Buffalo Bulletin 2013 Vol.32 (Special Issue 2): 1151-1154
Changes in Insulin-Like Growth Factor-Binding Proteins in Swamp Buffalo
Uterus during Estrous Cycle
Sirima THONGRUAY,a* Kitiya SRISAKWATTANA,a Wanvipa SUTHIKRAI,a Mariem
YUSUKSAWADb and Prakong TANGPRAPRUTIGULc
a
Research and Development Center for Livestock Production Technology, Faculty of Veterinary
Science, Chulalongkorn University, Henri Dunant Street, Phathumwan, Bangkok 10330, Thailand
b
Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330,
Thailand
c
Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
*Corresponding email: [email protected]
ABSTRACT
Insulin-like growth factor-binding proteins (IGFBPs) play a role in regulating insulin-like
growth factor (IGFs) action in a wide variety of cell types. The present study characterized the
changes in concentration of IGF binding protein-2 (IGFBP-2) and IGF binding protein-3 (IGFBP-3)
in uterine obtained from swamp buffalo during the estrous cycle. The uteri of 36 slaughtered swamp
buffaloes were collected, the stages of estrous cycle were considered by macroscopic examination
of the ovaries and were classified to be early, mid, late luteal and follicular phase. Western blot
analysis of endometrial tissue showed that IGFBP-2 was affected by the stage of estrous cycle. The
concentration of endometrial IGFBP-2 protein was increased at early and peak at mid luteal phase
of the cycle. However, endometrial IGFBP-3 protein was faintly detectable and did not change
significantly. Plasma progesterone by radioimmunoassay was also harmonized with endometrial
IGFBP-2 protein. Results suggest that increased in endometrial IGFBP-2 at the early and mid luteal
phase of the estrous cycle may regulate the activity of the IGFs which may be required to promote
the development of the uterus at this stage.
Keywords: insulin-like growth factor binding protein, uterus, swamp buffalo, estrous cycle
INTRODUCTION
The Insulin-like growth factors (IGFs) system has been reported in the uterus during the
oestrus cycle and play important role in regulating the development, differentiation and function in
the uterus and placenta of many species. The actions of IGF-I and IGF-II are mediated through the
IGF receptor and modulated by specific proteins called insulin-like growth factor-binding proteins
(IGFBPs) which there are at least six different proteins (IGFBP-1,-2,-3,-4,-5 and -6). IGFBPs
prolong the half-life of the IGFs and have been shown to either inhibit or stimulate the growth
promoting effects of the IGFs. The expression of mRNA and protein for IGFBPs has been
demonstrated in uterine tissue from several species. The synthesis of IGFBPs is regulated by steroid
hormones (Giudice et al., 1991). The changes in IGFBPs in uterine tissues according to the stages
of estrous cycle have been reported in rat (Davenport et al., 1992, Ghahary et al., 1993), ovine
(Osgerby et al., 1999), ewe (Gadds et al., 2000) and bovine (Robinson et al., 2000). However, there
is no information concerning IGFBPs in swamp buffalo uterus, therefore the objective of this study
was to determined the changes in IGFBP-2 and IGFBP-3 in swamp buffalo uterus during the
estrous cycle.
MATERIALS AND METHODS
Sample Collection
Blood samples were collected from the jugular vein for progesterone analysis. Immediately
after slaughtered, the healthy uteri of 36 slaughtered swamp buffaloes were removed and placed on
iced, the endometrium tissues were dissected and frozen immediately in liquid nitrogen and stored
at -80°C until processed for western blot analysis. The stages of estrous cycle were considered by
Accepted April 10, 2013; Online February 24, 2014.
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Buffalo Bulletin 2013 Vol.32 (Special Issue 2): 1151-1154
macroscopic examination of the ovaries and were classified to be early, mid, late luteal and
follicular phase.
RIA
Plasma progesterone concentrations were measured by non-extracted method as previously
described by Kamonpatana et al. (1983).
Western blot analysis
IGFBPs were extracted from frozen endometrium, after homogenization 30 μg of protein
extract was subjected to 12% SDS polyacrylamide gels followed by electrotransfer onto
nitrocellulose membranes. The membrane was incubated with primary antibody, a specific rabbit
anti-bovine IGFBP-2 (1:1000, US Biological), goat polyclonal antibody to IGFBP-3 (1:600,
Abcam, Cambridge, UK), mouse anti-β-actin monoclonal antibody (1:4000, Sigma Chemical) and
then incubated in the secondary antibody conjugated to horseradish peroxidase. Bands were
visualized by chemiluminescence and exposed to X-Omat film (Kodak ). For data analysis of each
band corresponding to IGFBP-2, IGFBP-3 and β-actin was analysed by measuring changes in
optical density using image analysis.
Statistical analysis
Results were expressed as means + standard deviation (SD). Statistical analysis was made
by analysis of variance (ANOVA) using the least significant difference (LSD). A probability (P)
less than 0.05 was considered to be significantly different.
RESULTS AND DISCUSSIONS
In the present study, plasma concentrations of progesterone were determined to ranged
between 0.34+0.19 to 3.15+0.76 ng/ml during the estrous cycle in swamp buffalo. Plasma
progesterone concentrations were increased during early luteal phase and then further increased to a
maximum concentration during mid luteal phase and then declined during late luteal phase and were
lowest during the follicular phase (Figure 1). The pattern of progesterone was similar to those
reported by other investigations (Kamonpatana et al., 1976, Arora and Pandey, 1982).
Western Blot analysis of endometrial IGFBP-2 and IGFBP-3 protein in the swamp buffalo
throughout the estrous cycle was shown in Figure 2. There was a different pattern of endometrial
IGFBP-2 and IGFBP-3. The concentration of IGFBP-2 increased at early and peak at mid luteal
phase of the cycle and declined at the late luteal and follicular phase while IGFBP-3 was low
throughout the estrous cycle and there was no difference in concentration of IGFBP-3 during the
different stages of cycle. The timing of increased endometrial IGFBP-2 coincided with plasma
progesterone concentrations. These results supported the previous study that IGFBP-2 was
regulated by progesterone since it increased similarly during the luteal phase of estrous cycle in cow
(Geisert et al., 1991) and pig (Simmen et al., 1990). Moreover, the expression of IGFBP-2 was
increased by elevated progesterone (McCarthy et al., 2012).
In the present study, endometrial IGFBP-2 was greater than IGFBP-3 throughout the estrous
cycle. IGFBP-2 was the most abundant in cow endometrium (Kirby et al., 1996) thus IGFBP-2 may
play a role in modulate the action of IGFs in the endometrium. The important role of IGFBP-2 was
to modulate endometrial epithelial cell mitogenesis (Badinga et al., 1999).
Endometrial IGFBP-3 protein was low and there was no significant difference in
concentration of IGFBP-3 throughout the estrous cycle. The present result was similar to the study
in cow which there was no significant change in the expression of IGFBP-3 throughout the oestrous
cycle (Robinson et al., 2000). This result suggested that endometrial IGFBP-3 was not regulated by
progesterone.
Although the expression of IGFBP-3 protein in myometrium was not shown in this study,
the previous study reported that IGFBP-3 was more prevalent in myometrium than in endometrium
and myometrial IGFBP-3 production had a role in regulating transport of circulating IGFs into the
myometrium and subsequent distribution of IGFs to the surround tissues (Keller et al., 1998).
In summary, the present study demonstrated that endometrial IGFBP-2 protein may be
regulated by progesterone and an increase in endometrial IGFBP-2 in the luteal phase may regulate
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Buffalo Bulletin 2013 Vol.32 (Special Issue 2): 1151-1154
the activity of the IGFs which may be required to promote the development of the uterus at this
stage.
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Figure 1 Plasma progesterone concentrations in swamp buffalo during the different
stages of the estrous cycle; early luteal(EL), mid luteal(ML), late luteal
(LL) and follicular phase (F). Values are mean+SD (n=9 in each stage).
Different letters above each column indicate significant difference at
p<0.05.
A
B
Figure 2 Endometrial IGFBP-2 and IGFBP-3 concentrations in swamp buffalo
during the different stages of the estrous cycle. (A) Western blots for
endometrial IGFBP-2, IGFBP-3 and β-actin. (B) The ratios of
endometrial IGFBP-2 and IGFBP-3 to β-actin. Representative data are
mean+SD which obtained from at least 6 experiments in each stage.
Different letters above each column indicate significant difference at
p<0.05.
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