Testicular teratomagenesis from primordial germ cells with overexpression of germinal center-associated nuclear protein.

Testicular teratomas are the major histologic type of testicular germ cell tumors and their incidence continues to grow. Moreover, teratomas can develop from undifferentiated cells in induced pluripotent stem (iPS) cell transplantation therapy, seriously hampering the progress of regenerative medicine. Germinal center-associated nuclear protein (GANP) is thought to be important to the biogenetic control of primordial germ cells and is among the genes susceptible to testicular germ cell tumors. Thus, we analyzed the expression of GANP in human testicular postpubertal-type teratomas and established a novel mouse model to reveal the association between GANP and teratomagenesis. We analyzed 31 cases of human testicular postpubertal-type teratomas and, in all cases, GANP was overexpressed. The aberrant expression was also detected in germ cell neoplasia in situ accompanied by the teratoma. GANP expression was particularly high in the epithelia of the epidermis, cutaneous appendages, and trachea-like ciliated epithelium. To further clarify the association between GANP and teratomagenesis, we established a novel teratomagenesis mouse model (CAG-ganpTg mice). In the GANP-teratoma mice, GANP-overexpressing teratomas were more frequent at the testes and the middle portion of the uterus than has been seen in the previously established mouse models. In conclusion, GANP is overexpressed in testicular postpubertal-type teratomas and is an essential teratomagenic factor. We also found that CAG-ganpTg mice are useful mouse models of teratomagenesis that mimics human midline teratomas and that teratomas may originate from the overexpression of GANP in primordial germ cells.

Viable offspring obtained from Prm1-deficient sperm in mice.

Protamines are expressed in the spermatid nucleus and allow denser packaging of DNA compared with histones. Disruption of the coding sequence of one allele of either protamine 1 (Prm1) or Prm2 results in failure to produce offspring, although sperm with disrupted Prm1 or Prm2 alleles are produced. Here, we produced Prm1-deficient female chimeric mice carrying Prm1-deficient oocytes. These mice successfully produced Prm1(+/-) male mice. Healthy Prm1(+/-) offspring were then produced by transferring blastocysts obtained via in vitro fertilization using zona-free oocytes and sperm from Prm1(+/-) mice. This result suggests that sperm lacking Prm1 can generate offspring despite being abnormally shaped and having destabilised DNA, decondensed chromatin and a reduction in mitochondrial membrane potential. Nevertheless, these mice showed little derangement of expression profiles.

Histochemical evaluation of postnatal lectin-binding sites in the mouse prostate.

The prostate is a male accessory genital gland that plays an essential role in reproductive function. To understand the cytological characteristics of differentiating prostatic cells, we used lectin histochemistry combined with immunohistochemistry to examine the distribution of lectin-binding sites on prostatic cells during postnatal development in the mouse. During postnatal development, Hippeastrum Hybrid Lectin (HHL) lectin reacted consistently with the luminal cells of all prostatic lobes (regions), whereas the Ricinus Communis Agglutinin I (RCA-I) and Soybean Agglutinin (SBA) lectins showed remarkable differences with age, region, and cell type. We found that the lectin-binding pattern in differentiating prostatic cells acquired adult characteristics around 3 weeks after birth. The results indicate that prostatic cell differentiation during postnatal development in mice is characterized by the presence of cell- and region-specific lectin-binding sites in the prostate, suggesting that there may also be cellular and regional differences in their function. Furthermore, some lectins (HHL, RCA-I, and SBA) could provide useful markers for research into cell differentiation and for the pathological evaluation of prostatic diseases or in the diagnosis of male infertility.

Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells.

Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5-5 µM induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 µM, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10-20 µM), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM.

Cytokeratin localization and basal cell differentiation in the epididymal epithelium during postnatal development of the mouse.

The epididymis is a male genital organ that has plays various functions, including sperm concentration, maturation, and storage. The epididymal epithelium consists of principal cells, clear cells, and basal cells. To comprehensively understand the occurrence and morphological differentiation of basal cells, we examined the expression and localization of cytokeratins (CKs) in the epididymal epithelium during postnatal development of the mouse. Immunohistochemical staining showed that, in adult mice, CK5 and CK14 were exclusively expressed in the cytoplasm of basal cells. During postnatal development, basal cells that stained positive for CK5 and CK14 first appeared in immature columnar epithelial cells in mice aged 1 week. The immunoreactivity became progressively stronger in mice aged 2-3 weeks. In mice aged 3 weeks, the immunoreactivity was strong in regions IV and V. In mice aged ≥ 4 weeks, strong immunoreactivity was observed in all epididymal regions. CK5 and CK14 could be useful markers of differentiation in epididymal basal cells. These basal cells originate from immature columnar epithelial cells and are of two types­dome-shaped and flask-shaped­. The flask-shaped cells are mainly located in the initial segment of the mouse epididymis.

Lectin-binding sites in epithelial cells of the mouse prostate gland.

The prostate is an exocrine gland in the male reproductive tract that secretes seminal fluids. To gain insight into the cytochemical properties of prostatic epithelial cells, the characteristics of glycoconjugates in mouse prostate sections were examined by lectin histochemistry and immunohistochemistry. Characteristic staining patterns were observed, depending on the type of lectins present in the epithelia. Luminal cells reacted specifically with mannose-binding lectins (Galanthus nivalis lectin, Hippeastrum hybrid lectin, Narcissus pseudonarcissus lectin) and Maclura pomifera lectin in all lobes of the prostate. Luminal cells also expressed galactose, N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), and fucose residues in the lateral and ventral lobes. Basal cells expressed GlcNAc and fucose, and reacted with Datura stramonium lectin and Aleuria aurantia lectin in all lobes. These results indicate that in the mouse prostate, the selectivity of lectin-binding sites for distinct cell types and lobe-dependent staining may relate to cellular and regional differences in function. Furthermore, some lectins selectively bound to prostatic epithelial cells, indicating their potential use as markers for the histopathological evaluation of prostatic diseases, cancer diagnosis, or male infertility.

Cell- and region-specific expression of sugar chains in the mouse epididymal epithelium using lectin histochemistry combined with immunohistochemistry.

To understand the cytochemical properties of epididymal epithelial cells, the characteristics of glycoconjugates in the mouse epididymis were examined using the technique of lectin histochemistry combined with immunohistochemistry. Characteristic staining patterns depending on the type of lectins were observed in the epididymal epithelium. Principal cells expressed N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), and Fucose in the proximal region of the epididymis and Mannose, Glucose, and Galactose in the distal region of the epididymis. Basal cells expressed Mannose, Glucose, Galactose, and GlcNAc in the proximal region and Galactose in the distal region. On the other hand, clear cells expressed various sugar residues and differences among regions were not observed. Interestingly, principal cells, clear cells, and basal cells specifically reacted with Ulex Europaeus-Agglutinin I (UEA-I lectin), Maackia Amurensis-Lectin I (MAL-I lectin), and Griffonia simplicifolia Lectin I-B4 (GS-I B lectin), respectively. These findings indicate that the selectivity in lectin reactivity for distinct cell types and segment-dependent staining in the epididymis may be related to cellular and regional differences in function. Furthermore, because some lectins stain particular cells or cellular compartments selectively, these lectins could be useful markers for histopathological evaluation of diseases or diagnosis of male infertility.

Changes in lectin-binding sites on epididymal cells during postnatal development of the mouse.

Using lectin histochemistry combined with immunohistochemistry, we recently demonstrated that the principal, clear, and basal cells in the adult mouse epididymis specifically react with UEA-I, MAL-I, and GS-IB lectins, respectively. The present study examined the distribution of the lectin-binding sites for some lectins on the epididymal epithelium during postnatal development. Galactose staining with GS-IB was first detected in some of the undifferentiated epithelial cells in mice aged 1 week, and this characteristic became specific to basal cells in mice aged 2 weeks and above. Fucose staining with UEA-I was first detected in the principal cells in mice aged 3 weeks. Staining of sialic acid with MAL-I was first detected in all undifferentiated epithelial cells in mice aged 1 week, and this characteristic became specific to the narrow and clear cells in all regions and to the principal cells in regions IV and V in mice aged 3 weeks. The results indicate that epididymal differentiation is characterized by the expression of cell- and region-specific sugar chains that appear early during postnatal development before the sperm arrives in the epididymis.

Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation.

The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;AR(flox/Y) mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.

Regulation of ER molecular chaperone prevents bone loss in a murine model for osteoporosis.

Endoplasmic reticulum (ER) stress response is important for protein maturation in the ER. Some murine models for bone diseases have provided significant insight into the possibility that pathogenesis of osteoporosis is related to ER stress response of osteoblasts. We examined a possible correlation between osteoporosis and ER stress response. Bone specimens from 8 osteoporosis patients and 8 disease-controls were used for immunohistochemical analysis. We found that ER molecular chaperones, such as BiP (immunoglobulin heavy-chain binding protein) and PDI (protein-disulfide isomerase) are down-regulated in osteoblasts from osteoporosis patients. Based on this result, we hypothesized that up-regulation of ER molecular chaperones in osteoblasts could restore decreased bone formation in osteoporosis. Therefore, we investigated whether treatment of murine model for osteoporosis with BIX (BiP inducer X), selective inducer BiP, could prevent bone loss. We found that oral administration of BIX effectively improves decline in bone formation through the activation of folding and secretion of bone matrix proteins. Considering these results together, BIX may be a potential therapeutic agent for the prevention of bone loss in osteoporosis patients.

Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation.

Eukaryotic cells have signalling pathways from the endoplasmic reticulum (ER) to cytosol and nuclei, to avoid excess accumulation of unfolded proteins in the ER. We previously identified a new type of ER stress transducer, OASIS, a bZIP (basic leucine zipper) transcription factor, which is a member of the CREB/ATF family and has a transmembrane domain. OASIS is processed by regulated intramembrane proteolysis (RIP) in response to ER stress, and is highly expressed in osteoblasts. OASIS(-/-) mice exhibited severe osteopenia, involving a decrease in type I collagen in the bone matrix and a decline in the activity of osteoblasts, which showed abnormally expanded rough ER, containing of a large amount of bone matrix proteins. Here we identify the gene for type 1 collagen, Col1a1, as a target of OASIS, and demonstrate that OASIS activates the transcription of Col1a1 through an unfolded protein response element (UPRE)-like sequence in the osteoblast-specific Col1a1 promoter region. Moreover, expression of OASIS in osteoblasts is induced by BMP2 (bone morphogenetic protein 2), the signalling of which is required for bone formation. Additionally, RIP of OASIS is accelerated by BMP2 signalling, which causes mild ER stress. Our studies show that OASIS is critical for bone formation through the transcription of Col1a1 and the secretion of bone matrix proteins, and they reveal a new mechanism by which ER stress-induced signalling mediates bone formation.

Decreased abundance of urinary exosomal aquaporin-1 in renal ischemia-reperfusion injury.

Urinary exosomes, secreted into urine from renal epithelial cells, are known to contain many types of renal functional membrane proteins. Here, we studied whether renal ischemia-reperfusion (I/R) affects urinary exosomal aquaporin-1 (AQP1) excretion in rats subjected to renal I/R and patients who underwent renal transplantation. Immunoblotting studies demonstrated reduction of the urinary exosomal AQP1 level even at 6 h after renal I/R, and the level continued to be low over 96 h after I/R. Renal AQP1 mRNA and protein analyses revealed that the decreased excretion of urinary exosomal AQP1 is associated with renal AQP1 protein retention in the early phase and with a decreased expression level of renal AQP1 in the later phase of renal I/R injury. Decreased abundance of urinary exosomal AQP1 in a recipient patient was also observed at 48 h after renal allograft transplantation. No significant decrease in urinary exosomal AQP1 was observed in a rat model of nephropathy or in patients with proteinuria. Our studies suggest that the renal AQP1 expression level is possibly controlled by its urinary exosomal excretion and indicate that urinary exosomal AQP1 is a novel urinary biomarker for renal I/R injury.

Nerve fiber analysis on the so-called accessory subscapularis muscle and its morphological significance.

A rare muscular anomaly, so-called accessory subscapularis muscle, was found in the left axillary fossa of a 95-year-old male cadaver during a student dissection practise. The muscle arose near the lateral margin of the scapula from the surface of the subscapularis muscle and ran upward to fuse with the capsule of the shoulder joint via a tendon. It measured 1.0 cm in width, 7.0 cm in length and 1.5 mm in thickness, and was separated from the underlying subscapularis muscle by the axillary and inferior subscapular nerves. Macroscopically, the anomalous muscle received its nerve supply from a branch arising from the lower root of the radial nerve near the origin of the thoracodorsal nerve and entered the muscle from its ventral surface. Nerve fiber analysis showed that the supplying nerve originated from fibers of the dorsal element of C7 immediately cranial to the thoracodorsal nerve. These findings indicate that the present anomalous muscle might be close to the formation of the latissimus dorsi muscle in its derivation rather than the subscapularis muscle.

Preferential localization of rat GAPDS on the ribs of fibrous sheath of sperm flagellum and its expression during flagellar formation.

The proper assembly of sperm flagellar proteins is fundamental for sperm motility. The sperm- and spermatid-specific isoform of glyceraldehyde 3-phosphate dehydrogenase, GAPDS, is a flagellar protein indispensable for sperm flagellar movement. To obtain information on the assembly of the glycolytic enzyme into the flagellum, the precise localization of rat GAPDS in the flagellum and the stage of incorporation into the flagellum were examined using a monoclonal antibody. Immunolocalization of rat GAPDS was restricted to the fibrous sheath (FS) in the sperm flagellum, and was predominant in the circumferential ribs rather than the longitudinal columns. Immunoreactivity was first detected in the cytoplasm and flagella of the step-16 spermatids during the final step of FS formation. Together with the expression of other FS proteins, the present results indicate the sequential assembly of FS components, suggesting that the expression and transport of GAPDS is regulated in a coordinated manner during sperm flagellar formation.

BBF2H7, a novel transmembrane bZIP transcription factor, is a new type of endoplasmic reticulum stress transducer.

Endoplasmic reticulum (ER) stress transducers IRE1 (inositol requiring 1), PERK (PKR-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6) are well known to transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. Recently, we identified OASIS (old astrocyte specifically induced substance) as a novel ER stress transducer expressed in astrocytes. We report here that BBF2H7 (BBF2 human homolog on chromosome 7), an ER-resident transmembrane protein with the bZIP domain in the cytoplasmic portion and structurally homologous to OASIS, is cleaved at the membrane in response to ER stress. The cleaved fragments of BBF2H7 translocate into the nucleus and can bind directly to cyclic AMP-responsive element sites to activate transcription of target genes. Interestingly, although BBF2H7 protein is not expressed under normal conditions, it is markedly induced at the translational level during ER stress, suggesting that BBF2H7 might contribute to only the late phase of unfolded protein response signaling. In a mouse model of focal brain ischemia, BBF2H7 protein is prominently induced in neurons in the peri-infarction region. Furthermore, in a neuroblastoma cell line, BBF2H7 overexpression suppresses ER stress-induced cell death, while small interfering RNA knockdown of BBF2H7 promotes ER stress-induced cell death. Taken together, our results suggest that BBF2H7 is a novel ER stress transducer and could play important roles in preventing accumulation of unfolded proteins in damaged neurons.

Autophagy is activated for cell survival after endoplasmic reticulum stress.

Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.

A middle meningeal artery which arises from the internal carotid artery in which the first branchial artery participates.

A middle meningeal artery arising from the internal carotid artery was found in the right half of the head of an 85-year-old male cadaver during student dissection practice. It arose from the lateral aspect of the internal carotid artery in the carotid canal, arrived at the foramen lacerum after running forward. It then ran backward under the trigeminal ganglion and took the usual course after passing its posterior margin. On one hand, the maxillary artery did not issue the middle meningeal artery, gave off only a small twig supplying the lateral pterygoid muscle at the corresponding position. It was corroborated by the fact that the foramen spinosum was absent in this example. During usual development, the middle meningeal artery primarily springs from the supraorbital branch of the stapedial artery that arises from the dorsal part of the second branchial artery. Later, by the formation of the external carotid artery connecting with the common trunk of the infraorbital and mandibular branches (maxillomandibular division) of the stapedial artery and by the atrophy of the proximal part of it, the middle meningeal artery is finally supplied by the external carotid artery. But in this example, it is supposed that the middle meningeal artery arose from a more distal position of the internal carotid artery owing to the persistence of the anastomosis between the dorsal part of the first branchial artery and the supraorbital branch and the interruption of the connection between the supraorbital branch and maxillomandibular division of the stapedial artery.

An aberrant axillary artery penetrating the origin of the radial nerve from deep to superficial.

An aberrant axillary artery that penetrated the radial nerve from deep to superficial during its course, was observed. The brachial plexus in the present case was classified as the Adachi's C-type brachial plexus. Further, an accessory radial root existed, which was a nerve bundle branching from the deep aspect of the inferior trunk and communicating with the radial root from the posterior cord to form the radial nerve. The axillary artery went on along the lower border of the brachial plexus and passed between the radial root and the accessory radial root from deep to superficial at its third section. As the axillary artery penetrated the origin of the radial nerve from deep to superficial, it was judged to reach deep under the posterior cord, hence deeper than the brachial plexus.

Superficial brachial artery crossing over the ulnar and median nerves from posterior to anterior: embryological significance.

A rare variation of the superficial brachial artery was found in the right arm of an 82-year-old male cadaver in student dissection practice. In this case, the axillary artery passed normally between the medial and lateral roots of the median nerve and then bifurcated into a large superficial brachial artery and the deep brachial artery (A. brachialis profunda). The superficial brachial artery passed medially to the ulnar nerve and then crossed over to take a lateral course to the median nerve at the midpoint of the upper arm. It finally divided into the radial and ulnar arteries at the cubital fossa. The deep brachial artery terminated as the inferior ulnar collateral artery. These findings indicate that the present variant was a well-developed medial type of the superficial brachial artery that gave off the ulnar and radial arteries. The anatomical and embryological significance of the findings are discussed.

Rare variation of the arm artery: coexistence with the superficial brachial and superficial subscapular arteries in the absence of the normal brachial artery.

A rare variation in the arterial pattern was found in the right arm of an 87-year-old male cadaver in a student dissection practice. In this case, the superficial brachial and superficial subscapular arteries coexisted in the absence of the normal brachial artery (A. brachialis profunda). After branching off a large-sized superficial subscapular artery, the axillary artery did not penetrate the brachial plexus and gave rise to a superficial brachial artery, which arose from the axillary artery at the point between the ansa pectoralis and ansa mediana, and descended ventral to the median nerve branching off the profunda brachii and superior and inferior ulnar collateral arteries. The superficial brachial artery finally divided into the radial and ulnar arteries in the cubital fossa. The superficial subscapular artery passed inferior and dorsal to the medial cord of the brachial plexus, giving off the lateral thoracic artery, and then branched off into the thoracodorsal, circumflex scapular and posterior circumflex humeral arteries. Thus the main nerves of the brachial plexus were sandwiched between the superficial brachial artery and the superficial subscapular artery system. The morphological and clinical significance of this variant are discussed.