• 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.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.96-96
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• 2003

Nek2 (NIMA-related protein) is a mammalian cell cycle-regulated kinase that involves in chromosome condensation and centrosome regulation and NuMA (nuclear mitotic apparatus protein) is involved in spindle assembly during a cell cycle. The cellular distribution and organization of the centrosomal components is completely unknown during fertilization and embryonic development. We examined distribution of two well-known centrosomal proteins, Nek2 and NuMA in mouse gametes and embryos to get an insight in the reorganization of centrosomal proteins during germ cell development and early fertilization. Spermatogenic cells, gametes, and embryos were analyzed with anti-Nek2 or -NuMA antibodies by immunological assay, RT-PCR, and overexpression through gene transfection. Mitotically or meiotically active spermatogenic cells were intensively stained with these antibodies in both centrosomes and cytoplasm, whereas the oocytes showed different staining patterns depending on the meiotic stages. During maturation, GV, GVBD, and MI stage were clearly stained with NuMA antibody in the nucleus or cytoplasm at MII. Also, Nek2 was detectable in cytoplasm as scattered spots or chromosome associated at MII. In early developmental embryo, NuMA was detected in nucleus of each blastomere, while Nek2 was detected in cytoplasm. In contrast to previously reported results, Nek2 and NuMA were detected in both decondensing head, and the centriole of demembranated and decondensed sperm or whole body of trypsin-treated sperm for Nek2. During meiotic progress in oocytes, transcripts levels were the highest in MI stage and then downregulated in MII. Also, it shows dramatically change in early developmental embryos, firstly, it was increased until 4 cell stage and reduced in 8 cell stage, and finally, transcript levels were upregulated until blastoscyst. This finding suggests that cnetrosomal component may play an important role in reorganizing of functional centrosome during fertilization process and subsequent development.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.145-147
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• 2005

Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of $2{\~}4$ embryos/${\mu}L$ significantly improved development to the blastoryst stage ($72{\%}{\leq}$) compared with culture at the lower ($0.2{\~}1$e mbryos/${\mu}L,\;0\~37.5\%$) and the higher ($5{\~}6$ embryos/${\mu}L,\;30\~53\%$) concentration for 120 h when the oocytes were cultured in a 5 ${\mu}L$ drop under mineral oil In experiment 2, the embryos cultured at a concentration of $2{\~}4$ embryos/${\mu}L$ in a 10 ${\mu}L$ drop ($81.1{\%}$) showed significantly higher blastocyst rates than those in a 5 ${\mu}L$ drop ($68.5{\%}$). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.285-297
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• 1995

The mammalian oviduct is a place where ontogeny of an animal begins. Nowadays, however, it is possilbe to manipulate a part of physiological events occurring in the oviduct so that fertilization of gametes and early embryonic development of zygotes could proceed outside oviductal environment. Rabbit zygotes readily develop to blastocysts in a conventional culture condition. Most of the mouse fertilized eggs do so when cultured under a specific environment, e.g., in a medium containing ethylenediamine tetraacetic acid. Similarly, a significant number of zygotes from rat, sheep, pig or cattle can develop to blastocysts if they are cultured in the presence of particular component which appear to be somewhat species-specific. Instead of changing the components of medium, somatic cells including oviductal epithelial cells, have widely been used to improve mammalian embryonic development in vitro. Many investigators have reported that mammalian zygotes, whether fertilized in vivo or in vitro, could develop to blastocysts when they were cultured on a monolayer of various kinds of somatic cells or even in a somatic cell-conditioned medium. While little is known about the nature of embryotrophic factor(s) produced in vitro by somatic cells, the existence fo oviduct-specific protein(s) has consistently been demonstrated in many laboratories. Some of these proteins are reported to be associated with oviductal eggs. However, the physiological role of these proteins has still to be determined. Recently we observed that the perivitelline space of mouse oocytes was fluorescently stained with various fluorochrome-protein conjugates following ovulation into the oviducts or upon their expossure to oviductal extracts. Furthermore, it was also found that cattle or pig oviductal fluid gave similar results when examined using mouse ghost ZP. These observations lead to suggest that mammalian oviduct induces changes of biochemical properties of oocytes. Further studies are needed to clarify the nature of oviductal factor(s) and the physiological meaning of the reaction.

• Korean Journal of Animal Reproduction
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• v.18 no.3
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• pp.217-228
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• 1994

These experiments were carried out to establish the optimal condition of electrostimulatin inducing cell fusion and oocyte activation for nuclear transplantation in mouse embryos. Eggs selected for cell fusion or activation by electrostimulation were equilibrated for 5~10 min. in 0.3M sucrose solution and electrostimulated for 60$\mu$sec using 1 pulse of 60, 70, 80, 90 or 100 volts DC with electrodes 0.2 mm apart. Then they were cultured in 20${mu}ell$ dropsof Tyrode's solution. The results of these experiments are as follows : 1. When one pulse of 60, 70, 80, 90 or 100 volts DC for 60$\mu$sec were applied to 2-cell embryos for fusion of blastomeres, fusion rates were 50.0, 81.7, 91.7, 100 and 100%, respectively ; and developmental rates of fused embryos to blastocyst were 76.7 to 81.5%. Higher fusion rates were observed in 90V and 100V. 2. The average cell number in fused embryos developed to blastocyst was about half of the cell number in diploid controls; and the cell number decreased with increasing of voltages. 3. When pulse numbers were increased, fusion rates improved, but developmental rates were not signficiantly different from the group for which the number of pulse was not increased. And the cell number of blastocyst decreased even more. 4. Oocytes aged for 6hrs after ovulation were electrostimulated for oocyte activation by the same method used for cell fusion. Rates of oocyte activated by electrostimulation were 45.3 to 60.4%, and fragmentation rates were 7.5~15.1%. The lysis rates were 17.0~34.0%. The results of these experiments indicate that the optimal condition for achieving cell fusion and activation is 1 pulse, duration 60$\mu$sec in 90 Volt. The results also show that this condition is suitable for nuclear transplantation using mouse eggs.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.125-131
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• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• Clinical and Experimental Reproductive Medicine
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• v.22 no.2
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• pp.163-170
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• 1995

Growth factors (GFs) produced by the embryo or by the maternal reproductive tract have been reported to regulate the embryonic development and differentiation. Among GFs, EGF as a mitogen plays a role in mitosis and functional differentiation of trophectoderm cells in mouse. The present study was carried out to investigate the effect of EGF on development of mouse embryos and to localize EGF in the mouse oocytes and embryos, which has been reported to be detected in the reproductive tract in mammals. To investigate the effect of EGF on the development of the embryo, mouse 2-cell embryos were cultured to blastocysts stage in Ham's F10 medium, treated with EGF(10-50 ng/ml) for 72 hrs. Immunocytochemistry was performed from oocyte to blastocyst stage with anti-EGF and anti-Mouse IgG, in order to determine the stage which EGF would be expressed in mouse. Exogenous EGF (more than 10 ng/ml) in the culture medium improved the developmental and hatching rates in the mouse embryos. As a result of immunocytochemistry, the embryonic EGF was expressed after the late 4-cell stage. EGF is thought to enhance preimplantation embryonic development and hatching. Exogenous EGF in the culture medium is thought to activate EGF receptor in the late 4-cell embryos and to enhance blastulation and hatching in mouse embryos. It is concluded that EGF enhances the developmental and hatching rates in the mouse embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.8
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• pp.1190-1195
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• 2007

This study was conducted to improve efficiency of a follicle culture system without reducing developmental competence of intrafollicular oocytes. Preantral follicles (100 to $125{\mu}m$ in diameter) of F1 hybrid (B6CBAF1) mice were cultured singly for 216 h in modified ${\alpha}$-MEM-glutamax medium, to which 2.5 IU/ml hCG and epidermal growth factor was added 16 h prior to the end of culture. Medium change was either performed three times (54 h interval), twice (72 h interval), once (108 h interval), or not at all (216 h interval). Maturation (progression to the metaphase II stage) of intrafollicular oocytes was detected from 4 days after culture in the three-times change treatment, while all treatments yielded mature oocytes from day 5 of culture. Compared with the three-times change, decreasing the change frequency to once did not reduce the capacity to begin maturation (germinal vesicle breakdown of 82 to 86%), to mature (78 to 79%) and to develop into blastocysts after parthenogenetic activation (29 to 32%). Morphological parameters were similar among these treatments. Except for the no medium change treatment, similar colony-forming activity of inner cell mass cells after culturing of blastocysts in leukemia inhibitory factor-containing medium was detected, while the morphology of the colony-forming cells deteriorated in the change-once treatment compared with the change twice or three-times. In conclusion, the efficiency of the preantral follicle culture system could be improved by reducing frequency of medium change up to a 72 h interval (three times in total 216 h culture) without decreasing developmental competence of oocytes.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.353-360
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• 2002

Although successful pronuclear formation and apposition were seen in porcine oocytes following mouse sperm injection, little is known on the morphology of male and female pronuclei following sperm injection. The objective of this study is to describe the ultrastructure of porcine zygote following murine sperm injection in relation to the chronology of pronuclear S phase. At 40h ~ 44h following in vitro maturation, Cumulus cells were removed in TCM-HEPES with 0.1% hyaluronidase. Then, spermatozoa was injected into the cytoplasm of oocytes. After. injection, all oocytes were transferred to NCSU23 medium and cultured at 39$^{\circ}C$ under 5% $CO_2$ in air. Oocytes were fixed in 2% glutaraldehyde in Dulbeccos phosphate-buffered saline and observed by Transmission Electron Microscopy. Nuclear precursor bodies were observed in each pronucleus. A cluster of large and small granules was attached in the nucleolus precursor body. After the apposition of male and female chromatin, chromatin condensation was observed throughout the nucleoplasm and nucleolus precursor bodies and condensed chromatin in contact with clusters of small and large granules and the nuclear envelope were found in apposed pronuclear regions. These results suggest that non-species specific nuclear cytoplasmic interactions take place during pronuclear formation and apposition following sperm injection.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.163-169
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• 2001

OSBA(oocytes-sperm binding assay) is a tool developed for rapid test of optimal condition of IVF medium and protein source by binding ability of mouse sperm and egg. Mouse oocyte-cumulus complexes were prepared by removing of the cumulus cells with 0.1% hyaluronidase. 10$\pm$2 oocytes per 30 ${mu}ell$ medium drop were inseminated with 3 ${mu}ell$ sperm suspension and were cultured f3r 3 hours and 24 hours, respectively. And the oocytes were recovered gently and the No. of sperm bound on oocytes were counted. In the Exp. 1, the ratio of oocytes bound with one sperm at least were 60.2%(50/83), 2%(2/77) and 100%(79/79) in the medium with no protein, FBS(15%, v/v) and BSA(0.4%. w/v), respectively, Fetal bovine serum(FBS) seriously inhibited sperm binding on oocyte, although bovine serum albumin(BSA) promoted the binding ability. The inhibiting effect of FBS was dependent on the concentration of FBS. The sperm binding ability according to oocyte maturity was tested in the Exp. 2. There was no significant difference between Met. II (mature) and Met. I (intermediate mature) oocytes in the number of oocytes bound with sperm and the number of sperm bound on oocytes. Finally, in Exp. 3, two batches of Ham's F10 medium with good and poor quality by OSBA were tested (The ratios of embryos developed from PN 1-cell stage to hatched blastocyst; 25% vs. 70%). In the medium with good quality, sperm binding ability was significantly increased (P ＜ 0.05). The ratio of oocytes bound with one sperm at least was 66% and 90% in the medium with poor and good quality, respectively. Conclusively, It was possible to test IVF medium condition rapidly and easily by OSBA.