Study On The Molecular Mechanism Of Energy Metabolism Regulation And Induction Of Ferroptosis In Dairy Goat Sperm During The Freezing-Thawing Process

Semen cryopreservation technology plays a pivotal role in modern livestock artificial insemination.It facilitates the exchange of germplasm resources without being limited by temporal,geographical,or species constraints.Moreover,it promotes the effective utilization of male breeding stock and accelerates breed enhancement efforts.As an essential element contributing to the success of semen cryopreservation,semen cryopreservation diluent effectively mitigates the energy depletion and oxidative damage inflicted on sperm during the freeze-thaw process,thereby extending sperm viability,enhancing the post-thaw semen quality,and increasing the conception rate of females after artificial insemination.Sperm is a highly specialized cell capable of swimming within the female reproductive tract and possessing the ability to bind with ovum for fertilization.The substrates of energy metabolism are pivotal components of the necessary metabolic activities for spermatogenesis,maturation,and fertilization processes.However,the role played by semen diluent metabolic substrates in the energy metabolism of dairy goat sperm,as well as the specific mechanism of their involvement in motility pattern,remains unclear.Furthermore,the plasma membrane of goat sperm is rich in polyunsaturated fatty acids(PUFAs),making the sperm highly susceptible to low temperatures.The freezing-thawing process can lead to an excessive accumulation of reactive oxygen species(ROS),causing oxidative stress damage to the sperm.This can disrupt the sperm membrane structure,potentially leading to the loss of normal physiological functions and fertilization ability.However,the precise mechanism through which lipid ROS affects dairy goat sperm and causes oxidative damage remains uncertain.To address these questions,this study initially investigates the energy supply metabolic pathways and the mode of action of oxidative stress-induced damage in dairy goat sperm.Proteomics and metabolomics sequencing techniques are employed to explore potential regulatory mechanisms underlying energy metabolism and oxidative stress induction during the freezing-thawing process in dairy goat sperm.Additionally,a "low glucose + high glucose" dilution model is utilized to analyze the relationship between metabolites in dairy goat semen dilution and sperm motility.Furthermore,an oxidative damage model of sperm dilution is established to analyze how lipid ROS affects oxidative damage in dairy goat sperm during the freezing-thawing process.This study aims to uncover the relationship between energy metabolism and sperm motility in dairy goat sperm during the freezing-thawing process and reveal the mechanisms of sperm oxidative damage.The research has critical implications for developing efficient dilution solutions for dairy goat semen cryopreservation,establishing effective artificial insemination techniques for frozen semen,and contributing to genetic improvement and enhanced reproductive efficiency in dairy goats.The main findings of this study are as follows:1.The freezing-thawing process of dairy goat semen can result in sperm death or sub-lethal damage,with sperm motility,average path velocity,and curvilinear velocity decreasing by 32.05%,8.05%,and 11.39%,respectively.Utilizing TMT-labeled quantitative proteomics analysis,a total of 3015 proteins were identified in both fresh and frozen dairy goat semen.Among these,65 differentially expressed proteins were identified,comprising34 upregulated and 31 downregulated proteins.GO annotation analysis revealed that the differentially expressed proteins were primarily associated with cellular processes,biological regulation,metabolic processes,and other biological functions.These proteins were involved in molecular functions such as binding activity and catalytic activity.KEGG pathway enrichment analysis demonstrated that the differentially expressed proteins were notably enriched in 15 signaling pathways,with significant involvement in pathways such as oxidative phosphorylation,ribosome function,and cell apoptosis.Sperm-related proteins,including ND3,COXVB,RANBP9,FBXL4,TRIM36,and FTH1,were identified within these pathways.Furthermore,subsequent to freezing-thawing,the protein levels of ND3,RANBP9,CAPN11,and FTH1 were observed to decrease,suggesting their potential as biomarker candidates indicative of disrupted energy metabolism and oxidative damage in dairy goat sperm during the freezing-thawing process.2.Through untargeted metabolomics analysis,discrepancies in metabolites between fresh and frozen-thawed dairy goat semen were identified,leading to the detection of a total of 388 distinct metabolites.Among these were glucose-6-phosphate,phosphoenolpyruvate,flavin adenine dinucleotide,cis-aconitate,and itaconic acid.These metabolites were primarily associated with metabolic pathways including fructose and mannose metabolism,pyruvate metabolism,fatty acid biosynthesis,riboflavin metabolism,and the citrate cycle(TCA cycle),encompassing a total of 55 metabolic pathways.The integration of proteomics and metabolomics analyses unveiled the involvement of 9 signaling pathways,notably linked to glutathione metabolism,purine metabolism,and fatty acid biosynthesis.This included differentially expressed proteins such as ACTB,ACSBG1,LOC102189813,and differentially regulated metabolites like FA 18:00 and L-Gamma-Glutamyl-L-Amino Acid.Furthermore,following freezing-thawing,there was a downregulation of ACTB protein and an upregulation of ACSBG1 protein and FA 18:00,suggesting that the changes in differential proteins and metabolites might be involved in maintaining energy and oxidative homeostasis in dairy goat sperm.3.To investigate whether the changes in differential proteins like ND3 and COXVB,as well as differential metabolites like glucose-6-phosphate,cis-aconitate,and itaconic acid,following freezing-thawing are involved in maintaining the energy metabolism stability of dairy goat sperm,a "low glucose + high glucose " dilution model was used for co-incubation with sperm to analyze the effects of the glycolysis and oxidative phosphorylation pathways on sperm motility.The results showed that compared to high glucose dilution(370 m M glucose),low glucose dilution(75 m M glucose)promoted sperm progressive motility(68.31%±2.67% vs 47.13%±2.46%,P < 0.0001)and straight-line velocity(73.83±2.435μm/s vs 37.01±1.68 μm/s,P < 0.0001)of dairy goat sperm by activating mitochondrial oxidative phosphorylation.According to the differential protein ND3 and COXVB in mitochondria before and after freezing-thawing,the addition of 20 μmol/m L rotenone,10μmol/m L Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone,and 40 μmol/m L tigecycline to the low glucose dilution significantly reduced sperm motility,opening fluorescence intensity of mitochondrial m PTP,and ATP content(P < 0.05),and significantly reduced the protein expression levels of mitochondrial ND3,COX-1,COXVB,NRF1,and TFAM(P < 0.01).This suggests that low glucose dilution activates sperm mitochondrial activity and promotes mitochondrial oxidative phosphorylation to generate ATP,thereby maintaining the linear motion of sperm.Compared to low glucose dilution,high glucose dilution can induce sperm ROS levels(34.79% vs 27.3%,P < 0.001)and MDA content(0.66±0.009 nmol/mg vs 0.39±0.01 nmol/mg,P < 0.01)accumulation,and through mediating ferroptosis,significantly reduces dairy goat sperm motility(50.46%±1.8% vs76.45%±0.95%,P < 0.0001),ATP content(1.665±0.17 nmol/mg vs 5.1±0.21 nmol/mg,P <0.0001),and MMP(0.91±0.03ΔΨm vs 3.7±0.06ΔΨm,P < 0.0001)levels.This suggests that low glucose dilution maintains sperm energy stability through the activation of the LKB1/AMPK pathway,preventing oxidative damage caused by sperm ferroptosis.4.To investigate whether the upregulation of the differential protein FTH1 in frozen semen and the generation of lipid ROS during the freezing-thawing process induce sperm ferroptosis,this study established an iron overload oxidative damage model to explore the mechanism of sperm ferroptosis.The results revealed that incubating sperm with 5.0 mg/m L Iron Dextran in the dilution medium of goat semen at room temperature for 5 hours led to sperm nuclear membrane swelling and damage,accompanied by the appearance of a few vacuoles.Additionally,disruption,absence,and abnormal location of sperm mitochondria outer membranes were observed.This resulted in a significant increase in ROS level(36.47%VS 8.2%,P < 0.0001),LPO level(34.98±1.62 μmol/L VS 5.63±0.31 μmol/L,P < 0.0001),and MDA content(3.02±0.13 nmol/mg VS 2.01±0.12 nmol/mg,P < 0.0001).Moreover,a pronounced reduction in sperm motility(26.78%±2.60% VS 82.87%±2.63%,P < 0.0001)and MMP level(2.332±0.22 ΔΨm VS 2.98±0.34ΔΨm,P < 0.01)was observed,while GSH content(128.3±1.42 μg/mg VS 3.33±0.05 μg/mg,P < 0.0001)and SOD activity(0.811±0.08U/mg VS 0.54±0.04 U/mg,P < 0.001)significantly increased.This resulted in the excessive activation of the Nrf2/HO-1 signaling pathway and significant upregulation of ACSL4,DMT1,FTL,and NCOA4 protein expression levels(P < 0.05),while significantly decreasing the expression levels of SLC7A11,GPX4,DHODH,and FSP1 proteins(P <0.05).Moreover,compared with the iron overload injury group,incubating sperm with 2μmol/L Fer-1 and 150 nmol/L Mito Q in 5.0 mg/m L Iron Dextran diluted solution significantly reduced ROS levels(38.44% VS 20.99%,38.44% VS 24.88%,P < 0.0001)and MDA content(1.75±0.03 nmol/mg VS 1.073±0.05 nmol/mg,1.75±0.03 nmol/mg VS1.215±0.05 nmol/mg,P < 0.0001),and markedly promoted the expression levels of SLC7A11,GPX4,DHODH,and FSP1 proteins(P < 0.01),effectively preventing sperm ferroptosis.Furthermore,compared to the room temperature storage of goat semen,the freezing-thawing process led to reductions of 30.53%,23.1%,34.5%,32%,and 26.5% in sperm motility,plasma membrane integrity,acrosome integrity,ATP content,and mitochondrial membrane potential(MMP),respectively.Meanwhile,ROS and LPO levels increased by 33.9% and 44.6% respectively,and promoted the accumulation of ACSL4,DMT1,NCOA4,and FTH1 proteins,suggesting that the freezing-thawing process induced sperm NCOA4/FTH1 binding,triggering ferritin autophagic degradation and leading to autophagy-dependent ferroptosis,consequently impairing sperm functionality.In conclusion,this study conducted a proteomic and metabolomic analysis of key molecular markers in goat sperm before and after the freezing-thawing process.It elucidated the potential involvement of critical differentially expressed proteins(ND3,COXVB,CAPN11,FTH1,etc.)and metabolites(glucose-6-phosphate,phosphoenolpyruvate,cis-aconitate,and itaconic acid,etc.)in regulating sperm energy metabolism transition during freezing-thawing to maintain energy and oxidative balance.By employing the "low glucose + high glucose " dilution solution,this research clarified that sperm motion can be regulated according to the varying metabolic substrate environment.The study also demonstrated that low glucose dilution solution maintains sperm energy homeostasis by activating the LKB1/AMPK signaling pathway and inhibiting sperm ferroptosis.Using an iron overload oxidative damage model,the freezing-thawing process was found to cause accumulation of lipid ROS in sperm,promoting the accumulation of ACSL4,DMT1,NCOA4,and FTH1 proteins.This subsequently triggered the binding of NCOA4/FTH1,leading to the autophagic degradation of ferritin and inducing autophagy-dependent ferroptosis.The study definitively illustrated that the low glucose dilution solution facilitates mitochondrial oxidative phosphorylation for energy supply and participates in sperm linear motion.Additionally,it unveiled that the freezing-thawing process activates the Nrf2/HO-1antioxidant system,promoting NCOA4/FTH1 binding,which consequently triggers autophagy-dependent ferroptosis in sperm.This research provides a theoretical foundation for the development of efficient dilution solutions for dairy goat semen cryopreservation and enhancing the quality of frozen semen.Furthermore,it establishes a basis for the construction of an effective artificial insemination system for dairy goats.