Reprod Dom Anim doi: 10.1111/j.1439-0531.2012.01995.
x
ISSN 0936-6768
Conversion of Cortisone to Cortisol and Prostaglandin F2a Production by the
Reproductive Tract of Cows at the Late Luteal Stage In Vivo
HT Duong1,*, DJ Skarzynski2,*, KK Piotrowska-Tomala2, MM Bah2, K Jankowska2, P Warmowski2, K Łukasik2, K Okuda1 and
TJ Acosta1
1
Laboratory of Reproductive Physiology, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan;
2
Department of Reproductive Immunology and Pathology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences,
Olsztyn, Poland
Contents
Previous in vitro studies demonstrated that bovine endome-
trium has the capacity to convert inactive cortisone to
biologically active cortisol (Cr) and that Cr inhibits cytokine-
stimulated prostaglandin F2a (PGF) production. This study
was carried out to test the hypothesis that bovine reproductive
tract has the capacity to convert cortisone to Cr in vivo and to
evaluate the effects of intravaginal application of exogenous
cortisone on uterine PGF secretion during the late luteal stage.
The temporal relationships between PGF and Cr levels in
uterine plasma were also determined. Catheters were inserted
into jugular vein (JV), uterine vein (UV), vena cava caudalis
(VCC) and aorta abdominalis (AA) of six cows on Day 15 of
the oestrous cycle (ovulation = Day 0) for frequent blood
collection. On Day 16, the cows were divided randomly into
two groups and infused intravaginally with vaseline gel (10 ml;
control; n = 3) or cortisone dissolved in vaseline gel (100 mg;
n = 3). Blood samples were collected at )2, )1, )0.5, 0, 0.5, 1,
1.5, 2, 3, 4, 5 and 6 h after treatments (0 h). Intravaginal
application of cortisone increased plasma concentrations of Cr
between 0.5 and 1.5 h in UV, at 0.5 h in VCC, at 1 h in JV and
at 1.5 h in AA. The plasma concentrations of PGF in UV and
of PGF metabolite in JV were greater at 0.5 and 1 h in the
cortisone-treated animals than in control animals. The levels of
PGF in UV blood plasma decreased after Cr reached its
highest levels. The overall findings suggest that the female
reproductive tract has the capacity to convert cortisone to Cr
in vivo. Based on the temporal changes of PGF and Cr levels in
the uterine plasma, a biphasic response in PGF secretion was
found to be associated to the Cr increase induced by the
cortisone treatment at the late luteal stage in non-pregnant
cows.
Introduction
Glucocorticoids (GCs), synthesized from cholesterol in
the adrenal cortex, are involved in the regulation of a
variety of physiological processes, including metabolism
(Wang 2005), immunological response (McKay and
Cidlowski 1999) and female reproductive function
(Brann and Mahesh 1991; Andersen 2002). Cortisol
(Cr), an active GC, is an anti-inflammatory agent that
acts to modulate the production and action of cytokines
and prostaglandins required for ovulation, luteolysis,
embryo implantation, foetal growth and placenta
development as well as parturition (Goppelt-Struebe
1997; Hillier and Tetsuka 1998; Andersen 2002). The
effects of GCs on target tissues are modulated by
11b-hydroxysteroid dehydrogenases (HSD11Bs) (Bush
*These authors contributed equally to this work.
 2012 Blackwell Verlag GmbH
et al. 1968; Lee et al. 2007). Two isoforms of the enzyme
have been identified. The type 1 enzyme (HSD11B1)
mainly converts cortisone to Cr (the active form), while
the type 2 isoform (HSD11B2) inactivates Cr by
metabolizing it to cortisone (Krozowski et al. 1999).
We recently demonstrated that HSD11B1 mRNA
expression in the bovine endometrium changes through-
out the oestrous cycle and that endometrial tissue has
the capacity to convert inactive cortisone to biologically
active Cr in vitro (Lee et al. 2007). However, it remains
unknown whether the bovine reproductive tract has the
capacity to convert cortisone to Cr in vivo.
Prostaglandin F2a (PGF), a hormone synthesized and
secreted from the uterus (Kim and Fortier 1995;
McCracken et al. 1999; Skarzynski et al. 2000), has the
capacity to stimulate conversion of cortisone to Cr by
increasing the expression and enzymatic activity of
HSD11B1 in bovine endometrial stromal cells (Lee et al.
2009). We recently showed that injection of PGF in vivo
induced acute increases in the levels of circulating Cr
(Duong et al. 2011b). On the other hand, Cr can
modulate tumour necrosis factor a-regulated PGF
production and may reduce basal PGF production in
non-pregnant bovine endometrium stromal cells in vitro
(Lee et al. 2007). These findings indicate that Cr has a
role in regulating uterine PGF production. However, it
is unclear whether exogenous cortisone, an inactive GC,
can be converted into active Cr and consequently affects
uterine PGF production at the late luteal phase in cows.
Moreover, the temporal interrelationship between PGF
and Cr in the uterine blood plasma of cows at the late
luteal stage remains unknown.
This study was carried out to test the hypothesis that
bovine reproductive tract has the capacity to convert
cortisone to Cr in vivo and to evaluate the effects of
intravaginal application of exogenous cortisone on
uterine PGF secretion during the late luteal stage. The
temporal relationships between PGF and Cr levels in
uterine blood plasma were also determined.
Materials and Methods
All animal procedures were approved by the Local
Animal Care and Use Committee in Olsztyn, Poland
(Agreement No. 06 ⁄ 2007 ⁄ N).
Animals and surgical procedures
Healthy, normally cycling Polish Holstein Black and
White cows (n = 18) were used for this studies. The
2 HT Duong, DJ Skarzynski, KK Piotrowska-Tomala, MM Bah, K Jankowska, P Warmowski, K Łukasik, K Okuda
and TJ Acosta
animals were culled by their owners (the Farm of Polish
Academy of Sciences in Baranowo, and a private
agriculture farm in Cieszymowo, Poland) from dairy
cows herds because of low milk production. The oestrus
was synchronized using two injections of an analogue of
PGF (dinoprost, Dinolytic; Upjohn – Pharmacia
N.V.S.A., Puurs, Belgium) with an 11-day interval, as
described and recommended in our previous study
(Skarzynski et al. 2009b). Oestrus was determined by
observing external signs (i.e., vaginal mucus, standing
behaviour). Before surgery, the ovaries were examined
daily by transrectal ultrasonography (USG) using a
Draminski Animalprofi Scanner (Draminski Electronics
in Agriculture, Olsztyn, Poland) to determine the day
and side of ovulation and corpus luteum (CL) develop-
ment. The day of ovulation was defined as Day 0 of the
oestrous cycle.
To determine the effective dose of cortisone on Cr and
PGF output, a polyvinyl catheter was inserted into the
jugular vein (JV) of 12 cows on Day 15 of the cycle for
collection of the blood samples as described previously
(Skarzynski et al. 2003).
In the main experiment, the cows (n = 6) were
premedicated with an intramuscular (i.m.) injection of
xylazine at a dose of 25–30 mg per cow (Sedanzin;
Biolwet, Pulawy, Poland). Local anaesthesia was in-
duced by s.c. and i.m. injections of 2% procaine
hydrochloride (Polocainum Hydrochloricum; Biowet,
Drwelew, Poland) in the paralumbar fossa on the side of
ovary with CL. On Day 15, catheters (Medicut Catheter
Kit; Argyle, Japan Sherwood, Tokyo, Japan) were
inserted into JV uterine vein (UV), vena cava caudalis
(VCC) and aorta abdominalis (AA) for frequent blood
collection. A lateral laparotomy was performed for
cannulation of the UV. At surgery, 18-gauge catheters
were inserted into the UV 3–5 cm from uterine horn
before joining the utero-ovarian vein ipsilateral to the
functional CL (Woclawek-Potocka et al. 2010; Duong
et al. 2011b), the VCC and AA and fixed to the
surrounding connective tissue (Skarzynski et al.
2009a). After surgery, the cows were moved to a barn,
where they were fed with grass hay twice daily and were
given free access to water.
Determination of cortisone doses
Twelve cows were used to choose the effective doses of
cortisone. All doses were applied to the vagina via a
catheter near the cervix of the uterus on Day 16 of the
oestrous cycle. The cows were divided randomly into
four groups (n = 3 cows ⁄ group) and then infused with
vaseline gel (10 ml; control group) or three different
doses of cortisone (1, 10, 100 mg; cortisone groups;
Sigma – Aldrich, Chemie Gmbh, Munich, Germany;
No. C2755) dissolved in vaseline gel. The blood samples
were collected from JV at )2, )1, )0.5, 0, 0.5, 1, 1.5 and
2 h and then at 1-h intervals until 6 h after vaseline or
cortisone infusions. The time of cortisone or vaseline
infusion was defined as 0 h. Plasma concentrations of Cr
and 13, 14-dihydro, 15-keto-PGF (PGFM) in plasma
samples were measured. For further examination of
cortisone action at the late luteal stage in cows, a dose of
100 mg cortisone was used.
Effects of cortisone applications on prostaglandin F2a and
Cr concentrations
To examine the possible influence of cortisone on PGF
and Cr release from the reproductive tract, cows were
divided randomly into two groups (n = 3 cows ⁄ group)
and infused intravaginally with vaseline gel (control) or
100 mg of cortisone dissolved in vaseline gel (cortisone)
on Day 16 of the oestrous cycle. Blood samples were
collected from the JV, UV, VCC and AA at )2, )1,
)0.5, 0, 0.5, 1, 1.5 and 2 h and then 1-h intervals until
6 h after treatments. The time of vaseline gel or
cortisone infusion was defined as 0 h. For Cr, PGF
and PGFM determination, blood samples were collected
into sterile 10-ml tubes containing 200 ll of stabilizer
solution (0.3 M EDTA, 1% acetyl salicylic acid, pH 7.4).
All tubes were immediately chilled on ice for 10 min,
centrifuged at 2000 · g for 10 min at 4C, and the
obtained plasma was stored at )30C until further
analysis.
13, 14-dihydro, 15-keto-prostaglandin F2a (PGFM)
determination
The concentrations of 13,14-dihydro,15-keto- PGF
(PGFM) in the plasma samples were determined with
direct EIAs following the method described previously
(Skarzynski et al. 2003) by using horseradish peroxidase
enzyme-labelled PGFM as a tracer (1 : 40 000 final
dilutions) and PGFM antibody (1 : 10 000 final dilu-
tions). The anti-PGFM serum (WS4468-5) was kindly
donated Dr. W.J. Silvia, University of Kentucky,
Lexington, USA and characterized before (Skarzynski
et al. 2003). The cross-reactivity of the PGFM first
antibody with PGF at 50% binding was 2.8%. The
sensitivity of PGFM assays was 50 pg ⁄ ml. The PGFM
standard curve ranged from 32.5 to 8000 pg ⁄ ml, and the
median effective dose (ED50) of the assay was
315 pg ⁄ ml. The intra- and inter-assay coefficients of
variation (CVs) were on average 7.6% and 10.4%,
respectively.
Prostaglandin F2a determination
The concentrations of PGF in the plasma was deter-
mined directly with a double-antibody enzyme immu-
noassay as described previously (Skarzynski et al. 2000)
by using horseradish peroxidase enzyme-labelled PGF
as a tracer (1 : 75 000 final dilutions) and PGF antibody
(kindly donated by Dr. Seiji Ito of Kansai Medical
University, Osaka, Japan; 1 : 100 000 final dilutions).
The cross-reactivity of PGF first antibody with PGFM
at 50% binding was 0.1%. The sensitivity of PGF assays
was 30 pg ⁄ ml. The standard curve ranged from 15.6 to
4000 pg ⁄ ml, and the ED50 of the assay was 400 pg ⁄ ml.
The intra- and inter-assay CVs were 7.3% and 13.2%,
respectively.
Cortisol determination
The concentrations of Cr in the plasma were determined
in duplicate after diethyl ether extraction by second
antibody EIA as described previously (Acosta et al.
2002) by using horseradish peroxidase enzyme-labelled
 2012 Blackwell Verlag GmbH
Cortisone Conversion in Bovine Reproductive Tract 3

Cr as a tracer (1 : 80 000 final dilutions) and Cr (a) 10 Cortisone *b

antibody (raised in a rabbit against Cr-3-CMO; Cosmo *b
Bio Co., Tokyo, Japan; 1 : 400 000 final dilutions). The 8 *b
cross-reactivity of Cr first antibody with cortisone *

Cr (ng/ml)
a
treated at 50% binding was 0.6%. The sensitivity of 6 ab
a
Cr assays was 0.5 ng ⁄ ml. Each plasma sample (200 ll)
4 a
was extracted by diethyl ether as described previously
(Acosta et al. 2000). The residue was evaporated and 2 a a
a
then dissolved in 200 ll assay buffer (40 mM PBS 0.1%
BSA, pH 7.2). To estimate the recovery rate, Cr was 0
added to plasma (1 ng ⁄ ml), and the obtained values
were on average 75% (n = 5). The standard curve (b) 250
*b *b
ranged from 0.4 to 400 ng ⁄ ml, and the ED50 of the assay
200
was 1.6 ng ⁄ ml. The intra- and inter-assay CVs were on c

PGFM (pg/ml)
a c
average 5.4% and 6%, respectively. 150
ac ac
100 a
Statistical analysis a

Experimental data are shown as the mean ± SEM 50 ab ab

(n = 3). The concentrations of PGF, PGFM and Cr in
the blood collected at )0.5 and 0 h were used to
calculate the individual baseline. The data were not
normally distributed (no Gaussian distributions). The
analyses of Cr, PGF and PGFM in plasma samples were
performed using a repeated measures design approach
with treatments and time of sample collection (hours)
being fixed effects with all interactions included, as
described before (Skarzynski et al. 2007, 2009a). The
nonparametric Freidman and Kruskal–Wallis tests
with post hoc test (repeated measurement test ) multi-
ple comparisons of mean ranks) has been used
(GRAPHPAD PRISM Version 5.00; San Diego, CA,
USA; p < 0.05 was considered significant). Least
adjusted means and standard errors, as well as median
and quartiles, were determined. The correlation between
PGF and Cr concentrations after Cs application
in different blood vessels was additionally measured
using linear regression and Pearson correlation (GRAPH-
PAD PRISM).
Results
Determination of cortisone dose
The concentrations of Cr and PGFM in JV plasma in
four groups are shown in Fig. 1. For the PGF and
PGFM concentrations in JV plasma, the main effects of
hour and group and the group · hour interaction were
significant (p < 0.05). The Cr concentration increased
(p < 0.05) between hours 0 and 2, decreased (p < 0.05)
between hours 2 and 4, and did not change thereafter in
the 100 mg cortisone-treated animals. There was not
temporal change in Cr levels in control and animals
infused with lower doses of cortisone (1 and 10 mg;
Fig. 1a). Furthermore, the plasma concentration of
PGFM in JV first increased between hours 0 and 1,
decreased between hours 1 and 2, and again increased
between hours 2 and 3, then decreased until hour 6 in
the 100 mg cortisone-treated animals. However, the Cr
level did not change in the control and animals treated
with lower doses of cortisone (1 and 10 mg; Fig. 1b).
The Cr and PGFM levels were greater in the 100 mg
cortisone-treated animals than in the control and
a a
a ac ac
0
–2 0 2 4 6
Time after cortisone treatment (hours)
Control Cortisone 10 mg
Cortisone 1 mg Cortisone 100 mg
Fig. 1. Plasma concentration of cortisol (Cr; upper panel, a) and
prostaglandin F2a metabolite (PGFM; lower panel, b) in jugular vein
blood after intravaginal infusion of vaseline gel (Control; n = 3) or
cortisone at doses of 1, 10, 100 mg dissolved in vaseline gel
(n = 3 ⁄ dose) on Day 16 of the oestrous cycle. Data are the
mean ± SEM for three samples ⁄ time point. Asterisks indicate signif-
icant differences (p < 0.05) compared with baseline (before cortisone
or vaseline gel treatment). Different superscript letters indicate
significant differences (p < 0.05) among groups at the same time
point. Bars show Cr concentration in jugular venous plasma of the
cows infused intravaginally with vaseline gel (Control, n = 3)
animals treated with lower doses of cortisone (1 and
10 mg cortisone; p < 0.05) at 1 and 3 h post-treatment.
Based on these results, a cortisone dose of 100 mg was
used for further studies to investigate the local effect of
cortisone on release of Cr and PGF during the late luteal
phase in cows.
Effects of cortisone application on Cr concentrations
during the late luteal phase
For the Cr concentrations, the hour effect was signifi-
cant (p < 0.05). Intravaginal application of cortisone
on Day 16 of oestrous cycle induced a significant
(p < 0.05) increase in the plasma concentrations of Cr
between 0.5 and 1.5 h in UV, at 0.5 h in VCC, at 1 h in
JV and at 1.5 h in AA (Fig. 2). Furthermore, plasma Cr
levels were highest in UV.
Effects of cortisone application on prostaglandin F2a and
PGFM concentrations during the late luteal phase
For the concentrations of PGF in UV and PGFM in JV
plasma, the main effect of hour and groups and the
group · hour interaction were significant (p < 0.05).
Concentrations of PGF in UV and PGFM in JV (Fig. 3)

 2012 Blackwell Verlag GmbH

4 HT Duong, DJ Skarzynski, KK Piotrowska-Tomala, MM Bah, K Jankowska, P Warmowski, K Łukasik, K Okuda
and TJ Acosta

25 (p < 0.05) at 0.5 and 1 h in cortisone-treated animals

Cortisone
*
than in control animals on Day 16 of the oestrous cycle.
20 * * * The increase in the concentration of Cr (Fig. 2) and
*
* PGF (Fig. 3) in UV plasma occurred at the same time,
Cr (ng/ml)

15 and a high correlation between both measurements was

found (Pearson r = 0.76; p < 0.001). However, the
10 levels of UV PGF decreased after Cr reached its highest
levels.
5

0 Discussion
–2 0 2 4 6
Time after cortisone treatment (h)
The present study clearly demonstrated that the bovine
reproductive tract (uterus or ⁄ and vagina) has the
Uterine vein Aorta abdominalis
Vena cava caudalis Jugular vein
capacity to convert cortisone to Cr at the late luteal
stage in vivo. Moreover, intravaginal application of
Fig. 2. Plasma concentrations of cortisol (Cr) in uterine vein, vena exogenous cortisone (converted to endogenous Cr) on
cava caudalis, aorta abdominalis and jugular venous blood in cortisone- Day 16 of oestrous cycle significantly increased uterine
treated group (n = 3) on Day 16 of the oestrous cycle. Data are the PGF output in cows. These findings support that Cr
mean ± SEM for three samples ⁄ time point. Asterisks indicate signif- plays a role in regulating uterine PGF secretion at the
icant differences (p < 0.05) in Cr concentrations compared with the
baseline (before cortisone application) late luteal phase in cows.
Cr synthesized in the adrenal cortex affects many
organs. The circulating peripheral levels of Cr are
(a) 4 relatively constant throughout the oestrous cycle in
Cortisone *a cattle (Garverick et al. 1971; Roussel et al. 1983; Lyimo
et al. 2000). However, local Cr concentrations have been
3 shown to be modulated by HSD11B1 and HSD11B2 in
*a different organs of several species (Krozowski et al.
PGF (ng/ml)

1999). HSD11B1 acts predominantly as a NADP(H)-

2 dependent reductase to generate active Cr from inactive
cortisone. Our previous in vitro study demonstrated that
bovine endometrial explants, as well as cultured endo-
1 metrial cells, have the capacity to convert cortisone to
Cr (Lee et al. 2007). It has been shown that the
HSD11B1 is expressed in liver and adipose tissue, with
0 the highest activity normally observed in the liver (Yang
et al. 1992; Kushnir et al. 2004). In addition to liver,
(b) 150 Cortisone Control HSD11B1 was previously found to be expressed in the
Cortisone ovine and bovine uterus during the oestrous cycle (Yang
et al. 1996; Lee et al. 2007; Simmons et al. 2010). It was
a *
a
PGFM (pg/ml)

100 * of interest to know whether the bovine reproductive

50
0
–4 –2 0 2
Time after cortisone treatment (h)
Fig. 3. Plasma concentrations of prostaglandin F2a (PGF) in uterine
venous blood; (upper panel, a) and prostaglandin F2a metabolite
(PGFM) in jugular vein (lower panel, b) of cortisone-treated group
(cortisone, n = 3) or vaseline-treated group (control, n = 3) on Day
16 of the oestrous cycle. Data are the mean ± SEM for three
samples ⁄ time point. Asterisks indicate significant differences
(p < 0.05) compared with baseline (before cortisone or vaseline gel
treatment). Superscript letters indicate significant differences
(p < 0.05) between cortisone and control groups at the same time
point
increased between hours 0 and 1, decreased between
hour 1 and 1.5 and did not change thereafter. The levels
of PGF in UV and PGFM in JV plasma were greater
tract has the ability to convert cortisone to Cr in vivo at
the late luteal stage. In the present study, the concen-
trations of Cr increased in UV earlier than in VCC, JV
and AA; and plasma Cr levels were highest in UV. These
findings indicated that the increase in the levels of Cr
detected in the UV plasma is mainly because of the
4 6
conversion of cortisone to Cr by the bovine reproductive
tract at the late luteal stage and that Cr converted by the
reproductive tract enters into the UV and then is diluted
in the systemic circulation. Therefore, more time is
required to detect a significant increase in Cr concen-
trations in AA.
Furthermore, intravaginal application of cortisone
increased the concentrations of PGF and its metabolite–
PGFM within 3 h after treatment. Presently, intravaginal
administration is commonly used to administer antimi-
crobials, labour-inducing agents, prostaglandins and
steroids (Vermani and Garg 2000; Nyende et al. 2004).
The vaginal mucosa has good absorption potential
(Baloglu et al. 2009). Many vaginal formulations are
applied in the form of suppositories (Norman et al. 1991;
Pasquale et al. 1997), gelatin capsules (Miles et al. 1994)
and recently as bio-adhesive gels (Ross et al. 1997).

 2012 Blackwell Verlag GmbH

Cortisone Conversion in Bovine Reproductive Tract
Intra-vaginal administration of progesterone avoids liver
first-pass metabolism and has no systemic side-effects
(Levy et al. 2000). Recently, vaginal drug delivery has
gained further interest as a result of investigations
showing the existence of uterine first-pass effect (Bulletti
et al. 1997). The findings indicate that drugs adminis-
tered through the vaginal route are transported into the
uterus achieving higher tissue concentrations than if
administered orally or intramuscularly (Devroey et al.
1989). Thus, in our experiment, we have used the
intravaginal route to introduce the cortisone dissolved
in vaseline gel into the cows to study its local biological
effects.
PGF was found to stimulate HSD11B1 in human
placenta (Alfaidy et al. 2001) and increased the local
conversion of cortisone to Cr by increasing HSD11B1 in
the cow (Lee et al. 2009). GCs stimulated PG synthesis in
foetal membranes of human (Sun et al. 2003; Mirazi et al.
2004) and sheep (Zhang et al. 2006). These findings imply
the presence of a positive feedback loop between local
PGF and Cr synthesis during late pregnancy and labour
(Alfaidy et al. 2001, 2003). In cattle, our previous in vitro
study showed that Cr reduces basal PGF production in
non-pregnant bovine endometrial stromal cells, whereas
it did not affect epithelial cells PGF production (Lee et al.
2007). However, stromal cells are the main source of
PGE2 production (Murakami et al. 2001) while epithelial
cells are the main source of PGF synthesis (Kim and
Fortier 1995; Skarzynski et al. 2000). Until now, it has
been unclear whether exogenous cortisone, which is
converted to endogenous Cr, affects uterine PGF secre-
tion in cow at the late luteal stage in vivo. The increased
levels of HSD11B1 mRNA and bioactivity were tempo-
rally coincident with the increase in the basal release of
PGF during the oestrous cycle (Miyamoto et al. 2000;
Murakami et al. 2001; Lee et al. 2007). Furthermore,
treatment of metyrapone, an inhibitor of HSD11B1, to
reduce the local availability of Cr converted from
cortisone in the bovine reproductive tract at the late
luteal stage prolonged the luteal phase in cows (Duong
et al. 2011a). Thus, Cr may play a role in regulating PGF
secretion in vivo. A recent in vitro study using HSD11B1
inhibitor to block the conversion of cortisone to Cr
showed that cortisone does not affect directly PGF
production in non-pregnant endometrial tissue at late
luteal stage (Doung HT, Okuda K & Acosta TJ, unpublished
data). In the present study, we found that cortisone
infusion increased PGF and PGFM concentrations in the
UV and JV blood plasma, respectively, and that the
increase in uterine levels of PGF and Cr occurred
synchronously. These findings suggest that Cr converted
from exogenous cortisone can stimulate PGF secretion
from the bovine uterus. In fact, PGF treatment has been
shown to increase the levels of Cr in vitro (Lee et al. 2009)
and in vivo (Shrestha et al. 2010; Duong et al. 2011b). The
above results suggest that a positive feedback loop
between local Cr and PGF could play a role during
luteolysis in the bovine uterus. However, the exact role of
Cr, as well the mechanisms of it action on PGF output
from the bovine uterus, is not clarified yet.
Regulation of the pulsatile PGF release could be a
possible role of Cr during luteolysis. The pulsatile
 2012 Blackwell Verlag GmbH
5
release of PGF from the uterus in the late luteal phase
induces luteolysis in many species including cattle
(Poyser 1995). Many local, uterine factors may serve as
signals or triggers of PGF output from uterus
(Miyamoto et al. 2000; Okuda et al. 2002). Recent
in vivo studies (Shrestha et al. 2010; Duong et al.
2011b) have shown that Cr plays a role in regulating
PGF secretion in the bovine uterus during PGF-
induced luteolysis. In addition, it has been demon-
strated the temporal association between a PGFM
pulse and a Cr pulse during spontaneous luteolysis in
mare (Ginther and Beg 2009). Thus, Cr may be also one
of the factors responsible for the generation of PGF
pulses during luteolysis (Duong et al. 2011b). However,
it is unknown whether Cr directly regulates PGF
secretion at the late luteal stage. Lee et al. (2007) showed
that HSD11B1 mRNA expression and activity are
highest during luteolysis and the follicular phase. The
frequent – pulsatile release of PGF with high amplitude
was also observed during spontaneous luteolysis and
later (Days 17–20 of the cycle) (Parkinson and Lamming
1990; Kotwica et al. 1998; Ginther et al. 2010). Further-
more, a concomitant elevation of uterine PGF and Cr
concentrations was observed after an analogue of PGF
injection and then concentration of PGF decreased after
Cr reached its highest levels (Duong et al. 2011b).
Interestingly, in the present study, the levels of PGF in
UV blood plasma also decreased after Cr reached its
highest levels. Thus, Cr may prevent excessive uterine
PGF secretion within a short time period. The above
findings support our previous hypothesis (Duong et al.
2011b) that Cr is one of the factors responsible for the
generation of PGF pulses in cattle. However, future
studies are needed to clarify how Cr induces an acute
elevation of PGF concentration and prevents long-
lasting excessive PGF secretion in the bovine uterus
resulting in successive PGF pulses.
In conclusion, the bovine reproductive tract has the
capacity to convert inactive cortisone to bioactive Cr
in vivo. Based on the temporal changes of PGF and Cr in
the uterine circulation, a biphasic response in PGF
secretion was found to be associated to the Cr increase
induced by the cortisone treatment at the late luteal
stage in non-pregnant cows.
Acknowledgements
We thank Messrs. Henryk Jablonski and Maciej Baurycza for
providing animals for this study, Draminski Electronics in Agriculture
for providing a USG scanner, Dariusz Kiński, Jarosaw Kowiel,
Mateusz Sekua and Mateusz Wisniewski for technical assistance, and
Drs. Seiji Ito, Kansai Medical University, and W.J. Silvia, University
of Kentucky, for antisera of PGF and PGFM, respectively. This work
was supported by the Research Grant of the Polish Ministry of
Sciences and Higher Education (Grant No. 308327933) and Grant-in-
Aid for Scientific Research from the Japan Society for the Promotion
of Science (JSPS; No. 22580318). H.T. Duong is supported by a
scholarship from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. K.K.P-T is supported by the European Union
with the European Social Fund (Dr.INNO2, Olsztyn, Poland).
Conflict of interest
None of the authors have any conflict of interest to declare.
6 HT Duong, DJ Skarzynski, KK Piotrowska-Tomala, MM Bah, K Jankowska, P Warmowski, K Łukasik, K Okuda
and TJ Acosta
Author contributions
H.T. Duong-designed study, performed experiments, analysed results,
drafted paper; D.J. Skarzynski-designed study, reviewed manuscript,
K.K. Piotrowska-Tomala-performed experiments, analysed results;
M.M. Bah-designed study, performed surgery; K. Jankowska-analysed
References
Acosta TJ, Ozawa T, Kobayashi S, Hayashi
K, Ohtani M, Kraetzl WD, Sato K,
Schams D, Miyamoto A, 2000: Periovu-
latory changes in the local release of
vasoactive peptides, prostaglandin F2a,
and steroid hormones from bovine
mature follicles in vivo. Biol Reprod 63,
1253–1261.
Acosta TJ, Yoshizawa N, Ohtani M, Mi-
yamoto A, 2002: Local changes in blood
flow within the early and midcycle corpus
luteum after prostaglandin F2a injection
in the cow. Biol Reprod 66, 651–658.
Alfaidy N, Xiong ZG, Myatt L, Lye SJ,
MacDonald JF, Challis JR, 2001: Prosta-
glandin F2a potentiates cortisol produc-
tion by stimulating 11b-hydroxysteroid
dehydrogenase 1: a novel feedback loop
that may contribute to human labor.
J Clin Endocrinol Metab 86, 5585–5592.
Alfaidy N, Li W, MacIntosh T, Yang K,
Challis J, 2003: Late gestation increase in
11b-hydroxysteroid dehydrogenase 1
expression in human fetal membranes: a
novel intrauterine source of cortisol.
J Clin Endocrinol Metab 88, 5033–5038.
Andersen CY, 2002: Commentary – possible
new mechanism of cortisol action in
female reproductive organs: physiological
implications of the free hormone hypoth-
esis. J Endocrinol 173, 211–217.
Baloglu E, Senyigit ZA, Karavana SY,
Bernkop-Schnurch A, 2009: Strategies to
prolong the intravaginal residence time of
drug delivery systems. J Pharm Pharm Sci
12, 312–336.
Brann DW, Mahesh VB, 1991: Role of
corticosteroids in female reproduction.
FASEB J 5, 2691–2698.
Bulletti C, deZiegler D, Giacomucci E, Polli
V, Rossi S, Alfieri S, Flamigni C, 1997:
Vaginal drug delivery: the first uterine
pass effect. In: Bulletti C, deZiegler D,
Guller S, Levitz M (eds), Uterus: Endo-
metrium and Myometrium, Vol. 828. New
York Academy of Sciences, New York.
pp. 285–290.
Bush IE, Hunter SA, Meigs RA, 1968:
Metabolism of 11-oxygenated steroids.
Metabolism in vitro by preparations of
liver. Biochem J 107, 239–258.
Devroey P, Palermo G, Bourgain C, Van
Waesberghe L, Smitz J, Van Steirteghem
AC, 1989: Progesterone administration in
patients with absent ovaries. Int J Fertil
34, 188–193.
Duong HT, Piotrowska-tomala KK, Acosta
TJ, Bah MM, Sinderewicz E, Majewska
M, Jankowska K, Okuda K, Skarzynski
DJ, 2011a: Effects of cortisol on preg-
nancy rate and corpus luteum function in
heifers: an in vivo study. J Reprod Dev 58,
(doi:10.1262/jrd.11-122T).
Duong HT, Vu HV, Bah MM, Woclawek-
Potocka I, Dam TV, Skarzynski DJ,
Okuda K, Acosta TJ, 2011b: Acute
changes in the concentrations of prosta-
results; P. Warmowski-analysed results, K. Łukasik-performed exper-
iments, analysed results, K. Okuda-reviewed paper; T.J. Acosta-
designed study, performed surgery, reviewed paper. All authors have
approved the final article.
glandin F2a (PGF) and cortisol in uterine
and ovarian venous blood during PGF-in-
duced luteolysis in cows. Reprod Domest
Anim 47, (doi: 10.1111/j.1439-0531.2011.
01835.x).
Garverick HA, Erb RE, Niswender GD,
Callahan CJ, 1971: Reproductive steroids
in the bovine. 3. Changes during the
estrous cycle. J Anim Sci 32, 946–956.
Ginther OJ, Beg MA, 2009: Concentrations
of circulating hormones normalized to
pulses of a prostaglandin F2a metabolite
during spontaneous luteolysis in mares.
Theriogenology 72, 1111–1119.
Ginther OJ, Shrestha HK, Fuenzalida MJ,
Shahiduzzaman AK, Beg MA, 2010:
Characteristics of pulses of 13,14-dihy-
dro-15-keto-prostaglandin F2a before,
during, and after spontaneous luteolysis
and temporal intrapulse relationships
with progesterone concentrations in
cattle. Biol Reprod 82, 1049–1056.
Goppelt-Struebe M, 1997: Molecular mecha-
nisms involved in the regulation of prosta-
glandin biosynthesis by glucocorticoids.
Biochem Pharmacol 53, 1389–1395.
Hillier SG, Tetsuka M, 1998: An anti-
inflammatory role for glucocorticoids in
the ovaries? J Reprod Immunol 39, 21–27.
Kim JJ, Fortier MA, 1995: Cell type spec-
ificity and protein kinase C dependency
on the stimulation of prostaglandin E2
and prostaglandin F2a production by
oxytocin and platelet-activating factor in
bovine endometrial cells. J Reprod Fertil
103, 239–247.
Kotwica J, Skarzynski D, Jaroszewski J,
Williams GL, Bogacki M, 1998: Uterine
secretion of prostaglandin F2a stimulated
by different doses of oxytocin and
released spontaneously during luteolysis
in cattle. Reprod Nutr Dev 38, 217–226.
Krozowski Z, Li KX, Koyama K, Smith RE,
Obeyesekere VR, Stein-Oakley A, Sasano
H, Coulter C, Cole T, Sheppard KE, 1999:
The type I and type II 11b-hydroxysteroid
dehydrogenase enzymes. J Steroid Bio-
chem Mol Biol 69, 391–401.
Kushnir MM, Neilson R, Roberts WL,
Rockwood AL, 2004: Cortisol and corti-
sone analysis in serum and plasma by
atmospheric pressure photoionization
tandem mass spectrometry. Clin Biochem
37, 357–362.
Lee HY, Acosta TJ, Tanikawa M, Sakum-
oto R, Komiyama J, Tasaki Y, Piskula
M, Skarzynski DJ, Tetsuka M, Okuda K,
2007: The role of glucocorticoid in the
regulation of prostaglandin biosynthesis
in non-pregnant bovine endometrium.
J Endocrinol 193, 127–135.
Lee HY, Acosta TJ, Skarzynski DJ, Okuda
K, 2009: Prostaglandin F2a stimulates
11b-hydroxysteroid dehydrogenase 1
enzyme bioactivity and protein expression
in bovine endometrial stromal cells. Biol
Reprod 80, 657–664.
Levy T, Yairi Y, Bar-Hava I, Shalev J,
Orvieto R, Ben-Rafael Z, 2000: Pharma-
cokinetics of the progesterone-containing
vaginal tablet and its use in assisted
reproduction. Steroids 65, 645–649.
Lyimo ZC, Nielen M, Ouweltjes W, Kruip
TAM, van Eerdenburg F, 2000: Relation-
ship among estradiol, cortisol and inten-
sity of estrous behavior in dairy cattle.
Theriogenology 53, 1783–1795.
McCracken JA, Custer EE, Lamsa JC, 1999:
Luteolysis: a neuroendocrine-mediated
event. Physiol Rev 79, 263–323.
McKay LI, Cidlowski JA, 1999: Molecular
control of immune ⁄ inflammatory res-
ponses: interactions between nuclear fac-
tor-kappa B and steroid receptor-signaling
pathways. Endocr Rev 20, 435–459.
Miles RA, Paulson RJ, Lobo RA, Press MF,
Dahmoush L, Sauer MV, 1994: Pharma-
cokinetics and endometrial tissuel levels
of progesterone after administration by
intramuscular and vaginal routes – a
comparative-study. Fertil Steril 62, 485–
490.
Mirazi N, Alfaidy N, Martin R, Challis JR,
2004: Effects of dexamethasone and sul-
fasalazine on prostaglandin E2 output by
human placental cells in vitro. J Soc
Gynecol Investig 11, 22–26.
Miyamoto Y, Skarzynski DJ, Okuda K,
2000: Is tumor necrosis factor a a trigger
for the initiation of endometrial prosta-
glandin F2a release at luteolysis in cattle?
Biol Reprod 62, 1109–1115.
Murakami S, Miyamoto Y, Skarzynski DJ,
Okuda K, 2001: Effects of tumor necrosis
factor a on secretion of prostaglandins E2
and F2a in bovine endometrium through-
out the estrous cycle. Theriogenology 55,
1667–1678.
Norman TR, Morse CA, Dennerstein L,
1991: Comparative bioavailability of or-
ally and vaginally administered progester-
one. Fertil Steril 56, 1034–1039.
Nyende L, Towobola OA, Mabina MH,
2004: Comparison of vaginal and oral
misoprostol, for the induction of labour
in women with intra-uterine foetal death.
East Afr Med J 81, 179–182.
Okuda K, Miyamoto Y, Skarzynski DJ,
2002: Regulation of endometrial prosta-
glandin F2a synthesis during luteolysis
and early pregnancy in cattle. Domest
Anim Endocrinol 23, 255–264.
Parkinson TJ, Lamming GE, 1990: Interre-
lationships between progesterone, 13,14-
dihydro-15-keto F2a (PGFM) and LH in
cyclic and early pregnant cows. J Reprod
Fertil 90, 221–233.
Pasquale SA, Foldesy RG, Levine JP, Bach-
mann GA, Blackwell RE, 1997: Peripheral
progesterone (P) levels and endometrial
response to various dosages of vaginally
administered P in estrogen-primed women.
Fertil Steril 68, 810–815.
Poyser NL, 1995: The control of prostaglan-
din production by the endometrium in
relation to luteolysis and menstruation.
Prostaglandins Leukot Essent Fatty Acids
53, 147–195.
 2012 Blackwell Verlag GmbH
Cortisone Conversion in Bovine Reproductive Tract
Ross D, Cooper AJ, PryseDavies J, Berger-
on C, Collins WP, Whitehead MI, 1997:
Randomized, double-blind, dose-ranging
study of the endometrial effects of a
vaginal progesterone gel in estrogen-trea-
ted postmenopausal women. Am J Obstet
Gynecol 177, 937–941.
Roussel JD, Clement TJ, Aranas TJ, Seybt
SH, 1983: Changes in circulating plasma
levels of cortisol in lactating and non-
lactating dairy cattle during the estrous
cycle. Theriogenology 19, 535–539.
Shrestha HK, Beg MA, Imam S, Ginther
OJ, 2010: Luteal blood flow and concen-
trations of circulating progesterone and
other hormones associated with a
simulated pulse of 13,14-dihydro-15-
keto-prostaglandin F2a in heifers. Repro-
duction 139, 673–683.
Simmons RM, Satterfield MC, Welsh TH Jr,
Bazer FW, Spencer TE, 2010: HSD11B1,
HSD11B2, PTGS2, and NR3C1 expres-
sion in the peri-implantation ovine uterus:
effects of pregnancy, progesterone, and
interferon tau. Biol Reprod 82, 35–
43.
Skarzynski DJ, Miyamoto Y, Okuda K,
2000: Production of prostaglandin F2a by
cultured bovine endometrial cells in
response to tumor necrosis factor a: cell
type specificity and intracellular mecha-
nisms. Biol Reprod 62, 1116–1120.
Skarzynski DJ, Jaroszewski JJ, Bah MM,
Deptula KM, Barszczewska B, Gaw-
ronska B, Hansel W, 2003: Administra-
tion of a nitric oxide synthase inhibitor
counteracts prostaglandin-F-induced
luteolysis in cattle. Biol Reprod 68,
1674–1681.
 2012 Blackwell Verlag GmbH
Skarzynski DJ, Woclawek-Potocka I, Kor-
zekwa A, Bah MM, Piotrowska K, Bars-
zczewska B, Okuda K, 2007: Infusion of
exogenous tumor necrosis factor dose
dependently alters the length of the luteal
phase in cattle: differential responses to
treatment with indomethacin and
L-NAME, a nitric oxide synthase inhib-
itor. Biol Reprod 76, 619–627.
Skarzynski DJ, Piotrowska KK, Bah MM,
Korzekwa A, Woclawek-Potocka I, Sawai
K, Okuda K, 2009a: Effects of exogenous
tumour necrosis factor a on the secretory
function of the bovine reproductive tract
depend on tumour necrosis factor a con-
centrations. Reprod Domest Anim 44,
371–379.
Skarzynski DJ, Siemieniuch MJ, Pilawski
W, Potocka IW, Bah MM, Majewska M,
Jaroszewski JJ, 2009b: In vitro assessment
of progesterone and prostaglandin E2
production by the corpus luteum in cattle
following pharmacological synchroniza-
tion of estrus. J Reprod Dev 55, 170–176.
Sun K, Ma RL, Cui XL, Campos B,
Webster R, Brockman D, Myatt L,
2003: Glucocorticoids induce cytosolic
phospholipase A(2) and prostaglandin H
synthase type 2 but not microsomal pros-
taglandin E synthase (PGES) and cyto-
solic PGES expression in cultured
primary human amnion cells. J Clin
Endocrinol Metab 88, 5564–5571.
Vermani K, Garg S, 2000: The scope and
potential of vaginal drug delivery. Pharm
Sci Technolo Today 3, 359–364.
Wang M, 2005: The role of glucocorticoid
action in the pathophysiology of the
metabolic syndrome. Nutr Metab 2, 1–14.
7
Woclawek-Potocka I, Kowalczyk-Zieba I,
Skarzynski DJ, 2010: Lysophosphatidic
acid action during early pregnancy in the
cow: in vivo and in vitro studies. J Reprod
Dev 56, 411–420.
Yang K, Smith CL, Dales D, Hammond
GL, Challis JRG, 1992: Cloning of an
ovine 11b-hydroxysteroid dehydrogenase
complementary deoxyribonucleic-acid –
tissue and temporal distribution of its
messenger-ribonucleic-acid during fetal
and neonatal development. Endocrinol-
ogy 131, 2120–2126.
Yang K, Fraser M, Yu M, Krkosek M,
Challis JR, Lamming GE, Campbell LE,
Darnel A, 1996: Pattern of 11 b-hydrox-
ysteroid dehydrogenase type 1 messenger
ribonucleic acid expression in the ovine
uterus during the estrous cycle and preg-
nancy. Biol Reprod 55, 1231–1236.
Zhang Q, Collins V, Chakrabarty K, Wolf
RF, Unno N, Howe D, Rose JC, Wu
WX, 2006: Regulation of membrane-
associated prostaglandin E2 synthase 1
in pregnant sheep intrauterine tissues by
glucocorticoid and estradiol. Endocrinol-
ogy 147, 3719–3726.
Submitted: 7 Oct 2011; Accepted: 21 Jan
2012
Authors’s address (for correspondence):
Dr Tomas J. Acosta, Laboratory of Repro-
ductive Physiology, Graduate School of
Natural Science and Technology, Okayama
University, Tsushima Naka, Kita-ku 1-1-1,
Okayama 700-8530, Japan. E-mail: acosta@
cc.okayama-u.ac.jp