Heifers and cows that were culled from the herd due to failure to become pregnant were categorized into groups with low (\u3c 2 year), moderate (\u3e2 and \u3c 6 year) or high (≥ 6 year) fertility. Antral follicle counts were numerically lower in the low group and increased in the moderate- and high-fertility group. Granulosa cells from dominant follicles in moderate- and high-fertility cows had a greater ratio of Vascular Endothelial Growth Factor 164 (VEGF164) to VEGF164B compared to the low-fertility cows. Furthermore, there was more CARTPT in granulosa cells from subordinate follicles in moderate- and high-fertility cows than low. Gene expression is altered in granulosa cells from cows differing in fertility, suggesting these are candidate genes that may be used as markers to assist in determining reproductive longevity in beef cows

Beavers, Kevin A.

Brauer, Vanessa M.

Cupp, Andrea

Cushman, Robert A.

Kerl, Jill G.

Lu, Ningxia

McFee, Renee, Fee

Pohlmeier, William E

Sargent, Kevin M.

Slattery, Racheal G.

Smith, Jacqueline

Summers, Adam F.

Weber, Stetson P.

Wood, Jennifer R

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Nebraska Beef Cattle Reports Animal Science Department 2011 Granulosa Cell Gene Expression is Altered in Follicles from Cows with Differing Reproductive Longevity Andrea Cupp University of Nebraska-Lincoln, acupp2@unl.edu Jennifer R. Wood University of Nebraska-Lincoln, jwood5@unl.edu Renee McFee Fee University of Nebraska-Lincoln, rmcfee3@unl.edu Racheal G. Slattery University of Nebraska-Lincoln Kevin A. Beavers 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 Cupp, Andrea; Wood, Jennifer R.; McFee, Renee Fee; Slattery, Racheal G.; Beavers, Kevin A.; Pohlmeier, William E.; Sargent, Kevin M.; Lu, Ningxia; Smith, Jacqueline; Kerl, Jill G.; Brauer, Vanessa M.; Summers, Adam F.; Weber, Stetson P.; and Cushman, Robert A., "Granulosa Cell Gene Expression is Altered in Follicles from Cows with Differing Reproductive Longevity" (2011). Nebraska Beef Cattle Reports. 605. https://digitalcommons.unl.edu/animalscinbcr/605 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 Andrea Cupp, Jennifer R. Wood, Renee McFee Fee, Racheal G. Slattery, Kevin A. Beavers, William E. Pohlmeier, Kevin M. Sargent, Ningxia Lu, Jacqueline Smith, Jill G. Kerl, Vanessa M. Brauer, Adam F. Summers, Stetson P. Weber, and Robert A. Cushman This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/animalscinbcr/605 © The Board of Regents of the University of Nebraska.  All rights reserved. 2011 Nebraska Beef Cattle Report  — Page 13 Granulosa Cell Gene Expression is Altered in Folliclesfrom Cows with Differing Reproductive LongevityAndrea S. CuppJennifer R. WoodRenee McFeeRacheal G. SlatteryKevin A. BeaversWilliam E. PohlmeierKevin M. SargentNingxia LuJacqueline E. SmithJill G. KerlVanessa M. BrauerAdam F. SummersStetson P. WeberRobert A. Cushman1SummaryHeifers and cows that were culled from the herd due to failure to become pregnant were categorized into groups with low (< 2 year), moderate (>2 and < 6 year) or high (≥ 6 year) fertility. Antral follicle counts were numerically lower in the low group and increased in the moderate- and high-fertility group. Granulosa cells from dominant follicles in moderate- and high-fertility cows had a greater ratio of Vascular Endo thelial Growth Factor 164 (VEGF164) to VEGF164B compared to the low-fertility cows. Furthermore, there was more CARTPT in granulosa cells from sub-ordinate follicles in moderate- and high-fertility cows than low. Gene expression is altered in granulosa cells from cows differing in fertility, suggesting these are candidate genes that may be used as markers to assist in determining repro-ductive longevity in beef cows.IntroductionCows that stay in the herd longer and continue to produce a calf have greater reproductive longevity. While we may be able to predict reproductive longevity by a combination of number of antral follicles on the ovary, ovar-ian size, and reproductive tract score, there are no conclusive genetic or phenotypic markers of reproductive fertility in beef cattle. The long-term goal is to develop markers of repro-ductive longevity that may be imple-mented prior to selecting replacement heifers. A first step for this goal is to determine what genes are altered in granulosa cells (cells that communi-cate with and support egg maturation) to determine how ovarian follicle development (and development of the egg) may differ in cows culled from the herd at different ages due to preg-nancy failure.ProcedureAll procedures were approved by the University of Nebraska–Lincoln Institutional Animal Care and Use Committee (IACUC).Beef cows ranging in age from 1.5 to 11 years were synchronized with a modified Co-Synch proto-col, and upon CIDR removal were injected with Lutalyse, and ovaries were removed by flank laparotomy (surgical incision) 36 hours later to obtain dominant and subordinate follicles prior to ovulation. Ovaries were weighed, measured for length and width, and all visible surface fol-licles were counted. Granulosa cells were collected and extracted for RNA. Quantitative Polymerase Chain Reac-tion (QPCR) was conducted to deter-mine expression of genes known to influence follicle growth (VEGF164), follicle arrest (VEGF164B), and atresia (CARTPT). The expression of these genes was also correlated to follicle diameter (another potential biomark-er of fertility).ResultsThe number of antral follicles (follicles with fluid-filled cavities) present on the ovaries of cows from each fertility group is presented in Figure 1. While the numbers were numerically lower in the low-fertility group and increased to the high-fertility group, none of the groups were statistically different from each other. Figure 2 depicts the percent-age of low (< 30), medium (30-60), and high (> 60) antral follicle counts (AFC) on the ovaries of cows for each (Continued on next page) < 2-years > 2 < 6 years > 6-yearsFigure 1. Total number of antral follicles in different fertility groups. These values were not signifi-cantly different from each other.50403020100Total # of Antral Follicles(surface counts)Page 14 — 2011 Nebraska Beef Cattle Report  © The Board of Regents of the University of Nebraska.  All rights reserved.fertility classification. Because there are still low antral follicle count cows present in the high-fertility group, we cannot use antral follicle counts as the only method to predict reproductive longevity. Low antral follicle count appears to be only a risk factor for in-fertility, not a sole determining factor. Furthermore, because our analysis of antral follicle count is retrospective, and antral follicle count decreases as animals get older, the low antral follicle count cows in the older cow groups may be due to them getting closer to the end of their reproductive lifespan. Thus, we decided to evalu-ate expression of genes that have been shown to influence follicle develop-ment to determine if we could detect any differences that may result in less than optimal follicle development and egg maturation.Our laboratory has previously determined in rodents that treatment with VEGF164 stimulates follicle development, promoting early-stage follicles to later stages of develop-ment. Furthermore, treatment with VEGF165b inhibited follicle progres-sion and arrested follicles at early stages of development. Therefore, our objective was to determine if the ratio of VEGF164 angiogenic to VEGF164B anti-angiogenic isoforms in granu-losa cells from dominant follicles was greater in high-fertility cows and reduced in the low-fertility group. In Figure 3, the ratio of VEGF164 to VEGF164B is reduced in cows culled from the herd at less than or equal to 2 years of age when compared to either the moderate- or high-fertility group. Thus, the dominant follicles in the low-fertility group had less VEGF164 and more VEGF164B mes-senger RNA (mRNA) in their granu-losa cells. Furthermore, the diameter of the dominant follicles in the low-fertility group was numerically less but not significantly different (data not shown). This suggests that the dominant follicles in the < 2 years of age group are not growing optimally. This may affect egg maturation and development, resulting in less viable or fertile eggs. Figure 2. Percentage of cows that were categorized as high, medium or low antral follicle counts (AFC) in each fertility group.high moderate low100806040200AFC Classification(% of total) < 2-years > 2 < 6 years > 6-yearsFigure 3. Quantitative PCR for VEGF164/VEGF164B ratio in granulosa cells from dominant follicles in cows with different fertility. Different subscripts indicate significance at P < 0.05.Dominant Follicles2520151050VEGFA_164:VEGFA_164B/GAPDH(arbitrary units)abb < 2-years-old > 2 < 6-years-old > 6-years-old© The Board of Regents of the University of Nebraska.  All rights reserved. 2011 Nebraska Beef Cattle Report  — Page 15 expressed in subordinate follicles from the moderate- and high-fertility group than the low (Figure 4). This further supports our hypothesis that gene expression profiles are altered and may not be optimal in the low-fertility group, which possibly reflects why they failed to become pregnant. ImplicationsFinding genes that predict fertil-ity and reproductive longevity would allow for selection of heifers prior to their development to puberty and would increase profitability of cow/calf producers.1Andrea S. Cupp, associate professor, University of Nebraska–Lincoln Department of Animal Science; Jennifer R. Wood, assistant professor; Renee McFee, technician, Department of Animal Science; Racheal G. Slattery, graduate student, UNL Department of Veterinary and Biomedical Sciences; Kevin Beavers, technician, U.S. Meat Animal Research Center; William E. Pohlmeier, research lab manager; Kevin M. Sargent and Ningxia Lu, graduate students; Jacqueline E. Smith, research analyst; Jill G. Kerl, physiology lab manager; Vanessa M. Brauer, technician; Adam F. Summers and Stetson P. Weber, graduate students, UNL Department of Animal Science; Robert Cushman, physiologist, U.S. Meat Animal Research Center. < 2-years-old > 2 < 6-years-old > 6-years-oldFigure 4. Quantitative PCR for CARTPT in granulosa cells from subordinate follicles taken from cows with different fertility. Different subscripts indicate significance at P < 0.05.Subordinate Follicles201612840CARTPT/GAPDH(arbitrary units)abbInterestingly, subordinate follicle diameters among the fertility groups were different, with the cows in low fertility being the smallest, the mod-erate were greater than the low, and the high having the largest follicle diameter (data not shown). The dif-ferences in subordinate follicle growth may be directly related to factors be-ing produced by the dominant follicle. Also, when CARTPT, a gene expressed in subordinate and dominant fol-licles undergoing atresia, was mea-sured, there was dramatically more 

Granulosa Cell Gene Expression is Altered in Follicles from Cows with Differing Reproductive Longevity

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Nebraska Beef Cattle Reports Animal Science Department 
2011 
Granulosa Cell Gene Expression is Altered in Follicles from Cows 
with Differing Reproductive Longevity 
Andrea Cupp 
University of Nebraska-Lincoln, acupp2@unl.edu 
Jennifer R. Wood 
University of Nebraska-Lincoln, jwood5@unl.edu 
Renee McFee Fee 
University of Nebraska-Lincoln, rmcfee3@unl.edu 
Racheal G. Slattery 
University of Nebraska-Lincoln 
Kevin A. Beavers 
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 
Cupp, Andrea; Wood, Jennifer R.; McFee, Renee Fee; Slattery, Racheal G.; Beavers, Kevin A.; Pohlmeier, 
William E.; Sargent, Kevin M.; Lu, Ningxia; Smith, Jacqueline; Kerl, Jill G.; Brauer, Vanessa M.; Summers, 
Adam F.; Weber, Stetson P.; and Cushman, Robert A., "Granulosa Cell Gene Expression is Altered in 
Follicles from Cows with Differing Reproductive Longevity" (2011). Nebraska Beef Cattle Reports. 605. 
https://digitalcommons.unl.edu/animalscinbcr/605 
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 
Andrea Cupp, Jennifer R. Wood, Renee McFee Fee, Racheal G. Slattery, Kevin A. Beavers, William E. 
Pohlmeier, Kevin M. Sargent, Ningxia Lu, Jacqueline Smith, Jill G. Kerl, Vanessa M. Brauer, Adam F. 
Summers, Stetson P. Weber, and Robert A. Cushman 
This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/
animalscinbcr/605 
© The Board of Regents of the University of Nebraska.  All rights reserved. 2011 Nebraska Beef Cattle Report  — Page 13 
Granulosa Cell Gene Expression is Altered in Follicles
from Cows with Differing Reproductive Longevity
Andrea S. Cupp
Jennifer R. Wood
Renee McFee
Racheal G. Slattery
Kevin A. Beavers
William E. Pohlmeier
Kevin M. Sargent
Ningxia Lu
Jacqueline E. Smith
Jill G. Kerl
Vanessa M. Brauer
Adam F. Summers
Stetson P. Weber
Robert A. Cushman1
Summary
Heifers and cows that were culled 
from the herd due to failure to become 
pregnant were categorized into groups 
with low (< 2 year), moderate (>2 and 
< 6 year) or high (≥ 6 year) fertility. 
Antral follicle counts were numerically 
lower in the low group and increased in 
the moderate- and high-fertility group. 
Granulosa cells from dominant follicles 
in moderate- and high-fertility cows had 
a greater ratio of Vascular Endo thelial 
Growth Factor 164 (VEGF164) to 
VEGF164B compared to the low-fertility 
cows. Furthermore, there was more 
CARTPT in granulosa cells from sub-
ordinate follicles in moderate- and high-
fertility cows than low. Gene expression 
is altered in granulosa cells from cows 
differing in fertility, suggesting these are 
candidate genes that may be used as 
markers to assist in determining repro-
ductive longevity in beef cows.
Introduction
Cows that stay in the herd longer 
and continue to produce a calf have 
greater reproductive longevity. While 
we may be able to predict reproductive 
longevity by a combination of number 
of antral follicles on the ovary, ovar-
ian size, and reproductive tract score, 
there are no conclusive genetic or 
phenotypic markers of reproductive 
fertility in beef cattle. The long-term 
goal is to develop markers of repro-
ductive longevity that may be imple-
mented prior to selecting replacement 
heifers. A first step for this goal is to 
determine what genes are altered in 
granulosa cells (cells that communi-
cate with and support egg maturation) 
to determine how ovarian follicle 
development (and development of the 
egg) may differ in cows culled from 
the herd at different ages due to preg-
nancy failure.
Procedure
All procedures were approved by 
the University of Nebraska–Lincoln 
Institutional Animal Care and Use 
Committee (IACUC).
Beef cows ranging in age from 
1.5 to 11 years were synchronized 
with a modified Co-Synch proto-
col, and upon CIDR removal were 
injected with Lutalyse, and ovaries 
were removed by flank laparotomy 
(surgical incision) 36 hours later to 
obtain dominant and subordinate 
follicles prior to ovulation. Ovaries 
were weighed, measured for length 
and width, and all visible surface fol-
licles were counted. Granulosa cells 
were collected and extracted for RNA. 
Quantitative Polymerase Chain Reac-
tion (QPCR) was conducted to deter-
mine expression of genes known to 
influence follicle growth (VEGF164), 
follicle arrest (VEGF164B), and atresia 
(CARTPT). The expression of these 
genes was also correlated to follicle 
diameter (another potential biomark-
er of fertility).
Results
The number of antral follicles 
(follicles with fluid-filled cavities) 
present on the ovaries of cows from 
each fertility group is presented in 
Figure 1. While the numbers were 
numerically lower in the low-fertility 
group and increased to the high-
fertility group, none of the groups 
were statistically different from each 
other. Figure 2 depicts the percent-
age of low (< 30), medium (30-60), 
and high (> 60) antral follicle counts 
(AFC) on the ovaries of cows for each 
(Continued on next page)
 < 2-years > 2 < 6 years > 6-years
Figure 1. Total number of antral follicles in different fertility groups. These values were not signifi-
cantly different from each other.
50
40
30
20
10
0
To
ta
l #
 o
f A
n
tr
al
 F
ol
lic
le
s
(s
ur
fa
ce
 c
ou
n
ts
)
Page 14 — 2011 Nebraska Beef Cattle Report  © The Board of Regents of the University of Nebraska.  All rights reserved.
fertility classification. Because there 
are still low antral follicle count cows 
present in the high-fertility group, we 
cannot use antral follicle counts as the 
only method to predict reproductive 
longevity. Low antral follicle count 
appears to be only a risk factor for in-
fertility, not a sole determining factor. 
Furthermore, because our analysis of 
antral follicle count is retrospective, 
and antral follicle count decreases 
as animals get older, the low antral 
follicle count cows in the older cow 
groups may be due to them getting 
closer to the end of their reproductive 
lifespan. Thus, we decided to evalu-
ate expression of genes that have been 
shown to influence follicle develop-
ment to determine if we could detect 
any differences that may result in less 
than optimal follicle development and 
egg maturation.
Our laboratory has previously 
determined in rodents that treatment 
with VEGF164 stimulates follicle 
development, promoting early-stage 
follicles to later stages of develop-
ment. Furthermore, treatment with 
VEGF165b inhibited follicle progres-
sion and arrested follicles at early 
stages of development. Therefore, our 
objective was to determine if the ratio 
of VEGF164 angiogenic to VEGF164B 
anti-angiogenic isoforms in granu-
losa cells from dominant follicles 
was greater in high-fertility cows and 
reduced in the low-fertility group. 
In Figure 3, the ratio of VEGF164 to 
VEGF164B is reduced in cows culled 
from the herd at less than or equal 
to 2 years of age when compared to 
either the moderate- or high-fertility 
group. Thus, the dominant follicles 
in the low-fertility group had less 
VEGF164 and more VEGF164B mes-
senger RNA (mRNA) in their granu-
losa cells. Furthermore, the diameter 
of the dominant follicles in the low-
fertility group was numerically less 
but not significantly different (data 
not shown). This suggests that the 
dominant follicles in the < 2 years of 
age group are not growing optimally. 
This may affect egg maturation and 
development, resulting in less viable 
or fertile eggs. 
Figure 2. Percentage of cows that were categorized as high, medium or low antral follicle counts (AFC) 
in each fertility group.
high moderate low
100
80
60
40
20
0
A
FC
 C
la
ss
ifi
ca
ti
on
(%
 o
f 
to
ta
l)
 < 2-years > 2 < 6 years > 6-years
Figure 3. Quantitative PCR for VEGF164/VEGF164B ratio in granulosa cells from dominant follicles 
in cows with different fertility. Different subscripts indicate significance at P < 0.05.
Dominant Follicles
25
20
15
10
5
0
V
E
G
FA
_1
64
:V
E
G
FA
_1
64
B
/G
A
P
D
H
(a
rb
it
ra
ry
 u
n
it
s)
a
b
b
 < 2-years-old > 2 < 6-years-old > 6-years-old
© The Board of Regents of the University of Nebraska.  All rights reserved. 2011 Nebraska Beef Cattle Report  — Page 15 
expressed in subordinate follicles 
from the moderate- and high-fertility 
group than the low (Figure 4). This 
further supports our hypothesis that 
gene expression profiles are altered 
and may not be optimal in the low-
fertility group, which possibly reflects 
why they failed to become pregnant. 
Implications
Finding genes that predict fertil-
ity and reproductive longevity would 
allow for selection of heifers prior to 
their development to puberty and 
would increase profitability of cow/
calf producers.
1Andrea S. Cupp, associate professor, 
University of Nebraska–Lincoln Department 
of Animal Science; Jennifer R. Wood, assistant 
professor; Renee McFee, technician, Department 
of Animal Science; Racheal G. Slattery, graduate 
student, UNL Department of Veterinary and 
Biomedical Sciences; Kevin Beavers, technician, 
U.S. Meat Animal Research Center; William 
E. Pohlmeier, research lab manager; Kevin M. 
Sargent and Ningxia Lu, graduate students; 
Jacqueline E. Smith, research analyst; Jill G. Kerl, 
physiology lab manager; Vanessa M. Brauer, 
technician; Adam F. Summers and Stetson P. 
Weber, graduate students, UNL Department of 
Animal Science; Robert Cushman, physiologist, 
U.S. Meat Animal Research Center.
 < 2-years-old > 2 < 6-years-old > 6-years-old
Figure 4. Quantitative PCR for CARTPT in granulosa cells from subordinate follicles taken from 
cows with different fertility. Different subscripts indicate significance at P < 0.05.
Subordinate Follicles
20
16
12
8
4
0
C
A
R
T
P
T
/G
A
P
D
H
(a
rb
it
ra
ry
 u
n
it
s)
a
b
b
Interestingly, subordinate follicle 
diameters among the fertility groups 
were different, with the cows in low 
fertility being the smallest, the mod-
erate were greater than the low, and 
the high having the largest follicle 
diameter (data not shown). The dif-
ferences in subordinate follicle growth 
may be directly related to factors be-
ing produced by the dominant follicle. 
Also, when CARTPT, a gene expressed 
in subordinate and dominant fol-
licles undergoing atresia, was mea-
sured, there was dramatically more 


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Granulosa Cell Gene Expression is Altered in Follicles from Cows with Differing Reproductive Longevity

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