Mechanisms Of Prolactin Affecting Growth And Development Of Secondary Hair Follicles In Cashmere Goats

Prolactin(PRL)is associated with the cyclic growth of secondary hair follicle(SHF)and affects the growth and shedding of cashmere by regulating the growth and development of SHF,but the molecular mechanism by which PRL regulates the cyclic growth of SHF is unknown.In this study we investigated the effects of inhibition of PRL secretion on cashmere growth,hair follicle development,related hormone levels and gene expression in cashmere goats during the cashmere growing and non-growing periods on cashmere growth by vivo assays;we also performed transcriptome sequencing on skin tissues to screen key signaling pathways and investigated the effects of PRL on SHF and dermal papilla cell(DPC)growth and mechanisms through cellular assays.The theoretical basis for the improvement of cashmere production and quality in cashmere goats was developed.Effects of inhibition of PRL secretion in cashmere goats during the cashmere growing period and non-growing period on cashmere growth,hair follicle traits,hormone levels and gene expression:Twenty goats of similar weight and health at 3 months old were randomly assigned to either a bromocriptine treatment(0.06 mg/kg)or control group.The test was carried out from September to next March(cashmere growing period).Cashmere fibre,blood and skin samples are collected monthly and analysed for cashmere quality,serum hormones,hair follicle traits,skin tissue gene and protein expression and transcriptome sequencing data.The results showed that 1)BCT treatment increased cashmere growth rate from September to October and cashmere fineness in November and March(P<0.05).2)BCT treatment increased the number of SHF and S/P values and increased the ratio of active SHF in October and November.BCT treatment increased primary and secondary hair follicle hair bulb width,and epidermal thickness in October and November,but in December epidermal thickness was significantly less than the control group(P<0.05).3)In October,November and March,BCT treatment reduced serum PRL levels,reduced IGF1 levels in October,increased MT levels in October(P<0.05)and had no effect on serum COR,GH,T4 and T3 levels.4)BCT treatment decreased skin tissue PRL and MTNR1a expression levels in October and November,increased ROR a and VEGF expression levels in October,increased PF4 expression levels in October and November(P<0.05),and had no effect on LPRLR,SPRLR and IGF-1 expression;BCT treatment increased p-STAT5 protein expression and decreased p-STAT3 protein expression in skin tissue in October(P<0.05).Effects of inhibition of PRL secretion in cashmere goats during the non-growing period on hair follicle traits,hormone levels and gene expression:Twenty goats of similar weight and health at 6 months old were randomly assigned to either a bromocriptine treatment(0.06 mg/kg)or control group.The test was carried out from April to next June(cashmere non-growing period).Cashmere fibres were collected in June,and blood and skin samples were taken in April,May and June and analysed for cashmere quality,serum hormones,hair follicle traits,skin tissue gene and protein expression and transcriptome sequencing data.The results showed that 1)BCT treatment did not affect cashmere length and diameter.2)BCT treatment reduced SHF activity,secondary follicle hair bulb diameter and secondary follicle dept(P<0.05).3)BCT treatment reduced serum PRL level(P<0.05)and had no effect on GH level.4)BCT treatment reduced skin PRL,SPRLR,Kit and Fos expression(P<0.05)and did not affect skin LPRLR,RORa and MTNR1a expression;BCT treatment increased skin p-STAT5 protein expression and decreased skin p-STAT3,Kit and Fos protein expression(P<0.05).Effects of PRL on the growth of SHF and DPCs and mechanisms:1)Transcriptome analysis of skin tissues by inhibition of PRL secretion in cashmere goats.The skin tissue transcriptome(October)identified 160 DEGs,including 101 up-regulated genes and 59 down-regulated genes.Using DEGs enriched to GO items for directed acyclic plots,DEGs in BP eventually aggregate into immune responses,redox processes and regulation of tissue structure size;DEGs in CC eventually aggregate into intermediate fibrils;and DEGs in MF eventually aggregate into structural molecular activity,cofactor binding and chemokine activity.Protein network interaction(PPI)analysis of DEGs revealed that FGF21 and PF4 could interact with RORα,BCO2,FABP4,VNN2,PROCR,CYP1A1,MT3,TLCD4,CCL27 and CCL26.The skin tissue transcriptome(May)identified 691 DEGs,including 192 up-regulated genes and 499 down-regulated genes.Using DEGs enriched to GO items for directed acyclic plots,DEGs in BP eventually aggregated into skin development,epithelial cell migration and vascular development,DEGs in CC eventually aggregated into stromal membrane and plasma membrane rafts,DEGs in MF eventually aggregated into RNA polymerase II transcription factor activity,sequencespecific DNA binding,sequence-specific DNA binding of the RNA polymerase Ⅱregulatory region and proximal promoter sequence-specific DNA binding.Protein network interaction analysis was performed on DEGs to extract proteins that interacted with PRLR and FOS proteins and plot PPI.17 DEGs had proteins that interacted with both PRLR and Fos,with PDGFB scoring highest with PRLR and NR3C1 scoring highest with FOS,and PDGFB,IGF2 and Kit significantly enriched in the MAPK pathway.2)Isolated cashmere non-growing period SHF and DPCs were co-cultured with PRL.400 ng/μL PRL promoted the growth of isolated SHF;150 ng/μL PRL promoted DPC proliferation and migration,reduced apoptosis,and was able to inhibit DPC PRL and PF4 expression,promoted Kit,Fos,SPRLR,IGF-1 and VEGF expression in DPCs(P<0.05)and did not affect LPRLR expression.3)The most significantly differentially expressed Fos overexpression and interference vectors were constructed,lentivirally encapsulated,and infected DPCs.oFos was found to increase Fos expression level,sh3-Fos decreased Fos gene expression,and affected DPC proliferation by regulating Bax,Caspase-3 and Bcl-2 expression.3)Kit overexpression and interference vectors were constructed,lentivirally encapsulated,and infected with DPCs in the cashmere non-growing period.o-Kit increased Kit gene and Kit protein expression and promoted Fos expression;shl-Kit decreased Kit gene and Kit protein expression and decreased Fos expression,and affected DPC proliferation by regulating Bax,Caspase-3 and Bcl-2 expression(P<0.05).Subsequent treatment with PRL reduced DPC apoptosis caused by sh3-Fos and shl-Kit,and treatment with PRL antagonists attenuated DPC proliferation caused by o-Fos and o-Kit.In conclusion,PRL secretion in cashmere growing period inhibited SHF activity,and inhibition of PRL secretion increased hair follicle activity by regulating intermediate fibrillar cell structural factor activity through the MAPK pathway;The secretion of PRL in cashmere non-growing period increased SHF activity,and inhibition of PRL secretion decreased hair follicle activity by regulating skin cell matrix membrane transcription factor activity through the MAPK pathway;PRL stimulated Fos expression by increasing Kit expression,which promoted secondary hair follicle papilla cell proliferation and thus secondary hair follicle growth in cashmere goats.