Immunohistochemical localization of the calcium/calmodulin-dependent protein phosphatase,calcineurin, in the mouse testis: its unique accumulation in spermatid nuclei

Immunohistochemical localization of a calmodulin-dependent protein phosphatase, calcineurin, was studied in the mouse testis in relation to previous observations showing that calmodulin is unusually rich in spermatogenic stages from mid-pachytene spermatocytes to elongating spermatids. The antibodies raised against calcineurin from scallop testis reacted with subunit B, but not subunit A, of calcineurin isoforms from mouse brain and testis. Indirect immunofluorescence using these antibodies on the mouse testis revealed positive reactions only in the nuclei of round or elongating spermatids: calcineurin started to accumulate in nuclei from the acrosomal cap phase, peaked at the initial stage of nuclear elongation, and decreased thereafter. There was almost no signal in the cytoplasm; spermatogenic cells at other stages, including spermatogonia, spermatocytes, mature sperm, and other somatic cells in the seminiferous tubules were totally negative. Immuno-electron microscopy gave the same result, on the basis of measuring the density of immunogold particles. These results suggest a role for calcineurin in remodeling of the nuclear chromatin in metamorphosing spermatids.     

Biochemical and immunological characterization of a histone variant associated with spermatogenesis in the mouse

A chromosomal histone, H2S, specific to the mouse testis has been purified. Amino acid analysis indicated lack of cysteine and a high basic amino acid content typical of histones. Specific antibodies against histones H2S have been generated in rabbits and partially purified using (NH₄)₂SO₄ precipitation and ion-exchange chromatography. Protein transfer experiments indicate presence of antigenically similar histones in the rat and rabbit testes but not in the guinea pig and dog testes. In addition, histone complement of somatic tissues such as lung, kidney, liver and spleen lacked antigenically similar proteins. Immunocytochemical studies using peroxidase-antiperoxidase complex indicated presence of immunoreactive cells in the seminiferous epithelium which were lacking in the interstitium. These data demonstrate histone H2S to be a unique histone associated with spermatogenesis in the mouse.     

Metastasis tumor antigen 1 is involved in the resistance to heat stress-induced testicular apoptosis

Our previous study documented the expression of Mta1 during spermatogenesis. Here, we present evidence for a possible involvement of Mta1 in the regulation of testicular function, possibly by interacting with p53. A notable decrease of Mta1 expression was revealed at postsurgical day 6, consistent with the previously reported upregulation of p53 in mouse cryptorchidism. Furthermore, in vitro over-expression of Mta1 could remarkably elevate the resistance capability of spermatogenic tumor cells against heat-induced apoptosis with a marked impairment of p53 expression. These findings indicate that Mta1 may operate as a negative modifier of apoptosis by interacting with p53 during gametogenesis.     

A novel protein,sperm head and tail associated protein (SHTAP), interacts with cysteine‐rich secretory protein 2 (CRISP2) during spermatogenesis in the mouse

Background information. CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca²⁺ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results. Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (～20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ～26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions. The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.     

Correlation of extracellular matrix components with the cytoarchitecture of mouse Peyer's patches

Summary The distribution patterns of extracellular matrix elements were determined to ascertain whether they play a role in the localization of lymphocytes in discrete T-cell, B-cell and dome antigen-processing domains within Peyer's patches. Antibodies against collagen types I, III and IV, laminin and fibronectin were applied to cryosections of mouse Peyer's patches and localized by direct or indirect immunoperoxidase methods. T-cell domains were identified with a monoclonal antibody against Thy-1.2. Labeled reticular fibers in distinctive patterns were more numerous in parafollicular and dome areas than within follicles. Germinal centers contained few such fibers. In parafollicular areas, fibers were oriented predominantly toward follicle domes; their distribution corresponded to T-cell zones and lymphocyte traffic areas, with their orientation being parallel to the migration pathways of lymphocytes from high endothelial venules to the antigen-processing domes. Subepithelial and subendothelial basal laminae were immunopositive for type-IV collagen, laminin and fibronectin. The dome subepithelial basal lamina had pore-like discontinuities through which lymphocytes migrated to and from the epithelium. The correspondence of the distribution patterns of extracellular matrix to specific functional domains of Peyer's patches suggests that this matrix provides a structural framework for lymphocyte migration and localization.     

Distribution of lymphatic vessels in mouse thymus: immunofluorescence analysis

Thymic blood and lymphatic vessels in humans and laboratory animals have been investigated in morphological studies. However, occasionally a clear distinction between blood vessels and lymphatic vessels cannot be made from morphological characteristics of the vasculature. To visualize thymic lymphatics in normal adult BALB/c mice, we used antibodies against specific markers of lymphatic endothelial cells. Expression of vascular endothelial growth factor receptor–3 (VEGFR–3) was detected throughout the thymus, i.e., the capsule, cortex, and medulla. Most thymic lymphatics were present in capillaries of ~20 μm in caliber. The plexuses of lymphatic capillaries were occasionally detectable. Lymphatic vessels were frequently adjacent to CD31–positive blood vessels, and some lymphatic vessels were seen in the immediate vicinity of or within the perivascular spaces around postcapillary venules. The identity of VEGFR–3–positive vessels as lymphatics was further confirmed by staining with additional markers: LYVE–1, Prox–1, neuropilin–2, and secondary lymphoid tissue chemokine (SLC). The distributions of LYVE–1 were similar to those of VEGFR–3. Most lymphatic vessels were also identified by Prox–1. Neuropilin–2 was restricted to lymphatic vessels in the thymus. The most abundant expression of SLC in the thymus was in medullar epithelial cells; SLC was also expressed in lymphatic vessels and blood vessels. Thus, lymphatic endothelium in mouse thymus was characterized by positive staining with antibodies to VEGFR–3, LYVE–1, Prox–1, neuropilin–2, or SLC, but not with an antibody to CD31. Our results suggest the presence of lymphatic capillary networks throughout the thymus.     

Expression analysis of the NDRG2 gene in mouse embryonic and adult tissues

N-myc downstream-regulated gene 2 (NDRG2) is believed to be involved in cell growth events. However, its exact function is still unknown. To elucidate the role of this gene, we used an anti-Ndrg2 monoclonal antibody in immunohistochemistry and immunofluorescence assays to analyze the expression pattern of Ndrg2 protein in mouse embryos at various gestational ages and in a variety of adult mouse tissues. Ndrg2 immunoreactivity was generally localized to the cytoplasm. During mouse development, Ndrg2 expression was observed in many developing tissues and organs including the heart, brain, lung, gut, liver, kidney, skeletal muscle, cartilage, chorion, epidermis, and whisker follicles. Ndrg2 expression was developmentally dynamic, being generally lower in the early stages of development and markedly increasing during later stages. Ndrg2 expression was also observed in a variety of adult mouse tissues, particularly in the heart and brain. This is the first demonstration of Ndrg2 protein expression in both embryonic and adult mouse tissues. Our results suggest that NDRG2 plays important roles in histogenesis and organogenesis.This study was supported by grants from the National Key Basic Research and Development Program (no. 2002CB513007), the National Natural Science Foundation of China (nos. 30370315 and 30171044) and PCSIRT04-59.     

The secretion of two sperm maturation-related glycoproteins in BALB/c mouse epididymis

Summary A well-developed Golgi apparatus and rough and smooth endoplasmic reticulum in the principal cells of the mouse epididymis indicate active protein synthesis. Studies have shown that epididymal secretions are essential for sperm maturation. In a previous study, two wheat-germ agglutinin (WGA)-binding glycoproteins, GP-49 and GP-83, were identified on the surface of mature mouse sperm. In this study, synthesis and secretion of these two glycoproteins were investigated. Apparent WGA-binding was found on the stereocilia and in the apical region of principal cells in the corpus and cauda of epididymis. Post-fixation and pre-embedding cytochemical localization revealed that WGA-binding sites were situated in the Golgi apparatus, multivesicular bodies and stereocilia of principal cells. GP-49 and GP-83 were identified in the Nonidet P-40 homogenates of corpus and cauda epididymidis. In the epididymides of which ductuli efferentes had been ligated for more than 4 weeks, no sperm were found in the lumina of epididymal tubules. WGA-binding sites were present in the corpus and cauda; GP-49 and GP-83 were identified in tissue homogenates of the corpus and cauda as well. These findings suggest that GP-49 and GP-83 of mature sperm may be secreted by the principal cells of the corpus and cauda. These two molecules apparently conjugate to sperm whilst sperm transit through the epididymis.     

Developmental expression of MNAR mRNA in the mouse brain

During the development of the central nervous system, estrogen influences cellular differentiation and determines the functional connectivity of distinct neural networks. Estrogens generally act through nuclear estrogen receptors (ERs). Recent research has additionally revealed rapid estrogen effects requiring the binding of estrogen to membrane/cytoplasmic ERs and the activation of intracellular signaling systems such as the Src/MAPK cascade. The scaffold protein MNAR/PELP1 appears to be the designated functional mediator of such non-genomic estrogen effects between non-nuclear ERs and Src/MAPKs. In this study, we demonstrate the expression and differential regulation of MNAR mRNA in the developing male and female mouse brain by quantitative polymerase chain reaction. In the midbrain and hypothalamus, a gradual decline in MNAR mRNA levels has been observed prenatally with the highest values at embryonic day 15 and lowest at postnatal day 15. In the cortex, mRNA levels do not fluctuate until postnatal day 7 but decrease thereafter. No differences in MNAR expression between sexes have been detected. Analysis of neuronal and astroglia-enriched cell cultures has revealed the presence of MNAR in both cell types.     

Expression and distribution of trihydrophobin 1 in postnatal developing mouse testis

The human trihydrophobin 1 (TH1) is a highly conserved and widely expressed protein. It is clear that TH1 serves as a new specific negative regulator of A-Raf kinase. In this study, we found that TH1 associated with A-Raf in mouse testis by using coimmunoprecipitation analysis. Then we characterized the gene expression of TH1 in mouse testis and analyzed the changes of TH1 protein during postnatal development. The protein expression of TH1 in mouse testis was further analyzed by immunohistochemistry staining. Strong signals were detected in the seminiferous tubules and the distribution patterns varied with the different ages of postnatal mouse testis. TH1 was distributed in spermatocytes and Sertoli cells at 2 weeks postnatal, and was abundant in spermatogonia at 8 weeks postnatal. Leydig cells were positive to TH1 throughout testicular development. A high expression of TH1 in both Leydig cells and mouse Leydig tumor cells (mLTC-1cells) was found to be concentrated in the cytoplasm. The colocalization of TH1 and A-Raf in mLTC-1 cells or in adult testis was also observable.     

Changes in endoplasmic reticulum during spermiogenesis in the mouse

Summary Changes in the endoplasmic reticulum of mouse spermatids during spermiogenesis were examined by scanning electron microscopy, applying the OsO₄-DMSO-OsO₄ method, which permits 3-dimensional observation of cell organelles. At the same time, the endoplasmic reticulum was stained selectively by the Ur-Pb-Cu method, and 0.5 m-thick sections were prepared for observation by transmission electron microscopy. The results demonstrated stereoscopically the mode of disappearance of the endoplasmic reticulum. In spermatids of the early maturation phase, the endoplasmic reticulum was of uniform diameter, branched and anastomosed, forming a complicated three-dimensional network throughout the cytoplasm. A two-dimensional net was also noted to have formed just beneath the plasma membrane and about Sertoli cell processes invaginating the spermatid cytoplasm. As spermiogenesis progressed, the spread-out endoplasmic reticulum gradually aggregated to form a condensed, glomerulus-like structure consisting of a very thin endoplasmic reticulum connected to the surrounding endoplasmic reticulum. This structure corresponds to the so-called radial body. Thus, the endoplasmic reticulum may aggregate, condense, be transformed into a radial body, and be removed from the cytoplasm. The two-dimensional endoplasmic reticulum-net, just beneath the plasma membrane and surrounding processes of Sertoli cells, disappeared in spaces where the three-dimensional endoplasmic reticulum network was scarce. Both the two-dimensional endoplasmic reticulum-net structure and the three-dimensional endoplasmic reticulum network disappeared at the same time, indicating that they may be closely related.     

Characterisation and high-resolution distribution of a matrix attachment region-binding protein (MFP1) in proliferating cells of onion

The first matrix attachment region (MAR)-binding protein sequenced in plants, MFP1, has been characterised in two dicot species. Based on their antigenic relationship, we report here the conservation of MFP1-like proteins in proliferating root cells of onion (Allium cepa L). Two MFP1-like proteins with different molecular masses and solubilities were detected. The most abundant was a 90-kDa basic protein, presenting several separate spots in two-dimensional blots. The MFP1 was partially soluble and, similar to the proliferating cell nuclear antigen (PCNA)-labelled replication factories in the nucleus and nuclear matrix, was localised at discrete foci as detected by confocal microscopy. High-resolution immunolocalisation of MFP1 by electron microscopy identified the foci as nuclear structures, some of them containing PCNA, which are ultrastructurally similar to the replication factories described in animal cells. Our data provide the first report on MFP1-like proteins in the Alliaceae. In addition, we present evidence of the presence of AcMFP1 in the putative replication factories. Received: 12 May 2000 / Accepted: 13 September 2000     

RNA interference of metastasis-associated gene 1 inhibits metastasis of B16F10 melanoma cells in a C57BL/6 mouse model

Background information. MTA1 (metastasis‐associated gene 1) has been reported to be overexpressed in cancers with high potential to metastasize. Studies of the molecular mechanisms revealed that MTA1 plays an important role in the process of metastasis of many types of cancer. However, the role of MTA1 in melanoma development is unclear. Results. We have investigated the therapeutic value of MTA1 in the B16F10 melanoma cell line with the C57BL/6 mouse model. Studies in vitro showed that MTA1 promoted the metastatic ability of B16F10 cancer cells. MTA1 down‐regulation by RNA interference greatly reversed the malignant phenotypes of cancer cells. Immunohistochemical staining of MTA1 in human melanoma samples confirmed the up‐regulation of MTA1 in the process of carcinogenesis. Studies in vivo confirmed down‐regulation of MTA1 suppressed the growth and experimental metastasis of B16F10 melanoma cells. Conclusions. MTA1 plays an important role in melanoma development and metastasis. It has a promising potential as a target for in cancer gene therapy or chemotherapy.     

Ontogeny of ppp(A2′p)nA-binding protein in mouse brain

Mouse brain tissue extracts at various stages of development show a drastic change in the specific activity of pp(A2′p)₂A-[³²P]pCp binding protein. Identification of the ppp(A2′p)₃A-[³²P]pCp binding protein was established by (i) binding to the specific ligand ppp(A2′p)₃A-[³²P]pCp, (ii) displacement of binding by nanomolar concentration of pppA(pA)₃, and (iii) affinity labeling techniques in which periodate oxidized ppp(A2′p)₃A-[³²P]pC was specifically cross-linked to a protein with a molecular weight of 86 000. These data suggest that the ppp(A2′p)₃A-[³²P]pCp protein is closely associated with the process of cellular proliferation and differentiation.     

Morphogenesis of the photoreceptor outer segment during postnatal development in the mouse (BALB/c) retina

Summary Disc formation of rod photoreceptor cells in developing BALB/c mice retinas was studied by rapid freeze, freeze-substitution, freeze-etching, immunocytochemistry, and myosin S-1 decoration methods. Freeze-substituted photoreceptor cells contained variously shaped vesicles in the apical swelling of the connecting cilium or the base of the outer segment during postnatal development. Rapid freezing successfully arrested pinocytosis; the fusion of small vesicles to give large ones, and the compression of certain vesicles (0.3–0.6 m) appears to lead gradually to the formation of the so-called discs. We therefore propose that membranous discs are formed by the fusion of small pinocytotic vesicles and their subsequent compression. Discs formed in this way were partially stacked, but were ordered at random during the early developmental stages. During development, a partial stack of discs was progressively rearranged to a regular form as seen in mature outer segments. Cytoskeletal actin was expected to be involved in the disc formation; it was demonstrated in the distal axoneme of the connecting cilium during development and showed no change in its distribution. However, the polarity of the actin filaments, as revealed by myosin S-1 decoration in early developmental stages, was much more variable than in the adult. Barbed ends of actin filaments were associated with the plasma membrane or the membrane of vesicles. We also found actin filaments coiled up helically on ciliary microtubules.     

Differential expression of caveolin-3 in mouse smooth muscle cells in vivo

Expression of caveolin-1 and -3 in mouse smooth muscle cells in vivo was examined by immunohistochemistry. Caveolin-1 was detected in almost all smooth muscles examined, except for the pupillary dilator muscle, whereas caveolin-3 was present only in smooth muscles of some specific tissues. In the eye, the pupillary sphincter muscle was intensely positive for caveolin-3, whereas the ciliary muscle and pupillary dilator muscle were negative. In the gastrointestinal tract, caveolin-3 was detected in the inner circular layer, but not in the outer longitudinal layer. Vascular smooth muscle cells of the resistance-sized artery in the uterus and corpus cavernosum were intensely positive for caveolin-3, whereas those of the aorta were only weakly positive and those of the vena cava were negative. Caveolin-3 was also detected in smooth muscle cells of the urinary bladder, ureter, prostatic vas deferens, and seminal vesicle. The different levels of caveolin-3 expression among various smooth muscle tissues were confirmed by Western blot analysis. Even within the same muscle, the relative expression levels of caveolin-1 and -3 were variable among neighboring cells, suggesting distinct fine regulation of expression of these two caveolins. Moreover, even in the same cell, caveolin-1 and -3 showed different distributions. These results indicate that the two caveolins form distinct caveolae in smooth muscles, and that caveolin-1 and -3 serve different functions. Their differential expression may therefore be related to the functional diversity of smooth muscles. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of the Japanese Government.