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Journal of Andrology, Vol. 26, No. 2, March/April 2005
Copyright © American Society of Andrology

Overexpression of Endothelial Nitric Oxide Synthase in Transgenic Mice Accelerates Testicular Germ Cell Apoptosis Induced by Experimental Cryptorchidism

TOMOMOTO ISHIKAWA*,{dagger}, YUTAKA KONDO{ddagger}, KAZUMASA GODA{dagger} AND MASATO FUJISAWA{dagger}

From the * Center for Biomedical Research, The Population Council, New York, New York; {dagger} Division of Urology, Department of Organs Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe, Japan; and {ddagger} Department of Urology, Kawasaki Medical School, Kurashiki, Japan.

Correspondence to: Dr Tomomoto Ishikawa, Center for Biomedical Research, The Population Council, 1230 York Ave, New York, NY 10021 (e-mail: tishikaw{at}popcbr.rockefeller.edu).
Received for publication June 12, 2004; accepted for publication September 13, 2004.

   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Surgical induction of cryptorchidism in experimental animals causes testicular germ cell apoptosis and infertility. The mechanisms of germ cell apoptosis have been associated with oxidative stress or testicular exposure to elevated temperature. Nitric oxide (NO) has been associated with apoptosis in a number of cell types. The objective of this study was to investigate whether overexpression of endothelial NO synthase (eNOS) could accelerate apoptosis of germ cells in the testes of transgenic mice. There are 3 NOS isoforms, and we restricted the analysis to eNOS at this time. For the colocalization of eNOS, staining in degenerating germ cells that were apoptotic cells suggested that eNOS may be related to germ cell apoptosis. eNOS overexpression in the testes of eNOS transgenic (eNOS-Tg) mice was examined using Western blot analysis. Unilateral cryptorchidism was surgically induced in both eNOS-Tg and wild-type (WT) adult mice. The testes were evaluated 1, 3, 5, 7, and 14 days after the operation by weighing the testes and examining histopathologic features and cell apoptosis using in situ microscopic analysis of DNA fragmentation. Immunoblotting for eNOS protein demonstrated increases in eNOS protein expression in testes, as well as the lung and aorta. In eNOS-Tg mice, weight reduction of cryptorchid testis was significantly increased on days 3, 5, and 7 (P = .02, .02, and .04, respectively). The numbers of spermatocytes and spermatids of eNOS-Tg cryptorchid testis significantly decreased compared with those of WT cryptorchid testis from day 3 (spermatocytes: P = .04; spermatids: P = .02). Moreover, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling demonstrated that eNOS-Tg mice significantly accelerate germ cell apoptotic changes induced by experimental cryptorchidism compared with WT mice from day 3 (P = .03). We have provided evidence that eNOS plays a functional role in mouse spermatogenesis in cryptorchidism-induced apoptosis.

     Key words: Immunoblotting, TUNEL, spermatocytes, spermatids, spermatogonia


Nitric oxide (NO) is a free radical gaseous molecule with a well-described role as a signal transduction messenger molecule in several biological processes (Ignarro et al, 1987). The enzyme nitric oxide synthase (NOS) is the critical rate-limiting step in the production of NO through oxidation of the guanidine nitrogen of arginine. One isoform of the enzyme, endothelial NOS (eNOS), is a constitutive Ca2+-dependent NOS. Studies suggest that the generation of NO, which functions as an endothelium-derived relaxing factor, plays an important physiologic role in the control of vascular tone (Ignarro et al, 1987; Palmer et al, 1987; Kelm et al, 1988). Recently, evidence has emerged that supports a role for NOS in modulating sexual and reproductive functions in mammalian species. NO has been implicated in the apoptotic death of a number of cell types, including neurons (Bonfoco et al, 1995), chondrocytes (Blanco et al, 1995), and macrophages (Sarih et al, 1993). Zini et al (1996) have shown that eNOS may similarly be involved in germ cell apoptosis in the human testis and that the colocalization of eNOS staining in degenerating germ cells that were apoptotic cells suggested that eNOS may be related to germ cell apoptosis. In addition to its probable role in controlling vascular wall tone, the functions of eNOS in the testes remain unknown. eNOS immunostaining was colocalized to degenerating germ cells in normal and cryptorchid testes. We have also shown that the potential bioactivity of eNOS protein shown with immunohistochemical analysis has been supported by enzyme studies that confirm eNOS activity in Sertoli cells of infertile male patients (Fujisawa et al, 2001). Such a broad distribution illustrates that NO may have an important role in both normal and abnormal testicular function. The objective of the present study was to determine whether eNOS overexpression caused by eNOS transgenic (eNOS-Tg) mice could influence the apoptosis of germ cell in experimentally cryptorchid testes.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Animal Preparation

We have generated eNOS-Tg mice, which are transgenic mice that overexpress the bovine eNOS gene in endothelial cells by use of the endothelium-specific preproendothelin-1 promoter (Ohashi et al, 1998) (Figure 1). Heterozygous transgenic mice and their litter mate wild-type (WT) mice (C57BL/6; Clea, Tokyo, Japan) were used at 8 weeks of age. Founder mice that harbored the transgene were identified by polymerase chain reaction analysis of genomic DNA isolated from tail biopsy specimens at 4 weeks of age. Tail DNA was extracted by proteinase K (2.0 mg/mL) digestion and subsequent phenolization and purification using a Genomix DNA extraction kit (Talent, Trieste, Italy). Polymerase chain reaction detection was performed using transgene-specific oligonucleotide primers, ie, sense primer at the 3' end of preproendothelin-1 promoter (5'-GAAGTTAGCCGTGATTTCCTCTAGAGCCGGGTC) and antisense primer at the 5' end of eNOS complementary DNA (5'-TTGATGAAGTCCCTGGCCTGGCTCAGCAG). All animal experiments were conducted according to the Guidelines for Animal Experiments at Kobe University School of Medicine. To induce unilateral cryptorchidism, mice were anesthetized and a small incision was made in the upper abdomen. General anesthesia was given with sodium pentobarbital, 50 mg/kg intraperitoneally, and the abdomen was shaved and prepared with Betadine. The right testes were softly brought up into the abdominal cavity and fixed on the upper abdominal wall. The testes were taken as samples at 1, 3, 5, 7, and 14 days after surgery (n = 4, 4, 4, 4, and 3, respectively). The weight of the testis was determined before histologic staining.



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  		Figure 1. Schematic map of the murine preproendothelin-1 promoter and bovine endothelial nitric oxide synthase (eNOS) complementary DNA fusion gene used to generate eNOS transgenic mice. The preproendothelin-1 promoter (9.2 kb) is depicted as a shaded bar and eNOS complementary DNA (4.1kb) as an open bar. pA indicates SV40 intron/poly A signal; P1 and P2, oligonucleotide primers used to screen genomic DNA for the presence of the transgene; Sp, SpeI; S, SalI; H, HindIII; B, BamHI; N, NotI; and Sc, SacI.

 

Immunoblotting of eNOS

Crude homogenates of pooled tissues in a homogenizing buffer of 50-mM Tris hydrochloride, pH 7.4, 1-mM ethylene glycol bis (2-aminoethyl)-tetra-acetic acid (EGTA), 1-mM dithiotreitol (DTT), 1-µM pepstatin A, 2-µM leupeptin, and 1-µM (p-amidinophenyl) methane sulfonyl fluoride were ultracentrifuged at 100 000 x g to collect cytosolic fractions. The pellets were solubilized in the homogenizing buffer, which contained 10% glycerol and 20-mM 3-[(3-chol-amidopropyl) dimethylammonio]-1-propanesulfonate, and ultracentrifuged to extract the cytosolic fractions. Proteins were concentrated using Microcon Centrifugal Filter Devices (Millipore Corp, Bedford, Mass) as recommended by the manufacturer. Protein concentrations were determined by the method of Bradford (Bio-Rad Laboratories, Hercules, Calif) with bovine serum albumin fraction V as a standard protein. A total of 30 µg of protein samples from the cytosolic fraction was separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4% to 20% Tris-glycine gel (Novex, San Diego, Calif) under reducing conditions, transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and probed with a rabbit polyclonal (BD Transduction Laboratories, San Jose, Calif; dilution, 1:1000) or a murine monoclonal anti-bovine eNOS antibody (clone H32, IgG2a, 1:2500). Immunoreactive bands were visualized with horseradish peroxidase-conjugated anti-rabbit IgF (ab')2 fragment or anti-mouse IgG using an electrochemiluminescent detection kit (Amersham International PLC, Buckinghamshire, England) and quantified by densitometry.

Histopathologic Analysis

Testicular tissues were fixed overnight in Bouin solution, dehydrated, and embedded in paraffin. Sections (4-µm thick) stained with periodic acid-Schiff reagent and hematoxylin were observed under a light microscope. Twenty cross-sections of seminferous tubules were randomly selected and analyzed. Nuclei of Sertoli and germ cells present in cross-sections of seminferous tubules were counted. The number of germ cells were counted and defined by the following categories: spermatogonia, spermatocytes, and spermatids. The frequency of germ cells was expressed as the number of germ cells per single Sertoli cell. Since the distribution in the number of Sertoli cells per tubule cross-area was homogenous, each germ-Sertoli cell ratio was used as a measure to compare the extent of spermatogenic activity in each tubule.

TUNEL Method

In situ analysis of DNA fragmentation (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP-biotin nick end labeling [TUNEL] method): The ApopTag kit (Serologicals Corporation, Norcross, Ga) was used to detect the DNA fragmentation following the procedure recommended by the supplier. Sections were deparaffinized, rinsed in distilled water, and reacted with proteinase K (20 µg/mL). After rinsing in distilled water, sections were treated with 3% H2O2 to inactivate the endogenous peroxidase. DNA nick end labeling included the following steps: 1) react with biotinylated dUTP in the TdT-reacting solution, 2) incubate with streptavidin-peroxidase conjugate (Histostain-SP kit; Zymed Labs, Nagoya, Japan), and 3) visualize with 3,3'-diaminobenzidine tetrahydrochloride as a chromogen. The sections were lightly counterstained with methyl green and evaluated microscopically. For the negative control, sections were treated by the same procedure without TdT. Twenty cross-sections of seminferous tubules were randomly selected and observed, and the rate of germ cell apoptosis was expressed as the number of germ cells apoptosis per 1 Sertoli cell.

Statistical Analysis

Student's t test for unpaired observations was used to determine the significance of differences between eNOS-Tg mice and WT litter mates. Statistical analysis for multiple comparisons was performed using 1-way analysis of variance with Bonferroni correction. All values are given as means ± SEM, and statistical significance was set at P less than .05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Expression of eNOS in Transgenic Mice Testis

To initially determine whether eNOS overexpressed in eNOS-Tg testis, Western blot analyses were performed. Proteins were isolated as indicated in the "Materials and Methods" Western analyses showed a significant increase of eNOS protein expression (140 kDa) in testes, as well as the lung and aorta (Figure 2).



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  		Figure 2. Immunoblotting for endothelial nitric oxide synthase (eNOS) protein, demonstrating increases in eNOS protein expression. The particulate fractions (30 µg per lane) were electrophoresed, transferred, and probed with a polyclonal anti-eNOS antibody. Size markers in kilodaltons are shown to the left of the blot.

 

Time-Dependent Changes of Testicular Weights

The weights of scrotal testes were unchanged throughout the study period. In WT mice, the weight of the cryptorchidism testis was decreased to 81%, 59%, and 42% of that of the contralateral scrotal testis on days 3, 5, and 7, respectively. However, in eNOS-Tg mice, weight reduction of cryptorchid testis was significantly increased (62%, 44%, and 33% of the contralateral scrotal testis on days 3, 5, and 7, respectively; P = .02, .02, and .04, respectively, vs WT mice) (Figure 3). At least 3 testes each day and each type of mice were examined for this study.



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  		Figure 3. Time course of weight changes in cryptorchid and scrotal testis from endothelial nitric oxide synthase transgenic (eNOS-Tg) and wild-type (WT) mice. A time course of cryptorchid testis weight changes was presented as the percentages of the contralateral scrotal testis weight. The weights of scrotal testes were unchanged throughout the study period. Data are presented as mean ± SEM. *P < .05 (eNOS-Tg vs WT mice).

 

Histologic Analysis After Surgical Induction of Unilateral Cryptorchidism

In the cryptorchid testes removed on day 5 from the eNOS-Tg mice, many germ cells had disappeared from the seminiferous tubules, whereas in the cryptorchid testes removed on day 7 from WT mice, a considerable number of germ cells were still present in most seminiferous tubules (Figure 4A and B). Scrotal testes in both WT and eNOS-Tg mice showed normal histologic features throughout the study period. On day 1 after inducing unilateral cryptorchidism, the number of germ cells in eNOS-Tg seminferous tubules did not differ significantly from that in WT mice. However, on day 3, the number of germ cells per 1 Sertoli cell had started to decrease significantly. We observed a significant decrease in spermatocytes (6.3 ± 0.4 vs 7.5 ± 0.3 for eNOS-Tg vs WT mice, P = .04) and spermatids (8.3 ± 0.6 vs 10.5 ± 0.6, P = .02) but not spermatogonia (1.5 ± 0.4 vs 1.6 ± 0.2, P = .20) (Figure 5). There were also significant differences observed in spermatocytes and spermatids on days 5 and 7. However, there is no significant difference in spermatogonia shown in this experiment. On day 14, there was a significant difference observed in only spermatocytes.



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  		Figure 4. Histologic findings of the cryptorchid testes from wild-type mice (A) and endothelial nitric oxide synthase transgenic mice (B) on day 7. Sections were stained with hematoxylin-eosin (original magnification, x100).

 


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  		Figure 5. Germ cell index, expressed as germ cells per 1 Sertoli cell, at various stages of spermatogenesis of cryptorchid testes in wild-type (WT) and endothelial nitric oxide synthase transgenic (eNOS-Tg) mice. *P < .05 (eNOS-Tg vs WT mice).

 

Quantitative Analysis of Testicular Apoptosis in Cryptorchid Testis

As for the in situ staining of the cryptorchid testes, in the eNOS-Tg mice the number of apoptotic cells per 1 Sertoli cell was remarkably increased from day 3 and reached 0.52 ± 0.07 cells per 1 Sertoli cell on day 5 (Figure 6). The incidence of germ cell apoptosis was detected at all stages, but apoptotic cells were mainly primary spermatocytes and round spermatids. On day 1 after inducing unilateral cryptorchidism, the number of apoptotic germ cells in eNOS-Tg mice did not differ significantly from that in WT mice. However, on day 3, the number of apoptotic germ cells per 1 Sertoli cell had started to increase significantly (0.24 ± 0.07 vs 0.09 ± 0.02 for eNOS-Tg vs WT mice, P = .03). There are also significant differences between the cryptorchidism-induced testes of eNOS-Tg and WT mice on days 5 and 7 (P = .05 and .04, respectively). These results also demonstrated that testicular germ cell apoptosis was delayed by 2 days in WT mice compared with eNOS-Tg mice (Figure 7).



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  		Figure 6. Histologic analysis of the cryptorchid testes from endothelial nitric oxide synthase transgenic (eNOS-Tg) and wild-type (WT) mice after surgical induction of cryptorchidism. Sections were stained by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method from day 5 of WT mice (A) and from day 5 of eNOS-Tg mice (B). Arrows indicate apoptotic cells (original magnification, x100).

 


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  		Figure 7. Quantitative analysis of testicular apoptosis in cryptorchid testes. The number of positive staining cells per 1 Sertoli cell was counted, and mean ± SEM values were presented. *P < .05 (endothelial nitric oxide synthase transgenic vs wild-type mice).

 


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
This is the first study, to our knowledge, to report that overexpression of eNOS accelerates testicular germ cell apoptosis induced by experimental cryptorchidism. The testicular germ cell loss induced by cryptorchidism may be due to a change in the testicular temperature and apoptosis, but this mechanism is still unclear. To investigate the role of NO in apoptosis, we used the experimental unilateral cryptorchidism model in eNOS-Tg and WT mice.

It is well established that NO plays an important role in a variety of processes in the cardiovascular, neuronal, and immune systems (Monacada et al, 1991; Schmidt and Walter, 1994). Recently, it was demonstrated that NO production and NOS expression is localized in the testis. eNOS was expressed in myofibroblasts of the peritubular lamina propria, endothelial and smooth muscle cells of large blood vessels, Sertoli cells, and Leydig cells in the testes (Zini et al, 1996; Middendorff et al, 1997; Fujisawa et al, 2001). The extensive expression of eNOS in multiple cells of the testis strongly suggests a potential role for NO in germ cell differentiation.

The function of NO in Sertoli cells is still unknown. It has been reported that NO can modulate gene expression and cell differentiation in leukemic cells (Schmidt and Walter, 1994) and macrophages (Monacada et al, 1991), respectively. We previously demonstrated that NO production and inducible NOS messenger RNA expression in the Sertoli cells were increased following exposure to conditioned medium from primary round spermatid not pachytene spermatocyte cultures (Tatsumi et al, 1997). On the other hand, the fact that the colocalization of eNOS staining in degenerating germ cells that were apoptotic cells suggested that eNOS may be related to germ cell apoptosis (Zini et al, 1996). In addition, eNOS was positive using immunohistochemical analysis in degenerating germ cells in normal and cryptorchid testes (Zini et al, 1999). Therefore, NO may play an important role in controlling testicular function on the normal and abnormal conditions, including apoptotic change. In support of this hypothesis, there is strong evidence that links NO with apoptosis in chondrocyte (Blanco et al, 1995), macrophage (Sarih et al, 1993), and pancreatic B cells (Kaneto et al, 1995).

In the present study, which used a mouse model of experimental unilateral cryptorchidism, it was observed that testicular weight reduction, germ cell loss, and DNA fragmentation all began in the cryptorchid testes on day 5 in WT mice. In contrast, these changes were accelerated by 2 days in eNOS-Tg mice. These results suggest that abdominal heat stress induces germ cell loss through the NO-dependent pathway responsible for germ cell apoptosis. Bonfoco et al (1995) proposed that the NO-induced apoptosis might be mediated by peroxynitrite, which is generated by the reaction between NO and superoxide.

The degeneration of seminiferous epithelium induced by experimental cryptorchidism has been extensively studied. We previously showed that the number of spermatids and spermatocytes decreased significantly as a result of cryptorchidism (Fujisawa et al, 1988). Vigodner et al (2003) suggested that spermatic arrest occurred at stages IX and X in the early stages of cryptorchidism and the population of spermatogonia was characterized as stable for increased temperature effect in WT golden hamsters. The present study showed correlation with this study in both eNOS-Tg and WT mice. We proposed that overexpression of eNOS should mediate apoptosis and maturation arrest might be associated with apoptosis accelerated by NO during meiosis or before entrance into meiosis in severe cases. In addition to the general vasodilation that can be expected, other nonspecific effects may ensue (eg, tissue degeneration, reduction in the weight of scrotal testes). The general pathologic state of the mice could be the underlying cause for the observed results. Our results could be interpreted as a nonspecific response to a large increase in NO production, not necessarily due to overexpression of eNOS in testis. The alternations could be observed only 3 days after the beginning of the experimental procedure, a fact that may not be associated with a constitutive enzyme such as eNOS. However, we believe that the difference must be caused by the overexpression of eNOS. We think other nonspecific effects contribute little to these differences.

Yin et al (2002) showed that p53 and Fas were involved in testicular germ cell apoptosis induced by heat; however, single or double knockout mutations were not enough to prevent germ cell apoptosis in a cryptorchid testis. Ohta et al (2003) suggested that the role of p53 in heat stress is to control germ cell apoptosis in cells that differentiate from type A spermatogonia to spermatocytes but not in haploid cells or in cells undergoing meiotic division from spermatocytes. We suggest that cell type-specific and NO-dependent apoptotic systems also control germ cell apoptosis that is induced by heat stress during meiosis. In this study, which uses eNOS-Tg mice, we were able to examine the causality of eNOS in the experimental cryptorchidism-induced germ cell apoptosis and demonstrated that eNOS overexpression contributes to heat-induced germ cell apoptosis during spermatogenesis.

Although the function of NO in the seminiferous tubule requires further characterization, local effects of NO are involved in regulating spermatogenesis. Lue et al (2003) provided evidence that inducible NOS through its product, NO, participates in the induction of heat-induced germ cell apoptosis. Shiraishi et al (2001) provided the evidence that iNOS expression was markedly increased 1 hour after ischemia and was accompanied by a huge NO production, with a peak at 48 hours of reperfusion in experimental torsion model. Thus, inducible NOS may also have an important function in experimental cryptorchidism; however, there is no publication about inducible NOS in this condition. Wang et al (2002) demonstrated the important function of neuronal NOS in Leydig cells. Further studies will be needed to clarify the mechanism of these synthases in various testicular conditions (ie, cryptorchidism, varicocele, torsion, vasectomy, and spermatogenesis). This eNOS-Tg mouse model could also provide new insights into the various mechanisms in testis and eNOS through NO, which had an apoptotic effect on spermatogenesis.


   Conclusion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
The transgene expression of eNOS increased testicular germ cell apoptosis induced by experimental cryptorchidism. We have provided evidence that eNOS plays a functional role in mouse spermatogenesis in cryptorchidism-induced apoptosis. eNOS reduction may be a useful therapeutic strategy for male infertility associated with heat stress.


   References
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 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
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Fujisawa M, Matsumoto O, Kamidono S, Hirose F, Kojima K, Yoshida S. Changes of enzymes involved in DNA synthesis in the testes of cryptorchid rats. J Reprod Fertil. 1988; 84: 123 -130.

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