• Clinical and Experimental Reproductive Medicine
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• 제19권1호
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• pp.15-29
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• 1992

In the present study, it was aimed to find the role of calcium on the maturation of mouse follicular oocytes as well as for the role of calcium inhibitors, $Ni^{2+}$ and $La^{3+}$. Mouse follicular oocytes were cultivated in different media at $37^{\circ}C$, in 100% humidified $CO_2$ incubator for 3 and 17 hrs. The results were as follows; 1. There was no differences in GVBD between the control and experimental groups during the 3 hr culture. 2. Mouse oocytes were matured to higher rate in MHBS rather than HTF for 17 hr culture. 3. Maturation rate was significantly lower in $Ca^{2+}$-free and $Ca^{2+}$ 0.4 mM which were tested, compared to other calcium concentration used in the present study. 4. Calcium inhibitor, $Ni^{2+}$, it showed highest degeneration rate at all calcium concentrations and additionally in $Ni^{2+}$ $100{\mu}M$ treated group next. Maturation rate was significantly decrease as the $Ca^{2+}$ inhibitor concentration increased. 5. In all Lanthanum treated groups of calcium-free, degeneration were significantly high treated groups at 0.4 mM $Ca^{2+}$ concentrations degeneration rates of all group were significantly lower than that of the control but maturation rates were not significantly different in any group. In lanthanum $100{\mu}M$ treated group at 0.4 mM and 0.8 mM calcium concentration, its maturation rate was significantly higher than that of the control. Maturation rates of all groups of lanthanum treated at 1.71 mM calcium concentration were not significantly different among groups. 6. In the calcium treated group (0.4mM-1.7 mM), the presence of phosphate does not seem to be needed for oocyte maturation. However, the presence of phosphate at $Ca^{2+}$ 0.8 mM only seems to stimulated maturation.

• Clinical and Experimental Reproductive Medicine
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• 제44권3호
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• pp.126-131
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• 2017

Objective: Oocyte degeneration often occurs after intracytoplasmic sperm injection (ICSI), and the risk factor is low-quality oocytes. The follicular fluid (FF) provides a crucial microenvironment for oocyte development. We investigated the relationships between the FF volume aspirated from individual follicles and oocyte retrieval, oocyte maturity, oolemma stretchability, fertilization, and development. Methods: This retrospective study included data obtained from 229 ICSI cycles. Ovarian stimulation was performed according to a gonadotropin-releasing hormone antagonist protocol. Each follicle was individually aspirated and divided into six groups according to FF volume ( < 1.0, 1.0 to < 2.0, 2.0 to < 3.0, 3.0 to < 4.0, 4.0 to < 5.0, and ${\geq}5.0mL$). Oolemma stretchability during ICSI was evaluated using a mechanical stimulus for oolemma penetration, that is, the stretchability was assessed by oolemma penetration with aspiration (high stretchability) or without aspiration (low stretchability). Results: Oocyte retrieval rates were significantly lower in the < 1.0 mL group than in the ${\geq}1.0mL$ groups (46.0% [86/187] vs. 67.5%-74.3% [172/255 to 124/167], respectively; p< 0.01). Low oolemma stretchability was significantly more common in the < 1.0 mL group than in the ${\geq}1.0mL$ groups during ICSI (22.0% [13/59] vs. 5.8%-9.4% [6/104 to 13/139], respectively; p= 0.018). There was a relationship between FF volume and oolemma stretchability. However, there were no significant differences in the rates of fertilization, cleavage, ${\geq}7$ cells at day 3, and blastocyst development among all groups. Conclusion: FF volume is potentially associated with the stretchability of metaphase II oolemma during ICSI. Regarding oolemma stretchability, ensuring a uniform follicular size during ovarian stimulation is crucial to obtain good-quality oocytes.

• Journal of Animal Science and Technology
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• 제63권2호
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• pp.272-280
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• 2021

Cumulus-oocyte complexes (COCs), which contain immature oocytes, are matured in vitro for in vitro embryo production. Oocyte and cumulus cells are then separated using hyaluronidase. To date, there have only been a few reported cases of the toxic effects of hyaluronidase on porcine oocytes. The aim of this study was to compare the effects of bovine testis-derived hyaluronidase and recombinant human hyaluronidase on oocyte denudation and quality. Porcine COCs were matured for 44 h and denuded using different hyaluronidase concentrations and exposure times. Then, oocytes were activated by electrical parthenogenesis. In experiment 1, COCs were denuded using bovine-derived, ovine-derived (Hirax), and human recombinant (ALT-BC4) hyaluronidases for 10 and 20 min. In experiment 2, bovine-derived and human recombinant (ALT-BC4 and ICSI Cumulase®) hyaluronidases were used to denude the COCs for 2 and 20 min. In both experiments the oocytes were all completely denuded, and there was no degeneration. Rate of embryo development was significantly increased in group treated ALT-BC4 for 2 min and not significantly different in other treatment groups. In general it slightly decreased with longer exposure times. These results have confirmed that different sources of hyaluronidase do not have detrimental effects on the quality of porcine oocytes and suggest that the human recombinant hyaluronidase ALT-BC4 is suitable for oocyte denudation with an increased blastocyst rate.

• 한국동물학회지
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• 제31권4호
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• pp.273-282
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• 1988

생쥐에 PMSG와 hOG를 주사한 후 난자-난구복합체의 미세구조의 변화를 환찰함으로세 난구세포의 분산현상을 규명하고자 본 실험을 행하였다. 난자는 PMSG 주사후 48시간까지 별 다른 변화가 없었고 다만 표면막에 miGrOVilli와 Coaled pit의 수가 감소하는 경향을 보였다. 그러나 PMSG-hCG주사 12시간 후에 배란된 난자의 표면은 microvilli와 coated pit가 사라져서 평평하게 되었다. 방사관세포는 PMSG주사 48시간 후메 밀착해 있던 투명대와 간격이 생기기 시작하였고, 투명대를 통관하여 난자의 표면막과 desmosome으로 연결되어 있던 세포질돌기도 퇴화의 징후를 보였다. PMSG-hCG주사 후에는 급속히 격리, 분산되고 세포질돌기는 퇴화하였으며 dermo-some도 사라겼다. 난구세포들은 대조군에서 밀집되어 있었고 거의 gap junction으로 연결되어 있었는데, PMSG주사 24시간 후에는 모양이 등글게 되고 더욱 밀집되었으며, 48시간 후에는 거의 loose junction으로 연결되었고 분산되기 시작하였다. 결국 PMSG-hCG주사 If시간 후에는 완전히 분산되었고 거의 모두 핵응축과 괴사현상을 보였다. 난자- 난구 복합체의 분산은 배란전에 PMSG에 의하여 시작되고 hCG에 의하여 촉진 완결된다는 것이 확실하다. The ultrastructural changes of the oocyte-cumulus complexes of mouse alter injection of PMSG and hOG have been investigated in order to elucidate expansion phenomenon of the cumulus cells. The oocytes until 48 hours after PMSC injection showed no change except a tendency of decrease in numbers of microvilli and the coated pelts on surface membrane. However, surface membrane of the ovulated oocytes 12 hours after PMSC-hCC injection changed to be smooth due to disapperance of microvilli and coated pits. Corona radiate cells tightly attaching to zona pe]lucida 48 hours after PMSC injection began to be detached and their cytoplasmic processes connected by desmosome to oocyte surface membrane showed a degeneration symptom. Thereafter the detachment and degeneration were accelerated by hCG injection and followed by disappearence of desmosome. The cumulus cells in control group were compacted and connected by almost 9aP junction each another. Ite cumulus cells 24 hours after PMSG injection were changed to be round form and more tightly compacted. However, the cumulus cells 48 hours after PMSG injection were connected by almost loose junction and showed the beginning of expansion. Eventuallv, the cumulus cells 12 hours a%or PMSG-hCG injection were completely expanded, and became pvknotic and necrotic in most It is clear that the expansion of oocyte-cumulus complex were initiated by PMSC, then accelerated and completed by hCG before ovulation.

• Journal of Radiation Protection and Research
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• 제24권2호
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• pp.87-92
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• 1999

본 연구는 감마선 조사 후 원시반포의 퇴화과정에서 나타나는 형태학적인 변화를 알아보기 위하여 시행되었다. ICR 계통의 생후 3 주된 미성숙 생쥐에 $LD_{80(30)}$의 선량인 8.3 Gy를 2 시간 동안 전신조사하였다. 방사선 조사 후 0 시간, 3 시간, 6 시간, 그리고 12 시간에 난소를 적출하여 조직절편을 제작한 후 가장 큰 절편을 관찰하였다. 정상 대 퇴화 난포의 비율은 조사 후 6 시간에 유의하게 감소하였다. 6 시간에 퇴화된 원시난포의 수가 증가하였으며, germinal vesicle이 관찰되지 않거나 지질방울이 증가하였으며, 난막이 사라진 것을 알 수 있었다. 과립세포의 모양은 둥글게 변하였으며, 세포자연사하는 세포가 관찰되었다. 대조군의 경우 정상 대 퇴화 원시난포의 비율이 62.50% 이었으나, 이 비율은 0 시간, 3 시간, 6 시간, 12 시간에 각각 51.61 %, 48.97 %, 11.11 %, 7.14 %로 감소하였다. 결론적으로 방사선에 의해서 윈시난포에 퇴화가 유발되는데 그 양상은 세 가지 유형을 나타내었다. 과립세포의 모양은 정상적이나 난자의 세포자연사, 난자의 형태는 정상적이나 한 개 혹은 그 이상의 과립세포가 세포자연사하는 경우, 혹은 난자와 과립세포 모두가 세포자연사 과정을 겪는 경우를 확인할 수 있었다. 본 연구를 통해 원시난포 퇴화를 형태학적으로 규명할 수 있는 단서가 제시되었다.

• 한국수정란이식학회지
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• 제33권2호
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• pp.69-73
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• 2018

This study was conducted to examine the influences of two human chorion gonadotrophins (hCGs) being injected into young or aged (45- to 65-week old) outbred (ICR) mice on developmental capacity of oocytes retrieved. In vitro-culture and parthenogenetic activation of oocytes retrieved were employed for the assessment. Superovulation was determined as being induced when more than 25 oocytes were retrieved. No aged mice were superovulated, while in contrast, 67-100% were superovulated in the 6- to 8-week-old (young) mice. In the aged, hCG injection yielded better retrieval (5 vs. 13 to 14.8 oocytes/mouse). Overall, no significant difference between two hCGs was detected but between the young and aged, significant differences in maturational arrest (0% vs. 39% MI arrest and 46% vs. 15% degeneration) and developmental capacity (24% vs. 46% 8-cell embryo development) were detected. In conclusion, hCG injection contributes to increasing oocyte retrieval from aged outbred mice, but the kinds of gonadotrophin influenced the efficiency of hyperstimulation induction in specific ages.

• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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• pp.105-116
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• 1992

The present study was examined to clarify the role of calcium ion as a factor for the maturation of mouse oocytes. Follicles and cumulus-enclosed oocytes were isolated with two sharp needles under a stereomicroscope from female mouse (ICR) ovaries which were treated PMSG 5 IU 45-46 hours previously. Isolated follicles and cumulus-enclosed oocytes were cultured for 14-16 hours in an organ culture system at $37^{\circ}C$, 5% $CO_2$ in air and 100% humudified in incubator. MHBS was the basic medium used from which A23187, verapamil, $NiCl_{2.}$ $6H_2O$ and $LaCl_{3.}$ $7H_2O$ were added depending on the experimental groups. In follicle- or cumulus-enclosed oocytes wre cultured in these differently treated media. Following results were obtained from the present study. 1. The calcium ionophore A23187 directly or indirectly seems to stimulate GVBD of follicle-enclosed mouse oocytes. Increasing concentration of ionophore A23187 1ed to an increase in oocytes degeneration from the cumulus-enclosed mouse oocytes. 2. The organic $Ca^{++}$ channel blocker, verapamil does not induce GVBD of follicle-enclosed mouse oocytes. Specially, higher dose of 1 mM verapamil induced GVBD of follicle-enclosed mouse oocytes. However, cytoplasm of GVBD oocytes in 1 mM verapamil treated groups appeared shrunk. In the cumulus-enclosed oocytes, polar body formation was reduced in verapamil treated groups and degeneration increased. Verapamil inhibit oocyte maturation (polar body formation). 3. The $Ca^{++}$ inhibitor, Nickel ($NiCl_{2.}$ $6H_2O$) inhibits maturation of the follicle-enclosed oocytes. In the cumulus-enclosed oocytes the progression to MII (PB formation) was reduced and degeneration of mouse oocytes increased as the concentration of $Ni^{++}$ increase. The results indicates that nickel act as an inhibitor of calcium. 4. The $Ca^{++}$ inhibitors, Lanthanum ($LaCl_{3.}$ $7H_2O$) has shown different effect from that of nickel. In follicle-enclosed oocytes, 0.01mM lanthanum induced maturation of mouse oocytes. Polar body formation was reduced in the cumulus-enclosed oocytes all lanthanum treated group.

• 한국발생생물학회지:발생과생식
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• 제10권1호
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• pp.33-39
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• 2006

아무므불가사리의 생식소 발달과 생식주기를 밝히기 위하여 2003년 11월부터 2005년 2월까지 경상남도 고성 연안 해역에서 채집된 개체들을 대상으로 생식소 숙도지수 (GSI)의 월별변화, 생식소 발달과정 및 생식소 발달 단계별 난경 변화를 조사하였다. 생식소숙도지수의 월별 변화는 암컷과 수컷이 유사한 경향을 보였으며 암컷은 $3.88{\pm}3.04$, 수컷은 $0.87{\pm}0.57$의 값으로 3월에 연중 최대값을 가지다가 이후 서서히 감소하였다. GSI의 월별 변화와 생식소 발달의 조직학적 관찰을 근거로 생식 주기는 회복기($6{\sim}9$월), 성장기($10{\sim}1$월), 성숙기($2{\sim}3$월), 방출기($3{\sim}4$월), 퇴화 및 흡수기($4{\sim}5$월)의 연속적인 주기로 구분되었다. 아무르불가사리의 난발달 양상은 동시발달형이고 년 1회 산란하는 것으로 보인다.

• 한국수정란이식학회지
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• 제28권2호
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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

• 한국수정란이식학회지
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• 제28권3호
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• pp.193-198
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• 2013

This study was designed to evaluate the effect of L-cysteine on sperm characteristics and oocyte cleavage in vitro in Korean native cattle. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 5, 10 and 20 mM L-cysteine before cryopreservation. The viability in frozen-thawed sperm were estimated by SYBR14/PI double stain, acrosome damage with FITC-PNA, mitochondria intact with Rhodamin123 and hydrogen peroxide($H_2O_2$) level with carboxy-DCFDA by flow-cytometry. The developmental capacity was also assessed with cleavage rates in oocytes fertilized in vitro by frozen-thawed sperm. In results, the sperm viability was significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). In addition, acrosome damage was significantly decreased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). The mitochondria intact was also significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). On the other hand, the cleavage rates were significantly increased in 0 mM, 5 mM and 10 mM groups than 20 mM concentration of L-cysteine (p<0.05). The oocyte degeneration of oocytes were significantly decreased in 0 mM, 5 mM and 10 mM groups than in 20 mM L-cysteine group (P<0.05). However, there are no significantly differences among the L-cysteine treatment groups. We suggest that concentration of 10 mM L-cysteine have beneficial impact for sperm cryopreserved in Korean native cattle. This result also could be recommended for artificial insemination program if supported by an improvement in the fertility results and required further study.