(Received for publication, April 19, 1996, and in revised form, October 30, 1996)
From The Population Council, New York, New York 10021
Testin, a Sertoli cell secretory protein whose mRNA is predominantly expressed in the testis, was shown to become tightly associated with Sertoli cell membrane upon its secretion whose solubilization requires the use of a detergent such as SDS. In the in vitro studies using Sertoli cells cultured at high cell density, where specialized junctions were being formed, the concentration of "soluble" testin in the spent media was greatly reduced versus monolayer cultures at low cell density, where specialized junctions were absent. Conversely, the concentration of "membrane-bound" testin from detergent-solubilized Sertoli cell membrane extract was positively correlated to the existence of specialized junctions in these cultures. In normal rat testes, the level of radioimmunoassayable soluble testin in the cytosol was low. However, when the inter-testicular cell junctions were disrupted either by a drug treatment such as lonidamine in vivo or by a physical treatment in vitro such as exposing Sertoli-germ cell co-cultures where specialized junctions were formed to a hypotonic treatment, a drastic surge in the testin gene expression was noted. Thus, testin can become tightly associated with Sertoli cell membrane upon its secretion when intercellular junctions are formed. It is also a marker to monitor the integrity of inter-testicular cell junctions.
During spermatogenesis a sequence of cytological changes gives
rise to four mature spermatids from a single precursor cell (spermatogonium). Throughout these processes, developing germ cells
such as type B spermatogonia and preleptotene spermatocytes must
traverse from the basal compartment of the seminiferous epithelium, through the specialized junctions between adjacent Sertoli cells at the
basal lamina, to the seminiferous tubular lumen where fully developed
spermatids (spermatozoa) are released (for reviews, see Byers et
al. (1993) and Enders (1993)
). Therefore, specialized inter-Sertoli and Sertoli-germ cell junctions must be constantly disrupted and regenerated in a highly coordinated fashion to allow germ
cell migration while maintaining the integrity of the testis. However,
the molecules that are involved in these biochemical events and the
mechanism(s) by which germ cells migrate through the seminiferous
epithelium are largely unknown. Recent immunohistochemical studies have
demonstrated the presence of cell adhesion molecules in the testis
which include vinculin (Pfeiffer and Vogl, 1991
), zonula occludens 1 (ZO-1) (Byers et al., 1991
), and cadherins (Byers et
al., 1994
; Cyr et al., 1992
). It is known that
cadherins interact with each other via their extracellular domain in
the presence of calcium (Takeichi, 1991
). The cytoplasmic domain of cadherins has been shown to interact with a variety of cell adhesion molecules including actin,
-,
- and
-catenins, talins,
vinculin, and
-actinin (Burridge et al., 1988
). However,
Northern blot analysis has revealed that the cell adhesion molecules
such as
- and
-catenins as well as E-cadherin were expressed
predominantly in Leydig instead of Sertoli cells (Byers et
al., 1994
).
Earlier studies from this laboratory have shown that Sertoli cells
cultured in vitro in monolayer at low cell density (about 5 × 104 cells/cm2), where specialized
tight junctions did not form, actively synthesize and secrete a novel
protein designated testin which consists of two molecular variants of
35 kDa (testin I) and 37 kDa (testin II) (Cheng and Bardin, 1987; Cheng
et al., 1989
; Grima et al., 1992
). Nucleotide
sequence analysis of the full-length cDNA coding for testin has
revealed that it is a unique protein (Grima et al., 1995
).
Studies using Northern blots, RIA,1 and
immunoblots to examine the distribution of testin mRNA in multiple
tissues have revealed that testin is predominantly expressed in the
gonad whose mRNA level in other tissues cannot be visualized without the use of RT-PCR (Grima et al., 1995
). When the
pattern of localization of testin in the testis was examined by
immunohistochemistry and immunofluorescent microscopy, testin was
mainly localized at the inter-Sertoli and Sertoli-germ cell junctions
(Zong et al., 1992
, 1994
). In the non-gonadal tissues such
as the epididymis, the convoluted collecting tubules in kidney, small
intestine, and liver, immunoreactive testin can be found between
epithelial cells at sites where specialized junctions are known to
occur (Zong et al., 1994
; Grima et al., 1995
).
More important, the pattern of localization of testin in the testis and
multiple tissues is similar to that of ZO-1 and cadherins in the testis
and other epithelia (Stevenson et al., 1986
; Gumbiner
et al., 1988
; Byers et al., 1994
). However, it is
not clear if testin is indeed an integral component of specialized cell
junctions.
In this report, we present biochemical findings to demonstrate that testin is tightly associated with the testicular cell membrane upon its secretion. Interestingly, the concentration of the "soluble" form of testin secreted by Sertoli cells in vitro or its presence in the testicular cytosol in vivo is inversely correlated to the presence of intact testicular cell junctions. Both in vitro and in vivo studies demonstrate that a disruption of testicular cell junctions can lead to a surge in the concentration of the soluble form of testin as well as the steady-state testin mRNA level.
Biochemicals
Tris, acrylamide, N,N-diallytartardiamide,
bisacrylamide, TEMED, ammonium persulfate, Staphylococcus
aureus cells (formalin fixed), and agarose were purchased from
Life Technologies, Inc. (Gaithersburg, MD). Sodium chloride, citric
acid, and formamide were obtained from Aldrich (Milwaukee, WI).
Formaldehyde solution (37%, w/v) was from VWR Scientific (Piscataway,
NJ). Glycine, 2-mercaptoethanol, and SDS were from Bio-Rad.
Nitrocellulose filters (0.45 µm pore size) and NytranTM
plus nylon membranes were obtained from Schleicher and Schuell, Inc.
(Keene, NH). Ficoll (Type 70, Mr 70,000),
polyvinylpyrrolidone, dextran sulfate, salmon sperm DNA, sodium
phosphate, sodium acetate, MOPS, Ham's F-12/DMEM medium (1:1 mixture),
EDTA, and tRNA were obtained from Sigma. MatrigelR
(basement membrane matrix) was from Collaborative Biomedical Products
(Bedford, MA). RNA STAT-60TM was from Tel Test "B,"
Inc. (Friendswood, TX). Eosin Y and hematoxylin were from Zymed
Laboratories, Inc. (South San Francisco, CA). L-[35S]Methionine (specific activity, 1175 Ci/mmol), [
-32P]ATP (specific activity, 6000 Ci/mmol),
and [
-32P]dCTP (specific activity, 3000 Ci/mmol) were
obtained from DuPont NEN (Boston, MA). Nick translation and 5
-end
labeling kits were obtained from Boehringer Mannheim.
X-OmatTM AR autoradiographic films were obtained from Kodak
(Rochester, NY). 14C-Methylated protein molecular weight
markers were obtained from Amersham.
Seminiferous Tubular Cultures
Rats at 10, 20, 45, and 60 days of age were killed by
CO2 asphyxiation. At least three animals per age group were
used. Testes were carefully removed and seminiferous tubules were
isolated immediately by collagenase/dispase treatment as described
previously (Zwain and Cheng, 1994). The tubules were washed extensively
by sedimentation under gravity to remove the interstitial cells. Tubules were trimmed into 2-mm segments, and tubules from 1 g of
testicular tissue was suspended in 25 ml of serum-free F-12/DMEM supplemented with bovine insulin (20 µg/ml), transferrin (20 µg/ml), gentamycin (100 µg/ml), and penicillin (100 IU/ml). Tubules
(3 ml each from the above suspension) were cultured in 5-cm Petri dishes for 24 h at 35 °C in a humidified atmosphere of 95%
air, 5% CO2 (v/v). At the end of the culture period, the
seminiferous tubular culture medium was collected, centrifuged at
1000 × g for 20 min to remove cellular debris, and
stored at
20 °C. The Leydig cell contamination was judged to be
minimal by the following criteria: (i) lack of specific binding of
125I-luteinizing hormone by the tubules that had been
washed extensively by sedimentation under gravity versus 2 ng of luteinizing hormone/culture dish derived from tubules that were
washed twice by centrifugation at 2000 × g for 10 min.
(ii) The basal testosterone levels in the spent media from tubules
cultured with or without contaminating interstitial cells were 2.2 ± 1 and 0.54 ± 0.02 ng/ml, respectively. (iii) Addition of
luteinizing hormone (10 ng/ml) did not affect the testosterone level in
the media from tubules without interstitial cells, whereas it induced a
5-fold increase in testosterone level in the media from tubules with
contaminating interstitial cells. Thus, the seminiferous tubules used
for the culture experiments had negligible Leydig cell contamination.
Immunoreactive testin was later quantified from the seminiferous
tubular culture medium by RIA (Cheng et al., 1989
). Total
RNA was extracted from the remaining tubules as described below.
Preparation of Testicular Cell Cultures
Sertoli CellsSertoli cells were prepared from 20-day-old
Harlan Sprague Dawley rats as described (Skinner and Fritz, 1985).
Cells (0.45 × 106 cells/ml) were plated at a density
of 4 × 106 cells/9 ml/100-mm dish (about 5 × 104 cells/cm2) to allow the formation of
monolayer without specialized tight junctions in F-12/DMEM supplemented
with gentamycin (20 mg/liter), sodium bicarbonate (1.2 gm/liter), 15 mM HEPES, bovine insulin (10 µg/ml), human transferrin (5 µg/ml), epidermal growth factor (5 ng/ml), and bacitracin (5 µg/ml,
a protease inhibitor), and incubated at 35 °C in a humidified
atmosphere of 95% air, 5% CO2 (v/v). In experiments where
Sertoli cells were plated at high cell density (about 1 × 106 cell/cm2) to allow the formation of
specialized junctions which mimics the in vivo morphology of
the Sertoli cell, cells were cultured on Matrigel-coated wells or
dishes at 50 µl/cm2 diluted 1:2 with F-12/DMEM. For
Sertoli cells cultured at low (5 × 104
cells/cm2) and high cell density, medium was replaced with
fresh F-12/DMEM every 48 h and daily, respectively. To obtain
Sertoli cell cultures with greater than 95% purity, cultures were
hypotonically treated with 20 mM Tris-HCl, pH 7.4, for 2.5 min to lyse any residual germ cells (Galderi et al., 1981)
48 h after plating. The wells were washed two times with F-12/DMEM
and the cells were allowed to recover for an additional 24 h prior
to their experimental use. These germ cell-free Sertoli cell cultures
(day 0) were then used for all subsequent experiments.
Germ cells were prepared from 90-day-old Harlan
Sprague Dawley rats (about 300 g body weight) by a
mechanical procedure without any enzymatic treatment as detailed
elsewhere (Aravindan et al., 1990, 1996
). The proportion of
different germ cell types obtained by this isolation procedure is
comparable to the cell ratio in the testis in vivo
(Aravindan et al., 1990
) except that elongate spermatids
were removed by a glass wool filtration step. This mechanical isolation
procedure produced germ cells with a purity of greater than 95% with
minimal somatic cell contamination (Aravindan et al., 1996
).
Germ cells, largely round spermatids, spermatocytes, and some
spermatogonia were cultured in F-12/DMEM supplemented with 2 mM sodium pyruvate and 6 mM
DL-lactate at a density of 22.5 × 106
cells/9 ml in 100-mm Petri dishes for 20 h at 35 °C to obtain germ cell conditioned medium (GCCM) as detailed elsewhere (Pineau et al., 1993
). The cell viability was judged to be greater
than 95% at the end of an 18-20-h culture period when examined by
erythrosine red dye exclusion test (Pineau et al., 1993
).
For co-culture experiments with Sertoli cells, freshly isolated germ
cells were used immediately after their isolation. It was noted that
the viability of germ cells was extended up to 6 days in Sertoli-germ
cell co-cultures similar to previously published results (Tres and
Kierszenbaum, 1983
).
Sertoli cells (4 × 106 cells/100-mm
dish/9 ml) isolated from 20-day-old rats were cultured in serum-free
F-12/DMEM for 72 h at 35 °C as described to form a monolayer.
Thereafter different numbers of freshly prepared germ cells were added
and co-cultured with the Sertoli cells for specific time points. Before
the experiments were terminated, cultures were subjected to a hypotonic
treatment using sterile Tris buffer (20 mM, pH 7.4, at
22 °C) for 2.5 min to lyse germ cells, and the cultures were
immediately washed twice to remove the released cellular content before
total RNA was extracted by RNA STAT-60TM (Grima et
al., 1995, 1996
) so that the total RNA isolated from these
cultures were largely of Sertoli cell origin.
To examine the correlation between the
Sertoli cell testin steady-state mRNA level and the formation and
disruption of Sertoli-germ cell junctional complexes, Sertoli cells
were plated at high density (1 × 106
cells/cm2) on Matrigel-coated culture dishes for four days
to allow the formation of specialized tight junctions in F-12/DMEM in
the presence of both 1 × 107 M
testosterone and 100 ng/ml follicle-stimulating hormone since it is
known that follicle-stimulating hormone can enhance the formation of
junctional complexes between testicular cells in vitro
(Cameron and Muffly, 1991
). The formation of tight junctions between
Sertoli cells was monitored in parallel diffusion experiments using
either [3H]inulin or 125I-bovine serum
albumin where the cells were plated on Matrigel-coated bicameral
culture chambers as described previously (Grima et al., 1992
). It was noted that when 2-5 × 106 cpm of
either [3H]inulin or 125I-bovine serum
albumin was added to the apical chamber, more than 80% of the counts
were retained in the apical chamber when the radioactivity was assessed
36 h later indicating specialized tight junctions were indeed
formed. Thereafter, germ cells, consisting of round spermatids,
spermatocytes, and spermatogonia, were added to the above Sertoli cell
culture using a Sertoli:germ cell ratio of 1:2. Junctional complexes
between Sertoli and germ cells were allowed to form for 2 and 4 days.
In one set of wells at the specified time points, cultures were
hypotonically treated with 20 mM Tris, pH 7.4, for 2.5 min
to lyse all the germ cells (Galderi et al., 1981), thereby
disrupting the junctional complexes formed between Sertoli and germ
cells. The wells were washed and incubated for an additional 24 h.
RNA Extraction and Northern Blot Analysis
Total RNA was extracted from cell cultures or tissues using RNA
STAT-60TM as described previously (Grima et al.,
1995). The RNA concentration was quantified by UV spectrophotometry at
254 nm, and its integrity was assessed by gel electrophoresis and
ethidium bromide staining. Northern blot analysis was performed using
either a
-32P-labeled antisense testin RNA probe or a
-32P-nick translated testin cDNA probe (Grima
et al., 1995
) for hybridization. To ensure that equal
amounts of RNA were loaded into each lane, some blots were rehybridized
with a
-32P-labeled
-actin cDNA probe (Grima
et al., 1996
).
Quantitative RT-PCR
Due to the low abundance of testin mRNA transcript in the
non-gonadal tissues whose expression could not be visualized readily by
Northern blot, the changes of steady-state testin mRNA level in the
kidney during development and the uterus at different stages of the
estrus cycle were analyzed by quantitative RT-PCR. Testin was
co-amplified with rat ribosomal S16 which was used as an internal control to monitor the sample-to-sample variation in the RT and PCR. In
a series of preliminary experiments (data not shown), increasing
concentrations of template RNA were used to verify the linearity of the
assay with respect to the amount of products. The amount of PCR
products was also examined over a range of 10-45 amplification cycles
for a given amount of template RNA to select the optimal number of
amplification cycles in the linear range. In all experiments, the
amplification of the template RT-cDNA, as well as the S-16
RT-cDNA, were in the linear range. All samples within the same
treatment group were processed simultaneously for RT and PCR
in order to eliminate inter-assay variation. Briefly, about 4 µg of
total RNA was reverse transcribed to cDNAs using 2 µg of
oligo(dT)-15 primer and avian myeloblastosis virus reverse transcriptase in a final reaction volume of 25 µl as described previously (Grima et al., 1995). The RT reaction was
terminated by heating at 95 °C for 5 min. About 10 µl of this RT
product was then utilized for PCR using 1 µg each of the testin sense primer (5
-TTTCTTTATGTCCCAAAACGTGTG-3
corresponding to nucleotides 283-306), and antisense primer (5
-TACAATGGGGTATGTGGAATATGT-3
corresponding to nucleotides 928-951), together with 2.5 units of
Taq DNA polymerase, 16 µl of dNTPs (200 µM
each of dGTP, dATP, dCTP, and dTTP), 10 µl of 10 × PCR buffer,
and sterile water to a final reaction volume of 100 µl. The cycling
parameters for the PCR were: denaturation at 94 °C for 1 min,
annealing at 56 °C for 2 min, and extension at 72 °C for 3 min. A
total of 25 cycles were performed during which the production of testin
cDNA was linearized in the samples derived from the testes.
Specific amplification of testin mRNA with these primers yielded a
669-base pair product in the testicular samples that was not detectable
in the kidney or uterus samples due to is low abundance. To further
increase the detection sensitivity, the first PCR products were
separated from the primers and concentrated to about 20 µl in TE
buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
using QIAquick spin columns (Qiagen Inc., Chatsworth, CA). A 5-µl
aliquot of this reaction product served as a template for hot nested
PCR (Simmonds et al., 1990
) using a nested testin primer
from an internal nucleotide sequence (sense primer,
5
-GGTCATTGTGCCTCTAGTTGGGCTTTTAGTGCA-3
corresponding to
nucleotides 349-381). This internal primer was 5
-end-labeled by T4
polynucleotide kinase with [
-32P]ATP and about
1-10 × 106 cpm was used per PCR tube. About 1 µg
of the testin antisense primer (nucleotides 928-951) as described
above was used as another primer in this second PCR. To ensure that the
amplified testin DNA could be quantified between samples, an
endogenously and ubiquitously expressed RNA, rat ribsomal S16, was used
as an internal control that was co-amplified along with the testin to
act as a control for sample-to-sample variations in this second PCR.
The S16 primer pair was prepared based on the known rat ribosomal S16
gene (Chan et al., 1990
) as follows: sense primer
5
-TCCGCTGCAGTCCGTTCAAGTCTT-3
corresponding to nucleotides 15-38;
antisense primer, 5
-GCCAAACTTCTTGGATTCGCAGCG-3
corresponding to
nucleotides 376-399, where the antisense primer was 5
-end labeled
with [
-32P]ATP using polynucleotide kinase as
described above. About 1-10 × 106 cpm was used per
reaction tube and about 0.1 µg each of the unlabeled primers were
used. These testin and S16 primers, together with 5 µl from the first
PCR product which acted as a template, were amplified as described
above. A total of 22 cycles were performed. The cycles were followed by
a 15-min extension period at 72 °C. Under these conditions, the
production of both testin and S16 cDNA in this second PCR in the
kidney samples were linearized. An aliquot of 10 µl was resolved onto
a 5% polyacrylamide gel in 0.5 × TBE buffer and exposed to x-ray
film to visualize the PCR products. Specific amplification of the
testin and S16 mRNAs with these primers yield 603- and 385-base
pair products, respectively.
Determination of Estrus Cycle and Collection of Ovaries and Uteri
In the rat, the mean length of the estrus cycle is 5.4 days
which can be divided into four distinctive phases: (i) proestrus, 12-14 h; (ii) estrus, 25-27 h; (iii) metestrus, 6-8 h; and (iv) diestrus 55-57 h. These phases were monitored by daily vaginal smears
(Long and Evans, 1922; Mandl, 1951
) using adult female Harlan Sprague
Dawley rats and only those animals which exhibited two consecutive
estrus cycles were used for RNA extraction. Rats were killed by
CO2 asphyxiation, ovaries and uteri were removed immediately and stored at
70 °C until RNA extraction.
Induction of Tissue Restructuring in the Testis by Lonidamine
Lonidamine (Corsi et al., 1976; Silvestrini et
al., 1984
), 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid,
is known to disrupt the specialized junctions between Sertoli and germ cells by inducing premature release of germ cells, in particular elongate and round spermatids, into the seminiferous tubular lumen (De
Martino et al., 1981) possibly by inducing rearrangements of
the subcellular actin microfilaments, microtubules, and intermediate filaments (Malorni et al., 1992
). Adult male Harlan Sprague
Dawley rats weighing between 250 and 300 g body weight were orally
fed a single dose of lonidamine (5 or 50 mg/kg body weight) suspended in 0.25% methylcellulose (w/v). Animals, 5-6 rats for each treatment group, were sacrificed at specific time points. Testicular RNA was
isolated for Northern blot analysis using RNA-STAT-60TM.
Tissue homogenates of the testes were also prepared in the absence of
detergent to quantify the testin concentration by RIA as described (Cheng et al., 1989
).
Morphological Study of Rat Testis Treated with Lonidamine
At specified time points, rats treated with lonidamine were
sacrificed by CO2 asphyxiation. The testes were removed and
immediately embedded in Tissue-Tek® O.C.T. compound (Miles Inc.,
Elkhart, IN) and frozen in liquid nitrogen. All tissue blocks were then stored at 70 °C until sectioned. Five micron sections were cut at
20 °C with a cryostat (Hacker, Fairfield, NJ) and mounted on glass
slides coated with poly-L-lysine (Mr
>150,000). Sections were air-dried at room temperature and then fixed
in Bouin's fixative for 10 min and washed thoroughly with
phosphate-buffered saline. Sections were immunostained with anti-testin
antibody as detailed elsewhere (Zong et al., 1994
; Zhu
et al., 1994
; Grima et al., 1995
) and
counterstained with hematoxylin.
Sertoli Cell and Testicular Membrane Preparation
Sertoli cells were plated at high density (1 × 106 cells/cm2) or low density (5 × 104 cells/cm2) as described above for 4 days to allow the establishment of specialized junctions. Cells were harvested using a cell scraper and centrifuged at 800 × g for 10 min. Cell pellets were resuspended in buffer A (phosphate-buffered saline, 10 mM sodium phosphate, 0.15 M NaCl, pH 7.4, at 22 °C), frozen in liquid nitrogen, and thawed at room temperature. This freeze-thawing procedure was repeated three times to damage the cell membrane and to release the cytosol. The sample was then centrifuged at 15,000 × g for 2 min and the pellet washed in buffer B (10 mM Tris, 1 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride containing 0.25 M sucrose at pH 7.4 at 22 °C) to remove cytosol and soluble testin. The membrane proteins were then solubilized in SDS sample buffer containing 0.125 M Tris, 10% glycerol, 1% SDS, 0.1% Triton X-100, and 1.6% 2-mercaptoethanol (pH 6.8 at 22 °C), 20 min at room temperature, and 10 min at 100 °C.
General Methods
Protein estimation was performed by the Coomassie Blue
dye-binding assay (Bradford, 1976) using bovine serum albumin as a standard. Rat luteinizing hormone used for the receptor binding experiments and the ovine follicle-stimulating hormone used for Sertoli
cell cultures were obtained from the National Hormone and Pituitary
Program, the National Institute of Diabetes and Digestive and Kidney
Diseases, the National Institute of Child Health and Human Development,
and the U. S. Department of Agriculture. Rat testin RIA was performed
as described previously (Cheng et al., 1989
). Pulse-chase
labeling analysis using [35S]methionine was performed as
described previously (Grima et al., 1992
).
Studies by immunohistochemistry revealed that testin is
localized at the inter-Sertoli and Sertoli-germ cell junctions in the
testis. It can also be found in specialized junctions of multiple epithelia in several organs such as the epididymis, kidney, small intestine, and liver (Grima et al., 1995), but it is not
known if testin is indeed associated with Sertoli cell and/or
testicular membrane. To identify if it is a membrane bound component,
we sought to solubilize immunoreactive testin from both Sertoli cell membrane extracts derived from high (1 × 106
cells/cm2, SC-SC) and low (5 × 104
cells/cm2, SCM) cell density cultures, with or without the
presence of specialized junctions, respectively. The establishment of
tight junctions between Sertoli cells at high cell density on
Matrigel-coated dishes was monitored in parallel experiments using
bicameral culture chamber as described under "Experimental
Procedures." It was noted that the detergent-solubilized testin from
Sertoli cell membrane had an apparent molecular mass of 33 kDa
versus 35 and 37 kDa for testin I and testin II,
respectively (Fig. 1A). When Sertoli cells
were cultured in monolayer at 5 × 104
cells/cm2 where tight junctions did not form (SCM), they
secreted 3-5-fold more soluble testin into the spent media (Fig.
1B) than Sertoli cells cultured at high density alone
(SC-SC, 1 × 106 cells/cm2) or with germ
cells (SC-GC; SC:GC ratio, 1:2) where specialized junctions did form
(Fig. 1B). More important, the amount of soluble testin in
testicular cytosol prepared by routine homogenization without the use
of detergent (TC) or in rete testis fluid obtained by micropuncture
technique from rats with intact testes was virtually undetectable (Fig.
1B). In contrast, when the concentration of testin was
quantified in the membrane extract from Sertoli cell monolayer cultures
where tight junctions did not form (SCM) (Fig. 1C), it was
about 50-fold less than Sertoli cells cultured at high cell density
alone or co-cultured with germ cells where specialized junctions did
form (Fig. 1C). In addition, a significant amount of testin
was solubilized from intact testicular membrane with the use of
detergents including both SDS and Triton X-100; however, in the absence
of these detergents, testin was unable to solubilize from the
testicular membrane extracts (Fig. 1C). Moreover, testin was
almost non-detectable in the testicular cytosol from rats with intact
testes (Fig. 1, C versus B).
Characterization and quantification of testin
in Sertoli cell and testicular membrane extracts, spent media, and
reproductive tract fluids. A, immunoblot of Sertoli cell
membrane extracts and its comparison to crude Sertoli cell-enriched
culture medium using an anti-testin II antibody. Lane 1 is
crude Sertoli cell-enriched culture medium (10 µl) containing 0.6 µg of total protein; lanes 2 and 3 are Sertoli
cell membrane extracts containing about 20 and 100 µg of total
protein, respectively. Testin I and testin II were clearly visible in
the crude Sertoli cell-enriched culture medium (lane 1),
whereas the testin identified in the Sertoli cell membrane extract had
an apparent molecular mass of 33 kDa. B, the concentration
of immunoreactive testin in the spent media of Sertoli cells cultured
in low (SCM, 5 × 104 cells/cm2) and high
(SC-SC, 1 × 106 cells/cm2) cell density.
Tight junctions did and did not form in SC-SC and SCM, respectively.
The formation of tight junctions in these experiments was monitored by
[3H]inulin diffusion using Matrigel-coated bicameral
culture inserts (Grima et al., 1992). Rete testis fluid
(RTF) was obtained by micropuncture technique (Howards
et al., 1975
) and testicular cytosol (TC) was
prepared as described previously (Cheng and Bardin, 1987
). SC-GC are
Sertoli-germ cell co-cultures as described under "Experimental
Procedures" where specialized junctions were formed. C,
the concentration of immunoreactive testin in the membrane extracts of Sertoli cells prepared from SCM, SC-SC, and SC-GC cultures
and in the membrane extracts from intact testes solubilized with or
without detergents. Samples were dialyzed extensively against
phosphate-buffered saline before radioimmunoassay.
Changes of Steady-state Testin mRNA Level in Seminiferous Tubules during Testicular Development
The predominant expression
of testin mRNA in the gonad (Grima et al., 1995),
including the testis and the ovary, is likely the result of extensive
tissue restructuring due to germ cell and follicle development since
testin appears to be a marker protein for tissue restructuring. If such
an hypothesis is correct, one would expect a higher expression of
testin in the testis during postnatal development because of the
extensive tissue restructuring associated with testicular growth and
the formation of the blood-testis barrier. Seminiferous tubules were
used instead of whole testes since earlier studies by Northern blot and
RT-PCR have shown that Leydig cells isolated from adult rats expressed
testin mRNA in contrast to germ cells which did not express any
testin (Grima et al., 1995
; Zong et al., 1994
).
Therefore, intact tubules in the absence of interstitial cells were
used. The Leydig cell contamination was judged to be minimal by the
criteria outlined under "Experimental Procedures." It was noted
that the steady-state testin mRNA level decreased steadily in the
seminiferous tubules during post-natal development (Fig.
2A). Fig. 2B is the same blot
shown in Fig. 2A but re-hybridized with a
-actin cDNA
probe indicating that a similar amount of RNA was loaded for each lane.
The level of testin mRNA reduced by as much as 3-fold at 60 days of
age versus immature rats at 10 days of age when the testin
mRNA level was normalized against
-actin (Fig. 2C).
When the levels of testin in the spent media from these cultures were
quantified by RIA, it was noted that the secretion of testin by these
tubules was reduced drastically during postnatal development (Fig.
2D), consistent with the Northern blot data shown in Fig.
2A. Although the testin steady-state mRNA level
decreases dramatically in the testis during development, its level in
the adult testis is still the highest when compared to all other organs
in the adult male rat possibly due to the continual assembly and
disassembly of junctional complexes between Sertoli and germ cells
throughout spermatogenesis (Grima et al., 1995
).
Effect of Germ Cells or GCCM on Testin Synthesis, Secretion, and Steady-state mRNA Level
Durng postnatal development, there is
a drastic increase in germ cell:Sertoli cell ratio in the testis. Since
germ cells do not express testin mRNA (Zong et al.,
1994) nor do they secrete testin in vitro (Aravindan
et al., 1996
), the reduction of testin steady-state mRNA
in the testis during development could be the result of a reduced
contribution of total RNA by Sertoli cells in the samples being
analyzed. To examine the effects of germ cells on Sertoli cell testin
mRNA level, increasing numbers of germ cells (0-10 × 106 cells/dish) or GCCM (0-15 µg/dish) were co-cultured
with Sertoli cells for a 20-h period, neither germ cells nor GCCM
produced a significant reduction of Sertoli cell steady-state testin
mRNA (data not shown). We next investigated the effect of germ
cells or GCCM on the synthesis and/or secretion of testin by Sertoli cells in vitro by pulse-chase analysis. When germ cells
(10 × 106 cells) or GCCM (10 µg of protein/dish)
were co-cultured with Sertoli cells, it was found that neither germ
cells nor GCCM had any effect on Sertoli cell testin secretion that
could mimic the developmental changes shown in Fig. 2.
The decline in steady-state mRNA level in the tubules
during development is likely a reflection of reduced cell junction
turnover since the results described above are consistent with the
notion that germ cells do not play a major regulatory role. Therefore, we sought to determine whether the expression of testin correlates to
increased junctional complex turnover during development in other
organs. The kidney was selected for parallel examination because testin
was previously shown to be localized in the specialized epithelial cell
junctions in the collecting tubules (Grima et al., 1995).
Fig. 3A is the Northern blot using testicular
RNA derived from rats at 3, 10, and 45 days of age showing a reduction of steady-state testin mRNA level during development consistent with the data shown in Fig. 2. Fig. 3B is the eithidium
bromide staining of the same blot shown in Fig. 3A
indicating similar amounts of total RNA was used for each lane. Fig.
3C is the densitometrically scanned data normalized against
the
-actin level indicating a drastic reduction in testin mRNA
level during post-natal development. Since testin mRNA was not
detectable in the kidney by Northern blot due to its low abundance,
hot-nested RT-PCR was used. Fig. 3D is the RT-PCR results
using kidney RNA derived from rats at different ages ranging from 3 days before birth (
3) to 60 days of age (60). There is a drastic
reduction in the steady-state testin mRNA level in the kidney
during development whose level in the adult rat is virtually
undetectable (Fig. 3D). We have also detected a small
decline in S16 expression in postnatal development. In some preliminary
RT-PCR experiments we co-amplified testin with
-actin as an internal
control and noted that the
-actin level also declined slightly
during postnatal development as reported earlier (Liou et
al., 1994
).
Changes of Testin Steady-state mRNA Level in the Ovary and Uterus during the Estrus Cycle
In the ovary, extensive disruption
and regeneration of cell junctions take place between ovarian cells due
to follicle development and during ovulation. It has been shown that
the expression of testin mRNA is the highest in the ovary in the
female rat compared to other female organs (Grima et al.,
1995), suggesting the rapid turnover of cell junctions in the ovary is
one of the key determining factors of testin mRNA expression. We
have therefore examined whether the steady-state testin mRNA level
is correlated with the maturation of intrafollicular ova during the
proestrus phase and the eventual rupturing of the follicle at ovulation
in the estrus phase when the turnover of the specialized cell junctions is the highest. Fig. 4A is the Northern blot
indicating the level of the ovarian testin steady-state mRNA level
was high at proestrus, estrus, and metestrus, but reduced drastically
to an almost undetectable level at diestrus during which functional
regression of the corpora lutea occurs (Fig. 4A). Fig.
4B is the same blot shown in Fig. 4A but stained
with ethidium bromide indicating a similar amount of total RNA was used
for each lane. Since the testin mRNA is not readily detectable in
the uterus by Northern blot (Fig. 4A), the change of testin
steady-state mRNA level was monitored by hot-nested RT-PCR (Fig.
4C). It was noted that the level of S16 mRNA
co-amplified with testin remained relatively unchanged at different
stages of the estrus cycle (Fig. 4C). In contrast, the level
of testin is comparatively higher at diestrus versus other stages of the estrus cycle (Fig. 4C) when myometrial cells
are lost from the epithelial linings. These results thus support the postulate that the expression of testin mRNA is indeed correlated with the rate of turnover of specialized junctions when the maturing follicles migrate toward the ovarian surface by disrupting cell junctions between granulosa cells.
Changes in the Testin mRNA Level and Its Concentration in the Testis following a Disruption of the Specialized Junctions between Sertoli and Germ Cells by Lonidamine Treatment
If the high level
of testin mRNA expression detected in the gonad compared to other
tissues is indeed correlated with an increase in tissue restructuring
and remodeling due to germ cell and follicle development, a change in
the testicular testin mRNA level and its protein concentration are
expected when the testis is induced to undergo extensive tissue
restructuring. Lonidamine is a drug known to disrupt the junctional
complexes between Sertoli and germ cells (De Martino et al.,
1981), possibly by rearranging the actin microfilaments, microtubules,
and intermediate filaments (Malorni et al., 1992
) which in
turn induce premature release of elongate and round spermatids, and
spermatocytes from the seminiferous epithelium. Therefore, we have
examined the effect of lonidamine on the morphology of the testis and
the localization of testin by immunohistochemistry using an anti-testin
antibody. Fig. 5, A-F, show the
cross-sections of adult rat (about 300 g body weight) testes
treated with lonidamine at 50 mg/kg body weight for 1- (Fig.
5C), 2- (Fig. 5D), 6- (Fig. 5E), and
15-day (Fig. 5F) compared to control rat (Fig. 5,
A and B). Fig. 5, B-F, were sections
immunostained with anti-testin antibody whereas Fig. 5A is
the control section using preimmune serum. Virtually all elongate
spermatids were depleted from the seminiferous epithelium by day 2 after lonidamine treatment (Fig. 5D) and almost all round
spermatids were depleted by days 6-15 (Fig. 5, E and
F). By day 15, only spermatogonia and a few spermatocytes
were visible (Fig. 5F). When the steady-state testin
mRNA level in these testes was quantified by Northern blot (Fig.
6A) using animals which had received two
different doses of lonidamine at 5 and 50 mg/kg body weight, the
depletion of germ cells was associated with a drastic increase in
testin mRNA level (Fig. 6A). Fig. 6B is the
same blot shown in Fig. 6A but stained with eithidium
bromide indicating similar amount of RNA was used for each lane. Fig.
6, C and D, demonstrate that there was a surge in
the concentration of soluble testin in the testicular cytosol following
germ cell depletion when animals were treated with lonidamine at 5 and
50 mg/kg body weight, respectively. Since there is a drastic reduction
in the testicular weight following lonidamine treatment due to germ
cell loss, the testicular testin content from rats treated with
lonidamine at 50 mg/kg body weight was shown in Fig. 6E to
reflect the change in testicular weight. It was noted that the soluble
testin content indeed increased during lonidamine treatment (Fig.
6E). When examined by immunohistochemistry, the level of
testin at the sites between testicular cells where specialized
junctions are known to occur did not increase appreciably (Fig. 5,
C, D, E, F versus B), suggesting that the increase in testin
mRNA expression (Fig. 6A) only led to an increase in the soluble form of testin in the cytosol (Fig. 6, C-E).
Immunohistochemical examination of the epididymis indeed revealed a
drastic increase of testin accumulated in the epididymal lumen (data
not shown).
We next sought to examine whether the observed drastic effect of
lonidamine on the testicular testin mRNA level in vivo
is a direct toxic effect of the drug on Sertoli cells (Fig.
7). When Sertoli cells at low cell density (4 × 106 cells/9 ml/100-mm dish) were cultured for 3 days alone
(lane 1) or with vehicle containing ethanol (lane
2), its steady-state testin mRNA level is similar to cells
that were exposed to different concentrations of lonidamine at 0.1 (lane 3), 0.5 (lane 4), 1 (lane 5), 5 (lane 6), 10 (lane 7), 100 (lane 8),
and 1000 (lane 9) ng/ml. Fig. 7B is the same blot
shown in Fig. 7A stained with eithidium bromide indicating
similar amounts of total RNA was used for each lane. When the cell
viability was monitored by trypan blue exclusion test, lonidamine at
any of the above doses did not induce a significant change in cell
viability suggesting the observed effect shown in Fig. 6 is not the
result of cell toxicity. Similar observations were achieved in a
separate set of experiments when the cells were cultured with
increasing doses of lonidamine for 24 h alone (data not
shown).
Correlation between the Disruption of Sertoli-Germ Cell Junctional Complexes and the Steady-state Testin mRNA Level in Vitro
It
is known that when Sertoli and germ cells were co-cultured in
vitro, specialized junctional complexes similar to desmosome-like junctions were formed between these cells within 48 h (Cameron and
Muffly, 1991; Enders and Millette, 1988
). We have cultured Sertoli
cells (1 × 106 cells/cm2) on
Matrigel-coated dishes with freshly isolated germ cells (2 × 106 cells/cm2) consisting of round spermatids,
spermatocytes, and spermatogonia for 2 and 4 days to allow the
formation of Sertoli-germ cell junctional complexes. In the control
samples (Fig. 8A, lanes 1 and 2),
cultures were hypotonically treated for 2.5 min in 20 mM
Tris, pH 7.4, washed twice in F-12/DMEM about 10 min prior to their
termination to lyse germ cells to eliminate RNA contributed by germ
cells. In the test samples (Fig. 8A, lanes 3 and
4), these Sertoli-germ cell co-cultures were hypotonically
treated 24 h prior to termination to lyse germ cells to induce
disruption in Sertoli-germ cell junctional complexes. An obvious
increase in the Sertoli cell steady-state testin mRNA level was
noted when the junctions between Sertoli and germ cells were disrupted
(Fig. 8A, lanes 3 and 4 versus lanes 1 and
2). Fig. 8B is the same blot shown in Fig.
8A but stained with ethidium bromide indicating similar
amount of RNA was used for each lane.
Testin was initially purified from Sertoli cell-enriched culture
medium (Cheng and Bardin, 1987; Cheng et al., 1989
).
Northern blots and RT-PCR have shown that the testin mRNA is highly
expressed in the adult rat testis and ovary (Grima et al.,
1995
). The testin mRNA level in the non-gonadal tissues is so low
that its presence is not detectable without the use of hot-nested PCR
(Grima et al., 1995
). The fact that testin is viewed as a
secretory product of Sertoli cells, the "soluble form" of testin,
was established by immunoprecipitation experiments using pulse-chase
labeling analysis performed in this (Cheng et al., 1989
;
Grima et al., 1992
) and other (Onoda and Djakiew, 1990
)
laboratories utilizing primary cultures of Sertoli cells. Subsequent
immunofluorescent (Zong et al., 1992
) and
immunohistochemical (Zong et al., 1994
; Grima et
al., 1995
) studies, however, have revealed that testin is, in
fact, localized at sites between Sertoli cells, and Sertoli-germ cells
in the testis, as well as between epithelial cells in several tissues
at sites where specialized cell junctions are known to occur. When a
survey of the immunoreactive testin localization at different stages of
the spermatogenic cycle was completed, it was found that testin was
localized predominantly at stage VIII of the cycle between Sertoli
cells and elongate spermatids just prior to the release of mature
spermatids (spermatozoa) into the seminiferous tubule consistent with
its localization in the ectoplasmic specialization, a unique
specialized anchoring junction between Sertoli cells and elongate
spermatids (Zong et al., 1994
). In addition, the
localization pattern of testin in the testis, epididymis, small
intestine, and kidney (Zong et al., 1994
; Grima et
al., 1995
) is similar to other known junctional complex components such as ZO-1 and cadherins (Stevenson et al., 1986
; Hirano
et al., 1987
; Gumbiner et al., 1988
; Byers
et al., 1994
). Thus, these other studies suggest that there
is a "membrane-associated form" of testin. Yet, the biological
function of testin in the testis is not known.
In this report, we have quantified the concentrations of testin derived from Sertoli cell membrane extracts where tight junctions did or did not form, and the spent media from corresponding Sertoli cell cultures at high and low cell density, respectively. It was noted that the amount of testin secreted in vitro, the soluble form, is inversely correlated to the existence of specialized junctions between Sertoli cells since Sertoli cells cultured at low cell density (5 × 104 cells/cm2) secrete 12 ± 2 µg of testin/mg of protein when tight junctions did not form versus 3.4 ± 1 µg of testin/mg of protein when tight junctions did form at high cell density (1 × 106 cells/cm2). However, the concentration of detergent-solubilized testin from the Sertoli cell membrane, the membrane-associated form, is positively correlated to the presence of specialized junctions between these cells. Moreover, solubilization of testin from Sertoli cell or testicular membrane extracts requires the use of detergents suggesting that testin is indeed tightly associated with the cell membrane. Interestingly, the apparent molecular mass of the "membrane-bound" form of testin is 33 kDa versus 35-37 kDa of the secreted protein suggesting that a peptide fragment has been removed. Direct N-terminal sequence analysis of the purified membrane-bound form recovered by high performance electrophoresis chromatography indicated that its N terminus was blocked. It remains to be determined whether testin is indeed a structural component of the cell membrane or its attachment to the cell surface can elicit other biochemical events.
The in vivo studies described in this report are also
consistent with the in vitro studies that the amount of the
soluble form of testin secreted by Sertoli cells is inversely related to the extent of junctional complex formation. In fluids recovered from
the rat rete testis and seminiferous tubular lumen, obtained by
micropuncture technique, and from testicular cytosol, obtained by
routine homogenization (without the use of detergents), it was shown
that the level of radioimmunoassayable testin (the soluble form) in
these fluids is virtually undetectable. This was drastically different
from androgen-binding protein, transferrin, and testibumin (SGP-1)
which were found at high concentrations in the intra-testicular fluids
and testicular cytosol (Cheng and Bardin, 1987; Bardin et
al., 1988
). Yet, Sertoli cells cultured at low cell density when
tight junctions did not form secreted measurable amounts of soluble
testin into the spent medium whose concentration was comparable to
androgen-binding protein, transferrin, and testibumin (Cheng and
Bardin, 1987
; Cheng et al., 1989
; Rossi et al.,
1989
). Thus, under in vivo conditions when specialized
junctions between testicular cells are present and not impaired, very
little of the soluble form of testin is found in testicular cytosol and virtually none is secreted into the tubular lumen. But in these instances, testin can be found in the testicular membrane extract, the
membrane-bound form, whose solubilization requires the use of
detergents. In addition, when specialized junctions between testicular
cells are disrupted by lonidamine, a surge of soluble testin in the
testicular cytosol by as much as 50-fold is noted.
The proposal of testin as a "membrane-associated" component and a
soluble secretory product of the Sertoli cell that can be found in the
spent medium is indeed a provocative concept. In this connection, it is
of interest to note that Ross (1976) and Russell et al.
(1980)
have put forth a hypothesis based on their eminent morphological
findings (for review, see de Kretser and Kerr, 1988
) suggesting pools
of free hemi-junctions can be recycled for inter-cellular junctions in
the testis. Thus, it is feasible that testin may be one of the
free-floating and recyclable junctional complex components. Such a
postulate is not entirely unlikely considering that the ratio of
Sertoli:germ cells in the adult Harlan Sprague Dawley rats is about
1:50 when estimated by morphometric (Wong and Christensen, 1982
; Sinha
Hikim and Swerdloff, 1994) or morphological analysis using serial
sections (Russell et al., 1983
; Weber et al.,
1983
), indicating a single Sertoli cell must provide most of the
necessary junctional complex components to regenerate the continuously
disrupted Sertoli-germ cell junctional complexes to about 50 developing
germ cells simultaneously throughout spermatogenesis. Having a soluble
and recyclable junctional complex component that allows rapid
junctional complex formation at the inter-Sertoli and Sertoli-germ cell
junctions wherever and whenever it is needed without replacing all of
the junctional complex components constantly is an efficient and
perhaps an inevitable cellular event.
The fact that a secretory protein can become tightly associated with
the cell surface following its secretion is not without precedence.
Wnt, a growing class of multifunctional factors that constitutes a set of 15 or more proteins involving both tumorigenesis and patterning events during development and tissue differentiation (Nusse and Varmus, 1992), are shown to be secreted proteins which then
become tightly associated with the cell surface or extracellular matrix
(Papkoff and Schryver, 1990
; Bradley and Brown, 1990
; Smolich et
al., 1993
). The most extensively studied mammalian Wnt
gene is mouse Wnt-1. The mRNA expression of
Wnt-1 in normal adult mice is restricted only in spermatids
and in the embryonic neural tube during mid-gestation (Shackleford and
Varmus, 1987
; Wilkinson et al., 1987
). The protein products
of mouse Wnt-1 are cysteine-rich glycoproteins of 36-44 kDa
(Brown et al., 1987
). The soluble form of Wnt-1
protein which is present at low abundance has recently been shown to
have mitogenic activity on quiescent cultures of C57MG or RAC311 target
cells, two mammary epithelial cells (Bradley and Brown, 1995
). Work is
now in progress to examine whether the soluble form of testin which is
present at low abundance in intact testis and in Sertoli cells cultured
at high cell density when specialized junctions are formed possesses
other biological functions.
During postnatal development, a decline in the testicular steady-state testin mRNA level was noted. In adult rats at 45 days of age, there is 4-fold less testin mRNA than in immature rats at 3 days of age. Results of the current study suggest that the decrease of testin mRNA is not likely the result of germ cell modulation. We postulate that the high expression of the testin steady-state mRNA level in the immature rat testes is a reflection of rapid junctional complex turnover due to extensive tissue restructuring in postnatal testicular development. This hypothesis is further supported by the high level of testin steady-state mRNA expression in pre- and neonatal kidney which was drastically reduced in adult rats. It must be noted that there are no germ cells in the kidney. Furthermore, the changes in testin steady-state mRNA level in the ovary at different stages of the estrus cycle seem to support this postulate since there is extensive junctional complex assembly and disassembly between granulosa cells due to follicle development. Therefore, the decrease in testin steady-state mRNA level in the testis seems to be an age-dependent process and is probably proportional to the decline of junctional complex turnover during development. In addition, the testin steady-state mRNA level in adult organs including the brain, kidney, liver, small intestine, and spleen is extremely low and is undetectable without the use of hot nested RT-PCR (Grima, et al., 1995). Within these adult organs there is little cell renewal and tissue restructuring. However, in the adult rat testis, there is extensive junctional assembly and disassembly throughout spermatogenesis which is why the level of steady-state testin mRNA is still the highest in this organ.
In summary, we have shown that testin, a secretory product of Sertoli cells, can become tightly associated with the testicular cell membrane whose solubilization from Sertoli or testicular membrane requires the use of detergents. In addition, both the Sertoli cell testin secretory activity and the mRNA level is tightly coupled to the integrity of specialized junctions between testicular cells. When the Sertoli-germ cell junctions are disrupted by an exogenous agent such as lonidamine to deplete germ cells, a surge in both the testin mRNA level and its protein secretory activity are detected. Moreover, the steady-state testin mRNA is also correlated with an extensive renewal of cell-cell junctions during development, suggesting testin is a marker in monitoring germ cell-Sertoli cell interactions throughout spermatogenesis.
A recent study using cell surface labeling in conjunction with affinity chromatography, a receptor-like binding protein for testin was extracted from Sertoli cell membrane (Grima, J., Wong, C. S., Lee, W. M., and Cheng, C. Y. (1996) Sixth International Congress on Cell Biology, San Francisco, CA, December 7-11, Special Poster Session, Abstr. H122). The demonstration of a receptor-like binding protein suggests that testin may act as a signaling molecule which may explain why there is a transient but acute expression of testin when the Sertoli-germ cell junctions are being disrupted.