• Development and Reproduction
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• v.25 no.3
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• pp.173-183
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• 2021

The incidence of infertility among individuals of reproductive age has been growing due to genetic and environmental factors, and considerable research efforts are focused on solving this issue. Ovarian development is an overly complex process in the body, involving the interaction between primordial germ cells and gonad somatic cells. However, follicles located in the center of the in vitro ovary are poorly formed owing to ovarian complexity, nutrient deficiency, and signaling deficiency. In the present study, we optimized methods for dissociating gonads and culture conditions for the in vitro generation of miniaturized ovaries. The gonads from embryos were dissociated into cell masses and cultured on a Transwell-COL membrane for 3-5 weeks. Approximately 12 follicles were present per in vitro ovary. We observed that miniaturized ovaries successfully matured to MII oocytes in vitro from 150 to 100 ㎛ gonad masses. This method will be useful for investigating follicle development and oocyte production.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.71-79
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• 2008

Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) is one of the recently developed proteomic technologies which is based on capturing proteins and peptides by chemically modified surfaces and highly sensitive for the analysis of complex biological samples. In the present study, 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 porcine oocytes at the in vitro matured metaphase II (MIIl) and germinal vesicle (GV) stages. By selected CM10 chip, 16 candidates were found to be up-regulated in GV stage oocytes compared with in MII stage oocytes, their molecular weights were 8,180 (2 candidates), 10,226 (5 candidates), 15,767 (5 candidates) and 16,770 (4 candidates) Da respectively. And the expression of 29 candidates were higher in MII than in GV stage oocytes, their molecular weight were 10,832 (3 candidates), 17,743 (8 candidates), 20,122 (3 candidates), 22,131 (3 candidates), 24,857 (7 candidates) and 33,507 (5 candidates) Da, respectively. The expression of selected 13 candidates (0.2 and 1.0 % error tolerances) were analyzed using real time RT-PCR. The proteins that differentially regulated during oocyte in vitro maturation in the pigs may be potential biomarkers of oocyte maturation and quality.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.287-293
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• 2000

This study was undertaken to access the effects of collection method, room temperature at oocyte recovery and culture media on the oocyte quality, fertilization and cleavage rates of in vitro matured and fertilized oocytes of Korean native goats. Ovaries obtained from a slaughterhouse were transported to the laboratory and were divided into 2 groups. One group of ovaries was maintained at 30 to 35$^{\circ}C$ of the room temperature and another group was remained at 20 to $25^{\circ}C$ during oocyte recovery. The oocytes were recovered by follicle aspiration, slicing and aspiration+slicing methods from 3 groups of follicles according to size; <2 mm, 2 to 6 mm and >6 mm. The matured oocytes were inseminated with buck epididymal spermatozoa at a concentration of 3~3.5$\times$10$^{6}$ m1 and fertilization was identified when 2 pronuclei were present in the cytoplasm. Although the recovery rate per ovary obtained by the combination of follicle aspiration + slicing(19.6$\pm$2.2) method was higher than aspiration(11.7$\pm$1.1) and slicing(14.8$\pm$1.8) collection, optimal recovery according to oocyte grades resulted form ovarian slicing compared to aspiration or combined methods(P<0.05). However, no significant differences were found in the mean number(2.5$\pm$1.8; 3.3$\pm$3.3; 2.9$\pm$2.4) and the proportion of favorable oocytes(Grades I, II and III) recovered(31.6%, 36.0%, 36.4%,) according to follicle size(<2 mm; 2 to 6 mm; >6 mm). Fertilization rate was 60.0%, 67.7%, 70.6% and 56.4% and the proportion of embryos/zygotes was 11.1%, 7.1%, 5.0% and 2.8% in 20~$25^{\circ}C$/BO, 30~35$^{\circ}C$/BO, 20~$25^{\circ}C$/TALP and 30~35$^{\circ}C$ /groups, respectively.

• 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.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.95-99
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• 1998

Three experiments were conducted with follicular oocytes, to compare some somatic cells, growth factors and media for in vitro maturation of bovine oocytes. In the first experiment, the type of somatic cells had no effects on in vitro maturation of bovine follicular ooctyes. In the second experiment, oocytes were matured in TCM199 su, pp.emented with growth factors on IVM of bovine follicular oocytes, then all were co-cultured with cumulus cells. The proportion of used oocytes that developed to expanding blastocysts was 22.2%, 20.2%, 17.7%, 22.2%, 24.4% and 20.2% after maturation in TCM199 su, pp.emented with control, insulin, IGF-I, IGF-Ⅱ, FGF and EGF, respectively. In the third experiment, oocytes were matured in BO, Ham's F10 and TCM199, then all were fertilized in BO, and embryos cultured in BO, Ham's F10 and TCM199, respectively. Cleavage rates in BO were 90%, had higher than in Ham's F10(80%) or in TCM199(64%). But production of expanding blastocysts in TCM199(21%) or Ham's F10(20.6%), had higher than in BO(4.6%).

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.111-122
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• 2017

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in $75{\mu}M$ of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.127-132
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• 2013

The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21~22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05

• Journal of Animal Science and Technology
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• v.63 no.2
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• pp.272-280
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• 2021

Cumulus-oocyte complexes (COCs), which contain immature oocytes, are matured in vitro for in vitro embryo production. Oocyte and cumulus cells are then separated using hyaluronidase. To date, there have only been a few reported cases of the toxic effects of hyaluronidase on porcine oocytes. The aim of this study was to compare the effects of bovine testis-derived hyaluronidase and recombinant human hyaluronidase on oocyte denudation and quality. Porcine COCs were matured for 44 h and denuded using different hyaluronidase concentrations and exposure times. Then, oocytes were activated by electrical parthenogenesis. In experiment 1, COCs were denuded using bovine-derived, ovine-derived (Hirax), and human recombinant (ALT-BC4) hyaluronidases for 10 and 20 min. In experiment 2, bovine-derived and human recombinant (ALT-BC4 and ICSI Cumulase®) hyaluronidases were used to denude the COCs for 2 and 20 min. In both experiments the oocytes were all completely denuded, and there was no degeneration. Rate of embryo development was significantly increased in group treated ALT-BC4 for 2 min and not significantly different in other treatment groups. In general it slightly decreased with longer exposure times. These results have confirmed that different sources of hyaluronidase do not have detrimental effects on the quality of porcine oocytes and suggest that the human recombinant hyaluronidase ALT-BC4 is suitable for oocyte denudation with an increased blastocyst rate.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.234-237
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• 2011

Objective: During stimulated IVF cycles, up to 15% of oocytes are recovered as immature. The purpose of this study was to investigate the trend of oocyte maturity in repeated ovarian stimulation for IVF. Methods: One hundred forty-eight patients were selected who underwent two consecutive IVF cycles using same stimulation protocol during 2008 to 2010. Ovarian stimulation was performed with FSH and human menopausal gonadotropin and flexible GnRH antagonist protocol in both cycles. Oocyte maturity was assessed according to presence of germinal vesicle (GV) and the first polar body. Immature oocyte was defined as GV stage or metaphase I oocyte (GV breakdown with no visible polar body) and cultured up to 48 hours. If matured, they were fertilized with ICSI. Results: Percentages of immature oocytes were 30.8% and 32.9% ($p$=0.466) and IVM rates of immature oocytes were 36.2% and 25.7% ($p$=0.077), respectively. A significant correlation was noted between percentage of immature oocytes in the two cycles (R=0.178, $p$=0.03). Women with >40% immaturity in both cycles (n=21) showed lower fertilization rate of $in$ $vivo$ matured oocytes (56.4% vs. 72.0%, $p$=0.005) and lower pregnancy rate (19.0% vs. 27.1%, $p$=0.454) after the second cycle when compared with women with <40% immaturity (n=70). In both groups, female age, number of total retrieved oocyte and embryos transferred were similar. Conclusion: In repeated ovarian stimulation cycles for IVF, the immature oocyte tended to be retrieved repetitively in consecutive IVF cycles.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.145-151
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• 2002

The aim of these experiments was to investigate in vitro nuclear maturation of canine oocyte collected from various stages of estrus cycles, Ovaries were obtained from 1 to 4 year-old mongrel bitch and minced for oocyte collection in phosphate buffered saline with 100 iu penicillin-G $m\ell$／sup -1／, 50 $\mu\textrm{g}$ streptomycin sulphate $m\ell$／sup -1／ and 0.1％ (w／v) polyvinyl alcohol. Cumulus-oocyte-complexes (COCs) were washed in HEPES buffered tissue culture medium (TCM)199 and in vitro matured in TCM-199 culture medium supplemented with sodium pyruvate 0.028mg/$m\ell$, L-glutamine 0.146mg／$m\ell$, penicillin G 10,000 IU／$m\ell$, streptomycin 0.031mg／$m\ell$ and 10％ (v／v) fatal calf serum. COCs were in vitro matured for 48～72 hrs at 39$^{\circ}C$ in humidified 5％ $CO_2$ in air atmosphere. In vitro matured oocytes were remove the cumulus cells using 0.2％ (v／v) hyaluronidase. After denuding, oocyte were placed in acetic acid : methanol : chlorform solusion (3:6 : 1.5 v／v) for 30 sec and acetic acid: ethanol(1:3 v／v) for 48hrs fixation. Nuclear maturation was classified to GV, GVBD, MI, MII and degenerate oocyte under microscopy after 1％ aceto-orcein stain. In vitro maturation rates at 48hrs were not significantly difference among the oocytes collected from different stage of estrus at 15.9％, 16.3％, 23.7％ and 18.2％ for anestrus, proestrus, estrus and diestrus. However, the oocytes maturation(36.6％) of collected from estrus ovaries were significantly different from oocytes derived from proesturs, diestrus and anestrus ovaries(30.8％, 17.5％ and 22.1％; p＜0.05). The overall in vitro maturation rates were significantly higher (p＜0.05) in 72hrs culture than 48hrs culture system. In summary, there was a tendency for higher in vitro maturation rates with the oocyte collected from estrus ovary than other stages of estrus. Also, for nuclear maturation, in vitro culture of oocyte for 72hrs was better than 48hrs culture.