Fertilization Promoting Peptide가 사람 정자의 운동양태, 수정능력획득 및 첨체반응에 미치는 영향

강희규,김묘경,김동훈,한성원,최도연,이호준,김문규,Kang, Hee-Gyoo,Kim, Myo-Kyung,Kim, Dong-Hoon,Han, Sung-Won,Choi, Do-Hyun,Lee, Ho-Joon,Kim, Moon-Kyoo 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.2

Objective: This study has been carried out to evaluation the effect of fertilization promoting peptide (FPP) on the kinematic parameters, capacitation and acrosome reaction of the frozen-thawed human spermatozoa. Methods: After FPP treatment, we examined kinematic parameters, capacitation and acrosome reaction, using the methods of computer-aided sperm analysis (CASA) and chlortetracycline (CTC) fluorescence analysis. Results: We have obtained the evidence that FPP can promote the capacitation and inhibit the spontaneous acrosome reaction of frozen-thawed human spermatozoa in vitro. FPP ($25{\sim}100$ nM) induced a significant increase in the proportion of B-pattem capacitated spermatozoa, and a significant decrease in the proportion of F-pattem uncapacitated ones without significant stimulation of acrosomal exocytosis. In the kinematic parameters treatment, FPP treated groups maint3ined higher LIN, BCF and STR than those of control. The VAP, VSL, VCL and ALH were not different. Therefore it is suggested that FPP in human seminal plasma may play a positive role in promoting human sperm function.

반응성 산소족이 사람 정자의 운동성에 미치는 영향

강희규,김동훈,한성원,김묘경,권혁찬,이호준,김문규,Kang, Hee-Gyoo,Kim, Dong-Hoon,Han, Sung-Won,Kim, Myo-Kyung,Kwon, Hyuck-Chan,Lee, Ho-Joon,Kim, Moon-Kyoo 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.4

Objective: To investigate the effects of ROS on kinematic parameters in human spermatozoa. To verify the changes in above parameters, human spermatozoa were incubated with xanthine-xanthine oxidase (X-XO), $H_2O_2$, sodium nitroprusside (SNP) or lymphocyte. Otherwise, spemlatozoa were incubated under low $O_2 (5%) condition. Methods: CASA was employed to analyze sperm motion parameters. Results: Under $H_2O_2 treatment, all kinematic parameters of spermatozoa were dramatically increased during 30 min, but gradually decreased thereafter. Under the low concentration of $H_2O_2 (125 ${\mu}M$ and 250 ${\mu}M$), the movement velocity (VAP, VCL, VSL) decreased, but forward movement increased. Under the 1mM $H_2O_2, sperm showed reduced kinematic parameters except during first 30 min of incubation. In the cases of X-XO and SNP treatment, the movement velocity increased but the forward movement reduced. After incubation for 3 hr treatment, the kinematic parameters gradually decreased in high concentration of X-XO. However these parameters maintained or increased in low concentration of X-XO. There was no obvious changes in the above parameters in the high concentration of SNP. In the presence of high concentration of lymphocytes, all parameters decreased. Under the 5% $O_2 condition, the parameters of the movement velocity and movement pattern increased, but forward movement decreased. Taken togethers, it suggested that ROS increased the movement velocity but decreased the forward movement and lateral head replacement. $H_2O_2, X-XO, SNP and lymphocyte treatment significantly increased capacitated spermatozoa within I h of incubation. There was no significant difference in capacitation between low- and high $O_2 group. Conclusion: The early onset of capacitation in the presence of ROS suggest that ROS might be a positive regulator of sperm capacitation and hyperactivation. These results demonstrate that low concentration ROS facilitates the movement velocity but high concentration ROS was inhibitory.

A Study on the Decondensation and Pronucleus Formation of Sperm Nucleus in the Mouse Oocyte

강희규,김태전,배형준,문희주,이호준,양혜영,김문규 대한의생명과학회 2001 Biomedical Science Letters Vol.7 No.4

To investigate the ability to decondense sperm head penetrated into cytoplasm of the oocytes and the relationship between this ability and the level of glutatione (GSH) in mouse oocyte at various maturing stages. The fertilizability of oocytes at various stages of maturation, the decondensation of sperm nucleus and the formation of male pronucleus, were observed and the levels of GSH were measured in oocyte at same stages. Besides, the relation between fertilizability and level of GSH in oocyte cytoplasm treated with L-buthionine-S, R-sulfoxmine (L-BSO), the inbitor of biosynthesis of GSH, was determined. The decondensation of sperm head was not found in GV stage and L-BSO treated oocytes. In maturing oocytes (GVBD, MI), the decondensation was found, but the formation of male pronucleus was not. The levels of GSH in oocyte cytoplasm were measured; 2.2 pmol per oocyte in the ovulated and the matured in vitro each, 1.0 pmol in GV intact oocyte, 1.3 pmol in GVBD, and 1.5 pmol in MI phase oocyte. In L-BSO treated oocytes the levels of GSH were measured 0.08∼0.09 pmol per oocyte, slightly lower than GV stage oocyte. In conclusion, GSH in oocyte is supposed to be synthesized and storaged in cytoplasm during maturation. The failure of decondensation in the cytoplasm of GV stage and L-BSO treated is suggested that GSH is an essential factor in decondensing the sperm head and that the a certain level of GSH, more than in GV oocyte cytoplasm, is required in de condensation.

중증 뇌동맥류 파열 환자의 조기수술 예후

강희규,문병관,이승진,김주승 凡石學術奬學財團 2000 凡石學術論文集 Vol.4 No.-

반응성 산소족이 사람 정자의 수정능력 획득과 첨체반응에 미치는 영향

강희규,김동훈,한성원,김묘경,권혁찬,이호준,윤용달,김문규 한국발생생물학회 2000 발생과 생식 Vol.4 No.2

본 연구에서는 반응성 산소족이 수정능력획득, 칩체반응에 미치는 영향을 알아보고자 반응성 산소족으로 superoxide anion은 xanthine (X) -xanthine oxidase (XO) system을, hydroperoxide는 $H_2O$$_2$를 농도별로 처 리하였으며, nitric oxide (NO)는 NO donor인 sodium nitroprusside (SNP)를 처리하였다 또한 남성불임요인의 하나로 알려진 leukocytospermia에 대한 영향을 알아보기 위해 lymphocyte를 농도별로 처리하였고, 일반적인 배양기내 산소농도인 20% $O_2$농도를 생체내 농도와 유사한 5% $O_2$ 농도로 낮추었을 때 의 결과를 알아보고자 하였다. 수정능력 획득 정도와 첨체반응률을 알아보기 위해 chlortetracycline (CTC) 염색방법을 이용하였다. 지질과산화 정도는 정자내 malondialdehyde (MDA)의 생성량을 흡광기를 이용하여 정량하였다. $H_2O$$_2$, X-XO, SNP와 lymphocyte 처리군은 1시간 배양시에 수정능력획득률이 유의하게 증가하였으나, 저산소처리군에서는 차이가 없었다. 저 농도의 $H_2O$$_2$를 처리한 경우에는 지질과산화 정도가 감소하였으나, 고 농도에서는 대조군에 비해 유의하게 증가하였다. 고 농도의 Iymphocyte를 처리한 경우에는 1시간 처리시에 지질과산화가 유의하게 증가하였으나, 처리된 산소농도에 따른 지질과산화의 차이는 없었다. 첨체반응률의 경우, 처리한 모든 반응성 산소족에서 대조군에 비해 높은 첨체반응률을 확인하였다. X(100 $\mu$M)-XO(100mIU)의 경우가 가장 높은 첨체반응률을 나타내었다. 이러한 결과들은 반응성 산소족이 수정능력획득, 지질과산화 그리고 첨체반응에 영향을 미치는 것을 확인하여 주었다. 또한 반응성 산소족이 생성된 경우에 수정능력획득이 보다 빠르게 진행되어지는 것은 반응성 산소족이 정자의 과운동성과 수정능력획득의 중요한 조절자임을 시사한다고 사료된다. To investigate the effects of reactive oxygen species (ROS) on capacitation, acrosome reaction in human spermatozoa. Human spermatozoa were incubated with xanthine-xanthine oxidase (X-XO), $H_2O$$_2$, sodium nitroprusside (SNP) or lymphocyte. Otherwise, spermatozoa were incubated under low $O_2$ (5 %) condition. Chlortetracycline (CTC) staining was conducted to assess capacitation and acrosome reaction. Analysis of lipid peroxidation was done by spectrophotometric determination of malondialdehyde (MDA) production in spermatozoa. $H_2O$$_2$, X-XO, SNP and lymphocyte treatment significantly increased capacitated spermatozoa within 1 h of incubation. There was no significant difference in capacitation between low- and high $O_2$ groups. In the presence of low concentration of $H_2O$$_2$, lipid peroxidation decreased significantly. However, under the high concentration of $H_2O$$_2$, lipid peroxidation significantly increased at the end of incubation compared to control. In the presence of high concentration of lymphocytes, lipid peroxidation significantly increased compared to control at 1hr of incubation. There was no significant difference in lipid peroxidation according to $O_2$ concentration examined. Acrosome reaction (AR) was evaluated by CTC staining after the progesterone challenge. In all ROS groups, AR increased compared to control. The X(100 $\mu$M) - XO (100mIU) system was the most potent to induce AR. Taken together, it suggested positive control of AR by ROS and the positive relationship between the lipid peroxidation and AR. The early onset of capacitation in the presence of ROS suggest that ROS might be a positive regulator of sperm capacitation and hyperactivation.

일반연제 발표 : 불임 및 생식내분비 ; 체외수정시술시 채취된 난자의 성숙도와 배 발달속도가 임신에 미치는 영향

강희규,김동훈,이명섭,김묘경,한성원,박원일,정지학,연혜정,이호준,홍서유 대한산부인과학회 1999 대한산부인과학회 학술대회 Vol.84 No.-

Alteration of Spindle Formation and Chromosome Alignment in Post-Ovulatory Aging of Mouse Oocytes

강희규,차병헌,전진현 한국발생생물학회 2011 발생과 생식 Vol.15 No.3

The objective of this study was to elucidate the dynamics of microtubules in post-ovulatory aging in vivo and in vitro of mouse oocytes. The fresh ovulated oocytes were obtained from oviducts of superovulated female ICR mice at 16 hours after hCG injection. The post-ovulatory aged oocytes were collected at 24 and 48 hours after hCG injection from in vivo and in vitro, respectively. Immunocytochemistry was performed on β-tubulin and acetylated α-tubulin. The microtubules were localized in the spindle assembly, which was barrel-shaped or slightly pointed at its poles and located peripherally in the fresh ovulated oocytes. The frequency of misaligned metaphase chromosomes were significantly increased in post-ovulatory aged oocytes after 48 hours of hCG injection. The spindle length and width of post-ovulatory aged oocytes were significantly different from those of fresh ovulated oocytes, respectively. The staining intensity of acetylated α-tubulin showed stronger in post-ovulatory aged oocytes than that in the fresh ovulated oocytes. In the aged oocytes, the spindles had moved towards the center of the oocytes from their original peripheral position and elongated, compared with the fresh ovulated oocytes. Microtubule organizing centers were formed and observed in the cytoplasm of the aged oocytes. On the contrary, it was not observed in the fresh ovulated oocytes. The alteration of spindle formation and chromosomes alignment substantiates the poor development and the increase of disorders from the post-ovulatory aged oocytes. It might be important to fertilize on time in ovulated oocytes for the developmental competence of embryos with normal karyotypes

Molecular Analysis of Uncultured Chorionic Villi Cells and Amniocytes by Fluorescence in Situ Hybridization

강희규,심효남,심재선,차영범,이승재,박종민 대한산부인과학회 1995 대한산부인과학회 학술대회 Vol.76 No.-

Efficacy of Intracytoplasmic Sperm Injection for Leukocytospermia

강희규,--,--,--,--,--,--,--,-- THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 2004 Journal of biomedical laboratory sciences Vol.10 No.1

White blood cells (WBCs) are present in most human ejaculates, but abnormally high concentration of seminal leukocytes map reflect an underlying pathological condition. The World Health Organization (WHO) has defined leukocytospermia as status of more than 10^(6) WBC/mL of semen. The purpose of this study was firstly, to compare the outcomes between conventional IVF and ICSl in leukocytospermia, and secondly, to investigate whether ICSI may be an alternation treatment for patients with leukocytospermia. Total 346 cycles of conventional IVF and ICSI candidates underwent IVF cycles at Eulji Hospital Infertility Clinic. Semen Parameters including concentration, motility, morphology of spermatozoa and concentration of leukocytes were assessed from the raw ejaculates. There was no difference in sperm concentration, motility and morphology. The rates of fertilization and good embryo development from ICSI were significantly higher than those from conventional IVF in leukocytospermia (60.4% & 32.5% respectively for ICSI group and 44.4% & 28.5%, respectively for IVF group, P<0.001). The pregnancy rate after ICSI was also higher than that from conventional IVF (34.0% vs 29.1%. P<0.05). These results indicate that the presence of seminal leukocyes (>1×10^(6) WBC/mL of semen) is adversely related with fertilization, embryo development and pregnancy rate. Therfore the measurement of seminal leukocytes in routine semen analysis appears to be of prognostic value with regard to male fertilizing potential. In conclusion. it is suggested that ICSI is an alternative choice of treatment for patients with leukocytospermia.

반응성 산소족이 생쥐 정자의 첨제반응과 지질 과산화 및 수정에 미치는 영향 : (1) Superoxide Anion과 Hydroxideyl Radical의 영향

강희규,김묘경,김동훈,한성원,이호준,김성례,김문규 한국발생생물학회 1999 발생과 생식 Vol.3 No.2

반응성 산소족이 생쥐정자의 세포막을 지질과 산화시키고 첨체반응 동의 일련의 수정과정에 미치는 영향과 반응성 산소족의 억제제로서 superoxide dismutase (SOD)를 처리하였을 때 위의 현상들이 개선되어지는가를 생쥐정자를 이용하여 알아보았다. 반응성 산소족을 생성하기 위하여 Fe^(2+)/Ascorbic acid (Fe^(2+)/Asc)와 xanthine-xanthine oxidase (X-XO)계의 반응을 이용하였다. 반응성 산소족올 처리한 군들에서는 총 운동성이 180분까지 대조군보다 감소하였으나, SOD 처리하였을 때는 통계적으로 유의하게 운동성이 개선되었다 (p<0.05). 첨체반응은 농도 의존적으로 모든 처리군에서 대조군보다 유의하게 감소하였다. 정자세포막의 지질 과산화 정도는 모든 처리군에서 대조군에 비해 증가하였으며, 수정율은 반응성 산소족 처리군에서 낮게 나타났다. 이상의 결과들을 종합해 보면, 반응성 산소족은 정자의 세포막을 지질 과산화 시킴으로 정자와 난자간의 수정을 위한 일련의 과정에 해로운 영향을 끼치는 것으로 사료된다. This study was to investigate whether reactive oxygen species (ROS) could trigger lipid peroxidation of spermatozoa and inhibit the acrosome reaction and whether superoxide dismutase (SOD; ROS scavenger) could inhibit lipid peroxidation and enhance acrosome reaction of spermatozoa. Spermatozoa were treated with ROS generated by addition of Fe^(2+)/Ascorbic acid (Fe^(2+)/Asc) and xanthine-xanthine oxidase (X-XO). Fe^(2+)/Asc and X-XO without SOD groups had significantly decreased total motility from 0 minute to 180 minute, but SOD treated groups were significantly improved motility. Acrosome reaction was significantly reduced by addition of Fe^(2+)/Asc or X-XO in dose dependent manner, but SOD treated groups were increased in lower concentrations but not significant. Lipid peroxidation rates in all treated groups were higher than control. Fertilization rates in ROS treated groups were significantly tower than control. These results are consistent with a causative role of ROS for lipid peroxidation in the etiology of defective sperm function and also suggest a possible physiological role for the oxygen species generated by spermatozoa in mediating sperm-zona fusion.