Analysis of Regulation of Key Genes of Ovarian and Uterine Cell Function in Postpartum Uterine Infected Buffalo.

After parturition, uterine infection is one of the most common endemic diseases of dairy cattle and buffalo, leading to infertility by disruption of uterine and ovarian function. Recent studies revealed that uterine tract infection affect day of first service, service period, service per conception and calving interval in cattle and buffalo. In uterine infection, not only is the uterus damaged, but there is perturbation of ovarian follicle. Similarly, metritis and endometritis is associated with slower growth of the dominant follicle in the ovary, fewer ovulations and lower peripheral plasma estradiol concentrations compared with clinically normal cattle. To understand the effect of postpartum uterine infection on uterine and ovarian function in buffalo, we characterized postpartum uterine infection and studied the effect of LPS and or inflammation on uterine and ovarian genes expression in buffalo. For morphological and biochemical characterization of postpartum infection, we collected cervical mucus and blood. Based on the presence or absence of flecks in cervical mucus, animals were classified to two categories i.e. morphologically normal and morphologically infected animals. In morphologically normal animals there is high amount of estradiol and low amount of LPS, whereas, in infected animals, there was low amount of estradiol and high amount of LPS. Due to variation of Estradiol (E2) and LPS levels among individuals, morphologically animals were further classified into subclinical endometritic and normal animals. Results showed that content of LPS and estradiol were inversely related to each other in normal, subclinical endometritic and clinical endometritic animals. To understand further the effect of LPS and its mode of action on granulosa cells, studies were done in vitro using serum free granulosa cells culture. In vitro resu showed maximum effect on CYP19 expression and E2 secretion. LPS induced the expression of IL-1 -6 at 2hr, but their expression came to nearly normal levels at 6hr after LPS treatment. LPS has been found to down regulate the expression of 3PPARand RUNEX-1 genes; while LPS induced the expression of CEBPCYP-11, GAPDH, BCL-2 and iNOS. Analysis of global gene expression by microarray in normal and endometritis uterine tissues showed that 157 genes were differentially expressed at 2 fold level in endometritic animals as compared with the normal animals. Out of which, 85 genes were found to be down regulated while 72 genes were up regulated. Analyses of these genes showed that majorly these genes are involved in the immunity and developmental processes. Real time PCR, immunoblotting, immunostaining and ChIP studies showed that LPS mediated the negative regulation of CYP19 expression by inducing expression and translocation of CEBPand binding on CYP19PII. The immuno-precipitation of histones with anti-acetyl Histone H3 (Lys 9/14) antibodies showed that translocated CEBPs to CYP19 promoter and negatively regulated by reducing the acetylation of CYP19PII. Similarly, LPS induced the acetylation at promoter of ILwhere NFbinds and induced proinflammatory gene expression. Conjugated linoleic acid (CLA) is an anti-inflammatory agent. In the present study, we studied LPS induced immune response in buffalo granulosa cells and whether CLA can attenuate LPS induced proinflammatory gene expression in buffalo granulosa cells in vitro. inflammatory gene expression. While granulosa cells pre-exposed with CLA for 24 h showed reduced LPS induced protreatment also reduced the levels of nuclear NFnflammatory gene expression. The present findings raised the possibility of using dietary CLA in preventing LPS induced granulosa cells dysfunction underlying infertility. , ! "! #! , , ! #! #! , "! #! , ! #! "! #! #! #! #! "! #! " ! $! %&! '()*+,! -. /&0! 12! $! #+3& "! 4563 ! #+37! "! 7! $! 83 9:;"! :4<="! 6:9="! +9="! //<= ! =>51? 3&! "! -1@/3 "! -. /3&&"! A</;9"! @-+32! B5C:! $! 2 157 ! | 85 72 ! | | " , ! -1@/3 ! "! -. /&0/##! -. /&0! $! 3 ! 98%+DE! 0)&F,! -1@/3 ! -. /&0! -. /&0! $! #+3 ! 4563 ! 563 ! #! 3 ! $! $! $! 2F! $! %&G ,! 563 ! $! //<= ! %AH0772,! $! $!