• Asian-Australasian Journal of Animal Sciences
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• 제15권12호
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• pp.1695-1701
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• 2002

The purposes of this study were to optimize the in vitro maturation (IVM) and culture (IVC) systems of rabbit oocytes. Cytoskeletal structures in the germinal vesicle stage (GV) and during IVM are also investigated. Ovaries were transported from local slaughterhouses and the cumulus-oocyte complexes (COCs) were collected from ovarian follicles (${\geq}1mm$). COCs were randomly allocated to TCM199-based medium ($T_1$, TCM-199) supplemented with $NaHCO_3$, glucose, sodium pyruvate and FSH ($T_2$), $T_2+E_2+LH$ ($T_3$), $T_3+FBS$ ($T_4$), or $T_1+E_2+LH+FSH+FBS$ ($T_5$), for IVM. In Experiment 1, COCs were retrieved from the follicles and 51 GV oocytes were fixed in the fixative (MTSB-XF) for nuclear and cytoplasmic examinations. In Experiment 2, progressive changes of both the nucleus and the cytoskeleton were examined at 0, 6, 16, and 20 h after IVM. Maturation (MR) and developmental rates were assessed in Experiment 3. Cytoplasmic microtubules (MT) were clearly observed in rabbit GV oocytes. To our knowledge, this is the first report that describes the appearance of MT structures in the GV stage ooplasm. Tremendous variations in cytoskeletal alterations were observed among treatments with the exception of the vitelline ring (VR), which is constantly visible and unchanged during maturation. Germinal vesicle breakdown (GVBD) does not occur at 6 h after onset of maturation culture. When the oocytes for IVM were collected within 2 h, results from Experiment 3 showed that rates of nuclear maturation were 42, 8, 42, 37 and 65% at 16 h of IVM for $T_1$ through $T_5$, respectively, in which $T_1$, $T_4$ and $T_5$ had significantly greater MR than those in other groups (p<0.05). Morula/blastocyst development after parthenogenetic activation ranged from 20 to 63% with significantly greater rates in $T_3$, $T_4$ and $T_5$ (p<0.05). These results suggested that oocytes recovered from slaughterhouse ovaries can be matured and parthenogenetically activated in vitro, but the MR remained low in this study. Addition of $E_2$ and LH in the medium may be beneficial for cytoplasmic maturation, but FBS exerts a nega- tive role in the subsequent development of parthenogenetic embryos when energy substrates are provided in the IVC media. More studies are required for improving the MR and further development of the GV stage rabbit oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제40권4호
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• pp.143-147
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• 2013

Oocyte in vitro maturation (IVM) is an assisted reproductive technology in which oocytes are retrieved from the antral follicles of unstimulated or minimally stimulated ovaries. IVM of human oocytes has emerged as a promising procedure. This new technology has advantages over controlled ovarian stimulation such as reduction of costs, simplicity, and elimination of ovarian hyperstimulation syndrome. By elimination or reduction of gonadotropin stimulation, IVM offers eligible infertile couples a safe and convenient form of treatment, and IVM outcomes are currently comparable in safety and efficacy to those of conventional in vitro fertilization. IVM has been applied mainly in patients with polycystic ovary syndrome or ultrasound-only polycystic ovaries, but with time, the indications for IVM have expanded to other uncommon situations such as fertility preservation, as well as to normal responders. In this review, the current clinical experiences with IVM will be described.

• Clinical and Experimental Reproductive Medicine
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• 제27권4호
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• pp.387-392
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• 2000

Objectives: 1) To compare the efficacy of urofollitropin (Follimon) to that of recombinant human FSH (rhFSH) on the growth and maturation of mouse early preantral follicles in vitro, and 2) effect of vitrification on the growth and maturation of preantral follicles and oocytes . Methods: Isolated early preantra1 follicles (100-130 ${\mu}m$ diameter) were cultured for 12 days in 20 ${\mu}l$ ${\alpha}$-MEM media drop under the mineral oil. Follimon or rhFSH was added to the culture medium at various concentrations (0, 10, 100, and 1000 mIU/ml). Results: With Follimon, the dose of 10 mIU/ml showed the best follicle survival, growth, and MIl rate of oocyte than the other concentrations. Whereas the optimal dose of rhFSH was 100 mIU/ml. Despite the different optimal doses, the efficacy of two different FSHs on the follicle growth and maturation was similar. Isolated mouse preantral follicles were cryopreserved by vitrification and cultured in vitro for 12 days with 100 mIU/ml rhFSH. Despite the decreased follicular survival rate after thawing, the follicular growth and maturation rate of its oocyte were comparable to those of the fresh follicle. Conclusion: Results from the present study revealed that 1) the optimal doses of Follimon and rhFSH for in-vitro culture of mouse follicles are different, and 2) the frozen-thawed follicles develop normally after vitrification.

• 한국수정란이식학회지
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• 제13권2호
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• pp.139-145
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• 1998

In the present study, effects of interleukin-2 (IL-2), a differentiator and proliferator of T-cells, on nuclear maturation and sperm penetration of bovine oocytes was examined in a serum-free or serum-containing medium. Basic medium was used TCM-199 supplemented with 2.2g / ι sodium bicarbonate, 100 i.u. /rnl penicillin. 100$\mu$g /ml streptomycin, 0.25$\mu$g/ml Fungizone, this medium treated with FCS and IL-2. In experiment 1, we examined the effect of the addition of 0, 1, 5, 10 or 15nM /ml IL-2 to tissue culture medium (TCM-199) on nuclear maturation of oocytes Development of oocytes to the Metaphase II (M II) stage (%) was significantly (P<0.05) higher at 1, 5,10 and 15 nM /ml IL-2(54.2, 73.5, 80.0 and 69.6%, respectively) than at 0 nM /ml IL-2(35.7%). In experiment 2, we examined the effect of the addition of l0nM /ml IL-2 or 5% FCS in oocyte maturation. Nuclear maturation rates were significantly(P<0.05) higher l0nM /ml IL-2(80%) than non-treatment(35.7%) and 5% FCS(63.6%) treatment. On the other hand, there were no significant difference in the proportion of oocytes developed to the 2-cell stage after addition of IL-2 and/or FCS. These results suggest that IL-2 supports nuclear maturation of bovine immature oocytes in vitro. Serum-free maturation system using IL-2 might be useful for evaluation of various factors on oocyte maturation.

• 한국수정란이식학회지
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• 제27권3호
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• pp.163-169
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• 2012

The 3-isobutyl-1-methylxanthine (IBMX) is non-selective phosphodiesterase and is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte. The present study was conducted to analyze: (1) nuclear maturation (examined by the Hoechst staining), (2) whether cytoplasmic maturation (examined by the intracellular glutathione (GSH) concentration) of porcine oocytes is improved during meiotic arrest after prematuration (22 h) with IBMX. Before in vitro maturation (IVM), oocytes were treated with 1 mM IBMX for 22 h. After 22 h of pre-maturation, the higher rate of IBMX treated group oocytes were arrested at the germinal vesicle (GV) stage (42.3%) than control IVM oocytes (10.1%). It appears that the effect of IBMX on the resumption of meiosis has shown clearly. In the end of IVM, the reversibility of the IBMX effect on the nuclear maturation has been corroborated in this study by the high proportions of MII stage oocytes (72.5%) reached after 44 h of IVM following the 22 h of inhibition. However, intracellular GSH concentrations were lower in the oocytes treated with IBMX than the control oocytes (6.78 and 12.94 pmol/oocyte, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pre-treated with IBMX for 22 h did not equal that of control oocytes in the current IVM system. These results indicate that pre-maturation with IBMX for 22 h may not be beneficial in porcine IVM system.

• 한국발생생물학회지:발생과생식
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• 제22권4호
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• pp.297-307
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• 2018

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and $100{\mu}M$ ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in $50{\mu}M$ ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by $50{\mu}M$ ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and $50{\mu}M$ ALA groups (p<0.05), especially, treatment of $50{\mu}M$ ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and $50{\mu}M$ ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

• 한국발생생물학회지:발생과생식
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• 제25권2호
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• pp.75-82
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• 2021

We studied steroid metabolites produced from red-spotted grouper ovarian follicles during maturation. Oocytes with 350-500 ㎛ diameter were in vitro incubated in the presence of [³H] 17α-hydroxyprogesterone as a precursor. Steroid metabolites were extracted from incubated media and oocytes. The extracts were separated and identified using thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry. The identified metabolites were androstenedione (A₄), testosterone (T) and estrone (E₁). The metabolites of A₄ was dominant in all size of oocytes and it was the highest in 480 ㎛ diameter oocytes. The metabolites of two progestins, 17α,20β-dihydroxy-4-pregnen-3-one and 17α,20α-dihydroxy-4-pregnen-3-one were detected in the oocytes less than 480 ㎛ diameter although they were not identified definitely. In the oocytes of 480 ㎛ diameter, metabolite of progestin was the highest, and germinal vesicle (GV) was still in the middle of cytoplasm. In the oocytes of 500 ㎛ diameter, GV was began to migrate and the major metabolites were A₄ and E₁. The metabolite of E₁ was detected in all size of oocytes and it was higher than that of E₂. These results suggest that oocytes of 480 ㎛ diameter are the transitional stage involving steroidogenic shift to final oocyte maturation and potential function of E₁ during maturation process.

• Asian-Australasian Journal of Animal Sciences
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• 제22권1호
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• pp.35-41
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• 2009

This study was undertaken to evaluate how ${\beta}$-mercaptoethanol (bME), an exogenous antioxidant, interacts with preantral follicles cultured in vitro. Mouse primary or secondary follicles were cultured in glutathione (GSH)-free or GSH-containing medium supplemented with bME of various concentrations, and the growth of preantral follicles, the maturation of intrafollicular oocytes and preimplantation development after parthenogenesis were monitored. In experiment 1, 0, 25, 50 or 100 ${\mu}M$ bME was added to culture medium supplemented with 100 ${\mu}M$ GSH or not. When secondary follicles were cultured in GSH-free medium, no significant change in follicle growth was detected after bME addition. However, exposure to bME in the presence of GSH significantly inhibited both follicle growth and oocyte maturation. Such detrimental effect became prominent in primary follicles and bME strongly inhibited follicle growth in the absence of GSH. In conclusion, there are stage-dependent effects of bME on follicle growth and oocyte maturation, and selective use of antioxidants contributes to establishing an efficient follicle culture system.

• 한국발생생물학회지:발생과생식
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• 제27권3호
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• pp.127-135
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• 2023

Effects of changes in photoperiod on the reproductive events in fish are suggested to be mediated mainly via the action of melatonin (MEL). Changing levels of plasma MEL throughout the day and year are suggested to influence the hypothalamus-pituitary-gonadal axis in fish. Therefore, in this study, we aimed to investigate the effects of MEL on oocyte maturation and germinal vesicle breakdown (GVBD) in the marine fish, Chaenogobius annularis, in vitro. Oocytes at three different stages (pre-, mid-, and late-vitellogenesis) were incubated with (a) only MEL (5, 10, 50, 100, 500, and 1,000 pg/mL) and (b) 50 pg/mL of 17α,20β-dihydroxy-4-pregnen-3-one (17α20βP), maturation-inducing hormone (MIH) of this species, and MEL (4-h incubation before addition of MIH). Any single MEL treatment did not significantly induce GVBD. However, treatment with 50 pg/mL MEL or MIH significantly induced GVBD. These results suggest that preincubation with MEL accelerates the effect of MIH on longchin goby oocyte maturation.

• 한국산학기술학회논문지
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• 제20권9호
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• pp.333-340
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• 2019

본 연구는 염소 난자의 체외배양 시 혈청과 호르몬 첨가의 효과를 알아보기 위하여 체외성숙 효율, 유전자발현 및 체외수정 후 배발달율을 조사하였다. 본 실험에서는 기본 체외성숙 배양액에 10% gBS, 10% gFF를 첨가하여 혈청유무에 따른 효과와 FSH를 조합하여 체외성숙 효율을 조사한 결과, 처리구 모두 대조구보다 유의적으로 높은 성숙률을 보였다. 특히 gBS와 FSH를 조합하였을 때 처리구 중에서도 77.8%로 유의적으로 가장 높은 체외성숙 결과를 보였다. 또한, 각 처리구로부터 얻은 성숙 난자를 이용하여 체외수정 배발달 효율을 조사한 결과 모두 유의적으로 향상되었으며 특히 gBS와 gFF에 FSH를 혼합 처리한 두 그룹이 우수한 결과를 보였다. 난자의 성숙과 관련된 BMP15와 GDF9의 mRNA 발현량을 비교한 결과, 처리구간 특이점이나 유의적 차이가 나타나지 않았다. 본 연구의 결과 염소 난자의 체외성숙에 관한 추가적인 실험이 필요할 것으로 생각되나, gBS, gFF 및 FSH 첨가가 성숙 효율 향상에 도움이 됨을 알 수 있었다.