• The Korean Journal of Zoology
• /
• v.31 no.4
• /
• pp.265-272
• /
• 1988

세포융합방법을 사용하여 성장증인 포유동물의 난자에 들어있는 성숙억제요인(maturation inhibiting activity, 1연Al에 대해 조사하였다. 성장중인 생쥐난자와 성장한 미성숙난자를 1:1로 융합하여 배양했을 서 (14-17시간)에는 거의 모두 핵붕괴를 일으키었으나(90oyo), 2:1로 융합했을 때는 대부분(약 64%) 3개의 핵을 모두 간직하고 있었다. 돼지난자의 경우는 성장중인 것깎 성장한 것을 1:1로 융합하여 배양했을 때에도 융합체들은 모두 핵을 간직하고 있었으며 돼지의 성장중인 난자와 생쥐의 성장한 난자를 융합했을 때에도 모두 핵을 보존하고 있었다. 이에 반하여 돼지와 생쥐 모두에서 성장한 난자끼리 융합했을 때에는 예외없이 핵붕괴가 일어났다. 이러한 결과는 성장중인 생쥐나 돼지의 난자에 각IA가 존재한다는 열과 이종간에도 효과가 있다는 것을 보여주고 있다. 또한 이는 MIA와 성숙촉진요인(maturation promoting factor, MPH의 상대적인 양의 변화가 난자의 성숙조절에 증요한 9f할을 한다는 것을 시사해주고 있다.In an attempt to elucidate the nature of maturation inhibiting activity (MIA) in growing mamma-lian oocvtes, growing mouse and pig oocytes incompetent to resume meiosis were fused with fully grown immature oocvtes in various combinations and cultured for 14-17 hours. In slant cells composed of two mouse growing ooh임es and one large immature oocyte (2:기, their GVs remained well conserved (about 64%) after culture, but not in the ceils composed of one by one pairs. In giant cells of pig composed of one growing and onto large immature oocytes, both GVs remained conserved. In the cells composed of one pig growing and one mouse large oocytes, both GVs were also conserved. In contrast to this, pairs of large mouse oocvtes or those of large pig oocvtes had no CVs after culture. Thus, we could acertain the existEnce of MIA and none-pecificty of it in the mouse and pig growing oocvtes. The results also suggest that the relative amount of substances showlns MfA or MPF activity may be important in the regulation of oocyte amount of substances showing MIA or MPF activity may be important in the regulation of oocyte maturation.

• The Korean Journal of Zoology
• /
• v.34 no.2
• /
• pp.188-195
• /
• 1991

The effect of lanthanum ion (La3 +), which is associated with the mobilization of internal calcium, on the regulation of oocyte maturation was investigated with Rana dybowskii follicles. Follicular oocytes matured (germinal vesicle breakdown, GVBD) dose dependently when they were exposed to La3+ (O.O1-1.O mM) and the maturation occurred in 9-12 hours after the la3+(0.33 mM) stimulation. lanthanum also accelerated the onset of maturation of the lollicular oocytes exhibiting spontaneous maturation. Three hours of exposure to La3+ was enough to induce the maturation. The La3 + -induced maturation was not associated with progesterone production by follicle cells, and the maturation was inhibited by forskolin (9 $\mu$ M), and cyclobeximide (0.01 - 1.0 - $\mu$g/2 ml) in the medium. The La3+ and hormone stimulated maturation showed the same patterns of protein phosphorylation and dephosphorylation during the maturation. The data suggest that the oocyte maturation by La3+ stimulation is very similar to that by progesterone. Thus, it seems that internal mobilization of Ca2+ plays a key role in the initiation of oocyte maturation in amphibia.

• The Korean Journal of Zoology
• /
• v.33 no.1
• /
• pp.53-62
• /
• 1990

Fully grown amphibian oocytes undergo their maturation (germinal vesicle breakdown, GVBD) during in vitro follicle culture when they are stimulated with frog pituitary homogenate (FPH) or progesterone. Present experiments were designed to determine whether proteolytic enzymes are involved in the regulation of the matunation process. Treatment of a $\alpha$ -chymoiyypsin inhibitor, N a -tosyl-L-phenylalanine-chloromethyl-ketone(TP) to the oocytes exhibited a biphasic phenomenon, the induction of the maturation without added hormone at relatively low doses (0.001-1 $\mu$M) and inhibition of the hormone induced oocyte maturation at a high dose (100 $\mu$M). Treatment of a trypsin inhibitor, N a -tosyl-L-lysine-chloromethyl ketone(TLCK) to the oocytes did not induce the maturation, but rather suppressed the hormone induced oocyte maturation in a high dose(100 $\mu$ M). Treatment of exogenous iyypsin to the oocyte induced their maturation without added hormone in a dose dependent manner (0.001-1 $\mu$ M). The data presented here indicate that some proteolytic enzymes play a role in the regulation of the maturation(meiotic arrest or reinitiation) in amphibians.

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

• Korean Journal of Animal Reproduction
• /
• v.21 no.2
• /
• pp.111-116
• /
• 1997

We determined the effects of follicular fluid fractions in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on number of cells in blastocysts following culture. Follicular fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. Follicular fluid was separated into different molecular weight fractions by untrafiltration through a membrane using a centrifuge at 500$\times$g, for 2h. For the maturation medium, follicular fluid fractions (30%, v/v), whole fluid (30%) or PVP(3mg/ml) were added to TCM 199(0.1$\mu\textrm{g}$/ml estradiol-17$\beta$, 100IU hCG). After maturation for 24h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 days after fertilization. There were no differences in maturation rates or fertilization rates among any maturation conditions. The rates of development to >2-cell stage of the oocytes were significantly decreased when fraction of follicular fluid below 10,000 MW were added into maturation medium, compared with control and fraction above 10,000 MW(26.0% vs 40.8% to 64.0%, respectveily. p<0.01). Likewise, the rates of development to blastocysts of fertilized oocytes were significantly decreased in maturation medium containing fraction of follicular fluid (<10,000 MW). The average cell number of blastocysts derived from oocytes that matured in the fraction(>10,000 MW) of follicular fluid was 154.7$\pm$13.7. These embryos contained more cells than those matured in whole follicular fluid, or the fraction(<10, 000 MW) of follicular fluid or control(107.0$\pm$8.4, 91.8$\pm$11.8 and 95.8$\pm$6.2, respectively). In conclusion, we found that fractions of follicular fluid contained factors stimulating or inhibiting oocyte cytoplasmic matruation. These suggest that a factor(s) inducing cytoplasmic maturation of oocytes may exist in >10,000 MW fraction of follicular fluid.

• The Korean Journal of Zoology
• /
• v.36 no.2
• /
• pp.277-284
• /
• 1993

A possible involvement of maturation promoting factor (nfPF) in the two-cell block phenomenon was studied by fusion experiments. Germinal vesicle (GlF) ooeyte was fused with a blastomore from late or blocked 2-cell mouse embryos. and germinal vesicle breakdoum (GVBD) of fused GV oocvtes in the presence of dbcAMP (100$\mu$g/ml) was scored as an index of MPF aniviD. GnD was induced approximately 30% by fusion of a blastomere derived from late 2-cell embryos, but not from blocked 2-cell embryos. The rate of GVBD was changed when GV oocyte was fused with a blastomere from late 2-cell embryos which were treated with u-amanitin, puromvcin or colcemid before and after hsion: Treatment of late 2-cell embryos with puromycin (50 Is/mll but not with u-amanitin (100 Is/ml) clearly inhibited GVBD, indicating that do novo protein synthesis maw be required for the appearance of MPF activity in late 2-cell embryos. Treatment of late 2-cell embryos w기h colcemid (0.1 Is/mll doubled GVBD, presumably due to the maintenance of metaphase or mitotic phase. SDS-PAGE and twoiimensional electrophoresis revealed that there was no difference in protein synthetic pattern in late and blocked 2-cell embryos, but three phosphoproteins with 27, 35 and 46 M)a, presumsblv M-phase components were phosphorylated in late 2-cell embryos but not in blocked 2-cell embryos. It seems then that MPF activity is closely related to phosphorylstion of M-phase components in late 2-cell embryos.

• The Korean Journal of Zoology
• /
• v.31 no.2
• /
• pp.87-94
• /
• 1988

Korean frogs (R. dybowskii and R. nigromaculara) were collected from chonnam area and their oocyte maturation was induced by using in ultro follicle culture system. Follicles were isolated from the frog ovary and cultured for 24 hr in (amphibian Ringer's soluion AR) at 22 C in the presence or absence of hormones. Follicular cocytes of R. dybowskii were induced to mature (germinal vesicle breakdown, GVBD) by the presence of progesterone, 0.1 $\mu$g/2 ml and that of R. nigromaculata by 1 $\mu$g/2 ml of progesterone. Follicles of the frogs were also responded to (frog pituitary homogenate FPH) in terms of their cocyte maturation. Follicular cocytes of R. dybowskii were induced to mature by FPH at concentration of 0.01 pituitary equivalent/2 ml and that of R.nigromaculata at 0.1 pit equiv./2 ml. The culture time required for the maturation of bath frog follicles was 915 hr. The responsiveness of the follicles of korean frogs to hormones (progesterone or FPH) was nearly the same as that of R. pipiens which are most commonly used amphibians. Particularly, follicular cocytes of R. dybowskii used from February matured spontaneously without stimulation of hormones during in vitro culture. Furthermore, those cocytes were spontaneous- ly ovulted when the ovarian fragments were cultured in a flask.

• Korean Journal of Animal Reproduction
• /
• v.21 no.1
• /
• pp.19-23
• /
• 1997

This present study was carried out to examine the effect of maturation media and liquid boar semen on in vitro maturation and feritilization of pig oocytes. The results obtained were as follows : When the oocytes were cultured for 36∼42 hours in mTCM-199, Waymouth MB 725/1 and mTLP-PVA medium, the maturation rates were 90%, 92% and 88%, respectively. The sperm penetration rates of pig oocyte matured in vitro were 87%(mTCM-199), 90%(Waymouth MB 725/1) and 86%(mTLP-PVA), respectively. The rates of nuclear maturation and fertilization of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 725/1(91%) than oocytes matured in mTCM-199(66%) and mTLP-PVA(62%) medium (P<0.05). When the collected sperm-rich fraction without diluent was used fro in vitro fertilization in mTCM-199 fertilization medium, the fertilization rate was 87.9%. However, when the liquid boar semen diluted with B tschwiler diluent was used at day 3 and 5 after dilution, the fertilization rate was 40.8% and 0.0%, respectively.