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Title: ENHANCEMENT OF OVINE TROPHOBLAST INTERFERON BY GRANULOCYTE MACROPHAGE- COLONY STIMULATING FACTOR: POSSIBLE INVOLVEMENT OF PROTEIN KINASE C

Author
item IMAKAWA, KAZUHIKO - UNIV KS MED CTR, KS CITY
item CARLSON, K - WICHITA STATE UNIV, KS
item MCGUIRE, WILLIAM - 5438-01-10
item Christenson, Ronald
item TAYLOR, A - WICHITA STATE UNIV, KS

Submitted to: Journal of Molecular Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/20/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Reproduction failure is one of the most costly and limiting factors in the livestock industry. A large majority of reproductive losses in sheep occur during early pregnancy as a result of failed embryo-maternal signaling at the time of pregnancy recognition. The mechanism by which uterine endometrial growth factors increase the embryo produced signaling protein is not defined. Day 16 sheep embryos were aseptically collected and cultured for 24 hrs with uterine endometrial growth factors. Protein kinase C (PKC) pathway inducers and inhibitors were added to the culture and production of signaling protein measured. The results indicate that the stimulatory effect of the growth factor is mediated through the PKC pathway. Local manipulation of this pathway may improve embryo survival and lambing rates for the U.S. sheep industry.

Technical Abstract: Interferon tau (oIFNtau), the major secretory product of ovine conceptuses between days 13 and 21 (day 0 = day of estrus) of pregnancy, is implicated in maternal recognition of pregnancy. However, the molecular mechanisms by which endometrial GM-CSF and IL-3 upregulate oIFNtau production have not been defined. In this study, day 16 conceptuses were treated with an inducer, phorbol 12-myristate 13-acetate (PMA) and inhibitor (calphostin C of the protein kinase C (PKC) pathway. Treatment with either 150 units/ml hGM-CSF (P<0.05) or 10 nM PMA (P<0.1) resulted in a significant increase in oIFNtau mRNA expression. Pretreatment of conceptuses with 1 uM PMA for 12 hr to produce PKC deficient tissues or treatment with 50 mM calphostin C abolished the hGM-CSF induced increase in oIFNtau mRNA. An in vitro expression system has been established for the analysis of oIFNtau gene regulatory sequences. The oIFNtau010 gene has been isolated previously and found to be the principal oIFNtau gene upregulated during the pre-implanta eriod. 5'-flanking regions of the oIFNtau010 gene, -2 kb and -0.8 kb, have been cloned into a basic chloramphenicol acetyltransferase reporter plasmid. These oIFNtau010 promoter/enhancer constructs were transfected into human choriocarcinoma cells (JAR and JEG3) and their responsiveness to hGM-CSF and second messenger system activators including PMA, calcium ionophore (A23187) and 8-br-cAMP were characterized. The oIFNtau010 promoter/enhancer constructs were upregulated by hGM-CSF and PMA treatments (p<0.0l). Treatment with PMA and A23187 prevented the promoter/e er activation seen with PMA alone. Combined results from culture data and transfection data suggest that the stimulatory effect of GM-CSF on oIFNtau is mediated through the PKC pathway.