Study On Histological Characteristics Of Hair Follicle And Expression Pattern Of Related Factors In The Skin During Hair Cycle In Yak

ObjectsYak（Bos grunniens）is a special plateau mammal that lives in the extreme environments of the Tibetan highlands,which has the basic features of extreme cold,high altitudes with reduced oxygen content in the air,and high ultraviolet radiation.Yaks play an important role in normal life for people living in the plateau,such as providing meat,milk and wool,riding and packing goods and materials.This experiment was conducted to study the histological characteristics of hair follicle and expression patterns of related factors in skin during the hair cycle in yak.Histological study was used to observe the histological characteristics of hair follicle during the hair cycle.To determine the expression pattern and function of HSP27,HSP70,HSP90 and GSDMA in skin during the hair cycle in yak,real-time RT-PCR,Western blot and immunohistochemistry methods were used.These data provided molecular and morphological evidence that HSPs and GSDMA existed in yak skin and may play key roles in hair follicle biology.Our study may lead to a better understanding of the physiological functions of HSPs and GSDMA in yak skin.Methods1.Histological method was used to observe the histological characteristics of hair follicle during the hair cycle.Biopsy samples were taken from the scapula regions of the skin from the same five approximately 1-year-old yaks during the hair cycle（telogen,anagen and catagen）.Skin samples from the yaks were fixed on the paperboard to prevent shrinkage,stored in 4% paraformaldehyde solution,softened,dehydrated,embedded in paraffin,sectioned at a thickness of 6μm and deparaffinized.The sections were stained using Sacpic method for hair follicle structures.The IPP6.0 software was used to measure the depth,diameter,density and activity of secondary follicle.2.RT-PCR method was used to clone the c DNA of GSDMA and software was used to analyze the sequence.Then the relative expression levels of GSDMA m RNAs in yak major tissues were detected using q RT-PCR method.3.q RT-PCR,western blot and immunohistochemistry methods were used to detect the expression in both m RNA and protein levels of HSP27,HSP70,HSP90 and GSDMA in yak skin during the hair cycle.Results1.Tissue types were clearly defined and consistent stain intensities with the Sacpic method.The Sacpic stain was well suited for visual assessment of follicle activity because it accentuated the inner root sheath.The structure of hair follicle group changed over hair growth cycle.In telogen,the structure of hair follicle group was loosened,hair shaft falled off,nearly no inner root sheath structure was observed.In anagen,the structure of hair follicle group was obvious,the number of primary follicles and secondary follicles was increased,connective tissue sheath and outer root sheath thickened gradually,structure of inner root sheath was complete gradually.In catagen,hair follicle group became incomplete,the number of hair follicles was decreased,the secondary follicle atrophied and became smaller,and inner root sheath moved up and became shorter.The size,quantity,activity and deep within the skin of the secondary follicle also changed with hair growth cycles.Anagen was the most active period during secondary follicle developmental process.The development of the secondary follicle was not a complete synchronization.2.RT-PCR method was used to clone and sequence the c DNA of GSDMA.Results showed that GSDMA gene sequence containing a complete coding sequence,with the coding region length of 750bp（Gen Bank accession No: XM₁995113）.Nucleotide sequence analysis and the phylogenetic tree revealed that the GSDMA gene of yak was close to Bos indicus（100%）,Bos mutus（99.75%）and Bos Taurus（99.58%）,far from Sus scrofa（97.8%）,Equus caballus（97.59%）and Homo sapiens（97.17%）.The relative expression levels of GSDMA m RNAs in yak tissues were detected using q RT-PCR method.The results showed that GSDMA m RNA expressed at highest level in skin,and high levels of the GSDMA m RNA were detected in ovary and oviduct.There were nearly no GSDMA m RNA expression in other tissues such as heart,liver,and spleen.GSDMA m RNA was also detected in gastrointestinal tract,the results showed that GSDMA m RNA expression was high specific.In yak stomach,the highest level of GSDMA m RNA was found in abomasum,followed by omasum and reticulum,the lowest level of GSDMA m RNA was found in rumen.In yak small intestine,GSDMA m RNA expressed at highest level in jejunum,followed by duodenum,which the lowest level in ileum.In large intestine,the highest expression was found in colon,followed by cecum,and the lowest expression was in rectum.GSDMA m RNA and protein showed the same expression pattern in yak skin during the hair cycle.The expression of GSDMA m RNA and protein in the anagen stage was the highest,followed by the catagen stage,and the expression in the telogen stage was the lowest.There was no significant difference among three stages（P>0.05）in GSDMA m RNA expression level.GSDMA protein expression levels were significantly different among hair cycle（P<0.05）.GSDMA protein expression was observed in all stages of hair cycle.The distribution of GSDMA was found in the stratum basale of epidermis during whole hair cycle and varied with different stage in hair follicle.In anagen stage,GSDMA was found in outer root sheath,hair matrix and forming inner root sheath of secondary follicle in the skin.In catagen stage,GSDMA was mainly found in inner root sheath and residual hair matrix.In telogen stage,GSDMA was found in hair bud and sebaceous gland.3.HSP27,HSP70 and HSP90 showed different expression patterns during the hair cycle using RT-PCR,western blot and immunohistochemistry methods.The expression of HSP27 m RNA and protein in the anagen stage was the highest,followed by the catagen stage,and the expression in the telogen stage was the lowest.The expression of HSP70 m RNA and protein in the telogen stage was the highest,followed by the anagen stage,and the expression in the catagen stage was the lowest.The expression of HSP90 m RNA and protein in the anagen stage was the highest,followed by the telogen stage,and the expression in the catagen stage was the lowest.Each of the three HSPs showed same expression patterns in both m RNA and protein level,however,the discrepancy were different.The expression level of HSP27 m RNA showed significant difference between anagen and telogen stages（P<0.05）as well as between anagen and catagen stages（P<0.05）,but there was no difference between telogen and catagen stages（P>0.05）.HSP27 protein expression levels were significantly different among hair cycle（P<0.05）.There was no significant difference among three stages（P>0.05）in HSP70 and HSP90 m RNA expression level.However,there was significant difference of HSP70 protein expression levels among three stages（P<0.05）.The expression levels of HSP90 protein showed significant difference between anagen and telogen stages（P<0.05）as well as between anagen and catagen stages（P<0.05）,but there was no difference between telogen and catagen stages（P>0.05）.HSPs were mainly expressed in the outer root sheath of hair follicle during the hair cycle,also expressed in epidermis,sebaceous gland and sweat gland in the skin of yak.Conclusion1.The structure of hair follicle group changed over hair growth cycle.And the size,quantity,activity and deep within the skin of the secondary follicle also changed over hair growth cycles.Anagen was the most active period during secondary follicle developmental process.The development of the secondary follicle was not a complete synchronization.2.In this study,we clone yak GSDMA gene sequence（Gen Bank accession No: XM₁995113）.Nucleotide sequence analysis and the phylogenetic tree revealed that the GSDMA gene of yak was close to Bos indicus,Bos mutus and Bos Taurus,far from Sus scrofa,Equus caballus and Homo sapiens.It showed that the gene was both highly conservative and highly specific in evolution3.Yak GSDMA m RNA mainly expressed in skin and gastronintestinal tract and showed tissue specific,which was related to the specific epithelial expression pattern.There were nearly no GSDMA m RNA expression in other tissues such as heart,liver,and spleen.4.In the hair cycle,the changes of GSDMA expression indicated that it was involved in the regulation of apoptosis and proliferation.The persistent expression of GSDMA on the epidermis of the skinwas linked to the proliferation of epidermal stem cells.5.In hair cycle,HSP27,HSP70 and HSP90 were all involved in the hair follicle cell cycle activity,and may also be involved in cell apoptosis.The different expression patterns and expression levels indicated that each heat shock protein had different functions.