Synthesis of ß-tubulin occurs within chromatoid body of round spermatids.

mRNAs for proteins required in elongated spermatids are considered to be transcribed at an early stage and stored in cytoplasm, presumably in chromatoid body (CB), one type of nuage component (a unique structure that appears and disappears during spermatogenesis), because transcription of genes does not occur at late stages. In elongated spermatids, a large amount of tubulin molecules is required to form microtubules of manchette and flagellum. To investigate the possible role of CB in translation of tubulin mRNA, we performed immunofluorescence and immunoelectron microscopic localization studies of α- and ß-tubulin in rat spermatogenic cells. ß-tubulin was detected in CB, but α-tubulin was not. Other nuage components present in pachytene spermatocytes (ISPG, IMC, SB) were negative for both α- and ß-tubulin. Our findings suggest that: (i) ß-tubulin in round spermatids is translated within the CB, whereas α-tubulin is not; (ii) αß-heterodimers are formed outside CB and incorporated into microtubules of manchette and flagellum.

Preparation of Colloidal Gold Particles and Conjugation to Protein A/G/L, IgG, F(ab')2, and Streptavidin.

Colloidal gold probes, including protein A/G/L, IgG, F(ab')2, and streptavidin labeled with gold particles, are useful tools to localize antigens in cells and tissues by immunoelectron microscopy (IEM). This chapter describes different methods for the preparation of colloidal gold and conjugation of colloidal gold to protein A/G/L, IgG, and streptavidin.

Argonaute2 Protein in Rat Spermatogenic Cells Is Localized to Nuage Structures and LAMP2-Positive Vesicles Surrounding Chromatoid Bodies.

Localization of Argonaute2 (AGO2) protein--an essential component for the processing of small interfering RNA (siRNA)-directed RNA interference (RNAi) in RNA-induced silencing complex (RISC) in nuage of rat spermatogenic cells--was evaluated by immunofluorescence microscopy (IFM) and immunoelectron microscopy (IEM). AGO2 was shown, for the first time, to be localized to four previously classified types of nuage: irregularly shaped perinuclear granules (ISPGs), intermitochondrial cement (IMC), satellite bodies (SBs), and chromatoid bodies (CBs). Dual IEM staining for AGO2/Maelstrom (MAEL) protein or AGO2/MIWI protein demonstrated that AGO2 is colocalized with MAEL or MIWI proteins in these types of nuage. Dual IFM and IEM staining of AGO2/lysosomal-associated membrane protein 2 (LAMP2) showed that CB in round spermatids are in contact with and surrounded by LAMP2-positive vesicles, whereas nuage in pachytene spermatocytes are not. Taken together, our findings indicate that: (i) AGO2 in pachytene spermatocytes functions in ISPGs, IMC, and SBs; (ii) AGO2 in round spermatids functions in CBs, and that CBs are associated with lysosomal compartments.

Disruption of mitochondrial fission in the liver protects mice from diet-induced obesity and metabolic deterioration.

AIM/HYPOTHESIS: Mitochondria and the endoplasmic reticulum (ER) physically interact by close structural juxtaposition, via the mitochondria-associated ER membrane. Inter-organelle communication between the ER and mitochondria has been shown to regulate energy metabolism and to be central to the modulation of various key processes such as ER stress. We aimed to clarify the role of mitochondrial fission in this communication. METHODS: We generated mice lacking the mitochondrial fission protein dynamin-related protein 1 (DRP1) in the liver (Drp1LiKO mice). RESULTS: Drp1LiKO mice showed decreased fat mass and were protected from high-fat diet (HFD)-induced obesity. Analysis of liver gene expression profiles demonstrated marked elevation of ER stress markers. In addition, we observed increased expression of the fibroblast growth factor 21 (FGF21) gene through induction of activating transcription factor 4, master regulator of the integrated stress response. CONCLUSIONS/INTERPRETATION: Disruption of mitochondrial fission in the liver provoked ER stress, while inducing the expression of FGF21 to increase energy expenditure and protect against HFD-induced obesity.

Localization of aristolochic acid in mouse kidney tissues by immunohistochemistry using an anti-AA-I and AA-II monoclonal antibody.

Aristolochic acids (AAs) are found in herbal medicines of Aristolochiaceae plants, including Aristolochia and Asarum species. AAs are associated with a rapidly progressive interstitial nephritis, which is called aristolochic acid nephropathy (AAN). However, the in-situ localization of AAs in the target organ, the kidney, has not been investigated yet. In the present study, the accumulation of aristolochic acid I (AA-I) in mouse kidney was revealed by immunoperoxidase light microscopy as well as colloidal gold immunoelectron microscopy (IEM) based on an anti-AA-I and AA-II monoclonal antibody (mAb). Male BALB/c mice were treated with 1.25 or 2.50 mg kg(-1) of AA-I per day for 5 days. Paraffin sections and ultra-thin sections of kidney tissue were respectively prepared. Under light microscopy, the apical surface of proximal tubules was strongly stained for AA-I, whereas no obvious immunostaining was found in the distal tubules and glomerulus, which remained relatively intact. Under electron microscopy, epithelial cells of the proximal tubules, distal tubules and collecting tubules were broken to various degrees. Gold labeling in the proximal and distal tubules was stronger than that in the collecting tubules. In renal tubules, immunogold signals of AA-I tended to accumulate in the mitochondria and peroxisomes, though the signals could be observed all over the cell. Gold signals were also found in the erythrocytes of glomeruli. The MAb against AA-I and AA-II provides a clue for the identification of proteins or factors which might interact with AA-I and thus induce targeted damage of kidney.

Mitochondrial fission factor Drp1 maintains oocyte quality via dynamic rearrangement of multiple organelles.

Mitochondria are dynamic organelles that change their morphology by active fusion and fission in response to cellular signaling and differentiation. The in vivo role of mitochondrial fission in mammals has been examined by using tissue-specific knockout (KO) mice of the mitochondria fission-regulating GTPase Drp1, as well as analyzing a human patient harboring a point mutation in Drp1, showing that Drp1 is essential for embryonic and neonatal development and neuronal function. During oocyte maturation and aging, structures of various membrane organelles including mitochondria and the endoplasmic reticulum (ER) are changed dynamically, and their organelle aggregation is related to germ cell formation and epigenetic regulation. However, the underlying molecular mechanisms of organelle dynamics during the development and aging of oocytes have not been well understood. Here, we analyzed oocyte-specific mitochondrial fission factor Drp1-deficient mice and found that mitochondrial fission is essential for follicular maturation and ovulation in an age-dependent manner. Mitochondria were highly aggregated with other organelles, such as the ER and secretory vesicles, in KO oocyte, which resulted in impaired Ca(2+) signaling, intercellular communication via secretion, and meiotic resumption. We further found that oocytes from aged mice displayed reduced Drp1-dependent mitochondrial fission and defective organelle morphogenesis, similar to Drp1 KO oocytes. On the basis of these findings, it appears that mitochondrial fission maintains the competency of oocytes via multiorganelle rearrangement.

Inulavosin and its benzo-derivatives, melanogenesis inhibitors, target the copper loading mechanism to the active site of tyrosinase.

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure-activity relationship of inulavosin and its benzo-derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper-binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo-derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo-tyrosinase that has a conformational defect.

DDX6 localizes to nuage structures and the annulus of mammalian spermatogenic cells.

The localization of DEAD (Asp-Glu-Ala-Asp) box helicase 6 (DDX6) in spermatogenic cells from the mouse, rat, and guinea pig was studied by immunofluorescence (IF) and immunoelectron microscopy (IEM). Spermatogenic cells from these species yielded similar DDX6 localization pattern. IF microscopy results showed that DDX6 localizes to both the nucleus and cytoplasm. In the cytoplasm of spermatogenic cells, diffuse cytosolic and discrete granular staining was observed, with the staining pattern changing during cell differentiation. IEM revealed that DDX6 localized to the five different types of nuage structures and non-nuage structures, including small granule aggregate and late spermatid annuli. Nuclear labeling was strongest in leptotene and zygotene spermatocytes and moderately strong in the nuclear pocket of late spermatids. DDX6 also localized to the surface of outer dense fibers, which comprise of flagella. The results show that DDX6 is present in nuage and non-nuage structures as well as nuclei, suggesting that DDX6 has diverse functions in spermatogenic cells.

Expression of MAEL in nuage and non-nuage compartments of rat spermatogenic cells and colocalization with DDX4, DDX25 and MIWI.

The functions of MAELSTROM protein (MAEL) in spermatogenesis are gradually being identified but the precise distribution of MAEL in spermatogenic cells during spermatogenesis has not yet been mapped. We studied the expression of MAEL in rat testis by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence staining showed that MAEL was localized in intermitochondrial cement, irregularly-shaped perinuclear granules and satellite bodies of pachytene spermatocytes, and in chromatoid bodies of spermatids. The SBs appeared exclusively in pachytene spermatocytes at stages IX-X and were stained strongly for MAEL. In step 12-19 spermatids, many granules stained for MAEL but not DDX4. These granules were confirmed to be non-nuage structures, including mitochondria-associated granules, reticulated body, granulated body by IEM. In the neck region of late spermatids and sperm, MAEL-positive small granules were found. MAEL is colocalized with MIWI in nuage and non-nuage. The results suggest that MAEL seems to function in nuage and non-nuage structures and interacts with MIWI.

Gold nanoparticles for immunogold localization of antigens in plant tissues.

Here we describe postembedding immunoelectron microscopic technique applied to ultrathin sections of plant material. The method relies on the use of gold nanoparticles. The methods include tissue fixation, dehydration, embedding of plant specimens, and staining of semithin and ultrathin sections. The described method is suitable for localization of antigens including proteins (peroxisomal enzymes) and a low molecular substance such as ginsenoide-Rb1.

Nuage proteins: their localization in subcellular structures of spermatogenic cells as revealed by immunoelectron microscopy.

Chromatoid body (CB) was identified as granules stained by basic dye 130 years ago and called by various names. Electron microscopy revealed that the CB belonged to nuage (cloud in French) specific for germ cells. We described the localization of several proteins, including RNA helicases, in the nuage compartments classified into six types and in several spermatogenic cell-specific structures. All the proteins examined were detected in the nuage, including the CB with different staining intensities. Several proteins were localized to non-nuage structures, suggesting that these nuage proteins structures are related to nuage function.

Expression of DDX25 in nuage components of mammalian spermatogenic cells: immunofluorescence and immunoelectron microscopic study.

The localization of DDX25/GRTH and gonadotropin-stimulated RNA helicase was studied in the spermatogenic cells of rat, mouse, and guinea pig by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence studies identified four kinds of granular staining: (1) fine particles observed in meiotic cells; (2) small granules associated with a mitochondrial marker, appearing in pachytene spermatocytes after stage V; (3) short strands lacking the mitochondrial marker in late spermatocytes; and, (4) large irregularly shaped granules in round spermatids. IEM identified DDX25 signals in nine compartments: (1) fine dense particles in the meiotic cells; (2) intermitochondrial cement; (3) loose aggregates of 70-90 nm particles; (4) chromatoid bodies; (5) late chromatoid bodies; (6) satellite bodies; (7) granulated bodies; (8) mitochondria-associated granules; and, (9) reticulated bodies. Compartments (1) to (6) were previously classified into nuage while (7) to (9) were classified as nuage components by the present study. The results suggest that DDX25 functions in these nine compartments.

Proteome of ubiquitin/MVB pathway: possible involvement of iron-induced ubiquitylation of transferrin receptor in lysosomal degradation.

Ubiquitylation of membrane proteins triggers their endocytosis at the plasma membrane and subsequent lysosomal degradation through multivesicular bodies (MVBs). A dominant-negative mutant SKD1/Vps4B caused an accumulation of ubiquitylated membrane proteins in MVBs. We have identified 22 membrane proteins whose trafficking is potentially regulated by ubiquitylation. Nine of them, including transferrin receptor (TfR), are indeed ubiquitylated and/or accumulated in MVBs in the cells expressing mutant Vps4. While the recycling route and iron-regulated expression of TfR are well characterized, the mechanism by which the degradation of TfR is regulated is largely unknown. We show that an excess of iron enhances both TfR's ubiquitylation and degradation in lysosomes. Probably, the up-regulated expression of ferritin, an endogenous iron-chelating molecule, attenuated the iron-induced degradation of TfR. Exogenously introduced lysine-less TfR, compared to the wild-type one, showed resistance to the iron-induced ubiquitylation and degradation, when endogenous TfR, which most certainly heterodimerizes with exogenous ones, was depleted with siRNA. These data suggest that the iron-induced ubiquitylation and degradation of TfR along with MVB pathway physiologically plays an important role in iron homeostasis.

Immunofluorescence and immunoelectron microscopic localization of medicinal substance, Rb1, in several plant parts of Panax ginseng.

It is important to know the localization of medicinal substance, Rb1, of Ginseng, Panax ginseng, in this plant in order to achieve efficient extraction of Rb1 or to culture producing cells. In this report, we describe the localization of Rb1 in various parts of the plant as determined by immunofluorescence (IF) and immunoelectron microscopies (IEM). Using IF, we show that Rb1 is localized to chloroplasts, peroxisomes and cytoplasm but not to vacuoles of leaf parenchymal cells. In the leaf stem, Rb1 is localized to the vascular bundles as well as vacuoles. In the root, vacuoles of parenchymal cells are stained at various intensities. Using IEM, gold particles showing Rb1 antigenic sites are present in the compartments stained by IF technique. In addition, Rb1 is localized in the sieve elements of the phloem and degrading primary cell wall of xylem, and in the root parenchymal cells Rb1 is associated with electron dense polymorphic materials but not in starch granules. Translocation and storage of Rb1 and effective utilization of leaves are discussed.

Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells.

The cytoplasmic dynamin-related guanosine triphosphatase Drp1 is recruited to mitochondria and mediates mitochondrial fission. Although the mitochondrial outer membrane (MOM) protein Fis1 is thought to be a Drp1 receptor, this has not been confirmed. To analyze the mechanism of Drp1 recruitment, we manipulated the expression of mitochondrial fission and fusion proteins and demonstrated that (a) mitochondrial fission factor (Mff) knockdown released the Drp1 foci from the MOM accompanied by network extension, whereas Mff overexpression stimulated mitochondrial recruitment of Drp1 accompanied by mitochondrial fission; (b) Mff-dependent mitochondrial fission proceeded independent of Fis1; (c) a Mff mutant with the plasma membrane-targeted CAAX motif directed Drp1 to the target membrane; (d) Mff and Drp1 physically interacted in vitro and in vivo; (e) exogenous stimuli-induced mitochondrial fission and apoptosis were compromised by knockdown of Drp1 and Mff but not Fis1; and (f) conditional knockout of Fis1 in colon carcinoma cells revealed that it is dispensable for mitochondrial fission. Thus, Mff functions as an essential factor in mitochondrial recruitment of Drp1.

Preparation of colloidal gold particles and conjugation to protein A, IgG, F(ab')(2), and streptavidin.

Colloidal gold probes, including protein A-, IgG-F(ab')(2)-, and streptavidin-labeled gold particles, are useful tools for localization of antigens in cells and tissues by immunoelectron microscopy (IEM). This chapter describes different methods for the preparation of colloidal gold and conjugation of colloidal gold to protein A, IgG, and streptavidin.

Localization of mouse vasa homolog protein in chromatoid body and related nuage structures of mammalian spermatogenic cells during spermatogenesis.

The localization of vasa homolog protein in the spermatogenic cells of mice, rats, and guinea pigs was studied by immunofluorescence and electron microscopies with the antibody against mouse vasa homolog (MVH) protein. By immunofluorescence microscopy, four types of granular staining patterns were identified: (1) fine particles observed in diplotene and meiotic cells, (2) small granules associated with a mitochondrial marker and appearing in pachytene spermatocytes after stage V, (3) strands lacking the mitochondrial marker in late spermatocytes, and (4) large irregularly shaped granules in round spermatids. Immunoelectron microscopy defined the ultrastructural profiles of these MVH protein-positive granules: the first type consisted of small dense particles, the second had intermitochondrial cement (IMC), the third type, consisting of strands, had loose aggregates of either material dissociated from IMC or 70-90-nm particles, and the fourth had typical chromatoid bodies (CBs). The results suggest that MVH proteins function in these components of nuage. MVH protein-positive structures other than CBs disappeared during meiosis and CB appeared first in early spermatids. The results suggest that the formation of nuage is discontinued between spermatocytes and spermatids. The formation of nuage in spermatocytes and of CB in spermatids is discussed.

[Hormone excretion and peroxisomes of human immortalized extravillous trophoblast cells (TCL-2) derived from first-trimester placenta].

We studied the hormone excretion of human immortalized extravillous trophoblast cells (TCL-2, first-trimester cells) and determined whether peroxisomes are present in TCL-2. The results of TCL-2 were compared with those of TCL-1 (third-trimester cells). Morphologically, TCL-2 cells were fibroblast-like, and the growth rate of TCL-2 was slower than that of TCL-1 during 3 days culture. Progesterone was detected in the medium of TCL-2, and its concentration was approximately one-tenth of that in TCL-1. The activity of the peroxisomal marker enzyme catalase was detected in the TCL-2 homogenate, and it was about one-third the level of that in TCL-1. Fatty acyl-CoA oxidase activity was detected in TCL-2, and it was about one-seventh the level of that in TCL-1. On the other hand, human chorionic gonadotropin (hCG) was detected in the medium of TCL-2, and its concentration after 3 days of culture was about 2-fold that in TCL-1. Using the diaminobenzidine (DAB) method, peroxisomes were found in TCL-2, but only a very small amount of catalase was detected. These results indicate that human immortalized extravillous trophoblast cells (TCL-2) synthesize, secrete hCG and progesterone, and may contain peroxisomes. Because extravillous trophoblast cells are difficult to obtain from the first-trimester placenta, TCL-2 cells are useful for the study of the physiologic functions (including peroxisomal function) of first-trimester cells.

OAZ-t/OAZ3 is essential for rigid connection of sperm tails to heads in mouse.

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. Although considerable amounts of polyamines are synthesized and stored in the testes, their roles remain unknown. Ornithine decarboxylase antizymes (OAZs) control the intracellular concentration of polyamines in a feedback manner. OAZ1 and OAZ2 are expressed ubiquitously, whereas OAZ-t/OAZ3 is expressed specifically in germline cells during spermiogenesis. OAZ-t reportedly binds to ornithine decarboxylase (ODC) and inactivates ODC activity. In a prior study, polyamines were capable of inducing a frameshift at the frameshift sequence of OAZ-t mRNA, resulting in the translation of OAZ-t. To investigate the physiological role of OAZ-t, we generated OAZ-t-disrupted mutant mice. Homozygous OAZ-t mutant males were infertile, although the polyamine concentrations of epididymides and testes were normal in these mice, and females were fertile. Sperm were successfully recovered from the epididymides of the mutant mice, but the heads and tails of the sperm cells were easily separated in culture medium during incubation. Results indicated that OAZ-t is essential for the formation of a rigid junction between the head and tail during spermatogenesis. The detached tails and heads were alive, and most of the headless tails showed straight forward movement. Although the tailless sperm failed to acrosome-react, the heads were capable of fertilizing eggs via intracytoplasmic sperm injection. OAZ-t likely plays a key role in haploid germ cell differentiation via the local concentration of polyamines.

Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice.

Mitochondrial morphology is dynamically controlled by a balance between fusion and fission. The physiological importance of mitochondrial fission in vertebrates is less clearly defined than that of mitochondrial fusion. Here we show that mice lacking the mitochondrial fission GTPase Drp1 have developmental abnormalities, particularly in the forebrain, and die after embryonic day 12.5. Neural cell-specific (NS) Drp1(-/-) mice die shortly after birth as a result of brain hypoplasia with apoptosis. Primary culture of NS-Drp1(-/-) mouse forebrain showed a decreased number of neurites and defective synapse formation, thought to be due to aggregated mitochondria that failed to distribute properly within the cell processes. These defects were reflected by abnormal forebrain development and highlight the importance of Drp1-dependent mitochondrial fission within highly polarized cells such as neurons. Moreover, Drp1(-/-) murine embryonic fibroblasts and embryonic stem cells revealed that Drp1 is required for a normal rate of cytochrome c release and caspase activation during apoptosis, although mitochondrial outer membrane permeabilization, as examined by the release of Smac/Diablo and Tim8a, may occur independently of Drp1 activity.