O-GlcNAcylation orchestrates porcine oocyte maturation through maintaining mitochondrial dynamics and function.

O-linked ß-N-acetylglucosamine (O-GlcNAc) modification exists widely in cells, playing a crucial role in the regulation of important biological processes such as transcription, translation, metabolism, and the cell cycle. O-GlcNAc modification is an inducible reversible dynamic protein post-translational modification, which regulates complex cellular activities through transient glycosylation and deglycosylation. O-GlcNAc glycosylation is specifically regulated by O-GlcNAc glycosyltransferase (O-GlcNAc transferase, OGT) and O-GlcNAc glycoside hydrolase (O-GlcNAcase). However, the mechanisms underlying the effects of O-GlcNAc modification on the female reproductive system, especially oocyte quality, remain unclear. Here, we found that after OGT was inhibited, porcine oocytes failed to extrude the first polar body and exhibited abnormal actin and microtubule assembly. Meanwhile, the mitochondrial dynamics and function were also disrupted after inhibition of OGT function, resulting in the occurrence of oxidative stress and autophagy. Collectively, these results inform our understanding of the importance of the glycosylation process for oocyte maturation, especially for the maturation quality of porcine oocytes, and the alteration of O-GlcNAc in oocytes to regulate cellular events deserves further investigation.

Zinc deficiency compromises the maturational competence of porcine oocyte by inducing mitophagy and apoptosis.

Zinc, an essential trace mineral, plays a pivotal role in cell proliferation, maintenance of redox homeostasis, apoptosis, and aging. Serum zinc concentrations are reduced in patients with polycystic ovary syndrome (PCOS). However, the underlying mechanism of the effects of zinc deficiency on the female reproductive system, especially oocyte quality, has not been fully elucidated. Thus, we established an in vitro experimental model by adding N,N,N',N'-Tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) into the culture medium, and to determine the potential regulatory function of zinc during porcine oocytes maturation. In the present study, we found that zinc deficiency caused aberrant meiotic progress, accompanied by the disrupted cytoskeleton structure in porcine oocytes. Zinc deficiency impaired mitochondrial function and dynamics, leading to the increase of reactive oxygen species (ROS) and acetylation level of the antioxidative enzyme superoxide dismutase 2 (SOD2), eventually induced the occurrence of oxidative stress and early apoptosis. Moreover, zinc deficiency perturbed cytosolic Ca2+ homeostasis, lipid droplets formation, demonstrating the aberrant mitochondrial function in porcine oocytes. Importantly, we found that zinc deficiency in porcine oocytes induced the occurrence of mitophagy by activating the PTEN-induced kinase 1/Parkin signaling pathway. Collectively, our findings demonstrated that zinc was a critical trace mineral for maintaining oocyte quality by regulating mitochondrial function and autophagy in porcine oocytes.

Zinc deficiency deteriorates ovarian follicle development and function by inhibiting mitochondrial function.

Zinc (Zn) is a crucial trace element essential for human growth and development, particularly for reproductive health. Previous research has shown a decrease in serum zinc concentration with age and individuals with conditions such as polycystic ovary syndrome (PCOS) and diabetes mellitus. However, the specific effects of zinc deficiency on the female reproductive system, especially ovarian function, are not fully understood. In our study, we observed a significant reduction in the total number of follicles and mature follicles in the zinc deficiency group. This reduction correlated with decreased level of anti-Mullerian hormone (AMH) and abnormal gene expression affecting hormone secretion regulation. Furthermore, we found that zinc deficiency disrupted mitochondrial dynamics, leading to oxidative stress in the ovaries, which further inhibited autophagy and increased ovarian apoptosis. These changes ultimately resulted in the failure of germinal vesicle breakdown (GVBD) and reduced oocyte quality. Meanwhile, administration of zinc glycine effectively alleviated the oocyte meiotic arrest caused by dietary zinc deficiency. In conclusion, our findings demonstrated that dietary zinc deficiency can affect hormone secretion and follicle maturation by impairing mitochondrial function and autophagy.

WITHDRAWN: Clearance of lysosomal glycogen accumulation by Transcription factor EB (TFEB) in muscle cells from lysosomal alpha-glucosidase deficient mice.

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Expression of cochlin mRNA splice variants in the inner ear.

Proteomic analysis of inner ear proteins revealed unique properties of cochlin, encoded by the COCH gene. We detected 3 cochlin isoforms, p63s, p44s and p40s, in the inner ear tissue and a short 16-kDa isoform, cochlin-tomoprotein (CTP), in the perilymph. The role of the cochlin isoforms has not been elucidated. To improve our understanding of the mechanism of cochlin isoform expression, we investigated rat cochlin mRNA expression in the inner ear and other organs. We performed RNA-ligation-mediated amplification of cDNA ends (RLM-RACE) using RNA isolated from the inner ear and spleen of rats, which are known to express abundant cochlin mRNA. We also examined the expression profile of full-length cochlin mRNA by nested RT-PCR in the cerebrum, cerebellum/brain stem, eye, inner ear, thyroid gland, thymus gland, lung, heart, liver, spleen, adrenal gland, kidney and blood. We verified CTP expression in rat perilymph by Western blot. By RLM-RACE, alternately spliced variants of cochlin mRNA with 3 different lengths were detected (2442, 2008 and 724 bp). The two longer mRNAs encode full-length cochlin with different polyadenylation signals in the 3'-untranslated region, which are expressed both in the ear and spleen. The short variant encodes the limulus factor C, cochlin, late gestation lung protein (LCCL) domain and the N-terminal sequence of the von Willebrand factor A (vWFA1) domain, and this variant was detected only in the ear. All 3 variants have the same transcriptional start site. By RT-PCR, we found that full-length cochlin was expressed in all organs examined, with a splice variant in the heart. By Western blot, we detected short isoforms (11-17 kDa) in the perilymph. Cochlin isoform formation is regulated, at least in part, by alternative splicing at the transcriptional level. The short mRNA was detected only in the inner ear, and this variant may provide a clue to understanding the formation and function of cochlin isoforms.

Cochlin-tomoprotein: a novel perilymph-specific protein and a potential marker for the diagnosis of perilymphatic fistula.

BACKGROUND: Perilymphatic fistula (PLF) is an abnormal connection between the inner and middle ear. A procedure for obtaining definite proof of a PLF remains elusive, and methods of diagnosis remain controversial. To date, there is no clinically relevant biochemical marker for perilymph leakage. Using proteomic analysis of inner ear proteins, we have previously found unique properties of cochlin, encoded by the COCH gene. We detected 3 cochlin isoforms (p63s, p44s and p40s) in the inner ear tissue and a short 16-kDa isoform of cochlin-tomoprotein (CTP) in the perilymph. Since cochlin was found to be highly specific to the inner ear, we speculated that CTP might also be specific to the perilymph. The aim of this study was to determine whether CTP, a novel perilymph-specific protein, could be used as a marker for the diagnosis of PLF. METHODS: By Western blotting, we investigated the specificity of CTP expression in a range of body fluids that included perilymph, serum, saliva and cerebrospinal fluid. To elucidate the detection limit of CTP, serially diluted recombinant human (rh)CTP as well as human perilymph was tested. RESULTS: CTP was selectively expressed in all 20 perilymph samples tested, but not in 77 samples of the other body fluids. The detection limit of rhCTP was 0.27 ng or 0.022 microl of perilymph per well on Western blot analysis. CONCLUSION: The results strongly suggest that CTP can be a specific marker of perilymph leakage. Moreover, CTP has the potential to be a biochemical marker that allows a definitive diagnosis of the etiology of PLF-related hearing loss and vestibular disorders.

HT-2 toxin exposure induces mitochondria dysfunction and DNA damage during mouse early embryo development.

HT-2 toxin is one of the type A trichothecene mycotoxins existed in contaminated feed and has exerted various toxic effects on human and livestock, as it induces lesions in multiple tissues including reproductive system. However, till now it is still unclear about the toxicity of HT-2 on mammalian embryos. In this study, we showed that HT-2 treatment disrupted mouse early embryo development, and we also found the occurrence of oxidative stress, showing with the increased ROS level. This might be due to the mitochondria dysfunction, since the occurrence of aberrant mitochondria distribution was observed. Moreover, HT-2 exposure caused DNA damage, showing with the positive signal of γH2 A.X; and HT-2 treatment embryos showed increased LC3 positive signals, indicating the induction of autophagy, which further confirmed the occurrence of DNA damage. Thus, our results showed that HT-2 exposure impaired mouse embryo development by inducing oxidative stress, mitochondria dysfunction and DNA damage.

Spatiotemporal expression of cochlin in the inner ear of rats during postnatal development.

Cochlin (encoded by COCH) constitutes 70% of non-collagenous protein in the inner ear, and the expression of cochlin is highly specific to the inner ear. Eleven missense mutation and one in-frame deletion have been reported in the COCH gene, causing hereditary progressive sensorineural hearing loss and vestibular dysfunction, DFNA9. These data imply that cochlin should bear an essential and crucial role in the inner ear function. However, the role of cochlin has not been fully clarified. We have investigated the spatiotemporal expression of cochlin in the inner ear of rats during postnatal development to better understand the functional role of cochlin. By immunohistochemistry, cochlin expression was faint in the cochlea and vestibule on the 6th day after birth (DAB6). At DAB70, strong expression of cochlin was detected in the spiral limbus and spiral ligament within the cochlea, and in the stromata of the maculae of otolithic organs and crista ampullaris within the vestibule. Immunoreactivity for cochlin increased during the postnatal development. Western blot analysis also showed an increase in the expression of cochlin isoforms. Furthermore, the dominant isoform of cochlin expressed changed from p63s to p40s between DAB24 and DAB70. These results suggest that the expression of cochlin may be related to the maturation of inner ear function, and the change in isoforms of cochlin expressed will provide important insight into the understanding of both cochlin function and formation of cochlin isoforms. This is the first to report about the spatiotemporal expression of cochlin in the developing rat inner ear.

Modulation of mTOR signaling as a strategy for the treatment of Pompe disease.

Mechanistic target of rapamycin (mTOR) coordinates biosynthetic and catabolic processes in response to multiple extracellular and intracellular signals including growth factors and nutrients. This serine/threonine kinase has long been known as a critical regulator of muscle mass. The recent finding that the decision regarding its activation/inactivation takes place at the lysosome undeniably brings mTOR into the field of lysosomal storage diseases. In this study, we have examined the involvement of the mTOR pathway in the pathophysiology of a severe muscle wasting condition, Pompe disease, caused by excessive accumulation of lysosomal glycogen. Here, we report the dysregulation of mTOR signaling in the diseased muscle cells, and we focus on potential sites for therapeutic intervention. Reactivation of mTOR in the whole muscle of Pompe mice by TSC knockdown resulted in the reversal of atrophy and a striking removal of autophagic buildup. Of particular interest, we found that the aberrant mTOR signaling can be reversed by arginine. This finding can be translated into the clinic and may become a paradigm for targeted therapy in lysosomal, metabolic, and neuromuscular diseases.

M-mode color Doppler ultrasonic imaging of vertical tongue movement during articulatory movement.

To observe and estimate the movement of the tongue, ultrasonic investigation is the most harmless real-time monitoring procedure for analyzing articulatory movements. Color Doppler ultrasonic imaging is special in that it can only sample a moving target, and it can indicate the velocity and direction of the target by color and brightness in real time. This study assessed and demonstrated the validity of M-mode color Doppler ultrasonic imaging to observe the movements of the tongue during syllable repetition tasks performed by normal subjects and dysarthric patients, those affected by amyotrophic lateral sclerosis, cerebellar ataxia, Parkinsonism, and polymyopathy. When the transducer was set below the jaw, upward movement was indicated by a blue signal and downward movement was indicated by a red one on the screen of the ultrasound machine. We also measured the velocity of the tongue by contrast scale classified by 15 degrees. Thus, we could observe vertical tongue movement by a color-coded pattern after quantitative analysis. The Doppler signal patterns of normal subjects were verified by simultaneous video x-ray fluorography recordings. The findings for dysarthric patients corresponded well with previously reported features analyzed by other methods. Therefore, color Doppler ultrasonic imaging of the tongue is a useful procedure to researchers for clinical speech and voice studies.

Expression of full-length Cochlin p63s is inner ear specific.

OBJECTIVE: The COCH gene mutated in DFNA9, murine an autosomal dominant hereditary hearing impairment, encodes Cochlin. Cochlin is also suggested to be the self-antigen of autoimmune sensorineural hearing loss. We previously reported that Cochlin constitutes 70% of the inner ear proteins and is classified into three types of isoform, p63s, p44s, and p40s. To study the specificity of expression of Cochlin isoforms in various organs, here we have investigated expression of the COCH gene at both the transcriptional and translational level. METHODS: COCH gene expression was studied by RT-PCR and Southern blot analysis. Cochlin isoforms were studied by Western blot analysis using an isoform specific antibody. RESULTS: At the transcriptional level, COCH mRNA was detected only in the inner ear by RT-PCR. Southern blot analysis of RT-PCR products detected a high level of COCH mRNA in the inner ear, lower level in spleen, and very low levels in the cerebrum, cerebellum/brain stem, eye, liver and kidney. At the translational level, Western blot analysis showed that a set of isoform, p63s, p44s, and p40s was detected at high levels only in the inner ear. In contrast, multiple proteins were detected at much lower levels in other organs tested. Notably, full-length Cochlin p63s was detected only in the inner ear. CONCLUSION: Our findings demonstrate that the COCH gene is expressed preferentially in the inner ear and that expression of full-length Cochlin p63s is specific to the inner ear. These results will be central to understanding the function of Cochlin and its role in the pathophysiology of DFNA9.

Pompe disease: Shared and unshared features of lysosomal storage disorders.

Pompe disease, an inherited deficiency of lysosomal acid α-glucosidase (GAA), is a severe metabolic myopathy with a wide range of clinical manifestations. It is the first recognized lysosomal storage disorder and the first neuromuscular disorder for which a therapy (enzyme replacement) has been approved. As GAA is the only enzyme that hydrolyses glycogen to glucose in the acidic environment of the lysosome, its deficiency leads to glycogen accumulation within and concomitant enlargement of this organelle. Since the introduction of the therapy, the overall understanding of the disease has progressed significantly, but the pathophysiology of muscle damage is still not fully understood. The emerging complex picture of the pathological cascade involves disturbance of calcium homeostasis, mitochondrial abnormalities, dysfunctional autophagy, accumulation of toxic undegradable materials, and accelerated production of lipofuscin deposits that are unrelated to aging. The relationship of Pompe disease to other lysosomal storage disorders and potential therapeutic interventions for Pompe disease are discussed.

Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease.

Mitochondria-induced oxidative stress and flawed autophagy are common features of neurodegenerative and lysosomal storage diseases (LSDs). Although defective autophagy is particularly prominent in Pompe disease, mitochondrial function has escaped examination in this typical LSD. We have found multiple mitochondrial defects in mouse and human models of Pompe disease, a life-threatening cardiac and skeletal muscle myopathy: a profound dysregulation of Ca(2+) homeostasis, mitochondrial Ca(2+) overload, an increase in reactive oxygen species, a decrease in mitochondrial membrane potential, an increase in caspase-independent apoptosis, as well as a decreased oxygen consumption and ATP production of mitochondria. In addition, gene expression studies revealed a striking upregulation of the ß 1 subunit of L-type Ca(2+) channel in Pompe muscle cells. This study provides strong evidence that disturbance of Ca(2+) homeostasis and mitochondrial abnormalities in Pompe disease represent early changes in a complex pathogenetic cascade leading from a deficiency of a single lysosomal enzyme to severe and hard-to-treat autophagic myopathy. Remarkably, L-type Ca(2+)channel blockers, commonly used to treat other maladies, reversed these defects, indicating that a similar approach can be beneficial to the plethora of lysosomal and neurodegenerative disorders.

Legacies of past exploitation and climate affect mammalian sexes differently on the roof of the world - the case of wild yaks.

In polar environments, a lack of empirical knowledge about biodiversity prompts reliance on species distribution models to predict future change, yet these ignore the role of biotic interactions including the role of long past human exploitation. To explore how mammals of extreme elevation respond to glacial recession and past harvest, we combined our fieldwork with remote sensing and used analyses of ~60 expeditions from 1850-1925 to represent baseline conditions for wildlife before heavy exploitation on the Tibetan Plateau. Focusing on endangered wild yaks (Bos mutus), we document female changes in habitat use across time whereupon they increasingly relied on steeper post-glacial terrain, and currently have a 20x greater dependence on winter snow patches than males. Our twin findings­that the sexes of a cold-adapted species respond differently to modern climate forcing and long-past exploitation­indicate that effective conservation planning will require knowledge of the interplay between past and future if we will assure persistence of the region's biodiversity.

Establishment and application of minigene models for studying pre-mRNA alternative splicing.

The objective of the present study is to establish a minigene model for studying pre-mRNA alternative splicing. To prepare the minigene DNA constructs, with human or mouse genomic DNA as templates, GluR-B, FGF-2R and Zis "minigene" fragments were amplified using PCR and cloned to the eukaryotic expression vectors. The three constructed minigenes and the expression vectors of Tra2beta1 and Zis2 were co-transfected in Hela cells. RT-PCR analysis was performed to semi-quantitatively determine the spliced products from the minigenes. The results demonstrated that the constructed minigenes are useful in studying the pre-mRNA alternative splicing in cultured cells. With the established Zis minigene, we for the first time found that Zis2 isoform regulates the alternative splicing of Zis minigene.

Pompe disease: from pathophysiology to therapy and back again.

Pompe disease is a lysosomal storage disorder in which acid alpha-glucosidase (GAA) is deficient or absent. Deficiency of this lysosomal enzyme results in progressive expansion of glycogen-filled lysosomes in multiple tissues, with cardiac and skeletal muscle being the most severely affected. The clinical spectrum ranges from fatal hypertrophic cardiomyopathy and skeletal muscle myopathy in infants to relatively attenuated forms, which manifest as a progressive myopathy without cardiac involvement. The currently available enzyme replacement therapy (ERT) proved to be successful in reversing cardiac but not skeletal muscle abnormalities. Although the overall understanding of the disease has progressed, the pathophysiology of muscle damage remains poorly understood. Lysosomal enlargement/rupture has long been considered a mechanism of relentless muscle damage in Pompe disease. In past years, it became clear that this simple view of the pathology is inadequate; the pathological cascade involves dysfunctional autophagy, a major lysosome-dependent intracellular degradative pathway. The autophagic process in Pompe skeletal muscle is affected at the termination stage-impaired autophagosomal-lysosomal fusion. Yet another abnormality in the diseased muscle is the accelerated production of large, unrelated to ageing, lipofuscin deposits-a marker of cellular oxidative damage and a sign of mitochondrial dysfunction. The massive autophagic buildup and lipofuscin inclusions appear to cause a greater effect on muscle architecture than the enlarged lysosomes outside the autophagic regions. Furthermore, the dysfunctional autophagy affects the trafficking of the replacement enzyme and interferes with its delivery to the lysosomes. Several new therapeutic approaches have been tested in Pompe mouse models: substrate reduction therapy, lysosomal exocytosis following the overexpression of transcription factor EB and a closely related but distinct factor E3, and genetic manipulation of autophagy.

Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease.

A recently proposed therapeutic approach for lysosomal storage disorders (LSDs) relies upon the ability of transcription factor EB (TFEB) to stimulate autophagy and induce lysosomal exocytosis leading to cellular clearance. This approach is particularly attractive in glycogen storage disease type II [a severe metabolic myopathy, Pompe disease (PD)] as the currently available therapy, replacement of the missing enzyme acid alpha-glucosidase, fails to reverse skeletal muscle pathology. PD, a paradigm for LSDs, is characterized by both lysosomal abnormality and dysfunctional autophagy. Here, we show that TFEB is a viable therapeutic target in PD: overexpression of TFEB in a new muscle cell culture system and in mouse models of the disease reduced glycogen load and lysosomal size, improved autophagosome processing, and alleviated excessive accumulation of autophagic vacuoles. Unexpectedly, the exocytosed vesicles were labelled with lysosomal and autophagosomal membrane markers, suggesting that TFEB induces exocytosis of autophagolysosomes. Furthermore, the effects of TFEB were almost abrogated in the setting of genetically suppressed autophagy, supporting the role of autophagy in TFEB-mediated cellular clearance.

Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament.

CONCLUSIONS: We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. OBJECTIVE: To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. METHODS: The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. RESULTS: The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.

A pathological study of bamboo nodule of the vocal fold.

We examined pathologically a bamboo nodule of the vocal fold by means of immunohistochemical studies and scanning electron microscopic examination. A 38-year-old female showed a high index of antinuclear antibodies without any systemic symptoms but had complained of progressive voice disorder for 9 months. She had used her voice excessively in her occupation and for singing. Hematoxylin and eosin staining showed submucosal edema with lymphocyte and neutrocyte infiltrations and hyaline degeneration. Periodic acid methenamine silver staining showed hypertrophy of the basal lamina of the blood vessels. Immunohistochemical study showed IgG-positive cells in the blood vessel walls. Scanning electron microscopic study demonstrated immune complexes deposited as fine granules of high electrodense materials in the hypertrophic walls of the micro-blood vessels. After surgical resection of the bamboo nodules and advising her to avoid using her voice excessively, her voice improved gradually and the levels of IgG, immune complexes, and antinuclear antibody decreased for 1 year. These findings suggest that the bamboo nodules were not induced by an organ-specific reaction to an autoimmune disease, but mechanical damage to the micro-blood vessels induced by phonation injury of the vocal fold might have caused the deposit of high-molecular weight immune complexes in the damaged micro-blood vessels at the midportion of the vocal folds, which induced secondary inflammatory change at the midportion of the vocal fold.

A case of Creutzfeldt-Jacob disease with bilateral vocal fold abductor paralysis.

Bilateral vocal fold abductor paralysis was seen in a patient with Creutzfeldt-Jacob disease. After tracheotomy, the patient showed disappearance of reduced oxygen saturation with high-pitched inspiratory stridor and pulling phenomenon of the supraclavicular region and larynx. Electromyographic examinations of the intrinsic laryngeal muscles, including the thyroarytenoid and posterior cricoarytenoid muscles, demonstrated that there was no apparent action potential in those muscles during spontaneous respiratory movements, and there was no abnormal potential for those muscles at rest. By pushing the infrasternal region of the patient on the expiration, normal motor unit action potential could be seen in the posterior cricoarytenoid muscle on the next inspiration. Based on those findings, we concluded that bilateral vocal fold abductor paralysis in this case of Creutzfeldt-Jacob disease was not induced by disorders of the degeneration of motor nucleus in the ambiguus as in multiple system atrophy, but by a disorder of the upper motor neuron.