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Division of Endocrinology, Department of Medicine, Harbor-University of California-Los Angeles Medical Center and Research and Education Institute, Torrance, California 90509
Address all correspondence and requests for reprints to: Ronald S. Swerdloff, M.D., Division of Endocrinology and Metabolism, Harbor-University of California-Los Angeles Medical Center, Box 446, 1000 West Carson Street, Torrance, California 90509. E-mail: swerdloff{at}gcrc.rei.edu.
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Introduction |
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Inducible nitric oxide synthase (iNOS) is one of the three known mammalian nitric oxide synthase (NOS) isoforms responsible for nitric oxide (NO) production (5). The principal form of iNOS regulation appears to be at the level of genetic induction, hence its common appellation as an inducible enzyme. However, iNOS may also be constitutively expressed under physiological conditions in some tissues, such as pulmonary and bladder epithelia (6, 7). Unlike endothelial and neuronal isozymes, iNOS activity in the cell is largely independent of changes in intracellular Ca2+ concentration (8). NO, the end product of the enzyme NOS, is a potent biological mediator that functions at low concentrations as a signal in many diverse physiological processes but at high concentrations may cause DNA damage and cell death (9, 10). The characterization of mice with targeted disruption of iNOS genes has provided important insights into the physiological and pathophysiological roles of the iNOS in various tissues and organs (11, 12). Mice with a null mutation of the iNOS gene have been developed through homologous recombination. After homologous recombination, the calmodulin-binding domain of iNOS is replaced by the neomycin resistance gene, and therefore the reading frame of the modified gene is disrupted. Removing the calmodulin-binding domain resulted in an inactive iNOS (13). Mice with a disruption of the iNOS gene are grossly normal (14, 15).
We have previously demonstrated that in the hypothalamus of aging Brown Norway (BN) rats, NOS activity is increased due to increased iNOS but not neuronal NOS. The increased iNOS protein in the hypothalamus is associated with increased peroxynitrite formation and neuronal cell apoptosis (16). We have further shown that iNOS activation may be responsible for aging-related germ cell loss by apoptosis in BN rats (16, 17). We have observed by immunohistochemistry that iNOS protein appeared more abundantly in heat-induced apoptotic germ cells in Sprague Dawley rats. Studies also showed that iNOS protein was not only present in the rat testis but was also responsible for the seminiferous epithelium damage caused by lipopolysaccharide-induced inflammation or impairment of spermatogenesis induced by testicular torsion (18, 19). On the basis of the putative role of a high concentration of iNOS in NO-mediated toxicity in various tissues, we hypothesized that iNOS through its product, NO, may contribute to the induction of germ cell apoptosis. To verify the physiological role of iNOS in spermatogenesis, we studied adult iNOS-mutant mice. We found that iNOS-mutant mice had larger testes with increased sperm output and decreased germ cell apoptosis. To further substantiate the role of iNOS in the induction of germ cell apoptosis, we examined the involvement of iNOS in heat-induced germ cell apoptosis and found that inactivation of iNOS gene confers partial resistance to heat-induced testicular germ cell apoptosis.
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Materials and Methods |
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Study protocols
Experiment 1. Characterization of testicular phenotype of iNOS-mutant mice.
To examine the physiological role of iNOS in spermatogenesis, groups of 18 iNOS-mutant mice and their wild-type controls were studied. The reproductive endocrine function was evaluated by measuring plasma LH, FSH, and testosterone levels. Germ cell homeostatic balance was determined by quantitative assessment of the incidence of germ cell apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and the rate of germ cell proliferation by in vivo detection of bromodeoxyuridine (BrdU)-labeled preleptotene (PL) spermatocytes. The efficiency of spermatogenesis was characterized by testicular sperm count and histomorphometric analysis of the testes.
Experiment 2. Role of iNOS in heat-induced germ cell apoptosis.
To further examine the role of iNOS in induction of germ cell apoptosis, we examined the involvement of iNOS in heat-induced germ cell apoptosis. Groups of 12 iNOS-mutant and wild-type mice were studied. Scrota of both mutant and wild-type mice were exposed to a temperature of 22 C (control) or 43 C for 15 min and then killed 2 and 6 h after heat treatment. The quantitative assessment of the incidence of apoptosis at various stages of the spermatogenic epithelium cycles was performed by counting apoptotic germ cells per 100 Sertoli cells.
Blood collection and tissue preparation
Both wild-type and iNOS-mutant mice were injected with heparin (130 IU/100 g BW, ip) 15 min before being killed by a lethal injection of sodium pentobarbital (100 mg/kg BW ip) to facilitate testicular perfusion using a whole body perfusion technique (20). BW was recorded at autopsy. Blood samples were collected from the right ventricle of each animal immediately after death, and plasma was separated and stored at -20 C for subsequent hormone assays. Before perfusion, one testis from each mouse was removed, weighed, and after decapsulation the testicular parenchyma was used for determining the number of advanced (steps 13–16) spermatids by the homogenization technique (21). The contralateral testes were then fixed by vascular perfusion with either 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) or Bouin’s solution for 30 min, preceded by a brief saline wash. The seminal vesicles were carefully dissected out and weighed. The testes were removed, cut into small transverse slices, and placed in the same fixative overnight. One slice from the middle region of Bouin’s fixed testis was processed for routine paraffin embedding for detection of BrdU with immunostaining. One slice from the middle region of the testis fixed by glutaraldehyde was embedded in paraffin for in situ detection of apoptosis. The adjacent testicular slice from each mouse was further diced into small pieces (1 x 2 x 2 mm), postfixed in 1% osmium tetroxide/1.25% potassium ferrocyanide mixture, dehydrated in a graded series of ethanols, and embedded in Araldite (EM Media, Warrington, PA). Embedded testicular specimens were sectioned with an LKB (Rockville, MD) ultramicrotome at 2.05 µm and stained with 1% toluidine blue for light microscopic examination and for morphometric studies (22).
Hormone assays
Testosterone concentrations in plasma were measured by RIA, as reported previously (23). The minimal detection limit in the assay was 0.25 ng/ml. The intra- and interassay coefficients of variation were 8% and 11%, respectively. Plasma LH levels were measured by modified immunofluorometric assay as described previously (24, 25). Briefly, 96-well plates were coated with anti-h-LH antibody (TMA no. 5305, Medix Biomedica, Kauniainen, Finland) at 0.5 µg/well. Then, 20 µl of standard (0.02–25 ng/ml) or sample was added to each well, followed by the addition of 200 µl (66 µg/liter) biotinylated antibovine-LH antibody (MAB, no. 518B7, obtained from Dr. J. F. Roser, University of California, Davis, CA). The plates were sealed and shaken overnight at room temperature. After washing the plates with buffer [50 mM Tris-HCl, 0.15 M NaCl, 1 g/liter BSA, 0.5 g/liter sodium azide, and 0.2 ml/liter Tween 20 (pH 7.8)], Delfia streptavidin-Europium (Perkin-Elmer, Boston, MA) was added, and plates were incubated for 1 h. The plate was washed, and fluorescence was measured with a time-resolved fluorometer (model Victor 1420; Wallac, Inc., Turku, Finland). The minimal detection limit of the assay was 0.02 ng/ml. The intra- and interassay coefficients of variation were 6.9% and 12.3%, respectively. Plasma FSH levels were measured by immunofluorometric assay (25) by using reagents supplied by Organon (Oss, The Netherlands). The minimal detection limit in the assay was 0.04 ng/ml. The intra- and interassay coefficients of variation were 4.7% and 6.0%, respectively.
Assessment of apoptosis
In situ detection of cells with DNA strand breaks was performed in glutaraldehyde-fixed, paraffin-embedded testicular sections by the TUNEL technique using an Apop Tag-peroxidase kit (Intergen Co., Purchase, NY) as described earlier. Negative and positive controls were performed in every assay. As negative controls, tissue sections were processed in an identical manner, except that the terminal deoxynucleotidyl transferase enzyme was substituted with the same volume of PBS. Testicular sections from rats treated with 3-cm testosterone implant for 14 d were used as positive controls (22). Enumeration of the viable Sertoli nuclei with distinct nucleoli and apoptotic germ cell population was performed at stages I–IV, V–VI, VII–VIII, IX–X, and XI–XII using an American Optical microscope (Buffalo, NY) with a x100 oil immersion objective. For each testis, at least 10 tubules per stage group were used. These stages were identified according to the criteria proposed by Russell et al. (2) for paraffin sections. The rate of germ cell apoptosis [apoptotic index (AI)] was expressed as the number of apoptotic germ cells per 100 Sertoli cells (26).
BrdU labeling
To determine whether the increased testis weight in iNOS-mutant mice resulted from the increased rate of spermatogonial proliferation, groups of six iNOS-mutant and wild-type mice were given an ip injection of BrdU (50 mg/kg BW) 2 h before death (27). The testes were fixed by perfusion with Bouin’s fluid and processed for routine paraffin embedding. BrdU detection was performed using a commercial BrdU immunohistochemistry kit (Oncogene Research Products, Cambridge, MA). The germ cell proliferation was evaluated by a ratio of BrdU-labeled PL spermatocytes per 100 Sertoli cells at stages VII–VIII of spermatogenic epithelium cycle.
Immunohistochemical analysis
Bouin-fixed, paraffin-embedded testicular sections were deparaffinized, hydrated by successive series of ethanol, rinsed in distilled water, and then incubated in 2% H2O2 to quench endogenous peroxidases. Sections were blocked with 5% normal goat serum for 20 min to suppress nonspecific binding of IgG and subsequently incubated with a 1:100 dilution of affinity-purified rabbit polyclonal iNOS antibody (Refs. 28 and 29 ; BD Transduction Laboratories, San Diego, CA). Immunoreactivity was detected using biotinylated goat antirabbit IgG secondary antibody followed by avidin-biotinylated horseradish peroxidase complex visualized with diaminobenzidine tetrahydrochloride according to the manufacturer’s instructions (rabbit United Immunohistochemistry Detection System; Oncogene, Boston, MA). Slides were counterstained with hematoxylin and reviewed with the Olympus BH-2 light microscope.
Morphometric procedures
The volume densities (Vv) of seminiferous tubules, tubular lumens, interstitium, and Leydig cells were determined by point-counting method (20, 22). Five randomly selected sections per animal in each group were examined by an American Optical Microscope with a x40 objective and a x10 eyepiece fitted with a square lattice containing 121 intersections. The results were expressed as a percentage of the testis volume. The absolute volume of each of the testis components was then obtained by multiplying its Vv by fresh testis volume (Vv%). The diameters of 20 randomly selected transverse sections of seminiferous tubules were measured across the minor axis of their profiles with an ocular micrometer calibrated by means of a stage micrometer.
Numerical densities (Nv) of Sertoli and germ cells (number per unit volume of the seminiferous tubule) at stage VII-VIII of the cycle were determined by accepted stereological techniques as described previously (20, 22). For each testis sample, 10 round cross-sections of seminiferous tubules were used. The Floderus equation Nv = NA/(T + D - 2 h) was used to calculate the Nv of germ cell nuclei and Sertoli cell nucleoli, where NA is the number of nuclei or nucleoli counted per unit area of the seminiferous tubule profile, T is the section thickness, D is the average diameter of a given germ cell nucleus or the Sertoli cell nucleolus, and h is the height of the smallest recognizable nuclear or nucleolar profile in the section. The nuclear profiles of each of the germ cells [A1 spermatogonia, PL and pachytene (P) spermatocytes, and step 7 and 8 spermatids] and the number of Sertoli cell nucleoli (thereby cells, because only one typical nucleolus is present per nucleus or per cells) in the seminiferous tubules were counted under a x1000 magnification using an oil-immersion objective. The seminiferous tubule profile area (a) was determined by point counting using the equation a = p x u2, where p is number of points per tubular profile and u is the distance between two neighboring points in terms of the magnification used to measure the area. The mean diameters of Sertoli cell nucleoli and germ cell nuclei were obtained by direct measurements of their largest cross-sectioned profiles in serial sections. The height of the smallest recognizable nuclear or nucleolar profile was assumed to be one tenth of the diameter of the structure. The absolute number of these cells was then determined by multiplying their Nv by the fresh volume of the testis. Cell counts were finally expressed as the number of germ cells per Sertoli cell (germ cell/Sertoli cell ratios).
Statistical analyses
Statistical analyses were performed using the SigmaStat 2.0 Program (Jandel Corp., San Rafael, CA). Results were tested for statistical significance using t test or Student-Newman-Keuls method test after one-way repeated measures ANOVA. Differences were considered significant if P value was less than 0.05.
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Results |
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. Testes weights and testicular sperm content are shown in Fig. 1. No significant differences in the mean BW and seminal vesicle weight were observed between wild-type and iNOS-deficient mice. No significant changes were apparent in plasma LH, FSH, and testosterone levels between these animals. In contrast, 1) a significant (P < 0.05) increase in testis weight by 30.77% was noted in iNOS-mutant mice compared with wild-type animals, and 2) mean testicular sperm content (determined by counting the number of homogenization-resistant advanced steps 13–16 spermatids) was also significantly increased by 65.51% (16.67 ± 2.09 x 106/testis) over the values measured in the wild-type animals (5.57 ± 0.25 x 106/testis; Fig. 1).
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. iNOS-mutant mice had a significant increase in seminiferous tubular diameter and the volume of the seminiferous tubules compared with those in the wild-type animals. No apparent differences in volume of the interstitium and total Leydig cells were observed between wild-type and iNOS-mutant mice.
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). No significant changes in the number of spermatogonia and PL spermatocytes were apparent between wild-type and mutant animals. There was, however, a significant increase in the number of P spermatocytes and round spermatids in iNOS-mutant mice compared with wild-type animals (Table 3). Notably, the number of P spermatocytes and round spermatids in iNOS-mutant mice was increased by 39.63% and 33.79% when compared with those in wild-type animals, respectively.
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). There was no significant change in the rate of germ cell proliferation [proliferation index (PI) expressed as number of BrdU-labeled PL spermatocytes per Sertoli cell] in wild-type (PI = 4.60 ± 0.19) compared with iNOS-deficient mice (PI = 5.48 ± 0.43).
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shows the representative examples of location and frequency of TUNEL-positive apoptotic germ cells at stages XII of the seminiferous tubule in mutant and wild-type mice. Variable incidence of germ cell apoptosis (AI; expressed as numbers of apoptotic germ cells per 100 Sertoli cells) at various stages are summarized in Fig. 4. The incidence of germ cell apoptosis was detected at all stages, with a low rate at stages V–VI, VII–VII, and XI–X (AI = 1.17–4.68) and a higher level at stages I–IV and XI–XII (AI = 24.61–52.27) in wild-type mice. In contrast, the incidence of germ cell apoptosis was significantly decreased at stages I–IV and XI–XII (AI = 6.17–18.52) but not at stages V–VI, VII–VII, and XI–X (AI = 0.58–3.17) in iNOS-mutant mice. The spontaneous germ cell apoptosis was observed in spermatogonia, spermatocytes, and round spermatids in both wild-type and mutant mice. iNOS deficiency induced decreased apoptosis of P spermatocytes, early round spermatids at stages I–IV and diplotene, and dividing spermatocytes at stages XI–XII.
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). Importantly, 2 h after heat treatment, the rate of apoptosis in iNOS-mutant mice was lower to 58.4% at stages I–IV and 52.42% at stages XI–XII when compared with values measured in the wild-type animals (Fig. 5A). Similarly, 6 h after heat exposure, the rate of apoptosis in the mutant mice was decreased by 66.18% at stages I–IV and 43.08% at stages XI–XII when compared with wild-type levels (Fig. 5B). The most susceptible cells to heat-induced apoptosis are P spermatocytes and early round spermatids at stages I–IV and diplotene, and dividing spermatocytes at stages XI–XII in both wild-type and iNOS knockout mice. By immunohistochemistry, we found iNOS was localized in P spermatocytes, Sertoli cells, and prominently in Leydig cells in untreated wild-type mice (Fig. 6A). Although no apparent change in iNOS expression was noted in the nonsusceptible stages, iNOS immunostaining was markedly increased in Leydig cells and apoptotic germ cells (Fig. 6B).
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Discussion |
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We found in this study that iNOS-mutant mice had larger testes than wild-type males. The larger testes in iNOS-mutant mice were associated with increased sperm output, demonstrating that iNOS may play a physiological role in regulation of sperm production. Adams et al. (32) demonstrated that testosterone secretion was decreased in male rats by the NO donor isosorbide dinitrate, which is known to release exogenous NO in vivo, and the NOS substrate L-arginine methyl ester, which acts as a NOS substrate for endogenous NO production. However, we were not able to find any significant differences in plasma LH, FSH, and testosterone levels between wild-type and mutant mice, suggesting that increased sperm production perhaps might not be due to overstimulation of spermatogenesis by either intratesticular testosterone or FSH. This led us to suspect that iNOS may have an intrinsic effect on spermatogenesis by acting on suppression of apoptosis. Our results clearly demonstrated that there were no significant changes in germ cell proliferation based on the rate of BrdU labeling in PL spermatocytes. This was substantiated by our viable germ cell counting data in which the number of spermatogonia and PL spermatocytes showed no significant difference between iNOS-mutant and wild-type mice. In contrast, the incidence of apoptosis occurring mainly in P, diplotene, and dividing spermatocytes was significantly decreased at early and later stages. The decreased rate of spermatocytes apoptosis may be responsible for the increased number of P spermatocytes and round spermatids in iNOS-mutant mice. Seminiferous tubules consist of Sertoli cells and different classes of germ cells, and the diameter of seminiferous tubule depends on the number and the volume of Sertoli cells, germ cells, and amount of fluid in the lumen (2). In this study, we found no significant difference in the number of Sertoli cells, and lumen volume of seminiferous tubules (Table 2
) between wild-type and iNOS knockout mice indicated that increased diameter of seminiferous tubules in the iNOS knockout mice was due to an increased number of germ cells and the Sertoli cell volume. It may be relevant in this connection to note that seasonal transition from active to regressed states of spermatogenesis was accompanied by a marked reduction in the tubule diameter (61%) and the number of germ cells, with no change in the number of Sertoli cells (33). The Sertoli cells were markedly reduced in size that was significantly and positively correlated with the testicular weight, tubular diameter, and germ cell numbers (34).
The molecular mechanisms of the decreased spermatocytes apoptosis induced by iNOS inactivation are unclear. We have demonstrated that p53 deficiency in mice suppresses spermatocytes apoptosis and stimulates spermatogonial proliferation in vivo (35). Studies in p53 knockout mice have demonstrated that p53 mediates germ cell quality control in spermatogenesis (36). Studies in hepatocytes have shown that endogenous NO induces DNA damage and results in p53 accumulation (37). This evidence allows us to speculate that iNOS inactivation may down-regulate p53 expression, and p53 further decreases Bax, a proapoptotic protein, expression (38) resulting in decreased apoptosis of spermatocytes during meiosis. There have been a number of reports of knockout of apoptosis-regulating elements in the testis. Knockouts of proapoptotic Bax (39), antiapoptotic Bcl-w (40, 41), and meiosis-specific chaperone HSP70–2 (42) in mice are all present as infertile males with increased germ cell apoptosis. iNOS deficiency decreases germ cell apoptosis without affecting proliferation, indicating the essential role of apoptosis in determining the sperm output during spermatogenesis. We concluded from this study that iNOS indeed plays an essential role in eliminating some spermatocytes by apoptosis to maintain homeostasis of the germ cells.
We have previously demonstrated that mild testicular hyperthermia induces stage-specific and germ cell-specific apoptosis of germ cells in rat (43) and mouse (44). In agreement with our results on murine models of testicular hyperthermia (45, 46), we recently demonstrated that heat induces transient and reversible damage to the seminiferous epithelium through increased germ cell apoptosis in monkeys (47). Similar to rats, the data from this study demonstrated that a single exposure of mouse testes to heat (43 C for 15 min), as early as 2 and 6 h after treatment, induced increased germ cell apoptosis predominantly at early (I–IV) and late (XI–XII) stages in which P, diplotene, and dividing spermatocytes and early round spermatids were most susceptible to heat. The precise mechanisms by which germ cells die in response to heat stress are not fully understood (48). We have provided evidence showing that redistribution of proapoptotic Bax protein is the early step in an apoptotic pathway leading to germ cell death induced by mild testicular hyperthermia in rats (49). We have also observed that iNOS, an enzyme that produces toxic level of NO, is overexpressed in the heat-induced apoptotic germ cells in wild-type mice. In this study, using iNOS gene mutation mice, we were able to examine the causality of iNOS in heat-induced germ cell apoptosis and demonstrated that iNOS deficiency confers partial resistance to heat-induced germ cell apoptosis at early and late stages of spermatogenesis. We have provided evidence that iNOS plays a functional role in mouse spermatogenesis in spontaneous apoptosis and also participates in the induction of heat-induced germ cell apoptosis. The finding that iNOS functionally deficient mutant mice have larger than normal testicular size suggests an important physiological role of iNOS in spermatogenesis, the determination of sperm number and testes size.
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Footnotes |
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Results from this work were presented in part at the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000.
Abbreviations: AI, Apoptotic index; BN, Brown Norway; BrdU, bromodeoxyuridine; BW, body weight; iNOS, inducible NOS; NO, nitric oxide; NOS, NO synthase; Nv, numerical density; P, pachytene; PL, preleptotene; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; Vv, volume density.
Received December 13, 2002.
Accepted for publication April 3, 2003.
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