Within the UNL physiology herd, a group of cows have been identified with excess androgen (androstenedione, A4) in their dominant follicle (30 fold higher than controls) and a 17% reduction in calving rate, suggesting subfertility. Th e objective was to identify altered granulosa cell gene expression that could be preventing these cells from converting excess androgen into estrogen. Microarray analysis suggests these granulosa cells experience inhibited proliferation resulting in a reduced total population of cells. Improved understanding of the causes of this phenotype may provide beef producers with tools to identify potentially subfertile cattle and improve reproductive efficiency

Cupp, Andrea S.

Cushman, Robert A.

Davis, John S.

Kurz, Scott G.

McFee, Renee M.

McNeel, Anthony K.

Pohlmeier, William E.

Romereim, Sarah

Spuri- Gomes, Renata

Summers, Adam F., Summers

Wood, Jennifer R.

English

UNL | Libraries

University of Nebraska - Lincoln
DigitalCommons@University of Nebraska - Lincoln
Nebraska Beef Cattle Reports Animal Science Department
2016
Granulosa Cell Exposure to Excess Androgens
Inhibits Their Ability to Proliferate in the Cow
Which May Cause or Perpetuate Androgen Excess
Andrea S. Cupp
University of Nebraska-Lincoln, acupp2@unl.edu
Sarah Romereim
University of Nebraska-Lincoln, sromereim2@unl.edu
Adam F. Summers Summers
University of Nebraska-Lincoln
William E. Pohlmeier
University of Nebraska-Lincoln, bill.pohlmeier@unl.edu
Renee M. McFee
University of Nebraska-Lincoln, rmcfee3@unl.edu
See next page for additional authors
Follow this and additional works at: https://digitalcommons.unl.edu/animalscinbcr
Part of the Meat Science Commons
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.
Cupp, Andrea S.; Romereim, Sarah; Summers, Adam F. Summers; Pohlmeier, William E.; McFee, Renee M.; Spuri- Gomes, Renata;
Kurz, Scott G.; McNeel, Anthony K.; Cushman, Robert A.; Davis, John S.; and Wood, Jennifer R., "Granulosa Cell Exposure to Excess
Androgens Inhibits Their Ability to Proliferate in the Cow Which May Cause or Perpetuate Androgen Excess" (2016). Nebraska Beef
Cattle Reports. 873.
https://digitalcommons.unl.edu/animalscinbcr/873
Authors
Andrea S. Cupp, Sarah Romereim, Adam F. Summers Summers, William E. Pohlmeier, Renee M. McFee,
Renata Spuri- Gomes, Scott G. Kurz, Anthony K. McNeel, Robert A. Cushman, John S. Davis, and Jennifer R.
Wood
This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/animalscinbcr/873
2016 Nebraska Beef Cattle Report · 25 
©  Th e Board Regents of the University of 
Nebraska. All rights reserved.
al, ovaries were measured and dominant 
follicles collected. Follicular fl uid was 
aspirated from these follicles, and granu-
losa cells were removed via microdissec-
tion and messenger RNA was extracted. 
Messenger RNA was sent to University of 
Nebraska Medical Center to their microar-
ray core and placed on Aff ymetrix chips to 
determine diff erences in genes expressed in 
High A4 classifi ed cows (n = 5; excess A4 
where A4 greater than 40 ng/ml in follic-
ular fl uid) vs. Low A4 classifi ed cows (n- 4; 
control; A4 less than 40 ng/ml in follicular 
fl uid of dominant follicle).
Results
Statistics were performed (Analysis of 
Variance) and genes increased or decreased 
in granulosa cells from High A4 cows vs. 
Low A4 cows were selected based on sta-
tistical criteria (P < 0.005, False Discovery 
Rate < 0.05, fold- change > 1.5 or < - 1.5). 
Th ese criteria ensure the diff erences in 
expression of selected genes are not due to 
random variation between measurements 
or sampling error. Th e messenger RNAs 
for 166 genes were decreased and 90 genes 
were increased in granulosa cells from 
High A4 cows compared with Low A4.
To determine the biological relevance 
of these diff erences in gene expression, a 
soft ware package called Ingenuity Pathway 
Analysis was used to categorize the genes 
and how they may aff ect normal cellular 
functions. Overwhelmingly, the most 
inhibited functions in High A4 granulo-
sa cells involve cell cycle regulation. Th e 
analysis indicated granulosa cells from the 
High A4 cows experienced inhibition of 
proliferation. Th e expected decrease in to-
tal numbers of granulosa cells may explain 
why the follicle as a whole is not effi  ciently 
converting androgens to estrogens.
Th e major categories of genes with 
decreased expression included cell cycle, 
cell proliferation, and cellular growth and 
fertility in cattle. Ovarian steroidogenesis, 
the process of creating steroid hormones 
within the ovary, occurs within the theca 
and granulosa cells of the follicle. Th ese cell 
layers surround the oocyte; therefore, in-
creased concentrations of steroid hormones 
likely aff ect oocyte quality (2014 Nebraska 
Beef Cattle Report, pp. 11– 13), impacting 
fertility rates. Site specifi c enzymes within 
the granulosa cells are responsible for the 
conversion of androstenedione to estrogen, 
thus gene expression patterns within the 
granulosa cells could aff ect these enzymes 
or the genes responsible for producing 
these enzymes resulting in altered steroid 
hormone concentrations, creating an 
adverse environment for the developing 
follicle and oocyte. Th e objective was to 
determine if granulosa cell gene expression 
or function was altered in cows classifi ed as 
High A4 compared with Low A4 cows and 
assess how these changes impact fertility.
Procedure
Estrus was synchronized in cows 
utilizing a Co- Synch + CIDR protocol for 
timed AI, with ovariectomy performed 
aft er. Cows received a single injection (100 
μg/cow, i.m.) of GnRH (Cystorelin, Merial 
Limited, Duluth, GA) on treatment d 0 to 
induce ovulation and thus, initiate a new 
follicular wave. Also on d 0, an intravaginal 
insert (controlled internal drug release 
device [CIDR], Zoetis, Florham Park, NJ) 
containing 1.38 g of progesterone (P4) 
was inserted. Approximately 84 h prior to 
ovariectomy, cows were transported to the 
University of Nebraska- Lincoln Animal 
Science building for holding and surgery. 
Th e CIDR was removed on day 7 and 
cows received a single injection (25 mg/
cow; i.m.) of prostaglandin F2α (PGF2α; 
ProstaMate, AgriLabs, St. Joseph, MO). 
Th irty- six h aft er CIDR removal and PGF2α 
administration, ovaries were removed via 
right fl ank laparotomy. Following remov-
Summary
Within the UNL physiology herd, a group 
of cows have been identifi ed with excess 
androgen (androstenedione, A4) in their 
dominant follicle (30 fold higher than con-
trols) and a 17% reduction in calving rate, 
suggesting subfertility. Th e objective was to 
identify altered granulosa cell gene expres-
sion that could be preventing these cells from 
converting excess androgen into estrogen. 
Microarray analysis suggests these granulosa 
cells experience inhibited proliferation re-
sulting in a reduced total population of cells. 
Improved understanding of the causes of this 
phenotype may provide beef producers with 
tools to identify potentially subfertile cattle 
and improve reproductive effi  ciency.
Introduction
Profi tability of a beef herd is linked to a 
heifer or cow’s ability to become pregnant 
within the fi rst 21 d of the breeding season, 
allowing her to maintain a 365- d calving 
interval and wean a marketable calf each yr 
(1988 Journal of Animal Science, Hohen-
boken, pp. 1885– 1891). Achieving this 
timing is largely dependent on cows ovu-
lating each estrous cycle, which is largely 
dependent on the ovarian environment. In 
the UNL Physiology herd a group of cows 
with increased androgen production have 
been identifi ed (2012 Nebraska Beef Cattle 
Report, pp. 28– 29). Th is population of 
cattle produces calves with greater weaning 
weights (26 lb heavier) but tends to have 
reduced pregnancy rates (17% lower) com-
pared with cows with low androstenedione 
(A4) concentrations.
Most steroid hormone production 
(steroidogenesis) occurs in the gonad. In-
creased follicular androgens are associated 
with decreased ovulation effi  ciency and 
 Granulosa Cell Exposure to Excess Androgens 
Inhibits Th eir Ability to Proliferate in the Cow Which 
May Cause or Perpetuate Androgen Excess
Andrea S. Cupp, Sarah Romereim, Adam F. Summers, William E. Pohlmeier, Renee M. McFee, 
Renata Spuri- Gomes, Scott G. Kurz, Anthony K. McNeel, Robert A. Cushman, John S. Davis and Jennifer R. Wood
26 · 2016 Nebraska Beef Cattle Report
proliferation (Table 1). A wide variety of 
growth factor gene potential networks were 
down regulated in granulosa cells from 
the High A4 cows including Epidermal 
Growth Factor (EGF), Platelet Derived 
Growth Factor BB (PDGF BB), Leukocyte 
Inhibitory Factor (LIF), Vascular Endo-
thelial Growth Factor A (VEGFA), and 
Hepatocyte Growth Factor (HGF). A major 
function of these factors is to stimulate 
growth and proliferation by regulating the 
cell cycle and promoting increases in cell 
size and number.
Understanding what is diff erent about 
the granulosa cells from High A4 cows 
compared with Low A4 cows might allow 
us to develop techniques to enhance the 
fertility of aff ected cattle. Th is could be 
accomplished by treatments to increase 
granulosa cell proliferation survival to 
ensure conversion of A4 to estrogen thus 
preventing androgen excess. Better estrous 
synchronization techniques to ensure ovu-
lation occurs in the aff ected females may 
also be developed.
Andrea S. Cupp, professor Animal Science, 
Lincoln
Sarah Romereim, postdoctoral research 
associate
Adam F. Summers, former postdoctoral 
research associate
William E. Pohlmeier, research technician
Renee M. McFee, graduate student
Renata Spuri- Gomes, graduate student
Scott G. Kurz, research technician
Anthony K. McNeel, former US Meat Ani-
mal Research Center postdoctoral research 
associate
Robert A. Cushman, research physiologist 
U.S. Meat Animal Research Center, Clay 
Center, NE
John S. Davis, Professor, Department of 
OBGYN, University of Nebraska Medical 
Center, Omaha, NE
Jennifer R. Wood, associate professor Ani-
mal Science, Lincoln
Table 1.  Categories of genes that are either increased or decreased in 
granulosa cells from High A4 vs. Low A4 cows
Categories Diseases or functions 
annotation
P- value Predicted 
activation state
Number 
of genes
Cancer Incidence of malignant 
tumor
2.50E−04 Increased 9
Cancer Incidence of tumor 3.63E−04 Increased 12
Cell cycle Ploidy 6.01E−05 Increased 9
Embryonic 
development, 
organismal survival
Death of embryo 3.04E−05 Increased 9
Organismal survival Organismal death 2.43E−05 Increased 54
Cancer Cell transformation 4.68E−04 Decreased 15
Cancer Transformation of 
fi broblasts
9.56E−03 Decreased 5
Cell cycle M phase 1.14E−16 Decreased 24
Cell cycle Cell cycle progression 2.78E−18 Decreased 52
Cell cycle M phase of tumor cell lines 1.12E−15 Decreased 16
Cell cycle Mitosis 2.13E−19 Decreased 37
Cell cycle Interphase 3.23E−07 Decreased 25
Cell cycle Entry into mitosis 1.48E−05 Decreased 5
Cell cycle M phase of cervical cancer 
cell lines
1.99E−12 Decreased 12
Cell cycle, cellular 
movement
Cytokinesis 1.18E−10 Decreased 16
Cell cycle, cellular 
movement
Cytokinesis of tumor cell 
lines
1.33E−11 Decreased 11
Cell death and 
survival
Cell survival 2.67E−03 Decreased 30
Cell death and 
survival
Cell viability 3.15E−03 Decreased 28
Cell death and 
survival
Cell viability of tumor cell 
lines
8.80E−04 Decreased 20
Cell death and 
survival
Cell viability of myeloma 
cell lines
9.12E−03 Decreased 4
Cellular assembly 
and organization
Organization of 
cytoskeleton
6.23E−04 Decreased 32
Cellular assembly 
and organization
Organization of cytoplasm 1.41E−03 Decreased 33
Cellular assembly 
and organization
Microtubule dynamics 3.63E−04 Decreased 29
Cellular assembly 
and organization
Formation of microtubules 7.77E−04 Decreased 5
Cellular growth and 
proliferation
Proliferation of tumor cell 
lines
1.76E−08 Decreased 45
Cellular growth and 
proliferation
Proliferation of breast 
cancer cell lines
2.47E−05 Decreased 16
Cellular growth and 
proliferation
Proliferation of fi broblasts 2.28E−03 Decreased 11
Cellular growth and 
proliferation
Proliferation of cells 4.70E−05 Decreased 73
DNA replication, 
recombination, and 
repair
Alignment of chromosomes 5.49E−16 Decreased 11


Granulosa Cell Exposure to Excess Androgens
Inhibits Their Ability to Proliferate in the Cow Which
May Cause or Perpetuate Androgen Excess

DigitalCommons@University of Nebraska

University of Nebraska - LincolnDigitalCommons@University of Nebraska - LincolnNebraska Beef Cattle Reports Animal Science Department2016Granulosa Cell Exposure to Excess AndrogensInhibits Their Ability to Proliferate in the CowWhich May Cause or Perpetuate Androgen ExcessAndrea S. CuppUniversity of Nebraska-Lincoln, acupp2@unl.eduSarah RomereimUniversity of Nebraska-Lincoln, sromereim2@unl.eduAdam F. Summers SummersUniversity of Nebraska-LincolnWilliam E. PohlmeierUniversity of Nebraska-Lincoln, bill.pohlmeier@unl.eduRenee M. McFeeUniversity of Nebraska-Lincoln, rmcfee3@unl.eduSee next page for additional authorsFollow this and additional works at: https://digitalcommons.unl.edu/animalscinbcrPart of the Meat Science CommonsThis Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It hasbeen accepted for inclusion in Nebraska Beef Cattle Reports by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln.Cupp, Andrea S.; Romereim, Sarah; Summers, Adam F. Summers; Pohlmeier, William E.; McFee, Renee M.; Spuri- Gomes, Renata;Kurz, Scott G.; McNeel, Anthony K.; Cushman, Robert A.; Davis, John S.; and Wood, Jennifer R., "Granulosa Cell Exposure to ExcessAndrogens Inhibits Their Ability to Proliferate in the Cow Which May Cause or Perpetuate Androgen Excess" (2016). Nebraska BeefCattle Reports. 873.https://digitalcommons.unl.edu/animalscinbcr/873AuthorsAndrea S. Cupp, Sarah Romereim, Adam F. Summers Summers, William E. Pohlmeier, Renee M. McFee,Renata Spuri- Gomes, Scott G. Kurz, Anthony K. McNeel, Robert A. Cushman, John S. Davis, and Jennifer R.WoodThis article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/animalscinbcr/8732016 Nebraska Beef Cattle Report · 25 ©  Th e Board Regents of the University of Nebraska. All rights reserved.al, ovaries were measured and dominant follicles collected. Follicular fl uid was aspirated from these follicles, and granu-losa cells were removed via microdissec-tion and messenger RNA was extracted. Messenger RNA was sent to University of Nebraska Medical Center to their microar-ray core and placed on Aff ymetrix chips to determine diff erences in genes expressed in High A4 classifi ed cows (n = 5; excess A4 where A4 greater than 40 ng/ml in follic-ular fl uid) vs. Low A4 classifi ed cows (n- 4; control; A4 less than 40 ng/ml in follicular fl uid of dominant follicle).ResultsStatistics were performed (Analysis of Variance) and genes increased or decreased in granulosa cells from High A4 cows vs. Low A4 cows were selected based on sta-tistical criteria (P < 0.005, False Discovery Rate < 0.05, fold- change > 1.5 or < - 1.5). Th ese criteria ensure the diff erences in expression of selected genes are not due to random variation between measurements or sampling error. Th e messenger RNAs for 166 genes were decreased and 90 genes were increased in granulosa cells from High A4 cows compared with Low A4.To determine the biological relevance of these diff erences in gene expression, a soft ware package called Ingenuity Pathway Analysis was used to categorize the genes and how they may aff ect normal cellular functions. Overwhelmingly, the most inhibited functions in High A4 granulo-sa cells involve cell cycle regulation. Th e analysis indicated granulosa cells from the High A4 cows experienced inhibition of proliferation. Th e expected decrease in to-tal numbers of granulosa cells may explain why the follicle as a whole is not effi  ciently converting androgens to estrogens.Th e major categories of genes with decreased expression included cell cycle, cell proliferation, and cellular growth and fertility in cattle. Ovarian steroidogenesis, the process of creating steroid hormones within the ovary, occurs within the theca and granulosa cells of the follicle. Th ese cell layers surround the oocyte; therefore, in-creased concentrations of steroid hormones likely aff ect oocyte quality (2014 Nebraska Beef Cattle Report, pp. 11– 13), impacting fertility rates. Site specifi c enzymes within the granulosa cells are responsible for the conversion of androstenedione to estrogen, thus gene expression patterns within the granulosa cells could aff ect these enzymes or the genes responsible for producing these enzymes resulting in altered steroid hormone concentrations, creating an adverse environment for the developing follicle and oocyte. Th e objective was to determine if granulosa cell gene expression or function was altered in cows classifi ed as High A4 compared with Low A4 cows and assess how these changes impact fertility.ProcedureEstrus was synchronized in cows utilizing a Co- Synch + CIDR protocol for timed AI, with ovariectomy performed aft er. Cows received a single injection (100 μg/cow, i.m.) of GnRH (Cystorelin, Merial Limited, Duluth, GA) on treatment d 0 to induce ovulation and thus, initiate a new follicular wave. Also on d 0, an intravaginal insert (controlled internal drug release device [CIDR], Zoetis, Florham Park, NJ) containing 1.38 g of progesterone (P4) was inserted. Approximately 84 h prior to ovariectomy, cows were transported to the University of Nebraska- Lincoln Animal Science building for holding and surgery. Th e CIDR was removed on day 7 and cows received a single injection (25 mg/cow; i.m.) of prostaglandin F2α (PGF2α; ProstaMate, AgriLabs, St. Joseph, MO). Th irty- six h aft er CIDR removal and PGF2α administration, ovaries were removed via right fl ank laparotomy. Following remov-SummaryWithin the UNL physiology herd, a group of cows have been identifi ed with excess androgen (androstenedione, A4) in their dominant follicle (30 fold higher than con-trols) and a 17% reduction in calving rate, suggesting subfertility. Th e objective was to identify altered granulosa cell gene expres-sion that could be preventing these cells from converting excess androgen into estrogen. Microarray analysis suggests these granulosa cells experience inhibited proliferation re-sulting in a reduced total population of cells. Improved understanding of the causes of this phenotype may provide beef producers with tools to identify potentially subfertile cattle and improve reproductive effi  ciency.IntroductionProfi tability of a beef herd is linked to a heifer or cow’s ability to become pregnant within the fi rst 21 d of the breeding season, allowing her to maintain a 365- d calving interval and wean a marketable calf each yr (1988 Journal of Animal Science, Hohen-boken, pp. 1885– 1891). Achieving this timing is largely dependent on cows ovu-lating each estrous cycle, which is largely dependent on the ovarian environment. In the UNL Physiology herd a group of cows with increased androgen production have been identifi ed (2012 Nebraska Beef Cattle Report, pp. 28– 29). Th is population of cattle produces calves with greater weaning weights (26 lb heavier) but tends to have reduced pregnancy rates (17% lower) com-pared with cows with low androstenedione (A4) concentrations.Most steroid hormone production (steroidogenesis) occurs in the gonad. In-creased follicular androgens are associated with decreased ovulation effi  ciency and  Granulosa Cell Exposure to Excess Androgens Inhibits Th eir Ability to Proliferate in the Cow Which May Cause or Perpetuate Androgen ExcessAndrea S. Cupp, Sarah Romereim, Adam F. Summers, William E. Pohlmeier, Renee M. McFee, Renata Spuri- Gomes, Scott G. Kurz, Anthony K. McNeel, Robert A. Cushman, John S. Davis and Jennifer R. Wood26 · 2016 Nebraska Beef Cattle Reportproliferation (Table 1). A wide variety of growth factor gene potential networks were down regulated in granulosa cells from the High A4 cows including Epidermal Growth Factor (EGF), Platelet Derived Growth Factor BB (PDGF BB), Leukocyte Inhibitory Factor (LIF), Vascular Endo-thelial Growth Factor A (VEGFA), and Hepatocyte Growth Factor (HGF). A major function of these factors is to stimulate growth and proliferation by regulating the cell cycle and promoting increases in cell size and number.Understanding what is diff erent about the granulosa cells from High A4 cows compared with Low A4 cows might allow us to develop techniques to enhance the fertility of aff ected cattle. Th is could be accomplished by treatments to increase granulosa cell proliferation survival to ensure conversion of A4 to estrogen thus preventing androgen excess. Better estrous synchronization techniques to ensure ovu-lation occurs in the aff ected females may also be developed.Andrea S. Cupp, professor Animal Science, LincolnSarah Romereim, postdoctoral research associateAdam F. Summers, former postdoctoral research associateWilliam E. Pohlmeier, research technicianRenee M. McFee, graduate studentRenata Spuri- Gomes, graduate studentScott G. Kurz, research technicianAnthony K. McNeel, former US Meat Ani-mal Research Center postdoctoral research associateRobert A. Cushman, research physiologist U.S. Meat Animal Research Center, Clay Center, NEJohn S. Davis, Professor, Department of OBGYN, University of Nebraska Medical Center, Omaha, NEJennifer R. Wood, associate professor Ani-mal Science, LincolnTable 1.  Categories of genes that are either increased or decreased in granulosa cells from High A4 vs. Low A4 cowsCategories Diseases or functions annotationP- value Predicted activation stateNumber of genesCancer Incidence of malignant tumor2.50E−04 Increased 9Cancer Incidence of tumor 3.63E−04 Increased 12Cell cycle Ploidy 6.01E−05 Increased 9Embryonic development, organismal survivalDeath of embryo 3.04E−05 Increased 9Organismal survival Organismal death 2.43E−05 Increased 54Cancer Cell transformation 4.68E−04 Decreased 15Cancer Transformation of fi broblasts9.56E−03 Decreased 5Cell cycle M phase 1.14E−16 Decreased 24Cell cycle Cell cycle progression 2.78E−18 Decreased 52Cell cycle M phase of tumor cell lines 1.12E−15 Decreased 16Cell cycle Mitosis 2.13E−19 Decreased 37Cell cycle Interphase 3.23E−07 Decreased 25Cell cycle Entry into mitosis 1.48E−05 Decreased 5Cell cycle M phase of cervical cancer cell lines1.99E−12 Decreased 12Cell cycle, cellular movementCytokinesis 1.18E−10 Decreased 16Cell cycle, cellular movementCytokinesis of tumor cell lines1.33E−11 Decreased 11Cell death and survivalCell survival 2.67E−03 Decreased 30Cell death and survivalCell viability 3.15E−03 Decreased 28Cell death and survivalCell viability of tumor cell lines8.80E−04 Decreased 20Cell death and survivalCell viability of myeloma cell lines9.12E−03 Decreased 4Cellular assembly and organizationOrganization of cytoskeleton6.23E−04 Decreased 32Cellular assembly and organizationOrganization of cytoplasm 1.41E−03 Decreased 33Cellular assembly and organizationMicrotubule dynamics 3.63E−04 Decreased 29Cellular assembly and organizationFormation of microtubules 7.77E−04 Decreased 5Cellular growth and proliferationProliferation of tumor cell lines1.76E−08 Decreased 45Cellular growth and proliferationProliferation of breast cancer cell lines2.47E−05 Decreased 16Cellular growth and proliferationProliferation of fi broblasts 2.28E−03 Decreased 11Cellular growth and proliferationProliferation of cells 4.70E−05 Decreased 73DNA replication, recombination, and repairAlignment of chromosomes 5.49E−16 Decreased 11

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Granulosa Cell Exposure to Excess Androgens Inhibits Their Ability to Proliferate in the Cow Which May Cause or Perpetuate Androgen Excess

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