Antifertility Effect Of S-Alpha-chlorohydrin On Male Rats

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.Partâ… The effect of S-ACH on fertility of male SD rats40 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(G₀). Another female was added when spermatozoa were not founded in the vaginal smear after one week mating.The mated females were autopsied on G₁₂-G₁₅ 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.Partâ…¡Study on the stages of fertility of male SD rats witch impacted by(S)-α-chlorohydrinTo 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 2×10⁶ cell/ml,and incubated at 37℃,5%CO₂.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 2×10⁶ 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%CO₂ 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. Partâ…¢Study on the basis of the antifertility effect of (S)-α-chlorohydrin on male SD ratsTo 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.