Expression Of ISG15 And Its Conjugating Enzymes In Bovine PBMC During Early Pregnancy And Mechanism Of HSP70 Regulation By FSH In Bovine Cumulus Cells

Pregnancy recognition is associated with the upregulation of the great number of genes in ruminants. Among which the Interferon-stimulatory gene (ISG15) which is an ubiquitin-like-protein, conjugates many different proteins through its ISGlyation enzymes UBEIL and UBCH8 system is of importance. We hypothesized that the ISG15 and its ISGlyation Enzymes UBEIL and UBCH8 are differentially activated by the maternal recognition of the pregnancy in pregnant (n=13) and non-pregnant (17) cows. In-vitro experiments the PBMC’s cells were treated with different concentration of the interferon-tau (IFNT) and see the regulation of the ISG15, UBEIL and UBCH8. The results shows the ISG15, UBEIL and UBCH8 were significantly (p<0.05) up-regulated by IFNT. Simultaneously 30 cows were synchronized, and artificially inseminated (AI) at day-0. At the same time 12 cyclic healthy cows were randomly selected as control group, which were not inseminated (NON-AI group). The blood were collected from the AI cows on day-18 and the expression of the ISG15, UBEIL and UBCH8 were analyze in PBMC’s and compare to the NON-AI group (n=12). After 45 days the pregnancy was diagnosed through rectal palpation. The results showed that the ISG15, UBEIL and UBCH8 were significantly up-regulated in the pregnant group (n=13) of animals as compared non-pregnant group (n=17) as well as to NON-AI group (n=12). In conclusion, the IFNT which is secreted by the conceptus acts on PBMC’s and up-regulates the interferon stimulated gene ISG15, its activating UBEIL and its conjugating UBCH8 enzymes can be a possible marker for the early pregnancy diagnosisSimilarly, Oocyte competence is the ability of the oocyte to fulfil maturation, undergo successful fertilization, reach the blastocyst stage and yield a healthy baby. Cumulus cells are indispensable for this process. Their removal significantly impairs oocyte maturation and therefore the development outcome. Moreover, the properties and functions of cumulus cells are regulated by the oocyte and thought to reflect its degree of maturation. We believe that the cumulus compartment of oocyte is the site of specific signaling and gene expression events which support and reflect the gamete’s quality. Using state of the art molecular biology technologies as siRNA knockdown technique and quantitative real time PCR, as well as gene pathways software analysis, we have focused on the study of cumulus cells gene expression under stress condition and genes involved in the regulation of this stress.The first objective was how heat shock regulates the expression of the HSP70. Our results indicate that after keeping the cumulus cells at 42 degree Celsius for the 30 minutes (HS) the HSP70 was upregulated. But at the same time if we treat the cells with FSH the expression of the HSP70 is down-regulated showing FSH may involve in the regulation of the HSP70. Secondly, irrespective of the heat shock treatment the FSH have up-regulated the IFNT gene expression. Combining these two results we assume that the IFNT gene involves in regulation of the HSP70 or the IFNT covalently involve in the expression of HSP70. To investigate further we design the siRNA to knockdown the IFNT gene in cumulus using the transfection technique. After transfection and knock down of the IFNT gene as compare to negative control (si-C), the HSP70 expression was not down-regulated by the FSH after heat shock treatment. Hence it is concluded that FSH not only induce the expression of the IFNT gene in cumulus cells. But also regulates the HSP70 through IFNT gene.The healthy cumulus cells are very important for the oocyte competence. Ultimately, these biochemical markers and their associated signaling pathways in bovine, increased the knowledge of gene expression patterns in cumulus cells, which would yield insights into the molecular pathways controlling oocyte competence and would serve as a preamble to the understanding of crucial events in subsequent fertilization and early embryonic development.