Ultracytochemical and biochemical evidence for guanylate cyclase in guinea pig testis.код для вставкиСкачать
THE ANATOMICAL RECORD 212277-281 (1985) Ultracytochemical and Biochemical Evidence for Guanylate Cyclase in Guinea Pig Testis RITA PASCOLINI, ANTONIO SPRECA, SERGIO LORVIK, OLIVIERO FAGIOLI, AND GIORGIO FAN0 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 ABSTRACT 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 differentiation. 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). MATERIALS AND METHODS 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., 1979). 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. 278 R.PASCOLINI ET AL. 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. RESULTS 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 2 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. 10 GUANYLATE CYCLASE IN GUINEA PIG TESTIS 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- 279 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. 280 R.PASCOLINI ET AL. 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, GUANYLATE CYCLASE IN GUINEA PIG TESTIS 281 DISCUSSION ACKNOWLEDGMENTS 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, 1982). 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. LITERATURE CITED 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. Brooker, G., J.F. Harper, W.L. Terasaki, and R.D. Moyalan (1979) Radioimmunoassay of cyclic AMP and cyclic GMP. Adv. Cyclic Nucleotide Res., 10:l-34. Conti, M., R. Geremia, and V. 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