Phthalate esters have been extensively used as a plasticizer of synthetic polymers. Previous studies have revealed that some phthalate esters including di(n-butyl) phthalate (DBP) induce spermatogenic cell apoptosis, although its mechanism is not yet clear. The present study describes that disruption of Sertoli cell vimentin filaments by DBP administration may relate to spermatogenic cell apoptosis. The present histopathological study revealed that a single oral administration of 500 mg/kg DBP caused progressive detachment and displacement of spermatogenic cells away from the seminiferous epithelium and sloughing of them into the lumen. Degenerative spermatogenic cells characterized by chromatin condensation were frequently observed in DBP-treated rats. Ultrastructurally, the degenerative spermatogenic cells were separated from their neighbours, and a collapse of Sertoli cell vimentin filaments was recognized in DBP-treated rats. Sertoli cell cultures showed the increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, vimentin filaments clearly showed a gradual collapse in DBP-exposed Sertoli cells in vitro. These in vivo and in vitro experiments indicate that DBP-induced collapse of Sertoli cell vimentin filaments may lead to detachment of spermatogenic cells, and then detached cells may undergo apoptosis because of loss of the support and nurture provided by Sertoli cells.

The effects of mono(2-ethylhexyl) phthalate (MEHP) on 21-day-old C57Bl/6N mice and their Sertoli cell cultures were studied. Mice were given a single dose of 800 mg/kg MEHP by oral gavage and sacrificed 24 h later. At the same time, testes were harvested from another batch of mice for Sertoli cell cultures. Cultures were subsequently exposed to 0, 1, and 100 nmol/ml MEHP for 0, 3, 6, 12, and 24 h. An antivimentin antibody was used to detect intermediate filament changes in Sertoli cells. Meanwhile, detection of preapoptotic signals and presence of apoptotic cells were done using annexin V-FITC (fluorescein isothiocyanate) and TUNEL (deoxynucleotidyltransferase-mediated dUTP nick end labeling) analyses, respectively. In vivo results showed a correlation between the increase in TUNEL-positive cells and the vimentin disruption in treated mice. Toluidine blue staining of the Sertoli cell cultures showed the increased number and size of vacuoles in Sertoli cell cytoplasm. Vimentin immunohistochemistry showed gradual disappearance of vimentin in Sertoli cell cultures as time and dose increased. Some Sertoli cells were found to be annexin V-FITC positive, but no TUNEL-positive cells were found. Taken together, these results show that the appearance of vacuoles and the vimentin disappearance caused by MEHP in the Sertoli cells are related with each other and can be observed in relation to time. This can be used as an indicator of the loss of mechanical support for spermatogenic cells, which in the end causes apoptosis of spermatogenic cells.

The object of this study was to determine the details of morphological dynamics of spermatogenesis in Syrian hamsters exposed to both short photoperiod and low ambient temperature. Eight-week-old male hamsters, kept in a long photoperiod (14 h L, 10 h D), were transferred to a short photoperiod (6 h L, 18 h D) and kept there for 13 weeks to induce testicular regression. Some hamsters were then transferred from the room at 23 degrees C to that at 5 degrees C (5 degrees C group). Remaining hamsters were continuously kept at 23 degrees C (23 degrees C group). Thereafter, the morphology was examined. As a result, it took only 8 weeks until spermatogenesis recovered in the 23 degrees C group. However, it was not until 20 weeks that spermatogenesis was recognized in the 5 degrees C group. As the regulation of seasonal testicular activity is characterized by coordinated shifts in the relationships among mitosis, meiosis, and apoptosis, the changes in the proliferative and apoptotic activities were examined. Although no significant difference in proliferative activity of spermatogonia between the 5 degrees C and the 23 degrees C groups was confirmed, a notable increase in the rate of apoptosis was observed in the 5 degrees C group. Furthermore, this increase was more salient during the hibernation period. These findings suggest that both cold ambient temperature and hibernation caused the delay of testicular recrudescence and this delay arose from the increase of apoptotic activity but not the change in proliferative activity in spermatogonia in the 5 degrees C group.

No abstract

Summary: Di-iso-butyl phthalate (DiBP), a special plasticizer, is used as a substitute for di(n-butyl) phthalate(DBP). The effects of DiBP on testes in prepubertal rodents still remain to be obscure. Testicular toxicity of DiBP was investigated in 21-day-old Sprague-Dawley rats and C57BL/6N mice, using with in situ TUNEL method. For an acute exposure experiment, animals were once given DiBP at various concentrations by oral gavage. For a subchronic exposure experiment, they were daily given DiBP at various concentrations for consecutive 7 days. Controls were treated with corn oil under the same condition. For a recovery experiment, rats were once given DiBP (1000 mg/kg), and were sacrificed at day 1 to 8 after administration. Furthermore, the disorder of vimentin filaments in Sertoli cells after daily administration of DiBP (500 mg/kg) for consecutive 7 days in rats also identified by immunohistochemistry using anti-vimentin antibody. As a result, the present study demonstrated that DiBP can induce testicular atrophy in rats due to the increase of TUNEL-positive spermatogenic cells in both acute and subchronic exposure experiments. At the same time, the disorder of vimentin filaments in Sertoli cells was recognized. However, no such damages could be found in mouse testis. For the recovery experiment, the testis weight and testicular morphology returned to normal at day 6 after administration. In conclusion, the present study indicates that DiBP causes the significant increase of TUNEL-positive spermatogenic cells and the disorder of vimentin filaments in Sertoli cells in rats and that DiBP shows a species-specific toxicity.

No abstract

Mono(2-ethylhexyl) phthalate (MEHP) is a well-characterized testicular toxicant. In this study, morphological alterations of mice testes caused by repeated administrations of MEHP were examined by light and transmission electron microscopy. Prepubertal male mice were given a range of MEHP doses (600-900 mg/kg/day) for 3 consecutive days in corn oil by oral gavage. Control animals were given only corn oil. Thereafter, the testes were excised, fixed in 4% paraformaldehyde for light microscopy and/or 5% glutaraldehyde for transmission electron microscopy. Then, they were embedded, and sectioned. TUNEL analysis was done to quantify the occurrence of apoptosis in the testis. Cellular damages were also observed. Results showed that administration of 700 mg/kg of MEHP caused a significant increase in TUNEL-positive cells. At the same time, mice treated with higher doses of MEHP showed presence of degenerating (apoptotic and necrotic) spermatogenic cells. Appearance of small vacuoles in the Sertoli cell cytoplasm and displacement of spermatogenic cells were also observed. Sloughed and shed spermatogenic cells found in the tubular lumen were identified to be necrotic and apoptotic in appearance, respectively.

Summary: The phthalate esters have been used as plasticizers for various plastic products, and their testicular toxicity has been reported. In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of the phthalate esters, on prepubertal rat testes in vitro were examined. The testes of 20-day-old Sprague Dawley (SD) rats were cut into smaller pieces and seeded in medium, and then the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, TUNEL-positive spermatogenic cells were identified, and they gradually increased in number in time-and dose-dependent manners. Ultrastructurally, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still-functioning cell organelles, and packed cell contents in membrane-bounded bodies), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), apoptotic Sertoli cells (highly condensed nuclei and nuclear membrane lysis) and necrotic Sertoli cells (marginated chromatins along the nuclear membrane, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, based on transmission electron microscopic observations, MEHP treatment may affect spermatogenic cells, and lead them to necrosis. Thus, testicular tissue cultures and cell cultures are of advantageous for screening testicular toxicity of chemicals.Phthalic acid esters have already been proved as a potential compound to reduce fertility, and induce testicular atrophy in laboratory animals (Thomas and Thomas, 1984). One of the most widely-studied male reproductive toxicants in rats is di(2-ethylhexyl) phthalate (DEHP) (Richburg and Boekelheide, 1996). Albro et al. (1973), Rowland et al. (1977), Lake et al. (1977), and Thomas and Thomas (1984) have reported that after oral administration, phthalates are rapidly hydrolyzed in gut and other tissues by nonspecific esterases to produce the corresponding monoester. Pollack et al. (1985) also have reported that mono(2-ethylhexyl) phthalate (MEHP) is one of the most potently occurring monoester and an ultimate testicular toxic metabolite.Recently, research interest tends have changed from DEHP to MEHP. Although Richburg andBoekelheide (1996), andSuominen et al. (2003) have conducted MEHP experiments using laboratory animals (in vivo), there is still lack of MEHP testing using the testicular tissue culture (in vitro). The tissue culture is advantageous to clarify the direct effects of chemicals on target cells. Therefore, in this study, prepubertal Sprague-Dawley (SD) rat testes were used to ascertain the effects of MEHP on rat testicular tissue culture (in vitro), with specific focus on the morphological alterations of Sertoli and spermatogenic cells. Materials and MethodsBiochemical effects of mono(2-ethylhexyl) phthalate (MEHP) on Sertoli and spermatogenic cells in prepubertal male Sprague-Dawley rats (at