title: Regulation of ovulatory genes in bovine granulosa cells: lessons from siRNA silencing of PTGS2.
abstract: Prostaglandin endoperoxide synthase-2 (PTGS2), tumour necrosis factor-alpha-induced protein-6 (TNFAIP6), pentraxin-3 (PTX3), epidermal growth factor-like factors: amphiregulin (AREG) and epiregulin (EREG) are essential for successful ovulation. In this study, we compared the induction of these ovulatory genes in bovine granulosa cells (GCs) in vivo (after LH surge) and in vitro (forskolin (FRS) treatment). These genes were markedly stimulated in GCs isolated from cows 21 h after LH-surge. In isolated GCs, FRS induced a distinct temporal profile for each gene. Generally, there was a good agreement between the in vivo and in vitro inductions of these genes except for PTX3. Lack of PTX3 induction in isolated GCs culture suggests that other follicular compartments may mediate its induction by LH. Next, to study the role of PTGS2 and prostaglandins (PGs) in the cascade of ovulatory genes, PTGS2 was silenced with siRNA. PTGS2 siRNA caused a marked and specific knockdown of PTGS2 mRNA and PGE2 production (70% compared with scrambled siRNA) in bovine GCs. Importantly, PTGS2 silencing also reduced AREG, EREG and TNFAIP6 mRNA levels but not PTX3. Exogenous PGE2 increased AREG, EREG and TNFAIP6 mRNA levels, further confirming that these genes are prostanoid dependent. A successful and specific knockdown of PTGS2 was also achieved in endometrial cells (EndoCs) expressing PTGS2. Then, cholesterol-conjugated PTGS2 (chol-PTGS2) siRNA that facilitates cells' entry was investigated. In EndoCs, but not in GCs, chol-PTGS2 siRNA succeeded to reduce PTGS2 and PGE2 levels even without transfection reagent. PTGS2 knockdown is a promising tool to critically examine the functions of PTGS2 in the reproductive tract.
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, IsraelDepartment of Reproductive Immunology and PathologyInstitute of Animal Reproduction and Food Research, Polish Academy of Science, Olsztyn, PolandDepartment of Reproductive BiologyLeibniz Institute of Farm Animal Biology, Dummerstorf, GermanyDepartment of Ruminant ScienceAgricultural Research Organization, Bet-Dagan, Israel.
authors.forename: Ketan
authors.initials: K
authors.lastname: Shrestha
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, IsraelDepartment of Reproductive Immunology and PathologyInstitute of Animal Reproduction and Food Research, Polish Academy of Science, Olsztyn, PolandDepartment of Reproductive BiologyLeibniz Institute of Farm Animal Biology, Dummerstorf, GermanyDepartment of Ruminant ScienceAgricultural Research Organization, Bet-Dagan, Israel.
authors.forename: Karolina
authors.initials: K
authors.lastname: Lukasik
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, IsraelDepartment of Reproductive Immunology and PathologyInstitute of Animal Reproduction and Food Research, Polish Academy of Science, Olsztyn, PolandDepartment of Reproductive BiologyLeibniz Institute of Farm Animal Biology, Dummerstorf, GermanyDepartment of Ruminant ScienceAgricultural Research Organization, Bet-Dagan, Israel.
authors.forename: Anja
authors.initials: A
authors.lastname: Baufeld
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, IsraelDepartment of Reproductive Immunology and PathologyInstitute of Animal Reproduction and Food Research, Polish Academy of Science, Olsztyn, PolandDepartment of Reproductive BiologyLeibniz Institute of Farm Animal Biology, Dummerstorf, GermanyDepartment of Ruminant ScienceAgricultural Research Organization, Bet-Dagan, Israel.
authors.forename: Jens
authors.initials: J
authors.lastname: Vanselow
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, IsraelDepartment of Reproductive Immunology and PathologyInstitute of Animal Reproduction and Food Research, Polish Academy of Science, Olsztyn, PolandDepartment of Reproductive BiologyLeibniz Institute of Farm Animal Biology, Dummerstorf, GermanyDepartment of Ruminant ScienceAgricultural Research Organization, Bet-Dagan, Israel.
authors.forename: Uzi
authors.initials: U
authors.lastname: Moallem
authors.affiliation: Department of Animal SciencesThe Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, IsraelDepartment of Reproductive Immunology and PathologyInstitute of Animal Reproduction and Food Research, Polish Academy of Science, Olsztyn, PolandDepartment of Reproductive BiologyLeibniz Institute of Farm Animal Biology, Dummerstorf, GermanyDepartment of Ruminant ScienceAgricultural Research Organization, Bet-Dagan, Israel rina.meidan@mail.huji.ac.il.
authors.forename: Rina
authors.initials: R
authors.lastname: Meidan
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D051546:Cyclooxygenase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015232:Dinoprostone / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000494:pharmacology; D010060:Ovulation / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014665:Vasodilator Agents / Q000494:pharmacology
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014665:Vasodilator Agents; D005576:Colforsin; D007986:Luteinizing Hormone; D051546:Cyclooxygenase 2; D015232:Dinoprostone