• 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.

• Development and Reproduction
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• v.2 no.1
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• pp.69-74
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• 1998

The early gonadal development and sexual differentiation of Rhynchocypris oxycephalus are described from the stage of hatching to 150 days after hatching. During this peroid, the average length of the body grew from 0.64 cm to 5.96 cm. the primordial germ cells (PGCs), which could be recognized at the time of hatching, began to protrude into peritoneal cavity at a standard total length of 1.91 cm. At a standard total length of 2.29cm, initial ovarian differentiation wasidentified by the transformation of PGCs to meiotic oocytes. Finally, at the standard total length of 5.96 cm, the female gonads gradually developed towards migratory nucleus oocytes, characterisiing the maturation. Oocytes proliferated rapidly after sex differentiation while the testis entered a period of quiescence, as they continued to multiply but did not undergo growth until the standard total length of 4.00 cm. At a standard total length of 4.00 cm, spermatocytes arrested in thephase of interkinesis, Sertoli-like cells and sperm duct formation, with signs of meiotic activity, were observed. Therefore it may be concluded that R. oxycephalus belongs to the differentiated type of gonochoristic teleosts.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.3
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• pp.328-335
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• 2009

This study was undertaken to investigate the influence of exposure and removal of four different cryoprotectants (CPAs) on the ATP content of cumulus cell-enclosed (COs) and cumulus cell-denuded (DOs) immature porcine oocytes. The in vitro nuclear maturation of the COs, exposed to and removed from the CPAs was also assessed. Both COs and DOs were exposed to 1.5 M concentrations of each CPA (ethylene glycol (EG); propylene glycol (PG); dimethyl-sulfoxide (DMSO); and glycerol (G)) for durations of 5, 15, and 30 minutes at room temperature ($23.5{\pm}1.5^{\circ}C$), and immersed in physiological saline supplemented with 20% (v/v) fetal bovine serum for 5 minutes ($39^{\circ}C$) to remove each CPA. Before, during and after exposure to each CPA, the ATP content of both the COs and the DOs was measured. After removal from each CPA an aliquot of the COs was matured for 44${\pm}$2 h, and their nuclear maturation rates were measured up to the metaphase stage of the second meiotic division (the M-II stage). The maturation rates up to the M-II stage were not significantly different between all the groups that were exposed to each CPA for 5 minutes. For 15 and 30 minute exposures, the maturation rates of the COs exposed to PG, DMSO and EG were almost the same as those of the control groups; however, the rates of G group exposed for 15 and 30 minutes were significantly lower (p<0.05) than the control group. These groups were also found to have a decrease in the ATP content of COs and DOs during and after exposure for the same periods (p<0.05). In addition, although the ATP contents of the COs after exposure to EG for any period were the same as the controls, the ATP content of the DOs exposed to EG for any period were significantly lower (p<0.05) than those of the controls. When the ATP content of the COs and DOs of each CPA were compared, the DOs exposed to PG were found to have a significantly greater level (p<0.05) than DOs exposed to G for any duration. In addition, the ATP content of DOs exposed to PG for 30 min and removal was also higher (p<0.05) than when exposed to DMSO for the same period. These findings indicate that PG may be a useful CPA for the cryopreservation of immature porcine oocytes.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

• The Korean Journal of Zoology
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• v.30 no.1
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• pp.1-9
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• 1987

Cyclic AMP (cAMP) was known to play a key role in the regulation of cumulus expansion and oocyte maturation of mammalian cumulus-oocyte complexes (COC's) in vivo and in vitro. The present experiments were conducted to know how intracellular level of cAMP in these cells is controlled. Intracellular cAMP level was modulated by culturing mouse CGC's with an adenylate cyclase stimulator, forskolin, a phosphodiesterase inhibitor, 3-isobutyl-1-methyixanthine (IBMX), human chorionic gonadotrophin (HCG), or follicle stimulating hormone (FSH). The rate of cumulus expansion and germinal vesicle break-down (GVBD) was checked after culture and used as a biological end point. Forskolin in the medium began to stimulate the expansion of the complexes at 1 nM and induced maximum expansion (80~90%) at 0 1~10 $\mu$M. The expansion rate was reduced to 60% when forskolin concentration was increased to 100 $\mu$M. Oocyte GVBD occurred normally (75~82%) in the presence of 10 $\mu$M of forskolin, but partial suppression was appeared at 100 pM of the drug (40%). IBMX also stimulated the expansion from the concentration of 0.01 pM and induced full expansion (81~89%) between the concentration of 1-1000 $\mu$M. Meiotic resumption was occurred normally under 10 $\mu$M of IBMX, but suppressed drastically from the concentration of 100 $\mu$M. The minimum exposing time to hormone or drugs required to trigger cumulus expansion was two minutes with HCG, 15~30 minutes with FSH and fors kolin, and two hours with IBMX. The data presented here seemed to imply that intracellular cAMP level in cumulus cells is regulated by both adenylate cyclase and phosphodiesterase and cumulus expansion is induced by a peak of cAMP while meiotic arrest is maintained by continuous presence of cAMP.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.101-111
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• 2001

The present study was conducted to develop an in vitro culture system that would support bovine follicle growth from preantral to antral stage, oocyte maturation, fertilization, and embryonic development. Bovine preantral follicles (150$\pm$1.2 ${\mu}{\textrm}{m}$) surrounded by theca cell were isolated ezymatically and mechanically from ovarian cortical slides in Leibovitz L-l5 medium containing 1 mg/$m\ell$ collagenase and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium. The survival and growth rates of follicles cultured in the presence of FSH (10~150 ng/$m\ell$) were significantly higher than those of control group (P ＜ 0.001), but no significant differences were observed in survival and growth rates of follicles between the LH treatment groups (1~125 ng/$m\ell$) and the control. The survival (40%) and growth (244 $\pm$ 0.5 $\mu\textrm{g}$) of follicles cultured with FSH (90 ng/$m\ell$) and LH (25 ng/$m\ell$) were higher than those of control (25%, 160 $\pm$1.0 $\mu\textrm{g}$). Finally, 50% percent of healthy antral follicles were obtained, and almost 60% of them has complete meiotic division with 1st polar body (18.1%) and 10.0% have developed to the cleaved embryo and blastocyst stage. These results suggest that bovine preantral follicle with intact theca cell can grow to the antral stage using these culture conditions, and that oocytes from in vitro-matured bovine preantral follicle may acquire meiotic competence and can undergo fertilization and development.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.30-30
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• 2001

The culture of preantral follicles has important biotechnological implications through its potential to produce the large quantity of oocytes for embryo production, transgenesis research, conservation of rare breed, and a potential source of ovarian genetic material. The present study was conducted to establish the optimal conditions of in vitro culture for intact bovine preantral follicles; and to examine the developmental ability of oocytes derived from the in vitro-grown preantral follicles; and to investigate the effects of various concentrations of FSH and LH on these processes. Bovine preantral follicles (150 $\pm$ 1.2${\mu}{\textrm}{m}$), surrounded by theca cell, were isolated enzymetically and mechanically from ovarian cortical slides in Leibovitz L-15 medium containing 1 mg/$m\ell$ collagens and 0.2 mg/$m\ell$ DNase I and cultured for 25 days in the presence of different concentrations of bovine FSH and LH in $\alpha$MEM medium with insulin, transferrin, and selenite. The survival was tested by frypan Blue and Hematoxylin. The survival and growth rates of follicles were higher in FSH treatment groups than these in control (P<0.001), but there were no significant differences between the LH treatment groups and the control. In 25 days, the survival and growth rates of follicles in FSH and LH treatment group (50%, 300$\pm$1.0${\mu}{\textrm}{m}$) were higher than in FSH treatment group (40%, 244$\pm$0.5${\mu}{\textrm}{m}$) and the control group (25%, 160$\pm$ 1.0${\mu}{\textrm}{m}$). Fifty-five percent of healthy antral follicles were obtained, and 60% of the oocytes complete meiotic maturation to the metaphase II stage. Twenty-two percent of the mature oocytes underwent cleavage, and 9% developed to the blastocyst stage. In this study, in vitro-grown oocytes (111 $\pm$ $1.5mutextrm{m}$), under our culture conditions, were not equivalent in size to the in vivo-grown oocytes (130$\pm$1.3${\mu}{\textrm}{m}$). Therefore, these results suggest that bovine preantral follicles with intact theca cell can grow to the antral stage in 25days, and that oocytes from those follicles can acquire the meiotic competence and normally undergo fertilization and development to the blastocyst stage. However, the developmental capacity of in vitro-grown oocytes is presumably not comparable to those of the in vivo counterparts.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.375-383
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• 2000

The nuclear maturation and developmental competence of immature, oocytes collected from donors at various morphology of corpus luteum (CL) and fertilized in vitro was investigated by comparing the meiotic activity and the yields of embryos. Ovaries were divided and classified into 4 groups as the following criteria : Group 1 ; ovaries showed evidence of recent ovulation (corpus hemorragicum). Group 2 ; apex of CL was red or brown. Vasculization was limited to periphery of CL. Group 3 ; apex of CL was orange or tan. Vasculization was covered over apex of CL. Group 4 ; CL was light yellow to white and firm in texture and the vascular network on the surface of CL had disappeared. Modified TCM 199 was used for maturation in vitro of immature oocytes and development was induced by using TLP-PVA as a basic medium. When oocytes collected from each group of donors had been matured for 4, 14, and 24 hours in vitro, the proportion of oocytes reaching metaphase I and metaphase II were not different among oocytes from 4 group of ovaries. Mature metaphase II stage of oocytes in each group was first observed at 14 hours, whereas completion of maturation of. oocytes in each group was at 24 hours. Luteal morphology of ovaries had little effect on the proportion of embryos reached 2 cells and 8 cell stage. However, the proportion of embryos cleaved to morula and blastocyst stage was significantly higher in the oocytes obtained from group 1 and 3 than in the oocytes from group 2 and 4 (p＜0.05). This data suggest that reproductive status of the donor significantly influence the yield of in vitro embryos.

• The Korean Journal of Zoology
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• v.29 no.2
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• pp.79-85
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• 1986

The purpose of the present experiment is to make certain the exsistence of cAMP phosphodiesterase (PDE) in mouse oocytes and confirm its possible role on meiotic resumption. The results showed two types of cAMP in the oocytes with different Michaelis constants (Km) with specific maximum (Vmax); The Km and Vmax of one of two types of PDE were estimated at $0.14 \\pm 0.01 \\muM$ and $0.42 \\pm 0.07$ fmol cAMP hydrolyzed/oocyte/minute, and the other at $14.5 \\pm 2.0 \\muM$ and $2.2 \\pm 0.5$ fmol cAMP hydrolyzed/oocyte/minute. cAMP hydrolysis by PDE was reversibly inhibited in vitro by presence of theophylline or isobutyl-methyl-xanthine (IBMX), which is well known as an inhibitor of oocyte maturation. Consequently, it can be assumed that maturation of oocyte is affected by the high level of intracellular cAMP, and its level is well maintained by presence of PDE inhibitor, such as theophylline and IBMX.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.347-356
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• 2020

Gangliosides are glycolipids in which oligosaccharide is combined with sialic acids. Our previous studies have suggested an interplay between ganglioside GD1a/GT1b and meiotic maturation capacity in porcine oocyte maturation. Furthermore, ganglioside GD1a and GT1b are known for its antioxidant activity, but it is still unclear whether possible antioxidant role of GD1a and GT1b is involved in porcine embryos development competence during in vitro culture (IVC). Here, the effects of ganglioside GD1a and GT1b on the embryonic developmental competence during in vitro culture of porcine were investigated. The effects of ganglioside GD1a and GT1b on the expression of ST3GAL2 were confirmed during embryos development (2-cell, 4-cell, 8-cell and blastocyst) using immunofluorescent staining (IF). As a result, the fluorescent expression of ST3GAl2 was higher in embryos at 4-8 cells stage than blastocysts. Blastocyst development rate significantly increased in only 0.1 μM GD1a and GT1b treated groups compared with control group. To investigate the cellular apoptosis, we analyzed TUNEL assay. In case of only 0.1 μM GD1a and GT1b treated groups, the total number of cells in blastocyst compared with control group, but there was no significant difference in the rate of apoptotic cells. We identified the intracellular ROS levels using DCF-DA staining. According to the result, ROS production significantly decreased in blastocysts derived from the 0.1 μM GD1a and GT1b treated groups. These results suggest that ganglioside GD1a and GT1b improve the developmental competence of porcine embryos via reduction of intracellular ROS during preimplantation stage.