MSD Finca Productiva Reproduccion

1. Animal Reproduction Science 82–83 (2004) 281–294
Steroidal regulation of uterine immune defenses
G.S. Lewis∗
United States Department of Agriculture, Agricultural Research Service, U.S. Sheep Experiment Station,
HC 62 Box 2010, Dubois, ID 83423, USA
Abstract
Progesterone suppresses uterine immune defenses and predisposes postpartum animals to non-
specific uterine infections. Progesterone can also suppress uterine eicosanoid synthesis. This ef-
fect of progesterone seems to be an important factor in the onset of uterine infections because
eicosanoids can enhance uterine immune defenses. In fact, exogenous prostaglandin F2 (PGF2 ),
an eicosanoid that stimulates uterine PGF2 production, enhances uterine immune defenses and
promotes the ability of ewes and sows to resolve uterine infections, even when progesterone is
maintained at luteal phase concentrations. Prostaglandin F2 is also a proinflammatory molecule
that stimulates the production of proinflammatory cytokines and may enhance uterine production of
leukotriene B4 (LTB4 ), which stimulates various neutrophil functions. Neutrophils seem to mount
the initial response to bacteria that enter the uterus, and proinflammatory cytokines and LTB4 en-
hance phagocytic activity of neutrophils. Even though there are clear associations among PGF2 ,
LTB4 , proinflammatory cytokines, phagocytosis, and the ability of the uterus to resist or resolve
infections, the mechanisms of action of exogenous PGF2 in mitigating the immunosuppressive
effects of progesterone have not yet been defined. However, defining the PGF2 mechanisms should
yield important new information that can be used to develop novel prevention and treatment strate-
gies that do not rely on antibiotic and antimicrobial compounds for managing uterine infections.
Published by Elsevier B.V.
Keywords: Uterus; Infections; Progesterone; Prostaglandins; Eicosanoids
1. Introduction
Progesterone suppresses uterine immune defenses and predisposes the uterus to nonspe-
cific infections. This occurs most commonly in postpartum animals, and postpartum uterine
infections may reduce the reproductive performance of livestock (Arthur et al., 1989; Lewis,
∗ Tel.: +1-208-374-5306; fax: +1-208-374-5582.
E-mail address: glewis@pw.ars.usda.gov (G.S. Lewis).
0378-4320/$ – see front matter. Published by Elsevier B.V.
doi:10.1016/j.anireprosci.2004.04.026

2. 282 G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294
1997; Dhaliwal et al., 2001). Even though the immunosuppressive effects of progesterone
have been recognized for at least 50 years (Black et al., 1953; Rowson et al., 1953), the
complete mechanisms of action of progesterone are not known. However, recent research
has revealed important clues about how progesterone suppresses uterine immune defenses,
and those clues are critical for developing new prevention and treatment strategies that do
not rely on antibiotic and antimicrobial compounds for managing uterine infections.
Uterine infections are called nonspecific when numerous potentially pathogenic bacteria
can be isolated from infected uteri; the initial colonizing bacteria are not known; and the
specific bacteria that cause the signs of infection are not known (Griffin et al., 1974a,b; Del
Vecchio et al., 1994; Lewis, 1997). Despite that, Arcanobacterium pyogenes and Escherichia
coli are most often associated with spontaneous uterine infections in livestock (Griffin et al.,
1974a,b; Del Vecchio et al., 1994; Dhaliwal et al., 2001). Thus, A. pyogenes and E. coli are
often used to induce uterine infections for studies designed to define the permissive role of,
for example, progesterone (Lewis, 2003; Wulster-Radcliffe et al., 2003).
The annual incidences of uterine infections in postpartum animals range from 10 to 50% of
the dairy cattle (Arthur et al., 1989; Lewis, 1997), 20 to 75% of the dairy buffaloes (Usmani
et al., 2001), and 5 to 10% of the dairy sheep (Tzora et al., 2002) in a given herd or flock. Pub-
lished incidences of similar uterine infections, rather than the mastitis–metritis–agalactica
syndrome, in postpartum sows are not available. However, based on personal communi-
cations with swine herd managers and veterinarians and scientists with swine genetics
companies, uterine infections seem to be “common,” but the specifics are often considered
to be proprietary. Nevertheless, one study indicates that uterine infections during the luteal
phase in pigs are associated with increased embryonal deaths (Scofield et al., 1974), and
another report indicated that 42% of the gilts and 39% of the sows in Finland were culled
because of impaired fertility, which was the most common reason for culling female pigs
(Heinonen et al., 1998). Even though numerous authors have reported the incidences of post-
partum uterine infections for cattle, buffaloes, and sheep, the reports are rough estimates.
The “true” incidence of uterine infections for any livestock species is not known. This is
because detection and diagnosis are often inaccurate; most postpartum animals are not eval-
uated for signs of uterine infections; and uterine infections are not considered contagious,
as is brucellosis, for example, so reporting is not mandatory (Lewis, 1997).
Intramuscular (i.m.) injections of prostaglandin F2 (PGF2 ) are an efficacious treatment
for pyometra in cattle (Lewis, 1997; Sheldon and Noakes, 1998). Pyometra seems to be the
most common type of uterine infection in dairy cattle, and pyometra is the type typically
associated with impaired reproductive performance (Lewis, 1997). Pyometra is defined
simply as pus in the uterus, but, without extensive clinical and histological evaluations,
one cannot determine whether all layers of the uterine wall are involved and whether the
pathogens that caused the pus to form in the uterus have escaped into the body cavity and
the circulatory system. Thus, the term can be applied to a condition with a wide range of
consequences. Progesterone plays a permissive role in the onset of pyometra, which usually
develops coincidently with luteal function during the postpartum period (Lewis, 1997). The
belief is that potentially pathogenic bacteria that reside in livestock environments enter the
uterus during or after calving. Cows with assisted births and cows in which retained fetal
membranes are removed manually seem to be the most vulnerable because attendants, in
effect, inoculate the uterus with bacteria (Lewis, 1997). In cows that develop pyometra, the

3. G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 283
introduced bacteria apparently reside in the uterus, without proliferating into an infection,
until luteal progesterone suppresses uterine immune functions. The bacteria are then able
to proliferate and produce the signs of infection. Pyometra usually persists until luteolysis,
when reduced progesterone concentrations no longer suppress uterine immune defenses
and the uterus is able to resolve the infection. Because it is luteolytic, exogenous PGF2
is injected to reduce progesterone concentrations and promote the resolution of uterine
infections. However, the true mechanism of action of PGF2 in resolving uterine infections
is not known. In fact, PGF2 has effects that are not related to its effects on luteal function.
Despite the fact that exogenous PGF2 is efficacious, intrauterine and systemic antibiotic
treatments are still common, and new antibiotic treatments are being introduced (Lewis,
1997; Sheldon and Noakes, 1998; Chenault et al., 2001). Genuine concerns about antibiotic
use in livestock and the potential for creating antibiotic-resistant strains of bacteria have
focused our research on determining whether nonantibiotic, native compounds will enhance
host immunity and prevent or resolve uterine infections. Because of the role of progesterone
in making the uterus susceptible to infections, determining its mechanisms of action is
essential for formulating methods to enhance the ability of the uterus to control pathogenic
bacteria. Therefore, this article is a brief review of the role of progesterone in converting
the uterus from an organ that is resistant to one that is susceptible to infections and of how
eicosanoids may be used to mitigate the immunosuppressive effects of progesterone.
2. Role of progesterone
The uterus in cattle, sheep, and pigs is susceptible to infections when progesterone con-
centrations are increased, and it is resistant to infections when progesterone concentrations
are decreased. Numerous authors have reported this during the last 50 years (Black et al.,
1953; Rowson et al., 1953; Hawk et al., 1961, 1964; Lander Chacin et al., 1990; Seals
et al., 2002a; Wulster-Radcliffe et al., 2003). Spontaneous uterine infections in dairy cows,
for example, are not typically detected until after the first postpartum corpus luteum forms
and begins producing progesterone (Lewis, 1997). However, the uterine bacterial load can
be great enough to cause puerperal metritis shortly after calving, before progesterone con-
centrations increase (Arthur et al., 1989; Lewis, 1997; Seals et al., 2002a). In postpartum
beef cows, intrauterine infusions of A. pyogenes and E. coli did not produce infections,
unless progesterone concentrations had started to increase (Del Vecchio et al., 1992). After
progesterone concentrations had begun to increase, all of the cows developed infections
after intrauterine infusions of A. pyogenes and E. coli (Del Vecchio et al., 1992). Intrauter-
ine infusions of A. pyogenes and E. coli into postpartum ewes did not produce infections
when progesterone concentrations were basal (Seals et al., 2002b; Lewis, 2003). However,
infections developed in all postpartum ewes that had spontaneous luteal function and all
postpartum ewes that had been ovariectomized and treated i.m. with progesterone before
intrauterine infusions of A. pyogenes and E. coli (Seals et al., 2002b; Lewis, 2003). Further-
more, ewes and gilts that received intrauterine infusions of A. pyogenes and E. coli during
estrus did not develop uterine infections, whereas all ewes and gilts receiving intrauterine
infusions of A. pyogenes and E. coli during the luteal phase developed infections (Ramadan
et al., 1997; Seals et al., 2003; Wulster-Radcliffe et al., 2003).

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Neutrophils seem to mount the initial response to bacteria that enter the uterus, and the
ability of neutrophils to respond to intrauterine bacteria may be the most critical component
of the uterine immune defense mechanism (Hussain, 1989; Saad et al., 1989; Hussain and
Daniel, 1991). Despite that, lymphocyte proliferation in vitro is commonly used as a general
measure of uterine immune function (Hussain, 1989; Saad et al., 1989; Hussain and Daniel,
1991; Slama et al., 1991; Cai et al., 1994). Unstimulated, concanavalin A (Con A)-stimulated
(stimulates T-cells), and lipopolysaccharide (LPS)-stimulated (stimulates B-cells) lympho-
cyte proliferation were all greater for cells collected from vena caval blood from postpartum
ewes that were ovariectomized before luteal function was detected than they were for cells
collected from vena caval blood from ovary-intact postpartum ewes (Lewis, 2003). (Unless
stated otherwise, the number of lymphocytes added to each culture well was fixed in each
study cited. For the purposes of this article, words such as greater, increased, decreased,
etc. refer to comparisons with P-values of less than 0.05.) We use procedures described
in Benoit and Dailey (1991) to collect vena caval blood through catheters that are posi-
tioned just cranial to the entry of uteroovarian blood; blood from this site is enriched with
uteroovarian blood. Moreover, exogenous progesterone, compared with sesame oil treat-
ment, reduced unstimulated and Con-A stimulated lymphocyte proliferation in postpartum
ewes (Lewis, 2003). In addition, unstimulated, Con A-stimulated, and LPS-stimulated lym-
phocyte proliferation were greater when cells were collected from ewes during estrus than
when they were collected during the luteal phase, and unstimulated and Con A-stimulated
lymphocyte proliferation were greater when cells were collected from gilts during estrus
than when cells were collected from gilts during the luteal phase (Ramadan et al., 1997;
Wulster-Radcliffe et al., 2003). Other authors have reported similar data (Segerson and
Gunsett, 1993; Hansen, 1998; Szekeres-Bartho et al., 2001; Par et al., 2003). These effects
of exogenous and endogenous progesterone on lymphocyte proliferation were associated
with the inability of the uterus to prevent the development of infections (Ramadan et al.,
1997; Seals et al., 2002b, 2003; Lewis, 2003; Wulster-Radcliffe et al., 2003).
Progesterone clearly changes the uterus from an organ that is resistant to one that is
susceptible to infections. The literature cited above can be used to support the hypothesis
that the uterus “defaults” to “resistant to infections” when progesterone concentrations
are basal and bacterial contamination is not severe enough to overwhelm uterine immune
defenses, as it seems to do with puerperal metritis. We call this a “protected period,” even
though we recognize that the protection is not absolute and some unknown load of bacteria is
likely to overwhelm uterine immune defenses during this period. We have used postpartum
and seasonally anestrous ewes to test hypotheses about the so called protected period.
To test hypotheses about the so called protected period, autumn-lambing ewes were
ovariectomized on day 9 or day 14 postpartum, which was before detection of spontaneous
luteal function and increased progesterone concentrations, treated i.m. with canola oil or
safflower oil, and given intrauterine infusions of A. pyogenes and E. coli (Seals et al., 2002b;
Lewis, 2003). None of these ewes developed uterine infections (Seals et al., 2002b; Lewis,
2003). However, all of the ewes in the same experiments with spontaneous luteal function
and all of the ewes given exogenous progesterone i.m. in canola oil or safflower oil devel-
oped infections in response to intrauterine A. pyogenes and E. coli infusions (Seals et al.,
2002b; Lewis, 2003). Concentrations of PGF2 in vena caval blood collected from the ewes
ovariectomized on day 14 were inversely related to progesterone concentrations: PGF2

5. G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 285
was greatest in ewes with the least vena caval progesterone; and lowest in ewes with the
greatest progesterone (Lewis, 2003). The inverse relationship between PGF2 and proges-
terone is consistent with results from a study in which 6 -methyl-17 -hydroxyprogesterone
acetate, which is commonly used to control the estrous cycle, reduced uterine PGF2 pro-
duction (Fort´n et al., 1994). For the day 14 postpartum ewes, ovariectomy increased Con A-
ı
and LPS-stimulated lymphocyte proliferation, but exogenous progesterone decreased Con
A-stimulated proliferation (Lewis, 2003). Even though exogenous progesterone suppressed
lymphocyte proliferation somewhat for ewes ovariectomized on day 9, the results were not
as clear as they were for day-14 postpartum ewes (Seals et al., 2002b). Overall, susceptibil-
ity to uterine infections was associated with increased progesterone concentrations, reduced
PGF2 production, and reduced lymphocyte proliferation in vitro. By contrast, resistance to
uterine infections was associated with basal progesterone concentrations, increased PGF2
production, and increased lymphocyte proliferation in vitro. Thus, the “default” uterine
immune defenses were adequate to prevent infections, and the idea of a protected period is
not inappropriate.
We have also used seasonally anestrous ewes to determine whether the uterus is “protected”
from infections in the long-term absence of ovarian progesterone and estradiol (Mink et al.,
2003). These ewes had not been detected in estrus for at least three months, had basal
endogenous progesterone concentrations, and had no ovarian follicles large enough to be
estrogen active. The ewes were treated with either progesterone in safflower oil or safflower
oil alone and given intrauterine infusions of A. pyogenes and E. coli. Control ewes did not
develop infections after intrauterine infusion of A. pyogenes and E. coli, but all ewes treated
with progesterone developed uterine infections after intrauterine A. pyogenes and E. coli
infusions (Mink et al., 2003). The A. pyogenes and E. coli were infused 2 days before pro-
gesterone injections were initiated to determine whether the bacteria would be eliminated
shortly after they were introduced or whether the bacteria could reside in the uterus without
proliferating into an infection. Enough of the bacteria were clearly able to survive in the
uterus until exogenous progesterone suppressed the uterine immune defenses and permitted
them to proliferate into uterine infections.
Data from our studies with postpartum and seasonally anestrous ewes, in which proges-
terone concentrations were basal, seem to support the idea that the uterus “defaults” to being
resistant to A. pyogenes and E. coli infections, unless a compound, such as progesterone,
actively suppresses uterine immune defenses. Even though default uterine immune defenses
seemed sufficient to prevent frank A. pyogenes and E. coli infections, they did not eliminate
the bacteria from the uterus during the 2-day interval from bacteria infusion to initiation
of progesterone treatments. The ability of A. pyogenes and E. coli to remain “quiescent”
in the uterus until progesterone suppresses the uterine immune defenses seems consistent
with the scenario that has been described for postpartum dairy cows that develop uterine
infections coincident with the onset of luteal function (Lewis, 1997). Thus, the seasonally
anestrous ewe model would seem to offer the opportunity to test a number of hypotheses
about the onset of uterine infections in livestock.
The temporal relationships between reductions in progesterone, increases in estradiol, or
phase of the estrous cycle before hormone assays became routine, and resistance to uterine
infections have been recognized for more than 40 years (Hawk et al., 1961, 1964; Brinsfield
et al., 1964, 1967; Ramadan et al., 1997; Wulster-Radcliffe et al., 2003). However, a direct

6. 286 G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294
effect of estradiol on the resistance or susceptibility of the uterus to infections has not been
clearly established for cattle, sheep, or pigs. In fact, in two recent studies, estradiol did
not affect uterine involution in postpartum cattle and sheep, and the authors speculated
that estradiol treatment would not improve uterine health (Sheldon et al., 2003a,b). Thus,
because progesterone unequivocally suppresses uterine immune defenses and the role of
estradiol has not been established, progesterone seems to be the primary ovarian steroid
governing the susceptibility of the uterus to pathogenic bacteria.
3. Role of eicosanoids
Prostaglandin F2 and its various analogues have been used to resolve uterine infections in
livestock, but, as already stated, its true mechanism of action is not known. Other eicosanoids
have also been evaluated, and some may promote uterine health. Eicosanoids, which include
prostaglandins and leukotrienes, are members of a large family of compounds that are
synthesized from arachidonic acid through the cyclooxygenase and lipoxygenase pathways
(Pace-Asciak and Granström, 1983; Müller-Peddinghaus and Kast, 1996). Because the
family is so large, only eicosanoids that seem to have an obvious role in regulating uterine
immune defenses will be discussed.
Relationships among jugular progesterone, jugular 13,14-dihydro-15-keto-PGF2
(PGFM), which is a metabolite of PGF2 , and onset of uterine infections have been charac-
terized in postpartum dairy cows (Del Vecchio et al., 1992, 1994; Nakao et al., 1997; Seals
et al., 2002a). The half-life of PGFM is approximately 15 min, compared with approxi-
mately 1 min for PGF2 , and jugular PGFM concentrations closely reflect uterine PGF2
production during the postpartum period, but not during the estrous cycle when the uterus
produces considerably less PGF2 (Williams et al., 1983; Guilbault et al., 1984; Fort´n et al.,
ı
1994; Wade and Lewis, 1996). Jugular PGFM concentrations were reduced in postpartum
dairy cows that subsequently developed uterine infections, compared with PGFM concen-
trations in cows that did not develop uterine infections (Nakao et al., 1997; Seals et al.,
2002a). Small increases in progesterone, probably luteal, preceded the onset of uterine in-
fections (Seals et al., 2002a), and PGFM concentrations increased at the onset of uterine
infections (Del Vecchio et al., 1992; Seals et al., 2002a). Increased PGFM concentrations
were probably due to uterine inflammation in response to the growth of bacteria and release
of endotoxin (Roitt et al., 1998; Leung et al., 2001).
The studies with dairy cows and sheep seem to indicate that progesterone–PGF2 in-
teractions, and not just progesterone, are important for the regulation of uterine immune
defenses and the ability of the uterus to prevent infections. Indeed, uterine PGF2 pro-
duction seems to be related to the ability of the uterus to prevent or resolve infections.
One hypothesis that combines those ideas is that progesterone suppresses uterine immune
defenses and prevents the uterus from resisting infections, but PGF2 , and most likely
other eicosanoids such as leukotriene B4 (LTB4 ), can enhance uterine immune defenses
and mitigate the effects of progesterone. In vitro experiments lend support to that hypoth-
esis; PGF2 , LTB4 , 5-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, and
lipoxin B4 are chemoattractant to neutrophils (Hoedemaker et al., 1992). Indeed, neutrophils
seem to mount the initial defense against intrauterine pathogens, and suppressed neutrophil

7. G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 287
functions seem to make the uterus susceptible to infections (Hussain, 1989; Hussain and
Daniel, 1991; Cai et al., 1994). Another eicosanoid, PGE2 , can suppress immune func-
tions, including neutrophil functions, and predispose cows to uterine infections (Slama
et al., 1991). Progesterone–eicosanoid interactions are clearly broad and are likely to in-
volve a variety of compounds in addition to PGF2 . Thus, because numerous compounds
can interact with progesterone and affect immune functions, our research has focused on
progesterone–PGF2 interactions.
As is widely known, exogenous PGF2 induces luteolysis, which reduces circulating
progesterone, and permits the uterus to clear infections. However, exogenous PGF2 has
helped resolve uterine infections in cows without luteal function (Del Vecchio et al., 1994).
Clinical veterinarians often speculate that PGF2 injections stimulate uterine contractions
that expel bacteria from the uterus (personal communications). But this idea ignores the
facts that uterine contractions per se are not likely to kill the bacteria causing the infection;
bacteria are likely to remain in the uterus and proliferate again with the next bout of luteal
function; and published literature does not support the idea that uterine contractions cleanse
the uterus. Indeed, a recent study with cows indicates that a PGF2 analogue, cloprostenol,
enhances uterine contractions for perhaps 45 min after injection, and intrauterine pressure
was increased for only 15 min after injection (Hirsbrunner et al., 2003). Moreover, a study
of uterine infections in mares indicates that uterine contractions may reduce the volume
of fluid in the uterus, but they do not eliminate the bacteria (Nikolakopoulos and Watson,
1999). However, based on a good deal of literature, one may speculate that exogenous PGF2
has direct effects on uterine immune defenses, which can eliminate bacteria. Nevertheless,
studies are needed to separate the effects of uterine contractions from the direct effects of
PGF2 on complete resolution of uterine infections.
A critical issue that is often overlooked in discussions about the role of exogenous PGF2
in uterine infections is the fact that the direct effects of exogenous PGF2 on uterine immune
defenses and the effects of PGF2 on luteal function and progesterone concentrations are
completely confounded. We have conducted studies with sheep and pigs to separate the
effects of exogenous PGF2 on luteal function and uterine immune defenses and address the
questions: Is exogenous PGF2 effective because it enhances uterine immune defenses and
mitigates the effects of progesterone, or is exogenous PGF2 effective because it decreases
progesterone concentrations?
We have used pigs and sheep to answer those questions. We selected pigs because PGF2
is not luteolytic in pigs until after approximately day 12 of the estrous cycle (Guthrie and
Polge, 1976). Sows were assigned to a 2×2 factorial array of treatments (n = 6 sows/group)
to determine whether PGF2 had direct effects on uterine immune defenses (unpublished
research). The two main effects were intrauterine infusion of A. pyogenes and E. coli (i.e.,
bacteria versus phosphate-buffered saline (PBS)) and PGF2 (i.e., 10 mg of PGF2 versus
saline). Bacteria or PBS was infused on day 7 of the estrous cycle, and PGF2 or saline
was injected i.m. on day 9 of the same cycle. Uteri were collected on day 11 of the cy-
cle, two days after PGF2 or saline injections. Progesterone concentrations in vena caval
blood did not differ among groups and averaged 64 ng/mL during the study, indicating that
PGF2 did not affect luteal function. Vena caval estradiol-17 concentrations did not dif-
fer among groups and averaged 1 ng/mL. Injection of PGF2 increased vena caval PGF2
concentrations, which is consistent with data for sheep after exogenous PGF2 (Wade and

8. 288 G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294
Lewis, 1996). Unstimulated and LPS-stimulated lymphocyte proliferation also increased
in sows that received exogenous PGF2 , even though exogenous PGF2 increased vena
caval PGE2 concentrations. The bacteria-treated sows developed uterine infections, but the
PBS-treated sows did not develop uterine infections. Based on criteria used to determine
whether an experimental animal has a uterine infection (i.e., volume of sediment, which
contains leucocytes, bacteria, and cellular debris from the uterus, in uterine flushings col-
lected postmortem and the ability to culture of A. pyogenes and E. coli from the flushings),
PGF2 -treated sows were resolving the uterine infections at the time of slaughter (sedi-
ment volume, as a percentage of total flushing volume: 70% for bacteria-saline, 30% for
bacteria-PGF2 , and <5% for PBS-saline and PBS-PGF2 sows). These results indicate that
exogenous PGF2 enhanced uterine immune defenses and allowed the uterus to begin re-
solving the infections, despite luteal phase progesterone concentrations and basal estradiol
concentrations.
In another study to test the hypothesis that PGF2 has effects on uterine immune defenses
that are independent of progesterone concentrations, we used ovariectomized, progesterone-
treated ewes (unpublished research). This model was necessary because PGF2 is luteolytic
in sheep after approximately day 4 of the estrous cycle. The treatment groups were in a 2×2×
2 factorial array (n = 8 ewes/group). The main effects were ovariectomy (i.e., ovariectomy
versus sham procedure), progesterone (5 mg of progesterone at 12-h intervals versus sesame
oil diluent at the same times), and PGF2 (15 mg of PGF2 versus saline). Ewes were either
ovariectomized or a sham procedure was performed on day 0 of the estrous cycle (i.e., day
of estrous onset). Progesterone or sesame oil was injected i.m. from day 0 through day 11.
A. pyogenes and E. coli were infused intrauterine on day 6, and PGF2 or saline was injected
i.m. on day 9. Uteri were collected on day 12. All days are relative to day 0. Progesterone
concentrations in vena caval blood were as anticipated. Progesterone concentrations in sham
ovariectomy–sesame oil–saline ewes were typical for luteal phase sheep. Exogenous PGF2
induced luteolysis and reduced progesterone concentrations in ewes that did not receive
exogenous progesterone. Ovariectomy reduced progesterone to basal concentrations, and
exogenous progesterone maintained or increased progesterone concentrations. Exogenous
PGF2 increased vena caval PGF2 concentrations. All sham–ovariectomy ewes developed
uterine infections, but sham–oil–saline ewes and sham–oil–PGF2 ewes were resolving the
infections by day 12 (sediment volume approximately 8%). The sham–progesterone–saline
ewes had severe uterine infections on day 12 (sediment volume of 28%, which is much
greater than usual for sheep with uterine infections), but sham–progesterone–PGF2 ewes
seemed to be resolving their infections on day 12 (sediment volume of approximately
15%). The ovariectomy–oil–saline ewes did not have uterine infections on day 12 (sedi-
ment volume of 5%), but the ovariectomy–progesterone–saline ewes had typical infections
(sediment volume of approximately 16%) on day 12. The ovariectomy–oil–PGF2 ewes
did not have uterine infections on day 12 (sediment volume of approximately 2%), and
the ovariectomy–progesterone–PGF2 ewes had nearly resolved the infections (sediment
volume of approximately 4%) by day 12. Progesterone reduced unstimulated lymphocyte
proliferation, and PGF2 increased unstimulated, Con A-stimulated, and LPS-stimulated
lymphocyte proliferation. Based on the data, we reasoned that exogenous PGF2 enhanced
the ability of the uterus to resolve infections, regardless of progesterone concentrations.
The sows and ewes in the two experiments selected as examples received only one PGF2

9. G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 289
injection, and this seemed to be enough to initiate the resolution of the uterine infections.
A second injection of PGF2 , 12–24 h after the first, might have resolved all of the induced
infections, but we have not yet conducted experiments to test that possibility.
Data from recent studies support the idea that exogenous PGF2 has direct effects on
uterine immune defenses: effects that are independent of luteal function and progesterone
concentrations. In addition, the ability of the uterus to secrete PGF2 , and perhaps other
eicosanoids, may govern the ability of the uterine immune defenses to resist or resolve
uterine infections. Thus, it seems likely that PGF2 can enhance uterine immune defenses
and mitigate the immunosuppressive effects of progesterone.
4. Eicosanoid–progesterone relationships
Eicosanoids and progesterone seem to have many independent effects on the activity
of immune cells. Progesterone is typically immunosuppressive, and that phenomenon has
been studied and reviewed extensively. Progesterone regulation of the synthesis of immuno-
suppressants and blocking factors has received particular attention, so those data will not
be reviewed in this article (Segerson and Gunsett, 1993; Hansen, 1998; Szekeres-Bartho
et al., 2001). Rather, this section of this article will contain information about eicosanoid–
progesterone relationships. Indeed, understanding these relationships at the cellular or
molecular level and using the information to develop methods to mitigate the immuno-
suppressive effects of progesterone has considerable potential for preventing or resolving
uterine infections in livestock environments.
The uterus is normally able to prevent potentially pathogenic bacteria introduced dur-
ing estrus from proliferating into infections (Ramadan et al., 1997; Wulster-Radcliffe et al.,
2003). During estrus, when progesterone concentrations are decreased and estradiol concen-
trations are increased, uterine PGF2 and endometrial leukotriene production are increased
(Ottobre et al., 1980; Kindahl et al., 1984; Zarco et al., 1988; Vagnoni et al., 2001). As luteal
function develops and progesterone concentrations begin to increase, uterine PGF2 and
LTB4 production decrease to basal levels within a few days after estrus, and the uterus be-
comes susceptible to infections (Kindahl et al., 1984; Zarco et al., 1988; Slama et al., 1993;
Vagnoni et al., 2001). In vitro studies indicate that PGF2 enhances neutrophil chemotaxis
and the ability of neutrophils to ingest bacteria, and LTB4 enhances chemotaxis, random
migration, and antibody-independent cell-mediated cytotoxicity (Hoedemaker et al., 1992).
These effects alone, assuming they occur in vivo, on neutrophil functions should help the
uterus manage pathogens. But, in addition to direct effects on neutrophils, PGF2 is a
proinflammatory molecule that may stimulate production of proinflammatory cytokines
that enhance phagocytosis and lymphocyte functions (Kelly et al., 2001; Seals et al., 2003).
Furthermore, a study with cows indicates that LTB4 promotes uterine involution and reduces
the risk of uterine infections (Slama et al., 1993).
Prostaglandin F2 injections increase uterine PGF2 and luteal LTB4 production
(Steadman and Murdoch, 1988; Wade and Lewis, 1996). Even though definitive data are not
available, one may speculate that PGF2 could enhance uterine LTB4 production because
nordihydroguaiaretic acid, which inhibits lipoxygenase activity and LTB4 production, pro-
longed the luteal phase in cattle and sheep and the uterus seems to have been the mediator

10. 290 G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294
(Milvae et al., 1986; Cooke and Ahmad, 1998). Injections of PGF2 that increase uterine
PGF2 production probably increase uterine phospholipase A2 (PLA2 ) and cyclooxygenase
2 activities, which would produce free arachidonic acid and then convert the arachidonic
acid to PGF2 (Binelli et al., 2000; Diaz et al., 2002; Narayansingh et al., 2002). The free
arachidonic acid could also be used to produce additional cyclooxygenase products and
lipoxygenase (e.g., LTB4 ) products. In addition, tumor-necrosis factor (TNF ) mediates
inflammatory and cytotoxic responses (Roitt et al., 1998) and stimulates endometrial PGF2
production; PLA2 seems to be the mediator (Miyamoto et al., 2000; Skarzynski et al., 2000).
Thus, based on a considerable amount of literature, methods for promoting uterine PGF2
and LTB4 production should enhance immune defenses and enable the uterus to prevent
or resolve infections. In fact, a long-acting PGF2 analogue, fenprostalene, injected some-
time between days 7 and 10 postpartum reduced the incidence of endometritis in cows with
dystocia and/or retained fetal membranes (Nakao et al., 1997). Fenprostalene should have in-
creased uterine PGF2 production (Wade and Lewis, 1996), but the sampling frequency and
site of sample collection were not adequate to determine whether that was the case (Nakao
et al., 1997). Moreover, a single subcutaneous fenprostalene injection on the day of en-
dometritis detection reduced the interval from parturition to conception (Nakao et al., 1997).
Despite the evidence that exogenous PGF2 can promote uterine health in livestock, the com-
plete mechanisms of action of exogenous PGF2 in uterine health have not yet been reported.
If imposing treatments to increase uterine PGF2 and LTB4 production mitigates the im-
munosuppressive effects of progesterone on uterine immune defenses and promotes uterine
health in livestock environments, the mechanism for this is likely to be quite complex be-
cause progesterone does more that just stimulate the production of immunosuppressants
and blocking factors. Progesterone also decreases the activity of a number of proinflamma-
tory molecules and stimulates PGE synthase activity; PGE2 inhibits immune cell functions
in vitro (Segerson and Gunsett, 1993; Hunt et al., 1997; Hansen, 1998; Szekeres-Bartho
et al., 2001; Arosh et al., 2002; Seals et al., 2002b). In addition, progesterone suppresses
interleukin (IL)-8 production, which stimulates chemotaxis, superoxide release, and gran-
ule release from phagocytic cells, in reproductive tissues (Ito et al., 1994; Kelly et al., 1994;
Mitchell et al., 2002, 2003; Loudon et al., 2003). Progesterone also suppresses the produc-
tion of IL-6, which promotes B-cell differentiation and production of acute-phase proteins
(Montes et al., 1995), and inhibits IL-12 production (Par et al., 2003). Interleukin-12 induces
interferon- production and enhances natural killer-cell cytotoxicity, and PLA2 , presum-
ably via increased free arachidonic acid, may mediate the effects of IL-12 (Par et al., 2003).
Even though a variety of immunosuppressive effects have been ascribed to progesterone,
the effects vary among reports, much of the research has been conducted with in vitro, and
not in vivo, models, and very little of the research has been conducted to understand the
relationship between production of various proinflammatory molecules and the ability of
the uterus in livestock to resistant or resolve infections.
5. Conclusions
Based on available literature, progesterone is the ovarian steroid that primarily governs the
ability of the uterus in livestock to resist infections. Progesterone typically suppresses im-

11. G.S. Lewis / Animal Reproduction Science 82–83 (2004) 281–294 291
mune defenses and makes the uterus susceptible to infections. Injections of PGF2 increase
uterine production of PGF2 , and probably LTB4 . These two eicosanoids seem to promote
uterine immune defenses and uterine health. Eicosanoids and progesterone affect several
proinflammatory molecules that may modulate uterine immune defenses, but the roles of
these molecules in determining whether the uterus is resistant or susceptible to infections
have not yet been defined. Determining how uterine PGF2 mitigates the immunosuppres-
sive effects of progesterone and stimulates the uterus to resolve infections should clearly be
important to scientists and clinicians working to understand the underlying causes of uterine
infections. This area of research should yield important new strategies for preventing and
treating uterine infections that do not include antibiotic and antimicrobial compounds.
Acknowledgements
The author is grateful for the contributions from his trainees and collaborators, partic-
ularly R.P. Del Vecchio, I. Matamoros, A.A. Ramadan, R.C. Seals, S. Wang, and M.C.
Wulster-Radcliffe.
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