Vascular Endothelial Growth Factor (VEGF) is produced by cells surrounding the egg in the follicle prior to ovulation. If VEGF is inhibited, ovulation does not occur. The VEGF gene can be spliced to produce different protein isoforms which have specific functions. Our objective was to determine if VEGF 120 and 164 mRNA isoforms are differentially regulated in the preovulatory follicle. From our studies, VEGF isoforms are differentially regulated during both CL regression and after a simulated LH surge. Differences observed in VEGF isoform regulation may allow for manipulation of ovulation in the beef cow

Bergman, Jeffrey W.

Bott, Rebecca C.

Clopton, Debra T.

Cupp, Andrea S.

Martin, Jeremy L.

Moline, Karl V.

Ten Broeck, Robin A.

English

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Nebraska Beef Cattle Reports Animal Science Department 
January 2006 
Vascular Endothelial Growth Factor mRNA Isoforms 120 and 164 
are Differentially 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 
Rebecca C. Bott 
University of Nebraska-Lincoln 
Karl V. Moline 
University of Nebraska-Lincoln, kmoline1@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.; Bott, Rebecca C.; Moline, Karl V.; Bergman, 
Jeffrey W.; and Cupp, Andrea S., "Vascular Endothelial Growth Factor mRNA Isoforms 120 and 164 are 
Differentially Regulated Prior to Ovulation" (2006). Nebraska Beef Cattle Reports. 108. 
https://digitalcommons.unl.edu/animalscinbcr/108 
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, Rebecca C. Bott, 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/108 
Page 18 — 2006 Nebraska Beef Report  © 2005, The Board of Regents of the University of Nebraska.  All rights reserved.
Vascular Endothelial Growth Factor mRNA Isoforms 120 
and 164 are Differentially Regulated Prior to Ovulation
Robin A. Ten Broeck
Debra T. Clopton
Jeremy L. Martin
Rebecca C. Bott
Karl V. Moline
Jeff W. Bergman
Andrea S. Cupp1
Summary
Vascular Endothelial Growth Factor 
(VEGF) is produced by cells surround-
ing the egg in the follicle prior to ovula-
tion. If VEGF is inhibited, ovulation 
does not occur. The VEGF gene can be 
spliced to produce different protein iso-
forms which have specific functions. Our 
objective was to determine if VEGF 120 
and 164 mRNA isoforms are differen-
tially regulated in the preovulatory fol-
licle. From our studies, VEGF isoforms 
are differentially regulated during both 
CL regression and after a simulated LH 
surge. Differences observed in VEGF iso-
form regulation may allow for manipu-
lation of ovulation in the beef cow. 
Introduction
Follicular development within the 
bovine ovary is a dynamic process. It 
begins prior to birth and continues 
throughout the cow =s reproductive 
lifespan. Angiogenesis, the formation 
of new blood vessels, is crucial in the 
ovulatory follicle. The blood vessels 
supply the follicle with necessary nu-
trients for development and growth 
prior to ovulation. VEGF is expressed 
by granulosa cells (cells surrounding 
the egg) prior to ovulation in the bo-
vine follicle and is an important factor 
in the regulation of normal angiogen-
esis in the developing follicle and cor-
pus luteum. Several VEGF isoforms 
exist, the most common isoforms are 
VEGF 164 and VEGF 120. In other tis-
sues such as the heart and lung VEGF 
isoforms have specific functions in 
vascular development. VEGF 120 is 
highly diffusible and recruits endo-
thelial cells (precursor to blood vessel 
cells) in the establishment of blood 
vessels. VEGF 164 recruits endothelial 
cells that form the large blood ves-
sels. However, it is not known how 
these isoforms function in the ovarian 
preovulatory follicle nor how inhibi-
tion of VEGF might alter ovulation. 
The objective of the current study is 
to determine if the two major VEGF 
mRNA isoforms, 120 and 164, are 
differentially regulated prior to ovu-
lation in the granulosa cells of the 
dominant follicle.
Procedure
Trial 1
Two injections of PGF
2α (5mg/cow) 
were administered 14 days apart. 
PGF
2α induces regression of the cor-
pus luteum initiating the LH surge 
and ovulation. A vaginal probe with 
needle attachment was used to collect 
follicular fluid and granulosa cells 
from the dominant follicle. Follicular 
aspirates were collected at 12, 18, 24, 
30, 36, 48, 54, 60, 66, and 72 hours 
(n=average of 8) after the second 
injection of PGF
2α. Messenger RNA 
was extracted from granulosa cells 
and samples were reversed transcribed 
to cDNA. Progesterone and estrogen 
concentrations were measured in fol-
licular fluid. A ratio greater than 1 of 
estrogen to progesterone indicated 
that the follicle was still dominant. 
The mRNA expression of VEGF iso-
forms 120 and 164 were determined 
using real-time quantitative poly-
merase chain reaction (PCR) at each 
follicular aspirate time point. Data 
were analyzed using an ANOVA with 
SAS. Comparisons of means were 
analyzed using a Tukey-Kramer test. 
In 12 animals, blood samples were 
collected after the second injection 
of PGF
2α at two-hour intervals from 
0 to 72 hours to determine when the 
LH surge occurred. The LH surge 
was detected in six of the 12 cows and 
occurred between 54 and 72 hours. 
Due to this variation, a second trial 
was conducted to obtain follicle aspi-
rates after a simulated surge of LH by 
injecting GnRH (0.1mg/cow).
Trial 2
Two injections of PGF
2α (5mg/cow) 
were administered 14 days apart with 
an injection of GnRH (0.1mg/cow) 
administered 48 hours after the sec-
ond injection of PGF
2α. Follicular 
aspirates were collected via vaginal 
probe with needle attachment at 
3, 6, 12, and 24 hours after GnRH 
(n=average of 3). Messenger RNA was 
extracted as described previously and 
expression of VEGF mRNA isoforms 
120 and 164 were analyzed using 
quantitative real time PCR. Data were 
analyzed using an ANOVA with SAS. 
Comparisons of means were tested us-
ing a Tukey-Kramer test.
V
E
G
F 
Is
of
or
m
s/
G
ap
D
H
2
1.5
1
0.5
 12 18 24 30 36 48 54 60 66 72
Hours after PG
VEGF 120
VEGF 164b,c
a
a,b b
b,c
a
b,c
a
b,c
a
b,c
a
c
a c
a
c a c
a
Figure 1. VEGF mRNA isoforms expression of 120 and 164 at different time points after second 
injection of PG. Different letters within each isoform at collection time points denote 
differences of P < 0.01.
© 2005, The Board of Regents of the University of Nebraska.  All rights reserved. 2006 Nebraska Beef Report  — Page 19 
also was an increase of VEGF 120 (P < 
0.01) compared to time points 54, 60, 
66, and 72 hour, but not compared to 
all other time points (Figure 1). 
In the 12 cows bled to determine 
timing of the LH surge, 50% of the 
cows had an LH surge between 54 and 
72 hours (Figure 2). In the other half 
of the cows, the LH surge did not oc-
cur within the 72-hour time period 
and presumably had occurred after 72 
hours (Figure 3). Due to this variation 
in occurrence of the LH surge, data 
collected after the 54-hour time point 
was deemed not representative of all 
cows in the study. Luteinizing hor-
mone has been shown to affect VEGF 
mRNA expression, therefore cows 
that had LH surges would have differ-
ent VEGF mRNA expression profiles 
when compared to cows that did not 
have an LH surge at the 54 to 72 hour 
period. Thus a second trial was con-
ducted to more accurately synchronize 
the LH surge by an injection with 
GnRH to determine how the LH surge 
would affect VEGF mRNA isoforms. 
In trial 2, an increase in VEGF 
120 mRNA expression (P < 0.05) was 
detected at 3 hours post GnRH (Fig-
ure 4). VEGF 120 is responsible for 
recruiting endothelial cells to develop 
initial blood vessels. The increase 
in VEGF 120 mRNA at 3 hours may 
represent recruitment of endothelial 
cells to develop blood vessels in the 
developing corpus luteum. There was 
no difference in VEGF 164 at any of 
the collections after GnRH. Thus, it 
appears that VEGF 164 and 120 iso-
forms maybe independently regulated 
at corpus luteum regression (trial #1) 
and after the LH surge (trial #2). De-
termining the role of VEGF isoforms 
during the preovulatory period may 
allow for better 1) synchronization of 
ovulation for timed-AI; and 2) corpus 
luteum formation which may reduce 
embryonic mortality in beef cattle.
1Robin A. Ten Broeck, graduate student; 
Debra T. Clopton, research technician; Jeremy 
L. Martin, graduate student; Rebecca C. Bott, 
graduate student; Karl V. Moline, cow/calf man-
ager; Jeff W. Bergman, agriculture technician; 
Andrea S. Cupp, assistant professor, Animal 
Science, Lincoln.
LH
 (
n
g/
m
L)
160
140
120
100
80
60
40
20
0
14 4308
5098 7063
7117 9066
 0 8 16 24 32 40 48 56 64 72
Time (hour)
Figure 2. A subset of 12 cows were bled every two hours for 72 hours to determine timing of LH 
surge. This graph represents cows that displayed an LH surge within 72 hours.
Figure 3. A subset of 12 cows were bled every two hours for 72 hours to determine timing of LH 
surge. This graph represents cows that did not display an LH surge within 72 hours.
160
140
120
100
80
60
40
20
0
LH
 (
n
g/
m
L)
307 6031
8052 8196
9012 9208
 0 6 12 18 24 30 36 42 48 54 60 66 72
Time (hour)
Figure 4. VEGF mRNA isoforms expression of 120 and 164 after injection of GnRH. Different letters 
within each isoform at collection time points denote differences of P < 0.05.
V
E
G
F 
Is
of
or
m
s/
G
ap
D
H
VEGF 120
VEGF 164
 3 6 12 24
Hour after GnRH
b
a
a
a
a
a
a
0.60
0.50
0.40
0.30
0.20
0.10
0.00
a
Results
In the first trial, the greatest con-
centration of VEGF 164 mRNA was 
observed at 18 hours post second in-
jection of PGF
2α� (P < 0.01; Figure 1). 
VEGF 164 is the predominant VEGF 
isoform responsible for the forma-
tion of large blood vessels. A peak in 
164 isoform at 18 hour post-PGF2α 
may contribute to the development of 
vasculature in the theca layer of the 
largest follicle on the ovary to estab-
lish its dominance. At 18 hours there 


Vascular Endothelial Growth Factor mRNA Isoforms 120 and 164 are Differentially Regulated Prior to Ovulation

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Nebraska Beef Cattle Reports Animal Science Department January 2006 Vascular Endothelial Growth Factor mRNA Isoforms 120 and 164 are Differentially 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 Rebecca C. Bott University of Nebraska-Lincoln Karl V. Moline University of Nebraska-Lincoln, kmoline1@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.; Bott, Rebecca C.; Moline, Karl V.; Bergman, Jeffrey W.; and Cupp, Andrea S., "Vascular Endothelial Growth Factor mRNA Isoforms 120 and 164 are Differentially Regulated Prior to Ovulation" (2006). Nebraska Beef Cattle Reports. 108. https://digitalcommons.unl.edu/animalscinbcr/108 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, Rebecca C. Bott, 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/108 Page 18 — 2006 Nebraska Beef Report  © 2005, The Board of Regents of the University of Nebraska.  All rights reserved.Vascular Endothelial Growth Factor mRNA Isoforms 120 and 164 are Differentially Regulated Prior to OvulationRobin A. Ten BroeckDebra T. CloptonJeremy L. MartinRebecca C. BottKarl V. MolineJeff W. BergmanAndrea S. Cupp1SummaryVascular Endothelial Growth Factor (VEGF) is produced by cells surround-ing the egg in the follicle prior to ovula-tion. If VEGF is inhibited, ovulation does not occur. The VEGF gene can be spliced to produce different protein iso-forms which have specific functions. Our objective was to determine if VEGF 120 and 164 mRNA isoforms are differen-tially regulated in the preovulatory fol-licle. From our studies, VEGF isoforms are differentially regulated during both CL regression and after a simulated LH surge. Differences observed in VEGF iso-form regulation may allow for manipu-lation of ovulation in the beef cow. IntroductionFollicular development within the bovine ovary is a dynamic process. It begins prior to birth and continues throughout the cow =s reproductive lifespan. Angiogenesis, the formation of new blood vessels, is crucial in the ovulatory follicle. The blood vessels supply the follicle with necessary nu-trients for development and growth prior to ovulation. VEGF is expressed by granulosa cells (cells surrounding the egg) prior to ovulation in the bo-vine follicle and is an important factor in the regulation of normal angiogen-esis in the developing follicle and cor-pus luteum. Several VEGF isoforms exist, the most common isoforms are VEGF 164 and VEGF 120. In other tis-sues such as the heart and lung VEGF isoforms have specific functions in vascular development. VEGF 120 is highly diffusible and recruits endo-thelial cells (precursor to blood vessel cells) in the establishment of blood vessels. VEGF 164 recruits endothelial cells that form the large blood ves-sels. However, it is not known how these isoforms function in the ovarian preovulatory follicle nor how inhibi-tion of VEGF might alter ovulation. The objective of the current study is to determine if the two major VEGF mRNA isoforms, 120 and 164, are differentially regulated prior to ovu-lation in the granulosa cells of the dominant follicle.ProcedureTrial 1Two injections of PGF2α (5mg/cow) were administered 14 days apart. PGF2α induces regression of the cor-pus luteum initiating the LH surge and ovulation. A vaginal probe with needle attachment was used to collect follicular fluid and granulosa cells from the dominant follicle. Follicular aspirates were collected at 12, 18, 24, 30, 36, 48, 54, 60, 66, and 72 hours (n=average of 8) after the second injection of PGF2α. Messenger RNA was extracted from granulosa cells and samples were reversed transcribed to cDNA. Progesterone and estrogen concentrations were measured in fol-licular fluid. A ratio greater than 1 of estrogen to progesterone indicated that the follicle was still dominant. The mRNA expression of VEGF iso-forms 120 and 164 were determined using real-time quantitative poly-merase chain reaction (PCR) at each follicular aspirate time point. Data were analyzed using an ANOVA with SAS. Comparisons of means were analyzed using a Tukey-Kramer test. In 12 animals, blood samples were collected after the second injection of PGF2α at two-hour intervals from 0 to 72 hours to determine when the LH surge occurred. The LH surge was detected in six of the 12 cows and occurred between 54 and 72 hours. Due to this variation, a second trial was conducted to obtain follicle aspi-rates after a simulated surge of LH by injecting GnRH (0.1mg/cow).Trial 2Two injections of PGF2α (5mg/cow) were administered 14 days apart with an injection of GnRH (0.1mg/cow) administered 48 hours after the sec-ond injection of PGF2α. Follicular aspirates were collected via vaginal probe with needle attachment at 3, 6, 12, and 24 hours after GnRH (n=average of 3). Messenger RNA was extracted as described previously and expression of VEGF mRNA isoforms 120 and 164 were analyzed using quantitative real time PCR. Data were analyzed using an ANOVA with SAS. Comparisons of means were tested us-ing a Tukey-Kramer test.VEGF Isoforms/GapDH21.510.5 12 18 24 30 36 48 54 60 66 72Hours after PGVEGF 120VEGF 164b,caa,b bb,cab,cab,cab,caca cac a caFigure 1. VEGF mRNA isoforms expression of 120 and 164 at different time points after second injection of PG. Different letters within each isoform at collection time points denote differences of P < 0.01.© 2005, The Board of Regents of the University of Nebraska.  All rights reserved. 2006 Nebraska Beef Report  — Page 19 also was an increase of VEGF 120 (P < 0.01) compared to time points 54, 60, 66, and 72 hour, but not compared to all other time points (Figure 1). In the 12 cows bled to determine timing of the LH surge, 50% of the cows had an LH surge between 54 and 72 hours (Figure 2). In the other half of the cows, the LH surge did not oc-cur within the 72-hour time period and presumably had occurred after 72 hours (Figure 3). Due to this variation in occurrence of the LH surge, data collected after the 54-hour time point was deemed not representative of all cows in the study. Luteinizing hor-mone has been shown to affect VEGF mRNA expression, therefore cows that had LH surges would have differ-ent VEGF mRNA expression profiles when compared to cows that did not have an LH surge at the 54 to 72 hour period. Thus a second trial was con-ducted to more accurately synchronize the LH surge by an injection with GnRH to determine how the LH surge would affect VEGF mRNA isoforms. In trial 2, an increase in VEGF 120 mRNA expression (P < 0.05) was detected at 3 hours post GnRH (Fig-ure 4). VEGF 120 is responsible for recruiting endothelial cells to develop initial blood vessels. The increase in VEGF 120 mRNA at 3 hours may represent recruitment of endothelial cells to develop blood vessels in the developing corpus luteum. There was no difference in VEGF 164 at any of the collections after GnRH. Thus, it appears that VEGF 164 and 120 iso-forms maybe independently regulated at corpus luteum regression (trial #1) and after the LH surge (trial #2). De-termining the role of VEGF isoforms during the preovulatory period may allow for better 1) synchronization of ovulation for timed-AI; and 2) corpus luteum formation which may reduce embryonic mortality in beef cattle.1Robin A. Ten Broeck, graduate student; Debra T. Clopton, research technician; Jeremy L. Martin, graduate student; Rebecca C. Bott, graduate student; Karl V. Moline, cow/calf man-ager; Jeff W. Bergman, agriculture technician; Andrea S. Cupp, assistant professor, Animal Science, Lincoln.LH (ng/mL)16014012010080604020014 43085098 70637117 9066 0 8 16 24 32 40 48 56 64 72Time (hour)Figure 2. A subset of 12 cows were bled every two hours for 72 hours to determine timing of LH surge. This graph represents cows that displayed an LH surge within 72 hours.Figure 3. A subset of 12 cows were bled every two hours for 72 hours to determine timing of LH surge. This graph represents cows that did not display an LH surge within 72 hours.160140120100806040200LH (ng/mL)307 60318052 81969012 9208 0 6 12 18 24 30 36 42 48 54 60 66 72Time (hour)Figure 4. VEGF mRNA isoforms expression of 120 and 164 after injection of GnRH. Different letters within each isoform at collection time points denote differences of P < 0.05.VEGF Isoforms/GapDHVEGF 120VEGF 164 3 6 12 24Hour after GnRHbaaaaaa0.600.500.400.300.200.100.00aResultsIn the first trial, the greatest con-centration of VEGF 164 mRNA was observed at 18 hours post second in-jection of PGF2α� (P < 0.01; Figure 1). VEGF 164 is the predominant VEGF isoform responsible for the forma-tion of large blood vessels. A peak in 164 isoform at 18 hour post-PGF2α may contribute to the development of vasculature in the theca layer of the largest follicle on the ovary to estab-lish its dominance. At 18 hours there 

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Vascular Endothelial Growth Factor mRNA Isoforms 120 and 164 are Differentially Regulated Prior to Ovulation

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