• Clinical and Experimental Reproductive Medicine
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• v.32 no.1
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• pp.65-70
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• 2005

본 연구는 이식 후 남은 잉여의 포배기 배아를 두 번의 냉동과 융해 과정을 반복적으로 실시한 후 이식한 결과에 관한 보고이다. 사람 포배기 배아의 동결보존에서 높은 생존율과 성공적인 임신율이 보고되고 있으나 미성숙 난자로부터 발달한 포배기 배아에 두 번의 초급속 냉동 방법을 실시한 후 이식한 보고는 되어 있지 않다. 이에 본 연구에서는 다낭성 난소 증후군 환자에게서 얻은 미성숙 난자로부터 발달한 포배기 배아를 artificial shrinkage 후 초급속 냉동함으로써 생존율을 높이는 방법을 이용하여 재냉동 이식하였을 때 임신에 성공한 증례를 보고하고자 한다. 29세의 환자로부터 채취한 55개의 미성숙 난자들(germinal vesicle stage oocytes)을 체외배양 하여 성숙한 37개의 난자들로부터 30개의 수정란을 얻을 수 있었다. 12개의 배아가 포배기 배아까지 발달하였으며 이 중 3개의 양질의 포배기 배아를 선별하여 이식하였고, 이식을 한 후에 남은 9개의 포배기 배아들은 artificial shrinkage의 과정을 마친 후에 초급속 냉동 방법을 이용하여 동결보존 하였다. 그 중, 4개의 포배기 배아들을 융해한 후 이식을 하지 않고 다시 재냉동을 하여 보관하였고 이 후 재냉동 되었던 4개의 포배기 배아들을 다시 융해 하여 이식을 한 결과 임신이 되어 건강한 남아를 분만하였다. 이로써 미성숙 난자로부터 얻은 포배기 배아가 두 번의 냉동과 융해의 과정을 통해 크게 손상을 입지 않고 생존할 수 있다는 것을 알 수 있었다. 그러므로 융해이식 후 남은 잉여의 포배기 배아를 다시 냉동 보관하여 다음 주기에 이용함으로써 축적된 임신율을 증가시킬 수 있을 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• v.14 no.2
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• pp.109-118
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• 1987

Ninety-one patients with irreparable tubal disease underwent in Vitro Fertilization-Embryo Transfer (IVF-ET) in Seoul National University Hospital. Ovulation was stimulated in 104 cycles by human menopausal gonadotropin (HMG) or follicular stimulating hormone (FSH)/human chorionic gonadotropin (HCG). The patients were classified as high (>900 pg/ml), intermediate (400-900 pg/ml), or low (<400pg/ml) responder according to preovulatory $E_2$ response and four $E_2$ patterns were found. The overall pregnancy rate per cycle in this consecutive series was 11.5% (n=12). The number of preovulatory oocytes per cycle was higher significantly in intermediate and high responder group than in low responder group (P<0.01), While the number of immature oocytes per cycle significantly higher in low responder group than high and intermediate responder group (P<0.01). The pregnancy rate in each responder group was not signigicant. According to the $E_2$ pattern of response, there was no significant difference in number of the immature and preovulatory oocytes recovery per cycle. There was a apparently direct relationship between the preovulatory $E_2$ pattern and pre gnancy rate was noted.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.345-353
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• 1999

In the studies on the hatching mechanisms in mammals, many investigators focused on the embryonic intrinsic factor(s) in in vitro culture, but the uterine environment as the extrinsic factor(s) is thought to play an important role in hatching mechanism. Therefore, to evaluate the effect of uterine environment on the hatching event in vivo, the immature(GV) and ovulated(MII) oocytes, and the late 2-cell embryos of mouse were transferred to pseudopregnant foster mother's uterus during peri-implantation period. So it was verified whether there would happen hatching by only uterine environment independently on embryonic stage. The ultrastructural changes of the zona surface of transferred group were compared with those 01 in vivo and vitro group by SEM. 36 hrs after transfer, the immature and ovulated oocytes almost degenerated, and the late 2-cell embryos developed to various embryonic stages. However, the embryos which didn't develop to blastula stage did not hatch. The ultrastructural network of ZP in transferred group seemed to be smoothed uniformly, which was different from in vitro group. In conclusion, it is suggested that the uterine environment during peri-implantation period enhances the embryo hatching by provoking the structural change of ZP.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.1-5
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• 2014

Recent 2 decades, including in vitro maturation (IVM), assisted reproductive technologies (ARTs) achieved noteworthy development. However the efficiency of ARTs with in vitro matured oocytes is still lower than that with in vivo oocytes. To overcome those limitations, many researchers attempted to adapt co-culture system during IVM and consequently maturation efficiency has been increased. The beneficial effects of applying co-culture system is contemplated base on communication and interaction between various somatic cells and oocytes, achievement of paracrine factors, and spatial effects of extracellular matrix (ECM) from somatic cell surface. The understanding of co-culture system can provide some information to narrow the gap between in vitro and in vivo. Here we will review current studies about issues for understanding cu-culture system with various somatic cells to improve in vitro maturation microenvironment and provide bird view and strategies for further studies.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.105-113
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• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.237-241
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• 2012

Embryonic genome activation (EGA) is the first major transition that occurs after fertilization, and entails a dramatic reprogramming of gene expression that is essential for continued development. Although it has been suggested that EGA in porcine embryos starts at the four-cell stage, recent evidence indicates that EGA may commence even earlier; however, the molecular details of EGA remain incompletely understood. The RNA polymerase II of eukaryotes transcribes mRNAs and most small nuclear RNAs. The largest subunit of RNA polymerase II can become phosphorylated in the C-terminal domain. The unphosphorylated form of the RNA polymerase II largest subunit C-terminal domain (IIa) plays a role in initiation of transcription, and the phosphorylated form (IIo) is required for transcriptional elongation and mRNA splicing. In the present study, we explored the nuclear translocation, nuclear localization, and phosphorylation dynamics of the RNA polymerase II C-terminal domain in immature pig oocytes, mature oocytes, two-, four-, and eight-cell embryos, and the morula and blastocyst. To this end, we used antibodies specific for the IIa and IIo forms of RNA polymerase II to stain the proteins. Unphosphorylated RNA polymerase II stained strongly in the nuclei of germinal vesicle oocytes, whereas the phosphorylated form of the enzyme was confined to the chromatin of prophase I oocytes. After fertilization, both unphosphorylated and phosphorylated RNA polymerase II began to accumulate in the nuclei of early stage one-cell embryos, and this pattern was maintained through to the blastocyst stage. The results suggest that both porcine oocytes and early embryos are transcriptionally competent, and that transcription of embryonic genes during the first three cell cycles parallels expression of phosphorylated RNA polymerase II.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.123-130
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• 1997

Normal maturation of the mammalian oocytes is prerequisite for the fertilization and the early embryonic development. We have been tested the effects of purine and its de novo synthetic inhibitor, azaserine(Aza) on the maturation of germinal vesicle(GV) and germinal vesicle breakdown(GVBD) mouse oocytes. Denude-immature oocytes were cultivated in the media containing adenosine, guanosine, and/or azaserine, and checked the matruation stage by monitoring the prominent morphological changes. In GV stage oocytes, GV was arrested temporarily by the adenosine(1.0%) and protractedly by the guanosine(65.9%, P<0.001). The regression was increased significantly at the adenosine(90%, P<0.001) but decreased at the guanosine(1.6%, P<0.05). Inhibiting the de novo synthesis of purine, nuclear maturation rate was increase(90.4% : 96.7%), but GV arrest was significantly increased by cotreatment with guanosine(P<0.001). Polar body extraction significantly was increased at the Aza(P<0.05), but not in others. In GVBD oocytes, adenosine itself did not affect GVBD arrest. Guanosine, on the other hand, elevated GVBD arrest rate(P<0.001), but co-treated with Aza, decreased GVBD arrest(P<0.001). Aza increased GVBD arrest rate(20.2%, P<0.05) compared with control. From those results, we know that guanosine shows more prominent effect on the inhibition of nuclear maturation at the GV stage, and of the 1st polar body extrusion at the GVBD stage. Adenosine showed the cytoplasmic toxicity at GV stage oocyte. Our data speculate that cytoplasmic cAMP level is auto-regulated by endogenous adenylate cyclase while GVBD is inhibited by guanosine, since purine toxicity is not observed in the GVBD stage. And it is showed that purine metabolism is concerned with nuclear maturation, that the amounts of purine metabolism is not even during the oocyte maturation.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.127-132
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• 2013

The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21~22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.71-77
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• 2012

The objective of this study was to examine the effect of thymidine treatment during $in$ $vitro$ maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group ($p$<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group ($p$<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group ($p$<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups ($p$<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.97-104
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• 2009

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 77${\sim}$82%) and embryo cleavage (75% vs. 86${\sim}$90%) after SCNT than those matured in other media but blastocyst formation was not altered (13${\sim}$27%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.