Life Sciences 67 (2000) 133Ð145

In vitro regulation of extracellular superoxide dismutase

in sertoli cellsq
Dolores D. Mruk, C. Yan Cheng*
Population Council, Center for Biomedical Research, New York, New York, USA

Abstract
Rat Sertoli and germ cells express extracellular superoxide dismutase (SODEX), however, the rela-
tive level of SODEX expressed by these cells was not known. We report herein germ cells consisting
largely of spermatogonia, spermatocytes, and round spermatids expressed only one-third SODEX as
that of Sertoli cells when examined by semi-quantitative RT-PCR. While cocultures of germ cells with
Sertoli cells failed to induce any changes in SODEX expression possibly due to the limited number
of cells that can be supported by the in vitro culture system dissimilar to the in vivo condition, incuba-
tion of total germ cell-conditioned medium with Sertoli cells was able to signiÞcantly inhibit Sertoli
cell SODEX expression dose-dependently suggesting a germ cell-derived soluble factor(s) may regulate
SODEX in the testis. On the other hand, cytokines such as TGF-b1, b-NGF, or FGF and steroid
hormones such as estradiol-17b, progesterone, testosterone, and DHT were unable to effect the ex-
pression of Sertoli cell SODEX. However, FSH at 100 ng/dish was able to induce a signiÞcant increase
in Sertoli cell SODEX expression. While cytokines, the known mediators of the inßammatory response,
were unable to affect Sertoli cell SODEX expression, the induction of generalized inßammation in vivo
was able to cause a 2- to 2.5-fold increase in testicular SODEX expression concomitant with a transient
increase in the liver but not in the brain. Taken collectively, these results demonstrate that while
SODEX is an important antioxidant enzyme protecting the testis from reactive oxygen species,
the mechanism(s) regulating its expression may involve an array of molecules and is a complicated
cellular event. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Extracellular superoxide dismutase; Sertoli cells; Germ cells; Testis

q
This work was supported in part by grants from the CONRAD Program (CICCR, CIG-96-05-A), Noopolis
Foundation, National Institutes of Health (HD-13541), and the Rockefeller Foundation (PS-9601, PS-9721, PS-
9815). This work was performed as part of a dissertation submitted to the Hong Kong University Higher Degree
committee by D.D.M. for the partial fulÞllment for the requirements of the Degree of Doctor of Philosophy.
Ovine FSH was a gift from the National Hormone and Pituitary Program, NICHD, NIH.
* Corresponding author. Tel.: 212-327-8738; fax: 212-327-7678.
E-mail address: yan@popcbr.rockefeller.edu (C. Yan Cheng)

0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 0 9 -3
134 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145

Introduction
In the rat testis, the blood-testis barrier formed by specialized tight junctions between ad-
jacent Sertoli cells at the basal compartment of the seminiferous epithelium creates a unique
microenvironment in which spermatogenesis and spermiogenesis take place. Developing
germ cells, in addition to undergoing complex morphological, molecular, and biochemical
changes, migrate progressively from the basal to the adluminal compartment of the seminif-
erous epithelium where they are eventually released into the tubular lumen during spermia-
tion (for reviews, see 1, 2). This extensive tissue restructuring in the seminiferous epithelium
involves the active participation of numerous molecules. As such, these biochemical events
also result in the production of reactive oxygen and nitrogen species such as superoxide, hy-
droxyl, peroxyl, hydroperoxyl, alkoxyl, nitric oxide, and nitrogen dioxide radicals which
when left unchecked can lead to cell damage and death in the testis (3, 4).
Consequently, organisms have evolved numerous sophisticated antioxidant defense mech-
anisms to protect themselves. Among the presently known antioxidant enzymes protecting
cells from various forms of reactive oxygen species are superoxide dismutases (SODs), cata-
lases (CATs), and peroxidases (Pxs)(for reviews, see 5, 6). SODs speciÞcally scavenge su-
peroxide radicals and convert them into hydrogen peroxide and oxygen which in turn are bro-
ken down into water by catalase in peroxisomes and glutathione peroxidase in the cytosol
and mitochondria.
A recent study has demonstrated in addition to producing cytosolic and mitochondrial
SODs, (3, 4) Sertoli cells are also capable of synthesizing and secreting an extracellular
form of SOD (SODEX) (7). Certainly, this is not surprising if one considers the relatively
large surface area of the Sertoli cell which remains in contact with developing germ cells in
all phases of spermatogenesis suggesting the net output of energy and ßow of metabolites is
enormous. These result in the generation of reactive oxygen and nitrogen species. More-
over, SODEX expression was also detected in germ cells (7) demonstrating they, too, have
intricate antioxidant defense mechanisms (3, 4). More important, germ cells when cocul-
tured with Sertoli cells for a short period of time using a germ:Sertoli cell ratio of 1:1 failed
to induce any changes in the expression of SODEX in the cocultures (7) suggesting germ
cell dependence on Sertoli cell metabolic factors in vitro does not result in increased oxida-
tive stress.
Previous studies have demonstrated there are several changes in both cytosolic and mito-
chondrial SOD expression due to various experimental conditions which include factors such
as hormones and cytokines, and the induction of the inßammatory response. For instance,
studies have illustrated the incubation of various cytokines such as interleukin-1a (IL-1a)
and tumor necrosis factor-a (TNF-a) was able to induce remarkable changes in mitochon-
drial SOD expression whereas cytosolic SOD expression remained relatively unchanged in
the rat corpus luteum (8). Since limited studies have been performed on the regulation of
SODEX in the testis, we sought to examine the modulation of SODEX under various experi-
mental conditions such as the induction of inßammatory response and the role of cytokines in
the expression of SODEX in an attempt to better understand the regulation of this molecule
in the testis.
 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145 135

Materials and Methods

Sertoli cell-enriched cultures
Primary Sertoli cell cultures were prepared from 20-day-old Sprague-Dawley rats by se-
quential enzymatic treatments as previously described (7, 9, 10). Isolated cells were plated on
MatrigelTM-(diluted 1:7 with serum-free HamÕs F12 nutrient mixture and DulbeccoÕs modi-
Þed EagleÕs medium [F12/DMEM, 1:1, v/v]) coated 12-well dishes at a density of 0.53106
cells/cm2 in F12/DMEM supplemented with gentamicin (20 mg/L), sodium bicarbonate
(1.2 gm/L), 15 mM HEPES, bovine insulin (10 mg/ml), human transferrin (5 mg/ml), bacitracin
(5 mg/ml), and epidermal growth factor (2.5 ng/ml) in a dish volume of 1 ml. Cells were in-
cubated at 35 8C in a humidiÞed atmosphere of 95% air and 5% CO2 (v/v). Cultures were hy-
potonically treated 48 hr after plating with 20 mM Tris, pH 7.4 at 22 8C for 2.5 min to lyse
contaminating germ cells (11) followed by two successive washes with F12/DMEM to re-
move cellular debris. These Sertoli cells were then incubated for an additional 5 days to al-
low the formation of specialized occluding, anchoring, and communicating junctions. Media
were replaced every 24 to 48 hr thereafter. The establishment of tight junctions was assessed
as previously described using bicameral units by the measurement of trans-epithelial resis-
tance, polarized secretion of transferrin, and maintenance of non-equilibrium of media be-
tween the basal and apical compartments (12Ð14). Thereafter, these Sertoli cells were cul-
tured in either the presence of germ cell-conditioned media (GCCM)-derived proteins,
cytokines, or hormones for 0 to 24 hr and subsequently processed for RNA extraction.

Germ cell cultures and preparation of GCCM

Germ cells were isolated from adult Sprague-Dawley rats (250 to 300 gm b.w.) by a me-
chanical procedure without the use of trypsin since trypsinization was shown to affect the
functional and cell adhesional properties of these cells (15, 16). These germ cells consisting
largely of spermatogonia, pachytene spermatocytes, and round spermatids (elongate sperma-
tids and spermatozoa were removed by passing cells through glass wool) were greater than
95% pure when examined by DNA ßow cytometry and direct microscopic examination. Fur-
thermore, RNA extracted from these germ cells for RT-PCR failed to amplify testin cDNA
(17, 18), a Sertoli and Leydig cell product, demonstrating negligible somatic cell contamina-
tion. The resulting germ cells were plated in 100-mm dishes at a cell density of 0.33106
cells/cm2 supplemented with gentamicin (20 mg/L), sodium bicarbonate (1.2 gm/L), 15 mM
HEPES, 2 mM sodium pyruvate, 6 mM sodium DL-lactate, bovine insulin (10 mg/ml), hu-
man transferrin (5 mg/ml), bacitracin (5 mg/ml), and epidermal growth factor (2.5 ng/ml) in a
dish volume of 9 ml and cultured as described above. Media were collected at the end of the
18-hr culture period as GCCM. In addition, germ cell viability was greater than 90% at
the end of the 18-hr culture period as determined by the erythrosine red-dye exclusion test
(19). A batch of 5 L of GCCM containing approximately 500 mg protein was pooled, con-
centrated at 4 8C, and equilibrated against F12/DMEM using a Millipore Minitanª tangen-
tial ultraÞltration unit equipped with eight Minitanª plates with a Mr cut-off at 10,000. The
sample was then centrifuged at 45,000 g for 45 min to remove cellular debris, Þltered
through a 0.2 mm Þlter unit, and stored at Ð20 8C until use.
136 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145

Treatment of Sertoli cell cultures with cytokines

Sertoli cells were isolated essentially as described above and cultured on Matrigelª-
coated 12-well dishes at a density of 0.53106 cells/cm2. Cultures were hypotonically treated
as described above. Thereafter, Sertoli cells were cultured for 0 to 24 hr in the presence of
either human recombinant transforming growth factor-b1 (TGF-b1, 10 ng/dish), b-nerve
growth factor (b-NGF, 10 ng/dish), interleukin-1a (IL-1a, 10 ng/dish), and Þbroblast growth
factor (FGF, 50 ng/dish) (Calbiochem, La Jolla, CA) in a dish volume of 1 ml. Total RNA
was subsequently extracted from these cells for RT-PCR. Control experiments included Ser-
toli cells cultured alone in duplicate under the same conditions as described above without
the addition of any cytokines.
Treatment of Sertoli cell cultures with hormones
Sertoli cells were isolated essentially as described above and cultured on Matrigelª-
coated 12-well dishes at a density of 0.53106 cells/cm2. Cultures were hypotonically treated
as described above. Thereafter, Sertoli cells were cultured in either the presence of FSH (100
ng/dish for 0 to 24 hr), estradiol-17b (131027 M for 0 to 24 hr; 131025 M to 131029 M for
4 hr), progesterone (131027 M for 0 to 24 hr; 131025 M to 131029 M for 4 hr), testosterone
(131025 M to 131029 M for 4 hr) or 17b-hydroxy-5a-androstan-3-one (DHT, 131027 M
for 0 to 24 hr) (Sigma, St. Louis, MO) in a dish volume of 1 ml. Total RNA was subsequently
extracted from these cells for RT-PCR. Control experiments included Sertoli cells cultured
alone in duplicate under the same conditions as described above either without the addition
of any hormones or with vehicle (ethanol) only.
Induction of generalized inßammation
An inßammatory response was induced in adult male Sprague-Dawley rats (250 to 300 gm
b.w.) by injection with fermented yeast as previously described (20). Brießy, rats were injected
subcutaneously at multiple sites with 10 ml/kg b.w. of 10% (gm/ml) fermented brewerÕs yeast
suspended in sterile water. Animals were killed by CO2 asphyxiation, and testes, liver, and
brain were removed at 2, 6, 24, 48, and 96 hr after the injection of yeast. Tissues were imme-
diately frozen in liquid nitrogen and stored at 280 8C until use for RNA extraction.
Hepatectomy
Adult male Sprague-Dawley rats were anesthetized with Metofaneª. A small abdominal
incision was made, approximately 70% of the liver removed as previously described (21),
and the incision closed by a suture. Sham operated animals consisted of rats in which an ab-
dominal incision was made, the liver lobes manipulated without resection, and the incision
closed by a suture. Animals were killed by CO2 asphyxiation, and the liver was removed at 6,
18, and 24 hr after the operation. Tissue was immediately frozen in liquid nitrogen and stored
at 280 8C until use for RNA extraction.
RT-PCR
Total RNA was extracted from either cells or tissues using RNA STAT-60ª (Tel-test ÒBÓ
Inc., Friendswood, TX) according to the manufacturerÕs instructions. RT-PCR was performed
 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145 137

essentially as previously described (7, 10, 14). Brießy, 2 mg of total RNA was reverse tran-
scribed into cDNAs using 5 mg of oligo (dT)15 and an M-MLV reverse transcriptase kit
(Promega, Madison, WI) in a Þnal reaction volume of 25 ml. From this reaction product, 3 ml
was used and served as a template for PCR in combination with 0.3 mg each of the SODEX
sense and antisense primers coampliÞed with the rat cytoplasmic b-actin primers. The primers
used for the ampliÞcation of SODEX (22) and b-actin (23) were as follows: 59-ATGGTGGCCT
TCTTGTTCTGC-39 (SODEX, sense, nucleotides 109 to 129), 59-GTGCTGTGGGTGCGGCA
CACC-39 (SODEX, antisense, nucleotides 535 to 555), 59-TCACCGAGGCCCCTCTGAACCCTA-
39 (b-actin, sense, nucleotides 314 to 337), and 59-GGCAGTAATCTCCTTCTGCATCCT-39
(b-actin, antisense, nucleotides 931 to 954). CoampliÞcation with b-actin was included to
ensure that equal amounts of RNA were reverse transcribed and ampliÞed in each reaction
tube. The cycling parameters for the PCR reaction were as follows: denaturation at 948C for
1 min, annealing at 618C for 2 min, and extension at 728C for 3 min. A total of 22 cycles
were performed. The cycles were followed by an extension period at 728C for 15 min. Ali-
quots of 5 ml were resolved onto 5% T polyacrylamide gels in 0.5-strength TBE buffer (45
mM Tris; 45 mM boric acid; 1 mM EDTA, pH 8.0 at 228C). In some instances, an aliquot
from the RT product was used as a template for hot-nested PCR as previously described (7,
10, 14). About 0.2 mg of the antisense SODEX primer was 59-end labeled with g-[32P]-ATP
(speciÞc activity, 6000 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) by using
T4 polynucleotide kinase (Promega). The antisense b-actin primer was also 59-end labeled
for coampliÞcation as described above. Under these conditions, the ampliÞcations of SODEX
and b-actin were both in the linear range, as veriÞed in preliminary experiments when an ali-
quot of 5 ml of PCR product was withdrawn from each of the PCR reaction tubes in cycles
18, 20, 22, 25, 27, and 30 for gel analysis. PCR products were visualized by ethidium bro-
mide staining and autoradiography. The 447 bp cDNA generated by RT-PCR was previously
conÞrmed to be authentic SODEX when it was electroeluted and subcloned into pGEM Tª
vector (Promega) for nucleotide sequencing (7).

General methods
Densitometric scanning of autoradiograms was performed using an UltroScan XL En-
hanced Laser Densitometer (Amersham Pharmacia Biotech) at 600 nm. Statistical analysis
was performed by StudentÕs t-test using the GB Statistical Analysis Software package (ver-
sion 3.0, Dynamic Microsystems, Inc., Silver Spring, MD).

Results
Expression of SODEX by Sertoli and germ cells
In our earlier study, Sertoli and germ cells were both found to express SODEX (7). How-
ever, the relative contribution of SODEX expression by germ cells in comparison to Sertoli
cells in the seminiferous epithelium was not known. Using multiple batches of cell prepara-
tions, we demonstrated by semi-quantitative RT-PCR that germ cells isolated from adult rats
consisting largely of spermatogonia, spermatocytes, and round spermatids expressed only
138 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145

Fig. 1. Comparative examination of SODEX expression by germ and Sertoli cells. (A) An autoradiogram of RT-
PCR showing germ cells consisting largely of spermatogonia, spermatocytes, and round spermatids are capable of
only one-third of SODEX expression as that detected by Sertoli cells. Co-ampliÞcation was performed using a
b-actin primer pair. (B) Densitometric scannings of at least three different autoradiograms of RT-PCR such as the
one shown in (A) normalized against b-actin. (C) Ethidium bromide stained gel of the RT-PCR experiment shown
in (A) illustrating that the relative expression of b-actin in germ and Sertoli cells is comparable between these two
different cell types. * SigniÞcantly different from germ cells, p,0.001.

about one-third of SODEX when compared with Sertoli cells (Fig. 1A and 1B). Fig. 1C de-
monstrates the relative expression of b-actin in germ cells and Sertoli cells is comparable
between these two different cell types.
Effects of germ cells or GCCM-derived proteins on Sertoli cell SODEX expression
In a previous study, we reported that the coculture of germ cells with Sertoli cells using a
germ:Sertoli cell ratio of 1:1 for up to 24 hr at the time germ cells attached to Sertoli cells but
before the establishment of any specialized junctions was unable to cause any signiÞcant
changes in the expression of SODEX (7). Since there is a drastic increase in the germ:Sertoli
cell ratio in the testis in vivo during maturation, we used a germ:Sertoli cell ratio of 5:1 (Ser-
toli cell density of 0.53106 cells/cm2) in the present study for coculture experiments. How-
ever, the increase in germ cell number still failed to affect SODEX expression (data not
shown). Since it is known that the germ:Sertoli cell ratio increases to as much as 50:1 in the
adult rat testis (24Ð26), we felt that an in vitro coculture experiment at this cell ratio should
be performed. However, cells cultured at this cell density underwent rapid cell death due to
insufÞcient diffusion of metabolites and metabolic wastes from the cocultured cells as ob-
served in preliminary experiments when the cell viability was assessed by trypan blue staining
 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145 139

Fig. 2. Regulation of Sertoli cell SODEX expression by increasing concentrations of GCCM-derived proteins in
vitro. (A) An autoradiogram of RT-PCR showing a dose-dependent decrease in SODEX expression when Sertoli
cells were incubated with 20 to 1000 mg total GCCM-derived proteins/dish for 4 hr. Co-ampliÞcation was per-
formed using a b-actin primer pair. (B) Densitometric scannings of at least three different autoradiograms of
RT-PCR such as the one shown in (A) normalized against b-actin. * SigniÞcantly different from control at time 0 hr
without the addition of GCCM-derived proteins, p,0.01; ** signiÞcantly different from control at time 0 hr with-
out the addition of GCCM-derived proteins, p,0.001; ns, not signiÞcantly different from control at time 0 hr
without the addition of GCCM-derived proteins.

(Wong and Cheng, unpublished observations). To circumvent this difÞculty, proteins derived
from concentrated GCCM were used instead to examine whether germ cells are indeed capa-
ble of modulating Sertoli cell SODEX expression. The addition of GCCM-derived proteins at
100 to 300 mg total protein/dish in vitro was able to signiÞcantly reduce Sertoli cell SODEX
expression at 4 hr whereas the addition of 500 to 1000 mg total protein/dish was able to re-
duce Sertoli cell SODEX expression by about 50% (Fig. 2A and 2B). Three different batches
of concentrated GCCM-derived proteins were assessed for their dose-dependent inhibitory
effects on Sertoli cell SODEX expression each yielding similar results.
Effects of cytokines on Sertoli cell SODEX expression
Germ cells are capable of producing and/or expressing various cytokines such as bFGF
(27, 28), NGF (29Ð31), and TGF-b1 (32). Since it has also been demonstrated that these mole-
cules are capable of inßuencing cytosolic, mitochondrial, and extracellular SOD expression in
vitro (8, 33, 34), we proceeded to examine the expression of SODEX in Sertoli cells incubated
with various cytokines. Several cytokines such as b-NGF (10 ng/dish), TGF-b1 (10 ng/dish),
and FGF (50 ng/dish) were unable to signiÞcantly affect SODEX expression when cultured
with Sertoli cells for up to 24 hr in vitro (data not shown). The only cytokine that had a positive
stimulatory effect was IL-1a (data not shown) which is consistent with our earlier report (7).
Effects of hormones on Sertoli cell SODEX expression
In addition to the involvement of cytokines, androgens, estrogens, and progestins also play
fundamental roles in the regulation of spermatogenesis (for reviews, see 35Ð37). Further-
more, there are differences in the activity of SOD measured in several organs in the male and
female suggesting SODEX may be under the inßuence of sex steroids and hormones. There-
fore, we examined the expression of SODEX in Sertoli cells incubated with various hormones.
FSH (100 ng/dish) was capable of causing a 2- to 4-fold increase in Sertoli cell SODEX ex-
140 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145

Fig. 3. Regulation of SODEX expression by FSH (100 ng/dish from 0 to 24 hr, A, B) and testosterone (1 3 1025 M to
1 3 1029 M at 4 hr, C) in vitro. (A) An autoradiogram of RT-PCR showing there was a 2- to 4-fold increase in SODEX
expression from 1 to 24 hr when Sertoli cells were incubated with FSH. Co-ampliÞcation was performed using a
b-actin primer pair. (B) Densitometric scannings of at least three different autoradiograms of RT-PCR such as the
one shown in (A) normalized against b-actin. (C) Densitometric scannings of at least three different autoradiograms
of RT-PCR normalized against b-actin illustrating no signiÞcant changes in SODEX expression were detected when
cells were cultured in the presence of testosterone. Sertoli cells (0.5 3 106 cells/cm2) were cultured on Matrigel-
coated 12-well dishes with 1 ml F12/DMEM in each well. * SigniÞcantly different from control at 0 hr without
the addition of either FSH or testosterone or with vehicle (ethanol) only, p,0.01; ** signiÞcantly different from
control at 0 hr without the addition of either FSH or testosterone or with vehicle only, p,0.001; ns, not signiÞ-
cantly different from control at 0 hr without the addition of either FSH or testosterone or with vehicle only.

pression from 2 to 24 hr (Fig. 3A and 3B) whereas neither estradiol-17b (131027 M from 0
to 24 hr or 131025 M to 131029 M at 4 hr, data not shown), progesterone (131027 M from
0 to 24 hr, data not shown), testosterone (131025 M to 131029 M at 4 hr, Fig. 3C), DHT
(131027 M from 0 to 24 hr, data not shown), nor vehicle (ethanol, data not shown) were able
to signiÞcantly affect Sertoli cell SODEX expression. Furthermore, the increase in SODEX ex-
pression is speciÞc to FSH since the incubation of Sertoli cells alone without the presence of
FSH failed to elicit any changes in the expression of SODEX (data not shown).
Effects of the inßammatory response on SODEX expression
Since SOD has been implicated in inßammation (for reviews, see 5, 38, 39) but cytokines
that regulate the inßammatory response failed to elicit any changes in the expression of Ser-
toli cell SODEX, we examined SODEX expression utilizing an in vivo model of inßammation.
 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145 141

Fig. 4. Changes in SODEX expression in the testis (A,B), liver (C,D), and brain (E) when adult male rats were
induced with inßammation by the injection of fermented yeast in vivo. (A,C) Autoradiograms of RT-PCR showing
there was a 2- to 2.5-fold and 1.5-fold increase in SODEX expression in the testis and liver, respectively, whereas
no signiÞcant changes in expression were detected in the brain (E) during induced inßammation. Co-ampliÞcation
was performed using a b-actin primer pair. (B,D) Corresponding densitometric scannings of at least three different
autoradiograms of RT-PCR normalized against b-actin. * SigniÞcantly different from control at time 0 hr without
the induction of inßammation, p,0.05; ns, not signiÞcantly different from control at time 0 hr without the induc-
tion of inßammation.

The subcutaneous injection of fermented yeast into the adult male rat thereby inducing in-
ßammation was able to cause about a 2- to 2.5-fold increase in SODEX expression in the testis
(Fig. 4A and 4B) and a transient increase in SODEX expression in the liver (Fig. 4C and 4D),
whereas no changes in SODEX expression were detected in the brain (Fig. 4E). In contrast,
hepatectomy failed to elicit any changes in SODEX expression in the liver when examined by
RT-PCR (data not shown).
142 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145

Discussion
Since a previous report demonstrated that coculture of germ cells with Sertoli cells in vitro
for a short period of time when there are intricate interactions involving numerous molecules
was unable to elicit any signiÞcant changes in the expression of SODEX, we presumed the
function and/or regulation of this molecule in the testis to be extremely complex (7). The un-
derlying rationale is that these extensive cell-cell interactions will lead to an increase in me-
tabolism that in turn will change the content of the free oxygen radicals in the testis. In this
report, we demonstrate that germ cells are capable of modulating Sertoli cell SODEX expres-
sion in vitro via a secretory factor(s) since incubation of crude GCCM-derived proteins at
100 to 1000 mg total protein/dish were able to effect SODEX expression. It is not surprising
that germ cells are also capable of expressing SODEX since their membranes are very abun-
dant in polyunsaturated fatty acids, and they possess few repair mechanisms making them
highly susceptible to radical-induced damage. However, the level of SODEX expression in
germ cells is only a fraction of that expressed by Sertoli cells. In addition, a previous report
demonstrated that there was a drastic increase in testicular SODEX expression during devel-
opment at the onset of spermatogenesis suggesting the possible involvement of germ cells in
contributing to the steady-state SODEX mRNA level in the testis (7). Furthermore, the depen-
dence of germ cells on Sertoli cells for metabolic factors such as sodium lactate and pyruvate
in vivo and vitro generates reactive oxygen species resulting in a subsequent increase in
SODEX. The failure of germ cells cultured at either a 1:1 or 1:5 cell ratio to affect Sertoli cell
SODEX expression may be due to the fact that under these experimental conditions the exact
in vivo conditions are not being mimicked since in vivo the Sertoli cell in the adult rat testis
has contact with approximately 50 germ cells (24Ð26). These data, in addition to the fact that
SODEX expression surged drastically in the testis upon maturation, in which there was an in-
crease in Sertoli-germ cell interactions following the onset of spermatogenesis, seemingly
suggests that increased cellular interactions may be required to switch on the expression of
SODEX in the testis. It is also possible that germ cells and/or other testicular cells are also ca-
pable of stimulating Sertoli cell SODEX expression in vivo via a soluble factor(s) which in
turn protects Sertoli, germ, and/or other cell types in the seminiferous epithelium from reac-
tive oxygen species. Nevertheless, we do not immediately understand the physiological con-
sequence of a germ cell-derived factor(s) that can inhibit SODEX expression as illustrated in
this report since conventional wisdom seems to suggest a stimulation instead. Perhaps other
antioxidants participate in these events leading to an increase in overall antioxidant activity.
In addition, a comparative study examining the relative expressions of the other antioxidant
enzymes such as cytosolic and mitochondrial SODs and catalase in Sertoli and germ cells has
not been demonstrated.
The observation that cytokines were unable to signiÞcantly effect Sertoli cell SODEX ex-
pression is quite unexpected being that many cellular processes such as cell movement, in-
ßammation, tissue repair and remodeling depend upon the intricate interactions between
cells and the extracellular matrix. For instance, TGF-b whose mRNA was detected in both
Sertoli and germ cells (32) is known to affect the metabolic activities of testicular somatic
cells (40, 41). Marklund has shown that cytokines which included INF-g, TNF-a, and TGF-b
were able to signiÞcantly affect SODEX expression when incubated with human dermal Þbro-
 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145 143

blasts (33). However, the responses reported in this earlier study were quite slow, generally
taking several days to develop. Therefore, this may be a possible reason for the failure of
cytokines to inßuence SODEX expression since the Sertoli cells used in our experiments were
cultured in the presence of various cytokines for only up to 24 hr. Furthermore, rat SODEX,
unlike its mammalian counterpart, is a dimer of about 68 kDa that possesses little, if any, af-
Þnity for heparin and does not bind to heparan sulfate in vivo suggesting that the mechanism
of action and/or regulation of this molecule in the rat is different from the other mammals
studied thus far.
In addition to the involvement of cytokines in the inßammatory response, it is also known
that the immune system is regulated by gonadal steroids such as androgen, estrogen, and
progesterone via the hypothalamic-pituitary-gonadal-thymic axis (for reviews, see 42, 43).
Many investigators have reported differences in male-female antioxidant enzyme activities in
various tissues suggesting that these discrepancies are due to the involvement of gonadal hor-
mones. For instance, it was reported that the activity of glutathione peroxidase and catalase in
the rat liver was higher in males (44Ð46) whereas the reverse was demonstrated in another
study (47). Nevertheless, it is known that Sertoli cells isolated from immature rats are capa-
ble of metabolizing testosterone to 5a-reduced androgens (DHT) and 5a-androstanediols
(DIOL) and to estradiol-17b and estrone (for reviews, see 48Ð51). However, data presented
herein unequivocally demonstrate that estradiol-17b, progesterone, testosterone, and DHT
were unable to signiÞcantly effect SODEX expression when cultured with Sertoli cells illus-
trating that SODEX expression in the testis is not regulated by steroid hormones. It is not
known from this study whether natural or synthetic glucocorticoids such as cortisone or
dexamethasone can affect Sertoli cell SODEX expression.
On the other hand, a striking increase in SODEX expression was detected when Sertoli
cells were cultured in the presence of FSH (100 ng/dish). The physiological role of FSH in
modulating SODEX expression is not immediately known. The binding of FSH onto its recep-
tors on the Sertoli cell membrane causes a surge in cAMP levels which subsequently stimu-
lates protein kinase activity resulting in increased rates of RNA, DNA and protein synthesis
(52Ð55). For instance, FSH stimulates the production of numerous proteins, among them an-
drogen binding protein (ABP), plasminogen activator, and transferrin (for reviews, see 56,
57). In addition, FSH has also been shown to modulate glucose transport by Sertoli cells
(58). While the reasoning for the observed effect of FSH on SODEX expression is not clear,
the enhanced expression of SODEX by FSH must play an essential role in protecting the testis
from free radical damage.
We also examined the expression of SODEX in the testis, liver, and brain induced with gen-
eralized inßammation by the injection of fermented yeast. It is known that the response of
mononuclear phagocytes to infection and immunological stresses such as inßammation is ex-
tremely rapid, involving the production of several cytokines. Yet, we have demonstrated
herein that SODEX expression in the Sertoli cell, the major phagocytic component in the sem-
iniferous epithelium, was unresponsive to treatments with various cytokines. Unexpectedly, a
surge in SODEX expression was detected in the testis and liver but not in the brain when rats
were induced with inßammation suggesting that SODEX may be an acute-phase protein in the
testis and liver. In contrast, hepatectomy failed to elicit any changes in the expression of liver
SODEX. The failure to detect any changes liver SODEX in hepatectomized rats may be due to
144 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145

the fact that this model of inßammation is extremely complicated in the sense that in addition
to inßammation, there is also extensive tissue regeneration and restructuring. These pro-
cesses involve a host of molecules that may indeed be masking SODEX expression at the time
points examined in this study. Collectively, these results suggest that the regulation of SODEX
in the Sertoli cell is complex but more important, that the mechanism of action of SODEX in
the testis remains to be elucidated.

References
1. B.P. SETCHELL and G.M.B. WAITES, The Handbook of Physiology, D.W. Hamilton and R.O. Greep (Eds),
143Ð172, Williams and Wilkens, Baltimore (1975).
2. D.M. DE KRETSER and J.B. KERR, The Physiology of Reproduction, E. Knobil, J. Neill, L.L. Ewing, G.S.
Greenwald, C.L. Markert, and D.W. Pfaff (Eds), 837Ð932, Raven Press, New York (1988).
3. V. PELTOLA, I. HUHTANIEMI, and M. AHOTUPA, J. Androl. 13 450Ð455 (1992).
4. F. BAUCHE, M.H. FOUCHARD, and B. JEGOU, FEBS Letts. 349 392Ð396 (1994).
5. J.M. MC CORD, New Horiz. 1 70Ð76 (1993).
6. H. SIES, Exp. Physiol. 82 291Ð295 (1997).
7. D.D. MRUK, C.H. CHENG, Y.H. CHENG, M.Y. MO, J. GRIMA, B. SILVESTRINI, W.M. LEE, AND C.Y.
CHENG, Biol. Reprod. 59 298Ð308 (1998).
8. N. SUGINO, C.M. TELLERIA, and G. GIBORI, Biol. Reprod. 59 208Ð215 (1998).
9. C.Y. CHENG, J.P. MATHER, A.L. BYERS, and C.W. BARDIN, Endocrinol. 118 480Ð488 (1986).
10. D.D. MRUK and C.Y. CHENG, J. Biol. Chem. 274 27056Ð27068 (1999)
11. M. GALDIERI, E. ZIPARO, F. PALOMBI, M.A. RUSSO, and M. STEFANINI, J. Androl. 5 249Ð259 (1981).
12. J. GRIMA, C. PINEAU, C.W. BARDIN, and C.Y. CHENG, Mol. Cell Endocrinol. 89 127Ð140 (1992).
13. J. GRIMA, C.C.S. WONG, L.J. ZHU, S.D. ZONG, and C.Y. CHENG, J. Biol. Chem. 273 21040Ð21053
(1998).
14. D.D. MRUK, L.J. ZHU, W.M. LEE, B. SILVESTRINI, and CHENG CY, J. Androl. 8 612Ð622 (1997).
15. G.R. ARAVINDAN, C.P. PINEAU, C.W. BARDIN, and C.Y. CHENG, J. Cell. Physiol. 168 123Ð133 (1996).
16. G.R. ARAVINDAN, D.D. MRUK, W.M. LEE, and C.Y. CHENG, Endocrinol. 138 3259Ð3268 (1997).
17. C.Y. CHENG, J. GRIMA, M.S. STAHLER, R.A. LOCKSHIN, and C.W. BARDIN, Endocrinol. 118 480Ð
488 (1989).
18. J. GRIMA, L.J. ZHU, S.D. ZONG, J.F. CATTERALL, C.W. BARDIN, and C.Y. CHENG, Biol. Reprod. 52
340Ð355 (1995).
19. H.J. PHILLIPS AND J.E. TERRYBERRY, Exp. Cell. Res. 13 341Ð347 (1957).
20. B. SILVESTRINI, A. GUGLIELMOTTI, L. SASO, and C.Y. CHENG, Clin Chem 35 2207Ð2211 (1989).
21. G.M. HIGGINS and R.M. ANDERSON, Arch. Pathol. Lab Med. 12 186Ð202 (1931).
22. A.C.F. PERRY, R. JONES, and L. HALL, Biochem. J. 293 21Ð25 (1993).
23. U. NUDEL, R. ZAKAT, M. SHANI, S. NEUMAN, Z. LEVY, and D. YAFFE, Nucleic Acids Res. 11 1759Ð
1771 (1983).
24. L.D. RUSSELL, D.M. TALLON, J.E. WEBER, V. WONG, and R.N. PETERSON, Am. J. Anat. 167 181Ð192
(1983).
25. J.E. WEBER, L.D. RUSSELL, V. WONG, and R.N. PETERSON, Am. J. Anat. 167 163Ð179 (1983).
26. V. WONG and L.D. RUSSELL, Am. J. Anat. 167 143Ð161 (1983).
27. A. MAYERHOFER, L.D. RUSSELL, C. GROTHE, M. RUDOLF, and M. GRATZL, Endocrinol. 129 921Ð
924 (1991).
28. I.S. HAN, S.R. SYLVESTER, K.H. KIM, M.E. SCHELLING, S. VENKATESWARAN, V.D. BLANC-
KAERT, M.P. MC GUINNESS, and M.D. GRISWOLD, Mol. Endocrinol. 7 889Ð897 (1993).
29. L. OLSON, C. AYER-LELIEVRE, T. EBENDAL, and A. SEIGER, Cell Tissue Res. 248 275Ð286 (1987).
30. C. AYER-LELIEVRE , L. OLSON, T. EBENDAL, F. HALLBROOK, and H. PERSSON, Proc. Natl. Acad.
Sci. USA 85 2628 (1988).
 D.D. Mruk, C. Yan Cheng / Life Sciences 67 (2000) 133Ð145 145

31. M. ONODA, B. PFLUG, and D. DJACKIEW, J. Cell Physiol. 149 536Ð543 (1991).
32. K.J. TEERDS and J.H. DORRINGTON, Biol. Reprod. 48 40Ð45 (1993).
33. S.L. MARKLUND, J. Biol. Chem. 267 6696Ð6701 (1992).
34. P.L. JONES, D. PING, and J.M. BOSS, Mol. Cell Biol. 17 6970Ð6981 (1997).
35. C. DESJARDINS, J. Ani. Sci. 47 56Ð79 (1978).
36. M.K. SKINNER, J.N. NORTON, B.P. MULLANEY, M. ROSSELLI, P.D. WHALEY, and C.T. ANTHONY,
Ann. NY Acad. Sci. 637 354Ð363 (1991).
37. I. HUHTANIEMI and J. TOPPARI, Adv. Exp Med. Biol. 377 33Ð54 (1995).
38. S.L. MARKLUND, Free Radicals, Aging, and Degenerative Diseases, J.E. Johnson, R. Walford, D. Harman,
and J. Miguel, (Eds), 509Ð526, Alan R. Liss, New York (1986).
39. J.M. MC CORD, Molecular Basis of Oxidative Damage by Leukocytes, A. Jesaitis and E. Dratz, (Eds), 225Ð
239, CRC, Boca Raton, (1992).
40. G. ESPOSITO, M. KERAMIDAS, C. MAUDUIT, J.J. FEIGE, A.M. MORERA, and M. BENAHMED,
Endocrinol. 128 1441Ð1449 (1991).
41. P. GRASSO, L.E. REICHERT JR, M.B. SPORN, and T.A. SANTA-COLOMA Endocrinol. 132 1745Ð1749
(1993).
42. C.J. GROSSMAN, Science 227 257Ð261 (1985).
43. E.J. GOLYSTEYN and M.J. FRITZLER, J. Rheumatol. 14 982Ð990 (1987).
44. R.E. PINTO and W. BARTLEY, Biochem J 109 34P (1968).
45. R.E. PINTO and W. BARTLEY, Biochem. J. 112 109Ð115 (1969).
46. R.E. PINTO and W. BARTLEY, Biochem. J. 115 449Ð456 (1969).
47. I.D. CAPEL and A.E. SMALLWOOD, Res. Commun. Chem. Pathol. Pharmacol. 40 367Ð378 (1983).
48. E. STEINBERGER, Physiol. Rev. 51 1Ð22 (1971).
49. J.H. DORRINGTON and I.B. FRITZ, Endocrinol. 96 879Ð889 (1975).
50. E. STEINBERGER, A. STEINBERGER, and M. FICHER, Recent Prog. Horm. Res. 26 547Ð588 (1970).
51. M.J. WELSH and J.P. WIEBE, Endocrinol. 103 838Ð844 (1978).
52. M.D. GRISWOLD, E.R. MABLY, and I.B. FRITZ, Mol. Cell Endocrinol. 4 139Ð149 (1976).
53. M.D. GRISWOLD, A. SOLARI, P.S. TUNG, AND I.B. FRITZ, Mol. Cell Endocrinol. 7 151Ð165 (1977).
54. D.J. LAMB, Y.H. TSAI, A. STEINBERGER, and B.M. SANBORN, Endocrinol. 108 1020Ð1026 (1981).
55. M.H. ERRARD, J.M. SAEZ, and A. DAZORD, J. Steroid Biochem. 22 281Ð284 (1985).
56. V. HANSSON, O. DJOSELAND, O. TORGERSEN, E.M. RITZEN, F.S. FRENCH, and S.N. NAYFEH,
Andrologia 8 195Ð202 (1976).
57. C.W. BARDIN, C.Y. CHENG, N.A. MUSTO, and G.L. GUNSALUS, The Physiology of Reproduction. E.
Knobil, J. Neill, L.L. Ewing, G.S. Greenwald, C.L. Markert, and D.W. Pfaff, (Eds), 933Ð974, Raven Press,
New York, (1988).
58. P.F. HALL and M. MITA, Biol. Reprod. 31 863Ð869 (1984).