• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.65-72
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• 2001

Objectives: This study was to investigate the fertilization rate after intracytoplasmic sperm injection (ICSI) or partial zona dissection (PZD) of human and hamster sperm into hamster oocyte in in vitro fertilization (IVF). In addition, the possibility of clinical application was evaluated by the comparison of usefulness and difference of these method. Materials and Methods: Hamster immature oocytes were obtained from oviducts superovulated by PMSG and hCG, and hamster sperms were obtained from epididymis. The freezed human sperms were thawed before use. Fertilization were confirmed by two pronuclei, one pronucleus, swollen sperm head or/and two polar bodies at $7{\sim}8$ hour after ICSI or PZD. Results: The fertilization rates after ICSI and PZD of human sperm to hamster oocyte were 3.6%, 64.2%,73.6%, and 55.6% for negative control, positive control, ICSI, and PZD respectively, suggesting that ICSI only showed improved fertilization rate (p<0.01). The fertilization rates after ICSI and PZD of hamster sperm to hamster oocyte were 11.1%, 51.2%, 39.6%, and 72.7% for negative control, positive control, ICSI, and PZD respectively, suggesting that PZD only showed improved fertilization rate (p<0.01). PZD showed significantly higher fertilization rate than ICSI (p<0.05). Conclusions: As for the fertilization rate by ICSI and PZD using hamster oocyte in IVF, ICSI technique was considered to be more useful for human sperm and PZD technique for hamster sperm. Therefore, ICSI technique was considered more appropriate for experimental application using human sperm and hamster oocyte.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.2
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• pp.173-177
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• 1989

Defective or inadequate semen quality, usually presenting as low sperm count or poor sperm motility , is recognizable by semen analysis. However, the ability of spermatozoa to fertilize an ovum is not determined used in various experiments. In this study, hamster oocyte sperm penetration assay was used to determine the fertilizing capacity of sperms in 20 subjects which divided into two groups, group A with 10 normal fertile men, and group B with 10 infertile men. The % penetration in group A and group B were 61% and 35% respectively, which showed statistically not significant but fertilization index was significantly different between group A(FI=2.24) and group B(FI=O.05). Additionally it seemed that the percentage of sperm penetraton was influenced more by the motility of spermatozoa than by the number.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.1
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• pp.15-21
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• 1989

An in vitro fertilization assay employing zona-free hamster embryos was used to investigate human spermatozoal fertilitzing ability. Yanaghimarchi et al.(1976) first introduced this cross species fertilzation technique, with its application as a diagnostic tool for male infertility. Human spermatozoa were preincubated for 3 to 4 hrs in B W W medium at concentration of $4{\times}10^6$ sperm/ml prior to the addition to zona-free hamster embryos. After 3 hrs, human sperm was evaluated for fertilizing potential by the presence of swelling or decondencing sperm head in the cytoplasm. The results of penetration rates for sperm were as follow : 1. The average penetration rate of a 7 fertile donor group was $47.8{\pm}27.67%$(Range 14.3-98.0%) 2. The average penetration rate of 12 infertile patients with normal semen analysis was $21.7{\pm}26.9%$(Range 0-38.8%) 3. The average penetration rate of 10 infertile patients with semen abnormalities was $6.1{\pm}8.1%$(Range 0-25%)

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.89-99
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• 2004

Stretch-activated channels (SACs) responds to membrane stress with changes in open probability (Po). They play essential roles in regulation of cell volume and differentiation, vascular tone, and in hormonal secretion. SACs highly present in Xenopus oocytes and Ascidian oocytes are suggested to be involved in the regulation of pH and fluid transport to balance the osmotic pressure, but remain unclear in mammanlian oocytes. This study was investigated to find the presence of SACs in hamster oocytes and to examine their electrophysiological properties. To infer a role of SAC in relation to the development of early stage, we followed up to the stage of two-cell zygote with patch clamp techniques. Single channels were elicited by negative pressure (lower than ­15 cm$H_2O$). Interestingly, SACs were dependent on permeable cations such as $Na^+$ or $K^+$. As permeable cation removed from both sides across the membrane, SAC activity completely disappeared. When permeable cations present only in intracellular compartment, outward currents appeared at positive potentials. In contrast to this, inward currents occurred only at the negative voltage when permeable cation absent in cell interior. These result suggests that SAC carry cations through the nonselective cation channel (NSC channel). Taken together, we found that stretch activated channels present in hamster oocyte and the channel may carry cations through NSC channels. This stretch activated-NSC channels may play physiological role(s) in oocyte growth, maturation, fertilization and embryogenesis in fertilized oocytes to two-cell zygotes of hamster.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.385-391
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• 1997

The purpose of this study was to examine the effects of anti-sperm antibody (ASA) on the fertilization processes using conventional IVF and ICSI procedure in human and hamster oocytes. In human IVF, we have observed restricted fertilization with sperm testing positive for ASA. ($23{\sim}90%$ IgA, 60-97 % IgG). However, if ICSI was perform in the next IVF cycle with the same patients, we could successfully fertilize the oocytes (37%; p<0.001), thus achieving pregnancy and delivery. When the sperm were cocultured in medium containing ASA, there were binding of ASA to sperm surface. In addition, the mean rate of the acrosomal reaction in an in vitro acrosome reaction test was lower for Ab-bound sperm (43.5%) than for Ab-free sperm group (51.3%, p<0.05). We used human sperm and hamster oocytes to confirm the negative effects of the ASA on fertilization. The sperm and/or oocytes have been expose to medium containing ASA before IVF and ICSI. In this experiment, the ASA was bound to the oocyte and sperm surface. The following results were obtain by using various combinations of ASA free or ASA bound sperm with ASA free or ASA bound oocytes for IVF. When ASA free sperm were inseminate with ASA free and ASA bound hamster oocytes, the fertilization rates are 89.6% and 74.3% respectively. However, when ASA bound human sperm were use the results were 62.5% and 55.6% respectively. These shows the fertilization rate was significantly decreased in both ASA bound and ASA free oocytes when using ASA bound sperm. No difference found when ASA are present on the oocyte surface. When the hamster oocytes was treated by ICSI with ASA free or ASA bound human spermatozoa, no significant difference was found. These results showed that ICSI is the most promising method for couples who fertilization was not possible by conventional IVF because of ASA.

• 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.

• 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.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.57-66
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• 2002

Objective : This study was designed to investigate the interrelationship and clinical usefulness of sperm morphology by strict criteria (SM), acrosome reaction following ionophore challenge test (ARIC) and sperm penetration assay (SPA) using zona-free hamster ova as prognostic factors in in vitro fertilization. Materials and Methods: Semen samples were provided by 83 patients undergoing IVF. We first evaluated the differences between normal fertilization group and poor fertilization group on three andrologic tests. Secondly, we analyzed the relationship between the three andrologic tests and in vitro fertilization on IVF settings. Finally, we evaluated the effectiveness of the three andrologic tests as the prognostic indicators for fertilizing ability. Results: The fertilization rate of all men in the poor fertilization group was less than 30%; but there was no evidence that this poor fertilization was due to oocyte defects. The results of three andrologic tests were significatly higher in normal fertilization group. Fertilization rate (%) in vitro was highly correlated (p<0.001) with % normal sperm by SM, ARIC value (%), and SPA result. By using Receiver-Operator-Characteristic curve (ROC), we evaluated the effectiveness of these three tests. The sensitivity and specificity of SM, ARIC test and SPA in predicting fertilization potential in IVF setting were 76% and 75%, 84% and 90%, and 76% and 95%, respectively. Conclusion: Our data suggest that the three andrologic tests can be reliable tools as prognostic factors of sperm fertilizing ability. Among these test, ARIC test and SPA gave more accurate information on fertilizing capacity. ARIC test was shown to have a predictive value for fertilizing ability comparable to that of SPA that appears to be a simple and cost-effective addition to current andrology laboratory. Combined application of these three tests may give more information on predicting sperm fertilizing capacity.