• Clinical and Experimental Reproductive Medicine
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• v.15 no.1
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• pp.17-24
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• 1988

Follicular fluid(FF) prostaglandin $E_2$(PG$E_2$) and $PGF_{2{\alpha}}$ levels were compared in 3 groups of spontaneously ovulatory women undergoing ovulation induction with clomiphene citrate (CC) alone or with human menopausal gonadotropin(hMG) (14 patients), hMG(9 patients), or pure FSH/hMG(11 patients) for the purpose of in vitro fertilization. FF volume aspirated did not differ significantly according to the maturity of the oocyte. According to hyperstimulation regimens, the volume of FF from which preovulatory occytes were obtained was significantly less in the hMG-treated group than in the other groups. In follicles of preovulatory oocytes, FF PG$E_2$ values were significantly lower in the FSH treated group than in the Cc.treated or hMG-treated group, and FF $PGF_{2{\alpha}}$ values were significantly higher in the hMG-treated group than in the CC-treated or FSH-treated group. In follicles of immature or atretic oocytes, there was no significant difference in FF PG$E_2$ and PG$F_{2{\alpha}}$ concentrations of the similar morphology of the oocyte according to hyperstimulation regimens. In all cycles, FF PG$E_2$ and PG$F_{2{\alpha}}$ values of preovulatory oocytes were not significantly different from those of immature oocytes, but those of atretic oocytes were relatively lower than those of fertilizable oocytes and it was statistically signifincant in PG$E_2$ values of CC-treated group. In all treatment groups, FF PG$E_2$ and PG$F_{2{\alpha}}$ levels did not show and close relationship with the success of fertilization in vitro and of pregnancy after embryo transfer. Above results suggested that FF PG$E_2$ and PG$F_{2{\alpha}}$ be involved in oocyte maturation and ovulation, but their relationship with the success of in vitro fertilization was not found.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.297-304
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• 2017

This study was designed to determine the effect of monosodium glutamate (MSG) on in vitro maturation (IVM) of oocytes and early development of parthenogenesis (PA) embryos in pigs. Each IVM and IVC medium was supplemented with various concentrations (0, 0.1, 0.5 and 5 mM) of MSG and non-essential amino acids (NEAA) depending on the experimental design. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II (MII) stage were electrically activated to induce parthenogenesis (PA). When immature oocytes were treated with MSG in the absence of NEAA during IVM, nuclear maturation (83.1-87.1%), intra-oocyte glutathione content, cumulus expansion, and cleavage (91.4-93.4%) of PA embryos were not influenced by MSG treatment at all concentrations. However, blastocyst formation of PA embryos was significantly increased by 5.0 mM MSG ($45.3{\pm}6.2%$) compared to control ($25.6{\pm}3.4%$). MSG treatment during IVM in the presence of NEAA did not show significant effect on nuclear maturation of oocytes and blastocyst formation after PA while 0.5 mM MSG ($89.3{\pm}1.9%$) decreased (P < 0.05) cleavage of PA embryos compared to 0.1 mM MSG ($94.6{\pm}1.1%$). When PA embryos were treated for 7 days with MSG during IVC, 5.0 mM MSG significantly decreased blastocyst formation ($27.8{\pm}4.9%$) compared to no treatment ($41.4{\pm}1.9%$) while no decrease in blastocyst formation was observed in 0.1 and 0.5 mM ($37.4{\pm}3.4%$ and $34.4{\pm}2.6%$, respectively). Our results demonstrated that 5 mM MSG in a NEAA-free chemically defined maturation medium showed positive effect on PA embryonic development while 5 mM MSG treatment during IVC was deleterious to PA embryonic development in pigs.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.11
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• pp.1541-1546
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• 2011

This study was carried out to examine the influence of minimum essential medium (MEM) vitamins supplementation to in vitro maturation medium and in vitro culture medium on the development of porcine embryos. Porcine embryo development was investigated following cultivation in both in vitro maturation and culture medium with the supplementation of MEM vitamins (0, 0.1, 0.2 and 0.4%) using immature oocytes collected from the ovary of prepubertal gilts. Embryo development was observed and the total cell number in each blastocyst generated under the culture conditions was quantified following supplementation of the medium. The embryonic development rate of the blastocyst and hatched blastocyst was higher, but not significantly so, when 0.4% MEM vitamins were supplemented to the in vitro maturation medium of the porcine oocyte. Interestingly, the total number of cells in the blastocyst was significantly higher in the in vitro maturation MEM vitamins supplemented group compared to either the untreated group or the group which had MEM vitamins supplemented to both in vitro maturation and in vitro culture medium simultaneously (p<0.05). Therefore, the supplementation of 0.4% MEM vitamins to the in vitro mature medium has a beneficial effect on the embryonic development of in vitro produced blastocysts derived from the immature porcine oocytes.

• Journal of Embryo Transfer
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• v.20 no.1
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• pp.63-69
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• 2005

This study were carried out to evaluate the possibility of nuclear progression of canine immature oocytes, of which was cultured in a reproductive tract, such as oviduct, ovarian bursa and uterus of estrus bitch for 4, 5 and 6 days following immediately collection. Cumulus intact oocytes(COC) fore collected from domestic dog following ovariohysterectomy at local veterinary clinics. In Exp. 1, COCs $of>110\;{\mu}m$ diameter were selected and cultured in vitro at $39^{\circ}C$, $5\%\;CO\_{2} $ in air atmosphere. The nuclear progression of canine oocytes checked at 24, 48 or 72 h after in vitro maturation. There was not increased the nuclear progression to the M II stage depending on culture periods at 24, 48 and 72h $(1.3\%,\;3.7\%\;and\;4.7\%)$. In Exp. 2, to evaluate of nuclear progression of immature oocytes, collected or in vitro cultured oocytes were transfer into a canine reproductive tract (oviduct, ovarian bursa and uterus). The recovery rates of canine oocytes from a reproductive tract after 4 days $(33.7\%)$ in vivo culture were significantly higher than those 5 $(17.7\%)$ 6 day $(3.4\%)$ (P<0.05). The survival rates of collected oocytes after 4 days $(60.0\%)$ were also significantly higher than those of 5 days $(30.2\%)$ and 6 days $(38.9\%)$ (P<0.05). The meiotic resumption rates of canine oocytes were not significantly difference among the culture periods at 4 days $(5.9\%)$, 5 days $(0.0\%)$ and 6 days $(0.0\%)$. These results show that the nuclear progression of canine immature oocytes from in in vivo culture was not affect the nuclear resumption of oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.2
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• pp.82-89
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• 2016

Objective: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. Methods: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. Results: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (${\pm}2.66$) vs. 75.40 (${\pm}4.92$); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (${\pm}4.79$) vs. 67.79 (${\pm}5.17$); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. Conclusion: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.4
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• pp.331-336
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• 2005

Objective: To investigate the effects of female age on in vitro maturation and fertilization of immature oocytes from controlled ovarian hyperstimulation (COH) in human IVF-ET program. Method: A total of 96 immature oocytes (GV & metaphase I) obtained from 40 cycles of IVF-ET (29 patients). The mean age of female patients was $31.8{\pm}3.1years$. Ovulation was triggered by urinary or recombinant hCG. Immature oocytes were cultured with YS medium containing 30% of patients' human follicular fluids, LH (1 IU/mL), FSH (1 IU/mL) and EGF (10 ng/mL), and then matured oocytes were fertilized by ICSI. In vitro maturation and fertilization of immature oocytes were analyzed according to age of female (< 34 or ${\geq}34years$). Results: The maturation rate was similar between two groups (68% vs 64%). The fertilization rate of in?vitro-matured oocytes was higher in patients < 34 years old, but there was no statistical significance (64% vs 50%, p=0.347). The fertilization rate of in-vitro-matured oocytes was significantly lower compared with those of in-vivo-matured oocytes in both age groups (64% vs 79%, p=0.035, 50% vs 86%, p=0.007). Conclusion: In older female group, fertilization rate of in-vitro-matured oocytes seems to be decreased. Further investigations should be warranted to increase fertilization potential of in-vitro-matured oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.1
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• pp.23-28
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• 2003

The present study was undertaken to investigate the effects of PVP concentration and exposure temperature to vitrification solution on the post-thaw survival, in vitro maturation and development of immature bovine oocytes (germinal vesicle stage). The vitrification solution (VS) consisted of 40% ethylene glycol (EG)+0.5 M sucrose (S)+10% FBS. PVP was added to VS: 0%, 5% or 10%. The cumulus-oocyte complexes (COCs) were diluted in VS as one step, after 2 min the COCs were loaded in straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were plunged into $30^{\circ}C$ water bath for 10s. After thawing, the oocytes were diluted in 0.5 M (in DPBS with 10% FBS) sucrose solution for 5 min. The survival rate (FDA-test and trypan blue) of immature bovine oocytes was measured. The survival rate was higher in 5% PVP (91.5%) than in 0% (64.2%) or in 10% PVP (79.7%). The proportion of metaphase II formation was 69.35% in control (no vitrified COCs), 9.3% in 40% EG+0.5 M S+0% PVP and 21.05% in 40% EG+0.5 M S+5% PVP (p<0.05). The effect of room temperature ($25^{\circ}C$ for 10 min) and cold temperature ($4^{\circ}C$ for 10 min) on COCs were determined in this study. After IVF, the cleavage and blastocysts rate of oocytes exposed to room temperature and cold temperature in VS+5% PVP was significantly different (2 cell: 63.20% vs 37.97%, blastocysts: 18.40% vs 2.53%). The cleavage rates of frozen-thawed oocytes were 20.53% with PVP and 22.13% without PVP (p>0.05). Two out of 151 oocytes (1.32%) developed to blastocyst stage after frozen-thawed with 5% PVP (p>0.05). Development of oocytes after frozen-thawing to the 2 cell were not significantly affected with or without PVP following IVF. However, the vitrification of immature bovine oocytes with PVP maintained the ability to develop to the blastocyst stage after IVM-IVF and IVC, while no blastocysts were obtained from oocytes vitrified without PVP. These results suggested that PVP has a protective role for vitrification of immature bovine oocytes as far as survival is concerned, however, the protection was not sufficient enough to support blastocyst formation.

• Korean Journal of Veterinary Research
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• v.57 no.2
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• pp.89-95
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• 2017

This study was conducted to determine the effects of biophoton treatment during in vitro maturation (IVM) and/or in vitro culture (IVC) on oocyte maturation and embryonic development in pigs. An apparatus capable of generating homogeneous biophoton energy emissions was placed in an incubator. Initially, immature pig oocytes were matured in the biophoton-equipped incubator in medium 199 supplemented with cysteine, epidermal growth factor, insulin, and gonadotrophic hormones for 22 h, after which they were matured in hormone-free medium for an additional 22 hr. Next, IVM oocytes were induced for parthenogenesis (PA) or provided as cytoplasts for somatic cell nuclear transfer (SCNT). Treatment of oocytes with biophoton energy during IVM did not improve cumulus cell expansion, nuclear maturation, intraoocyte glutathione content, or mitochondrial distribution of oocytes. However, biophoton-treated oocytes showed higher (p < 0.05) blastocyst formation after PA than that in untreated oocytes (50.7% vs. 42.7%). In an additional experiment, SCNT embryos produced from biophoton-treated oocytes showed a greater (p < 0.05) number of cells in blastocysts (52.6 vs. 43.9) than that in untreated oocytes. Taken together, our results demonstrate that biophoton treatment during IVM improves developmental competence of PA- and SCNT-derived embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.496-500
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• 2007

Holstein cow ovaries obtained at a slaughterhouse were used to study the influence of the oocyte collection methods (slicing, puncture, aspiration I and II) on recovery efficiency and subsequent in vitro maturation and embryonic development competence of immature oocytes recovered. In the slicing method, the whole ovarian was chopped into small pieces with a surgical blade. In the puncture method, the whole ovarian surface was punctured by 18-g needle. In other 2 aspiration methods, collected oocytes by aspirating from the visible follicles using an 18-g needle attached to a 5 ml syringe (aspiration I) or using a constant negetive pressure (-80 mmHg) with a vacuum pump (aspiration II). The oocytes were classified into 4 classes on the basis of the morphology of cumulus cells and cytoplasmic appearance of oocyte. Slicing ($9.6{\pm}0.4$) and puncture ($9.7{\pm}0.4$)yielded a larger number of oocytes per ovary than other two aspiration methods (aspiration I and II were $5.8{\pm}0.3$and $5.6{\pm}0.4$, respectively) (p<0.05). The number of the highest quality oocytes (grade A) per ovary was significantly higher in slicing ($4.2{\pm}0.2$) and puncture ($4.6{\pm}0.1$) methods than in other methods (aspiration I and II were $1.2{\pm}0.2$ and $1.4{\pm}0.2$, respectively) (p<0.05). The rate of nuclear maturation of the highest and higher quality oocytes (grade A and grade B, respectively) was not affected by the oocytes collection methods. The oocytes collection methods also did not influence subsequent embryonic developmental competence after in vitro fertilization with M II stage oocytes. It is concluded that slicing and puncture methods of the ovaries can be used as an alternative techniques to aspiration by the syringe or vacuum pump.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.97-104
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• 2009

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 77${\sim}$82%) and embryo cleavage (75% vs. 86${\sim}$90%) after SCNT than those matured in other media but blastocyst formation was not altered (13${\sim}$27%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.