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

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011.
A057 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION
EXPRESSION OF MRNA ENCODING FGFR1B AND FGFR2B IN BOVINE FETAL OVARIES DURING GESTATION
TION
Rubia Bueno Da Silva 1 , Ester Siqueira Caixeta 2 , Christopher Price 3 & José Buratini Junior 4
1,2,4
UNESP, BOTUCATU, SP, BRAZIL. 3 UNIVERSITÉ DE MONTREAL, MONTREAL, CANADÁ.
Recent findings suggest the involvement of fibroblast growth factor 10 (FGF-10) in regulation of preantral follicle development.
During gestational ovarian development, expression of RNAm FGF-10 is progressively upregulated with a significant increase at 120 days of
gestation, coincident with the increase in primary follicles number (Castilho, data not published). FGF-10 signaling requires activation of FGFs
receptors 1B or 2B (FGFR1B, FGFR2B). In the present study, we assessed mRNA expression patterns of FGFR1B and FGFR2B in bovine
fetal ovaries during gestation. Bovine fetal ovaries were obtained in a local slaughterhouse, fetal age was estimated by the crown-rump length and
samples were grouped according with days of gestation as follows: 60 (n = 7), 75 (n = 7), 90 (n = 7), 120 (n = 7), 150 (n = 7) and 210 (n = 7).
Expression of mRNA encoding FGFR1B and FGFR2B was determined by semi-quantitative real time RT-PCR using bovine specific primers,
and cyclophilin A as the endogenous control. Reverse transcription was performed with SuperScriptIII (Invitrogen, São Paulo, Brazil) and
PCR with Power SYBR green master mix (Applied Biosystems, Foster City, USA) in an ABI Prism ® 7500. Relative expression values were
determined by the Pfaffl equation, analyzed by ANOVA and groups were compared by orthogonal contrast test (JMP software). Expression of
mRNA encoding FGFR1B and FGFR2B was detected throughout gestation. mRNA abundance of FGFR1B increased from 90 to 120 days of
gestation, when we previously observed an increase in FGF-10 expression and in the number of primary follicles. In contrast, FGFR2B mRNA
levels decreased from 60 to 90 days of gestation, around the time primordial follicle activation begins. In conclusion, both receptors of FGF-10
were shown to be developmentally regulated during gestation. The temporal association between gene expression patterns and fetal follicular
dynamics suggests that FGFR1B and FGFR2B signalling may be involved in the control of follicle activation.
Keywords: fgfr1b, fgfr2b, bovine.
A058 FOLLICULOGENESIS, OOGENESIS AND SUPEROVULATION
LEUKEMIA INHIBITOR
ORY FACT
CTOR STIMULATES
TES THE IN VITRO DEVEL
VELOPMENT OF SHEEP PREANTRAL AL FOLLICLES
AND THE PRODUCTION OF EMBRYOS
Valesca Barreto Luz 1 , Ticiana Franco Pereira da Silva 1 , Valdevane Rocha Araújo 1 , Ana Beatriz Graça Duarte 1 , Juliana Jales de
Hollanda Celestino 2 , Deborah de Melo Magalhães Padilha 1 , Roberta Nogueira Chaves 1 , Ivina Rocha Brito 1 , Anderson Pinto
Almeida 1 , Cristiano Feltrin 3 , Marcelo Bertolini 3 , Gerlane Modesto da Silva 1 & José Ricardo de Figueiredo 1
1
UECE, FORTALEZA, CE, BRAZIL. 2 UNILAB, REDENÇÃO, CE, BRAZIL. 3 UNIFOR, FORTALEZA, CE, BRAZIL.
The aim of this study was to investigate the effects of leukemia inhibitory factor (LIF), with or without FSH on the development of
sheep preantral follicles (PAF) and the influence embryo production. Ovaries of sheeps were dissected, and follicles (> 200 µm) were selected
and cultured individually in drops of 100 µL of base medium, that consisted of α-MEM supplemented with 3 mg/mL of BSA, ITS (insulin 10
µg/mL, transferrin 5.5 µg/mL and selenium 5 ng/mL), 2 mM of glutamine, 2 mM of hypoxantine, 50 µg/mL of ascorbic acid and LIF (50 ng/
mL) in the presence or absence of sequential rFSH (day 0-6: 100 ng/mL, days 6-12: 500 ng/mL and days 12-18: 1000 ng/mL). In vitro culture
(IVC) was performed at 39°C and 5% CO2 for 18 days, and all medium was replenished every six days. At the end of the IVC period, all healthy
follicles were carefully opened and oocytes (>110 µm) with a homogeneous cytoplasm and surrounded by at least one compact layer of cumulus
cells were selected for in vitro maturation (IVM). The selected cumulus-oocyte complexes were washed three times in maturation medium:
TCM199B (TCM199 + sodium bicarbonate) supplemented with BSA (1 mg/mL), pyruvate (1 mM/mL), rFSH (0.5 µg/mL), LH (5 µg/mL),
17β-estradiol (1 ug/mL), EGF (10 ng/mL), IGF-1 (50 ng/mL) and cysteamine (100 µM/mL). After washing, the oocytes were transferred to 50
µL drops of maturation medium under mineral oil and then incubated for 40 h at 39ºC in 5% CO2. Mature oocytes from treatment with the highest
rate of MII were activated by exposure to 5 µM ionomycin in TCM-HEPES supplemented with 0.4% BSA for 5 min followed by 3.5 h
incubation in 2 mM 6-DMAP in SOFaa + 0.4% BSA at 39°C. Then, activated embryos were cultured in SOFaa + 10% SFB for 5 days.
Cleavage and embryo rates were determined on days 2 and 5, respectively (Day 0: embryo activation). The results were compared by Chi-square
test and were expressed as percentages. There were no significant differences between treatments regarding oocytes for IVM (= 110 µm) and
meiosis resumption. In the group of follicles cultured with LIF in the absence of FSH showed a rate of MII of 29.63%, however in the presence
of FSH this rate was of 16.66%. After parthenogenesis, which was only performed in treatment without FSH, were obtained 7 embryos (8 cels)
from 12 mature oocytes. We concluded that the LIF associated or not to FSH stimulates the in vitro development of sheep PAF. Furthermore,
embryos can be obtained from PAF cultured in the presence of LIF.
Keywords: leukemia inhibitory factor, follicule, embryo.
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