• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.155-161
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• 2012

Germ cell-specific hyaluronidases such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5) are in part responsible for dispersal of the cumulus cell mass, which is a critical step in establishing fertilization in mammals. In this study, we identified two testis-hyaluronidases, SPAM1 and Hyal5, in hamster and rat. These two genes were expressed specifically in the testis. At the protein level, hamster SPAM1 and Hyal5 display 78.7% and 75.4% identity with mouse SPAM1 and Hyal5. Further, the activity of the enzymes with respect to cumulus cell dispersion did not differ, although we observed that the enzymatic activity differed in pH range. These studies suggest that different sperm hyaluronidases are capable of dispersing the cumulus cell mass despite differences in enzyme activity.

• The Korean Journal of Zoology
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• v.31 no.2
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• pp.81-86
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• 1988

The relationship between cumulus cell expansion, cocyte maturation and metabolic cooperativitiy was investigated by using mouse and pig cocyte-cumulus complexes in vitro. Cocyte germinal vesicle breakdown (GVBD) and cumulus expansion were manipulated with hormones or reagents which increase intracellular cAMP leveL Metabolic cooperativity between oocyte and cumulus cells was assessed by determination of the fraction of radiolabelled uridine marker that was transferred from the cumulus mass to the oocyte. Uptake of uddine marker by mouse and pig cumulus mass was increased by about fourfold of basal level with the stimulation of hormones (human choriononic gonadotrophin, HCG; follicle stimulating hormone, FSH) or cyclic AMP sttmulators (3-isobutyl-1-methylxanthine, IBMX; forskolin) during culture. However, the fraction of uridine that was transferred from the cumulus mass to the cocyte (transfer ratio) was gradually decreased during culture, irrespective with the presence of hormones or stimulators. The decrease of the transfer ratio was not correlated with the state of occyte whether they have GV or not, or with the degree of cumulus expansion. In mouse complexes, HCG induced more significant reducton of transfer ratio than other treatments. These results do not support the idea that modulations of metabolic cooperativity between cumulus cells and oocytes are important for the regulation of meiotic resumption in mammals.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.233-240
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• 1996

This study was carried out to determine the effect of cumulus cell attachment and various factors on in vitro maturation of pig foflicular oocytes. Oocytes with various configuration of cumulus cell mass were collected ftom ovaries of mature gilts by asperating with syringe equipped with needles of different gauges, follicle size and with or without cumulus cells. They were cultured in TCM-199 mediun containing FGS(fetal calf serum) for 30~48 hours in incubator with air containing 5% $CO_2$ at 38.5$^{\circ}C$. Mter orcein staining at in vitro maturation condition, GV, GVBD, anaphase, telophase and M II were observed. Results are surumarized as follows: 1. Recovery rates were 55.8, 55.5 and 34.4% when the cumulus-compacted oocytes were collected with 18, 21, 26 gauge needles of syringes, respectively. 2. 79% of oocytes with compacted cumulus cells were at GV stage and most of the oocytes with partially denuded and denuded cumulus cells were from GVBD to M- II stages. 3. Percentage of mature oocytes among those which are follicular diameter of 1~2, 3~6 and over 6 mm was 42.6, 53.2 and 60.8%, respectively. 4. Percentage of mature oocytes among those which are compacted, partially denuded and denuded was 60.5, 46.2 and 35.4% respectively. 5. Percentage of mature oocytes in co-cultured with monolayers of cumulus cells was higher (57.1%) than that found with oocytes cultured alone (53.4%).

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.491-498
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• 2011

During mammalian fertilization, germ cell-specific hyaluronidases, such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5), are important for the dispersal of the cumulus mass. In this study, we demonstrated that bull Hyal5 is a single copy gene on chromosome 4 that is expressed specifically in the testis. In addition, we expressed recombinant bull SPAM1 and Hyal5 in human embryonic kidney 293T cells and showed that these enzymes possessed hyaluronidase activity. We also demonstrated that a polyclonal antibody against bull sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggested that bull Hyal5 may have a critical role in bull fertilization.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.333-339
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• 1996

Cumulus cells have possibly influence on fertilization of mouse oocytes and their subsequent development in vitro, because they readily produce lactate and pyruvate and can modify the concentration of substrates in the medium. In vitro fertilization of mouse oocytes with cumulus mass and their developments in five media which were differently composed in concentrations of glucose, lactate and pyruvate were observed. In the absence of glucose (CZ2 medium) decreased (p<0.01) the percentage of fertilization and embryos reaching the blastocyst stage. But, in the same concentration of glucose, lactate and pyruvate as mouse oviductal fluid with (MT1 medium) and without (MT2 medium) cumulus mass and modified CZB medium containing glucose (CZ1 medium) had no effects (p>0.05). These studies indicate that the adjustments of energy substrates concentration to the physiological level did not improve the fertilization of mouse oocytes with cumulus mass and their development in vitro, and the deletion of glucose showed adverse effects.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.26-31
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• 2004

This work was undertaken in order to study the developmental competence of nuclear transfer cat embryo with fetal fibroblast and adult skin fibroblast as donor nuclei. Oocytes wererecovered by mincing the ovaries in Hepes-buffered TCM199 and selected the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark. Homogenous ooplasm were cultured for maturation in TCM199 + 10% fetal bovine serum (FBS) for 12 hours and used as a source of recipient cytoplast for exogenous somatic nuclei. In Experiment 1, we evaluated the effect donor cell types on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate was not different between fetal fibroblast and adult skin cell (71.2 vs. 66.8; 71.0 vs. 57.6; 4.0 vs. 6.1 %, P<0.05). In Experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of seven recipient queens was delivered naturally 2healthy cloned cats and 1 stillborn from fetal fibroblast cell of male origin after 65 days embryo transfer. One of three recipient queens was delivered naturally 1 healthy cloned cat from adult skin cell of female after 65 days embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.9-12
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• 2016

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors that regulate many critical processes involved in early folliculogenesis and oocyte maturation. In this study, effects of GDF9 and BMP15 treatment during in vitro maturation of porcine oocytes upon development after parthenogenetic activation were investigated. Neither GDF, BMP15 alone nor in combination affects the number and viability of cumulus cells or the rates of oocyte maturation and blastocyst development. However, the treatment of GDF9 on porcine oocytes increased the number of trophectodermal (TE) cells of blastocysts derived from activated oocytes (P<0.05). The treatment of BMP15 increased the cell numbers of both inner cell mass (ICM) and TE cells (P<0.05). The treatment with the combination of GDF9 and BMP15 further increased the numbers of ICM and TE cells, compared with GDF9 or BMP15 treatment alone (P<0.05). In conclusion, the treatment of GDF9 or BMP15 (or both) enhanced the quality of blastocysts via the increased number of ICM and/or TE cells.

• Korean Journal of Animal Reproduction
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• v.15 no.1
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• pp.41-47
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• 1991

The ability of fertilization in vitro and subsequent development of superovulated oocytes was assessed in a controlled environment using an in vitro fertilization technique. The in vitro fertilization percentage of oocytes with cumulus mass declined significantly(P<0.05) with increased doses from 4~10 to 16~40IU of PMSG for superovulation. However, the porportion of polyspermic penetration varied from 2.3 to 9.7% and there was no significant difference between treatments in incidence of polyspermy. When morphologically normal ova with cumulus mass were cultured for 66~72h in a plastic mini-straw to undergo fertilization in vitro, the mean percentage of embryos developed to 2-16 and 4-16cell stage was 61.8 and 17.6%; it was slightly(P<0.05) superior to the corresponding results from petri dish. A total of 52 two-cell embryos fertilized in a mini-straw were transferred to seven pseudopregnant rat. Among these recipients, two normal young were born from one recipient which received a total of six embryos. These results suggest that superovulated oocytes are proportionately less competent than normally ovulated oocytes to undergo fertilization in a controlled environment using an in vitro fertilization technique. Also, a plastic mini-straw designed was slightly superior to petri dish as a culture vessel for fertilization and embryo development in vitro.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.119-124
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• 2006

The present study compared the developmental potential of cloned porcine embryos with mesenchymal stem cells (MSCs), fetal fibroblasts (FFs) and cumulus cells (CCs) by assessing the cleavage and blastocyst rate, total cell number, inner cell mass (ICM) ratio and apoptosis. MSCs were isolated by ficoll gradients from femur of -6 month old female pig, and maintained for primary cultures. FFs from a female fetus at ${\sim}30$ day of gestation were established, and CCs were obtained from cumulus oocyte complexes (COCs) aspirated from $3{\sim}6$ mm follicles in diameter. Donor cells at $3{\sim}4$ passage were employed for nuclear transfer (NT). COCs were matured and fertilized in vitro(IVF) as control. Cleavage rate was significantly (P<0.05) higher in IVF than in NT embryos with MSCs, FFs and CCs ($82.7{\pm}8.9%\;vs\;70.6{\pm}5.4,\;68.7{\pm}5.1\;and\;63.4{\pm}5.6%$, respectively). However, blastocyst rates in IVF and NT embryos derived from MSCs ($24.5{\pm}2.8\;and\;20.4{\pm}8.3%$) did not differ, but were significantly (P<0.05) higher than NT derived from FFs and CCs ($10.6{\pm}2.7\;and\;9.8{\pm}2.1%$). Total cell number and the ratio of ICM to total cells among blastocysts cloned from MSCs ($35.4{\pm}5.2\;and\;0.40{\pm}0.09%$, respectively) were significantly (P<0.05) higher than those from FFs and CCs ($24.9{\pm}6.2%\;vs\;0.19{\pm}0.16,\;23.6{\pm}5.5\;and\;0.17{\pm}0.16%$, respectively). Proportions of TUNEL positive cells in NT embryos from FFs and CCs ($6.9{\pm}1.5\;and\;7.4{\pm}1.7%$, respectively) were significantly (P<0.05) higher than in MSCs ($4.8{\pm}1.4%$) and IVF ($2.3{\pm}0.9%$). The results demonstrate that MSCs have a greater potential as donor cells than FFs and CCs in achieving enhanced production of cloned porcine embryos.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.1
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• pp.26-32
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• 2022

Objective: We investigated the effect of supplementing fertilization medium and/or culture medium with astaxanthin (AST) on the two phases of in vitro fertilization: gamete fertilization and embryo development. Methods: Mouse cumulus-oocyte complexes were divided into four groups with 5 µM AST added to the fertilization medium (group 3, n=300), culture medium (group 2, n=300), or both media (group 4, n=290). No AST was added to the control group (group 1, n=300). Results: The fertilization rate was significantly higher (p<0.001) in the groups using AST supplemented fertilization medium (group 3, 79.0%; group 4, 81.4%) than those without AST (group 1, 56.3%; group 2, 52.3%). The blastocyst rate calculated from the two-cell stage was significantly lower (p<0.001) in the groups using AST-supplemented embryo culture medium (group 2, 58.0%; group 4, 62.3%) than in those without AST (group 1, 82.8%; group 3, 79.8%). The blastocyst rate calculated from the number of inseminated oocytes was highest in group 3 (189/300, 63.0%) and lowest in group 2 (91/300, 30.3%) with statistical significance compared to other groups (p<0.001). There were significantly higher numbers of cells in the inner cell mass and trophectoderm, as well as significantly higher total blastocyst cell counts, in group 3 than in the control group. Conclusion: An increased blastocyst formation rate and high-quality blastocysts were found only in the fertilization medium that had been supplemented with AST. In contrast, AST supplementation of the embryo culture medium was found to impair embryo development.