• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.201-207
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• 2002

Objective: To observe the capability of fertilization and embryo development including blastocyst formation of the oocytes in simple media after thawing of the cryopreserved cumulus-free mouse oocytes by vitrification method. Methods: Oocytes were collected from 5 to 6 weeks old ICR female mice, and were denuded from the cumulus cells by 0.1% hyaluronidase. Recovered mature oocytes in study group were cryopreserved by vitrification method using EM grid for $5{\sim}7$ days. In brief, oocytes were exposed in dPBS containing 1.5 M EG and 5.5 M EG+1 M sucrose for 2.5 minutes and 20 seconds each, and then executed vitrification by plunging in LN2 after loading on EM grid. Thawing treated by exposure of 1, 0.5, 0.25 and 0.125 M sucrose solution for 2.5 minutes each in order and used for experiments. Spermatozoa aspirated form the epididymis of 12 weeks old ICR male mice were used for insemination after capacitation. T6 media containing 0.4% BSA were used for fertilization and development. Results: Survival and fertilization rates after thawing were 76.9% and 79.6% respectively. Fertilization rate was lower (p<0.005) than that of control group (92.9%). There was no difference in embryo developmental rates from 2-cell to morula, however, the blastocyst formation rate and mean cell numbers of blastocysts in study group (63.3%, $58.9{\pm}9.2$) were lower compared with those of control group (76.1%, $63.5{\pm}8.9$). Conclusion: Vitrification is an effective method for mouse mature oocyte cryopreservation with high survival and fertilization rate after thawing. And in simple media, fertilization rates and embryo development of frozen-thawed mouse oocytes are satisfactory.

• Clinical and Experimental Reproductive Medicine
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• v.41 no.4
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• pp.158-164
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• 2014

Objective: To assess whether an early GnRH antagonist start leads to better follicular synchronization and an improved clinical pregnancy rate (CPR). Methods: A retrospective cohort study. A total of 218 infertile women who underwent IVF between January 2011 and February 2013. The initial cohort (Cohort I) that underwent IVF between January 2011 and March 2012 included a total of 68 attempted IVF cycles. Thirty-four cycles were treated with the conventional GnRH antagonist protocol, and 34 cycles with an early GnRH antagonist start protocol. The second cohort (Cohort II) that underwent IVF between June 2012 and February 2013 included a total of 150 embryo-transfer (ET) cycles. Forty-three cycles were treated with the conventional GnRH antagonist protocol, 34 cycles with the modified early GnRH antagonist start protocol using highly purified human menopause gonadotropin and an addition of GnRH agonist to the luteal phase support, and 73 cycles with the GnRH agonist long protocol. Results: The analysis of Cohort I showed that the number of mature oocytes retrieved was significantly higher in the early GnRH antagonist start cycles than in the conventional antagonist cycles (11.9 vs. 8.2, p=0.04). The analysis of Cohort II revealed higher but non-significant CPR/ET in the modified early GnRH antagonist start cycles (41.2%) than in the conventional antagonist cycles (30.2%), which was comparable to that of the GnRH agonist long protocol cycles (39.7%). Conclusion: The modified early antagonist start protocol may improve the mature oocyte yield, possibly via enhanced follicular synchronization, while resulting in superior CPR as compared to the conventional antagonist protocol, which needs to be studied further in prospective randomized controlled trials.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.9
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• pp.1239-1246
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• 2016

The breeding of yaks is highly seasonal, there are many crucial proteins involved in the reproduction control program, especially in follicular development. In order to isolate differential proteins between mature and immature follicular fluid (FF) of yak, the FF from yak follicles with different sizes were sampled respectively, and two-dimensional gel electrophoresis (2-DE) of the proteins was carried out. After silver staining, the Image Master 2D platinum software was used for protein analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed for differential protein identification. The expression level of transferrin and enolase superfamily member 1 (ENOSF1) was determined by Western blotting for verification analysis. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development.

• Development and Reproduction
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• v.20 no.4
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• pp.289-296
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• 2016

This report describes the sex differentiation of the Korean rose bitterling, Rhodeus uyekii, from hatching to 170 days post-hatch (DPH) in relation to total length (TL), body weight (BW), and integral water temperature (IWT). The growth curve of TL from just hatching to 83 DPH was $5.144e^{0.045t}$ ($R^2=0.961$; t, time), and that of BW was $2.398e^{0.086t}$ ($R^2=0.725$). Primordial germ cells (PGCs) were observed at 17 DPH (7.9 mm TL, 3.74 mg BW, $374^{\circ}C$ IWT), and thereafter began to protrude into the peritoneal cavity. At 21 DPH ($9.2{\pm}0.14mm$ TL, $4.8{\pm}0.07mg$ BW, $462^{\circ}C$ IWT), some PGCs contained condensed chromatin and oocyte were observed in meiotic prophase. In contrast to the ovaries, which grew gradually after sexual differentiation, testes began multiplying at 25 DPH (10.1 mm TL, 5.42 mg BW, $550^{\circ}C$ IWT), when testicular differentiation was first identified, and multiplied continuously thereafter. At 33 DPH (11.2 mm TL, 10.5 mg BW, $726^{\circ}C$ IWT), the developing testes contained spermatogonia that exhibited mitotic activity. No spermatocyte or sperm cell was observed until 83 DPH (18.9 TL, 48.2 mg BW, $1,826^{\circ}C$ IWT). At 170 DPH (32.5 mm TL, 270.1 mg BW, $3,740^{\circ}C$ IWT), which was the end point of this study, the mature ovaries showed germinal vesicle breakdown, while the mature testes contained observable spermatocytes and sperm cells. These results allow us to identify the sex differentiation type of the Korean rose bitterling as differentiated gonochoristic.

• Applied Microscopy
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• v.33 no.2
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• pp.117-130
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• 2003

This research investigates the fine structural as well as the morphological changes of the mouse ovarian tissue after irradiation of various dose rates of 6 MeV LINAC radiation. The normal structure of the ovarian tissue is consisted of various stages of follicles including primordial and growing follicles, and ovarian stromal connectives. When we observed the ovarian tissues irradiated with a dose rate of 200 cGy/min using light and electron microscopes, granular cells in growing follicles are in irregular shape unlike normal follicles. Small segments of cells scattered in follicular antrum among granular cells. We could observe neutrophils and macrophages around the segments, which means the cells already got in the process of decease owing to the effects radiation. With coincident to the increase of the dose rate of x-ray irradiation as 400 or 600 cGy/min, the mature follicles appeared as an irregular form and the granular cells surrounding oocyte also deformed comparing to their normal counterparts. The granulosa cells within mature follicle are already occurred necrotic change and apoptosis. The nuclei in some cells got so fragmented that the segments formed the shape of a horseshoe or scattered in small and condensed pieces. All the cells at a granular layer irradiated with a dose rate of 600 cGy/min show typical characteristics of apoptosis. The neutrophils involved in inflammatory reaction appear evidently in follicular antrum of growing follicles, and macrophage scattered with residual and apoptotic bodies.

• The Korean Journal of Malacology
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• v.22 no.1 s.35
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• pp.13-22
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• 2006

Gonadosomatic index (GSI), oogenesis and reproductive cycle of Glossaulax didyma were investigated by the cytological histological obserbations and morphometric data. Samples were collected monthly from the intertidal zone of Biin Bay, Seocheon, Korea, for one year. Monthly variations in the GSI showed similar patterns with gonadal development. In the early vitellogenic oocyte, the Golgi complex and mitochondria were invovled in the formation of lipid droplet and yolk precursor. In the late vitellogenic oocytes, the rough endoplasmic reticulum and multivesicular bodies were involved in the formation of profeid yolk granules in the cytoplasm. A mature yolk granule was composed of three components: main body (central core), superficial layer, and the limiting membrane. The spawning season was from early June to late August, and the main spawning occurred between July and August when the seawater temperature was above $19^{\circ}C$. The female reproductive cycle can be classified into five successive stages: early active stage (December to February), late active stage (February to March), ripe stage (April to July), spawning stage (June to August), recovery stage (August to November). Fully mature oocytes were approximately $250-270{\mu}m$ in diameter.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.237-241
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• 2012

Embryonic genome activation (EGA) is the first major transition that occurs after fertilization, and entails a dramatic reprogramming of gene expression that is essential for continued development. Although it has been suggested that EGA in porcine embryos starts at the four-cell stage, recent evidence indicates that EGA may commence even earlier; however, the molecular details of EGA remain incompletely understood. The RNA polymerase II of eukaryotes transcribes mRNAs and most small nuclear RNAs. The largest subunit of RNA polymerase II can become phosphorylated in the C-terminal domain. The unphosphorylated form of the RNA polymerase II largest subunit C-terminal domain (IIa) plays a role in initiation of transcription, and the phosphorylated form (IIo) is required for transcriptional elongation and mRNA splicing. In the present study, we explored the nuclear translocation, nuclear localization, and phosphorylation dynamics of the RNA polymerase II C-terminal domain in immature pig oocytes, mature oocytes, two-, four-, and eight-cell embryos, and the morula and blastocyst. To this end, we used antibodies specific for the IIa and IIo forms of RNA polymerase II to stain the proteins. Unphosphorylated RNA polymerase II stained strongly in the nuclei of germinal vesicle oocytes, whereas the phosphorylated form of the enzyme was confined to the chromatin of prophase I oocytes. After fertilization, both unphosphorylated and phosphorylated RNA polymerase II began to accumulate in the nuclei of early stage one-cell embryos, and this pattern was maintained through to the blastocyst stage. The results suggest that both porcine oocytes and early embryos are transcriptionally competent, and that transcription of embryonic genes during the first three cell cycles parallels expression of phosphorylated RNA polymerase II.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.42 no.1
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• pp.41-47
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• 2009

The seasonal reproductive cycle of Gomphina melanaegis collected in the coastal area of Jumunjin, between April 2006 and March 2007, was analyzed. Maturation cycle parameters such as the gonad index (GI), ovarian egg diameter, frequency of developmental stages, protein content, and RNA/DNA variation in the gonads were analyzed monthly for the 40 samples. According to the indices from histological sections, the frequency of gonad developmental stages, and the oocyte diameter, this clam has a long-term partial spawning pattern from March to October. However, GI and nucleic acid values showed that the mature stage is from March to July and that the main spawning season is August. The peak RNA and DNA contents were good indicators of sexual maturation in females and males, respectively. The variation in protein content corresponded with the RNA/DNA ratios.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.2
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• pp.83-89
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• 2013

Objective: To investigate outcomes of stimulated IVF cycles in which GnRH antagonist was omitted on the ovulation triggering day. Methods: A total of 86 women who underwent controlled ovarian hyperstimulation with recombinant FSH and GnRH antagonist flexible multiple-dose protocols were recruited and prospectively randomized into the conventional group (group A) or cessation group (group B). The GnRH antagonist, 0.25 mg/day of cetrorelix, was started when the leading follicle reached 14 mm in diameter and was continuously administered until the hCG triggering day (group A, 43 cycles) or until the day before hCG administration (group B, 43 cycles). The maturity of oocytes, fertilization rate, embryo quality, and implantation and clinical pregnancy rates were evaluated. Results: The duration of ovarian stimulation, total dose of gonadotropins, serum estradiol levels on hCG administration day, and number of oocytes retrieved were not significantly different between the two groups. The total dose of GnRH antagonist was significantly lower in group B than group A ($2.5{\pm}0.9$ vs. $3.2{\pm}0.8$ ampoules, p<0.05). There was no premature luteinization in any of the subjects. The proportion of mature oocytes and fertilization rate were not significantly different in group B than group A (70.7% vs. 66.7%; 71.1% vs. 66.4%, respectively). There were no significant differences in the implantation or clinical pregnancy rates. Conclusion: Our prospective randomized study suggested that cessation of GnRH antagonist on the hCG administration day during a flexible multiple-dose protocol could reduce the total dose of GnRH antagonist without compromising its effects on pregnancy rates.

• The Journal of Korean Obstetrics and Gynecology
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• v.31 no.4
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• pp.89-109
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• 2018

Objectives: The purpose of this systematic review is to assess the effects of herbal medicine with in vitro fertilization-embryo transfer (IVF-ET) based on the current evidence. Methods: Eligible randomized controlled trials (RCT) searched from Pubmed which compared a combination of herbal medicine and IVF with IVF alone were included. Results: Sixteen trials, in which 2025 women involved, were included in this review. The review results showed that the effect of traditional herbal medicine can improve the clinical pregnancy rate (herbal medicine intervention: 30.36~63.64%, Control: 9.38~47.5%) and rate of high quality oocytes and embryos. The mechanism may be through regulating follicular fluid to improve microenvironment for oocytes which would lead to a successful embryo implantation. Conclusions: This analysis showed that combination of IVF and traditional herbal medicine used in the included trials improve clinical pregnancy rate and IVF success. During in vitro fertilization, TCM can regulate the microenvironment in the follicular fluid to mature the oocyte, improve the quality of the embryo, and help the development and implantation of the embryo. Further large randomized placebo controlled trials are needed to confirm the effectiveness of traditional herbal medicine with concurrent IVF.