Gonadotropin-Releasing Hormone Induction of Apoptosis in the Testes of Goldfish (Carassius auratus)
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内分泌学杂志 2005年第3期
Department of Biological Sciences (C.V.A.-V., A.G.B., H.R.H.) and Mucosal Inflammatory Research Group (A.G.B.), University of Calgary, Calgary, Alberta, Canada T2N 1N4
Address all correspondence and requests for reprints to: Dr. H. R. Habibi, Department of Biological Sciences, University of Calgary, Biosciences 282, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4. E-mail: habibi@ucalgary.ca.
Abstract
Apoptosis, or programmed cell death, can occur via death receptor or mitochondrial pathways. Normal spermatogenesis in mammals involves apoptosis mediated, in part, by the death receptor fas and its ligand. The regulation of programmed cell death in the gonads has been shown to be dependent on a number of locally produced factors, including GnRH. Whereas the role of GnRH in the control of apoptosis and follicular atresia has been documented in the mammalian ovary, GnRH regulation of testicular apoptosis remains obscure. A previous study in our laboratory demonstrated the involvement of GnRH on the induction of DNA fragmentation in mature, perispawning testis. In this study, we tested the hypothesis that GnRH plays a differential regulatory role during male gamete maturation by studying the effect of GnRH on the induction of apoptosis during goldfish spermatogenesis. Treatment with GnRH resulted in DNA fragmentation only during late stages of spermatogenesis as assessed by oligonucleotide detection and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assays. The GnRH-induced apoptosis in the goldfish testis was found to be mediated by increased levels of fas and fas ligand-like proteins as well as elevated activity of caspase-3 (an executioner caspase) and -8 (a death receptor-activated caspase). The results suggest the involvement of the death receptor pathway in GnRH-induced apoptosis, providing support for the hypothesis that GnRH plays an important role in the control of spermatogenesis in the goldfish testis.
Introduction
APOPTOSIS IS A FORM of cell death characterized by morphological and biochemical changes including DNA fragmentation, formation of apoptotic bodies, and a minimal inflammatory response (1, 2). In most cases, the apoptotic process converges into the activation of caspases, a group of aspartyl-directed cysteine proteases that are present in the cell as proenzymes (3). In receptor-mediated apoptosis, the interaction of a death receptor such as fas with its ligand (fasL) induces the recruitment and activation of procaspase-8, which in turn, cleaves several substrates including procaspase-3. The resultant caspase-3 activates an endonuclease (caspase-activated deoxyribonuclease), which is partially responsible for the low-molecular-weight pattern of DNA fragmentation observed during apoptosis (4, 5).
Testicular apoptosis occurs during normal spermatogenesis in mammals and cartilaginous fish (6), and it is thought to be essential for the maintenance of the correct ratio between Sertoli cells and gametes (7). Caspases are well-conserved proteins (8, 9, 10), and their presence in teleosts has also been demonstrated, although their distribution in adult tissues remains unknown (8, 9). Several components of the apoptotic machinery have been shown to be present in the male gonads. For instance, the expression of caspases and the fas/fasL system has been demonstrated in the mammalian testes (11, 12, 13, 14, 15, 16, 17). Furthermore, the interaction between fas and fasL has been shown to regulate germ cell numbers during spermatogenesis (15, 16, 17). Caspases are well-conserved proteins (8, 9, 10), and their presence in teleosts has also been demonstrated, although their distribution in adult tissues remains unknown (8, 9, 14). Fas and fasL-like proteins have been shown to be expressed in birds, amphibians, and teleost fishes (18, 19, 20); however, little information is available about the potential role of these proteins in the regulation of fish gametogenesis. Moreover, despite our current knowledge about the occurrence of apoptosis in the fish ovary and testis (6, 21, 22, 23, 24), the molecular mechanisms that mediate programmed cell death in these tissues are still unknown.
In teleost fish, testes are bilateral elongated lobes located in the abdominal cavity, and spermatogenesis occurs in cysts within the seminiferous lobules (25). The majority of teleost fish, including goldfish, display a seasonal pattern of reproduction, and the testes undergo variations in size and histological appearance (25, 26). During spermatogenesis, all cells within a cyst develop synchronously (26). To spawn, teleosts rely heavily on environmental cues, such as temperature and light (26); lack of these cues will lead to gonadal regression, a process that may occur via the induction of apoptosis.
The regulation of testicular apoptosis, at least in mammals, appears to be mediated by both gonadotropins and locally produced factors such as gonadal steroids (27). GnRH and its receptors (GnRH-Rs) are among the hormone receptor systems that are locally expressed in the testes of mammals (28, 29, 30), amphibians (31), teleost fish (32), and lamprey (33). Although for years GnRH has been proposed to play a role in the regulation of the gonadal function, the exact mechanisms underlying its actions has remained elusive. For example, GnRH has been shown to exert both stimulatory and inhibitory effects on steroidogenesis in the testis of different species (34, 35). The observed variation in responses after GnRH treatment may be attributed to species differences, gonadal status, experimental approach, period of exposure to the hormone, and hormone concentration. However, the basis for these paradoxical effects of GnRH is poorly understood. We previously reported that GnRH induces apoptosis in the mature goldfish testis (36). The present study expands our earlier observations by investigating the role of GnRH in the regulation of testicular apoptosis at different stages of spermatogenesis and the mechanism of GnRH-induced apoptosis in the goldfish testis. The results demonstrate that, in the goldfish testis, GnRH-induced apoptosis occurs only during the late stage of spermatogenesis, mediated by increased levels of fas and fasL-like proteins as well as activation of caspases-3 (executioner caspase) and -8 (death receptor activated caspase). The results suggest the involvement of the death receptor pathway in the testicular response to GnRH, providing support for the hypothesis that GnRH plays an important role in the control of spermatogenesis in goldfish.
Materials and Methods
Animals
Male goldfish (Carassius auratus) of the comet variety (length 8–10 cm) were purchased from Aquatic Imports (Calgary, Alberta, Canada). Fish were maintained in a 1500-liter semirecirculating aquarium (60% replacement per day) at 18 C on a 16-h light, 8-h dark photoperiod and fed a commercial fish diet (Nutrafin floating pellets; Rolf C. Hagen Inc., Montreal, Québec, Canada).
Hormones and chemicals
sGnRH (salmon GnRH; pGlu, His, Trp, Ser, Tyr, Gly, Trp, Leu, Pro, Gly-NH2), and cGnRH-II (chicken GnRH; pGlu, His, Trp, Ser, His, Gly, Trp, Tyr, Pro, Gly-NH2) were purchased from American Peptide Co. (Sunnyvale, CA). GnRH antagonist (Ac-D-2-Nal-p-Chloro-D-Phe-?-(3-pyridyl)-D-Ala-Ser-Lys(nicotinyl)-D-Lys(nicotinyl)-Leu-Lys-(isopropyl)-Pro-D-Ala-NH2) was purchased from Peninsula Laboratories (San Carlos, CA). GnRH peptides (10 μg/20 μl) were solubilized in 0.1 M acetic acid. Hormones were stored at –20 C until use. Appropriate concentrations of the hormones were prepared by diluting the stock solution in sterile culture medium immediately before use. Caspase-3 colorimetric substrate (Ac-DEVD-pNA; pNA: p-nitroanilide), caspase-3/7 inhibitor (Ac-DEVD-CHO), and p-nitroanilide were purchased from Sigma (St. Louis, MO). Caspase-8 colorimetric substrate (substrate I, Ac-IETD-pNA, 368057) was purchased from EMD Biosciences, Inc. Calbiochem (San Diego, CA). The peptides and p-nitroanilide were solubilized in dimethyl sulfoxide (Sigma) to prepare a 20 mM, 200 μM, and stock solutions that were stored protected from light at –20 C until use.
Testis incubation
Animals were killed in accordance with the principles and guidelines of the Canadian Council of Animal Care. Mature testes [spermiating testis with gonadosomato index (GSI): 3.5–4.5%], immature testis (nonspermiating testis with GSI: 1.9–2.5%) were dissected out and incubated as previously described (36). GnRH was added immediately and the tissue slices were incubated for different times at 18 C. A time course for caspase-3 activity, which is upstream of DNA fragmentation, was performed with time points at 1, 2, 5, 8, and 24 h after GnRH treatment. No caspase-3 activity was observed at 1, 2, and 5 h after the treatment, and the increases at 8 h were not statistically significant. Therefore, further experiments were carried out at 24 h post GnRH treatment. In experiments carried out in the presence of GnRH antagonist, 1 h preincubation with the antagonist was performed, followed by coincubation with GnRH for a further 24 h. The experiments were carried out three to five times, and in each experiment, gonads from one fish were used for all treatments. At the end of the incubation period, testes were kept at –20 C for determination of DNA fragments or were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay, caspase activity, or Western blot analysis.
Detection of DNA fragments
DNA fragmentation in testicular tissues was assessed by using a cell death detection ELISA kit (Roche Diagnostics, Laval, Québec, Canada), according to the manufacturer’s instructions. This quantitative sandwich ELISA specifically measures the histone region (H1, H2A, H2B, H3, and H4) of mono- and oligonucleosomes, which are released during apoptosis. Photometric readings were obtained after 10 min of reaction in a double-wavelength plate reader at 405 nm (Molecular Devices, Menlo Park, CA). Apoptosis was measured in duplicate from each sample and expressed as the absorbance ratio of treated tissues vs. the absorbance from untreated control tissues, arbitrarily set as 1.0, as previously described (37). To correct for differences in tissue content per slice, protein quantification was carried out by the bicinchoninic acid assay (Pierce, Rockford, IL), using BSA (Roche Diagnostics) as standard and a U2000 spectrophotometer (Hitachi, Tokyo, Japan).
TUNEL assay and hematoxylin-eosin staining
After GnRH treatment, testis slices were fixed in 4% formalin-buffered solution (Sigma) for 18 h and embedded after standard histological methods. Five-micrometer sections were stained with hematoxylin-eosin (Gill-formulation, Fisher Scientific, Fair Lawn, NJ) and mounted in Permount (Fisher Scientific).
Tissue sections (5 μm) were also stained for nick end labeling by using a commercial assay kit (in situ cell death detection kit-AP, Intergen Co., Purchase, NY). Sections were incubated as previously described (21). Slides were counterstained with 4',6'-diamino-2-phenylindole (DAPI) for 10 min at room temperature, mounted in antifade mounting media (Aqua Poly/Mount, Polysciences Inc.) and examined under a fluorescence microscope (Leica DMR, Québec, Canada).
Caspase-3 and caspase-8 activity assays
After the incubation period, tissue samples were rinsed with PBS. Three 20-μm slices were pooled and homogenized through different gauze needles in 250 μl extraction/substrate caspase Buffer [50 mM Pipes-KOH (pH 7.4), 220 mM mannitol, 68 mM sucrose, 50 mM KCl, 5 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, 10 μg/ml proteinase inhibitor phenylmethylsulfonyl fluoride] for 30 min on ice. Samples were frozen at –80 C for 3–4 h after which they were thawed and refrozen overnight at –80 C. Samples were quick thawed, spun at 14,000 x g at 4 C for 15 min, and the supernatant assessed for caspase-3 activity. Total protein content was determined by a Bradford assay (Bio-Rad Laboratories, Hercules, CA), using BSA as the standard. Samples (20 μg total protein) were read in duplicate on 96-well-plates (Falcon). The concentration of caspase-3 or caspase-8 substrates was 2 mM in a final volume of 100 μl/well of extraction/substrate buffer. In some experiments, a caspase inhibitor was coincubated with the substrate at a final concentration of 200 μM. Samples were incubated at 37 C for 2 h and read immediately in a plate reader at 405 nm (Molecular Devices), using p-nitroanilide as standard. Results were expressed as caspase-3 activity (milligrams of product per milligram total protein per hour) on correction for protein content and incubation time. HeLa cells treated with 1 μM staurosporine and 1 mM ethanol-treated HepG2 cells were used as positive control for the induction of caspase-3 and -8 activities, respectively (38, 39).
Western blot analysis
Western blot analysis was performed on tissue lysates that were obtained by extraction with a buffer containing 1% (vol/vol) Triton X-100, 50 mM Tris-HCl (pH 8), 10 mM CaCl2, 200 mM NaCl, and 10 μg/ml phenylmethylsulfonyl fluoride, followed by Bradford assay, as previously described (40). Lysates were subjected to electrophoresis through a 15% sodium dodecyl sulfate-polyacrylamide gel at 100 V. Immunoblotting was performed using a 1:1000 dilution of antihuman fas (sc7886) or antihuman fasL (sc834) polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Anti-fasL antibody was blocked by incubating a 1:1000 dilution of anti-fasL antibody with 5 x fasL-blocking peptide for 2 h at room temperature. Blots were reprobed with a 1:7000 dilution of mouse anti-?-tubulin (Sigma) to verify equivalent loading of samples. Bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) and quantified by densitometry. After quantification, the concentration of each target protein was normalized vs. ?-tubulin and plotted as a ratio. For each gel, lysates from phorbol ester-treated HL-60 cells were used as positive controls (41).
Data analysis
Statistical significance was determined by one-way ANOVA and the test for multiple comparison analysis Newman-Keul’s (Student-Newman-Keuls’s); P values < 0.05 were considered to be significant.
Results
sGnRH and cGnRH-II induction of DNA fragmentation in mature testes is stage dependent
We previously demonstrated the induction of DNA fragmentation by GnRH in the mature goldfish testes. In this study, we expanded these results by investigating the possible role of GnRH during fish spermatogenesis. Mature (spermiating testis, GSI: 3.5–4.5–5%) or immature testicular fragments (nonspermiating testis, GSI: 1.9–2.5%) were incubated in presence of the two native goldfish GnRH forms (sGnRH or cGnRH-II). In mature testes, treatment with sGnRH and cGnRH-II for 8 and 24 h significantly increased DNA fragmentation, compared with the control. The GnRH-induced apoptosis expressed as DNA fragmentation became very clear after 24 h of treatment for both sGnRH and cGnRH-II (Fig. 1, A and B; P < 0.05). After 8 h, however, DNA fragmentation induced by sGnRH and cGnRH-II were significantly higher than that of control but not at all doses tested. The effect of both sGnRH and cGnRH-II on immature testis was clearly different. Treatments with GnRH resulted in either no significant effect or small decrease in apoptosis in immature testis (Fig. 1, C and D). In the case of sGnRH, a small but significant decrease in DNA fragmentation was observed in immature testis for all concentrations tested with the exception of the highest level (10–7 M) (Fig. 1C; P < 0.05). As for cGnRH-II, a small decrease in apoptotic ratio was significant only at 10–9 M, compared with the control (Fig. 2B; P < 0.05). These results indicate that GnRH induces DNA fragmentation in mature perispawning but not in immature (early reproductive season) testes. Therefore, only mature testes were used in the following experiments to further investigate the onset of apoptosis induced by GnRH.
FIG. 1. GnRH induction of DNA fragmentation goldfish testis. Testicular fragments were incubated for 8 or 24 h with sGnRH (A and C) or cGnRH (B and D), followed by analysis of DNA fragmentation. A and B, Mature testes (GSI 3.5–4.5%). C and D, Immature testes (GSI 1.9–2.5%). Values represent the mean ± SEM of results obtained from three to five different experiments, expressed as a ratio of apoptosis with respect to the control. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
FIG. 2. Hematoxylin-eosin and TUNEL stainings in the mature goldfish testis. A and B, Hematoxylin-eosin staining for mature goldfish testes (mature, GSI 3.5–5%, spermiating testis). Original magnification, x400. Arrowheads, Spermatogonials; arrows, Sertoli-like cells. sp, Spermatids; sc, spermatocytes. C–I, Testicular fragments were treated with 10–7 M sGnRH during 24 h, fixed in 4% formalin for 18 h and processed for TUNEL staining and DAPI nuclear counterstaining. Note that DNA fragments were detected in seminiferous tubules and interstitial tissue. Original magnification, x400 and x1000.
Testis morphology and in situ TUNEL analysis
Routine staining revealed the coexistence of more than one germ cell stage within a seminiferous tubule in mature goldfish testes (Fig. 2A). The characteristics of these cells are similar to those described for Characiformes (42). Large spermatogonias were observed in the interstitial connective tissue (Fig. 2B) (42). Stage I spermatocytes, with compacted chromatin masses in the nuclear periphery, and spermatids were present within the cysts (Fig. 2, A and B). In addition, Sertoli-like cells were observed in the lining of the cysts (Fig. 2B). Spermatozoa were also present at this stage of gonadal maturation because they were released when testicular tissues were washed during the tissue culture procedures.
To investigate whether somatic or germinal cells underwent apoptosis in response to GnRH, DNA fragmentation was evaluated using the TUNEL assay. Saline-treated testis slices (control) were shown to be negative for TUNEL staining, whereas DAPI nuclear staining was positive (Fig. 2, C–I). Increased apoptosis was found in the GnRH-treated testis, compared with the control (Fig. 2, D and F). In the sGnRH treated testis, both germ cells and interstitial cells stained positive for DNA fragments (Fig. 2, F and H). Whereas complete identification of the stage of spermatogenesis affected by GnRH was not possible, the results suggest that spermatocytes may be among the cells sensitive to GnRH-induced apoptosis (Fig. 2H). Negative controls (no terminal dideoxy transferase enzyme) displayed no staining for DNA fragments. Similar results were obtained after treatment with cGnRH-II (data not shown).
sGnRH and cGnRH-II-induction of caspase-3 activity in mature testis
Caspase-3 has been shown to be present and activated in mammalian testes in response to several proapoptotic stimuli (11, 13). In addition, there is evidence that caspase-3 is conserved in fish (8) and involved in the induction of apoptosis through generation of ceramide. To further confirm the GnRH induction of apoptosis in mature goldfish testes, we investigated the activation of procaspase-3 in response to sGnRH or cGnRH-II (10–9 to 10–7 M). In these experiments, staurosporine-treated cells were used as positive control (control: 0.35 ± 0.038; staurosporine: 1.3 ± 0.10 mg product per milligram protein per hour).
Significant increases in caspase-3 activity were observed after treatment with either sGnRH or cGnRH-II at all concentrations tested (Fig. 3, A and B; P < 0.05). Samples were coincubated with the substrate and the caspase-3/7 inhibitor, Ac-DEVD-CHO, to test the specificity of the caspase assay. The presence of the inhibitor significantly decreased caspase-3 activity induced by both sGnRH and cGnRH-II at all the concentrations tested (Fig. 4, A and B; P < 0.05). Testis samples were also preincubated for 1 h with 10–6 M antide, a GnRH antagonist known to block GnRH activity in the goldfish ovarian follicles (43), to test the specificity of the GnRH response. The GnRH antagonist significantly inhibited sGnRH-induced caspase-3 activity (Fig. 4C; P < 0.05), without affecting basal level.
FIG. 3. sGnRH (A) and cGnRH-II (B) activation of caspase-3 in the mature goldfish testis. Fragments from mature testes were incubated for 24 h in the presence of sGnRH or cGnRH-II and analyzed for caspase activity. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
FIG. 4. Effect of the inhibitor Ac-DEVD-CHO on sGnRH- (A) and cGnRH-II (B)-induced caspase-3 activity in a cell-free system. A and B, Caspase-3 activity in cell lysates from mature testis, treated 24 h with sGnRH or cGnRH-II in the presence or absence of the caspase-3 inhibitor Ac-DEVD-CHO. C, Caspase-3 activity in mature testis fragments preincubated for 3 h with a GnRH antagonist and cultured for 24 h with or without sGnRH. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with corresponding controls (P < 0.05).
Effect of GnRH on fas and fasL-like proteins in the mature testes
The fas/fasL system has been shown to be involved in the induction of apoptosis in the several mammalian tissues, including testes (15, 16, 17). Experiments were carried out to investigate the effects of sGnRH and cGnRH-II on fas and fasL protein levels in the goldfish testes by Western blot analysis. HL-60 promyelocytic cell line extract was used as positive control, and tubulin level was used to correct for loading errors. Figure 5, A–C, shows representative immunoblots for fasL and quantitative results from three separate experiments. Treatment for 24 h with either sGnRH or cGnRH-II significantly increased fasL-like protein level, compared with the control (Fig. 5, A and C; P < 0.05). To test the specificity of binding, the anti-fasL antibody was preincubated overnight with its commercially available blocking peptide. As shown in Fig. 5B, the blocking peptide completely abrogated the anti-fasL antibody binding in the gonadal tissues, and partially blocked the antibody binding to the positive control (HL-60 cell lysate). Two bands corresponding to fas were detected in both fish and human extracts. The two immunoreactive bands may represent alternative splicing variants of fas, which have been previously reported to exist in humans (44). Both positive bands were shown to increase in response to GnRH treatment in the testes samples (Fig. 6A). Treatment with both sGnRH and cGnRH-II significantly increased fas expression in the goldfish testis, although the cGnRH-II-induced increase in fas levels at 10–9 M was not statistically significant (Fig. 6B; P < 0.05).
FIG. 5. Effect of GnRH on FasL protein levels in the mature goldfish testis. Western blot analysis of total lysates from mature testes treated with sGnRH or GnRH-II for 24 h. Protein extracts from HL-60 cells were used as positive controls. A, FasL. B, The specificity of the FasL antibody was tested by preincubating the antibody overnight at 4 C with 5x blocking peptide. Membranes were reblotted with an anti-?-tubulin antibody, which was used as an internal standard. C, Densitometry analysis for FasL, normalized vs. ?-tubulin. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
FIG. 6. Effect of GnRH on Fas (receptor) protein levels in the mature goldfish testis. Western blot analysis of total lysates from mature testes treated with sGnRH or GnRH-II for 24 h. Protein extracts from HL-60 cells were used as positive controls. A, Fas protein levels. Membranes were reblotted with an antitubulin antibody, which was used as an internal standard. B, Densitometry analysis for Fas, normalized. C, Densitometry analysis for Fas, normalized vs. ?-tubulin. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
sGnRH and cGnRH-II-induced caspase-8 activity in mature goldfish testis
We also investigated the activity of caspase-8 in response to GnRH in the goldfish testes because increased levels of fas and fasL may lead to the recruitment of procaspase-8 to the death receptor complex (45). In these experiments, ethanol-treated HepG2 cells were used as positive controls (control, 0.71 ± 0.05; ethanol, 1.76 ± 0.17 mg product per milligram protein per hour). A significant increase in caspase-8 activity was observed after 24 h of treatment with sGnRH and cGnRH-II for all concentrations tested (Fig. 7; P < 0.05).
FIG. 7. GnRH-induced activation of caspase-8 in the mature goldfish testis. Fragments from mature testis were incubated for 24 h with sGnRH or cGnRH-II and analyzed for caspase activity. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
Discussion
This study investigated the role of GnRH in control of apoptosis during spermatogenesis in goldfish. The results clearly demonstrate a stage-dependent variation in sGnRH- and cGnRH-II-induced apoptosis in the goldfish testes as well as providing information on the mechanism of GnRH-induced testicular apoptosis. Both sGnRH and cGnRH-II, which are expressed in the goldfish testis (32), significantly increased apoptosis in the perispawning goldfish testis containing germ cells at different stages of differentiation. The same GnRH peptides either have no effect or exert small protective action in immature nonspermiating testis. The apoptotic effect of GnRH can be seen more clearly after 24 h of treatment than 8 h, particularly for cGnRH-II. After 8 h of treatment and at lower concentrations, both sGnRH and cGnRH-II peptides displayed comparable activity. However, cGnRH-II was without a significant effect at 10–7 M, the highest concentration tested. A contributing factor could be down-regulation of GnRH-R at higher concentrations of cGnRH-II. Similar patterns of concentration-dependent desensitization have been previously observed for sGnRH and cGnRH-II in the goldfish pituitary (46, 47). The fact that only cGnRH-II seems to cause down-regulation may indicate that more than one receptor is involved in the response to sGnRH and cGnRH-II or that testicular receptors display different sensitivity to the two molecular forms of GnRH. In this regard, the presence of GnRH receptor subtypes with different ligand selectivity has been suggested in the goldfish pituitary (48) and ovary (49).
After 24 h of treatment, DNA fragmentation was significantly increased for all doses of sGnRH and cGnRH-II tested. In contrast, GnRH peptides did not induce DNA fragmentation in immature testes, composed largely of spermatogonia. Instead, a small but significant decrease was observed for some doses of GnRH: 10–11 to 10–8 M for sGnRH and 10–9 M cGnRH-II. Contributing factors were probably receptor desensitization, possible differential ligand selectivity, and variability, which become particularly important when differences are small. Our results are in agreement with previous studies showing that in vivo GnRH treatment stimulates spermatogonia cell proliferation in amphibians and mice (50, 51). The differential response to GnRH during gametogenesis cannot be attributed to the lack of GnRH expression because studies done in our laboratory suggest that sGnRH and cGnRH-II are expressed at both stages of gonadal maturation (data not shown). More likely reasons could be differential expression of GnRH-R subtypes or the coupling of the same GnRH-R to different intracellular messenger pathways during gametogenesis as suggested before for the goldfish pituitary and ovary (48, 49, 52). Another important contributing factor could be the interaction of local growth factors and steroid hormones that could modulate GnRH effects during gametogenesis because such interactions have been previously reported in mammalian gonads and the goldfish testes (53, 54, 55).
DNA fragmentation in response to GnRH treatment was further confirmed by performing in situ TUNEL staining. Results from these experiments show that both germ and somatic cells are affected by GnRH treatment. This is particularly relevant because the distribution pattern of GnRH-R in the teleost testis has not been investigated. The present results demonstrate DNA fragmentation in the germinal cells at the spermatocyte stage, suggesting that these cells are likely to be a target of GnRH-induced apoptosis in the goldfish testis. In support of this hypothesis, spermatocytes were shown to be susceptible to both spontaneous and induced apoptosis in mammals and cartilaginous fish (6, 54, 56, 57), and there is evidence for the expression of GnRH-Rs in the mammalian spermatocytes (54). In the present study, DNA fragmentation was also observed in interstitial cells, which could contain Leydig cells. However, no positive staining for DNA fragmentation was observed in the Sertoli-like cells. It should be noted that mammalian Leydig cells express GnRH-Rs (58) and undergo cell death in response to agents such as ethanol dimethanesulfonate and glucocorticoid treatment (11, 59). Furthermore, exposure to endocrine-disrupting compounds with suspected estrogen-like activity was shown to induce apoptosis in Leydig cells of Japanese Medaka (60). Moreover, previous studies demonstrated that mammalian Sertoli cells undergo apoptosis under certain experimental conditions, but there is no evidence for GnRH induction of apoptosis in this cell type (61, 62). Clearly, more studies would be necessary to investigate the effect of GnRH on Sertoli cell apoptosis in mammals and nonmammalian vertebrates.
To investigate the mechanism of GnRH-induced apoptosis, the activity of the effector procaspase-3 was measured because there is a possibility for caspase-independent cell death pathways (63). To determine the specificity of the assay, a caspase inhibitor was used that completely blocked the enzyme activity, indicating that the specificity of the goldfish caspase-3 active site is similar to that of the mammalian and zebrafish enzymes (8). We also used a GnRH antagonist to demonstrate the specificity of GnRH-mediated response. The sGnRH-induced caspase-3 activity was blocked in the presence of the GnRH antagonist, indicating that GnRH-mediated induction of apoptosis in the goldfish testis is specific. This is clear despite some variability in the control groups treated with GnRH antagonist alone. The results demonstrate a clear increase in the activity of caspase-3 after 24 h treatments with sGnRH and cGnRH-II, demonstrating the involvement of an executioner caspase pathway. The involvement of this pathway was further investigated by measuring fas and fasL protein levels in the GnRH-treated testes. The fas/fasL system has been previously shown to mediate apoptosis in normal and injured mammalian testes (15, 17).
Recently fas and fasL have been shown to participate in GnRH induction of apoptosis in ovarian and endometrial tumor cells as well as uterine leiomyomas (64). To date, however, no information is available on the regulation of fasL and/ or fas by GnRH in the male gonad. The present results provide evidence for the first time that fas and fasL-like proteins mediate GnRH induction of apoptosis in the goldfish testes. Up-regulation of fas has been shown to be a prerequisite for mammalian germ cell apoptosis (15). Furthermore, because Sertoli cells constitutively express fasL, apoptosis may still occur in the absence of fasL up-regulation, if there is an increase in target cell sensitivity due to increased fas expression (65). Induction of fas and fasL expression in the mammalian testis in association with apoptosis have been demonstrated in response to a variety of agents/factors including ionizing radiation, ischemia/perfusion damage, deprivation of serum and growth factors, heat treatment, and androgen withdrawal (65, 66, 67, 68). The evidence presented here suggests that GnRH induction of apoptosis may also involve increased activation of caspase-3 and DNA fragmentation by increasing activity of the fas/FasL system. However, the mechanism by which GnRH may induce an increase in fas and fasL proteins remains unknown. A similar trend for down-regulation of GnRH activity could be seen in the sGnRH- and cGnRH-II-induced fas and fasL production at the highest concentration of the peptides (10–7 M) tested. This is consistent with the response observed for the DNA fragmentation assay.
Procaspase-8 is recruited to fas after ligand interaction, inducing both caspase-3 cleavage and mitochondrial dysfunction (45). Thus, we investigated caspase-8 activation in response to GnRH. The results demonstrate GnRH mediated activation of caspase-8, indicating that the observed increase in fas and fasL levels in response to GnRH could lead to the recruitment of this caspase and the activation of the apoptotic cascade.
In summary, the present results demonstrate a stage-dependent regulation of apoptosis in the goldfish testis by native forms of GnRH peptides. Both sGnRH and cGnRH-II stimulate apoptosis via a death receptor-mediated pathway in the mature spermiating testis but not in the immature gonads. The findings provide a support for the hypothesis that native GnRH peptides play an autocrine/paracrine role in the regulation of testicular development in goldfish.
Acknowledgments
The authors thank Drs. M. Lohka and M. Cavey (Department of Biological Sciences, University of Calgary) for their invaluable advice in microscopy matters.
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Address all correspondence and requests for reprints to: Dr. H. R. Habibi, Department of Biological Sciences, University of Calgary, Biosciences 282, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4. E-mail: habibi@ucalgary.ca.
Abstract
Apoptosis, or programmed cell death, can occur via death receptor or mitochondrial pathways. Normal spermatogenesis in mammals involves apoptosis mediated, in part, by the death receptor fas and its ligand. The regulation of programmed cell death in the gonads has been shown to be dependent on a number of locally produced factors, including GnRH. Whereas the role of GnRH in the control of apoptosis and follicular atresia has been documented in the mammalian ovary, GnRH regulation of testicular apoptosis remains obscure. A previous study in our laboratory demonstrated the involvement of GnRH on the induction of DNA fragmentation in mature, perispawning testis. In this study, we tested the hypothesis that GnRH plays a differential regulatory role during male gamete maturation by studying the effect of GnRH on the induction of apoptosis during goldfish spermatogenesis. Treatment with GnRH resulted in DNA fragmentation only during late stages of spermatogenesis as assessed by oligonucleotide detection and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assays. The GnRH-induced apoptosis in the goldfish testis was found to be mediated by increased levels of fas and fas ligand-like proteins as well as elevated activity of caspase-3 (an executioner caspase) and -8 (a death receptor-activated caspase). The results suggest the involvement of the death receptor pathway in GnRH-induced apoptosis, providing support for the hypothesis that GnRH plays an important role in the control of spermatogenesis in the goldfish testis.
Introduction
APOPTOSIS IS A FORM of cell death characterized by morphological and biochemical changes including DNA fragmentation, formation of apoptotic bodies, and a minimal inflammatory response (1, 2). In most cases, the apoptotic process converges into the activation of caspases, a group of aspartyl-directed cysteine proteases that are present in the cell as proenzymes (3). In receptor-mediated apoptosis, the interaction of a death receptor such as fas with its ligand (fasL) induces the recruitment and activation of procaspase-8, which in turn, cleaves several substrates including procaspase-3. The resultant caspase-3 activates an endonuclease (caspase-activated deoxyribonuclease), which is partially responsible for the low-molecular-weight pattern of DNA fragmentation observed during apoptosis (4, 5).
Testicular apoptosis occurs during normal spermatogenesis in mammals and cartilaginous fish (6), and it is thought to be essential for the maintenance of the correct ratio between Sertoli cells and gametes (7). Caspases are well-conserved proteins (8, 9, 10), and their presence in teleosts has also been demonstrated, although their distribution in adult tissues remains unknown (8, 9). Several components of the apoptotic machinery have been shown to be present in the male gonads. For instance, the expression of caspases and the fas/fasL system has been demonstrated in the mammalian testes (11, 12, 13, 14, 15, 16, 17). Furthermore, the interaction between fas and fasL has been shown to regulate germ cell numbers during spermatogenesis (15, 16, 17). Caspases are well-conserved proteins (8, 9, 10), and their presence in teleosts has also been demonstrated, although their distribution in adult tissues remains unknown (8, 9, 14). Fas and fasL-like proteins have been shown to be expressed in birds, amphibians, and teleost fishes (18, 19, 20); however, little information is available about the potential role of these proteins in the regulation of fish gametogenesis. Moreover, despite our current knowledge about the occurrence of apoptosis in the fish ovary and testis (6, 21, 22, 23, 24), the molecular mechanisms that mediate programmed cell death in these tissues are still unknown.
In teleost fish, testes are bilateral elongated lobes located in the abdominal cavity, and spermatogenesis occurs in cysts within the seminiferous lobules (25). The majority of teleost fish, including goldfish, display a seasonal pattern of reproduction, and the testes undergo variations in size and histological appearance (25, 26). During spermatogenesis, all cells within a cyst develop synchronously (26). To spawn, teleosts rely heavily on environmental cues, such as temperature and light (26); lack of these cues will lead to gonadal regression, a process that may occur via the induction of apoptosis.
The regulation of testicular apoptosis, at least in mammals, appears to be mediated by both gonadotropins and locally produced factors such as gonadal steroids (27). GnRH and its receptors (GnRH-Rs) are among the hormone receptor systems that are locally expressed in the testes of mammals (28, 29, 30), amphibians (31), teleost fish (32), and lamprey (33). Although for years GnRH has been proposed to play a role in the regulation of the gonadal function, the exact mechanisms underlying its actions has remained elusive. For example, GnRH has been shown to exert both stimulatory and inhibitory effects on steroidogenesis in the testis of different species (34, 35). The observed variation in responses after GnRH treatment may be attributed to species differences, gonadal status, experimental approach, period of exposure to the hormone, and hormone concentration. However, the basis for these paradoxical effects of GnRH is poorly understood. We previously reported that GnRH induces apoptosis in the mature goldfish testis (36). The present study expands our earlier observations by investigating the role of GnRH in the regulation of testicular apoptosis at different stages of spermatogenesis and the mechanism of GnRH-induced apoptosis in the goldfish testis. The results demonstrate that, in the goldfish testis, GnRH-induced apoptosis occurs only during the late stage of spermatogenesis, mediated by increased levels of fas and fasL-like proteins as well as activation of caspases-3 (executioner caspase) and -8 (death receptor activated caspase). The results suggest the involvement of the death receptor pathway in the testicular response to GnRH, providing support for the hypothesis that GnRH plays an important role in the control of spermatogenesis in goldfish.
Materials and Methods
Animals
Male goldfish (Carassius auratus) of the comet variety (length 8–10 cm) were purchased from Aquatic Imports (Calgary, Alberta, Canada). Fish were maintained in a 1500-liter semirecirculating aquarium (60% replacement per day) at 18 C on a 16-h light, 8-h dark photoperiod and fed a commercial fish diet (Nutrafin floating pellets; Rolf C. Hagen Inc., Montreal, Québec, Canada).
Hormones and chemicals
sGnRH (salmon GnRH; pGlu, His, Trp, Ser, Tyr, Gly, Trp, Leu, Pro, Gly-NH2), and cGnRH-II (chicken GnRH; pGlu, His, Trp, Ser, His, Gly, Trp, Tyr, Pro, Gly-NH2) were purchased from American Peptide Co. (Sunnyvale, CA). GnRH antagonist (Ac-D-2-Nal-p-Chloro-D-Phe-?-(3-pyridyl)-D-Ala-Ser-Lys(nicotinyl)-D-Lys(nicotinyl)-Leu-Lys-(isopropyl)-Pro-D-Ala-NH2) was purchased from Peninsula Laboratories (San Carlos, CA). GnRH peptides (10 μg/20 μl) were solubilized in 0.1 M acetic acid. Hormones were stored at –20 C until use. Appropriate concentrations of the hormones were prepared by diluting the stock solution in sterile culture medium immediately before use. Caspase-3 colorimetric substrate (Ac-DEVD-pNA; pNA: p-nitroanilide), caspase-3/7 inhibitor (Ac-DEVD-CHO), and p-nitroanilide were purchased from Sigma (St. Louis, MO). Caspase-8 colorimetric substrate (substrate I, Ac-IETD-pNA, 368057) was purchased from EMD Biosciences, Inc. Calbiochem (San Diego, CA). The peptides and p-nitroanilide were solubilized in dimethyl sulfoxide (Sigma) to prepare a 20 mM, 200 μM, and stock solutions that were stored protected from light at –20 C until use.
Testis incubation
Animals were killed in accordance with the principles and guidelines of the Canadian Council of Animal Care. Mature testes [spermiating testis with gonadosomato index (GSI): 3.5–4.5%], immature testis (nonspermiating testis with GSI: 1.9–2.5%) were dissected out and incubated as previously described (36). GnRH was added immediately and the tissue slices were incubated for different times at 18 C. A time course for caspase-3 activity, which is upstream of DNA fragmentation, was performed with time points at 1, 2, 5, 8, and 24 h after GnRH treatment. No caspase-3 activity was observed at 1, 2, and 5 h after the treatment, and the increases at 8 h were not statistically significant. Therefore, further experiments were carried out at 24 h post GnRH treatment. In experiments carried out in the presence of GnRH antagonist, 1 h preincubation with the antagonist was performed, followed by coincubation with GnRH for a further 24 h. The experiments were carried out three to five times, and in each experiment, gonads from one fish were used for all treatments. At the end of the incubation period, testes were kept at –20 C for determination of DNA fragments or were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay, caspase activity, or Western blot analysis.
Detection of DNA fragments
DNA fragmentation in testicular tissues was assessed by using a cell death detection ELISA kit (Roche Diagnostics, Laval, Québec, Canada), according to the manufacturer’s instructions. This quantitative sandwich ELISA specifically measures the histone region (H1, H2A, H2B, H3, and H4) of mono- and oligonucleosomes, which are released during apoptosis. Photometric readings were obtained after 10 min of reaction in a double-wavelength plate reader at 405 nm (Molecular Devices, Menlo Park, CA). Apoptosis was measured in duplicate from each sample and expressed as the absorbance ratio of treated tissues vs. the absorbance from untreated control tissues, arbitrarily set as 1.0, as previously described (37). To correct for differences in tissue content per slice, protein quantification was carried out by the bicinchoninic acid assay (Pierce, Rockford, IL), using BSA (Roche Diagnostics) as standard and a U2000 spectrophotometer (Hitachi, Tokyo, Japan).
TUNEL assay and hematoxylin-eosin staining
After GnRH treatment, testis slices were fixed in 4% formalin-buffered solution (Sigma) for 18 h and embedded after standard histological methods. Five-micrometer sections were stained with hematoxylin-eosin (Gill-formulation, Fisher Scientific, Fair Lawn, NJ) and mounted in Permount (Fisher Scientific).
Tissue sections (5 μm) were also stained for nick end labeling by using a commercial assay kit (in situ cell death detection kit-AP, Intergen Co., Purchase, NY). Sections were incubated as previously described (21). Slides were counterstained with 4',6'-diamino-2-phenylindole (DAPI) for 10 min at room temperature, mounted in antifade mounting media (Aqua Poly/Mount, Polysciences Inc.) and examined under a fluorescence microscope (Leica DMR, Québec, Canada).
Caspase-3 and caspase-8 activity assays
After the incubation period, tissue samples were rinsed with PBS. Three 20-μm slices were pooled and homogenized through different gauze needles in 250 μl extraction/substrate caspase Buffer [50 mM Pipes-KOH (pH 7.4), 220 mM mannitol, 68 mM sucrose, 50 mM KCl, 5 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, 10 μg/ml proteinase inhibitor phenylmethylsulfonyl fluoride] for 30 min on ice. Samples were frozen at –80 C for 3–4 h after which they were thawed and refrozen overnight at –80 C. Samples were quick thawed, spun at 14,000 x g at 4 C for 15 min, and the supernatant assessed for caspase-3 activity. Total protein content was determined by a Bradford assay (Bio-Rad Laboratories, Hercules, CA), using BSA as the standard. Samples (20 μg total protein) were read in duplicate on 96-well-plates (Falcon). The concentration of caspase-3 or caspase-8 substrates was 2 mM in a final volume of 100 μl/well of extraction/substrate buffer. In some experiments, a caspase inhibitor was coincubated with the substrate at a final concentration of 200 μM. Samples were incubated at 37 C for 2 h and read immediately in a plate reader at 405 nm (Molecular Devices), using p-nitroanilide as standard. Results were expressed as caspase-3 activity (milligrams of product per milligram total protein per hour) on correction for protein content and incubation time. HeLa cells treated with 1 μM staurosporine and 1 mM ethanol-treated HepG2 cells were used as positive control for the induction of caspase-3 and -8 activities, respectively (38, 39).
Western blot analysis
Western blot analysis was performed on tissue lysates that were obtained by extraction with a buffer containing 1% (vol/vol) Triton X-100, 50 mM Tris-HCl (pH 8), 10 mM CaCl2, 200 mM NaCl, and 10 μg/ml phenylmethylsulfonyl fluoride, followed by Bradford assay, as previously described (40). Lysates were subjected to electrophoresis through a 15% sodium dodecyl sulfate-polyacrylamide gel at 100 V. Immunoblotting was performed using a 1:1000 dilution of antihuman fas (sc7886) or antihuman fasL (sc834) polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Anti-fasL antibody was blocked by incubating a 1:1000 dilution of anti-fasL antibody with 5 x fasL-blocking peptide for 2 h at room temperature. Blots were reprobed with a 1:7000 dilution of mouse anti-?-tubulin (Sigma) to verify equivalent loading of samples. Bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) and quantified by densitometry. After quantification, the concentration of each target protein was normalized vs. ?-tubulin and plotted as a ratio. For each gel, lysates from phorbol ester-treated HL-60 cells were used as positive controls (41).
Data analysis
Statistical significance was determined by one-way ANOVA and the test for multiple comparison analysis Newman-Keul’s (Student-Newman-Keuls’s); P values < 0.05 were considered to be significant.
Results
sGnRH and cGnRH-II induction of DNA fragmentation in mature testes is stage dependent
We previously demonstrated the induction of DNA fragmentation by GnRH in the mature goldfish testes. In this study, we expanded these results by investigating the possible role of GnRH during fish spermatogenesis. Mature (spermiating testis, GSI: 3.5–4.5–5%) or immature testicular fragments (nonspermiating testis, GSI: 1.9–2.5%) were incubated in presence of the two native goldfish GnRH forms (sGnRH or cGnRH-II). In mature testes, treatment with sGnRH and cGnRH-II for 8 and 24 h significantly increased DNA fragmentation, compared with the control. The GnRH-induced apoptosis expressed as DNA fragmentation became very clear after 24 h of treatment for both sGnRH and cGnRH-II (Fig. 1, A and B; P < 0.05). After 8 h, however, DNA fragmentation induced by sGnRH and cGnRH-II were significantly higher than that of control but not at all doses tested. The effect of both sGnRH and cGnRH-II on immature testis was clearly different. Treatments with GnRH resulted in either no significant effect or small decrease in apoptosis in immature testis (Fig. 1, C and D). In the case of sGnRH, a small but significant decrease in DNA fragmentation was observed in immature testis for all concentrations tested with the exception of the highest level (10–7 M) (Fig. 1C; P < 0.05). As for cGnRH-II, a small decrease in apoptotic ratio was significant only at 10–9 M, compared with the control (Fig. 2B; P < 0.05). These results indicate that GnRH induces DNA fragmentation in mature perispawning but not in immature (early reproductive season) testes. Therefore, only mature testes were used in the following experiments to further investigate the onset of apoptosis induced by GnRH.
FIG. 1. GnRH induction of DNA fragmentation goldfish testis. Testicular fragments were incubated for 8 or 24 h with sGnRH (A and C) or cGnRH (B and D), followed by analysis of DNA fragmentation. A and B, Mature testes (GSI 3.5–4.5%). C and D, Immature testes (GSI 1.9–2.5%). Values represent the mean ± SEM of results obtained from three to five different experiments, expressed as a ratio of apoptosis with respect to the control. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
FIG. 2. Hematoxylin-eosin and TUNEL stainings in the mature goldfish testis. A and B, Hematoxylin-eosin staining for mature goldfish testes (mature, GSI 3.5–5%, spermiating testis). Original magnification, x400. Arrowheads, Spermatogonials; arrows, Sertoli-like cells. sp, Spermatids; sc, spermatocytes. C–I, Testicular fragments were treated with 10–7 M sGnRH during 24 h, fixed in 4% formalin for 18 h and processed for TUNEL staining and DAPI nuclear counterstaining. Note that DNA fragments were detected in seminiferous tubules and interstitial tissue. Original magnification, x400 and x1000.
Testis morphology and in situ TUNEL analysis
Routine staining revealed the coexistence of more than one germ cell stage within a seminiferous tubule in mature goldfish testes (Fig. 2A). The characteristics of these cells are similar to those described for Characiformes (42). Large spermatogonias were observed in the interstitial connective tissue (Fig. 2B) (42). Stage I spermatocytes, with compacted chromatin masses in the nuclear periphery, and spermatids were present within the cysts (Fig. 2, A and B). In addition, Sertoli-like cells were observed in the lining of the cysts (Fig. 2B). Spermatozoa were also present at this stage of gonadal maturation because they were released when testicular tissues were washed during the tissue culture procedures.
To investigate whether somatic or germinal cells underwent apoptosis in response to GnRH, DNA fragmentation was evaluated using the TUNEL assay. Saline-treated testis slices (control) were shown to be negative for TUNEL staining, whereas DAPI nuclear staining was positive (Fig. 2, C–I). Increased apoptosis was found in the GnRH-treated testis, compared with the control (Fig. 2, D and F). In the sGnRH treated testis, both germ cells and interstitial cells stained positive for DNA fragments (Fig. 2, F and H). Whereas complete identification of the stage of spermatogenesis affected by GnRH was not possible, the results suggest that spermatocytes may be among the cells sensitive to GnRH-induced apoptosis (Fig. 2H). Negative controls (no terminal dideoxy transferase enzyme) displayed no staining for DNA fragments. Similar results were obtained after treatment with cGnRH-II (data not shown).
sGnRH and cGnRH-II-induction of caspase-3 activity in mature testis
Caspase-3 has been shown to be present and activated in mammalian testes in response to several proapoptotic stimuli (11, 13). In addition, there is evidence that caspase-3 is conserved in fish (8) and involved in the induction of apoptosis through generation of ceramide. To further confirm the GnRH induction of apoptosis in mature goldfish testes, we investigated the activation of procaspase-3 in response to sGnRH or cGnRH-II (10–9 to 10–7 M). In these experiments, staurosporine-treated cells were used as positive control (control: 0.35 ± 0.038; staurosporine: 1.3 ± 0.10 mg product per milligram protein per hour).
Significant increases in caspase-3 activity were observed after treatment with either sGnRH or cGnRH-II at all concentrations tested (Fig. 3, A and B; P < 0.05). Samples were coincubated with the substrate and the caspase-3/7 inhibitor, Ac-DEVD-CHO, to test the specificity of the caspase assay. The presence of the inhibitor significantly decreased caspase-3 activity induced by both sGnRH and cGnRH-II at all the concentrations tested (Fig. 4, A and B; P < 0.05). Testis samples were also preincubated for 1 h with 10–6 M antide, a GnRH antagonist known to block GnRH activity in the goldfish ovarian follicles (43), to test the specificity of the GnRH response. The GnRH antagonist significantly inhibited sGnRH-induced caspase-3 activity (Fig. 4C; P < 0.05), without affecting basal level.
FIG. 3. sGnRH (A) and cGnRH-II (B) activation of caspase-3 in the mature goldfish testis. Fragments from mature testes were incubated for 24 h in the presence of sGnRH or cGnRH-II and analyzed for caspase activity. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
FIG. 4. Effect of the inhibitor Ac-DEVD-CHO on sGnRH- (A) and cGnRH-II (B)-induced caspase-3 activity in a cell-free system. A and B, Caspase-3 activity in cell lysates from mature testis, treated 24 h with sGnRH or cGnRH-II in the presence or absence of the caspase-3 inhibitor Ac-DEVD-CHO. C, Caspase-3 activity in mature testis fragments preincubated for 3 h with a GnRH antagonist and cultured for 24 h with or without sGnRH. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with corresponding controls (P < 0.05).
Effect of GnRH on fas and fasL-like proteins in the mature testes
The fas/fasL system has been shown to be involved in the induction of apoptosis in the several mammalian tissues, including testes (15, 16, 17). Experiments were carried out to investigate the effects of sGnRH and cGnRH-II on fas and fasL protein levels in the goldfish testes by Western blot analysis. HL-60 promyelocytic cell line extract was used as positive control, and tubulin level was used to correct for loading errors. Figure 5, A–C, shows representative immunoblots for fasL and quantitative results from three separate experiments. Treatment for 24 h with either sGnRH or cGnRH-II significantly increased fasL-like protein level, compared with the control (Fig. 5, A and C; P < 0.05). To test the specificity of binding, the anti-fasL antibody was preincubated overnight with its commercially available blocking peptide. As shown in Fig. 5B, the blocking peptide completely abrogated the anti-fasL antibody binding in the gonadal tissues, and partially blocked the antibody binding to the positive control (HL-60 cell lysate). Two bands corresponding to fas were detected in both fish and human extracts. The two immunoreactive bands may represent alternative splicing variants of fas, which have been previously reported to exist in humans (44). Both positive bands were shown to increase in response to GnRH treatment in the testes samples (Fig. 6A). Treatment with both sGnRH and cGnRH-II significantly increased fas expression in the goldfish testis, although the cGnRH-II-induced increase in fas levels at 10–9 M was not statistically significant (Fig. 6B; P < 0.05).
FIG. 5. Effect of GnRH on FasL protein levels in the mature goldfish testis. Western blot analysis of total lysates from mature testes treated with sGnRH or GnRH-II for 24 h. Protein extracts from HL-60 cells were used as positive controls. A, FasL. B, The specificity of the FasL antibody was tested by preincubating the antibody overnight at 4 C with 5x blocking peptide. Membranes were reblotted with an anti-?-tubulin antibody, which was used as an internal standard. C, Densitometry analysis for FasL, normalized vs. ?-tubulin. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
FIG. 6. Effect of GnRH on Fas (receptor) protein levels in the mature goldfish testis. Western blot analysis of total lysates from mature testes treated with sGnRH or GnRH-II for 24 h. Protein extracts from HL-60 cells were used as positive controls. A, Fas protein levels. Membranes were reblotted with an antitubulin antibody, which was used as an internal standard. B, Densitometry analysis for Fas, normalized. C, Densitometry analysis for Fas, normalized vs. ?-tubulin. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
sGnRH and cGnRH-II-induced caspase-8 activity in mature goldfish testis
We also investigated the activity of caspase-8 in response to GnRH in the goldfish testes because increased levels of fas and fasL may lead to the recruitment of procaspase-8 to the death receptor complex (45). In these experiments, ethanol-treated HepG2 cells were used as positive controls (control, 0.71 ± 0.05; ethanol, 1.76 ± 0.17 mg product per milligram protein per hour). A significant increase in caspase-8 activity was observed after 24 h of treatment with sGnRH and cGnRH-II for all concentrations tested (Fig. 7; P < 0.05).
FIG. 7. GnRH-induced activation of caspase-8 in the mature goldfish testis. Fragments from mature testis were incubated for 24 h with sGnRH or cGnRH-II and analyzed for caspase activity. Values represent the mean ± SEM of results obtained from three different experiments. Results were analyzed by ANOVA and Student-Newman-Keuls’s test; asterisks indicate significant difference compared with control (P < 0.05).
Discussion
This study investigated the role of GnRH in control of apoptosis during spermatogenesis in goldfish. The results clearly demonstrate a stage-dependent variation in sGnRH- and cGnRH-II-induced apoptosis in the goldfish testes as well as providing information on the mechanism of GnRH-induced testicular apoptosis. Both sGnRH and cGnRH-II, which are expressed in the goldfish testis (32), significantly increased apoptosis in the perispawning goldfish testis containing germ cells at different stages of differentiation. The same GnRH peptides either have no effect or exert small protective action in immature nonspermiating testis. The apoptotic effect of GnRH can be seen more clearly after 24 h of treatment than 8 h, particularly for cGnRH-II. After 8 h of treatment and at lower concentrations, both sGnRH and cGnRH-II peptides displayed comparable activity. However, cGnRH-II was without a significant effect at 10–7 M, the highest concentration tested. A contributing factor could be down-regulation of GnRH-R at higher concentrations of cGnRH-II. Similar patterns of concentration-dependent desensitization have been previously observed for sGnRH and cGnRH-II in the goldfish pituitary (46, 47). The fact that only cGnRH-II seems to cause down-regulation may indicate that more than one receptor is involved in the response to sGnRH and cGnRH-II or that testicular receptors display different sensitivity to the two molecular forms of GnRH. In this regard, the presence of GnRH receptor subtypes with different ligand selectivity has been suggested in the goldfish pituitary (48) and ovary (49).
After 24 h of treatment, DNA fragmentation was significantly increased for all doses of sGnRH and cGnRH-II tested. In contrast, GnRH peptides did not induce DNA fragmentation in immature testes, composed largely of spermatogonia. Instead, a small but significant decrease was observed for some doses of GnRH: 10–11 to 10–8 M for sGnRH and 10–9 M cGnRH-II. Contributing factors were probably receptor desensitization, possible differential ligand selectivity, and variability, which become particularly important when differences are small. Our results are in agreement with previous studies showing that in vivo GnRH treatment stimulates spermatogonia cell proliferation in amphibians and mice (50, 51). The differential response to GnRH during gametogenesis cannot be attributed to the lack of GnRH expression because studies done in our laboratory suggest that sGnRH and cGnRH-II are expressed at both stages of gonadal maturation (data not shown). More likely reasons could be differential expression of GnRH-R subtypes or the coupling of the same GnRH-R to different intracellular messenger pathways during gametogenesis as suggested before for the goldfish pituitary and ovary (48, 49, 52). Another important contributing factor could be the interaction of local growth factors and steroid hormones that could modulate GnRH effects during gametogenesis because such interactions have been previously reported in mammalian gonads and the goldfish testes (53, 54, 55).
DNA fragmentation in response to GnRH treatment was further confirmed by performing in situ TUNEL staining. Results from these experiments show that both germ and somatic cells are affected by GnRH treatment. This is particularly relevant because the distribution pattern of GnRH-R in the teleost testis has not been investigated. The present results demonstrate DNA fragmentation in the germinal cells at the spermatocyte stage, suggesting that these cells are likely to be a target of GnRH-induced apoptosis in the goldfish testis. In support of this hypothesis, spermatocytes were shown to be susceptible to both spontaneous and induced apoptosis in mammals and cartilaginous fish (6, 54, 56, 57), and there is evidence for the expression of GnRH-Rs in the mammalian spermatocytes (54). In the present study, DNA fragmentation was also observed in interstitial cells, which could contain Leydig cells. However, no positive staining for DNA fragmentation was observed in the Sertoli-like cells. It should be noted that mammalian Leydig cells express GnRH-Rs (58) and undergo cell death in response to agents such as ethanol dimethanesulfonate and glucocorticoid treatment (11, 59). Furthermore, exposure to endocrine-disrupting compounds with suspected estrogen-like activity was shown to induce apoptosis in Leydig cells of Japanese Medaka (60). Moreover, previous studies demonstrated that mammalian Sertoli cells undergo apoptosis under certain experimental conditions, but there is no evidence for GnRH induction of apoptosis in this cell type (61, 62). Clearly, more studies would be necessary to investigate the effect of GnRH on Sertoli cell apoptosis in mammals and nonmammalian vertebrates.
To investigate the mechanism of GnRH-induced apoptosis, the activity of the effector procaspase-3 was measured because there is a possibility for caspase-independent cell death pathways (63). To determine the specificity of the assay, a caspase inhibitor was used that completely blocked the enzyme activity, indicating that the specificity of the goldfish caspase-3 active site is similar to that of the mammalian and zebrafish enzymes (8). We also used a GnRH antagonist to demonstrate the specificity of GnRH-mediated response. The sGnRH-induced caspase-3 activity was blocked in the presence of the GnRH antagonist, indicating that GnRH-mediated induction of apoptosis in the goldfish testis is specific. This is clear despite some variability in the control groups treated with GnRH antagonist alone. The results demonstrate a clear increase in the activity of caspase-3 after 24 h treatments with sGnRH and cGnRH-II, demonstrating the involvement of an executioner caspase pathway. The involvement of this pathway was further investigated by measuring fas and fasL protein levels in the GnRH-treated testes. The fas/fasL system has been previously shown to mediate apoptosis in normal and injured mammalian testes (15, 17).
Recently fas and fasL have been shown to participate in GnRH induction of apoptosis in ovarian and endometrial tumor cells as well as uterine leiomyomas (64). To date, however, no information is available on the regulation of fasL and/ or fas by GnRH in the male gonad. The present results provide evidence for the first time that fas and fasL-like proteins mediate GnRH induction of apoptosis in the goldfish testes. Up-regulation of fas has been shown to be a prerequisite for mammalian germ cell apoptosis (15). Furthermore, because Sertoli cells constitutively express fasL, apoptosis may still occur in the absence of fasL up-regulation, if there is an increase in target cell sensitivity due to increased fas expression (65). Induction of fas and fasL expression in the mammalian testis in association with apoptosis have been demonstrated in response to a variety of agents/factors including ionizing radiation, ischemia/perfusion damage, deprivation of serum and growth factors, heat treatment, and androgen withdrawal (65, 66, 67, 68). The evidence presented here suggests that GnRH induction of apoptosis may also involve increased activation of caspase-3 and DNA fragmentation by increasing activity of the fas/FasL system. However, the mechanism by which GnRH may induce an increase in fas and fasL proteins remains unknown. A similar trend for down-regulation of GnRH activity could be seen in the sGnRH- and cGnRH-II-induced fas and fasL production at the highest concentration of the peptides (10–7 M) tested. This is consistent with the response observed for the DNA fragmentation assay.
Procaspase-8 is recruited to fas after ligand interaction, inducing both caspase-3 cleavage and mitochondrial dysfunction (45). Thus, we investigated caspase-8 activation in response to GnRH. The results demonstrate GnRH mediated activation of caspase-8, indicating that the observed increase in fas and fasL levels in response to GnRH could lead to the recruitment of this caspase and the activation of the apoptotic cascade.
In summary, the present results demonstrate a stage-dependent regulation of apoptosis in the goldfish testis by native forms of GnRH peptides. Both sGnRH and cGnRH-II stimulate apoptosis via a death receptor-mediated pathway in the mature spermiating testis but not in the immature gonads. The findings provide a support for the hypothesis that native GnRH peptides play an autocrine/paracrine role in the regulation of testicular development in goldfish.
Acknowledgments
The authors thank Drs. M. Lohka and M. Cavey (Department of Biological Sciences, University of Calgary) for their invaluable advice in microscopy matters.
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