Dairy J. Sci. 89:33753385
American Dairy Science Association, 2006.
Pregnancy, Bovine Somatotropin, and Dietary n-3 Fatty Acids
in Lactating Dairy Cows: II. Endometrial Gene Expression
Related to Maintenance of Pregnancy
T. R. Bilby,* A. Guzeloglu, L. A. MacLaren, C. R. Staples,* and W. W. Thatcher*1
*Department of Animal Sciences, University of Florida, Gainesville 32611-0910
Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Selcuk University, Konya, Turkey 42075
Department of Plant and Animal Sciences, Nova Scotia Agricultural College, Truro, Nova Scotia, Canada
ABSTRACT
The objectives were to examine the effects of bovine
somatotropin (bST), pregnancy, and dietary fatty acids
on expression of key endometrial genes and proteins
regulating prostaglandin synthesis in lactating dairy
cows. Two diets were fed, at about 17 d in milk (DIM),
in which oil of whole cottonseed (control diet) was compared with calcium salts of sh oil-enriched lipid (FO).
Ovulation was synchronized in cows with a presynchronization plus Ovsynch protocol and cows were inseminated articially or not inseminated on d 0 (d 0 = time
of synchronized ovulation; 77 12 DIM). On d 0 and
11, cows received bST (500 mg) or no bST, and were
slaughtered on d 17 to recover uterine secretions and
endometrial tissue. Number of cows in the control diet:
5 bST-treated cyclic (bST-C), 5 non-bST-treated cyclic
(no bST-C), 4 bST-treated pregnant (bST-P), and 5 nonbST-treated pregnant (no bST-P) cows and in the FO
diet: 4 bST-treated FO-cyclic (bST-FO-C) and 5 nonbST-treated cyclic (no bST-FO-C) cows. The FO diet
increased progesterone receptor (PR) mRNA, and treatment with bST increased PR mRNA concentration in
endometrium of no bST-C, but not in no bST-FO-C or
no bST-P cows. Concentrations of estrogen receptor-
(ER) mRNA and protein, and oxytocin receptor (OTR)
mRNA were decreased in no bST-P cows compared with
no bST-C cows. Treatment with bST tended to increase
OTR and ER mRNA concentrations in cyclic cows fed
control or FO diets. Immunohistochemistry demonstrated effects of bST, FO, and pregnancy on distributions of ER and PR proteins in endometrium. Pregnancy and FO feeding decreased ER abundance in
luminal epithelium. Prostaglandin H synthase-2
(PGHS-2) protein was elevated in pregnant cows and
localized to the luminal epithelium. Both FO and bST
treatments reduced staining intensity of PGHS-2 pro-
Received October 21, 2005.
Accepted April 5, 2006.
1
Corresponding author: [e-mail]
tein. Concentrations of prostaglandin E synthase
mRNA were elevated in either cyclic or pregnant cows
in response to bST, whereas bST decreased prostaglandin F synthase mRNA in pregnant cows. Uterine lumen
uids had more PGF2 and prostaglandin E2 in pregnant than cyclic cows. Uterine lumen uids of bST-P
cows contained more prostaglandin E2 than those from
no bST-P cows. In summary, both pregnancy and bST
altered endometrial gene expression, and cyclic cows
responded differently to bST than pregnant cows. Feeding FO modulated PR, ER, and PGHS-2 expression
and distribution among endometrial cell types in a manner that may favor establishment and maintenance
of pregnancy.
Key words: pregnancy, bovine somatotropin, fatty acid
INTRODUCTION
Early pregnancy loss in lactating dairy cattle can
have devastating effects on the economic success of
dairy farms (Santos et al., 2004a). Nearly 40% of pregnancy losses occur in association with the period of 15
to 17 d following estrus. This is the critical period during
which the conceptus must produce sufcient quantities
of IFN- to prevent pulsatile prostaglandin (PG) secretion and maintain the corpus luteum (CL). Changing
from a cyclic to a pregnant state not only depends on the
production of antiluteolytic signals from the developing
conceptus, but also on the capacity of the endometrium
to respond to these signals, thus blocking pulsatile
PGF2 production. Such communications between the
conceptus and maternal units are not always successful, thus leading to early embryonic loss.
The endometrium plays a critical role in regulating
the estrous cycle and establishment of pregnancy. Elevated concentrations of plasma progesterone during the
late luteal phase of the estrous cycle caused down regulation of progesterone receptors (PR) in the uterus
(Wathes and Lamming, 1995). In pregnant ewes, the
expression of estrogen receptor- (ER) is suppressed
during early pregnancy, and it has been hypothesized
that IFN- inhibits oxytocin receptor (OTR) upregula-
3375
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BILBY ET AL.
tion by inhibiting a preceding increase in ER expression (Spencer and Bazer, 1995). Although the role of
PR and ER in regards to OTR regulation is obscure,
OTR certainly are suppressed by IFN- secreted from
the conceptus (Wathes and Lamming, 1995). Intrauterine infusions of recombinant IFN- in cyclic ewes from
d 11 to 16 postestrus had no effect on prostaglandin
H synthase-2 (PGHS-2) expression in the endometrial
epithelium (Kim et al., 2003). In this latter study, it
was suggested that antiluteolytic effects of IFN- are
to inhibit ER and OTR gene transcription, thereby
preventing endometrial production of luteolytic pulses
of PGF2.
In the uterine luminal epithelium, arachidonic acid
is released from phospholipids by hydrolysis and acted
upon by PGHS-2 to form prostaglandin H2 (PGH2),
which is converted to either PGF2 and (or) prostaglandin E2 (PGE2) through 2 reductases, prostaglandin F
synthase (PGFS) and prostaglandin E synthase
(PGES), respectively. Kim et al. (2003) reported that
PGHS-2 mRNA concentrations were greater in endometrium from pregnant ewes than in that from cyclic ewes
by d 16 postestrus. It is unknown whether relative expression of the 2 synthetic enzymes, PGFS and PGES,
changes during the period of CL maintenance in pregnant lactating dairy cattle.
Programs to optimize reproductive performance in
dairy cattle have received considerable attention. Recently, recombinant bST, a commercially available
product used to increase milk production, was shown
to increase pregnancy rates when given as part of a
timed AI protocol in lactating dairy cows (Moreira et al.,
2001). Santos et al. (2004a) showed that bST increased
pregnancy rate through reducing pregnancy loss. Evidence exists for cross-talk between hormone signaltransduction systems such as ER with IGF-I (Lee et
al., 1997; Klotz et al., 2002), or direct effects such as bST
increasing IFN- effectiveness (Badinga et al., 2002).
Effects of bST on fertility may involve an interaction
between bST and IFN- signaling pathways to regulate
PG secretion or other components of the PG cascade
critical for maintenance of pregnancy. Little is known,
however, about the molecular and cellular effects of
bST on endometrial gene expression at the time of pregnancy recognition.
Another commercially available product that may
benet fertility of lactating dairy cows is a calcium salt
of lipid-enriched sh oil (FO) supplement containing n3 fatty acids such as eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA). Previous studies indicated that n-3 fatty acids could decrease PGF2 secretion by bovine endometrial cells in vitro (Mattos et
al., 2003).
Journal of Dairy Science Vol. 89 No. 9, 2006
Little is known about the effects of a FO-enriched
supplement and its interaction with bST treatment on
endometrial function at the molecular level. The objectives of the present study were to examine the effects
of pregnancy, bST treatment, and a FO-supplemented
diet on the regulatory enzymes of the PG cascade and
how they may regulate genes and proteins in the uterine environment that are known to inuence pregnancy recognition.
MATERIALS AND METHODS
Materials
Gonadotropin-releasing hormone (Fertagyl; Intervet
Inc., Millsboro, DE), PGF2 (Lutalyse; Pzer Animal
Health, Kalamazoo, MI), and bST (Posilac; Monsanto
Co., St. Louis, MO) were used for experimental treatments of cows. Other purchased materials included Trizol, cDNA Cycle kit, TOPO vector (TOPO TA Cloning
Kits), and Random Primers DNA Labeling System (Invitrogen Corp., Carlsbad, CA); Taq polymerase (M166A;
Promega, Madison, WI). Also purchased was biotinconjugated antirabbit IgG (Santa Cruz Biotechnology,
Santa Cruz, CA), normal horse serum, biotinylated
horse antimouse IgG, horseradish peroxidase-avidinbiotin complex, 3, 3-diaminobenzidille (DAB kit; Vectastain; Vector Laboratories, Burlingame, CA); enhanced chemiluminescence (ECL) kit (Renaissance
Western Blot Chemiluminescence Reagent Plus; NEN
Life Science Products, Boston, MA); ultrasensitive hybridization buffer (ULTRAhyb;Cat # 8670; Ambion Inc.,
Austin, TX); dCTP -32P and Biotrans nylon membrane
(MP Biomedicals, Irvine, CA); isotopically labeled [5, 6,
8, 11, 12, 14, 15-3H]-PGF2 and PGE2, nitrocellulose
membranes (Hybond-ECL), horseradish peroxidaselinked antimouse IgG (NA931V), and antirabbit IgG
(NA934V; Amersham Biosciences Corp., Piscataway,
NJ). All other laboratory materials were from Fisher
Scientic (Pittsburgh, PA) and Sigma Chemical Co. (St.
Louis, MO).
Cows and Experimental Diets
A more detailed description of cows, management,
and collection of samples is given in a companion article
(Bilby et al., 2006b). Briey, 40 multiparous Holstein
cows in late gestation were fed diets formulated to contain 1.51 Mcal of NEL/kg, 13.1% CP, and a cation-anion
difference of 90 mEq/kg (DM basis) beginning approximately 3 wk before expected calving. Upon calving, cows
were fed 2 dietary treatments containing none or 1.9%
calcium salt of a sh oilenriched lipid product (EnerGII Reproduction formula, Virtus Nutrition, Fairlawn,
OH). The fatty acid prole of the fat source as given by
PREGNANCY, SOMATOTROPIN, AND FATTY ACIDS ON ENDOMETRIUM
the manufacturer was 2.2% C14:0, 41.0% C16:0, 4.2%
C18:0, 30.9% C18:1, 0.2% C18:1 trans, 8.0% C18:2, 0.5%
C18:3, 0.4% C20:4, 2.0% C20:5, 2.3% C22:6, and 2.7%
unknown. The control diet contained a greater concentration of whole cottonseed, and therefore, was similar
in concentration of ether extract and NEL to that containing FO (Bilby et al., 2006b). The control diet was
fed to all cows during the rst 9 DIM. From 10 to 16
DIM, 10 cows were assigned to consume an FO diet
containing half the nal concentration of the fat product
(0.95% of dietary DM) to adjust the cows to a new fat
source. Starting at 17 DIM, these cows were switched
to the 1.9% FO diet and continued on that diet until
the end of the study. Cows fed the ruminally protected
FO consumed approximately 14.8 g/cow per day of EPA
and DHA. Thirty cows were assigned to the control diet
for the duration of the study. Cows were milked thrice
daily and calibrated electronic milk meters recorded
milk weights at each milking. Body weights were measured and BCS assigned weekly by the same 2 individuals.
Estrus Synchronization and Tissue Collection
Detailed description of the estrus synchronization is
described in Bilby et al. (2006b). The number of cows
used for analyses on d 17 in the control diet was 5 bSTtreated cyclic (bST-C), 5 non-bST-treated cyclic (no
bST-C), 4 bST-treated pregnant (bST-P), and 5 nonbST-treated pregnant (no bST-P) cows; and in the FO
diet: 4 bST-treated FO-cyclic (bST-FO-C) and 5 nonbST-treated cyclic (no bST-FO-C) cows. Conceptuses
and uterine secretions were recovered and endometrial
tissue collected as described by Bilby et al. (2004).
RNA Isolation and Northern Blotting
Total RNA was isolated from endometrial tissues (n =
28) and the Northern blotting procedure was performed
as described by Bilby et al. (2004). The specic bovine
cDNA used were ER, OTR, PR, PGHS-2, PGES, PGFS,
or glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). Probes were obtained using a reverse transcription PCR procedure as described in Guzeloglu et al.
(2004a). Complementary DNA was reverse-transcribed
from total RNA (5 g) prepared from the bovine CL
following the manufacturers protocol with the cDNA
Cycle kit, utilizing established primer sets for bovine
OTR, PR, and PGES (Guzeloglu et al., 2004a).
The ER cDNA was a gift from N. H. Ing (Texas A&
M University, College Station, TX). The PGFS (Xiao et
al., 1998) and PGHS-2 (Liu et al., 2001) cDNA were a
gift from J. Sirois (University of Montreal, St. Hyacinthe, Canada).
3377
Immunohistochemical Analyses
Parafn sections (5 m) from the antimesometrial
border of the uterus from 27 cows (5 no bST-C, 5 bSTC, 4 no bST-FO-C, 4 bST-FO-C, 4 no bST-P, and 5 bSTP) were prepared. After deparafnization, an antigen
retrieval procedure was performed by heating sections
in a microwave oven at high power for 5 min in 0.01 M
sodium citrate buffer (pH 6.0). Sections were allowed
to cool in microwave for 28 min, and then washed in
distilled water and in PBS (0.01 M, pH 7.2). Nonspecic
endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in methanol for 10
min at room temperature. After a 10-min wash in PBS,
nonspecic binding was blocked using 2% BSA in PBS
for ER, 5% (vol/vol) normal horse serum in PBS for
PR, and 5% normal goat serum in PBS for PGHS-2 in
a humidied chamber at room temperature for 1 h.
Tissue sections were then incubated in the dark at room
temperature with the primary antibody: 1) monoclonal
anti-ER (NeoMarkers, Medicorp, Montreal, QC, Canada) or the negative-control mouse IgG at an equivalent
concentration diluted 1:500 in 2% BSA, and incubated
overnight; 2) monoclonal anti-PR (NeoMarkers, Lab Vision, Fremont, CA) or the negative-control mouse IgG
at equivalent concentration diluted 1:500 in 5% horse
serum and incubated for 2 h; 3) polyclonal anti-PGHS2 (Cayman Chemical, Cedarlane Lab., Hornby, ON,
Canada) or the negative control, anti-PGHS-2 preincubated with PGHS-2 blocking peptide (1:5 vol/vol ratio)
for 1 h, diluted 1:200 in PBS, and incubated for 2 h.
After three 5-min washes in PBS, the sections were
incubated in the dark for 1 h at room temperature with
a biotinylated horse antimouse IgG for ER (diluted
1:200 in 2% BSA) and PR (diluted 1:800 in 5% horse
serum) antibodies, or biotinylated goat antirabbit IgG
for the PGHS-2 antibody (diluted 1:200 in 5% goat serum). Thereafter, three 5-min washes in PBS were performed and tissue sections were incubated for 30 min
at room temperature with horseradish avidin-biotinperoxidase complex. Site of the bound enzyme was visualized by the application of 3,3-diaminobenzidine in
H2O2. Sections were counterstained with hematoxylin
and dehydrated before they were mounted with
Permount.
Microscopic Image Analysis
Subjective image analysis was performed to estimate
the relative abundance of ER, PR, and PGHS-2 staining in different cell types. One evaluator assessed immunostaining on 10 randomly selected elds of intercaruncular endometrium in 3 pieces of endometrium from
each cow. Caruncular endometrium was not evident in
all cows. Five uterine compartments were evaluated:
Journal of Dairy Science Vol. 89 No. 9, 2006
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BILBY ET AL.
luminal epithelium (LE), supercial glandular epithelium (SGE; close to the uterine lumen), deep glandular
epithelium (DGE; close to the myometrium), supercial
intercaruncular stroma (SS; just beneath the luminal
epithelium layer), and deep intercaruncular stroma
(DS; between supercial stroma and the myometrium).
Because specic staining for PGHS-2 protein was detectable exclusively in the cytoplasm of endometrial
luminal epithelial cells, only those cells were scored.
Intensity of staining was scored on a 4-point scale,
where 0 = no staining (no brown), 1 = less (light brown),
2 = moderate (brown), and 3 = heavy (dark brown), and
the staining intensities were expressed as percentage
of positively stained cells for each point in the scale
(Guzeloglu et al., 2004a).
Western Blotting for ER and PGHS-2 Proteins
Endometrial tissues (300 mg) from 27 cows were used
for Western blotting of ER and PGHS proteins, as described by (Guzeloglu et al., 2004a).
Radioimmunoasssay
Concentrations of PGF2 and PGE2 were measured
in uterine ushings by direct radioimmunoassay (RIA)
as described by Danet-Desnoyers et al. (1994) and Gross
et al. (1988), respectively. The anti-PGF2 antiserum
was diluted 1:5,000 and the anti-PGE2 antiserum was
diluted 1:1000 in Tris buffer. Intraassay coefcients of
variation for PGF2 and PGE2 assay were 11.8 and
8.7%, respectively.
Statistical Analyses
Abundances of ER and PGHS-2 proteins in Western
blots as well as ER, PR, OTR, PGFS, PGES, and
PGHS-2 mRNA in Northern blots were analyzed using
the least squares ANOVA, GLM procedure of SAS (SAS
Institute, Inc., Cary, NC). Main effects of treatment (no
bST-C, no bST-P, no bST-FO-C, bST-C, bST-FO-C, bSTP), gel, and treatment gel interaction were examined,
and for mRNA responses, band intensities of GAPDH
mRNA were used as a covariate to adjust for loading
differences. If treatment gel effects were not signicant they were removed from the model. Predesigned
orthogonal contrasts were used to compare treatment
means for: bST, pregnancy status and bST pregnancy
status; bST, FO, and bST FO.
Total contents of PGF2 and PGE2 in uterine ushings were analyzed using the GLM procedure of SAS.
The model included the effect of treatment (no bST-C,
no bST-P, no bST-FO-C, bST-C, bST-FO-C, and bSTP), and orthogonal contrasts were constructed among
Journal of Dairy Science Vol. 89 No. 9, 2006
treatments to examine effects of: bST, pregnancy status
and bST pregnancy status; bST, FO, and bST FO.
Data generated from immunohistochemistry of ER,
PR, and PGHS-2 were analyzed by the Mixed model
procedure of SAS (SAS Inst. Inc.) for each type of cell.
The model included treatment (no bST-C, no bST-P, no
bST-FO-C, bST-C, bST-FO-C, and bST-P), class (none,
less, moderate, and heavy staining intensity), and treatment class interaction. Cow within treatment was
the error term used to test for treatment effects. A
series of orthogonal contrasts were constructed to test
treatment effects (bST, pregnancy status and bST
pregnancy status; bST, FO, and bST FO), class (none,
less, moderate, and heavy staining intensity), and
treatment class interactions.
RESULTS
Endometrial PR Expression
Injections of bST increased (P < 0.05; bST FO interaction) steady-state concentrations of PR mRNA in cyclic cows only when FO was absent from the diet, because cows fed FO had similar elevated concentrations
to control cows given bST (Table 1). In addition, bST
increased (P < 0.01; interaction) steady state concentrations of PR mRNA in cyclic but not pregnant cows (Table 1).
Immunohistochemical staining of PR was exclusively
in the nuclei of the SGE and DGE, with little or no
staining in the LE, SS, and DS. Within the SGE, FO
increased (P < 0.05) the amount of moderate and heavy
staining (Figure 1C). The bST, as a main effect, reduced
(P < 0.01; Figure 2) moderate and heavy staining in the
DGE from cyclic and pregnant cows. In the endometrium of cyclic cows, however, no bST-FO-C alone reduced (bST FO interaction; P < 0.01) the moderate
and heavy staining and there was not a further reduction with bST (Figure 3).
Endometrial ER Expression
Steady state concentrations of ER mRNA in endometrial tissues tended (P < 0.10) to be reduced in pregnant cows compared with cyclic cows fed a control diet
(Table 1). Pregnancy decreased (P < 0.05) abundance
of ER protein in the endometrium (Table 1) as detected
by Western blotting.
Immunohistochemistry was used to localize ER in
the endometrium, and staining was detected exclusively in the nuclei of LE, SGE, DGE, and SS (Figure
1E). Pregnancy (P < 0.01; Figure 1F) and no bST-FOC decreased (P < 0.01; Figure 4) ER abundance in LE,
and pregnancy decreased (P < 0.05) ER abundance in
3379
PREGNANCY, SOMATOTROPIN, AND FATTY ACIDS ON ENDOMETRIUM
Table 1. Least squares means and pooled SE for uterine endometrial mRNA and protein, and uterine luminal ushings (ULF) protein
expression at d 17 after a synchronized estrus (d 0) in lactating cyclic (C) cows fed a control diet, pregnant (P) cows fed a control diet, and
cyclic cows fed a sh oil-enriched lipid (FO) diet and injected with or without bST on d 0 and 11 (n = 28)
Treatment2
Contrast3 cyclic
Contrast3 pregnant
Response1
No bST-C
bST-C
No bSTFO-C
bST-FO
No
bST-P
bST-P
SE
FO
bST
bST
FO
P
bST
bST
P
PR mRNA, AU
ER mRNA, AU
ER protein, AU
OTR mRNA, AU
PGHS-2 mRNA, AU
PGHS-2 protein, AU
PGFS mRNA, AU
PGF2, ng/ULF
PGES mRNA, AU
PGE2, ng/ULF
60
11
70
45
84
39
27
57
45
39
69
15
71
50
83
48
28
60
48
39
68
12
70
49
85
70
28
130
46
82
67
16
73
52
83
70
28
50
47
15
61
11
67
39
78
91
27
618
43
250
59
8
70
41
82
80
25
672
46
357
2.1
2.1
1.2
1.3
3.3
1.8
0.6
92
0.9
38
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
*
NS
*
NS
NS
NS
NS
NS
NS
NS
NS
NS
*
*
**
NS
*
*
**
**
NS
NS
NS
NS
NS
NS
NS
NS
**
NS
**
NS
NS
NS
NS
NS
**
NS
NS
NS
1
PR = Progesterone receptor; ER = estradiol receptor ; OTR = Oxytocin receptor; PGHS-2 = prostaglandin H synthase-2; PGFS =
prostaglandin F synthase; PGES = prostaglandin E synthase. Arbitrary units (AU) were generated by densitometry and mRNA results
were adjusted using glyceraldehyde-3-phosphate dehydrogenase as a covariate.
2
No bST-C= No bST cyclic, bST-C = bST-cyclic, No bST-FO-C = No bST-sh oil-cyclic, bST-FO-C = bST-sh oil cyclic, No bST-P = No bSTPregnant, bST-P = bST-pregnant.
3
Contrasts for cyclic cows were: FO = all FO-fed cows vs. all cyclic-control fed cows, bST = all cyclic cows injected with bST vs. all cylic
cows not given bST, and bST FO interaction. Contrasts for pregnant cows were: P = all pregnant cows vs. cyclic control-fed cows, and
bST = all pregnant and cyclic cows fed the control diet and injected with bST vs. all pregnant and cyclic cows fed the control diet without
bST, and bST P interaction.
P 0.10, *P 0.05, **P 0.01, NS = nonsignicant.
the SGE compared with cyclic control fed cows (Figure 1F).
decreased (P < 0.01) the percentage of PGHS-2 heavy
staining in cyclic cows (no bST-FO-C/bST-FO-C = 33.5%
< no bST-C/bST-C = 49.5%; 7% SE).
Endometrial OTR Expression
Among cyclic control and FO-fed cows, bST tended
(P < 0.10; Table 1) to increase OTR mRNA. Steadystate concentrations of OTR mRNA in pregnancy were
decreased (P < 0.01) compared with cyclic control fed
cows (Table 1).
Endometrial PGHS-2 Expression
No differences were detected in steady-state concentrations of PGHS-2 mRNA due to treatments (Table 1).
In contrast, PGHS-2 protein in pregnant endometrial
tissue was increased (P < 0.05) 2-fold compared with
cyclic cows as detected by Western blotting (Table 1).
Staining for PGHS-2 protein was localized specically to the cytoplasm of endometrial LE cells as detected by immunohistochemistry. When primary antibody was rst incubated with PGHS-2 blocking peptide,
the absence of staining demonstrated the specicity
for PGHS-2 (Figure 1G). Some light staining also was
detected in SGE, DGE, SS, and DS. Staining in these
endometrial cell types, however, was inconsistent and
not evaluated. Within the LE, pregnancy increased (P
< 0.01) percentage of heavy staining (Figure 1H) and
bST treatment blocked this response in pregnant cows
(no-bST-P = 72% > bST-P = 39%; 7% SE). Feeding FO
Endometrial PGFS and PGES mRNA Expression
An interaction was detected (P < 0.01) between cyclic
and pregnant control-fed cows with bST slightly increasing steady-state concentrations of endometrial
PGFS mRNA in cyclic (bST-C > No bST-C), but decreasing concentrations in pregnant (bST-P < no bST-P) control-fed cows (Table 1). Relative steady-state concentrations of PGES mRNA in endometrium were increased
in response to bST for cyclic control and FO-fed cows
(P < 0.05; Table 1) as well as for cyclic and pregnant
cows (P < 0.01; Table 1).
Total Contents of PGF2 and PGE2
in Uterine Luminal Flushings
Volumes of recovered ushing uids (36.2 mL) and
percentage recovered (90.4%) did not differ among
treatments. Total PGF2 (645 93 vs. 58 87 ng; P <
0.01) and PGE2 (303 37 vs. 39 35 ng; Table 1) contents were substantially greater (P < 0.01) in uterine
luminal ushings of pregnant cows (no bST-P/bST-P)
compared with cyclic control fed cows (no bST-C/bSTC), respectively. Treatment with bST or FO had no
effect on uterine ushing PG content in cyclic cows.
In addition, statistical analyses restricted to pregnant
Journal of Dairy Science Vol. 89 No. 9, 2006
3380
BILBY ET AL.
Figure 1. Expression of progesterone receptor (PR; panels A, B, C), estrogen receptor- (ER; panels D, E, F), and prostaglandin H
synthase-2 (PGHS-2; panels G, H, I) in bovine endometrium at d 17 following an induced ovulation. No immunopositive staining was detected
when primary antibody was replaced by mouse IgG (panel A for PR and panel D for ER) or when primary antibody was rst incubated
with PGHS-2blocking peptide (panel G for PGHS-2). Staining for ER and PR was detected exclusively in the nuclei of the epithelial and
stromal cells. Fish oil (panel C) increased (P < 0.05) abundance of PR in the supercial glandular epithelium compared with the cyclic
control-fed cows (panel B). Pregnancy decreased (P < 0.05) ER staining in the luminal and supercial glandular epithelium (panel F)
compared with the cyclic control-fed cows (panel E). Staining for PGHS-2 was observed in the cytoplasm of endometrial luminal epithelial
cells. Pregnancy (panel I) increased (P < 0.05) the intensity of staining for PGHS-2 protein in the luminal epithelial cells compared with
the cyclic control-fed cows (panel H). Magnication = 20.
cows ( bST), bST increased (P < 0.05) PGE2 (250 39
vs. 357 35 ng; P 0.05), but not PGF2.
Simple and Partial Correlations for the
Prostaglandin Cascade at d 17
A series of simple and partial correlations were detected in the uterus at d 17 (Table 2). Although correlations are not proof of causative effects, signicant associations were detected for endometrial gene expression,
protein abundance, and PG within the uterine ushings
at d 17. Endometrial PR mRNA concentration was correlated negatively with ER protein, PGHS-2 protein,
and correlated positively with PGES mRNA and PGFS
Journal of Dairy Science Vol. 89 No. 9, 2006
mRNA. These associations were detected both as simple
and partial correlations indicating an inherent association after adjustment for treatment effects (Table 2).
Endometrial ER mRNA expression was correlated
positively with OTR mRNA, PGHS-2 mRNA, and negatively correlated with PGF2 in the ULF; however, these
associations were not detected after adjustment for
treatments. In contrast, ER protein abundance was
correlated positively with PGHS-2 protein and negatively with both PGES and PGFS mRNA. The OTR
mRNA expression correlated was positively with
PGHS-2 mRNA and PGFS mRNA, and negatively correlated with both PGE2 and PGF2 in the ULF. However, after adjustment for treatments (e.g., pregnancy
PREGNANCY, SOMATOTROPIN, AND FATTY ACIDS ON ENDOMETRIUM
3381
Figure 2. Expression of progesterone receptor (PR) in bovine endometrium at d 17 following an induced ovulation. The bST injections
decreased (P < 0.01) abundance of PR in the deep glandular epithelium of both cyclic and pregnant cows compared with cyclic and pregnant cows not injected with bST. Staining intensity weighted average
(none = 0, less = 1 vs. moderate = 2, heavy staining = 3); no bST = cyclic
and pregnant control-fed cows; bST = bST-cyclic and bST-pregnant
control-fed cows.
status) these associations were not detected. The
PGHS-2 mRNA was correlated positively with PGHS2 protein. Abundance of PGHS-2 protein was correlated
negatively with both PGES and PGFS mRNA. All simple correlations involving uterine luminal concentrations of PGE2 and PGF2 were not detected when adjusted for treatments (e.g., high concentrations found
in pregnancy but not during the cycle).
DISCUSSION
An understanding of the differential regulation of PG
secretion in cyclic and pregnant animals is pivotal to
Figure 3. Expression of progesterone receptor (PR) in bovine endometrium at d 17 following an induced ovulation. The bST reduced
moderate and heavy staining in the deep glandular epithelium of
cyclic control-fed cows; however, FO alone reduced the moderate and
heavy staining and there was not a further reduction with bST (bST
FO interaction; P < 0.01). Staining intensity weighted average
(none = 0, less = 1 vs. moderate = 2, heavy staining = 3); no bST-C =
cyclic control-fed cows, bST-C = bST-treated, cyclic control-fed cows,
no bST-FO-C = cyclic FO-fed cows, bST-FO-C = bST-treated, cyclic
FO-fed cows.
Figure 4. Expression of estrogen receptor- (ER) in bovine endometrium at d 17 following an induced ovulation. The FO reduced
moderate and heavy staining in the luminal epithelium compared
with cyclic control-fed cows (P < 0.01). Staining intensity weighted
average (none = 0, less = 1 vs. moderate = 2, heavy staining = 3);
control = cyclic control and bST cyclic control-fed cows; FO = cyclicFO and bST-treated, cyclic FO-fed cows.
our understanding of pregnancy establishment and the
endometrial response to factors such as bST and FO.
In this study, bST tended to increase pregnancy rates
at d 17 (Bilby et al., 2006b), consistent with previous
studies utilizing large numbers of lactating dairy cows
(Moreira et al., 2001; Santos et al., 2004a).
Effects of pregnancy, bST, and FO on steady-state
concentrations of mRNA (ER, PR, and PGHS-2) and
their respective proteins were examined in uterine endometrium. A 4.3-kb PR mRNA transcript was detected
in the endometrium at d 17 following an induced ovulation, consistent with published reports of PR transcript
size (Meikle et al., 2001). Progesterone receptor mRNA
was elevated in FO-fed cows and bST did not further
stimulate PR mRNA expression compared with cyclic
control-fed cows. In contrast, bST stimulated PR mRNA
in cyclic control-fed cows, but had no effect in pregnant cows.
Consistent with Kimmins and MacLaren (2001), immunohistochemistry indicated that the PR was localized mainly in the stroma and the glandular epithelium.
More specically, immunohistochemistry showed that
the heaviest PR staining was in the SGE and DGE. In
the SGE, FO appeared to increase PR abundance, which
would agree with PR mRNA expression. Progesterone
not only prepares the uterus for implantation of the
embryo but also helps maintain pregnancy by stimulating uterine secretions for nourishment to the developing
Journal of Dairy Science Vol. 89 No. 9, 2006
3382
BILBY ET AL.
Table 2. Simple and partial correlations1 of uterine endometrial and uterine luminal ushing (ULF) variables2 at d 17 after a synchronized
estrus (d 0) in lactating cyclic cows fed a control diet, pregnant cows fed a control diet, cyclic cows fed a sh oilenriched lipid diet and
injected with or without bST on d 0 and 11
PR
mRNA
PR mRNA
ER mRNA
ER protein
OTR mRNA
PGHS-2 mRNA
PGHS-2 protein
PGES mRNA
PGFS mRNA
PGE2 ULF
PGF2 ULF
ER
mRNA
ER
protein
OTR
mRNA
PGHS-2
mRNA
0.41*
0.44*
0.34
0.34
0.71**
0.80**
0.56**
0.74**
PGHS-2
protein
0.35
0.34
0.42*
0.41*
0.54**
0.69**
0.73**
0.57**
0.51**
0.42*
PGES
mRNA
PGFS
mRNA
0.74**
0.58**
0.58**
0.45**
0.37*
0.45**
0.32
0.35
PGE2
ULF
0.41
PGF2
ULF
0.44*
0.49**
0.52**
0.40*
0.41*
0.77**
1
Simple correlations above the diagonal; partial correlations adjusted for treatments below the diagonal.
PR = Progesterone receptor; ER = estradiol receptor; OTR = oxytocin receptor; PGHS-2 = prostaglandin H synthase-2; PGES = prostaglandin
E synthase; PGFS = prostaglandin F synthase; PGE2 = prostaglandin E2.
P < 0.10; *P < 0.05; **P < 0.01.
2
conceptus. By FO elevating endometrial PR mRNA and
PR protein in the SGE, progesterone may have a more
profound effect on the uterus of cows fed FO.
The PR protein also was detected in the deep glands
on d 16 in the bovine endometrium of pregnant cows
(Robinson et al., 1999). Previous studies showed that
GH treatment in ovariectomized ewes receiving ovarian
steroid replacement therapy did not alter expression of
PR in the uterus (Spencer et al., 1999). However, in the
present study bST appeared to have differential effects
on PR protein localization staining intensity depending
on the pregnancy status and diet (Figures 2 and 3, respectively).
As observed in cattle (Meikle et al., 2001), a 6.8-kb
ER mRNA transcript was detected in the endometrium at d 17 following an induced ovulation. On d
17 after GnRH, ER mRNA and protein concentration
were reduced in pregnant compared with cyclic cows,
regardless of whether cows received bST or not. Immunohistochemistry indicated that ER receptor of the
LE, SGE, and DGE of the endometrium of cyclic cows
was greater than that of pregnant cows. Similarly, an
upregulation of ER mRNA and protein in the luminal
epithelium was detected around d 14 to 16 of the estrous
cycle in cyclic cows and expression was very low in
pregnant cows at the same stage (Kimmins and
MacLaren, 2001).
Pulsatile secretion of PGF2 from endometrial tissue,
that initiates luteolysis, is thought to be dependent
upon an increase in OTR concentration within the luminal epithelium. Four transcripts of OTR (5.6, 3.3, 2.1,
and 1.5 kb) were detected in endometrial tissue from a
cow in estrus (Guzeloglu et al., 2004a). All 4 transcripts
were present at relatively low levels in endometrial
tissue at d 17, and the most prominent (5.6 kb) transcript was quantied. In the present study, pregnancy
Journal of Dairy Science Vol. 89 No. 9, 2006
decreased steady-state concentrations of OTR mRNA,
and bST stimulated OTR mRNA in both cyclic control
and FO-fed cows. The decrease in the OTR mRNA concentration in pregnant cows treated with or without
bST may be due to a concurrent decrease in ER mRNA
and protein expression in pregnant cows, as reported
in sheep (Spencer et al., 1995). Indeed, OTR mRNA was
correlated with ER mRNA. The bST stimulation in
OTR mRNA was associated with a concurrent increase
in ER mRNA stimulation in cyclic control and FO-fed
cows. The OTR is undetectable in the bovine endometrium during much of the luteal phase, but increases
on or after d 15 of the estrous cycle (Robinson et al.,
1999). On d 17, at the time of expected initiation of
luteolysis, OTR concentrations were about 10% of what
was seen during estrus (Fuchs et al., 1990). Injection
of oxytocin into nonpregnant cows induced the release
of PGF2 on d 16 (Lamming and Mann, 1995). These
ndings indicate that only subtle increases of OTR concentrations are needed to induce a luteolytic release of
PGF2. Conversely, small but signicant reductions in
OTR mRNA due to pregnancy, as detected in the present study, may have profound inhibitory effects on pulsatile secretion of PGF2.
In the endometrium, PGHS-2 protein was localized
to the luminal epithelium. A PGHS-2 transcript of 4.4
kb, as shown by Liu et al. (2001), and a specic 72-kDa
PGHS-2 protein band were detected in all samples of
endometrium at d 17 following an induced ovulation.
Endometrial expression of PGHS-2 mRNA did not differ
in response to pregnancy, bST treatments, or FO. However, PGHS-2 protein was increased in endometrium of
pregnant cows. The increase in pregnancy was detected
both by Western blotting and immunohistochemistry.
Immunohistochemistry responses revealed an interaction between bST and pregnancy with bST injections
PREGNANCY, SOMATOTROPIN, AND FATTY ACIDS ON ENDOMETRIUM
attenuating the pregnancy increase in PGHS-2 protein
in the LE. This attenuation was not statistically signicant when quantied with Western blotting although there was a 22% reduction. Immunohistochemistry allows for a spatial evaluation of PGHS-2 protein
that was present mainly in the LE and showed inhibitory effects in response to bST in pregnant cows and to
FO feeding in cyclic cows. Such differences cannot be
detected in an endometrial protein homogenate comprised of all cell types. Emond et al. (2004) also reported
that endometrial expression of PGHS-2 protein was
increased during early pregnancy and in response to
intrauterine infusions of IFN-. Arosh et al. (2002)
showed that the PGHS-2 protein concentrations peaked
around d 16 to 18 of the cycle without any changes
in PGHS-2 mRNA concentrations. In sheep, PGHS-2
mRNA levels were similar in cyclic and pregnant ewes,
although PGHS-2 protein expression was maintained
at greater levels in pregnant rather than cyclic ewes
after about d 15 (Charpigny et al., 1997). The expression
of PGHS-2 mRNA was found to be greater in pregnant
ewes between d 10 and 18, with concentrations decreasing by d 16 in cyclic ewes (Kim et al., 2003). The present
study detected no changes in PGHS-2 mRNA; collectively, these ndings are consistent with an upregulation of endometrial PGHS-2 protein during pregnancy,
and support the hypothesis of Charpigny et al. (1997)
that pregnancy may be associated with increased translation efciency and (or) increased stability of PGHS2 protein in the absence of changes in steady-state concentrations of PGHS-2 mRNA. Interestingly, this increased expression of PGHS-2 protein in the endometrium of early pregnancy could explain the greater basal
concentrations of 13,14-dihydro 15-keto PGF2 (PGFM)
reported in pregnant cows (Williams et al., 1983). However, increased basal concentrations of PGFM reect a
secretion pattern that is not luteolytic or pulsatile in
nature. An absence of luteolytic pulses, but greater
basal concentrations, of plasma PGFM indicate that
pregnancy does not suppress completely the endometrias ability to synthesize PG, but alters the pattern
of secretions. Pregnancy-induced expression of PGHS2 protein in the uterus might indicate that the antiluteolytic action of IFN- to suppress pulsatile secretion
of PGF2 is not likely at the level of PGHS-2 expression.
A decline in the concentrations of ER mRNA, ER
protein, and OTR mRNA of pregnant cows treated with
or without bST may have caused a suppressive effect
on pulsatile secretion of PGF2 by endometrium that
would normally initiate luteolysis. In early pregnancy,
constant increases in production of IFN- by well-developed embryos would inhibit the luteolytic pulse generator for secretion of PGF2. The increase in PGHS-2 protein of pregnancy could contribute to the greater basal
3383
concentrations of PGFM detected in the circulation during early pregnancy (Williams et al., 1983). When poorly
developed embryos produce small amounts of IFN- at
the time of pregnancy recognition, pulsatile secretion
of PGF2 may occur, leading eventually to luteolysis
as a consequence of a lack in attenuation of oxytocin
receptor and estrogen receptor expressions.
Furthermore, pregnancy-associated events such as
regulation of localized immune function, angiogenesis,
regulation of blood ow, and development of implantation sites require presence of PGHS-2 protein (Matsumoto et al., 2002). Increased PGHS-2 protein in pregnancy may support these localized responses but the
afferent regulators of PGHS-2 for pulsatile secretion of
PGF2 have been suppressed. Low concentrations of
IFN- have been shown to be inhibitory to PGF2 secretion and PGHS-2 mRNA expression by endometrial
cells in vitro (Guzeloglu et al., 2004b). Therefore, underdeveloped embryos, which would produce reduced
amounts of IFN-, may not survive the process of implantation due to a possible negative effect of reduced
IFN- concentrations on PGHS-2 mRNA. However,
these responses would contribute to extended interestrus intervals for cows losing embryos.
It is important to determine the responses of PGFS
and PGES mRNA (i.e., relative gene expression that
may or may not be related to enzymatic protein or activity) regarding potential downstream metabolism of
PGH2. It is hypothesized that downstream metabolism
of PGH2 to PGF2 could be decreased or conversion of
PGH2 into PGE2 increased, because luteolytic peaks of
PGF2 are reduced in pregnancy. Transcripts of 1.3 kb
for PGES (Arosh et al., 2002) and 1.4 kb for PGFS (Xiao
et al., 1998) were detected in the endometrium at d 17
following an induced ovulation. An interaction between
bST and pregnancy occurred in which bST increased
PGFS mRNA expression in cyclic control-fed cows but
bST decreased PGFS mRNA expression in pregnant
cows. Also, bST increased PGES mRNA expression in
pregnant, cyclic control- and FO-fed cows. Because bST
decreased PGFS mRNA and increased PGES mRNA in
pregnant cows, this may be potentially associated with
more PGE2 and less PGF2 secretions creating a
stronger antiluteolytic signal to maintain pregnancy.
This may be another explanation for the bST-induced
increase in pregnancy rate, beyond that of increased
conceptus development, that contributes to a decrease
in early embryonic loss as previously reported (Moreira
et al., 2001; Santos et al., 2004a). Also the reduction in
PGFS and PGES mRNA due to pregnancy may be a
consequence of the overall reduction in the hormonal
afferent components regulating the PG cascade (i.e.,
ER mRNA and protein, and OTR mRNA). However,
presence of the conceptus differentially modulates enJournal of Dairy Science Vol. 89 No. 9, 2006
3384
BILBY ET AL.
dometrial responsiveness to bST in a manner to reduce
PGFS mRNA and increase PGES mRNA to support
pregnancy. When the conceptus was not present, bST
stimulated ER and OTR mRNA, and this was associated with an increase in PGFS and PGES. Indeed, the
amount of PGE2 in uterine ushings was elevated in
pregnant cows treated with bST. In our study, pregnant
cows had far greater concentrations of PGF2 and PGE2
in uterine ushings than cyclic animals. Bovine conceptuses were able to convert radiolabeled arachidonic acid
into PG (Lewis et al., 1982). Thus, high luminal content
of PG in pregnant ruminants may be due, at least in
part, to PG synthesis and release by the conceptus. Also,
bST increased endometrial PGES mRNA expression in
pregnant cows, possibly accounting for some of the
PGE2 that was increased in bST-treated pregnant cows
compared with non-bST-treated pregnant cows.
Effect of bST treatment on uterine OTR, ER, and
PR genes and ER and PGHS-2 proteins of lactating
Holstein cows was signicant and diverse. Critical regulatory components such as mRNA for OTR, ER,
PGES, and PGFS were stimulated in cyclic cows due to
bST. However, different responses occurred in pregnant
cows, which may reect direct conceptus-induced alterations in regulation of gene expression due to such
agents as IFN-, or indirect conceptus-induced alterations within the endometrium, that effect responsiveness to bST. Spencer et al. (1999) demonstrated
that pretreatment of ewes with IFN- was necessary to
induce endometrial responsiveness to placental lactogen and GH. Badinga et al. (2002) demonstrated, in a
bovine endometrial cell line, that both bovine GH and
IFN- suppressed phorbol 12,13-dibutyrateinduced
PGF2 production. When added in combination there
was an additive effect in reducing PGF2 secretion. The
need for IFN- priming for a GH response also appears
to be true for responses of the GHIGF family (Bilby
et al., 2006b).
The FO had little effect on the endometrial components that directly regulate the PG cascade. This was
not unexpected because EPA and DHA exert their regulatory effects as alternative substrates that reduce the
lipid pool of arachadonic acid in the endometrium (Bilby
et al., 2006a). Such a displacement reduces the overall
amount of arachadonic acid precursor for PG of the 2
series and increases the precursor pools of EPA and
DHA for biosynthesis of 3-series PG (Mattos et al.,
2003).
CONCLUSIONS
At d 17 of the cycle, the potential luteolytic cascade
was affected as pregnancy decreased steady-state concentrations of ER mRNA, ER protein, OTR mRNA,
Journal of Dairy Science Vol. 89 No. 9, 2006
and increased PGHS-2 protein. A decline in the concentrations of ER and OTR would contribute to attenuation in the endometrial pulsatile secretion of PGF2
that would initiate luteolysis in cyclic lactating cows.
Increased PGHS-2 protein in early pregnancy could
contribute to the greater basal concentrations of PGFM
detected in the circulation and support developmental
processes of the conceptusendometrial unit.
Collectively, this study showed that the differential
responses (i.e., PGFS) to bST depend on pregnancy status. The bST may regulate enzymes (i.e., PGES) downstream of PGHS-2 to decrease PGF2 and increase
PGE2, thereby maintaining pregnancy. Pregnancy
seems to attenuate gene expression and protein secretion of hormonal components regulating the PG cascade
thereby reducing pulsatile release of PGF2. Feeding
FO not only increased PR mRNA expression, but also
had differential cellular responses with FO increasing
PR in the SGE that may be benecial for preparation
of the uterus to maintain pregnancy. In addition, feeding FO reduced localization of ER (i.e., LE, DGE, and
SS) and PGHS-2 (i.e., LE), and altered amount of substrate precursor available for PG synthesis (Bilby et al.,
2006a) that would contribute to a reduction in pulsatile
release of PGF2. Development of pharmaceutical (e.g.,
bST) and nutraceutical (e.g., selective fatty acids) management schemes that are integrated with reproductive
management may increase pregnancy rates and warrant further investigation.
ACKNOWLEDGMENTS
Authors thank the staff of the University of Florida
Dairy Research Unit for assistance in feeding and managing the experimental cows. Appreciation is extended
to Larry Eubanks and the staff of the University of
Florida Meats Laboratory for timely and careful sacrice of experimental cows. This journal article was supported by the Florida Agricultural Experiment Station.
Financial support also was received by the NRI Competitive Grant Program-USDA, grant no. 98-35203-6367.
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