• Title/Summary/Keyword: Follicular Oocyte

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Effects of Morphology, Reproductive Cycle, Incubation Time and Activation of Oocytes on Developmental Rate of Embryos Fertilized in vitro (난자의 형태, 번식주기, 배양시간 및 활성화 처리가 개 난자의 체외수정후 발생에 미치는 영향에 관한 연구)

  • 이동수;김상근
    • Journal of Embryo Transfer
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    • v.18 no.1
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    • pp.27-33
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    • 2003
  • The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes in vitro maturation of canine oocytes and development of canine IVM/IVF embryos. The results were summarized as follows: 1. The developmental rates to 16 cells of fresh, salts and 4$^{\circ}C$-stored oocytes with and without cumulus cells were 14.3%, 5.0% and 7.5%, 2.8% and 5.7%, 0.0%, respectively. The rate of oocytes with cumulus cells(5.7%~14.3%) was higher than that of denuded oocytes(0.0%~5.0%). 2. The developmental rate to If cells of in vitro cultured oocytes recovered from ovaries collected at different stages of the reproductive cycle were 0.0%, 10.7%, 1.5%, respectively. 3. The developmental rate to 16 cells of fresh oocytes with cumulus cell cultured for 24, 32 and 48 hrs in $CO_2$ incubator were 0.0%, 5.3%, 11.8%, respectively. The rate of oocytes cultured for 48 hrs was higher than that oocytes cultured for 24 and 32 hrs. 4. The development to If cells treated activation and non-activation oocytes were 15.0%, 6.7%, respectively. The rate of oocytes treated activation was higher than that oocyte treat non-activation.

Culture Conditions for In Vitro Maturation of Abattoir Derived Oocytes of Native Zebu Cows of Bangladesh

  • Morshed, S.M. Niyaz;Bhuiyan, Mohammad Musharraf Uddin;Rahman, Mohammad Moshiur;Singha, Joydev Kumer;Juyena, Nasrin Sultana
    • Journal of Embryo Transfer
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    • v.29 no.3
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    • pp.201-206
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    • 2014
  • The objectives of the study were to determine an effective culture dish, culture duration and protein supplementation in medium for in vitro maturation (IVM) of oocytes of native zebu cows in Bangladesh. The ovaries of cows were collected from local slaughterhouse followed by aspiration of follicular fluid. The cumulus-oocyte-complexes (COCs) with more than 3 compact cumulus cell layers were cultured in tissue culture medium (TCM) 199 for maturation. The maturation of oocytes was determined by observing polar body under microscope. To determine an effective culture dish, 130 COCs derived from 48 ovaries in a well of 4-well dish and 102 COCs derived from 36 ovaries in drops covered with mineral oil within 35 mm petri dish were cultured for 24 hours. The rate of maturation of oocytes did not vary between 4-well dish ($51.3{\pm}15.0%$) and drops in petri dish ($52.4{\pm}11.6%$). To determine the effective culture duration, 185 COCs derived from 62 ovaries were cultured in drops for 18, 21, 24 and 27 hours. The rate of maturation of occytes ranged from $51.9{\pm}9.4%$ (18 hours) to $59.0{\pm}17.1%$ (27 hours) and the difference in maturation rate among different culture durations was not significant (P>0.05). To determine an effective protein supplementation, 63 oocytes from 19 ovaries were cultured separately in TCM 199 supplemented with either fetal bovine serum (FBS) or bovine serum albumin (BSA). The rate of maturation was significantly (P<0.01) higher in medium supplemented with FBS ($55.63{\pm}16.19%$) than that of BSA ($14.82{\pm}9.36%$). In conclusion, COCs of native zebu cows can be cultured for IVM either in 4-well culture dish or droplets in petri dish for 18 to 27 hours in medium supplemented with FBS.

Characterization of Genes Related to the Cell Size Growth and CCN Family According to the Early Folliculogenesis in the Mouse (쥐의 초기 난포 발달에 관여하는 Cell Size Growth 및 CCN Family 유전자에 관한 연구)

  • Kim, Kyeoung-Hwa;Park, Chang-Eun;Yoon, Se-Jin;Lee, Kyung-Ah
    • Clinical and Experimental Reproductive Medicine
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    • v.32 no.3
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    • pp.269-277
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    • 2005
  • Objectives: Previously, we sought to compile a list of genes expressed during early folliculogenesis by using cDNA microarray to investigate follicular gene expression and changes during primordialprimary follicle transition and development of secondary follicles (Yoon et al., 2005). Among those genes, a group of genes related to the cell size growth was characterized during the ovarian development in the present study. Methods: We determined ovarian expression pattern of six genes related to the cell size growth (cyr61, emp1, fhl1, socs2, wig1 and wisp1) and extended into CCN family (${\underline{c}}onnective$ tissue growth factor/${\underline{c}}ysteine$-rich 61/${\underline{n}}ephroblastoma$-overexpressed), ctgf, nov, wisp2, wisp3, including cyr61 and wisp1 genes. Expression of mRNA and protein according to the ovarian developmental stage was evaluated by in situ hybridization, and/or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry, respectively. Results: Among 6 genes related to the cell size growth, cyr61 and wisp1 mRNA was detected only in oocytes in the postnatal day5 mouse ovaries. cyr61 mRNA expression was limited to the nucleolus of oocytes, while wisp1 was expressed in the cytoplasm and nucleolus of oocytes, except nucleus. cyr61 mRNA expression, however, was found in granulosa cells from secondary follicles. The rest 4 genes in the cell size growth group were detected in oocytes, granulosa and theca cells. Cyr61 and Wisp1 proteins were expressed in the oocyte cytoplasm from primordial follicle stage. Especially, Cyr61 protein was detected in pre-granulosa cells, Wisp1 protein was not. By using RT-PCR, we evaluated and decided that Cyr61 protein is produced by their own mRNA in pre-granulosa cells that was not detected by in situ hybridization. cyr61 and wisp1 genes are happen to be the CCN family members. The other members of CCN family were also studied, but their expression was detected in oocytes, granulose and theca cells. Conclusions: We firstly characterized the ovarian expression of genes related to the cell size growth and CCN family according to the early folliculogenesis. Cyr61 protein expression in the pre-granulosa cells is profound in meaning. Further functional analysis for cyr61 in early folliculogenesis is under investigation.

Influence of Media and Hormones on the In Vitro Development of Porcine Follicular Oocytes (배지 및 첨가호르몬이 돼지난포란의 체외발생능에 미치는 영향)

  • Park, Byung Kwon;Lee, Kyu Seung
    • Korean Journal of Agricultural Science
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    • v.26 no.2
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    • pp.19-24
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    • 1999
  • This study was conducted to investigate the influence of media and hormones on in vitro maturation and development of porcine follicular oocytes. Basic media were used to TCM-199, Waymouth MB751/1 and BMOC-II, and hormones were used to hCG and FSH in each medium. The results obtained were summarized as follows : 1. The maturation rates of oocytes cultured in TCM-199 medium containing hCG, FSH and hCG+FSH were 78.05, 72.50 and 67.50%, respectively. The maturation rates of oocytes with hormones were significantly (P<0.05) higher than those of oocytes cultured without hormone. However, the cleavage rate(hCG 46.88%, FSH 31.04%. hCG+FSH 37.04%) of embryo cultured in TCM-199 containing hormone was significantly(P<0.05) lower than that(89.47%) of oocytes cultured without hormone. 2. The maturation rates of oocytes cultured in Waymouth MB751/1 medium containing hCG. FSH and hCG+FSH were 69.77, 71.43 and 80.00%, respectively. The maturation rates of oocytes with hormones were significantly(P<0.05) higher than those of oocytes cultured without hormone. However. the cleavage rate(hCG 46.67%. FSH 36.00%, hCG+FSH 35.71%) of embryo cultured in Waymouth MB751/1 containing hormone was significantly(P<0.05) lower than that(60.00%) of oocytes cultured without hormone. 3. The maturation rates of oocytes cultured in BMOC-II medium containing hormone were 66.67(control). 66.67(hCG). 91.89(FSH) and 81.82(hCG+FSH)%. respectively. showing the highest rate in FSH treatment. And, the cleavage rates of oocytes cultured in BMOC-II medium containing hormone were 81.82 (control, 79.17(hCG), 50.00(FSH) and 66.67(hCG+FSH)%, respectively.

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Effects of Some Factors on In Vitro Production of Embryos from Antral Follicle-Derived Porcine Oocytes I. Effects of Maturation Media on In Vitro Maturation, Fertilization and Development (돼지 난포란 유래 체외수정란 생산에 대한 제요인의 영향 I. 체외성숙, 체외수정, 체외발달에 대한 체외성숙 배양액의 영향)

  • Yeon, S.-H.;Choi, S.-H.;Kim, C.-D.;Son, D.-S.;Han, M.-H.;Lee, K.-S.
    • Journal of Embryo Transfer
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    • v.19 no.2
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    • pp.165-172
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    • 2004
  • This study was carried out to examine the effects of maturation media on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-37 (mNCSU-37), modified NCSU-23 (mNCSU-23), or TCM-199 supplemented with 10% porcine follicular fluid (pFF). Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium(mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU-23. The results are as follows. 1. In the result of IVM, the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different among the media, though numeric value of them were slightly lower in TCM-199 than in mNCSU-37 or in mNCSU-23. 2. In the result of IVF, though the rate of sperm penetration was not significantly different among the maturation media, the percentage of oocytes with male pronucleus (MPN) of ones matured in mNCSU-37 (88.0%) was significantly higher than in TCM-199 (71.1%) (p<0.05). 3. In the result of IVD, the percentage of cleaved oocytes of ones matured in mNCSU-37 (52.3%) or in mNCSU-23 (53.7%) was significantly higher than in TCM-199 (43.1%) (p<0.05), but the rate of blastocysts at day 6 was not significantly different among the maturation media, though putative embryos from oocytes matured in mNCSU-37 or in mNCSU-23 were developed more than in TCM-199. These results suggested that mNCSU-37 or mNCSU-23 was more appropriate than TCM-199 as IVM medium for porcine immature oocytes.

Effects of Some Factors on In Vitro Production of Embryos from Antral Follicle-Derived Porcine Oocytes III. Effects of Fertilization Media and the Sperm Concentration during Fertilization on In Vitro Fertilization and Development (돼지 난포란 유래 체외수정란 생산에 대한 제요인의 영향 III. 체외수정배양액과 정자농도가 체외수정 및 체외발달에 미치는 영향)

  • Yeon S. H.;Son D. S.;Jean H. J.;Choi S. H.;Kim I. C.;Park C. S.;Lee K. S.
    • Journal of Embryo Transfer
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    • v.19 no.3
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    • pp.265-273
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    • 2004
  • This study was carried out to examine the effects of fertilization media and sperm concentration on in vitro fertilization (IVF) and development (IVD) of porcine oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10% porcine follicular fluid (pFF). After the fertilization by experimental scheme, putative embryos were developed in vitro in NCSU-23. The results are as follows. When the oocytes were fertilized in vitro in modified TBM or modified TLP-PVA by 1 ${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were not significantly different between two media. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were not different between two media, either. When the oocytes were fertilized in vitro in mTBM by 5${\times}$10$^4$, 1${\times}$10$^{5}$ or 5${\times}$10$^{5}$ sperm/$m\ell$, all of the fertilization parameters were significantly (P<0.05 or P<0.01) increased as sperm concentration was elevated. Subsequently, as these putative embryos were developed in vitro in NCSU-23, the percentage of oocytes cleaved and of blastocysts were significantly boosted (P<0.01) as sperm concentration at fertilization was elevated from 5${\times}$10$^4$ to 1${\times}$10$^{5}$ sperm/$m\ell$, but were not different between 1${\times}$10$^{5}$ and 5${\times}$10$^{5}$ sperm/$m\ell$.

Developmental Capacity of Bovine Follicular Oocytes after Ultra-Rapid Freezing by electron Microscope Grid II.Cryopreservation of In Vitro Matured Bovine Oocytes (Electron Microscopic Grid를 이용한 초급속 동결이 소 난포란의 발달능에 미치는 영향. II. 체외 성숙된 소 미수정란의 동결에 관한 연구)

  • 김은영;김남형;이봉경;윤산현;박세필;정길생;임진호
    • Korean Journal of Animal Reproduction
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    • v.22 no.1
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    • pp.1-9
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    • 1998
  • This study was carried out to confirm whether the developmental capacity of bovine mature oocytes frozen ultra-rapidly using electron microscopic(EM) grids and EFS30 can be obtained, and whether the cryoprotectants and the freezing method used in this study effect detrimentally to the bovine oocytes by indirect immunocytochemistry. As freezing solution, we used EFS30 which consisted of 30% ethylene glycol, 0.5 M sucrose, 18% ficoll and 10% FBS added in D-PBS. The results obtained in this experiment were summarized as follows: When the effects of cryoprotectant and freezing procedure on the microtuble, micrfilament and chromatin morphology of oocytes were evaluated using indirect immunocytochemistry, the results of freezing as well as exposure group were not different with that of the control oocytes. When the fertilization abnormality after ultrarapid freezing of bovine mature oocytes was examined by Hoechst staining, the rates of total penetration(96.7, 9.0%), normal two pronuclei formation(74.6, 68.9%) and mean number of sperm / oocyte(1.50, 1.44) were not different between control and freezing group. In addition, when the developmental capacity of frozen-thawed of oocytes(85.5%) was survived, 74.5% of them were cleaved and 31.4% of cleaved embryos were developed to blastocyst. These data were similar to those of the control(76.0%, 34.6%) and exposure(74.5%, 33.0%) except survival rates. Also, when the total cell number of blastocysts produced from the each treatment at day after IVF was examined by hoechst staining, there were not different among groups. There results demonstrate that developmental capacity of frozen-thawed bovine mature oocytes can be successfully obtained by ultra-rapid freezing method using EM grid and EFS30 solution.

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Effects of clomiphene citrate on ovarian function and embryo developmental capacity in the rat (랫드에 있어서 클로미펜 시트레이트가 난소기능 및 수정란 발육성에 미치는 영향)

  • Yun, Young-won;Kwun, Jong-kuk
    • Korean Journal of Veterinary Research
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    • v.32 no.1
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    • pp.15-24
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    • 1992
  • The effects of CC the ovulatory response, oocyte normality, ovarian steroidogenesis and subsequent embryo developmental potential were examined in PMSG-treated rats. On Days of 25~27 of age, immature female Sprague Dawley rats were treated with three different doses(0.05, 0.1 or 1.0mg /day) of clomiphene citrate or vehicle. The females subsequently received 4IU PMSG on Day 28 and/or 10IU hCG on Day 30, and were killed on Day 31. Some females given 0.1mg CC or vehicle with 4IU PMSG were then mated and killed on Days 2, 3, 4 and 5 of pregnancy. Compared to vehicle(control) group, by increasing the doses of CC, there were a significant decrease in the ovulatory response as judged by both the proportion of rats ovulating and the mean number of oocytes per rat and a marked reduction of ovarian weight. The increasing doses of CC substantially promoted the degeneration(%) of oocytes ovulating in a dose-dependent manner. The CC-mediated inhibitions of the ovulatory response and ovarian weight were oompletely overcome by a subsequent treatment of hCG. Increasing doses of CC resulted in a siginificant elevation of serum estradiol with the decreased levels of progesterone and androgens. The additive treatment with hCG was effective to reduce the elevation of estradiol and to increase the reduction of progesterone produced by high dose(1.0mg) of CC. The preimplantation embryos recovered from 0.1mg CC-treated pregnant rats demonstrated a progressive early loss from Day 3 of pregnancy with a significant increase in the percentage of degeneration during all periods examined, compared to controls. The rate of progressive embryo cleavage in the CC-treated rats were slower than that in controls from Day 3 of pregnancy. Additionally, the percentage of the cleaved embryos recovered from the CC-treated rats remained significantly lower consistently from Day 2 of pregnancy, compared to control regimen. These results demonstrate a possible mechanism of CC-mediated inhibition of ovulatory response in the rats which may include the attenuation or blockade of the endogenous secretion of gonadotropins and also suggest that its detrimental effects observed on oocyte normality and embryonic development may be caused by abnormal follicular steroidogenesis( especially elevated estradiol) preceding fertilization.

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GnRH Antagonist Versus Agonist Flare-up Protocol in Ovarian Stimulation of Poor Responder Patients (저 반응군의 체외수정시술을 위한 과배란유도에 있어 GnRH Antagonist 요법과 GnRH Agonist Flare Up 요법의 효용성에 관한 연구)

  • Ahn, Young-Sun;Yeun, Myung-Jin;Cho, Yun-Jin;Kim, Min-Ji;Kang, Inn-Soo;Koong, Mi-Kyoung;Kim, Jin-Yeong;Yang, Kwang-Moon;Park, Chan-Woo;Kim, Hye-Ok;Cha, Sun-Hwa;Song, In-Ok
    • Clinical and Experimental Reproductive Medicine
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    • v.34 no.2
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    • pp.125-131
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    • 2007
  • Objective: The aim of this study was to compare GnRH antagonist and agonist flare-up treatment in the management of poor responder patients. Methods: One hundred forty-four patients from Jan. 1, 2002 to Aug. 31, 2005 undergoing IVF/ICSI treatment who responded poorly to the previous cycle (No. of oocyte retrieved$\leq$5) and had high early follicular phase follicle stimulating hormone (FSH>12 mIU/ml were selected. Seventy-five patients received agonist flare-up protocol and 71 patients received antagonist protocol. We analyzed the number of oocytes retrieved, number of good embryos (GI, GI-1), total dose of hMG administered, implantation rate, cycle cancellation rate, pregnancy rate, live birth rate. Results: The cancellation rate was high in antagonist protocol (53.5% vs. 30.1%). The number of oocyte retrieved, the number of good embyos were high in agonist flare-up group. There was no statistical difference between GnRH agonist flare up protocol and GnRH antagonist protocol in implantation rate (14.5%, 10.1%), clinical pregnancy rate per transfer (29.4%, 21.2%) and live birth rate per transfer (21.6%, 18.2%). Although the result was not statistically significant, GnRH agonist flare up group showed a nearly doubled pregnancy rate and live birth rate per initial cycle than GnRH antagonist group. Conclusions: The agonist flare-up protocol appears to be slightly more effective than the GnRH antagonist protocol in implantation rate, pregnancy rate, live birth rate but shows statistically no significance. Agonist flare-up protocol improved the ovarian response in poor responders. However, based of the result of the study, we can expect improved ovarian response in poor responders by GnRH agonist flare up protocol.

Increase of Production Ratio of Pre-selected Superior Dairy Female Offspring by Combination of OPU derived Oocytes and X-bearing Semen

  • Kim, Seong-Su;Choi, Byung-Hyun;Lee, Kyeong-Lim;Jin, Jong-In;Suh, Tae-Kwang;Son, Cheol-Ho;Park, Chan-Ho;Shin, Seung-Oh;Han, Kwang-Jin;Lim, Hyun-Tae;Cho, Kyu-Woan;Kong, Il-Keun
    • Journal of Embryo Transfer
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    • v.30 no.2
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    • pp.73-82
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    • 2015
  • This study was designed to evaluate the possibility of increase through dairy female offspring's ratio by transfer of pre-selected transferrable blastocyst that was produced by pre-selected X-bearing semen with OPU derived oocytes. Elite dairy female cow is demanded strongly compared with male, the so called, farmer wants to produce only an elite female dairy offspring as a candidate female dairy cow for producing milk. In our study, we selected 2 elite dairy bull semen from National Agricultural Cooperative Federation to pre-select X-bearing semen and 5 elite dairy female cows as donor for collecting of OPU derived oocytes. OPU derived embryo production system was carried out an aspiration of immature oocytes from 5 donor cows 2 times per week, total 200 times for 2 to 7 months by an ultrasonographic guided follicular aspiration system and then produced in vitro-produced blastocysts by in vitro maturation, fertilization and culture. Dairy donor semen selected H-319, 320 bull in National Agricultural Cooperative federation was sorted X-bearing semen by flow-cytometer and frozen for using IVF with OPU derived oocytes. Donor cows were selected 5 elite dairy cows from Gyeongju Dairy Cow Community and then disease tests such as 4 kinds of disease before selecting was checked. Oocyte proportion of grade 1 to 3 from total collected oocytes was significantly lower in donor A and B than those in donor C, D and E (82.16 and 70.03% vs. 90.0, 91.78 and 93.57%), respectively (p<0.05). However, number of oocytes per session in donor A, C and E was significantly higher than those in donor B and D ($7.77{\pm}3.26$, $5.85{\pm}2.10$ and $7.03{\pm}2.14$ vs. $4.68{\pm}2.61$ and $5.21{\pm}1.97$ oocytes), but donor A was significantly higher than donor C (p<0.05). Development to blastocyst in donor B, C and E was significantly higher than those in donor A and D (31.0, 25.0 and 25.0% vs. 14.3 and 4.5%), but donor A was not different in donor C and E (p<0.05). Nine out of 10 blastocysts (90.0%) derived from OPU blastocysts were confirmed male embryos that was induced with Y-bearing semen to confirm sex ratio only. Total 96 blastocysts derived from female bearing semen were transferred into synchronized recipients and then confirmed 42 recipients (43.8%) pregnancy rate, 36 offspring (37.5%) and 91.7% female sex ratio (33 female vs. 3 male offspring). Taken together all data, elite dairy female offspring could be produced effectively by in vitro production system between pre-selected x-bearing semen and OPU derived oocytes that would be influential breeder in the breeding of dairy farm to increase effectively elite dairy offspring ratio as well as net income in the dairy farmer.