PubMed Health⌕ Search

Biomedical subjects

K Narimoto

Publications and source records attributed to K Narimoto.

17 recordsLinked to original sources
PubMed1993

Role of protein supplements in the culture of mouse embryos.

The effect of various types of proteins used in single protein supplements for Bigger-Whitten-Whittingham (BWW) medium on the in vitro development of mouse preimplantation embryos was evaluated. Thioredoxin, superoxide dismutase (SOD), and apotransferrin showed prominent growth-promoting activity, whereas bovine serum albumin (BSA), fatty acid-free BSA, and catalase showed moderate promoting effects. beta-lipoprotein, ovalbumin and hemoglobin were ineffective, and holo-type transferrin and ceruloplasmin were actually toxic to the embryos. These results suggest that the growth-promotive effect of proteins on mouse pronuclear stage embryos is due to their antioxidative action, or to the removal of some free metal ion(s) such as Fe(3+). The mild growth promoting effect of both BSA and fatty acid free BSA suggest that the effect mediated by BSA is not dependent on bound fatty acids, but more likely is due to their antioxidative effect or chelating effect.

Journal Article↗
PubMed1993

Development of hamster one-cell embryos recovered under different conditions to the blastocyst stage.

One-cell stage embryos, recovered from superovulated golden hamsters (8 to 12 weeks of age) 12 hours after egg activation, were cultured in HECM-1 medium at 37 degrees C and 5% CO(2) in air. The culture conditions investigated were the time and temperature required for embro recovery, the pH shift of the washing medium, and the oxygen concentration of the gas phase during and after embryo recovery. Each condition was assessed by the developmental efficiency of the embryo as determined by morphological criteria. As the time required for embryo recovery was reduced, the developmental rates of the embryos were improved: 2.3% (3 128 ) 26.9% (35 130 ) at 5 and 3 minutes, respectively, as determined by the number of embryos developed to the blastocyst stage. No blastocysts were obtained when more than 10 minutes were required for embryo recovery. As the oxygen concentration was reduced from 40 to 20% or to 5%, rather high developmental rates were obtained even when the time required for embryo recovery was prolonged: 6.9% (9 130 ) and 21.7% (28 129 ) of the embryos developed to the blastocyst stage when they were recovered under 5% oxygen within 10 and 5 minutes, respectively. Neither the temperature during embryo recovery (37 degrees C and 25 degrees C) nor the pH shift (pH 7.22 to 7.52) of the washing medium used in embryo recovery procedures influenced the development of the embryos. These findings suggest that the developmental block in hamster embryos may involve oxidative stress, which may result from exposure to high oxygen concentration and light during the manipulation of oocytes and embryos.

Journal Article↗
PubMed1992

Effects of oxygen toxicity on early development of mouse embryos.

To examine the effects of oxygen toxicity on embryonic development, mouse pronuclear embryos were cultured under low oxygen conditions with or without superoxide dismutase (SOD), and the blastulation rate was compared with that of embryos cultured under standard conditions. The blastulation rate of mouse pronuclear embryos cultured under standard conditions was only 1.5% (2/131). This rate was increased significantly, to 28.5% (43/151), when the embryos were cultured under low oxygen conditions; and to 31.0% (35/113) when SOD (500 micrograms/ml) was added to the medium under standard conditions; the rate was increased to 75.2% (115/153) when the embryos were cultured under low oxygen conditions in the presence of SOD. The minimum effective concentration of SOD in the culture medium was 50 micrograms/ml under conditions of 5% O2. The blastulation rate was significantly decreased after 1-hr exposure of pronuclear embryos to room atmospheric oxygen concentration (20% O2), and subsequent culture under 5% O2 with SOD did not result in an improved blastulation rate. Culture with SOD under 5% O2 promoted the development of two-cell stage embryos to the blastocyst stage. When two-cell stage embryos were collected 48 hr after hCG and cultured for 66 hr, their blastulation rate was similar to that of embryos collected from mice 114 hr after hCG. These results suggested that embryonic development in vitro is greatly affected by atmospheric oxygen throughout the early embryonic stages and that this harmful effect can be prevented by culturing embryos under low oxygen conditions and in the presence of SOD.

Animals↗
PubMed1992

Effects of superoxide dismutase on mouse in vitro fertilization and embryo culture system.

PURPOSE: We recently found that, for mouse embryos fertilized in vivo, the two-cell block could be attenuated by adding superoxide dismutase (SOD), a scavenger of superoxide radicals, to the culture medium. In this study, we evaluated the effects of SOD on the process of fertilization and on the further development of the embryos fertilized in vitro. METHODS: We performed incubation of mouse epididymal spermatozoa, in vitro fertilization, and further cultivation in Biggers-Whitten-Whittingham's medium supplemented with various concentrations of Cu.Zn-SOD. RESULTS: High concentrations (2000 micrograms/ml or more) of SOD prevented loss of motility in mouse sperm over time. The addition of SOD (less than 2000 micrograms/ml) to the basic medium showed no significant difference in the fertilization rate. Also, no significant difference was observed in the rate of polyspermy or parthenogenesis between the basic and the SOD-supplemented media. However, 18% of the two-cell-stage embryos developed to the expanded blastocyst stage in the 500 micrograms/ml SOD-supplemented medium, while no blastocysts were found in the basic medium. Furthermore, the addition of SOD 7 hr after insemination increased the expanded blastocyst rate (28%). CONCLUSIONS: These results indicate that the addition of SOD exerts a protecting effect from oxidative stress both on sperm viability and on the development of embryos fertilized in vitro as well as in vivo, while its addition showed no effect on the process of fertilization.

Animals↗
PubMed1992

Effect of visual light on in vitro embryonic development in the hamster.

The effect of light exposure during collection and culture of hamster embryos on their subsequent development in vitro was examined. When embryos were collected under dark conditions (70 lux) within 10 minutes and then cultured in a HECM-1 medium in 5% CO2 in air, the developmental rates of 1-cell embryos to the 4- and 8-cell stages were 88.6% (93/105) and 66.7% (70/105), respectively. These rates were significantly higher than those under light conditions (1600 lux): 51.9% (56/108) and 34.3% (37/108). Light irradiation during the culture of 1-cell embryos suppressed subsequent development. The degree of suppression correlated inversely with duration of light irradiation, and light irradiation of 30 minutes or more completely blocked development to the 2-cell stage. When 1-cell embryos were irradiated through a yellow filter, cutting the light wavelengths to less than 500 nm, embryonic development was still suppressed. However, the degree of the suppression varied and 45.7% (53/116), 6.0% (7/116), and 0.9% (1/116) of the embryos developed to the 2-, 4-, and 8-cell stages, respectively, under 30 minute light irradiation. Inhibitory effects of light irradiation on the development of 2- and 8-cell embryos were also observed, showing an inverse correlation with duration; the developmental rates of 2-cell embryos to the 8-cell stage under 0, 10, and 30 minutes of irradiation were 85.6% (107/125), 1.6% (2/122), and 0% (0/129), respectively, and those of 8-cell embryos to the blastocyst stage were 79.8% (91/114), 74.8% (86/115), and 0% (0/110), respectively. These findings indicate that early-stage embryos are sensitive to light exposure; however, severe light exposure adversely affects the development of embryos at any stage. Thus, the protection of embryos from light exposure at all stages of embryo manipulation, from collection to culture, is essential.

Journal Article↗
PubMed1992

Oxidative stress on mouse embryo development in vitro.

Oxygen radicals are involved in the in vitro block phenomenon of embryo development, because a low oxygen tension and superoxide dismutase (SOD) have been shown to promote the in vitro development of mouse embryos. One of the target molecules damaged by oxygen radicals may be the thiol (SH) group of proteins because it is readily oxidized. In this study, we evaluated the effects of thioredoxin, which is a powerful protein disulfide reductase, on mouse (Institute of Cancer Research, ICR) preimplantation embryo development. Culture of mouse pronuclear embryos recovered 17 h after human chorionic gonadotrophin (hCG) administration in the presence of thioredoxin (200 micrograms/mL) significantly increased the blastulation rate (75.3%) when compared to the control culture system (8.9%). The effects of thioredoxin were observed only from the pronuclear stage to the two-cell stage (17-48 h after hCG administration). An additive effect of thioredoxin and SOD, or thioredoxin and a low oxygen tension, was observed. These results suggest that the oxidation of the SH group of proteins is one of the causes of developmental blockage of embryos in vitro. The target protein for reduction by thioredoxin has not been identified yet, but thioredoxin will be a new clue for clarifying the mechanism of blocking development in vitro.

Amino Acid Sequence↗
PubMed1992

Localization of CuZn-superoxide dismutase in the human male genital organs.

We performed an immunohistochemical analysis using a polyclonal antibody to determine the localization of CuZn-superoxide dismutase (SOD), a scavenger of superoxide anion radicals, in the human male genital organs. In the testis, intense immunoreactivity of CuZn-SOD was shown in both the cytoplasm and nucleus of the spermatogonia of the seminiferous tubules. Spermatocytes, further differentiated germ cells and Sertoli cells showed no or weak immunoreactivity. In the ductus epididymis, the principal cells showed no or weak immunoreactivity except for the stereocilial region, while the basal cells showed relatively intense immunoreactivity. In the ductus deferens, the prostate and the seminal vesicles, columnar and cuboidal epithelia showed CuZn-SOD immunoreactivity. The immunoreactivity was more intense in the epithelia of the ductus deferens than in the prostate or the seminal vesicles. Basal cells in the prostate also showed intense immunoreactivity. Collectively, the present immunohistochemical results suggest that CuZn-SOD in the male genital organs is localized where it could play an important role in cell differentiation, including spermatogenesis. The CuZn-SOD could also play a role in local defence mechanisms against tissue damage mediated through superoxide anion radicals, as well as in providing SOD to the seminal plasma.

Adult↗
PubMed1992

Pregnancy achieved by transferring blastocysts into endometrial stroma in mice.

To prevent the extra-uterine discharge of transferred embryos, we directly inserted mouse embryos into the endometrial stroma (intra-endometrial embryo transfer). A 27G injection needle was inserted near the utero-tubal junction into the endometrial stroma. After removal of the needle, a glass micropipette was inserted and one embryo was transferred with a very small amount of culture medium. To determine the feasibility of this method, the uterine lumen was flushed with phosphate-buffered saline from the tubal ends immediately after transferring blastocysts into pseudopregnant mice on day 2 and day 4. The rates of recovery of embryos from the uterine lumen were 5.0% (1/20; day 4) and 15.0% (3/20; day 2). These results suggest that a high rate of intra-endometrial embryo transfer is possible. The embryonic viability rates (number of viable grown fetuses/number of blastocysts transferred) of this method were 50.0% (28/56; day 4) and 25.0% (5/20; day 2). Living offspring were delivered from both recipients which had received embryos on day 2 and day 4 of pseudopregnancy. In human in-vitro fertilization and embryo transfer, attempts have also been made to immobilize the embryos, and this method might be clinically applicable. Moreover, this method will be a good in-vivo model for studies on the mechanism of implantation.

Abortion, Veterinary↗
PubMed1992

Release of two-cell block by reduction of protein disulfide with thioredoxin from Escherichia coli in mice.

The development of mouse pronuclear-stage embryos in media containing various concentrations of thioredoxin was monitored and the influence of antithioredoxin immunoglobulin G (IgG) and heat-treated thioredoxin on the thioredoxin-induced effects was evaluated. A significant increase in the number of four-cell embryos (76.3%) and blastocysts (37.3%) was observed when embryos were cultured in the medium containing 50 micrograms thioredoxin ml-1 compared with the rates (55.8 and 3.8%, respectively) in the basic medium. The number of blastocysts increased significantly to a maximum of 70.2% at 500 micrograms ml-1. The biological activity of thioredoxin was evident after dialysis, but was markedly impaired by the addition of anti-thioredoxin IgG to the culture medium. Treatment at 60 degrees C for 5 min did not affect the enzymatic and biological activity of thioredoxin. More severe heat treatment (121 degrees C for 30 min) attenuated the enzymatic activity to 40% of its initial value and reduced the biological activity (number of blastocysts, from 77.8 to 51.6%). These results indicate that the effect of thioredoxin on the two-cell block is due to the thioredoxin molecule itself, and suggest that disulfide formation within or between proteins resulting from oxidative stress is one of the major causes of the two-cell block.

Animals↗
PubMed1991

Developmental potentiality of embryos cultured under low oxygen tension with superoxide dismutase.

A low-oxygen culture of preimplantation embryos with superoxide dismutase (SOD) has been demonstrated to produce a marked increase in the blastocyst formation. In this study, we examined the effects of low oxygen-SOD cultures of embryos on the potentiality of embryonic development after intrauterine transplantation and the reproductive ability of the offspring. Mouse pronuclear embryos were cultured in Biggers-Whitten-Whittingham's solution containing 0.3% bovine serum albumin and 500 micrograms/ml SOD under a low-oxygen condition (5% O2, 5% CO2, 90% N2), and 37.2% (110/296) of the embryos developed to blastocysts, with a significant difference from the 1.3% (3/231) under an atmospheric oxygen condition without SOD (P less than 0.01). The blastocysts obtained under a low oxygen-SOD culture condition were transplanted by direct insertion into the uterus of pseudopregnant females 3 days after infertile coitus. The implantation rate was 65.0% (65/100), and the embryo viability rate was 46.0% (46/100). In the control group, in which in vivo developed blastocysts were transplanted into 3-day pseudopregnant females, the implantation rate was 72.5% (81/112), and the embryo viability rate was 51.8% (58/112). The differences between the two groups were not significant. Significant differences also were not observed in either the body weight of pups at birth and 35 days after birth or the mean number of pups delivered by successive mating of the offspring between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Immunohistochemical localization of superoxide dismutase in the human ovary.

To elucidate the physiological function of superoxide dismutase (SOD) in the ovary, we examined the immunohistochemical distribution of CuZn-SOD in the human ovary. We also measured the CuZn-SOD concentration in human follicular fluid by enzyme-linked immunosorbent assay (ELISA). The germinal epithelium and tunica albuginea showed no or weak immunoreactivity. No or weak staining activity was also observed in the non-antral follicle. Once the follicle began to form the antral cavity, theca interna cells began to show intensive immunostaining of SOD, as compared with no staining in the granulosa and theca externa cells. In the gestational corpus luteum, theca and granulosa lutein cells showed intensive and moderate staining activity, respectively. The concentration of CuZn-SOD was 0.222 +/- 0.186 ng/mg protein (mean +/- SD) in the preovulatory follicular fluid. In the present study, the immunohistochemical distribution of SOD was confirmed in the human ovary for the first time. Taking into consideration the fact that SOD catalyses the dismutation reaction of superoxide anion radicals, the present results suggest that theca interna cells play an important role in the protection of the developing oocyte from oxygen radicals by acting as a blood-follicular barrier during follicle maturation.

Adult↗
PubMed1991

Block to development in cultured rat 1-cell embryos is overcome using medium HECM-1.

Rat pronuclear embryos were cultured in hamster embryo culture medium-1 (HECM-1) or a modified Krebs-Ringer bicarbonate solution (mKRB). Embryo cultures in HECM-1 were also challenged with low oxygen concentrations. In HECM-1, 57.9% (70/121) of the pronuclear embryos developed into the 4-cell stage after 48 h of culture. The rates of 8-cell, morula and blastocyst formation were 32.2% (39/121), 17.4% (21/121) and 9.9% (12/121), respectively. On the other hand, in mKRB, rat pronuclear embryos showed developmental blockages at the 2-cell and 4-cell stages, and never developed beyond the 4-cell stage. The rate of blastocyst formation under a low oxygen concentration was 20.1% (43/214), showing a significant difference from the value of 5.5% (11/201) obtained under a standard oxygen concentration (P less than 0.005). This is the first report of successful culture of rat pronuclear embryos to the blastocyst stage. Furthermore, it is suggested that protection from oxidation stress is a prerequisite for rat embryo development in vitro.

Animals↗
PubMed1991

Protection from oxidative stress by thioredoxin and superoxide dismutase of mouse embryos fertilized in vitro.

A low oxygen tension and superoxide dismutase (SOD) have been demonstrated to promote development of embryos in vitro. In this study, we evaluated the effects of thioredoxin (TRX), a powerful protein disulphide reductase, on the development of mouse embryos fertilized in vitro. Moreover, to clarify the difference between the embryo-protecting mechanisms of TRX and SOD, as well as the critical periods of their actions, we carried out experiments in which we transferred embryos among three different media: basic, TRX- and SOD-supplemented. The rates of 4-cell, blastocyst and expanded blastocyst formation in TRX- (53%, 40% and 34% respectively), or SOD- (59%, 29%, 22%) supplemented media were significantly higher than those (28%, 4%, 0%) in the basic medium. The results of the transfer of embryos using basic and TRX-supplemented media indicated an increase in the rate of expanded blastocysts when TRX was applied from 7 to 30 h, and especially from 18 to 24 h after insemination. In contrast, the results of the transfer of embryos using basic and SOD-supplemented media indicated that SOD, which was also most effective when applied from 7 to 30 h after insemination, had some effects after 30 h. These results indicate that TRX and SOD promote the in-vitro development of mouse embryos fertilized in vitro, which suggests that protection of embryos from oxidative stress is a prerequisite for their development in vitro. SOD and TRX provide protection from oxidative stress by different mechanisms.

Animals↗
PubMed1991

The expression of c-kit protein during oogenesis and early embryonic development.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and was shown to be allelic with the white-spotting locus (W) of the mouse. Mutations at the W locus have pleiotropic effects on the development of hematopoietic stem cells, melanoblasts, and primordial germ cells. In order to elucidate the role of c-kit protein in gametogenesis and oocyte maturation, we have examined immunohistochemically the expression of c-kit in the ovaries of mice at late fetal and postnatal stages, and in early embryos. By the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody, the c-kit protein was detected in ovaries after the time of birth, but not before. The expression of c-kit was observed mainly on the surface of oocytes, but not in granulosa cells nor in interstitial regions. Oocytes of primordial to fully grown Graafian follicles showed the c-kit protein. When ovulation was induced by hCG, the expression of c-kit in ovulated unfertilized oocytes was weaker than in oocytes of Graafian follicles. In 1-cell embryos the c-kit protein was still observed, but with cell division its expression further decreased, and it was not detected in embryos of 4-cell, 8-cell, and morula stages. In summary, the highest expression of c-kit was observed on the surface of oocytes arrested in the diplotene stage of meiotic prophase. With ovulation and the resumption of meiotic maturation, its expression declined. These results suggest that the c-kit protein may play some role in meiotic arrest, oocyte growth, and oocyte maturation.

Animals↗
PubMed1987

Functional differentiation in steroidogenesis of two types of luteal cells isolated from mature human corpora lutea of menstrual cycle.

Enriched small and large cell fractions were prepared from mature corpora lutea from 15 women in the midluteal phase by enzymatic dissociation, followed by Percoll gradient centrifugation. The steroidogenic function of each cell type was assessed by measuring the gonadal steroids released into the incubation medium. The large cell fraction was estimated to be 97% pure, with minimal contamination by small cells, whereas the small cell fraction was approximately 68% pure, being contaminated with 10% large cells and 22% nonsteroidogenic cells. In the unstimulated state, large cells were approximately 2-fold more potent in progesterone formation and aromatase activity, but only half as potent in androstenedione and testosterone formation as an equal number of small cells. When stimulated with hCG, the small cells responded with significant increases in progesterone, androstenedione, and testosterone release, but the large cells did not. Both cell types secreted estrone and 17 beta-estradiol in the presence of androgen substrate, but the addition of FSH significantly stimulated aromatization only in large cells. Thus, small and large human luteal cells have steroidogenic properties similar to those exhibited by follicular thecal and granulosa cells, respectively.

Androstenedione↗
PubMedDate not supplied

Analysis of oviduct-derived embryonic growth stimulator activity.

A co-culture study was carried out using primary cultures of hamster oviduct tissues to analyze oviduct-derived embryonic growth stimulating factor. In co-cultures of mouse pronuclear embryos and hamster oviduct tissue, the 4-cell rate after 48 hours (53.2%, 109/205) and blastocyst rate after 96 hours (22.4%, 46/205) were significantly higher than those of the standard culture group (4-cell rate, 41.8%, 76/182; blastocyst rate, 4.4%, 8/182), suggesting an embryonic growth stimulating effect of the co-culture. Similar effects were observed in co-cultures of both 2-cell mouse embryos collected 40 or 48 hours after the hCG administration and morulae collected 64 hours after hCG, but no such effect was observed in co-cultures of hamster embryos. When mouse embryos were cultured in a conditioned medium, which was the supernatant of the hamster oviduct tissue culture in Biggers-Whitten-Whittingham's (BWW) solution, no embryonic growth stimulating effect was observed. Further, no stimulating effect was observed in the conditioned medium obtained from 24-hour co-cultures of embryos and oviduct tissue. From these results, we considered the embryonic growth stimulating factor to be labile, with concentration-dependent effects. The absence of a stimulatory effect of co-culturing on the growth of hamster embryos also suggests the presence of species variations in the in vitro arrest of embryogenesis.

Animals↗