were detected,as well as genes in AMPK-mTOR pathway.The results showed that the SSCs viability decreased to about 60%with 80 nM HT-2 treatment,and the subsequent experiment was performed at a concentration of 80 nM.Compared with the control group,the ROS level in the treatment group was significantly increased(P<0.05),the activities of antioxidant enzymes GSH-px,SOD and CAT were significantly increased(P<0.05),and the content of MDA was significantly increased(P<0.05);mRNA expression levels of proliferation and differentiation-related genes PLZF,GFRA1 and DAZL in the treatment group were significantly decreased(P<0.05);p-AMPK/AMPK,p-Raptor/Raptor and p-ULK1/ULK1 ratio was significantly increased(P<0.05)and the p-mTOR/mTOR ratio was significantly decreased(P<0.05)with the treatment.The autophagy marker proteins Beclinl and the ratio of LC3II/I was significantly increased(P<0.05),p62 protein was significantly decreased(P<0.05),and the number of phagosome and autophagosomes increased.With the treatment in SSCs,the expression level of apoptosis-related protein BAX/Bcl-2 was significantly increased(P<0.05),and the mitochondrial membrane potential was decreased compared with control.In summary,HT-2 caused oxidative damage of SSCs and activated AMPK-mTOR-ULK1 pathway.Enventually,the fate of SSCs depend on the interaction in apoptosis and autophagy induced by AMPK-mTOR-ULK1 pathway. |
