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Ultracytochemical and biochemical evidence for guanylate cyclase in guinea pig testis.

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THE ANATOMICAL RECORD 212277-281 (1985)
Ultracytochemical and Biochemical Evidence for
Guanylate Cyclase in Guinea Pig Testis
Institutes of Comparative, Human, and Veterinary Anatomy (R.P., A.S., S.L., 0 . F ) and
Laboratory of Physiology (G.F), University of Perugia, 06100 Perugia, Italy
Guanylate cyclase activity has been studied biochemically and cytochemically in guinea pig testis. The results of the biochemical assays indicate a n
equal distribution of this enzyme between the soluble and particulate fractions,
which have a different sensitivity to adenosine triphosphate. The cytochemical
results demonstrate that the reaction product of guanylate cyclase is detectable in
the interstitial capillary endothelial cells and, in the seminiferous epithelium, mainly
a t the level of the adjacent surfaces of Sertoli and germ cells of the intermediate and
adluminal compartments. Guanylate cyclase activity appears at the level of pachytene spermatocytes and persists throughout subsequent stages of development. The
distribution in the seminiferous epithelium seems to indicate that guanylate cyclase
is involved in the interrelationships between Sertoli and germ cells during gamete
It is known that adenosine 3'5'-cyclic monophosphate
(CAMP)moderates the Sertoli cell response to follicle
stimulating hormone (FSH)(Fakunding et al., 1976)and
also plays a role in the differentiating processes of germ
cells (Conti et al., 1979). Although the precise role of
cyclic guanosine (cGMP) monophosphate in the testis is
not clear it seems to be involved in the adaption of this
organ to injury (Earp et al., 1980). The cGMP synthesis
enzyme guanylate cyclase (GC) exists in a soluble and
in a particulate form; their activities are approximately
equally distributed in the testis (Earp et al., 1980). Soluble GC reflects the cGMP levels directly, while particulate GC reflects them indirectly and responds to
gonadotropin stimulation (Spruill et al., 1979; Earp et
al., 1980).
In a previous work the distribution of adenylate cyclase (AC) activity was analyzed in guinea pig seminiferous epithelium (Pascolini et al., 1983) using a
cytochemical procedure. We now report the results of a
cytochemical and biochemical study on GC activity in
guinea pig testis. Biochemical study confirms the presence of GC in this organ (Earp et al., 19801, while the
cytochemical result localized the distribution pattern of
this enzyme in the seminiferous epithelium. The preliminary findings were reported at the Fifth International
Conference on Cyclic Nucleotides and Protein Phosphorylation (Fan6 et al., 1983).
The testes used in this study were taken from guinea
pigs weighing from 400 to 500 gm.
The biochemical parameters (Km and Vmax) were
carried out on homogenates of decapsulated testis previously tritiated in liquid Nz. Both soluble and particulate GC were obtained from single homogenate samples
(two to four testes) by two series of ultracentrifugations
0 1985 ALAN R. LISS, INC.
(105,000 g at 4°C for 1 hour) the second of which was
performed in the presence of Triton X-100 (2% vlv) after
12 hours of incubation with the same detergent.
The specific activity was measured in the supernatant
by the Garbers and Murad method (1979)in the presence
of Mn2' as cofactor, using guanosine triphosphate (GTP)Na as substrate and a creatinphosphatelcreatinphosphokinase mixture as the reformatting triphosphate nucleotide system. The concentration of cGMP was
determined as picomole per minute per milligram of
protein by a radioimmunological assay (Brooker et al.,
In a separate set of experiments the specific activity
was also measured in the presence of 10 mM adenosine
triphosphate (ATP).
Cytochemical Localization of GC
Pieces of decapsulated testis were immersed for 15
minutes in a 0.1 M cacodylate buffer (pH 7.4) containing
1% glutaraldehyde and 3.5% sucrose. Thereafter the
pieces were rinsed in the same buffer and sectioned a t
80 pm. Then tissue slices were incubated according to
the procedure of Kang et al. (1982) using GTP as substrate and NaN3 as a weak activator of GC and inhibitor
of GTPases (Murad et al., 1979).
For controls, tissue slices were incubated in the same
medium without a GTP substrate or in the presence of
GTP but inactivated at 70°C for 5 minutes before incubation. After incubation all specimens were rinsed in
proTRIS-maleate buffer, postfixed in 1%0 ~ 0 4 and
cessed for transmission electron microscopy (TEM) fol-
Received May 30, 1984;accepted March 1, 1985.
lowing the standard procedure. Ultrathin sections were
prepared, after a brief staining in a saturated solution
of uranyl acetate in 50% ethanol, and examined with a
Philips TEM 400 operating at a n accelerating voltage of
60 kV. The experiments were repeated several times
with similar results for each replicate.
Biochemical Analysis
Under standard condition the Km and Vmax, calculated by the Lineweaver-Burk equation, showed little
differences for the particulate and soluble fractions of
the enzyme. In fact the particulate fraction had a Km
value of 1.30 x 10- 5 M and a Vmax value of 19.3 pmoU
midmg. The corresponding values obtained from the
M and 25.1 pmoU
soluble fraction were 0.567 x
midmg, respectively.
In the presence of 10 mM ATP, known to be a n inhibiting factor of GC (Schultz, 19741, the difference between
the two forms of GC was more evident. Under these
conditions the nucleotide induced a different effect on
the biochemical patterns of the two enzymes. Figure 1
shows that the Vmax of the particulate form is slower,
up to 50% of the standard value (from 19.3 to 10.0 pmoU
midmg), while the soluble form shows a rise (loo%,
approximately, of the Vmax value obtained under standard conditions: from 25.1 to 45.4 pmolimidmg).
The Km value seemed to be less influenced by ATP
action. The Km value rose in both the particulate and
the soluble fractions: 1.86 x
M and 1.34 x lop3
M, respectively.
Cytochemical Analysis
In the interstitial tissue the reaction product of GC
was localized a t the level of the capillaries and lymphatic sinusoids (Figs. 2,3).
In the seminiferous epithelium the reaction product of
GC showed a clear distribution pattern. In the basal
aspect of the Sertoli cell there was no evidence of GC
activity (Figs. 2,3). The membrane-associated reaction
product was detectable, however, in the intermediate
and adluminal compartments of the Sertoli cell. Some
reaction product was present on the adjoining SertoliSertoli membranes (Fig. 4), but the precipitation sites
were mainly the adjacent plasma membranes of Sertoli
pachytene spermatocytes and of Sertoli spermatids (Figs.
5-8). Fingerlike processes of the Sertoli cell penetrate
the spermatid cytoplasm and their derived vesicles
showed the reaction product (Figs. 6,7).
The reaction product of GC on the surface of the spermatozoa was never observed in the lumen (Fig. 9).
Control specimens incubated in a substrate-free medium did not show any reaction product (Fig. 10). Heatinactivated tisue was also devoid of lead deposits.
2 1
Fig. 1 . Lineweaver-Burk plots of soluble (closed circles) and particulate (asterisks) GC in the
presence (right) or in the absence (left) of lOmM ATP. Each point represents the mean of nine
points carried out from three separate experiments.
Fig. 2. GC reaction product is localized on the surface of a capillary
endothelial cell @) and in its basement membrane (BM). Note that the
basal surface of the Sertoli cell (S) is devoid of reaction product. Lum,
lumen; P, pericyte; MC, myoid cell. ~28,000.
Fig. 3.Deposits of the reaction product are localized on the endothelial surface @) of a lymphatic sinusoid. The basal surface of the Sertoli
cell (S) and adjacent plasma membrances of Sertoli and young sper-
matocytes (YSt) lack the reaction product. MC, myoid cell; BM, basement membrane. X15,OOO.
Fig. 4. GC reaction product on the adjacent plasma membranes of
Sertoli cell ( S )and pachytene spermatwytes (PSt). Reaction product is
also detectable on the adjoining Sertoli-Sertoli surfaces (arrowheads).
X 10,000.
Fig. 5. GC reaction product on the adjacent surfaces of Sertoli (S)
pachytene spermatocytes (PSt) and of Sertoli spermatids (Sp) during
acrosome phase. ~8,500.
Fig. 6 . GC reaction product on the adjacent plasma membranes of
Sertoli cells (S) and spermatids (Sp) on the adjacent surfaces of a
the cytoplasm of spermatid show reaction product (arrowheads). CB,
chromatoid body. x 13,000.
Fig. 8. GC reaction product on the adjacent plasma membranes of
Sertoli cells (S)and spermatids (Sp). ~24,000.
fingerlike Sertoli process and spermatid cytoplasm and on the walls of
the derivate vesicle (arrowheads). x 11,000.
Fig. 9. The flagellar plasma membranes of spermatozoa in the lumen
(Lum) are devoid of reaction product. x 16,000.
Fig. 7.The adjacent plasma membranes of spermatids (Sp) and the
Sertoli cell (S) are stained for GC. Some reaction product is detectable
in the vesicles of the Sertoli cell; the double walls of the vesicles within
Fig. 10. The control adluminal compartment of seminiferous epithelium incubated in the cytochemical medium lacking the GTP substrate. Sp, spermatid; s. Sertoli cell. x 10,000,
The values of Km and Vmax obtained from the experiments performed seem to indicate, in the complete
guinea pig testis, the presence of two different forms of
GC with different sensitivities to ATP. These findings
are in agreement with those reported by other authors.
In particular, our data on GC kinetic features seem to
confirm those observed by Spruill et al. (1979) and Earp
et al. (1980) on rat testis.
The cytochemical analysis of the distribution of GC
activity in the heterogeneous cell population of guinea
pig testis revealed that this enzyme is localized a t the
level of the endothelial cells and, in the seminiferous
epithelium, mainly on the adjacent surfaces of Sertoli
pachytene spermatocytes and Sertoli spermatids.
The presence of GC activity in the endothelial cells
has already been demonstrated in rat brain capillaries
by Karnushina et al. (1980) and in the capillaries of rat
uterus and oviduct by Kang et al. (1982, 1983). The GC
activity in the testis can be related, according to Karnushina et al. (1980), to transendothelial transport.
The distribution of GC in the seminiferous epithelium
is of interest particularly when compared to the AC
distribution in the same epithelium (Pascolini et al.,
1983). The reaction product of AC was distributed in
both the basal and the adluminal compartments, while
GC activity was not detectable on the basal portion. It
is of significance that in the basal aspect of the Sertoli
cell, where FSH receptors are predominantly localized
(Desjardins et al., 1974), there was evidence of AC activity but no evidence of GC activity. This different localization of the two enzymes seems to reflect their different
behavior. It is known that a direct relationship between
agonist stimulation of AC and the regulation of biological processes exists, while GC does not seem to respond
directly to the hormone agonist (Goldberg and Haddox,
1977; Mittal and Murad, 1977). As stated above, GC
reaction product appeared in the tubular microenvironment on the adjacent surfaces of Sertoli and germ cells
at a well-defined differentiated stage (pachytene spermatocytes) and persisted in subsequent stages of development. Hence, it is likely that GC is involved in
modulating the communication system between Sertoli
and germ cell, a n important consideration for the gamete differentiation (Ziparo et al., 1982; Galdieri et al.,
1983; Boitani et al., 1983). An indirect confirmation of
this hypothesis seems to be the presence of GC activity
in the fingerlike processes of the Sertoli cells invading
the cytoplasm of spermatids, where a n evident morphological and functional relationship between Sertoli and
spermatids exists (Russell, 1983; Morales and Clermont,
It was not possible to determine cytochemically if GC,
which is detectable on the adjacent surfaces of Sertoli
and germ cells is associated with Sertoli cell or germ
cell membranes, but the presence of the reaction product
also on Sertoli-Sertoli membranes and the absence over
luminal spermatozoa might suggest the former possibility.
Finally, the absence in the lumen of the reaction product on the flagellar membrane of spermatozoa confirms
that in vertebrate spermatozoa GC is insignificant (Gray
et al., 1976; Herman et al., 1976) in contrast with sea
urchin and tube worm spermatozoa, whose flagellar
plasma membranes appear to be the area richest in GC
activity (Gray et al., 1976; Sano, 1976).
This research was supported by a grant from the Ministry of Public Instruction. We thank Dr. D. Boothman
for reviewing the manuscript.
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guinea, guanylate, cyclase, evidence, pig, testis, biochemical, ultracytochemical
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