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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.

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.

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.

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.

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.

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.

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.