Studies On Testis Development Atlas,Structure And X-chromosome Dose Compensation After Hu Sheep Birth

Testis is an important reproductive organ in male mammals,and it can produce sperm and secrete androgens.Testis is closely related to the fecundity of male animals.So,it has great scientific value to understand the biological process and molecular mechanism of testis development.At present,there is no report on the development altas and structural changes of the sheep testis after birth.In this study,we used single-cell RNA sequencing(sc RNA-seq)to study the differential gene expression,change of cell composition and the key signal pathway,pseudotime analysis of germ cell in Hu sheep testicular cells from birth to adult,and testis development altas were constructed in Hu sheep.On this basis,the tissue structure of testis,the expression localization of the important hormone receptors and the development changes of important cells were studied in testis and the X chromosome dose compensation of male germ cell was explored by calculating the content and expression of X chromosome and autosomal RNA.Finally,the testicular development was systematically analyzed after Hu sheep birth.The main results of this study were as follows:Testicular tissue of 0,30,90,180,270,360 and 540 d was performed after Hu sheep birth by sc RNA-seq,(1)There were differences in testicular cells among different days :there were 8 cell groups in P0 and P30 testicular tissue,including 2 germ cells and 6somatic cells.There were 10 cell groups in the testicular of P90 Hu sheep,including 4germ cells and 6 somatic cells.There were 13 cell groups in the testicular from P180 to P540,including 7 germ cells and 6 somatic cells;(2)DDX39A(DEx D-box helicase 39A)can be used as a specific marker for Early primary spermatocyte of Hu sheep testis,and HELLS(lymph specific helicase)can be used as a specific marker for Spermatogonia;(3)The main signaling pathways are not consistent in different stages of testicular development in different days of age after birth,PI3 K Akt signal pathway,Rap1 signal pathway,c AMP signal pathway and MAPK signal pathway mainly functioned in the testis from 0 to 90 days,and were related to the proliferation and development of testicular somatic cells.Estrogen signaling pathway and HIF-1 signaling pathway were functional at 90 to 180 days and were related to the differentiation and proliferation of spermatogonia.Hippo signaling pathway and TGF-beta signaling pathway were functional from 180 days to days,which were related to spermatogenesis and maturation;(4)The expression of ZBTB16 increased significantly from P30,which inhibited SSCs differentiation.The expression levels of MYH11,NANOS3 and TKTL1 increased significantly from P90.MYH11 was involved in the formation of blood testis barrier,and NANOS3 and TKTL1 were involved in the differentiation of SSCs.AFF4 expression increased significantly from P180,which was involved in sperm maturation.The expression level of APOA1 was stable after P30 and was involved in sperm development.The expression of INHA decreased significantly from P180,which inhibited spermatogenesis and FSH secretion;(5)The unsupervise pseudotime developmental trajectory of testis germ cells of Hu sheep was constructed,which was in line to the developmental of male germ cell : SSCs-Spermatogonia-Early primary spermatocyteLate primary spermatocyte-Round spermatid-Elongated spermatid-spermatozoa.2.The morphological analysis of the Hu sheep testicular structure were performed at seven different ages.The results showed that Spermatogonia and Sertoli cells were only observed in the seminiferous tubules from birth to 30 d,spermatocytes appeared at 90 d,and sperm appeared at 180 d,The results indicated that Hu sheep had reached sexual maturity at 180 d.The diameter of seminiferous tubules increased with age increasing with the fastest growth rate between 30 and 90 d.3.FSHR and LHR are mainly located in testis Sertoli cells,Leydig cells and Spermatogonia,while AR is mainly located in testis Sertoli cells.The expression level changes in different ages of testis,and the expression level is high in 0 ～ 30 d,and decreases to the lowest in 30-180 d,and the expression level is stable in 180 d to 540 d,which coordinate with testis development and spermatogenesis.4.SOX9 is the marker of Sertoli cells,PCNA is a specific marker of cell proliferation.SOX9/PCNA immunofluorescence staining showed that sertoli cells rapidly proliferated from 30 to 90 d and stopped proliferating after 180 d.AMH is a marker of immature sertoli cells.SOX9/AMH immunofluorescence staining showed that sertoli cells developed and matured after 90 d.UCHL1 is the marker of undifferentiated Spermatogonia.UCHL1/PCNA immunofluorescence staining showed that undifferentiated Spermatogonia gradually began to proliferate from 0 d,At 180 d,it increased rapidly,but at 270 d,360 d and 540 d,there was no significant change.VASA is the specific marker for male germ cells,KIT is specific marker for differentiated Spermatogonia.UCHL1/VASA and UCHL1/KIT immunofluorescence staining showed that undifferentiated Spermatogonia began to differentiate after 30 d,and spermatocytes appeared after 90 d.5.β-catenin(Catenin Beta 1)and ZO-1(Cytosolic Claudin-1)are the main components of the blood-testis barrier.By immunofluorescence staining of β-catenin and ZO-1,the results showed that the blood-testis barrier did not finish at 90 d,however,finished at 180 d.6.The relative RNA content and expression of X chromosome and autosomes in the Hu sheep testicular cells showed that there was no dose compensation of X chromosome in the germ cells at different Hu sheep testis developmental stages.