Editor-in-Chief
Prof. Yi-Fei WANG,
Cytokines
in the BALB/c mouse testis in various conditions
E.
Veräjänkorva1, M. Martikainen1,
P. Pöllänen1,2
Department
of Anatomy1 and Department of Obstetrics and Gynecology2,
University of Turku, FIN-20520 Turku, Finland
Asian J Androl 2001 Mar; 3: 9-19
Keywords:
testosterone;
vasectomy; estrogens; cryptorchidism; abdominal testis; varicocele;
cyokines; IL-10; macrophages; clonal anergy
Abstract
Aim: To investigate whether testosterone, estrogens, vasectomy, experimental cryptorchidism, varicocele or aging would induce changes in the cytokine environment of the mouse testis. Methods: In adult male BALB/c mice, testosterone implants, estradiol benzoate, vasectomy, unilateral cryptorchidism, unilateral varicocele were administered/performed. The mice were followed up for different periods of time and were then sacrificed with testes incised for examination. The control mice received the vehicle or sham-operation. Results: IL-10 was present in Leydig cells of nearly every testis and IL-10+ macrophages in 39% of testes. IL-6 was found in the testes of intact adult mice, mice treated with testosterone for 70 days, cryptorchid testes and sham-operated testes. Conclusion: Results suggest that IL-10 might be involved in the generation of the immunologically privileged microenvironment in the testis.
1 Introduction
Testis
is an immunologically privileged site where nearly every pathological
condition of the male reproductive tract is associated with an increased
prevalence of sperm
antibodies[1]. The cytokines have two major roles in the testis:
1) to mediate pathophysiological
outcomes of immune-endocrine interactions during inflammatory disease,
2) to work as growth and differentiation factors that help to orchestrate
cellular interactions during normal physiological functions[2].
It is known that the cells responsible for the maintenance of the blood-testis
barrier in the testis, the Sertoli cells, produce an IL-1α-like factor[3,4]
and IL-6[5-7], as well as TGFβ[8-10] and Fas Ligand[11].
In addition, it has been reported that IL-11[12] is expressed
in differentiating male germ cells. IFN-α and -γ[13] and
TNF-α[14] are produced by testicular cells. However, a comprehensive
study on the cytokine environment of the testis in physiological as well
as pathological conditions has not yet been made.
The
cells mainly responsible for protection of germ cells from autoimmune
reactions, the Sertoli cells and the principal cells of the epididymis,
are dependent on androgens[15,16], the concentration of which
is decreased in both the
testis and the epididymis during male hormonal contraception[17].
A decrease in androgen
level may result in the breakdown of blood-testis and blood-epididymis
barrier, the release of sperm autoantigens and the development of autoimmune
destruction of the male reproductive tract. Indeed, it has been reported
that the blood-testis barrier starts to break down in conditions of subnormal
testosterone production in the seasonal breeder mink[16] with
the formation of sperm antibodies[18]. In such a process, cytokines
should have a distinctive role. This is also true in case of disrupting
spermatogenesis in cryptorchidism[19] and
vasectomy[20,21]. In all these conditions, the microenvironment
of the testis might have been changed.
2 Materials and methods
2.1
Animals
BALB/c
male mice 2 (prepubertal), 4 (pubertal), 11 and 25 weeks of age were used as
donors of normal testicular tissue. Each group consisted of at least three
mice. The animals had free access to food and water and they were maintained
in a normal dark/light
cycle. Permissions for the experiments and to use organs from the
animals after sacrifice with CO2 (Permission № 659/96) were granted
by the local animal authorities.
2.2
Antibodies
Monoclonal
antibodies against mouse cytokines were used as primary antibodies. The
antibodies were as follows: rat-anti-mouse IL-2 (IgG2a; Pharmingen,
clone S4B6), rat-anti-mouse IL-4 (IgG1; Pharmingen, clone 11B11),
rat-anti-mouse IL-6 (IgG1; Pharmingen, clone MP5-20F3), rat-anti-mouse
IL-10 (IgG2b; Pharmingen, clone JES5-16E3), rat-anti-mouse
IL-12 (IgG1; Pharmingen, clone C15.6, reactive to the p40
subunit), rat-anti-mouse TNF-α (IgG1; Pharmingen, clone MP6-XT3),
rat-anti-mouse IFN-γ (IgG1; Pharmingen, clone R4-6A2) and
rat anti-mouse CD106 (VCAM-, c
2.3
Administration of testosterone
To
simulate the male hormonal contraception, a subcutaneous silastic (medical
grade tubing, ID 3.35 mm, OD 4.65 mm, Aromando Medizintechnik, Düsseldorf,
Germany; closed with Medical adhesive silicone type A, Dow Corning Medical
Products, Midland, Michigan, USA) testosterone implant (one cm in length)
or an empty (control) implant was introduced through a small incision
under the back skin of 12 normal, over 11-weeks old male mice under pentobarbital
anaesthesia (60 mg/kg, Mebunat, Orion, Finland). The wound was closed
with 5-0 Dermalon (American Cyanamid company, Wayne, NJ, USA) sutures.
The mice were followed for 17, 35, 52 and 70 days,
after which they were sacrificed with CO2. The testes were
collected and frozen in liquid nitrogen for later use.
2.4
Administration of estrogens
A
volume of 150 μL of an estrogen preparation (0.375 mg Estradiol. benz.,
1.5 mg Estradiol.
phenylpropion; Dimenformon ProlongatumR, N. V. Organon, Oss,
Holland) was injected ip of over 11 weeks old male mice. The mice were
followed for seven days, after which they were sacrificed with CO2.
The testes were collected and frozen in liquid nitrogen.
2.5
Vasectomy
Mice
(n=12) were vasectomized through a mid-abdominal incision under
pentobarbital anaesthesia. The vasa were exposed and ligated using 5-0
Dermalon sutures at two
sites 5 mm from each other and cut between the sutures with scissors.
Sham-operated animals were handled similarly except that the vasa were
left intact. The abdominal wound was closed using 5-0 Dermalon sutures.
The mice were followed for
17, 35, 52 and 70 days, after which they were sacrificed with CO2.
The testes were collected and frozen in liquid nitrogen.
2.6
Cryptorchidism
Mice
(n=3) were made unilaterally cryptorchid through mid-abdominal
incision under pentobarbital (MebunatR, Orion, Turku, Finland) anaesthesia.
The left testis was drawn from the scrotum to the abdominal cavity and
sutured to the parietal peritoneum with 5-0 Dermalon sutures. Sham*-operated
animals were handled similarly except that both testes were left intact.
The mice were followed for seven days, after which they were sacrificed
with CO2. The testes were collected and frozen in liquid nitrogen
for later use.
2.7
Varicocele
The
left spermatic vein of three mice was ligated via a mid-abdominal incision
under pentobarbital anaesthesia. Sham-operated mice were handled similarly
except that the spermatic
vein was left intact. The abdominal wound was closed in one layer using
5-0 Dermalon sutures. The mice were followed for 14 days, after which
they were sacrificed with CO2. The testes were collected and
frozen in liquid nitrogen for later use.
2.8
Indirect immunofluorescence (IF)
Frozen
sections 2-5 μm in thickness were cut in a cryostat. They were air-dried
briefly at room temperature and then fixed in cold acetone (-20℃)
for 1-2 minutes. The sections were stored at -20℃
and just before use, they were soaked
in PBS. The non-specific binding sites were blocked by incubating the
section in 5% normal rabbit serum in PBS (pH 7.4) for 15-20 minutes. After
washing in PBS, blocking of non-specific binding sites was continued with
5% normal rat serum in PBS for 15-20 minutes. The sections were washed
in PBS. Then they were incubated with the primary antibodies (diluted
into the concentration of 2.0 μg/mL in 1% BSA in PBS; Sigma, St. Louis,
MO) for 60 minutes. The sections were washed in PBS and incubated for
60 minutes with the secondary antibody (FITC-conjugated rabbit-anti-rat
Ig, Dako, cat no. F 0234, Copenhagen, Denmark, diluted in 5% normal mouse
serum in 1% BSA in PBS at the concentration of 10 μg total protean/mL).
After washing in PBS the sections were mounted in 1,4-diazabicyclo［2.2.2.］octane
(DABCO, Sigma, St. Louis, MO) -containing glycerol (50% glycerol in 2×PBS,
0.1% NaN3 and 100 mg/mL DABCO). The sections were examined
and photographed under an ultraviolet-microscope
equipped with an epi-illuminator and appropriate filters (Leitz, Wetzlar,
Germany).
2.9
Double immunocytochemistry
3 Results
3.1
IL-6
IL-6
was detected in the testes of postpubertal intact mice, mice treated for
70 days
with testosterone or empty implants and cryptorchid and sham-operated
mice. IL-6 is localised to the Leydig cells (Figs.
1a and b). Of the 77 mice studied, 15 (19.5%) had IL-6 expressing
cells at least in the other testis. There were no differences in the expression
of IL-6 between the testosterone and empty implant-treated mice or between
the cryptorchid and sham-operated mice.
3.2
IL-10
IL-10+
cells were present in all the studied testes except in two mice treated
with testosterone for 17 days, in all three mice vasectomized for 52 days,
in one mouse
each in the 14, 35 and 52 days sham-operated groups as well as in all
the three mice in
the varicocele group. Thus, 66 mice out of 77 (85.7%) investigated were
positive for IL-10. IL-10 was localised at the interstitial cells (Figures.
1c-h). Two types of IL-10+ cells were present, 1) morphologically
macrophage (mφ)-like cells, 2) other
interstitial cells, probably Leydig cells. The latter often showed weaker
staining intensity than the mφ-like
IL-10+ cells. The identity of the IL-10+ morphologically mφ-like cells
was investigated using double immunocytochemistry. The IL-10+ mφ-like
cells were found in several treatment groups: prepubertal intact mice,
postpubertal intact mice (11 weeks old), mice treated for 52 days with
testosterone or empty implant, mice treated for 35, 52 and 70 days with
an empty implant, mice treated 70 days with a testosterone implant, 35
and 70 days vasectomized and sham operated (vasectomy control) mice, mice
treated for 7 days
with estrogen, and 14 days sham operated (varicocele control) mice. In
30 mice out of 77 (39.0%) IL-10+ mφ cells were detected in the interstitial
tissue of the testis (Table 1). Double staining was made to identify the
IL-10+, morphologically mφ-like cells.
IL-10 colocalized with the macrophage-specific CD11b in the testes of
intact 9-11 weeks old mice (Figure 2).
As the normal mouse testis does not contain granulocytes, the IL-10+ interstitial
cells were thus most probably macrophages.
3.3
Other cytokines and controls
Figure
1. a) IL-6 in interstitial tissue of a mouse treated
with a testosterone implant for 35 days; immunocytochemistry on a frozen
section (×1267), b) the samefield as in Figure 1a, a phase contrast (×1267),
c) IL-10+ cells (arrows) in interstitial tissue of a mouse treated with
an empty implant for 52 days; immunocytochemistry on a frozen section
(×1267), d) the same field as in Figure 1c, phase contrast
(×1267), e) IL-10+ cells (arrows) in interstitial tissue of amouse treated
with an empty implant for 52 days; immunocytochemistry on a frozen section
(×633), f) the same field as in Figure 1e, phase contrast (×633), g)
IL-10 in the interstitial
tissue of a mouse treated with a testosterone implant for 70 days; immunocytochemistry
on a frozen section (×1267), h) the same field as in Figure 1g,
phase contrast (×1267), i) CD106 in the interstitial tissue of a mouse
testis 52 days after vasectomy; immunocytochemistry on a frozen section
(×1267),j) the same field as in figure 1i, phase contrast (×1267), k)
IL-4 staining of a
testis of a mouse treated with an empty implant for 52 days, IL-4 is not
present; immunocytochemistry
on frozen section (×1267), l) the same field as in Figure1k, phase contrast
(×1267).
Figure 2. IL-10 immunocytochemistry
on frozen section (a and d) as well as CD11b (b and
e), the same field in c as in a and b, the same field in f as in d and
e. Figure 1a-c ×1267,
Figure 1d-f ×633.
Figure
3. A simplified illustration of immunological microenvironment of
testis. If a T cell
migrating into testicular interstitial tissue meets its antigen there
on MHC-II of testicular macrophages (MΦ), it should go anergic because
of lack of necessary co-stimulating factors CD80 and CD86[60]. In addition
to lack of necessary co-stimulation, clonal anergy is promoted by secretion
of IL-10 by test icular
interstitial cells as shown in present study. Furthermore, TGFβ-1 produced by
Sertoli cells affects Tcell activation in this site. FasL affects only
activated T cells.
Table 1. Presence of IL-10+ Leydig cells, IL-10+ macrophage-like cells, IL-6, IL-2, IL-4, IL-12, TNF-α and IFN-γ in testes of mice after various treatments.
|
Treatment
groups |
IL-10+ |
IL-10+
mΦ |
IL-6 |
IL-2 |
IL-4 |
IL-12 |
TNF-α |
IFN-γ |
|
Intact
mice: |
||||||||
|
Prepubertal.
(10 d) |
4/4 |
2/4 |
0/4 |
0/4 |
0/4 |
0/4 |
0/4 |
0/4 |
|
Pubertal.
(4 w) |
3/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
Postpub.
(9-11 w) |
3/3 |
3/3ad |
3/3adg |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
Postpub.
(26 w) |
4/4 |
0/4 |
0/4 |
0/4 |
0/4 |
0/4 |
0/4 |
0/4 |
|
Testosterone
implant: |
||||||||
|
17
d |
1/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
35
d |
3/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
52
d |
3/3 |
0/3v |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
70
d |
3/3 |
3/3jmp |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
Empty
implant: |
||||||||
|
17
d |
3/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
35
d |
3/3 |
1/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
52
d |
3/3 |
3/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
70
d |
3/3 |
3/3j |
3/3jmp |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
Vasectomized: |
||||||||
|
17
d |
3/3p |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
35
d |
3/3p |
2/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
52
d |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
70
d |
3/3p |
3/3jp |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
Sham
operated: |
||||||||
|
17
d |
3/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
35
d |
2/3 |
1/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
52
d |
2/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
70
d |
3/3 |
3/3jp |
0/3jp |
0/3 |
0/3 |
0/3 |
0/3 |
0/3 |
|
Varicocele
(14 d) |
||||||||