• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.87-94
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• 1996

For evaluating the suitability of human follicular fluid(HFF) as protein supplement in ART, this preliminary study was performed to examine the maturation promoting activity of HFF on mouse follicular oocytes in vitro. Mouse follicular oocytes were collected from ovaries of 21-28 day old ICR mice by puncturing the antral follicles with fine needle at 48 hours after PMSG injection. The oocytes were rinsed and cultured in modified Whittingham's $T_6$ medium containing purines or dbcAMP to maintain meiotic arrest, and different concentrations of HFF were added into the culture medium to examine the effect of HFF on releasing the oocytes from the suppressive influence of the meiotic inhibitors. As a control for HFF, the maturation promoting activity of human fetal cord serum(HFCS) was investigated and compared with the activity of HFF. While HFF was separated, by molecular weight(M.W), into high M.W. fraction(M.W>30,000) and low M.W. fraction(M.W<30,000) and the effects of the fractions on meiotic resumption were investigated in the presence of the meiotic inhibitors. Also hormone analysis was performed to compare the content of hormones in HFF with that in HFCS. Same concentrations of HFF and HFCS induced similar germinal vesicle break down(GVBD) rates of the oocytes meiotic arrested by purines(4mM hypoxanthine+0.75mM adenosine), but the extrusion rate of 1st polar body(PB) of the oocytes cultured in HFF(65.0%, P<0.05) was significantly higher than that(51.6%) in HFCS. While, in the presence of 200 M dbcAMP, the maturation promoting activity of HFF (GVBD: 70.5%, $p<10^{-6}$; 1st PB extrusion: 67.1%, $p<10^{-3}$) was significantly greater than that of HFCS(GVBD: 35.2%; 1st PB extrusion: 41.1%). The oocytes cultured in the fraction of HFF containing high M.W. components showed higher meiotic maturation rates than the oocytes cultured in the low M.W. fraction of HFF. Gonadotropins and $E_2$ were known to improve the completion of maturation changes, and the levels of these hormones were higher in HFF than in HFCS. Therefore, HFF was more effective than HFCS to use for promoting meiotic resumption of mouse oocytes in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.95-102
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• 1996

This study was performed to investigate the effect of human follicular fluid (HFF) on development of mouse embryos, for evaluating the suitability of HFF as a substitutive material of human fetal cord serum in ART program. The various concentrations of HFF were added into the culture medium and the effects of HFF concentrations were examined to identify the optimal concentration of HFF for embryo development. The potency of HFF in improving embryo development was compared to that of other protein supplement. Collected HFFs were classified with the maturity of the containing oocytes; mature, immature, atretic, and then the effects of the classified HFFs on embryo development were examined. Also, HFF was separated into the low (<30,000 Da) and high (>30,000 Da) molecular weight fractions and the effects of the fractions on embryo development were investigated. The highest development rate was found in culture medium supplemented with 20% HFF, bnt this rate was reversely reduced at the concentrations of HFF higher than 20%. The development rates to the blastocyst, hatching blastocyst, attachment and outgrowth cultured in mature HFF was significantly higher than those in immature and atretic HFF, and mean cell number in blastocyst was higher in mature HFF than in immature and atretic HFF. The development rates of mouse embryos according to protein sources were significantly higher in HFF than in fetal cord serum (FCS), maternal serum (MS) and bovine serum albumin (BSA), and mean cell number in blastocyst cultured in HFF was higher than that in FCS, MS and BSA. The development rates of embryo and mean cell number in blastocyst cultured in high molecular weight fraction of HFF were higher than those in low molecular weight fraction, but the results of high molecular weight fraction were lower than those of whole HFF. Therefore, these results indicated that human mature follicular fluid was useful for improving the development of mouse embryos, which suggests a possibility that HFF also may be used efficiently for improving the culture condition in human ART program as a protein supplement.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.103-108
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• 1996

Through the previous studies(I,II), it was observed that human follicular fluid(HFF) was more effective than human fetal cord serum(HFCS) on promoting meiotic resumption of oocytes and improving embryonic development of mouse in vitro. On the basis of these results, we have gradually exchanged HFCS with HFF as protein supplement in human ART. This study was performed to investigate the efficiency of HFF on improving the pregnancy rate in ART. Oocytes were retrieved transvaginally from patients treated with pituitary suppression with GnRH-agonist and ovarian stimulation with human menopausal gonadotro-pin(HMG) and pure follicle stimulating hormone(FSH). Aspirated oocytes were rinsed and cultured in TCM-199 containing HFF, and the concentrations of HFF were adjusted to 10, 20, and 30% according to the use for insemination, embryo growth and embryo transfer, respectively. As possible as, we tried to do embryo transfer into fallopian tube to mimic the coincidence of the cell stage with the place of sojourn in vivo, so we performed various ART programs(IVF & ET; in vitro fertilization, ZIFT; zygote intra fallopian-tube transfer, ZIFT & ET) according to the tubal conditions of patients. On the while, intra cytoplasmic sperm injection(ICSI) was used to assist IVF of the patients who had shown poor standard IVF results by immunological or severe male factor. Of the 255 cycles of ART programs using HFF as protein supplement, 118 cycles were turn out to be succeeded in pregnancy(46.2%, per cycle, p<0.05), while 21 pregnancies were achieved in the 69 cycles using HFCS(30.4%). The 255 cycles using HFF were subdivided into cycles with the type of ART programs, and each pregnancy rate of the ART programs were 44.7% (IVF & ET, 76/170 cycles), 53.4%(ZIFT, 31/58 cycles) and 40.7% (ZIFT & ET, 11/27 cycles), respectively. In the 61 ICSI cycles using HFF, 28 cycles succeed in pregnancy(45.9%), while 7 pregnancies were obtained in the 17 ICSI cycles using HFCS. Also the ongoing pregnancy rate in the group using HFF(78.8%, 93/118 cycles) was higher than that in the group using HFCS(61.9%). Therefore, we found that the use of HFF as protein supplement was more suitable and effective than the use of HFCS to improve the pregnancy rate in ART.

• Development and Reproduction
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• v.3 no.1
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• pp.39-44
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• 1999

In the present study, we investigated the effect of maturity and motility of spermatozoa on the formation of pronuc-leus and subsequent developmental capacity of the human embryo in vitro. The fertilization was performed by means of intracytoplasmic sperm injection (ICSI) in HEPES-buffered m-TCM-199 medium. In the first part of the experiment, motile or im-motile human spermatozoa ejaculated were injected into cumulus-enclosed human oocytes matured in vivo. Significantly (p<0.002) higher proportion of oocytes that was injected with motile spermatozoa formed 2 pronuclei than the oocytes injected with immotile spermatozoa (79.8% vs 51.7%). In the second part of the experiment, cumulus-enclosed human oocytes matured in vivo were injected with motile or immotile spermatozoa collected from testes. There was no difference between motile and immotile spermatozoa. In the third part of the experiment, using modified Tyrode's medium containing 10.0 mM lactate, 0.5 mM pyruvate, 0.2 mM taurine, 1.0 mM glutamine, 2.22 mM MEM amino acids, vitamin and 10% human follicular fluid, we found that the development of oocytes that formed 2 pronuclei were able to develop to 9-16 cells regardless of maturity and motility of spermatozoa.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.9
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• pp.333-340
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• 2019

This study investigated the effects of goat blood serum (gBS), goat follicular fluid (gFF) and gonadotropin (FSH) on the in vitro maturation, fertilization and development of Korean native black goat oocytes. Our results indicate that the gBS combined with FSH treated group showed significantly higher maturation rate than the other groups. Furthermore, blastocyst formation rate was significantly increased in all treated groups, and gBS and gFF combined with FSH treated groups were higher than other groups. However, gene expression levels of BMP15 and GDF9 in COC, both oocyte maturation related genes, remained unaffected after 24 h maturation. The results of the present study indicate that supplementation of the maturation medium with gBS, gFF and FSH is efficacious in improving the in vitro maturation, fertilization and development of Korea native black goat oocytes.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.193-201
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• 2001

This study was designed to examine the factors affecting in fertilization and development of embryos in vitro, and to examine whether zone drilling by laser irradiation can improve the hatching rate of IVF embryos from DNA marker-proved Hanwoo. DNA markers related to marbling score were identified using DNA fingerprinting with Ml3 probe and restriction enzyme Hae III. Oocytes were aspirated from immature ovarian follicles using a combined method of rectal ovarian-palpation and transvaginal ultrasound-guidance(6.5MHz) under local anesthesia. The aspirated oocytes were washed twice with fresh D-PBS containing 5% FBS and were rewashed 4 to 5 times with TCM-199 containing 5% FBS. A morphological grade of I to IV was assigned to each oocyte. Data were analyzed using the GLM procedure of SAS. Sperm separation methods did not have any significant effect on cleavage or developmental abilities of IVF embryos. Significantly(P<0.05) higher cleavage rate was observed in embryos from GI(60.0%, 3/5), GII(69.2%, 18/26) and GIII(62.1%, 59/95) compared to embryos from GIV oocytes(36.2%, 25/69). And the developmental rate to blastocyst stage was higher(P<0.05) in embryos from GI(33.3%, 1/3) and GII oocytes(38.9%, 7/18) than those from GIII(16.9%,10/59) and GIV oocytes(4.0%, 1/25). There was no significant difference in development of IVF embryos to blastocyst by media for in vitro culture. Proportion of hatched blastocyst was significantly(P<0.05) higher in embryos received zona drilling by laser than those of non-drilled.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.81-81
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• 2002

$\beta$-Mercaptoethanol($\beta$-ME)은 일반적으로 황화합물(thiol compounds)의 일종으로, 배양액 중에서 이황화결합(disulfide bonds)을 분해하여 일정한 물질의 산화.환원반응에 관여하며, 특히 cysteine이 cystine으로 산화되는 것을 차단함으로서 cysteine의 이용능력을 증대시키고, GSH의 합성을 촉진 및 증대시키는 것으로 알려져 있고, 각종 활성산소로부터 세포를 보호하는 역할을 수행하는 것으로 보고되었다. 특히, 돼지수정란의 체외배양체계에 유의적인 영향을 미치는 것으로 보고되었다(Abebydeera 등, Theriogenol., 50:747-756, 1998). 따라서 본 실험에서는 돼지난포란의 체외성숙시 $\beta$-ME의 첨가배양이 체외수정과 배발달에 미치는 영향에 관하여 조사하였다. 돼지난포란을 10% PFF, 0.1mg/ml cysteine, 10IU/m1 PMSG, 10IU/m1 hCG 및 10ng/m1 EGF가 첨가된 NCSU23 배양액에 $\beta$-ME를 각각 0, 25, 50 및 100uM을 처리하여 22시간 동안 배양을 실시하고, 성선자극호르몬이 배제된 배양액에서 추가로 22시간을 배양하여 체외성숙을 유도하였다. 체외성숙이 유기된 난자는 난구세포를 제거하고, 2.5mM caffeine과 0.1% BSA가 첨가된 mTBM배양액에 정자를 1.25 $\times$ $10^{5}$cells/ml의 농도로 5-6시간 동안 공동배양을 실시하여 체외수정을 유도하였다. 체외수정후 일부의 수정란은 12시간에 난자 급속 염색방법으로 염색하여 다정자침입률 및 자.웅전핵형성률 등을 확인하였다. 그리고 나머지1-세포기의 수정란은 0.4mg/ml BSA가 함유된 NCSU23 배양액에 30 embryos/50ul 소적으로하여 38.8$^{\circ}C$, 5% $CO_2$의 탄산가스 배양기에서 각각 7일간 배양을 실시하였다. 조사된 결과는 SAS/STAT를 이용하여 통계분석을 실시하였다. 체외수정 12시간 후에 난자 급속 염색법으로 염색을 실시한 결과, 모든 처리구에서 핵성숙률(76.4~95.2%), 정자침투율(51.1~66.9%), 웅성전핵형성률(95.2~100%), 다정자침입률(18.2~25.6%) 및 평균침입정자수(1.2~l.4개)에서 유의적인 차이가 인정되지 않았다. 체외배양 48시간 난할률을 조사한 결과, 처리구별 차이(53.9~67.9%)는 인정되지 않았으나, 배양 7일째 배반포형성률은 각각 14.5, 25.4, 17.3 및 12.4%로서 25uM의 $\beta$-ME처리구가 유의적(P<0.05)으로 높은 배발달률을 나타내었고, 총세포수에 있어서는 대조구와 처리구간 유의적인 차이가 인정되지 않았다. 따라서 돼지 난포란을 성숙배양할 때, 25uM $\beta$-ME를 첨가배양하는 것이 양질의 돼지체외수정란을 생산하는 하나의 방법으로 조사되었다.다.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.17-23
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• 2002

The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.87-87
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• 2001

도살장에서 구입한 소의 난소로부터 회수한 미성숙 난포란을 체외에서 성숙, 수정시킨 후, 2~8세포기 수정란을 CR$_1$aa 체외배양액에 일정량의 aesculetin, taurine을 첨가하여 체외배양을 실시한 두 처리구간의 항산화 효과를 비교 검토하고, aesculetin과 taurine에 growth factor(EGF, PDGF)를 첨가배양하여 체외수정란의 체외발육에 항산화제와 growth factor의 상호작용 효과를 검토하였다. 대조구, aesculetin(1$\mu\textrm{g}$/$m\ell$) 및 taurine(2.5mM)을 첨가하여 체외수정란을 체외배양시킨 결과 상실배이상 발육된 체외발육율은 각각 46.5%, 64.3% 및 60.5%로서 대조구보다 aesculetin과 taurine 첨가구가 유의하에 높은 성적을 얻어, aesculetin과 taurine이 체외수정란의 체외발육에 높은 효과를 나타냈다. 대조구, aesculetin (1$\mu\textrm{g}$/$m\ell$) ＋ PDGF (1ng/$m\ell$) 첨가구, aesculetin (l$\mu\textrm{g}$/$m\ell$) ＋ EGF (10ng/$m\ell$)첨가구, taurine (2.5mM) ＋PDGF (1ng/$m\ell$) 첨가구 및 taurine (2.5mM)＋EGF (10ng/$m\ell$) 첨가구에서 배반포기 발육율은 각각 46.0%, 70.0%, 58.0%, 56.0 및 66.0%로써 처리구가 무첨가구에 비해 유의하게 높은 성적을 얻어(P<0.05), 항산화제와 growth factor의 첨가 배양이 체외수정란의 체외발육에 상승효과가 있음이 입증되었다. 배양액에 천연추출 aesculetin과 상품화된 aesculetin을 첨가하여 체외배양을 실시한 결과 배반포기 발육율은 대조구, 천연추출 aesculetin 및 상품화된 aesculetin 첨가구에서 각각 38.6%, 54.6% 및 53.5%로써 첨가구가 무첨가구보다 높은 성적을 얻어, aesculetin의 항산화 효과가 입증되었다(P<0.05). 그러나 모든 처리구에서 배반포까지 발육된 체외수정란의 세포수에는 커다란 차이가 인정되지 않았다.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.239-246
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• 1992

Capacitated and acrosome~reacted spermatozoa were microinjected into the perivitelline space of bovine oocytes matured in vitro. Oocytes obtained from the ovaries of slaughtered heifers and cows were cultured in vitro in the TCM-199 supplemented with 20% FCS for 24 hr at 39$^{\circ}C$ under an atmosphere of 5% CO$_2$ 8% O$_2$. Fresh or frozen spermatozoa were incubated for 2 hr at 39°C under an atmos-phere of 5% CO$_2$, 8% O$_2$ in Ham's F-lO medium containing 0.75% BSA for capacitation, and kept for 30 min in culture medium containing 12 mM of dbcGMP and lOmM of immidazol for acrosome resction. One motile spermatozoon was injected into the perivitelline space of each oocyte. The 2nd polar body and the pronuclei were observed in 9.5% and 5.4% of oocytes, respectively. The rate of cleavage of oocyte over 2-cell stage was 4.1%(10 of 242), These results indicate that the microinjection may be a useful technique to study sperm-oocyte interaction.