The Bombyx mori G protein ß subunit 1 (BmGNß1) gene inhibits BmNPV infection.

G protein ß subunit 1 (GNß1) has several functions, including cell growth regulation, the control of second messenger levels, and ion channel switching. Previous transcriptome analyses in our laboratory have shown that BmGNß1 transcription is reduced following infection with Bombyx mori nucleopolyhedrovirus (BmNPV), but it is unknown what role this gene may have in the host response to BmNPV infection. In this study, the BmGNß1 gene was cloned using the RACE method. After BmNPV infection, BmGNß1 was downregulated in Baiyu strains in tissues such as the hemolymph and midgut. Indirect immunofluorescence showed that BmGNß1 was localized to the cytoplasm. We further constructed a BmGNß1-pIZ/V5-His-mCherry overexpression plasmid and designed siRNA to evaluate the role of BmGNß1 in host response to infection. The results showed that BmGNß1 overexpression inhibited BmNPV proliferation, while knockdown of BmGNß1 was correlated with increased BmNPV proliferation. The siRNA-mediated reduction of BmGNß1 was correlated with an increase in BmNPV infection of BmN cells, increased BmNPV vp39 transcription, and reduced survival time of BmNPV-infected B. mori. Overexpression of BmGNß1 in BmN cells was also correlated with apoptosis and a modification in transcript levels of genes involved in host response to BmNPV infection (PI3K, AKT, Bmp53, BmFOXO, Caspase-1, Bmp21, BmPKN and BmCREB), suggesting that BmGNß1 may influence the apoptotic host response of infected B. mori through the PI3K-AKT pathway. This study provides potential targets and theoretical support for breeding BmNPV-resistant silkworm varieties.

Complementary transcriptomic and proteomic analyses elucidate the toxicological molecular mechanisms of deoxynivalenol-induced contractile dysfunction in enteric smooth muscle cells.

Deoxynivalenol (DON) is one of the frequent Fusarium mycotoxins and poses a serious threat to public health worldwide. DON-induced weight loss is tightly connected with its ability to decrease feed intake by influencing gastrointestinal tract (GIT) motility. Our previous reports indicated that DON interfered with intestinal motility by injuring the contractility of enteric smooth muscle cells (SMC). Here, we further explored the potential mechanisms by employing a complementary method of transcriptomics and proteomics using the porcine enteric smooth muscle cell line (PISMC) as an experimental model. The transcriptomic and proteomic data uncover that the expression of numerous extracellular matrix (ECM) proteins and multiple integrin subunits were downregulated in PISMC under DON exposure, suppressing the ECM-integrin receptor interaction and its mediated signaling. Furthermore, DON treatment could depress actin polymerization, as reflected by the upregulated expression of Rho GTPase-activating proteins and cofilin in PISMC. Meanwhile, the expression levels of downstream contractile apparatus genes were significantly inhibited after challenge with DON. Taken together, the current results suggest that DON inhibits enteric SMC contractility by regulating the ECM-integrin-actin polymerization signaling pathway. Our findings provide novel insights into the potential mechanisms behind the DON toxicological effects in the GIT of humans and animals.

Knockdown of ANGPTL4 inhibits adipogenesis of preadipocyte via autophagy.

It has been demonstrated that angiopoietin-like protein 4 (ANGPTL4) plays an important regulatory role in lipid metabolism and backfat deposition appears to vary in different pig breeds. However, the correlation between ANGPTL4 and backfat deposition have not been well characterized and the role of ANGPTL4 in regulating adipogenesis remains unclear. Therefore, this study aimed to investigate correlation between ANGPTL4 and backfat deposition and to explore the effects of ANGPTL4 on preadipocyte differentiation and the underlying mechanism. Our results showed that the backfat thickness and the ANGPTL4 gene expression of Laiwu pigs were significantly higher than those in DLY pigs and the ANGPTL4 gene expression was positively correlated with backfat thickness both in DLY pigs and Laiwu pigs. Moreover, an increase in ANGPTL4 expression and activation of autophagy were observed during the differentiation of stromal vascular fraction cells. In addition, knockdown of ANGPTL4 inhibited the differentiation of 3T3-L1 cells with decreased expression of LC3-II and ATG5 and increased expression of SQSTM1, suggesting the involvement of autophagy in ANGPTL4-mediated adipogenesis. In conclusion, these results suggested that ANGPTL4 is positively correlated with backfat deposition in pigs and knockdown of ANGPTL4 inhibits adipogenesis of preadipocyte via autophagy, providing new insights into the regulation of fat deposition and to improve the carcass quality and meat quality of porcine.

Heat Stress Modulates a Placental Immune Response Associated With Alterations in the Development of the Fetal Intestine and Its Innate Immune System in Late Pregnant Mouse.

The placenta is critical for the regulation of fetal innate immune function. Maternal heat stress (HS) impairs the immune function and the intestinal barrier in the offspring. However, the effects of maternal HS on the placental immune response and the development of the fetal intestine and its innate immune system remain unclear. Fetal mice were divided into the utero control (IUTN) and heat stress (IUHS) groups according to the maternal ambient temperature. Transcriptome analysis revealed that the expressions of placental immune response-related genes such as macrophage antigen CD68 and Fc gamma receptors 1 and 3 (fcgγ1 and fcgγ3) were increased, but the mRNA expression and protein levels of colony-stimulating factor-1 (Csf1) were decreased in the HS group compared with the TN group (p < 0.05). Furthermore, there was no significant difference in the intestinal length normalized to pup weight between the IUTN and IUHS groups. The expression of genes (such as alpi and ttr) involved in fetal duodenum and jejunum development was downregulated by maternal HS, whereas the expression of genes enriched in the cell cycle was increased. The mRNA expression and protein levels of cell division cycle 6 (Cdc6) in the fetal duodenum and jejunum were much higher in the IUHS group than in the IUTN group (p < 0.05). Maternal HS also down-regulated the expression of genes enriched in the innate immune system in the fetal duodenum and jejunum. The mRNA expression and protein levels of interleukin 1 alpha (IL1a) were reduced in the IUHS group compared with the IUTN group (p < 0.05). Taken together, these data demonstrated that maternal HS modulated the expression of genes in the placenta related to the immune response and inhibited the development of the fetal intestine and its innate immune system.

Pathogenicity Detection and Genome Analysis of Two Different Geographic Strains of BmNPV.

The pathogenicity of different concentrations of Bombyx mori nuclear polyhedrosis virus- Zhenjiang strain (BmNPV ZJ) and Yunnan strain (BmNPV YN) was assessed in Baiyu larvae. The structures of the two viral strains were observed by negative-staining electron microscopy, and their proliferation was examined by quantitative polymerase chain reaction (qPCR). The genomic sequences of these two viruses were obtained to investigate the differences in their pathogenicity. The lethal concentration 50 (LC50) of BmNPV ZJ against Baiyu larvae was higher than that of BmNPV YN, indicating a relatively more robust pathogenicity in BmNPV YN. Electron microscopic images showed that the edges of BmNPV YN were clearer than those of BmNPV ZJ. The qPCR analysis demonstrated significantly higher relative expressions of immediately early 1 gene (ie-1), p143, vp39, and polyhedrin genes (polh) in BmNPV ZJ than in BmNPV YN at 12-96 h. The complete genomes of BmNPV ZJ and BmNPV YN were, respectively, 135,895 bp and 143,180 bp long, with 141 and 145 coding sequences and 40.93% and 39.71% GC content. Considering the BmNPV ZJ genome as a reference, 893 SNP loci and 132 InDel mutations were observed in the BmNPV YN genome, resulting in 106 differential gene sequences. Among these differential genes, 76 (including 22 hub genes and 35 non-hub genes) possessed amino acid mutations. Thirty genes may have been related to viral genome replication and transcription and five genes may have been associated with the viral oral infection. These results can help in understanding the mechanisms of pathogenicity of different strains of BmNPV in silkworms.

Heat stress affects fetal brain and intestinal function associated with the alterations of placental barrier in late pregnant mouse.

High ambient temperature-induced heat stress (HS) during pregnancy may affect the placental function and fetal development. Late gestation is a critical period of the developing fetal brain and intestine. The study aimed to investigate the effects of HS during late pregnancy on the function of placenta, fetal brain and intestine in a mouse model. We found that the number of stillborn fetal mice were increased due to maternal HS. Transcriptome analysis revealed that the expression of genes enriched in nutrients transport and metabolism of HS group were up-regulated in the placenta, but down-regulated in the fetal duodenum and jejunum. Interestingly, the concentration of triglyceride (TG) in the HS group was raised in the placenta, but reduced both in the fetal duodenum and jejunum compared with the thermal-neutral (TN) group. Additionally, maternal HS also reduced total cholesterol (TC) contents in the fetal duodenum. The mRNA expression and protein levels of placental fatty acid binding protein 2 and 4 (fabp2 and fabp4) were not affected by maternal HS, but the mRNA expression and protein levels of cluster of differentiation 36 (CD36) and diacylglycerol acyltransferase-2 (Dgat2) were decreased in the fetal intestine. Furthermore, maternal HS reduced the mRNA expression and protein levels of the placental 11beta-hydroxysteroid dehydrogenase type 2 (Hsd11b2) and 5-hydroxytryptamine receptor 1D (Htr1d). The concentrations of corticosterone and the expression of heat shock protein 90 beta family member 1 (hsp90b1), hypoxia up-regulated 1 (hyou1) and corticotropin releasing hormone receptor 1 (crhr1) enriched in response to glucocorticoids in the fetal brain were increased by maternal HS. Taken together, our findings demonstrated that maternal HS disrupted the placental glucocorticoid barrier and serotonin system associated with the raised corticosterone levels in the fetal brain, which might contribute to the decreased capacity of nutrients transport and metabolism in the fetal intestine.

High ambient temperature exposure during late gestation disrupts glycolipid metabolism and hepatic mitochondrial function tightly related to gut microbial dysbiosis in pregnant mice.

As global warming intensifies, emerging evidence has demonstrated high ambient temperature during pregnancy negatively affects maternal physiology with compromised pregnant outcomes; however, little is known about the roles of gut microbiota and its underlying mechanisms in this process. Here, for the first time, we explored the potential mechanisms of gut microbiota involved in the disrupted glycolipid metabolism via hepatic mitochondrial function. Our results indicate heat stress (HS) reduces fat and protein contents and serum levels of insulin and triglyceride (TG), while increases that of non-esterified fatty acid (NEFA), ß-hydroxybutyric acid (B-HBA), creatinine and blood urea nitrogen (BUN) (P < 0.05). Additionally, HS downregulates both mitochondrial genes (mtDNA) and nuclear encoding mitochondrial functional genes with increasing serum levels of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) (P < 0.05). Regarding microbial response, HS boosts serum levels of lipopolysaccharide (LPS) (P < 0.05) and alters ß-diversity (ANOSIM, P < 0.01), increasing the proportions of Escherichia-Shigella, Acinetobacter and Klebsiella (q < 0.05), while reducing that of Ruminiclostridium, Blautia, Lachnospiraceae_NK4A136_group, Clostridium VadinBB60 and Muribaculaceae (q < 0.05). PICRUSt analysis predicts that HS upregulates 11 KEGG pathways, mainly including bile secretion and bacterial invasion of epithelial cells. The collective results suggest that microbial dysbiosis due to late gestational HS has strong associations with damaged hepatic mitochondrial function and disrupted metabolic profiles.

Heat stress during late gestation disrupts maternal microbial transmission with altered offspring's gut microbial colonization and serum metabolites in a pig model.

Heat stress (HS) during gestation has been associated with negative outcomes, such as preterm birth or postnatal metabolic syndromes. The intestinal microbiota is a unique ecosystem playing an essential role in mediating the metabolism and health of mammals. Here we hypothesize late gestational HS alters maternal microbial transmission and structures offspring's intestinal microbiota and serum metabolic profiles. Our results show maternal HS alters bacterial ß-diversity and composition in sows and their piglets. In the maternal intestine, genera Ruminococcaceae UCG-005, [Eubacterium] coprostanoligenes group and Halomonas are higher by HS (q < 0.05), whereas the populations of Streptococcus, Bacteroidales RF16 group_norank and Roseburia are decreased (q < 0.05). In the maternal vagina, HS mainly elevates the proportions of phylum Bacteroidetes and Fusobacteria (q < 0.05), whereas reduces the population of Clostridiales Family XI (q < 0.05). In the neonatal intestine, maternal HS promotes the population of Proteobacteria but reduces the relative abundance of Firmicutes (q < 0.05). Moreover, the core Operational taxonomic units (OTU) analysis indicates the proportions of Clostridium sensu stricto 1, Romboutsia and Turicibacter are decreased by maternal HS in the intestinal and vaginal co-transmission, whereas that of phylum Proteobacteria and Epsilonbacteraeota, such as Escherichia-Shigella, Klebsiella, Acinetobacter, and Comamonas are increased in both the intestinal and vaginal co-transmission and the vagina. Additionally, Aeromonas is the only genus that is transmitted from environmental sources. Lastly, we evaluate the importance of neonatal differential OTU for the differential serum metabolites. The results indicate Acinetobacter significantly contributes to the differences in the adrenocorticotropic hormone (ACTH) and glucose levels due to HS (P < 0.05). Further, Stenotrophomonas is the most important variable for Cholesterol, low-density lipoprotein (LDL), diamine oxidase (DAO), blood urea nitrogen (BUN) and 5-hydroxytryptamine (5-HT) (P < 0.10). Overall, our data provides evidence for the maternal HS in establishing the neonatal microbiota via affecting maternal transmission, which in turn affects the maintenance of metabolic health.

Responses of intestinal morphology and function in offspring to heat stress in primiparous sows during late gestation.

Late gestation is a key period for intestinal development. Maternal heat exposure may induce intestinal dysfunction of offspring. To investigate the responses of intestinal morphology and function of offspring to the maternal heat stress (HS), twelve first-parity Landrace × Large White sows were assigned to thermoneutral (TN) (18-22 °C; n = 6) or HS (28-32 °C; n = 6) treatment groups at 85 d of gestation until natural farrowing. Twenty-four newborn piglets (two piglets at medium body weight from each litter) were randomly selected and divided into in utero thermoneutral (IUTN, n = 12) and heat-stressed (IUHS, n = 12) groups according to the sow's treatment. Blood and intestinal samples were harvested to evaluate stress hormone levels, intestinal morphology, integrity and barrier function in the newborn piglets. Our results showed that maternal HS piglets exhibited increased serum adrenocorticotropic hormone (ACTH) concentration compared with that observed in the IUTN group. IUHS piglets showed lower lactase activities in the jejunum and ileum, whereas no significant differences were found between the two groups in the length of intestine, villus length or crypt depth. Serum diamine oxidase (DAO) activity was increased in IUHS piglets. IUHS piglets also exhibited decreased ZO-1, ZO-2 and MUC2 mRNA expression in the jejunum, while the protein levels were not affected. Additionally, IUHS piglets had a lower apoptotic percentage and FAS mRNA expression in the jejunum than those in the IUTN group. Taken together, these results demonstrate that high ambient temperature during late gestation of primiparous sows causes stress response in neonatal piglets, compromising intestinal permeability and mucosal barrier function, which may be partly mediated by inducing intestinal apoptosis.

Heat stress affects fecal microbial and metabolic alterations of primiparous sows during late gestation.

BACKGROUND: Heat stress (HS) jeopardizes intestinal barrier functions and augments intestinal permeability in pigs. However, whether HS-induced maternal microbial and metabolic changes in primiparous sows during late gestation remains elusive. We present here, a study investigating the fecal microbial and metabolic responses in late gestational primiparous sows when exposed to HS. METHODS: Twelve first-parity Landrace × Large White F1 sows were randomly assigned into two environmental treatments including the thermoneutral (TN) (18-22 °C; n = 6) and HS (28-32 °C; n = 6) conditions. Both treatments were applied from 85 d of gestation to farrowing. The serum and feces samples were collected on d 107 of gestation, for analyses including intestinal integrity biomarkers, high-throughput sequencing metagenomics, short-chain fatty acid (SCFA) profiles and nontargeted metabolomics. RESULTS: Our results show that HS group has higher serum Heat shock protein 70 (HSP70), lipopolysaccharide (LPS) and lipopolysaccharide-binding protein (LBP) levels. The gut microbial community can be altered upon HS by using ß-diversity and taxon-based analysis. In particular, the relative abundance of genera and operational taxonomic units (OTUs) related to Clostridiales and Halomonas are higher in HS group, the relative abundance of genera and OTUs related to Bacteroidales and Streptococcus, however, are lower in HS group. Results of metabolic analysis reveal that HS lowers the concentrations of propionate, butyrate, total SCFA, succinate, fumarate, malate, lactate, aspartate, ethanolamine, ß-alanine and niacin, whereas that of fructose and azelaic acid are higher in HS group. These metabolites mainly affect propanoate metabolism, alanine, aspartate and glutamate metabolism, phenylalanine metabolism, ß-alanine metabolism, pantothenate and CoA biosynthesis, tricarboxylic acid cycle (TCA) and nicotinate and nicotinamide metabolism. Additionally, correlation analysis between significant microbes and metabolites indicated that the HS-induced microbiota shift is likely the cause of changes of intestinal metabolism. CONCLUSIONS: Taken together, we reveal characteristic structural and metabolic changes in maternal gut microbiota as a result of late gestational HS, which could potentially provide the basis for further study on offspring gut microbiota and immune programming.

Extracellular ATPs produced in seminal plasma exosomes regulate boar sperm motility and mitochondrial metabolism.

Artificial insemination using diluted semen with reduced presence of seminal plasma has been applied worldwide. Sperm stored in seminal plasma rather than diluted or removed show improvement in survival and motility. However, the link between seminal plasma and sperm remains poorly understood. This study focuses on the effect of extracellular adenosine triphosphate (exATP) produced by boar seminal plasma exosomes on sperm motility, as well as the underlying molecular mechanisms. The seminal plasma exosomes had an average diameter of 86.6 nm and showed universal exosome markers, such as heat shock protein 70 (HSP70) and CD63. Production of net ATP increased when exosomes were incubated with glucose and partly inhibited by a glycolytic inhibitor such as iodoacetate. Fresh boar sperm incubated with exATP significantly increased sperm motility and reduced apoptotic rate. Ser21 phosphorylation of glycogen synthase kinase 3α (inactivation) also significantly increased, consistent with the increase in mitochondrial transmembrane potential in the exATP-treated sperm. Moreover, exATP treatment increased the intracellular ATP (inATP) concentration and decreased the ADP/ATP ratio in boar sperm. Lactate content in the incubation medium was decreased, whereas lactate dehydrogenase activity in sperm was increased. This finding suggested that exATP could prompt lactate to produce inATP in order to sustain motility. The combined results indicate that exATP produced in seminal plasma exosomes may finely modulate mitochondrial metabolism to control sperm motility. The results can provide insights into semen dilution and artificial insemination.

Effects of Stress on the Mucus-microbial Interactions in the Gut.

Stress shows both direct- and indirect-effects on the functions of the gastrointestinal tract, in particular on the mucus physiology and the composition of microbiota. Mucus mainly consists of heavily glycosylated proteins called mucins, which are secreted by goblet cells. The gut mucus layer is a pivotal part of the intestinal protection and colonized by commensal microbes, essential for the development and health of the host. There is a symbiotic interaction between intestinal microbiota and the host cells. On the one hand, mucus provides nutrients for the growth and adhesion of microbes; on the other hand, mucin-degrading bacteria generate energy sources for the host epithelium. However, the mucusmicrobial interaction has rarely been considered in the context of stress exposure. Therefore, this paper principally reviews the effects of stress on both mucus secretion and gut microbiota and is hoped to provide a new perspective for future study.

Heat stress induces distinct responses in porcine cumulus cells and oocytes associated with disrupted gap junction and trans-zonal projection colocalization.

Cumulus cells (CCs), the granulosa cells surrounding the oocytes, play critical roles in oocytes maturation through intercellular communication by extending trans-zonal projections (TZPs) to contact oocytes via gap junctions (GJs). The adverse effect of heat stress (HS) on oocyte maturation has been well documented, whereas the HS responses of CCs and the oocytes in association with GJ/TZP colocalization remain unclear. In this study, porcine cumulus-oocyte complexes (COCs) were subjected to HS at 41.5°C for 24 hr during in vitro maturation. Cumulus expansion was impaired and oocyte quality was reduced with lower survival rate, polar body extrusion rate, and early embryo developmental potentials. CCs and oocytes isolated from COCs demonstrated distinct responses to HS. The messenger RNA abundance of heat shock protein-related genes and mitochondrial DNA-encoded genes, together with ATP content, were significantly increased in CCs, yet decreased in oocytes, despite activation of caspase 3 detected in both CCs and oocytes. Similar changes were observed when denuded oocytes and isolated CCs subjected to HS separately, except mitochondria reactive oxygen species (mROS). In heat-stressed COCs, mROS was significantly increased only in oocytes. However, when isolated CCs and denuded oocytes were heat-stressed separately, mROS was significantly increased only in CCs. Moreover, F-actin, a TZP marker, and its colocalization with a GJ protein connexin-45, were significantly reduced in heat-exposed COCs. These results indicate that HS induces distinct responses in porcine CCs and oocytes in association with disrupted GJ and TZP colocalization.

Melatonin-induced increase of lipid droplets accumulation and in vitro maturation in porcine oocytes is mediated by mitochondrial quiescence.

Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA-encoded genes was significantly down-regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin-treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up-regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin-induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence.

Heat Stress Reduces Sperm Motility via Activation of Glycogen Synthase Kinase-3α and Inhibition of Mitochondrial Protein Import.

The adverse effects of high environmental temperature exposure on animal reproductive functions have been concerned for many decades. However, the molecular basis of heat stress (HS)-induced decrease of sperm motility has not been entirely elucidated. We hypothesized that the deteriorate effects of HS may be mediated by damage of mitochondrial function and ATP synthesis. To test this hypothesis, we use mature boar sperm as model to explore the impacts of HS on mitochondrial function and sperm motility. A 6 h exposure to 42°C (HS) induced significant decrease in sperm progressive motility. Concurrently, HS induced mitochondrial dysfunction that is indicated by decreased of membrane potential, respiratory chain complex I and IV activities and adenosine triphosphate (ATP) contents. Exogenous ATP abolished this effect suggesting that reduced of ATP synthesis is the committed step in HS-induced reduction of sperm motility. At the molecular level, the mitochondrial protein contents were significantly decreased in HS sperm. Notably, the cytochrome c oxidase subunit 4, which was synthesized in cytoplasm and translocated into mitochondria, was significantly lower in mitochondria of HS sperm. Glycogen synthase kinase-3α (GSK3α), a negative regulator of sperm motility that is inactivated by Ser21 phosphorylation, was dephosphorylated after HS. The GSK3α inhibitor CHIR99021 was able to abolish the effects of HS on sperm and their mitochondria. Taken together, our results demonstrate that HS affects sperm motility through downregulation of mitochondrial activity and ATP synthesis yield, which involves dephosphorylation of GSK3α and interference of mitochondrial remodeling.

Lipopolysaccharide-induced mitochondrial dysfunction in boar sperm is mediated by activation of oxidative phosphorylation.

Lipopolysaccharide (LPS) has been reported to exert detrimental effects on boar sperm viability. In the present study, LPS was detected in boar semen samples at an average level of 0.62 ± 0.14 µg/mL. We treated boar sperm with 1 µg/mL LPS for 6 hours and examined alterations in sperm motility and apoptosis, together with mitochondrial functionality and mitochondrial reactive oxygen species generation. The expression and the location of toll-like receptor 4 (TLR4) and mitochondrial transcription factor A (TFAM) were determined to reveal possible mechanisms. LPS-treated sperm showed significant reduction in motility (P < 0.05) and viability (P < 0.05). LPS induced sperm mitochondrial damage via oxidative stress which is indicated by marked ultrastructural changes in the mitochondria including swelling, disorientation and vacuole, a decrease of mitochondrial membrane potential (ΔΨm; P < 0.05), as well as an increase of malondialdehyde levels (P < 0.01). Moreover, the production of mitochondrial reactive oxygen species through oxidative phosphorylation (OXPHOS) was significantly (P < 0.05) increased, which leads to oxidative stress. The copy number of mitochondrial DNA was significantly (P < 0.05) higher in LPS-treated sperm. Moreover, cytochrome c oxidase subunit IV (COXIV), an important subunit in mitochondrial electron transport chain and OXPHOS, was significantly (P < 0.05) upregulated after LPS treatment. TFAM, the key transcription factor that activates mitochondrial DNA replication and transcription, was translocated from the head to the midpiece of sperm where mitochondria are distributed in LPS-treated sperm. Taken together, these results indicate that LPS-induced decrease of motility and viability in boar sperm is mediated by abnormal activation of OXPHOS and mitochondrial membrane lipid peroxidation. These findings may provide new insights in understanding the mechanisms underlying the bacterial infection-induced sperm damage.

Relationships between mitochondrial DNA content, mitochondrial activity, and boar sperm motility.

Energy produced by mitochondria via oxidative phosphorylation (OXPHOS) is essential for mammalian sperm motility. Mammalian mitochondrial DNA (mtDNA)-encoded proteins are subunits of the OXPHOS system. Paradoxically, there are less mitochondrial and mtDNA contents in motile sperm than less motile sperm. Here, mature boar sperm was used as a model to investigate the relationships between mtDNA content, mitochondrial activity, and sperm motility. Motile and less motile sperm were separated by centrifugation on a discontinuous percoll density gradient. The contents and expression of mtDNA as well as mitochondrial activity and biosynthesis were determined to reveal possible mechanisms. Motile sperm showed less mitochondrial (P < 0.01) and mtDNA (P < 0.05) contents as compared to less motile sperm. Higher mitochondrial activity in motile sperm indicated by mitochondrial ultrastructure, higher mitochondrial transmembrane potential (P < 0.01), as well as higher mitochondrial respiratory chain complex I activity (P < 0.05). Moreover, more mitochondrial reactive oxygen species (P < 0.01) suggested higher mitochondrial biosynthesis in motile sperm. Although less mtDNA contents in motile sperm, accompanied by the higher expression of transcription factors, the level of mtDNA-encoded protein (cytochrome c oxidase subunit 1) which play pivotal role in OXPHOS was higher in motile sperm. The results are helpful to interpret why mtDNA-less sperm have higher mitochondrial activity and better motility.