• 한국가축번식학회지
• /
• 제18권2호
• /
• pp.141-150
• /
• 1994

전자현미경에 의해 자궁부착 전후의 돼지 수정란의 형태형성 및 분화에 따른 배발생 과정을 검토하였다. 돼지 초기배는 자궁이주후 균일하게 자궁에 배분되기전 약 2~3일간은 자궁각의 proximal portion에 존재하며, 임신 4일째에 할구와 할구의 경계를 상실하는 tight한 gap junction을 가진 상실배로 발달한다. 배반포를 형성하는 시기에 estradiol 17$\beta$는 compact한 상실배를 cavitated blastocyst로 발달을 촉진시키면서, steroid hormone이 이후의 배발생을 지배한다. Hatching의 시기는 교배후 6~7일경 zona pellucida을 둘러사고 있는 glycoprotein의 thinning과 lysis에 의해 이루워지는데, hatching 과정은 embryo의 세포수와 무관하였으며, 이때의 embryo의 직경은 0.5~1.0mm인 것을 본 실험에서 확인하였다. 12일경부터는 embryo는 prostaglandins, IGF-binding protein, retinol binding protein, plasminogen activator등의 단백질이 풍부해 이들 인자가 elongation 개시 후보로 고려될 수 있었다. 또한 이 시기의 embryo는 embryonic disc로 발달시 progesterone과 estrogen을 estradiol 17$\beta$로 전활할 수 있으며, 이러한 변화와 함께 spherical stage로부터 tubular 혹은 filamentous form으로 변형되었다. Estrogen이 임신을 통해 prostagladins의 분비를 uterine lumen에 지시하는지는 알 수 없으나 13일 경을 전후해 conceptus estrogen이 uterine arterial blood flow, uterine vasular permeability을 증가시키는 것으로 나타났으며, 자궁에서 protein과 calcium, PGF2$\alpha$, plasminogen inhibitor를 증가시키는 것으로 나타났다. 이 시기의 자궁 변화와 함께 embryo의 attachment는 trophoblast와 uterine membrane사이의 느슨한 결합에 의해 개시되었으며, 18일경 uterine과 trophoblastic microvili의 interdigitation에 의해 완성된다. 이 시기에 conceptus attachment를 위해 필요한 uterine microvili에서의 glycocalyx의 형성과 endometrial epithelium의 erosion을 야기하기 위해 plasminogen activator을 분비하였으며, 반면 자궁에서 plasminogen 역할을 하는 것은 estrogen이며, blastocyst cell 표면의 lectin binding이 attachment에 중요한 역할을 한다. 이상과 같은 일련의 과정을 거친 초기배는 성공적인 임신으로 유도된다고 본다. 따라서, 본 연구는 이상과 같이 착상을 전후한 시기의 배를 전자현미경에 의해 형태형성의 변화를 특히 착상을 전후해 배 취사율이 높은 시기를 대상으로 분석하였다. 이 분석 시기중 성공적인 착상성공율은 56%(71/126)였다.

• 한국수정란이식학회지
• /
• 제25권3호
• /
• pp.179-187
• /
• 2010

Embryonic compaction is essential for normal preimplantation development in mammals. The present study was to investigate the effects of compaction patterns on developmental competence of pig embryos. The proportion of blastocyst formation derived from compacted morula was higher than those of compacting and pre-compacting morula (P<0.01). Nuclei numbers of inner cell mass (ICM), trophectoderm (TE), and total of blastocysts derived from compacted group were also superior to those of compacting and pre-compacting groups (P<0.05). Then, compaction patterns, developmental ability and structural integrity were compared between mono- and poly-spermic embryos. The rate of compacted morula in mono-spermic embryos was higher than that of poly-spermic embryos (P<0.05). Especially, the rate of blastocyst formation derived from compacted embryos in mono-spermic embryo group was higher than that of poly-spermic embryo group (P<0.05), although no difference was detected between the two groups in the structural integrity. Finally, we confirmed that beta-catenin was differentially expressed according to compaction patterns in morula and blastocyst stage embryos. In conclusion, our results suggest that the compaction patterns during preimplantation development play a direct role in developmetal competence and quality of pig embryos.

• 한국수정란이식학회지
• /
• 제23권2호
• /
• pp.101-108
• /
• 2008

This study investigated the developmental ability and gene expression of somatic cell nuclear transfer embryos using ear skin fibroblast cells derived from miniature pig. When miniature pig (m) and landrace pig (p) were used as donor cells, there were no differences in cleavage (79.2 vs. 78.2%) and blastocyst rates (27.4 vs. 29.7%). However, mNT blastocysts showed significantly higher apoptosis rate than that of pNT blastocysts (6.1 vs. 1.7%) (p<0.05). The number of nuclei in pNT blastosysts was significantly higher than that of mNT (35.8 vs. 29.3) (p<0.05). Blastocysts were analyzed using Realtime RT-PCR to determine the expression of Bax-${\alpha}$, Bcl-xl, H19, IGF2, IGF2r and Xist. Bax-${\alpha}$ was higher in mNT blastocyst than pNT blastocyst (p<0.05). There was no difference in Bcl-xl between two NT groups. Bax-${\alpha}$/Bcl-xl was, however, significantly higher in mNT blastocyst compared to pNT. The expression of imprinting genes were aberrant in blastocysts derived from NT compared to in vivo blastocysts. H19 and IGF2r were significantly lower in mNT blastocysts (p<0.05). The expression of IGF2 and Xist was similar in two NT groups. However, imprinting genes were expressed aberrantly in mNT compared to pNT blastocysts. The present results suggest that the NT between donor cells derived from miniature pig and recipient oocytes derived from crossbred pig might affect reprogramming of donor cell, resulting in high apoptosis and aberrant expression patterns of imprinting genes.

• 한국수정란이식학회지
• /
• 제28권2호
• /
• pp.133-139
• /
• 2013

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitro-matured pig oocytes and treated for 4 h after electric activation with $0.5{\mu}M$ latrunculin A (LA), $10.4{\mu}M$ cytochalasins B (CB), and $4.9{\mu}M$ cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8~88.6%), blastocyst formation (30.7~ 32.4%), and mean cell number of blastocysts (33.5~33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5~78.0%) and mean blastocyst cell number (33.6~35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5~85.7%) and blastocyst cell number (41.1~43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.

• 한국수정란이식학회지
• /
• 제27권2호
• /
• pp.121-126
• /
• 2012

The objective of this study was to examine the effect of in vitro culture media on embryonic development of in vitro-matured (IVM) oocytes after parthenogenetic activation (PA) in pigs. Immature pig oocytes were matured in TCM-199 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 22~26 h. IVM oocytes were activated by electric pulses and cultured in porcine zygote medium-3 (PZM-3) and North Carolina State University-23 supplemented with essential and non-essential amino acids (NCSU-23aa). These media were further modified by supplementing 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and $10{\mu}M$ ${\beta}$-mercaptoethanol (designated as mPZM-3 and mNCSU-23aa, respectively). Culture of PA embryos in mPZM-3 significantly increased development to the blastocyst stage than culture in NCSU-23aa (36.2% vs. 24.8%, p<0.05). Modified PZM-3 showed a significantly higher blastocyst formation than NCSU-23aa in both groups of embryos that were activated at 44 h and 48 h of IVM (51.0% vs. 35.5% and 49.0% vs. 34.2% in oocytes activated at 44 h and 48 h of IVM, respectively). Irrespective of the follicle diameter where oocytes were collected, embryonic development to the blastocyst stage was increased (p<0.05) by the culture in mPZM-3 compared to culture in NCSU-23aa (25.9% vs. 34.2% and 32.9% vs. 44.8% in embryos derived from small and medium size follicles, respectively). Our results demonstrated that culture media had significant effect on preimplantation development PA embryos and that mPZM-3 was superior to mNCSU-23 in supporting development to the blastocyst stage in pigs. This beneficial effect of mPZM-3 on embryonic development was not impaired by other factors such as time of oocyte activation and origin of immature oocytes (small and medium size follicles).

• 한국발생생물학회:학술대회논문집
• /
• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
• /
• pp.108-108
• /
• 2003

The purpose of this is to investigate the effects of vitrification in open pulled straws (OPS) on in vitro survival of porcine embryos. Blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed boar semen, and subsequent culture on granulosa cell monolayer. After frozen-thawing, embryos were culture in NCSU-23 medium with 5 mM hypotaurine, 4 mg/$m\ell$ BSA and 10 ng/$m\ell$ for 48 hrs to survival tests. When blastocysts were frozen-thawed by OPS methods, the embryos with normal morphology were 32.1, 34.5 and 38.9 % in early blastocyst, blastocyst and expanded blastocyat stages. The rates of partial damaged embryos were significantly (P<0.05) higher in early biastocysts than expanded blastocysts. In another experiment, the embryos frozen by OPS methods were cultured for 48 hrs for survival and developmental rates in vitro. The proportions of embryos hatched were 11.8, 20.2 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. On the other hand, The proportions of embryo with normal morphology after culture were 23.5, 25.0 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. These finding indicate the possible broader application for OPS methods that this procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages.

• 한국수정란이식학회지
• /
• 제25권1호
• /
• pp.1-7
• /
• 2010

The objective of this study was to examine the effect of vitamin B (pantothenic acid, folic acid, and myo-inositol) that was supplemented to embryo culture medium on in vitro development of parthenogenetically activated (PA) pig embryos. Cumulus-oocyte complexes derived from slaughtered ovaries were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (hCG and eCG) for the first 22 h and then further cultured in hormone-free medium for an additional 22 h. After maturation culture, metaphase II oocytes that extruded 1st polar body were electrically activated and treated with $5.0\;{\mu}g/ml$ cytochalasin B for 4 h. Then, PA embryos were cultured for 7 days in a modified NCSU-23 that was supplemented with pantothenic acid, myo-inositol, or folic acid at different concentrations ($3{\sim}300\;{\mu}M$) according to the experimental design. Myo-inositol added to culture medium did not show any beneficial or inhibitory effects on embryo cleavage and blastocyst formation. However, $300\;{\mu}M$ pantothenic acid significantly inhibited blastocyst formation compared to control (no addition) (24% vs. 36%, p<0.05). Folic acid ($300\;{\mu}M$) significantly (p<0.05) increased blastocyst formation (56%) compared to control (41%). Our results demonstrated that in vitro development of PA embryos was significantly influenced by vitamin B and addition of $300\;{\mu}M$ folic acid to culture medium improved in vitro development of pig PA embryos.

• 한국수정란이식학회지
• /
• 제17권3호
• /
• pp.239-249
• /
• 2002

Embryos derived from pig oocytes matured in mSOF are able to develop to blastocysts after IVF. Experiment 1 evaluated the effects of two maturation media (TCM-199 vs mSOF) on maturation rate, fertilization parameters, including penetration, polyspermy, male pronuclear formation, and the mean number of sperm penetrated per oocyte. Experiment 2 and Experiments 3 examined the effects of two maturation media on zona pellucida solubility and cortical granule distribution by transmissible electron microscopy, respectively. Experiment 4 assessed the effects of two maturation media on the in vitro embryo cleavage rate and development to blastocyst. Lastly, experiment 5 examined the cell number of blastocyst. An effect of media (P<0.05) was detected for mSOF on the mean number of sperm per oocyte. In TCM group, zona digestion time (196.5$\pm$15.5 vs 131.6$\pm$20.1 before IVF, 397.5$\pm$30.3s vs 185.3$\pm$16.4s after IVF, p<0.05) was higher in TCM-199 group. No significant effects of media was observed on cortical granule distribution between two groups by TEM. An effect (P<0.05) was observed on embryo development to blastocyst (16% vs 8%) but not on cleavage rates. No significant effects of media was observed on total cell number of blastocyst. We found that the high mean number of sperm penetrated per oocyte and the weaker zona pellucida on the basis of the digestion time was shown in pig oocytes matured in mSOF, however, porcine oocyte maturation with supplemented synthetic oviduct fluid medium (mSOF) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199.

• 한국수정란이식학회지
• /
• 제32권4호
• /
• pp.287-295
• /
• 2017

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

• 한국수정란이식학회지
• /
• 제24권2호
• /
• pp.97-104
• /
• 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.