• 한국가축번식학회지
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• 제16권3호
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• pp.247-260
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• 1992

This study was carried out to investigate the histomorphological changes of ovaries obtained from 100 of 1-year-old female Korean loach(misgurnus anguillicaudatus). The light microscopic and ultrastructural changes ofooplasm and follicular membranes of oocytes, were observed by lightand transmission electron microscope during the reproductive cycle. All data were collected from November in 1991 to May in 1992. The results obtained in this study were as follows: The size of the nucleoli and number of the yolk granules increased as the oocytes grown. Yolk granules were loosely deposited in the oocytes as crystalline granules. Due to the presence of large early and late maturing oocytes, their ovaries were enlarged, transparent, granular and yellowish in color. The lattice was broken down at hydration, leaving the egg transparent. As the percentages of fish in LMO and RO stage increased from March to April, mean gonadosomatic index(GSI) values(18.49%) increased. Zona radiata change a squamous into cuboid shape in EMO stage. Processes from the granulosa cells and from the oocyte, microvilli grow and make contact with other in the pore canals of the zona radiata during vitellogenesis, but are withdrawn as the zona radiata becomes more compact and devoid of pore canals during oocyte maturation. Seasonal changes in the microscopic appearance of the ovaries were well correlated with those in both GSI and macroscopic appearance.

• 한국동물생명공학회지
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• 제34권1호
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• pp.35-39
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• 2019

This study aimed to recover the ovarian function through in vitro culture of preantral follicles from aged mice. First, we isolated the preantral follicles from ovaries of sixty-seven-week old B6D2F1 mice with decreased fecundity to know how many follicles were present in them, which was 6 preantral follicles including 2 primary, 2 early secondary and late secondary follicles from 8 aged mice. It was confirmed that a few follicles (~2) were present in aged mice through histological analysis compared to adult mice as control. The 9 days of in vitro culture of preantal follicles showed in vitro growth and induced maturation after treatment with hCG (2.5 IU/mL) and EGF (5 ng/mL). Cumulus cells in the cumulus-oocyte complexes (COCs) were removed using hyaluronidase and oocytes at the germinal vesicle (GV) and GV breakdown (GVBD) were obtained from preantral follicle culture of aged mice in vitro. In conclusion, these observations demonstrated that there still were a few preantral follicles in the ovaries of 67 week-old mice, which we were able to culture in vitro and oocytes were obtained from them. This study proposed an in vitro culture system using preantral follicle as a therapeutic strategy for fertility preservation in humans for assisted reproductive medicine.

• Clinical and Experimental Reproductive Medicine
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• 제14권1호
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• pp.7-17
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• 1987

It appears that a major determinant of the success of in vitro fertilization is the selection of the optimal follicle containing an oocyte capable of being fertilized and producing a normal pregnancy. However, the hormonal basis of oocyte maturation is not well substantiated by the as yet available informations. It has been suggested that prolactin(PRL) may stimulate the formation of an oocyte maturation inhibitor and thus inhibit the maturation of oocyte. During the hyperstimulated menstrual cycles serum estradiol($E_2$) levels are markedly elevated, and it seems justified to assume that serum prolactin levels may be elevated since estrogens are potent stimulators of prolactin secretion. This study was carried out to ascertain the effect of the elevated serum estradiol levels on the serum prolactin levels in women undergoing ovarian hyperstimulation with either hMG and/or clomiphene citrate. Serum estradiol and prolactin profiles were measured from third menatrual cycle day to ovulation or ovum aspiration day in 11 normal menstruating women and 30 women who underwent an in vitro fertilization procedure with ovarian hyperstimulation by hMG, clomiphene citrate/hMG, clomiphene citrate. Ovum aspiration was performed 36 hours after hCG administration. The day of ovum aspiration or ovulation was designated Day 0. Serum estradiol levels increased progressively during the follicular phase and this rise peaked on Day-1 at a mean concentration of 1,204${\pm}$189.0pg/ml in Group II(hMG), 1,194${\pm}$167.9pg/ml in Group III(clomiphene citrate/hMG), 1,035${\pm}$195.1pg/ml in Group IV(clomiphene citrate) respectively and on Day -2 of 336${\pm}$34.5pg/ml in Group I(normal control). The elevated estradiol levels fen rapidly after ovulation or ovum aspiration. Serum estradiol values of hyperstimulated groups(Group II, III, IV) were significantly higher than that of control group(Group I) from Day -6 to Day +1, but there was no significant difference of estradiol values among the hyperstimulated groups. Serum prolactin levels increased and peaked on Day +1 at a mean concentration of 60.8${\pm}$14.4ng/ml in Group II, 34.2${\pm}$7.0ng/ml in Group III, 30.1${\pm}$5.7ng/ml in Group IV respectively, but no significant elevation was observed in Group I. Levels of estradiol and prolactin can be positively and significantly correlated in the hyperstimulated groups. However, the increase of serum prolactin levels in hMG group was significantly higher than those in clomiphene citrate/hMG or clomiphene citrate group.

• 한국발생생물학회지:발생과생식
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• 제12권3호
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• pp.275-281
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• 2008

본 연구에서는 해산어를 이용하여 bisphenol A(BPA)와 nonylphenol(NP)이 난모세포 성숙 과정에 어떤 영향을 미치는지 조사하기 위해 성숙단계에 있는 노래미(Hexagrammos agrammus) 난모세포(난경 약 1.88 mm)를 대상으로 in vitro에서 BPA와 NP 처리에 의한 난모세포의 성스테로이드 생성농도를 조사하였다. 난모세포에 BPA와 NP를 농도구별(0.1, 1, 10, 100, 1,000 ng/$m{\ell}$)로 첨가하고, 50 IU의 human chorionic gonadotropin(HCG)를 농도구별 BPA 또는 NP와 함께 첨가하거나 하지 않고 48시간 동안 배양하였다. 배양 후 배양액 내의 $17{\alpha},20{\beta}$-dihydroxy-4-pregnen-3-one($17{\alpha}20{\beta}OHP$), estradiol-$17{\beta}(E_2)$ 그리고 testosterone(T)의 농도를 방사면역측정법(RIA)을 통해 정량하였다. BPA 처리구에서는 100 ng/$m{\ell}$의 농도구에서 HCG 처리 유무에 상관없이 $E_2$ 생성이 촉진되었다. HCG 처리하에서 0.1 ng/㎖의 농도구에서 T 생성은 촉진 되었으나, HCG를 처리하지 않은 실험구의 모든 농도구에서 T 생성은 저해되었다. NP 처리구에서는 HCG를 처리하지 않은 실험구의 10 ng/$m{\ell}$의 농도구에서 $17{\alpha}20{\beta}OHP$와 T 생성이 촉진되었고, 1 ng/$m{\ell}$의 농도구에서는 $E_2$ 생성이 억제되었다. 이상의 결과를 종합하면, 노래미의 성숙단계의 난모세포에서 BPA는 약한 estrogen-agonistic 효과를, NP는 estrogenantagonistic 효과를 지니는 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제43권2호
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• pp.119-125
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• 2016

Objective: The aim of this study was to report a case series of in vitro matured (IVM) oocyte freezing in gynecologic cancer patients undergoing radical surgery under time constraints as an option for fertility preservation (FP). Methods: Case series report. University-based in vitro fertilization center. Six gynecologic cancer patients who were scheduled to undergo radical surgery the next day were referred for FP. The patients had endometrial (n=2), ovarian (n=3), and double primary endometrial and ovarian (n=1) cancer. Ex vivo retrieval of immature oocytes from macroscopically normal ovarian tissue was followed by mature oocyte freezing after IVM or embryo freezing with intracytoplasmic sperm injection. Results: A total of 53 oocytes were retrieved from five patients, with a mean of 10.6 oocytes per patient. After IVM, a total of 36 mature oocytes were obtained, demonstrating a 67.9% maturation rate. With regard to the ovarian cancer patients, seven IVM oocytes were frozen from patient 3, who had stage IC cancer, whereas one IVM oocyte was frozen from patient 4, who had stage IV cancer despite being of a similar age. With regard to the endometrial cancer patients, 15 IVM oocytes from patient 1 were frozen. Five embryos were frozen after the fertilization of IVM oocytes from patient 6. Conclusion: Immature oocytes can be successfully retrieved ex vivo from macroscopically normal ovarian tissue before radical surgery. IVM oocyte freezing provides a possible FP option in patients with advanced-stage endometrial or ovarian cancer without the risk of cancer cell spillage or time delays.

• Clinical and Experimental Reproductive Medicine
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• 제50권4호
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• pp.270-276
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• 2023

Objective: This study investigated the clinical and laboratory factors associated with the presence of dysmorphic oocytes in intracytoplasmic sperm injection (ICSI) cycles. Methods: The study involved 200 ICSI cycles, performed from 2020 to 2021, that yielded at least one mature oocyte. Clinical characteristics and ovarian stimulation methods were compared between 68 cycles with at least one dysmorphic oocyte (the dysmorphic group) and 132 cycles with normal-form oocytes only (the non-dysmorphic group). Dysmorphic oocytes were characterized by dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body. Results: The ages of the women, indications for in vitro fertilization, serum anti-Müllerian hormone levels, and rates of current ovarian endometrioma were similar between the dysmorphic and non-dysmorphic groups. In both groups, the three ovarian stimulation regimens, two types of pituitary suppression, and total gonadotropin dose were employed similarly. However, the dual-trigger method was used more frequently in the dysmorphic group (67.6% vs. 50%, p=0.024). The dysmorphic group contained significantly more immature oocytes and exhibited significantly lower oocyte maturity (50% vs. 66.7%, p=0.001) than the non-dysmorphic cycles. Within the dysmorphic group, significantly lower oocyte maturity was found in the cycles using a dual-trigger, but not in those with a human chorionic gonadotropin trigger. Conclusion: ICSI cycles with dysmorphic oocytes are closely associated with reduced oocyte maturity. This association was observed exclusively in dual-trigger cycles.

• 한국동물학회지
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• 제31권2호
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• pp.87-94
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• 1988

전라남도 일대에서 서식하는 북방상개구리(R. dybowskii)와 참개구리(R. nigromaculara)를 채집하는 생체외배양에 의한 여포난자의 성숙을 유도하였다. 북방산개구리의 여포난자는 배양액(amphibian Ringer's soluion AR)에 첨가한 progesterone, 0.1 $\mu$g/2 ml에 의해 난자의 성숙(핵붕괴)이 유도 되었으며 참개구리의 난자는 1 $\mu$g/2 ml (frog pituitary homogenate FPH)을 얻어서 그 효과를 조사해본 결과 북방산개구리에서는 0.01 pituitary equivalent/2 ml에서, 참개구리는 0.1 pit equiv./2 ml에서부터 여포난자의 성숙이 일어났다. 난자의 성숙에 요하는 시간은 두 개구리에서 모두 9-15시간이었으며 호르몬에 대한 반응성, 성숙기간 등은 개구리 재료로 가장 많이 사용되는 법개구리(R. pipiens)와 거의 일치하였다. 특이하게 2개월 이후에 사용한 북방산개구리의 여포난자는 호르몬의 도움없이도 성숙이 일어났으며 성숙기간도 3시간으로 매우 빨라졌다. 난소조각을 배양했을 때 자발적으로 성숙을 일으키는 여포들은 자발적인 배란까지도 일으키는 것을 발견하였다.

• Reproductive and Developmental Biology
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• 제32권3호
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• pp.147-152
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• 2008

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 $56^{\circ}C$ and $97^{\circ}C$, 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 $56^{\circ}C$ 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.

• Asian-Australasian Journal of Animal Sciences
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• 제26권11호
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• pp.1545-1552
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• 2013

This study was conducted to investigate the effects of Trichostatin A (TSA) on cumulus expansion during mouse oocyte maturation. TSA treatment inhibited cumulus expansion and significantly reduced the cumulus expansion index (CEI) (p<0.05). To determine the underlying mechanism, the expression levels of several key factors that play crucial roles in cumulus expansion including components of extracellular matrix (ECM) (Has2, Ptgs2, Ptx3, and Tnfaip6) and Growth differentiation factor 9 (GDF9) were measured in control and TSA treated samples by real-time PCR. The effect of TSA on ERK phosphorylation (p-ERK1/2) in cumulus cells and GDF9 protein level in fully grown oocytes (FGOs) were detected by Western blotting. The expression levels of the ECM genes were significantly decreased (p<0.05) by TSA treatment while GDF9 expression did not response to TSA (p>0.05). TSA treatment blocked the activation of ERK1/2 (p<0.05) and had no significant effect on GDF9 protein expression (p>0.05). Collectively, these results suggested that TSA treatment altered ECM gene expression and blocked ERK1/2 activation to inhibit cumulus expansion in the mouse.

• Clinical and Experimental Reproductive Medicine
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• 제21권2호
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• pp.177-182
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• 1994

ICR female mice aged 3 to 4 weeks, were stimulated with 7.5 IU PMS injection. At 48-52h post-PMS injection, ovaries were dissected out and oocytes-cumulus complexes(OCCs) were divided into three groups, cumulus-free oocytes(O), cumulus-free oocyte cocultured with cumulus cells(O+C) and OCC. The oocyte were cultured in TCM199 containing various protein sources, FCS, BSA or PVP with gonadotropins(Gns) for 24h. Spermatozoa were collected from cauda epididymis and capacitated in T6 + BSA for 2h. After oocyte maturation in vitro(IVM) in different experimental groups, matured oocytes were inseminated with the capacitated spermatozoa in T6 + BSA for 6h. In the groups of IVM in TCM + BSA or PVP, fertilization(IVF) did not occur efficiently. However, increased fertilization was found in TCM+ FCS group. The oocytes groups, with cumulus cells showed decreased polyspermy in FCS group (O; 31.8 %, O + C; 12.2 %, OCC; 16%), the addition of Gns did not prevent polyspermy in all three groups. The rates of fertilization increased in zona-free oocytes in PVP group. This results showed that culture system for IVM and IVF could be improved. Furthermore, PVP can be used for the substitution of protein source during maturation, and its low rate of fertilization has been found due to zona hardening which occurred in FCS-free medium.