Marion G. Miller

Fluorescent probes and flow cytometry to assess rat sperm integrity and mitochondrial function

Fluorescent assessment of cellular integrity and mitochondrial function by flow cytometry can provide a rapid and precise means of determining the functional status of large numbers of spermatozoa. In the present study, rat sperm viability was assessed with SYBR-14 and PI and sperm mitochondria were differentially labeled with JC-1. Sperm samples of variable viability were prepared using varying proportions of fresh and frozen spermatozoa. SYBR-14 stained sperm correlated well with expected sperm viability (r = 0.98). Motile sperm stained with JC-1 appeared orange in the midpiece indicating a high mitochondrial membrane potential whereas immotile sperm with a low membrane potential stained green. The percentage of spermatozoa staining orange was highly correlated (r = 0.99) with expected sperm viability. Flow cytometry using specific fluorescent probes is a useful technique for detecting changes in rat sperm plasma membrane integrity and mitochondrial function in large numbers of spermatozoa.

Germ cell apoptosis in rat testis after administration of 1,3-dinitrobenzene.

The present study investigated the occurrence of apoptosis in rat testis 6, 12, 24, or 48 h after administration of 1,3-dinitrobenzene (DNB)(25 mg/kg, i.p.). Apoptotic cells were visualized using TUNEL in situ detection. In untreated control animals, occasional tubules contained a few apoptotic nuclei. After DNB treatment, more tubules were apoptotic and more germ cells within individual tubules were affected. The increase in incidence and severity of apoptosis was statistically significant 24 and 48 h after exposure. Spermatocytes were the primary cell population affected. Apoptosis was primarily present in Stages VI-VIII and IX-XIII. DNA ladders, characteristic of apoptosis, were clearly present by 24 h after DNB administration, faint at 12 h, and not present in the control or at 6 h. Although initiating mechanisms may be very different for different chemicals, apoptosis is a common and late manifestation of the testicular response to numerous toxicants.

Metabolism and Testicular Toxicity of 1,3-Dinitrobenzene in Rats of Different Ages

Susceptibility to 1,3-dinitrobenzene (1,3-DNB)-induced testicular damage is known to increase with age. The present study investigated the possibility that age-dependent differences in metabolism and disposition could account for differences in toxicity. [14C]1,3-DNB (25 mg/kg, ip) was administered to Sprague-Dawley rats which were 31, 75, or 120 days of age. Levels of 1,3-DNB and 1,3-DNB metabolites were determined in blood and urine. As animal age increased, peak blood concentrations of 1,3-DNB were lower and declined more slowly indicating an age-dependent decrease in rate of metabolism and a possible increase in volume of distribution. In younger animals, faster elimination rates were associated with higher blood levels of metabolites. Urinary metabolites were generally similar for all age groups with the exception of the diacetamidobenzene metabolite which was significantly lower in the urine of 31 days old rats. There were clear differences in the toxicokinetic profile for 1,3-DNB between the 31 day old rats and the other two age groups. However, differences between the 75 and 120 day old animals were less marked. Testicular damage induced by 1,3-DNB (25 mg/kg, ip) was hardly detectable in the youngest animals, while the intermediate age group showed a moderate lesion particularly in later stages of spermatogenesis. For the oldest animals, testicular damage was more severe, particularly in the earlier stages of spermatogenesis. Overall, the rapid elimination rate could account for the lack of 1,3-DNB toxicity in very young animals. However, simple metabolic differences were less likely to adequately explain the increase in testicular damage found as animal age increased from 75 to 120 days.

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

Abstract 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 μM); 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.

α-Chlorohydrin induced changes in sperm fertilizing ability in the rat: association with diminished sperm ATP levels and motility☆

In the present study, alpha-chlorohydrin (ACH) (5, 10, 25, 50 and 75 mg/kg, po) was administered to rats and the effects on sperm ATP levels, sperm motility, and the ability of sperm to bind and penetrate rat oocytes were determined. Groups of rats were killed 5 days and 3 h following treatment. At both time points, sperm from ACH-treated rats (>/=10 mg/kg) had significantly lower levels of ATP when diluted in media containing glucose. No diminution of ATP was seen in sperm diluted in phosphate-buffered saline (PBS). Computer analysis of sperm motility indicated that straight-line velocity (VSL) was the most sensitive parameter to ACH treatment and was significantly decreased in rat sperm three hours after ACH exposure (25 mg/kg). A clear drop in percent penetration (35% vs. 85% in control) of zona-free rat oocytes by rat sperm of both ACH groups was observed at 10 mg/kg. Higher dose levels produced no significant further decrease in percent penetration. Overall, the fertilizing ability of sperm was highly sensitive to ACH doses that caused minor but significant changes in sperm ATP levels and no significant changes in motility. These data are consistent with the spermatozoans need for an uncompromised energy supply to maintain its ability to bind and penetrate the oocyte.

Automated analysis of toxicant-induced changes in rat sperm head morphometry☆

An automated sperm morphometry analysis (ASMA) instrument was developed to obtain measurements of toxicant-induced changes in rat sperm head morphometry. 1,3-dinitrobenzene (1,3-DNB), a testicular toxicant known to affect sperm parameters, was used. Twelve-week-old Sprague-Dawley rats were allocated to a control (C) and to two 1,3-DNB treatment groups (T1 = 15 mg/kg; T2 = 25 mg/kg). 1,3-DNB was administered as a single dose by gavage, and animals were sacrificed 22 days after exposure. Sperm were collected, and morphology smears were made by a standard method. One hundred sperm heads were digitized on each slide, and 8 metric measurements were automatically reported. All measurements tended to decrease in a dose-dependent manner with increasing doses of 1,3-DNB. All values for total width (Wa) and interior width (W(e)) were significantly different from one another. Wa/L was significantly larger in the control than in T1 or T2, and symmetry (S = Wb/Wa) was significantly smaller in the control than in T1 or T2. Multivariate cluster analysis revealed three subpopulations that were also visually distinct. Subpopulation no. 1 was normal, based on published descriptions of normal rat sperm; subpopulation no. 2 was abnormal with a flattened curvature and a normal length; subpopulation no. 3 was abnormal with a foreshortened length and a flattened curvature. T1 and T2 contained significantly more sperm from subpopulation no. 2 and no. 3 than C (T1 = 22% and T2 = 34% vs. C = 8% by cluster analysis). C had 93% normal sperm, while the treatments had 78% and 66%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Male Reproductive Toxicity of Trichloroethylene: Sperm Protein Oxidation and Decreased Fertilizing Ability

Abstract The objective of the present study was to characterize and investigate potential mechanisms for the male reproductive toxicity of trichloroethylene (TCE). Male rats exposed to TCE in drinking water exhibited a dose-dependent decrease in the ability to fertilize oocytes from untreated females. This reduction in fertilizing ability occurred in the absence of treatment-related changes in combined testes/epididymides weight, sperm concentration, or sperm motility. In addition, flow cytometric analysis showed that there were no treatment-related differences in sperm mitochondrial membrane potential or acrosomal stability. TCE caused slight histological changes in efferent ductule epithelium, coinciding with the previously reported ductule localization of cytochrome P450 2E1. However, no alterations were noted in the testis or in any segment of the epididymis. Because there were no treatment-related changes to sperm indices and no clear pathological lesions to explain the reduced fertilization, the present study investigated TCE-mediated sperm oxidative damage. Oxidized proteins were detected by immunochemical techniques following the derivatization of sperm protein carbonyls with dinitrophenyl hydrazine. Immunochemical staining of whole, intact sperm showed the presence of halos of oxidized proteins around the head and midpiece of sperm from TCE-treated animals. The presence of oxidized sperm proteins was confirmed by Western blotting using in vitro-oxidized sperm as a positive control. Thiobarbituric acid reactive substances analyses showed a dose-dependent increase in the level of lipid peroxidation in sperm from treated animals, as well. Oxidative damage to sperm may explain the diminished fertilizing capacity of exposed animals and provide another mechanism by which TCE can adversely affect reproductive capabilities in the male.

Stage-specific effects of the fungicide carbendazim on Sertoli cell microtubules in rat testis

The aim of the present study is to provide a morphological explanation of carbendazim (CBZ)-induced sloughing of germ cells that occurs in a stage-specific manner. Therefore, very early alterations in the seminiferous tubule epithelium were examined histologically in the rat testis after oral administration of CBZ (400mg/kg). Gaps between the elongated and round spermatids, the first indication of germ cell sloughing (pre-sloughing), were observed in stage late VI-early VII seminiferous tubules at 90-min post-treatment. Tubulin immunoreaction in the Sertoli cells was reduced in intensity in tubules with pre-sloughing. However, electron microscopy demonstrated that there were some intact microtubules in these cells. At 120 min, sloughing was seen in stage late VI-early VII and XIII-XIV. Tubulin immunoreaction in the Sertoli cells was greatly decreased in intensity in tubules where cell sloughing was observed. Electron microscopy showed that there were few microtubules in the body region of these cells. Stages II-V and mid-VII-VIII were exempt from the sloughing effect at 180 min. These changes in microtubules were not observed in Sertoli cells that did not exhibit sloughing characteristics, regardless of the post-treatment intervals. The present results suggest that stage specificity of sloughing is due to the stage-specific susceptibility of Sertoli cell microtubules to CBZ.

Role of Testis Exposure Levels in the Insensitivity of Prepubertal Rats to Carbendazim-Induced Testicular Toxicity☆☆☆

Our recent studies have indicated that benomyl (BNL)-induced testicular toxicity is mediated by its major metabolite carbendazim (CBZ). The present study has used CBZ to investigate hypotheses that could explain prepubertal insensitivity to BNL. When CBZ (164 mg/kg intraperitoneally) was administered to postpubertal and prepubertal rats, it caused little testicular damage in prepubertal rats, but in adult rats, sloughing of the seminiferous epithelium resulted. When the inhibitory effect of CBZ on prepubertal testicular microtubule assembly was compared with that on postpubertal assembly, the IC50 values were very similar. Pharmacokinetic studies revealed that blood levels of CBZ were comparable in the two age groups; however, higher levels of CBZ were found in the adult testes (210.52 nmol/g wet wt) in comparison with young testes (67.77 nmol/g wet wt). These data suggest that delivery to and/or retention of CBZ in the testis may play a role in the age-dependent differences in susceptibility to CBZ toxicity. When CBZ was administered intratesticularly to reach levels sufficient to cause damage, the young animals did show an increased incidence of vacuolization and detachment of the seminiferous epithelium; however, in contrast to the older animals, sloughing of the seminiferous epithelium was not observed in the prepubertal animals. Overall, the low levels of CBZ measured in the testes of prepubertal animals offer a partial explanation for the insensitivity of young animals to CBZ-induced testicular toxicity following intraperitoneal administration. A differential responsiveness between the two age groups is also likely, however, since prepubertal animals lack elongated spermatids and it is sloughing of this cell type that characterizes CBZ-induced testicular toxicity in the adult.

In vitro fertilization after in vivo treatment of rats with three reproductive toxicants.

One objective of these experiments was to establish a sensitive assay to evaluate fertilizing potential of rat gametes in vitro. A second objective was to evaluate this in vitro fertilization (IVF) assay as a method to detect in vivo effects of reproductive toxicants on male and female gametes using three known reproductive toxicants as model systems. The IVF assay with zona-free oocytes was more precise than the assay with cumulus-intact oocytes in these studies (coefficients of variation of 8.7 and 14.4%, respectively). Sperm fertilizing potential for zona-free oocytes was reduced by treatment of rats with m-dinitrobenzene (10-10 000 microg/kg) and ethylene glycol monomethyl ether (50-100 mg/kg) that had no effect on sperm motility. Molinate (60 mg/kg for 5 days) reduced sperm fertilizing potential concurrently with reductions in sperm motility. Neither molinate (60 mg/kg for 5 days) nor dinitrobenzene (0.002% in the drinking water for 14 days) administered to females seemed to affect the fertilizability of their oocytes. Ethylene glycol monomethyl ether treatment (0.15-0.25% in the drinking water for 14 days) reduced the number of ovulated oocytes. IVF is a means to evaluate toxicant effects on female gametes and demonstrates sperms ability to interact with the oocyte plasma membrane.