Analysis of absolute amounts of two meiotic cohesin subunits, RAD21L and REC8, in mouse spermatocytes.

RAD2lL and REC8, meiosis-specific paralogs of the canonical cohesin subunit RAD21, are essential for proper formation of axial/lateral elements of the synaptonemal complex, synapsis of homologous chromosomes, and crossover recombination in mammalian meiosis. However, how many meiotic cohesins are present in germ cells has not been investigated because of the lack of an appropriate method of analysis. In the present study, to examine the intracellular amount of meiotic cohesins, we generated two strains of knock-in (KI) mice that expressed a 3×FLAG-tag at the C-terminus of RAD21L or REC8 protein using the CRISPR/Cas9 genome editing system. Both KI mice were fertile. Western blot analyses and immunocytochemical studies revealed that expression levels and localization patterns of both RAD21L-3×FLAG and REC8-3×FLAG in KI mice were similar to those in wild-type mice. After confirming that tagging of endogenous RAD21L and REC8 with 3×FLAG did not affect their expression profiles, we evaluated the levels of RAD21L-3×FLAG and REC8-3×FLAG in the testes of 2-week-old mice in which only RAD21L and REC8 but little RAD21 are expressed in the meiocytes. By comparing the band intensities of testicular RAD21L-3×FLAG and REC8-3×FLAG with 3×FLAG-tagged recombinant proteins of known concentrations in western blot analysis, we found that there were approximately 413,000 RAD21L and 453,000 REC8 molecules per spermatocyte in the early stages of prophase I. These findings provide new insights into the role played by cohesins in the process of meiotic chromosome organization in mammalian germ cells.

Oocyte-derived growth factors promote development of antrum-like structures by porcine cumulus granulosa cells in vitro.

Oocytes communicate with the surrounding somatic cells during follicular development. We examined the effects of two oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the development of porcine oocyte-cumulus cell complexes (OCCs) in vitro. We collected OCCs from early antral follicles (1.2-1.5 mm) and prepared oocytectomized cumulus cell complexes (OXCs), which were then cultured in a growth medium supplemented with 0-100 ng/ml GDF9 and/or BMP15 for 7 days. In the medium without GDF9 or BMP15, OCCs developed during culture, and approximately 30% of them formed antrum-like structures. GDF9 promoted OCC development and structure formation in a dose-dependent manner. However, OXCs did not form antrum-like structures without growth factors. GDF9 promoted the development of OXCs, and 50 and 100 ng/ml GDF9 promoted the formation of the structures by 8% and 26%, respectively; however, BMP15 did not promote the formation of these structures. OXCs were then cultured with 100 ng/ml GDF9 and various concentrations of BMP15 to investigate their cooperative effects on the formation of antrum-like structures. BMP15 promoted the formation of antrum-like structures in a dose-dependent manner. In conclusion, GDF9 derived from oocytes is probably important for the formation of antrum-like structures in porcine OXCs, and BMP15 cooperates with GDF9 to form these structures.

Reestablishment of transzonal projections and growth of bovine oocytes in vitro.

Transzonal projections (TZPs) that maintain bidirectional communication between oocytes and granulosa cells or cumulus cells are important structures for oocyte growth. However, whether TZPs develop between TZP-free oocytes and granulosa cells, and whether reestablished TZPs support oocyte growth, is unknown. We first examined changes in TZPs after denudation of bovine oocytes collected from early antral follicles (0.5-0.7 mm). Twenty-four hours after denudation, almost all the TZPs disappeared. We also examined the reestablishment of TZPs by coculturing TZP-free denuded oocytes (DOs) with mural granulosa cells (MGCs) collected from early antral follicles. In addition, to confirm if the reestablished TZPs were functional, the reconstructed complexes (DO+MGCs) were subjected to in vitro growth culture and found that the MGCs adhered to TZP-free DOs and TZPs were reestablished. During in vitro growth culture, DO+MGCs developed and formed antrum-like structures. After culture, the number of TZPs in DO+MGCs increased, and the oocytes grew fully and acquired meiotic competence. These results suggest that reestablished TZPs are able to support oocyte growth.

Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte-cumulus cell complexes in vitro.

During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. We examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes collected from early antral follicles (1.2-1.5 mm). First, we confirmed that GDF9 and BMP15 mRNAs were expressed almost exclusively in the oocytes. Oocyte-cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with 0-100 ng/ml of GDF9 or BMP15 for 5 days. GDF9 dose-dependently increased the OCC diameter, while BMP15 did not. GDF9 and BMP15 had no significant effects on oocyte growth (P > 0.05). When OCCs that had been cultured with 50 and 100 ng/ml BMP15 were subjected to a subsequent maturation culture, they expanded fully by gonadotropic stimulation and 49% and 61% of oocytes matured to metaphase II (MII), respectively. In contrast, GDF9 did not promote cumulus expansion, and < 10% of oocytes matured to MII. Based on the difference in cumulus expansion, we compared the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) mRNAs in cumulus cells. The level of LHCGR mRNA was increased in cumulus cells of the BMP15 group, although there were no significant differences in FSHR mRNA levels among the groups. These results suggest that GDF9 promotes the growth of OCCs and that BMP15 promotes LHCGR mRNA expression in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.

Is age-related increase of chromosome segregation errors in mammalian oocytes caused by cohesin deterioration?

BACKGROUND: Mammalian oocytes initiate meiosis in fetal ovary and are arrested at dictyate stage in prophase I for a long period. It is known that incidence of chromosome segregation errors in oocytes increases with advancing age, but the molecular mechanism underlying this phenomenon has not been clarified. METHODS: Cohesin, a multi-subunit protein complex, mediates sister chromatid cohesion in both mitosis and meiosis. In this review, molecular basis of meiotic chromosome cohesion and segregation is summarized. Further, the relationship between chromosome segregation errors and cohesin deterioration in aged oocytes is discussed. RESULTS: Recent studies show that chromosome-associated cohesin decreases in an age-dependent manner in mouse oocytes. Furthermore, conditional knockout or activation of cohesin in oocytes indicates that only the cohesin expressed before premeiotic S phase can establish and maintain sister chromatic cohesion and that cohesin does not turnover during the dictyate arrest. CONCLUSION: In mice, the accumulating evidence suggests that deterioration of cohesin due to the lack of turnover during dictyate arrest is one of the major causes of chromosome segregation errors in aged oocytes. However, whether the same is true in human remains elusive since even the deterioration of cohesin during dictyate arrest has not been demonstrated in human oocytes.

GDF9 and BMP15 induce development of antrum-like structures by bovine granulosa cells without oocytes.

The role of oocytes in follicular antrum formation is not well understood. We examined the effect of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the formation of antrum-like structures by cultured bovine oocyte-granulosa cell complexes (OGCs). OGCs containing growing oocytes (105‒115 µm in diameter) were collected from early antral follicles (1.2‒1.8 mm) and used to prepare oocytectomized complexes (OXCs) and granulosa cell complexes (GCs). The mRNAs of GDF9 and BMP15 were expressed in the oocytes, but not in the granulosa cells. The complexes were cultured for five days with or without GDF9 and BMP15 either alone or in combination. The OGCs maintained their complex integrity and developed antrum-like structure, whereas OXCs and GCs neither maintained their integrity nor developed any antrum-like structure without growth factors. GDF9 or BMP15 alone increased the integrity of these complexes and induced antrum-like structures in OXCs and GCs. Moreover, the combination of GDF9 and BMP15 was more potent for both phenomena in all types of complexes. In OXCs and GCs cultured without GDF9 and BMP15 or with BMP15 alone, outgrowing granulosa cells differentiated into fibroblast-like cells. The combination of GDF9 and BMP15 suppressed the appearance of fibroblast-like cells in OXCs and GCs during incubation. Instead, the granulosa cells appeared rhomboid and pebble-like in shape, similar to those in OGCs cultured without supplementation of GDF9 and BMP15. These results suggest that oocytes maintain complex integrity by preventing granulosa cell differentiation and participate in follicular antrum formation via GDF9 and BMP15.

Inhibition of PDE3A sustains meiotic arrest and gap junction of bovine growing oocytes in in vitro growth culture.

Bovine growing oocytes with a diameter of 105-115 µm from early antral follicles (1.2-1.8 mm) are able to resume meiosis, but lack the competence to mature to metaphase II. To confer full maturation competence onto the oocytes, culture systems which can support their growth and prevent their meiotic resumption during culture are needed. In this study, we cultured growing oocytes for 5 days to examine the effects of different phosphodiesterase (PDE) inhibitors on meiotic arrest and acquisition of full maturation competence of growing oocytes, and their gap junctional communication with cumulus cells. Growing oocyte-cumulus complexes (OCCs) were cultured with 3-isobutyl-1-methylxanthine (IBMX; broad-spectrum PDE inhibitor), rolipram (PDE4D inhibitor), cilostamide and milrinone (PDE3A inhibitors). The mean diameters of oocytes increased similarly in all groups. IBMX, cilostamide and milrinone induced antrum formation by OCCs and maintained meiotic arrest of oocytes during culture, whereas rolipram neither promoted antrum formation nor maintained oocyte meiotic arrest. Gap junctional communication between oocytes and cumulus cells was maintained by IBMX and cilostamide, but not by rolipram as judged by the transfer of injected lucifer yellow dye from oocytes to cumulus cells. In subsequent in vitro maturation, oocytes grown with IBMX, cilostamide and milrinone showed full maturation competence. These results suggest that PDE3A inhibition maintains the meiotic arrest of bovine growing oocytes and sustains their gap junctional communication with cumulus cells for 5 days, thereby contributing to their acquisition of full maturation competence.

The Regulation and Function of Cohesin and Condensin in Mammalian Oocytes and Spermatocytes.

Germ cells, such as oocytes and spermatocytes, produce haploid gametes by a special type of cell division called meiosis. The reduction of chromosome number is achieved in meiosis I, in which homologous chromosomes (homologs) are paired and recombined with their counterparts and finally segregated from each other. How meiotic chromosomes behave in a different manner from mitotic chromosomes has been a fascinating problem for cellular and developmental biology. Cohesin and condensin are multi-subunit protein complexes that play central roles in sister chromatid cohesion and chromosome condensation (also segregation), respectively. Recent studies investigating the expression and function of cohesin and condensin in mammalian germ cells greatly advance our understanding of the molecular mechanism underlying the meiotic chromosomal events. Furthermore, accumulating evidence suggests that reduction of cohesin during prophase I arrest in mammalian oocytes is one of the major causes for age-related chromosome segregation error. This review focuses on the regulation and functions of cohesins and condensins during mammalian meiosis.

Ectopic expression of meiotic cohesin RAD21L promotes adjacency of homologous chromosomes in somatic cells.

Pairing, synapsis, and crossover recombination of homologous chromosomes (homologs) are prerequisite for the proper segregation of homologs during meiosis I. The meiosis-specific cohesin subunit, RAD21L, is essential for such meiotic chromosomal events, but it is unknown to what extent RAD21L by itself contributes to the process since various meiotic genes are also involved. To reveal the exclusive contribution of RAD21L to the specific regulation of homologs, we examined the effects of ectopic RAD21L expression on chromosome dynamics in somatic cells. We found that expression of GFP-fused RAD21L by plasmid transfection significantly shortened the distance between two FISH signals representing a pair of homologs for chromosome X or chromosome 11 in the nuclei compared to that in control (non-transfected) cells whereas expression of GFP-fused RAD21, a mitotic counterpart of RAD21L, showed no detectable effects. This indicates that RAD21L, when ectopically expressed in somatic cells, can promote homolog adjacency, which resembles the homolog pairing normally seen during meiosis. Furthermore, deletion of the N-terminal winged helix domain from RAD21L, prevented its association with another cohesin subunit, SMC3, and abolished the phenomenon of homolog adjacency upon ectopic expression. Our findings suggest that RAD21L-containing cohesin can promote homolog adjacency independently of other meiotic gene products, which might be central to the process of meiotic homolog paring.

Meiotic cohesin subunits RAD21L and REC8 are positioned at distinct regions between lateral elements and transverse filaments in the synaptonemal complex of mouse spermatocytes.

Cohesins containing a meiosis-specific α-kleisin subunit, RAD21L or REC8, play roles in diverse aspects of meiotic chromosome dynamics including formation of axial elements (AEs), assembly of the synaptonemal complex (SC), recombination of homologous chromosomes (homologs), and cohesion of sister chromatids. However, the exact functions of individual α-kleisins remain to be elucidated. Here, we examined the localization of RAD21L and REC8 within the SC by super-resolution microscopy, 3D-SIM. We found that both RAD21L and REC8 were localized at the connection sites between lateral elements (LEs) and transverse filaments (TFs) of pachynema with RAD21L locating interior to REC8 sites. RAD21L and REC8 were not symmetrical in terms of synaptic homologs, suggesting that the arrangement of different cohesins is not strictly fixed along all chromosome axes. Intriguingly, some RAD21L signals, but not REC8 signals, were observed between unsynapsed regions of AEs of zygonema as if they formed a bridge between homologs. Furthermore, the signals of recombination intermediates overlapped with those of RAD21L to a greater degree than with those of REC8. These results highlight the different properties of two meiotic α-kleisins, and strongly support the previous proposition that RAD21L is an atypical cohesin that establishes the association between homologs rather than sister chromatids.

Condensin confers the longitudinal rigidity of chromosomes.

In addition to inter-chromatid cohesion, mitotic and meiotic chromatids must have three physical properties: compaction into 'threads' roughly co-linear with their DNA sequence, intra-chromatid cohesion determining their rigidity, and a mechanism to promote sister chromatid disentanglement. A fundamental issue in chromosome biology is whether a single molecular process accounts for all three features. There is universal agreement that a pair of Smc-kleisin complexes called condensin I and II facilitate sister chromatid disentanglement, but whether they also confer thread formation or longitudinal rigidity is either controversial or has never been directly addressed respectively. We show here that condensin II (beta-kleisin) has an essential role in all three processes during meiosis I in mouse oocytes and that its function overlaps with that of condensin I (gamma-kleisin), which is otherwise redundant. Pre-assembled meiotic bivalents unravel when condensin is inactivated by TEV cleavage, proving that it actually holds chromatin fibres together.

Demands for carbohydrates as major energy substrates depend on the preimplantation developmental stage in pig embryos: differential use of fructose by parthenogenetic diploids before and after the 4-cell stage in the pig.

The embryo culture technique has been improving, but the detailed demands for energy substrates such as glucose, fructose, pyruvate and lactate of preimplantation embryos are still unclear. In the present study, the demands of pig preimplantation embryos at each different developmental stage were investigated by use of parthenogenetic diploids as a model of pig preimplantation embryos. Pig parthenogenetic diploids showed different use of glucose and fructose before and after the 4-cell stage. Although glucose supported the development of pig embryos throughout the preimplantation stages and even maintained the expansion and hatching of blastocysts, it suppressed development to the blastocyst stage when glucose coexisted with pyruvate and lactate from 4 h after activation, but not after 48 h (early 4-cell stage). Since ketohexokinase that metabolizes fructose was not expressed in 2-cell and 4-cell diploids, a medium that included only fructose as a major energy substrate did not support early cleavage of pig diploids beyond the 4-cell stage, and almost no diploids developed to the morula stage just as in a medium without carbohydrates. These results may explain the different suppressive effects on pig preimplantation development between glucose and fructose when pyruvate and lactate were present in a medium. In addition, 4-cell diploids that had been cultured in a medium with pyruvate and lactate developed to the expanded blastocyst stage without any carbohydrates as a major energy substrate. These results show that the demands for carbohydrates are different depending on the developmental stage in pig preimplantation embryos.

Roles of cohesin and condensin in chromosome dynamics during mammalian meiosis.

Meiosis is a key step for sexual reproduction in which chromosome number is halved by two successive meiotic divisions after a single round of DNA replication. In the first meiotic division (meiosis I), homologous chromosomes pair, synapse, and recombine with their partners in prophase I. As a result, homologous chromosomes are physically connected until metaphase I and then segregated from each other at the onset of anaphase I. In the subsequent second meiotic division (meiosis II), sister chromatids are segregated. Chromosomal abnormality arising during meiosis is one of the major causes of birth defects and congenital disorders in mammals including human and domestic animals. Hence understanding of the mechanism underlying these unique chromosome behavior in meiosis is of great importance. This review focuses on the roles of cohesin and condensin, and their regulation in chromosome dynamics during mammalian meiosis.

Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids.

Expression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α- was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α- throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.

Condensins I and II are essential for construction of bivalent chromosomes in mouse oocytes.

In many eukaryotes, condensins I and II associate with chromosomes in an ordered fashion during mitosis and play nonoverlapping functions in their assembly and segregation. Here we report for the first time the spatiotemporal dynamics and functions of the two condensin complexes during meiotic divisions in mouse oocytes. At the germinal vesicle stage (prophase I), condensin I is present in the cytoplasm, whereas condensin II is localized within the nucleus. After germinal vesicle breakdown, condensin II starts to associate with chromosomes and becomes concentrated onto chromatid axes of bivalent chromosomes by metaphase I. REC8 "glues" chromosome arms along their lengths. In striking contrast to condensin II, condensin I localizes primarily around centromeric regions at metaphase I and starts to associate stably with chromosome arms only after anaphase I. Antibody injection experiments show that condensin functions are required for many aspects of meiotic chromosome dynamics, including chromosome individualization, resolution, and segregation. We propose that the two condensin complexes play distinctive roles in constructing bivalent chromosomes: condensin II might play a primary role in resolving sister chromatid axes, whereas condensin I might contribute to monopolar attachment of sister kinetochores, possibly by assembling a unique centromeric structure underneath.

RAD21L, a novel cohesin subunit implicated in linking homologous chromosomes in mammalian meiosis.

Cohesins are multi-subunit protein complexes that regulate sister chromatid cohesion during mitosis and meiosis. Here we identified a novel kleisin subunit of cohesins, RAD21L, which is conserved among vertebrates. In mice, RAD21L is expressed exclusively in early meiosis: it apparently replaces RAD21 in premeiotic S phase, becomes detectable on the axial elements in leptotene, and stays on the axial/lateral elements until mid pachytene. RAD21L then disappears, and is replaced with RAD21. This behavior of RAD21L is unique and distinct from that of REC8, another meiosis-specific kleisin subunit. Remarkably, the disappearance of RAD21L at mid pachytene correlates with the completion of DNA double-strand break repair and the formation of crossovers as judged by colabeling with molecular markers, γ-H2AX, MSH4, and MLH1. RAD21L associates with SMC3, STAG3, and either SMC1α or SMC1ß. Our results suggest that cohesin complexes containing RAD21L may be involved in synapsis initiation and crossover recombination between homologous chromosomes.

Evidence of the existence of adenylyl cyclase 10 (ADCY10) ortholog proteins in the heads and connecting pieces of boar spermatozoa.

The aim of this study is to provide evidence of the existence of the adenylyl cyclase 10 (ADCY10) ortholog proteins in boar spermatozoa. Experiments with RT-PCR techniques, nucleotide sequence analyses and Northern blot analyses revealed that boar testes exclusively express approximately 5.1-kbp RNA, the nucleotide sequence of which is highly similar to that of human ADCY10. Database analyses with CDART suggested that pig ADCY10 ortholog proteins conserve two catalytic domains of adenylyl cyclase. Western blot techniques and indirect immunofluorescence with a specific antiserum to pig recombinant ADCY10 ortholog proteins showed that 48-kDa and 70-kDa truncated forms of pig ADCY10 ortholog proteins are localized in the equatorial segments and connecting pieces of boar ejaculated spermatozoa. Finally, cell imaging techniques with fluo-3/AM indicated that incubation with sodium bicarbonate (an ADCY10 activator) can initiate the calcium influx in the boar sperm heads that is controlled via the cyclic AMP signaling cascades. These results are consistent with the suggestion that functional ADCY10 ortholog proteins exist in the heads of boar spermatozoa. This is the first direct evidence of the existence of ADCY10 proteins in the heads of mammalian spermatozoa.

Knockdown of FOXO3 induces primordial oocyte activation in pigs.

Mammalian ovaries are endowed with a large number of primordial follicles, each containing a nongrowing oocyte. Only a small population of primordial oocytes (oocytes in primordial follicles) is activated to enter the growth phase throughout a female's reproductive life. Little is known about the mechanism regulating the activation of primordial oocytes. Here, we found that the primordial oocytes from infant pigs (10- to 20-day-old) grew to full size at 2 months after xenografting to immunodeficient mice, whereas those from prepubertal pigs (6-month-old) survived without initiation of their growth even after 4 months; thereafter, they started to grow and reached full size after 6 months. These results suggest that the mechanism regulating the activation of primordial oocytes in prepubertal pigs is different from that in infant pigs. In this regard, the involvement of FOXO3, a forkhead transcription factor, was studied. In prepubertal pigs, FOXO3 was detected in almost all (94+/-2%) primordial oocyte nuclei, and in infant pigs, 42+/-7% primordial oocytes were FOXO3 positive. At 4 months after xenografting, the percentage of FOXO3-positive primordial oocytes from prepubertal pigs had decreased to the infant level. Further, siRNA was designed to knock down porcine FOXO3. FOXO3-knockdown primordial follicles from prepubertal pigs developed to the antral stage accompanied by oocyte growth at 2 months after xenografting. These results suggest that primordial oocytes are dormant in prepubertal pigs by a FOXO3-related mechanism to establish a nongrowing oocyte pool in the ovary, and that a transient knockdown of the FOXO3 activates the primordial oocytes to enter the growth phase.

Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells.

Reductional chromosome segregation in germ cells, where sister chromatids are pulled to the same pole, accompanies the protection of cohesin at centromeres from separase cleavage. Here, we show that mammalian shugoshin Sgo2 is expressed in germ cells and is solely responsible for the centromeric localization of PP2A and the protection of cohesin Rec8 in oocytes, proving conservation of the mechanism from yeast to mammals. However, this role of Sgo2 contrasts with its mitotic role in protecting centromeric cohesin only from prophase dissociation, but never from anaphase cleavage. We demonstrate that, in somatic cells, shugoshin colocalizes with cohesin in prophase or prometaphase, but their localizations become separate when centromeres are pulled oppositely at metaphase. Remarkably, if tension is artificially removed from the centromeres at the metaphase-anaphase transition, cohesin at the centromeres can be protected from separase cleavage even in somatic cells, as in germ cells. These results argue for a unified view of centromeric protection by shugoshin in mitosis and meiosis.

Evidence for existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa.

Cyclic adenosine 3',5'-monophosphate (cAMP) signaling regulates the expression of fertilizing ability in mammalian spermatozoa. Many articles indicate that this signaling is mediated mainly via protein kinase A. Recently, a guanine nucleotide exchange factor for small G protein Rap1 (an exchange protein directly activated by cAMP: Epac) was discovered as a new mediator of cAMP signaling in somatic cells. The aim of this study was to reveal the existence of cAMP-Epac signaling in mouse spermatozoa. Northern blot analysis and in situ hybridization suggested that Epac1 and Epac2 mRNAs were transcribed in the seminiferous epithelia of the testis. This shows that expression of Epac mRNAs is present in mouse testicular germ cells. Indirect immunofluorescence with specific polyclonal antibodies suggested possible co-localization of Epac1 and Rap1 proteins in the heads of epididymal spermatozoa. Moreover, treatment of epididymal spermatozoa with an Epac-specific cAMP analog, 8-pMeOPT-2'-O-Me-cAMP, induced activation of Rap1, as revealed with a commercial kit for pull-down assay. These results indicate the existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa.