VEGF normally acts to stimulate vascular development (angiogenesis) and is necessary for ovulation of the dominant follicle. The inhibitory isoform blocks the actions of VEGF angiogenic isoforms; therefore, the objectives of the experiments were to identify the bovine inhibitory isoform, VEGF164b and to determine its expression prior to and after the LH surge. VEGF164b mRNA was upregulated prior to but did not change after the LH surge. Therefore, VEGF164b may be necessary for preparation of the dominant follicle prior to ovulation

Bergman, Jeffrey W.

Clopton, Debra T.

Cupp, Andrea S.

Martin, Jeremy L.

Moline, Karl V.

Ten Broeck, Robin A.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Nebraska Beef Cattle Reports Animal Science Department January 2007 Vascular Endothelial Growth Factor Inhibitory Isoform Is Regulated Prior to Ovulation Robin A. Ten Broeck University of Nebraska-Lincoln Debra T. Clopton University of Nebraska-Lincoln, dclopton1@unl.edu Jeremy L. Martin University of Nebraska-Lincoln Karl V. Moline University of Nebraska-Lincoln, kmoline1@unl.edu Jeffrey W. Bergman University of Nebraska-Lincoln, jbergman1@unl.edu See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/animalscinbcr  Part of the Animal Sciences Commons Ten Broeck, Robin A.; Clopton, Debra T.; Martin, Jeremy L.; Moline, Karl V.; Bergman, Jeffrey W.; and Cupp, Andrea S., "Vascular Endothelial Growth Factor Inhibitory Isoform Is Regulated Prior to Ovulation" (2007). Nebraska Beef Cattle Reports. 97. https://digitalcommons.unl.edu/animalscinbcr/97 This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Nebraska Beef Cattle Reports by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Robin A. Ten Broeck, Debra T. Clopton, Jeremy L. Martin, Karl V. Moline, Jeffrey W. Bergman, and Andrea S. Cupp This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/animalscinbcr/97 Page 114 — 2007 Nebraska Beef Report  © The Board of Regents of the University of Nebraska.  All rights reserved.Vascular Endothelial Growth Factor Inhibitory Isoform Is Regulated Prior to OvulationRobin A. Ten BroeckDebra T. CloptonJeremy L. MartinKarl V. MolineJeff W. BergmanAndrea S. Cupp1SummaryVEGF normally acts to stimulate vascular development (angiogenesis) and is necessary for ovulation of the dominant follicle. The inhibitory isoform blocks the actions of VEGF angiogenic isoforms; therefore, the objectives of the experiments were to identify the bovine inhibitory isoform, VEGF164b and to determine its expression prior to and after the LH surge. VEGF164b mRNA was upregulated prior to but did not change after the LH surge. Therefore, VEGF164b may be necessary for preparation of the dominant follicle prior to ovulation. IntroductionDuring deviation of the dominant follicle from the subordinate follicles, the dominant follicle develops an extensive vasculature network in the theca layer that surrounds the basement membrane. The granulosa cell layer surrounding the oocyte within the basement membrane of the follicle is avascular at this time point. VEGF is expressed by granulosa cells prior to ovulation in the bovine follicle and working via the theca cells to increase vacularization, providing nutrients to the developing follicle. VEGF aids in cell migration, proliferation, and increased microvascular permeability through the stimulation of endothelial cells which are cells that form blood vessels.Recent studies have identified an anti-angiogenic inhibitory VEGF splice variant, VEGF164b. Vascular Endothelial Growth Factor 164b is the inhibitory isoform of bovine VEGF164. The VEGF inhibitory isoform blocks actions of VEGF pro-angiogenic isoforms.The objective of the current study was to identify VEGF164b and to determine the expression pattern of VEGF164b in granulosa cells of the bovine follicle prior to and after the LH surge. The hypothesis was that VEGF164b isoform would be differently expressed and may be used to regulate folliculogenesis during the bovine estrous cycle.ProcedureIn the first trial, cows were administered two injections of PGF2α 14 days apart and follicular aspirates were collected at 12, 18, 24, 0, 6, 48, 54, 60, and 72 hours (n=average of 8) after the second injection of PGF2α to obtain granulosa cells prior to the LH surge. Messenger RNA was extracted from granulosa cells and samples were reversed transcribed to cDNA. Progesterone and estrogen concentrations were measured in follicular fluid. A ratio greater than 1 of estrogen to progesterone indicated the follicle was still dominant. The mRNA expression of VEGF164b isoform was determined using real-time quantitative polymerase chain reaction (PCR) at each follicular aspirate time point.Blood samples were collected every two hours from 12 cows to determine when the cows had an LH surge. Since all the cows had an LH surge from 56 hours to greater than 72 hours, the granulosa cells collected prior to 56 hours were used in the analysis.A second trial was conducted to obtain granulosa cells after a GnRH induced LH surge. Cows were injected with two injections of PGF2α (25 mg/cow) and 48 hours after the second injection of PGF2α  administered GnRH (100 μg/cow). Follicular aspirates were collected at , 6, 12, 18, and 24 hours after GnRH (n=average of 10). Messenger RNA was extracted as described previously and expression of VEGF mRNA isoform 164b was analyzed using quantitative real time PCR.Quantitative RT-PCR data for both trials were analyzed using an ANOVA with SAS. Comparisons of means were tested using a Tukey-Kramer test.ResultsAnalysis of newly identified bovine VEGF164bThe VEGF164b inhibitory isoform was subcloned and sequenced in our laboratory. Conventional RT-PCR confirmed the presence of VEGF164b in bovine granulosa cells from dominant follicles (Figure 1). We speculate that every angiogenic isoform of VEGF has an anti-angiogenic isoform that regulates their functions.Quantitative Real-time PCR (QRT-PCR) for granulosa cellsIn the first trial, mRNA expression was measured at 12, 18, 24, 0, 6, 48, and 54 hours post second injection of PGF2α (PG). In Trial 1, the inhibitory isoform, VEGF164b, increased at 18 hours (P<0.05); Figure 2.A second trial was conducted to more accurately simulate the LH surge by an injection with GnRH and to © The Board of Regents of the University of Nebraska.  All rights reserved. 2007 Nebraska Beef Report  — Page 115 collect follicle aspirates after the LH surge. In the second trial, there was no difference in VEGF164b at any of the time points after GnRH (Figure ). Thus, it appears that the inhibitory isoform may be regulated after corpus luteum regression (Trial 1) but not after the LH surge (Trial 2).ConclusionThe current studies demonstrate VEGF mRNA isoform expression patterns prior to and after the LH surge. VEGF164b increases 18 hours after CL regression. From these results, we speculate that VEGF mRNA isoform expression is finely regulated by ovarian growth factors and steroid hormones to provide for necessary vascular development and follicle progression. These studies establish a role for VEGF anti-angiogenic isoforms prior to ovulation in the bovine follicle. It is possible that we may be able to use VEGF anti-angiogenic isoforms to manipulate follicle development to more accurately time ovulation in synchronization or superovulation protocols of beef females.1Robin A. Ten Broeck, graduate student; Debra T. Clopton, research technician; Jeremy L. Martin, graduate student; Karl V. Moline, cow/calf manager; Jeff W. Bergman, agriculture technician; and Andrea S. Cupp, associate professor, Animal Science, Lincoln.500 bp200 bpVEGF164b224 bpGene RulerGnRH (0)GnRH ()GnRH (6)GnRH (12)GnRH (24)PGF 2α (12)PGF 2α (24)PGF 2α (6)PGF 2α (54)No Template ControlFigure 1. Conventional reverse transcription PCR to detect VEGF164b isoform in bovine granu-losa cells collected at 0, 3, 6, 12 and 24 hours after GnRH injection (Trial 2) or 12, 24, 36 and 54 hours after a second injection of PGF2α (Trial 1).Figure 2. Expression of mRNA for anti-angiogenic VEGF164b isoform using QRT-PCR hours after second injection of PGF2α. VEGF isoforms were normalized to GapDH. Messenger RNA expression at each time point is denoted by different letters. 12 18 24 0 6 48 54Hour after PGF2α4.0.5.02.52.01.51.00.50.0VEGF164b/GapDHabaaaa aa 0  6 12 18 24Hour at GnRHaaaaa654210VEGF164b/GapDHFigure 3. Expression of mRNA for anti-angiogenic VEGF164b isoform using QRT-PCR hours after second injection of GnRH (Trial 2). VEGF isoforms were normalized to GapDH. There were no differences in expression of VEGF164b mRNA as denoted by the same letter for each time point.

Vascular Endothelial Growth Factor Inhibitory Isoform  Is Regulated Prior to Ovulation

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University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Nebraska Beef Cattle Reports Animal Science Department 
January 2007 
Vascular Endothelial Growth Factor Inhibitory Isoform Is 
Regulated Prior to Ovulation 
Robin A. Ten Broeck 
University of Nebraska-Lincoln 
Debra T. Clopton 
University of Nebraska-Lincoln, dclopton1@unl.edu 
Jeremy L. Martin 
University of Nebraska-Lincoln 
Karl V. Moline 
University of Nebraska-Lincoln, kmoline1@unl.edu 
Jeffrey W. Bergman 
University of Nebraska-Lincoln, jbergman1@unl.edu 
See next page for additional authors 
Follow this and additional works at: https://digitalcommons.unl.edu/animalscinbcr 
 Part of the Animal Sciences Commons 
Ten Broeck, Robin A.; Clopton, Debra T.; Martin, Jeremy L.; Moline, Karl V.; Bergman, Jeffrey W.; and Cupp, 
Andrea S., "Vascular Endothelial Growth Factor Inhibitory Isoform Is Regulated Prior to Ovulation" (2007). 
Nebraska Beef Cattle Reports. 97. 
https://digitalcommons.unl.edu/animalscinbcr/97 
This Article is brought to you for free and open access by the Animal Science Department at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Nebraska Beef Cattle 
Reports by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Authors 
Robin A. Ten Broeck, Debra T. Clopton, Jeremy L. Martin, Karl V. Moline, Jeffrey W. Bergman, and Andrea 
S. Cupp 
This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/
animalscinbcr/97 
Page 114 — 2007 Nebraska Beef Report  © The Board of Regents of the University of Nebraska.  All rights reserved.
Vascular Endothelial Growth Factor Inhibitory Isoform 
Is Regulated Prior to Ovulation
Robin A. Ten Broeck
Debra T. Clopton
Jeremy L. Martin
Karl V. Moline
Jeff W. Bergman
Andrea S. Cupp1
Summary
VEGF normally acts to stimulate 
vascular development (angiogenesis) 
and is necessary for ovulation of the 
dominant follicle. The inhibitory 
isoform blocks the actions of VEGF 
angiogenic isoforms; therefore, the 
objectives of the experiments were 
to identify the bovine inhibitory 
isoform, VEGF164b and to determine 
its expression prior to and after the 
LH surge. VEGF164b mRNA was 
upregulated prior to but did not 
change after the LH surge. Therefore, 
VEGF164b may be necessary for 
preparation of the dominant follicle 
prior to ovulation.
 
Introduction
During deviation of the dominant 
follicle from the subordinate follicles, 
the dominant follicle develops an 
extensive vasculature network in 
the theca layer that surrounds the 
basement membrane. The granulosa 
cell layer surrounding the oocyte 
within the basement membrane of 
the follicle is avascular at this time 
point. VEGF is expressed by granulosa 
cells prior to ovulation in the bovine 
follicle and working via the theca 
cells to increase vacularization, 
providing nutrients to the developing 
follicle. VEGF aids in cell migration, 
proliferation, and increased 
microvascular permeability through 
the stimulation of endothelial cells 
which are cells that form blood 
vessels.
Recent studies have identified an 
anti-angiogenic inhibitory VEGF 
splice variant, VEGF164b. Vascular 
Endothelial Growth Factor 164b is 
the inhibitory isoform of bovine 
VEGF164. The VEGF inhibitory 
isoform blocks actions of VEGF pro-
angiogenic isoforms.
The objective of the current study 
was to identify VEGF164b and to 
determine the expression pattern 
of VEGF164b in granulosa cells of 
the bovine follicle prior to and after 
the LH surge. The hypothesis was 
that VEGF164b isoform would be 
differently expressed and may be used 
to regulate folliculogenesis during the 
bovine estrous cycle.
Procedure
In the first trial, cows were 
administered two injections of PGF
2α 
14 days apart and follicular aspirates 
were collected at 12, 18, 24, 0, 6, 
48, 54, 60, and 72 hours (n=average 
of 8) after the second injection of 
PGF
2α to obtain granulosa cells prior 
to the LH surge. Messenger RNA was 
extracted from granulosa cells and 
samples were reversed transcribed 
to cDNA. Progesterone and estrogen 
concentrations were measured in 
follicular fluid. A ratio greater than 1 
of estrogen to progesterone indicated 
the follicle was still dominant. The 
mRNA expression of VEGF164b 
isoform was determined using real-
time quantitative polymerase chain 
reaction (PCR) at each follicular 
aspirate time point.
Blood samples were collected every 
two hours from 12 cows to determine 
when the cows had an LH surge. Since 
all the cows had an LH surge from 56 
hours to greater than 72 hours, the 
granulosa cells collected prior to 56 
hours were used in the analysis.
A second trial was conducted to 
obtain granulosa cells after a GnRH 
induced LH surge. Cows were injected 
with two injections of PGF
2α (25 mg/
cow) and 48 hours after the second 
injection of PGF
2α  administered 
GnRH (100 μg/cow). Follicular 
aspirates were collected at , 6, 12, 18, 
and 24 hours after GnRH (n=average 
of 10). Messenger RNA was extracted 
as described previously and 
expression of VEGF mRNA isoform 
164b was analyzed using quantitative 
real time PCR.
Quantitative RT-PCR data for both 
trials were analyzed using an ANOVA 
with SAS. Comparisons of means 
were tested using a Tukey-Kramer 
test.
Results
Analysis of newly identified bovine 
VEGF164b
The VEGF164b inhibitory isoform 
was subcloned and sequenced in 
our laboratory. Conventional RT-
PCR confirmed the presence of 
VEGF164b in bovine granulosa cells 
from dominant follicles (Figure 1). 
We speculate that every angiogenic 
isoform of VEGF has an anti-
angiogenic isoform that regulates 
their functions.
Quantitative Real-time PCR (QRT-PCR) 
for granulosa cells
In the first trial, mRNA expression 
was measured at 12, 18, 24, 0, 6, 48, 
and 54 hours post second injection of 
PGF
2α (PG). In Trial 1, the inhibitory 
isoform, VEGF164b, increased at 18 
hours (P<0.05); Figure 2.
A second trial was conducted to 
more accurately simulate the LH surge 
by an injection with GnRH and to 
© The Board of Regents of the University of Nebraska.  All rights reserved. 2007 Nebraska Beef Report  — Page 115 
collect follicle aspirates after the LH 
surge. In the second trial, there was 
no difference in VEGF164b at any of 
the time points after GnRH (Figure 
). Thus, it appears that the inhibitory 
isoform may be regulated after corpus 
luteum regression (Trial 1) but not 
after the LH surge (Trial 2).
Conclusion
The current studies demonstrate 
VEGF mRNA isoform expression 
patterns prior to and after the LH 
surge. VEGF164b increases 18 hours 
after CL regression. From these 
results, we speculate that VEGF 
mRNA isoform expression is finely 
regulated by ovarian growth factors 
and steroid hormones to provide 
for necessary vascular development 
and follicle progression. These 
studies establish a role for VEGF 
anti-angiogenic isoforms prior to 
ovulation in the bovine follicle. It is 
possible that we may be able to use 
VEGF anti-angiogenic isoforms to 
manipulate follicle development to 
more accurately time ovulation in 
synchronization or superovulation 
protocols of beef females.
1Robin A. Ten Broeck, graduate student; 
Debra T. Clopton, research technician; Jeremy 
L. Martin, graduate student; Karl V. Moline, 
cow/calf manager; Jeff W. Bergman, agriculture 
technician; and Andrea S. Cupp, associate 
professor, Animal Science, Lincoln.
500 bp
200 bp
VEGF164b
224 bp
G
en
e 
R
ul
er
G
n
R
H
 (
0)
G
n
R
H
 (
)
G
n
R
H
 (
6)
G
n
R
H
 (
12
)
G
n
R
H
 (
24
)
P
G
F 2
α 
(1
2)
P
G
F 2
α 
(2
4)
P
G
F 2
α 
(
6)
P
G
F 2
α 
(5
4)
N
o 
Te
m
pl
at
e 
C
on
tr
ol
Figure 1. Conventional reverse transcription PCR to detect VEGF164b isoform in bovine granu-
losa cells collected at 0, 3, 6, 12 and 24 hours after GnRH injection (Trial 2) or 12, 24, 36 
and 54 hours after a second injection of PGF
2α
 (Trial 1).
Figure 2. Expression of mRNA for anti-angiogenic VEGF164b isoform using QRT-PCR hours after 
second injection of PGF
2α. VEGF isoforms were normalized to GapDH. Messenger RNA 
expression at each time point is denoted by different letters.
 12 18 24 0 6 48 54
Hour after PGF
2α
4.0
.5
.0
2.5
2.0
1.5
1.0
0.5
0.0
V
E
G
F1
64
b/
G
ap
D
H
a
b
a
a
a
a a
a
 0  6 12 18 24
Hour at GnRH
a
a
a
a
a
6
5
4

2
1
0
V
E
G
F1
64
b/
G
ap
D
H
Figure 3. Expression of mRNA for anti-angiogenic VEGF164b isoform using QRT-PCR hours after 
second injection of GnRH (Trial 2). VEGF isoforms were normalized to GapDH. There 
were no differences in expression of VEGF164b mRNA as denoted by the same letter for 
each time point.


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Vascular Endothelial Growth Factor Inhibitory Isoform Is Regulated Prior to Ovulation

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