Apoptosis and Molecular Pathways in the Seminiferous Epitheliumof Aged and Photoinhibited Syrian Hamsters(Mesocricetus auratus)

EVA MORALES,* CONCEPCION FERRER,* ADELINA ZUASTI,* JOSE C. GARCIA-BORRON,{MANUEL CANTERAS,{ AND LUIS M. PASTOR*

From the Departments of *Cell Biology (Aging Institute), �Biochemistry and Molecular Biology, and `Statistics,

Faculty of Medicine, University of Murcia, Murcia, Spain.

ABSTRACT: Aging and short photoperiod exposure induce germ

cell apoptosis in the Syrian hamster; however, the specific germ cells

affected and the molecular pathways triggered have not been

elucidated. We analyzed germ cell apoptosis and the expression of

the Fas/Fas-L system, Bcl-2 family, and p53 in aged and

photoinhibited hamsters and compared with those young maintained

in natural photoperiod. Aging increased apoptosis in spermatogonia

and spermatocytes, but in photoinhibited hamster testes only an

increase in apoptotic spermatocytes was observed. Apoptosis was

higher in aged hamsters in stages I–IV, V–VI and VII–VIII. Aging

increased apoptosis of spermatogonia in stages I–IV and V–VI.

Apoptotic pachytene spermatocytes were significantly higher in

stages I–IV, V–VI, and VII–VIII in aging. Apoptotic preleptotene

and pachytene spermatocytes were higher in aging, but no

differences were observed in leptotene-zygotene. Fas-L was

expressed by Sertoli cells, of young, aged, and photoinhibited

hamsters. Bcl-xL was strongly expressed in germ cells on young

hamsters and slightly in aging and after short photoperiod exposure.

Spermatocytes of photoinhibited hamsters were intensively stained

with Fas, Bax, Bcl-xs/L, and p53. In conclusion, aging increases

apoptosis in spermatogonia and spermatocytes, depending on the

stage of the seminiferous epithelium cycle, whereas after a short

photoperiod exposure only an increase in apoptotic spermatocytes is

observed. The results suggest that Fas, Bcl-xL, Bax, and p53

participate in germ cell apoptosis induction after short photoperiod

exposure, whereas only Bcl-xL is involved in aging.

Key words: Aging, Bcl-2, Fas, photoperiod, testis.

J Androl 2007;28:123–135

G erm cell death occurs via apoptosis, whether

spontaneously during normal spermatogenesis or

triggered by different stimuli (Blanco-Rodrıguez and

Martınez-Garcıa, 1996, 1998; Cai et al, 1997; Sinha

Hikim et al, 1997; Lee et al, 1999). In the Syrian hamster

both the short photoperiod exposure, which is charac-

terized by a sharp fall in testosterone (Calvo et al, 1997),

and aging, characterized by normal androgen levels

(Horn et al, 1996; Calvo et al, 1999), are physiological

situations of germ cell loss and atrophy of the

seminiferous epithelium (Horn et al, 1996; Morales et

al, 2002, 2003, 2004).

Apoptosis has been involved in testicular germ cell

loss during aging in numerous species, including humans

(Brinkworth et al, 1997; Kimura et al, 2003) and

rodents, such as the mouse (Barnes et al, 1998), rat

(Wang et al, 1999; Barnes et al, 1999) and Syrian

hamster (Morales et al, 2003). In aged rats germ cell

apoptosis has been related to specific stages of the

seminiferous epithelium cycle (Wang et al, 1999),

although this aspect has not been studied in other

species. Also, short photoperiod exposure induces germ

cell apoptosis in white-footed mouse (Young et al, 1999,

2000) and Syrian hamster (Morales et al, 2002).

However, the specific germ cell population affected has

not sufficiently been documented in both models of

seminiferous epithelium atrophy.

Apoptosis can be triggered by 3 mechanisms: 1) the

binding of Fas-L to Fas receptor expressing cells (Itoh et

al, 1991; Oehm et al, 1992; Suda et al, 1993); 2) the

activation of Bcl-2 family members (Yang and Kors-

meyer, 1996; Green and Reed, 1998); and 3) the endo-

plasmic reticulum pathway (Nakagawa et al, 2000; Bitko

and Barik, 2001; Yoneda et al, 2001). Fas/Fas-L system

seems to play an important role in testicular germ cell

apoptosis regulation (Pertikainen et al, 1999). Fas is

expressed in spermatogonia, spermatocytes, and sperma-

tids, all apoptotic cells (Lee et al, 1997). Fas expression

Supported by grant PI-56/00866/FS/01 from the Fundacion Seneca,

Comunidad Autonoma de la Region de Murcia.

Correspondence to: Prof Dr Luis M. Pastor, Department of Cell

Biology, Faculty of Medicine, Campus de Espinardo, University of

Murcia, E-30071 Murcia, Spain (e-mail: bioetica@um.es).

Received for publication September 13, 2005; accepted for

publication August 24, 2006.

DOI: 10.2164/jandrol.106.000778

A portion of this report has been communicated in abstract form

for the 44th American Society of Cell Biology Annual Meeting.

Journal of Andrology, Vol. 28, No. 1, January/February 2007Copyright E American Society of Andrology

123

has also been related to germ cell degeneration in arrest of

spermatogenesis (Eguchi et al, 2002; Francavilla et al,

2002). There is some discrepancy as to the preciselocalization of Fas-L in the seminiferous tubules, with

some groups showing Fas-L in the Sertoli cells (Bellgrau

et al, 1995; French et al, 1996; Lee et al, 1997; Koji, 2001),

while others have reported that Fas-L is also expressed in

the germ cells (Woolveridge et al, 1999; Francavilla et al,

2000). Bcl-2 and the long form Bcl-x (Bcl-xL) promote cell

survival by inhibiting apoptosis. However, other mem-

bers of the Bcl-2 family (Bax, Bak, Bcl-xS, and Bad)promote cell death (Oltvai et al, 1993). Members of the

Bcl-2 family also regulate spermatogenesis, inducing

apoptosis in spermatogonia, primary spermatocytes,

and spermatids (Oldereid et al, 2001) and participating

in the differentiation process (Oldereid et al, 2001). Also,

p53 has been involved in the elimination of damaged

germ cells as well as those produced in excess (Stephan et

al, 1996), as well as in the control of proliferation andapoptosis in spermatogonia (Beumer et al, 1998).

Although germ cell apoptosis was implicated in aging

and was seen to occur after short photoperiod exposure in

a previous study in our laboratory (Morales et al, 2002,

2003), the specific cell types that display apoptosis, the

relationship of apoptosis with specific stages of the

seminiferous epithelium cycle, and the molecular path-

ways involved have not been sufficiently studied. Theobjectives of the present study were: 1) to study how the

different populations of germ cells are affected by

apoptosis caused by aging and short photoperiod

exposure; 2) to analyze the induction of germ cell

apoptosis during the different stages of the seminiferous

epithelium cycle in aging Syrian hamster; and 3) to

examine the expression of some of the Bcl-2 family

members, the Fas/Fas-L system, and p53 in the seminif-erous epithelium of Syrian hamsters and to investigate the

roles of these proteins in the induction of testicular germ

cell apoptosis in aging and short photoperiod exposure.

Materials and Methods

Animals and Tissue Preparation

A total of 16 male Syrian hamsters (Mesocricetus auratus) were

used in the present study. They were distributed in 3 groups:

a first group of 5 control animals that were 6 months old;

a second group of 6 hamsters that were 24 months old; and

a third group of five 6-month-old animals that were exposed to

short photoperiod. All the animals were maintained at

a temperature of 20uC and given food and water ad libitum.

The illumination was regulated by a programmer so that the 6-

and 24-month-old groups were maintained in a natural

photoperiod of 14:10 (L:D), whereas the third group was

subjected to a short photoperiod cycle of 8:16 (L:D) for

2 months. Animals were sacrificed by an intraperitoneal

overdose of sodium pentobarbital, and both testes were

removed. Right testes were processed for Western blot

analysis, and left testes were processed for in situ detection

of apoptosis and immunohistochemistry. This study was

performed according to Spanish ethical and legal standards

regarding animal protection.

In Situ Germ Cell Apoptosis Detection and Quantification

Left testes were fixed in methacarn (methanol:chloroform:

acetic acid 6:3:1), and representative samples, which were

chosen randomly, were dehydrated, immersed in toluene, and

embedded in Paraplast Plus (Panreac Quımica SA, Barcelona,

Spain). Germ cell apoptosis was examined following the

protocol of TACS TdT in situ Apoptosis Detection kit

(TUNEL reaction; R&D Systems Inc, Minnesota, Minn).

For this, 5-mm-thick sections were deparaffinized, hydrated,

washed, and incubated with proteinase K (1 mg/mL). The

peroxidase activity was quenched with 10% H2O2. The samples

were immersed in 16 TdT labeling buffer (1 mol TACS Safe-

TdT Buffer, 0.5 mg/mL BSA, 0.6 mmol 2-mercaptoethanesul-

fonic acid) at 18u–24uC for 5 minutes and incubated for 1 hour

at 37uC with TdTdNTP Mix (0.25 mmol biotinylated dNTP),

506 Mn+2 (1 mL), TdT Enzyme (1 mL), and 16 TdT labeling

buffer. The reaction was stopped with TdT stop buffer

(0.1 mol EDTA, pH 8.0). Subsequently, the samples were

washed in PBS and incubated with streptavidin-horseradish

peroxidase for 10 minutes at 18u–24uC. After washing in PBS,

the samples were stained with TACS Blue Label and incubated

with Contrast C solution. Negative control sections, processed

without TdT, did not show positive labeling.

The apoptotic germ cells were identified according to their

position within the seminiferous epithelium, the cell size and

nuclear morphology, and stage of the seminiferous epithelium.

The quantity of apoptosis and the percentage of apoptotic

germ cells were recorded in the populations of spermatogonia

(SG) and spermatocytes (SC). For this purpose, 5-mm-thick

sections were collected every 50 mm of tissue and processed for

in situ germ cell apoptosis detection. Of the sections processed,

4 randomly chosen testis cross-sections were counted per

animal. Within each section, 25 random fields were chosen by

systematically moving the microscope lens across the tissue

section without overlap and selecting every second field and

were analyzed (1 field 5 0.018 mm2). In each field of study the

number of TUNEL-positive and negative germ cells was

scored in the populations of spermatogonia and spermato-

cytes. The following apoptotic indices of each population of

germ cells, expressed as the percentage of TUNEL-positive

cells, were calculated: 1) the total apoptotic index (% of

TUNEL-positive SG+SC); 2) the total apoptotic index in

spermatogonia (% of TUNEL-positive SG); and 3) the total

apoptotic index in primary spermatocytes (% of TUNEL-

positive primary SC).

Also, in 6- and 24-month-old hamsters the apoptotic activity

was examined during the different stages of the seminiferous

epithelium cycle. The identification of stages was based on the

description of Leblond and Clermont (1952) and Tiba et al

(1992). Similarly to previous studies (Wang et al, 1999; Sinha

Hikim et al, 2003), the stages were grouped into 4 groups:

124 Journal of Andrology N January �February 2007

I–IV, V–VI, VII–VIII, and IX–XIII. For quantification of

apoptosis during the seminiferous epithelium cycle, at least 100

randomly selected perpendicular seminiferous tubule cross

sections on 4 sections from each animal of each group were

analyzed. In each tubular section, the number of TUNEL-

positive and negative germ cells was scored in the populations

of spermatogonia, preleptotenes, pachytenes, and leptotene-

zygotene spermatocytes. The apoptotic index of each popula-

tion of germ cells during the different stages of the

seminiferous epithelium cycle was expressed as the percentage

of TUNEL-positive cells.

Immunohistochemistry

Five-mm-thick sections fixed in methacarn and embedded in

paraffin were deparaffinized in xylene, hydrated, and trans-

ferred to PBS for 10 minutes. Endogenous peroxidase was

blocked with 1% H2O2 in PBS for 30 minutes. After washing

in PBS, samples were blocked with 1.5% normal rabbit or goat

serum (Jackson ImmunoResearch, West Grove, Pa), depend-

ing on the origin of the secondary antibody. Subsequently, the

samples were washed in PBS and incubated overnight at 4uC,

with the primary antibody diluted in PBS/BSA. The primary

antibodies were purchased from Santa Cruz Biotechnology

(Santa Cruz, Calif), and the concentrations used were the

following: anti-Fas (1:30) (sc-7886/ FL-335), anti-Fas-L (1:50)

(sc-834/N-20), anti-Bcl-xL (1:100) (sc-8392/H-5), anti-Bcl-xS/L

(1:100) (sc-1041/L19), anti-Bcl-2 (1:100) (sc-492/N-19), anti-

Bax (1:100) (sc-526-P19), and anti-p53 (1:20) (sc-6243/FL-393).

The samples were washed in PBS and then incubated for

45 minutes with the corresponding biotinylated secondary

antibody (CHEMICON International, Temecula, Calif) di-

luted 1:500 in PBS/BSA. Sections were again washed in PBS

and subsequently incubated for 45 minutes with HRP-strepta-

vidin (Dako, Glostrup, Denmark) diluted 1:300 in PBS/BSA.

Samples were washed again in PBS; bound antibody was

visualized after the addition of 0.05% solution of 3,39-di-

aminobenzidine tetrachloride in TBS, to which 0.03% H2O2

was added. The slides were subsequently counterstained with

hematoxylin. Control sections, in which the primary antibody

was replaced by PBS, were similarly processed. For all the anti-

bodies analyzed, no immunostaining was detected in control

sections from which the primary antibody had been omitted.

Western Blot Analysis

The right testis was snap frozen in liquid nitrogen and stored at

270uC until required for protein extraction. Tissue was

homogenized in 1% SDS in PBS and a tablet of protease

inhibitors Complete Mini, EDTA-free (Roche, Mannheim,

Germany) for each 10 mL of homogenization solution

(Pentikainen et al, 1999). One mL of homogenization solution

was used for each 100 mg of fresh tissue. After centrifugation

at 17 000 6 g for 30 minutes at 4uC, the supernatants were

collected and their protein concentrations determined by BCA

method (Pierce, Rockford, Ill). The equivalent amounts of

protein were mixed with sample buffer (0.5 mol Tris-HCl

pH 6.8, 20% glycerol, 10% SDS, 0.1% bromophenol blue, and

0.5% ß-mercaptoethanol) and incubated at 95uC for 5 min-

utes. Proteins (10–15 mg) were loaded in 8%–12% SDS-

polyacrylamide gels, and electrophoresis was performed in

the presence of marker standards with molecular weights

between 200 and 6.5 kd (Bio-Rad Laboratories, Hercules,

Calif) at 15 mA during stacking and 25 mA during the

separation. The proteins were transferred to Immobilon-P

membranes (Millipore, Bedford, Mass) by semidry electro-

phoretic transfer for 1 hour at room temperature in transfer

buffer (50 mmol Tris, 40 mmol glycine, 10% SDS and 20%

methanol, pH 9.2) at 22 V. Subsequently, the membrane was

blocked with 5% nonfat milk in PBS for at least 1 hour at

room temperature. Before blocking, the marker standards

were separated and labeled with Amido Black solution (0.1%

Amido Black, 45% methanol, and 10% acetic acid) to check

the protein molecular weight. After 3 washes of 10 minutes in

PBS containing 0.1% Tween 20 (PBST), the membrane was

incubated overnight at 4uC using the same primary antibodies

used for immunohistochemistry diluted 1:1000 in a solution

containing 5% albumin in PBST. After 3 washes of 10 minutes

in PBST, the membranes were incubated for 1 hour at room

temperature with the same biotinylated secondary antibodies

used for immunohistochemistry diluted 1:10 000 PBST. Sub-

sequently, the membranes were washed with PBST and

incubated with HRP-streptavidin (Dako) diluted 1:5000.

Immunoreactive bands were located with the enhanced

chemiluminescence detection Kit (Amersham Biosciences,

Buckinghamshire, United Kingdom) and Hyperfilm ECL

(Amersham Biosciences). The semiquantitative study was

performed for each antibody with an automatic image

analyzer (MIP version 4.5; Consulting Image Digital, Barce-

lona, Spain). After digitalization and inversion of grey level

image, the bands were delimited out manually, and the area

and medium grey were calculated for each band in a range of

0–255. Labeling density was obtained by multiplying the area

times medium grey and was taken as an index of band

intensity. The densitometrical analysis is present as a bar

diagram with arbitrary unit (the densitometrical minor value

was the unit in each bar diagram).

Statistical Analysis

The mean values obtained in aged and photoinhibited

hamsters were compared with data obtained in young

hamsters (control group) using Student’s t test and, for

measurements lacking equal variance, by a Welch test. The

tests were performed using log-transformed data. Data were

back-transformed following analyses and are presented as

means 6 SEM. Statistical evaluation of apoptotic indices

during the different stages of the seminiferous epithelium was

performed by analysis of variance in conjunction with a least

significant difference (LSD) test. Mean differences were

considered statistically significant when P is less than .05.

Results

In Situ Detection of Apoptosis

In young and aged hamsters the germ cells undergoing

apoptosis were predominantly spermatogonia and sper-

Morales et al N Apoptotic Pathways in Aged and Photoinhibited Testes 125

matocytes in different meiotic phases, and occasionally

round spermatids. A study of apoptosis during the

seminiferous epithelium cycle showed that apoptotic

spermatogonia were present in stages I–IV (Figure 1A)

and V–VI (Figure 1F) of young and aged hamsters, but

were rarely observed in stages VII–VIII and IX–XIII.

Both in young and aged hamsters, apoptotic spermato-

cytes were observed in different phases of meiosis,

Figure 1. TUNEL-positive germ cells in 6-month-old (A–D) and 24-month-old (E–I) hamsters during the different stages of the seminiferousepithelium cycle. SG indicates spermatogonia; P, pachytene spermatocyte; PL, preleptotene spermatocyte; LZ, leptotene-zygotenespermatocyte; and PD, pachytene-diplotene spermatocyte. (J–K) TUNEL-positive spermatocytes (arrows) in photoinhibited hamsters.Magnification: A, scale bar 5 10.4 mm; B, scale bar 5 15.6 mm; C, scale bar 5 12.5 mm; D, scale bar 5 14.3 mm; E, scale bar 5 6.7 mm; F,scale bar 5 55 mm; G–I, scale bar 5 14.8 mm; J, scale bars 5 60.6 mm ; K, scale bar 5 55.5 mm.

126 Journal of Andrology N January �February 2007

including preleptotenes in stages VII–VIII of the

seminiferous epithelium cycle (Figure 1C), early pachy-

tenes in stages I–IV (Figure 1A and E), middle

pachytenes in stages V–VI (Figure 1B and F) and VII–

VIII (Figure 1G), late pachytenes-diplotene in stagesIX–XIII (Figure 1I), and also leptotene-zygotenes in

stages IX–XIII (Figure 1D and H).

In photoinhibited hamsters the germ cells undergoing

apoptosis were predominantly early spermatocytes and,

especially, pachytene spermatocytes (Figure 1J and K)

and, on some occasions, spermatogonia and early round

spermatids. Apoptotic spermatocytes were observed in

different meiotic phases: preleptotenes, early, middle,and late pachytenes (Figure 1J and K), and leptotene-

zygotenes.

Aging and Short Photoperiod Exposure IncreaseApoptotic Indexes in the Seminiferous Epithelium ofSyrian Hamsters

Quantitatively, aging induced an increase in the

percentage of both spermatogonia and spermatocytes

in apoptosis. During the cycle of the seminiferous

epithelium, germ cell apoptosis (% of apoptotic SG+SC)

was higher in aging in stages I–IV, V–VI, and VII–VIII

(P , .05) (Table 1). The percentage of apoptoticspermatogonia was significantly increased in stages

I–IV and V–VI (P , .05), with no differences in stages

VII–VIII and IX–XIII (Table 1). The percentage of

spermatocytes in their preleptotene phase and pachytene

in apoptosis was significantly higher in aged than in

young hamsters (P , .05), with no differences in

leptotene-zygotene spermatocytes. The percentage of

pachytene spermatocytes in apoptosis was significantly

increased in stages I–IV, V–VI, and VII–VIII (P , .05),

with no differences in stages IX–XIII.

After 8 weeks of short photoperiod exposure, the

percentage of apoptotic spermatocytes was significantly

increased with respect to animals maintained in a normal

photoperiod (P , .05). But no differences in the

percentage of apoptotic spermatogonia were found

between groups (Table 2).

Immunohistochemistry

Fas/Fas-L System—Immunohistochemistry showed the

presence of Fas-L in cytoplasmic prolongations of

Sertoli cells (Figure 2A) and in Leydig cells of young,

aged (Figure 2B), and photoinhibited hamsters (Fig-

ure 2C). Also, spermatozoon tails were positive for

Fas-L in young (Figure 2A) and aged hamsters. Fas

expression was observed in Leydig cells of young and

aged hamsters (Figure 2D and E), and a slight immu-

noreactivity was also present in the tails of mature

spermatozoa (Figure 2D and E). However, in photo-

inhibited hamsters a strong expression of Fas was

observed in spermatocytes (Figure 2F) and occasionally

in Leydig cells and early round spermatids.

p53—Testicular germ cells of young and aged

hamsters were negative for p53 staining (Figure 2G

and H). However, photoinhibited hamsters showed an

intense immunoreactivity for p53, predominantly in the

spermatocytes (Figure 2I) and occasionally in the

spermatogonia and early round spermatids.

Bcl-2 Family Proteins—Staining for Bax was observed

in round spermatid acrosomes of young and aged

hamsters (Figure 2J and K). In photoinhibited hamsters

an intense immunoreactivity for Bax was observed in

spermatocytes (Figure 2L) and some staining was

observed in Leydig cells. In young and aged hamsters,

the seminiferous tubules were negative for Bcl-2 staining

Table 1. Apoptotic indexes in control (6 months) and agedhamsters (24 months)*

Control Aged hamsters

Total apoptotic index (SG+SC) (%) 0.77 6 0.04 1.51 6 0.233

Stages I–IV 0.23 6 0.08 0.58 6 0.233

Stages V–VI 0.06 6 0.03 0.60 6 0.203

Stages VII–VIII 0.38 6 0.11 0.95 6 0.463

Stages IX–XIII 0.16 6 0.10 0.21 6 0.084

Total apoptotic index in SG (%) 0.24 6 0.03 0.76 6 0.023

Stages I–IV 0.66 6 0.28 1.56 6 0.313

Stages V–VI 0.12 6 0.05 0.61 6 0.053

Stages VII–VIII 0.00 0.00

Stages IX–XIII 0.00 0.00

Total apoptotic index in SC (%) 0.20 6 0.01 0.55 6 0.063

Preleptotenes 0.65 6 0.08 1.53 6 0.343

Leptotenes-zygotenes 0.21 6 0.05 0.18 6 0.054

Pachytenes 0.12 6 0.007 0.45 6 0.063

Stages I–IV 0.18 6 0.06 0.41 6 0.133

Stages V–VI 0.02 6 0.03 0.59 6 0.333

Stages VII–VIII 0.08 6 0.05 0.39 6 0.163

Stages IX–XIII 0.10 6 0.04 0.24 6 0.054

* Values represent mean 6 SEM. SG indicates spermatogonia;

SC, spermatocytes.

3 Significant differences between control group and aged hamsters

(P , .05).

4 No significant differences between groups.

Table 2. Apoptotic indexes in control (6 months) andphotoinhibited hamsters*

Control Photoinhibited

Total apoptotic index (SG+SC) 0.77 6 0.03 2.84 6 0.163

Apoptotic index in SG 0.24 6 0.03 0.76 6 0.174

Apoptotic index in SC 0.19 6 0.01 2.62 6 0.283

* Values represent mean 6 SEM. SG indicates spermatogonia;

SC, spermatocytes.

3 Significant differences between control group and aged hamsters

(P , .05).

4 No significant differences between groups.

Morales et al N Apoptotic Pathways in Aged and Photoinhibited Testes 127

Figure 2. Immunohistochemical localization of Fas-L, Fas, p53, and Bax in testes of young, aged and photoinhibited Syrian hamsters.Contrasted with hematoxylin. (A–C) Fas-L expression was observed in cytoplasmic prolongations of Sertoli cell (arrows), spermatozoon tails(asterisk), and Leydig cells (arrowhead). (D–F) Leydig cells (arrowhead) and spermatozoon tails (asterisks) were positive to Fas. Inphotoinhibited hamsters numerous spermatocytes were positive to Fas (arrows). (G–I) In young (G) and aged (H) hamsters a slight staining ofp53 was observed in spermatid acrosomes (asterisks, nonspecific stained). In photoinhibited hamsters (I) numerous spermatocytes (arrows)showed a strong immunoreactivity for p53. (J–L) Staining for Bax was observed in round spermatid acrosomes of young (J) and aged (K)hamsters (asterisks). In photoinhibited hamsters (L) spermatocytes (arrows) were intensively stained with Bax. Magnification: A and C; scalebar 5 16.6 mm; B; scale bar 5 20.8 mm; D; scale bar 5 35.7 mm; E; scale bar 5 166.6 mm; F; scale bar 5 13.8 mm; G and H; scale bar 568 mm; I ; scale bar 5 50 mm; J and K; scale bar 5 55.5 mm; L; scale bar 5 14.2 mm.

128 Journal of Andrology N January �February 2007

(Figure 3A and B). However, in photoinhibited ham-

sters Bcl-2 was intensively expressed by Sertoli and

Leydig cells (Figure 3C).

Germ cells of young hamsters, including spermato-

gonia, spermatocytes, spermatids, and spermatozoa,

showed an reactivity to Bcl-xL during the different

stages of the seminiferous epithelium (Figure 3D). Also,

some immunoreactivity was observed in Sertoli cells.

However, both in aged and photoinhibited hamsters the

Bcl-xL staining declined in germ cells and Sertoli cells

(Figure 3E and F). Bcl-xS/L was present in germ cells

and Leydig cells of young and aged hamsters (Fig-

ure 3G). Also, in aged hamsters, an intense immunore-

activity for Bcl-xS/L was observed in spermatocytes of

the seminiferous tubules which were in maturation

arrest of spermatogenesis (Figure 3H). In photoinhib-

ited hamsters a strong immunoreactivity for Bcl-xS/L

was observed, especially in spermatocytes (Figure 3I).

Western Blot Analysis

Similar levels of Fas-L (38 kd) were found in the testes

of young, aged, and photoinhibited hamsters (Fig-ure 4A). A slight expression of Fas (48 kd) was observed

in young and aged animals. However, in hamsters

Figure 3. Immunohistochemical localization of Bcl-2, Bcl-xL, and Bcl-xS/L in the testis of young, aged, and photoinhibited Syrian hamsters.Contrasted with hematoxylin. (A–C) No immunostaining for Bcl-2 was observed in testes of young and aged hamsters. Sertoli (asterisks) andLeydig cells of photoinhibited hamsters were strongly positive to Bcl-2. (D–F) A strong immunoreactivity to Bcl-xL was observed in germ cells ofyoung hamsters (D). Bcl-xL staining decreased in aged (E) and photoinhibited hamsters (F). (G–I) Germ cells of young and aged hamsters werepositive to Bcl-xS/L (asterisks). In photoinhibited hamsters spermatocytes (arrows) were intensively stained with Bcl-xS/L. Magnification: A–C;scale bar 5 50 mm; D; scale bar 5 23.5 mm; E and F; scale bar 5 22.4 mm; G; scale bar 5 66.6 mm; H; scale bar 5 100 mm; I; scale bar 535.7 mm.

Morales et al N Apoptotic Pathways in Aged and Photoinhibited Testes 129

Figure 4. Western blot analysis of Fas/Fas-L system, p53, and proteins of Bcl-2 family in whole testicular lysates of young (6 m), photoinhibited(sp), and aged hamsters (24 m). The densitometric analysis is presented as a bar diagram. Blots included K-562 whole cell lysate (Fas-L),Jurkatt whole cell lysate (Fas and Bcl-2), and BJAB whole cell lysate (Bax and Bcl-xS/L) as positive controls.

130 Journal of Andrology N January �February 2007

exposed to a short photoperiod a strong expression of

Fas was observed (Figure 4B). In hamsters exposed to

a short photoperiod, the levels of p53 were higher than

in young hamsters maintained in a normal photoperiod

(Figure 4C). Aged hamsters showed levels of p53 similar

to young hamsters. Bax (23 kd) levels were similar in

young, aged, and photoinhibited hamsters (Figure 4D).

Western analysis demonstrated similar levels of Bcl-2

(28 kd) in the testes of young and aged hamsters, but

a strong expression was found in photoinhibited

hamsters (Figure: 4E). In aged hamsters and those

exposed to a short photoperiod, the levels of Bcl-xL

(31 kd) showed lower intensity than young hamsters

maintained in normal conditions (Figure 4F).

Discussion

Testicular Germ Cell Apoptosis in Aged Hamsters andAnimals Exposed to a Short Photoperiod

In young hamsters maintained in normal photoperiod

spontaneous apoptosis was observed in spermatogonia

and primary spermatocytes and occasionally in round

spermatids. Apoptotic spermatogonia were found pre-

dominantly in stages I–IV and V–VI of the seminiferous

epithelium cycle. In primary spermatocytes apoptosis

was detected predominantly in preleptotenes, and to

a lesser extent in leptotene-zygotene and pachytene. In

the Syrian hamster, apoptosis constitutes a cellular

mechanism that allows the seminiferous epithelium to

control the number of germ cells supported by Sertoli

cells, eliminating aberrant cells and regulating sperm

production (Hsueh et al, 1996). This control takes place

in stages when spermatogonia are more numerous and

during the beginning of meiosis (preleptotenes), sup-

porting the idea that apoptosis affects spermatogonia in

areas where these are too numerous, and is absent from

areas of low spermatogonia density (De Rooij and

Janssen, 1987; De Rooij and Lok, 1987).

Aging and exposure to a short photoperiod provoked

a significant increase in apoptosis in the seminiferous

epithelium of Syrian hamsters. However, aging induced

increase in apoptotic spermatogonia and spermatocytes,

whereas short photoperiod exposure encouraged an

increase in apoptotic spermatocytes without affecting

the population of spermatogonia.

Aging increased germ cell apoptosis in stages I–IV, V–

VI, and VII–VIII of the seminiferous epithelium cycle of

Syrian hamsters. In previous studies in rats, an increase

in germ cell apoptosis was observed in all the stages of

the seminiferous epithelium cycle, although the increase

was only statistically significant in stages XII–XIV

(Wang et al, 1999). Other experimental situations,

including heat exposure and testosterone deprivation,

have led to differences in the incidence of germ cell

apoptosis during the stages of the seminiferous epithe-lium cycle. After gonadotrophin deprivation and in-

tratesticular testosterone in the rat, apoptosis occurred

in stages VII–VIII, whereas hyperthermia induced germ

cell apoptosis in stages I–IV and XII–XIV (Sinha Hikim

et al, 2003). These studies indicate that apoptosis

depends on the stages of the seminiferous epithelium

cycle and that it differs according to the trigger stimuli

even in the same animal species.In the Syrian hamster, aging increased apoptosis in

the spermatogonia in stages I–IV and V–VI, while

apoptosis was absent in stages VII–VIII and IX–XIII,

similar to the results observed in young animals. As

mentioned above, these results probably reflect the

existence of an optimal and constant regulation of germ

cell density by an increasing apoptosis of spermatogonia

in areas where these cells are too numerous (De Rooijand Janssen, 1987; De Rooij and Lok, 1987). Also, aging

induced an increase in apoptotic pachytene spermato-

cytes over to the low rate found in young hamsters. It is

known that control points are not confined to cells that

divide by mitosis, but also operate during meiosis. Of

particular note is the control checkpoint at the end of the

pachytene phase of the meiotic prophase, when the

meiotic recombination and chromosome synapse areincomplete (Roeder, 1997), preventing the production of

aneuploid gametes (Roeder and Bailis, 2000). Our results

in aging hamsters suggest that this increase in apoptosis

in pachytene spermatocytes provides a control mecha-

nism of meiosis that could eliminate aberrant aged germ

cells. The strong coincidence between spontaneous and

age-induced apoptosis in the same germ cell types and in

the same stages of the seminiferous epithelium cyclesuggests that aging causes an exacerbation of apoptosis at

checkpoints where the germ cells are eliminated sponta-

neously. It is reasonable to think that the high degree of

synchronization required during the spermatogenic pro-

cess imposes a strict control over germ cell apoptosis,

both in normal situations and in situations that imply

a deteriorated spermatogenic capacity of the epithelium,

such as aging.

Germ cell types that suffer apoptosis during testicularregression after exposure to a short photoperiod may

differ between species. In photoinhibited Syrian hamster

the main apoptotic germ cells observed are the

spermatocytes and occasionally spermatogonia and

round spermatid. Similar results to these presented have

been reported in the mouse and birds (Sinha Hikim et al,

1997; Young et al, 1999, 2001). The present study is the

first to demonstrate this phenomenon quantitatively,and to indicate that apoptosis does not increase in the

population of spermatogonia. Testis exposed to short

Morales et al N Apoptotic Pathways in Aged and Photoinhibited Testes 131

photoperiod shows a pattern of cell death similar to

pharmacological deprivation of gonadotrophins (Sinha

Hikim et al, 1997; Young and Nelson, 2001). This

indicates that spermatocytes are more susceptible to

perturbations of the seminiferous epithelium environ-

ment during short photoperiod exposure, which is

characterized by severe testosterone deprivation (Furuta

et al, 1994; Calvo et al, 1997).

Role of Fas/Fas-L System, Bcl-2 Family, and p53 in GermCell Apoptosis in Aging and After ShortPhotoperiod Exposure

In the Syrian hamster Fas-L expression was observed in

Sertoli and Leydig cells as well as in spermatozoon tails

of young and aged animals. Similar results have been

documented in humans (Francavilla et al, 2000) and

rodents, including the mouse (French et al, 1996) and rat

(Lee et al, 1997). Fas-L expression in the normal testis

may be related to the fact that the testis is an

immunologically privileged organ, and Fas-L would be

involved in the elimination of active and infiltrated T

cells that express Fas (Bellgrau et al, 1995). Other

authors have described Fas-L expression in postmeiotic

germ cells, such as mature spermatozoa in the rat and

mouse (D’Alessio et al, 2001) and even in humans

(Francavilla et al, 2002), which is consistent with the

present results obtained in the Syrian hamster. These

results have led us to propose an alternative hypothesis

concerning the role of Fas-L in the reproductive system,

whereby Fas-L may represent an important molecule in

the complex mechanism developed by male gametes to

escape immunological reaction both in the male genital

tract (autoimmune reaction against auto antigens of

sperm cells) and the female genital tract (D’Alessio et al,

2001; Riccioli et al, 2003).

In young and aged Syrian hamsters, a slight expres-

sion of Fas was observed, mainly in Leydig cells, which

agrees with previous results reported in rodents, such as

mouse (Koji et al, 2001) and rat (Lee et al, 1999), as well

as in humans (Francavilla et al, 2000, 2002). These

results suggest that there is no evidence to support

a relationship between the Fas system and germ cell

apoptosis in the testis of young and aged hamsters, as

was found in the mouse (Koji, 2001). Although the Fas

system is essential for germ cell apoptosis in several

pathological situations, it is irrelevant in normal

conditions (Koji, 2001). However, Western blot analysis

revealed a significant increase in Fas expression in

photoinhibited testes. Also, immunohistochemically Fas

was strongly expressed by the spermatocytes of hamsters

exposed to a short photoperiod, which is coincident with

the predominantly TUNEL-positive germ cells observed

in the spermatocytes of these animals. In Fas-mediated

apoptosis, the binding of Fas-L to Fas antigen is

a prerequisite (Koji et al, 2001). So the temporal and

spatial association between Fas and Fas-L expression inhamsters exposed to short photoperiod suggests that the

Fas system is a mediator of germ cell apoptosis

induction after short photoperiod exposure. Although

the Fas system has been correlated with germ cell

degeneration in situations of meiotic and postmeiotic

arrest of spermatogenesis in man (Eguchi et al, 2002;

Francavilla et al, 2002), our immunohistochemical

results in aged hamsters suggest that there is noconnection between TUNEL-positive germ cells and

Fas expression. Also, Western blot analysis revealed no

changes in the expression of Fas and Fas-L in aged

animals, which suggests that the Fas/Fas-L system is not

involved in apoptosis induction of germ cells in the

testes of aged Syrian hamsters.

Another widely known regulatory system of apoptosis

is the Bcl-2 family composed of anti- and proapoptotic

proteins which regulate apoptosis by controlling therelease of cytochrome c and other mitochondrial

changes (Yang and Korsmeyer, 1996; Green and Reed,

1998). In the present study, Western blot analysis and

the immunohistochemical study demonstrated an ex-

pression of the antiapoptotic form of Bcl-xL in germ

cells of young hamsters, whereas aged and photoinhib-

ited hamsters showed a lower degree of expression. On

the other hand, proapoptotic form Bcl-xS was expressedin all the groups (young, aged, and photoinhibited).

With respect to the localization of both proteins, Bcl-xL

was expressed in all the populations of germ cells in

young hamsters. Based on the decrease of Bcl-xL

expression in germ cells of photoinhibited and aged

hamsters, as shown by Western blot analysis and

immunohistochemistry, we can affirm that when Bcl-

xS/L antibody is used, what we see predominantly is theexpression of Bcl-xS in germ cells in these 2 groups of

animals. Accordingly, and based on the immunohisto-

chemical results, in animals exposed to a short photo-

period using Bcl-xS/L antibody we found a strong

expression of Bcl-xS predominately in the spermato-

cytes, which is concordant with TUNEL-positive germ

cells as well as with other proapoptotic proteins studied

in the present report. Also the seminiferous epitheliumof aged hamsters showed germ cells positive for Bcl-xS.

As suggested by other authors, a balance of anti- and

proapoptotic members of the Bcl-2 family is critical for

regulating the survival of testicular germ cells (Oltvai et

al, 1993; Yang and Korsmeyer, 1996; Yamamoto et al,

2001; Sakamaki, 2003). Our results indicate that the

lower expression of the anti-apoptotic form Bcl-xL in

germ cells of aged and photoinhibited hamsters may bedue to a predominance of proapoptotic Bcl-xS forms,

similar to that observed by immunocytochemistry in

132 Journal of Andrology N January �February 2007

aging human testes (Kimura et al, 2003). This imbalance

between members of the Bcl-2 family, with a predomi-

nance of proapoptotic forms in germ cells, seems to beresponsible for the increase in germ cell apoptosis found

in aged hamsters activating the mitochondrial pathway

of apoptosis; the Fas/Fas-L system and p53 also

participate in the increase of germ cell apoptosis in

hamsters exposed to a short photoperiod.

The present results indicate that the levels of Bcl-2 are

low in the testis both of young and aged hamsters. These

results are congruent with previous studies obtained inmouse (Hockenbery et al, 1991; Knudson et al, 1995)

and humans (Beumer et al, 2000; Sakamaki, 2003),

which suggested that Bcl-2 plays no function in the male

gonad in normal conditions, unlike in the ovary, where

this protein is critical for primordial ovarian follicle

formation (Ratts et al, 1995). On the contrary, using

Western blot an overexpression of Bcl-2 by Sertoli and

Leydig cells was observed in hamsters exposed to shortphotoperiod compared with young animals. Studies in

transgenic animals have indicated that the overexpres-

sion of Bcl-2 in somatic cells of the testis produces

alterations in spermatogenesis, including the inhibition

of gamete formation, spermatid malformations, vacuo-

lization of the epithelium, loss of germ cells, and

increased apoptosis, while seminiferous tubules are

characterized by an accumulation of spermatogonia,Sertoli cells, and apoptotic germ cell in the meiotic

prophase (Knudson et al, 1995; Furuchi et al, 1996;

Rodrıguez et al, 1997; Yamamoto et al, 2001). These

spermatogenic defects found in transgenic animals are

comparable with the modifications observed in hamsters

exposed to a short photoperiod, which suggests that

Bcl-2 plays an important role in the atrophy of the

seminiferous epithelium of Syrian hamsters after expo-sure to the short photoperiod.

Previous studies have reported that Bax is expressed

predominantly in primary spermatocytes and sperma-

tids (Oldereid et al, 2001). Our immunohistochemical

study revealed that Bax was expressed by the spermatid

acrosomes of young and aged hamsters, which suggests

that Bax plays a role in the maturation and differenti-

ation process of the acrosome in the hamster, as has

been suggested in other species (Oldereid et al, 2001).Also, Bax has been classified as a proapoptotic member

of the Bcl-2 family and has been related to the induction

of apoptosis of spermatocytes and spermatids in hu-

mans (Oldereid et al, 2001). Based on our immunohis-

tochemical results, Bax does not seen to be involved in

germ cell apoptosis in young or aged hamsters.

However, in hamsters exposed to short photoperiod,

the expression of Bax by the spermatocytes wasconcordant with the expression of Fas and p53 as well

as with TUNEL-positive cells. These results suggest that

Bax is involved in germ cell apoptosis induction after

exposure to a short photoperiod.

Strong immunostaining with p53 was found in

spermatocytes and occasionally in spermatogonia of

photoinhibited hamsters. These results indicate that p53

expression is concordant with Fas and Bax expression as

well as with TUNEL-positive germ cells in photoinhib-

ited hamsters, which suggests that p53 is involved in

germ cell apoptosis induction after short photoperiod

exposure.

In summary, aging of the seminiferous epithelium in

the Syrian hamster is characterized by an increase in

germ cell apoptosis in the populations of spermatogonia

and spermatocytes and is dependent on the stage of the

cycle. After short photoperiod exposure, however, the

increase in apoptosis is only observed in the spermato-

cytes. Different molecular pathways are triggered toinduce germ cell apoptosis in aged animals and those

exposed to a short photoperiod. The results obtained

show both the intrinsic and extrinsic pathways being

activated after short photoperiod exposure, but only the

intrinsic pathway during aging.

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