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Dietary antioxidant supplementation did not affectdeclining sperm function with age in the mouse but did

increase head abnormalities and reduced spermproduction

J Ten, Fj Vendrell, A Cano, Jj Tarín

To cite this version:J Ten, Fj Vendrell, A Cano, Jj Tarín. Dietary antioxidant supplementation did not affect decliningsperm function with age in the mouse but did increase head abnormalities and reduced sperm produc-tion. Reproduction Nutrition Development, EDP Sciences, 1997, 37 (5), pp.481-492. <hal-00900046>

Original article

Dietary antioxidant supplementationdid not affect declining sperm function with agein the mouse but did increase head abnormalities

and reduced sperm production

J Ten FJ Vendrell, A Cano, JJ Tarín*

Department of Paediatrics, Obstetrics, and Gynaecology, Faculty of Medicine,University of Valencia, Avda Blasco Ibanez 17, 46010 Valencia, Spain

(Received 7 April 1997; accepted 18 September 1997)

Summary - The present study aims to ascertain whether dietary administration of a mixture of vita-mins C and E may prevent the negative effects of paternal age on male fertility variables in themouse. Experimental males were fed a standard diet supplemented with either a low or high dose ofvitamins C and E. Oocytes enclosed in cumulus masses were inseminated using a 2 x 2 factorialdesign established according to whether males were young (3-4 months of age) or aged (22-24months of age) and whether they were fed a control or antioxidant diet. Aged males showed a sig-nificant decrease in number of spermatozoa/mg epididymis when compared to young males. Dietarysupplementation with low doses of vitamins C and E did not have any effect on sperm quality, fer-tilization and conceptus development in vitro. However, high doses of dietary vitamins C and Edecreased the number of spermatozoa/mg epididymis, and increased the percentage of spermatozoawith misshapen heads distended in the distal part. These findings suggest that dietary supplementa-tion with pharmacological doses of vitamins C and E may disturb spermatogenesis. The mechanisminvolved may be associated with either the antioxidant properties of vitamins C and E or theirinhibitory action on steroidogenesis by Leydig and/or Sertoli cells.

dietary supplementation / in vitro fertilization / paternal age / sperm quality / vitamins Cand E

Résumé - Un régime supplémenté en antioxydant n’empêche pas la décroissance de la sper-matogenèse en fonction de l’âge de la souris, mais induit des anomalies de la tête et mêmediminue la production du sperme. L’étude suivante a été entreprise pour savoir si l’administrationd’un mélange de vitamines C et E sur la diète peut éviter les effets négatifs de l’âge paternel sur les

* Correspondence and reprintsTel: (34) 6 386 41 70; fax: (34) 6 386 4815; e-mail: tarinjj@uv.es

paramètres de fertilité masculine sur la souris. Les mâles du groupe expérimental ont été alimentésavec un régime standard, augmenté d’une dose faible ou forte de vitamines C et E. Les femelles ontété inséminées en utilisant un schéma factoriel 2 x 2, établi en fonction de l’âge des mâles (jeunes :3-4 mois, ou âgés : 22-24 mois) et ont été alimentés avec un régime témoin ou additionné d’anti-oxydant. Les mâles âgés ont montré une diminution significative du nombre de spermatozoïdes/mgd’épididyme par rapport aux jeunes mâles. La supplémentation du régime avec une dose faible de vita-mines C et E n’a eu aucun effet sur la qualité du sperme, la fécondation, et le développement duconceptus in vitro. Pourtant, une dose élevée de vitamines C et E a diminué le nombre de spermato-zoïdes/mg d’épididyme et augmenté le pourcentage de spermatozoïdes malformés à têtes disten-dues dans la partie distale. Ces résultats indiquent que des doses pharmacologiques de vitamines Cet E peuvent altérer la spermatogenèse grâce à leurs propriétés antyoxydantes potentielles ou à leuraction inhibitrice sur la stéroïdogenèse par des cellules de Leydig ou de Sertoli.

âge paternel / fécondation in vitro / qualité du sperme / supplément diététique / vitamines Cet E

INTRODUCTION

There are many studies that show negativeeffects of paternal age on male fertility. Age-ing in human testes is associated with vas-cular degeneration, and a reduction in num-ber and function of Leydig cells, intersticialcells, myoid cells and Sertoli cells. Thesechanges are accompanied by reduction inthe capability to produce testosterone, andrising plasma levels of luteinizing hormone(LH), follicle stimulating hormone (FSH)and oestradiol (Johnson, 1986; see Cum-mins et al, 1994 for a review). Increases inLH and FSH plasma levels, together withreduction in the number of Sertoli cells, arecorrelated with a decrease in daily spermproduction which, in turn, is associated witha major degeneration of germ cells (Johnsonet al, 1984; Johnson, 1986, 1989). Further-more, as testicular vascularity decreases,spermatogenic failure increases, which mayresult in a reduction in sperm output in rats(Auroux et al, 1985) and humans (Johnson,1989). The study of age-related histologicchanges in the adult mouse testis reveal theappearance of vacuoles in the seminiferous

epithelium after 6 months of age, with atro-phied tubules that initially appear in patchesand then spread throughout the testis. Thethickness of the basement membrane sur-

rounding the atrophied tubules is positively

correlated with the degree of atrophy(Takano and Abe, 1987). With increasingpaternal age, spermatozoa may show mor-phological abnormalities during their devel-opment. They may degenerate and bephagocytozed by Sertoli cells, which showage-associated lipid accumulations withintheir cytoplasm (Lynch and Scott, 1950;Paniagua et al, 1987).

The study of the reproductive perfor-mance of C57BL/6NNia male mice (Parken-ing et al, 1988), revealed that only 42% ofaged males mated versus 100% in youngmales, with 65% siring young versus 96% inyoung males. Aged males that failed to matehad atrophied testes, fewer and less motilesperm, lower testosterone levels and a highpercentage of degenerating epithelium liningseminiferous tubules. A longitudinal analy-sis of 2- to 30-month-old CBF1 male miceshowed progressive losses in mating suc-cess and sperm production between 18 and30 months, with decreased levels of serumLH and testosterone (Bronson and Des-jardins, 1977). In another study, Parkening(1989) found that aged males that did notmate or mated without producing offspring,showed a reduced number of spermato-zoa/mg epididymis, a decreased spermmotility, increased percentage of abnormalspermatozoa and decreased fertilization rate

and number of concepti reaching the blas-tocyst stage.

According to the ’free radical theory ofageing’ (Harman, 1956), free radicals, gen-erated metabolically or accidentally by ion-izing radiation, are the main factors forexplaining the degenerative ageing process(Harman, 1981 ). Miquel and co-workersestablished an important variation to thistheory, assigning a key role in the senes-cent process to the damage exerted by oxy-gen radicals to mitochondria (Miquel andFleming, 1986). In this way, reactive oxygenspecies (ROS) such as the superoxide anion(OZ ), hydrogen peroxide (H202) and thehighly toxic hydroxyl free radical (OH.),are produced continuosly in mitochondriabecause of the ’leakage’ of high energy elec-trons along the electron transport chain.Mitochondrial proteins and lipids can bedamaged, but mitochondrial DNA (mtDNA)is the main target for oxidative attackbecause of its location near the inner mito-chondrial membrane sites where the oxi-dants are formed. Furthermore mtDNA lacks

protective histones and has no DNA repairactivity (for a review, see Shigenaga et al,1994). ).

Age-associated oxidative stress may beprevented by antioxidants such as vitaminsC and E. Fraga et al (1991), reported a pro-tective effect of dietary intake of vitamin Cagainst endogenous oxidative damage tohuman sperm DNA. Dawson et al (1987)reported an improvement in sperm quality inmen with sperm agglutination in excess of25% after supplementing their diets withvitamin C. An improvement in sperm qual-ity of heavy smokers was also reported afterascorbic acid supplementation (Dawson etal, 1992). Furthermore, oral administrationof vitamin E significantly improved spermfunction as assessed by the zona bindingtest (Kessopoulou et al, 1995) and fertiliza-tion rate (Geva et al, 1996).

In the present study, we aim to ascertainwhether dietary administration of a mixture

of vitamins C and E may prevent the nega-tive effects of paternal age on sperm quality,fertilization, and conceptus development invitro in the mouse.

MATERIALS AND METHODS

Animals, dietary supplementsand oocyte retrieval

F1 hybrid male and female mice (C57BL/6JIcofemale X CBA/Jlco male) were given a standarddiet (containing 590 g carbohydrates, 30 g lipidsand 160 g protein/kg diet) and tap water ’ad libi-tum’. In the experimental group, males were fedthe same standard diet but supplemented withan antioxidant mixture of vitamin C and vitaminE. In a first set of experiments, diet was supple-mented with 0.025 g vitamin C/kg and 0.06 gvitamin E/kg. These supplements resulted in anaverage daily intake of 0.1 mg vitamin C and0.24 mg vitamin E/kg body weight. These dosesare based on several studies of human beings atthese concentrations (Fraga et al, 1991;Kessopoulou et al, 1995). Males were fed thisdiet from the second month of life until theirdeath at 4 months of age (young males) or duringthe last 3 months before their death at 22-24months of age (aged males). In a second set ofexperiments, males were fed an antioxidant mix-ture containing 10 g vitamin C/kg diet and 0.6g vitamin E/kg diet. These supplements resultedin an average daily intake of 1 600 mg vitamin Cand 96 mg vitamin E/kg body weight. This par-ticular mixture of vitamins was chosen based ona previous study demonstrating a protective effectof this mixture against GSH oxidation in bloodand mitochondria from livers and brains of agedrats (De-la-Asunci6n et al, 1996). Young maleswere fed this diet from the second month of lifeuntil their death at 4 months of age, and agedmales during the 7 months before their death at22-24 months of age. Females fed a control dietwere superovulated at 2 months of age by anintraperitoneal injection of 10 0 IU pregnant mare’s sserum gonadotrophin (PMSG) (Folligon; Inter-vet-Intemational, Boxmeer, Holland) followed48 h later by 10 IU of human chorionic

gonadotrophin (hCG) (Chorulon; Intervet-Inter-national). They were killed by cervical disloca-tion 14 h after the hCG injection. Their oviductswere excised and placed into 2 mL medium M2(Quinn et al, 1982) where they were supple-

mented with 4 mg bovine serum albumin (BSA;Fraction V, Sigma Chemical Co, St Louis, MO,USA)/mL. Oocytes enclosed in cumulus masseswere released from the ampullae, washed andtransferred directly to insemination droplets.

Fertilization and conceptusdevelopment in vitro

In each experiment, four males (two young andtwo aged, fed a standard or antioxidant diet) werekilled by cervical dislocation 2 h before insemi-nation. Cauda epididymes were weighed, excisedand placed into 0.5 mL medium T6 (Quinn et al,1982) supplemented with IS mg BSA/mL. Tenminutes later, the caudae were discarded and thesperm solution adjusted to a concentration of 5 x106 motil spermatozoa/mL. Spermatozoa werethen incubated until insemination in 20 pLdroplets overlaid with mineral oil in an atmo-sphere of 5% C02 in air at 37 °C. Oocytesenclosed in cumulus masses from four differentfemales were inseminated using a 2 x 2 facto-rial design. At 8 h after insemination, oocytes ofeach group were washed, transferred, and incu-bated in groups of ten into 20 pL droplets ofmedium M 16 (Whittingham, 1971 At this time,oocytes were assessed for fertilization, by observ-ing if they extruded a second polar body (PB)and had two pronuclei. Cleavage was monitoredevery 24 h for 5 days.

Sperm vitality

To evaluate sperm vitality, sperm was stained ina 1:1 aqueous solution containing 7.23 mM yel-low eosin (Sigma Chemical Co) and 154 mMNaCI. A minimum of 100 spermatozoa per groupwas observed using a x40 objective. Eosin doesnot pass through intact membranes and so liv-ing spermatozoa remain colourless.

Sperm morphology

Ten microlitres of sperm suspension wereextended on microscope slides and were air dried.They were then fixed for 1 h in Carnoy (abso-lute ethanol/glacial acetic acid, 3:1, v/v) andstained for 5 min with an aqueous solution con-

taining 5% yellow eosin. Slides were then washed

with running water and air dried. A minimum of200 spermatozoa per group was observed (usingx100 immersion objective) to assess their mor-phology. Spermatozoa were considered abnor-mal if they contained head malformations cor-responding to classification 1-5 as reported byKrzanowska (1981) or if they contained morethan one flagellum or an unusually shaped flag-ellum. According to Krzanowska’s sperm clas-sification, class 1 corresponds to spermatozoawith almost normal heads but with a changedcurvature in the distal part of their heads (la) orwith slightly bent acrosomes (lb). Class 2 cor-responds to spermatozoa with heads of com-pletely normal shape, except that they possesscanals of low stainability, situated transversely,obliquely or longitudinally to the long axis oftheir heads (2a). The most typical form of thisclass has a narrow, condensed head and containsa longitudinal canal of very low stainability (2c);sometimes the canal is not visible (2b). Class 3corresponds to misshapen and flat heads dis-tended in the apical part (3a) or in the distal part(3b). Spermatozoa of class 4 have severely mis-shapen heads showing abnormalities in distri-bution of chromatin (4a); some of these headspossess vacuoles or short canals of less stainablematerial (4b). Finally, spermatozoa of class 5have severely misshapen ’thread-like heads’.

Fluorescence staining and countingof nuclei

In some experiments, concepti that had reachedthe 16-cell or early blastocyst stage at 81 h post-insemination were fixed and their nuclei labelled.After removing their zona pellucidae with Tyrodeacid (pH 2.4), concepti were fixed and labelledovernight in absolute alcohol containing 94 pMbisbenzimide (Hoechst 33258; Sigma ChemicalCo) at 4 °C. They were then washed for 1 h inabsolute alcohol and mounted on microscopeslides in glycerol beneath a coverslip and care-fully squashed using a pencil rubber. The num-bers of nuclei in each conceptus were thencounted. Nuclei showing metaphase chromo-somes were counted as single cells. The mitoticindex of each conceptus was defined as the per-centage of the number of metaphases/total num-ber of cells.

Statistical analysis

Two-way analysis of variance (Anova) wasapplied for comparisons of means. Significancewas defined as P < 0.05. The entire statistical

analysis was carried out using the StatisticalPackage for Social Sciences (SPSS). ).

RESULTS

Tables I and II show the effect of paternalage and dietary supplementation with lowdoses of vitamins C and E on sperm quality,fertilization and conceptus development invitro. The number of spermatozoa/mg epi-didymis (P < 0.05) (table I), percentage ofspermatozoa with misshapen heads dis-tended in the apical part (3a) (P S 0.005)(table I), and mitotic index (P < 0.05) (tableII) decreased significantly with paternal age.Dietary supplementation with low doses ofvitamins C and E did not have any effecton seminal, fertilization or conceptus devel-opment parameters.

Tables III and IV show the effect of

paternal age and dietary supplementationwith high doses of vitamins C and E onsperm quality, fertilization and conceptusdevelopment in vitro. Paternal age was asso-ciated with decreased number of spermato-zoa/mg epididymis (P 5 0.05) (table III).Dietary supplementation with high doses ofantioxidants decreased the number of sper-matozoa/mg epididymis (P < 0.05) andincreased the percentage of spermatozoawith misshapen heads distended in the dis-tal part (3b) (P < 0.05) (table III). A similarincrease in number of spermatozoa withmisshapen ’thread-like heads’ (type 5) wasobtained, but it was ignored because ofuncertainty in the data due to the small num-ber of misshapen spermatozoa recorded. Noeffect of diet on fertilization and conceptusdevelopment was observed (tables III andIV).

DISCUSSION

The present study shows a decrease in num-ber of spermatozoa/mg epididymis withpaternal age. This result is in agreement witha previous study by Parkening (1989) whichalso demonstrated an age-associateddecrease in number of spermatozoa/mg epi-didymis.

A reduction in number of spermato-zoa/mg epididymis may be due to a decreasein number and function of Sertoli cells, sinceeach of them can support only 28 germ cells(28:1 ) (Johnson, 1986, 1989). Gosden et al(1982) reported differences in structure andgametogenic potential of seminiferoustubules in ageing mice, which affected thespermatogenesis process. Auroux et al(1985), in their work on testicular ageing inthe Wistar rat, found a decrease in testicular

vascularity with age. This, in turn, mayincrease spermatogenic failure and reducesperm output (Johnson, 1989). Patemal-age-associated oxidative stress may explain also,at least in part, the decreased number ofspermatozoa/mg epididymis found in thepresent study.

The mitochondrial genome of post-mitotic somatic cells is a key target for freeradicals and thus for the ageing process(Miquel and Fleming, 1986). Accumulationof oxygen radical damage to mitochondriamay be especially patent in post-mitotic dif-ferenciated cells such as Sertoli cells and

Leydig cells. There are many studies sug-gesting the presence of oxidative stress inthese cells. Ageing of Leydig cells is asso-ciated with defective mitochondrial func-tion and steroid metabolism (Kinoshita etal, 1985). Moreover, in humans and micethere is age-associated accumulation of lipo-fuscin pigment (insoluble end-product ofROS-induced lipid peroxidation) in Leydigand Sertoli cells. Elastin deposits may alsoappear in the lamina propia of the ductuliefferentes and epididymis as well as lipo-fuscin accumulations in the principal cells of

the epididymis (see Cummins et al, 1994for a review).

The present study showed no effects onsperm quality, fertilization and conceptusdevelopment in vitro when the diet was sup-plemented with low doses of vitamins C andE. However, high doses of vitamins hadnegative effects on sperm quality. In fact,the number of spermatozoa/mg epididymisdiminished and the percentage of sperma-tozoa with misshapen heads distended inthe distal part increased in males fed a diet

supplemented with pharmacological dosesof vitamins C and E.

Vitamins C and E, are considered ’antiox-idants’ but in fact they are redox (reducing-oxidizing) agents, and, in some circum-stances, they can act as pro-oxidants(Herbert, 1994, 1996; Schwartz, 1996). Nor-mally, an antioxidant effect appears whencells are exposed to physiological concen-trations found in food, whereas a pro-oxi-dant effect appears at higher concentrations(Herbert, 1994). Thus, the high doses ofantioxidants used in this work may have

produced a pro-oxidant effect on the Ser-toli and/or Leydig cells and so have dis-turbed the process of spermatogenesis. How-ever, it is necessary to bear in mind that this

particular ’cocktail’ of vitamins protectsagainst GSH oxidation in blood and mito-chondria from livers and brains of aged rats(De-la-Asunci6n et al, 1996). Furthermore,we have demonstrated that this mixture ofvitamins C and E prevents age-associateddecrease in ovulation rate after exogenousovarian stimulation and precludes the con-comitant increase in percentage of aneu-ploid and diploid oocytes in the mouse(Tarin JJ et al, unpublished). This body ofevidence suggests, therefore, that dietarysupplementation with pharmacological dosesof vitamins C and E may have an antioxidanteffect on sperm and/or Sertoli and/or Leydigcells. However, this antioxidant mixturemay act by other mechanisms rather thankeeping cells in a reduced state. In fact, the

presence of high concentrations of vitaminC in the testes may disturb steroidogenesisby the Leydig and Sertoli cells and so inducea drop in number of spermatozoa/mg epi-didymis as well as an increase in percent-age of spermatozoa with misshapen headsdistended in the distal part. This notion is

supported by several studies reporting thathigh concentrations of ascorbic acid mayinhibit ovarian and adrenal steroidogenesis(for a review, see Levine and Morita, 1985).

In humans, there exists a negative corre-lation between sperm morphology, fertil-ization rate (Zenzes et al, 1985; Kruger etal, 1988) and pre-implantation conceptusdevelopment (Ron-El et al, 1991; Parinaudet al, 1993; Janny and Menezo, 1994). How-ever, in mice treated with high doses ofvitamins C and E, we did not find any asso-ciation between the presence of spermatozoawith misshapen heads distended in the dis-tal part, fertilization and conceptus devel-opment. This may be due to the relativelyhigh concentration of spermatozoa used atinsemination together with the relativelylow incidence of abnormal forms observedin the present study. This effect would bereinforced by the fact that misshapen sper-matozoa are selected against at fertilizationby the zona pellucida (Kot and Handel,1987; Menkveld et al, 1991) and/or cumuluscells (Krzanowska and Lorenc, 1983).

The present study shows that paternalage does not affect the percentage of fertil-ized oocytes that reach the blastocyst stagein vitro. The only significant difference withrespect to conceptus development in vitrobetween young and aged males is the nega-tive effect of paternal age found in themitotic index of concepti 81 h post-insemi-nation in the first set of experiments withlow doses of vitamins C and E. This effect,however, may be an artefact because it wasnot found in the experiments made with highdoses of vitamins C and E.

In summary, the present study i) con-firmed previous studies demonstrating that

paternal age decreased the number of sper-matozoa/mg epididymis; and ii) showed thatdietary supplementation with high doses ofvitamins C and E decreased the number of

spermatozoa/mg epididymis and increasedthe percentage of misshapen spermatozoawith distended heads in the distal part. Itremains to elucidate whether the negativeeffects of high doses of vitamins C and Eon spermatozoa are due to their potentialantioxidant effect or their inhibitory actionon steroidogenesis by Leydig and/or Ser-toli cells.

ACKNOWLEDGEMENTS

This study was supported by a grant from theMinisterio de Sanidad y Consumo to AC (FIS93/0680).

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