Parkin interacts with Klokin1 for mitochondrial import and maintenance of membrane potential.

Parkin is a multifunctional protein, including maintaining mitochondrial homeostasis. Recent evidence suggests that Parkin is recruited from the cytoplasm to damaged mitochondria with low membrane potential. We found that intracellular localization of Parkin changed with cellular growth phase. Parkin was preferentially localized in the mitochondria of cultured cells. The mitochondria with large amounts of Parkin showed preserved membrane potentials even during treatment with carbonyl cyanide m-chlorophenylhydrazone. Here we report a novel protein named Klokin 1 that transports Parkin to the mitochondria. Klokin 1 was localized to the mitochondria and enhanced mitochondrial expression of Parkin. Klokin 1 enhanced cell viability in Parkin-silenced cells. Klokin 1 expression was enhanced in the brains of Parkin-deficient mice but not in an autopsied PARK2 brain. Our findings indicate that mitochondrial Parkin prevents mitochondrial depolarization and that Klokin 1 may compensate for Parkin deficiency.

Prevention of mitochondrial disease inheritance by assisted reproductive technologies: prospects and challenges.

BACKGROUND: Mitochondrial diseases are caused by the mutations in both nuclear and mitochondrial DNA (mtDNA) and the treatment options for patients who have mitochondrial disease are rather limited. Mitochondrial DNA is transmitted maternally and does not follow a Mendelian pattern of inheritance. Since reliable and predictable detection of mitochondrial disorders in embryos and oocytes is unattainable at present, an alternative approach to this problem has emerged as partial or complete replacement of mutated mtDNA with the wild-type mtDNA through embryo manipulations. Currently available methods to achieve this goal are germinal vesicle transfer (GVT), metaphase chromosome transfer (CT), pronuclear transfer (PNT) and ooplasmic transfer (OT). SCOPE OF REVIEW: We summarize the state of the art regarding these technologies and discuss the implications of recent advances in the field for clinical practice. MAJOR CONCLUSIONS: CT, PNT and GVT techniques hold promise to prevent transmission of mutant mtDNA through ARTs. However, it is clear that mtDNA heteroplasmy in oocytes, embryos and offspring produced by these methods remains as a legitimate concern. GENERAL SIGNIFICANCE: New approaches to eliminate transmission of mutant mtDNA certainly need to be explored in order to bring the promise of clinical application for the treatment of mitochondrial disorders. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.

Oophorectomy for fertility preservation via reduced-port laparoscopic surgery.

BACKGROUND: For fertility preservation of women patients scheduled to undergo chemotherapy or radiotherapy, unilateral oophorectomy was performed, and the ovary was cryopreserved. METHODS: Two-port surgery was conducted in 3 patients, and single-port surgery using a single-incision laparoscopic surgery port in 3. An 18-G Cathelin needle equipped with a syringe was directly inserted transabdominally to reach the small follicle on the ovarian surface; then, follicular fluid was recovered by aspiration through the syringe as with in vitro fertilization procedures, and immature oocytes were collected from the resulting culture medium under microscopy and cryopreserved. Vitrification of the ovarian tissue was performed using the cryotissue method. RESULTS: The operative time and estimated blood loss were 39.7 minutes (17-57) and 8.6 mL (2-20), and the numbers of ovarian cortical tissues and immature oocytes collected were 10.1 (5.5-15) and 16.3 (0-36), respectively. CONCLUSIONS: It is suggested that fertility preservation operations before chemotherapy or radiotherapy can be safely done using reduced-port surgery.

Uterine autotransplantation in cynomolgus macaques: the first case of pregnancy and delivery.

BACKGROUND: For women with congenital uterine infertility, or for those who have undergone hysterectomy, uterine transplantation is one of the potential treatments to regain fertility. In this study, we utilized a primate model of uterine transplantation, and evaluated the patency of our microsurgical anastomoses, and the perfusion of the transplanted uterus. METHODS: Two female cynomolgus monkeys underwent surgery. We anastomosed two arteries and one vein in Case 1 and two arteries and two veins in Case 2. The arteries used were the uterine arteries and the anastomosis was done to the external iliac artery. We used one of the ovarian veins in both animals, but resected the ovary from the Fallopian tube. Uterine arterial blood flow and uterine size were determined by intraoperative indocyanine green (ICG) angiography and ultrasonography. The biopsy of the uterine cervix was performed after surgery. RESULTS: ICG angiography showed that the unilateral uterine artery perfused the bilateral uterine bodies and cervix. In Case 1, ICG angiography showed the occlusion of one of the anastomosed arteries during the operation and the uterus appeared atrophied 2 months after operation. In Case 2, the transplanted uterus survived and normal menstruation occurred. The animal achieved a natural pregnancy and was delivered by the Caeserean section due to early separation of the placenta. The newborn suffered fetal distress. CONCLUSIONS: These results show the anastomosis of at least the bilateral uterine arteries and the unilateral ovarian vein is required for uterus transplantation. This is the first report of a natural pregnancy in a primate following uterine autotransplantation.

Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos.

This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.

Successful vitrification of bovine and human ovarian tissue.

Ovariectomy and ovarian tissue cryopreservation has the potential to preserve the natural fertility of cancer patients prior to sterilizing chemo- and radiotherapies. Ovarian tissue cryopreservation with the conventional slow-freezing method has yielded limited success, partly because of oocyte loss during freeze-thaw and subsequent transplant. Based on the high-efficiency vitrification Cryotop method, a practical vitrification procedure for murine, bovine and human ovarian tissue was developed. A Cryotissue method was designed for cryopreservation of ovarian tissue, and vitrification experiments were performed in a bovine animal model with ovarian size and structure similar to the human. There was no difference in oocyte viability (>89%) between fresh and vitrified ovarian cortical tissue in either bovine or human samples. Ovarian tissue was successfully autotransplanted to six cattle. Autotransplantation of vitrified-warmed tissue back to the cattle resulted in no loss of oocyte viability. In addition, human ovarian tissue from cancer patients, and from ovary transplant donors was also vitrified by the Cryotissue method. After warming, high oocyte survival in human tissue (similar to bovine tissue) was obtained. These results indicate that an ultra-rapid cooling vitrification method has the potential for clinical use in human ovarian tissue cryopreservation.

Early outcomes of a municipally funded oocyte cryopreservation programme in Japan.

One factor explaining the declining birth rate in Japan is the social advancement of women. Women are delaying marriage and childbirth, with many then facing so-called 'social infertility'. Advanced infertility treatment options, such as in vitro fertilization, are available, but the costs are high. Further, the success rates for 'older' women are only around 10%. We report the preliminary results of an oocyte cryopreservation programme promoted and subsidized by our city government. Citywide seminars were conducted to generate awareness of issues surrounding fertility. Among the total 81 attendees were women considering oocyte retrieval and the current practice of oocyte retrieval and cryopreservation and its associated risks were explained. Fifty-seven attendees, women under 34 years of age, were considered potential candidates for the procedure. These women wished to delay pregnancy for specific reasons, such as occupational demands. Twenty-six of these women expressed a definite desire for oocyte cryopreservation, and 19 have thus far completed the oocyte retrieval and cryopreservation procedure. Frozen MII oocytes have ranged in number from 3 to 22 per patient (mean ± SD, 8.3 ± 5.2). Outcomes thus far indicate that women whose fertility is at risk can be assisted by this fertility preservation method and that it will help address the problem of the declining birth rate in Japan.

Reduction of GSTP1 expression by DNA methylation correlates with clinicopathological features in pituitary adenomas.

Pi-class glutathione-S-transferase (GSTP1) located on chromosome 11q13 encodes a phase II metabolic enzyme that detoxifies reactive electrophilic intermediates. GSTP1 plays an important role in the protecting cells from cytotoxic and carcinogenic agents and is expressed in normal tissues at variable levels in different cell types. Altered GSTP1 activity and expression have been reported in many tumors and this is largely due to GSTP1 DNA hypermethylation. The role of GSTP1 in pituitary tumorigenesis has not been investigated. In this study, we evaluated the GSTP1 expression level and GSTP1 DNA methylation status in a series of pituitary adenomas. Using immunohistochemistry, we identified expression of GSTP1 in all of the various normal hormone-producing adenohypophysial cell types. In pituitary adenomas, loss or reduced expression of GSTP1 was detected in 27 of 53 tumors (50.9%). Expression of GSTP1 was significantly lower in invasive adenomas than in noninvasive adenomas (P<0.05). Using methylation-specific polymerase chain reaction (MS-PCR), GSTP1 DNA promoter hypermethylation was detected in adenomas (38 of 53, 71.7%) but not in normal tissues. GSTP1 methylation was more frequent in grade II, III, and IV tumors (66.7, 85, and 83%, respectively) than in grade I tumors (33%, P<0.05). In addition, the frequency of GSTP1 methylation was higher in invasive tumors (85%) than in noninvasive tumors (59%; P<0.05). Methylation status correlated with significant downregulation of GSTP1 expression; the frequency of GSTP1 methylation was higher in tumors with reduced-GSTP1 expression (85%) than in tumors with normal or high GSTP1 expression (54%; P<0.05). These data indicate that GSTP1 inactivation through CpG hypermethylation is common in pituitary adenomas and may contribute to aggressive pituitary tumor behavior.

Morules with optically clear nuclei in ovarian borderline endometrioid tumor.

Optically clear nuclei (OCN) have been observed in morules of some neoplasms and in some conditions unrelated to the development of the morules. We first report a case of ovarian borderline endometrioid tumor (BET) showing the morules associated with OCN. The patient was a 47-year-old premenopausal woman with a left ovarian cystic tumor, atypical endometrial hyperplasia, and elevated serum levels of FSH, LH, estradiol, and CA 125. The resected ovarian tumor measured 6 cm in diameter, and showed a papillary growth. Histologically, the ovarian tumor was consistent with BET, and the morules with OCN were scattered. Immunohistochemically, OCN were proven to be rich in biotin. An aberrant nuclear expression of beta-catenin was observed in both the tumor cells and the morular cells. Our case may suggest the possibility that the appearance of OCN with or without morules in ovarian tumors is related to endometrioid differentiation of the tumor cells, and should be recognized as a diagnostic clue of ovarian endometrioid tumors. Although female sex hormones have been reported to play a role in the occurrence of OCN, the participation of beta-catenin mutation has also been suggested.

In vitro fertilization and polyspermy in the pig: factors affecting fertilization rates and cytoskeletal reorganization of the oocyte.

Polyspermy is a common phenomenon in the pig. Extensive information has become available from in vitro studies on not only the quality of oocytes but also the quality of spermatozoa. However, little information is available on the relative penetration rates of fresh and frozen spermatozoa from the same ejaculate from boars of different breeds. The present results, based on a total of 15 boars of three different breeds, revealed that the inter-breed variation in fertilization and polyspermic rates is larger than intra-breed variation. It was also shown that the incidence of polyspermy as well as penetration rate was greatly decreased by freezing and thawing, even if a higher number of sperm was coincubated with cumulus-free oocytes for a longer period compared to fresh sperm of the same ejaculate. This study focuses on the cytoskeletal organization of the oocyte with respect to the status of cumulus investment, and monospermic and polyspermic fertilization. The status of cumulus cells correlated with the density of transzonal cumulus-cell processes and with the maturation rate of oocytes and, to some degrees, the incidence of polyspermy. Polyspermic zygotes formed multiple microtubule domains in association with individual male pronuclei (PN), but in a high degree of polyspermy (more than trispermy), the pronuclear apposition did not proceed. The effect of multiple PN of paternal and maternal origin on the cytoskeletal reorganization is also discussed.

Down-regulation of E-cadherin and catenins in human pituitary growth hormone-producing adenomas.

Growth hormone (GH)-producing pituitary adenomas can be ultrastructurally divided into two major types: densely granulated and sparsely granulated. The latter type of adenoma characteristically exhibits globular accumulations of cytokeratin filaments known as fibrous bodies, which are immunohistochemically identifiable as juxtanuclear dot-like immunoreactivity. We hypothesize that the formation of fibrous body might be related to dysfunction of adhesion molecules, because of the functional relationship between intermediate filaments and the cadherin-catenin complex and frequent observation of loss of cohesiveness of the adenoma cells. Our recent immunohistochemical study showed that expression of E-cadherin and its undercoat proteins, alpha-, beta- and gamma-catenin, in GH cell adenomas with prominent fibrous bodies was significantly reduced compared with GH cell adenomas without fibrous bodies and the normal adenohypophysial cells. Although no mutation of exon 3 of the beta-catenin gene was found in any GH cell adenomas with fibrous bodies, methylation-specific polymerase chain reaction analysis revealed that the E-cadherin promoter region was methylated in 37.5% of these adenomas, two of which displayed total methylation, but not in GH cell adenomas without fibrous bodies. We conclude that the decreased expression of the E-cadherin-catenin complex and methylation of the E-cadherin gene promoter region are events associated with the formation of fibrous bodies in GH cell adenomas. It remains to be clarified to explain the mechanism by which down-regulation of adhesion molecules is involved in the abnormal assembly of intermediate filaments.

Spectrum of different types of hypophysitis: a clinicopathologic study of hypophysitis in 31 cases.

Hypophysitis has been histologically classified into five types: lymphocytic hypophysitis (LYH), granulomatous hypophysitis (GRH), xanthogranulomatous hypophysitis (XGH), xanthomatous hypophysitis (XH), and necrotizing hypophysitis. The present study evaluated 31 cases of hypophysitis to clarify their characteristic clinicopathologic features. The lesions were histologically classified into four groups: LYH (22 cases) including lymphocytic adenohypophysitis (LAH) (19 cases) and lymphocytic infundibuloneurohypophysitis (LINH) (3 cases), GRH (5 cases), XGH (2 cases), and XH (2 cases). In each group, the pituitary gland showed lymphocytic infiltration associated with focal or diffuse hypophysial destruction of variable severity and fibrosis. Histologic and clinical overlap among different types of hypophysitis, especially between LAH and LINH, suggest that these entities may have similar etiologic background and/or represent different stages of the same lesion. Considering the sampling sites and clinical manifestations, LAH may not usually involve the neurohypophysis, but LINH may often extend to the adenohypophysitis. A selective loss of adrenocorticotropic hormone-positive cells was seen in two patients with LAH despite only very slight lymphoplasmacytic infiltration. This suggests that there may be at least two causative mechanisms for hypopituitarism in hypophysitis: nonspecific destruction of all types of adenohypophysial cells by severe inflammation and selective destruction of specific adenohypophysial cells.

[A clinicopathological investigation of two autopsy cases of calpainopathy (LGMD2A)].

In this study, we compared the clinicopathological findings of two autopsy cases of patients with calpainopathy (LGMD2A) from different families. The patient in case 1 was a 72-year-old man with a history of type 2 diabetes mellitus. He exhibited recent memory impairments from the age of 70. ECG revealed an incomplete right bundle branch block. A homozygous frameshift mutation c.1796dupA was found in the CAPN3 gene. Cause of death was respiratory insufficiency and heart failure. The patient in case 2 was a 70-year-old man with a history of hypertension. ECG revealed an incomplete right bundle branch block. A homozygous missense mutation c.1080G>C (p.Trp360Cys) in CAPN3 gene was identified. Cause of death was ischemic cardiomyopathy and systemic circulatory failure. In both cases, muscle pathology revealed severe dystrophic changes. In case 2, cardiac hypertrophy and old myocardial infarcts with stenosis of coronary arteries were observed. Histological examination of the sinoatrial node showed fatty infiltration with ischemic changes in case 2. In both cases, the patients' brains showed cerebral atrophy and well preserved neurons. Calpain 3 abnormality was correlated with skeletal muscle involvement. It should be considered that LGMD2A might be complicated by dysfunction of the cardiac conduction system.

A pedunculated submucosal lesion in the stomach with inverted downgrowth.

A 70-year-old man with a gastric lesion was referred to our hospital because of an unusual pedunculated lesion in the gastric body. Endoscopic ultrasound showed scattered cystic lesions within a heterogeneous area confined to the submucosal layer. Endoscopic mucosal resection was performed to obtain a precise diagnosis, as well as for removal. The lesion was histopathologically diagnosed as a heterotopic submucosal gland. We herein describe this rare type of gastric polyp and provide a literature review.

Precisely designed layered silicate as an effective and highly selective CO2 adsorbent.

A layered silicate co-modified with amino and octadecyl groups exhibited efficient and very selective adsorption for CO2 over N2 and H2O at ambient pressure and temperature.

Sunlight-induced effective heterogeneous photocatalytic decomposition of aqueous organic pollutants to CO2 assisted by a CO2 sorbent, amine-containing mesoporous silica.

Photocatalytic mineralization of aqueous formic acid and phenol on pure TiO(2) under sunlight irradiation was substantially accelerated to give a reliable photocatalytic efficiency by conducting the reactions in the presence of a CO(2) sorbent, amine-containing SBA-15, placed in the gas phase of the reactor.

Effective and selective bisphenol A synthesis on a layered silicate with spatially arranged sulfonic acid.

The silylated derivatives of a layered alkali silicate, magadiite, modified with propylsulfonic or arylsulfonic acid were synthesized and used as catalysts for an acid-catalyzed condensation of phenol with acetone. The propylsulfonated magadiites with a different amount of the attached silyl group were synthesized by the silylation of the dodecylammonium-exchanged magadiite with the tuned amount of 3-(mercaptopropyl)trimethoxysilane and the subsequent oxidation of the attached thiol to sulfonic acid. The arylsulfonated magadiite was synthesized by the silylation of the dodecylammonium-exchanged magadiite with 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane and the subsequent hydrolysis of the attached sulfonyl chloride to sulfonic acid. The X-ray diffraction (XRD) patterns and elemental mappings of the products, and the photoluminescent spectra of the Eu(3+)-exchanged products suggested that propylsulfonic or arylsulfonic acid was homogeneously distributed in the interlayer space. When all the sulfonated materials were used as an acid catalyst for condensation between phenol and acetone, p,p' bisphenol A selectively formed over the o,p' isomer, and higher yield and selectivity were attained on the catalysts with larger amount of the attached sulfonic acid. When the interlayer space of the propylsulfonated magadiite was expanded by the co-attachment of octadecylsilyl group, lower selectivity was obtained. The arylsulfonated magadiite showed considerably higher p,p' bisphenol A yield than the propylsulfonated magadiites.

Changes in the expression of decoy receptor 3 in granulosa cells during follicular atresia in porcine ovaries.

During follicular development in mammalian ovaries, the majority of follicles undergo atresia. One of the characteristics of this process is apoptotic cell death in granulosa cells. Several death ligands and receptors, including Fas ligand (FasL) and Fas, have been detected in ovarian follicles and also demonstrated to be capable of inducing apoptosis in follicular cells. Decoy receptor 3 (DcR3) competes with Fas to bind FasL but lacks intracellular death domains, thus inhibiting the induction of apoptosis by the FasL/Fas system. In the present study, we examined the expression of putative porcine DcR3 (pDcR3) mRNA in porcine ovarian follicles. Total RNA was extracted from granulosa cells and thecal layer cells of tertiary follicles at healthy, early atretic and progressed atretic stages, and the expression of pDcR3 mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR). The nucleic acid sequence in the coding region had 80% homology to that of human DcR3, and the deduced amino acid sequence was 73% identical to that of human DcR3. In an in situ hybridization experiment, pDcR3 mRNA expression was confirmed in granulosa and thecal layers, in both healthy and atretic follicles. Quantitative real time RT-PCR analysis showed that the expression of pDcR3 mRNA was weaker in granulosa cells of atretic follicles than those of healthy follicles. No notable changes were seen in the thecal layer cells. These results suggest that DcR3 plays a significant role in the regulation of apoptosis in granulosa cells, but not in thecal layer cells, during atresia.

Production of the first offspring from oocytes derived from fresh and cryopreserved pre-antral follicles of adult mice.

Although mammalian ovaries contain hundreds of thousands of pre-antral follicles, fewer than 1% of these reach maturity and ovulation. Obtaining immature eggs from the pre-antral follicles of ovarian tissue could increase the possibility of preserving fertility in women undergoing anti-cancer treatment, and in women who wish to delay pregnancy and child raising until they are older. This study reports the birth of 10 healthy mouse pups derived from oocytes obtained from pre-antral follicles after adult ovary tissue cryopreservation and allotransplantation. High in-vitro maturation (55.1%), fertilization (76.3%) and cleavage (98.3%) rates were achieved using these oocytes, and there was no significant difference between the vitrified and control samples except in maturation rate (55.1 versus 72.8%, P < 0.05). After an ultra-rapid vitrification procedure, the warmed tissue fragments were transplanted beneath the kidney capsule of severe combined immunodeficient mice for onward in-vivo culture. Within 10 days of culture, 138 full size oocytes developed from the 456 transplanted pre-antral follicles. In-vivo growth of follicles was followed by in-vitro oocyte maturation, in-vitro fertilization and subsequent embryo transfer, leading to the birth of 10 healthy pups. These results may lead to increasing the possibility of preserving fertility by cryopreservation of ovarian tissue.