Despite of the presence of estradiol-17β (E2) in ovarian follicles, its role(s) in in vitro maturation (IVM) is still largely unknown, especially in pigs. Thus, the current study was conducted to investigate the effect of E2 on in vitro maturation (IVM) of porcine oocytes and subsequent preimplantation development using in vitro fertilization (IVF)- or somatic cell nuclear transfer (SCNT)-derived embryos. To define the effects of E2 on IVM and early embryogenesis, porcine oocytes were matured in the presence or absence of E2, fertilized in vitro and cultivated to blastocyst stage. Compared to control group, the production of MII oocytes was significantly increased by treatment with E2, accompanying with the increase in MPF content and ERK phosphorylation, and monospermic fertilization and blastocyst development rates were also greatly elevated in the E2-treated oocytes. In addition, the advantageous role of E2 was also found in blastomere survival, which was further evidenced by both elevation of anti-apoptotic transcript Bcl-XL and decrease of pro-apoptotic transcript Bax. Furthremore, these positive effects of E2 were highly reproducible in early development of SCNT embryos. Collectively, the current study strongly suggests that E2 can be used as a efficient IVM supplement leading to successful nuclear/cytoplasmic maturatioin in pigs.

X‐box binding protein‐1 (XBP‐1) is an important regulator of a subset of genes active during endoplasmic reticulum (ER) stress. In the present study, we analyzed XBP‐1 level and location to explore the effect of ER stress on oocyte maturation and developmental competency of porcine embryos in an in vitro culture system. First, we examined the localization of XBP‐1 at different meiotic stages of porcine oocytes and at early stages of parthenogenetic embryo development. Fluorescence staining showed that expression of functional XBP‐1 was weak in mature oocytes and at the one‐cell, two‐cell, and eight‐cell stages of embryos, but abundant at the GV oocyte, four‐cell, morula, and blastocyst stages. In addition, RT‐PCR revealed that both spliced XBP‐1 (XBP‐1s ) and unspliced XBP‐1 (XBP‐1u) were expressed at the GV oocyte, four‐cell, morula, and blastocyst stages. Tunicamycin (TM), an ER stress inducer, blocked porcine embryonic development at the four‐cell stage, exhibiting the effect on embryonic genome activation. Next, porcine embryos cultured in the presence of tauroursodeoxycholate (TUDCA), an ER stress inhibitor, were studied. Total cell numbers and the extent of the ICM increased (p<0.05), whereas the rate of nuclear apoptosis decreased (p<0.05). Moreover, expression of the anti‐apoptotic gene Bcl‐2 increased whereas expression of the pro‐apoptotic genes Bcl‐xl and p53 decreased. The results indicated that inhibition of ER stress enhanced porcine oocyte maturation and embryonic development by preventing ER stress‐mediated apoptosis in vitro.

Polar body was usually used as a determinant of oocyte's maturation. Polar body morphology could reflect the embryo quality and implantation competence. This review only focuses on morphology of the first polar body and embryo developmental rate in the presence or absence of polar body. However, it is very difficult to describe whether polar body has any effects on embryo development in vitro or in vivo. Further intensive research is needed to determine its function on embryo development.

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ) of porcine FSH on nuclear maturation and 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 matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes () was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage () and mean number of cells in blastocyst ( cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 FSH,i numectivelo. In SCNT, fusion () of cell-cytoplast couplets and siosequent embryo cleavage () were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 FSHr(25% vs. ). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

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. 7782%) and embryo cleavage (75% vs. 8690%) after SCNT than those matured in other media but blastocyst formation was not altered (1327%) 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.

The aim of this study was to investigate what components of porcine epididymal fluid (pEF) influences the nuclear maturation of porcine germinal vesicle oocytes. Porcine cumulus-oocytes complexes from follicles were cultured in TCM 199 containing pEF. After 48 h cultures, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. Maturation rate of oocytes was significantly increased in media supplemented with 10% pEF during in vitro maturation (IVM) than in those without pEF. When lipid component of pEF was removed by treating n-heptane, no significant difference was observed in maturation of oocytes between n-heptane treatrment and intact pEF group. However, the proportion of oocytes reaching at metaphase II (M II) was significantly (p<0.05) decreased in the oocytes cultured in media containing trypsin-treated pEF compared to those in media with intact pEF. When porcine GV oocytes were matured in the medium supplemented with intact pEF or pEF heated at 56'C and 97'C, rates of oocytes remained at GV stage were 11.7%, 29.4% and 42.0%, respectively. However, there were no difference in proportion of oocytes reaching at MII stage among intact pEF group and 56'C group. Present study suggests that 1) pEF contains an enhancing component(s) for nuclear maturation in vitro of oocytes, 2) protein(s) of pEF may be capable to promote nuclear maturation in vitro, and 3) enhancing component for nuclear maturation may consist of two factors, which are responsible for germinal vesicle breakdown (GVBD) and promotion of MII stage.

개 난자의 체외성숙율을 높이기 위하여 다양한 방법들이 시도되고 있지만 여전히 그 효율성은 낮다. 본 연구는 개 난자의 체외성숙 시, 성선 자극 호르몬인 황체형성호르몬(LH)과 난포자극호르몬(FSH), 상피세포성장인자(Epidermal growth factor, EGF) 그리고 시스테인(cysteine)을 각각 첨가하여 72시간 동안 체외성숙시킨 후 핵성숙율(GV: germinal vesicle, GVBD: germinal vesicle break down, MI: metaphase I, MII: metaphase II, UK: unknown stage)을 확인하였다. LH와 FSH를 첨가하였을 때 첨가하지 않은 군과 GV, MI 및 MII율에는 유의적인 차이는 없었다. 하지만 GVBD율은 첨가군이 유의적으로(p<0.05) 높았다. 성선 자극 호르몬을 첨가한 배지에 10 ng/ml의 EGF를 첨가하였을 때 MII율이 첨가하지 않은 군보다 유의적으로(p<0.05) 높았다(4.54% vs. 7.06%). cysteine을 첨가하였을 경우, 핵성숙율에 유의적인 차이는 보이지 않았지만 전반적으로 핵성숙율이 향상된 것을 확인할 수 있었다. 따라서 개 난자의 체외성숙 시, 10 μg/ml의 LH와 FSH, 10 ng/ml의 EGF 그리고 0.57 mM의 cysteine을 첨가하는 것이 핵성숙율을 향상시키는 것으로 사료된다.

This study was conducted to investigate the effects of oocyte maturational age and activation condition on in vitro development of porcine parthenogenetic embryos (parthenotes). Porcine follicular oocytes were matured in vitro for 30 to 44 hr. Maturation rate was examined during in vitro maturation (IVM) every 2 hr interval. The cdc2 kinase activity was measured at 36 and 44 hr of IVM. Some oocytes were activated at 36 or 44 hr of IVM by three different conditions; 1) single electric stimulation (1.5 kV/cm for 30 sec; ES), 2) double electric stimulations (1.5 kV/cm for 30 sec, followed by 1.0 kV/cm for 50 sec after 1 hr; ES+ES) or 3) ES+ES followed by culture in 6-dimethlyaminopurine (6-DMAP) for 4 hr (ES+ES+D), and cultured for 6～7 days. Maturation rate was significantly increased as culture period was increased to 36 hr (66.9%, p<0.05), and then gradually increased to 87.1% at 44 hr of IVM. The cdc2 kinase activity was decreased (p<0.05) with culture period prolonged from 36 hr to 44 hr. Lower blastocyst formation rate (4.3%, p<0.05) were obtained by ES in 36 hr-matured oocytes compared to other treatments (16.5 and 20.5%) in the same age and the same treatment in 44 hr-matured oocytes (15.0%). High blastocyst formation rate (23.6%) was obtained by ES+ES+D in 44 hr-matured oocytes (p<0.05). These results demonstrate that porcine oocyte activation and in vitro development of parthenotes can be affected by interactions between oocyte maturational age and activation condition.

Previous studies have shown that BMI-1026 is a potent inhibitor of the cyclin-dependent kinases (cdk). In cell culture, the compound also arrests G2/M strongly and G1/S and S weakly. Two key kinases, cdk1 (p34cdc2 kinase) and mitogen-activated protein (MAP) kinase (erk1 and 2), perform crucial roles during oocyte maturation and, later, metaphase II (MII) arrest. In mammalian oocytes, both kinases are activated gradually around the time of germinal vesicle breakdown (GVBD) and maintain high activity in eggs arrested at metaphase II. In this study, we examined the effects of BMI-1026 on GVBD and MII arrest in mouse oocytes. BMI-1026 inhibited GVBD of immature oocytes and activated MII-arrested oocytes in a concentration-dependent manner, with more than 90% of oocytes exhibiting GVBD inhibition and MII activation at 100 nM. This is approximately 500～1,000 times more potent than the activity reported for the cdk inhibitors roscovitine (~50 M) and butyrolactone (～100 M). Based on the results of previous in vitro kinase assays, we expected BMI-1026 to inhibit only cdk1 activation in oocytes and eggs, not MAP kinase. However, in our cell-based system, it inhibited the activity of both kinases. We also found that the effect of BMI-1026 is reversible. Our results suggest that BMI-1026 inhibits GVBD and activates MII-arrested oocytes efficiently and reversibly and that it also inhibits both cdk1/histone H1 kinase and MAP kinase in mouse oocytes.

본 연구는 북방산개구리의 난자 및 배아를 활용하여 난자성숙현상과 배아발달과정에 미치는 Cd2+의 독성효과를 조사하였다. 결과 Cd2+ 0.1ppm에서 난자의 성숙현상을 억제하였으며 Cd2+ 작용의 가역성을 조사하기 위해 3시간 동안 난자들을 Cd2+에 노출시킨 후 보통배양액으로 옮겨 17시간 배양한 결과 1ppm에서는 가역성을 나타내었으나 2.5ppm에서는 비가역적 인 손상을 주었다. 발달 중인 2세포 배아를 Cd2+의 여러 농도에 노출시킨 결과 0.1ppm에서 발달이 억제되었으며 노출시간이 길어진 32세포 시기에는 세포붕괴현상을 유발하였다. 한편, 포배기 배아를 Cd2+의 여러 농도에 노출시 켜 96시간 배양한 후 유생의 치사율 및 기형율을 대상으로 probit 분석법으로 조사한 결과 LC50은 0.1ppm, EC50은 0.08ppm, TI는 5.0을 나타내어 Cd2+은 높은 치사율을 나타내는 물질로 나타났다. 기형 양상은 척추기형이 0.05ppm에서 14.3%,꼬리기형이 0.1ppm에서 75.0%,복부기형 이 0.01ppm에서 66.7%를 나타내었고 profound형 기형이 0.1ppm에서 25.0%를 각각 나타냈으며 Cd2+ 0.1ppm에서 머리에서 꼬리까지의 성장을 억제하였다. 결론적으로 본 연구의 결과들은 Cd2+이 난자성숙, 난할 및 배아의 발달과정에 높은 독성의 효과를 가짐을 나타낸다.