In this study we investigated the epigenetic modifications elicited by treatment with the CB2‐specific agonist JWH133 [3‐(1‘,1‘‐dimethylbutyl)‐1‐deoxy‐8‐THC] in isolated SPG. Selective activation of CB 2 induced expression of Prdm9 , the gene encoding a meiosis‐specific histone methyltransferase involved in the definition of meiotic recombination sites, thus determining an enrichment of the global level of H3K4me3 ( 20 – 22 ). A similar effect was exerted by RA. Furthermore, both JWH133 and RA triggered specific histone modifications that are associated with transcriptionally active chromatin in the promoter regions of premeiotic genes, such as c‐Kit and Stra8. We present evidence that alterations of CB 2 function by administration of its specific agonist (JWH133) or antagonist [AM630(6‐iodopravadoline)] to prepubertal mice disrupt the normal progression of spermatogenesis in vivo , probably by altering the fine‐tuned timing of germ cell differentiation. In conclusion, our findings indicate a physiologic role of CB 2 in regulating the onset of meiosis and in maintaining the homeostasis of spermatogenesis.

Spermatogenesis is a complex and highly coordinated process that involves germ cell proliferation, differentiation, and morphogenesis. Male germ cells are unique as they divide mitotically [spermatogonia (SPG)], undergo meiosis [spermatocytes (SPCs)], and give rise to haploid cells [spermatids (SPTs)] that differentiate into sperm through a complex morphogenetic process. In adult testis, spermatogonia stem cells (SSCs), known as A single (As), proliferate and divide asymmetrically to produce As cells and undifferentiated SPG (Apr and Al), which then differentiate into A1–4, In, and type B SPG ( 1 ). Differentiating SPG express the tyrosine kinase receptor (c‐KIT) and undergo a definite number of proliferative cycles before entering meiosis prophase I as preleptotene SPCs ( 2 ). Retinoic acid (RA) is known to induce germ cells to enter meiosis in both males and females ( 3 , 4 ). RA promotes differentiation of SPG and entry into meiosis of male germ cells by inducing expression of c‐KIT and of the meiosis inducer STRA8, both in vivo ( 5 – 7 ) and in vitro ( 8 ). However, RA may not be the only inducer of meiosis in male germ cells, and other endogenous effectors are most likely involved, to ensure the homeostasis of the complex process of spermatogenesis. For instance, we have reported that the endocannabinoid (eCB) system (ECS) is a potential key inducer of the mitosis‐meiosis switch in male germ cells ( 9 ). The ECS is formed by eCBs, their receptors, and the enzymes involved in their biosynthesis and degradation ( 10 ). eCBs are endogenous lipid molecules that, similarly to exogenous cannabinoids, bind to cannabinoid receptors (CBs) and modulate their biologic activities. To date, 2 main eCBs, anandamide [ N ‐arachidonylethanolamine (AEA)] and 2‐arachidonylglycerol (2‐AG), have been characterized, and 2 CBs, types 1 (CB 1 ) and 2 (CB 2 ), have been cloned ( 11 ). CB 1 is the most abundant G protein‐coupled receptor expressed in the brain, whereas CB 2 is mainly expressed in immune cells ( 12 ). Male mitotic germ cells possess high levels of 2‐AG and express CB 2 , whose activation promotes cell progression into meiosis ( 9 ). Identification of the ECS in reproductive cells and tissues in mammals, as well as in other vertebrates and invertebrates, highlights the key role played by this system in the control of reproductive function during evolution ( 13 , 14 ).

Enriched SPG fractions were obtained from testes of immature 7‐d‐old Swiss CD‐1 mice, according to a published method ( 9 ). After dissection of the albuginea, the testes were digested with collagenase and trypsin. The cell suspension was plated in Petri dishes for 4 h in minimum essential medium (MEM), supplemented with 1 mM dl‐lactic acid, 2 mM sodium pyruvate, and 10% fetal calf serum (FCS), to promote adhesion of somatic cells. SPG were recovered and stimulated for 24 h with 1 μM JWH133 or 0.3 μM RA (Sigma‐Aldrich, Milan, Italy) and then processed for chromatin immunoprecipitation (ChIP), Western blot (WB), or mRNA analysis. Where indicated, cells were incubated with 5 μM AM630 (Tocris Bioscience) 15 min before addition of JWH133.

After 3 wk of treatment (P28), as expected, seminiferous tubules at stages I–VI were increased (52%) in the control testes (Fig. 6 E ), and a fraction of these tubules (40%) contained elongating SPTs, but not beyond steps 13–14. However, in JWH133‐treated testis, spermatogenesis proceeded to more advanced stages, with most of the tubules being at stages VII–VIII (47%) containing elongating SPTs up to step 16 (Fig. 6 F ). By contrast, AM630 administration slowed down spermatogenesis, and we observed that a high percentage of tubules were at stages XI‐XII (40%) and I‐VI (40%) (Supplemental Fig. S2 C, D ). These findings suggest that exposure to an exogenous CB 2 agonist induces germ cell differentiation to start and proceed faster (Fig. 5 ). To validate this hypothesis, we more precisely staged the seminiferous tubules by staining testis sections with DAPI and anti‐SCP3 antibody. Analysis of the cellular association in representative seminiferous tubules confirmed the accelerated maturation of the testicular cells in treated testis vs. the control (Supplemental Fig. S3). A summary of the distribution of the percentage of seminiferous tubule stages in control and JWH133‐ and AM630‐treated mice at P21 (2 wk treatment) and P28 (3 weeks treatment) is presented in Fig. 6 G .

JWH133‐treated mice show acceleration of germ cell differentiation in the first wave of the spermatogenic cycle. Testis histology from control ( n = 5) and JWH133‐treated ( n =5) mice at ages P7, after 5 h of acute treatment ( A, B ); at P21, after 2 weeks of treatment ( C, D ); and at P28, after 3 wk of treatment ( E, F ). Representative seminiferous tubules at different ages are shown. The roman numeral inside each seminiferous tubule indicates the spermatogenesis stage. Arrowheads: somatic cells and developing germ cells: SCs, SPG, pachytene SCT (P), rounded SPTs (rSPTs), and elongated SPTs (eSPT). H&E staining was used. ( G ) Distribution of spermatogenesis stages (percentage per stage of the total) at P21 (2 wk of treatment) and P28 (3 wk of treatment) in control, JWH133‐ and AM630‐treated mice. Seminiferous tubule cross sections were randomly chosen in 3 nonserial sections per animal, totaling 100 tubules/animal ( n = 5 for each treatment). Grouped stages I‐VI,VII‐VIII, IX‐X, and XI‐XII are reported, and the frequency of tubules according to these stages is indicated. Spermatogenesis stages were identified according to the criteria of Russell et al. ( ). Enlarged images of specific stages are shown in each panel.

Spermatogenic cycle of the mouse. The 12 possible cell associations (stages) that can be observed in mouse seminiferous tubules of the testis during spermatogenesis are shown. These associations arise from both the precise timing of the production of new generations of differentiating germ cells at the base of the seminiferous tubules and the precise timing of the complex events of the differentiation process along the mitotic, meiotic, and spermiogenic processes. In the mouse, the 12 stages, which constitute a cycle of the seminiferous tubule, repeat in a given section at 8.6 d intervals, and the total spermatogenic process lasts 35 d. Continuous lines: the more advanced germ cell types of the first wave of spermatogenesis at P21 (red) and P28 (blue) in control mice. Dashed lines: treatment with JWH133, which causes an acceleration of germ cell differentiation at both P21 and P28, leading to the appearance of the more advanced cell differentiation steps of spermatogenesis. Details of symbols used in the staging maps shown can be found in Russell et al. ( ).

To understand the role of CB 2 in the progression of spermatogenesis in vivo , we investigated the effect of acute and chronic administration ofJWH133 to immature mice, and we evaluated the kinetics of germ cell progression during the first wave of spermatogenesis. Mice were injected daily with JWH133 from d 7 after birth, and the testes were harvested 5 h after administration (acute treatment) or 2 and 3 wk (chronic treatment). The expectation is that, if the timing of spermatogenic progression is normal, the most advanced stage at postnatal day (P)21 (2 wk treatment) would be at the end of meiosis II and steps 1–3 of SPT differentiation, whereas at P28 (3 wk treatment) most differentiated cells should be at steps 13–14 ( 32 ) ( Fig . 5 ). At the end of each time of treatment, testis morphology was analyzed by staining with H&E, and the percentage of each stage of the seminiferous tubules was determined. No histologic defects were detected in the testis of mice after acute treatment (P7) with JWH133, as indicated by the presence of only SPG and SC as in the testis of the vehicle‐treated mice ( Fig . 6 A, B ). Morphologic analysis of control testes after 2 wk of treatment (P21) showed a high percentage (42%) of tubules at stages VII‐VIII of the seminiferous epithelium cycle (Fig. 6 C ), in which pachytene SPCs was identified. More advanced cell types corresponding to haploid germ cells were detected in ~27% of control tubules at stages I–VI, and their differentiation did not progress beyond step 1, according to the precisely timed cycle of events leading to spermatozoa maturation ( 32 ). Treatment with JWH133 for 2 wk determined an increase in the percentage of tubules at stages I‐VI up to 49%, in which haploid germ cells reached more advanced stages of differentiation, up to step 6 of round SPT differentiation (Fig. 6 D ). On the contrary, treatment for 2 wk with the CB2‐specific antagonist AM630 determined a significant decrease of tubules at stages I‐VI containing haploid germ cells (19%) and a concomitant accumulation of metaphase cells at previous stages XI‐XII (24%) (Supplemental Fig. S2 A, B ).

RA induction of entry into meiosis of cultured SPG appears to be mediated, at least in part, by activation of the PI3K‐AKT pathway ( 8 ). Thus, we investigated whether AKT activation is necessary for induction of SPG entry into meiosis by JWH133 and whether cotreatment with RA exerts an additive effect. WB analysis revealed that treatment with JWH133 alone determined a gradual and transient increase in phospho‐AKT level, similar in magnitude to that observed with RA ( Fig . 4 ). Nevertheless, costimulation with both agents did not cause an additive effect on AKT phosphorylation.

Additive effect of RA and JWH133 on entry into meiosis of SPG. A ) Representative immunofluorescence images show SCP3 (green) organization on nuclear spreads at the early‐leptotene, leptotene, and zygotene stages of meiosis prophase I, observed in cultured SPG treated with RA or JWH133 for 24 h. B ) Percentage of nuclei with SCP3 staining in control SPG or in cells stimulated with JWH133, RA, or both agents for 24 h. C ) Percentage of leptotene and zygotene nuclei in control cultures of germ cells or in cells treated for 24 h with RA, JWH133, or both agents. D ) Immunoblot analysis of CB2 in SPG cultured for 24 h in the absence or presence of RA, JWH133, or both agents. Densitometric analysis of values of CB 2 normalized to values for actin are reported as fold induction over the control (graph). E ) Immunoblot analysis of c‐KIT, STRA8, and SCP3 in SPG, SPCs, and SPTs showing premeiotic germ cell—specific expression. F ) Immunoblot analysis of c‐KIT, STRA8, SCP3, and actin expression in SPG cultured for 24 h in the absence or presence of RA, JWH133, or both agents, showing the additive effect. Densitometric analysis of c‐KIT and STRA8 is shown in the graphs. In each case, the values for c‐KIT and STRA8 were normalized by reference to values for actin and shown as fold induction over control. Bars represent the sd . ∗ P < 0.05; ∗∗ P < 0.01.

After finding that independent activation of CB 2 and RA receptors increases the number of meiotic cells in culture, we evaluated their possible interaction in SPG, by treating them for 24 h in the absence or presence of JWH133, or RA, or a combination of both. At the end of the treatment, nuclear spreads were prepared and stained with DAPI and with the SCP3 antibody, which marks a synaptonemal complex protein specifically expressed during meiosis prophase I. SCP3‐positive cells corresponding to meiotic nuclei of early‐leptotene, leptotene, and zygotene SPCs were identified and counted ( Fig . 3 A ). In cells treated with JWH133 or RA as single agents, the percentage of meiotic SCP3‐positive cells increased up to 16.7 ± 0.6 and 15.72 ± 2.63%, respectively, over the control untreated cells (7.13 ± 0.77%) (Fig. 3 B ). Simultaneous addition ofbothJWH133 and RA produced a further significant increase of SCP3‐positive nuclei (22.91 ± 1.78%), indicating an additive effect. This increase was caused by the accumulation of the cells at both the leptotene and zygotene stages, suggesting that the combined treatment increased the percentage of cells entering meiosis and accelerated their progression through prophase I (Fig. 3 C ). We excluded that the additive effect was caused by an induction of CB 2 expression, because we did not observe an increase in CB 2 protein level after treatment with both agents (Fig. 3 D ). To confirm the additive effect of JWH133 and RA at molecular level, we investigated the expression of the premeiotic and meiotic protein markers c‐KIT, STRA8, and SCP3, which are specifically expressed in SPG (Fig. 3 E ). RA significantly increased the c‐KIT and STRA8 protein levels (9.3 ± 0.44‐ and 1.98 ± 0.05‐fold, respectively) (Fig. 3 F ), whereas treatment with JWH133 increased the c‐KIT protein level (6.32 ± 0.3‐fold) but did not significantly increase STRA8 protein expression, although it induced STRA8 mRNA expression (1.76 ± 0.26‐fold) as evaluated by qRT‐PCR (Supplemental Fig. S1). The combination of both agents caused a further significant upregulation of the c‐KIT (13.2 ± 1.2‐fold vs. control) and STRA8 (2.5 ± 0.2‐fold vs. control) proteins (Fig. 3 F ). The increased percentage of meiotic nuclei was not associated with the increased SCP3 protein level, which was assembled in a pattern typical of the organization on the meiotic chromosomes (Fig. 3 A ).

Actively transcribed genes are also characterized by demethylation of the repressive H3K9me3 and ‐me2 marks. H3K9me2 is mainly associated with facultative heterochromatin, whereas H3K9me3 is observed at constitutive heterochromatin. JWH133 determined a decrease of H3K9me2 level of ~4.7‐ and ~2.7‐fold in the c‐Kit and Stra8 promoter regions, respectively. The effect of RA mirrored that of JWH133, reducing the H3K9me2 level ~2.44‐ and ~2.64‐fold in the promoter regions of c‐Kit and Stra8 , respectively. By contrast, JWH133 or RA treatment elicited an enrichment in H3K9me2 level in the Gfra1 promoter of ~2.01‐ and 5.50‐fold, respectively, indicating that these agents repressed its transcription in differentiating SPG. Our results suggest that CB 2 activation regulates transcription of the c‐Kit and Stra8 genes at meiotic entry through specific alterations of histone modifications.

H3K4me3 and ‐9me2 levels in the promoters of early meiotic genes after stimulation with JWH133 or RA. Histone modification of H3K4me3 and H3K9me2 levels were examined by ChIP, followed by qPCR in the indicated sequences of 5' flanking regions of Stra8 and c‐Kit genes, and in the 5'UTR of Gfra1. Chromatin from SPG, untreated or treated with RA or JWH133, was immunoprecipitated with the indicated antibodies. Purified DNA from enriched chromatin fragments was amplified by qRT‐PCR with specific primers (Supplemental Table S1). Values indicated as fold enrichment over control are the average of the results of 3 independent ChIP experiments. Error bars represent means ± sd . ∗ P < 0.05, significantly different vs. control.

Expression of the c‐Kit and Stra8 genes is induced by treatment of SPG with JWH133 or RA ( 8 , 9 ). Given that H3K4me3 typically marks regions of transcriptionally active chromatin in the genome ( 30 ), we performed ChIP analysis using anti‐H3K4me3 antibody on chromatin extracts from SPG. Treatment of SPG for 24 h with JWH133 increased H3K4me3 levels in the promoter regions of Stra8 of ~3.11 ± 0.26‐fold and of c‐Kit of ~1.47 ± 0.2‐fold ( Fig . 2 ). A similar effect was observed after treatment with RA, which increased the H3K4me3 level in the Stra8 promoter of ~4.43 ± 0.36‐fold and of the c‐ Kit promoter of ~2.35 ± 0.34‐fold. Conversely, H3K4me3 levels in the promoter region of the Gfra1 gene, which is expressed in SSCs but not in differentiating SPG ( 31 ), was not modulated by JWH133 nor by RA (increase of 1.06 ± 0.12‐ and 0.86 ± 0.11‐fold, respectively, over the control), indicating that treatment with these agents specifically affect premeiotic and meiotic genes and does not elicit an overall open chromatin state in mouse SPG.

Histone modification of the H3K4me3 level and histone methyltransferase Prdm9 expression are modulated by JWH133 in mouse SPG. A ) WB analysis of histone extracts from SPG, treated or not with JWH133 or AM630, or pretreated with AM630 and then with JWH133 for 24 h. H3K9me3 is reported to be a marker of constitutive heterochromatin. B ) WB analysis on histone extracts from control SPG and SPG treated with 0.3 μM RA or 1 μM JWH133 for 24 h. JWH133 and RA increased the global level of H3K4me3 ~1.68 ± 0.30‐ and 1.57 ± 0.36‐fold, respectively. Densitometric analyses of histone modifications in ( A ) and ( B ) are shown on the right. In each case, the values for modified histones were normalized to values for [ 3 H]histone. C ) Semiquantitative RT‐PCR evaluation of mRNA levels of H3K4me3 methyltransferases, as reported in Table , in SPG cultured for 24 h in the absence or presence of JWH133 or RA. D ) qRT‐PCR evaluation of the PRDM9 mRNA level on SPG cultured for 24 h in the absence or presence of JWH133 or RA. The expression levels in control SPG were set at 1. Standard deviations were calculated by real‐time PCR. ∗ P < 0.05, significant difference vs. the control. E ) Semiquantitative RT‐PCR evaluation of PRDM9 mRNA levels in mouse male germ cells at different stages of differentiation. SPG were isolated from 7 dpp testis and SPCs and SPTs from adult testis by using elutriation methods ( ).

During testis development, H3K4me3 globally increases when type B SPG enter meiosis and reach the leptotene stage of prophase I ( 22 ). We investigated H3K4me3 modification during in vitro meiotic entry of SPG, induced by the CB 2 agonist JWH133. SPG from 7 d postpartum (dpp) were cultured for 24 h in presence or absence of JWH133, to induce meiotic entry. Global changes of H3K4me3 were evaluated by WB analysis of histone extracts. JWH133 induced a significant increase in the global level of H3K4me3 ( Fig . 1 A ), whereas the levels of H3K9me3 and ‐me2 were unaffected. The effect on the H3K4me3 level induced by JWH133 was abolished by addition of the CB 2 antagonist AM630, indicating that it is specifically associated with CB 2 activation. A similar effect on H3K4me3 was observed after treatment of SPG with RA (Fig. 1 B ), suggesting that both agents induce a meiosis‐related change in histone modification. We were interested in identifying the H3K4me3 methyl transferases that may be involved in the modulation of H3K4me3 global level induced by JWH133 at entry into meiosis. A list of his tone‐modifying enzymes with a role in progression of meiosis is reported in Table 1 ( 25 – 29 ). Semiquantitative RT‐PCR analysis of SPG showed that the expression of histone methyltransferase PRDM9 was modulated by JWH133 and RA, whereas other H3K4me3 methyltransferases/demethylases were not affected (Fig. 1 C ), suggesting that upregulation of Prdm9 expression is a specific event induced by JWH133. qRT‐PCR demonstrated that JWH133 increased Prdm9 mRNA expression of ~1.81 ± 0.12‐fold over control cells, similar to that observed after treatment with RA (2.04 ± 0.04‐fold increase) (Fig. 1 D ). Analysis of Prdm9 expression on isolated germ cell populations at different stages of differentiation showed that it is expressed in SPG, whereas it is barely detectable in pachytene SPCs and SPTs (Fig. 1 E ), in accordance with its role in H3K4me3 methylation at meiotic recombination sites ( 22 ). This result underlines a correlation between Prdm9 expression and H3K4me3 enrichment in SPG at meiotic entry and indicates that CB 2 activation induces epigenetic changes in cultured SPG that mimic those occurring during the physiological meiotic transition of germ cells in vivo. Thus, our results identified the Prdm9 gene as a specific target of both CB 2 and RA signaling in male germ cells.

DISCUSSION

The current study was designed to investigate the physiological role of CB 2 in male germ cells. Through the comparison of molecular events activated by CB 2 and those induced by RA, we offer new insights on the molecular mechanisms involved in male germ cell differentiation and on the role of CB 2 in spermatogenesis. RA is considered the master physiological inducer of meiosis in germ cells, and it drives SPG differentiation and meiosis onset, both in vivo and in vitro (7). We reported recently that the ECS is an additional positive regulator of the mitosis—meiosis switch in male germ cells and that CB 2 plays a pivotal role in this event (9). Because CB 2 promotes entry into meiosis of SPG by inducing expression of premeiotic and early meiotic genes, we asked whether epigenetic modifications account for enhanced gene expression. We showed that CB 2 activation causes specific histone modifications that are hallmarks of increased gene expression (33). Similar epigenetic modifications were elicited by RA treatment of SPG. Indeed, both JWH133 and RA increased H3K4me3 levels in the 5'‐flanking region of the c‐Kit and Stra8 genes, concomitant with a decrease in methylation of H3K9me2. Thus, our study represents the first evidence that CB 2 activation triggers histone modifications that modulate the expression of key meiotic genes.

Previous reports suggested a role for CB 1 signaling in global epigenetic modification of the chromatin status in late stages of spermiogenesis (34, 35). Our study provided evidence that CB 2 activation, as well as RA treatment, elicit a global increase in H3K4me3 at the onset of meiosis. This effect is somewhat selective, as we did not detect global changes in H3K9me2. The specificity of the effect is probably linked to the well‐characterized role of the H3K4me3 modification in meiosis (21). In mice, a significant global enrichment in H3K4me3 occurs when B‐type SPG enter meiosis and reach the leptotene stage of prophase I, concomitant with an increase in the expression of methyltransferase Prdm9 which trimethylates H3K4, thus specifying the recombination hotspots. Prdm9 is a meiosis‐specific Krüppel‐associated box (KRAB) domain zinc finger methyltransferase that is essential for progression through prophase I (19). In our study, JWH133 increased Prdm9 mRNA expression in SPG, whereas it did not affect the expression of other histone methyltransferases. Hence, we suggest that the observed global enrichment in H3K4me3 level, after exposure to JWH133, is mainly caused by the increased expression of the Prdm9 gene. However, Prdm9 activity is probably not involved in H3K4me3 enrichment in the promoter regions of c‐Kit and Stra8. In fact, no direct evidence of a role for PRDM9 as a transcription factor has been reported. Moreover, PRDM9 binding sites are not detected in the 5‘‐flanking regions of c‐Kit and Stra8. Finally, recent genomewide analysis of distribution of recombination initiation sites in the mouse male genome showed that most of them are associated with testis‐specific H3K4me3 marks that are distinct from those associated with transcription (36). Hence, we suggest that a methyltransferase, different from PRDM9 and not yet identified, mediates H3K4me3 methylation at the 5‘‐flanking region of c‐Kit and Stra8 genes. One important outcome of our finding is the possibility of reproducing in vitro some of the molecular events occurring at the onset of meiosis in in vivo spermatogenesis, including the specification of hotspots. Moreover, our studies identify the Prdm9 gene as a novel target of both CB 2 and RA in male germ cells.

We found that signaling pathways triggered by CB 2 activation and exposure to RA have similar effects on the mitosis‐meiosis switch of male germ cells and that costimulation with both agents exerted an additive effect on meiotic entry of SPG. This result was supported, at the morphologic level, by the higher percentage of SCP3‐positive cells and, at the molecular level, by a further increase in the c‐KIT and STRA8 proteins in comparison with single treatments. However, we noticed that JWH133 treatment increased STRA8 expression only at the mRNA level without affecting the protein level. One possibility is that the low increase in STRA8 expression is detectable by the higher sensitivity of qPCR with respect to WB. Alternatively, RA may have a role in translation of STRA8 mRNA, as it was recently suggested for c‐KIT (37). On the other hand, RA and CB 2 do not seem to interact at the level of the PI3K/AKT signaling cascade, their downstream target, which plays an essential role in meiosis progression and male fertility (38). Indeed, we observed no additive effect in the phosphorylation of AKT with the combined treatment. Thus, further studies are necessary to elucidate the nature of the molecular interaction between the CB 2 and RA pathways in male meiotic germ cells.

Given the effects elicited by CB 2 activation on the meiotic transition of male germ cells in vitro (9), we set out to investigate the effects caused by administration of the CB 2 agonist and antagonist drugs on the first wave of spermatogenesis. Our results in vivo are in agreement with the in vitro studies and show an effect of CB 2 activation on germ cell differentiation. Prolonged activation of the receptor by administration of JWH133 caused an acceleration of the onset of spermatogenesis in immature mice, whereas pharmacologic blockade of CB 2 (AM630) slowed germ cell differentiation. These observations confirm the role of CB 2 in regulating the correct progression of spermatogenesis. It is noteworthy, however, that although our in vitro work was focused on the role of CB 2 in germ cells, we cannot exclude a role of Sertoli cells (SCs) in mediating these effects on spermatogenesis in vivo. SCs are known to be critical for the coordination of the spermatogenetic program, by fulfilling nutritional and structural roles that are indispensable in male germ cell development (39). Mouse SCs express a functional CB 2 (40), whose activation by an exogenous agonist may also affect germ cell maturation and timing of differentiation through the production of growth factors and cytokines. Moreover, SCs are the only targets of follicle‐stimulating hormone (FSH) and androgens, the main male reproductive hormones, within the testis. Of interest, recent studies indicate the existence of an interplay between ECS and sex hormones (41), and further studies should be performed to characterize this cross‐talk in spermatogenesis. On the other hand, we speculate that the CB 2 agonist induces an acceleration of the onset of spermatogenesis by acting directly on the gonad and not indirectly on the pituitary—gonadal axis, because CB 2 is not expressed in the pituitary gland, which instead express CB 1 (42). The collective results of our work indicate that CB 2 signaling contributes to the physiology of mouse spermatogenesis and suggest that eCBs, such as 2‐AG, and, at a lower level, AEA, which are present within the testis (9), may regulate the correct progression of the spermatogenetic process in vivo through its activation.