Identification And Expression Analysis Of Key Genes In Secondary Follicle Development Of Chinese Merino Sheep

Morphogenesis of hair follicles begins at the embryonic stage and is completed before birth.The primary wool follicles(PF)of Merino sheep are initiated firstly and followed by initiation of secondary wool follicles(SF),then secondary derived follicles are branched from the SF.It is known that morphogenesis of hair follicles determine the wool yield and quality.However,the mechanism underlying the SF development and branching is still unclear.It has been reported that malnutrition during pregnancy can reduce the density of fetal hair follicles.In order to study the morphogenesis of Chinese Merino wool follicle and to estimate the effect of nutrition on hair follicles development,in the study,30 pregnant ewes were used and randomly divided into5 periods according to the gestation stage(55 to 85,85 to 105,and 105 to 135 of gestation,day135 of gestation to 7 days after birth,and 7 to 35 days after birth).In each period,3 ewes were offered sub-maintenance(Sub-M)nutrition and another 3 ewes were offered maintenance(M)nutrition.Daily feed intake of Sub-M ewes was 75% of the M group.During the feeding restriction,the changes of blood biochemicals were detected.After the feeding restriction,the fetuses of each group were obtained by caesarean operation.Then the fetuses body size,chest circumference,abdominal circumference,body weight and organs weight were measured respectively.Moreover,the number of PF and SF in each group was estimated and the ratio of secondary / primary hair follicle density(S/P)was calculated.Then the skin samples with significant difference in wool follicle density were collected and the transcriptome and proteome sequencing were performed.The key genes related to hair follicle development were identified by GO,KEGG enrichment,IPA regulatory network and protein interaction analysis.The expression of these genes in fetal follicles at different gestation stages was tested by immunohistochemistry and real-time PCR.The results were showed as following:(1)Concentration of Free fatty acids(NEFA)in the blood was significantly higher(p<0.05)in the sub-M group than that in the M group.There was no significant difference in fetus weight,body size,and organ development between the Sub-M group and the M group.The morphogenesis and development of wool follicle were similar in each group,but the SF density and S/P ratio in the Sub-M group were significantly lower than those in the M group(P<0.05).It is indicated that short-term Sub-M(75% control nutrition)has no significant effect on ewe fetal development.However,SF density and S/P ratio were reduced after Sub-M during the on D105-135 gestation.(2)At D135 gestation stage,there were significant differences in SF density and S/P ration between the Sub-M group and the M group.According to the result,3 independent skin samples were selected respectively from the M group(D105 and D135)and the Sub-M group(DR135)for the transcriptome and proteome sequencing.Functional annotation analysis enriched several pathways involving in the regulation of intermediate filament,intermediate filament cytoskeleton and keratin filament.Differentiatialy expressed genes(DEGs)were enriched involving in the regulation of estrogen signaling pathway,protein digestion and absorption,cell adhesion molecules(CAMs)and Mucin type O-glycan biosynthesis.These results showed that Wnt pathway and TGF-?/ BMP pathway played an important role in hair follicle development during D105 to D135 gestation stage.Genes including SFRP4,PITX1,COL9A1,COL6A5,COL2A1,BAMBI,KRT16 and KRT20 may be involved in SF branching and development of Merino sheep.(3)The expressions of SFRP2,SFRP4,BMP2,NOG and KRT16 at D105,D135 and D135 gestation stage were detected by q PCR and immunohistochemistry.The results showed that these genes(with the exception of SFRP2)were expressed in the inner and outer root sheath cells of the fetuses hair follicle.The q PCR results were consistent with the transcriptome sequencing results.This study suggested that these genes were involved in the development regulation of SF.(4)During D105-135 gestation stage,differentially expressed proteins were enriched in the pathways related to regulation of intermediate filament,intermediate filament cytoskeleton and keratin filament,which involved in hair follicle development.Moreover,genes related to estrogen signaling pathway,MAPK signaling pathway and Wnt signaling pathway were significantly enriched by KEGG analysis.We found that TCHH,CAV3,DCN,CST6,COL6A1,COL6A3,KAP3.2,KRTAP6-1,KRT34,KRT2.11,KRTAP11-1 were involved in hair follicle development during D105 to 135 gestation stage,among which TCHH,CAV3,DCN and KAP3.2 may play an important role in SF branching and development.In this study,we observed the morphogenesis and development of PF and SF in Chinese Merino sheep.The expression of genes related to hair follicle development were evaluated by transcriptome and proteome sequencing.Our results provide a theoretical basis for the morphogenesis and development of Merino wool follicle,and these results help us to explore the mechanisms underlying SF development and branching in Merino sheep.