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The endocannabinoid system and pivotal role of the
CB
2
receptor in mouse spermatogenesis
Paola Grimaldi
a,1,2
, Pierangelo Orlando
b,c,1,2
, Sara Di Siena
a
, Francesca Lolicato
a
, Stefania Petrosino
c,d
, Tiziana Bisogno
c,d
,
Raffaele Geremia
a
, Luciano De Petrocellis
c,e
, and Vincenzo Di Marzo
c,d
aDepartment of Public Health and Cellular Biology, University of Rome ‘‘Tor Vergata’’, 00133 Rome, Italy; bInstitute of Protein Biochemistry (PO), Consiglio
Nazionale delle Ricerche, via P. Castellino 111, Napoli, Italy; and Institutes of dBiomolecular Chemistry and eCybernetics, and cEndocannabinoid Research
Group, Consiglio Nazionale delle Ricerche, via Campi Flegrei 34, Pozzuoli, Italy
Edited by Ryuzo Yanagimachi, University of Hawaii, Honolulu, HI, and approved April 16, 2009 (received for review December 16, 2008)
The exact role of the endocannabinoid system (ECS) during sper-
matogenesis has not been clarified. We used purified germ cell
fractions representative of all phases of spermatogenesis and
primary cultures of spermatogonia. This approach allowed the
precise quantification of the cannabinoid receptor ligands, anan-
damide and 2-arachidonoylglycerol, and of the expression at
transcriptional and transductional levels of their metabolic en-
zymes and receptors. Our data indicate that male mouse germ cells
possess an active and complete ECS, which is modulated during
meiosis, and suggest the presence of an autocrine endocannabi-
noid signal during spermatogenesis. Mitotic cells possess higher
levels of 2-arachidonoylglycerol, which decrease in spermatocytes
and spermatids. Accordingly, spermatogonia express higher and
lower levels of 2-arachidonoylglycerol biosynthetic and degrading
enzymes, respectively, as compared to meiotic and postmeiotic
cells. This endocannabinoid likely plays a pivotal role in promoting
the meiotic progression of germ cells by activating CB
2
receptors.
In fact, we found that the selective CB
2
receptor agonist, JWH133,
induced the Erk 1/2 MAPK phosphorylation cascade in spermato-
gonia and their progression toward meiosis, because it increased
the number of cells positive for SCP3, a marker of meiotic prophase,
and the expression of early meiotic prophase genes.
cannabinoid receptors 兩meiosis 兩TRPV1
Since its discovery, the endocannabinoid system (ECS) has been
shown to be implicated in several fundamental physiological
functions as well as in many pathological conditions (1, 2). The ECS
is modulated during cell proliferation, differentiation, and apopto-
sis through alterations of the expression levels of cannabinoid
receptors (CNRs) of type 1 (CB
1
) and 2 (CB
2
), and of the enzymes
involved in the biosynthesis and degradation of the 2 main CNR
agonists: anandamide (AEA) and 2-arachidonoylglycerol (2-AG)
(1). It has been demonstrated that AEA and synthetic agonists of
CNRs exert antitumoral and antimetastatic activities by inhibiting
cell proliferation, angiogenesis, and tumor cell migration (2).
A central role of CB
1
receptors in the regulation of the pituitary–
gonad axis has been described by Wenger et al. (3), demonstrating
the involvement of CB
1
in testosterone production by Leydig cells.
The presence of an active ECS has been described both in testis and
isolated spermatozoa of mammals, sea-urchin, and Rana esculenta
(4–9). In particular, it has been demonstrated (6, 7) that activation
of CB
1
receptors by AEA in both human and boar spermatozoa
reduces their motility and the acrosomal reaction (10).
Spermatogenesis is a highly coordinated complex process char-
acterized by mitotic (spermatogonia), meiotic (spermatocytes), and
differentiative haploid (spermatids) phases. Spermatogenesis is
initiated in the basal compartment of the seminiferous epithelium,
by spermatogonial stem cells that proliferate and differentiate into
type A1 spermatogonia. Type A1 spermatogonia undergo a series
of synchronized mitotic divisions, giving rise to type B spermato-
gonia, which enter the meiotic phase of spermatogenesis as primary
spermatocytes (11). Meiosis is characterized by two consecutive cell
divisions, following a single DNA duplication, and by genetic
exchange (crossing-over) between homologous chromosomes, end-
ing up with 4 haploid round spermatids (12). Gye et al. (5), using
both cryostat sections and extracts of mouse testes obtained during
postnatal development, demonstrated CB
1
immunoreactivity in
proliferating gonocytes and spermatozoa. Cobellis et al. (9) de-
scribed the immunolocalization of CB
1
receptors in testes from the
R. esculenta that, on the basis of seasonal considerations, the
authors ascribed mainly to elongated spermatids. Another well
established molecular target of AEA, the transient receptor po-
tential vanilloid type-1 (TRPV1) channel (13), which is activated
also by temperatures higher than 42 °C (14, 15), was suggested to
play a role in the stabilization of the plasma membranes in
capacitated sperm (7), and to confer heat resistance to male germ
cells (16). Because the molecular and cellular bases of the involve-
ment of the ECS in controlling spermatogenesis and male fertility
in mammals remain unclear, the aim of this work was to investigate
the presence and functional role of the ECS in germ cells at
different stages of differentiation using purified germ cell fractions
representative of each spermatogenesis phase (17, 18).
Results
Expression of CB
1
,CB
2
, and TPRV1 Receptors in Male Mouse Differ-
entiating Germ Cells. Reverse transcription quantitative PCR (qRT-
PCR) analysis (Fig. 1) showed that CB
1
receptor mRNA levels
gradually increase in purified meiotic spermatocytes (SPC) and
postmeiotic spermatids (SPT). CB
2
receptors showed elevated
transcriptional levels in all stages of spermatogenesis with a relative
peak of expression (⬇10-fold increase) in SPC. The expression
levels of TRPV1 showed a strong (⬇100-fold) increase in meiotic
cells. Protamine, a well known specific target of postmeiotic cells
(19), was also evaluated as a marker of cross-contamination in
isolated germ cell fractions. Protamine mRNA levels were ⬇1,000
fold higher in SPC with respect to spermatogonial ger m cell fraction
(SPG) and showed a dramatic increase, ⬇25,000-fold, in SPT (data
not shown).
Because Sertoli cells (SRT) constitute the most relevant somatic
contaminant (5–10%) of the mitotic germ cell fractions, we com-
pared the transcriptional levels of CB
1
,CB
2
, and TRPV1 receptors
in SPG fractions with those of a purified preparation of SRT. The
RNA levels of all of the receptors resulted significantly lower in
purified SRT (Fig. 1).
The expression of the receptors at the protein level was analyzed
by Western blot analysis (WB) in cell extracts. The CB
1
receptor
Author contributions: P.G., P.O., L.D.P., and V.D.M. designed research; P.G., P.O., S.D.S., F.L.,
S.P., T.B., and L.D.P. performed research; P.G., P.O., R.G., L.D.P., and V.D.M. analyzed data;
and P.G., P.O., R.G., L.D.P., and V.D.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1P.G. and P.O. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: grimaldi@uniroma2.it or
p.orlando@ibp.cnr.it.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0812789106/DCSupplemental.
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CELL BIOLOGY
band was very faint in SPG and gradually increased in meiotic and
postmeiotic germ cell extracts (Fig. S1 A), it was detectable in
cauda-epididymis spermatozoa and was absent in SRT, in agree-
ment with previous data (4, 9). The CB
2
receptor protein was
detected in all differentiative stages, and its amount was lower in
SRT and undetectable in spermatozoa (SPZ) (Fig. S1B). The
TRPV1 receptor protein was detectable in SPC and showed
relatively higher levels in SPT (Fig. S1C). SPZ and SRT also
expressed the TRPV1 receptor.
The presence of CB
2
in differentiating SPG was confirmed by
immunofluorescence microscopy. Fig. 2Ashows a strong immuno-
staining for this receptor, which was localized as intense punctuate
fluorescence in peripheral regions of the cells and on the bridges of
interconnected dividing cells as shown in Fig. 2 B. A strong, more
diffuse staining was present in SPC, whereas round SPT showed
weaker staining (Fig. 2 A). Interestingly, in elongating SPT, the
immunoreactive materials accumulate in the caudal pole of the
sperm where the cytoplasm is localized. At the end of spermio-
genesis, when SPZ are released, the cytoplasm forms the residual
bodies in which immunofluorescence was observed (Fig. 2 A).
These latter findings explain the aforementioned absence of CB
2
receptors in epididymial SPZ.
Functional CB
2
Receptors in Primary Cultures of Spermatogonia. One
of the signal transduction pathways triggered by G proteins coupled
to CB
1
and CB
2
receptors involves activation of the MAPK
phosphorylation cascade (20, 21). The presence of a functional CB
2
receptor in SPG was evaluated by treating primary cultures of these
cells with the potent selective CB
2
agonist, JWH133 (22). Treat-
ment with different c oncentrations of this compound and for 15 min
indicated that the optimal concentration to obtain the phosphor-
ylation of extracellular regulated kinases (Erk1/2) MA PK was 1
M
(Fig. 3A). Erk1/2 activation was maintained for 30 min and then
decreased to return to basal after 1 h (Fig. 3B). By contrast, when
SPG were treated in parallel with the CB
1
specific agonist, ACEA
(22) (Fig. 3C), Erk1/2 phosphorylation was not observed. Erk1/2
activation was also induced by the c-kit ligand (KL) (23). Preincu-
bation of SPG with AM630 (10
M), a specific CB
2
antagonist (22),
antagonized the effect of JWH133 (Fig. 3D).
To evaluate the contribution of SRT contaminant on Erk1/2
phosphorylation induced by JWH133, we isolated pure c-kit ex-
pressing SPG by magnetic microbeads conjugated with CD117 and
compared this preparation to a purified preparation of SRT
(Fig. S2). JWH133-induced Erk1/2 phosphorylation over vehicle-
treated cells was clearly lower in purified SRT, thus suggesting the
little SRT contaminant could not have affected the results obtained
in SPG.
JWH133 Induces Meiotic Prophase in Spermatogonia Primary Cell
Cultures. To investigate whether the MAPK signal transduction
pathway activated in SPG by JWH133 is related to mitotic cell cycle,
we performed a morphological examination of cell nuclei f rom SPG
treated with 1
M JWH133 for 1 and 24 h. The number of mitotic
figures (nuclei showing condensed metaphase chromosomes) did
not change in presence of the agonist, suggesting that the activation
of CB
2
receptors in SPG does not have a mitogenic effect (data not
shown). To demonstrate whether CB
2
-activated signal transduction
in SPG could correlate with cell differentiation toward the meiosis
prophase, we prepared nuclear spreads from these cells that were
then probed with antibodies against a synaptonemal complex
protein (SCP3) and a marker of meiotic nuclei (24). Fig. 4Ashows
that in control cell cultures ⬇7.82 ⫾1.94% of SPG naturally
undergo meiotic progression after 24 h of culture. Treatment with
JWH133 induced a ⬇2-fold increase in the percentage of nuclei
showing nuclear organization of SCP3 on meiotic chromosome
figures (16.60 ⫾5.54%). This effect was reversed by pretreatment
of the cells with the CB
2
antagonist AM630, which did not modify
the basal progression of meiosis per se. SCP3 distribution onto
condensing chromatin allowed to identify different meiotic stages
of prophase, as shown in Fig. 4B. The relative distribution of these
stages in control and in agonist-treated cells are reported in Fig. 4C,
showing that treatment of cells with JWH133 induces a ⬇2-fold
increase of leptotene and zygotene figures with respect to control
cells, whilst inducing also the appearance of a low but consistent
number of zygo-pachytene stages.
This increase in meiotic prophase was confirmed by analyzing the
transcriptional levels of selected premeiotic and meiotic markers by
qRT-PCR (Fig. 4D). The c-Kit, a protein expressed in SPG com-
mitted to meiotic prophase (25), increased by ⬇2-fold following
treatment with JWH133. Dmc1, which encodes a meiosis specific
recombinase expressed during the first meiotic prophase (23),
showed an increase of 1.4-fold vs. control, whereas Stra8, a protein
essential for progression through the early stages of meiosis (23),
increased by 1.6-fold. Lhx8, a LIM homeobox gene upregulated in
KL treated spermatogonia (25), and Spo11, which is responsible for
the initiation of meiotic recombination at pachytene stage through
the formation of DNA double-strand breaks (23), increased not
significantly.
Endocannabinoid Levels and Expression of Endocannabinoid Biosyn-
thetic and Degradative Enzymes. SPG showed high levels of 2-AG
that dramatically decreased by ⬇20-fold in postmeiotic SPT,
whereas AEA levels were substantially unchanged (Table 1). The
transcriptional expression levels of the enzymes involved in the
biosynthesis and degradation of endocannabinoids were evaluated
Fig. 1. CB1,CB2, and TRPV1 mRNA levels in mitotic, meiotic, and postmeiotic male mouse germ cells. qRT-PCR was performed as described in Methods using
enriched fractions of premeiotic (SPG), meiotic (SPC), postmeiotic (SPT) germ cells (A), and Sertoli cells (SRT). The expression levels in SPG, i.e., the reference
condition (*), were considered as 1 for all of the targets. The means of threshold-cycles for the reference condition were 28.9, 27.2, and 28.5 for CB1 (hatched
bars), CB2 (gray bars), and TRPV1 (black bars), respectively. Standard deviations were calculated by the Gene expression module of iQ5 real-time PCR. All
differences were significant (P⬍0.05) as evaluated according to Pfaffl et al. (40). A typical experiment (R.I.N. ⬎8.5, see Methods) is depicted.
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by qRT-PCR. The mRNA levels of the two 2-AG biosynthetic
diacylgycerol lipase isoenzymes (DAGL alpha and beta) (26),
decreased in meiotic and postmeiotic germ cells in comparison to
premeiotic germ cells (Fig. 5A). These data were paralleled by a
relevant increase in meiotic germ cells of the 2-AG degrading
enzyme, monoacylglycerol lipase (MAGL) (1) (Fig. 5B). The
ab-hydrolase domain containing (ABDH) 6 and 12 enzymes (1), for
which a role in 2-AG hydrolysis was recently proposed, were
expressed in SPG at amounts comparable to MAGL levels, as
judged by comparing the threshold cycles and the efficiency (see
Methods) of the 3 PCRs (data not shown). The ABHD 12 hydrolase
exhibited a modest increase of expression in meiotic and postmei-
otic germ cells, whereas the levels of ABHD 6 hydrolase were
unchanged.
Fig. 5Aalso shows a transcriptional increase during meiosis of
N-acyl phosphatidylethanolamine-phosholipase D (NAPE-PLD)
and fatty acid amide hydrolase (FAAH), the enzymes involved,
respectively, in the biosynthesis and degradation of AEA (1). We
also evaluated by enzymatic assays the DAGL, FAAH and MAGL
activities during meiotic differentiation, and in SRT as a control
(Table S1). Because during meiotic differentiation the size of germ
cells and the content of protein and DNA varies dramatically, the
most appropriate way to express specific activities is to use the cell
number. The enzymatic data substantially confirmed the mRNA
expression data. Comparisons of the amounts of the degrading
enzymes among a mouse control tissue, SRT and the various germ
cells were performed at both the protein level, by WB (Fig. S3), and
by qRT-PCR (Fig. S4).
Discussion
The importance of the ECS in male reproductive functions, attested
to at the phylogenetic level by its discovery in male reproductive
organs of both vertebrates and invertebrates, has been pointed out
in previous papers (4 – 8, 10, 27–30). AEA reduces sperm-fertilizing
capacity in sea urchin SPZ by inhibiting the acrosome reaction (8,
28) and inhibits key fertilization fuctions such as sperm motility,
capacitation, and acrosome reaction, in both boar and human (6, 7,
Fig. 2. CB2immunofluorescence staining of differentiating male germ cells.
Immunofluorescence staining for CB2receptors (red signal), Hoechst nuclear
counterstain (blue signal) and merged images of isolated testicular germ cells
at different stages of differentiation. (A) Immunodetection of CB2receptors
in: spermatogonia (Top), pachytene spermatocytes (Middle) and round (r-spt)
and elongated (e-spt) spermatids (Bottom). Note that the immunofluores-
cence is also localized in vescicles containing only the cytoplasm that is
released from the cell during spermiogenesis (residual bodies, rb); (B) High-
magnification of CB2immunofluorescence pattern in dividing and intercon-
nected spermatogonia.
Fig. 3. MAPK signaling is activated by the CB2selective agonist JWH133 in
spermatogonia.(A) Dose-dependent activation of ERK phosphorylation by
JWH133 in mouse spermatogonia. Isolated mouse spermatogonia were incu-
bated with or without 10⫺8–5 ⫻10⫺6M JWH133 for 15 min at 37 °C. At the end
of the incubation, cell extracts were prepared and the immunoblot was
performed as described in Methods.(B) Time-course analysis of JWH133-
stimulated phosphorylation of ERKs in spermatogonia. Isolated spermatogo-
nia were incubated with 10⫺6M JWH133, for 0 –60 min at 37 °C. At the end of
the incubation, cell extracts were prepared for Western blot analysis with
anti-phosphoERKs antibody. Densitometric analysis of phosphoERKs is shown
on Right.(C) Isolated spermatogonia were treated with the CB2-selective
agonist JWH133, or with the CB1-selecive agonist ACEA, or with c-Kit ligand
(KL), as positive control, and pERKs activation was evaluated. Densitometric
analysis is shown in Right.(D) The effects of preincubation (15 min) with the
CB2-selective antagonist AM630 (1
M) on the phosphorylation of ERKs by
JWH133 (1
M) in mouse spermatogonia was tested at different times (5 min
to 24 h).
Grimaldi et al. PNAS
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29, 30). Ectopic synthesis of AEA and immunolocalization of the
CB
1
receptor have been demonstrated in boar, human, mouse, and
rat SPZ as well as in SRT and Leydig cells (4–7, 9, 29). These
findings attest to the ubiquity of the ECS in the male reproductive
organ and support its role in physiological functions such as the
control of testosterone production and the regulation of the pitu-
ary-gonad axis (3).
Despite these several previous studies, the involvement of the
ECS in spermatogenesis has been only partially explored. Cobellis
et al. (9) described an increase of the expression of the CB
1
cannabinoid receptor and of FAAH in SPT of R. esculenta. Gye et
al. (5) utilized a postpartum selection of newborn mice to obtain a
selective enrichment of premeiotic, meiotic, and postmeiotic germ
cells in testes. They described relatively high CB
1
expression levels
in mouse testes at 1 week postpartum, which were ascribed mainly
to proliferating gonocytes. CB
1
expression decreased during pre-
pubertal development (2 week), increased during puberty (4 week),
and reached a peak in adult testis, a picture compatible with a
decrease of CB
1
mRNA transcriptional expression during meiosis,
followed by an increase in postmeiotic SPT during the last tran-
scriptional phase. Our present data confirmed an increase of CB
1
expression at both transcriptional and transductional levels in the
postmeiotic fractions, although, in terms of absolute expression, low
transcriptional levels were observed in premeiotic cells. However,
all of the data reported so far agree with the absence of CB
1
receptors in SPG and with an increase of these receptors in mature
SPT. Regarding TRPV1 channels, which are expressed in both
sperm germ cells and SPZ (7, 16), we observed here a strong
increase of mRNA expression in SPC and SPT, paralleled by
increased TRPV1 protein levels from meiotic germ cells to differ-
entiating round SPT. This result is important in view of the recent
observation that TRPV1 plays a crucial role in the defense of testis
against heat stress (16), and suggests for this receptor a protective
role in meiotic progression, in addition to its regulatory function in
sperm capacitation (7).
The present study, however, was mostly focused on the possible
roleofCB
2
receptors in spermatogenesis, a possibility that was
recently hypothesized (31) but not yet investigated. Our findings
clearly show, at both transcriptional and transductional levels, the
presence of CB
2
receptors in male germ cells from mitotic SPG to
haploid SPT. Interestingly, at the end of spermiogenesis, the CB
2
protein is released into the residual body and is not detectable in
mature SPZ, in agreement with previous observations (6, 32). CB
2
exhibited high transcriptional levels, as evaluated on the basis of
threshold cycles, in all spermatogenesis fractions analyzed, with a
relative peak of activity in meiotic fractions, possibly reflecting an
Fig. 4. JWH133 promotes differentiation of male
germ cells from 7 days post partum (dpp) mice.(A)
Histogram representing the percentage of nuclei with
meiotic SCP3 staining in control testicular germ cells
from 7 dpp or in cells stimulated with JWH133, AM630,
or pretreated with AM630 and then treated with
JWH133, after 24 h of culture. Bars represent s.d.. (B)
Representative immunofluorescence images showing
SCP3 (green) organization on nuclear spreads at the
stages of early leptotene, leptotene, zygotene and
zygo-pachytene of meiotic prophase, observed in 24 h
cultured spermatogonia. (C) Percentage of leptotene,
zygotene and zygo-pachytene nuclei in control cul-
tures of germ cells from 7 dpp or in cells treated for 24 h
with JWH133. Bars represent s.d. (D) qRT-PCR were
performed as described in Methods in JWH133-treated
premeiotic germ cells (JWH133) and untreated cells
(Ctr). The expression levels in Ctr, i.e., the reference
condition (*), were considered as 1 for all of the tar-
gets. The means of threshold cycles for the reference
condition were 25.76, 23.21, 26.25, and 29.79 for c-Kit
(first bar from the left), Stra-8 (second bar from the
left) DMC-1 (third bar from the left), Lhx8 (fourth bar
from the left), and SPO11 (last bar from the left),
respectively. Standard deviations were calculated by
the Gene expression module of iQ5 real-time PCR. The
differences (except for Spo11 target) were significant
(P⬍0.05) as evaluated according to Pfaffl et al. (40). A
typical experiment (R.I.N. ⬎8.5, see Methods)is
depicted.
Table 1. Endocannabinoid levels in pre-meiotic, meiotic and
post-meiotic male mouse germ cells
AEA (pmoles ⫾SD) 2-AG (pmoles ⫾SD)
Spermatogonia 0.68 ⫾0.13 100.65 ⫾6.83
Spermatocytes 0.51 ⫾0.23 44.13 ⫾16.14
Spermatids 0.22 ⫾0.11 5.84 ⫾1.72
AEA and 2-AG were evaluated by LC-MS on enriched fractions of pre-
meiotic (Spermatogonia), meiotic (Spermatocytes), and postmeiotic (Sperma-
tids) male mouse germ cell fractions, as described in Methods. The data are the
mean of 4 independent determinations in duplicate experiments. pmol values
were normalized for 1 ⫻107cells. s.d., standard deviation.
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accumulation of the transcript during meiosis to be translated at
later stages, a well known process occurring during spermatogenesis
(33). These data seem to indicate the existence of a continuous
transcriptional activity directed to assure high levels of CB
2
recep-
tors in all phases of differentiation process.
A functional involvement of CB
2
receptors in cell differentiation
was previously described in HL-60 cells transfected with CB
2
(34).
CB
2
receptors and their main agonist 2-AG (AEA is less efficacious
than 2-AG at these receptors) were also shown to be critical for
haematopoietic cell differentiation (35). Therefore, we investigated
here the effect of CB
2
activation on SPG differentiation. First of all,
our data indicate the presence of a functionally active CB
2
receptor
in these cells. We found that treatment of SPG with a CB
2
-selective
agonist activates the MAPK pathway by increasing Erk1/2 phos-
phorylation in a way attenuated by a CB
2
-selective antagonist.
Furthermore, we showed that CB
2
activation exerts a prodifferen-
tiative effect in isolated SPG cell fractions by increasing the
percentage of meiotic nuclei at all prophase stages (from leptotene
to zygo-pachytene spermatocytes) and by increasing the mRNA
levels of c-kit (23, 25), Stra8 (27, 36) and Dmc1 (23), i.e., genes that
are all involved in meiotic prophase commitment and/or in meiotic
progression. This study reports a prodifferentiative effect of CB
2
receptors in male germ cells.
These findings prompted us to analyze, during spermatogenesis,
the levels of the main physiological CB
2
receptor ligands, AEA and
2-AG, and of the enzymes involved in their metabolism. Our data
show a dramatic decrease of 2-AG levels in meiotic and postmeiotic
germ cells that was paralleled by a decrease of the transcriptional
and enzymatic levels of biosynthetic 2-AG enzymes, DAGL-alpha
and -beta, and an increase of 2-AG degradation enzymes: MAGL,
ABHD 12, and FAAH. It is noteworthy that MAGL underwent a
stronger expression increase in meiotic and postmeiotic fractions
with respect to ABHD 12 and FA AH, thus indicating a main role
for this enzyme in 2-AG inactivation in this biological setting.
We observed a transcriptional increase during meiosis of both
NAPE-PLD, one of the enzymes involved in the biosynthesis of
AEA, and of the AEA-degrading FAAH. Possibly as a result, we
found that the levels of AEA remained constant during spermat-
ogenesis. It is possible that the up-regulation of AEA metabolic
enzymes could ser ve to maintain an appropriate ‘‘anandamide
tone’’ during meiosis as seen in mouse embryos (37). Because AEA
is an agonist for TRPV1 channels, this tone would ensure the
proposed TRPV1 protective action against temperature increase at
the pachytene stage (16). An alternative explanation for these
findings could be that NAPE-PLD mRNA is stockpiled and
translated into protein in the mature SPT to ensure adequate levels
of AEA that could inhibit SPZ motility during the passage through
the epididymal tract (30).
In conclusion, our data demonstrate that mouse germ cells
possess an active and complete ECS, which is modulated during
spermatogenesis. They suggest the involvement of an autocrine
endocannabinoid signal in the mitotic and meiotic phases of sper-
matogenesis, and indicate a pivotal role of CB
2
receptors in this
process.
Methods
Male Mouse Germ Cells Isolation and Culture. Enriched SPG fractions were
obtained from testes of immature 7-day-old Swiss CD-1 mice, as reported (23).
Briefly, after dissection of the albuginea membrane, testes were first digested
with collagenase to remove interstitial cells and then with hyaluronidase and
trypsin. The cell suspension was plated in Petri dishes for4hinMEMsupple-
mented with 1 mM DL-lactic acid, 2 mM sodium pyruvate, and 10% FCS to
promote adhesion of somatic cells. After this preplating treatment, enriched SPG
suspension was recovered, and the purity was monitored morphologically after
Giemsa staining. Homogeneity of the SPG population was ⬇85–90%, including
all stages: undifferentiated and differentiating (type A1-A4, intermediate, and
type B) SPG, 4.5% of preleptotene SPC (last S phase before meiosis), and rare
leptotene SPC (23). The 10–15% contaminating cells were somatic cells (fibro-
blasts, myoid, and Sertoli cells).
Isolation of Kit positive SPG was performed by using magnetic-activated
cell sorting (MACS) with CD117 conjugated microbeads (Miltenyi Biotec). SPG
were stimulated with either JWH133 (Tocris Bioscience), or ACEA (Tocris
Bioscience), or with the vehicle at different time points and then they were
processed for immunofluorescence, WB or mRNA preparation. Where indi-
cated, cells were also incubated with AM630 (Tocris Bioscience) 15 min before
JWH133 addition. Testes of adult CD1 mice were used to prepare meiotic (SPC)
and haploid germ cells (SPT). Germ cell suspensions were obtained by sequen-
tial collagenase-trypsin digestions of freshly withdrawn testes. Germ cells at
pachytene SPC, round SPT, and elongated SPT stages were separated on the
basis of their size by centrifugal elutriation in PBS containing 0.5% BSA, as
described (38). The obtained SPC fraction was enriched in cells at pachytene
stages (90%) and the 10% contaminating cells were round SPT, early meiotic
cells, and somatic cells. The purity of the round SPT fraction was ⬇90% and the
10% contaminating cells included meiotic cells, elongated SPT, and residual
bodies. The elongated SPT (45–50%) were recovered in the same fraction of
residual bodies (⬇50%). Mature SPZ were obtained from the cauda of the
epididymis of mature mice. SRT cultures were prepared as described (23).
Endocannabinoid Measurement. AEA and 2-AG were quantified by liquid chro-
matography–mass spectroscopy in the selected ion monitoring mode using
mouse germ cell fractions containing 5– 8 ⫻1010 cells, as described (26). Data were
means of 4 independent determinations in duplicate experiments normalized as
pmols per 1 ⫻107cells. Statistical analysis was performed by ANOVA followed by
Bonferroni’s test (StatMost ).
Quantitative RT-PCR. Total RNA was extracted, analyzed by a 2100 Bionalyzer
(Agilent) RNA Integrity Number (R.I.N.) ⬎7.0 and retrotranscribed as described
(39). qRT-PCR analysis was performed essentially as described by an iCycler-iQ5
Fig. 5. Evaluationof the mRNA levels of the enzymes involved in AEA and 2-AG
biosynthesis in premeiotic, meiotic and postmeiotic male mouse germ cells.
qRT-PCR analyses were performed as described in Methods using enriched frac-
tions of mitotic (SPG), meiotic (SPC), and postmeiotic (SPT) germ cells. The expres-
sion levels in SPG, i.e., the reference condition (*), were considered as 1 for all
targets. The means of threshold cycles for the reference condition were 25.0, 25.6,
26.0, and 25.8 for DAG-alpha (light gray bar), DAG-beta (hatched bar), NAPE-PLD
(stippled bar), and FAAH (black bar), respectively (A), and 23.09, 24.33 and 24.32
for ABHD12 (light gray bar), ABHD6 (medium gray bar), and MGLL (black bar),
respectively (B). Standard deviations were calculated by the Gene expression
module of iQ5 real-time PCR. All differences were significant (P⬍0.05), as
evaluated according to Pfaffl et al. (40). A typical experiment (R.I.N. ⬎8.5, see
Methods) is depicted.
Grimaldi et al. PNAS
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July 7, 2009
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vol. 106
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11135
CELL BIOLOGY
(Bio-Rad) in a 25-
l reaction mixture containing 10–50 ng of cDNA Optimized
primers for SYBR-green analysis (GenBank accession nos.: CB1,NM㛭007726; CB2,
NM㛭009924; TPRV1, NM㛭001001445; FAAH, NM㛭010173; NAPE-PLD, AB112350;
MAGL, NM㛭011844; ABHD6, NM㛭025341; ABHD12, NM㛭024465; DAGL alpha,
NM㛭198114; DAGL beta, BC016105; Stra8, NM㛭009292; SPO11, NM㛭012046;
DMC1, NM㛭010059; c-kit,NM㛭021099; Lhx8, NM㛭010713) and optimum annealing
temperatures were designed by Allele-Id software version 6.0 (Biosoft Interna-
tional) and were synthesized (HPLC-purification grade) by MWG-Biotech. Assays
were performed in quadruplicate (⌬from threshold cycle of replicate samples
⬍0.3) and a standard curve from consecutive 5-fold dilutions (100– 0.16 ng) of a
cDNA pool representative of all samples was included, for PCR-efficiency deter-
mination. Relative gene expression analysis, correct for PCR efficiency and nor-
malized respect to reference genes

-actin (GenBank accession no. NM㛭007393)
and glyceraldehyde-3-phosphate dehydrogenase (GenBank accession no.
NM㛭008084) was performed by the iCycler-iQ5 software Gene expression module.
Significance probability was evaluated according to Pfaffl et al. (40).
Western Immunoblotting and Immunofluorescence. Germ cells were lysed in 1%
Triton X-100, 150 mM NaCl, 15 mM MgCl2, 15 mM EGTA, 10% glycerol, 50 mM
Hepes (pH 7.4) with protease inhibitors. Proteins were separated by 10% SDS/
PAGE and transferred to nitrocellulose membrane (Amersham). The membrane
was blocked in PBS–5% skim milk powder for 1 h. Incubation of the membrane
with the primary antibody was carried out at 4 °C overnight in PBS–5% BSA and
then with the appropriate HRP-conjugated secondary antibody (SantaCruz). First
antibody incubation was carried out with 1:1,000 dilution of anti-CB1rabbit
polyclonal antibody (Abcam, ab3559), 1:1,000 dilution of anti-CB2rabbit poly-
clonal antibody (Abcam, ab3561), 1:1,000 anti-TRPV1 rabbit polyclonal antibody
(Abcam, ab6166), 1:1,000 anti-phospho Erk1/2 rabbit polyclonal antibody (Cell
Signaling Technology, 9101), 1:500 anti-FAAH rabbit polyclonal antibody (Cay-
man, 101600), 1:500 anti MAGL rabbit polyclonal antibody (Cayman, 100035),
anti-actin rabbit polyclonal (New England Biolabs, sc-7210). The HRP conjugate
was detected by a chemioluminescence ECL kit (Amersham) and autofluorogra-
phy. For immunofluorescence, germ cells were let to adhere onto polyL-lysine
coated slides and permeabilized for 10 min in 0.1% Triton X-100 in PBS. After 1 h
in blocking solution (5% BSA in PBS), antibodies were added at 1:100 dilution in
0.5% BSA in PBS and incubated overnight at 4 °C. Anti-rabbit Cyanin-3 secondary
antibodies were added to the cells for an additional 1 h. Nuclei were labeled with
Hoechst 33349 (1
g/ml). For meiotic cell spreads, SPG were prepared and stained
essentially as described (25). Slides were washed twice in PBS and incubated with
anti-SCP3 rabbit polyclonal antibody (Novus NB 730F) in blocking solution (10%
serum from goat, 3% BSA, 0.05% Triton X-100 in PBS), overnight at 4 °C. After
washing, anti-rabbit FITC secondary antibody (Novus NB300 –231) was added for
1 h at 37 °C. The slides were washed and allowed to dry. Vectashield Mounting
Medium with DAPI (Vector Laboratories) was added and the slides were viewed.
Enzymatic Assays. Cells were resuspended in Tris–HCl buffer, pH 7.4 and homog-
enized in a Dounce homogenizer. The homogenates were centrifuged at 800 ⫻
g(5 min) and then at 10,000 ⫻g(25 min), at 4 °C. The supernatant (cytoplasm
fraction) was used for the MAGL activity assay, whereas the pellet (membrane
fraction) was resuspended in the same buffer and used for DAGL and FAAH
activity assays. Assays were carried out as described (26).
ACKNOWLEDGMENTS. We thank M. Pellegrini for assistance with nuclear
spread preparation; P Rossi and A. Ligresti for helpful suggestions on the manu-
script; and S. Piantedosi for artwork. This work was supported by the Italian
Ministry of University (Grant Prin 2007 200788TPYE㛭002) and Ricerca Scientifica
d’Ateneo (Grant RSA 2007).
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0812789106 Grimaldi et al.
