Chlortetracycline Fluoresence 분석을 통한 수정능 획득 과정에서의 $Ca^{2+}$-ATPase 역할

박경식,Park, Kyoung-Sik 대한생식의학회 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.3

It has been reported that the $Ca^{2+}$-ATPase and the $Ca^{2+}-Na^+$ exchanger play an important role for the regulation of intracellular $Ca^{2+}$ in somatic cells, the $Ca^{2+}$-ATPase located in the plasma membrane helps the $Ca^{2+}$ concentration in maintain low $[Ca^{2+}]_i$. Roldan & Fleming reported that the spermatozoan $Ca^{2+}$-ATPase plays an important role in the capacitation and acrosome reaction. We used to assess $Ca^{2+}$ changes by chlortetracycline (CTC) patterns in the capacitation and acrosome reaction of human and hamster spermatozoa. In the present study applying quercetin which has been known as an ATPase antagonist, the enzymatic effect of $Ca^{2+}$-ATPase on capacitation and acrosome reaction was found to be remarkable: a significant increase of the transformation from the original type to the B type and the AR type of spermatozoa. This finding suggests that $Ca^{2+}$-ATPase play an important role in the efflux and the influx of the $Ca^{2+}$ which have been known to be an essential factor for the capacitation and acrosome reaction, and that the inhibitory action of the $Ca^{2+}$-ATPase might be a prerequsit step toward the capacitation and acrosome reaction. In conclusion, this study suggest the considerable evidence as follows: the increment of the intracellular $Ca^{2+}$ concentration occurred by controlling the slope of $Ca^{2+}$ concentration through $Ca^{2+}$-ATPase activites in both the intracellular and extracellulr fluid may be important procedures for the capacitation and the acrosome reaction, and finally for fertilization of the sperm and ovum.

T-형 $Ca^{2+}$ 채널 길항제인 Mibefradil을 첨가한 인간 정자의 첨체반응 관찰

이재호,손원영,이정하,이인선,김영찬,한징택,Lee, Jae-Ho,Son, Weon-Young,Lee, Jung-Ha,Lee, In-Sun,Kim, Young-Chan,Han, Ching-Tack 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.1

Objective: The sperm acrosome reaction is a $Ca^{2+}$-dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies suggested a role of $Ca^{2+}$ channels in acrosome reactions. This study was conducted to investigate the T-type calcium channel is operated in acrosome reaction of human spermatozoa. Method: Human semen samples were obtained from healthy donors with normal criteria. The spermatozoa were divided into five groups: Group 1 were non-treated as a control; Group 2 where spermatozoa were exposed to 5 ${\mu}M$ $Ca^{2+}$ A23187 $(Ca^{2+}i)$; Group 3 where spermatozoa were exposed 5 ${\mu}M$ $Ca^{2+}i$ and mibefradil; Group 4 where spermatozoa were exposed 5 ${\mu}M$ $Ca^{2+}i$ and nifedipine, and Group 5 where spermatozoa were treated with 5 ${\mu}M$ $Ca^{2+}i$ and both of mibefradil and nifedipine. Spermatozoa in all groups were retrieved after incubation for 15 and 30 minutes at $37^{\circ}C$. After staining with PSA-FITC, fluorescence was observed under a fluorescence microscope, and AR was evaluated on a total>100 spermatozoa/side. Result and Conclusion: We observed on acrosome reaction inhibition rate in human spermatozoa the various of concentration of mibefradil, nifedipine. Maximum response was noted with 1.0 ${\mu}M$ mibefradil and the decrease of acrosome reaction inhibition rate 45%. Nifedipine in acrosome reaction inhibition rate was only about 25%. The $Ca^{2+}i$-induced AR of spermatozoa was significantly suppressed by mibefradil. Incidence of the suppression was depending on concentration of mibefradil. Results from the present study suggest that the human spermatozoa possess T-type channel. The observation that reversible inhibitor of T channels in male germ cells provides a new mechanism of contraceptive action.

Effect of Extracellular Ca<SUP>2+</SUP> and Ca<SUP>2+</SUP>-ATPase on the Acrosome Reaction of Spermatozoa

Yung-Keun Oh(오영근),Jae-Ho Chang(장재호),In-Ho Choi(최인호),Noh-Pal Jung(정노팔),Hyung-Cheul Shin(신형철),Byoung-Ju Kwak(곽병주) 대한의생명과학회 1998 Biomedical Science Letters Vol.4 No.1

세포내, 외 Ca²? 농도구배 유지에는 Ca²?ATPase와 Ca²?-Na? exachangers가 주요한 기능을 한다고 알려져 있는데 특히 Ca²?-ATPase의 기능에 대해 많은 연구가 행해지고 있다. Ca²?-ATPase는 체세포에서 세포막에 위치하고 있으며 Ca²?을 세포외부로 배출하는 기능을 함으로써 세포내부의 Ca²?농도를 낮게 유지할 수 있도록 하는 기능을 담당하고 있다. 이러한 Ca²?-ATPase는 포유동물의 정자에도 존재하고 있지만 그 기능에 대해서는 아직 많은 설명이 되어있지 않다. 본 연구에서 정자가 수정을 하기위한 기능적인 능력이 Ca²? 농도와 관련된 변화와 얼마나 연관되어 있는가를 규명하고, 이러한 Ca²?농도 조절이 원형질막의 중요인자인 Ca²?-ATPase와는 어떠한 연관성이 있는가를 알기위해 시도한 결과, Ca²?-ATPase는 세포내, 외 Ca²?의 농도구배를 조절함으로써 세포내 Ca²?의 농도를 증가시켜 정자가 수정능 획득과정으로 빨리 전환하도록 유도하고 첨체반응에 중요한 역할을 하는 것으로 판단되며, 세포외 Ca²?농도가 높게 유지될 경우에도 정자의 첨체반응이 유도됨으로써 난자와 용이하게 수정을 할 수 있는 생리적 환경이 제공될수 있다고 사료된다. This study has been designed in order to examine a physiological role of Ca²? which has been known as an essential factor for capacitation, to confirm whether the enzyme activity of Ca²?-ATPase on capacitation is important or not, and to clarify relationship between various levels of the Ca²? concentration and Ca²?-ATPase which has been known to be an important factor of the plasma membranes. In the present study applying quercetin, a Ca²?-ATPase inhibitor, the enzymatic effect of Ca²?-ATPase on capacitation was found to be remarkable: a significant increase of the transition from the original type (type A) to the type B and the type AR of the spermatozoa. This finding suggests that Ca²?-ATPase plays an important role in the efflux and the influx of the Ca²? which has been known to be an essential factor the capacitation and acrosome reaction, and that the inhibitory action of the Ca²?-ATPase might be a prerequsite step toward the acrosome reaction. The conclusion reached can be deduced as follows: increment of the intracelluar Ca²? concentration occurred by controlling the slope of Ca²? concentration through Ca²?-ATPase activities in both the intra- and extracelluar fluid may be an important procedure for capacitation and acrosome reaction, and ultimately for fertilization of the spermatozoa and the ova.

Effect of Extracellular Ca²+ and Ca²+-ATPase on the Acrosome Reaction of Spermatozoa

Kwak,Byoung-Ju,Shin,Hyung-Cheul,Chang,Jae-Ho,Jung,Noh-Pal,Choi,In-Ho,Oh,Yung-Keun THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1998 Journal of biomedical laboratory sciences Vol.4 No.1

세포내, 외 Ca²+ 농도구배 유지에는 Ca²+-ATPase와 Ca²+-Na+ exachangers가 주요한 기능을 한다고 알려져 있는데 특히 Ca²+-ATPase의 기능에 대해 많은 연구가 행해지고 있다. Ca²+-ATPase는 체세포에서 세포막에 위치하고 있으며 Ca²+을 세포외부로 배출하는 기능을 함으로써 세포내부의 Ca²+농도를 낮게 유지할 수 있도록 하는 기능을 담당하고 있다. 이러한 Ca²+-ATPase는 포유동물의 정자에도 존재하고 있지만 그 기능에 대해서는 아직 많은 설명이 되어있지 않다. 본 연구에서 정자가 수정을 하기위한 기능적인 능력이 Ca²+농도와 관련된 변화와 얼마나 연관되어 있는가를 규명하고, 이러한 Ca²+농도 조절이 원형질막의 중요인자인 Ca²+-ATPase와는 어떠한 연관성이 있는가를 알기위해 시도한 결과, Ca²+-ATPase는 세포내, 외 Ca²+의 농도구배를 조절함으로써 세포내 Ca²+의 농도를 증가시켜 정자가 수정능 획득과정으로 빨리 전환하도록 유도하고 첨체반응에 중요한 역할을 하는 것으로 판단되며, 세포외 Ca²+농도가 높게 유지될 경우에도 정자의 첨체반응이 유도됨으로써 난자와 용이하게 수정을 할 수 있는 생리적 환경이 제공될수 있다고 사료된다. This study has been designed in order to examine a physiological role of Ca²+ which has been known as an essential factor for capacitation, to confirm whether the enzyme activity of Ca²+-ATPase on capacitation is important or not, and to claarify relationship between various levels of the Ca²+ concentration and Ca²+-ATPase which has been known to be an important factor of the plasma membranes. In the present study applying quercetin, a Ca²+-ATPase inhibitor, the enzymatic effect of Ca²+-ATPase on capacitation was found to be remarkable: a significant increase of the transition from the original type (type A) to the type B and the type AR of the spematozoa. This finding suggests that Ca²+-ATPase plays an important role in the efflux and the influx of the Ca²+ which has been known to be an essential factor the capacitation and acrosome reaction, and that the inhibitory action of the Ca²+-ATPase might be a prerequsite step toward the acrosome reaction. The conclusion reached can be deduced as follows: increment of the intracelluar Ca²+ concentration occurred by controlling the slope of Ca²+ concentration through Ca²+-ATPase activities in both the intra- and extracelluar fluid may be an important procedure for capacitation and acrosome reaction, and ultimately for fertilization of the spematozoa and the ova.

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.

정자 미세주입에 의한 생쥐 난포난의 체외수정과 발달 : 미세 주입정자수의 효과 The effect of microinjected sperm numbers

조병욱,신택순,이길왕,강한석,김선구 密陽産業大學校 農業技術開發硏究所 1999 農業技術開發硏究所報 Vol.3 No.2

정자의 미세주입이 생쥐 난자의 체외수정과 배발달에 미치는 영향을 조사하기 위하여 수정능 획득 후 첨체반응시킨 정자를 1~3개씩 무작위로 난자의 위난강내 주입하였다. 난자는 PMSG 와 HCG로 과배란 시킨 생쥐의 난관에서 채취하였고 정자는 lactate와 albumin이 함유된 Ham's F-10 배양액에서 2시간 배양후 12mM 의 abc GMP와 10mM 의 imidazole 포함된 배양액에서 10분간 배양하였다. 난자의 수정율은 31.4%이었고, 2세포기 28.5%, 3~6세포기 11.9%, 8세포기 5.3%로 발달하였다. 난자에 주입된 정자수는 체외수정과 배발달에 영향을 미치지 않았다. This study was conducted to investigate the in vitro fertilization of mouse oocytes by microinjection of sperm and in vitro development of mouse embryos. Capacitated and acrosome reacted sperm were injected into the perivitelline space of a mouse oocyte. Oocytes were collected from the oviduct of the mouse treated with PMSG and HCG. Sperm were incubated for 2 hrs at 37℃ under an atmosphere of 5% CO₂in Ham's F-10 medium containing lactate and albumin for capacitation and kept for 10 minutes in culture medium containing 12mM of dbeGMP and 10mM of immidazol for acrosome reaction. Single or multiple sperm were injected into the perivitelline space of each occyte. Fertilization was recognized by presence of second polar body or two prounclei. The rat of fertilization was 31.4%. The rates of embroys developed into 2-cell, 3~6cell and 8 cell stage were 28.5%, 11.9% and 5.3% respectively. The number of sperm injected into perivitelline space of occytes did not affect in vitro fertilization of oocytes and development of embryos.

The Role of Mercury in the Etiology of Sperm Dysfunction in Holstein Bulls

Arabi, M. Asian Australasian Association of Animal Productio 2006 Animal Bioscience Vol.19 No.3

A large number of toxicological substances and pharmacological and physical agents can cause reproductive intervention at the cellular and molecular level. The present study was designed to assess the effect of mercury ($HgCl_2$) at 50 to $550{\mu}M$ concentration ranges, in vitro, on the sperm membrane and DNA integrity, viability, and acrosomal status of normal bull spermatozoa. The samples were processed for sperm analyses using semen-diluting fluid (PBS, pH 7.2). We recorded a sharp increase in the lipid peroxidation/LPO rate; the highest was at $550{\mu}M$ mercury concentration, indicating a deleterious effect of mercury on the sperm membrane intactness. There was also a strong negative correlation between LPO rate and % viable spermatozoa (R = 0.987, p<0.001). Data obtained from a comet assay technique revealed that mercury is capable of inducing DNA breaks in the sperm nuclei. Interestingly, 92% of DNA breaks were double-stranded. The correlation between LPO rate and % DNA breaks was 0.984. Performing the gelatin test indicates that mercury is able to alter the integrity of acrosomal membranes showing an abnormal acrosome reaction. In this regard, a strong link was found between LPO rate and % halos (R = 0.990, p<0.001). Collectively, mercury proved to be a potent oxidant in the category of environmental factors affecting bull spermatozoa. Hence, considering the wide spread use of mercury and its compounds, these metals should be regarded with more concern.

RESEARCH : Capacitation and acrosome reaction differences of bovine, mouse and porcine spermatozoa in responsiveness to estrogenic compounds

( Do Yeal Ryu ),( Ye Ji Kim ),( June Sub Lee ),( Md Saidur Rahman ),( Woo Sung Kwon ),( Sung Jae Yoon ),( Myung Geol Pang ) 한국동물자원과학회(구 한국축산학회) 2014 한국축산학회지 Vol.56 No.26

Background: Endocrine disruptors are exogenous substance, interfere with the endocrine system, and disrupt hormonal functions. However, the effect of endocrine disruptors in different species has not yet been elucidated. Therefore, we investigated the possible effects of 17ß-estradiol (E2), progesterone (P4), genistein (GEN) and 4-tert-octylphenol (OP), on capacitation and the acrosome reaction in bovine, mouse, and porcine spermatozoa. In this in vitro trial, spermatozoa were incubated with 0.001-100 μM of each chemical either 15 or 30 min and then assessed capacitation status using chlortetracycline staining. Results: E2 significantly increased capacitation and the acrosome reaction after 30 min, while the acrosome reaction after 15 min incubation in mouse spermatozoa. Simultaneously, capacitation and the acrosome reaction were induced after 15 and 30 min incubation in porcine spermatozoa, respectively. Capacitation was increased in porcine spermatozoa after 15 min incubation at the lowest concentration, while the acrosome reaction was increased in mouse spermatozoa after 30 min (P <0.05). E2 significantly increased the acrosome reaction in porcine spermatozoa, but only at the highest concentration examined (P <0.05). P4 significantly increased the acrosome reaction in bovine and mouse spermatozoa treated for 15 min (P <0.05). The same treatment significantly increased capacitation in porcine spermatozoa (P <0.05). P4 significantly increased capacitation in mouse spermatozoa treated for 30 min (P <0.05). GEN significantly increased the acrosome reaction in porcine spermatozoa treated for 15 and 30 min and in mouse spermatozoa treated for 30 min (P <0.05). OP significantly increased the acrosome reaction in mouse spermatozoa after 15 min (P <0.05). Besides, when spermatozoa were incubated for 30 min, capacitation and the acrosome reaction were higher than 15 min incubation in E2 or GEN. Furthermore, the responsiveness of bovine, mouse and porcine spermatozoa to each chemical differed. Conclusions: In conclusion, all chemicals studied effectively increased capacitation and the acrosome reaction in bovine, mouse, and porcine spermatozoa. Also we found that both E2 and P4 were more potent than environmental estrogens in altering sperm function. Porcine and mouse spermatozoa were more responsive than bovine spermatozoa.

Capacitation and acrosome reaction differences of bovine, mouse and porcine spermatozoa in responsiveness to estrogenic compounds

Ryu, Do-Yeal,Kim, Ye-Ji,Lee, June-Sub,Rahman, Md. Saidur,Kwon, Woo-Sung,Yoon, Sung-Jae,Pang, Myung-Geol Korean Society of Animal Sciences and Technology 2014 한국축산학회지 Vol.56 No.7

Background: Endocrine disruptors are exogenous substance, interfere with the endocrine system, and disrupt hormonal functions. However, the effect of endocrine disruptors in different species has not yet been elucidated. Therefore, we investigated the possible effects of $17{\beta}$-estradiol (E2), progesterone (P4), genistein (GEN) and 4-tert-octylphenol (OP), on capacitation and the acrosome reaction in bovine, mouse, and porcine spermatozoa. In this in vitro trial, spermatozoa were incubated with $0.001-100{\mu}M$ of each chemical either 15 or 30 min and then assessed capacitation status using chlortetracycline staining. Results: E2 significantly increased capacitation and the acrosome reaction after 30 min, while the acrosome reaction after 15 min incubation in mouse spermatozoa. Simultaneously, capacitation and the acrosome reaction were induced after 15 and 30 min incubation in porcine spermatozoa, respectively. Capacitation was increased in porcine spermatozoa after 15 min incubation at the lowest concentration, while the acrosome reaction was increased in mouse spermatozoa after 30 min (P < 0.05). E2 significantly increased the acrosome reaction in porcine spermatozoa, but only at the highest concentration examined (P < 0.05). P4 significantly increased the acrosome reaction in bovine and mouse spermatozoa treated for 15 min (P < 0.05). The same treatment significantly increased capacitation in porcine spermatozoa (P < 0.05). P4 significantly increased capacitation in mouse spermatozoa treated for 30 min (P < 0.05). GEN significantly increased the acrosome reaction in porcine spermatozoa treated for 15 and 30 min and in mouse spermatozoa treated for 30 min (P < 0.05). OP significantly increased the acrosome reaction in mouse spermatozoa after 15 min (P < 0.05). Besides, when spermatozoa were incubated for 30 min, capacitation and the acrosome reaction were higher than 15 min incubation in E2 or GEN. Furthermore, the responsiveness of bovine, mouse and porcine spermatozoa to each chemical differed. Conclusions: In conclusion, all chemicals studied effectively increased capacitation and the acrosome reaction in bovine, mouse, and porcine spermatozoa. Also we found that both E2 and P4 were more potent than environmental estrogens in altering sperm function. Porcine and mouse spermatozoa were more responsive than bovine spermatozoa.

Effect of bicarbonate and progesterone on plasma membrane integrity, acrosome reaction and proportion of fatty acids in boar sperm

박춘근,이상희 사단법인 한국동물생명공학회 2022 Journal of Animal Reproduction and Biotechnology Vol.37 No.3

This study investigated the influence of sodium bicarbonate (NaHCO3) and progesterone on acrosome reaction and proportion of polyunsaturated fatty acid (PUFA) composition boar sperm. The sperm were diluted with semen extender and incubated with NaHCO3 and progesterone at 38℃, 5% CO2 for 6 h. Plasma membrane integrity and acrosome reaction were analyzed using SYBR14/propidium iodide (PI) and FITC-PNA/PI doubling staining method, and proportion of PUFA was analyzed using gas chromatography. In results, Plasma membrane integrity was significantly decreased in 50 mM NaHCO3 group and acrosome reaction was significantly increased by over the 100 mM NaHCO3 group compared to control group (p < 0.05). In addition, progesterone significantly increased decreased plasma membrane integrity at 100 mM progesterone and acrosome reaction at over the 5.0 µM progesterone (p < 0.05), but there was no difference among the 5.0 to 100 µM groups. PUFAs were significantly decreased in 100 mM NaHCO3 and 50 µM progesterone treatments compared to control group. In summary NaHCO3 and progesterone induce acrosome reaction and reduce PUFA composition in boar sperm, therefore, the results maybe help to understand basically knowledge for the acrosome reaction and PUFA composition in boar sperm.