2011 (SBTE) 25th Annual Meeting Proceedings - International ...

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A193 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION
BOVINE EMBRYO MODULATES GENE EXPRESSION OF UTERINE GROWTH FACT
CTORS
Alan Diego Bezerra Lira 1 , Abimael Estevam Silva Júnior 1 , Luciana Alves Fátima 2 , Lawrence de Oliveira Barros 3 , Paula de Carvalho Papa 2 & Danila Barreiro
Campos 1
1
UFPB, AREIA, PB, BRAZIL. 2 USP, SAO PAULO, SP, BRAZIL. 3 UFRPE, RECIFE, PE, BRAZIL.
In most species the molecular basis of maternal-fetal communication are not well defined, and studies related to the modulation of
uterine events by the embryo are limited. Kashiwagi et al. (2007, Kashiwagi, A., Endocrinology 148, 4173-4184) showed that the mouse
embryo has an active role in establishing the uterine environment during implantation, regulating development and differentiation of maternal
endometrium. The observation that caruncles of the pregnant uterine horn are more developed than those from the non-pregnant horn in cattle,
and that in pregnancies from in vitro-produced embryos several anomalies are detected, including insufficient caruncular development and
vascularization, led us to hypothesize that not only the ovarian hormones control caruncular development, but also fetal factors are involved. This
study aimed to investigate the role of bovine embryo in the modulation of VEGF and bFGF system gene expression in uterine caruncles.
Caruncles from pregnant and non-pregnant uterine horns were collected at 35 days of gestation and total RNA was isolated using Trizol ® reagent
(Invitrogen, Carlsbad, USA) and specific columns for RNA extraction. Samples were treated with DNase and reverse-transcription was
performed using the Superscript III kit (Invitrogen, Carlsbad, USA) and 1µg of RNA. Expression of VEGF and its receptors Flt-1 and KDR,
and bFGF and its receptors R1, R2 and R4 was determined by real-time PCR, using tubulin as the endogenous control. Data were expressed
relative to tubulin and calculated by ÄÄCt method with correction by the amplification efficiency. Results showed that expression levels of bFGF
and its R2 receptor were significantly lower in the caruncles of the pregnant horn when compared with caruncles of the non-pregnant horn, with
no variation in the abundance of receptors R1 and R4, as well as VEGF and its receptors Flt-1 and KDR. VEGF and bFGF growth factors are
described as the most important factors controlling angiogenesis in the placenta, however, Mingju et al. (2006, Mingju, C., Matrix Biology 25,
342-354) have demonstrated that bFGF does not alter levels of cellular proliferation and inhibits expression of genes related to tissue remodeling
in granulosa cells. Thus, the reduced expression of bFGF and its receptor R2 in caruncles of pregnant uterine horn could be associated with
intense tissue remodeling occurring during placentation. Results suggest that the bovine embryo may be involved in the process of caruncular
development through modulation of uterine growth factors gene expression.
Keywords: growth factors, caruncle, embryo.
A194 EMBRYOLOGY, BIOLOGY OF DEVELOPMENT AND PHYSIOLOGY OF REPRODUCTION
NEW MEDIUM FOR THE USE OF BRILLIANT CRESYL BLUE IN OOCYTE SELECTION FOR IN VITRO SWINE
EMBRYOS PRODUCTION - PRELIMINARY DATA
Elisa Caroline da Silva Santos 1 , Jorgea Pradieé 1 , Alexander Oliveira Gonçalves 1 , Rafael Gianela Mondadori 1 , Thomaz Lucia Jr. 1 ,
Arnaldo Diniz Vieira 1 & Ligia Margareth Cantarelli Pegoraro 2
1
UFPEL, PELOTAS, RS, BRAZIL. 2 EMBRAPA/CPACT, PELOTAS, RS, BRAZIL.
N
Selection of competent oocytes improves the in vitro embryo production outcomes. Among the available methods of selection,
Brilliant Cresyl Blue (BCB) is a cell viability dye, which determines the presence of glucose-6-phosphate dehydrogenase. This enzyme is active
in growing oocytes and inactive in the fully grown oocytes. Thus, BCB stained oocytes are considered more competent, because they already
reached their full growth. This selection method was previously tested in several species, however, the results of its efficiency remains
contradictory. Some studies in which exposure to the dye resulted in a decrease of oocyte viability suggested that the maintenance medium (PBS),
and time of exposure during the staining process may be relevant for the decrease of oocyte viability. This study tested the modified “porcine
zygote medium” (mPZM-4) for the staining of porcine oocytes with BCB as well as reducing the dye exposure time from 90 to 60 min. We used
388 cumulus oocyte complexes (COCs) obtained from abattoir ovaries, distributed on media containing 26 µM BCB on mPZM-4 (n = 133),
PBS (n = 142) and control (mPZM-4 without BCB, n = 113). The COCs were maintained for 60 min in BCB solution at 39°C. Subsequently,
they were separated in five groups according to the dye reaction: mPZM-4 dyed and not-dyed, PBS dyed and not-dyed and control. After the
exposure to the dye and selection, all COCs were submitted to in vitro maturation. In the first 24 h IVM was conducted in mPZM-4 medium
containing EGF, LH, FSH, c-AMP and porcine follicular fluid. The final 24h maturation period was conducted in the same medium, without
hormones and cAMP. The nuclear maturation was evaluated with Hoechst (Sigma ® H33342) using epifluorescence microscopy. Metaphase II
(MII) oocytes were considered matured. Data were compared using Chi-square test. The rate of MII was 38% in the control group (n = 113),
23% in dyed mPZM-4 (n = 91), 7% in not dyed mPZM-4 (n = 42), 24% in dyed PBS (n = 82) and 19% in not dyed PBS (n = 60). The results
show that mPZM-4 and PBS can be used for BCB staining. The rate of IVM in the not dyed mPZM-4 group was lower than control (P < 0.05).
These preliminary results show that the BCB diluted in mPZM-4 and an exposure period of 60 min, can be used to select more competent
structures to in vitro swine embryo production. To confirm the data more repetitions will be done.
Keywords: brilliant cresyl blue, porcine oocytes, maintenance médium.
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