Article

Keywords

Ethanol

Apoptosis

Testis

iNOS

TUNEL

Nabil EidDepartment of Anatomy andCell Biology, Division of lifesciences, Osaka MedicalCollege, Osaka, Japan

Yuko ItoDepartment of Anatomy andCell Biology, Division of lifesciences, Osaka MedicalCollege, Osaka, Japan

Yoshinori OtsukiMD&PhD.Department of Anatomy andCell Biology, Division of lifesciences, Osaka MedicalCollege, 2–7 Daigaku-machi,Takatsuki, Osaka 569–8686,JapanTel.: +81 726 831221;Fax: +81 726 846511

E-mail address:an1001@art.osaka-med.ac.jp(Y. Otsuki).

Involvement of inducible nitric oxide synthase inDNA fragmentation in various testicular germ cellsof ethanol-treated rats

Nabil Eid, Yuko Ito and Yoshinori OtsukiAbstract

Chronic alcohol intake is associated with testicular damage, hypogonadism and infertility. Previously, we reportedenhancement of testicular germ cell apoptosis in ethanol-treated rats (ETR) associated with upregulation of apoptosisrelated genes, Fas, FasL, p53, and activation of caspases. Here, we investigated the involvement of inducible nitric oxidesynthase (iNOS) which is amajor mediator of oxidative stress in ethanol-inducedgerm cell apoptosis.Moreover, we exploredthe various germ cell types undergoing apoptotic DNA fragmentation and their relationship to phagocytosing Sertoli cells.Adult Wistar rats were fed either 5% ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 12 weeks. iNOSexpression in control and ETR was investigated using immunohistochemistry. DNA fragmentation in various germ cells ofETR was detected using TUNEL method under both light and electron microscopy. Damaged spermatids with apoptoticfeatures were observed by transmission electron microscopy (TEM). Compared to very weak expression of iNOS in controltestis, marked increase of iNOS was detected in Sertoli, germ cells and some interstitial cells in ETR. Phagocytosed retainedelongated spermatids undergoing DNA fragmentation was detected by TUNEL method in ETR, in addition to spermatidsundergoing nuclear vacuolization and chromatin degeneration using TEM. The upregulation of iNOS in the testes of ETRmay induce apoptosis of germ cells through the generation of excessive nitric oxide and androgen suppression. IncreasedDNA fragmentation in spermatids of ETR may be involved in infertility and offspring diseases. We recommend for furtherresearch to detect and treat oxidative stress-mediatedpaternalDNA damage in alcoholics. © 2011WPMHGmbH. Publishedby Elsevier Ireland Ltd. All rights reserved.

Introduction

DNA damage in the male germ line has

been linkedwith impaired fertility. The major

mechanisms of paternal DNA damage include

chromatin remodelling, oxidative stress and

abortive apoptosis. The terminal deoxynu-

cleotidyl transferase-mediated dUDP nick-end

labeling (TUNEL) assay has been reported to

be an efficient method for detection of DNA

damage in sperm and prediction of male

fertility [1,2]. This method depends on the

assessment of fragmented DNA and quantifies

the incorporation of dUTP at breaks in double-

stranded DNA in a reaction catalyzed by

terminal deoxynucleotidyl transferase.

Activation of the iNOS generates large

amount of NO inducing apoptosis by direct

DNA damage or indirectly via mitochondrial

pathway by p53-dependent and independent

mechanisms. Unlike the constitutive forms

of NOS (eNOS, nNOS), iNOS produces a large

amount of NO over a prolonged period of time

after its activation by cytokines or bacterial

endotoxins [3]. Immunohistochemical studies

in mammalian testis revealed that increased

iNOS in germ cells was involved with

apoptosis [4,5].

Previously, we found that chronic ethanol

intake enhanced apoptosis of testicular germ

cells in adult rats. This apoptogenic effect

of ethanol was associated with increased

expression of protein levels of p53, Fas

and active caspase-3 in germ cells and FasL

in Sertoli cells. Ethanol-induced testicular

damage was reported to be mediated mainly

byDNAdamage, oxidative stress and androgen

suppression, resulting in testicular atrophy

and male infertility in humans and experi-

mental animals [6].

Although chronic ethanol-induced neu-

ronal apoptosis has been associated with

upregulation of iNOS in rat cerebral cortex and

various tissues [7], the possible involvement

of iNOS in ethanol-induced testicular germ

cell apoptosis is not reported yet. Moreover,

the various germ cell types which undergo

apoptosis in ETR and their relationship to

the phagocytosing Sertoli cells in particular

elongated spermatids are not clearly known.

Using TUNEL method under both light and

electron microscopy in addition to TEM,

we investigated the various types of germ

cells undergoing DNA fragmentation in ETR.

Additionally, we investigated the expression

of iNOS using immunohistochemistry.

Methods

Animals and experimental design

Sixteen adult male Wistar rats with an

approximate average weight of 300 g were

purchased from SLC Japan Co. (Shizuoka,

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Japan). The rats were divided equally into

two groups, i.e., group one was fed ethanol

in a Lieber–DeCarli liquid diet (36% of total

calories as ethanol) while the other group was

fed with a control diet, which was replaced

isocalorically with maltodextrin instead of

ethanol for 12 weeks [6]. The rats were

anaesthetized by an intraperitoneal injection

of pentobarbital and killed by a stab wound in

the right atrium. After perfusion through the

left ventricle, first with physiological saline,

theywere re-perfusedwith either 4% formalin

for light microscopic examination (5 animals

in each group) or 2% paraformaldehyde

and 2.5% glutaraldehyde in 0.1M phosphate

buffer (PB) for electron microscopy (3 animals

in each group).

Preparation of tissue sections

Paraffin embedded sections for immuno-

histochemistry and TUNEL method were

prepared.

TUNEL light microscopic (TUNEL/LM)immunolabeling

A commercially available kit (ApopTag perox-

idase kit; Intergen Co., New York, USA) was

used for the detection of the 3'-OH ends of

damaged DNA.

Statistical analysis of testicular apoptosis

The percentage of apoptotic seminiferous

tubules (STs) was determined by counting and

comparing a total of 100 STs from each of

the five rats in each group. An apoptotic ST

is defined as the one containing three or more

apoptotic germ cells, using cross-sections of

STs as reported previously [6]. Data were an-

alyzed using Student’s t-test for quantitative

analysis of testicular apoptosis between the

control and ETR. A p -value of <0.05 was

considered statistically significant.

TUNEL by transmission electron microscopy(TUNEL/EM)

The procedure of TUNEL/EM is fundamentally

the same as that of the light microscopic

TUNEL (TUNEL/LM), except for the incor-

poration of immunogold particles, as a

substitution for the immunoperoxidase in

LM/TUNEL [8].

Immunohistochemistry of iNOS

Rabbit polyclonal antibody for iNOS (a gift

from Prof. Hiroyasu Esumi, National Cancer

Center, Tokyo) was used in dilution of 1:200.

The secondary antibody was peroxidase-

labelled goat anti rabbit antibody with a

dilution of 1:100. The details of immunohis-

tochemistry were described previously [6].

Transmission electron microscopy (TEM)

After post-fixation with 1% osmium tetroxide

in 0.1M PB for 2h, specimens were routinely

dehydrated in ethanol and then embedded in

epoxy resin [6]. Ultra-thin sections (60–80nm

thick) were double-stainedwith uranyl acetate

for 15min, then with lead citrate for 5min

and examined under an H-7100 transmission

electron microscope (Hitachi Co., Ltd, Tokyo,

Japan).

Results

Upregulation of iNOS in the testes of ETR

The expression of iNOSwas veryweak (arrows)

in the ST of control testis (Fig.1A). On the

other hand, marked increase in the intensity

of iNOS expression was detected in germ

and Sertoli cells in ST of ETR (Fig.1B-D).

Increased expression of iNOS was marked in

primary spermatocytes and in both apical and

basal regions of Sertoli cells. Some cells in

the interstitium were reacted, although their

identity was not clearly known.

Enhanced apoptosis in germ cells of ETR: TUNELanalysis and statistical study

Figure 2A-2C demonstrates TUNEL/LM find-

ings while Figure 2D indicates TUNEL/EM

study. In the control testis (Figure 2A), a few

apoptotic cells were observed in the basal part

of ST; however, in ETR (Figure 2B), marked

depletion of germinal epithelium associated

with increased number of apoptotic germ

cells was observed in both the basal and

adluminal parts of ST. Marked increase in

the frequency of TUNEL positive elongated

spermatids (based on morphology) within the

cytoplasm of Sertoli cells was observed in

ETR (Figure 2C). Apoptotic germ cell with

marked chromatin condensation associated

with fragmented DNA immunolabeled with

gold particles using TUNEL/EM is shown in

Figure 2D.

Statistical analysis revealed that the per-

centage of apoptotic STs in ETR (13.6) was

significantly higher than those of control

testis (3.8) (p< 0.001).

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Figure 1. Induction and upregulation of iNOS in the testes of ETR. (A) Control testis; (B–D) ETR testes.The framed area in (B) is magnified in (C). Compared to very low expression of iNOS ingerm cells (arrows) of control testis (A), overexpression of iNOS was observed in ETR (B). Theimmunolabeling of iNOS was observed frequently in primary spermatocytes (arrow heads in C),and Sertoli cells (black arrows in D). Red arrows in D mark Sertoli cell nuclei. ES, elongatedspermatids; v, vacuole. Note the close association of ES to iNOS expressing Sertoli cells in ETR.(A,B) ×100; D, ×200 (original magnification).

Figure 2. TUNEL positivegerm cells in control (A) and ETR testes (B–D). (A) a few TUNEL positive cells (brownstaining) in control testis. (B) Increase in the frequencyof TUNEL positive cells in ETR. (C) Apoptoticelongated spermatids (arrows)are observed in ETR. A, B, ×100; C ×200 original magnification.(D) Apoptotic germ cells using TUNEL/EM. The area marked by arrow is magnified in the inset toshow gold particles. ×4000.

Ultrastructural features of damaged spermatidsin ETR

Figure 3A showed retained elongated sper-

matid heads partially surrounded by vacuolar

space and phagocytosed within Sertoli cells.

In Figure 3B, a damaged spermatid head

with decondensed granular chromation was

clearly seen. In Figure 3C, a round spermatid

with small nucleus containing two vacuoles

was demonstrated, whereas in Figure 3D, a

phagocytosed apoptotic body inside cytoplasm

of Sertoli cell was demonstrated.

Discussion

The major findings in the current study

were the induction and upregulation of iNOS

mainly in Sertoli and germ cells of ETR and

the associated DNA fragmentation in various

germ cells including elongated spermatids

as assessed by TUNEL/LM. In addition, DNA

fragmentation of apoptotic germ cells was

correlated with condensed nuclear chromatin

using TUNEL/EM. Finally, chromatin damage

in elongated and nuclear vacuolization of

round spermatids was frequently observed

in the testes of ETR. Immunohistochemical

observations in the current study revealed

a very weak expression of iNOS in control

testis compared to a marked increase in the

expression of iNOS in Sertoli cells, germ cells

and some cells in the interstitium of ETR.

The factors that induce and upregulate iNOS

expression in ETR could be due to increase

in the levels of inflammatory cytokines

associated with alcohol intake [7,9]. Therefore,

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Figure 3. Electron micrographs showing damaged spermatids in ETR. (A) Elongated spermatids (arrowheads) surrounded by vacuole (star) and partially phagocytosed by Sertoli cell. (B) Damagedelongated spermatid with granular decondensed chromatin and a few masses of condensedchromatin (white arrow). (C) Damaged round spermatids (Rs) with 2 nuclear vacuoles (arrow).(D) Apoptotic germ cell body (AB) phagocytosed by Sertoli cell. A, ×1200; B, ×3000; C, ×1200;D, ×2500 (original magnification). S, Sertoli cell nucleus.

the upregulation of iNOS in germ cells of ETR

was most probably involved in their apoptosis

through the generation of high levels of

NO directly inducing DNA fragmentation or

indirectly through mitochondrial pathway as

suggested by others [3–5]. TUNEL/LM analysis

in the current study revealed increased

apoptosis in all types of germ cells of ETR

compared to few apoptotic germ cells in

control testis. The use of testicular sperm in

assisted reproduction as ICSI was reported

to improve pregnancy rates in infertile

patients [10]. Therefore, based on TUNEL,

method avoiding the selection of spermatids

with DNA damaged from the testes of

alcoholics has to be considered in infertility

treatment in particular ICSI.

Correlating ultrastructural features of apo-

ptosis with the DNA cleavage has been

reported to be a major advantage of TUNEL/

EM [8]. Our results showed the first time

evidence of the presence of apoptotic sper-

matogonia partially phagocytosed by Sertoli

cell and characterized by marked chromatin

condensation and fragmented nuclear DNA

immunolabeled with gold particles. TEM

findings in the testes of ETR of granular

decondensed chromation in elongated sper-

matids and nuclear vacuolization in round

spermatids may be considered advanced stage

of apoptosis as reported by others [2,10].

Conclusions

Ethanol-induced DNA fragmentation and

apoptosis of germcells are,most probably,me-

diated by induction and upregulation of iNOS.

Increased DNA fragmentation in the sper-

matids of alcoholics has to be considered in the

treatment of male factor infertility especially

assisted reproduction techniques. This re-

quires careful selection of spermatid or sperm

with intact DNA through application of some

techniques like TUNEL method in addition to

the use of high doses of anti-oxidants to im-

prove testicular oxidative stress in alcoholics.

Conflict of interest statement

The authors have no conflict of interest to

report.

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