They Can Handle the Stress: MPK17 and PMD1 act in a salt-specific pathway.

Arabidopsis MAP KINASE17 (MPK17) was recently identified as a novel regulator of peroxisome division in response to salt stress. Further, the known peroxisome division factor PEROXISOME AND MITOCHONDRIAL DIVISION FACTOR1 (PMD1) genetically acts downstream of MPK17. We previously showed that mutants defective in either MPK17 or PMD1 fail to proliferate peroxisomes in response to NaCl stress. Here, we show that, unlike their abnormal NaCl responses, mpk17 and pmd1 mutants display wild type responses to other stresses known to alter peroxisome proliferation, suggesting that plants distinguish among peroxisome division-inducing stresses and alter the peroxisome division pathway based on the stress applied.

Next-generation sequencing as a tool to quickly identify causative EMS-generated mutations.

The advent of next generation sequencing has influenced every aspect of biological research. Many labs are now using whole genome sequencing in Arabidopsis thaliana as a means to quickly identify EMS-generated mutations present in isolated mutants. Following identification of these mutations, examination of T-DNA insertional alleles defective in candidate genes or complementation of the mutant phenotype with a wild type copy of candidate genes can be used to verify which mutation is causative for the phenotype of interest. Here, we discuss the benefits and pitfalls of using this method to identify mutations underlying phenotypes.

Impact of international laboratory partnerships on the performance of HIV/sexually transmitted infection testing in five resource-constrained countries.

To review a quality control and quality assurance (QC/QA) model established to ensure the validity and reliability of collection, storage and analysis of biological outcome data, and to promote good laboratory practices (GLPs) and sustained operational improvements in international clinical laboratories, we conducted a two-arm randomized community-level HIV behavioural intervention trial in five countries: China, India, Peru, Russia and Zimbabwe. The trial was based on diffusion theory utilizing a Community Popular Opinion Leaders (CPOLs) intervention model with behavioural and biological outcomes. The QC/QA model was established by the Biological Outcome Workgroup, which collaborated with the Data Coordinating Center and John Hopkins University Reference Laboratory. Five international laboratories conducted chlamydia/gonorrhoea polymerase chain reaction (PRC)-based assays, herpes simplex virus type 2 enzyme immunoassay (EIA), syphilis serology (rapid plasma regain and Treponema pallidum particle agglutination assay, HIV serology (EIA/Western blot) and Trichomonas vaginalis culture. Data were collected at baseline, 12 and 24 months. Laboratory performance and infrastructure improved throughout the trial. Recommendations for improvement were consistently followed. Quality laboratories in resource-poor settings can be established, operating standards can be improved and certification can be obtained with consistent training, monitoring and technical support. Building collaborative partnership relations can establish a sustainable network for clinical trials, and can lead to accreditation and international laboratory development.

Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.).

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.

Rat epididymal sperm quantity, quality, and transit time after guanethidine-induced sympathectomy.

Guanethidine, a chemical that selectively abolishes peripheral noradrenergic nerves, was used to investigate the role of sympathetic innervation in the maintenance of epididymal sperm quantity and quality. Four groups of 10 adult male rats each were treated daily for 21 days, by i.p. injections, with either 0 (saline vehicle), 6.25, 12.5, or 25 mg/kg guanethidine. Norepinephrine content was reduced to undetectable levels in the cauda epididymidis in all guanethidine groups after 3 wk of treatment and was reduced to 7.4% of the control values after 1 wk of 6.25 mg/kg treatment. While body weight gain was significantly decreased at 12.5 and 25 mg/kg compared to that in controls, there was a significant increase in the weights of the seminal vesicles/coagulating glands in all treated groups. The number of homogenization-resistant spermatids per testis and the daily sperm production per testis remained unchanged. The weight of the epididymis was significantly increased at 6.25 and 12.5 mg/kg. Moreover, the number of cauda epididymal sperm and the transit time were increased significantly at 6.25 mg/kg (10.2 days) compared to values in the control cauda (6.3 days). Neither serum testosterone levels nor LH was affected in a dosage-related manner. There were no effects of guanethidine treatment on cauda epididymal sperm motility or morphology. A quantitative analysis of detergent-extracted cauda epididymal sperm proteins by SDS-PAGE revealed no differences, but there were diminutions in seven proteins in homogenates of caput/corpus tissue. Histologic analysis of testis and epididymis sections revealed no differences between control and denervated animals. In a subsequent experiment the lowest effective dosage (6.25 mg/kg) was given to rats for 1 wk, and an increased number of cauda epididymal sperm and a delay in sperm transit were observed. Our results indicate that low-dosage guanethidine exposure denervates the epididymis within 1 wk, thereby delaying epididymal transit; however, neither 1- nor 3-wk exposure produces qualitative changes in the sperm.

Fertility of rat epididymal sperm after chemically and surgically induced sympathectomy.

Guanethidine, a chemical that selectively blocks sympathetic noradrenergic neurons, was used to investigate the role of sympathetic innervation in the fertility of rat epididymal sperm, using both natural mating and in utero insemination protocols. This animal model correlates, at least in part, with spinal cord injury (SCI) in men. Adult male rats were treated daily by i.p. injections, for 21 or 42 days, with 0 or 6.25 mg/kg guanethidine. To compare the effects of guanethidine-induced sympathectomy with those following surgically induced sympathectomy, the inferior mesenteric ganglion and the proximal hypogastric nerves were removed in another group of rats. Both chemically and surgically induced sympathectomy increased the weight of the epididymis and seminal vesicles/coagulating glands as well as the number and the transit time of cauda epididymal sperm. Neither serum testosterone levels nor LH was affected by treatment with guanethidine. Using natural mating, no litters were produced by guanethidine-treated rats. Chemically denervated rats failed to produce copulatory plugs or ejaculate into the uterus. However, distal cauda epididymal sperm from chemically or surgically denervated rats displayed normal fertilization ability (80%) using in utero inseminations. In addition, the sperm of denervated rats did not show abnormal sperm chromatin structure using an assay that detects DNA damage. We conclude that sympathectomy delays the transit of sperm through the cauda epididymidis and produces ejaculatory dysfunction but does not compromise sperm quality in the distal cauda epididymidis. Moreover, these data provide compelling evidence that there is no association between the prolonged transit time of sperm within the epididymis, i.e., pre-ejaculatory sperm aging, and the fertility of those sperm, which has important implications for artificial insemination using sperm from men with SCI.

Spermatotoxicity of dichloroacetic acid.

The testicular toxicity of dichloroacetic acid (DCA), a disinfection byproduct of drinking water, was evaluated in adult male rats given both single and multiple (up to 14 d) oral doses. Delayed spermiation and altered resorption of residual bodies were observed in rats given single doses of 1500 and 3000 mg/kg; these effects persisted to varying degrees on post-treatment days 2, 14, and 28. Delayed spermiation and formation of atypical residual bodies also were observed on days 2, 5, 9, and 14 in rats dosed daily with 1440, 480, 160, and 54 mg/kg. Distorted sperm heads and acrosomes were observed in step 15 spermatids after 14 doses of 480 and 1440 mg/kg. Decreases in the percentage of motile sperm occurred after 9 doses of 480 and 1440 mg/kg and 14 doses of 160 mg/kg. Increased numbers of fused epididymal sperm were observed on days 5, 9, and 14 in rats dosed with 1440, 480, and 160 mg/kg, respectively; other morphologic abnormalities occurred at 160 mg/kg and higher. On day 14, a significant decrease in epididymis weight was observed at 480 and 1440 mg/kg, and epididymal sperm count was decreased at 160 mg/kg and higher. These studies demonstrate that the testicular toxicity induced by DCA are similar to those produced by the analogue, dibromoacetic acid. However, the testicular toxicity of DCA is less severe at equal molar concentrations. Moreover, the DCA-induced testicular lesions occur with greater potency as the duration of dosing increases, indicating the importance of using low-dose subchronic exposures to assess the health risk of prevalent disinfection byproducts.

Histopathologic changes in the testes of rats exposed to dibromoacetic acid.

The present report details histopathologic changes in the testis and epididymis of rats gavaged daily for 2 to 79 d with a by-product of water disinfection, dibromoacetic acid (DBAA). On treatment day 2 abnormal retention of Step 19 spermatids was observed in animals given the highest dosage of 250 mg/kg. Additional changes on day 5 included the fusion of mature spermatids and the presence of atypical residual bodies (ARB) in the epithelium and lumen of Stage X-XII seminiferous tubules. By day 9, ARB were seen in most stages of the seminiferous epithelial cycle and in the caput epididymidis. On day 16 distorted sperm heads were recognized in Step 12, and older spermatids, and luminal cytoplasmic debris was found throughout the epididymis. On day 31, there was vacuolation of the Sertoli cell cytoplasm, extensive retention of Step 19 spermatids near the lumen of Stage IX and X tubules, and vesiculation of the acrosomes of late spermatids. Marked atrophy of the seminiferous tubules was present 6 months after 42 doses of 250 mg/kg. ARB and retention of Step 19 spermatids were observed after 31 and 79 doses of 50 mg/kg and increased retention of Step 19 spermatids was seen in several rats dosed with 10 mg/kg. No abnormalities were detected at the dosage of 2 mg/kg. The changes suggest that the testicular effects of DBAA are sequelae to structural and/or functional changes in the Sertoli cell.

Comparison of rat epididymal sperm counts by IVOS HTM-IDENT and hemacytometer.

Epididymal sperm counts, a common measurement in male reproductive toxicity studies, are routinely determined using a hemacytometer. Recently, computer assisted methods for automated sperm counts have been developed. In the present study we evaluated an automated system, the TOX IVOS (Hamilton Thorne Research, Beverly, MA) HTM-IDENT option, that utilizes a DNA-specific stain and fluorescence illumination to identify sperm for enumeration. Cauda and caput epididymal sperm counts were determined in 48 adult male Sprague-Dawley rats, using both the hemacytometer and HTM-IDENT. The mean hemacytometer and HTM-IDENT counts (+/- SD) were 250 +/- 43 and 254 +/- 52 million, respectively, for cauda sperm, and 123 +/- 13 and 127 +/- 18 million, respectively, for caput sperm. The average coefficient of variation using the hemacytometer was 13.8% as compared to 17.3% for the HTM-IDENT. Comparison of the machine count and a visual count from the Display Statics screen of the HTM-IDENT indicated that when two or more sperm heads touched or overlapped, the machine counted them as one. Manual (visual) and machine counts when compared over a range of nine concentrations from 3.7 to 47.8 million/mL differed by 4 to 12% at the lowest to highest concentration. The concentration of epididymal sperm samples used in comparing the two counting methods ranged from 5.8 to 17.7 million/mL. Therefore, the HTM-IDENT undercounting error attributable to sperm heads touching was less than 6%. Overall the data indicate good agreement between the HTM-IDENT and the hemacytometer counts. Furthermore, both counting time and technician fatigue were markedly reduced. Thus the HTM-IDENT option improves the efficiency of epididymal sperm counting without loss of precision.

Dibromoacetic acid affects reproductive competence and sperm quality in the male rat.

Recently, we demonstrated with short-duration tests that dibromoacetic acid (DBAA), a commonly occurring by-product of water disinfection, alters sperm morphology and motility in the male rat. These results suggested that the effects of DBAA on sperm quality were likely to compromise reproductive competence of the male rat early in subchronic exposure. The present studies were undertaken to investigate the dose response and time course of alterations in fertility and sperm quality. Proven breeder male rats were gavaged daily with 0, 2, 10, 50, or 250 mg DBAA/kg for up to 79 days; interim and terminal measurements of sperm quality and reproductive outcome were made. Because of the known neurotoxicity of the analogue, dichloroacetic acid, both natural breeding and artificial inseminations were evaluated in untreated females to distinguish between possible behavioral and spermatogenic effects. DBAA compromised male fertility during the second treatment week in naturally bred rats dosed with 250 mg/kg. The early antifertility effect appeared to be the result of behavioral changes since females artificially inseminated with sperm collected on Day 9 successfully produced offspring. However, sperm morphology and motility also were rapidly affected by DBAA treatment so that no offspring via natural insemination and only one litter via artificial insemination were produced subsequent to Day 15. Through 31 days, substantial effects on sperm motility, sperm morphology, and epididymal sperm numbers were observed, but there was no demonstrable effect on serum testosterone or sperm production. Because severe toxicity developed in the group given 250 mg/kg, exposure of these animals was prematurely terminated after 42 doses and their recovery was monitored through a 6-month posttreatment period; decreased testis weights and only limited recovery of reproductive performance were observed. Exposure to 50 mg/kg resulted in moderate changes in sperm morphology and motility and moderate decreases in epididymal sperm counts in rats dosed for 31 or 79 days. However, these males remained fertile, litter size was unaffected, and no paternally mediated developmental defects were noted in their offspring. No effects on sperm quality were detected at dosages of 2 or 10 mg/kg. However, compared to controls, naturally bred DBAA-treated rats tended to have fewer inseminations, fewer copulatory plugs, and fewer multiple litters, suggesting that DBAA may have altered mating behavior at dosages as low as 10 mg/kg.

Spermatotoxicity of dibromoacetic acid in rats after 14 daily exposures.

Halogenated acetic acids are major disinfection by-products of water chlorination and ozonation. Limited data in experimental animals indicate that repeated doses of dichloroacetic acid (DCA) or single doses of dibromoacetic acid (DBAA) cause testicular damage. In the present study, spermatotoxic effects were investigated in rats given oral doses of 0, 10, 30, 90, or 270 mg DBAA/kg/day for 14 days. In rats dosed with 270 mg/kg/day, there were marked effects on epididymal sperm motility and morphology including the flagellar fusion of 2 or more sperm. Testis weight, epididymis weight, and testicular sperm head counts were mildly reduced relative to control, whereas epididymal sperm counts were substantially decreased. Histologic changes in the testis included retention of Step 19 spermatids in Stages IX to XII, abnormal development of late spermatids, and the formation of atypical structures resembling residual bodies that were observed predominantly in Stages X to XIV and I of the cycle of the seminiferous epithelium. At the dosage of 90 mg/kg/day, effects on spermiation, spermatid development, epididymal sperm counts, sperm motility, and sperm morphology were less severe than at the higher dosage. Reduced caput sperm counts and mild effects on spermiation also occurred at 30 and 10 mg/kg/day. These studies indicate that subchronic exposure to DBAA has the potential to affect reproductive outcome in the rat. Compared to previous studies of DCA (12), DBAA, on a molar basis, appears to be a stronger testicular toxicant than the dichloro analogue.

Acute spermatogenic effects of bromoacetic acids.

Chlorine and bromine can react with natural organic substances in source waters to form haloacetic acids, major disinfection by-products of water chlorination. Several toxic effects including testicular damage have been attributed to the chloroacetic acids but little information is available on the bromine analogues. In this report we present the results of acute toxicity and acute spermatotoxicity studies of monobromoacetic acid (MBAA) and dibromoacetic acid (DBAA). In adult male rats the acute oral toxicity of MBAA was 10-fold that of DBAA (LD50 177 vs 1737 mg/kg). No reproductive-related endpoints were affected in rats given a single dose of 100 mg MBAA/kg or 14 daily doses of 25 mg MBAA/kg/day. In rats dosed with DBAA, serum testosterone fell to 17% of control 2 days after a single dose of 1250 mg/kg but returned to control levels by Day 14. Marked effects on sperm motion were seen on post-treatment Days 14 and 28. Degenerative flagellar changes in cauda sperm were present on Day 14 while abnormal sperm head shapes and flagellar degeneration were observed in both caput and cauda sperm on Day 28. Histopathology indicated altered spermiation at all time-points as evidenced by retention of Step 19 spermatids beyond Stage VIII of the cycle of the seminiferous epithelium. Disorganization, distortion, and degeneration of late spermatids were also observed. On Day 14 structures resembling residual bodies were rarely seen in the testis but were numerous in the epididymis. Caput sperm counts were decreased on Day 2 and cauda sperm counts were decreased on Days 14 and 28.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimization of the Hamilton-Thorn computerized sperm motility analysis system for use with rat spermatozoa in toxicological studies.

To optimize the Hamilton-Thorn Motility Analyzer (HTM; Hamilton-Thorn Research, Beverly, MA) for use in reproductive toxicology studies with rat spermatozoa, the accuracy and precision of the instrument were assessed under a variety of instrument settings. Videotapes of both fast- and slow-swimming sperm were analyzed repeatedly to obtain data across a range of sperm velocities as might be encountered as a consequence of exposure to reproductive toxicants. Acquisition rates were varied across the HTM menu choices (30, 19, 10, or 7 frames/sec) as were the number of frames analyzed (5 to 20) at each framing rate. For fast-swimming samples (mean straight-line velocity (VSL) approximately 130 microns/sec) generally good agreement between computer-assisted sperm analysis (CASA) and manually obtained data was found for percentage of motile sperm and straight-line velocity; i.e., CASA values were within 10% of manual values for most frame/rate combinations. The accuracy of these measures held true over a wide range of sperm concentrations and percentage motilities. However, CASA measures were less accurate for sperm samples of lower velocities (mean VSL approximately 50 microns/sec and mean VSL approximately 30 microns/sec) in that the velocity of very slow sperm was overestimated (particularly at 30 frames/sec). A soft-ware change (6.5R) and performing analyses at 19 instead of 30 frames/sec improved straight-line accuracy for the slow sperm and enhanced the discrimination between fast (presumably control) and slow (presumably treated) sperm samples. These data show that this motility analyzer could be successfully configured to evaluate rodent sperm samples. The use of such CASA systems in toxicology studies will provide valuable information that may improve human reproductive risk assessment.

Acute and long-term effects of a single dose of the fungicide carbendazim (methyl 2-benzimidazole carbamate) on the male reproductive system in the rat.

The effects of carbendazim (methyl 2-benzimidazole carbamate) on the testis, efferent ductules, and sperm were determined in the adult rat after a single oral dose. Two experimental trials were performed: a time response between 2 hours and 32 days after exposure using 0 and 400 mg/kg, and a dose response at 2 and 70 days after exposure using 0 to 800 mg/kg doses. In experiment 1, effects were seen throughout the 32-day period, beginning 8 hours after exposure; the effects included first an increase in testis weight, then decreases in testicular spermatid numbers and in the percentage of morphologically normal cauda sperm. In experiment 2, significant testicular and efferent ductal alterations occurred in animals treated with doses of 100 mg/kg or greater. A dose-dependent increase in testicular weight 2 days after treatment was accompanied by increases in seminiferous tubular diameter and excessive loss of immature germ cells in a stage-dependent manner. There was also a dose-dependent increased incidence of occlusions in the efferent ductules. The occluded ductules were characterized by severe inflammation and exhibited disorganization of the epithelium. At 70 days, there were dose-dependent decreases in mean testis weight and mean seminiferous tubular diameter; however, only minimal long-term effects were seen at 50 mg/kg. In testes exhibiting seminiferous tubular atrophy of greater than 25% (100 mg/kg or greater doses), all of the testes were associated with efferent ductules containing occlusions. Caput sperm numbers were significantly reduced in these testes. Occlusions, abnormal ductules, fibrosis, spermatic granulomas, and mineralization were observed in the ductuli efferents. Long-term effects of carbendazim on the testis were induced primarily by ductal occlusions. Results show that carbendazim produces more severe short- and long-term effects on the male reproductive system than the fungicide benomyl.

Endpoints of spermatotoxicity in the rat after short duration exposures to fourteen reproductive toxicants.

Multiple endpoints of spermatotoxicity in short duration tests (1-5 days exposure; 2.5-week assay interval) were investigated in a number of chemicals reported to produce minimal to severe reproductive effects when administered subchronically. Six of these chemicals (boric acid, dinoseb, 2,5-hexanedione, methoxychlor, metronidazole, ornidazole) produced substantial spermatotoxicity after 1 to 5 doses. Spermatotoxic effects of chlordimeform were equivocal while p,p'-DDT, n-hexane, and sodium chlorite were judged negative. Four chemicals with known acute effects (benomyl, busulfan, ethylene glycol monomethyl ether, nitrobenzene) elicited expected histopathologic responses after a single dose. Testicular histology, testicular sperm head counts, cauda sperm counts, sperm morphology, and sperm velocity proved to be the most toxicologically sensitive endpoints in one or more of the studies, but histopathology of the testis and epididymis was the most consistent indicator of reproductive damage. The percentage of motile sperm and sperm concentration in the epididymal fluid were the least sensitive measurements. The data suggested that most chemicals with the potential to produce moderate to severe sperm damage are detectable with a short duration test. Complementary multiple endpoints enhanced the interpretation of results, often identified cellular targets, and provided insight on possible mechanisms. Specific responses were often similar to specific effects reported for subchronic exposures. A short duration test could be of value as a screen in structure-activity studies or to set priorities for chemicals requiring further evaluation. As a supplement to breeding studies, the data generated in the short test could also be used to enhance the design and interpretation of the longer tests.

Effect of acute exposure to boric acid on the male reproductive system of the rat.

Adult male rats were dosed orally on d 0 with 0 or 2000 mg/kg of boric acid and killed on posttreatment d 2, 14, 28, and 57, or dosed with 0, 250, 500, 1000, or 2000 mg/kg of boric acid and killed on posttreatment d 14. At d 14, atypical structures that appeared to be enlarged irregular cytoplasmic lobes of Step 19 spermatids were observed in Stage VIII seminiferous tubules of rats dosed with 1000 and 2000 mg/kg. Abnormal retention of Step 19 spermatids and residual bodies was also observed in Stage IX-XIII tubules of these rats. The retained spermatids and residual bodies were seen in both the luminal and basal regions of the epithelium. A substantial increase in the testicular sperm head count occurred in animals dosed with 2000 mg/kg. Abnormal caput epididymal sperm morphology and reduced caput epididymal sperm reserves were observed at 1000 mg/kg and higher. Serum LH, FSH, TSH, and prolactin values were not affected at any dosage. At d 28, rats dosed with 2000 mg/kg exhibited continued retention of Step 19 spermatids into Stage X, abnormal caput and cauda sperm morphology, and decreased percentages of motile cauda spermatozoa with reduced straight-line swimming velocities. By d 57 substantial recovery was apparent; some retention of Step 19 spermatids into Stage X tubules was still present in two out of six rats but the sperm parameters were comparable to controls. The study indicated that acute oral exposure to boric acid adversely affected spermiation and sperm quality in the adult male rat. At the dosages used the effects appeared reversible. The no-effect level was 500 mg/kg.

Reproductive toxicity of a single dose of 1,3-dinitrobenzene in two ages of young adult male rats.

These studies evaluated the reproductive response and the possible influence of testicular maturation on the reproductive parameters, in male rats treated with 1,3-dinitrobenzene (m-DNB). Young adult male rats (75 or 105 days of age) were given a single oral dose of 0, 8, 16, 24, 32, or 48 mg/kg of m-DNB and killed at 14 days post-treatment. Mortality and neurotoxicity were observed at 48 mg/kg, but only in the older animals. Epididymis weight, testicular sperm head counts, cauda sperm reserves, and sperm morphology were affected at 16 and 24 mg/kg and higher in the older and younger animals, respectively. Testis weight and sperm motility were affected at 24 mg/kg and higher in both age groups. Histologic changes included maturation depletion of mid and late spermatids at 16 mg/kg and higher, atrophy of a few to many seminiferous tubules at 24 mg/kg and higher, and immature germ cells in the epididymis. The movement and/or mixing of luminal elements in the epididymis appeared to be influenced by severe testicular effects. In separate groups given only the 48 mg/kg dosage, fertilizing ability was lost by 5-6 weeks post-treatment and several animals failed to recover in 5 months. In the breeder males, minimal to extensive degrees of seminiferous tubule atrophy and sloughed germ cells in the epididymis were still present after 175 days. The studies indicated that the lowest dosage to produce reproductive changes was 16 mg/kg with a no-effect level of 8 mg/kg. A few animals suffered protracted or permanent reproductive damage. Since the older animals were more susceptible to both the general and the reproductive toxicity of m-DNB, the less severe reproductive changes in the younger animals cannot be attributed solely to maturational differences in the testis.

Unilateral depletion of testicular glutathione levels in the rat following intratesticular injections of diethylmaleate and buthionine sulfoximine.

A method was developed to selectively deplete glutathione (GSH) in a single rat testis. Using intratesticular injections of a mixture of two GSH-depleting agents, diethylmaleate and buthionine sulfoximine, testicular GSH levels were decreased to 33-54% of control 2 hr after injection and remained suppressed for 24 hr. GSH levels in the contralateral testis and liver were not affected by this treatment. Comparisons between GSH-depleted and vehicle-injected (contralateral) testes, evaluated 2 weeks later, showed that although testis and epididymal weights and cauda epididymal sperm reserves were slightly reduced (to greater than or equal to 90% of controls), no changes were seen in testicular spermatid counts or in the morphology or motility of cauda epididymal sperm. An increase in histologically abnormal tubules localized to the injection site occurred in some GSH-depleted testes; however, the proportion of normal tubules containing step 19 spermatids was not affected. Thus, intratesticular injections of GSH-depleting agents selectively lowered GSH levels in the treated testis, with minimal adverse effects. This protocol can now be applied to investigate specific roles of GSH in the testes, particularly with regard to the possible modulation of the effects of testicular toxicants.

The value of multiple endpoint data in male reproductive toxicology: revelations in the rat.

To explore the relationship between sperm measures and reproductive success in the laboratory rat, a review has been conducted of results of several male reproductive toxicology studies containing both fertility and multiple endpoint data. Comparisons are made between subchronic and acute exposure studies, emphasizing for each approach the value of multiple endpoint data. Further, the choice of endpoints in male reproductive toxicology is discussed with select examples of endpoints for which an understanding is evolving. We conclude that sperm measures are far more sensitive indicators of reproductive organ impairment than is the measurement of fertility, and that evaluation of multiple endpoints in acute studies is a key step in determining mechanisms of toxicant action that are critical for accurate interspecies risk extrapolation.

Acute effects and long-term sequelae of 1,3-dinitrobenzene on male reproduction in the rat. I. Sperm quality, quantity, and fertilizing ability.

Groups of eight adult male rats were given a single oral dose of 0 or 48 mg/kg of 1,3-dinitrobenzene and sacrificed at 1, 2, 4, 8, 16, 24, 32, 72, and 175 days posttreatment. The groups killed at 175 days were bred to untreated females during weeks 3, 4, 6, 9, 13, and 24. Decreased testis weight and testicular sperm numbers were observed by day 4; decreased cauda sperm reserves and epididymis weight occurred by day 8 and day 16, respectively. Reduced numbers of motile spermatozoa and abnormal sperm morphology were seen in spermatozoa from the cauda epididymidis by day 16. Fertilizing ability, as indicated by the presence of two pronuclei and a sperm tail in eggs flushed from the oviducts of inseminated females, was slightly reduced by week 4 and declined to zero by week 6. Group means for reproductive organ weights, sperm production, and sperm reserves failed to return to control levels although some individual animals approached full recovery. Normal fertilizing ability was restored in most animals by week 13, but two of seven remained infertile. Occlusion of some efferent ductules was observed in three of seven animals at 175 days. This study indicates that 1,3-dinitrobenzene is a potent testicular toxicant in the rat, capable of producing marked testicular damage, infertility, and possibly sterility from a single exposure.