ABSTRACT: Oligonucleotide microarrays (GeneChip
Bovine Genome Arrays, Affymetrix Inc., Santa Clara,
CA) were used to evaluate gene expression profiles in
anterior pituitary glands collected from 4 anestrous and
4 cycling postpartum primiparous beef cows to provide
insight into genes associated with transition from an
anestrous to a cycling status. Tissues were collected
40 to 61 d after calving from anestrous cows and from
cyclic cows between d 7 and 13 of the estrous cycle
(luteal phase) from d 54 to 77 after calving. Hybridiza-
tion signals were normalized across arrays, and genes
with mean differences in expression that were at least
1.5-fold apart and with a minimum difference in mean
signal intensity of 10 were compared. Based on these
criteria, 47 transcripts were increased (P < 0.025) and
31 transcripts were decreased (P < 0.025) in pituitary
gland tissue from cycling compared with anestrous
cows. Few transcripts identified in this analysis were
associated previously with reproductive function. To
provide greater detail on the influence that stage of the
estrous cycle (i.e., collection during the luteal phase)
might have on the differences detected in gene expres-
sion, quantitative real-time PCR was used to compare
gene expression in anterior pituitaries of anestrous cows
with an additional independent set of anterior pituitary
glands collected at 4 different stages of the estrous cycle:
0.5 to 2 d (n = 9), 5 to 6.5 d (n = 5), 11.4 to 13.7 d (n
= 5), and 17.9 to 19 d (n = 6) after the onset of estrus.
Gastrin-releasing peptide, the gene that exhibited the
largest fold increase in expression in the microarray ex-
periment, and IGFBP3 mRNA were expressed at great-
er (P < 0.004) amounts in samples from the different
stages of the estrous cycle than in samples from ane-
strous cows. In addition, expression of IGFBP3 mRNA
was proportional to serum progesterone concentrations
throughout the estrous cycle (P < 0.05). Expression of
versican mRNA was decreased (P = 0.03) in samples
from the different stages of the estrous cycle compared
with anestrous cow samples. Results identified numer-
ous genes that may be involved in the transition from
anestrous to cycling status, providing novel insight into
mechanisms regulating reproductive function.
Key words: anterior pituitary gland, cattle, gene expression, postpartum anestrus
©2011 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2011. 89:1035–1041
A major limitation to successful reproduction is the
failure of cows to resume estrus after parturition. Mech-
anisms that control resumption of estrus after parturi-
tion have been the subject of study for decades (Short
et al., 1990). It is now well documented that prolonged
periods of postpartum anestrus are associated with in-
sufficient endocrine signal (GnRH) from the hypothala-
mus to the anterior pituitary gland (Dunn and Moss,
1992; Schillo, 1992; Wettemann et al., 2003). However,
a growing list of peptides and proteins have been shown
to act within the anterior pituitary gland to influence
gonadotropin secretion (Evans, 1999; Schwartz, 2000).
The complex nature of the potentially numerous inter-
actions among this growing list of factors presents a
challenge for making progress in this area of research.
The development of microarrays specific for cattle pro-
vides an opportunity to perform near genome-wide eval-
uations of gene expression. The objective of the pres-
ent research was to compare gene expression profiles in
anterior pituitary glands collected from anestrous and
cycling postpartum beef cows to provide insight into
Differential gene expression in anterior pituitary glands from anestrous
and cycling postpartum beef cows1
A. J. Roberts*†2 and D. J. McLean†‡
*USDA-ARS, Fort Keogh Livestock and Range Research Laboratory (LARRL), Miles City, MT 59301;
and †Center for Reproductive Biology, and ‡Department of Animal Sciences,
Washington State University, Pullman 99164
1 Mention of a proprietary product does not constitute a guarantee
or warranty of the product by USDA or the authors and does not
imply its approval to the exclusion of other products that may be
also suitable. The authors thank B. Shipp, USDA-ARS, Fort Ke-
ogh Livestock and Range Research Laboratory, Miles City, MT, and
Derek Pouchnik, Center for Reproductive Biology, Washington State
University, Pullman, for contributions in conducting the experiment
and laboratory analysis of samples used in this manuscript.
2 Corresponding author: firstname.lastname@example.org
Received August 16, 2010.
Accepted December 5, 2010.
Published December 4, 2014
gland gonadotropes in response to GnRH. The down-
stream result of JUN induction has been investigated in-
tensively in anterior pituitary gland cell lines (Salisbury
et al., 2009). As a component of transcription factor
complexes, JUN induces multiple genes as a secondary
response to GnRH, including the GnRH receptor gene,
and stimulates its own transcription (Salisbury et al.,
2009). The finding that transcript expression of JUN
in cycling cows is greater than that in anestrous cows
when using a microarray approach to identify anterior
pituitary gland-expressed genes associated with repro-
ductive cycling supports the concept that depressed
GnRH secretion may contribute to the anestrous state
observed in this subgroup of animals.
With the exception of some of the genes discussed
above, the majority of genes that exhibited differen-
tial expression have not been implicated previously in
regulating reproduction. These results indicate that
the methodology used in this study may provide op-
portunities to identify novel mechanisms regulating re-
production and other traits of importance for livestock
Of the 78 transcripts exhibiting differences in expres-
sion attributable to cycling status, 21 (27%) represent
transcripts that are not yet annotated and do not ex-
hibit homology with sequences from other species. The
number of unannotated transcripts exhibiting differen-
tial patterns of expression is representative of the total
number of unannotated transcripts without homology
to other species that are found on the GeneChip (31%).
This large number of uncharacterized probes on the
bovine GeneChip is indicative of the current status
of the field of bovine genomics and suggests that the
data generated continually need to be reappraised. For
example, the original UniGene cluster containing the
sequence encoding GRP has been retired. Sequences
originally assigned to this UniGene are now distributed
in 5 other clusters or no longer reside in a cluster. Thus,
the original UniGene cluster is no longer informative,
and the original reference sequence used in generating
the probe is required to determine proper annotation.
In conclusion, although further verification of the re-
sults of this research is required, the majority of genes
identified by this study have not previously been asso-
ciated with processes regulating reproductive function,
or the differentially expressed transcripts represent cur-
rently uncharacterized genes. Results from the present
study indicate that microarray analysis of gene expres-
sion profiles in bovine reproductive tissues collected at
different physiological stages will undoubtedly result in
the identification of novel mechanisms involved in regu-
Clapper, J. A., J. L. Snyder, A. J. Roberts, D. L. Hamernik, and G.
E. Moss. 1998. Estradiol increases relative amounts of insulin-
like growth factor binding protein (IGFBP)-3 in serum and
expression of IGFBP-2 in anterior pituitaries of ewes. Biol.
Dunn, T. G., and G. E. Moss. 1992. Effects of nutrient deficiencies
and excesses on reproductive efficiency of livestock. J. Anim.
Evans, J. J. 1999. Modulation of gonadotropin levels by peptides act-
ing at the anterior pituitary gland. Endocr. Rev. 20:46–67.
Freund, R. S., and R. C. Littell. 1981. SAS for Linear Models. SAS
Inst. Inc., Cary, NC.
Funston, R. N., G. E. Moss, and A. J. Roberts. 1995. Insulin-like
growth factor-1 (IGF-1) and IGF-binding proteins in bovine
sera and pituitaries at different stages of the estrous cycle. En-
Liu, D., Z. Zhang, and C. T. Teng. 2005. Estrogen-related receptor-α
and peroxisome proliferator-activated receptor-γ coactivator-1α
regulate estrogen-related receptor-α gene expression via a con-
served multi-hormone response element. J. Mol. Endocrinol.
McLean, D. J., P. J. Friel, D. Pouchnik, and M. D. Griswold. 2002.
Oligonucleotide microarray analysis of gene expression in FSH-
treated rat Sertoli cells. Mol. Endocrinol. 16:2780–2792.
Morel, G., M. Priam, and A. Enjalbert. 1994. Evidence for direct
action of gastrin-releasing peptide (GRP) in rat lactotrophs,
somatotrophs, and gonadotrophs. Endocrine 2:669–674.
Puigserver, P., and B. M. Spiegelman. 2003. Peroxisome proliferator-
activated receptor-γ coactivator 1α (PGC-1α): Transcriptional
coactivator and metabolic regulator. Endocr. Rev. 24:78–90.
Roberts, A. J., R. N. Funston, and G. E. Moss. 2001. Insulin-like
growth factor binding proteins in the bovine anterior pituitary.
Roberts, A. J., J. Klindt, and T. G. Jenkins. 2005. Effects of vary-
ing energy intake and sire breed on duration of postpartum
anestrus, insulin-like growth factor-1, and growth hormone in
mature crossbred cows. J. Anim. Sci. 83:1705–1714.
Salgado, R. M., L. P. Capelo, R. R. Favaro, J. D. Glazier, J. D. Ap-
lin, and T. M. Zorn. 2009. Hormone-regulated expression and
distribution of versican in mouse uterine tissues. Reprod. Biol.
Salisbury, T. B., A. K. Binder, J. C. Grammer, and J. H. Nilson.
2009. GnRH-regulated expression of Jun and JUN target genes
in gonadotropes requires a functional interaction between
TCF/LEF family members and β-catenin. Mol. Endocrinol.
Santos, C. V., C. C. Pazos-Moura, and E. G. Moura. 1995. Effect
of gastrin-related peptide GRP and GRP antagonists on TSH
secretion from rat isolated pituitaries. Life Sci. 57:911–915.
Schillo, K. K. 1992. Effect of dietary energy on control of luteinizing
hormone secretion in cattle and sheep. J. Anim. Sci. 70:1271–
Schwartz, J. 2000. Intercellular communication in the anterior pitu-
itary. Endocr. Rev. 21:488–513.
Short, R. E., R. A. Bellows, R. B. Staigmiller, J. G. Berardinelli,
and E. E. Custer. 1990. Physiological mechanisms controlling
anestrus and infertility in postpartum beef cattle. J. Anim.
Snyder, J. L., J. A. Clapper, A. J. Roberts, D. W. Sanson, D. L.
Hamernik, and G. E. Moss. 1999. Insulin-like growth factor-I,
insulin-like growth factor-binding proteins, and gonadotropins
in the hypothalamic-pituitary axis and serum of nutrient-re-
stricted ewes. Biol. Reprod. 61:219–224.
Wang, W., G. L. Xu, W. D. Jia, J. L. Ma, J. S. Li, Y. S. Ge, W. H.
Ren, J. H. Yu, and W. B. Liu. 2009. Ligation of LR2 by versi-
can: A link between inflammation and metastasis. Arch. Med.
Wettemann, R. P., C. A. Lents, N. H. Ciccioli, F. J. White, and I.
Rubio. 2003. Nutritional- and suckling-mediated anovulation in
beef cows. J. Anim. Sci. 81(E. Suppl. 2):E48–E59.
Gene expression in anestrous and cycling cows