Respiratory chain complexes and membrane fatty acids composition

in rat testis mitochondria throughout development and ageing

Martha E. Vazquez-Memijea,*, Marıa J. Cardenas-Mendeza, Adela Tolosaa,

Mohammed El Hafidib

aInstituto Mexicano del Seguro Social, Unidad de Investigacion Medica en Genetica Humana, Centro Medico Nacional Siglo XXI-IMSS.

Apdo Postal 73-032, Mexico, DF CP 06725, MexicobDepartamento de Bioquımica, Instituto Nacional de Cardiologıa, Mexico, DF, Mexico

Received 20 December 2004; received in revised form 11 March 2005; accepted 11 March 2005

Available online 7 April 2005

Abstract

Throughout the maturation of germ cells, a morphological, biochemical and functional differentiation of mitochondria has been shown to

occur. Ageing is known to cause changes involved in energy metabolism. These changes have been related to molecular and functional

alterations in the properties of biological membranes. Variations in membrane lipid composition and lipid–protein interactions occur with

ageing in several tissues. The present paper describes the relationship between these membrane alterations and the activities of lipid-

dependent enzymes of isolated testis mitochondria in rats of from 10 days of age to 24 months. The specific activities of these enzymes are

lower in preparations from adult and aged rats as compared to those from young rats. Temperature breaks of Arrhenius plots show age-

dependent shifts to higher temperatures for the NADH-dehydrogenase, succinate-dehydrogenase, cytochrome c oxidase, and ATPase in

senescent animals. Analysis of the membrane fatty acid composition reveals a distinct age-dependent fall in the content of polyunsaturated

fatty acids accompanied by an increase in the proportion of saturated fatty acids and a decrease in polyunsaturated fatty acid percentage. The

results suggest that during spermatogenesis and the ageing process some changes in the composition of the fatty acids in the surrounding

membrane affect the protein–lipid interactions, producing a decrease in mitochondrial enzyme activities.

q 2005 Elsevier Inc. All rights reserved.

Keywords: Ageing; Fatty acids composition; Spermatogenesis; Testis mitochondria; Respiratory chain complexes

1. Introduction

Mitochondria from various animal tissues or organs

show considerable differences in morphology and function.

The morphology and energy metabolism of testicular

mitochondria change markedly during spermatogenesis.

Morphologically, at least three different types of mitochon-

dria are present: the usual cristae orthodox-type mitochon-

dria in Sertoli cells, spermatogonia, preleptotene and

leptotene spermatocytes; the intermediate form of mito-

chondria in leptotene and zygotene spermatocytes; and the

condensed mitochondria form in pachytene spermatocytes,

0531-5565/$ - see front matter q 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.exger.2005.03.006

* Corresponding author. Tel.: C52 555627 6941; fax: C52 555588 5174.

E-mail address: elisavazquezm@prodigy.net.mx (M.E. Vazquez-

Memije).

spermatids and spermatozoa (Seitz et al., 1995). The mature

testis, as compared with the immature, has a lower oxygen

consumption and a different rate of aerobic and anaerobic

glycolysis. ATP production is primarily dependent upon

glucose metabolism in the mature but not in the prepuberal

testis (Grotegoed and Den Boer, 1990). On the other hand,

there is a marked change of ATPase (Ca2C-Mg2C) activity

in rat testis from birth to maturity (Delhumeau et al., 1973);

adult rat testis mitochondria are partially uncoupled and the

mitochondrial ATPase exhibits peculiar features, in that part

of the F1 portion is loosely bound to the membrane and its

sensitivity to oligomycin is relatively low (Vazquez-Memije

et al., 1984). This ATPase is not stimulated by either DNP or

FCCP. However, it is capable of performing ATP synthesis

(Vazquez-Memije et al., 1988).

The ageing process is the accumulation of oxidative

damage to cells and tissues associated with a progressive

increase in the chance of morbidity and mortality (Harman,

Experimental Gerontology 40 (2005) 482–490

www.elsevier.com/locate/expgero

M.E. Vazquez-Memije et al. / Experimental Gerontology 40 (2005) 482–490 483

1956, 1972; Beckman and Ames, 1998; Junqueira et al.,

2004). It is a complex process exhibiting a large variety of

changes at each level of biological organization. Structural

and functional alterations of mitochondria have been

proposed to play a critical role in the cellular ageing process,

since mitochondria provides energy for basic metabolic

processes. It is a common observation that aged humans and

animals tend to be less energetic. Several studies have

demonstrated an age-related decrease of respiratory chain

enzymes (Kwong and Sohal, 2000; Navarro, 2004) and ATP

synthase activity (Guerrieri et al., 1992). The phospholipid

composition (Paradies et al., 1992), as well as the structure

and function of some enzymes of the inner mitochondrial

membrane, seem to be altered also in mitochondria of aged

rats (Paradies et al., 1992; Muller-Hocker et al., 1997;

Okuyama et al., 1998; Pepe et al., 1999; Chvojkova et al.,

2001; Bourre, 2004).

The membrane lipid composition has long been known to

play a regulatory role in determining the activities of

mitochondrial membrane bound enzymes (Daum, 1985;

Crider and Xie, 2003). The special requirement of most of

these enzymes for specific phospholipids with a defined

degree of unsaturation, emphasizes the sensitivity of those

functional proteins to biochemical events that bring about

the disruption of unsaturated fatty acid composition

(Parenti-Castelli et al., 1979; Daum, 1985; Ostrander

et al., 2001; Gohil et al., 2004).

This paper attempts to evaluate the role of unsaturated

fatty acids in age-dependent activity changes of mitochon-

drial enzymes associated with the inner membrane in

mitochondria of rat testis. The results show a marked

changes in specific activities of enzymes from rat testis

mitochondria throughout development and ageing, that

could be attributable to alterations in the physical state of

the surrounding phospholipids.

2. Materials and methods

Reagents of the highest purity available were purchased

from Sigma (St Louis, MO). Sprague–Dawley strain male

albino rats of different ages were used. Rat testis

mitochondria were isolated in 250 mM sucrose, 3 mM

TEA, 1 mM EDTA, pH 7.4, by a method previously

described (Vazquez-Memije et al., 1988) and stored in

250 mM sucrose at 30–40 mg/ml atK70 8C.

2.1. Enzyme activities

NADH-dehydrogenase (NADH-DH) activity was

measured following the oxidation of 0.2 mM NADH at

340 nm in a medium consisting of 35 mM potassium buffer,

pH 7.5 and 1.7 mM potassium ferricyanide. The extinction

coefficient of 6.22 mMK1 cmK1 for NADH was used.

NADH-cytochrome c oxidoreductase (NADH-CCR)

activity was determined in a medium containing: 25 mM

potassium phosphate buffer, pH 7,5; 0.1 mM cytochrome c,

0.2 mM NADH, 0.5 mM KCN in the presence of 1 mM

rotenone.

Succinate dehydrogenase activity (SDH). The reaction

mixture consisted of 50 mM potassium phosphate buffer pH

7.0, 1 mM KCN, 0.05 mM dichlorophenolindophenol and

16 mM succinate. Absorbance changes were followed at

600 nm, using an extinction coefficient of 19.1 mMK1cmK1

for dichlorophenolindophenol.

Succinate-cytochrome c oxido-reductase (SCCR). The

activity was measured in the presence of 0.1 mM cyto-

chrome c, 0,5 mM KCN, 3 mM succinate and potassium

phosphate buffer at pH 7.5. Absorbance changes were

followed at 550 nm.

The cytochrome c oxidase (COX) activity was deter-

mined by a spectrophotometric method, using 0.1% reduced

cytochrome c in 10 mM potassium buffer, pH 7.0.

Absorbance changes were followed at 550 nm, using an

extinction coefficient of 18 mMK1 cmK1 for cytochrome c.

In some experiments the activity of cytochrome c

oxidase (COX) was determined by the polarographical

measurement of oxygen consumption using a micro Clark-

type electrode in a phosphate buffer (50 mM), pH 7.2,

containing 30 mM cytochrome c, 5 mM sodium ascorbate,

and 0.5 mM N,N,N 0,N 0-tetramethyl-p-phenylenediamine

dihydrochloride (TMPD).

ATPase activity was assayed either by a spectrophoto-

metric method in which ATP is regenerated through the

action of pyruvate kinase and phosphoenolpyruvate, or by

determining Pi released from ATP. Protein was estimated

according to Lowry et al. (1951).

2.2. Lipid isolation and analysis

Lipids were extracted by the method of Folch et al.

(1957). Briefly, 10 mg of mitochondrial protein in the

presence of 100 mg diheptadecanoylphsphatidylcholine, as

an internal standard, was extracted with a mixture of CHCl3/

MeOH (2/1 v/v) containing 0.005% of BHT (Butylated

hydroxy toluene) to avoid the peroxidation of polyunsatu-

rated fatty acids. The bottom layer was evaporated until dry

and the lipids were dissolved in a known volume of CHCl3.

Neutral lipids were separated from phospholipids by thin

layer chromatography on silica gel 60G (Merck, Mexico)

using a mixture of hexane-ethyl acetate-formic acid (20/80/

1 v/v) as an elution solvent. The phospholipids were trans-

esterified by mixing them with 2% anhydrous H2SO4 in

MeOH for 1 h at 80 8C. The fatty acid composition of testis

mitochondria was determined by gas chromatography using

a CarloErba fractovap 2301 gas chromatograph provided

with a 30 m!0.25 mm SP3320 capillary column (Supelco,

PA, USA) and equipped with a flame ion detection system

(FID). The analysis was performed at an isotherm

temperature of 200 8C. The injection temperature was

250 8C. Fatty acid methyl esters were identified comparing

M.E. Vazquez-Memije et al. / Experimental Gerontology 40 (2005) 482–490484

their retention time with commercial standards as described

elsewhere (El Hafidi et al., 2001).

2.3. Statistical analysis

Statistical analysis was performed with the SPSS

statistical software. Data are expressed as meanGSD.

Statistical significance was assessed by using one-way

ANOVA. Differences were considered statistically signifi-

cant at p!0.05. Pearson’s correlation analysis was used to

examine the relationship between fatty acid percentage and

enzyme activity.

NA

DH

-DH

(nm

oles

/min

/mg

port

ein)

0

200

400

600

800

1000

1200

SD

H (

nmol

es/m

in/m

g pr

otei

n)

0

20

40

60

80

CO

X (

nmol

es/m

in/m

g pr

otei

n)

0

200

400

600

800

1000

Citr

ate

synt

hase

(nm

ol/m

in/m

g pr

otei

n)

0

100

200

300

400

500

A

C

E

G

10 d

ays

16 d

ays

21 d

ays

12-1

4 m

onth

s

24 m

onth

s

3 m

onth

s

Fig. 1. Activities of respiratory-chain enzymes: NADH dehydrogenase (A), NA

cytochrome c reductase (D), cytochrome c oxidase (E), oligomycin sensitive ATP

different ages. Activities were measured as described in Section 2. MeansGSD o

3. Results

To examine the role of mitochondria throughout sperma-

togenesis and during the ageing process, we determinated

whether the activity of some complexes of the oxidative

phosphorylation are affected and follow a common pattern.

To do this we measured the activity of NADH-DH

(Cx:complex I), NADH-CCR (Cx I/III), SDH (Cx II),

SCCR (Cx II/III), COX (Cx IV) and oligomycin-sensitive

ATPase (Cx V) in rat testis mitochondria at different ages.

We also measured, as control of non-membrane enzyme, the

activity of citrate synthase (Fig. 1G), an enzyme from

NA

DH

-CC

R

(nm

oles

/min

/mg

prot

ein)

0

50

100

150

200

250

300

SC

CR

- (n

mol

es/m

in/m

g pr

otei

n)

0

10

20

30

40

50

60

ATP

ase

(nm

oles

Pi/m

in/m

g pr

otei

n)

0

100

200

300

400

500

600

700

B

D

F

10 d

ays

16 d

ays

21 d

ays

12-1

4 m

onth

s

24 m

onth

s

3 m

onth

s

DH-cytochrome c reductase (B), succinate dehydrogenase (C), succinate

ase (F), and citrate synthase (G) in mitochondria isolated from rat testis at

f at least five experiments.

3.1 3.2 3.3 3.4 3.51.5

2.0

2.5

3.0

3.5D

37.0°

34.8°

1/T x 10-3

3.0 3.2 3.4 3.6

1.0

1.5

2.0B

36.53°

27.23°

1/T x 10-33.0 3.1 3.2 3.3 3.4 3.5

1.5

2.0

2.5

3.0

3.5A

31.80°

30.95°

Log(

spec

ific

activ

ity)

Log(

spec

ific

activ

ity)

Log(

spec

ific

activ

ity)

Log(

spec

ific

activ

ity)

1/T x 10-3

3.1 3.2 3.3 3.4 3.51.0

1.5

2.0

2.5

3.0C

34.63o

30.66o

1/T x 10-3

Fig. 2. Arrhenius plots of NADH-DH (A), SDH (B), COX (C) and ATPase (D) activities in mitochondrial preparations from adult (B) and senescent rats (C).

The specific activity is calculated as nmoles/min/mg protein. The arrows indicate the break (transition temperature in 8C) in the Arrhenius plots. The x axis is

the reciprocal of assay temperature in absolute degrees.

M.E. Vazquez-Memije et al. / Experimental Gerontology 40 (2005) 482–490 485

the mitochondrial matrix. As shown in Fig. 1, the young rats

(21 day-old) exhibited the highest activity of the respiratory

chain enzymes, with the exception of complex IV and

complex I/III, where maximal activities were observed at 10

days of age. The activity decreased between 11–30% in the

adult age and even more in the 24 month-old animals.

Complexes IV and V showed a higher reduction (50%) from

young to adult age as compared to the other complexes. The

complex IV activity, determined by polarographic (unpub-

lished data) and spectrophotometric methods, followed the

same behaviour; the prepubertal rats (10 day-old) showed

maximal activity which readily decreased with ageing

(Fig. 1E). Citrate synthase activity remained unchanged

Table 1

Comparison of the activation energies of some mitochondrial enzymes from adul

Enzymes Activation energies (kcal/mol)

Adult

Below Above

Transition

NADH-dehydrogenase 6.22G0.6 5.19G0.81

Succinato dehydrogenase 2.56G0.58 5.94G0.79

Cytochrome c oxidase 2.67G0.5 7.84G0.57

ATPase 7.65G0.63 10.17G1.28

Data represent the mean of 3 different determinationsGSD.

until the adulthood. In aged animals, the activity decreased

around 50%. (Fig. 1G).

It has been reported that membrane fluidity may have a

direct influence on the activity of some membrane-

associated enzymes. Since Arrhenius plots of the activity

of these enzymes may reflect changes in lipid–protein-

interaction and membrane fluidity (Gorgani et al., 1986;

Baracca et al., 1986), the behaviour of some enzymes at

different temperatures was studied. Fig. 2 shows the

Arrhenius plots of complexes I, II, IV and ATPase in

adult and senescent rats. Sharp changes in activation

energies became apparent for all enzymes studied

(Table 1). The temperature breaks of mitochondria from

t and old rats

Old

Below Above

Transition

10.70G0.34 1.45G0.41

2.51G0.43 4.90G1.05

10.24G0.33 2.32G0.64

6.36G0.66 11.80G0.45

M.E. Vazquez-Memije et al. / Experimental Gerontology 40 (2005) 482–490486

either 3 month-or 24 month-old rats were specific for each

particular enzyme. Furthermore, the discontinuities in the

Arrhenius plots of the corresponding enzymes from

preparations of adult and old rats exhibited significant

differences, with shifts to higher temperatures in the

mitochondria from old rats, indicating a more rigid state

of the membrane. The differences were less apparent in the

NADH-dehydrogenase complex (DtZ0.85 8C), whereas

discontinuities in the Arrhenius plot of complex II, complex

IV and complex V were found to be quite distinct

(DtZ9.30, 3.97 and 2.20 8C, respectively) in aged animals.

Besides these changes, specific activities of the particular

enzymes were lower. ATPase and COX activities in

mitochondria from 24 month-old rats decreased by

50–60%, while the activities of NADH dehydrogenase and

succinate dehydrogenase fell by 21 and 44%, respectively.

The analysis of the fatty acid composition by gas liquid

chromatography exhibited distinct changes in the degree of

fatty acid unsaturation. As shown in Table 2, fatty acid

composition of membrane phospholipids changed markedly

during the different stages of development. The most

abundant saturated fatty acid, palmitic acid, increased

from 24.74% in young animals to 35.24% in senile rats,

whereas the polyunsaturated fatty acid, linoleic acid,

decreased from 21.24% in young animals to 4.08% in

senile animals. Dihomo-g-linolenic acid (C20:3nK6) and

arachidonic acid increased from 0.76 and 13.50% in young

rats to 1.10 and to 17.26% in senile rats, repectively.

Docosapentaenoic acid (C22:5nK6) increased from 13.66

to 17 45%. No significant changes were found in the

proportion of monounsaturated fatty acids, such as oleic

Table 2

Fatty acid composition of membrane phospholipids from rat testis mitochondria

Fatty acid 16 days 3 months

C16:0 24.74G3.87 26.77G0.85

C16:1nK7 0.90G0.54 0.31G0.27

C18:0 6.52G1.06 5.83G0.12

C18:1nK9 11.36G2.24 11.24G0.84

C18:2nK6 21.42G2.55 23 04G1.48

g-C18:3nK6 0.18G0.21 0.27G0.11

C20:0 0.38G1.99 0.34G0.16

a-C18:3nK3 2.24G2.02 0.86G0.82

C20:3nK6 0.76G0.32 0.78G0.13

C20:4nK6 13.50G1.38 13.54G0.13

C20:5nK3 0.64G0.50 0.28G0.13

C22:5nK6 13.66G1.11 15.08G0.2

C22:5nK3 1.72G1.05 1.04G0.66

C22:6nK3 1.06G0.40 1.26G0.10

SFA 34.47G3.97 32 61G0.72

MUFA 12.77G2.80 11.55G1.09

PUFA (nK6) 49.53G3.15 53.18G1.11

PUFA (nK3) 5.82G2.51 2.58G0.71

Data represent the weight of each individual FA /weight of total FA as percentage

palmitic acid; C16:1nK7, palmitoleic acid; C18:0, stearic acid;C18:1nK9, oleic a

a-linolenic acid; C20:3nK6, dihomo-g-linolenic acid; C20:4nK6, arachidonic a

SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsa

day-old animals.

and palmitoleic. These changes in the percentage of

individual fatty acids were reflected in the total saturated

fatty acids (SFA) and polyunsaturated fatty acids

(PUFA(nK6)). It was observed also that total SFA

increased significantly, whereas total PUFA(nK6)

decreased with age. No change in total monounsaturated

fatty acid and PUFA (nK3) was observed with ageing.

Taking into account all mitochondrial preparations inde-

pendent of age, there was a significant negative correlation

between total SFA and enzyme activities studied (Fig. 3).

NADH-DH, SDH and COX correlated negatively with SFA

(rZK0.89, rZK0.69 and rZK0.80, respectively). In the

same way, a significant positive correlation was found

between enzyme activities and total PUFA(nK6). No such

correlations were found between monounsaturated and

PUFA(nK3) and enzyme activities.

4. Discussion

Numerous reports have documented that the mitochon-

dria of germ cells modify their morphology, organization,

number and location during the spermatogenesis processes

(Andre, 1962; Machado de Domenech et al., 1972;

Meinhardt et al., 1999). In the first month of life, the

developing testis has increasing energy requirements for

cellular growth and the maturation of complex physiologi-

cal processes (Meinhardt et al., 1999). In agreement with

this, our results showed that the complexes of oxidative

phoshorylation follow a common pattern of activity

throughout spermatogenesis and the ageing process. During

at different ages

12–14 months 24 months

28.9G2.97 35.24G1.43**

0.52G0.19 0.42G0.14

7.32G0.64 8.84G1.34

11.80G2.80 13.02G1.97

15.66G1.13 4.08G0.58*

0.45G0.39 0.66G0.15

0.28G0.06 0.06G0.01

0.53G0.05 0.02G0.01

1.40G0.42 1.10G0.04**

15.77G0.96 17.26G2.33*

0.61G0.47 0.65G0.18

15.71G0.34 17.45G3.05*

0.95G0.46 0.65G0.36

1.34G0.10 0.72G0.27*

35 41G2.69 42.14G2.48**

12.32G2 069 13.44G1.98

49.01G1.99 41.54G3.97**

3.19G0.39 2.91G0.30*

(mean of % wtGSD, nZ4 different animals). Nomenclature of FA: C16:0,

cid; C18:2nK6, linoleic acid; g-C18:3nK6, g-linolenic acid; a-C18:3nK3,

cid; C20:5nK3 eicosapentaenoic acid; C22:5nK6, docosapentaenoic acid.

turated fatty acids. *p!0.05 and **p%0.01. Significantly different from 16

% of total SFA

26 28 30 32 34 36 38 40 42 44 46 48

nmol

/mg/

min

nmol

/mg/

min

nmol

/mg/

min

nmol

/mg/

min

nmol

/mg/

min

nmol

/mg/

min

0

200

400

600

800

1000

1200

% of total SFA

26 28 30 32 34 36 38 40 42 44 46 480

10

20

30

40

50

60

70

% of total SFA

26 28 30 32 34 36 38 40 42 44 46 480

200

400

600

800

1000

% of total PUFA (n-6)

36 38 40 42 44 46 48 50 52 54 560

200

400

600

800

1000

1200

% of total PUFA (n-6)

36 38 40 42 44 46 48 50 52 54 560

10

20

30

40

50

60

70

% of total PUFA (n-6)

36 38 40 42 44 46 48 50 52 54 560

200

400

600

800

1000

r=-0.89P<0.001

r=-0.90P<0.001

r=-0.79P<0.001

r=0.87P<0.001

r=0.88P<0.001

r=0.75P<0.001

A

B

C

D

E

F

Fig. 3. Correlation between total SFA and NADH-DH (A), SUC-DH (B) and COX (C) activities and between total PUFA (nK6) and NADH-DH (D), SUC-DH

(E) and COX (F) activities. Pearson’s correlation analysis was used to examine the relationship between fatty acid and enzyme activity. The ages included in

this analysis were 16 days, 3, 12–14, and 24 months.

M.E. Vazquez-Memije et al. / Experimental Gerontology 40 (2005) 482–490 487

mitosis and first meiosis of the germ cell there is an increase

in the activity of most of the complexes (Fig. 1), probably

due to both an increase in the number of mitochondria and in

the respiratory chain enzyme concentration per mitochon-

drion. From adult to aged rats, a diminution of the activity,

between 30–65% in all complexes, was observed. The

general decline of the oxidative phosphorylation capacity is

associated with the appearance in senescent tissues of

marked structural changes in mitochondria, such as

enlargement, matrix vacuolization, shortened cristae, etc.

(Wilson and Franks, 1975). It is well known that the

oxidative phosphorylation capacity of mammalian cells

changes during their life-span. At birth, a clear onset of

oxidative phosphorylation occurs as a result of the rapid

induction of mitochondrial biogenesis (Pollak, 1975; Papa,

1996). Oxidative phosphorylation activity attains a maxi-

mum level in young and adult mammals. However, this

activity can be different in response to changes in

energy demand in the different tissues. Morphological

(Muller-Hocker, 1989, 1992) and biochemical observations

(Cardellach et al., 1989; Cooper et al., 1992; Boffoli et al.,

1994, 1996) indicate a progressive decline with age of the

mitochondrial respiratory systems in most mammal tissues,

and rat testis is no exception.

In this work we tried to establish a relation between age-

dependent changes in the functioning of oxidative

phosphorylation enzymes and alterations in associated

membrane lipids. It is well known that the enzymes studied

require specific phospholipids for full activity (such as

cardiolipin) which stabilize and increase the respiratory

chain complex activities (Paradies et al., 1997; Pfeiffer

et al., 2003).

The degree of unsaturation and the length of the fatty

acid chain play important roles in determining the influence

of membrane lipids with respect to the specific enzyme

activities (Brenner, 1984). The Arrhenius plot of

M.E. Vazquez-Memije et al. / Experimental Gerontology 40 (2005) 482–490488

the enzymes we studied showed that age-dependent changes

exist in the functioning of the enzymes embedded within the

inner membrane.

Ageing has an effect on enzyme activity mainly by

modifying the physico-chemical properties of biological

membranes. Among the factors shown to affect membrane

fluidity are cholesterol/phospholipid molar ratio, phospho-

lipid composition, degree of fatty acid unsaturation, and

lipid/protein ratio. Analysis of the testis mitochondrial

membrane lipids revealed substantial changes in the fatty

acid composition in young, adult, and aged rats. These

changes are associated with loss of fluidity of the membrane

and altered lipid–protein interaction that, in turn, would

lead to a hindered orientation and /or lower mobility of the

enzymes in the membrane. An increase in the percentage

of total SFA and a decrease in the proportion of

PUFA(nK6) might reflect changes in the physical state of

the membrane that could account for the reduced enzyme

activities observed. The decrease in unsaturated(nK6)/

saturated ratio with ageing in testis might be associated

with a reduction of membrane fluidity, as implied by

the temperature breaks of Arrhenius plots showing age-

dependent shifts to higher temperatures for NADH dehy-

drogenase, succinate dehydrogenase, cytochrome c oxidase

and ATPase in senescent animals.

The physical state of the membrane was not determined

in this study; however, it is known that levels of SFA and

PUFA are the major factors in determining membrane

fluidity (Stubbs et al., 1981). In addition, the correlation

between the activity of some enzymes studied and the

proportion of total SFA or PUFA(nK6), in all ages reported

in this work, indicated that ageing, accompanied by a

variety of changes in fatty acid composition, could alter the

activity of the mitochondrial respiratory chain. The

increased total SFA proportion was associated with a high

accumulation of palmitic acid (Table 1), substrate of delta 9

desaturase, a key enzyme for the biosynthesis of MUFA,

whose expression in testis has been found to decrease with

ageing (Saether et al., 2003). However, arachidonic acid

content in old rats was slightly higher than in young rats,

whereas linoleic acid content was lower throughout the

analyzed life span, suggesting the increased capacity of

testis to convert dihomo-gamma-linolenic acid to arachi-

donic acid by delta 5 desaturation, as described in the liver

(Maniongui et al., 1993; Laganiere et al., 1993). The high

proportion of arachidonic and docosapentaenoic acid

(C22:5nK6) we observed in testis mitochondria is

concordant with results obtained in whole testis (Coniglio

et al., 1977). This suggests another function of these acids

such as maintaining the adequate biophysical state of the

membrane, which influences protein activity. In addition,

the importance of C22 polyunsaturated fatty acid in relation

to male fertility has been illustrated by studies in avians and

humans, demonstrating that the amount of C22:6nK3 in

spermatozoa is positively correlated with sperm motility

(Surai et al., 2000). On the other hand, arachidonic acid, as

a free fatty acid, interacts with the mitochonrial electron

transport chain and promotes the generation of reactive

oxygen species (Cocco et al., 1999). It has a dual effect on

mitochondrial respiration, a decrease in state 3 and

uncoupled state and an increase in state 4 (Takeuchi,

et al., 1991). Arachidonic acid may inhibit mitochondrial

ATP production during brain ischaemia and may act on the

sites closely related to NAD-linked respiration, but not the

FAD-linked one, in addition to its uncoupling effect. Some

mechanisms have been proposed, including decoupling

(Rottenberg, 1983; Rottenberg and Hashimoto, 1986),

protophoric effect (Luvisetto et al., 1987; Pietrobon et al.,

1987), and the inhibition of ATP/ADP carrier (Andreyev

et al., 1989). It is well recognized that ageing promotes

formation of reactive oxygen species (ROS) in mitochon-

dria. The involvement of mitochondria both as producers

and as targets of reactive oxygen species has been the basis

for the mitochondrial theory of ageing (Linnane et al.,

1989). It proposes that accumulation of somatic mutations

of mtDNA, induced by exposure to ROS, leads to errors in

the mtDNA-encode polypeptides; these errors are stochastic

and randomly transmitted during mitochondrial and cell

division. Such alterations, which affect the mitochondrial

complexes involved in energy conservation, would result in

defective electron transfer and oxidative phosphorylation.

Respiratory chain defects may further increase ROS

production, thus establishing a vicious circle (Ozawa,

1995, Cadenas 2004) since any damage to the respiratory

chain may enhance ROS production. Damage by oxidative

stress to mitochondrial components include mtDNA

mutations, protein oxidation, lipid peroxidation and changes

in the fatty acid pattern of mitochondrial membrane-lipids

thereby causing a reduction in activities of membrane bound

enzymes. It has been described that oxidative stress affects

respiratory chain enzymes (complex I is particularly

sensitive), ATPase, the adenine nucleotide translocator

and transhydrogenase (Lenaz, 1998, 2000). Compatible

with this, complex I and oligomycin sensitive ATPase from

aged rat testis, showed the higher reduction of specific

activity (around 80%, both).

Spermatogenesis is a complex process, hormonally

regulated, that involves mitotic cell division, meiosis and

the process of spermiogenesis. Besides other events, germ

cell differentiation is characterized by a gradual structural

modification of many organelles, including mitochondria,

which play a unique role in all stages of testis development.

The morphological and functional maturity of germ cell

mitochondria are a reflection of the permanent change in the

testicular microenvironment. Each of these steps represents

a key element in the spermatogenic process. Defects which

occur in any of them can result in the failure of the entire

process and lead to the production of defective spermatozoa

and reduction or absence of sperm production, as occurs in

ageing.

The results presented here are consistent with the concept

that ageing affects the association of lipid–protein which, in

M.E. Vazquez-Memije et al. / Experimental Gerontology 40 (2005) 482–490 489

turn, modifies the activities of the membrane bound-

enzymes. In addition, our data revealed characteristic

patterns of age-related changes in membrane levels of

long-chain polyunsaturated fatty acids, such as arachidonic

(C20:4) and docosapentaenoic acid (C22:5) which increased

progressively with age, making the peroxidizability index

increase accordingly. The radical-induced peroxidation of

the surrounding membrane lipids in testis mitochondria is

currently being studied in our laboratory.

Acknowledgements

This study was supported in part by grant FP-0038-445

from Fondo Fomento de la Investigacion del IMSS. Mexico.

MJ Cardenas and A. Tolosa received a scholarship from

CONACyT, Mexico.

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