FEBS Letters 580 (2006) 6109–6114

c-FlipL is expressed in undifferentiated mouse male germ cells

Claudia Giampietria,*, Simonetta Petrungaroa, Pierpaolo Colucciab, Fabrizio Antonangelia,Alessio Paonea, Fabrizio Padulaa, Paola De Cesarisc, Elio Ziparoa, Antonio Filippinia

a Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Histology and Medical Embryology, University of Rome ‘‘La Sapienza’’,00161 Rome, Italy

b Department of Surgery ‘‘P. Valdoni’’, University of Rome ‘‘La Sapienza’’, 00161 Rome, Italyc Department of Experimental Medicine, University of L’ Aquila, 67100 L’ Aquila, Italy

Received 25 July 2006; revised 22 September 2006; accepted 9 October 2006

Available online 16 October 2006

Edited by Francesc Posas

Abstract Apoptosis represents a fundamental process duringfetal/post-natal testis development. Therefore pro- and anti-apoptotic proteins are essential to regulate testis physiology.c-FlipL is a known inhibitor of caspase 8/10 activity; in this studyits perinatal expression in mouse male germ cells was investi-gated. In testis sections and seminiferous tubule whole mountc-FlipL was found to be expressed in undifferentiated spermato-gonia and to co-localize with germ stem cells markers. In vivoinvestigations in the vitamin-A deficient mouse, lacking differen-tiated germ cells, confirmed c-FlipL expression in undifferenti-ated spermatogonia. Further analyses showed Fas expressionbut no significant caspase 8/10 activity when c-FlipL was highlyexpressed. Altogether these data suggest that c-Flip may controlthe survival rate of undifferentiated spermatogonia.� 2006 Federation of European Biochemical Societies. Pub-lished by Elsevier B.V. All rights reserved.

Keywords: Testis; Apoptosis; Stem cells; Caspases

1. Introduction

Apoptosis is a form of controlled and highly ordered cell

death and occurs in several physiological conditions including

spermatogenesis. In testicular germ cells at the puberal age, an

early germ cells apoptotic wave occurs, likely aimed at remov-

ing abnormal germ cells and maintaining a proper ratio be-

tween maturing germ cells and somatic Sertoli cells [1–3].

Fas-receptor stimulation through Fas-ligand binding is one

of the several signals inducing apoptosis. It determines activa-

tion of the caspase cascade via caspase-8 or 10 ultimately trig-

gering effector caspases and leading to apoptosis [4]. This

process may be interfered by the caspase-8/10 inhibitor named

c-Flip (cellular Flice inhibitory protein), which blocks

Fas-mediated apoptosis [5,6] by preventing the proteolytic

activation of pro-caspase-8 and pro-caspase-10 [6,7]. c-Flip

expression is known to be regulated by RNA alternative splic-

ing leading to different protein isoforms, such as c-Flip long

isoform (c-FlipL) and c-Flip short isoform (c-FlipS) [8]. c-FlipL

has been recently reported to induce both an inhibitory and an

activating effect on caspases-8 and -10 [9].

*Corresponding author. Fax: +39 06 4462854.E-mail address: claudia.giampietri@uniroma1.it (C. Giampietri).

0014-5793/$32.00 � 2006 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2006.10.010

Prespermatogenesis represents the prepuberal stage of sper-

matogenesis. At this stage germ cells, named gonocytes,

acquire spermatogonial stem cell (SSC) properties by attaching

to the basement membrane [10]. How gonocytes undergo the

transition to SSCs is still under investigation. SSCs belong to

the undifferentiated spermatogonia type mostly represented

in the perinatal mouse testis, which include: (i) A-single (likely

the SSC) (ii) A-paired (iii) A-aligned; all exhibiting distinct

characters from the more advanced differentiating spermato-

gonia (A1, A2, A3, A4, Intermediate and B spermatogonia)

[11].

We have previously investigated c-FlipL and Fas expression

in mouse adult testis and fetal gonocytes [12,13]. Although cell

death and Fas expression have been reported in germ cells dur-

ing the first days after birth, apoptosis has not been found [14].

We therefore analyzed c-Flip expression in mouse testis during

the first days post birth (dpb) in order to investigate whether c-

Flip may play a protective role in Fas-mediated apoptosis in

undifferentiated spermatogonia.

2. Materials and methods

2.1. AnimalsCD1 strain mice were obtained from Charles River Laboratories,

(Como, Italy). The day of birth was considered 0 dpb. All animalexperiments were conducted according to the EU guidelines for Careand Use of Laboratory Animals.

2.2. Immunohistochemical stainingImmunohistochemical staining was performed on testis sections

using the rabbit polyclonal anti-c-FlipL (Upstate Biotechnology, LakePlacid, NY, USA) according to previous reports [12].

2.3. Whole-mount immunofluorescenceSeminiferous tubule segments were dissociated and fixed for 1 h at

4 �C in 4% paraformaldehyde. Tubules were then treated with glycine(1 M, 30 min) and permeabilized with 0.1% Triton X-100. Tubuleswere incubated with anti-c-FlipL (Abcam, Cambridge, UK) overnightat 4 �C and with Cy-3-conjugated donkey anti-rabbit IgGs (JacksonImmunoresearch, Cambridgeshire UK) for 1 h. After four washes tu-bules were stained for 30 min with the nucleic acid fluorescent dye TO-PRO-3 (Molecular Probes, Inc. Eugene, OR, USA) and mounted inVectashield mounting medium (Vector Laboratories, Burlingame,CA, USA).

2.4. Germ cells preparation and flow cytometric analysisSpermatogonia-enriched germ cells were collected as previously de-

scribed [15] and flow cytometric analysis was performed as previouslyreported [16]. c-FlipL expression was determined by the rabbit

blished by Elsevier B.V. All rights reserved.

Fig. 1. c-FlipL immunohistochemical staining on testis sections from0 to 5 dpb (magnification 200·). Data are representative of fourindependent experiments.

6110 C. Giampietri et al. / FEBS Letters 580 (2006) 6109–6114

polyclonal anti-c-FlipL (Upstate Biotechnology) and by a secondaryFITC-conjugated goat anti-rabbit antibody (Sigma); the GFRa-1expression was determined by a mouse anti-GFRa-1 (R&D Systems,Inc. Minneapolis, MN, USA) and by a secondary Alexafluor 680-R phyecoerythrin-conjugated goat anti-mouse (Molecular Probes);Thy-1 expression was followed with an anti-Thy-1-PE (BD Bioscience,Erembodegem, Belgium).

Cells from 4 dpb testes were plated to obtain an enriched populationof undifferentiated spermatogonia [17]. In this experimental condition,somatic cells adhered to plastic dishes whereas non-adhering undiffer-entiated spermatogonia population was collected to analyse Fasexpression by using the biotin conjugated anti Fas Jo2 and streptavi-din-PE (BD bioscience).

Analyses were performed on a Coulter Epics XL flow cytometer(Beckman Coulter, Fullerton, CA). Data were analyzed with the Win-MDI software.

2.5. Caspase activity assaysCaspase 8/10 activity was assayed on cytosolic extracts using the cas-

pase-8 and -10 fluorogenic substrate AEVD-AFC (Calbiochem, SanDiego, CA) as previously described [12].

2.6. Western blot analysisWestern blot analysis was performed as previously described using

anti-c-FlipS/L Dave-2 (Alexis Corporation, Lausen, Switzerland) andanti-caspase-8 (Calbiochem) [12].

2.7. Generation of vitamin A deficient mice2-Weeks-old male CD1 mice, were weaned onto a vitamin A defi-

cient (VAD) diet (Vitamin A deficient diet modified. MP Biomedicals,Irvine, CA, USA). Mice were maintained on this diet until testis reduc-tion was observed (about 12 weeks).

2.8. Statistical analysisStatistical evaluation was performed by t-student analysis and

P < 0.05 was considered statistically significant.

Fig. 2. Western blot and densitometric analysis of c-FlipL and c-FlipS

in testis extracts at 4 dpb. Data are representative of three independentexperiments.

3. Results

3.1. c-FlipL is expressed in gonocytes and undifferentiated

spermatogonia

In order to investigate c-FlipL expression during the transi-

tion from gonocytes to spermatogonial cells, we performed

immunohistochemical analyses on testis sections taken from

0 to 5 days post birth (dpb) (Fig. 1). c-FlipL-specific staining

was observed in the gonocytes cytoplasm at 0, 1 and 2 dpb;

c-FlipL signal was also detected at 3 and 4 days post partum,

in gonocytes near the basement membrane [18]. These data

indicate a strong c-FlipL expression during the transition from

gonocytes to undifferentiated spermatogonia and a weak, but

still present, expression at 5 dpb on undifferentiated spermato-

gonia [19]. To have a more detailed view of c-Flip expression

in perinatal testis, we performed Western blot analyses of both

isoforms (c-FlipL and c-FlipS) on 4 dpb testis extracts (Fig. 2).

Both isoforms were expressed and as revealed by densitometric

analysis, c-FlipS was weakly represented. Altogether these re-

sults indicate that both gonocytes and undifferentiated sperma-

togonia express high level of c-FlipL.

3.2. c-FlipL is expressed in both single and paired interconnected

cells

The topographical arrangement of interconnected germ cells

cannot be evaluated on two-dimension testis sections, there-

fore immunofluorescence on whole mount preparations of

seminiferous tubules was performed according to other

authors [18–20]. Pups testes were analyzed from 4 to 6 dpb.

The age was specifically chosen to exclude A1–A4 spermatogo-

nia or more differentiated germ cells in the seminiferous epithe-

lium and to maximize the number of undifferentiated cells

present in the germ cell pool [19]. c-FlipL staining by indirect

immunofluorescence was then carried out. In Fig. 3 Sertoli

cells (white arrowheads) as well as spermatogonia (red arrows)

are distinguished through nuclear morphology after nuclear

TOPRO-3 staining [19]. c-FlipL expression appears to occur

in spermatogonia but not in Sertoli cells. These studies on

whole mount preparations of seminiferous tubules support

the hypothesis that c-FlipL is expressed in a small testis sub-

population, namely in undifferentiated spermatogonia.

Fig. 3. Confocal laser scanning of seminiferous tubules whole mounts.Sertoli cells nuclei (white arrowhead) and spermatogonia nuclei (redarrow) are blue stained while c-FlipL expressing spermatogonia are redstained. Bar corresponds to 12 lm. The inset, representing a portion athigher magnification, shows a c-FlipL positive spermatogonium. Dataare representative of four independent experiments.

Fig. 4. Confocal laser scanning of seminiferous tubules whole mounts.Analysis revealed the presence of c-FlipL red stained A-singlespermatogonia (A) and c-FlipL red stained A-paired spermatogonia(B). Data are representative of four independent experiments. Barcorresponds to 13 lm.

C. Giampietri et al. / FEBS Letters 580 (2006) 6109–6114 6111

When an A-single spermatogonium divides to form A-paired

spermatogonia the two derived cells are connected by an inter-

cellular bridge. These cytoplasmic bridges form at any division

leading to large clones of interconnected germ cells. Spermato-

gonia cells with no intercellular bridge within 25 lm are

reported to be A-single spermatogonia [21]. To investigate

whether c-FlipL positive cells are A-single spermatogonia, a

confocal analysis was performed through the tubule through-

out several planes to exclude the presence of intercellular

bridges nearby. This analysis allowed to conclude that A-single

spermatogonia are c-FlipL positive (Fig. 4A) as well as A-

paired interconnected cells (Fig. 4B) while Sertoli cells do

not express c-FlipL in perinatal testes.

3.3. c-FlipL is expressed by a small subset of GFRa-1+/Thy-

1+germ cells

In order to better characterize cells expressing c-FlipL in

perinatal testis, flow cytometric analysis was performed and

undifferentiated spermatogonia were identified based on the

expression of specific markers. In particular GFRa-1, the

GDNF co-receptor was investigated. GDNF is produced by

Sertoli cells and regulates spermatogonial stem cells prolifera-

tion [19]. We also investigated Thy-1, an additional spermato-

gonial stem cells marker [22]. Flow cytometric analysis on

testis cell preparations from murine pups at 6 dpb showed c-

FlipL positive cells within GFRa-1/Thy-1 positive germ cells

(Fig. 5). In panel 5A Thy-1 and c-FlipL double positive cells

are represented (included in the ellipse) while in panel 5B

GFRa-1 positive cells within the double positive (c-FlipL and

Thy-1) population are shown. Panel 5C shows the GFRa-1

isotype staining. The percentage of triple GFRa-1/c-FlipL/

Thy-1 positive cells is around 0.04%, indicating that c-FlipL

is expressed in a small and specific subset of undifferentiated

spermatogonia.

3.4. Fas expression and caspase-8/10 activity in perinatal testis

To investigate the apoptotic machinery, Fas receptor and cas-

pase-8 were analysed. Fas was evaluated by flow cytometric

analysis in an enriched population of undifferentiated spermato-

gonia and was found to be significantly expressed (Fig. 6).

Caspase-8 precursor, a physiological target of c-FlipL, was iden-

tified by Western blot experiments on testis extracts at 4 and

10 dpb. Densitometric analysis revealed that caspase-8 precur-

sor expression was lower at 10 dpb than at 4 dpb (Fig. 7).

Accordingly, as shown in Fig. 8, caspase enzymatic activity

was evident at 10 dpb while it was not detected at 4 dpb, when

c-FlipL is highly expressed and there is no apoptosis [14]. Such

results, obtained both in testis extract and in an enriched undif-

ferentiated spermatogonia population, suggest that c-FlipL

expression may be related with protection from apoptosis and

lack of caspase activity in mouse testis at 4 dpb [14].

3.5. c-FlipL is expressed in undifferentiated spermatogonia from

vitamin-A deficient mouse

As a further approach, c-FlipL expression was investigated

in a particular rodent model, namely the vitamin-A deficient

(VAD) mouse model. Vitamin A deprivation in rodents results

Fig. 5. Flow cytometric analysis of 6 dpb germ cells preparations. (A)Double positive (c-FlipL and Thy-1) cells. (B) and (C) The GFRa-1positive cells within the c-FlipL and Thy-1 double positive population(ellipse) and the GFRa-1 isotype control. Data are representative ofthree independent experiments.

Fig. 6. Flow cytometric analysis of 4 dpb enriched undifferentiatedspermatogonia. Fas expression (white) vs isotype control (grey). Dataare representative of three independent experiments.

Fig. 7. Western blot analysis of caspase-8 at 4 and 10 dpb. Densito-metric analysis normalized versus b-actin is also shown (a.u.: arbitraryunits). Data are representative of three independent experiments.

6112 C. Giampietri et al. / FEBS Letters 580 (2006) 6109–6114

in mature germ cells loss in the testis [23]. Only A-single,

A-paired and A-aligned spermatogonia are observed in this

animal model, while a single retinol injection and vitamin A

re-inclusion in the diet resume spermatogenesis. This indicates

that when mature germ cells are lost, spermatogonial stem cells

remain and are capable of initiating spermatogenesis under

permissive conditions.

Fig. 9, representing c-FlipL immunostaining in VAD testis,

shows only few positive germ cells in some tubule sections,

confirming the expression in a specific subset of undifferenti-

ated spermatogonia.

4. Discussion

The machinery underlying apoptosis requires the hierarchi-

cal caspases activation, achieved by a cascade of proteolytic

processing steps. Specific inhibitors are available to block this

process at different levels. For instance c-IAP1 and c-IAP2

protect germ cells from Fas-mediated apoptosis by inhibiting

caspases in the seminiferous epithelium [14]. It has been dem-

onstrated c-IAPs role in B spermatogonia and hypothesized

the involvement of other caspases inhibitors in the homeostasis

of both undifferentiated and differentiated A spermatogonia. c-

Flip is a known caspase 8/10 inhibitor and its role in control-

ling apoptosis has been addressed in several studies by our and

others’ groups [8,12]. As c-FlipL is highly expressed in mouse

fetal and adult testis [12,13], we hypothesized that c-FlipL

Fig. 8. Caspase 8/10 activity (a.u: arbitrary units) evaluated in testisextracts at 4 and 10 dpb and in enriched undifferentiated spermato-gonia preparation at 4 dpb. The experiment was performed intriplicate. Average of three independent experiments ± S.D. isreported. � indicates significant difference vs control.

C. Giampietri et al. / FEBS Letters 580 (2006) 6109–6114 6113

may have a role as a caspase-8/10 inhibitor during the first

days post birth when spermatogonia are still undifferentiated.

Our previous studies on c-FlipL expression and function in

fetal mouse gonocytes [13] indicate that c-FlipL is directly

involved in the control of gonocytes apoptotic waves during

fetal life by inhibiting caspase activity after 13 days post coi-

tum. In the present study we investigated c-FlipL spatio-tem-

poral distribution and regulation within the mouse testis

during the first days after birth, when mainly gonocytes, A-sin-

gle, A-paired and A-aligned spermatogonia are found in the

seminiferous epithelium [19]. Strong c-FlipL expression was

found in perinatal germ cells until 4 dpb when both gonocytes

and undifferentiated spermatogonia are found; such expression

Fig. 9. Immunohistochemical c-FlipL staining in testis from VAD mousmagnification. The experiment was carried out on three different animals wi

appears to have a temporal control since it decreases, although

not completely, at 5 dpb, when undifferentiated spermatogonia

are the prevalent germ cell population. Testis whole mount

immunofluorescence confirmed c-FlipL staining in the

cytoplasm of undifferentiated spermatogonia particularly in

A-single and A-paired spermatogonia. A further evidence for

c-FlipL expression in undifferentiated spermatogonia was

achieved in vivo in the VAD mouse model [24] where the lack

of differentiated germ cells determines an enrichment in undif-

ferentiated spermatogonia. Flow cytometric analysis results re-

vealed c-FlipL positive cells within GFRa-1 and Thy-1 positive

spermatogonia. GFRa-1 is expressed both in A-single (pre-

sumably stem cell) and A-paired spermatogonia, while Thy-1

is a well described germ stem cell marker [19,22]. This allows

to hypothesize that c-FlipL is expressed in a subset of undiffer-

entiated spermatogonia including germ stem cells. Remarkably

other stem cells, namely human hematopoietic CD34+ cells,

have been recently shown to express high levels of c-Flip

mRNA and protein which may serve to protect CD34+ cells

from Fas-mediated apoptosis [25].

Previous works from our and other groups have elucidated

the role of Fas both in gonocytes and in differentiated germ cells

[12,13,26]. Mechanisms controlling apoptosis in undifferenti-

ated spermatogonia still need to be clarified. The present work,

showing Fas expression in undifferentiated spermatogonia,

indicates that these cells might be sensitive to Fas-dependent

apoptosis. The finding that undifferentiated spermatogonia

also express c-Flip, suggests its inhibitory role in controlling

apoptotic signalling possibly explaining the physiological ab-

sence of apoptosis in 4 dpb testis [14].

In our previous studies [12,27] we reported that within the

different male germ cells populations in the adult mouse, sper-

matogonia show the lowest c-FlipL expression and display

e (magnification 400·). The inset represents a particular at higherth similar results.

6114 C. Giampietri et al. / FEBS Letters 580 (2006) 6109–6114

caspase activation. These previous data well correlate with the

evidence that apoptosis occurs frequently during the first sper-

matogenic wave particularly in spermatogonia. The data pre-

sented in this manuscript may help elucidating why

apoptosis is absent at 4 dpb, as previously described by other

authors [14].

In conclusion c-Flip expression is strictly controlled in differ-

ent germ cell populations in perinatal testis suggesting that it

may have an important survival modulating role during the

transition from gonocytes to undifferentiated spermatogonia.

Acknowledgments: We gratefully thank Dr. A. Facchiano, Prof. F. Pa-lombi, Dr. A. D’Alessio, Dr A. Riccioli and Dr D. Starace for helpfuldiscussion. Skillful assistance in animal care was provided by Miss V.Di Dio and Miss L. Marzilli. This work was supported by Grants fromMIUR-COFIN 2005.

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