Alpha-chlorohydrin Research Articles

EVALUATING THE EFFICACY OF THE MALE CHEMOSTERILANT ALPHA-CHLOROHYDRIN ON THREE EGYPTIAN WILD RODENT PESTS UNDER LABORATORY CONDITIONS

The current study was designed to evaluate the efficacy of the male chemosterilant alpha-chlorohydrin (ACH) in the control of three Egyptian wild rodent pests (Order: Rodentia, Family: Muridae), namely; the Cairo spiny mouse, Acomys cahirinus (Desmarest, 1819) the lesser Egyptian gerbil, Gerbillus gerbillus (Olivier, 1801) and Shaw’s jird, Meriones shawi Rozet, 1833. The effect of ACH was tested by the examination of the relative weights of the reproductive organs, the sperm parameters, as well as the histological alterations in the testis and epididymis. The results of this study showed that ACH has no significant antifertility effect when given to A. cahirinus at doses of 50 mg/kg body weight for 5 days, 70 mg/kg for 4 days; to G. gerbillus at doses of 50 mg/kg for 3 days, and 60 mg/kg for 4 days; and to M. shawi at doses of 50 mg/kg for 5 days, and 80 mg/kg for 3 days. Taken together, it could be concluded that ACH has a species-specific action, and is not recommended to be used in the control of the rodent pests of the present study.

Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray

BackgroundAs one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals.MethodsRats were treated with ACH for ten consecutive days, and then each male rat copulated with two female rats in proestrus. Then sperm maturation and other fertility parameters were analyzed. Furthermore, we identified epididymal-specific genes that are associated with fertility between control and ACH groups using an Affymetrix Rat 230 2.0 oligo-microarray. Finally, we performed RT-PCR analysis for several differentially expressed genes to validate the alteration in gene expression observed by oligonucleotide microarray.ResultsAmong all the differentially expressed genes, we analyzed and screened the down-regulated genes associated with metabolism processes, which are considered the major targets of ACH action. Simultaneously, the genes that were up-regulated by chlorohydrin were detected. The genes that negatively regulate sperm maturation and fertility include apoptosis and immune-related genes and have not been reported previously. The overall results of PCR analysis for selected genes were consistent with the array data.ConclusionsIn this study, we have described the genome-wide profiles of gene expression in the epididymides of infertile rats induced by ACH, which could become potential epididymal specific targets for male contraception and infertility treatment.

Effects of low doses of alpha chlorohydrin on the dehydrogenases and oxidases of rat epididymal epithelium and sperms: a correlative histochemical and biochemical study.

Histochemical studies have been made of the isocitrate dehydrogenase, succinic dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, DPN diaphorase, TPN diaphorase, delta 5-3 beta-hydroxysteroid dehydrogenase and monoamine oxidase in the caput, corpus and cauda epididymides of normal and alpha chlorohydrin (6.5 mg/kg/9 days) treated rats. Administration of alpha chlorohydrin in a low dose caused a conspicuous decrease in all these enzymes except delta 5-3 beta-HSD, in various cell types of epididymal epithelium and sperms. Biochemical estimations of isocitrate dehydrogenase, succinic dehydrogenase, malate dehydrogenase and delta 5-3 beta-HSD have further supported and confirmed these histochemical observations. These changes in enzyme activities after treatment with low dose of alpha chlorohydrin strongly suggest that TCA cycle and amino acid metabolism of epididymis become defective, much earlier before any histological damage to the epididymis becomes visible.

Effect of low doses of alpha chlorohydrin on the dehydrogenases and oxidases of rat testis. A histochemical study.

Zusammenfassung Effekt einer low-dose Behandlung mit α-Chlorohydrin auf die Dehydrogenase und Oxidasen im Rattenhoden — Eine histochemische Untersuchung In dieser histochemischen Untersuchung wurden folgende Enzyme nach low-dose Behandlung mit α-Chlorohydrin (6,5 mg/kg/9 Tage) in den verschiedenen Zellarten des Rattenhodens untersucht: Isocitratdehydrogenase, Succinatdehydrogenase, Malatdehydrogenase, Glutamatdehydrogenase, Δ5- 3α-Hydroxysteroiddehydrogenase, NAD- und NADP-Diaphorase und Monoaminoxidase. Die Anwendung von α-Chlorohydrin fuhrt zu einer Verminderung oder dem Verschwinden all dieser Enzyme bis auf NADP-Diaphorase und Δ5 -3α-HSD. Diese Veranderungen der Enzymaktivitaten im Rattenhoden zeigen deutlich, das nach einer low-dose Behandlung mit α-Chlorohydrin lange bevor histologische Defekte auftreten, der Zitronensaurezyklus und Aminosaurestoffwechsel im Hoden geschadigt werden. Die Steroidbiosynthese bleibt jedoch unbeeintrachtigt, wie die gleichbleibenden Aktivitaten der Δ5 -3α-HSD und NADP-Diaphorase bewiesen.

1H NMR to investigate metabolism and energy supply in rhesus macaque sperm

Sperm ATP is derived primarily from either glycolysis or mitochondrial oxidative phosphorylation. In the present studies, 1H NMR spectroscopy was used to characterize the metabolite profile in primate sperm treated either with alpha-chlorohydrin (ACH), a known inhibitor of sperm glycolysis or pentachlorophenol (PCP), an uncoupler of oxidative phosphorylation. Sperm were collected from monkeys in the fall and spring, washed and incubated with either the media control, ACH (0.5 mM) or PCP (50 uM). Using principal components analysis, PC1 scores plot indicated that the greatest level of variance was found between fall and spring samples and not chemical-treated samples. However, PC4 scores plot did show a consistent effect of ACH treatment. From the PC1 loadings plot, metabolites contributing to the seasonal differences were higher levels of formate in the fall and higher levels of carnitine and acetylcarnitine in the spring as well as possible differences in lipoprotein content. The PC4 loadings plot indicated that ACH treatment decreased lactate and ATP consistent with inhibition of glycolysis. Carnitine also was decreased and acetylcarnitine increased although the latter was not statistically significant. With PCP-treated sperm, no difference between control and treated samples could be discerned suggesting either that primate sperm are insensitive to uncoupling agents or that glycolysis played the more important role in maintaining sperm ATP levels. Overall, NMR studies may prove useful in the development of metabolomic markers that signal sperm metabolic impairments and have the potential to provide useful biomarkers for reproductive health.

Sperm Mitochondrial Integrity Is Not Required for Hyperactivated Motility, Zona Binding, or Acrosome Reaction in the Rhesus Macaque1

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 microM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque.

Key parameters of sperm motion in relation to male fertility in rats given alpha-chlorohydrin or nitrobenzene.

This study was undertaken to detect key parameters of rat sperm motion in relation to male fertility by comparing the differences in sperm motion induced by treatment with alpha-chlorohydrin (ACH), known to produce spermatotoxicity, and nitrobenzene (NTB), known to produce testicular toxicity. Male rats received ACH (5 or 20 mg/kg/day) or NTB (60 mg/kg/day) for either 3 days or 18 days. Epididymal sperm was assessed for motility using a Hamilton-Thorne Sperm Analyzer (HTM-IVOS). Numerical data for statistical analysis and graphical renditions of sperm motion using parameters in radar charts and reconstructed sperm tracks were analyzed to evaluate sperm motion. Males were allowed to copulate with untreated females and cesarean sections were conducted in order to examine the effects of drug administration on male fertility. Linearity of sperm track (linearity (LIN) and/or straightness (STR)) decreased and/or beat cross frequency (BCF) increased only in ACH groups (5 or 20 mg/kg/day), although the percentage of motile sperm, sperm velocities (average path velocity (VAP), curvilinear (VCL), and straight line velocity (VSL)) and amplitude of lateral head displacement (ALH) decreased on Day 18 in both ACH and NTB (60 mg/kg/day) groups. Furthermore, from the individual reconstructed sperm tracks, it was clear that ACH-treated spermatozoa were characterized by abnormal motion ("jerking") with low vigor (low velocities) and little or no forward progression. Finally, only ACH treatment led to a reduction in pregnancy rate or infertility. Therefore, our results suggest that linearity (especially VSL, STR and LIN) in sperm motion is a key parameter for assessing a chemical's potential to induce male infertility.

Alpha-Chlorohydrin inhibits glyceraldehyde-3-phosphate dehydrogenase in multiple organs as well as in sperm.

Numerous studies have documented inhibitory effects of alpha-chlorohydrin (ACH) on glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in spermatozoa. A sperm-specific G3PDH isoform has been described. The possibility that ACH may inhibit G3PDH in cell types other than sperm was investigated in this work. In addition, the onset of ACH-induced epididymal toxicity was described. Changes to epididymal histology occurred 6 h following a single dose of ACH (50 mg/kg po) and were confined to the proximal initial segment. By 24 h, no epithelial cells lined the basement membrane of that region. Three h after ACH administration (50 mg/kg po), G3PDH activity was significantly decreased in sperm (85%) as well as in kidney (31%), liver (49%), and epididymis (35%). Enzyme activity remained inhibited at 6 and 24 h. G3PDH was immunolocalized in the epididymis and staining was highest in the efferent ducts and initial segment as well as in smooth muscle. Since G3PDH is a microtubule-associated protein and microtubule-dependent endocytosis occurs in the epididymis, beta-tubulin was also immunolocalized. beta-tubulin densely stained the apical region of initial segment and caput epithelial cells. Disruption of beta-tubulin immunostaining correlated with the localization and onset of the lesion. Co-localization of G3PDH and beta-tubulin immunostaining was not observed although both antibodies most densely stained the initial segment. Our data indicate that histologic changes to the proximal initial segment of the epididymis occur rapidly, but subsequent to G3PDH inhibition. Moreover, ACH inhibition of G3PDH is not confined to sperm, although the sperm enzyme is most sensitive to inhibition.

In vitro inhibition of rat cauda epididymal sperm glycolytic enzymes by ornidazole, alpha-chlorohydrin and 1-chloro-3-hydroxypropanone.

Chlorinated antifertility compounds are known to inhibit glycolysis of spermatozoa as they reside in the epididymis but new compounds need to be evaluated that retain antifertility action but do not exhibit side-effects. In this study, two known antifertility agents and a related compound were compared for their inhibition of rat sperm metabolism and motility in vitro. The dose-dependent inhibition in vitro of the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) of distal cauda epididymal rat spermatozoa by (R)-, (S)- and (R,S)-ornidazole (ORN), (R,S)-alpha-chlorohydrin (ACH) and 1-chloro-3-hydroxypropanone (CHOP) was compared. The direct inhibition of GAPDH by ORN suggests that it inhibits without prior conversion outside the cell but inhibition was not stereo-specific. The GAPDH, but not TPI, activity of spermatozoa incubated with ACH and CHOP was highly correlated with kinematic parameters of spermatozoa incubated in pyruvate- and lactate-free medium. ACH only inhibited the activity of intact spermatozoa and the inhibition was not reversed by washing the particulate sperm fraction after sonication. High concentrations of ACH (100 mmol/L) killed intact rat spermatozoa and decreased the extent of GAPDH inhibition. CHOP, unlike ACH, was an effective inhibitor of both intact and sonicated cells. Pre-CHOP, the dimethylketal precursor of CHOP, and its other hydrolysis product MeOH, were both ineffective in vitro. CHOP and related ketals may be more effective inhibitors of sperm glycolysis than ACH and may prove useful for investigating sperm-specific glycolytic inhibition, a prerequisite for the development of antiglycolytic, post-testicular acting contraceptives.

Sperm motion predicts fertility in male hamsters treated with alpha-chlorohydrin.

An understanding of the relationship between altered sperm motion and sperm function (fertility) is important when interpreting the biological significance of toxicant-induced changes in sperm velocity in rodent test species. Previous studies showed that a brief (4-day) exposure of male hamsters to the model chemical alpha-chlorohydrin (ACH) results in significant deficits in epididymal and uterine sperm velocity, which are associated with both a delay and a failure of fertilization in vivo. To characterize this effect in terms of fertility, similarly treated male hamsters were bred to untreated females and pups were counted the day before parturition. ACH treatment resulted in a dose-dependent decline in the percentage of sperm-positive females that were pregnant at the end of gestation (100, 78, 67, 22, and 0 where males were treated with 0, 33, 49, 66, and 83 mg ACH/kg/day, respectively). Cauda epididymal sperm from the same males were assayed for motion characteristics using computer-assisted sperm analysis (CASA), and for fertilizing ability in vitro. While the percentage of motile sperm was unaffected by ACH treatment, sperm velocity declined in a dose-dependent manner at all ACH treatment levels. Furthermore, the velocity of sperm from infertile males was shifted downward consistently across the entire velocity distribution. Since treated males tended to either be infertile (no pups) or have near normal litter size, the correlation between sperm velocity and litter size was nonlinear. Therefore, logistic regression models using velocity cut-off values were the most useful models for predicting fertility. These results support the contention that fertility relies on there being a sufficient number of sperm that exceed a velocity threshold. Sperm from treated males were also less likely to support in vitro fertilization (IVF), providing further evidence of impaired sperm function associated with acute exposure to ACH.

Synchronous assessment of sperm motility and fertilizing ability in the hamster following treatment with alpha-chlorohydrin.

To investigate the relationship between sperm motion parameters and fertilizing ability, a model was developed to assess both of these endpoints synchronously using a toxicant that inhibits sperm motion. alpha-Chlorohydrin (ACH) was administered daily for 4 days to male hamsters at 0, 33, 49, 66, and 83 mg/kg body weight. These males were then allowed a 45-minute breeding period with untreated estrus females on the morning of day 5. One hour after breeding, sperm samples were surgically recovered from the uteri of the females for motility analysis. Six hours later, eggs were flushed from the oviducts and evaluated for fertilization. Cauda epididymal sperm were also collected from the males shortly after breeding. Proportions of motile and progressively motile sperm were manually quantified, and overall sperm velocity and the velocity of representative vigorously swimming sperm in both the uterine and epididymal samples were measured by computer-aided sperm analysis. Significant decreases in in vivo fertilization rates and epididymal sperm motion parameters were observed at 66 and 83 mg/kg ACH, whereas uterine sperm motion was adversely affected at all ACH dosages used. All sperm motion parameters except the percentage of motile sperm in the epididymis were significantly correlated with fertilization rates by both linear and logistic regression. Overall, uterine and epididymal sperm endpoints predicted fertilizing ability comparably well. Stepwise multiple linear regression gave a model containing epididymal sperm velocity (EVCL) and uterine sperm percent motility (UMOT) with an R2 value of 0.649. Stepwise multiple logistic regression gave models containing EVCL alone and EVCL and UMOT in binary (fertile/infertile) and quantal models, respectively.

The male antifertility agents alpha chlorohydrin, 5-thio-D-glucose, and 6-chloro-6-deoxy-D-glucose interfere with sugar transport across the epithelium of the rat caput epididymidis.

The effects of the male antifertility agents alpha-chlorohydrin, 5-thio-D-glucose, and 6-chloro-6-deoxy-D-glucose on sugar transport (3H-3-O-methyl-D-glucose and 3H-2-deoxy-D-glucose) across the rat caput epithelium was studied in vivo and in vitro. The compound alpha-chlorohydrin reduced sugar transport uptake in vivo but not in vitro, whereas 5-thio-D-glucose and 6-chloro-6-deoxy-D-glucose were both effective in vivo and in vitro. The mechanism of action of these compounds on sugar movement across the caput epithelium is probably complex. Direct competition for the glucose carrier situated on the basolateral membrane and intratubular effects are suggested. Thirty-day injections of 5-thio-D-glucose or alpha-chlorohydrin did not have adverse effects on sugar transport or the permeability of the blood-testis and blood-epididymis barriers as assessed by an in vitro technique.

Effect of low doses of alpha chlorohydrin on the enzymes of glycolytic and phosphogluconate pathways in the rat testis and epididymis.

A biochemical study has been made of the effects of low doses of alpha chlorohydrin on all the glycolytic enzymes and two key enzymes of phosphogluconate pathway i.e. glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphogluconate dehydrogenase (6-PGDH) of rat testis and epididymis. All the glycolytic enzymes of testis and epididymis are decreased after treatment with alpha chlorohydrin. G-6-PDH and 6-PGDH are decreased only in epididymis and not in the testis. LDH, ADH and glucose-6-phosphatase were also studied histochemically to show that the drug affects the glycolytic enzymes of epididymal cells and various testicular cell types of testis. Possible significance of these results is discussed.

Lack of effect of alpha-chlorohydrin on the ATP content of rat, mouse and human spermatozoa.

The effect of alpha-chlorohydrin on the ATP content of rat mouse and human spermatozoa was investigated both in vivo and in vitro. In vivo alpha chlorohydrin 1) reduced fertility 2) shortened the life of motile sperm 3) reduced glycolytic activity in spermatozoa and significantly (p less than .02) increased the ATP content of rat spermatozoa. In vitro incubation of spermatozoa for 1 hour in the presence of .6M- alpha-chlorohydrin significantly (p less than .05) reduced the ATP content of rat and mouse spermatozoa in comparison with spermatozoa incubated in the presence of propylene glycol or buffer alone. Sperm motility was also markedly impaired. The results suggest that alpha-chlorohydrin inhibits some sperm enzymes essential for ATP regeneration.

Sequential histopathological and biochemical events in rat caput epididymis after alpha monochlorohydrin administration

This paper presents sequential histological, histochemical and biochemical changes in rat caput epididymis after a single 100 mg/kg oral dose of alpha chlorohydrin. Desquamation of caput epithelium occurs as early as two days after drug treatment. The caput epididymis is blocked because of accumulation of testicular fluid containing exfoliated immature testicular cells and caput epithelium. There was no effect of drug on motility and morphology of spermatozoa examined from different segments of epididymis and vas deferens. Marked decrease in acid and alkaline phosphatases, nucleic acids and proteins have been registered after drug treatment. On the contrary increase in SDH, cholesterol and glycogen was observed after drug treatment. Decrease in phospholipids in initial stages has also been observed.

Alpha chlorohydrin: studies on the mechanism of action in male swine.

The male antifertility agent 3-chloro-12-propanediol (alpha chlorohydrin) was investigated to determine whether the resultant infertility was caused by impaired sperm transport in the female. The approximate time for alpha chlorohydrin to become effective was also sought as was the site of action of the drug. Gilts were inseminated intratubally via laporotomy. When untreated semen was deposited 6 hours before ovulation 21 normal ova were recovered and 12 were fertilized. 11 gilts inseminated 4 hours postovulation had 25 of 34 recovered ova fertilized. Semen from boars fed 5 mg alpha chlorohydrin per kg for 5 days did not fertilize any of the 17 ova from gilts inseminated 6 hours preovulation or any of the 32 ova recovered from the group inseminated 4 hours postovulation. When semen from normal boars was mixed with alpha chlorohydrin no fertilized ova were recovered from the postovulatory inseminated gilts and only 2 ova were fertilized in the preovulatory gilts. 2 boars were fed 5mg/kg per day alpha chlorohydrin; before feeding 4 out of 6 inseminated gilts became pregnant. 7 hours after feeding semen was collected and 7 gilts inseminated; none became pregnant. Semen collected 31 hours after initial treatment and inseminated did not produce pregnancy. Caudal epidiymal semen from control and alpha chlorohydrin-fed boars was also used for insemination. None of 8 gilts inseminated with treated semen became pregnant while 4 out of 7 gilts who were inseminated with the control semen became pregnant. Direct exposure of the ova within the tube did not improve fertilization by treated semen.