Maintenance DNA methylation in pre-meiotic germ cells regulates meiotic prophase by facilitating homologous chromosome pairing.

Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 - two essential proteins for maintenance DNA methylation - are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.

Tsga8 is required for spermatid morphogenesis and male fertility in mice.

During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We have previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X-chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility.

Lack of whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (WFDC2) causes neonatal death from respiratory failure in mice.

Respiratory failure is a life-threatening problem for pre-term and term infants, yet many causes remain unknown. Here, we present evidence that whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (Wfdc2), a protease inhibitor previously unrecognized in respiratory disease, may be a causal factor in infant respiratory failure. Wfdc2 transcripts are detected in the embryonic lung and analysis of a Wfdc2-GFP knock-in mouse line shows that both basal and club cells, and type II alveolar epithelial cells (AECIIs), express Wfdc2 neonatally. Wfdc2-null-mutant mice display progressive atelectasis after birth with a lethal phenotype. Mutant lungs have multiple defects, including impaired cilia and the absence of mature club cells from the tracheo-bronchial airways, and malformed lamellar bodies in AECIIs. RNA sequencing shows significant activation of a pro-inflammatory pathway, but with low-quantity infiltration of mononuclear cells in the lung. These data demonstrate that Wfdc2 function is vitally important for lung aeration at birth and that gene deficiency likely causes failure of the lung mucosal barrier.

[A Case of Cowden Syndrome Associated with Lhermitte-Duclos Disease].

A 24-year-old woman slowly developed mild unsteadiness of gait. Neurological examination revealed mild dysmetria of the left upper and lower limbs. Standing and gait were unsteady, and tandem gait was impossible. Cranial magnetic resonance imaging (MRI) showed an enlarged left cerebellar hemisphere with striated lines, a characteristic finding of Lhermitte-Duclos disease. She also had papules on the forehead, goiter, lactating adenoma, glycogenic acanthosis in the esophagus, café-au-lait spot, and hemangioma and keratosis on the dorsum of foot. The diagnosis of Cowden syndrome was established by finding the mutation in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene. Cowden syndrome is an autosomal dominant disorder characterized by multiple hamartomas in a variety of tissues. Recognition of Lhermitte-Duclos disease as a neurological condition of Cowden syndrome is important, and once the diagnosis of Lhermitte-Duclos disease is made, a close physical investigation is necessary because the hamartomas tend to develop malignancies. (Received March 15, 2017; Accepted July 24, 2017; Published December 1, 2017).

[A case of MELAS associated with histochemical findings of muscles characteristic of MERRF].

We here report a 39-year-old woman of short stature with sensorineural deafness, who suddenly developed status epilepticus. T2-weighed image of brain magnetic resonance imaging (MRI) revealed a high signal lesion in the left temporal area, the distribution of which was not compatible with any particular arterial supply. Lactate and pyruvate were elevated in the serum and cerebrospinal fluid. As the mitochondrial gene analysis revealed the m.3243A>G mutation, diagnosis of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode (MELAS) was made. In the histochemical study of a biopsied muscle, the intramuscular blood vessels reacted strongly with SDH (SSV), but the SSV was negative for cytochrome c oxidase (COX), the findings characteristic of myoclonic epilepsy with ragged-red fibers (MERRF). This is the first case of MELAS in which the muscle histochemistry showed positive SSV unassociated with increased COX.

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells.

BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Plasmodium induced by SU6656, an Src family kinase inhibitor, is accompanied by a contractile ring defect.

We have shown that SU6656, a potent Src family kinase inhibitor, has the ability to induce multinucleation at a high frequency in diverse cells: rat skin fibroblasts, bone marrow adherent cells, 5F9A mesenchymal stem cell-like clones, 2C5 tracheal epithelial cells and MDCK epithelial cells from dog kidney. To gain insight into the mechanism of multinucleation, we observed the process by time-lapse and confocal microscopy. These multinuclei generally seem to exist independently in one cell without any connections with each other. By time-lapse microscopy, multinucleated cells were found to be formed through the mechanism of plasmodium: karyokinesis without cytokinesis. The observation of EGFP-actin transfected cells by time-lapse confocal laser scanning microscopy suggested that plasmodium occurred with deficient contractile ring formation. Although we examined the differentiation of these cells, the multinucleated cells could not be categorized into any type of cell in vivo known to exhibit multinuclei.

Ultrastructure of tracheal epithelial cells migrating in an in vivo environment.

The tracheal epithelium can be induced to move as a cellular sheet by heterotopic transplantation, which offers the opportunity to observe migrating cells as a group in an in vivo environment. We therefor investigated the ultrastructural characteristics of migrating tracheal epithelial cells with special reference to the moving front using this transplantation. The migrating epithelial cells underwent squamous metaplasia and lost their differentiated characteristics such as cilia or secretory granules. Several unique observations were made concerning the mechanism of mobility: one is that epithelial cells in the front were elongated in a direction perpendicular to the course of movement, different from previous reports in vitro. The second is that lamellipodia, which are regarded as the major locomotive machinery in the adult wound epithelium, did not make up the major part of the front; the major portion of the anterior fringe of the moving front was usually smooth and gently curved, and actin cables parallel to the elongated cells were observed by confocal laser microscopy, indicating that the purse-string mechanism of epithelial wound healing takes place. The third finding is that the cells in the front had irregular bleb-like structures on their antero-basal surface, which were formed even in the portion where the cells did not attach to the matrix. Few organelles were recognized in these structures. From their location, one might propose that these bleb-like structures play a role in the recognition of the substrate and thus the movement of the cell sheet.

Oligopotent mesenchymal stem cell-like clone becomes multinucleated following phorbol ester, TPA stimulation.

We established a mesenchymal stem cell clone, 5F9A, from rat bone marrow substrate adherent cells by repeated limiting dilutions. The cells have a fibroblastic shape and form intimate contacts with adjacent cells with interdigitations and junctions similar to adherence and tight junctions in a semi-confluent culture. Analysis of the phenotypes of these cells by RT-PCR and FACS demonstrated that they resembled mesenchymal stem cells, and the cells could differentiate into adiopocytes and osteoblasts under appropriate conditions in vitro showing their oligopotency. Furthermore, the cells were induced to become multinuclear cells by TPA (12-o-tetradecanoylphorbol 13-acetate) stimulation.

TPA-induced multinucleation of a mesenchymal stem cell-like clone is mediated primarily by karyokinesis without cytokinesis, although cell-cell fusion also occurs.

The 5F9A cell, which is a mesenchymal stem cell-like clone established from rat bone marrow substrate adherent cells, can differentiate into adipocytes and osteoblasts in vitro under the appropriate conditions. Multinucleated cells could be also induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in 5F9A cells. This effect was mediated by protein kinase C. Possible mechanisms of multinucleation by TPA were hypothesized to be either karyokinesis without cytokinesis or cell-cell fusion. By observation using time-lapse phase-contrast microscopy, we determined that the multinucleated cells were generated mainly by karyokinesis without cytokinesis. Cell fusion was studied using time-lapse photography, and confocal laser scanning microscopy using two differentially labeled cells. These techniques demonstrated that multinucleated 5F9A cells could be produced by cell fusion, albeit at a low frequency. We conclude that multinucleated 5F9A cells are formed primarily by karyokinesis without cytokinesis, although some cells are also formed by cell-cell fusion.

Identification and characterization of a novel Delphilin variant with an alternative N-terminus.

Delphilin is identified as a Glutamate receptor delta2 (GluRdelta2) subunit interacting protein, consisting of a PDZ domain and formin homology (FH) domains 1 and 2, in addition to a C-terminal coiled-coil structure. Delphilin has been shown to be selectively expressed in cerebellar Purkinje cells where it co-localizes with the GluRdelta2 subunit at the Purkinje cell-parallel fiber synapses. Although Delphilin specifically interacts with the GluRdelta2 C-terminus via its PDZ domain, the physiological role of the interaction is not yet understood. Here, we report that the Delphilin protein exhibits diversity at its N-terminus by variable usage of the first several exons. Interestingly, the two Delphilin mRNAs which correspond to the first one initially identified (now designated as Delphilin alpha) and the second that contains a newly identified first exon (designated as Delphilin beta), show different chronological expression profiles. Delphilin beta mRNA was not decreased throughout the cerebellar development in vivo and in vitro, while in vivo Delphilin alpha mRNA gradually decreases following the first postnatal week. Delphilins alpha and beta also revealed different subcellular distribution with some overlap. Specifically, the cerebellar synaptosomal membrane fraction contained the Delphilin beta protein. Both Delphilin alpha and beta localized at the dendritic spines with GluRdelta2; however, dendritic shafts in cultured Purkinje cells also included Delphilin beta. In MDCK cells upon becoming confluent, Delphilin alpha moved to the cell-cell junction area, whereas Delphilin beta maintained a diffuse distribution pattern throughout the cytoplasm. Taken as a whole, these two different Delphilins seemed to play functionally different roles in developing and matured cerebellar Purkinje cells.

Dynamic changes in intranuclear and subcellular localizations of mouse Prrp/DAZAP1 during spermatogenesis: the necessity of the C-terminal proline-rich region for nuclear import and localization.

Mouse Prrp (mPrrp)/DAZAP1 is a mouse ortholog of Xenopus Prrp, which is involved in vegetal pole localization of Vg1 mRNA in oocytes and is highly expressed in the testis. The mouse protein has been reported to be a shuttling protein which localizes in the nucleus of pre-meiotic spermatogenic cells and round spermatids, and shifts its location into the cytoplasm in elongating spermatids, suggesting that mPrrp may be involved in mRNA transport as well as that of the Xenopus ortholog. We reexamined immunohistochemical analyses of mPrrp/DAZAP1 during spermatogenesis utilizing a newly established monoclonal antibody and reconfirmed it to be a shuttling protein. We also carried out new observations that included remarkable intranuclear movement during spermatogenesis. In addition, we found that a long amino acid stretch which spanned over the C-terminal half of the protein was required for the nuclear import. These observations demonstrated dynamic changes in subnuclear and subcellular localization which might reflect specific functions during spermatogenesis.

Increment of murine spermatogonial cell number by gonadotropin-releasing hormone analogue is independent of stem cell factor c-kit signal.

Recent studies have demonstrated that GnRH-analogues can stimulate regeneration of spermatogenesis of rats when administered after testicular damages. Although the mechanism of this phenomenon has not been elucidated yet, stem cell factor (SCF) produced by Sertoli cells was proposed to mediate the effects of GnRH-analogues on spermatogonial proliferation and/or survival. In the present study, we quantitatively evaluated the proliferation of spermatogonia and addressed whether SCF mediates the effect of GnRH-analogue on spermatogonial proliferation, using a novel approach combining spermatogonial transplantation and laser confocal microscopic observation. In the first experiment, using wild-type mice as recipients for spermatogonial transplantation, the number of donor spermatogonia per 100 Sertoli cells in each spermatogenic colony was significantly higher in the experimental group of mice treated with leuprorelin, a GnRH-agonist, than that of the control group at 4 and 5 wk after transplantation. In the second experiment, Steel/Steeldickie (Sl/Sld) mutant mice, which lack expression of membrane bound form SCF, were used as recipients. As seen in the first experiment, the number of undifferentiated spermatogonia was significantly higher in leuprorelin-treated than in the control group. Since undifferentiated spermatogonia do not express the receptor of SCF, the present study clearly demonstrates that neither membrane-bound nor secreted forms of SCF are involved in the mechanism of GnRH-analogue's effect on spermatogonial proliferation and/or survival.

Bilateral persistent sciatic arteries in a Japanese man.

During student dissecting practice, a rare developmental anomaly showing persistent sciatic artery (PSA) was found in the bilateral lower limbs of a 74-year-old Japanese male cadaver. The PSA was a continuation of the internal iliac artery on both sides, did not anastomose with the perforating arteries and ended by anastomosing with the popliteal artery on both sides. The course and distribution of the PSA were relatively consistent with previous reports. In the present case, however, the PSA and inferior gluteal arteries existed simultaneously on both sides, despite the general assumption that the inferior gluteal artery is a remnant of sciatic artery regression.