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.

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.

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.

Multiple genes for G protein-coupled receptors and their expression in lingual epithelia.

Using the polymerase chain reaction (PCR), we identified a gene family including more than 60 members which encoded similar G protein-coupled seven-transmembrane receptors. Sequence analyses of six representatives out of the 60 PCR clones showed that they had significant structural similarity to olfactory and optic receptors. Their expression is restricted in the surface of lingual epithelia.

High dose rate brachytherapy for carcinoma of the uterine cervix: comparison of two different fractionation regimens.

PURPOSE: There is no consensus as to the best dose-fractionation regimen in high dose rate (HDR) brachytherapy for cervix cancer. Since 1983, two fractionation regimens have been used in different time periods at National Cancer Center Hospital, and their treatment results have been compared in terms of 5-year survival, local control, and complication rate to find the better therapeutic regimen. METHODS AND MATERIALS: From November 1983 to October 1990, 130 patients with uterine cervix carcinoma were treated with HDR intracavitary brachytherapy using a remote afterloading system. There were 21 Stage Ib patients, 5 Stage IIa, 29 Stage IIb, 2 Stage IIIa, 68 Stage IIIb, and 5 Stage IVa. The median age was 64 years. The median follow-up time was 50 months. Radiotherapy consisted of external beam irradiation to the pelvis (mean dose of 50 Gy), combined with HDR brachytherapy (mean dose of 20 Gy to point A) given 5 Gy per session twice weekly (group A: 54 patients) or 6 Gy once weekly (group B: 76 patients). RESULTS: The overall 5-year survival was 52% in group A and 72% in group B. Local recurrence rate was 11%, and distant failure rate was 21%, with no difference between the two groups. The complication rate was significantly lower in group B (37%) than in group A (55%). Multivariate analysis has shown that factors affecting survival were stage, brachytherapy dose, and local control status. No factor was predictive of local control, but the external beam radiation dose significantly influenced the risk of complications. CONCLUSION: The once-weekly HDR intracavitary applications combined with properly adjusted external beam pelvic irradiation is a safe and effective treatment for patients with uterine cervix cancer.

Carcinoma of the uterine cervix treated with external irradiation alone.

One hundred and four out of 2701 patients with carcinoma of the uterine cervix were treated with a curative intent by external irradiation alone at the National Cancer Center Hospital from 1962 to 1979. All patients were judged inappropriate for the combined treatment of intracavitary and external irradiation, which was the treatment of choice for patients with advanced carcinoma of the uterine cervix in the hospital. The 5-year survival rate was 17% overall and 36, 17, and 5% for patients with Stage II, III, and IV disease, respectively. The local control rate was 20%, at 2 years, for all patients. Major complications were observed in five patients. There were no major complications in patients given a total dose of less than 115 in the Time Dose Fractionation factor (TDF). External irradiation combined with interstitial irradiation and/or hyperthermia is being considered to improve the results.

High-dose-rate intracavitary irradiation in the treatment of carcinoma of the uterine cervix: early experience with 84 patients.

Eighty-four patients with previously untreated invasive carcinoma of the uterine cervix were treated by high-dose-rate intracavitary irradiation using a remotely controlled afterloading system (Ralstron) with or without external irradiation at the National Cancer Center Hospital, Tokyo, between 1977 and 1981. Survival rates and local control rates were comparable to those for 372 patients treated by low-dose-rate intracavitary irradiation with or without external irradiation from 1972 to 1981 at the hospital. The incidence of major complications was 5.1 and 2.4% for the patients treated by low-dose-rate intracavitary irradiation and by high-dose-rate irradiation, respectively. The results are comparable to those reported by other institutions. We have abandoned the conventional low-dose-rate intracavitary irradiation with the impression that the high-dose-rate remotely controlled afterloading system is a good alternative to the conventional one.

Peritoneal cytology in endometrial cancer - an investigation of the source of endometrial cancer-cells in the peritoneal-cavity.

In order to identify the source of endometrial cancer cells in the peritoneal cavity, the incidence of positive peritoneal cytology was assessed according to pTNM classification. In 74 cases free peritoneal fluid in the pelvic cavity was aspirated. In the absence of fluid smears of the cul-de-sac was made by scraping (45 cases). Five of 15 positive aspirated cases were pT1N0 or pT2N0. These five cases underwent surgery alone and are still alive with no evidence of disease. Malignant cells which supposedly gain access to the pelvic cavity via the fallopian tube have low potential for implantation.

Age-related changes in cytoskeletal components of the BDF1 mouse Sertoli cell.

Age-related changes in mouse Sertoli cell cytoskeletal components (F-actin, vimentin and cytokeratin) were investigated by light and transmission electron microscopy and immunofluorescence using BDF1 mice from 3-33 months of age. In old mice (30 and 33 months of age), the testicular seminiferous epithelia were extremely thin, containing scarce round spermatids and spermatocytes with no elongated spermatids. In these epithelia, the Sertoli cells had lost their polarity and had become flattened. F-actin was detectable at the junction between adjoining Sertoli cells and around the spermatid head in young mice. In old mice, F-actin was distributed at the junction between adjacent Sertoli cells, around the spermatid head, and at the luminal side of the Sertoli cell cytoplasm. Vimentin was detected around the Sertoli cell nucleus and extended into the Sertoli cell trunk towards the tubular lumen in young mice. In old mice testes, however, vimentin was recognized around the Sertoli cell nucleus, but not in the Sertoli cell trunk. Additionally, sheet-like reactions of vimentin, running parallel to the basement membrane, were detected near the luminal surface. Although cytokeratin was not detected in the Sertoli cells of mice until 27 months of age, it was obvious in the extremely thin seminiferous epithelia of older mice. Cytokeratin was randomly distributed within the Sertoli cell cytoplasm. In these Sertoli cells, the expression of vimentin was concurrently detected. Detection of cytokeratin in the extremely thin seminiferous epithelia is one of the most characteristic phenomena of age-related testicular changes in Sertoli cells of older mice.

Age-related morphological changes in the testis of the BDF1 mouse.

Age-related morphological changes in the testis of the BDF1 mouse were studied by light and transmission electron microscopy. No apparent changes were detected until 12 months of age. After 18 months of age, vacuoles firstly appeared in the seminiferous epithelium. These vacuoles were gradually increased in number and showed a tendency to cluster with each other in accordance with age. While, germ cells were decreased in number. The sloughing of germ cells caused a thin seminiferous epithelium. In the tubule with a thin epithelium, spermatogenesis was severely interrupted. After 30 months of age, extremely thin seminiferous epithelia were observed. In these epithelia, most of spermatids and spermatocytes disappeared, and most of Sertoli cells lost their polarity to be flattened. On the other hand, in the interstitial region, PAS-positive cells (mononuclear phagocytes) tended to increase in number after 24 months of age. PAS-positive extracellular matrix newly appeared at 27 months of age. In the cytoplasm of Leydig cells, a whorl of sER was frequently found. Degeneration of testes proceeded with age. The regressive tubules occupied only 2.2% at 18 months of age, but extended to 63.0% at 33 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.

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.

Production of term offspring by in vitro fertilization using old mouse spermatozoa.

We successfully produced offspring of old male BDF1 mice using in vitro fertilization. Although 7 old male mice (33 months of age) were infertile and revealed frequent degeneration in the seminiferous epithelia, 4 of them had spermatozoa in the caudae epididymides. The IVF rate of their sperm with eggs from young ICR mice was very high (82.0%). The production rate of offspring was also satisfactory (61.6%). The present study indicates that the IVF method is useful for producing offspring of aged male mice.