정자 운동성에 대한 Nitric Oxide와 Peroxynitrite의 효과
Effects of Nitric Oxide and Peroxynitrite Peroxynitrite on Sperm Motility and Viability
고혁제, 정은택, 송주흥,
소속 상세정보
고혁제 ( )
원광대학교 의과대학 비뇨기과학교실
정은택 ( )
원광대학교 의과대학 비뇨기과학교실
송주흥 ( )
원광대학교 의과대학 비뇨기과학교실
KMID : 0358319980390090896
Abstract
서론
nitric oxide(NO)는 nitric oxide synthase(NOS)의 촉매작용에 의하여 L-arginine
guanidine group의 terminal nitrogen이 산화될 때 cirtrulline과 동량으로 형성되어지는 유리
기 분자로서, 생체에서 발생해 쉽게 분해되는 가스성 물질이다. NO의 기능은 혈관내피세포
에서 유리된 후 혈관근육세포에 작용하여 혈관을 이완시키거나, 신경계에서 신경전달물질로
서 작용하기도 하며 그 외 평활근을 이완시키고 혈소판접착 및 응집을 방해하거나 염증 및
자가면역반응에 의한 조직손상에도 관여한다. 최근에는 NOS가 쥐의 고환과 부고환을 포함
해 여러 다른 세포들에서 발현되어진다고 밝혀져 NO의 역할이 매우 다양하다는 것을 암시
하고 있다.
superoxide 음이온(O2 )이 포유류에서 세포내 산소소모의 결
과로 생성된다는 사실은 잘 알려져 있다. 이 라디칼은 세포막의 인지질에 작용하여 지질과
산화성 손상을 일으키는데 정자는 지질과산화에 특히 예민하고 만약
(O2 ) 의 생성이 증가되면 정자세포막의 과산화성손상으로 인
해 정자의 기능저하가 초래될 수 있다.
최근에 알려진 바에 의하면 NO와 (O2 )의 상호작용에 의해
발생되는 강력한 산화성 물질의 음이온인 Peroxynitrite(ONOO-)도 대식세포
와 다른 세포계에서 세포막인지질에 지질과 산화성 손상을 야기하는 것으로 알려졌다.
이에 저자는 생체에서 생성되어지는 NO가 인간의 생식과 관련된 정자의 운동성과 생존
성에 영향을 미치고 있을 것으로 추론하고, 인간의 정자에 NO 공여물질인 sodium
nitroprusside(SNP)를 처리한 다음 정자 운동성 및 생존성에 어떠한 역할을 하는지를 알아
보고 그 기전에 Peroxynitrite의 관련성과 역할을 알아보고자 본 연구를 수행하였다.
#초록#
Purpose: Recently, in assisted reproductive technologies(ART) programs theme Is an
increasing Interest in the use of agents for the enhancement of sperm motility for
assisted fertilization. In an attempt to improve the motility at the cryopreserved human
semen and hence the fertilizing capacity of asthenospermic semen samples, different
semen preparation techniques have been attempted and the effects of chemical stimulants
as nitric oxide(NO) have been studied extensively.
Superoxide anions cause lipid peroxidative damage to cell membrane phospholipids, and
sperm are known to be particularly susceptible to lipid peroxidation. Such sperm with
damaged membranes are impaired functionally. Recently, peroxynitrite, an anion and a
potent oxidant, generated by the interaction of nitric oxide and superoxide anions has
been demonstrated In macrophages and other cellular systems. Also this anion cause
lipid peroxidative damage to cell membrane phospholiplds. We therefore Investigated
whether NO and peroxynitrite have the roles to modulate sperm motility and to affect
Its viability.
Materials and Methods: Normal human semen samples(as per World health
Organization (WHO) criteria) were obtained after 3day period of abstinence by donors.
The samples(n=5) were incubated with either sodium nitroprusside(SNP; 0.1, 05, 1 or
2mM) or peroxynitrite (10, 50 or 100μM) and the percent viability and motility were
assessed at various time inteval up to 4hr. The human semen samples were treated
with N-acetyl-L-cystein(NAC;10mM), SNP (0.5mM), phorbol myristate acetate(PMA;
100nM), of SNF plus PMA. Both superoxide and peroxynitrite release were measured
directly by chemiluminometer. Percent viability and motility were assessed at 4hr of
Incubation. A sample of each aliquot was placed in a Mauler chamber for
videomicrography Percent motility were analyzed by using the sperm analysis imaging
system. The sperm vlability was assessed by flow cytometer using LIVE/DEAD sperm
viability kIt. The production of superoxide and peroxynitrite were measured by the
method of chemiluminescence assay.
Result : All results represent a mean±SEM, n=5. Treatment of human semen
samples for 4hr with SNP, a NO generating agent, significantly decreased sperm
motility and viability in high concentration 〔relative motility(% of control); 38±4 and
30±5, relative viability; 42±4 and 30±3 by 1 and 2mM of SNP]. In the presence of low
concentration SNP(0.5mM), the sperm viability was not significantly affected(82±3),
whereas the sperm motility was affected(64 2). SNP(0.5mM) also decreased sperm
motility(80±2 at 2hr 64±3 at 4hr, 44±3 at 6hr, and 38±4 at 8hr) in a time dependent
manner. Since it was demonstrated that superoxide anions are one of the common
source of lipid peroxidation, we investigated whether superoxide anions produced by
human semens could Interact wlth NO to generate peroxynitrite. Adding
N-acetyl-L-cystein(NAC) to the human semen samples partially blocked spontaneous
release of superoxide, whereas PMA augmented the release of superoxide from human
semen samples (control:0.9 106 0.3, NAC: 0.5 106±0.4, and
PMA: 2.5 106±0.4photons/60min). The production of superoxide was
corresponded with the production of peroxynltrite(control: 1.0 104CPM,
SNP: 3.8 106CPM, SNP plus PMA. 12×106CPM). In
addition, SNP in combination with PMA(65±3) markedly decreased sperm motility than
that of SNP alone(77±2.5) at 4hr, implying that nitric oxide might inhibit sperm motility
via the formation of peroxynitrite In human semen samples. Exogenous peroxynitrite
also decreased sperm motility in a dose dependent manner(10μM: 64±2, 50rM: 53±3,
and 1 0μM of peroxynitrite: 23 4).
Conclusions: These results suggest that NO inhibits sperm motility via the formation
of peroxynltrite and further demonstrate that NO-induced inhibition of sperm motility is
depended on the production of superoxide from human semens because peroxynitrite is
generated by the interaction of NO and superoxide.
키워드
Sperm motility and viability; Nitric oxide; Superoxide anions; Peroxynitrite;
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