Granulosa cells express vascular endothelial growth factor A (VEGFA), and VEGFA mRNA levels increase as bovine follicles reach preovulatory status. To further evaluate the role of VEGFA isoforms in follicular development, cows were either synchronized with a modified Co- Synch protocol (CIDR) or treated with melengestrol acetate (MGA) with subsequent aspiration of the dominant follicles. Higher mRNA levels for the antiangiogenic isoform, VEGFA_164B, along with AMH and CARTPT in E2-inactive follicles suggest that these factors are markers for unhealthy, atretic follicles. In contrast, higher mRNA levels for the proangiogenic isoform, VEGFA_164, in E2-active follicles indicate that this isoform may help predict healthy ovulatory follicles

Artac, Robin A.

Brauer, Vanessa M.

Cupp, Andrea S.

Cushman, Robert A.

Kerl, Jill G.

McFee, Renee M.

Pohlmeier, William E., Pohlmeier

English

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Nebraska Beef Cattle Reports Animal Science Department 
2012 
Vascular Endothelial Growth Factor A (VEGFA) in Ovulatory 
Follicles 
Renee M. McFee 
University of Nebraska-Lincoln, rmcfee3@unl.edu 
Robin A. Artac 
University of Nebraska-Lincoln 
William E. Pohlmeier Pohlmeier 
University of Nebraska-Lincoln, wpohlmeier2@unl.edu 
Jill G. Kerl 
University of Nebraska-Lincoln 
Vanessa M. Brauer 
University of Nebraska-Lincoln 
See next page for additional authors 
Follow this and additional works at: https://digitalcommons.unl.edu/animalscinbcr 
 Part of the Animal Sciences Commons 
McFee, Renee M.; Artac, Robin A.; Pohlmeier, William E. Pohlmeier; Kerl, Jill G.; Brauer, Vanessa M.; 
Cushman, Robert A.; and Cupp, Andrea S., "Vascular Endothelial Growth Factor A (VEGFA) in Ovulatory 
Follicles" (2012). Nebraska Beef Cattle Reports. 654. 
https://digitalcommons.unl.edu/animalscinbcr/654 
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 
Renee M. McFee, Robin A. Artac, William E. Pohlmeier Pohlmeier, Jill G. Kerl, Vanessa M. Brauer, Robert A. 
Cushman, and Andrea S. Cupp 
This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/
animalscinbcr/654 
Page 26 — 2012 Nebraska Beef Cattle Report  © The Board of Regents of the University of Nebraska.  All rights reserved.
Vascular Endothelial Growth Factor A (VEGFA)  
in Ovulatory Follicles
Renee M. McFee
Robin A. Artac
William E. Pohlmeier
Jill G. Kerl
Vanessa M. Brauer
Robert A. Cushman
Andrea S. Cupp1
Summary
 Granulosa cells express vascular 
endothelial growth factor A (VEGFA), 
and VEGFA mRNA levels increase 
as bovine follicles reach preovulatory 
status. To further evaluate the role 
of VEGFA isoforms in follicular 
development, cows were either 
synchronized with a modified Co-
Synch protocol (CIDR) or treated with 
melengestrol acetate (MGA) with 
subsequent aspiration of the dominant 
follicles. Higher mRNA levels for the 
antiangiogenic isoform, VEGFA_164B, 
along with AMH and CARTPT in 
E2-inactive follicles suggest that these 
factors are markers for unhealthy, atretic 
follicles. In contrast, higher mRNA 
levels for the proangiogenic isoform, 
VEGFA_164, in E2-active follicles 
indicate that this isoform may help 
predict healthy ovulatory follicles.
Introduction
 
 Several factors are important 
for dominant follicle development. 
Granulosa cells express vascular 
endothelial growth factor A (VEGFA) 
and its receptors even though they 
are avascular follicular cells and 
VEGFA mRNA levels increase as 
bovine follicles reach preovulatory 
status. Follicular fluid anti-Müllerian 
hormone (AMH) levels decrease 
during antral follicle growth but 
then increase during early atresia of 
large follicles while granulosa cell 
expression of CART prepropeptide 
(CARTPT) is greater in estrogen 
(E2)-inactive follicles than E2-
active follicles. The current study 
evaluated granulosa cell expression 
of VEGFA_164 (proangiogenic) and 
VEGFA_164B (antiangiogenic) in 
dominant follicles in comparison with 
AMH and CARTPT expression.
Procedure
 All procedures were approved by 
the University of Nebraska–Lincoln 
Institutional Animal Care and Use 
Committee. Nonlactating beef cows 
that were 75% MARC III (1/4 Angus, 
1/4 Hereford, 1/4 Pinzgauer, 1/4 Red 
Poll) and 25% Red Angus/European 
Composite background crossbreds 
with an average age of 7.5 ± 2.7 years 
and weight of 1,200 lb were used in 
this study. Cows in the first treatment 
group (CIDR) were synchronized with 
the Co-Synch + CIDR timed artificial 
insemination (AI) protocol, except 
follicle aspiration was performed 
after synchronization rather than 
timed AI. The second group of cows 
was treated with melengestrol acetate 
(MGA) for 14 days and received 
three injections of ProstaMate (day 
0, 7, and 14) to eliminate any luteal 
tissue prior to follicle aspiration. 
Aspiration of dominant follicles was 
performed transvaginally with the use 
of caudal epidural anesthesia and an 
endovaginal ultrasound transducer 
with an attached needle guide. 
 Follicular fluid E2 and proges-
terone (P4) levels were measured to 
determine E2-activity for each follicle. 
Total RNA was extracted from granu-
losa cells for quantitative RT-PCR to 
evaluate mRNA abundance for VEG-
FA_164, VEGFA_164B, AMH, and 
CARTPT. The constitutively expressed 
gene, glyceraldehyde-3-phosphate 
dehydrogenase (GAPDH), was used as 
a control for RNA amplification. Data 
were analyzed by one-way ANOVA 
using JMP software, and differences 
were considered to be statistically sig-
nificant at P < 0.05 unless otherwise 
stated.
Results
 Using E2:P4 ratios, follicles were 
classified as E2-active (E2:P4 > 1; 
healthy) or E2-inactive (E2:P4 ≤ 1; 
atretic). In the CIDR group, mRNA 
levels for both AMH (P = 0.0015) and 
CARTPT (P = 0.0004) were greater 
in aspirated granulosa cells from 
E2-inactive follicles versus E2-active 
follicles (Figure 1C-D). Although 
VEGFA_164B mRNA levels were 
higher in E2-inactive follicles, and 
VEGFA_164 was more abundant in 
E2-active follicles, these differences 
were not significant (Figure 1A-B). For 
the MGA-treated cows, mRNA levels 
for VEGFA_164B (P < 0.0001), AMH 
(P = 0.007), and CARTPT (P = 0.0009) 
were more abundant in E2-inactive 
follicles compared to E2-active 
follicles (Figure 1B-D). In addition, 
mRNA levels for VEGFA_164  
(P = 0.02) were greater in E2-active 
follicles than E2-inactive follicles 
(Figure 1A). 
 Evaluation of E2-active follicles 
between CIDR and MGA-treated cows 
did not reveal differences in mRNA 
levels for VEGFA_164, VEGFA_164B, 
or AMH and although CARTPT  
(P = 0.11) levels were higher in follicles 
from MGA cows; this difference was 
not significant (Figure 1A-D). For 
E2-inactive follicles, mRNA levels 
for VEGFA_164 (P = 0.04) were more 
abundant in follicles from CIDR cows 
than MGA-treated cows (Figure 1A). 
Although not significant, mRNA 
levels for VEGFA_164B were higher 
in follicles from MGA-treated cows 
than CIDR cows (Figure 1B). No 
differences were seen in mRNA levels 
© The Board of Regents of the University of Nebraska.  All rights reserved. 2012 Nebraska Beef Cattle Report  — Page 27 
Figure 1.  Granulosa cell quantitative RT-PCR results for VEGFA_164 (A), VEGFA_164B (B), AMH (C), and CARTPT (D) for E2-active and E2-inactive 
dominant follicles from CIDR and MGA-treated cows. The mean ± SEM normalized values are presented. Different letters represent a statistically 
significant difference in means between E2-active and E2-inactive follicles for each treatment group (P < 0.05). Asterisks represent a statistically 
significant difference in means between CIDR and MGA-treated cows for each follicle type (P < 0.05). 
A Active Inactive
Active Inactive
Active Inactive
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 CIDR MGA
 CIDR MGA
 CIDR MGA
 CIDR MGA
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between the two treatment groups 
for either AMH or CARTPT (Figure 
1C-D). 
 Increased expression of AMH and 
CARTPT in E2-inactive follicles sup-
ports previous evidence that these fac-
tors are markers for unhealthy, atretic 
antral follicles. For VEGFA, higher 
levels of the antiangiogenic isoform 
(VEGFA_164B) were present in E2-in-
active follicles and higher levels of the 
proangiogenic isoform (VEGFA_164) 
were present in E2-active follicles. 
Furthermore, VEGFA_164 was more 
abundant in E2-inactive follicles from 
CIDR cows while VEGFA_164B was 
more abundant in E2-inactive follicles 
from MGA-treated cows. Treatment 
with MGA has been shown to pro-
mote the development of persistent 
dominant follicles and is associated 
with decreased ooctye viability. These 
data suggest that expression patterns 
for VEGFA isoforms may be used to 
predict the health status of dominant 
follicles. 
1Renee M. McFee, research technician; 
Robin A. Artac, former graduate student; 
William E. Pohlmeier, research technician; Jill 
G. Kerl, research technician; Vanessa M. Brauer, 
research technician; Robert A. Cushman, 
physiologist, University of Nebraska–Lincoln 
Meat Animal Research Center, Clay Center, Neb.; 
Andrea S. Cupp, professor, UNL Department of 
Animal Science, Lincoln, Neb.


Vascular Endothelial Growth Factor A (VEGFA)
in Ovulatory Follicles

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Nebraska Beef Cattle Reports Animal Science Department 2012 Vascular Endothelial Growth Factor A (VEGFA) in Ovulatory Follicles Renee M. McFee University of Nebraska-Lincoln, rmcfee3@unl.edu Robin A. Artac University of Nebraska-Lincoln William E. Pohlmeier Pohlmeier University of Nebraska-Lincoln, wpohlmeier2@unl.edu Jill G. Kerl University of Nebraska-Lincoln Vanessa M. Brauer University of Nebraska-Lincoln See next page for additional authors Follow this and additional works at: https://digitalcommons.unl.edu/animalscinbcr  Part of the Animal Sciences Commons McFee, Renee M.; Artac, Robin A.; Pohlmeier, William E. Pohlmeier; Kerl, Jill G.; Brauer, Vanessa M.; Cushman, Robert A.; and Cupp, Andrea S., "Vascular Endothelial Growth Factor A (VEGFA) in Ovulatory Follicles" (2012). Nebraska Beef Cattle Reports. 654. https://digitalcommons.unl.edu/animalscinbcr/654 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 Renee M. McFee, Robin A. Artac, William E. Pohlmeier Pohlmeier, Jill G. Kerl, Vanessa M. Brauer, Robert A. Cushman, and Andrea S. Cupp This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/animalscinbcr/654 Page 26 — 2012 Nebraska Beef Cattle Report  © The Board of Regents of the University of Nebraska.  All rights reserved.Vascular Endothelial Growth Factor A (VEGFA)  in Ovulatory FolliclesRenee M. McFeeRobin A. ArtacWilliam E. PohlmeierJill G. KerlVanessa M. BrauerRobert A. CushmanAndrea S. Cupp1Summary Granulosa cells express vascular endothelial growth factor A (VEGFA), and VEGFA mRNA levels increase as bovine follicles reach preovulatory status. To further evaluate the role of VEGFA isoforms in follicular development, cows were either synchronized with a modified Co-Synch protocol (CIDR) or treated with melengestrol acetate (MGA) with subsequent aspiration of the dominant follicles. Higher mRNA levels for the antiangiogenic isoform, VEGFA_164B, along with AMH and CARTPT in E2-inactive follicles suggest that these factors are markers for unhealthy, atretic follicles. In contrast, higher mRNA levels for the proangiogenic isoform, VEGFA_164, in E2-active follicles indicate that this isoform may help predict healthy ovulatory follicles.Introduction  Several factors are important for dominant follicle development. Granulosa cells express vascular endothelial growth factor A (VEGFA) and its receptors even though they are avascular follicular cells and VEGFA mRNA levels increase as bovine follicles reach preovulatory status. Follicular fluid anti-Müllerian hormone (AMH) levels decrease during antral follicle growth but then increase during early atresia of large follicles while granulosa cell expression of CART prepropeptide (CARTPT) is greater in estrogen (E2)-inactive follicles than E2-active follicles. The current study evaluated granulosa cell expression of VEGFA_164 (proangiogenic) and VEGFA_164B (antiangiogenic) in dominant follicles in comparison with AMH and CARTPT expression.Procedure All procedures were approved by the University of Nebraska–Lincoln Institutional Animal Care and Use Committee. Nonlactating beef cows that were 75% MARC III (1/4 Angus, 1/4 Hereford, 1/4 Pinzgauer, 1/4 Red Poll) and 25% Red Angus/European Composite background crossbreds with an average age of 7.5 ± 2.7 years and weight of 1,200 lb were used in this study. Cows in the first treatment group (CIDR) were synchronized with the Co-Synch + CIDR timed artificial insemination (AI) protocol, except follicle aspiration was performed after synchronization rather than timed AI. The second group of cows was treated with melengestrol acetate (MGA) for 14 days and received three injections of ProstaMate (day 0, 7, and 14) to eliminate any luteal tissue prior to follicle aspiration. Aspiration of dominant follicles was performed transvaginally with the use of caudal epidural anesthesia and an endovaginal ultrasound transducer with an attached needle guide.  Follicular fluid E2 and proges-terone (P4) levels were measured to determine E2-activity for each follicle. Total RNA was extracted from granu-losa cells for quantitative RT-PCR to evaluate mRNA abundance for VEG-FA_164, VEGFA_164B, AMH, and CARTPT. The constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as a control for RNA amplification. Data were analyzed by one-way ANOVA using JMP software, and differences were considered to be statistically sig-nificant at P < 0.05 unless otherwise stated.Results Using E2:P4 ratios, follicles were classified as E2-active (E2:P4 > 1; healthy) or E2-inactive (E2:P4 ≤ 1; atretic). In the CIDR group, mRNA levels for both AMH (P = 0.0015) and CARTPT (P = 0.0004) were greater in aspirated granulosa cells from E2-inactive follicles versus E2-active follicles (Figure 1C-D). Although VEGFA_164B mRNA levels were higher in E2-inactive follicles, and VEGFA_164 was more abundant in E2-active follicles, these differences were not significant (Figure 1A-B). For the MGA-treated cows, mRNA levels for VEGFA_164B (P < 0.0001), AMH (P = 0.007), and CARTPT (P = 0.0009) were more abundant in E2-inactive follicles compared to E2-active follicles (Figure 1B-D). In addition, mRNA levels for VEGFA_164  (P = 0.02) were greater in E2-active follicles than E2-inactive follicles (Figure 1A).  Evaluation of E2-active follicles between CIDR and MGA-treated cows did not reveal differences in mRNA levels for VEGFA_164, VEGFA_164B, or AMH and although CARTPT  (P = 0.11) levels were higher in follicles from MGA cows; this difference was not significant (Figure 1A-D). For E2-inactive follicles, mRNA levels for VEGFA_164 (P = 0.04) were more abundant in follicles from CIDR cows than MGA-treated cows (Figure 1A). Although not significant, mRNA levels for VEGFA_164B were higher in follicles from MGA-treated cows than CIDR cows (Figure 1B). No differences were seen in mRNA levels © The Board of Regents of the University of Nebraska.  All rights reserved. 2012 Nebraska Beef Cattle Report  — Page 27 Figure 1.  Granulosa cell quantitative RT-PCR results for VEGFA_164 (A), VEGFA_164B (B), AMH (C), and CARTPT (D) for E2-active and E2-inactive dominant follicles from CIDR and MGA-treated cows. The mean ± SEM normalized values are presented. Different letters represent a statistically significant difference in means between E2-active and E2-inactive follicles for each treatment group (P < 0.05). Asterisks represent a statistically significant difference in means between CIDR and MGA-treated cows for each follicle type (P < 0.05). A Active InactiveActive InactiveActive InactiveActive Inactive43210VEGFA_164/GAPDH(arbitrary units) CIDR MGA CIDR MGA CIDR MGA CIDR MGAB20151050VEGFA_164B/GAPDH(arbitrary units)Caaaaaaaaaabbbbbb*302520151050AMH/GAPDH(arbitrary units)D140120100806040200CARTPT/GAPDH(arbitrary units)between the two treatment groups for either AMH or CARTPT (Figure 1C-D).  Increased expression of AMH and CARTPT in E2-inactive follicles sup-ports previous evidence that these fac-tors are markers for unhealthy, atretic antral follicles. For VEGFA, higher levels of the antiangiogenic isoform (VEGFA_164B) were present in E2-in-active follicles and higher levels of the proangiogenic isoform (VEGFA_164) were present in E2-active follicles. Furthermore, VEGFA_164 was more abundant in E2-inactive follicles from CIDR cows while VEGFA_164B was more abundant in E2-inactive follicles from MGA-treated cows. Treatment with MGA has been shown to pro-mote the development of persistent dominant follicles and is associated with decreased ooctye viability. These data suggest that expression patterns for VEGFA isoforms may be used to predict the health status of dominant follicles. 1Renee M. McFee, research technician; Robin A. Artac, former graduate student; William E. Pohlmeier, research technician; Jill G. Kerl, research technician; Vanessa M. Brauer, research technician; Robert A. Cushman, physiologist, University of Nebraska–Lincoln Meat Animal Research Center, Clay Center, Neb.; Andrea S. Cupp, professor, UNL Department of Animal Science, Lincoln, Neb.

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Vascular Endothelial Growth Factor A (VEGFA) in Ovulatory Follicles

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