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Studies on the Rapid Freezing of Mouse Embryo II. Effects of the Development Stage and Seeding on the Mouse Embryo Survival of Rapid Freezing (Mouse 수정란의 급속동결에 관한 연구 제II보 Mouse수정란 급속동결에 있어서 수정란의 발육단계와 식빙(seeding)이 생존율에 미치는 영향)

  • 강만종;김영훈;문성호;김중규
    • Korean Journal of Animal Reproduction
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    • v.13 no.3
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    • pp.141-148
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    • 1989
  • The effects of seeding method and optimum time for freezing embryos according to the developmental stages on embryo survival rates after rapid freezing were determined using the FDA-test. The summarized results are as follows : 1. In the rapid freezing of embryos, the sucrose added medium together with Co-seeding or non-seeding showed the FDA scores of 4.67 and 4.20, respectively, but, raffinose addition obtained FDA scores of 4.27 and 3.97. 2. The developmental stage of embryos at freezing was most critical on the survival of embryos after thawing. Higher FDA scores were obtained in the order of blastocyst stage(4.94), morula stage(3.82) and ealy stage(2.65) in sucrose added medium. The same trend was observed in the raffinose added medium with an order or 4.91, 4.47 and 2.32. 3. Microscopic study of embryo before freezing and post-thawing indicated that the embryo showed shrinkage within 5 minutes after the embryo was transfer to the freezing medium. When thawed embryo was tranfered to the dilution medium, swelling of the embryo was observed and there after it reshrank indicating the removal of cryoprotectant from the embryo. The size of the embryo recovered to the original state when it was moved into a PBS-solution.

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Effect of Mature Human Follicular Fluid on the Development of Mouse Embryos in vitro (성숙난포액을 이용한 생쥐배아의 발달에 관한 연구)

  • Park, S.Y.;Lee, J.J.;Kim, S.H.;Ku, P.S.
    • Clinical and Experimental Reproductive Medicine
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    • v.19 no.2
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    • pp.125-131
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    • 1992
  • The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

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The Effect of Various Vitrification Methods on Developmental Rate of Mouse Pronuclear Embryos at Different Recovery Times (다양한 유리화 동결 방법이 각 시간대별 생쥐 전핵기 배아의 발달에 미치는 영향)

  • Kim, Ji-Chul;Seo, Byoung-Boo;Park, Sung-Baek;Kim, Jae-Myeoung
    • Journal of Embryo Transfer
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    • v.27 no.1
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    • pp.63-69
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    • 2012
  • The objective of this study was to investigate the effectiveness of cryopreservation methods for the effect of various vitrification containers, such as EM-grid, OPS, or cryo-loop on the survival and developmental rate of vitrified mouse pronuclear embryos, and mouse cleavage embryo, at 21, 24, 27 and 30 hr after hCG injection. Post-thaw cleavage was similar among treatments, while the developmental rates of mouse blastocyst and hatched blastocyst were higher ($p$ <0.05) in 27 hr and 30 hr than 21 hr. The developmental rate of hatched blastocyst at vitrified cleavage mouse embryos in cryo-loop was significantly higher than vitrified pronuclear embryos of control group as well as EM-grid and OPS ($p$ <0.05). The developmental rate using cryo-loop was higher than EM-grid, but in case of OPS at vitrified cleavage and mouse pronuclear embryos, no significant difference was noticed. These results of our study show that the developmental rates of mouse embryos were unaffected by various vitrification containers, but in case of mouse embryos and hatched blastocysts at late vitrified pronuclear embryos the developmental rates were higher than early vitrified pronuclear embryos. Moreover, the developmental rate of hatched blastocyst at vitrified cleavage mouse embryos was significantly higher than vitrified pronuclear embryos. For better execution of this study, it will be mandatory to include improvement of vitrification containers, cryopreservation methods and conditions, higher survival rate, safe preservation, contamination and embryo loss.

Effects of Glucose and IGF-I on Expression of Glucose Transporter 1 (Glut1) and Development of Preimplantation Mouse Embryo (생쥐의 착상전 배아의 발생과 Glucose Transporter 1 (Glut1) 발현에 대한 포도당과 IGF-I의 영향)

  • 전한식;계명찬;김종월;강춘빈;김문규
    • Development and Reproduction
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    • v.2 no.2
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    • pp.205-212
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    • 1998
  • A sodium-independent facilitative glucose transporter 1 (Glut1) is a major route by which glucose can be transported across the plasma membrane of mouse embryo. Although it has been known that insulin-like growth factor-I (IGF-I) promotes glucose transport into the mouse embryo, whether IGF-I directly regulates transcription of Glut1 has been uncovered in mouse preimplantation embryo. This study was aimed to elucidate the role of glucose and IGF-I in development and Glut1 expression in preimplantation mouse embryo. Two-cell embryos developed in blastocyst regardless of the glucose in the presence of pyruvate. IGF-I significantly increased the number of blastomeres in the mid-blastula. Deprivation of glucose did not affect the amount of Glut1 transcripts in morula cultured from 2-cell embryo. IGF-I potentiated Glut1 expression in morula cultured from 2-cell embryo even in the absence of glucose. Taken together, it is concluded that depletion of glucose does not promote Glut1 expression the in morula cultured form 2-cell embryo, and that increment of Glut1 expression possibly mediates embryotropic effect of IGF-I on preimplantation mouse embryo.

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Cryopreservation (Vitrification) of Mouse Embryos (마우스의 배의 동결보존)

  • 강민수
    • Journal of Embryo Transfer
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    • v.6 no.2
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    • pp.30-36
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    • 1991
  • The method of vitnilcation has various merits. It needs neither seeding nor slow freezing. It can freeze embryo by putting it directly into liquid nitrogen at the indoor temperature to $0^{\circ}C$. The operation process is quite easy. Moreover, higher promise of survival can be expected as there is no physical damage by any lumps of ice with the exception of cells. In Kasal's experiment (1990) using EFS liquid and Kang's experiment (1991) using GFS liquid the ratio of the damaged embryo was only 2-3%. But, the method of vitrification is now on the process of improvement, and the final or united method is not yet established. At the present time, most of the major institutes all over the world are using the traditional freezing method in the preservation of mouse embryo, but it is very likely that the vitrification will prevaIl in the near future considering the various merits of it. Calves can be begotten from the embryo by means of vitriilcated preservation in the cases of cow, rat, and rabbit as well as of mouse. In addition, recent experiments have shown that vitrificated preservation was successful in the case of drosophila embryo which was much bigger than mammalian embryo, which fact tells that this method is expected to be preferably used even in the preservation of living organs in the near future.

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Optimization of Embryo Density and the Volume of Culture Medium for an Improvement of Mouse Parthenogenetic Embryo Development

  • Roh Sangho;Choi Young-Joo;Min Byung-Moo
    • Reproductive and Developmental Biology
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    • v.29 no.3
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    • pp.145-147
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    • 2005
  • Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of $2{\~}4$ embryos/${\mu}L$ significantly improved development to the blastoryst stage ($72{\%}{\leq}$) compared with culture at the lower ($0.2{\~}1$e mbryos/${\mu}L,\;0\~37.5\%$) and the higher ($5{\~}6$ embryos/${\mu}L,\;30\~53\%$) concentration for 120 h when the oocytes were cultured in a 5 ${\mu}L$ drop under mineral oil In experiment 2, the embryos cultured at a concentration of $2{\~}4$ embryos/${\mu}L$ in a 10 ${\mu}L$ drop ($81.1{\%}$) showed significantly higher blastocyst rates than those in a 5 ${\mu}L$ drop ($68.5{\%}$). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

Studies on the Aggregation of H-Y Antibody-Sexed and Bisected Rabbit Embryo (H-Y항체에 의한 토끼배의 성 감별과 이등분 절단 토끼배의 융합에 관한 연구)

  • 최화식;임경순;진동일
    • Korean Journal of Animal Reproduction
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    • v.21 no.2
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    • pp.85-93
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    • 1997
  • These experiments were carried out to examine the development capacity of sexed and then bisected embryo from 8-cell to morula stage. Antisera to histocompatibility-Y(H-Y) antigen were prepared in inbred SD female rat by repeated immunization of spleen cell or testis supernatant from males of same strain. Male and female embryos were separated by delaying development of embryos against H-Y antibody. After sexing, rabbit embryos were bisected and aggregated. The results obtained from the these experiments were summuerized as follows: 1. When mouse and rabbit 8-, 16-cell and morular embryos were culature in H-Y antiserum, the ratio of embryo which has developed to hatching blastocyst was 53.4, 46.3 and 57.4% in mouse embryos, and 49.0, 52.0 and 61.0% in rabbit embryo, respectively. The ratio of mouse and rabbit embryos developed to hatching blastocyst showed nearly natural sex rate(50%), except rabbit mourla showed a little higher ratio(61.0%) as compared to natural sex ratio. 2. When rabbit demi-embryos from 8-, 16-cell embryo and morula were cultured, the percentage of demi-embryos was 70, 68 and 58% without zona pellucida removed, and 62, 69 and 51% with zona pellucida. The rate of aggregation was higher in 8- and 16-cell demi-embryos than in morula demi-embryo. 4. When sexed-demi-embryo was aggregated with another demi-embryo with demi-embryo with same sex, the rate of embryo developed to blastocyst was 60, 50 and 25%, respectively. Eight-cell demi-embryo showed highest rate. In conclusion, it showed that H-Y antiserum which was made by rat spleen cell enabled sexing rabbit embryos. And when rabbit sexed 8-, 16-cell and morula demi-embryo were aggregated, they were developed to eu-blastocyst which suggested the potential of sexed embryo manipulation.

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Study on the sexing of preimplantation mouse embryo exposed to H-Y antisera II. Sexing of mouse embryos by immunofluorescence assay (H-Y항체에 의한 생쥐초기배의 성판별에 관한 연구 II. 간접면역형광측정법에 의한 성판별)

  • 양부근;장정순;김정익
    • Korean Journal of Animal Reproduction
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    • v.12 no.1
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    • pp.37-41
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    • 1988
  • These studies were carried out to examine the sex of preimplantation mouse embryo. For the investigation of sex-ration of mouse embryos, morula and blastocysts stage embryos treated with H-Y antiserum (10%, v/v) and FITC anti-mouse-IgG were divided into the positive and negative embryos. Positive and negative identified embryos were observed the viability according to the in vitro cultured and the sex ratio was also investigated by chromosomal analysis. The results obtained in these studies were summarized as follows: 1. Two hundred sixty-seven recovered embryos of morula or blastocyst stage were incubated in medium containing H-Y antiserum and FITC anti-mouse-IgG. Positively or negatively identified embryos were 139 and 128. This trend indicated the approximal sex ratio was 1:1. 2. Sex ratio was measured using the embryos treated with indirect immunofluorescence assay to examine the relationship between embryo developmental stage and sex ratio. Sex ratio of morula stage embryos was 45.2% positive and 54.8% negative, on the other hand, the ratio switched to 56.4% positive and 43.6% negative embryo in blastocyst stage. 3. Fourty-seven positive and 57 negative embryos were obtained out of 104 morula stage embryos treated with indirect immunofluorescence assay. Survived positive or negative embryos during in vitro culture were 42 and 49, respectively out of 47 and 57 embryos. 4. The numbers of negative and positive embryos were 171 and 92 out of 163 blastocyst embryos which were incubated in the medium containing H-Y antiserum and FITC anti-mouse-IgG. The result of karyotype test showed the successful rate of sexing embryo is positive and negative embryos was63.0% (58/92) and 62.0% (44/71). The final female to male ratio within 58 positive embryos was 22.7:77.6, and the ratio of the 44 negative embryos was 77.3:22.7.

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Histological and Ultrastructural Differentiations of the Neuroepithelial Cells in the Mouse Embryo during Early Neurulation (마우스 초기 신경배형성과정에서 신경상피세포의 조직학적 및 미세구조적 분화)

  • Kim, Yul-A;Chung, Young-Wha
    • Applied Microscopy
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    • v.29 no.1
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    • pp.105-124
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    • 1999
  • Histological and ultrastructural differentiations of the neuroepithelial cells in the mouse embryo during neurulation were observed. The neural plates and grooves consisted of pseudostratified columnar epithelium in the embryonic day (ED) 8 embryo were developed. In the ED 9 embryo, the neural tube was developed in all body length of embryo except both the cephalic and caudal ends. Secondary neurulation was shown at the tail bud of the ED 10 embryo. In the ED 8 embryo, the primitive streak was shown in the posterior end of the embryonic disc. The neuroepithelium, notochord and mesenchyme were well differentiated in the cephalic and cervical portions. In the ED 9 and 10 embryos, the roof plates of neural tubes were constituted of the closing of the surface ectodermal cells in the hindbrain and the neuroepithelial cells in the spinal cord. The floor plate of neural tube were consisted of the low pseudostratified columnar epithelium. The spinal motor nerve fibers were initially differentiated in the ED 10 embryo. According to the electron density of the cell and the differentiation of tell organelles, the neuroepithelial cells in the ED 9 and 10 embryos were classified into three types: dark, intermediate and light types. All types in the ED 9 embryo were observed but the dark cell in the ED 10 embryo was not done. The free ribosomes and polysomes in all neuroepithelial cells were developed. The RER and lipid droplets in the dark cell and the Golgi complex in the intermediate and light cells were observed. Many microfilaments in the cytoplasmic processes of intermediate cell and the microfilaments and microtubules in the light cell processes were observed to be well differentiated.

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