Recent advances in ovulation synchronization and superovulation in ...

Abstracts. II International Symposium on Animal Biology of Reproduction, Nov. 19-22, 2008, São Paulo, SP, Brazil.
Hypoxia, glucose uptake and vascularization in CL of non-pregnant dogs
L.M.M.C. Sousa 1 , V.C. Amaral 1 , J. Buratini Jr 2 , B. Hoffmann 3 , P.C. Papa 1
1 Sector of Anatomy, Dept. of Surgery, FMVZ/USP, SP, Brazil, 05508-270.
2 Dept. of Physiology, IB/UNESP, Botucatu, SP, Brazil.
3 Klinik fuer Geburtshilfe, Gynaekologie und Andrologie der Gross- und Kleinetiere mit tierarztlicher Ambulanz, Justus-Liebig-
Universität Gieβen, Gieβen, Germany.
Introduction
Luteolysis in dogs, differently from farm animals, occurs independently of a uterine luteolysin (1). During canine
functional luteolysis, a decrease in progesterone (P4) concentrations occurs after day 20 after ovulation (Day 0 =
ovulation). Morphological features of luteal and endothelial cells degeneration are first observed after day 45 after
ovulation, concomitantly with a decrease in blood oxygen content in luteal tissue, which arises to hypoxic levels.
Whether hypoxia plays a role in the regulation of corpus luteum (CL) function in the bitch is not clear. Angiogenesis
and glycolysis are regulated by hypoxia through specific transcriptional factors up-regulation, notably hypoxia
inducible factor-1α (HIF-1α, 2). Thus, we hypothesized that HIF-1α is involved in angiogenesis and glucose
availability in different functional phases of canine ovarian cycle, especially during diestrus, contributing to local
regulation of CL function.
Materials and Methods
For that purpose, we assessed mRNA levels of HIF-1α, Facilitative Glucose Transporter 1 (GLUT1), vascular
endothelial growth factor (VEGF) and VEGF receptors - Flt-1 and KDR. Crossbred adult female dogs were
submitted to ovarysalpingohysterectomy each ten days from day 10 to 70 after ovulation (n = 4/group). Blood
samples were collected to P4 assessment by RIA. The day of ovulation was considered the day when plasma P4
levels reached ≥5ng/ml (3). CL were dissected and stored at – 80ºC until RNA was extracted, reversed transcribed
and cDNA submitted to Real time (TaqMan) RT-PCR analysis. GAPDH was used as housekeeping gene. Statistical
analysis was done using One-way analysis of variance followed by Newman-Keuls test. Correlations were done
using Spearman’s correlation test. Differences were considered significant when p ≤ 0.05.
Results and Discussion
P4 concentration was peaked at day 20 after ovulation (22.95 ± 3.84 ng/mL), and decreased continuously
towards day 70 (0.67 ± 0.51 ng/mL). HIF-1α and GLUT1 mRNA expression was differentially regulated over
diestrus showing a concomitant down regulation at day 40 after ovulation. and were positively correlated to
each other (r = 0.523, p = 0.01). VEGF, Flt-1 and KDR mRNA expression was also cycle dependent and higher
during the early and mid than in the late luteal phase. Although VEGF expression was highly positively correlated to
VEGFRs expression (r ≥ 0.62, p ≤ 0.007), nor VEGF either VEGFRs showed any correlation to HIF-1α and
GLUT1. P4 concentrations were correlated to VEGF and VEGFRs expression (r = 0.65, p < 0.05), however, nor
HIF-1α either GLUT1 showed any correlation to P4. These findings suggest that HIF-1α acts directly stimulating
GLUT-1 mRNA expression, apparently independent of P4 production. Humoral factors, as P4, may be involved in
angiogenic process regulating VEGF expression (4) or vice-versa (5). Thus, our data point towards direct
involvement of angiogenic factors in CL formation, lifespan and function. Additionally, our results provide evidence
for the role of HIF-1α in glucose uptake by CL, suggesting that HIF-1α time-dependent expression is essential for
luteal cells function maintenance under hypoxic stress.
References
(1) Hoffmann B, Hoveler R, et al. 1992. J Reprod Fertil, 96:837-845; (2) Nilsson I, Shibuya M, et al. 2004. Exp Cell
Res, 299:476-485; (3) Concannon PW, McCann JP, et al. 1989. J Reprod Fertil, 39:3-25. (4) Maybin JA, Duncan
WC. 2004. Reproduction, 128:423-431; (5) Yamashita H, Kamada D, et al. 2008. Mol Reprod Dev, DOI
10/102/mrd.
Financial support: CNPq
E-mail: ppapa@usp.br
Anim. Reprod., v.6, n.1, p.233, Jan./Mar. 2009 233