• Clinical and Experimental Reproductive Medicine
• /
• v.31 no.1
• /
• pp.75-81
• /
• 2004

Objective: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. Methods: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1, 2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-$\beta$-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). Results: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. Conclusion: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

• Korean Journal of Animal Reproduction
• /
• v.12 no.2
• /
• pp.112-119
• /
• 1988

These studies were conducted to investigate the effects of culture temperature and time on the in vitro maturation and semen type and media on the in vitro fertilization of bovine follicular oocytes, and to asses in vitro fertilization rate of oocytes cultured by extraffollicular method following fertilization in vitro, or transfer into the pseudopregnant rabbit oviduct or uterus. The bovine oocytes recovered from follicles were cultrued for 18 hrs or 72hrs at 38$^{\circ}C$ with 5% CO2 in moist air. Flesh-diluted(2 folds) and frozen-thawed semen in 0.5ml straw from a fertile bull were used. In order to obtain capacitation of spermatozoa were treated with bovine follicular fluids(BFF) and Inophore A(IA). The results obtained were summarized as follows: 1. The oocytes were classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 62.0%, 12.0%, 17.2%, 5.9% and 3.0% of the total oocytes harvested, respectively. 2. The oocytes matured to metaphase II were significantly increased between 24-48hrs of incubation and at 37-39$^{\circ}C$ with 5% CO2 in moist air. 3. The in vitro fertilization rate following transferred into rabbit oviduct or uterus with bull semen and in vitro matured oocytes were higher ligation than non-ligation of oviduct or uterus. 4. The in vitro fertilization rate of oocytes matured in vitro were higher neat than frozen semen and treatment of IA than BFF on the capacitation of spermatozoa. 5. The effects of semen types and media on in vitro fertilization of oocytes matured in vitro were higher fertilization rate of neat than friozen semen, and media was not significant.

• Journal of Embryo Transfer
• /
• v.25 no.1
• /
• pp.1-7
• /
• 2010

The objective of this study was to examine the effect of vitamin B (pantothenic acid, folic acid, and myo-inositol) that was supplemented to embryo culture medium on in vitro development of parthenogenetically activated (PA) pig embryos. Cumulus-oocyte complexes derived from slaughtered ovaries were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (hCG and eCG) for the first 22 h and then further cultured in hormone-free medium for an additional 22 h. After maturation culture, metaphase II oocytes that extruded 1st polar body were electrically activated and treated with $5.0\;{\mu}g/ml$ cytochalasin B for 4 h. Then, PA embryos were cultured for 7 days in a modified NCSU-23 that was supplemented with pantothenic acid, myo-inositol, or folic acid at different concentrations ($3{\sim}300\;{\mu}M$) according to the experimental design. Myo-inositol added to culture medium did not show any beneficial or inhibitory effects on embryo cleavage and blastocyst formation. However, $300\;{\mu}M$ pantothenic acid significantly inhibited blastocyst formation compared to control (no addition) (24% vs. 36%, p<0.05). Folic acid ($300\;{\mu}M$) significantly (p<0.05) increased blastocyst formation (56%) compared to control (41%). Our results demonstrated that in vitro development of PA embryos was significantly influenced by vitamin B and addition of $300\;{\mu}M$ folic acid to culture medium improved in vitro development of pig PA embryos.

• Journal of Embryo Transfer
• /
• v.26 no.4
• /
• pp.223-227
• /
• 2011

The objective of this study was to compare of different isolation method of mouse preantral follicles, and to examine in vitro development of mouse preantral follicles isolated by different method. Preantral follicles were mechanically or enzymatically extracted from mouse ovaries. Mechanical isolation method used fine gauge needles and enzymatic method of isolating follicles used collagenase. The recovered preantral follicles were cultured for 10 days in alpha-minimal essential medium (${\alpha}$-MEM) + 5% FBS + Insulin-Transferrin-Selenium (ITS) + 100 mIU/ml FSH. The collected primary follicles by enzymatic treatment were higher than mechanical method. Others stage preantral follicle by mechanical isolation were higher than enzymatic method. After 10 days of culture, no statistical differences were shown in survival rates of preantral follicle among the 2 culture groups. The metaphase II rates of the oocytes were significantly higher (p<0.05) in mechanical method (17.8%) than in enzymatic method (5.1%). These results suggest that the isolation method of choice depends on the target stage preantral follicles and mechanical isolation is an optimal method of preantral folliclesin a culture of mouse preantral follicle.

• Journal of Animal Reproduction and Biotechnology
• /
• v.34 no.2
• /
• pp.86-92
• /
• 2019

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.10 no.6
• /
• pp.1407-1413
• /
• 2009

The present study was conducted to gain insights into oocyte maturation and early embryo development, SELDI-TOF-MS was used to find the protein candidates that are specifically or prominently expressed in mouse oocytes at the in vitro matured metaphase II (MIl) and germinal vesicle (GV) stages. By using selected CM10 chip, found 16 candidates which were up-regulated in GV stage oocytes compared with in MIl stage oocytes, molecular weight are 8180 (2 candidates), 10226 (5 candidates), 15767 (5 candidates) and 16770 (4 candidates) Da respectively. And 29 candidates were higher in MIl than in GV stage oocytes, molecular weight are 10832 (3 candidates), 17744(8 candidates), 20122 (3 candidates), 22131 (3 candidates), 24857 (7 candidates) and 33507 (5 candidates) Da, respectively. All (45) candidate (0.2 and 1.0 % error tolerances) were performed real time RT-PCR analysis and further selected 13 more potential candidates.

• Clinical and Experimental Reproductive Medicine
• /
• v.42 no.3
• /
• pp.111-117
• /
• 2015

Objective: In order to increase the number of mature oocytes usable for intracytoplasmic sperm injection (ICSI), we aimed to investigate the effect of co-culturing granulosa cells (GCs) on human oocyte maturation in vitro, the fertilization rate, and embryo development. Methods: A total of 133 immature oocytes were retrieved and were randomly divided into two groups; oocytes that were cultured with GCs (group A) and oocytes that were cultured without GCs (group B). After in vitro maturation, only oocytes that displayed metaphase II (MII) underwent the ICSI procedure. The maturation and fertilization rates were analyzed, as well as the frequency of embryo development. Results: The mean age of the patients, their basal levels of follicle-stimulating hormone, and the number of oocytes recovered from the patients were all comparable between the two study groups. The number of oocytes that reached MII (mature oocytes) was 59 out of 70 (84.28%) in group A, compared to 41 out of 63 (65.07%) in group B (p=0.011). No significant difference between fertilization rates was found between the two study groups (p=0.702). The embryo development rate was higher in group A (33/59, 75%) than in group B (12/41, 42.85%; p=0.006). The proportion of highest-quality embryos and the blastocyst formation rate were significantly lower in group B than in group A (p=0.003 and p<0.001, respectively). Conclusion: The findings of the current study demonstrate that culturing immature human oocytes with GCs prior to ICSI improves the maturation rate and the likelihood of embryo development.

• Clinical and Experimental Reproductive Medicine
• /
• v.38 no.4
• /
• pp.210-215
• /
• 2011

Objective: This study was performed to identify whether growth and differentiation factor-9 (GDF-9) and transforming growth factor-${\beta}1$ (TGF-${\beta}1$) expressions would be lower in the follicular fluid (FF) of those over age 35 who underwent IVF than under age 35. Methods: A total of 24 IVF cycles (20 patients) were included in this study. All of patients were stimulated for IVF by the GnRH short protocol and divided into two groups for analysis, according to their age: <35 group (14 cycles, 11 patients) vs. ${\geq}35$ group (10 cycles, 9 patients). The expression levels of GDF-9 and TGF-${\beta}1$ were determined by western blotting and quantitative enzyme-linked immunosorbent assay. Results: The numbers of retrieved oocytes and metaphase II oocytes were significantly lower in the ${\geq}35$ group. Lower expression of GDF-9 and TGF-${\beta}1$ by western blotting in the ${\geq}35$ group were observed as well. The mean GDF-9 and TGF-${\beta}1$ levels by enzyme-linked immunosorbent assay were lower in the ${\geq}35$ group. The values were $6,850.5{\pm}928.4$ ng/L vs. $3,333.3{\pm}1,089.2$ ng/L of GDF-9 ($p$ <0.05) and $3,844.1{\pm}571.1$ ng/L vs. $2,187.7{\pm}754.0$ ng/L of TGF-${\beta}1$ ($p$ <0.05). A negative correlation between GDF-9 and age was observed (r=-0.546, $p$=0.006). Conclusion: GDF-9 and TGF-${\beta}1$ production from stimulated ovaries during IVF appears to decrease with age.

• Clinical and Experimental Reproductive Medicine
• /
• v.42 no.3
• /
• pp.118-125
• /
• 2015

Objective: The goal of this study was to evaluate the pregnancy potential of immature (metaphase I or germinal vesicle stage) oocytes retrieved in intracytoplasmic sperm injection (ICSI) cycles. Methods: A total of 1,871 couples with infertility underwent 2,984 ICSI cycles. Cycles in which three or fewer oocytes were retrieved were included in this study in order to evaluate the pregnancy potential of immature oocytes. Cycles were divided into five groups (group I-V), according to the maturation status of the oocytes at the time of cumulus cell removal and ICSI. The fertilization and pregnancy rates after ICSI were analyzed and compared among the study groups based on the maturation status of the retrieved oocytes. Results: The retrieval of only immature oocytes was associated with a significant decrease in the fertilization rate ($76.1%{\pm}37.3%$ vs. $49.0%{\pm}49.1%$, $66.7%{\pm}48.7%$; group I vs. group II, group III, respectively) and the average number of transferred embryos ($1.5{\pm}0.7$ vs. $1.1{\pm}0.4$, $1.1{\pm}0.6$). The cycle cancellation rate was significantly higher when only immature oocytes were retrieved. The clinical pregnancy rate decreased significantly when the transferred embryos had originated from immature oocytes (16.9% vs. 10.3%, 1.2%). Conclusion: In ICSI cycles, the fertilization potential and pregnancy potential of the immature oocytes retrieved in ICSI cycles were inferior to those of mature oocytes. Therefore, increasing the number of injectable oocytes and transferrable embryos by using immature oocytes after their spontaneous in vitro maturation does not necessarily improve pregnancy outcomes.

• Asian-Australasian Journal of Animal Sciences
• /
• v.33 no.10
• /
• pp.1579-1589
• /
• 2020

Objective: This study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction. Methods: Pig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3). Results: LIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and β-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos. Conclusion: The above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.