* Department of Histology and Medical Embryology, University of Rome "La Sapienza," 00161 Rome, Italy; and Howard
Hughes Medical Institute and Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas,
Texas 75235
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Abstract |
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The potent smooth muscle agonist endothelin-1 (ET-1) is involved in the local control of seminiferous tubule contractility, which results in the forward propulsion of tubular fluid and spermatozoa, through its action on peritubular myoid cells. ET-1, known to be produced in the seminiferous epithelium by Sertoli cells, is derived from the inactive intermediate big endothelin-1 (big ET-1) through a specific cleavage operated by the endothelin-converting enzyme (ECE), a membrane-bound metalloprotease with ectoenzymatic activity. The data presented suggest that the timing of seminiferous tubule contractility is controlled locally by the cyclic interplay between different cell types. We have studied the expression of ECE by Sertoli cells and used myoid cell cultures and seminiferous tubule explants to monitor the biological activity of the enzymatic reaction product. Northern blot analysis showed that ECE-1 (and not ECE-2) is specifically expressed in Sertoli cells; competitive enzyme immunoassay of ET production showed that Sertoli cell monolayers are capable of cleaving big ET-1, an activity inhibited by the ECE inhibitor phosphoramidon. Microfluorimetric analysis of intracellular calcium mobilization in single cells showed that myoid cells do not respond to big endothelin, nor to Sertoli cell plain medium, but to the medium conditioned by Sertoli cells in the presence of big ET-1, resulting in cell contraction and desensitization to further ET-1 stimulation; in situ hybridization analysis shows regional differences in ECE expression, suggesting that pulsatile production of endothelin by Sertoli cells (at specific "stages" of the seminiferous epithelium) may regulate the cyclicity of tubular contraction; when viewed in a scanning electron microscope, segments of seminiferous tubules containing the specific stages characterized by high expression of ECE were observed to contract in response to big ET-1, whereas stages with low ECE expression remained virtually unaffected. These data indicate that endothelin-mediated spatiotemporal control of rhythmic tubular contractility might be operated by Sertoli cells through the cyclic expression of ECE-1, which is, in turn, dependent upon the timing of spermatogenesis.
Key words: endothelin; endothelin-converting enzyme; spermatogenesis; peritubular myoid cells; seminiferous epithelium ![]() |
Introduction |
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ENDOTHELIN-1 (ET-1)1 is a 21-amino acid (aa) vasoconstrictive peptide originally isolated from the supernatant of cultured porcine aortic endothelial
cells (Yanagisawa et al., 1988). Subsequently, three distinct endothelin genes encoding three closely related peptides were identified: ET-1, ET-2, and ET-3 (Inoue et al., 1989
). These endothelin isopeptides are each produced
from corresponding preproETs of ~200 residues (Inoue et
al., 1989
) and act on two distinct subtypes of G-protein-
coupled receptors termed ETA and ETB (Arai et al., 1990
;
Sakurai et al., 1990
). Longer intermediates termed big endothelins (big ETs, 38-41 aa) are first excised from the
preproETs by dibasic pair-specific endopeptidases (Seidah et al., 1993
). Big ETs are then further cleaved at Trp21-Val/
Ile22 by the endothelin-converting enzyme (ECE) to produce the 21-residue mature peptides (Opgenorth et al.,
1992
). The fact that the biological activity of big ETs is
negligible (Kimura et al., 1989
) indicates that ECE is a key
enzyme for the production of biologically active ETs.
Complementary DNAs coding for two bovine ECEs have
been isolated recently and the corresponding proteins
have been termed ECE-1 (Xu et al., 1994
) and ECE-2
(Emoto and Yanagisawa, 1995
). Both enzymes are membrane zinc-binding metalloendopeptidases with a single
transmembrane domain, a short NH2-terminal cytoplasmic tail and a large extracellular COOH-terminal containing
the catalytic domain (Shimada et al., 1996
; Turner and
Tanzawa, 1997
). Analysis of the conversion of big ET-1
into ET-1 by ECE in vivo and in vitro (McMahon et al.,
1991
; Xu et al., 1994
) has demonstrated that the conversion takes place on the cell surface. Recently, the presence
of ECE on the plasma membrane has also been confirmed by ultrastructural immunolocalization showing that ECE
and angiotensin-converting enzyme colocalize on the luminal membrane of endothelial cells (Barnes et al., 1998
).
The abundance of ECE-1 mRNA in whole testis extracts favors the hypothesis that this enzyme plays an important role by mediating ET activation in the testis (Xu
et al., 1994). In the mammalian testis, seminiferous tubules
are ensheathed by a layer of smooth muscle-like cells, the
peritubular myoid cells. In the adult rat, myoid cells are arranged to form a squamous epithelioid layer in which no
major orientation is apparent (Hermo and Clermont, 1976
;
Palombi et al., 1992
). The main biological function of peritubular contractility is the generation of impulses for the
progression of spermatozoa (Hargrove et al., 1977
). The
transport of spermatozoa along the seminiferous tubule
lumen towards the rete testis, is thought to result from
forces that are not intrinsic to the sperm cells (Ellis et al.,
1981
; Eddy, 1988
). In fact, seminiferous tubules have been
reported to undergo rhythmic contraction; in the apparent
absence of nerve endings, the fine regulation of contractility is presumably subject to paracrine control.
Recently we demonstrated that ET-1 is specifically able
to induce contraction of rat myoid cells both in cell culture
and in peritubular tissue (Tripiciano et al., 1996). In addition, we demonstrated the simultaneous presence of ETA
and ETB endothelin receptors on individual myoid cells,
both of which mediate contraction through distinct regulation of calcium-mediated signaling (Filippini et al., 1993
;
Tripiciano et al., 1997
). The studies of Fantoni et al. (1993)
and Maggi et al. (1995)
have demonstrated that Sertoli cells produce and secrete ET-1 in rat and human testis.
Sertoli cells, somatic cells of the testis that provide the
structural framework of the seminiferous tubules and the
milieu for germ cell proliferation and differentiation, are
targets for the hormones (FSH and testosterone) responsible for the initiation and maintenance of spermatogenesis
(Bardin et al., 1988
). In the seminiferous epithelium, each
Sertoli cell maintains an extensive surface relationship, along its apical sides, with germ cells at various stages of
differentiation up to spermiation while the basal side faces
the peritubular myoid cells.
Seminiferous tubule contractility represents a fundamental and potentially critical function in male fertility, controlling testicular output of both fluid and sperm. Therefore, any level of regulation mediating seminiferous tubule contractility may represent a specific control mechanism regulating the timing of the contraction-relaxation cycle. In this study, we have examined the in vivo and in vitro specific expression of ECE in rat Sertoli cells as well as the biological activity of this enzyme from intact cultured Sertoli cells. Furthermore, we highlight the functional relevance of ECE by showing that it can mediate regional seminiferous tubule contraction by converting big ET-1 into fully biologically active ET-1. We provide evidence of differential expression of ECE-1 in the testis during spermatogenesis that underlies a pulsatile production of ET-1, accounting for a novel mechanism controlling contractility.
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Materials and Methods |
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Materials
Collagenase A from Clostridium histoliticum and DNase-I were obtained from Boehringer Mannheim. Trypsin was purchased from Difco Laboratories. MEM was obtained from GIBCO BRL. Percoll was purchased from Pharmacia. ET-1 and big ET-1 were obtained from Peninsula Laboratories, Inc.; other reagents, when not specified, were purchased from Sigma Chemical Co. Plastic culture dishes and multiwell plates were from Falcon.
Animals
The animals used were adult and three-week-old Wistar rats (Charles River), fed ad libitum until killed by CO2 asphyxia or cervical disarticulation. Animals were kept in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Alkaline Phosphatase Cytochemistry
Selective myoid cell identification through alkaline phosphatase cytochemistry was performed as previously described (Palombi and Di
Carlo, 1988), based on the method of Ackermann (Ackermann, 1962
). In
brief, the fixed cells were incubated in an alkaline solution containing 0.5 mg/ml Fast Blue RR in water and 40 µl/ml
-naphtol phosphate (0.25%
solution, pH 8.6). After 30 min incubation in the dark, a purple-blue precipitate appeared specifically on the surface of myoid cells (Palombi and
Di Carlo, 1988
).
Cell Isolation and Culture
Sertoli Cells.
Primary Sertoli cell cultures from 18-20-d-old Wistar rats
were prepared as previously described (Dorrington et al., 1975). Seminiferous tubules obtained by trypsin dispersion of testicular parenchyma
were subjected to collagenase digestion to remove the peritubulum. The
resulting fragments of seminiferous epithelium, mainly composed of Sertoli cells, were cultured at 32°C in a humidified atmosphere of 5% CO2
and 95% air in a chemically defined medium (MEM). After 3 d in culture,
germ cells contaminating the Sertoli cell monolayer were selectively removed through hypotonic shock (Galdieri et al., 1981
); the cells were used
one day after the treatment.
Myoid Cell Cultures and Sertoli Cell/Myoid Cell Cocultures.
The supernatant-mixed cell population resulting from the collagenase treatment of
seminiferous tubules (see above) was centrifuged at 40 g, yielding mostly
minute fragments of tubular wall (Sertoli cells and myoid cells): culturing
of this preparation in MEM for 3 d at 37°C results in a mixed monolayer in
which myoid cells can be identified (Tripiciano et al., 1996) by differences
in their morphology in phase contrast and through alkaline phosphatase cytochemistry after fixation. For pure myoid cell cultures, the tubular wall
fragments were digested in trypsin and EDTA to a single cell suspension,
subsequently fractionated on a discontinuous Percoll density gradient
(Palombi et al., 1988
; Filippini et al., 1993
). Percoll-purified myoid cells
were cultured under serum-free conditions at 37°C. The assessment of myoid cell purity, performed routinely for each preparation on the basis of
the presence of alkaline phosphatase activity, was never below 96%.
Germ Cell Preparations.
Seminiferous tubules from 35-60-d-old rats
were freed from interstitial tissue by collagenase treatment and dispersed
into single cells, as previously described (Geremia et al., 1977). The resulting cell suspension, highly enriched in germ cells, was used as such
("mixed germ cells") or fractionated into several cell classes by velocity
sedimentation at unit gravity in an albumin gradient (Lam et al., 1970
;
Boitani et al., 1983
). The two cellular fractions, composed, respectively, of
middle-late pachytene spermatocytes and of round spermatids (steps 1-8
of spermiogenesis), were found to be ~90% pure; the fraction composed
of intermediate spermatids (steps 9-14) was ~60% pure, also containing
late spermatids (~10%) and residual bodies (~30%).
RNA Isolation and Northen Blot Analysis
RNA was extracted from testicular cells and different organs using
the acid guanidine thiocyanate-phenol-chloroform extraction method
(Chomczynski and Sacchi, 1987). Sertoli cell poly(A)+ RNA was prepared
by means of a Quick Prep mRNA purification kit (Pharmacia Biotech).
Total RNA (10 µg) and mRNA (4 µg) were separated in a formaldehyde
and 1.1% agarose gel, transferred to a nitrocellulose membrane Gene
Screen Plus, and then hybridized in QuikHyb solution, as recommended
by the manufacturer (Stratagene). Random-primed 32P-labeled cDNA inserts (~4.7 and ~2 kb) encoding bovine ECE-1 and ECE-2 were used as
probes (Xu et al., 1994
). The membranes were washed in 2× SSC and
0.1% SDS at 55°C and were exposed to an x-ray film for 3 d at
80°C.
Measurement of [Ca2+]i
[Ca2+]i was measured by dual wavelength fluorescence in single cells
loaded with the Ca2+-sensitive indicator fura-2 (Grynkiewicz et al., 1985).
Testicular myoid cells were plated onto coverslips in serum-free MEM.
After 4 d in culture, the cells were incubated in MEM containing 3 mM fura-2-acetoxymethylester for 1 h at 37°C. The cells were then rinsed
with Krebs-Henseleit-Hepes (KHH) buffer (140.7 mM Na+, 5.3 mM K+,
132.4 mM Cl
, 0.98 mM PO42
, 1.25 mM Ca2+, 0.81 mM Mg2+, 5.5 mM
glucose, and 20.3 mM Hepes) supplemented with 0.2% fatty acid-free
BSA. Measurements were performed in single cells, at 340- and 380-nm
excitation wavelengths, with an AR-Sm microfluorimeter (Spex Industries) connected to a Diaphot TMD inverted microscope (Nikon Corp.)
equipped with a CF ×40 objective. Emission was collected by a photomultiplier carrying a 510-nm cut-off filter and recorded by an ASEM Desk
2010 computer (ASEM SpA), which automatically calculated real-time
340/380 ratios. Calibration of the signal was obtained at the end of each
observation by adding 5 µM ionomycin to saturate the dye maximal fluorescence, followed by 7.5 mM EGTA plus 60 mM Tris-HCl, pH 10.5, to
release Ca2+ from fura-2 and obtain minimal fluorescence. [Ca2+]i was
calculated according to previously described formulas (Grynkiewicz et al.,
1985
).
Preparation and Treatment of Seminiferous Tubule Segments for Contraction Assay in Scanning Electron Microscopy
Seminiferous tubules were prepared as previously described (Tripiciano
et al., 1996). In brief, testes from 2-mo-old rats were decapsulated and digested under gentle shaking at room temperature in MEM containing 1 mg/ml collagenase. After dispersion of the interstitium, the tubular mass
was rinsed in MEM, then stretches of tubules were dissected by means of
sharp needles and carefully transferred to 35-mm culture dishes in 300 µl
of medium. For the dissection of homogeneous samples at precise stages
of the seminiferous epithelium, the tubular segments were identified under transillumination (Parvinen and Ruokonen, 1982
). The tubules were
incubated for 10 min at 32°C in a humidified chamber under an atmosphere containing 5% CO2. At the end of the incubation time, the medium
was replaced by 600 µl of medium to be tested at different experimental
times, as detailed in figure legends. Samples were fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, dehydrated and critical point dried in ethanol, coated with gold, and then viewed in a Hitachi S-570 scanning electron microscope.
Evaluation of ECE Activity
ECE activity in Sertoli cell cultures was assayed through estimation of exogenous big-endothelin conversion (Little et al., 1994). The culture medium, conditioned for 30 min to 3 h in the presence of big ET (with or
without ECE inhibitors), was purified on a Sep-Pak C18 solid phase cartridge (Waters). After drying by vacuum centrifugation and reconstitution
in buffer, the samples were assayed for endothelin content by means of a
commercial enzyme immunoassay (EIA) kit (Cayman Chemical Co.) according to the manufacturer's instructions.
Preparation of 35S-labeled RNA Probes and In Situ Hybridization
Adult and 18-d-old Wistar rat testes were fixed in 4% paraformaldehyde
in PBS at 4°C overnight. The fixed testes were dehydrated with ethanol
and embedded in paraffin by standard procedures. 5-µm-thick paraffin
sections were placed on slides pretreated with 3-amino-propyltriethoxysilane. Sections were analyzed by in situ hybridization using the procedure
described by Davidson et al. (1988). In brief, before hybridization, sections were deparaffinized, rehydrated, partially digested with proteinase-K
(20 µg/ml), and then treated with acetic anhydride. These last two steps
were necessary to improve access of the probe to the mRNA and reduce nonspecific binding of the nucleic acid probes. Sections were dehydrated
and then incubated at 55°C for ~18 h with 35S-labeled RNA probes. For
generation of RNA probes, the 0.5 Kb 5' PstI-PstI fragment of bovine
ECE-1 cDNA, nucleotides 214-751, was subcloned in pBluescript vector
and transcribed in vitro with T7 (anti-sense) and T3 (sense) RNA polymerases. Unbound cRNA probe was removed by incubation in RNase solution (40 µg/ml) for 30 min at 37°C in 0.5 M NaCl, TE buffer and by two
20-min washes at 65°C in 2× saline sodium citrate (SSC). Autoradiography was performed with Ilford K2 liquid emulsion (Ilford). After exposure for the time periods indicated, sections were stained with carmalum and examined under a Zeiss microscope using dark- or brightfield illumination. The stages of the seminiferous epithelium were identified from adjacent sections using the criteria of Leblond and Clermont (1952)
.
Statistical Analysis
Data are presented as the mean ± SE of results from at least three independent experiments. Student's t test was used for statistical comparison between means where applicable.
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Results |
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Determination of Expression Levels of ECE-1 and ECE-2 mRNAs in Different Testicular Cells and in Extragonadal Tissue
RNA blot analysis with the bovine ECE-1 cDNA as probe
(Xu et al., 1994) showed that a ~4.7 kb ECE-1 mRNA is
expressed abundantly in cultured Sertoli cells (Fig. 1 a).
As shown, the expression of ECE-1 mRNA is much higher
in the testis from 20-d-old rats than in the adult. Since the
increase in weight of the testis depends mostly on germ
cell multiplication, the evidence that homogenous preparations of specific types of germ cells exhibit no expression
of ECE-1 indicates that the reported high expression of
ECE-1 mRNA in the testis (Xu et al., 1994
) could be attributed prevalently to Sertoli cells. Peritubular myoid
cells express much lower levels than Sertoli cells (Fig. 1 a).
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In addition, a 3.1-kb mRNA is expressed at a lower level. Since only the 4.7-kb ECE-1 mRNA is present in Northern blots of poly(A)+ RNA from cultured rat Sertoli cells, the two different sizes of mRNA are presumably generated by alternative poly(A)+ addition in the 3' noncoding region (Fig. 1 b). Conversely, Northern blot analysis of testicular cells with the bovine ECE-2 cDNA as probe (Emoto et al., 1995) revealed no expression of ECE-2 mRNA in the seminiferous epithelium cells, while a 3.3-kb mRNA was detected in the control neural tissue and adrenal gland (not shown).
Time-dependent Conversion of Big ET-1 and Big ET-3 by Intact Sertoli Cells
To analyze ECE activity, we examined whether cultured
Sertoli cells can convert synthetic rat big ET-1 exogenously added to the culture medium. We therefore assayed
the generation of mature ET-1 by means of a competitive
enzyme immunoassay (EIA) that does not cross-react with
the substrate big ET-1. As shown in Fig. 2, big ET-1 was
efficiently converted into ET-1 by intact Sertoli cells in a
time-dependent fashion. At a substrate concentration of 1 µM, up to 69% of the added big ET-1 was converted into
ET-1. The Sertoli cell ECE was more efficient in converting big ET-1 than big ET-3. When the metalloprotease inhibitor phosphoramidon (PR), known to specifically inhibit ECE activity (Xu et al., 1994), was present during
incubation, it completely inhibited the production of mature ET-1 (Fig. 2). The analogue of big ET-1, [D-Val22]big
ET-1 [16-38], an inhibitor of ECE (Morita et al., 1994
), strongly inhibited ECE activity and was as effective as PR
in completely inhibiting the production of ET-1 by Sertoli
cells incubated with big ET-1.
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Effect of Big ET-1 or Sertoli Cell-conditioned Medium on Calcium Mobilization in Isolated Myoid Cells
We have previously demonstrated that ET-1 is able to induce PI turnover and rapidly increase [Ca2+]i in testicular
myoid cells (Tripiciano et al., 1997). Cytofluorimetric analysis of intracellular calcium mobilization, measured by
dual wavelength fluorescence in single cells loaded with
the Ca2+-sensitive indicator fura-2, indicate that the inactive precursor of ET-1, big ET-1, does not induce calcium
mobilization in myoid cells; however, the same cells are
able to respond to the addition of ET-1 with an increase in
calcium levels (Fig. 3 a), which confirms the total biological inactivity of big ET-1. Conversely, when myoid cells
were stimulated with medium conditioned by Sertoli cells
for 30 min in the presence of 100 nM big ET-1 (SCMbig), a
rapid [Ca2+]i transient comparable to that induced by ET-1
was observed; subsequent stimulation with ET-1 was ineffective (Fig. 3 b). Therefore, medium conditioned by Sertoli cells in the presence of big ET-1 desensitizes myoid
cells to the actions of ET-1, which clearly indicates that the
biologically active molecule in SCMbig is ET-1 itself, converted from big ET-1 by ECE expressed in Sertoli cells. The observed slow calcium response is comparable to that
obtained in response to 0.5-1 nM ET-1 which is below
EC50, but sufficient to desensitize to 100 nM ET-1 (not
shown). When SCMbig were conditioned in presence of
phosphoramidon (PR), an inhibitor of ECE, no effect on
calcium response was observed even though myoid cells
were still responsive to ET-1 (Fig. 3 c). Fig. 3 d shows the
levels of ET-1-, SCMbig- (treated or untreated with phosphoramidon), and big ET-1-dependent [Ca2+]i increases
(both peak and plateau).
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Morphological Response of Cultured Myoid Cells
To corroborate the presence of fully functional ECE activity from intact Sertoli cells, we have treated cultured myoid cells with SCMbig one day after plating. Treatment at
this culture time with SCMbig resulted in an immediate
rounding-up with retraction of cytoplasm, which could be
directly followed in an inverted microscope (not shown).
To assess whether the observed myoid cell contraction was
specific for this cell type and whether Sertoli cells express
significant activity of ECE, which is able to process big
ET-1 into an amount of ET-1 sufficient to determine the
contraction of myoid cells, we used mixed cultures containing fragments of seminiferous epithelium and patches
of myoid cells. This mixed population of tubular and peritubular tissue was treated with 100 nM big ET-1 and shape
changes which occurred 10-20 min after treatment were
photographically recorded (Fig. 4). In these cultures, myoid cells patches were observed to undergo contraction in
response to big ET-1, while the morphology of adjacent
Sertoli cells remained unmodified. We processed the same
sample for the detection of alkaline phosphatase activity, a
specific marker for testicular myoid cells (Palombi and Di
Carlo, 1988), and found that the cells that contracted are
stained for alkaline phosphatase (Fig. 4, c and d). Inhibition of ECE activity by 2 mM PR resulted in the block of
contractile response to big ET-1 (Fig. 5). Since PR is a
metabolically stable phosphorylated sugar derivative and
is unlikely to enter cells at an appreciable rate within a
short incubation time, our observation indicates that the
conversion of big ET into ET is a plasma membrane event
that occurs on the extracellular side, analogous to the production of the vasoconstrictor angiotensin II from angiotensin I.
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Myoid Cell Contraction Induced by Big ET-1 in Peritubular Tissue
Fig. 6 a shows the surface of a seminiferous tubule as viewed in the scanning electron microscope. In the adult testis, the myoid cells appear arranged in a continuous monolayer of ephithelioid polygonal cells, particularly flat and wide and with bulging central nucleus. Addition of either 100 nM ET-1 (Fig. 6 b) or 100 nM big ET-1 (Fig. 6 c) results in dramatic contraction of the myoid peritubular cells, which display enhanced bulging of the central area and reduced distance between cell centers in most areas. From a morphological point of view treatment with either ET-1 or with its inactive precursor, big ET-1, induces a basically equal contraction of myoid cells; the only difference between these two treatments is in the timing required to achieve this effect. In fact, we observed myoid cell contraction within 15 s of ET-1 treatment, but only ~10 min after big ET-1 addition, presumably because more time is required for a sufficient amount of big ET-1 to be converted into biologically active ET-1 by ECE-1 expressed by adjacent Sertoli cells in the seminiferous tubule. When seminiferous tubules were challenged with big ET-1 in the presence of PR, we did not observe any contraction of myoid cells, which appeared as flat as in the control sample (Fig. 6 d). Furthermore, as a further control, we challenged seminiferous tubules with SCMbig. In this case, strong contraction of the myoid peritubular cells was observed within a few seconds (Fig. 7 a). When the seminiferous tubules were stimulated only with Sertoli cell- conditioned medium, the surface of myoid cells appeared to be unaffected, as in the control samples (Fig. 7 b).
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In Situ Hybridization of ECE-1 mRNA in the Rat Testis
To explore the possibility that the production of ET within the seminiferous epithelium is a discontinuous, cyclically regulated process, we studied the transcription of ECE-1 by in situ hybridization. Sense and antisense RNA for ECE-1 mRNA was prepared as detailed in Materials and Methods. Interestingly, the ECE-1 probe showed striking regional differences in the level of signal (Fig. 8, a and c). The density of the grains was maximal at stages IX-X of the cycle and at the background level in all other stages. The control samples, hybridized with sense ECE-1 probe, displayed a low level of background labeling, with no appreciable differences in grain density between seminiferous tubule profiles and interstitium, thus confirming the specificity of the hybridization signals. In tubules at stages IX-X, the bovine ECE-1 probe hybridized to a basal columnar region surrounding the germ cells (Fig. 8 c, left tubule). This indicates that the ECE-1 gene is expressed above all in Sertoli cells prevalently in the basal region. These findings are in agreement with and extend the above Northern blot analysis, indicating that ECE-1 is expressed in Sertoli cells from adult animal in a cyclical fashion during the seminiferous cycle in stages IX-X soon after spermiation. By contrast, testis from 20-d-old rats exhibits homogeneous labeling intensity in all seminiferous tubules (not shown).
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Peritubular Myoid Cell Contractility Is Controlled by ECE Expression at Specific Stages of the Seminiferous Epithelium Cycle
To investigate whether the restricted expression of ECE-1
could be functionally related to the regulation of peritubular contractility induced by big ET-1, seminiferous tubule
segments from adult testis were microdissected to isolate
specific "stages" of the seminiferous tubules (Parvinen
and Ruokonen, 1982) and their ability to respond to either
ET-1 or big ET-1 was studied at the scanning electron microscope. Fig. 9 a shows a transilluminated tubular segment in which the transition from stage VIII to stage IX is
very apparent. The hatched line indicates the level at
which the tubules were dissected. Two groups of specific
stages of the seminiferous tubule were tested: VII-VIII
and IX-XI, which showed low and high ECE expression,
respectively. Fig. 9 shows that treatment with big ET-1 is
able to induce a strong contraction of seminiferous tubules at stages IX-X in 10 min (Fig. 9 g); by contrast, the seminiferous tubule fragments containing stages VII-VIII are totally unaffected by the treatment with big ET-1 (Fig. 9 f).
Furthermore, ET-1 was still active in inducing an immediate contraction of the myoid peritubular cells in both
groups of seminiferous tubule fragments (Fig. 9, d and e).
These data suggest a direct correlation between the restricted expression of ECE-1 and the functional regulation of seminiferous tubule contraction.
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Discussion |
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Seminiferous tubule contractility is fundamental for sperm
progression towards the rete testis and its regulation represents, therefore, a key point in male fertility. In this
study, we focused on the paracrine communication between Sertoli cells and peritubular myoid cells as it represents an interesting model of cell-cell interactions between epithelial cells of the seminiferous tubule (which
play a crucial role during spermatogenesis) and a particular class of nonvascular smooth muscle cells (responsible
for seminiferous tubule contraction). Peritubular myoid
cells express -smooth muscle actin and desmin (Virtanen
et al., 1986
; Tung and Fritz, 1990
), and specifically respond
to endothelin undergoing cell contraction both in cell culture and in peritubular tissue (Filippini et al., 1995
; Tripiciano et al., 1996
, 1997
).
Given the cyclicity that characterizes seminiferous epithelium activity, we wondered whether endothelin production might be cyclically regulated at the level of the maturation of its precursor by ECE.
In this report we describe the distribution of ECE-1 during the seminiferous epithelium cycle and present evidence that differential expression of ECE-1 in the Sertoli cells during spermatogenesis results in specific and regional seminiferous tubule contraction.
It has been shown that cultured Sertoli cells exhibit a
basal production of ET-1 in the media (Fantoni et al.,
1993). Preliminary observations, which showed that Sertoli cells incubated with ECE-1 specific inhibitors strongly
reduced the secretion of ET-1 while increasing the accumulation of big ET-1 (not shown), prompted us to hypothesize a role for ECE-1 as a local regulator of ET-1 actions.
Furthermore, the occurrence of phosphoramidon-sensitive ECE activity on Sertoli cells suggests that some processing of secreted big ET-1 may occur on the surface of
ET-1-producing cells, adjacent to myoid cells. Since big
ET-1 appears to be much more stable than ET-1 to generic
proteolytic degradation (Murphy et al., 1994
), this targeted conversion may allow more effective delivery of the
active product in intact form to its receptors on myoid cells.
Since the prediction of its existence (Yanagisawa et al.,
1988), ECE has been considered to be a potential site of
regulation of endothelin production as well as a plausible target for therapeutic intervention in the endothelin
system. Recently, the existence of three distinct ECE-1
isoforms has been demonstrated (Shimada et al., 1995
;
Valdenaire et al., 1995
; Schweizer et al., 1997
). These three
isoforms (ECE-1a, ECE-1b, and ECE-1c) differ only in
their N-terminal regions and are derived from a single
gene through the use of alternative promoters. The three
isoforms show similar kinetic rate constants, processing
big ETs with similar velocities and have all been found to
cleave the three big endothelins, but with a clear preference for big ET-1, which is in agreement with our results
showing that intact Sertoli cell ECE-1 converts big ET-1
more efficiently than big ET-2 or big ET-3.
Recently, Yanagisawa et al. (1998) clearly demonstrated
that the activity of ECE-1 is essential and that a physiologically relevant endothelin-converting enzyme exists for
both big ET-1 and big ET-3 in vivo. In fact, ECE-1
/
mice (which all died within 30 min of birth) reproduced
the phenotype resulting from the defects in both ET-1/
ETA- and ET-3/ETB-mediated signaling pathways, which clearly shows that mature ET-1 and ET-3 are not synthesized in the relevant microenviroments without ECE-1 activity. Furthermore, a significant amount of mature ET-1/
ET-2 still existed in the serum of ECE-1
/
embryos despite the absence of ECE-1, which suggests that other peptidases are responsible for the production of mature ETs.
Intriguingly however, these remaining mature ETs completely failed to rescue the developmental phenotype of
ECE-1
/
mice, which indicates that defined mature ETs
must be produced at specific microenviroments in order to
achieve a biological effect. The present study provides evidence that the restricted expression of ECE-1 might play a
pivotal role in the control of peritubular contractility by
providing a fine local modulation of biologically active ET levels.
If ET acts as a local regulator of seminiferous tubule
contractility, it is conceivable that ECE is localized on the
basal side of the Sertoli cells. In fact, Northern blot analysis showed ECE-1 mRNA in cell extracts from purified
Sertoli cells. Our in situ hybridization studies indicate that
ECE-1 is predominantly localized in tubular areas where
Sertoli cell bodies reside, particularly in the basal region.
Sertoli cells are the only somatic cell type in the seminiferous epithelium; along the side of these elongated perennial
elements, it is possible to observe, at any given time, several generations of germ cells, which flow radially to be
eventually released as mature sperm into the tubular lumen. It has long been known that activities of the Sertoli
cell, among which FSH responsiveness, vary according to
the specific subset of differentiating germ cells with which
it is associated ("stages" of the seminiferous epithelium)
(Parvinen, 1982). In the prepuberal rat, in which the cyclicity of the epithelium has not been established yet, uniform
expression of ECE was observed; in the adult, by contrast, ECE expression appears to be regulated in a temporal and
spatial manner during spermatogenesis and the seminiferous epithelium cycle. Interestingly, expression of ECE-1 is
exclusively restricted, in the adult rat, to stages IX-X of
the cycle. These stages are characterized by the fact that
they immediately follow spermiation and represent ~5%
of the entire cycle length, which may explain why ECE expression was overlooked in a previous study (Takahashi et al., 1995
).
When segments of seminiferous tubule at precise stages of the seminiferous epithelium cycle were dissected and individually exposed to the inactive precursor big ET-1 to test their ability to induce myoid cell contraction through the generation of active ET-1, fragments containing stages preceding IX were found to be unresponsive to the precursor. By contrast, in segments from stages after spermiation, normal contraction of myoid cells was observed in response to the inactive precursor, which indicates efficient processing of big ET-1. In parallel samples, directly stimulated with ET, no difference in responsiveness to the active peptide was observed, which suggests that myoid cells are constantly capable of responding. These experiments demonstrate a direct correlation between a restricted expression of ECE-1 and its biological function.
A perspective that warrants exploration is the mechanisms that regulate the expression of ECE-1 and the developmental transition from the diffuse to the restricted
pattern of distribution of ECE-1, which may be connected
to the known cyclic (Parvinen, 1982) and developmental
changes in hormonal sensitivity Sertoli cells undergo (reviewed in Gondos and Berndston, 1993
). Moreover, alterations in the pattern of ECE-1 and ET production might
be involved in the pathogenesis of peritubular hyalinization, given the well-known role played by ET in fibrosis
and matrix overproduction in a number of tissues (Hahn
et al., 1993
; Hocher et al., 1999
).
In conclusion, our data could be used to outline a simplified model concerning the regulation of seminiferous tubule contractility, according to which the restricted expression pattern of ECE-1 would finely modulate local endothelin levels. In this model, ET-1 precursors produced by Sertoli cells are processed to biologically active ET-1 only in restricted areas of seminiferous tubule according to the spatiotemporal control of ECE-1 expression on the Sertoli cells (in turn, presumably dependent upon the spermatogenic cycle). Thus, seminiferous tubule contraction may originate in the specific tubular segments adjacent to those at which spermiation has just occurred, to be propagated as effective peristaltic waves by additional mechanisms that have yet to be identified.
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Footnotes |
---|
Address correspondence to Dr. Antonio Filippini, Department of Histology and Medical Embryology, University of Rome "La Sapienza," Via A. Scarpa, 14-00161 Rome, Italy. Tel.: 39-06-4976-6585. Fax: 39-06-446-2854. E-mail: filippini{at}uniroma1.it
Received for publication 1 December 1998 and in revised form 22 March 1999.
Research supported by grants from the Italian Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST).
The first two authors contributed equally to this work.
We wish to thank Dr. Elisabetta Dejana for her valuable discussion and critical reading of the manuscript. The skillful technical assistance of Mr. Quinto Giustiniani is gratefully acknowledged.
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Abbreviations used in this paper |
---|
aa, amino acid; ECE, endothelin-converting enzyme; EIA, enzyme immunoassay; ET, endothelin; FSH, follicle-stimulating hormone; KHH, Krebs-Henseleit-Hepes; PR, phosphoramidon.
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References |
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---|
1. | Ackermann, A.. 1962. Substituted naphtol AS phosphate derivatives for the localization of leukocytes alkaline phosphatase activity. Lab. Invest. 11: 563-566 . |
2. | Arai, H., S. Hori, I. Aramori, H. Ohkubo, and S. Nakanishi. 1990. Cloning and expression of a cDNA encoding an endothelin receptor. Nature. 348: 730-732 |
3. | Bardin, C.W., C. Yan Cheng, N.A. Musto, and G.L. Gunsalus. 1988. The Sertoli cell. In The Physiology of Reproduction. E. Knobil and J. Neill, editors. Raven Press, Ltd., New York. 1:933-974. |
4. |
Barnes, K.,
C. Brown, and
A.J. Turner.
1998.
Endothelin-coverting enzyme. Ultrastructural localization and its recycling from the cell surface.
Hypertension.
31:
3-9
|
5. | Boitani, C., F. Palombi, and M. Stefanini. 1983. Influence of Sertoli cell products upon the in vitro survival of isolated spermatocytes and spermatids. Cell Biol. Int. Rep. 7: 383-393 |
6. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenyl-chloroform extraction. Anal. Biochem 162: 156-159 |
7. | Davidson, D., E. Graham, C. Sime, and R. Hill. 1988. A gene with sequence similarity to Drosophila engrailed is expressed during the development of the neural tube and vertebrae in the mouse. Development. 104: 305-316 [Abstract]. |
8. | Dorrington, J.H., N.F. Roller, and I.B. Fritz. 1975. Effects of follicle-stimulating hormone on cultures of Sertoli cell preparation. Mol. Cell. Endocrinol. 3: 59-70 . |
9. | Eddy, E.M. 1988. The spermatozoon. In The Physiology of Reproduction. E. Knobil and J. Neill, editors. Raven Press, Ltd., New York. 1:27-68. |
10. | Ellis, L.C., M.D. Groesbeck, C.H. Farr, and R.J. Tesi. 1981. Contractility of seminiferous tubules as related to sperm transport in the male. Arch. Androl. 6: 283-294 |
11. |
Emoto, N., and
M. Yanagisawa.
1995.
Endothelin-converting enzyme-2 is a
membrane-bound, phosphoramidon-sensitive metalloprotease with acidic
pH optimum.
J. Biol. Chem
270:
15262-15268
|
12. |
Fantoni, G.,
P.L. Morris,
G. Forti,
G.B. Vannelli,
C. Orlando,
T. Barni,
R. Sestini,
G. Danza, and
M. Maggi.
1993.
Endothelin-1: a new autocrine/paracrine
factor in rat testis.
Am. J. Physiol
265:
E267-E274
|
13. | Filippini, A., A. Tripiciano, F. Palombi, A. Teti, R. Paniccia, M. Stefanini, and E. Ziparo. 1993. Rat testicular myoid cells respond to endothelin: characterization of binding and signal transduction pathway. Endocrinology. 133: 1789-1796 [Abstract]. |
14. | Filippini, A., A. Tripiciano, M. Stefanini, E. Ziparo, and F. Palombi. 1995. Endothelin as a potential stimulator of seminiferous tubule contractility. In Endothelins in Endocrinology: New Advances. E. Baldi, M. Maggi, I.T. Cameron, and M.J. Dunn, editors. Ares-Serono Symposia, Rome. 15:219-222. |
15. | Galdieri, M., E. Ziparo, F. Palombi, M.A. Russo, and M. Stefanini. 1981. Pure Sertoli cell cultures: new model for the study of somatic-germ cell interaction. J. Androl. 5: 249-254 . |
16. | Geremia, R., C. Boitani, M. Conti, and V. Monesi. 1977. RNA synthesis in spermatocytes and spermatids and preservation of meiotic RNA during spermiogenesis in the mouse. Cell Differ 5: 343-355 |
17. | Gondos, B., and W.E. Berndston. 1993. Postnatal and pubertal Sertoli cell development. In The Sertoli Cell. L.D. Russell and M.D. Griswold, editors. Cache River Press, Clearwater, FL. 115-154. |
18. | Grynkiewicz, G., M. Poenie, and R.Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450 [Abstract]. |
19. | Hahn, A.W., T.J. Resink, E. Mackie, T. Scott-Burden, and F.R. Buhler. 1993. Effects of peptide vasoconstrictors on vessel structure. Am. J. Med 94: 13S-19S |
20. | Hargrove, J.L., J.H. MacIndoe, and L.C. Ellis. 1977. Testicular contractile cells and sperm transport. Fertil. Steril. 28: 1146-1157 |
21. | Hermo, L., and Y. Clermont. 1976. Light cells within the limiting membrane of rat seminiferous tubules. Am. J. Anat. 145: 467-483 |
22. |
Hocher, B.,
I. George,
J. Rebstock,
A. Bauch,
A. Schwarz,
H.H. Neumayer, and
C. Bauer.
1999.
Endothelin system-dependent cardiac remodeling in
renovascular hypertension.
Hypertension.
33:
816-822
|
23. | Inoue, A., M. Yanagisawa, S. Kimura, Y. Kasuya, T. Miyauchi, K. Goto, and T. Masaki. 1989. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Natl. Acad. Sci. USA 86: 2863-2867 [Abstract]. |
24. | Kimura, S., Y. Kasuya, T. Sawamura, O. Shinimi, Y. Sugita, M. Yanagisawa, K. Goto, and T. Masaki. 1989. Conversion of big endothelin-1 to 21-residue endothelin-1 is essential for expression of full vasoconstrictor activity: structure-activity relationships of big endothelin-1. J. Cardiovasc. Pharmacol 13: S5-S7 . |
25. | Lam, D.M., R. Furrer, and W.R. Bruce. 1970. The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse. Proc. Natl. Acad. Sci. USA. 65: 192-199 [Abstract]. |
26. | Leblond, C.P., and Y. Clermont. 1952. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. NY Acad. Sci. 55: 548-573 . |
27. | Little, D.K., D.M. Floyd, and A.A. Tymiak. 1994. A rapid and versatile method for screening endothelin converting enzyme activity. J. Pharmacol. Toxicol. Methods. 31: 199-205 |
28. |
Maggi, M.,
T. Barni,
C. Orlando,
G. Fantoni,
G. Finetti,
G.B. Vannelli,
R. Mancina,
L. Gloria,
L. Bonaccorsi,
M. Yanagisawa, and
G. Forti.
1995.
Endothelin-1 and its receptors in human testis.
J. Androl.
16:
213-224
|
29. | McMahon, E.G., M.A. Palomo, W.M. Moore, J.F. McDonald, and M.K. Stern. 1991. Phosphoramidon blocks the pressor activity of porcine big endothelin-1-(1-39) in vivo and conversion of big endothelin-1-(1-39) to endothelin-1-(1-21) in vitro. Proc. Natl. Acad. Sci. USA. 88: 703-707 [Abstract]. |
30. | Morita, A., M. Nomizu, M. Okitsu, K. Horie, H. Yokogoshi, and P.P. Roller. 1994. D-Val22 containing human big endothelin-1 analog, [D-val22]Big ET-1 [16-38], inhibits the endothelin converting enzyme. FEBS Lett. 353: 84-88 |
31. | Murphy, L.J., R. Corder, A. Mallet, and A.J. Turner. 1994. Generation by the phorphoramidon-sensitive peptidases, endopeptidase-24.11 and thermolysin, of endothelin-1 and C-terminal fragment from big endothelin-1. Br. J. Pharmacol 113: 137-142 [Abstract]. |
32. |
Opgenorth, T.J.,
J.R. Wu-Wong, and
K. Shiosaki.
1992.
Endothelin-converting
enzymes.
FASEB J.
6:
2653-2659
|
33. | Palombi, F., and C. Di Carlo. 1988. Alkaline phosphatase is a marker for myoid cells in cultures of rat peritubular and tubular tissue. Biol. Reprod. 39: 1101-1109 [Abstract]. |
34. | Palombi, F., D. Farini, P. De Cesaris, and M. Stefanini. 1988. Characterization of peritubular myoid cells in highly enriched in vitro cultures. In Molecular and Cellular Endocrinology of the Testis. B.A. Cooke and R.M. Sharpe, editors. Serono Symp, Raven Press, New York. 50:311-317. |
35. | Palombi, F., D. Farini, M. Salanova, S. De Grossi, and M. Stefanini. 1992. Development and cytodifferentiation of peritubular myoid cells in the rat testis. Anat. Rec. 233: 32-40 |
36. | Parvinen, M.. 1982. Regulation of the seminiferous epithelium. Endocr. Rev. 3: 404-417 |
37. | Parvinen, M., and A. Ruokonen. 1982. Endogenous steroids in rat seminiferous tubules. Comparison of different stages of the epithelial cycle isolated by transillumination-assisted microdissection. J. Androl 3: 211-220 . |
38. | Sakurai, T., M. Yanagisawa, Y. Takuwa, H. Miyazaki, S. Kimura, K. Goto, and T. Masaki. 1990. Cloning of a cDNA encoding a non-isopeptide selective subtype of the endothelin receptor. Nature. 348: 732-735 |
39. | Schweizer, A., O. Valdenaire, P. Nelbock, U. Deuschle, J. Dumas, and Milne Edwards, J.G. Stumpf, and B.M. Loffler. 1997. Human endothelin-converting enzyme (ECE-1): three isoforms with distinct subcellular localizations. Biochem. J. 328: 871-877 |
40. | Seidah, N.G., R. Day, M. Marcinkiewicz, and M. Chretien. 1993. Mammalian paired basic amino acid convertases of prohormones and proproteins. Ann. NY Acad. Sci. 680: 135-146 |
41. | Shimada, K., M. Takahashi, M. Ikeda, and K. Tanzawa. 1995. Identification and characterization of two isoforms of an endothelin converting enzyme-1. FEBS Lett. 371: 140-144 |
42. | Shimada, K., M. Takahashi, A.J. Turner, and K. Tanzawa. 1996. Rat endothelin-converting enzyme-1 forms a dimer through Cys412 with a similar catalytic mechanism and a distinct substrate binding mechanism compared with neutral endopeptidase-24.11. Biochem. J. 315: 863-867 |
43. | Takahashi, M., K. Fukuda, K. Shimada, K. Barnes, A.J. Turner, M. Ikeda, H. Koike, Y. Yamamoto, and K. Tanzawa. 1995. Localization of rat endothelin-converting enzyme to vascular endothelial cells and some secretory cells. Biochem. J. 311: 657-665 |
44. | Tripiciano, A., A. Filippini, Q. Giustiniani, and F. Palombi. 1996. Direct visualization of rat peritubular myoid cell contraction in response to endothelin. Biol. Reprod. 55: 25-31 [Abstract]. |
45. |
Tripiciano, A.,
F. Palombi,
E. Ziparo, and
A. Filippini.
1997.
Dual control of
seminiferous tubule contractility mediated by ETA and ETB endothelin receptor subtypes.
FASEB J.
11:
276-286
|
46. |
Tung, P.S., and
I.B. Fritz.
1990.
Characterization of rat testicular peritubular
myoid cells in culture: ![]() |
47. |
Turner, A.J., and
K. Tanzawa.
1997.
Mammalian membrane metallopeptidases:
NEP, ECE, KELL, and PEX.
FASEB J.
11:
355-364
|
48. |
Valdenaire, O.,
E. Rohrbacher, and
M.G. Mattei.
1995.
Organization of the
gene encoding the human endothelin-converting enzyme (ECE-1).
J. Biol.
Chem
270:
29794-29798
|
49. | Virtanen, I., M. Kallayoki, O. Narvanen, J. Paranko, L.E. Thornell, M. Miettinen, and V.P. Lehto. 1986. Peritubular myoid cells of human and rat testis are smooth muscle cells that contain desmin-type intermediate filaments. Anat. Rec. 215: 10-20 |
50. | Xu, D., N. Emoto, A. Giaid, C. Slaughter, S. Kaw, D. deWit, and M. Yanagisawa. 1994. ECE-1: a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell. 78: 473-485 |
51. | Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto, and T. Masaki. 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 332: 411-415 |
52. |
Yanagisawa, H.,
M. Yanagisawa,
R.P. Kapur,
J.A. Richardson,
S.C. Williams,
D.E. Clouthier,
D. de Wit,
N. Emoto, and
R.E. Hammer.
1998.
Dual genetic
pathways of endothelin-mediated intercellular signalling revealed by targeted disruption of endothelin converting enzyme-1 gene.
Development.
125:
825-836
|