Study On The Anti-Heat Stress Mechanism Of Spermatogenesis In Turpan Black Sheep

With the development of farming intensification and scale and the general increase of global temperature,animal heat stress is also becoming more and more serious.Heat stress can directly or indirectly affect animal physiology,production and health,and bring certain economic losses to the farming industry.Currently,animal heat stress has been studied mainly in dairy cows,chickens and pigs,while little research has been reported on heat stress in sheep.Therefore,it is more urgent to conduct research on heat stress in sheep.When sheep are exposed to high temperature and high humidity,which genes and metabolic pathways play a regulatory role? In view of this,this experiment aimed to search for genes and regulatory pathways related to heat stress in sheep.The main findings are as follows:1.First,we counted the body temperature of sheep during the time period from no heat stress to strong heat stress,and found that the temperature of different parts of the sheep in both groups started to increase at the beginning of heat stress and then remained at high heat levels,especially in Suffolk sheep We also collected serum during heat stress and detected changes in reproductive hormones and oxidative stress indexes by radioimmunoassay,and the results showed that during the heat stress treatment The results showed that both groups of sheep showed fluctuations in the indexes tested during the heat stress treatment,with the Turpan black sheep showing a relatively stable level and the Suffolk sheep showing significant fluctuations.2.Through the detection of sperm motility,density and motility performance,it was found that with the increase of heat stress intensity,various indicators of Suffolk sheep decreased significantly(P<0.01),while Turpan black sheep showed a downward trend,but did not reach a significant level.Therefore,we hypothesized that Turpan black sheep were able to maintain normal testicular temperature and normal sperm survival during heat stress.Then,in this study,the tests of acrosome staining and DNA fragmentation revealed that there was no significant difference between Turpan black sheep after heat stress and before heat stress,while Suffolk sheep after heat stress treatment showed a large number of incomplete sperm acrosomes and a high percentage of DNA fragmentation(P<0.05).3.H&E staining of sections of the collected testicular tissues was performed.Morphological results showed that the sectioned tissues were morphologically intact and all types of germ cells including spermatogonia,spermatocytes,round spermatozoa and long spermatozoa had been present in the testicular tissues of each experimental animal with no significant differences.The supporting cells,spermatogonia and spermatocytes within the testicular varicoceles were quantified with the help of immunofluorescence technique.It was found that the number of spermatogonia and supporting cells in the varicose seminiferous tubules of Turpan black sheep and Suffolk sheep before and after heat stress were not meiotic,whereas a significant decrease in spermatocytes was observed in Suffolk sheep after heat stress.We then analyzed the entire meiotic prophase I using meiotic spreading films,in which both sheep contained cells from the entire period after heat stress treatment,indicating that both groups of sheep could complete meiosis I.However,by comparing the percentage of cells in each period,it was found that the percentage of cells in the meiotic lineage was significantly higher in Suffolk sheep than in Turpan black sheep.Therefore,we hypothesized that not all cells in Suffolk sheep could complete meiosis under heat stress,and a large number of cells underwent arrest in the coarse line phase and subsequently underwent apoptosis.4.RNA-Seq data analysis of differentially expressed genes,GO enrichment and KEGG pathway before and after heat stress in two types of sheep showed that the GO enrichment of Tulufan black sheep before and after heat stress included biological processes such as cellular response to stimulation,cell development process,cell cycle process,meiotic cell cycle process and doublestrand break repair,and the KEGG pathway included Rap1 KEGG pathways include Rap1,Ras,cell cycle,meiosis,Fox O,m TOR signaling pathway,Notch signaling pathway,etc.;Suffolk sheep GO enrichment before and after heat stress include apoptosis process,negative regulation of developmental process,programmed cell death,regulation of homeostatic process,etc.;KEGG pathways include platelet activation,choline metabolism in cancer,AMPK,etc.;common differential genes GO enrichment include mitochondria Respiratory chain complex I,histone acetyltransferase activity,ATPase activity,transcriptional co-activator activity,etc.,and KEGG pathway,such as oxidative phosphorylation,proteoglycan in cancer,PI3K-Akt signaling pathway,etc.By comparing the GO enrichment and KEGG pathways of differential genes,we found that the pathways between groups were mainly divided into 3 categories: 1.in Tulipan black sheep were related to regulation of spermatogenesis and meiosis;2.in Suffolk sheep were related to apoptosis and inflammatory response and adaptability;3.in common differential genes were related to oxidative stress adaptability.Also,we selected the signature genes and found that all of them were upregulated,such as: meiosis-related genes SYCP2,BRCA1,TEX15,SMC4,SMC3,SETX,RAD54 L,MEIOC,MCM8,MCM6,CENPF,TEX11,SMC5,RPA1,etc.Apoptosis-related genes CASP8,TBK1,CFLAR,FOXO1,ADORA1,CASP8,MAP3K1,etc.,and oxidative stress-related genes NDUFB3,NDUFA8,NDUFA6,NDUFB7,HLTF,MYO9 B,TFAP2A,MED13,MYSM1,STAT3,etc.The results of this study reveal that hormonal regulatory changes as well as spermatogonia transformation,meiotic cell and haploid germ cell development in Tulufan black sheep are regulated by a large number of genetic changes during heat stress.Transcriptome analysis provides candidate genes for spermatocytes and spermatogenesis that play important roles in heat stress.The upregulation of genes involved in spermatogenesis protects the normal production of sperm in the heat-stressed Turpan black sheep,thereby achieving normal reproductive function,which further proves that the Turpan black sheep has evolved an anti-heat stress ability in the long-term heat stress environment.