The Role Of Epithelial Polarity-driven Membrane Separation In Lumen Formation Of Eccrine Sweat Glands In SD Rats

Objective1.To investigated the differential distribution of eccrine sweat glands(ESGs)and hair follicles(HFs)in the volar skin of C57BL/6 mice and SD rats.2.To study the structural and functional development of the ESG lumen of Sprague Dawley(SD)rats,and to explore the key mechanisms(apoptosis/autophagy/membrane separation driven by epithelial polarity)of ESG lumen formation in SD rats.Methods1.The forefeet and hindfeet of C57BL/6 mice and SD rats(8-10 weeks old)were photographed under a stereomicroscope,and the hair on the volar skin was counted;afer iodine-starch sweat test,the number of ESGs on the volar skin was counted.The volar skin specimens were collected,fixed in 4%paraformaldehyde,embedded in paraffin and sectioned for HE and double immunofluorescence staining(NKCC1/LHX2 and NKA/LHX2).The epidermis of the volar skin was separated and stained with both Nile Blue A and Oil Red O for detecting the distribution of ESGs and HFs.The mRNA expression level of En1 and LHX2 in the footpads and inter-footpads(IFPs)was detected by qRT-PCR.2.(1)The hind-footpads of SD rats from E20.5,P1-P12,P21,P28 and P56 were collected,fixed,prepared into paraffin and frozen specimens,and then sectioned.The sections were used for HE staining,TUNEL staining,immunofluorescence staining of LC3B(autophagy marker),F-actin(apical membrane marker),Laminin(basal membrane marker),E-cadherin(basolateral membrane marker)and Na~+-K~+-ATPase(ouabain binding site),and double immunofluorescence staining of K14/α-SMA.The ESGs sweating function was detected by iodine-starch sweat test.(2)The experimental design was randomized block design and there were 3 litters of newbtorn rats.Each litter of neonatal rats were divided randomly into three groups at P1(4 rats/group):control group,1μg ouabain group(1μg/kg.d),10μg ouabain group(10μg/kg.d).After 3 weeks,the hind-footpads of rats in each group were fixed,made into paraffin specimens,and sectioned serially for HE staining.Then we measured the inner diameter of the ESG ducts by Image J software and statistical analysis was performed according to random block design.Results1.(1)Hair and sweat drops were observed on the C57BL/6 mice fore-and hind-IFPs where the structure of ESGs and HFs existed,and LHX2(HF marker),NKCC1 and α1-Na~+-K~+-ATPase(NKA,ESG marker)were expressed;only sweat droplets were observed on the footpads of C57BL/6 mice and SD rats where only the structure of ESGs existed,and NKA and NKCC1 were expressed but LHX2 not;no HFs and sweat droplets were observed on the IFPs of SD rats where no ESGs and HFs structures existed,and LHX2,NKA and NKCC1 were all not expressed.(2)In the footpads and IFPs of C57BL/6 mice and SD rats,the relative quantitative change of En1 and LHX2 mRNA was consistent with the differential distribution of ESGs and HFs,respectively.2.(1)During development of ESGs in SD rat hind-footpads,epidermal cells grew from the base into the dermis to firstly form ESG duct cells at E20.5,the ESG formed microlumen at P1,ESG secretory part began to develop at P2,some ESGs had a small relatively complete lumen-like structure at P3,and obvious central lumen can be seen in the ESGs at P5.ESGs did not start to sweat until day 19.(2)Apoptosis and autophagy: TUNEL staining(apoptosis marker)and LC3B(autophagy marker)immunofluorescence staining both were negative in the hind-footpads of SD rats at E20.5,P1,P2,P3,P4,P5,P7,P9,P12(before and after of lumen formation);apcal-basal polarity: F-actin began to be expressed on the apical membrane of ESGs in SD rats at P1,E-cadherin was expressed on the basolateral membrane at P3,and Laminin began to be expressed on the basement membrane at P3.(3)Na~+-K~+-ATPase,the binding site of ouabain,was expressed in the ESGs of SD rats since the embryonic stage.Compared with the control group,the inner diameter of ESG ducts in SD rats in 1μg ouabain group and 10μg ouabain group was significantly reduced(P<0.01),while the inner diameter of ESGs in 1μg ouabain group and10μg ouabain group was no significant difference(P>0.05).Conclusions1.C57BL/6 mice and SD rats had their own characteristic distribution of ESGs and HFs in the volar skin.In C57BL/6 mice,there were ESGs but no HFs in the footpads,and both ESGs and HFs in the IFPs.In SD rats,there were ESGs but no HFs in the footpads,and neither ESGs nor HFs in the IFPs.Therefore,according to different purposes,researchers should choose mice or rats,and even forefeet or hindfeet as their research object.To study the development and regeneration of ESGs,the volar feet of SD rats are the first choice.2.Epithelial polarity-driven membrane separation but not cavitation plays an essential role in ESG lumen formation of SD rats.Na~+-K~+-ATPase-regulated fluid movement drives lumen expansion.