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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
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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.
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Proceedings of the International Symposium on Canine and Feline Reproduction, 2016 - Paris, France