Mechanisms Underlying Energy Metabolism In Boar Sperm

Artificial insemination(AI)is widely used due to the developing of pig production around the world.AI is contributed to the improvements in pig production,utilization of superior boars and genetics selection.Most of the semen used in AI is the liquid diluted semen which stored at 15?17~oC.The quality of storage semen is the most important factor to limit the efficiency of AI in pig production.Sperm needs ATP to maintain motility patterns.Glycolysis and oxidative phosphorylation(OXPHOS)are two important energy metabolic pathways for ATP production in somatic cells.The utilization of different metabolic pathways for ATP production in cell is depending on the substrates in the microenvironment.Sperm shows linear motility patterns in the seminal plasma and uterine fluids;while hyperactivated motility in oviduct fluids,which indicating that sperm metabolism involved in the regulation of sperm motility patterns.However,limited information about the roles of sperm metabolism regarding sperm motility patterns.It is important for improving the composition of the extender via investigating sperm metabolism to establish an efficient extender.Therefore,this study aims to explore the sperm metabolism and OXPHS,and elucidate the mechanism of sperm OXPHOS and its regulation factors involved in maintaining high-speed motility pattern.Firstly,a low-glucose medium model was used to investigate the effects of glycolysis and oxidative phosphorylation on sperm motility pattern by examining the moving trajectories and sperm mitochondrial transcription and translation status.Secondly,the mechanism of sperm damage induced by reactive oxygen species(ROS),a by-product of oxidative phosphorylation,was evaluated using the low glucose extender and analysis of sperm mitochondrial function.In addition,the present study also explore how sperm maintained the mitochondrial OXPHOS by evaluating the glutathione(GSH)synthesis and pentose phosphate pathway(PPP).The main results were showed in the following:(1)Sperm made circle or circle-like tracks in high glucose medium;reduction of the glucose level made sperm with linear-like tracks.Low-glucose medium enhanced mitochondrial adenosine triphosphate(ATP)generation by activating sperm mitochondrial activity.The boar sperm linear motility depended on the ATP generated in mitochondria.(2)The mitochondrial transcription and translation in boar sperm were working during the incubation in low-glucose medium in vitro.Mitochondrial translation inhibitor decreased sperm linear motility patterns revealed that the sperm mitochondrial transcription and translation systems are involved in regulating the sperm motility via affecting the mitochondrial function.(3)The low-glucose glucose medium led to sperm produced a large amount of ROS which is a by-product in the OXPHOS.Excessive ROS induced sperm with oxidative damage and then reduction of sperm linear motility.(4)The targets of ROS were sperm mitochondrial transcription system and mitochondrial ATP generation system.The levels of sperm proteins(MT-ND1(NADPH dehydrogenase subunits 1),MT-ND6(NADPH dehydrogenase subunits 6),POLRMT(mitochondrial RNA polymerase)and TFAM(mitochondrial transcription factor-A))modified by 4-HNE(4-hydroxynonenal)were increased with the increasing of ROS level.In addition,the sperm mitochondrial DNA is also damaged by the ROS.(5)Addition of exogenous mitochondrial-targeted antioxidants(pyrroloquinoline quinone,PQQ;and coenzyme Q10,Co Q10)to the low-glucose medium enhanced sperm linear motility,but when the medium was in presence of the mitochondrial translation inhibitor,neither of those antioxidants recover the sperm motility,indicating that PQQ and Co Q10 could not directly reduce the proteins(MT-ND1 and MT-ND6)damaged induced by the ROS due to those proteins existed in the respiratory chain in which closed to the sites of ROS generation.PQQ and Co Q10 maintains the sperm transcription system(mt-DNA,TFAM,and POLRMT),which results in maintaining the system of sperm mitochondrial gene expression and protein synthesis,thus,new synthesis proteins(MT-ND1 and MT-ND6)are replaced with the submits in ETC complexes under high levels of oxidative stress,which affects mitochondrial ETC function to generate ATP to maintain linear motility.(6)The amino acids involved in the glutathione(GSH)synthesis was detected in boar seminal plasma.It was observed that the level of methionine,glycine,glutamate and serine were very high in seminal plasma,while it was low in terms of cysteine and cystine.Addition of methionine,glycine,glutamate and serine to the low-glucose medium significantly improved the sperm linear motility via promoting the GSH synthesis to reduce the ROS damage.Boar sperm could utilize methionine to synthesize GSH as the enzymes of cystathionine beta synthase(CBS),cystathionase(CTH),glutamate-cysteine ligase(GCLC)and glutathione synthetase(GSS)expressed in boar sperm.The levels of CBS and CTH were increased with the increasing of oxidative stress,but the levels of GCLC and GSS did not significantly change.ROS could induce sperm to synthesize CBS and CTH proteins to utilize methionine for GSH synthesis for decreasing the oxidative stress.(7)The ERK1/2-e IF4E-RSK signaling pathway cloud be activated by oxidative stress.The levels of p-ERK1/2,p-RSK,and p-e IF4E in sperm were increased by the ROS accumulation,indicating that sperm could reduce the ROS damage via its antioxidative system.(8)The pentose phosphate pathway(PPP)of sperm was involved in the regulation of sperm linear motility.The non-glucose medium could not maintain the sperm linear motion for long time,while the 20%glucose medium(low glucose medium)could maintain it.The glucose in the low-glucose medium was entered the PPP pathway for NADPH synthesis,rather than entering the glycolytic energy pathway.Itaconic acid is a key factor reprogramming the sperm glucose metabolism.The low-glucose medium produced the intermediate metabolite itaconic acid via promoting the OXPHOS.Itaconic acid cloud inactivate the sperm glycolysis pathway with modifying the enzyme of aldolase A(ALDOA),thereby reducing the sperm glycolysis pathway and allowing the glucose to enter the PPP for NADPH synthesis and then participated in the regulation of cellular redox homeostasis.