| Precocious puberty is an excellent trait with important economic value in livestock production.Chinese indigenous pig breeds are endowed with unique characters of precocious puberty.The research on the regulatory mechanisms that underlie puberty onset in pigs is of great significance to accelerate the breeding process of highly fertile pigs.This study used the Hezuo boar,a indigenous breed for its early maturity characters,as the materials and the Landrace boar as the control.The testicular tissues of 30,90,120,180 and 240days old（n=3）were collected and studied as follows:1)The physical morphological structure of testicular tissue and the changes of serum T,E2,FSH and LH concentrations in Hezuo and Landrace boars at different stages were compared;2)The testicular histomorphology of Hezuo boar and Landrace boar at different stages were observed by HE staining and TEM,the development of seminiferous tubules,Leydig cells,Sertoli cells and spermatogenic cells were analyzed,and the timing of sexual maturity of Hezuo boars was measured combined with the level of serum T,E2,FSH and LH,which provided the basis for the construction of high-throughput sequencing library of testicular tissue in Hezuo boars before and after sexual maturity;3)The expression profiles of lnc RNA,m RNA and mi RNA in testicular tissues of 30-day-old（immature）and 120-day-old（mature）Hezuo boars and Landrace boars of the same age were studied by RNA-Seq,small RNA-Seq and bioinformatics analysis,and differential expression analysis,GO function and KEGG signal pathway enrichment analysis were carried out in order to explore the regulatory mechanism of lnc RNA,m RNA and mi RNA in precocious of Hezuo boars;4)The ce RNA regulatory network of lnc RNA,mi RNA and m RNA was constructed,and the potential candidate molecules related to precocious puberty were identified and analyzed.The main results of the study were as follows:（1）With the increase of age,the weight and volume of testes in Hezuo and Landrace boars gradually increased,showing the pattern of rapid development in the early stage and slowing down gradually in the later stage,and the testicular growth intensity was the highest in 30～90 days old,indicating that this stage is a critical period for testicular growth and development.（2）The serum T level of Hezuo boars generally increased with the increase of age and was higher than that of Landrace boars at all ages;the serum E2 level was higher at 30 days of age,decreased significantly at 90 days of age,then increased,and decreased after 180 days of age,and was significantly higher than that of Landrace boars at all stages（P<0.05）;The serum FSH level of Hezuo boars was higher than that of Landrace boars until 90 days of age（P<0.05）,and reached the highest level at 90 days of age,but was extremely significantly lower than that of Landrace boars at 120,180 and 240 days of age（P<0.01）;the serum LH concentrations showed different ranges of increase and decrease with age,but the overall level tended to be stable,and it was extremely significantly higher than that of Landrace boars at all ages（P<0.01）.The above results suggested that high levels of T,E2,FSH and LH may be related to the precocity of Hezuo boars,and FSH may play an important role in testicular development before sexual maturity of Hezuo boars.（3）The results of HE staining showed that the diameter and lumen area of seminiferous tubules in the testes of Hezuo and Landrace boars gradually increased with increasing age,and the number of Sertoli cells,spermatogenic cells and Leydig cells also increased in different magnitudes.The seminiferous tubule diameter of 30 and 90 days old Hezuo boars was significantly higher than that of Landrace boars（P<0.05）,and it was extremely significantly higher than that of Landrace boars at 120 day old（P<0.01）;The growth trend of the number of spermatogenic cells was consistent with the diameter of seminiferous tubules.At the age of 30 days,there were only spermatogonia in the testis,with the increase of age,the types of spermatogenic cells were gradually enriched,and sperm appeared in the testes of Hezuo boars at the age of 120 days,while Landrace boars did not.The number of Sertoli cells also increased significantly until 120 days of age and was significantly higher in Hezuo boars than in Landrace boars at 120 days of age（P<0.05）,and the number of Leydig cells was also higher in Hezuo boars than Landrace boars to some extent at all stages.In summary,the diameter of seminiferous tubules and the number of Sertoli cells,spermatogenic cells and Leydig cells in testes of Hezuo boars were extremely significantly higher than those of Landrace boars at 120 days of age（P<0.01）,which indicated that testicular development of Hezuo boars was earlier than that of Landrace boars and showed sexual maturity at 120 days of age.TEM results showed that more mitochondria,lipid droplets and various organelles were observed around spermatogenic and Sertoli cells in Hezuo boars compared to Landrace boars,which may promote testicular development in Hezuo boars.（4）In the Ha（30-day-old Hezuo boars）,Hb（120-day-old Hezuo boars）,La（30-day-old Landrace boars）and Lb（120-day-old Landrace boars）group,10706 lnc RNA（including 9282known lnc RNA and 1424 novel lnc RNA）and 21674 m RNA（including 20666 known m RNA and1008 novel m RNA）were identified by RNA-Seq.There were 1984,2073,230 and 284 differentially expressed lnc RNA,and 9343,8647,1322 and 1277 differentially expressed m RNA were identified in the Ha vs Hb,La vs Lb,Ha vs La and Hb vs Lb groups,respectively.The results of GO function and KEGG signal pathway enrichment of differentially expressed lnc RNA target gene and differentially expressed m RNA showed that they were mainly involved in GO functions such as growth and development,reproductive process and metabolism,and significantly enriched in PI3K-Akt,Hippo and other KEGG signal pathways closely related to cell development and reproduction.Through further screening,three candidate lnc RNA（LOC102166140,LOC110259451 and MSTRG.15011.2）that may be related to precocious puberty traits in Hezuo boar were identified.（5）A total of 1452 mi RNA（including 359 exist mi RNA,761 known mi RNA and 332 novel mi RNA）was identified in Ha,Hb,La and Lb by small RNA-Seq.549,468,133 and 247differentially expressed mi RNA were identified in the Ha vs Hb,La vs Lb,Ha vs La and Hb vs Lb groups,respectively.The GO function and KEGG signaling pathway enrichment of target genes predicted by differentially expressed mi RNA showed that they were mainly involved in GO functions such as cell development,reproductive development,metabolism and immune system development,and were significantly enriched in KEGG signal pathways related to reproduction,testicular development and spermatogenesis,such as Wnt,Rap1 and m TOR.Through further screening,six candidate mi RNA（ssc-mir-29b,ssc-mir-199b,ssc-mir-383,ssc-mir-149,ssc-mir-615and ssc-mir-370）that may be related to the precocious puberty traits of Hezuo boars were found.（6）Based on the analysis of coexpression ce RNA network of lnc RNA,m RNA and mi RNA,it was found that the target genes of differentially expressed lnc RNA were mainly involved in GO functions such as reproductive system development,reproductive process and sperm motility,as well as KEGG signal pathways such as ECM-receptor interaction,glycolysis/gluconeogenesis,steroid synthesis,PI3K-Akt,Hippo and Wnt.On this basis,ten genes related to testis development and spermatogenesis were selected to construct lnc RNA-mi RNA-m RNA ce RNA regulatory network,and three pairs lnc RNA-mi RNA-m RNA important relationship pairs（LOC100736806-ssc-mi R-146b-SPATA3,MSTRG.12337.2-ssc-mi R-130b-5p-DMRTC2 and XR＿002346198.1-ssc-mi R-34c-SMAD4）were locked through further screening,which may play important roles in the regulation of precocious puberty in Hezuo boar through ce RNA mechanism.In conclusion,we determined that the Hezuo boar reached sexual maturity at 120 days of age by the physical morphology of testis,serum reproductive hormones and histomorphological analysis of Hezuo boars at different developmental stages.Based on this,the characteristics and expression of lnc RNA,m RNA and mi RNA in testicular tissues of immature（30-day-old）and mature（120-day-old）Hezuo boars and Landrace boars of the same age were studied,and some important molecules that may be involved in the regulation of precocious puberty process in Hezuo boars as well as the lnc RNA-mi RNA-m RNA relationship pairs through ce RNA mechanism were identified by bioinformatics methods and functional analysis.In the future,the specific regulatory mechanisms of these molecules need to be further verified at the cellular level.This study hopes to provide a scientific basis for the research on accelerating the breeding process of high-fertility pigs through the study of the prematurity of Hezuo boar. |
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