• Development and Reproduction
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• v.15 no.1
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• pp.31-39
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• 2011

Change in intracellular $Ca^{2+}$-concentration ($[Ca^{2+}]_i$) is an essential event for egg activation and further development. $Ca^{2+}$ ion is originated from intracellular $Ca^{2+}$-store via inositol 1,4,5-triphosphate receptor and/or $Ca^{2+}$ influx via $Ca^{2+}$ channel. This study was performed to investigate whether changes in $Ca^{2+}$/calmodulin dependent protein kinase II (CaM KII) activity affect $Ca^{2+}$ influx during artificial egg activation with ethanol using $Ca^{2+}$ monitoring system and whole-cell patch clamp technique. Under $Ca^{2+}$ ion-omitted condition, $Ca^{2+}$-oscillation was stopped within 30 min post microinjection of porcine sperm factor, and ethanol-induced $Ca^{2+}$ increase was reduced. To investigate the role of CaM KII known as an integrator of $Ca^{2+}$- oscillation during mammalian egg fertilization, CaM KII activity was tested with a specific inhibitor KN-93. In the eggs treated with KN-93, ethanol failed to induce egg activation. In addition, KN-93 inhibited inward $Ca^{2+}$ current ($I_{Ca}$) in a time-dependent manner in whole-cell configuration. Immunostaining data showed that the voltage-dependent $Ca^{2+}$ channels were distributed along the plasma membrane of mouse egg and 2-cell embryo. From these results, we suggest that $Ca^{2+}$ influx during fertilization might be controlled by CaM KII activity.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.145-152
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• 1998

Many trials have been made to produce transgenic animals using sperm cells as a vector transferring foreign DNA into eggs, but reliable results are yet to be obtained (Brinster et al., 1989; Lavitrano et al., 1989; Bachiller et al., 1991; Sato et al., 1994). Recently, one of author(SO) demonstrated that mouse blastocysts derived from eggs fertilized by spermatozoa of male mice single injected with liposome-DNA complexes within the testis expressed thegene (Ogawa et al., 1995.) Here we report that a single injection of liposome-encapsulated DNAs into the testis of either male rats or mice resulted in successfully gene transfer to the postpartum progeny. The expression of mRNA derived from transgenes was also demonstrated in transgenic animals thus obtained. Further, the transmission of the exogenous gene to the descedants was confirmed in one line of transgenic rat up to F4 generation, indicating that the gene was stably incorporated into the germ line. Thus, direct single injection of foreign DNA into the testis provides a novel and convenient means to generate transgenic animals.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.71-80
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• 1996

The envelope of the rnannnalian oocyte plays crucial roles in sperm-oocyte interactions by providing sperm receptors, inducing acrosome reaction and preventing polyspermy. Understanding of properties of the zona pellucida (ZP) is essential for the artificial control of fertility in mammals. This study was carried out to produce and characterize monoclonal antibodies(MAbs) to porcine ZP proteins. Approximately 8,000 ZPs were obtained from follicular oocytes and dissolved in 40$\mu$l of double distilled water. Following immunization through foot-pad injections of Balb /c mice with a ZP solution, the popliteal lymph nodes were recovered at 2 weeks after the last injection. Hybridoma cell lines were established by fusing lymph node cells with P3X63 myeloma cells through selection using HAT medium and screening by immunofluorescence(IF) microscopy on the isolated ZP. Secreted MAbs were found to consist k chains and different heavy chains as evidenced by isotyping. Some of the MAbs demonstrated high specificity to the ZP in IF. The Mabs also showed positive cross reactivity with hamster and mouse eggs, while negative with bovine eggs. The results implicate that the MAbs can be used not only for identification of functional regions of the ZP, but also for elucidation of mechanisms involved in fertilization of mammals. The MAbs will provide basic information on biochemical anatomy of the ZP as well as can be candidates for the future contraceptive vaccines.

• The Korean Journal of Physiology
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• v.27 no.1
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• pp.107-114
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• 1993

Most of voltage operated $Ca^{2+}$ channels can be divided into three types (T-, N-, and L-type), according to the electrical and pharmacological properties. Their distribution is closely related to cell specific functions. Properties of the voltage activated $Ca^{2+}$ current in mouse eggs were examined to classify channel types and to deduce the function by using whole cell voltage clamp technique. $Ca^{2+}$ currents appeared below -40 mV and reached a maximum at -15 mV (half maximum was -31 mV), then decayed rapidly (inactivation time constant ${\tau}=28.2{\pm}9.59$ ms at -10 mV within 50 ms after the onset of step depolarization. Activation and inactivation of the $Ca^{2+}$ channel was steeply dependent on voltage, in a relatively low range of $-70\;mV{\sim}-10 mV,$ half maximum of activation was -31 mV and that of inactivation was -39 mV, respectively. This current was not decreased significantly by nifedipine, a specific dihydropyridine $Ca^{2+}$ channel blocker in the range of $1\;{\mu}M\;to\;100{\mu}M.$ The inhibitory effect of $Ni^{2+}\;on\;Ca^{2+}$ current was greater than that of $Cd^{2+}.$ The conductance of $Ba^{2+}$ through the channel was equal to or lower than that of $Ca^{2+}$ These results implied that $Ca^{2+}$ current activated at a lower voltage in the mouse egg is carried via a $Ca^{2+}$ channel with similar properties that of the T-type channel.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.207-216
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• 1998

Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

• The Korean Journal of Zoology
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• v.29 no.1
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• pp.13-22
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• 1986

In order to investigate the 'In Vitro 2-Cell Block' phenomenon found in certain mouse strains such as ICR, the present studies have been done. Fertilized eggs (1-cell) and 2-cell embryos recovered from the oviducts of the ICR mouse at the various time intervals after hCG injection to induce ovulation were cultured for 3 or 4 days to examine the capability for further cleavage beyond 2-cell stage. Consequently, it was found that some proportions of the 1-cell or 2-cell embryos recovered at 30 hours post hCG showed their cleaving capability and if the embryos were obtained after 48 hours of hCG injection, they were all at 2-cell stage and most of them developed to the blastocysts in vitro. It was also found that the embryos obtained at 27 hours post hCG showed their stronger capacity of further development in the groups cultured for shorter period than 24 hours in vitro before transferring to the oviduct. Based on the results, it can be inferred that mouse fertilized eggs should be remained inside the oviduct for a certain length of period after fertilization, or they should be cultured for a short period than 12 hours before returning back to the oviduct in order to develop to blastocysts. It was also found that though the embryos under the 2-cell block in culture showed normal feature up to 24 hours under the microscopical observation, they had already lost their capacity for the normal development, and if the culture of the 2-cell embryos was extended to 48 hours, they showed nuclei with heteropyknosis, and the vacuoles were were detected in the cytoplasm of embryonic cell if they were cultured for 72 hours.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.287-296
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• 2011

Mammalian fertilization is a complex cascade process consisting of sperm migration through the female reproductive tract, physiological changes to sperm such as sperm capacitation and acrosome reaction, and sperm-egg interaction in the oviduct in vivo. On the other hand, in vitro fertilization (IVF) is a process by which egg cells are fertilized by sperm outside the body: in vitro. IVF has been used for a variety of purposes in reproductive biotechnology for human and animals. The discovery of sperm capacitation in 1951 promoted the development of IVF technology. In the initial stage of IVF, sperm capacitation in preincubation medium was shown to be essential to fuse with eggs. Besides, sperms should detour some of the in vivo regulations for IVF. This review introduces a general mammalian fertilization process, including sperm capacitation, removal of cumulus matrix, acrosome reaction, and sperm-egg fusion and focuses on the roles of key biochemical molecules, signal mechanisms, and genes involved during IVF and novel results of sperm-oocyte interaction elucidated in various gene-knockout mice models.

• Korean Journal of Poultry Science
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• v.19 no.1
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• pp.13-16
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• 1992

Avian infectious bronchitis virus(IBV) was propagated in SPF eggs and purified by sucrose density gradient centrifugation in order to prepare the antigen. Several fusions were made between mouse myeloma cells and spleen cells from BALB/c mouse immunized with IBV antigen and two hybridoma clones producing specific monoclonal antibody(MCA) against the IBV were established. The MCAs were classified as IgG type and revealed no neutralizing and hemagglutination inhibition activity. Using the MCA IBV antigen was detected by IFA method in tracheal smears made from chickens infected with IBV during the experimental period of 10 days.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.9-17
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• 2004

Objective: The aim of this study were to compare the effects of EG and PROH on cryopreservation of mouse and human embryos, and to find the optimal protocol for embryo freezing. Methods: Human embryos derived from fertilized eggs showing 3 pronuclei (PN) and mouse embryos were divided into two groups respectively: dehydrated with 1.5 M EG + 0.2 M sucrose or 1.5 M PROH + 0.2 M sucrose using the slow freezing method. Moreover mouse embryos were controlled the exposure time of cryoprotectant during dehydration or rehydration steps. Results: The survival rates of human embryos were 79.2% (84/106) in EG group and 77.9% (88/113) in PROH group. In mouse embryos, the survival and development rates up to blastocyst were 70.6% (245/347), 44.1% (123/279) in EG group and 62.1% (198/319), 45.1% (123/279) in PROH group, respectively. However, in EG group, partially damaged embryos after thawing were decreased compared to PROH group. In combination group, when the exposure time during dehydration and rehydration were reduced, the survival and embryonic developments were increased slightly, but not significant. Conclusion: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH exhibited no statistical difference in the survival rate and/or developmental rate to blastocyst. However, the use of EG for cryopreservation of embryos might reduce the exposure time of the cryoprotectant because of a high permeation of EG and result in lessen its toxic effects.

• The Korean Journal of Zoology
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• v.31 no.1
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• pp.21-28
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• 1988

The present study was carried out to examine the fertilizability of the mouse oocytes pre-ex-posed to dbcAMP which is a well-known inhibitor of the oocyte maturation. The oocytes once cultured in the dbcMP-containing medium for a certain length of, time were cultivated in the dbcMp-free medium to induced the maturation, then mixed with sperms, and observed following culture for 24 hours. The fertilization rate of cocytes was judged by the index of the number of 2-cell embryo developed 24hr following insemination. The fertilization rate of the oocyte previously incubated with dbcAMP (100 g/ml) for 2, 4, 8 16 hours was 32.3, 14.5, 4.7 and 8.8%, respectively, while that of the control was 53.3% indicating that the fertilizability was decreased as a function of time exposed to dbcAMP. The pretreatment of dbcMP, however, didn't affect the process of sperm penetration to egg. In addition, there is no prominent changes in the morphological architecture of fertielized eggs which has been exposed to dbcAMP as revealed by electron microscopic observation. Consequendy, it can be concluded that the mouse cocytes once inhibited their maturation by dbcMP may retain, in some extent, the fertilizability, although most of the fertilized egg may not proceed to further development because of the failure of pronucleus formation.