Expression of Protein Markers in Spermatogenic and Supporting Sertoli Cells Affected by High Abdominal Temperature in Cryptorchidism Model Mice.

Cryptorchidism is a congenital abnormality resulting in increased rates of infertility and testicular cancer. We used cryptorchidism model mice that presented with the translocation of the left testis from the scrotum to the abdominal cavity. Mice underwent the surgical procedure of the left testis at day 0 and were sacrificed at days 3, 5, 7, 14, 21, and 28 post-operatively. The weight of the left cryptorchid testis decreased significantly at days 21 and 28. The morphological changes were observed after 5 days and showed detached spermatogenic cells and abnormal formation of acrosome at day 5, multinucleated giant cells at day 7, and atrophy of seminiferous tubules at days 21 and 28. The high abdominal temperature disrupted the normal expression of cell adhesion molecule-1, Nectin-2, and Nectin-3 which are essential for spermatogenesis. In addition, the pattern and alignment of acetylated tubulin in cryptorchid testes were also changed at days 5, 7, 14, 21, and 28. Ultrastructure of cryptorchid testes revealed giant cells that had been formed by spermatogonia, spermatocytes, and round and elongating spermatids. The study's findings reveal that cryptorchidism's duration is linked to abnormal changes in the testis, impacting protein marker expression in spermatogenic and Sertoli cells. These changes stem from the induction of high abdominal temperature.

Reconstitution of functional tight junctions with individual claudin subtypes in epithelial cells.

The claudin family of membrane proteins is responsible for the backbone structure and function of tight junctions (TJs), which regulate the paracellular permeability of epithelia. It is thought that each claudin subtype has its own unique function and the combination of expressed subtypes determines the permeability property of each epithelium. However, many issues remain unsolved in regard to claudin functions, including the detailed functional differences between claudin subtypes and the effect of the combinations of specific claudin subtypes on the structure and function of TJs. To address these issues, it would be useful to have a way of reconstituting TJs containing only the claudin subtype(s) of interest in epithelial cells. In this study, we attempted to reconstitute TJs of individual claudin subtypes in TJ-deficient MDCK cells, designated as claudin quinKO cells, which were previously established from MDCK II cells by deleting the genes of claudin-1, -2, -3, -4, and -7. Exogenous expression of each of claudin-1, -2, -3, -4, and -7 in claudin quinKO cells resulted in the reconstitution of functional TJs. These TJs did not contain claudin-12 and -16, which are endogenously expressed in claudin quinKO cells. Furthermore, overexpression of neither claudin-12 nor claudin-16 resulted in the reconstitution of TJs, demonstrating the existence of claudin subtypes lacking TJ-forming activity in epithelial cells. Exogenous expression of the channel-forming claudin-2, -10a, -10b, and -15 reconstituted TJs with reported paracellular channel properties, demonstrating that these claudin subtypes form paracellular channels by themselves without interaction with other subtypes. Thus, the reconstitution of TJs in claudin quinKO cells is advantageous for further investigation of claudin functions.Key words: tight junction, claudin, paracellular permeability, epithelial barrier.

Quantitative evaluation of spermatogenesis by fluorescent histochemistry.

Identifying the types of spermatogenic cells that compose seminiferous tubules, as well as qualitative confirmation of the presence or absence of disorders, has been regarded as crucial in spermatogenesis. Sperm count and fertilizing capacity, both of which depend on the quality as well as quantity of spermatogenesis, are factors critical to fertilization. However, the quantitative assessment of spermatogenesis is not commonly practiced. Spermatogenesis has species-specific stages; when the specific stage in the seminiferous tubules is precisely determined, the types of spermatogenic cells in each stage can be spontaneously identified. Thereafter, a unique marker is used to classify the cells observed in each stage. Quantitative assessment of spermatogenesis has the potential to detect inapparent spermatogenesis disorders or numerically indicate the degree of the disorder. To this end, a histochemical approach using unique markers is indispensable for the quantitative assessment of spermatogenesis. Future developments in techniques to measure cell populations using computer software will further facilitate the establishment of quantitative assessment of spermatogenesis as a standard analysis method that can contribute significantly to advance our understanding of spermatogenesis.

Angulin-1 seals tricellular contacts independently of tricellulin and claudins.

Tricellular tight junctions (tTJs) are specialized tight junctions (TJs) that seal the intercellular space at tricellular contacts (TCs), where the vertices of three epithelial cells meet. Tricellulin and angulin family membrane proteins are known constituents of tTJs, but the molecular mechanism of tTJ formation remains elusive. Here, we investigated the roles of angulin-1 and tricellulin in tTJ formation in MDCK II cells by genome editing. Angulin-1-deficient cells lost the plasma membrane contact at TCs with impaired epithelial barrier function. The C terminus of angulin-1 bound to the TJ scaffold protein ZO-1, and disruption of their interaction influenced the localization of claudins at TCs, but not the tricellular sealing. Strikingly, the plasma membrane contact at TCs was formed in tricellulin- or claudin-deficient cells. These findings demonstrate that angulin-1 is responsible for the plasma membrane seal at TCs independently of tricellulin and claudins.

Angulin-2/ILDR1, a tricellular tight junction protein, does not affect water transport in the mouse large intestine.

Angulin-2/ILDR1 is a member of the angulin protein family, which is exclusively expressed at tricellular tight junctions in epithelia. Tricellular tight junctions are found where three cells meet and where three bicellular tight junction strands converge. Tricellular tight junctions are thought to be important for paracellular permeability of ions and water in epithelial tissues. It was recently reported that angulin-2/ILDR1 knockout mice have water transport abnormalities in the kidney. Since angulin-2/ILDR1 is the main tricellular tight junction protein in the large intestine, the goal of this research was to examine the effect of angulin-2/ILDR1 knockout on large intestinal paracellular water transport. We found that Ildr1 knockout mice showed no detectable phenotype other than deafness. In addition, paracellular transport as assessed by Ussing chamber was unchanged in Ildr1 knockout mice. However, we found that in the colon and the kidney of Ildr1 knockout mice, another tricellular tight junction protein, angulin-1/LSR, changes its expression pattern. We propose that with this replacement in tissue localization, angulin-1/LSR compensates for the loss of angulin-2/ILDR1 and maintains the barrier and function of the epithelia in the large intestine as well as the kidney.

The extracellular domain of angulin-1 and palmitoylation of its cytoplasmic region are required for angulin-1 assembly at tricellular contacts.

Tricellular tight junctions (tTJs) create paracellular barriers at tricellular contacts (TCs), where the vertices of three polygonal epithelial cells meet. tTJs are marked by the enrichment of two types of membrane proteins, tricellulin and angulin family proteins. However, how TC geometry is recognized for tTJ formation remains unknown. In the present study, we examined the molecular mechanism for the assembly of angulin-1 at the TCs. We found that clusters of cysteine residues in the juxtamembrane region within the cytoplasmic domain of angulin-1 are highly palmitoylated. Mutagenesis analyses of the cysteine residues in this region revealed that palmitoylation is essential for localization of angulin-1 at TCs. Consistently, suppression of Asp-His-His-Cys motif-containing palmitoyltransferases expressed in EpH4 cells significantly impaired the TC localization of angulin-1. Cholesterol depletion from the plasma membrane of cultured epithelial cells hampered the localization of angulin-1 at TCs, suggesting the existence of a lipid membrane microdomain at TCs that attracts highly palmitoylated angulin-1. Furthermore, the extracellular domain of angulin-1 was also required for its TC localization, irrespective of the intracellular palmitoylation. Taken together, our findings suggest that both angulin-1's extracellular domain and palmitoylation of its cytoplasmic region are required for its assembly at TCs.

Claudins and JAM-A coordinately regulate tight junction formation and epithelial polarity.

Tight junctions (TJs) establish the epithelial barrier and are thought to form a membrane fence to regulate epithelial polarity, although the roles of TJs in epithelial polarity remain controversial. Claudins constitute TJ strands in conjunction with the cytoplasmic scaffolds ZO-1 and ZO-2 and play pivotal roles in epithelial barrier formation. However, how claudins and other TJ membrane proteins cooperate to organize TJs remains unclear. Here, we systematically knocked out TJ components by genome editing and show that while ZO-1/ZO-2-deficient cells lacked TJ structures and epithelial barriers, claudin-deficient cells lacked TJ strands and an electrolyte permeability barrier but formed membrane appositions and a macromolecule permeability barrier. Moreover, epithelial polarity was disorganized in ZO-1/ZO-2-deficient cells, but not in claudin-deficient cells. Simultaneous deletion of claudins and a TJ membrane protein JAM-A resulted in a loss of membrane appositions and a macromolecule permeability barrier and in sporadic epithelial polarity defects. These results demonstrate that claudins and JAM-A coordinately regulate TJ formation and epithelial polarity.

L-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2.

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic ß-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.

Rab2A is a pivotal switch protein that promotes either secretion or ER-associated degradation of (pro)insulin in insulin-secreting cells.

Rab2A, a small GTPase localizing to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), regulates COPI-dependent vesicular transport from the ERGIC. Rab2A knockdown inhibited glucose-stimulated insulin secretion and concomitantly enlarged the ERGIC in insulin-secreting cells. Large aggregates of polyubiquitinated proinsulin accumulated in the cytoplasmic vicinity of a unique large spheroidal ERGIC, designated the LUb-ERGIC. Well-known components of ER-associated degradation (ERAD) also accumulated at the LUb-ERGIC, creating a suitable site for ERAD-mediated protein quality control. Moreover, chronically high glucose levels, which induced the enlargement of the LUb-ERGIC and ubiquitinated protein aggregates, impaired Rab2A activity by promoting dissociation from its effector, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in response to poly (ADP-ribosyl)ation of GAPDH. The inactivation of Rab2A relieved glucose-induced ER stress and inhibited ER stress-induced apoptosis. Collectively, these results suggest that Rab2A is a pivotal switch that controls whether insulin should be secreted or degraded at the LUb-ERGIC and Rab2A inactivation ensures alleviation of ER stress and cell survival under chronic glucotoxicity.

JNK1/2-dependent phosphorylation of angulin-1/LSR is required for the exclusive localization of angulin-1/LSR and tricellulin at tricellular contacts in EpH4 epithelial sheet.

Tricellular tight junctions (tTJs) are specialized structural variants of tight junctions within tricellular contacts of an epithelial sheet and comprise several transmembrane proteins including lipolysis-stimulated lipoprotein receptor (angulin-1/LSR) and tricellulin. To elucidate the mechanism of its formation, we carried out stepwise screening of kinase inhibitors followed by RNAi screening to identify kinases that regulate intracellular localization of angulin-1/LSR to the tTJs using a fluorescence image-based screen. We found that the activity of JNK1 and JNK2, but not JNK3, was required for the exclusive localization of angulin-1/LSR at the tTJs. Based on a bioinformatics approach, we estimated the potential phosphorylation site of angulin-1/LSR by JNK1 to be serine 288 and experimentally confirmed that JNK1 directly phosphorylates angulin-1/LSR at this site. We found that JNK2 was also involved in the phosphorylation of angulin-1/LSR. Furthermore, GFP-tagged angulin-1/LSR(S288A), in which serine 288 was substituted by alanine, was observed to be dispersed to bicellular junctions, indicating that phosphorylation of Ser288 is crucial for the exclusive localization of angulin-1/LSR and tricellulin at tTJs. Our fluorescence image-based screening for kinases inhibitor or siRNAs combined with the phosphorylation site prediction could become a versatile and useful tool to elucidate the mechanisms underlying the maintenance of tTJs regulated by kinase networks.

PKCδ and ε regulate the morphological integrity of the ER-Golgi intermediate compartment (ERGIC) but not the anterograde and retrograde transports via the Golgi apparatus.

The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.