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Embryonic stem (ES) cells have been known to differentiate into various progenitor cells. In this study, we investigated the differentiation capacity of mouse ES cells into pancreatic hormone-secreting cells, and insulin-secreting cells. ES cells were cultured in Dulbecco's modified Eagle medium (DMEM) after removal of leukemia inhibitory factor (LIF) for 3 days and then transferred in DMEM supplemented with retinoic acid or activin A. When the culture of embryoid bodies (EBs) derived from ES cells in DMEM added with activin A (2 × 10-9 M or 2 × 10-10 M) for 5 days was performed, endoderm marker genes, GATA4, Sox17 and Foxa2 were expressed in the EBs. However, at 6 days of culture, expression of Sox17 was not observed. When EBs were cultured with activin A (2 × 10-9 M) for 6 days, and followed by 6 days of culture with retinoic acid (10-6 M), expression of the pancreatic cell marker genes, GATA4 and Fxa2, were continued, and pancreatic islet genes, insulins 1 and 2, glucagons and somatostatin, w...

In order to examine the effect of sex hormones on diabetic pathogenesis in female Spontaneously Diabetic Torii (SDT) rats, we performed oophorectomy on female SDT rats, administered female hormones after the oophorectomy, and measured body weight changes, plasma glucose concentration, and pancreatic histopathology. At 26 weeks of age, the body weight was significantly heavier in the oophorectomized group and significantly lighter in the estradiol benzoate (EB) administration group than in the sham-operated control group. Although glucose concentration did not significantly change in the oophorectomized group, it was significantly lower in the EB administration group than in both control and the oophorectomized group. Severe histopathological change was observed in the oophoretomized group but not in the EB administration group. Thus, EB blocked weight gain and pancreatic islet change that was induced by oophoretomy and it also lowered glucose concentration. These results suggest that estrogen plays a preventive role for diabetic pathogenesis in female SDT rats.

Purpose To establish an in vitro culture system for mouse round spermatids that models spermiogenesis and enables the assessment of oocyte activation ability.

There is growing evidence that the human amnion contains various types of stem cells. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In the present study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into pancreatic islet cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of pancreatic cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that Pdx-1, Isl-1, Pax-4, and Pax-6 showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that insulin, glucagon, and somatostatin were present in HADFIL cells following the induction of differentiation. These results indicate that HADFIL cell populations have the potential to differentiate into pancreatic islet cells. Although further studies are necessary to determine whether such in vitro-differentiated cells can function in vivo as pancreatic islet cells, these amniotic cell populations might be of value in therapeutic applications that require human pancreatic islet cells.

Under organ culture, female fetal gonads in mice cannot develop beyond the preantral follicle stage unless the follicles are individually isolated and cultured again. In this study, we investigated the effect of in vitro culture of female fetal gonads before transplantation on subsequent in vivo development. The gonads derived from female fetuses 12.5 days postcoitum were organ-cultured for 0, 7 and 14 days, and then were grafted underneath the kidney capsules of severe combined immunodeficient mice and recovered at 21, 14 and 7 days post-transplantation, respectively. The histological analysis of the grafts showed that the in vitro culture of the fetal gonads restricted follicular development to the antral follicle stage post-transplantation. In the grafts cultured for 14 days, particularly, no antral follicle was observed. However, the oocytes in these follicles had grown to around 65 microm in diameter and had competence to resume meiosis in vitro. When the fetal gonads were grafted after culture for 7 and 14 days, 13.0% and 6.8% of the oocytes progressed to the metaphase II stage, respectively. These data showed significant differences (P < 0.05) in comparison with the control group (25.3%). Our results indicate that the in vitro culture of female fetal gonads before transplantation affects the subsequent in vivo development of both follicular cells and oocytes, and in vitro oocyte maturation. However, this effect seems to be more severe in terms of follicular development when compared with oocyte growth and maturation.

Excessive production of reactive oxygen species (ROS) lead to oxidative stress in tissue or organ dysfunction. To confirm ovarian dysfunction by ROS, ischemia-reperfusion (I/R) treatment of the ovarian blood vessels was performed using experimental rats in this study. Preventive effect of superoxide dismutase (SOD) as a radical scavenger against ovarian I/R injury was also examined. Hormonal sensitivities to equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) in the ovary decreased after the I/R. Ovarian tissue and ovum destruction were also observed. These changes could be prevented by SOD administration. Our data suggest that I/R injury related to ROS production causes ovarian dysfunction. The dysfunction is prevented by administration of a radical scavenger.

Production of cloned laboratory animals is helpful in the establishment of medical models. In this study, we examined to produce reconstituted embryos derived from somatic cell nuclei, and to establish embryonic stem (ES) cell lines from the embryo in rabbits. Metaphase II (M-II) oocytes from superovulated rabbit were used as nuclear recipients. Nuclear donor cells were fibroblasts collected from a Dutch Beleted rabbit. The M-II chromosome and the 1st polar body were aspirated, and a fibroblast was inserted into the perivitelline space of the enucleated oocyte. The pairs were electrofused for cell membrane fusion using a cell fusion apparatus, and reconstituted embryos were produced. The embryos were activated and cultured in modified HTF medium and DMEM. The embryos developed to the blastocyst stage were removed their zona pellucida, and they were cultured on the feeder cell layer. As a result of having observed development of reconstituted embryos, 21.2% of the embryos were developed to the blastocyst stage. In the embryos cultured on the feeder cells, the adhesion on feeder cells was observed. We obtained inner cell mass (ICM) colony derived from reconstituted embryos. At present, we are investigating to establish the ES cell lines derived from the embryos reconstituted by nuclear transfer.

We successfully established two cell lines, an adenocarcinoma cell line (designated as HIGS) and Epstein-Barr virus-free normal B-lymphocyte cell line (designated as HIGS-BL), derived from a moderately to poorly differentiated adenocarcinoma of the stomach, and examined their characteristics. The tumor delivered to our laboratory from an operating room was cut into small pieces and cultured on the dishes. HIGS and HIGS-BL were established from each individual dish after the onset of primary culture. Although their culture methods were the same, the HIGS cell line was not established from the dishes growing HIGS-BL cells. In addition, HIGS-BL cells were scarcely observed in the HIGS cell dishes. Because of these factors, we have considered until now that HIGS-BL cells may inhibit the growth of HIGS cells or cause damage to HIGS cells by unknown mechanisms. Injection of HIGS-BL cells, other B-lymphocyte cell lines, or the conditioned media of HIGS-BL cells into nude mice bearing HIGS-grafted tumors was performed individually. When HIGS and HIGS-BL cells were co-cultured in the same dishes, HIGS-BL cells inhibited the proliferation of HIGS cells. The inhibition of grafted tumor growth was confirmed by the injection of not only the HIGS-BL cells but also the B-lymphocytes. Furthermore, this inhibition was only observed when the conditioned medium of B-lymphocytes was injected into the nude mice. These results suggested that the secretory products by general B-lymphocytes (including HIGS-BL) have some ability to inhibit the proliferation of HIGS cells. In addition, susceptibility tests to anti-cancer drugs suggested that HIGS cells were sensitive to CDDP, ADM and MMC, and HIGS-BL cells were sensitive to CDDP. If CDDP was used for chemotherapy in the patient, the drug produced atrophy of HIGS-BL cells. The study about HIGS and HIGS-BL cells reported the necessity for novel therapeutic approaches in oncotherapy.

We investigated the mitochondrial behavior, localization and heteroplasmy in reconstituted oocytes derived from germinal vesicle (GV) transfers. The karyoplast containing the GV nucleus and the ooplast (enucleated oocyte) were prepared from GV oocytes derived from B6D2F1 mice. Mitochondria in the karyoplast and ooplast were labeled with MitoRed (Dojindo Laboratories, Kummoto, Japan) and MitoTracker Green (Molecular Probes, Eugene, OR, USA), respectively. After labeling the mitochondria, the karyoplast and ooplast were paired and fused. The mitochondrial behavior in fused (reconstituted) oocytes during in vitro maturation and preimplantation development were observed using confocal laser-scanning microscopy. In reconstituted oocytes that had reached to the M-II stage, mitochondria localized and concentrated in the hemispherical area of oocytes containing the M-II spindle. We showed that the two types of mitochondria derived from the GV donor and the recipient in reconstituted oocytes exhibit similar behavior to the normal oocyte during meiosis, and that the mitochondrial heteroplasmy of these oocytes did not influence their in vitro maturation and preimplantation development.

Recently, the spermatogenic cell has been focused in male sterility. There are some reports on fertilization and embryonic developmental ability of spermatid. Deficiency of fertilization or developmental arrest frequently appears due to the ability. Functional immaturity is also observed in spite of abnormality of karyotype in spermatids. However, a functional difference between spermatids and spermatozoa is not clarified. In this study, fertilization and developmental ability of the spermatid were examined by the microinjection using immature spermatid including the round spermatid, elongating spermatid, elongated spermatid and intratesticular spermatozoa derived from three different mice strains; ddY, ICR and B6D2F1. Mature female mice were superovulated with PMSG and hCG. Cumulus-oosytes complex were obtained from their oviducts, and cumulus cells were removed from MII oocytes by hyaluronidase and pippeting. Spermatids were collected from mature male testis, and were injected into MII oocytes using a micromanipulator. Manipulated embryos were cultured in CZB medium, and their ability for development was examined. Epididymal spermatozoa from the three strains were used as control group. The fertilization rate was correlated with the spermatogenic stage. In embryonic developmental rate, on the other hand, there were no significant difference between the control and experimental embryos. The data however showed that elongating, elongated and round spermatid slightly deteriorated the embryonic development. These results suggest that the ability of the embryonic activation depend on the spermatid metamorphose.

This study was conducted to induce immunotolerance to human chorionic gonadotropin (hCG) as a heterologous protein in neonatal mice. Ovulatory responses after repeated induction of superovulation were also investigated in the animals. Subcutaneous injection of hCG (100 IU) was performed in female mice within 24 hours after birth. Serum anti-hCG antibody titers were assayed and ovulatory responses were observed after the repeated superovulation with pregnant mare serum gonadotropin (PMSG) and hCG.As a result, the production of anti-hCG antibody was suppressed. Thus, tolerance induction to hCG was achieved, although it was incomplete. Compared with the control mice, the ovulatory rate was higher after the induction of immunotolerance to hCG. It was suggested that the production of anti-hCG antibody has a close relation with a decrease in the ovulatory response after the repeated induction of superovulation by gonadotropins.

Mammalian oocytes undergo growth and development through prenatal and postnatal periods. This is an investigation of development in vitro of oocytes and ovarian tissues from fetal gonads of the 12.5 days post coitus. Cultured tissues survived and the oocytes entered the growth phase, subsequently achieving long-term (14-30 days) culture. Growing oocytes were observed in their general morphology and were enclosed within 1-2 layers of granulosa cells, which resembled primary or secondary follicles. However, no follicular structures were also found in the cultures despite the existence of growing oocytes. The oocytes enclosed in very flattened cells were detected as in histological structure on day 18 of culture. The oocyte diameter became approximately 50 μm under culture conditions supplemented with 15% fetal bovine serum or Ehrlich ascites tumor fluid. These results indicate that the maximum culture period for oocyte growth in this organ culture is approximately 3 weeks and that the oocytes can grow in vitro to at least a medium size even if normal follicular structure is not formed.

To investigate the developmental competence in vitro of female germ cells in fetal gonads, gonadal ridges of 12.5 days post coitus (dpc) were organ-cultured for 18 days, and then oocyte-granulosa cell complexes (OGCs) were isolated from the cultures. Collected OGCs were further cultured for 11 days. The oocytes grown in vitro reached 64.1 ± 0.4 μm. Transmission electron microscopy analysis showed that oocytes grown in vitro had maintained connection with surrounding somatic cells. Some oocytes resumed meiosis by 1 μM okadaic acid or 10 ng/ml epidermal growth factor (EGF). The oocytes that underwent germinal vesicle breakdown reached 71.3 ± 0.5 μm. This study showed growth and meiotic resumption in vitro of oocytes through the support of ovarian tissue from fetal gonads.

Ovarian follicles constantly undergo selection and sifting in the process of their development, and many ovarian follicles fall into atresia. Follicular atresia can be prevented by superovulation induction with gonadotropin (GTH), but this treatment causes ovulation of cleaved-unfertilized ova. In this study, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) reaction was performed in the ovary of mice in which superovulation was induced repeatedly using PMSG/hCG. The TUNEL reaction was positive in oocytes that showed cleavage already in developing follicle and in surrounding granulosa cells. Ovarian follicles developed by superovulation using GTHs include abnormal oocytes with cell fragmentation. This change is remarkably similar to apoptosis.

In a previous report, we showed that follicles isolated from frozen/thawed mouse ovarian tissues reached the mature follicle stage on the 12th day of culture. However, the developmental ability was lower than that of fresh ovarian tissue. The purpose of this study was to define a culture system with some technical modification for preantral follicles isolated from frozen/thawed ovarian tissue and to confirm cell injury. Ovaries obtained from three-week-old female mice were cryopreserved by the rapid freezing method. Preantral follicles isolated from frozen/thawed ovarian tissues were cultured for 12–16 days. The follicles were then stimulated with human chorionic gonadotropin. In vitro fertilization was performed on the released cumulus–oocyte complexes (COCs). Preantral follicle viability was assessed by supravital staining using Hoechst 33258. Using this stain cell death was found in part of the granulosa cells of a follicle obtained from frozen/thawed ovarian tissue. On the 14th and 16th days of culture, the diameters of follicles isolated from frozen/thawed ovaries were larger than on the 12th day of culture. The released COCs were fertilized and developed to the blastocyst stage in 15.8% (12/76) of the oocytes taken from the fresh group, and in 0% (0/73), 2.9% (2/69) and 19.1% (22/115) of the oocytes taken from the frozen/thawed group that had been cultured for 12, 14 and 16 days respectively. The preantral follicles isolated from frozen/thawed mouse ovarian tissues developed slowly compared with the freshly prepared preantral follicles. During prolonged culture from 12 to 16 days, these follicles obtained the potential to fertilize and develop to the blastocyst stage.

The Spontaneously Diabetic Torii (SDT) rat, a newly established animal model for diabetes mellitus, presents nonobese type 2 diabetes with ocular complications. In the present study, oral glucose tolerance tests and biochemical and histopathological examinations were performed in female SDT rats at 16 and/or 25 weeks of age, before the onset of diabetes. At 25 weeks of age, glucose tolerance was significantly impaired, and plasma immunoreactive insulin levels at 120 min after glucose loading were significantly higher (P< 0.05). Body weight and fasting levels of plasma triglycerides and nonesterified fatty acids were significantly higher than those in control animals. Histopathologically, inflammatory cell infiltration and fibrosis were observed in and around the pancreatic islets. These results strongly suggest that female SDT rats are useful as a model to investigate impairment of glucose tolerance and hyperlipidemia prior to the onset of diabetes.

There are many reports about the in vitro culture of spermatogenic cells, but no-one has succeeded in inducing the differentiation from spermatogonia to intact sperm. Also the study of in vitro testicular tissue culture has hardly advanced. We studied the culture of mouse immature testicular tissue derived from 5-day-old mice. We aimed to achieve the differentiation of spermatogenic cells in order to observe spermatogenesis in testicular tissue in vitro. We also froze mature testicular tissue and immature testicular tissue cultured for 2 weeks. Furthermore, spermatogenic cells differentiated by culturing were injected into metaphase II oocytes to determine whether these differentiated cells and frozen-thawed testicular tissue have fertilising and developmental ability. Under the culture conditions employed, secondary spermatocytes and a few round spermatids differentiated from spermatogonia were observed in the immature testicular tissue cultured for 2 weeks. When spermatogenic cells derived from cultured immature testicular tissue, cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were injected into ooplasm, the oocytes were fertilised and fertilised oocytes developed to the 8-cell stage. We suggest that spermatogenic cells derived from cultured immature testicular tissue have fertilising and developmental abilities equivalent to that of sperm. Also these abilities of spermatogenic cells obtained from cultured frozen immature testicular tissue and frozen-thawed mature testicular tissue were better than those of the same cells before freezing.

Cryopreservation of ovarian tissues containing many immature oocytes occurs in both gamete/embryo research and clinical medicine. Using vitrification, we studied factors related to meiosis after cryopreservation using the COCs (cumulus oocyte complexes) and preantral follicles obtained from cryopreserved ovarian tissues. COCs were isolated and cultured for 17 approximately 19 hr. Thereafter, Metaphase II stage (MII stage) oocytes and fertilized oocytes after IVF were observed at a rate of 76.5% and 60.0%, respectively. Preantral follicles (100 approximately 130 microm in diameter) were isolated and cultured in alpha MEM containing hFSH, ITS, and FBS. HCG and EGF were added to the media to stimulate ovulation on the 12th day of culture. The survival rates of the follicles obtained from the frozen/thawed ovaries were 66.4%. After 12 days of culture, the diameter of the follicles isolated from fresh (620.2 +/- 11.3 microm) and frozen/thawed ovaries (518.7 +/- 15.1 microm) differed as did the estradiol concentrations (3474.2 +/- 159 pg/ml vs. 1508.2 +/- 134 pg/ml). After in vitro ovulation, MII stage oocytes were observed in 84.5% of the fresh group and 60.5% of the frozen/thawed group while the fertilization rate was 74.2% and 53.5%, respectively. These studies demonstrate that cryopreservation of mouse ovarian tissues by vitrification did not affect the oocyte's ability to undergo meiosis. Thus, this technique may become a powerful tool for the preservation of the female gamete.

Mitochondria (mt) have been reported to be closely related to the maturation of mammalian oocytes, but their function in oocyte maturation has not been elucidated. In this study, we examined the kinetics of mt and chromatin configuration during in vitro maturation of mouse oocytes to clarify the relationship between oocyte maturation and mitochondrial distribution morphologically. Oocytes were recovered from 6-to 8-wk-old ICR strain female mice. Germinal vesicle (GV) -stage oocytes were divided into 4 groups and cultured: group A, oocytes collected after pregnant mare serum gonadotropin (PMSG) injection; and group B, oocytes collected after PMSG-human chorionic gonadotropin injection. Groups A and B were subdivided into 2 groups: denuded oocytes (DO) and cumulus-enclosed-oocytes (CEO). At 0, 4, 8, 12 and 16 h from the onset of the culture, oocytes were fixed and stained to visualize alpha-tubulin, chromatin and mt using confocal laser scanning microscopy (CLSM). It was observed that mt aggregated around the nucleus from the GV-stage through progression to germinal vesicle breakdown (GVBD). With the movement of the nucleus, mt were concentrated around the nucleus and polarized. The maturation rate (the rate of the first polar body extrusion) and the fertilization rate of CEO were significantly higher than that of DO in both groups A (p

In nuclear transplantation, especially in enucleation, rotating the oocyte to a certain position has been an important work. Therefore, we developed an oocyte rotation system by using electrostatic force, which is known to be excellent in handling minute objects. This system has become the basic technology for the automated nuclear transplantation system. Mouse oocyles were rotated at a speed of about 60 deg/s by electrostatic phenomena (i.e. electrorotation and dielectrophoresis), and the direction of rotation could be reversed. In order to confirm the electrostatic effect on the fertilization and developmental ability of oocytes, we performed IVF and nuclear transplantation with rotated oocytes. The result was that the fertilization and developmental ability of the rotated oocytes was the same as that of the control, proving that rotation by electrostatic force does not influence the fertilization and development ability of oocytes.

We have previously reported that human amniotic epithelial (HAE) cells expressed neuronal and glial cell markers using immunostaining and western blotting. To study the expression system of these cell markers in HAE cells, we investigated the expression of mRNA for oligodendrocyte markers in HAE cells by reverse-transcriptase-polymerase chain reaction (RT-PCR) and northern blotting. Neural cell-type-specific expression system was used to examine the transcriptional activity of myelin basic protein (MBP). Oligodendrocyte markers were expressed such as CNPase, MBP and proteolipid protein (PLP and DM-20). PLP gene transcripts in the cells were in a lower level than DM-20, compared with those of human brain. Neural cell-type-specific expression system disclosed HAE cells were about 20% positive for β-Gal using AdexMBP-NL-LacZ. This strongly indicates that HAE cells have MBP-specific gene expressing cells.

A square case containing ova and culture medium was vibrated with a voice coil motor to concentrate the ova at the center of the case. Only those ova which conform to the memorized shape are then selected with a picture processing device and are positioned one by one at the point of a holding pipette. A system with the functions mentioned was experimentally constructed. This system can automatically position any ovum in a certain position, thereby shortening the time of the process to search out an ovum and position it at the point of the holding pipette.

Prolidase deficiency is an autosomal recessive inherited disease characterized clinically by frequent infections, mental retardation, and various skin lesions. Fundamental treatments for these manifestations have not been established. We performed adenovirus-mediated gene transfer of human prolidase cDNA into fibroblasts from patients with prolidase deficiency. Infection with the adenovirus vector carrying human prolidase cDNA increased prolidase activity in fibroblasts up to approximately 7.5 times of that of normal control fibroblasts. This indicates the feasibility of adenovirus-mediated gene therapy to treat patients with prolidase deficiency in the future.

Aim: Taxanes are regarded as key chemotherapy agents for breast cancer and gynecologic malignancies; therefore the ovarian toxicity of paclitaxel (PTX) is a matter of importance to younger women with malignancies. We examined the ovarian toxicity of PTX and its influence on fertility in rats. Methods: Female Wistar rats aged 6–8 weeks received five doses of PTX at 3-day intervals. Ovarian toxicity was assessed by counting follicles, corpora lutea and atretic follicles, as well as by detecting apoptosis and measuring serum levels of estradiol (E2) and progesterone (P4). Fertility was assessed by mating females immediately or 24 days after PTX treatment. The number of fetuses, implantations and resorption sites was counted in each group. Results: Exposure to PTX caused a decrease of antral follicles, but not primordial or pre-antral follicles. The number of corpora lutea showed a significant decrease, while follicular atresia was increased significantly. Apoptosis was only detected in antral follicles. Serum E2 levels were lower than in control rats, but not significantly, while P4 levels did not differ from those in control rats. Rats mated immediately after PTX treatment showed a significant decrease of fetuses and implantations, but these effects were not detected in rats mated at 24 days after treatment. Conclusion: Our findings suggest that the ovarian toxicity of PTX is mild and transient. Use of PTX may help to maintain the fertility of younger women because the fertility of rats was not influenced at 24 days after exposure to this drug.

This study was undertaken to determine the expression time of fertilization and oocytes activation abilities of spermatids in the mouse. When elongating or elongated spermatids isolated from fresh testes of adult males (B6D2F1) were injected into mature mouse oocytes, both spermatids could activate the mature oocytes and occur fertilization. On the one hand, the round spermatids could not activate mature oocytes, when microinjected into oocytes. In some experiments, recovered round spermatids were cultured under co-culture systems using Sertoli cells as a feeder cell. Under the co-culture system, developed elongating spermatids could stimulate and fertilized mature oocytes. These results indicate that the start of oocyte activation appearance is between the stage of round spermatid and elongating spermatids and the activation ability increases with the advance of spermiogensis. On the other hand, round spermatids isolated from males of ICR strain mouse already have the oocyte activation ability and the fertilizing ability. The result obtained suggests that the expression time of the oocyte activating ability is difficult between the mouse strain.

We successfully established a novel cell line (OS-1) derived from human ovarian small cell carcinoma, hypercalcemic type secreted PTH, PTH-rP and ACTH. The OS-1 cell line was established from metastatic focus of uterus. A patient was 25-year-old Japanese woman. The first she received left ovariectomy on April 2002. The histopathological diagnosis was ovarian small cell carcinoma, pT2c, Nx, Mx. Then on June 2003, metastatic focus of uterus was ectomied. A part of the recurrent tumor of uterus was cut into small pieces with razor blades, and dissociated with 0.1% trypsin-0.02% EDTA/ PBS(-) solution at room temperature. The single cells and small cell clusters were seeded into 60mm dishes and cultured in growth medium (GM: DMEM/F12 supplemented with 20% fetal bovine serum and 0.1% non-essential amino acids solution) at 37 degrees C, 4.7% CO2 in humidified air. Medium was exchanged twice a week. The OS-1 cells grew as floating cultures in the dishes. Radioimmunoassay of the conditioned media was revealed that the cultures secreted large amount of PTH, PTHrP and ACTH simultaneously. Susceptibilities of anti-cancer drugs to the OS-1 cells were examined using oxygen electrode meter (Daikin), and the results suggested VLB and TXL were effective, and CDDP, CPT-11, VP-16, VCR, CPA, MMC and CBDCA were not effective. In our knowledge, it is the first report that the cell line secreting PTH, PTHrP and ACTH was successfully established from ovarian small cell carcinoma, hypercalcemic type. We expect that OS-1 cell line contribute to study on the mechanism of ectopic hormone secretion and susceptibility of anti cancer drugs to the small cell carcinoma.

The optimum dose for establishing superovulation in mice of Fertirelin Acetate (FA), an LH-RH analogue, was examined. Mice were subcutaneously injected with 5 IU of hCG at 17:00 (Day 0), and with various doses of FA (0.001 to 1.0 microg) five times at 4 h intervals on and after 22:00 on Day 0. To induce ovulation, 5 IU of hCG was again injected subcutaneously at 17:00 on Day 2. In the groups administered with doses ranging from 0.01 to 0.5 microg of FA, the number of ovulated eggs was significantly (p0.05) larger than in the control group (12.9 +/- 5.9). The greatest number of ovulated eggs (22.6 +/- 7.3) was obtained in the group administered with 0.025 microg of FA. The results indicate that the effective dose of LH-RH analogue, FA, is between 0.1 and 0.5 microg for superovulation induction in mice.

A nerve cell line designated NC-HIMT was established from a HIMT cell line derived from a benign ovarian, three germ layer immature teratoma removed from a 21-year-old Japanese female. The HIMT cells were elongated, ellipsoid or spherical in shape, whose karyotype was on the high side of normal diploidy. Small amounts of retinoic acid enhanced differentiation and maturation of the HIMT cells into nervous tissue, and the NC-HIMT cell line was established by the colony isolating technique when the HIMT cell line was cultured in the presence of retinoic acid-supplemented medium. After establishment, the NC-HIMT cell line was cultured and maintained in retinoic acid-free growth medium. Even though these cells were cultured without retinoic acid, the phenotype of nerve cells remained and the cells were also maintained in a state of high normal diploidy. The nerve cells contacted each other with their long cell projections and formed networks. Immunocytochemical observations using anti-bovine NSE, alpha-internexin, neurofilament 200kD, peripherin and GFAP confirmed that the cells were either nerve cells or glia cells. These results assume that HIMT cells, which were derived from an immature teratoma, have progenitor and/or stem cells which can differentiate into nerve and/or glial cells.

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.

Regenerative medical treatment with embryonic stem cells (an ES cell) is a goal for organ transplantation. Structures that are tubular in nature (i.e. blood capillaries) were induced from early embryonic stem (EES) cells in vitro using embryotrophic factor (ETFs). In addition, cardiac muscle cells could be identified as well. However, differentiation of EES cells into a complete cardiovascular system was difficult because 3 germ layer primordial organs are directed embryologically in various ways and it is not possible to guide only cardiovascular organs. Thus, we introduced ETFs after the formation of an embryoid body and were successful in cloning cell clusters that beat, thus deriving only cardiovascular organs. The application of this to the treatment of various cardiovascular diseases is promising.

A cell line with the characteristics of hepatocytes was established from rat early embryonic stem cells (REES). This cell line was established using a new novel method of Ishiwata et al. from two cell embryos taken from the spontaneous dwarf rat (SDR). The hepatocyte cell line (REES-hep) was instituted from dark red colored tissue in embryos during embryogenesis using REES cell line cultured in the presence of embryotrophic factors. These cell lines were cultured with DMEM/F12 medium supplemented 10% FBS and 1 ng/ml of LIF. They were found to maintain their diploid state, were characterized with 42 normal chromosomes and proliferated to confluence; contact inhibition was also present. These cells produced albumin when cultured using a collagen sponge gel system and reconstructed in a funicular form resembling the cell cords of liver. The cells also produced albumin and bilirubin when transplanted into the spleen of SDR Reconstruction of a REES-hep cell line from early embryonic stem cells should help in treating hepatic insufficient patients. It will be valuable for further research, as an introduction to cell transplantation and application for use in a bio-hybrid typed liver apparatus.

During cryopreservation of ovarian tissue, the conditions of freezing and thawing are big factors controlling the survival rate of oocytes obtained. However, the conditions and procedures as they pertain to ovarian follicles and oocytes have not been established. Thus, we tried to determine the appropriate freeze — thaw times using the vitrification method with ethylene glycol and DMSO as cryoprotective agents and dd Y female mouse ovaries. The maturity rate from GV to the metaphase-II (MII) stage was 62.8% with ethylene glycol and 69.3% using DMSO, while the controls (GV oocytes obtained from a fresh ovary) showed a maturation rate of 83.6% (46/55). MII oocytes obtained by culturing GV oocytes in vitro showed a 64.3% (18/28) fertility rate via in vitro fertilization and a developmental rate into a 2 cell stage embryo of 35.7% (10/28) and into a 4-cell stage, 7.1% (2/28). However, development beyond the 8 cell stage embryo was not observed. A significant difference was not recognized between control (fresh) and ovarian tissues that had been frozen/thawed with respect to their ability to produce hormones. It is concluded that the vitrification method was effective for both freezing ovarian tissues and preserving its functional ability (maturation and capacitation).

This study was carried out to transform embryonic stem (ES) cells and to produce the reconstituted embryos derived from transgenic ES cell nuclei. Then, in vitro/vivo developmental potency of transgenic ES cells were compared to that of control ES cells (non-transgenic ES cells) in the reconstituted embryos. Unfertilized B6D2F1 ooplasm at metaphase II (M II) and two kinds of ES cell lines, 129SV and transgenic (tg) 129SV transformed by EGFP gene, were used as nuclear recipients and nuclear donors, respectively. The M II chromosome-spindle complex was aspirated into the pipette with a minimal volume of ooplasm. After enucleation, the ES cell nuclei was injected into the enucleated ooplasm directly. Then, reconstituted embryos were activated in SrCl2, and they were cultured in HTF medium. There was no difference of developmental rate between reconstituted embryos derived from the control (non-transgenic) and the tg ES cells. From this result, we indicated that transgenic ES cells might not change the property of peculiarity of the ES cell by gene transfer. The expression of GFP gene in the embryos was observed by fluorescence microscope at the 4-cell and more stage. As comparison with development of the embryos derived from the control and tg ES cells, the difference of the development could not be confirmed between the two cell groups. When the reconstituted embryos derived from the control and tg ES cells were transferred into oviduct or uterus of pseudopregnant females, fetuses were observed 13.5 days post coitum. However, in all fetuses, developmental arrest and regression were seen 19.5 days post coitum.

We developed a biological cell rotation system using electrostatic force, which is excellent for manipulating a minute object. This system is the important element of an automated nuclear transplantation system. This system could rotate a mouse's oocyte three dimensionally. And, it was confirmed that the rotation by electrostatic force did not influence fertilization and development ability of the oocyte.

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm, and ectoderm) of the organism, including the germline. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. In this study, we investigated the induction of mouse ES cell differentiation, using culture of embryoid bodies (EBs) into the diverse tissues. EBs were formed by culturing ES cells (129/SV strain) in DMEM supplemented with 10% FBS, in the absence of feeder cells and leukemia inhibitory factor (LF). EBs were induced to differentiate by treatment with retinoic acid (RA). In control medium (non-RA medium) beating muscles, blood vessels, hemocytes, and cartilages were frequently observed in EBs. Moreover, when EBs were cultured in medium including RA (5 x 10(-8) M, and 5 x 10(-9) M), differentiation of the optic vesicle, lens, retina, and neural groove was observed. In this study we demonstrated that an efficient system for inducing the differentiation of ES cells using EBs.

We cloned mouse prolidase cDNA from a mouse liver cDNA library. Homology to human prolidase is 83.2% at the nucleotide level and 87.2% at the amino acid level. Northern blot analysis showed that while prolidase mRNA was transcribed in brain, heart, liver, and muscle, it was predominantly transcribed in kidney.

We developed a new type 3-Dimensional Internal Structure Microscope (3D-ISM) for the observation of internal structures of samples. The internal structure of a sample is obtained by cutting it into thin slices and observing the cutting side continuously while cutting. The position of the camera, as well as the sample position are fixed. Therefore there is no shift between the sections, and this system can obtain a true color image of the sample, which is a high resolution and a high-quality three-dimensional image compared with X-ray CT and MRI. After repeatedly slicing a sample, the digital image data from the sectional views is transferred to a computer, where 3-dimensional images of the internal structure of the sample are reconstructed. Using this system we analyzed many biological organisms. In this paper, a mouse specimen has been cut and the 3-dimensional images are shown.This article presents the outline of the device and the principle of the observation. FIG.l shows a corresponding block diagram and Fig. 2 a schematic view of the 3D-ISM.

Experiments were designed to determine whether pmsg is essential for the continued maturation of follicles whose initial development is stimulated by the hormone in immature mice. Anti-pmsg serum (aps), prepared in rabbits, was used at various times after pretreatment with pmsg to determine the time required for pmsg to mature follicles. Follicular maturity was tested by the ovulatory response to hcg. When hcg was given 54 to 60 hr after pmsg, maximum ovulation was obtained. When aps was given 54 hr, followed by hcg 60 hr, after pmsg, the percentage ovulation rate decreased significantly from that of the corresponding control group in which aps and hcg were given simultaneously 54 hr after pmsg. When hcg was given 72 hr after pmsg, ovulation did not occur regularly. When a small amount of supplementary pmsg was given 58 hr after the initial dose, ovulation was induced regularly by hcg 72 hr after pretreatment with pmsg. From these experiments, it is concluded that a small amount of pmsg remains in the circulating blood up to 54 to 60 hr after the pretreatment with pmsg in immature mice. Blood levels of pmsg decrease below the threshold for the maintenance of follicles 72 hr after pretreatment.