• Korean Journal of Animal Reproduction
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• v.13 no.3
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• pp.188-194
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• 1989

These experiments were carried out to investigate fertilizable and developmental ability after zona drilling the unfertilized eggs and the eggs not fertilized by the 1st insemination. The results of in vitro fertilization of the mouse eggs treated by using micromanipulation and acid tyrode's solution with capacitated epididymal spermatozoa were as follows. In the case of ovulated unfertilized eggs, according to sperm count(106, 105, 104 and 103/ml) the rates of in vitro fertlilization treated by zona drilling were 86.0%, 82.0%, 70.0% and 54.0%, respectively, and those of control were 58.0%, 52.0%, 12.0% and 8.0%, respectively. The rates of in vitro fertilization of zona drilled eggs were significantly high compared with those of control, and there were no significant difference between two groups. According to the sperm count the zona drilled eggs developed to the blastocysts were 51.4%, 40.5%, 23.3% and 17.4% and those of control were 35.7%, 26.3%, 0% and 0%, respectively. Also, in the eggs not fertilized by 1st insemination, the fertilization rates of oocytes reinseminated after zona drilling was significantly higher(83.5%) than that of control(34.7%), and the rates of polyspermy were similar. The rates of development to the blastocysts was 18.6% in the zona drilling treated eggs, and that of control was 27.3%, there was no significant difference between two groups. These results indicated that oocytes not fertilized by 1st insemination as well as ovulated unfertilized eggs could be fertilized, improve fertilizing rates by zona drilling treatment, and development potential were normal.

• The Korean Journal of Physiology
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• v.25 no.2
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• pp.125-131
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• 1991

Inactivation properties of Ca current in the unfertilized eggs of mouse were studied by using the whole cell voltage clamp technique and single microelectrode voltage clamp technique. Membrane potential was held at -80 mV and step depolarization was applied from -50 mV to 50 mV for $200{\sim}500\;ms$. Peak of inward Ca currents was $-2{\sim}-4\;nA$ at a membrane Potentials from -20 mV to 0 mV and outward currents were not observed within the membrane voltage range studied $(-50{\sim}50\;mV)$. Inward currents were fully inactivated within 200 ms after the onset of step depolarization. As the membrane became depolarized, time constant of inactivation (${\tau}$) was decreased but remained around $20{\sim}30\;ms$ beyond 10 mV. When $Ca^{2+}$ was used as a charge earlier, inactivation of inward $Ca^{2+}$ current also occured and time course of inactivation was similar to that of $Ca^{2+}$ currents as charge carrier. In the bathing solution containing high potassium $(131\;mM\;K^+)$, process of inactivation was not changed except a parallel decrease of value for the entire range of membrane potential. Steady-state inactivation of the $current(h_{\infty})$ obtained from the double pulse experiment showed the voltage-dependent change. These results suggested that inactivation of Ca currents in the unfertilized eggs of mouse was voltage-dependent.

• The Korean Journal of Physiology
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• v.23 no.1
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• pp.35-42
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• 1989

The present study was performed to observe the effects of cations on resting membrane potential and pump activity in the unfertilized eggs of ICR strain mice. After an induction of superovulation, the fresh eggs with zona pellucida were collected and the membrane potentials were recorded. Recordings of membrane potential in this study was obtained from the physiological conditions ($37^{\circ}C$ and 4mM Ca in standard solution), differently from the another reports with unphysiological conditions (room temprature and high Ca in standard solution) for a stable and long-lasting observations. Presented data was obtained within 6 hours after collection from the oviduct. The results observed are as follows, 1) Resting potential of the unfertilized eggs was $-25.8{\pm}3.8mV$ $(Mean{\pm}Se,\;n=31)$. 2) As the K ion concentration was increased, resting membrane potential was depolarized but showed hyperpolarization with $K^{+}$ below 25mM. 3) Alteration of the resting membrane potential for the changes of $Na^{+}$ concentration were hardly observed, while resting potential was hyperpolarized as $Ca^{2+}$ concentration was increased. 4) Pump activity as transient or prolonged hyperpolarization was $-2.29{\pm}0.75mV$ $(Mean{\pm}Se,\;n=16)$, the hyperpolarization was increased in both amplitude and duration under the 10mM $Ca^{2+}$ solution. 5) Hyperpolarization due to pump activity was decreased or disappeared by $5{\times}10^{-5}\;M$ ouabain treatment and could not be observed under the both Na-free and Ca-free solutions. 6) Above results are likely to suggest that the resting potential of the mouse unfertilized eggs is affected to mainly by Ca-dependent K conductance and Na-Ca exchange mechanism and that there is pump activity coupling between $K{+}$, $Na^{+}$ and $Ca^{2+}$.

• Clinical and Experimental Reproductive Medicine
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• v.11 no.2
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• pp.51-57
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• 1984

The success of human in vitro fertilization (IVF) & embryo transfer (ET) has focused attention on the culture conditions that can provide optimal development of the preimplantation embryo. Studies of in vitro fertilization using mouse have direct implications to human IVF, since similar conditions are used for both species. Mouse IVF as a quality control system for human IVF & ET was studied since Feb., 1984. The results were as follows: 1. Egg retrieval following superovulation in IeR mice was l5.1${\pm}$5.3 eggs ovulated/mouse (Mean${\pm}$ S.D.) 2. In vitro cleavage rate was 61.7% (1146 eggs cleaved/l858 eggs inseminated) and % blastocyst was 42.6%. 3. In comparison with two media of Ham's F-10 and m-KRB, in vitro cleavage rate were 40.9%/63.l% and %blastocyst were 44.3%/61.2% (P<0.05). 4. It was concluded that mouse IVF system has a valuable place in human IVF & ET as a quality-control system and in human reproductive physiology as a research model.

• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.225-231
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• 1991

These experiments were carried out ot detect autoantiboies to zona pellucida in sera from infertile women using indirect ELISA and IFA and to investigate their effect on in vitro fertilization of mouse ova. In inidirect ELISA test, 12 of 116(10.3%) serum samples form infertile women gave positive reaction whereas all of 16 samples from fertile women and men were negative. Furthermore, in indirect IFA test, 17 of 116 (14.7%) serum samples from infertile women gave positive fluorescence whereas all of control sera were negative fluorescence. The fertilization rates(15.9%) of mouse eggs treated with positive sera were significantly lower than those(51.9%＋71.2%) autoantibodies to zonapellucida are responsible for infertility in unexplained infertile women, presumably by perventing sperm attachment and penetration.

• Korean Journal of Veterinary Research
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• v.30 no.2
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• pp.123-127
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• 1990

By nuclear transplantation technology twenty eight mice have been produced after transfer of heterozygous biparental eggs. Also heterozygous gynogenetic eggs with two female pronuclei and heterozygous androgenetic eggs with two male pronuclei have been obtained by injecting a male or female pronucleus with Sendai virus into the perivitelline space of enucleated haploid zygotes at pronuclear stage. The success rate of enucleation, karyoplast injection and fusion of both the pronuclei was 80.3, 83.4 and 81.8%, respectively. The overall pronuclei fusion rates by this technique were 56, 50 and 56% in biparental, gynogenetic and androgenetic eggs, respectively. The evidence was ascertained that the gynogenetic and androgenetic eggs were also able to develop in vitro up to blastocyst stage, even though their developmental potential was greatly diminished beyond 2-cell stage. The gynogenetic eggs were able to develop in vivo up to day 10 of pregnancy, while the androgenetic eggs failed to develop in vivo during the same period.

• The Korean Journal of Physiology
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• v.27 no.2
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• pp.175-183
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• 1993

There are many report suggesting that influx and intracellular calcium concentration $([Ca^{2+}]_i)$ are related to cell signalling in various cells. However, it has not been reported that calcium channel activation is affected by the substances involved in signal transduction pathways in the mouse eggs. In this study, the effects of isoprenaline (ISP) and cyclic AMP on calcium influx through calcium channels were investigated to show their relationship with the signal transduction process in unfertilized mouse eggs. Using whole cell voltage clamp techniques, calcium currents, elicited by the depolarizing pulses of 300 ms duration (from -50 mV to 50 mV in 10 mV increments) from a holding potential of -80 mV, were recorded. The current-voltage (I-V) relation of calcium currents was shown to be bell-shaped; the current began to activate at -50 mV and reached its maximum $(-1.33{\pm}0.16\;nA:\;mean{\pm}S.E.,\;n=7)$ at -10 mV, then decayed at around 50 mV. Calcium currents were fully activated within $7\;ms{\sim}20\;ms$ and completely inactivated 200 ms after onset of the step pulse. ISP within the concentration ranges of $10^{-8}\;M{\sim}10^{-4}\;M$ dose-dependently increased the amplitude calcium current. The permeable cyclic AMP analogue,8-bromocyclic AMP, also increased its maximal amplitude by 46ft at $10^{-5}\;M$, while protein kinase inhibitor (PKI), which is known to inhibit 0.02 phosphorylating units of cyclic AMP-dependent protein kinase (PKA) per microgram decreased calcium currents. Currents recorded in the presence of PKI were resistant to increase by the application of $10^{-5}\;M$. Also, PKI inhibited the calcium current increase elicited by ISP treatment. These results suggest that $\beta-adrenergic$ regulation of the calcium channel is mediated by the cAMP-dependent protein kinase. This signal transduction pathway might play a role in regulating $[Ca^{2+}]_i$, level due to the increase of calcium influx in mouse eggs.

• Applied Microscopy
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• v.43 no.4
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• pp.146-154
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• 2013

Capillaria hepatica (syn. Calodium hepatica) is a parasite found mainly in rodent liver. But, it has also been found in a wide variety of mammals, including humans. This worm is unique as it is the only nematode parasite that is embedded in the liver parenchyma of the host even during the adult stage of the life cycle. They produce eggs that elicit a marked granulomatous reaction that eventually destroys the worms. Fibrosis and lymphoplasmacytic inflammatory infiltration are often observed around adult nematodes embedded in the liver parenchyma of the host. For this reason, complete isolation of this slender worm and observation of the intact ultrastructure is very difficult. In this study, 10 intact whole worms (C. hepatica) were isolated from the liver of 3-week-old mouse after inoculation of artificially embryonated eggs collected from house rats (Rattus norvegicus). Their external structure of was observed with light and scanning electron microscopy. The length of the isolated female and male C. hepatica was approximately 69.60 mm and 36.92 mm, respectively. More detailed ultrastructure, including bacillary band, eggs and vulva in female and spicule and spicule sheath in male C. hepatica was also described.

• Development and Reproduction
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• v.24 no.4
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• pp.297-306
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• 2020

Repetitive changes in the intracellular calcium concentration ([Ca²⁺]i) triggers egg activation, including cortical granule exocytosis, resumption of second meiosis, block to polyspermy, and initiating embryonic development. [Ca²⁺]i oscillations that continue for several hours, are required for the early events of egg activation and possibly connected to further development to the blastocyst stage. The sources of Ca²⁺ ion elevation during [Ca²⁺]i oscillations are Ca²⁺ release from endoplasmic reticulum through inositol 1,4,5 tri-phosphate receptor and Ca²⁺ ion influx through Ca²⁺ channel on the plasma membrane. Ca²⁺ channels have been characterized into voltage-dependent Ca²⁺ channels (VDCCs), ligand-gated Ca²⁺ channel, and leak-channel. VDCCs expressed on muscle cell or neuron is specified into L, T, N, P, Q, and R type VDCs by their activation threshold or their sensitivity to peptide toxins isolated from cone snails and spiders. The present study was aimed to investigate the localization pattern of N and P/Q type voltage-dependent calcium channels in mouse eggs and the role in fertilization. [Ca²⁺]i oscillation was observed in a Ca²⁺ contained medium with sperm factor or adenophostin A injection but disappeared in Ca²⁺ free medium. Ca²⁺ influx was decreased by Lat A. N-VDCC specific inhibitor, ω-Conotoxin CVIIA induced abnormal [Ca²⁺]i oscillation profiles in SrCl₂ treatment. N or P/Q type VDC were distributed on the plasma membrane in cortical cluster form, not in the cytoplasm. Ca²⁺ influx is essential for [Ca²⁺]i oscillation during mammalian fertilization. This Ca²⁺ influx might be controlled through the N or P/Q type VDCCs. Abnormal VDCCs expression of eggs could be tested in fertilization failure or low fertilization eggs in subfertility women.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.207-211
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• 2015

Many pronuclear stage eggs were used to generate transgenic mice (Tg) by microinjection. In this study, we used in vitro fertilized mouse eggs, followed by ultrarapid freezing to establish a simple procedure for production of Tg mice. We produced in vitro fertilized mouse eggs and cryopreserved them by ultrarapid freezing method. A total of 139 cryopreserved-thawed pronuclear eggs, of which 101 (72.6%) were survived following microinjection of chicken ${\beta}-actin$ promoter-driven firefly improved luciferase cDNA (${\beta}-act/luc^+$) and were transferred into 5 recipients. All recipients became pregnant and gave birth to a total of 15 (14.8%) pups. As a control, same DNA construction (${\beta}-act/luc^+$) was also injected into 450 in vitro fertilized eggs, of which 338 (75.1%) were survived and then were transferred into 14 recipients. Eleven (78%) mice became pregnant and littered a total of 54 (19.1%) pups. Southern blotting analysis of Tg mice indicated that one (1/15, 6.6%) and three (3/54, 5.5%) transgenic mice were production from cryopreserved and in vitro fertilized eggs, respectively. All Tg mice produced from both eggs showed the expression of improved luciferase gene. These results indicated that efficiency of produced of Tg mice from cryopreserved eggs was comparable to that from in vitro fertilized eggs. Furthermore, it is suggested that microinjection of transgene into in vitro fertilized eggs cryopreserved by ultrarapid freezing is an easy and conveniently method for production of Tg mice.