S型氯代甘油醇抗雄性大鼠生育作用研究
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摘要
氯代甘油醇,(α-chlorohydrin,ACH),化学名3-氯-1,2-丙二醇(3-mono-chloro-propane-1,2-diol,3-MCPD),ACH是一种重要化工原料,普通人群可通过食用受污染的食物或以树脂生产的饮水暴露ACH。ACH被认为是经典的睾丸后毒物,短期低剂量经口染毒ACH可致多种雄性动物可逆性的不育。由于ACH抗雄性生育力作用环节及机制尚未阐明,故本研究以探察S-ACH抗雄性大鼠生育作用特点为切入点,探讨S-ACH的抗生育作用环节及可能的相关机制。
第一部分S型氯代甘油醇对雄性SD大鼠生殖能力的影响
成年雄性SD大鼠40只,随机分为4组,每组10只,对照组施以等体积双蒸水,实验组分别经口施予2.5,5.0,10.0mg/(kg·bw)S-ACH。持续52天染毒,染毒第10天起按雄:雌1:1合笼,连续合笼1周未发现交配则增添一只雌鼠合笼。交配成功后雌、雄鼠分开饲养,于交配后12-15天开腹检查雌鼠受孕状况。停止染毒14天后每组取5只雄鼠,按上述方法进行交配试验2周。分别计算雄鼠交配指数和生育力指数。染毒52天后检查睾丸生精能力和肝、肾、睾丸及附睾组织病理学变化,并观察停止染毒4周后各项受损指标的恢复情况。结果显示,染毒10天后各剂量组雄鼠生育力指数均为0,交配指数与对照组相比无显著差异(P＞0.05)。停止染毒14天后,2.5mg/kg剂量组雄鼠生育力指数明显恢复,在5mg/kg和10mg/kg剂量组则部分恢复,交配指数仍未受影响。各染毒剂量组的睾丸生精能力与对照无显著差异,肝、肾、睾丸组织病理学检查均未见异常。在10mg/kg剂量组大鼠附睾发现轻度的间质炎症反应。在5mg/kg和10mg/kg剂量组发现3例附睾精液囊肿。研究结果提示,S-ACH的作用靶点可能是发生后期的精细胞或处于成熟阶段的附睾内精子,附睾可能是S-ACH产生毒效应的靶器官。
第二部分S-型氯代甘油醇抗雄性大鼠生育的作用环节研究
为探讨S-型氯代甘油醇抗雄性大鼠生育的作用环节,本研究检测S-ACH染毒对精子运动和超活化运动的影响。1.S-ACH对大鼠精子运动的影响。雄性SD大鼠40只,随机分为4组,每组10只,采用灌胃染毒,对照组施以等体积双蒸水,实验组分别施予2.5,5.0,10.0mg/(kg·bw)S-ACH,染毒持续52天。染毒期满后每组留5只停止染毒,常规饲养4周。分别在染毒期满后及停止染毒4周后每剂量组取3只雄鼠检测精子运动。游出法收集大鼠附睾尾精子,移至支持大鼠精子获能的IVF培养液中,调整精子浓度为2×10~6个/ml,37℃,5%CO_2孵育。采用计算机辅助精子分析法(CASA)分别于孵育后的0,2,4,5h检测精子运动和4h时精子超活化状态。2.研究体外S-ACH染毒对精子受精的影响。用超排卵方法收集大鼠卵子,游出法收集雄鼠精子到IVF培养液,调整浓度为2×10~6精子/ml,平均分为4份,加入S-ACH,终浓度分别为0,0.1,1.0,10 mM。分别取含各浓度ACH的精子悬液50μl到35mm玻璃培养皿,矿物油封盖,将卵子平均分配到4份染毒精子悬液中,37℃,5%CO_2培养36h,镜下观察卵裂数,计算受精率。结果显示,S-ACH对精子运动影响与染毒剂量和孵育时间有关,S-ACH可显著抑制大鼠精子运动能力,其中VCL、VAP、VSL、ALH最为敏感,随着染毒剂量增加,运动速率下降和运动方式均发生改变。在10.0mg/(kg·bw)S-ACH精子,随着孵育时间延长精子运动显著下降。S-ACH可显著降低大鼠精子体外获能条件下超活化运动比例,其影响程度超过精子一般运动能力改变。停止染毒后运动和超活化运动均可恢复。S-ACH体外染毒后,体外受精率随剂量增大呈下降趋势。
第三部分S-型氯代甘油醇抗雄性SD大鼠生育的基础研究
为研究S-ACH抗雄性SD大鼠生育的基础,本研究检测S-ACH染毒对大鼠精子GAPDS活性、ATP和S-ACH对蛋白酪氨酸磷酸化信号转导通路的影响。雄性SD大鼠40只,灌胃染毒0,2.5,5.0,10.0mg/(kg·bw)S-ACH 52天,取5只停止染毒,常规饲养4周。分别检测染毒52天和停止染毒4周后大鼠附睾精子GAPDS活性和ATP水平。精子GAPDS活性通过检测该酶催化产物的340nm吸光度变化率计算,化学发光法进行测定ATP含量。检测获能条件下体外染毒0,0.1,1.0,10 mM S-ACH后精子酪氨酸磷酸化水平。结果显示,S-ACH可显著抑制大鼠精子GAPDS活性,GAPDS活性抑制和染毒剂量存在明显的剂量-效应关系。停止染毒后酶活性可恢复。染毒后ATP水平无显著改变。染毒期和恢复期的附睾精子cAMP水平随S-ACH染毒剂量增高附睾精子cAMP水平呈下降趋势,Pentoxifylline可增加精子cAMP水平。S-ACH体外染毒对获能条件下精子蛋白酪氨酸磷酸化无明显改变。
α-chlorohydrin,ACH,also called 3-mono-chloro-propane-1,2-diol,3-MCPD, is an important intermediate in chemical industry.Human may be exposed to it from contaminated foods and drinking water which was produced by resin.As a classic post-testicular toxicant,α-chlorohydrin can cause quick reversible infertility in males of many species at low oral dose.However,the anti-fertility mechanism of S-ACH remains unknown.In this research,the characteristics of S-ACH's anti-fertility action on male SD rats were investigated,and the possible mechanisms were explored and stages of anti-fertilization were analyzed.
PartⅠThe effect of S-ACH on fertility of male SD rats
40 adult male SD rats were randomly divided into 4 groups,each group contains 10 males,and 3 groups were administrated of S-ACH with daily oral doses of 2.5,5.0, 10.0mg/(kg·bw) for 52 days and control animals were given equal amount of distilled water.In the day 10th single males were caged with one females,and the day on which spermatozoa were found in the vaginal smear was regarded as Gestation(G_0). Another female was added when spermatozoa were not founded in the vaginal smear after one week mating.The mated females were autopsied on G_(12)-G_(15) to check pregnancy.In the 14th day after cessation of administration,5 males of each group were taken into mating tests as before.Mating indexes and male fertility indexes were calculated.After administration of S-ACH for 52 days,testicular sperm head counts was calculated from testis homogenates and histopathological changes in livers, kidneys,testes and epididymides were observed,and 4 weeks after cessation those injured endpoints were checked again.The results showed that none of the treated males could make female pregnant while their mating indexes and testicular sperm head counts did not decrease compared to the controls,and no obvious morphological injuries were observed in livers,kidneys,testes in treated rats.14 days after the cessation of administration,the male fertility indexes significantly recovered in 2.5mg/kg group,and partly regained in 5mg/kg and10mg/kg groups.At the dose of 10mg/kg,a slight inflammation was found in epididymides interstitial.3 spermatocoeles formation were observed at doses of 5mg/kg and 10mg/kg.These results suggested that the target site of S-ACH was the spermatids in thelate process of spermatogenesis or spermatozoa,and epididymis was the target organ of S-ACH.
PartⅡStudy on the stages of fertility of male SD rats witch impacted by(S)-α-chlorohydrin
To investigate the stages of rat fertility injured by S-ACH,sperm motility and hyperactivity and in vitro fertilization(IVF) were analyzed.40 adult male SD rats were randomly divided into 4 groups,each group contained 10 rats.Daily oral doses of 2.5,5.0,10.0mg/(kg·bw) S-ACH were administrated to rats for 52 days,and the controls were given the equal amount of distilled water.At the end of treatment,each group keeps 5 rats with normal feeding for another 4 weeks.3 rats' cauda epididymal sperm from dosed and recovered groups were collected by "swimming out" and then transferred to IVF medium,which supported rat sperm capacitation.The of concentration sperm suspension was adjusted to 2×10~6 cell/ml,and incubated at 37℃,5%CO_2.Then sperm motility and hyperactivity were determined by computer-assisted sperm analysis(CASA) at 0,2,4 and 5hrs,respectively.
To study the effects of S-ACH on sperm-eggs fusion,oocytes were collected by superovulation,sperm were collected by "swimming out" to IVF medium,sperm were adjusted to 2×10~6 cell/ml and evenly divided into 4 dishes,then S-ACH was added with final concentrations of 0,0.1,1.0,10 mM,respectively.50μl sperm suspensions with various S-ACH concentrations were transferred into fertilization dishes and covered with mineral oil.Oocytes were divided equally into 4 sperm suspensions of which covered with oil and incubated at 37℃,5%CO_2 for 36 hrs, and then two-cells were counted and IVF rates were calculated.The results showed that sperm motility related with the administrated dose of S-ACH and the incubated time of the sperm.S-ACH could significantly inhibited sperm motility,and VCL, VAP,VSL and ALH were sensitive to S-ACH.The rate of sperm motility and motion pattem changed with the increasing of dosage.With the extension of incubation time the sperm motility in 10mg/kg group was significantly reduced. Percentages of hyperactivity under capacitating condition were significantly reduced, which exceed to the reduction of motility.Both motility and hyperactivity recovered after withdrawal of S-ACH.The IVF rate showed an decreasing tendency with the doses of S-ACH.
PartⅢStudy on the basis of the antifertility effect of (S)-α-chlorohydrin on male SD rats
To explore the possible basis of the antifertility effect of S-ACH,GAPDS activity,ATP level and signaling pathway of protein tyrosine phosphorylation in rat sperm were determined after administration of S-ACH in vivo or in vitro.40 adult male SD rats were randomly divided into 4 groups,each group contained 10 rats. Daily oral doses of 2.5,5.0,10.0mg/(kg·bw) S-ACH were administrated to rats for 52 days,and the controls were given the equal amount of distilled water.At the end of treatment,each group kept 5 rats with normal feeding for another 4 weeks.GAPDS enzyme activity and ATP levels in the cauda epididymal sperm were determined both in dosed and recovered groups.Sperm ATP levels were assayed by luminometric method and sperm GAPDS enzyme activity was determined by detecting the changes of absorption of production concentration at 340nm.cAMP levels in sperm were measured by enzymeimmunoassay.Tyrosine phophorylation in sperm after administration of S-ACH at the concentrations of 0,0.1,1.0,10 mM respectively in vitro was detected by western blot.The results showed that S-ACH inhibited GAPDS enzyme activity with dose-dependent manner.Enzyme activity could recover after the cessation of S-ACH administration.ATP levels in different groups did not show significant difference,cAMP levels in administrating group showed a decreased tendency with dose increasing.Pentoxifylline could increase cAMP levels of sperm. Protein tyrosine phosphorylation had not interfered by the administration of S-ACH in vitro under capacitating condition.
第一部分S型氯代甘油醇对雄性SD大鼠生殖能力的影响
成年雄性SD大鼠40只,随机分为4组,每组10只,对照组施以等体积双蒸水,实验组分别经口施予2.5,5.0,10.0mg/(kg·bw)S-ACH。持续52天染毒,染毒第10天起按雄:雌1:1合笼,连续合笼1周未发现交配则增添一只雌鼠合笼。交配成功后雌、雄鼠分开饲养,于交配后12-15天开腹检查雌鼠受孕状况。停止染毒14天后每组取5只雄鼠,按上述方法进行交配试验2周。分别计算雄鼠交配指数和生育力指数。染毒52天后检查睾丸生精能力和肝、肾、睾丸及附睾组织病理学变化,并观察停止染毒4周后各项受损指标的恢复情况。结果显示,染毒10天后各剂量组雄鼠生育力指数均为0,交配指数与对照组相比无显著差异(P＞0.05)。停止染毒14天后,2.5mg/kg剂量组雄鼠生育力指数明显恢复,在5mg/kg和10mg/kg剂量组则部分恢复,交配指数仍未受影响。各染毒剂量组的睾丸生精能力与对照无显著差异,肝、肾、睾丸组织病理学检查均未见异常。在10mg/kg剂量组大鼠附睾发现轻度的间质炎症反应。在5mg/kg和10mg/kg剂量组发现3例附睾精液囊肿。研究结果提示,S-ACH的作用靶点可能是发生后期的精细胞或处于成熟阶段的附睾内精子,附睾可能是S-ACH产生毒效应的靶器官。
第二部分S-型氯代甘油醇抗雄性大鼠生育的作用环节研究
为探讨S-型氯代甘油醇抗雄性大鼠生育的作用环节,本研究检测S-ACH染毒对精子运动和超活化运动的影响。1.S-ACH对大鼠精子运动的影响。雄性SD大鼠40只,随机分为4组,每组10只,采用灌胃染毒,对照组施以等体积双蒸水,实验组分别施予2.5,5.0,10.0mg/(kg·bw)S-ACH,染毒持续52天。染毒期满后每组留5只停止染毒,常规饲养4周。分别在染毒期满后及停止染毒4周后每剂量组取3只雄鼠检测精子运动。游出法收集大鼠附睾尾精子,移至支持大鼠精子获能的IVF培养液中,调整精子浓度为2×10~6个/ml,37℃,5%CO_2孵育。采用计算机辅助精子分析法(CASA)分别于孵育后的0,2,4,5h检测精子运动和4h时精子超活化状态。2.研究体外S-ACH染毒对精子受精的影响。用超排卵方法收集大鼠卵子,游出法收集雄鼠精子到IVF培养液,调整浓度为2×10~6精子/ml,平均分为4份,加入S-ACH,终浓度分别为0,0.1,1.0,10 mM。分别取含各浓度ACH的精子悬液50μl到35mm玻璃培养皿,矿物油封盖,将卵子平均分配到4份染毒精子悬液中,37℃,5%CO_2培养36h,镜下观察卵裂数,计算受精率。结果显示,S-ACH对精子运动影响与染毒剂量和孵育时间有关,S-ACH可显著抑制大鼠精子运动能力,其中VCL、VAP、VSL、ALH最为敏感,随着染毒剂量增加,运动速率下降和运动方式均发生改变。在10.0mg/(kg·bw)S-ACH精子,随着孵育时间延长精子运动显著下降。S-ACH可显著降低大鼠精子体外获能条件下超活化运动比例,其影响程度超过精子一般运动能力改变。停止染毒后运动和超活化运动均可恢复。S-ACH体外染毒后,体外受精率随剂量增大呈下降趋势。
第三部分S-型氯代甘油醇抗雄性SD大鼠生育的基础研究
为研究S-ACH抗雄性SD大鼠生育的基础,本研究检测S-ACH染毒对大鼠精子GAPDS活性、ATP和S-ACH对蛋白酪氨酸磷酸化信号转导通路的影响。雄性SD大鼠40只,灌胃染毒0,2.5,5.0,10.0mg/(kg·bw)S-ACH 52天,取5只停止染毒,常规饲养4周。分别检测染毒52天和停止染毒4周后大鼠附睾精子GAPDS活性和ATP水平。精子GAPDS活性通过检测该酶催化产物的340nm吸光度变化率计算,化学发光法进行测定ATP含量。检测获能条件下体外染毒0,0.1,1.0,10 mM S-ACH后精子酪氨酸磷酸化水平。结果显示,S-ACH可显著抑制大鼠精子GAPDS活性,GAPDS活性抑制和染毒剂量存在明显的剂量-效应关系。停止染毒后酶活性可恢复。染毒后ATP水平无显著改变。染毒期和恢复期的附睾精子cAMP水平随S-ACH染毒剂量增高附睾精子cAMP水平呈下降趋势,Pentoxifylline可增加精子cAMP水平。S-ACH体外染毒对获能条件下精子蛋白酪氨酸磷酸化无明显改变。
α-chlorohydrin,ACH,also called 3-mono-chloro-propane-1,2-diol,3-MCPD, is an important intermediate in chemical industry.Human may be exposed to it from contaminated foods and drinking water which was produced by resin.As a classic post-testicular toxicant,α-chlorohydrin can cause quick reversible infertility in males of many species at low oral dose.However,the anti-fertility mechanism of S-ACH remains unknown.In this research,the characteristics of S-ACH's anti-fertility action on male SD rats were investigated,and the possible mechanisms were explored and stages of anti-fertilization were analyzed.
PartⅠThe effect of S-ACH on fertility of male SD rats
40 adult male SD rats were randomly divided into 4 groups,each group contains 10 males,and 3 groups were administrated of S-ACH with daily oral doses of 2.5,5.0, 10.0mg/(kg·bw) for 52 days and control animals were given equal amount of distilled water.In the day 10th single males were caged with one females,and the day on which spermatozoa were found in the vaginal smear was regarded as Gestation(G_0). Another female was added when spermatozoa were not founded in the vaginal smear after one week mating.The mated females were autopsied on G_(12)-G_(15) to check pregnancy.In the 14th day after cessation of administration,5 males of each group were taken into mating tests as before.Mating indexes and male fertility indexes were calculated.After administration of S-ACH for 52 days,testicular sperm head counts was calculated from testis homogenates and histopathological changes in livers, kidneys,testes and epididymides were observed,and 4 weeks after cessation those injured endpoints were checked again.The results showed that none of the treated males could make female pregnant while their mating indexes and testicular sperm head counts did not decrease compared to the controls,and no obvious morphological injuries were observed in livers,kidneys,testes in treated rats.14 days after the cessation of administration,the male fertility indexes significantly recovered in 2.5mg/kg group,and partly regained in 5mg/kg and10mg/kg groups.At the dose of 10mg/kg,a slight inflammation was found in epididymides interstitial.3 spermatocoeles formation were observed at doses of 5mg/kg and 10mg/kg.These results suggested that the target site of S-ACH was the spermatids in thelate process of spermatogenesis or spermatozoa,and epididymis was the target organ of S-ACH.
PartⅡStudy on the stages of fertility of male SD rats witch impacted by(S)-α-chlorohydrin
To investigate the stages of rat fertility injured by S-ACH,sperm motility and hyperactivity and in vitro fertilization(IVF) were analyzed.40 adult male SD rats were randomly divided into 4 groups,each group contained 10 rats.Daily oral doses of 2.5,5.0,10.0mg/(kg·bw) S-ACH were administrated to rats for 52 days,and the controls were given the equal amount of distilled water.At the end of treatment,each group keeps 5 rats with normal feeding for another 4 weeks.3 rats' cauda epididymal sperm from dosed and recovered groups were collected by "swimming out" and then transferred to IVF medium,which supported rat sperm capacitation.The of concentration sperm suspension was adjusted to 2×10~6 cell/ml,and incubated at 37℃,5%CO_2.Then sperm motility and hyperactivity were determined by computer-assisted sperm analysis(CASA) at 0,2,4 and 5hrs,respectively.
To study the effects of S-ACH on sperm-eggs fusion,oocytes were collected by superovulation,sperm were collected by "swimming out" to IVF medium,sperm were adjusted to 2×10~6 cell/ml and evenly divided into 4 dishes,then S-ACH was added with final concentrations of 0,0.1,1.0,10 mM,respectively.50μl sperm suspensions with various S-ACH concentrations were transferred into fertilization dishes and covered with mineral oil.Oocytes were divided equally into 4 sperm suspensions of which covered with oil and incubated at 37℃,5%CO_2 for 36 hrs, and then two-cells were counted and IVF rates were calculated.The results showed that sperm motility related with the administrated dose of S-ACH and the incubated time of the sperm.S-ACH could significantly inhibited sperm motility,and VCL, VAP,VSL and ALH were sensitive to S-ACH.The rate of sperm motility and motion pattem changed with the increasing of dosage.With the extension of incubation time the sperm motility in 10mg/kg group was significantly reduced. Percentages of hyperactivity under capacitating condition were significantly reduced, which exceed to the reduction of motility.Both motility and hyperactivity recovered after withdrawal of S-ACH.The IVF rate showed an decreasing tendency with the doses of S-ACH.
PartⅢStudy on the basis of the antifertility effect of (S)-α-chlorohydrin on male SD rats
To explore the possible basis of the antifertility effect of S-ACH,GAPDS activity,ATP level and signaling pathway of protein tyrosine phosphorylation in rat sperm were determined after administration of S-ACH in vivo or in vitro.40 adult male SD rats were randomly divided into 4 groups,each group contained 10 rats. Daily oral doses of 2.5,5.0,10.0mg/(kg·bw) S-ACH were administrated to rats for 52 days,and the controls were given the equal amount of distilled water.At the end of treatment,each group kept 5 rats with normal feeding for another 4 weeks.GAPDS enzyme activity and ATP levels in the cauda epididymal sperm were determined both in dosed and recovered groups.Sperm ATP levels were assayed by luminometric method and sperm GAPDS enzyme activity was determined by detecting the changes of absorption of production concentration at 340nm.cAMP levels in sperm were measured by enzymeimmunoassay.Tyrosine phophorylation in sperm after administration of S-ACH at the concentrations of 0,0.1,1.0,10 mM respectively in vitro was detected by western blot.The results showed that S-ACH inhibited GAPDS enzyme activity with dose-dependent manner.Enzyme activity could recover after the cessation of S-ACH administration.ATP levels in different groups did not show significant difference,cAMP levels in administrating group showed a decreased tendency with dose increasing.Pentoxifylline could increase cAMP levels of sperm. Protein tyrosine phosphorylation had not interfered by the administration of S-ACH in vitro under capacitating condition.
引文
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