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.

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.

The present study was performed to determine the ability of canine oocytes to achieve nuclear maturation according to oocyte diameter and different culture environments. All of the collected oocytes were classified by grade 1 to 3 and by their diameters such as <100㎛, <100㎛ to <110㎛, <110㎛ to <120㎛, >120㎛. Oocytes were cultured in culture medium supplemented with 10%FBS, 0.4%BSA,10% porcine follicular fluid (pFF), 10% canine serum (CS), or 10% canine estrus serum (CES). The mean number of oocytes recovered from estrus status ovaries was significantly higher than that of anestrus status ovaries (p<0.01). The maturation rate of grade 1 oocytes (>120㎛) was significantly higher than that of the other groups (p<0.05). Nuclear maturation to MI to MII in diameter of >110㎛ groups was significantly higher than that in <100㎛ group (p<0.05). The oocytes cultured in 10% FBS­supplemented group were significantly higher rate of GVBD compared to the other supplemented groups (p<0.05), and oocytes maturation to MI to MII in 10% FBS-, 0.4% BSA-, and 10% pFF-supplemented groups were significantly higher than those in 10% CS-supplemented group (p<0.05). Based on these results, the estrus status and the size of oocyte affect positively to improve nuclear maturation of canine immature oocytes in vitro. Among several protein sources, porcine follicular fluid was the most effective supplementation to culture medium to achieve higher in vitro maturation rate.

PCB가 산개구리 여포난자의 성숙과 프로제스테론 생성에 미치는 영향을 알아보기 위해 배양액에 일정 농도의 PCB(Arochlor 1248)를 농도별로 첨가한 후 난자들을 20시간 배양하였다. 난자의 성숙과 프로제스테론 생성을 유도하기 위하여 FPH(Frog pituitary homogenate: 0.01p.e/ml)를 사용하였으며 여포난자의 성숙율은 난자 내의 핵막 붕괴율로부터 구하였고 프로제스테론 생성은 배양액내로 분비되는 양을 조사하였다. 실험 결과 PCB는 10ppb의 농도부터 여포난자의 성숙과 프로제스테론 생성을 현저히 억제하였으며 PCB 작용의 가역성을 조사하기 위해 3시간 동안 여포난자들을 PCB에 노출시킨 후 보통 배양액으로 옮겨 계속 배양을 해 본 결과 PCB 2.5ppb에서는 가역성을 나타내었으나 5 ppb에서는 비가역적인 손상을 주었다. 이와 같이 PCB는 낮은 농도에서 난자의 성숙과 프로제스테론의 분비 등을 억제하였으며, 개구리 난자 배양계는 환경오염물질의 독성 검정에 요긴하게 사용할 수 있음을 시사하였다

The oocytes from most of animal species accumulate genetic information and other necessary materials during oogenesis for the later use in the early development. Over the years oocyte maturation has been studied extensively both in vitro and in vivo. Particularly, maturation of follicular oocyte in vitro becomes one of the important tools for the studies of basic cell biology, the in vitro technology of animal production, and in particular, the somatic cell cloning by nuclear transfer. We examined meiotic maturation and cumulus expansion in the presence of translation or transcription inhibitors for varying periods of in viかo maturation (IVM) of pig oocyte. In Experiment 1, the results revealed that translation and transcription inhibitors inhibited cumulus expansion and meiotic maturation during 35h of IVM. However, 50 to 60% of the oocytes underwent nuclear maturation without cumulus expansion during 75h of IVM. The rest of the oocytes were arrested at metaphase I (40-50%) in the presence of the inhibitors. In Experiment II, the OCCs were exposed to the drugs only for 15h to examine translation and transcription inhibitors on cumulus expansion and meiotic maturation. Transcription inhibitors for 15h did not arrest meiotic maturation when the oocytes were cultured for subsequent, necessary period of IVM, whereas cumulus expansion was completely inhibited, suggesting that initial 15h is critical transcription activity far cumulus expansion. Translation inhibitors for 15h exposure did not alter cumulus expansion and meiotic maturation during subsequent culture in the absence of the drugs. In Experiment III, the OCCs were exposed to the drugs only for later 30h to examine the influence of transcription and translation inhibitors on oocyte maturation. Interestingly, all meiotic maturation underwent normally with full expansion of cumulus. Similar results were obtained from Experiment IV where 5h of exposure from 15 to 20h of IVM culture to the drugs was performed and subsequently cultured for same period in fresh medium. Taken there results together, both transcription and translation are necessary for nuclear maturation and cumulus expansion, and first 15h IVM for cumulus expansion is critical. The arrested oocytes by the drugs were still capable of undergoing nuclear maturation, although cumulus expansion was affected.

This study was conducted to find out the effect of follicle size and oocyte type on in vitro maturation of poricine follicular oocytes. TCM-HEPEAS medium was used to basic medium, and the oocyte matured in vitro was stained with the Rapid staining method. The results obtained were summarized as follows; 1. The number of follicles an ovary was 20.5. The number of A-and B-typed oocytes an ovary was 2.34. The proportion of A-and b-types oocytes was 40% of the recovery oocytes. 2. Cumulus expanison indexes(CEI) by the follicle size were 1.62∼2.34(<2mm), 1.27∼2.28(2∼5mm) and 1.46∼2.75(>5mm). It was no differ to maturation rate by the follicle size. 3. The degree of oocyte maturation based on oocyte type did not differ for B-and C-typed oocyted but the index of oocyte type A was higher than that of b-and C-typed oocytes. 4. When follicluar oocytes were cultured for 42 hours, the proportion of the Met-II(second metaphase) stage were 22.5% (degree 1), 35.4%(degree 2) and 65.5% (degree 3).