Ovarian toxicity of paclitaxel and effect on fertility in the rat.

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.

Establishment and characterization of EB virus-free normal B-lymphocyte and interleukin-6-producing poorly differentiated adenocarcinoma cell lines derived from gastric tumor tissue.

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.

In vitro follicular development of cryopreserved mouse ovarian tissue.

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.

Optimum dose of LH-RH analogue Fertirelin Acetate for the induction of superovulation in mice.

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 (p<0.05) 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.

Development of reconstituted embryos derived from somatic cell nuclei in the rabbit.

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.

Establishment and characterization of a nerve cell line (NC-HIMT) from HIMT cells derived from a human ovarian immature teratoma with special reference to the induction of neuron differentiation by retinoic acid.

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.

Establishment and characterization of a human ovarian small cell carcinoma, hypercalcemic type, cell line (OS-1) secreting PTH, PthrP and ACTH--special reference to the susceptibility of anti-cancer drugs.

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.

Organogenesis of heart-vascular system derived from mouse 2 cell stage embryos and from early embryonic stem cells in vitro.

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.

New approach for the establishment of an hepatocyte cell line derived from rat early embryonic stem cells.

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.

In vitro differentiation of mouse embryonic stem cells after activation by retinoic acid.

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.

Development of reconstituted embryos derived from transgenic embryonic stem cell nuclei.

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.

Establishment of a novel method for cryopreservation and thawing of the mouse ovary.

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).