ORIGINAL ARTICLE

Alcohol inducedabstinence equal

O.O. Dosumu *, A.A.A

Department of Anatomy, Faculty of

Received 12 April 2013; accepted 2

Abstinence;

Testosterone;

Seminiferous epitheliumafter ethanol exposure.

Sexually mature male SpragueDawley rats were randomly divided into control, abstinent and

tological analysis of the seminiferous tubules of the animals in the non-abstinent group showed

count and motility, were also signicantly reduced (p< 0.001) while testicular malondialdehyde

hormone (FSH) remained unchanged. In the recovery or abstinent groups (group III), despite weeks

of abstinence from alcohol, the groups still demonstrated high levels of tMDA, low sperm count

ss sectional area

ferous epithelium

covery ten

glutathione; LH, luteinizing hormone; FSH, follicle stimulating

hormone; TT, testosterone; ROS, reactive oxygen species; TBARS,

thiobarbituric acid-reactive substances.* Corresponding author. Tel.: +234 803 370 1857.

E-mail address: olufunkedosumu@gmail.com (O.O. Dosumu).

Peer review under responsibility of Middle East Fertility Society.

Production and hosting by Elsevier

Middle East Fertility Society Journal (2014) xxx, xxxxxx

Middle East Fertility Society

Middle East Fertility Society Journal

www.mefsjournal.orgwww.sciencedirect.comAbbreviations: tMDA, testicular malondialdehyde; tGSH, testicularand motility and signicantly reduced (p< 0.001) testicular diameter and cro

values. However, increased TT levels and non-severe reduction in the semini

observed in these groups showed signs of epithelial regeneration and probable re1110-5690 2014 Production and hosting by Elsevier B.V. on behalf of Middle East Fertility Society.http://dx.doi.org/10.1016/j.mefs.2014.01.003

Please cite this article in press as: Dosumu OO et al. Alcohol induced testicular damage: Can abstinence equal recovery?, Middle East Fert(2014), http://dx.doi.org/10.1016/j.mefs.2014.01.003dencies.(tMDA) levels increased signicantly (p< 0.001). Hormonal assay showed signicant reductions

in the levels of testosterone (TT) (p< 0.05) while luteinizing hormone (LH) and follicle stimulatingsevere reduction of cells of the spermatogenic series, hypocellularity, tubular atrophy and signicant

reductions in the tubular diameter and cross-sectional areas (p< 0.001). Testicular weight, spermnon-abstinent groups. Alcohol was administered orally at 7 ml/kg body weight per day thrice in

a week for 2, 4 and 8 weeks. Control animals received an equivalent amount of distilled water. His-KEYWORDS

Alcohol;

Testis;

Oxidative stress;

ttesticular damage: Canrecovery?

. Osinubi, F.I.O. Duru

Basic Medical Sciences, College of Medicine, University of Lagos, Nigeria

8 January 2014

Abstract Drinking continues to be a major problem in many parts of the world. Signicant effects

on testicular morphology and function in animals as well as man have been well described. To

further explore the impact of chronic ethanol exposure on the testes, we designed this study specif-

ically to dene whether or not there was complete recovery after abstinence by examining reproduc-

ive hormones, testicular histomorphometry, testicular antioxidants as well as semen parametersil Soc J

tud

se

an

2.2. Animal experiments

Adult male SpragueDawley rats

used for the study. Animals were p

2.3.3. Hormone determination

The serum levels of TT, FSH and LH were measured using

2 O.O. Dosumu et al.

Please cite this article in press as: Dosum(2014), http://dx.doi.org/10.1016/j.mefsweighing (150170 g) were

rocured from the Nigerian

commercially available enzyme-linked immunoassay kit (Diag-nostic automation Inc, CA) according to the manufacturersinstructions.In conclusion, the present s

administration failed to rever

2014 Production

1. Introduction

The consumption of alcohol has long been part of everyday life

in many societies and it will continue to be so in the future.However, the World Health Organisation (67) has found thatalcohol consumption represents the third largest risk factor

for disease burden in high-income countries, behind only smok-ing and hypertension, both of which are also associated withalcohol misuse.

Ethanol has been reported to be among the most widelyabused drug which can suppress reproductive function and sex-ual behaviour in laboratory animals and humans. Alcohol abuse

has been considered as one of the problems associated with poorsemen production and sperm quality (1,57). Both chronic andacute consumption of alcohol has been reported to cause fertilitydisturbances such as low sperm count and motility, reduced ser-

um/plasma testosterone level, testicular atrophy and irregularityin the diameter of the seminiferous tubules in men and labora-tory animals (62,38,39,15). In addition, Martinez et al. (39) re-

ported histological abnormalities in testicular tissue ofalcoholic animals. These include intense intercellular spaces,irregular diameter of seminiferous tubules and high amount of

necrotic cells in the lumen compared with controls. Epididymalsperm motility also decreased in ethanol-treated rats.

In men, low levels of testosterone have been repeatedly asso-

ciated with both moderate consumption and chronic alcoholabuse (24,49,50,51,52). In addition, serum TT has negativelybeen associatedwith the duration of alcohol abuse (38). Forqueret al. (23) have also reported signicant reductions in androgen

levels following ethanol intoxication and withdrawal in males.Undoubtedly, ethanol consumption produces a signicant

decrease in the percentage of motility, concentration (38) and

normal morphology in human and animal spermatozoa(4,42). Previous studies have shown that alcohol ingestion fol-lowed by herbal treatment modalities showed good recovery

tendencies with testicular parameters almost restored to nor-malcy (15). However, it remains to be determined whether nor-malcy can be restored within the testicular milieu followingprolonged periods of abstinence without treatment. Hence,

the present study was carried out to determine the effects ofchronic administration of ethanol followed by abstinence onthe testes of adult rats.

2. Materials and method

2.1. Chemicals

Thirty percent ethanol (17) prepared from absolute ethanol

(99.86% v/v) with substance identication number 1170 man-ufactured by James Burrough (F.A.D. Ltd. UK) was used forthe study.u OO et al. Alcohol induced testic.2014.01.003y shows that total alcohol abstinence following chronic ethanol

completely alcohol-induced testicular damage.

d hosting by Elsevier B.V. on behalf of Middle East Fertility Society.

Institute of Medical Research (NIMR). The animals werehoused in the Anatomy Department Animal Control Room

in well ventilated plastic cages with 12:12 lightdark cycle at27 1 C. Rats were randomized into nine groups of veanimals each. The mode of administration for all groups was

through gastric intubation, and animals in the treatmentgroups received 7 ml/kg body weight of 30% ethanol perday, thrice in a week (17). All animals were largely divided intothree categories: I (control), II (abstinent) and III (non-absti-

nent). All group I rats served as control and received distilledwater; group II rats were subdivided into groups a, b, c andreceived ethanol for 2, 4 and 8 weeks respectively; group III

rats were also subdivided into groups a, b, c and fed ethanolfor 2, 4 and 8 weeks respectively followed by the same corre-sponding number of weeks of abstinence. At the end of the

treatment period, the rats were sacriced after which bloodand tissues (testes) were collected for the various assays. Allexperimental protocols followed the guidelines approved by

the Ethics Committee of the College of Medicine, Universityof Lagos, Nigeria.

2.3. Parameters investigated

2.3.1. Semen analysis

The cauda epididymis of the rats was incised and a drop of

epididymal uid delivered onto a glass slide, covered by a22 22 mm cover slip and examined under the light micro-scope at a magnication of 100 while evaluating differentelds (68). For the purpose of this study, motility was classiedas either motile or non-motile/dead (44). After assessing differ-ent microscopic elds, the relative percentage of motile sperm

was estimated and reported to the nearest 5% using the subjec-tive determination of motility (31).

The sperm count was determined using the Neubauer im-proved haemocytometer. Epididymal uid ratio of 1:20 was

prepared by adding 0.1 ml of uid to 1.9 ml of water. The dilu-tion was mixed thoroughly and both sides of the countingchamber were scored and the average taken. Spermatozoa

within ve of the red blood cell squares including those whichlie across the outermost lines at the top and right sides werecounted, while those at the bottom and left sides were left

out. The number of spermatozoa counted was expressed inmillions/ml (31).

2.3.2. Biochemical estimations

The lipid peroxidation products were estimated by measuringTBARS and were determined by (43). Nonenzymatic antioxi-dants such as reduced glutathione and catalase were estimated

by Ellman (19) and Sinha (54) respectively.ular damage: Can abstinence equal recovery?, Middle East Fertil Soc J

2.3.4. Histological studies

in testicular weight was however, signicant (p< 0.001) only

regime when compared with the control, while it reduced

signicantly (p< 0.05) in the alcohol treated non-abstinentgroup (Table 3).

a bV

SS

L

V

c

SS

L

I

d

SS

Lf

SS

e

Figure 1 (a) Cross-Section of the testis of control rat showing

the seminiferous tubules containing cells of the spermatogenic

series (SS) and the lumen (L) containing spermatozoa; Long arrow

represents spermatogonium; P represents primary spermatocytes;

Short arrow represents spermatids and spermatozoa. (bd) Cross-

Section of the testis of rat treated with alcohol for two, four and

eight weeks respectively showing hypocellularity, reduction in cells

of the spermatogenic series (SS) as a result of degeneration,

sloughing and shortening of seminiferous epithelium; The semi-

niferous tubules show a single layer of basal spermatogonia;

widened empty lumen (L); widened interstitium (I) due to tubular

atrophy as a result of degeneration, and V shows vascular

haemorrage. (eg) Cross-Section of the testes of rat treated with

alcohol for two, four and eight weeks respectively followed by the

same corresponding number of weeks for recovery showing slight

reduction in the cells of the spermatogenic series (SS). (H & E;

400).

Alcohol induced testicular damage 3in the non-abstinent group (Table 1).

3.2. Sperm parameters

In the treatment groups, sperm count and motility were signif-icantly reduced (p< 0.001). However, in the abstinent groupsthe sperm count values were still within the normal range (IIIa:

23.20 9.21; IIIb: 30.21 11.12; IIIc: 35.22 8.58) as thenormal range for sperm count is generally considered to bebetween 20 106/ml and 250 106/ml (31). Compared withthe control, abnormal spermatozoa with head only, tail only,

double heads, short tail increased signicantly (p< 0.05) inall treatment groups (Fig. 2; Table 2).

3.3. Biochemical parameters

When compared with the control, tMDA level increased signif-icantly (p< 0.05) in all the treated animals.

In the 2 & 4-week abstinent regime tGSH levels increasedsignicantly (p< 0.05) and reduced signicantly in the 8-weekAt the end of the experimental periods, animals were sacriced

by cervical dislocation. The testes were harvested, xed in 10%formaldehyde solution, passed through ascending series ofethanol baths, cleared in xylene and embedded in parafn.

Tissues were sectioned at 5 lm and stained with Haematoxylinand Eosin (H&E) to observe the structure (58,69). The slideswere then examined at magnications of 400 under opticalmicroscope.

2.3.5. Morphometric studies

Testicular Diameter and Cross-Sectional Areas of seminifer-

ous tubules were determined by using a rectangular measure-ment frame as described in the protocols of (16).

2.4. Statistical analysis

Data were expressed as means SD. Statistical signicance ofdata was analysed by analysis of variance plus Bonferronispost-hoc test. p< 0.05 was considered signicant.

3. Results

3.1. Effect on histology

Sections of the seminiferous tubules of the control rats were

moderately circular or oval in outline with normal stratiedseminiferous epithelium showing cells of the spermatogenicseries and spermatozoa within the lumen (Fig. 1a). Seminifer-

ous tubules of animals treated with alcohol alone showedsevere reduction of cells of the spermatogenic series, hypocell-ularity in the interstitium, widening of tubular lumen, tubular

atrophy and decreased spermatozoa in tubular lumen. Animalsin the abstinent groups showed decrease in the cells of thespermatogenic series and some degree of hypocellularity(Fig. 1bg). Testicular diameter and cross sectional area

reduced signicantly in all groups when compared with thecontrol group receiving distilled water (p< 0.05). ReductionPlease cite this article in press as: Dosumu OO et al. Alcohol induced testic(2014), http://dx.doi.org/10.1016/j.mefs.2014.01.003SS

Lular damage: Can abstinence equal recovery?, Middle East Fertil Soc J

Table 1 Effect of alcohol consumption followed by abstinence on

4 O.O. Dosumu et al.Treatment groups TW (g)

CTRL 1.18 0.11

NABT 2 WKS 0.63 0.12b

NABT 4 WKS 0.47 0.06b

NABT 8 WKS 0.73 0.23a

ABT 2 WKS 0.77 0.06

ABT 4 WKS 0.87 0.123.4. Hormonal assay

Compared with the control, animals in the non-abstinentgroup had signicantly reduced TT (p< 0.05) levels whileLH and FSH levels were not signicantly changed. In the

abstinent group, TT levels as well as FSH and LH levels werenot signicantly different from the values of control (Fig. 3).

ABT 8 WKS 1.00 0.20

Signicance, a: p< 0.05; b: p< 0.001

Key: CTRL, Control; NABT, Non-abstinence; ABT, Abstinence; TW,

sectional area of seminiferous tubules; WKS, Weeks.

0

10

20

30

40

50

60

70

80

90

100

C 2WK A 2WK N 2WK C 4WK A 4 WK N

% A

naly

sis

of m

olit

y &

mor

phol

ogy

Treatment group

Figure 2 Effect of alcohol consumption followed by abstinence on sp

signicant. Key: C 2WK, Control 2 Weeks; A 2WK, Abstinence 2 Week

4WK, Abstinence 4 Weeks; N 4WK, Non-abstinence 4 Weeks; C 8WK

abstinence 8 Weeks.

Table 2 Effects of alcohol consumption followed by absti-

nence on sperm count.

Treatment groups Sperm count (106/ml)

CTRL 162.50 17.68*

NABT 2 WKS 4.85 3.18**

ABT 2 WKS 23.20 9.21**

NABT 4 WKS 18.00 3.46**

ABT 4 WKS 30.21 11.12**

NABT 8 WKS 14.50 20.93**

ABT 8 WKS 35.22 8.58**

Key: CTRL, Control; NABT, Non-abstinence; ABT, Abstinence;

WKS, Weeks.* Signicance at p< 0.05.** Signicance at p< 0.001.

Please cite this article in press as: Dosumu OO et al. Alcohol induced testic(2014), http://dx.doi.org/10.1016/j.mefs.2014.01.003testicular weight, diameter and cross-sectional area.

D (lm) AC (103 lm2)

181.00 4.84 25.80 1.38

103.00 11.40b 8.42 1.92b

96.20 7.47b 7.30 1.14b

85.70 11.90b 5.87 1.70b

111.00 5.53b 9.77 0.95b

114.00 9.76b 10.30 1.72b

128.00 13.80b 13.00 2.86b

Testicular weight; D, Diameter of seminiferous tubules; AC, Cross-

Molity (%)

Morphology (%)4. Discussion

Alcohol consumption reportedly causes testicular injury (32).Both in vivo and in vitro studies indicate that ethanol hasadverse effects on Leydig cell morphology and function and

impairs spermatogenesis (65). Our study shows histologicalabnormalities in testicular tissue of animals in the non-absti-nent group (Fig. 1bd) such as sloughing and shortening of

seminiferous epithelium leading to reduction in cells of thespermatogenic series. Observed degeneration and atrophy ofseminiferous tubules leading to reduction in seminiferous tes-ticular diameter and cross-sectional areas are all consistent

with the ndings of Martinez et al. (39) and Adaramoye andArisekola (2) who reported histological abnormalities in thetesticular tissue of alcoholic animals.

One of the effects of ethanol consumption on the testes isprobably a change in the structure of the mitochondria (16).Kiessling and Tobe (33) have reported these effects in the liver

following chronic alcohol consumption. In their report, theystated that the mitochondria were often elongated and dis-torted, appearing either as swollen or elongated structures with

the cristae often distorted and without normal organization.These reported structural changes may suggest the possibilitythat testicular energy metabolism was compromised by chronic

4 WK C 8 WK A 8 WK N 8WK

s and period

erm motility and morphology. c: p< 0.05 signicant; a: p< 0.001s; N 2WK, Non-abstinence 2 Weeks; C 4WK, Control 4 Weeks; A

, Control 8 Weeks; A 8WK, Abstinence 8 Weeks; N 8WK, Non-

ular damage: Can abstinence equal recovery?, Middle East Fertil Soc J

Table 3 Effect of alcohol consumption followed by abstinence on testicular malondialdehyde, catalase and glutathione.

S,

Alcohol induced testicular damage 5Treatment groups tMDA (nmol/min)

CTRL 2 WKS 8.40 0.76

NABT 2 WKS 20.21 0.66**

ABT 2 WKS 11.25 2.95

CTRL 4 WKS 8.20 1.11

NABT 4 WKS 23.57 2.99**

ABT 4 WKS 20.03 1.30**

CTRL 8 WKS 10.68 1.04

NABT 8 WKS 29.24 2.51**

ABT 8 WKS 18.03 2.31*

Key: CTRL, Control; NABT, Non-abstinence; ABT, Abstinence; WK* Signicance at p< 0.05.** Signicance at p< 0.001.

6ethanol consumption. The ethanol-related decrease in sperma-

tozoa viability (Fig. 2) as observed in the large number of non-motile/dead spermatozoa in the treated groups is one of theindicators that chronic ethanol consumption may compromise

the structural integrity of the spermatozoa via the mitochon-drial pathway.

Ethanol reportedly elicits a decrease in the capacity of the

mitochondria to carry out mitochondrial protein synthesisdue to alterations in mitochondrial ribosomes which makesthem less functional (12). This results in a depression in thetranslation of the oxidative phosphorylation associated poly-

peptides (11) leading to enzyme inactivation (47). Ultimately,this result in a myriad of alterations within the mitochondriawhich may promote both apoptotic and necrotic cell death,

thus contributing to the progression of alcohol-induced testic-ular damage as observed in the sloughing and degeneration ofgerminal epithelium and interstitial cells of the alcohol-treated

groups with marked effects in the non-abstinent group

0

1

2

3

4

5

CTRL1 NABT1 ABT1 CTRL2 NABT2 A

Hor

mon

al le

vels

of t

reat

ed ra

ts

Treatment gr

Figure 3 Effects of alcohol consumption followed by abstinence on s

values of control. Key: CTRL, Control; NABT, Non abstinence; ABT

order.

Please cite this article in press as: Dosumu OO et al. Alcohol induced testic(2014), http://dx.doi.org/10.1016/j.mefs.2014.01.003tCAT (lmol/mg protein) tGSH (lmol/min)

6.24 0.27 0.36 0.02

4.30 0.37* 0.12 0.01**

7.50 0.41* 0.58 0.03*

6.33 0.30 0.37 0.02

7.29 0.23* 0.15 0.03**

12.17 0.35** 0.48 0.01*

6.60 0.44 0.38 0.01

8.74 0.28* 0.15 0.02**

9.89 1.00* 0.22 0.04*

Weeks.(Fig. 1bd). In addition, reactive oxygen species (ROS) pro-

motes the inappropriate activation of the mitochondrialpermeability transition, leading the cells to pro-apoptotic path-ways (28,48). Hence, ethanol-induced elevation of germ cell

apoptosis together with necrosis and suppression of cell prolif-eration may contribute to testicular atrophy (70). These effectswere observed in the reduced tubular diameter and cross sec-

tional areas of the treated animals.The toxicological signicance of CYP2E1 was rst appreci-

ated when it was shown that this enzyme was responsible forthe metabolism of many compounds to toxic products, and

the toxicity was increased after synthesis of the enzyme was in-duced (36). Ethanol has been found to induce the CYP2E1form of cytochrome P450 enzyme, which metabolizes and acti-

vates many toxicological substrates, to more toxic products.Ethanol ingestion causes an increase in free radical generationin the testes associated with increase in CYP2E1 activity (40).

CYP2E1 catalyses the conversion of ethanol to acetaldehyde

BT2 CTRL3 NABT3 ABT3oups

TT (ng/ml)

LH (mIU)

FSH (mIU)

erum TT, LH and FSH concentration. y: p< 0.05 signicant from

, Abstinence; 1,2, and 3 represent weeks 2, 4 & 8 in corresponding

ular damage: Can abstinence equal recovery?, Middle East Fertil Soc J

process, hence promoting TT generation.

6 O.O. Dosumu et al.and at the same time reduces dioxygen to a variety of ROS,including O2

(35). These enhanced O2 and other ROS

increase the degree of lipid peroxidation which has been impli-

cated as a mechanism for testicular injury during alcohol inges-tion (16,59). The excess lipid peroxidation in the alcohol-treated groups as measured by the formation of thiobarbituric

acid-reactive substances (TBARS) in the present study corrob-orates these ndings. As a result, MDA level used to measurethe degree of peroxidative damage sustained by the spermato-

zoa was signicantly increased in these groups (Table 3) lead-ing to impaired sperm function, decreased sperm motility(34,3,6) and ultimately increased number of non-motile/deadspermatozoa as observed in the present study. Indeed, loss of

motility has been highly correlated with the lipid peroxidationstatus of the spermatozoa (25).

GSH is a critical cellular antioxidant and is important in

limiting the toxicity of ethanol and other toxic chemicals(14). From the present study, ethanol severely depleted GSHlevels in the non-abstinent groups (0.12 0.01; 0.15 0.03;

0.15 0.02). Studies have suggested that ethanol depletesGSH levels via the generation of oxidants as well as by inhib-iting the mitochondrial glutathione transporter (9,66). Inhibi-

tion of the transport of GSH from the cytosol into themitochondria leads to depletion in the mitochondrial pool ofGSH after ethanol intake (10).

In addition, the depletion of mitochondrial glutathione

(mGSH) upon impairment of the mitochondrial transportactivity leaves the mitochondria unprotected from the damag-ing effects of ROS. Consequently, this results in a selective

decrease in the mGSH stores (14) and this is sufcient to sen-sitize spermatocytes to TNF-a-mediated cell death. This couldexplain the signicant increase in the number of non-motile/

dead spermatozoa observed in the treated groups (Fig. 2). Inthe abstinent groups tGSH levels increased signicantly in allthe groups compared with the non-abstinent groups, however,

tMDA levels remained high. Lieber (36) has suggested that thetestes supply of GSH may be exhausted by binding to carcin-ogens produced during alcohol detoxication, hence, theabsence of ethanol feeding allowed for the sustenance of

GSH levels within the cells. It is also possible that a changein the structure of the mitochondria such as the enlargementand distortion described by Kiessling and Tobe (32) compro-

mised mitochondrial glutathione transport (22) thus resultingin mitochondrial dysfunction (13,56). This may also explainthe high tGSH levels despite very high tMDA levels.

Alcohol has been reported to reduce serum/plasma TTlevels in experimental animals (38,15,30,59,45). In men, lowandrogen levels have also been repeatedly associated with bothmoderate consumption and with chronic alcohol abuse in alco-

holics (24,49,50,51,52,63). The present study corroboratesthese reports as TT levels were signicantly reduced in thenon-abstinent groups while no signicant change was observed

in the abstinent group when compared with control. Reportshave stated that TT concentrations may be elevated after alco-hol withdrawal (7,27,64). Similarly in rodents, plasma TT lev-

els have been reported to decline during withdrawal aftermoderate ethanol exposure (5,34,53) but rebound after ethanolcessation (46). In their study Emanuele et al. (20) reported a

signicant increase in TT level during a 3-month recovery per-iod. Similarly, (45) noted that abstention ameliorated the del-eterious effects of ethanol on testicular steroidogenesis andhistomorphology though not as effective as treatment withPlease cite this article in press as: Dosumu OO et al. Alcohol induced testic(2014), http://dx.doi.org/10.1016/j.mefs.2014.01.003In conclusion the present study demonstrates that absti-nence following chronic consumption of alcohol does notcompletely reverse the deleterious effects of alcohol on the

testes.

Conict of interest

The authors declare that there is no conict of interest.

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Alcohol induced testicular damage: Can abstinence equal recovery?1 Introduction2 Materials and method2.1 Chemicals2.2 Animal experiments2.3 Parameters investigated2.3.1 Semen analysis2.3.2 Biochemical estimations2.3.3 Hormone determination2.3.4 Histological studies2.3.5 Morphometric studies

2.4 Statistical analysis

3 Results3.1 Effect on histology3.2 Sperm parameters3.3 Biochemical parameters3.4 Hormonal assay

4 DiscussionConflict of interestReferences