Whole Transcriptome Sequencing For Screening Of Genes Related To Testicular Development And Spermatogenesis In Bactrian Camels

The Bactrian camel（Camelus bactrianus）can survive and produce normally in extreme climatic conditions compared to other domestic animals.However,the camel’s short breeding season and the late age of reaching puberty contribute to its low fecundity under natural pastoral conditions.Improving the fecundity of camels is essential for both improving their performance of production and genetic improvement.The normal development of the testis directly impacts the fecundity of males.Therefore,studying genes related to testicular development and spermatogenesis to elucidating their molecular mechanisms are of great importance for improving and protecting the fecundity of breeding stock and preventing and controlling male reproductive disorders.With the widespread use of transcriptome sequencing technology,comprehensive analysis of genes involved in testicular development and spermatogenesis has become possible.Based on this,this study investigated the effects of spermatogenesis in Bactrian camels at different ages using testicular histology and endocrinology and explored and predicted the genes involved in spermatogenesis to verify the impact of candidate genes on spermatogenesis at the molecular level.Follicle stimulating hormone（FSH）,Luteinizing hormone（LH）,testosterone（T）and estradiol（E2）concentrations were measured in the testes of Bactrian camels at different ages（three and six years old）by ELISA.The morphology of the epithelial cells of the seminiferous tubules was observed by HE staining.Based on whole transcriptome sequencing data from testis tissues,the expression profiles of m RNA,mi RNA and lnc RNA were analysed,and the competing endogenous RNAs（ce RNAs）network was constructed and screened for candidate target genes relevant to testicular development and spermatogenesis.A dual luciferase reporter gene method and fluorescent in situ hybridization were used to verify the effects of candidate genes on testicular development and spermatogenesis at the molecular level,providing a theoretical basis for enriching camel reproductive physiology,advancing camel genetic breeding,breeding improvement and improving fecundity.（1）By measuring testicular weight and size,results showed that adult male camels have significantly higher testicular weight,length,width,volume than pubertal male camels（P<0.05）.Observing testicular histomorphology,found that the number of Sertoli cells and elongated spermatids than pubertal male camels（P<0.05）.Spermatogonia,spermatocytes and round spermatids were not significantly different between adult and pubertal male camels（P>0.05）.Peripheral blood levels of reproductive hormones were measured by ELISA,with FSH,LH and T being significantly higher in adult males than in pubertal males（P<0.01）and E2 being significantly lower in adult male camels than in pubertal male camels（P<0.01）.The testicular tissues showed that FSH was significantly higher in adults than in pubertal camels（P<0.05）,LH and T were significantly higher in adults than in pubertal camels（P<0.01）,and E2 was significantly lower in adults than in young camels（P<0.05）.T concentrations in adult and pubertal male camel testes were significantly higher than in peripheral blood（P<0.01）.（2）Transcriptome sequencing was performed on adult and pubertal male camel testes,and 1538 differentially expressed m RNAs were screened at a threshold|log₂Fold change|≥1.5 and P<0.05.Enrichment analysis revealed that differential m RNAs were significantly enriched（P<0.05）in multiple testicular development and spermatogenesis-related signalling pathways.Eight m RNAs were selected in the spermatogenesis-related signalling pathways TGF-β,PI3K/Akt and Hippo.Including AMHR2,FGF1,ACTL7A,GATA4,WNT4,ID2,LAMA1 and IGF1,were validated by RT-q PCR and found that ACTL7A and GATA4 highly significantly up-regulated（P<0.01）,AMHR2 and LAMA1 significantly up-regulated（P<0.05）,and WNT4,DLG1,ID2 and IGF1 were highly significantly down-regulated（P<0.01）in the testes of pubertal male camels,with the opposite result in adult male camels.The RT-q PCR expression patterns were consistent with m RNA sequencing results.（3）Sequencing of lnc RNAs results showed that a large number of lnc RNAs were present in camel testes.Lnc RNA transcripts were characterized by significantly shorter lengths,fewer exons and lower expression than m RNA.Using the threshold|log₂Fold change|≥1.5 and P<0.05,702 differentially expressed lnc RNAs were screened,including 22 known lnc RNAs and 680 novel lnc RNAs.lnc RNAs were selected from PDE10A,LOC116157650,LOC105062954,LOC105072226,LOC116153106,and LOC116157147 differentially expressed lnc RNAs were validated by RT-q PCR and found that the expression of PDE10A was highly significantly up-regulated（P<0.01）in the testes of pubertal male camels and highly significantly down-regulated（P<0.01）in the rest,with the opposite result in adult male camels.The RT-q PCR expression patterns were consistent with lnc RNA sequencing results.（4）Sequences derived from small sequencing adult and pubertal male camel testes were compared to the Rfam and Repbase databases.Identified 1116 mi RNAs,a including838 known and 343 novel mi RNAs.Using the threshold|log2Fold change|≥1.5 and P<0.05,61 differentially expressed mi RNAs were screened.Targeted m RNA prediction was then performed on the differential mi RNAs,and 849 target m RNAs overlapped with the differentially expressed m RNAs.KEGG enrichment analysis showed that mi RNA target genes were mainly enriched in TGF-β,PI3K-AKT,Wnt,Gn RH,Hippo and other testicular development and spermatogenesis-related signalling pathways.Selected four differentially expressed mi RNAs were validated by RT-q PCR,and the results showed that mi R-11987,mi R-497-5p and mi R-513-3p were highly significantly up-regulated（P<0.01）,and mi R-486 were highly significantly down-regulated（P<0.01）in the testes of pubertal male camels,with the opposite result in adult male camels.The RT-q PCR expression patterns were consistent with mi RNA sequencing results.（5）The m RNAs negatively correlated with mi RNA expression were selected from849 target m RNAs to construct a network of Bactrian camel testicular development and spermatogenesis-related mi RNA-m RNA negative regulatory interactions.In comparison with adult male camels,six mi RNAs were down-regulated,12 mi RNAs were up-regulated,26 m RNAs were up-regulated,and 134 m RNAs were down-regulated in the testes of pubertal male camels.From these,mi R-362-3,mi R-140-5p,INHBB and TLR2 were selected for RT-q PCR validation,and it found that mi R-362-3p and mi R-140-5p were highly significantly up-regulated in young male camel testes（P<0.01）.At the same time,INHBB and TLR2 were extremely significantly down-regulated（P<0.01）.（6）Based on the above mi RNA-m RNA regulatory network,the binding sites of differential mi RNAs and lnc RNAs were predicted.Furthermore,the lnc RNA expression was negatively correlated with mi RNAs were selected to construct a ce RNA regulatory network of lnc RNA-mi RNA-m RNA related to Bactrain camel testicular development and spermatogenesis,which contained 30 differential lnc RNAs,13 differential mi RNAs and152 differential m RNAs.Compared with adult male camels,20 lnc RNAs,10 mi RNAs and 133 m RNAs were up-regulated,and 10 lnc RNAs,3 mi RNAs and 19 m RNAs were down-regulated in the testes of young male camels.（7）Validation of crucial ce RNA interactions in the lnc RNA-mi RNA-m RNA regulatory network associated with Bactrian camel testicular development and spermatogenesis using a dual-luciferase reporter gene assay revealed that the mi R-362-3p binds to the 3’UTR of INHBB and LOC105072226,and the mi R-140-5p binds to the3’UTR of TLR2 and LOC116153108.（8）Fluorescence in situ hybridization was used to detect the expression of the above six ce RNAs in testicular tissues.The results showed that TLR2 was mainly expressed on Leydig cells,Sertoli cells and spermatogonia of pubertal male camel testes,and to a lesser expression on spermatogonia and spermatocytes,and in adult male camel testes,TLR2 was expressed on spermatogonia,Sertoli cells and Leydig cells.mi R-140-5p was expressed on spermatogonia and Leydig cells of pubertal male camel testes.It was expressed on Leydig cells,Sertoli cells and germ cells of adult male camel testes.LOC116153108 was expressed on pubertal male camel testis Leydig cells and spermatogonia,and in adult camels testis,mainly expressed on Leydig cells.INHBB was expressed in Leydig cells,Sertoli cells and germ cells in the testes of pubertal and adult male camels.mi R-362-3p was mainly expressed on testicular Leydig cells,spermatogonia and Sertoli cells in pubertal male camels and on testicular Leydig cells in adulthood.LOC105072226 were mainly expressed on testicular Leydig cells in both adult and pubertal male camels.