Hanging drop monoculture for selection of optimal antioxidants during in vitro maturation of porcine oocytes.

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre-culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 µg/ml of L-carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non-presence of oxidant stress induced by H2O2 supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 µg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H2O2. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.

Formation of filamentous tau aggregations in transgenic mice expressing V337M human tau.

Formation of neurofibrillary tangles (NFTs) is the most common feature in several neurodegenerative diseases, including Alzheimer's disease (AD). Here we report the formation of filamentous tau aggregations having a beta-sheet structure in transgenic mice expressing mutant human tau. These mice contain a tau gene with a mutation of the frontotemporal dementia parkinsonism (FTDP-17) type, in which valine is substituted with methionine residue 337. The aggregation of tau in these transgenic mice satisfies all histological criteria used to identify NFTs common to human neurodegenerative diseases. These mice, therefore, provide a preclinical model for the testing of therapeutic drugs for the treatment of neurodegenerative disorders that exhibit NFTs.

New insights on how metals disrupt amyloid beta-aggregation and their effects on amyloid-beta cytotoxicity.

Amyloid-beta protein (A beta) aggregates in the brain to form senile plaques. By using thioflavin T, a dye that specifically binds to fibrillar structures, we found that metals such as Zn(II) and Cu(II) normally inhibit amyloid beta-aggregation. Another method for detecting A beta, which does not distinguish the types of aggregates, showed that these metals induce a non-beta-sheeted aggregation, as reported previously. Secondary structural analysis and microscopic studies revealed that metals induced A beta to make non-fibrillar aggregates by disrupting beta-sheet formation. These non-fibrillar A beta aggregates displayed much weaker Congo Red birefringence, and in separate cell culture experiments, were less toxic than self beta-aggregates, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The toxicity of soluble A beta was enhanced in the presence of Cu(II), which suggests the previously hypothesized role of A beta in generating oxidative stress. Finally, under an acidic condition, similar to that in the inflammation associated with senile plaques, beta-aggregation was robustly facilitated at one specific concentration of Zn(II) in the presence of heparin. However, because a higher concentration of Zn(II) virtually abolished this abnormal phenomenon, and at normal pH any concentrations strongly inhibit beta-aggregation and its associated cytotoxicity, including its anti-oxidative nature we suggest that Zn(II) has an overall protective effect against beta-amyloid toxicity.

Conjugated linoleic acid supplementation reduces adipose tissue by apoptosis and develops lipodystrophy in mice.

Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivatives of linoleic acid found in beef and dairy products. CLA has been reported to reduce body fat. To examine the mechanism(s) of CLA reduction of fat mass, female C57BL/6J mice were fed standard semipurified diets (10% fat of total energy) with or without CLA (1% wt/wt). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling (TUNEL) and DNA fragmentation analysis revealed that fat-mass decrease by CLA was mainly due to apoptosis. Tumor necrosis factor (TNF)-alpha and uncoupling protein (UCP)-2 mRNA levels increased 12- and 6-fold, respectively, in isolated adipocytes from CLA-fed mice compared with control mice. Because it is known that TNF-alpha induces apoptosis of adipocytes and upregulates UCP2 mRNA, a marked increase of TNF-alpha mRNA with an increase of UCP2 in adipocytes caused CLA-induced apoptosis. However, with a decrease of fat mass, CLA supplementation resulted in a state resembling lipoatrophic diabetes: ablation of brown adipose tissue, a marked reduction of white adipose tissue, marked hepatomegaly, and marked insulin resistance. CLA supplementation decreased blood leptin levels, but continuous leptin infusion reversed hyperinsulinemia, indicating that leptin depletion contributes to the development of insulin resistance. These results demonstrate that intake of CLA reduces adipose tissue by apoptosis and results in lipodystrophy, but hyperinsulinemia by CLA can be normalized by leptin administration.

Neuronal apoptosis inhibitory protein (NAIP) may enhance the survival of granulosa cells thus indirectly affecting oocyte survival.

In mammals, the postnatal loss of more than 99% of female germ cells is due mainly to the process of ovarian follicular atresia. Atresia is known to be mediated by apoptotic granulosa cell-death. Here we show the involvement of neuronal apoptosis inhibitory protein (NAIP) in ovarian folliculogenesis in which it prevents granulosa cell-death. NAIP has been isolated in association with a neurodegenerative disorder, spinal muscular atrophy (SMA), in which it has been shown to suppress mammalian cell-death induced by a variety of stimuli (Liston et al., 1996, Nature 379:349-353). In an in situ hybridization analysis with mouse ovaries, active expression of NAIP mRNA was localized in the granulosa cells of developing follicles from the primary stage to the Graafian stages, whereas the NAIP gene was not expressed or was weakly expressed in follicles that might be undergoing atresia. Gonadotropin, which is known to inhibit apoptosis in ovarian follicles, caused a 2.4-fold increase in NAIP gene expression in the ovary. To study the role of ovarian NAIP, antisense NAIP oligonucleotides were locally delivered into the ovarian bursa. Suppression of ovarian NAIP expression with antisense oligonucleotides evoked a decrease in the number of morphologically normal ovulated oocytes, implying an indirect involvement of NAIP in germ cell development by enhancing the survival of granulosa cells. These findings suggest that gonadotropin-inducible NAIP may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa cell-death during folliculogenesis.

Clinicopathological factors predicting retroperitoneal lymph node metastasis and survival in endometrial cancer.

BACKGROUND: By clarifying the significance of clinicopathological factors for retroperitoneal lymph node metastasis and survival of patients with endometrial cancer, we suggest ideas for optimal treatment of this disease. METHODS: A retrospective study was conducted in 310 women with endometrial cancer who underwent surgery with retroperitoneal lymphadenectomy. To evaluate retroperitoneal lymph node metastasis, age-adjusted and multivariable analyses were carried out for six clinicopathological factors including pathological grade, myometrial invasion, cervical invasion, peritoneal cytology, lymphatic permeation and vascular invasion. To evaluate survival, besides the above factors, a positive rate of metastasis of dissected retroperitoneal lymph nodes was included. RESULTS: In 40 patients (13%) with nodal metastasis, the average positive rate of metastasis of dissected retroperitoneal lymph nodes was 22%. For retroperitoneal lymph node metastasis, the odds ratio of deep myometrial invasion, cervical invasion and severe lymphatic permeation were 5.97, 2.72 and 12.01, respectively. For survival, the hazard ratios of the positive rates of metastasis of dissected retroperitoneal lymph nodes (both 25% and < 25%), positive peritoneal cytology and poor pathological grade were 7.10, 3.24, 3.82 and 3.27, respectively, and 5-year survival rates for them were 0, 50, 72 and 77%, respectively. CONCLUSIONS: For retroperitoneal lymph node metastasis, lymphatic permeation, deep myometrial invasion and cervical invasion were the independent prognostic factors. For survival, retroperitoneal lymph metastasis, poor pathological grades and positive peritoneal cytology were the independent prognostic factors. The positive rate of metastasis of dissected retroperitoneal lymph metastasis plays an important role in predicting survival of endometrial cancer. Lymph node biopsy is insufficient in treatment of this disease.

Identification of two alpha-subunit species of GTP-binding proteins, Galpha15 and Galphaq, expressed in rat taste buds.

We cloned cDNAs for two G protein alpha-subunits belonging to the Galphaq family, each capable of activating PLCbeta, from rat tongue. One is a Galphaq in the narrow sense, and the other, termed rat Galpha15, is a rat counterpart of mouse Galpha15, sharing an amino acid sequence similarity of 94%. RT-PCR and Northern blot analysis demonstrated that rat Galpha15 and Galphaq were distinctly expressed in tongue epithelia containing taste buds. Immunostaining also showed that rat Galpha15, together with the Galphaq, was localized mainly in taste buds. These studies suggest the possibility that these two Galpha proteins function for taste signal transduction in sensory cells.

Development of normal mice from metaphase I oocytes fertilized with primary spermatocytes.

Primary spermatocytes are the male germ cells before meiosis I. To examine whether these 4n diploid cells are genetically competent to fertilize oocytes and support full embryo development, we introduced the nuclei of pachytene/diplotene spermatocytes into oocytes that were arrested in prophase I (germinal vesicle stage), metaphase I, or metaphase II (Met II). Both the paternal and maternal chromosomes then were allowed to undergo meiosis synchronously until Met II. In the first and second groups, the paternal and maternal chromosomes had intermingled to form a large Met II plate, which was then transferred into a fresh enucleated Met II oocyte. In the third group, the paternal Met II chromosomes were obtained by transferring spermatocyte nuclei into Met II oocytes twice. After activation of the Met II oocytes that were produced, those microfertilized at metaphase I showed the best developmental ability in vitro, and three of these embryos developed into full-term offspring after embryo transfer. Two pups (one male and one female) were proven to be fertile. This finding provides direct evidence that the nuclei of male germ cells acquire the ability to fertilize oocytes before the first meiotic division.

Results of a qualitative and field study using the WHOQOL instrument for cancer patients.

The main purpose of the study was to evaluate quality of life (QOL) among cancer patients using the WHOQOL-100 instrument and to see if any significant differences were seen in cancer stages, treatment status and prognosis. This study consisted of two parts; qualitative and quantitative. For the qualitative study, two focus groups were conducted by medical professionals to establish the applicability of the WHOQOL instrument in evaluating the QOL of cancer patients, but most participants were negative about using a generic instrument such as WHOQOL. For the quantitative study, 197 cancer patients (average age 55.86) from eight medical centers using the WHOQOL instrument, in addition to each patient's information sheet filled in by their own physicians, were analyzed. The average overall QOL score was 3.39. There was high reliability (Cronbach's alpha = 0.9685) and a high correlation between the psychological and the environmental domains (r = 0.7021), the physical domain and the level of independence (r = 0.6031) and social relations and the environment (r = 0.6856) and between health conditions perceived by patients and QOL scores. In addition, differences by gender, treatments and cancer sites were also found to be significantly different at the 5% significance level. The results indicated that the WHOQOL core instrument was sensitive enough to evaluate the QOL of cancer patients.

Cerebral metastasis in patients with uterine cervical cancer.

We report on eight patients who developed brain metastases following uterine cervical cancer. The mean interval between diagnosis of the primary cancer and diagnosis of the brain metastasis was 28.4 months (range: 6.1-61.8 months). Nausea and vomiting due to increased intracranial pressure were the most frequent symptoms. Surgical excision of the brain lesions, followed by postoperative radiotherapy, was performed in three patients. The other five patients received only cranial radiotherapy. When the metastatic brain lesions were detected, other distant metastatic lesions were confirmed at the same time in five patients. The median survival time after diagnosis of the brain metastases was only 3.0 months.

Apoptosis-like cell death in experimentally-induced cryptorchidism in adult mice.

In order to elucidate the mechanism of germ cell degeneration in experimental cryptorchidism, we examined the testes of adult mice from a morphological standpoint. Adult ICR mice were made cryptorchid either unilaterally or bilaterally. In some mice, testes were surgically replaced back into the scrotum at 2 months after induction of cryptorchidism to observe the regenerative process. Morphological changes of cryptorchid and replaced testes have been studied by light and electron microscopy. Testes were also examined by the TUNEL (TdT-mediated dUTP-biotin nick end labelling) method to evaluate whether the degenerative cells, spermatocytes and spermatids, were dying by apoptosis or by any other process(es). At 8 weeks after the induction of cryptorchidism, the seminiferous epithelium consisted only of Sertoli cells, spermatogonia, and some spermatocytes of early meiotic stages. Soon after the replacement of testes to the scrotum, most of the seminiferous tubules resumed spermatogenic processes. Many degenerating cells, especially the spermatocytes, showed condensation of the nucleus and cytoplasm in cryptorchid testes. Although the cytoplasm was markedly eosinophilic under a light microscope to imply condensation of the cytoplasm, the extent of the condensation was not as pronounced under an electron microscope as reported in previous publications. The cytoplasm showed no expansion. By the TUNEL method, many of the degenerating cells, mainly the spermatocytes, have been shown as undergoing apoptosis. These data provide evidence that at least some of the cells die by apoptosis, or by a process similar to apoptosis.

Reinitiation of spermatogonial mitotic differentiation in inactive old BDF1 mouse seminiferous tubules transplanted to W/Wv mouse testis.

The seminiferous epithelia of old mice (33 mo of age) are composed of spermatogonia and Sertoli cells. Histochemical examination using the anti-c-kit monoclonal antibody demonstrated that the number of differentiating type A spermatogonia decreases with age. To elucidate the differential activity of old mouse spermatogonia, we transplanted extremely thin seminiferous epithelia of old BDF, mice into W/Wv mouse testes and examined whether or not they could reinitiate differentiation. Artificially cryptorchid mice were used as the control. At 2 wk after transplantation, spermatocytes and round spermatids were detected in transplanted seminiferous tubules of the control, whereas the most advanced spermatogenic cells in those of old mice were spermatocytes. At 4 wk after transplantation, although elongated spermatids were detected in transplanted tubules of the control, haploid cells (spermatids) were still undetectable in those derived from old mice. Thus, meiosis was never restored, although spermatogonia of old mice can reinitiate differentiation into spermatocytes under suitable testicular conditions. Since it has been reported in several mammalian species that age-related changes in the testicular microenvironment lead to the gerontal cessation of spermatogenesis, the present results suggest that both a defective extratubular environment and a defective intratubular environment may cause the cessation of spermatogenesis in old BDF, mice.

Testicular morphology of a greater Indian rhinoceros (Rhinoceros unicornis).

The testis of a greater Indian rhinoceros (Rhinoceros unicornis) was examined by naked eyes and light microscopy. The animal sampled was estimated to be 42 years old. Testis was ellipse-shaped and weighed 1,300 g. Although a number of elongated spermatids were distinguishable in some seminiferous tubules, the lumen of seminiferous tubules was closed and connective tissues conspicuously increased in amount in the intertubular space. These findings in testicular morphology of the animal may be due to ageing.

GUST27 and closely related G-protein-coupled receptors are localized in taste buds together with Gi-protein alpha-subunit.

Gustatory, like olfactory signalling is probably mediated by seven-transmembrane receptors and coupling GTP-binding proteins (G proteins). We investigated the expression of a subset of these receptors and the Gi protein alpha-subunit by using their specific antibodies. Based on our previous finding that the mRNA for GUST27, one of these receptors, is expressed in rat lingual epithelia, we first prepared an antibody to the synthetic nonapeptide, H-Ser-Tyr-Ser-Gln-Ile-Ala-Ser-Ser-Leu-OH, which corresponds to the third intracellular domain of GUST27 and also to those of a subset of related receptors whose occurrence can be predicted by PCR. Immunohistochemical studies with rat circumvallate papillae indicated that the anti-GUST27 antibody reacted with many of the taste buds examined, with strong signals appearing in particular taste cells. We then carried out a similar immunohistochemical experiment with an antibody to the Gi protein alpha-subunit and found that this subunit is also expressed in taste buds as demonstrated in the case of gustducin and transducin. Taken together, these results strongly suggest that GUST27 and closely related receptors, as well as Gi alpha proteins, are involved in intracellular taste signal transduction.

Prognostic value of p53 protein accumulation in cancer cell nuclei in adenocarcinoma of the uterine cervix.

Adenocarcinoma of the uterine cervix (CxAd) is one of the most distressing malignancies of the female reproductive system because of its tendency to spread aggressively and to be resistant to radiation and systemic therapies. To clarify the prognostic significance of p53 alteration in CxAd, we immunohistochemically examined the incidence of p53 nuclear accumulation, which is considered to be mostly parallel with p53 gene mutation, and its association with clinicopathological parameters in 26 patients with CxAd. The overall incidence of p53 nuclear accumulation was 46% (12 of 26), being higher in groups with clinically advanced disease, higher degrees of cellular atypia, and deeper myometrial invasion, but significantly lower in patients with integration of human papillomavirus (HPV) type 16 or 18 DNA. Nuclear p53 immunoreactivity as well as lymph node status, depth of invasion and the absence of HPV-DNA integration were significant indicators of a poor prognosis. Examination of p53 nuclear accumulation could be applied to biopsy material, and would be of practical assistance in predicting the prognosis of CxAd both preoperatively and postoperatively.

Frequent occurrence of c-Ki-ras gene mutations in well differentiated endometrial adenocarcinoma showing infiltrative local growth with fibrosing stromal response.

The frequency of c-Ki-ras mutation was examined by polymerase chain reaction (single-strand conformation polymorphism and direct sequencing analysis) and its association with histological parameters was analyzed in 110 endometrial adenocarcinoma hysterectomy specimens. The c-Ki-ras mutation was detected in 24 cases (22%). It occurred irrespective of tumor size, stage or histological grade, and was more frequent in tumors associated with endometrial hyperplasia (11 of 20, 55%) than in those without (10 of 73, 14%). In addition, the frequency of the mutation was significantly higher in tumors showing an infiltrative growth pattern accompanied by a stromal response consisting of edematous fibrous tissue (19 of 58, 33%), than in those which revealed an expansive growth pattern without such a stromal reaction (five of 52, 10%). Even among tumors showing the infiltrative growth pattern, the frequency of c-Ki-ras mutation was especially high (48%) in grade I cases in comparison with grade II or III cases (20% and 19%). Mutational activation of the c-Ki-ras gene may act at two points in the developmental pathway of endometrial adenocarcinoma by enhancing evolution of hyperplasia to adenocarcinoma, and by producing an infiltrative growth with a fibrosing stromal response.