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Close this window to return to IVIS www.ivis.org Proceedings of the 8th International Symposium on Canine and Feline Reproduction ISCFR June 22-25, 2016 Paris, France In a joint meeting with the XIX EVSSAR Congress Reprinted in IVIS with the permission of the ISCFR Organizers Reprinted in IVIS with the permission of the Organizers ISCFR VIII International Symposium on Canine and Feline Reproduction Close this window to return to IVIS PARIS, FRANCE JUNE 22-25, 2016 EVSSAR XIX Evaluation of hormone receptors and factors related to maternal –fetal interaction on canines embryos by qRT-PCR Ana Augusta Pagnano Derussi, Anthony Souza Castilho, Rodrigo Souza, Carlos Renato Guaitolini, Camila Ackermann, Rodrigo Volpato, Marilda Taffarel, Guilherme Cardoso, Maeli Dal Pai Silva, Maria Denise Lopes. Department of Veterinary Radiology and Animal Reproduction, FMVZ, UNESP- Botucatu. Sáo Paulo, Brazil [email protected] The maternal recognition of pregnancy in canine species is still unknown. Based on that, we investigate major hormone receptors (estrogen receptors (ER-α and ER-β), progesterone (PR) and oxytocin (OTR) and genes related to maternal –fetal interaction (leukemia inhibitory factor (LIF), its receptor (LIFR) and osteopontin (OPN) in canine embryos. For that, were obtained embryos through oviduct and uterine flushing from 10 bitches that were artificially inseminated (A.I) in their fertile period, being: 5 females inseminated and submitted to OHE on the 8 th day after A.I, which were collected 25 morulaes (MO) and 5 females inseminated and submitted to OHE on the 12 th day after AI, which were collected 25 blastocysts (BL). For flushing were using PBS (Nutricell, São Paulo). After the collected, the embryos were,frozen in cryogenic tubes at -80C with RNA later solution (Quiagen, UK). At the same day all embryos were thawed at room temperature slowly and washed in order to remove the RNA later. After this, they were analyzed on stereomicroscope (Leica Microsystems, Germany ) and separeted according development stage, thus forming 5 MO pools (containing 5 MO per pool) and 5 BL pools (containing 5 BL per pool). RNA extraction was performed using RNeasy micro Kit-Quiagen (Hildesheim, German) and RNA treatment using DNase I - Amplification Grade (Invitrogen Life Technologies, USA). Reverse transcription was used SuperScript III (Invitrogen) and for QRT-PCR was used Power SYBR Green (Applied Biosystems, EUA). Genes primers were designed with Primer Express ® software and GenBank information.Values obtained were normalized for the HPRT constitutive gene and mRNA Relative amount was determined using ΔΔct method and compared by t test. mRNA OTR was detected in both groups (MO and BL), mRNA PR, mRNA ER-, mRNA LIF were observed only in BL pools, while mRNA ER-, mRNA OPN and mRNA LIF- R were not expressed in any pool. The presence of mRNA OTR was significantly higher in morulae when compare to blastocysts, and It may indicate that OT has a relationship in cell proliferation mechanism in embryos, as well as in cancer1 cells and/or influenced induction of differentiation of embryonic cells as in mouse embryonic stem cells. There is few evidence of P4 direct effects on embryonic development during the preimplantation phase but maybe this hormone is required for sucessful of implantation as occurs in rodents .The difference of expression between mRNA ER and absence mRNA OPN and LIF expression , not discard the importance of these factors in canine gestation, but may indicate that action them occur in subsequent phases of gestation, as adherence process to lumen uterine since they has been detected in embryonic vesicles (unpublished results). In relation mRNA LIF expression we believed that LIF and your receptor plays an important role in maternal recognition this specie since was detected mRNA LIF-R in blastocysts and high incidence of mRNA LIF in canine uterus2. [1] Cassoni P, Marrocco T, Sapino A, Allia E, Bussolati G. Evidence of oxytocin/oxytocin receptor interplay in human prostate gland and carcinomas. Int. J. Oncol. 25: 899–904, 2004; [2] Derussi AAP, Souza, R.A, Volpato R, Guaitolini CR, ACKERMANN, CL, TAFFAREL, MO,CARDOSO, GS, DAL-PAI-SILVA, M, LOPES, MD. Metalloproteinase 2 (MMP-2), osteopontin (OPN) and leukemia inhibitory factor (LIF) genes expression in the oviduct and uterus of pregnant and nonpregnant bitches In: International Symposium on Canine and Feline Reproduction, ISCFR 2012 - EVSSAR 2012,, 2012, WHISTLER, B.C. CANADA.Annual Proceedings of the International Symposium on Canine and Feline Reproduction, 2012. 127 Proceedings of the International Symposium on Canine and Feline Reproduction, 2016 - Paris, France