Introduction {#Sec1} ============ During the process of spermatogenesis in mammals, the first spermatozoon of a new generation is formed within the seminiferous epithelium, which originates from differentiated germ cells, and the generation of new germ cells during the maturation of the testis is called spermatogenesis. In the seminiferous epithelium of an adult mouse, a single Sertoli cell is attached to and interacts with multiple germ cells via Sertoli--Sertoli and Sertoli--germ cell junctions (Scherf et al. [@CR48]; Nagano et al. [@CR38]; Hikosaka et al. [@CR16]; Hinrichs et al. [@CR17]; Houweling and Jockusch [@CR19]; Houweling et al. [@CR20]). A mature Sertoli cell has an elongated shape and its cytoplasmic processes extend longitudinally over the length of the seminiferous epithelium and attach to adjacent Sertoli cells (Geng et al. [@CR13]; Houweling and Jockusch [@CR19]). Several Sertoli--germ cell junctions are observed at the stage of the formation of the contact with a germ cell, whereas only a single Sertoli--Sertoli junction is present at the basal side of a mature Sertoli cell. Immunological and dye exclusion techniques have revealed the presence of junctional proteins and cell adhesion molecules between Sertoli and germ cells (Scherf et al. [@CR48]; Ootani et al. [@CR41]; Yamada et al. [@CR62], [@CR63]; Hikosaka et al. [@CR16]; Yagawa et al. [@CR61]; Houweling and Jockusch [@CR19]; Houweling et al. [@CR20]). Furthermore, cell-to-cell junctions between adjacent Sertoli cells are known to be regulated by extracellular factors including secreted proteins from the Sertoli cells and paracrine mediators secreted from germ cells (Cheng and Braun [@CR4]; Ootani et al. [@CR41]; Sato et al. [@CR47]; Nissen and Lovato [@CR39]; Sato et al. [@CR48]; Aittokallio et al. [@CR1]). In the course of spermatogenesis, Sertoli cells provide the microenvironment required for germ cell survival and development, and the development of the epididymis to ensure the passage of spermatozoa from the seminiferous epithelium to the vas deferens (Schweiger and Wartenberg [@CR49]; Yamagata et al. [@CR65]; Yamagata [@CR64]). During germ cell development, germ cells migrate across the seminiferous epithelium towards the adluminal compartment of the seminiferous tubule, from which they will be released into the epididymis (Scherf et al. [@CR48]; Nagano et al. [@CR38]; Hikosaka et al. [@CR16]; Hinrichs et al. [@CR17]; Houweling and Jockusch [@CR19]; Houweling et al. [@CR20]). In the present study, we examined cell adhesion molecule *N*-cadherin and connexin 31 (Cx31) protein expression in mouse testes at defined stages of spermatogenesis to understand their roles in testicular cell--cell interactions in vivo. We found that *N*-cadherin is transiently expressed during the initial stages of meiosis, and Cx31 is expressed during a longer period, consistent with its association with gap junctional communication (GJIC) in Sertoli--Sertoli and Sertoli--germ cell junctions. Materials and methods {#Sec2} ===================== Animals and drugs {#Sec3} ----------------- Adult (12- to 15-week-old) C57BL/6J male mice (CLEA Japan, Tokyo, Japan) were used for immunohistochemistry. Mice were maintained under a 12 h light:dark cycle at 23 ± 1°C with 55 ± 5% relative humidity. All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the Graduate School of Biostudies at Kyoto University, or were conducted in accordance with the Regulations for Animal Experimentation at the Tokyo University of Pharmacy and Life Sciences. All chemicals used in the study were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), unless otherwise stated. Antibodies {#Sec4} ---------- The mouse monoclonal anti-*N*-cadherin and anti-Cx31 antibodies were purchased from BD Biosciences Pharmingen (San Diego, CA). The mouse monoclonal anti-α-smooth muscle actin (SMA) antibody was purchased from Sigma-Aldrich (St. Louis, MO). The anti-mouse and anti-rabbit secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 488 and 568 phalloidin conjugates were purchased from Molecular Probes (Invitrogen, Carlsbad, CA). Hormone treatment {#Sec5} ----------------- For the hormone treatment experiments, mice were administered 15 mg/kg body weight of β-estradiol-17-β (E2; Sigma-Aldrich), or 7 mg/kg body weight of β-estradiol-17-β (E2) and 3 mg/kg body weight of progesterone (P4; Sigma-Aldrich) subcutaneously every other day between 1 and 30 days of age. Mice were housed in an autoclaved IVC cage. The temperature was maintained at 21--24°C and light cycle was 12-h light:12-h dark. Tissue preparation {#Sec6} ------------------ Mice were anesthetized with isoflurane and fixed by transcardial perfusion using 4% paraformaldehyde in phosphate-buffered saline (PBS). Testes were removed and post-fixed overnight in 4% paraformaldehyde/PBS at 4°C. The tunica albuginea was removed from the testicular tissue, followed by further fixation overnight at 4°C. Testes were dehydrated with increasing concentrations of ethanol (30, 50, 70, 80, 90 and 100%) and embedded in paraffin. Paraffin blocks were sectioned with a microtome at 7 μm. The sections were mounted on silane-coated glass slides (Matsunami Glass, Osaka, Japan) and stored at room temperature until further processing. Indirect immunofluorescence staining {#Sec7} ------------------------------------ Immunofluorescence staining of the mouse testicular sections was performed essentially as described previously (Fujikawa et al. [@CR12]; Mabuchi et al. [@CR30]). Briefly, paraffin-embedded sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol (100, 70, 50, 30% and water) and phosphate-buffered saline (PBS). The slides were pretreated in citrate buffer (0.01 M, pH 6.0) and cooled overnight at 4°C. Nonspecific binding sites were blocked by incubating the sections in a solution containing 5% skimmed milk, 1% bovine serum albumin, 0.1% cold water fish skin gelatin, 1% normal goat serum and 0.1% Triton X-100 for 30 min. Sections were then incubated for 90 min at room temperature with a mouse monoclonal anti-*N*-cadherin (1:50; BD Biosciences Pharmingen) or anti-Cx31 (1:100) antibody, followed by incubation for 90 min with an anti-mouse FITC-conjugated secondary antibody (1:100; Sigma-Aldrich). After washing in PBS, slides were incubated in Alexa Fluor 568 phalloidin (0.01 U/ml) for 90 min. After washing in PBS, slides were mounted in Fluoromount-G™ (Southern Biotechnology, Birmingham, AL) and examined by confocal laser scanning microscopy using an Olympus FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan) equipped with FV10-ASW 3.1 software (Olympus). All images were taken under the same conditions and analyzed using Adobe Photoshop software (Adobe Systems Inc., San Jose, CA). Immunostaining quantification {#Sec8} ----------------------------- Slides containing fluorescent-labeled nuclei in the spermatogonia, preleptotene/leptotene spermatocytes, zygotene/pachytene spermatocytes and step 2--5 round spermatids were identified and observed by confocal laser scanning microscopy using a confocal laser scanning microscope (FV1000; Olympus). Images were taken under the same conditions and analyzed using Adobe Photoshop software (Adobe Systems Inc., San Jose, CA). The relative intensity values were obtained from the intensity value of Alexa Fluor 568 phalloidin and DAPI (which stains nuclei) and then used for cell detection. The number of foci in each field was counted using ImageJ software (U.S. National Institutes of Health, Bethesda, MD). For detection of Cx31-positive spots, the regions in the images were adjusted to exclude spots that did not show Cx31-positive spots; the spot foci were detected and the ratio of Cx31 to the number of foci was evaluated. For detection of *N*-cadherin, the regions in the images were adjusted to exclude spots that did not show *N*-cadherin expression; the percentage of the *N*-cadherin-positive spot areas in total areas of each image was calculated. Data were collected