Expression,localization And Regulation Of UT-B And AQP3 Urea Transporters In The Rumen

The transfer of urea into the rumen has physiological and nutritional significance to ruminants.This is achieved through rapid transepithelial urea transport mediated by urea transporting proteins,namely UT-B urea transporters and possibly aquaglyceroporins.To better understand the cellular basis and the molecular adaptation mechanisms,four studies were conducted.The first study was to characterize the ruminal expression of aquaglyceroporins.Using RT-PCR,specific primers detected the gene expression of AQP3,AQP7 and AQP9 in the rumen epithelium of adult cattle,but not for AQP10.AQP3 was chosen for further analysis of protein expression and cellular distribution due to the apparently abundant gene expression.Using two anti-AQP3 antibodies,multiple AQP3 protein signals were detected at 25,32 and 42–45 k Da.Further immunolocalization studies showed that these AQP3 proteins located in the various cell layers of rumen epithelium,especially in the stratum spinosum and stratum basale.Using rumen epithelium samples of calves aged from birth to three months old,the second study performed immunolocalization to localize and semi-quantify UT-B and AQP3 development.The results revealed that the UT-B protein could accumulate in the basolateral membrane of stratum basale cells as early as the first one or two weeks after birth and spread to the whole cell membrane and further away to stratum spinosum in the later stages.Semi-quantification of the signal strength by expression score demonstrated that UT-B protein intensity increased with age.The time points of upregulation coincided with the events of solid feed intake and weaning,exhibiting a developing pattern similar to MCT1 and papillae growth.The calf AQP3 had much lower protein intensity than that of adults and did not present a clear developmental pattern.However,a few animals displayed a complementary or redundant organization to that of UT-B.In the third study,we examined the ruminal UT-B,AQP3 and MCT1 protein abundance of a semi-wild ruminant,fallow deer.The immunolocalization and immunoblotting analysis showed that the females,especially the adults,had significantly higher UT-B protein abundance than males.The AQP3proteins were only weakly expressed in the stratum corneum,with female adults and male juveniles having the highest abundance.No gender or age difference were revealed for MCT1 protein.The gender differences of UT-B abundance appear to be associated with gender differences of diet selection in fallow deer.The fourth study compared the UT-B protein expression level between yaks and indigenous cattle.No difference was revealed but the papillae surface area was smaller in yaks.In conclusion,this project confirmed the presence of AQP3 proteins in the rumen epithelium.Whist the exact physiological role of AQP3 remained unclear,the upregulation of UT-B protein closely corresponded to the elevated ruminal fermentation activity.The physiological significance of this adaption is likely to be enhanced ruminal urea entry thus,increasing both the buffering capacity against the higher level of H~+from SCFAs production and the levels of nitrogen available for bacterial growth.With the synchronization of energy and nitrogen metabolism,this ultimately contributes to the nitrogen balance of host ruminants.