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,
,
From the * State Key Laboratory of Reproductive
Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China;
the Beijing University of Chinese Medicine,
Beijing, China; and the Division of
Endocrinology, Department of Medicine, Harbor-University of California-Los
Angeles Medical Center and Los Angeles Biomedical Research Institute,
Torrance, California.
| Correspondence to: Dr Yi-Xun Liu, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Da Tun Lu, Chao Yang District, Beijing 100101, China (liuyx{at}ioz.ac.cn) or Dr Christina Wang, Division of Endocrinology, Department of Medicine, Harbor-University of California-Los Angeles Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA 90509 (wang{at}labiomed.org). |
| Received for publication March 25, 2008; accepted for publication September 23, 2008. |
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Abstract |
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Key words: Andrology, iNOS, 43°C water bath
It is obvious that a nonhuman primate (eg, monkey) represents the model of choice for contraceptive studies in humans (McLachlan et al, 2002; Narula et al, 2002). Similar to men, male monkeys exhibit profound suppression of serum gonadotropin levels but variable patterns of spermatogenic suppression, ranging from azoospermia to oligozoospermia, in response to exogenous administration of T. The regulation of apoptosis is complex and varies depending upon species, tissue type, cell lineage, and nature of the apoptotic stimuli. Thus, the use of the monkey as an experimental model, although expensive and demanding, will allow study of the signal transduction pathways leading to suppression of spermatogenesis in response to exogenous administration of T alone or in combination with mild testicular hyperthermia. This is important from a clinical standpoint because many aspects of the signal transduction pathways regulating human testicular germ cell apoptosis are not amenable to study directly. The upstream signaling pathways by which these hormonal and nonhormonal stimuli activate germ cell apoptosis are not well understood in the primate and are the topic of this article.
As a prelude to future experiments in men, we extended our experimental paradigm from rodents to monkeys (Lue et al, 2006; Jia et al, 2007) and more recently to humans (Wang et al, 2007) and demonstrated that indeed germ cell apoptosis plays an important role in the organized regression of spermatogenesis after hormone deprivation and/or testicular hyperthermia. In the present investigation, we examined the role of eNOS and iNOS in male germ cell apoptosis in monkeys after testicular hyperthermia, hormonal deprivation, or combined interventions. Our data indicate that iNOS may play a role in regulation of germ cell apoptosis in monkeys. Understanding the specific molecular components of the apoptotic pathway in germ cells is an essential step toward the development of novel therapeutic regimens to control accelerated apoptosis leading to abnormal spermatogenesis and infertility, as well as more targeted approaches to male contraception.
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Materials and Methods |
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At the end of the 12-week treatment, the Silastic capsules were removed under light anesthesia, and the monkeys were then allowed to complete an 8-week recovery phase. Three monkeys from each group were used for semen collection without any testicular surgical intervention throughout the study to ensure that the semen parameters were not affected by any surgical procedure (Zhang et al, 2005). The remaining 5 monkeys from each group were subjected to testicular biopsy either before or on days 3, 8, 28, and 84 during the treatment. Biopsies were taken alternatively on each side of the testes from a given monkey at various time points. Each testis was subjected to biopsy only twice throughout the study. Because of the limited tissue samples, Western blot analyses were carried out on days 3, 8, and 28 after treatments.
Each sample was divided into 2 portions. One portion was fixed in Bouin solution, embedded in paraffin, and sectioned for immunohistochemistry. The other portion was snap-frozen in liquid nitrogen for Western blotting and NOS activity assay.
Immunohistochemistry
Sections were deparaffinized, immersed in phosphate-buffered saline (PBS),
and treated with 0.3% hydrogen peroxide in PBS for 10 minutes to block
endogenous peroxidase activity. Sodium citrate buffer was used for antigen
retrieval. After 3 washes in PBS, the sections were exposed to 10% normal
horse serum to suppress nonspecific antigens. A mouse monoclonal iNOS antibody
(10432; BD Transduction Laboratories, San Diego, California;
Vera et al, 2006) and rabbit
polyclonal eNOS antibodies (BA0364, BOSTER, Wu-Han, China; 9572, Cell
Signaling Technology, Beverly, Massachusetts;
Chen et al, 1999) were used for
immunocytochemistry and Western blotting, respectively. The sections were
incubated with eNOS (1:50) or iNOS (1:100) antibody overnight at 4°C.
After 3 washes in PBS, the appropriate biotinylated secondary antibodies were
added and incubated for 30 minutes at room temperature, followed by addition
of the avidin-biotin-peroxidase complex. Immunostaining was developed with the
reagents of a 3,3'-diaminobenzidine kit (Sigma Chemical Co, St Louis,
Missouri). The sections were counterstained with hematoxylin, dehydrated, and
mounted. Negative controls, to which normal IgG was added instead of the
primary antibodies, were used to show specificity of the antibodies.
NOS Assay
The frozen testicular samples were homogenized separately in PBS, and
supernatants were collected after centrifugation (12 000 x g
for 15 minutes). After the total protein concentration in each sample was
determined, NOS activity in the tissue was measured using a colorimetric assay
kit (Nanjing Jiancheng Bioengineering Institute, Nanjing City, China), as
reported previously (Chen et al,
2006). Constitutively expressed NOS (cNOS) and iNOS activities
were measured by spectrophotometer. The principle of this assay is based on NO
content in the sample during a given timed reaction. NO is synthesized from
L-arginine in a reaction catalyzed by NOS, and then NO combines
with a nucleophilic substrate to produce a chromophore that absorbs light at
530 nm. One unit of NOS is defined as the amount of enzyme that produces 1.0
nmol of NO per minute at 37°C in 1.0 mg of protein from tissue homogenate.
The total NOS activity was calculated using a formula provided by the company.
The specific inhibitor of iNOS was added to the tissue homogenate, iNOS
activity was then obtained, and cNOS was calculated by subtracting iNOS
activity from total NOS activity (Chen et
al, 2006).
Western Blotting
Western blotting was performed using testicular lysates and subcellular
fractions as described previously (Sinha Hikim et al,
2003a,b).
In brief, 50 to 80 µg of protein per sample were electrophoresed on a 4% to
12% sodium dodecyl sulfate–polyacrylamide gel with
2-(N-morpholino)ethanesulfonic acid or
3-(N-morpholino)propanesulfonic acid buffer purchased from Invitrogen
(Carlsbad, California) at 200 V. Proteins from the gel were transferred
overnight at 4°C to an immunoblot polyvinylidene fluoride membrane
(Bio-Rad, Hercules, California). Membranes were incubated in blocking solution
(0.05% Tween-20 in Tris-buffered saline and 10% nonfat dry milk) for 1 hour at
room temperature and then probed using a rabbit polyclonal eNOS (1:2000; Cell
Signaling Technology), goat polyclonal actin (1:2000; Santa Cruz
Biotechnology, Santa Cruz, California), or mouse monoclonal iNOS (1:1000; BD
Transduction Laboratories) antibody for 1 hour at room temperature or
overnight at 4°C with constant shaking; the specificity of the antibodies
was described in a previous study (Chen et
al, 1999). Following three 10-minute washes in Tris-buffered
saline Tween-20 (TBS-T) buffer, membranes were incubated in anti-rabbit
(Amersham Biosciences, Piscataway, New Jersey), anti-goat, or anti-mouse
IgG-horseradish peroxidase (Santa Cruz Biotechnology) secondary antibodies at
a 1:2000 dilution. All antibodies were diluted in blocking buffer. For
immunodetection, membranes were washed 3 times in TBS-T buffer, incubated with
ECL solutions per the manufacturer's specifications (Amersham Biosciences),
and exposed to Fuji x-ray film (Fuji Medical Systems Inc, Stamford,
Connecticut). Cerebella from wild-type, used as a positive control
(Vernet et al, 1998), and
iNOS–/– mice were used as additional controls for iNOS
immunospecificity. Band intensities were determined using Quantity One
software from BioRad.
Statistical Analyses
Results are expressed as means ± SD (Figures
1 and
2) or SEM
(Figure 3). Statistical
analyses were performed using SigmaStat 2.0 program (Jandel Scientific, San
Rafael, California). Results were tested for statistical significance using
the Student-Newman-Keuls test after a 1-way analysis of variance. Differences
were considered significant at P < .05.
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Results |
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We also recently analyzed the temporal changes in the incidence of germ
cell apoptosis after these treatments (Lue
et al, 2006). In T group, the rate of germ cell apoptosis
increased by 2.8-fold as compared with control values at day 8 and remained
elevated at this level thereafter. Mild testicular hyperthermia for 2 days
resulted in a marked increase (
6.0- to 7.3-fold over control values) in
germ cell apoptosis at days 3 and 8, but germ cell death declined although not
fully returned to pretreatment levels by day 28. The number of dying cells in
the T+H group was markedly higher compared with either treatment alone on day
3 but began to decrease at day 8. The incidence of germ cell apoptosis in the
T+H group remained elevated over baseline values but similar to that induced
by T on day 28.
Expression of NOS in Monkey Testes after H and T
To investigate possible involvement of NOS in germ cell apoptosis induced
by T and/or H, we first examined eNOS expression in monkey testes by
immunocytochemistry (Figure 4)
as well as by Western blotting (Figure
5). In the control testes
(Figure 4A, panel A, and B, panel
A), eNOS was observed mainly in Sertoli cells and spermatogonia.
No obvious changes in eNOS production were detected after either intervention.
The profile of eNOS expression in the T+H group was similar to that in the
H-alone group (data not shown). Western blot analysis also revealed no obvious
alteration in eNOS levels in T, H, or T+H group
(Figure 5A). This was further
corroborated by densitometric evaluation
(Figure 3B). In contrast, as
shown in Figure 4A, T induced a
modest increase in iNOS expression in both germ cells involving mainly
pachytene spermatocytes and round spermatids and Sertoli cells on days 3
(Figure 1A, panel B), 8
(Figure 1A, panel C), and 28
(Figure 1A, panel D). The
expression of iNOS on day 84 was essentially similar to that of controls
(Figure 1A, panel E). No such
immunostaining was noted when the primary antibody was substituted with normal
IgG (Figure 1A, panel F). The
heat stress obviously increased testicular iNOS expression on day 3 and day 8
as detected by immunohistochemistry (Figure
1B, panels A through D). Increased iNOS immunoreactivity was noted
in heat-susceptible germ cells (pachytene spermatocytes and round spermatids)
3 days after treatment (Figure 1B, panel
B); on day 8. a few remaining germ cells showed strong iNOS
immunoreactivity (Figure 1C, panel
C). The expression of iNOS on day 28
(Figure 1B, panel D) and day 84
(data not shown) resembled that of controls
(Figure 1B, panel A).
Expression of iNOS in the T+H group on days 3 and 8 was similar to that
observed in the H-alone group (Figure 1C,
panels A through F). However, on day 28, intense iNOS expression
was noted in Sertoli cells (Figure 1C,
panel D) because the majority of apoptotic germ cells within this
time frame had been lost through phagocytosis
(Lue et al, 2006). iNOS
immunoreactivity seemed to be markedly reduced on day 84
(Figure 1C, panel E). No such
immunostaining was noted when the primary antibody was substituted with normal
IgG (Figure 1C, panel F). We
also analyzed iNOS expression by immunoblotting
(Figure 2). Cerebella from
wild-type, used as a positive control
(Vernet et al, 1998), and
iNOS–/– mice were used as additional controls for iNOS
immunospecificity. Western blot analysis showed an absence of iNOS in knockout
animals (Figure 2A). A
significant (P < .05) increase in iNOS levels was detected on days
8 and 28 after T and on days 3 and 8 after H
(Figure 2B and C). Increased
expression of iNOS was also noted at days 3, 8, and 28 after combined
intervention (Figure 2B), but
the difference was only significant on day 3
(Figure 2C).
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To further substantiate the immunoblotting and immunocytochemical data, we measured the activities of iNOS and cNOS, which includes both eNOS and neuronal NOS (Figure 3A through C). The cNOS activity measured in the samples should indirectly reflect the eNOS level. As shown in Figure 3A, no obvious alteration in cNOS activity was observed after T, whereas iNOS activity showed an increasing trend on days 3 and 8 and became obviously elevated on day 28 after T. iNOS activity, but not cNOS activity, after H increased significantly (P < .05) on day 3, reached maximum levels on days 8 and 28, and declined on day 84 (Figure 3B). We found similar changes in iNOS activity after T+H (Figure 3C).
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Discussion |
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NO is an important regulator in many physiologic processes. It has been reported that NOS at lower concentrations could regulate various physiologic activities via different signaling pathways, including the cGMP-PKG and MAPK pathway (Beck et al, 1999; Hofmann et al, 2000; Shao et al, 2002). At higher concentrations, the molecule could promote germ cell apoptosis through peroxynitrite, which is generated by a reaction between NO and superoxide (Bonfoco et al, 1995). Increased NO synthesis through up-regulation of iNOS has been implicated in cellular injury and apoptosis in various cell systems (O'Bryan et al, 2000a; Drew and Leeuwenburgh, 2002; Lee et al, 2002). Vera et al (2006) reported that aminoguanidine, a selective iNOS inhibitor, significantly prevented hormone deprivation–induced germ cell apoptosis.
We demonstrated in the present study that heat stress or exogenous T could significantly induce testicular iNOS production. The signaling for iNOS expression may be similar to that induced by cellular injury and apoptosis. This concept is supported by the evidence that up-regulation of testicular iNOS after treatment with endotoxin lipopolysaccharides caused a significant germ cell loss (O'Bryan et al, 2000a,b). Decisive evidence that iNOS plays an important role in testicular germ cell apoptosis derives from our earlier studies on iNOS knockout mice (Lue et al, 2003). These mice have enlarged testes and increased sperm number and exhibit stage-specific suppression of spontaneous germ cell apoptosis. These mice also confer partial resistance to heat-induced germ cell apoptosis. Furthermore, germ cell apoptosis was dramatically increased in a time-dependent manner after 43°C local treatment of monkey testes (Zhang et al, 2005) or experimental cryptorchidism (Tao et al, 2006). The number of apoptotic cells reached the maximum at day 8 and returned to the normal control level at the end of the recovery phase after 43°C warming (Zhang et al, 2005); this observation was consistent with the profile of changes in iNOS observed in the present study. We have also carried out experiments to examine a possible mechanism of heat-induced germ cell apoptosis by looking at expression of some apoptotic marker molecules such as Fas/FasL, Bcl-2/Bax, p53, or heat stress–related molecules (eg, Hsf and Hsps) in testes after H or T (Guo et al, 2000; Mu et al, 2000; Zhou et al, 2001a,b, 2002; Zhang et al, 2003a,b; Tao et al, 2006). The number of apoptotic germ cells increased on day 3, reaching a maximum on day 15 after experimental cryptorchidism (Tao et al, 2006). Studies by Lue et al (2006) have reported that the time course of increasing numbers of apoptotic germ cells is similar to that of the iNOS expression observed in the present study after implanting T, H alone, or their combination.
Evidence has shown that synthesis of NO through NO synthase may play an important role in regulation of testicular blood flow (Sharma et al, 1998) and serum level of T (Adams et al, 1994; Kostic et al, 1999, 2000; Weissman et al, 2005). We have measured serum (Lue et al, 2006) and testicular (unpublished data) T levels by radioimmunoassay after exogenous administration of T and/or heat. The serum T concentrations in the T and T+H groups were significant higher than that of the control animals during the T implanted period because of the release of the hormone from the exogenous T implants. On the other hand, no significant changes in testicular T levels were observed on day 3 after T treatment. However, a significant decrease in the hormone production was measured from 8 to 28 days after T treatment, suggesting that variation of T levels in the serum or in the testes is not obviously correlated to the changes in NOS expression observed in the present study.
We have also examined expression of eNOS and iNOS in monkey Leydig and vascular endothelial cells before and during T or H. No obvious changes in their expression were noted in such cells. The signal of iNOS mainly increased in Sertoli cells and apoptotic germ cells.
It is well known that Sertoli cells are important because they provide structural support, create an impermeable barrier, participate in germ cell development and apoptosis, and nourish germ cells via their secreted products (Bardin et al, 1988; Griswold et al, 1988, 1998). In our previous study, we demonstrated that Sertoli cells undergo dedifferentiation after experimental cryptorchidism (Zhang et al, 2006a) or 43°C local treatment of testes (Zhang et al, 2006b). All of these data showed that function of Sertoli cells is closely related to germ cell loss. iNOS up-regulation mediated by heat and/or exogenous T in Sertoli cells might be one of the important causes of germ cell apoptosis. One possible mechanism by which NO could induce germ cell apoptosis might be through perturbation of the Bax/Bcl-2 rheostat in the mitochondria, resulting in activation of the cytochrome c–mediated death pathway (Vera et al, 2006). Elucidation of the mechanisms by which a physical agent or well-studied hormone-based contraceptive induces germ cell death in a nonhuman primate model would fill a major gap in our knowledge about how the apoptotic program is controlled in the testis.
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Footnotes |
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These authors contributed equally to this article and share
coauthorship. 
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indicates constitutively expressed NOS; 
,
inducible NOS; *, significantly different (P < .05) than controls;
**, significantly different (P < .01) than controls; N, normal
testis; D, number of days after treatment.
