Androgen control of immunocompetence in the male house finch, Carpodacus mexicanus Müller
School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA
* Author for correspondence (e-mail: deviche{at}asu.edu)
Accepted 7 February 2004
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Summary |
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Key words: androgen, cell-mediated immunity, humoral immunity, immunocompetence handicap hypothesis, inflammation
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Introduction |
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Gonadal hormones exert multiple and complex influences on immune functions
(review: Ahmed and Talal,
1990). Lymphocytes in some species of fish have androgen receptors
(Slater et al., 1995
) and
consistent with the ICH hypothesis, T in mammals and fish is generally
immunosuppressive (Ahmed et al.,
1987
; Angele et al.,
1998
; Benten et al.,
1993
; Slater and Schreck,
1997
; but see Bilbo and Nelson,
2001
). By contrast, evidence for immunosuppressive effects of T in
birds, which have been used extensively to test the ICH hypothesis (e.g.
Lindström et al., 2001
;
Norris et al., 1994
;
Saino and Møller, 1994
;
Seutin, 1994
;
Zuk and Johnsen, 1998
),
remains equivocal. Testosterone treatment suppressed humoral and cell-mediated
immunity in European starlings, Sturnus vulgaris
(Duffy et al., 2000
) and
dark-eyed juncos, Junco hyemalis
(Casto et al., 2001
). This
treatment was also immunosuppressive in house sparrows, Passer
domesticus (Evans et al.,
2000
), song sparrows, Melospiza melodia
(Owen-Ashley et al., 2004
) and
captive superb fairy-wrens, Malurus cyaneus
(Peters, 2000
). Given to other
species, however, T had no detectable influence on immune responses
(red-winged blackbirds, Agelaius phoeniceus:
Hasselquist et al., 1999
;
greenfinch, Carduelis chloris:
Lindström et al.,
2001
).
Several factors may account for these differences. For example, the above
studies investigated various aspects of the immune system (antibody
production; local inflammatory response; number of circulating lymphocytes;
Nava et al., 2001) that may
have different latencies to respond and different sensitivities to T.
Additionally, heterologous antibody production, which is often used to
estimate humoral immunity, depends on the reproductive stage
(Nelson et al., 1998
;
Von Schantz et al., 1999
).
This production was measured at various times ranging from 5 to 13 days after
antigen administration (Evans et al.,
2000
; Casto et al.,
2001
; Peters,
2000
), but not all these studies examined the time course of
antibody production, in which cases antibody production may not have been
measured at its peak. Furthermore, the immunization protocol used in the above
studies often failed to induce detectable antibody concentrations in all
experimental subjects. In such cases, data for non-responsive individuals
either were retained in (Evans et al.,
2000
; Peters,
2000
) or were excluded from
(Casto et al., 2001
)
statistical analyses. Finally, effects of T on immunity may be indirect and
mediated by complex and poorly understood interactions between this and other
hormones, in particular corticosterone (CORT) as well as by changes in body
energy allocation (Evans et al.,
2000
; Owen-Ashley et al.,
2004
). If indirect, these effects likely vary as a function of
physiological parameters other than the reproductive status.
To address the above issues, we researched the influence of T treatment
concurrently on three rather than single aspects of the immune system:
cell-mediated immunity, humoral immunity and lymphocyte numbers. This research
was done on adult male house finches, Carpodacus mexicanus, a species
that has been used extensively to investigate the bases of mate selection
(Hill, 1990,
1991
), the mechanisms that
control a sexually selected trait (plumage coloration:
Hill et al., 1994
),
correlations between parasite infections and this trait
(Thompson et al., 1997
), the
bases of disease resistance (Duckworth et
al., 2004
) and the environmental control of reproductive
physiology (Hamner, 1968
).
Moreover, T plays a role in sexual signaling in the house finch, being
positively correlated with its most important sexual signal, plumage redness
(Duckworth et al., 2004
) and T
treatment to this species exacerbates coccidian infection
(Duckworth et al., 2001
).
Taken together, these studies suggest a role for T in maintaining honesty of
the plumage color through effects on immune responsiveness. To increase the
likelihood of birds producing measurable levels of antibody in response to
immunization, each subject received multiple rather than a single antigen
injections. In addition, we determined plasma antibody concentrations in
response to T administration several times during and after immunization
(Von Schantz et al., 1999
;
Peters, 2000
). Finally,
extraneous factors that potentially interact with the effects of the
experimental treatment were standardized by holding birds in identical
conditions and in a strictly controlled environment for the duration of the
study.
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Materials and methods |
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Testosterone capsule implantation and removal
On February 16, we randomly divided males into two groups. Ten males (T
males) received two subcutaneous T-filled Silastic capsules prepared as
described in Dloniak and Deviche
(2001) and 10 males (C males)
received two identical, but empty capsules. Each T-filled capsule was 11
mm-long and the actual portion filled with hormone approximated 9 mm. Capsules
were inserted under the skin through a 2 mm-long incision along one flank. In
previous studies, a similar T treatment increased plasma T concentrations in
house finches (Duckworth et al.,
2004
; Stoehr and Hill,
2001
) and other avian species of similar sizes (dark-eyed junco:
Ketterson and Nolan, 1992
) and
it maintained physiologically high circulating steroid concentrations that
were close to those measured at the beginning of the breeding season for up to
several months. We defined the day that capsules were administered as Day 0
(D0) and all data are reported with reference to this date
(Table 1). We weighed capsules
before implantation and again after removal (D66: five T males; D74: five
remaining T males) and oven drying at 37°C for several days and used the
difference between the initial and final weights of the capsules to calculate
that the average release rate of T was 112±2 µg
day1 bird1 (mean ±
S.D.). This rate compares favorably to that in another passerine of
similar size (dark-eyed junco: 92 µg day1
bird1; Deviche,
1992
).
|
Blood samples
We collected up to 350 µl of blood (<2% body mass) from a wing vein
of each finch into heparinized microhematocrit tubes on D-3, D10, D20, D39 and
D55. We used approximately 5 µl of blood to prepare thin smears on glass
microscope slides (Bennett,
1970; Deviche et al.,
2001a
) and kept the remainder of each sample on ice until it was
centrifuged at 4°C within 2 h of collection. Plasma was harvested and
stored at 20°C until assayed for T and antibody concentration.
Testosterone assay
We measured total circulating plasma T concentrations using a commercial
competitive immunoassay (ELISA; AssayDesigns, Inc., Ann Arbor, MI, USA). All
samples (5 µl plasma per assay well) were assayed simultaneously, in
duplicate and in a random order and according to the manufacturer's
specifications. Each ELISA plate included a complete standard curve and
positive and negative controls. We calculated plasma T concentrations using
Prism version 3.02 (Graphpad Software, Inc., San Diego, CA, USA). Validation
tests demonstrated that the assay measures T in house finch plasma accurately
and reliably (Results).
Morphological parameters
Immediately after blood collection, we weighed birds to the nearest 0.1 g
and measured their cloacal protuberance width (an androgen-dependent secondary
sexual characteristic: Deviche,
1992; Dloniak and Deviche,
2001
) to the nearest 0.1 mm using digital calipers.
Immunization and humoral immunity
We immunized finches against freshly (i.e. same day) washed sheep red blood
cells (SRBC; Hemostat, Dixon, CA, USA) using a modification of the Casto et
al. (2001) protocol. For this,
each male received a weekly 0.15 ml i.p. injection of 2% SRBC (D13, D20, D27
and D34). Injections on D20 were given after collection of blood samples and
morphological data. For the first injection (D13) washed SRBC were suspended
in 0.15 ml Freund's Complete Adjuvant (ICN Biomedicals, Inc., Aurora, IL,
USA). Cells used for subsequent injections (D20, D27 and D34) were suspended
in 0.15 ml of 0.1 mol l1 phosphate buffer. We found no
evidence for detrimental effects of the complete adjuvant injection as
measured by changes in body mass (see Results), signs of distress or
inflammation (P.D., personal observations) or mortality. We measured the
plasma concentration of antibodies to SRBC with an in vitro
hemagglutination test (Casto et al.,
2001
), using a commercial antiserum against SRBC (ICN Biomedicals,
Inc., Aurora, IL, USA). For this assay, plasma was diluted serially in 0.1 mol
l1 phosphate buffered saline (PBS) and all samples were
assayed in duplicate and in a random order. We collected data without
knowledge of the sample identity and expressed results as the number of wells
showing hemagglutination. Each assay plate included negative and positive
controls to ensure standardization.
Cell-mediated immunity
We assessed cell-mediated immunity by measuring the local inflammatory
response to a unilateral injection of the mitogenic plant protein
phytohemaglutinin (PHA; Sigma Chemical Co., St Louis, MO, USA;
Casto et al., 2001;
Lochmiller et al., 1993
).
Birds received a priming s.c. injection of PHA (0.25 mg in 50 µl PBS) into
the left wing web on D55 and a second, identical injection on D63. Wing web
thickness (mean of three consecutive determinations) was measured to the
nearest 25 µm with a pressure-sensitive dial thickness gauge (Mitutoyo,
model 7226) immediately before the D63 challenge injection and 24 h (D64), 48
h (D65) and 72 h (D66) later. Results are presented as the change in web
thickness relative to D63.
The Arizona State University Institutional Animal Use and Care Committee approved all experimental protocols and birds were caught under current federal and Arizona state scientific collecting permits.
Lymphocyte numbers
We stained blood smears using the May-Grunwald and Giemsa technique.
Stained smears were air-dried, dehydrated overnight under vacuum, cleared with
xylene and coverslipped using Cytoseal 60 (VWR). We studied smears with a
light microscope and digitized 20 randomly selected visual fields of each
smear at 400 x magnification with a color digital camera. Using Image
Pro (Media Cybernetics, Silver Spring, MD, USA), we automatically counted the
number of erythrocytes in each digitized image and manually counted the number
of lymphocytes in the same image. The average number of erythrocytes counted
per smear was 10,587±1290 (mean ± S.D.). We present
data as numbers of lymphocytes counted per 1000 erythrocytes.
Statistical analyses
Unless otherwise specified, we analyzed differences between C and T males
as a function of time using two-way analyses of variance for repeated measures
(2RANOVA) and, when appropriate, StudentNewmanKeuls multiple
pairwise comparison tests (StatSoft Statistica, version 5.1, Statsoft Inc.,
Tulsa, OK, USA). Data sets not complying with normality and/or
homoscedasticity requirements for 2RANOVA were ranked prior to analysis
(Conover and Iman, 1981).
Results from analyses of untransformed data are presented as mean ±
S.D. and results from analyses of ranked data are shown as medians
±0.5 interquartile intervals (Results and figure legends). We compared
the slopes of a T assay standard curve and a curve generated by serially
diluting house finch plasma using Graphpad Prism. The significance level of
all statistical tests was set at
=0.05.
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Results |
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Group differences
Plasma T levels varied as a function of time (upper panel,
Fig. 2; F4,68=28.2, P<0.0001) and were influenced by
the experimental treatment (F1,17=115.5,
P<0.0001). There also was a significant time x treatment
interaction (F4,68=13.1, P<0.0001). Before
capsule implantation plasma T levels were low and similar in C and T males.
Testosterone levels in C males remained low (less than 0.5 ng
ml1) throughout the study period. They were higher on D39
(0.41±0.20 ng ml1) and D55 (0.38±0.17 ng
ml1) than on D-3 (0.18±0.02 ng
ml1), D10 (0.29±0.12 ng ml1) and
D20 (0.22±0.16 ng ml1;
StudentNewmanKeuls tests: P<0.05), but remained
within the limits of levels measured outside the reproductive season in
non-breeding males (personal observations). The small increase (on average,
0.23 ng ml1) in plasma T in C males during the course of the
study may reflect slow maturation of the reproductive system despite short day
exposure, which may itself have resulted from C males hearing singing T males.
Testosterone levels after capsule administration were evenly higher in T than
C males. This difference persisted for the duration of the study.
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External morphology
Cloacal protuberance widths changed as a function of time (middle panel,
Fig. 2;
F4,72=9.6, P<0.0001) and were affected by T
administration (F1,18=27.9, P<0.0001) and
there was a time x treatment interaction
(F4,72=23.3, P<0.0001). On D-3 CPs in both
groups were small and undeveloped. They remained undeveloped in C males
throughout the study but gradually enlarged in T males, so that the two groups
differed starting on D10.
Body masses did altogether not differ between C and T males, but changed (F4,72=24.8; P<0.0001) in a group-dependent manner (lower panel, Fig. 2; group x time interaction: F4,72=3.15, P<0.02) during the course of the study. Testosterone-treated males had lower body masses than C males on D-3 and D10, but not at later sampling times.
Humoral immunity
We detected no antibody to SRBC in the plasma of any male prior to (D-3 and
D10) or 1 week after (D20) the first SRBC injection. However, all samples
collected 5 days after the fourth antigen injection (D39) and 80% of the
samples collected 2 weeks after this injection (D55), contained measurable
antibody concentrations. Concentrations on D39 and D55 were correlated
(Pearson product moment correlation: r=0.657, P<0.002;
N=20). Due to the general absence of detectable antibody before D39,
we compared groups of data with ANOVA only for D39 and D55.
Testosterone-treated and C males had similar antibody concentrations on D39.
Concentrations decreased in both groups between D39 and D55 (upper panel,
Fig. 3; date effect:
F1,18=49.3, P<0.0001) and the magnitude of
this decrease was larger in T than C males (date x treatment
interaction: F1,18=7.2, P=0.015). As a result, T
males had lower antibody concentrations than C males on D55.
|
Cell-mediated immunity
Wing web thickness of C and T males prior to the D63 challenge PHA
injection did not differ (C males: 0.598±0.049 mm; T males:
0.613±0.033 mm; medians ±0.5 interquartile intervals). Swelling
measured on D64, D65 and D66 changed from one day to another (lower panel,
Fig. 3;
F2,36=11.1, P<0.0002) in a hormone
treatment-related manner (time x treatment interaction:
F2,36=4.0, P<0.03). Swelling in C males did
not differ between D64 and D65, but decreased between D65 and D66. By
contrast, swelling in T males decreased between D64 and D65 and then did not
change between D65 and D66. This different time course resulted in less
swelling in T than C males on D65.
Lymphocyte numbers
Circulating lymphocyte concentrations changed between D-3 and D55
(F4,72=4.1, P<0.005;
Fig. 4), being higher 1 week
after the first SRBC injection (D20) than at other times
(StudentNewmanKeuls tests: P<0.05). Lymphocytes
numbers did not change between D-3, D10, D39 and D55 and were not influenced
by T administration.
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Discussion |
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Three independent measures confirmed the effectiveness of the T treatment.
First, T-filled Silastic capsules weighed less at the end than at the
beginning of the study, indicating T release. Second, T-treated males had
higher plasma T levels than C males for the duration of the experiment. These
levels (2530 ng ml1) in hormone-treated finches were
higher than those reported in free-ranging conspecific males
(Duckworth et al., 2004;
Stoehr and Hill, 2000
,
2001
). However, we found no
evidence suggesting that the T treatment induced pathological effects such as
a decrease in body mass or mortality. Furthermore, differences in T levels
between studies should be interpreted with caution as these levels were often
measured in different laboratories and using different types of assays (ELISA
vs radioimmunoassays). Finally, finches receiving T-filled capsules
had larger CPs, an androgen-dependent secondary sexual characteristic, than
control finches. This effect was specific as shown by the fact that T
treatment had no effect on body condition as estimated by body mass.
The methodology of the present investigation differed in several respects
from that of previous avian studies examining the production of heterologous
antibodies to assess the endocrine regulation of humoral immunocompetence
(Donker and Beuving, 1989;
Selvaraj and Pitchappan, 1985
)
and was designed to maximize immune responses of the experimental subjects. In
particular, house finches used here received an initial injection of SRBC in
Complete Freund's adjuvant followed with three additional injections of SRBC.
Multiple injections may have induced stronger and different antibody
responses, as well as a higher proportion of birds producing antibodies, than
a single (Casto et al., 2001
;
Donker and Beuving, 1989
;
Duffy et al., 2000
;
Peters, 2000
) or two
(Evans et al., 2000
;
Von Schantz et al., 1999
)
consecutive injections of antigen, especially in cases where no adjuvant was
used. Evidence in support of this hypothesis is provided by the fact that the
blood of all experimental finches contained antibodies to SRBC 5 days after
the fourth SRBC injection and antibodies were detected in 80% of the finches 2
weeks after this injection. At this time average antibody concentrations had
decreased, but were correlated with those 2 weeks earlier. Thus, the apparent
lack of antibody production by some individuals in previous studies may have
been related to the immunization protocol rather than from inherent lack of
response to the antigen. It is of interest that no anti-SRBC antibodies were
detected in the blood of C or T finches 1 week after the first SRBC injection.
This finding contrasts with that of Peters
(2000
) who reported maximal
antibody titers in superb fairy-wrens 9 days post-immunization. The difference
between studies may have resulted either from species differences in
immunocompetence, from differences in conditions experienced by the birds (see
below), or from the fact that immune responses to SRBC develop slower when
these cells are administered in adjuvant (present investigation) than in
phosphate-buffered saline solution
(Peters, 2000
).
Testosterone-treated males had evenly high circulating concentrations of the steroid for the duration of the study, yet the influence of this treatment on humoral and cell-mediated immunity was time-dependent. In particular, plasma anti-SRBC antibody concentrations were lower in T than C males on D55. Furthermore, local inflammatory response to PHA injection in C males persisted for 2 days post-injection and then subsided. By contrast, inflammation in T males decreased between 1 and 2 days after the injection. Thus, C and T males apparently mounted similar immune responses to SRBC and to PHA administration, but T administration compromised the maintenance of these responses.
Several mechanisms may account for the observed time-dependent effects of T on house finch immunity. As far as humoral immunity is concerned, T may have impaired the ability of birds to produce additional antibodies at the conclusion of the immunization period. Testosterone administration may also have accelerated the degradation or elimination of antibodies produced during and shortly after this period. Finally, the hemagglutination test used in the present study is not specific for antibody (immunoglobulins, Ig) types and detects IgG as well as IgM proteins. Testosterone may differently affect the production and degradation rates of these antibodies. Regardless of the mechanisms involved, the data emphasize the importance of measuring the dynamics of T effects on various immune responses rather than determining these responses at a single time point.
Previous studies investigated the mechanisms that mediate the
immunosuppressive influence of T (e.g.
Owen-Ashley et al., 2004), but
these mechanisms remain poorly understood. One hypothesis is that T directly
affects cells of the immune system. Supporting this hypothesis, lymphocytes in
fish (Slater et al., 1995
) and
the bursa of Fabricius in immature chickens
(Sullivan and Wira, 1979
) have
androgen receptors. In vitro studies using mammalian cells found T to
suppress the production of antibodies by blood mononuclear cells
(Kanda et al., 1997
), the
proliferation of lymphocytes induced by mitogens
(Ahmed et al., 1987
;
Kotani et al., 1974
;
Lehmann et al., 1988
) and the
survival of leukocytes (Slater and
Schreck, 1997
). Testosterone also reduced the in vitro
production of prostaglandin E2, an important mediator of local inflammatory
responses, by human monocytes (Miyagi et
al., 1993
). Evidence for similar effects of T in birds is,
however, currently lacking and little experimental supports the idea in these
organisms that T directly influences cells of the immune system.
Alternatively, effects of T on immune function in birds may be indirect and
mediated by the main adrenal glucocorticoid, CORT. Corticosterone in birds is
generally immunosuppressive (Marsh and
Scanes, 1994). This steroid is immunosuppressive also in some
(rodents: Collins and Deas,
1986
; Stewart et al.,
1988
; review: Sapolsky,
1992
) although not other vertebrates, in which it rather enhances
immunocompentence (side-blotched lizard, Uta stansburiana:
Svensson et al., 2002
).
Although CORT administration to chickens did not decrease their production of
heterologous antibodies in response to a single antigen injection
(Donker and Beuving, 1989
),
this treatment to mallards, Anas platyrhynchos, depressed innate as
well as humoral immunity (Fowles et al.,
1993
). Plasma T and CORT levels in intact male passerines are
seasonally correlated (Deviche et al.,
2000
; Klukowski et al.,
1997
; Johnsen,
1998
) and T treatment increased plasma CORT levels and/or the
concentration of plasma binding protein for this steroid in several avian
species (house sparrow: Evans et al.,
2000
; dark-eyed junco: Schoech
et al., 1999
; Deviche et al.,
2001b
; European starling:
Duffy et al., 2000
; song
sparrow: Owen-Ashley et al.,
2004
) including the house finch
(Deviche et al., 2004
). Taken
together, these findings are consistent with the hypothesis that
immunosuppressive effects of T in adult birds result from androgen-mediated
elevated CORT secretion as recently proposed by Evans et al.
(2000
). This mechanism may
involve a modulation by T of CORT actions on immune cells because in
vivo androgen treatment to immature chicks reduced their bursa of
Fabricius cell concentration of cytosolic glucocorticoid receptors
(Coulson et al., 1982
).
As already indicated (Introduction), studies examining effects of T on
avian immunity have provided equivocal results. In addition to procedural
differences, such as those described above, some of the observed variation may
relate to the fact that immunocompetence can be modulated by various
environmental and social factors (e.g. lizards: effect of cold:
Svensson et al., 1998; effect
of social factors: Svensson et al.,
2001
) that can in some cases themselves influence the activity of
the reproductive system (Leitner et al.,
2003
; Wingfield et al.,
2003
). In addition, immune responses have a genetic, heritable
component (blue tit, Parus coeruleus:
Råberg et al., 2003
;
lizards: Svensson et al.,
2001
). Thus, effects of T on immune functions, whether direct or
indirect (see above), may depend on complex interactions between endocrine and
non-endocrine factors including environmental and genotypic variables.
The present investigation found no influence of T treatment on lymphocyte
counts. Studies on this subject have yielded equivocal results. Testosterone
administration to red jungle fowl, Gallus gallus, decreased
lymphocyte counts (Zuk et al.,
1995), but increased these counts without affecting the numbers of
other leukocyte types in adult house sparrows
(Nava et al., 2001
). In intact
barn swallows, Hirundo rustica, T levels and lymphocyte counts were
unrelated (Saino and Møller,
1995
). Several factors including age and reproductive status may
account for differences between studies. For example, T administration
increased lymphocyte numbers in adult but not juvenile sparrows
(Nava et al., 2001
). Also,
lymphocyte counts and the size of the comb, a T-dependent
(Ligon et al., 1990
) and
sexually selected trait, were positively correlated in red jungle fowl before
the breeding season, but negatively correlated during this season
(Zuk and Johnsen, 1998
). All
finches used in the present study were adults that we exposed to short days to
prevent reproductive development. Regardless of whether finches had low or
high circulating T levels, lymphocyte counts were higher 1 week after the
first SRBC injection than at other times. We speculate that this increase was
in response to the solvent (Freund's Complete Adjuvant) itself or to the
presence of a foreign antigen (SRBC). The finding that T treatment did not
alter lymphocyte counts must be interpreted conservatively because the
sampling technique resulted in our counting relatively small numbers of
lymphocytes for each bird and counts within a group on a given date were quite
variable (see error bars on Fig.
4). Small lymphocyte counts and large inter-individual variation
in these counts may have hampered the detection of subtle effects of T
administration on this parameter.
To conclude, we found experimentally elevated circulating T levels in male house finches to suppress humoral immunity in response to a heterologous antigen and cell-mediated immunity in response to a foreign protein. These results confirm and extend previous findings and indicate that T can be immunosuppressive, a conclusion that supports the ICH hypothesis. The data add to the increasing evidence in birds that T exerts a great diversity of behavioral and physiological effects. Circulating T levels in most seasonally breeding male birds naturally undergo dramatic natural seasonal changes. In the future, these birds will likely continue to serve as choice models for investigating the environmental regulation of reproductive endocrinology, the role of gonadal hormones in the control of the immune functions and the mechanisms that mediate this role.
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Acknowledgments |
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