• Journal of Embryo Transfer
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• v.19 no.2
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• pp.101-112
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• 2004

Chloride($Cl^-$) channels play critical roles in cell homeostasis and its specific functions such as volume regulation, differentiation, secretion, and membrane stabilization. The presence of these channels have been reported in all kinds of cells and even in frog oocytes. These essential role of $Cl^-$­ channels in cell homeostasis possibly play any role in egg homeostasis and in the early stage of development, however, there has been no report about the presence of $Cl^-$­ channel in the mammalian oocyte. This study was performed to elucidate the presence of $Cl^-$­ channels in hamster eggs. When allowing only $Cl^-$­ to pass through the channel of the egg membrane by using impermeant cation such as N-methyl-D-glucamine(NMDG), single channel currents were recorded. These channel currents showed typical long-lasted openings interrupted by rapid flickering. Mean open $time({\tau}o)$ was 43${\pm}$10.14 ms(n=9, at 50 mV). The open probability(Po) was decrease with depolarization. The current-voltage relation showed outward rectification. Outward slop conductance(32${\pm}$5.4 pS, n=22) was steeper than the inward slop conductance(10${\pm}$1.3 pS). Under the condition of symmetrical 140 mM NaCl, single channel currents were reversed at 0 mV(n=4). This reversal potential(Erev) was shifted from 0 mV at 140 mM concentration of internal NaCl(140 mM [Na+]i) to ­9.8${\pm}$0.5 mV(n=4) at 70 mM [Na+]i and 11.5${\pm}$1.9 mV at 280 mM [Na+]i(n=4) respectively, strongly suggesting that these are single $Cl^-$­ channel currents. To examine further whether this channel has pharmacological property of the $Cl^-$­ channel, specific Cl­ channel blockers, IAA-94(Indanyloxyacetic acid-94) and DIDS(4, 4'-diisothiocyan ostillben- 2-2'disulfonic acid) were applied. IAA-94 inhibited the channel current in a dose-dependent manner and revealed a rapid and flickering block. From these electrophysiological and pharmacological resluts, we found the novel $Cl^-$­ channel present in the hamster oocyte membrane. The first identification of $Cl^-$­ channel in the hamster oocyte may give a clue for the further study on the function of $Cl^-$­ channel in the fertilization and cell differentiation.

• Development and Reproduction
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• v.22 no.3
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• pp.253-262
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• 2018

The spermatogenesis is the process by which spermatozoa are generated in the testes. The spermatozoa travel male reproductive tract during which they meet many substances secreted from reproductive organs. One of the substances is epididymal protease inhibitor (EPPIN) that is involved in the post-testicular maturation including capability of fertilizing the eggs. The expression of EPPIN gene was investigated in various tissues of sexually mature and regressed male Syrian hamsters by reverse transcription polymerase chain reaction (RT-PCR). The EPPIN gene was identified in the testis and epididymis of the male Syrian hamsters and compared to the genes reported previously. There was no expression of EPPIN gene in reproductively and completely regressed testes of Syrian hamster. These results suggest that the expressions of the EPPIN gene are associated with the reproductive capability in the Syrian hamsters.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.280-289
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• 1998

Voltage-sensitive ion channels contribute to establishment of the cell excitablity and the generation of the cellular function. At hamster oocytes in the primitive stage during developing process, an inward current elicited by voltage pulses was found to be carried mainly by $Ca^{2+}$. Even at present, $Ca^{2+}$ channels serve as the most probable route to pass this inward current but there is no evidence of the presence of this channels in eggs. To date, both the characteristic properties and the physiological role in the early stage of development remain unclear. Here we examined the characteristic properties of the inward current and changes in this currents at unfertilized oocytes, fertilized zygotes and two-cell embryos using whole-cell voltage clamp technique. The inward current carried reportedly by $Ca^{2+}$ was remained following removing external $Ca^{2+}$ but completely abolished by further replacement of impermeants such as tetramethylammonium ion ($TMA^+$) or $choline^+$ instead of $[Na^+]_0$. Tetrodotoxin did not affect on this inward current remained at $[Ca^{2+}]_0$-free condition. Removal of $Na^+$ ion out of the experimental solution clearly decreased the current. After adding 2mM $Ca^{2+}$ to the $Na^+$-free media, the inward current was restored. Interestingly, this current carried by either $Ca^{2+}$ or $Na^+$ was decreased by the reduction of intracellular $Cl^-$ concentration, or by $Cl^-$ channel blockers such as niflumic acid, DIDS and SITS. When $Cl^-$ concentration was lowered without changes in other ionic components, this inward current was reduced. At fertilized oocytes and two-cell embryos, the inward current carried by $Ca^{2+}$ and $Na^+$ was severely reduced. Also $Cl^-$ component could not be observed. From these results, the inward current is composed of $Ca^{2+}$, $Na^+$ and $Cl^-$ component, suggesting that the channel carrying this inward current is not selective specifically to $Ca^{2+}$. During early stage of development, the voltage-sensitive ion current seems not to contribute essentially to the cell cleavage and differentiation. The loss of $Cl^-$ component after fertilization suggests that $Cl^-$ may play a role in maintaining the viability of unfertilized ova.

• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.537-541
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• 2016

Stellantchasmus falcatus (Digenea: Heterophyidae) is first reported from Cambodia through recovery of the metacercariae from mullet fish and adult flukes from an experimentally infected hamster. We purchased 7 mullets, Chelon macrolepis, in a local market of Phnom Penh, Cambodia, and each of them was examined by the artificial digestion method on May 2010. The metacercariae of S. falcatus were detected in all mullets (100%) examined, and their average density was 177 per fish. They were elliptical, $220{\times}168{\mu}m$ in average size. They were orally infected to an hamster to obtain adult flukes. Adults recovered at day 10 post infection were observed with a light microscope and a scanning electron microscope (SEM). They were small, $450{\times}237{\mu}m$ in average size, had a small oral sucker ($41{\times}50{\mu}m$), subglobular pharynx ($29{\times}21{\mu}m$), slender esophagus ($57{\mu}m$), long and thick-walled expulsor ($119{\times}32{\mu}m$), spherical ovary ($58{\times}69{\mu}m$), and 2 ovoid testes (right: $117{\times}74{\mu}m$; left: $114{\times}63{\mu}m$). Eggs were small, yellow, and $23{\times}12{\mu}m$ in average size. In SEM observations, tegumental spines were densely distributed on the whole tegument, and single small type I sensory papillae were distributed around the lip of oral sucker. The small ventral sucker was dextrally located and had 8 type I sensory papillae on the left margin. It has been first confirmed in the present study that the mullet, C. macrolepis, is playing the role of a second intermediate host of S. falcatus in Cambodia.

• 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.

• Food Science and Preservation
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• v.16 no.5
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• pp.782-788
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• 2009

The genotoxicological safety of gamma-irradiated egg white and yolk was examined to ensure that required safety parameters were met, and in an effort to further apply gamma-irradiation for improvement of the hygienic qualities of eggs. Egg white and yolk were irradiated at 20 kGy, much higher than the legally approved dose (less than 5 kGy), and possible genotoxicity was evaluated using in vitro and in vivo tests. The SOS chromotest employing Escherichia coli PQ37, and a chromosomal aberration test in cultured Chinese hamster lung (CHL) cells, were performed in vitro with or without metabolic activation (S9). An in vivo micronucleus development test was conducted using mouse bone marrow cells. Negative results were obtained in the SOS chromotest. The incidence of chromosomal aberration in CHL cells and the frequency of micronuclear developmentin mouse bone marrow cells treated with irradiated samples were not significantly different from those of non-irradiated controls. Thus, it may be concluded that up to 20 kGy of gamma irradiation applied to egg white and yolk did not show any genotoxic effects under our experimental conditions.

• Korean Journal of Veterinary Research
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• v.36 no.2
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• pp.305-312
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• 1996

Cell volume regulatory mechanisms are usually disclosed by exposure of cell to anisotonic media. If a cell is suddenly exposed to hypotonic media, it swells initially like an osmometer but within minutes regains its original cell volume. This behavior has been labelled as regulatory cell volume decrease(RVD). RVD is believed to result from the loss of permeable ions through the membrane. In this study, we examined hypotonically induced changes in the membrance currents involved in RVD by using whole cell voltage clamp technique in the unfertilized hamster egg. At -40mV of the holding potential, the stationary current was maintained in the hamster egg exposed to isotonic solution composed of, mainly, 115mM NaCl and 40mM mannitol. Hypotonic solution was prepared by removing mannitol. Therefore, the concentrations of $Na^+$ and $Cl^-$ in this hypotonic media were the same as those in the isotonic solution. Following 30 to 60 sec after applying the hypotonic media to the egg, the inward current was evoked. This inward current was eliminated by $100{\mu}M$ 4-acetamido-4'-isothiocyanostil-bene-2,2'-disulfonic acid(SITS), an anion channel blocker, leaving the small outward current component. Further addition of 2mM $Ba^{2+}$, a broad $K^+$ channel blocker, completely abolished the small outward current left even in the presence of SITS during hypotonic stress. These results suggest that $K^+$ and $Cl^-$ move out of cells, resulting in RVD. To test the involvement of $Na^+$ in RVD, 20mM Na-isethionate was substituted for mannitol in isotonic media(135mM $Na^+$) and Na-isethionate (20mM) was freed the hypotonic solution. Only $Cl^-$ concentration in both isotonic and hypotonic media was kept constant at 115mM, whereas concentration of $Na^+$ was lowered in hypotonic solution to 115mM from 135mM in isotonic solution. Hypotonic medium induced the outward current in the egg equilibrated isotonically. This current was reduced by $100{\mu}M$ SITS but was augmented by 2 mM $Ba^{2+}$. In terms of RVD, these results imply that $Cl^-$ efflux is coupled with $K^+$, maybe for electroneutrality during hypotonic stress and/or with $Na^+$ via unknown transport mechanism(s). From the overall results, the hypotonic stress facilitates the movement of $Cl^-$ and $K^+$ out of the hamster egg to regain cellular volume with electroneutrality. If there exist a difference in $[Na^+]_0$ between isotonic and hypotonic solution, another transport mechanism concerned with $Na^+$ may, at least partly, participate in regulatory volume decrease.

• Korean Journal of Veterinary Research
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• v.34 no.3
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• pp.559-567
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• 1994

As a series of studies to investigate the effect of immunosuppression on Ascaris suum infection in undefinitive hosts, and a delicate relationship between host and parasite, in the present studies, SPF ICR mice were alloted to experiment 1(normal undefinitive host group) and experiment 2(immunosuppressive group treated with prednisolone acetate) and inoculated with a single dose of 1,100 embryonated A suum eggs. In normal group, the infection essentially terminates 4 days after inoculation(DAI) with the attainment of middle third-stage in the liver, although few larvae migrate to the lungs where a few advance to late third stage. In immunosuppressive group, significant numbers developed to late third-stage in liver 8 DAI. In general, increasing of the mast cells and the goblet cells in the jejunum mucosa, of T-cells in the spleen and of activity of peritoneal macrophages followed by expulsion of the worms in the both groups. Considering a series of the results, suitabilities for the host of the worm appeared the highest from rabbit, hamster and mouse in that order. In addition, patent infection of A suum in the mice was also not obviously observed in spite of immunosuppression by prednisolone acetate.

• Korean Journal of Veterinary Research
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• v.34 no.1
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• pp.127-134
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• 1994

As a series of studies to investigate the effect of immunosuppression on Ascaris suum infection in undefinitive hosts, a delicate relationship between host and parasite, in the present studies golden hamsters were alloted to experiment 1(normal undefinitive host group) and experiment 2(immunosuppressive group treated with prednisolone acetate) and inoculated with a single dose of 1,500 embryonated Ascaris suum eggs. The recovery rates, sizes and features of the larvae and immunological responses in the hamsters were chronologically monitored according to somatic migration. In both experiments, the larvae failed to develop into the adults, but the more and larger larvae were observed for a longer period from experiment 2 in comparison with experiment 1. The numbers of the mast cells in the small intestinal mucosa and mesenteric lymph nodes, of the goblet cells in the small intestinal mucosa and of T-cells in the mesenteric lymph nodes, spleens and cardiac blood from experiment 2 were fewer than those from the experiment 1. In general, increasing of these cells followed by expulsion of the worms in the both groups. Profound leukopenia due to lymphopenia was found through trial period in experiment 2. Considering the experimental results, development or expulsion mechanism of somatic migrant larvae may be related to lymphopenia and temporary increasing tendency of the mast cells, the goblet cells and T-cells. In addition, patent infection of A suum in the hamsters was not obviously observed in spite of immunosuppression by prednisolone acetate.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.311-323
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• 1994

This study was conducted to observe seasonal and individual changes in semen characteristics and sperm freezability, and sperm penetration into zona-free hamster eggs in Korean native goats. Buck response and change in semen characteristics to electrical stimulations was evaluated for four seasons throughout 2 years and percentage of motile sperm and normal apical ridge acrosome was investigated after equilibration and thawing for 4 seasons with 5 bucks. Sperm penetration rate was evaluated for 4 bucks. 1. Probe insertion at depth of 7cm and repeated stimulation for 3 sec was more effective(P<0.05) in buck response and semen collection than those of other conditions. 2. Semen characteristics from electrojaculation was signficantly(P<0.005) higher in spring and fall for semen volume, in spring and summer for sperm concentration and in fall for sperm motility than those in other seasons, respectively. However, there were no differences in total sperm among seasons. 3. Buck response to electrical stimulation showed significant difference(P<0.05) among individuals in all 3 seasons except winter. Significant individual difference in semen volume was only in spring and summer, but there was no indivudual difference in sperm concentration and total sperm in all season. 4. Washing of semen before freezing treatment was greatly(P<0.05) beneficial to sperm motility after thawing, no matter whether ejaculates exhibit egg yolk coagulation or not. 5. Sperm motility after glycerol equilibration was significantly(P<0.05) low in summer semen and motility after thawing was greatly(P<0.05) higher in winter semen than in other seasons. Freezability of unwashed sperm was significantly difference among bucks, but a yearly freezability of washed sperm after chilling and thawing were no differences among bucks and percentage of normal apical ridge acrosome were not different among seasons and bucks. 6. There was no significant difference in sperm motility after thawing between egg yolk levels in summer, although 20% level gave more higher motility than 5% level. 7. In summer, 3.2% glycerol and 3-h equilibration gave greatest percentage(P<0.05) of sperm motility and normal apical ridge acrosome after thawing. 8. Sperm penetration rate into zona-free hamster eggs was not different between bucks and seasons. Overall, it is concluded that to obtain maximum sperm output and successive semen freezing by electrojaculation method, buck selection with good response in all season could be basically considered and that seasonal effect on sperm freezability was more greater than that of individual bucks.