Effects of di-iso-butyl phthalate on testes of prepubertal rats and mice.

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.

An ultrastructural study on the effects of mono(2-ethylhexyl) phthalate on mice testes: cell death and sloughing of spermatogenic cells.

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.

An ultrastructural study on cytotoxic effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in Shiba goat in vitro.

In this study, the effects of mono(2-ethylhexyl) phthalate (MEHP), one of metabolites of di(2-ethylhexyl) phthalate, on immature Shiba goat testes in vitro were examined. The testes of 2-month-old Shiba goats were cut into smaller pieces, and seeded in medium. At 1, 3, 6 and 9 hr after administration of MEHP at various concentrations (0, 100 nmol ml(-1), 1 nmol ml(-1), and 1 x 10(-3) nmol ml(-1), respectively), the specimens were obtained for light and transmission electron microscopic observations. As a result, at 1 hr after exposure to MEHP, the vacuolization and nuclear membrane rupture appeared in Sertoli cells. Such alterations tended to gradually increase in number in time and dose-dependent manners. Moreover, by MEHP treatment, apoptotic spermatogenic cells (characterized with chromatin condensation, cytoplasm shrinkage without membrane rupture, still functioning cell organelles, and packed cell contents in membrane-bounded bodies), apoptotic Sertoli cells (characterized with nuclear membrane lysis, nuclear condensation), necrotic spermatogenic cells (characterized with swollen and ruptured mitochondria, plasma membrane lysis, spilt cell contents, and chromatin clumps), and necrotic Sertoli cells (characterized with marginated chromatins along the nuclear membrane, ruptured vesicles within the MNB, some swollen and ruptured cell organelles, e.g. mitochondria) could be identified. Conclusively, ultrastructurally the treatment with MEHP at low concentration tends to lead spermatogenic and Sertoli cells to apoptosis, whereas that at high concentration tends to lead spermatogenic and Sertoli cells to necrosis. Thus, the testicular tissue culture is advantageous for screening testicular toxicity of chemicals.

Effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in rats in vitro.

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.

Bisphenol A-induced morphological alterations in Sertoli and spermatogenic cells of immature Shiba goats in vitro: An ultrastructural study.

Background and aims: There is no information currently available regarding the effects of bisphenol A (BPA) on testes in ruminants. Therefore, to establish and clarify the effects of BPA in ruminants, testicular tissue cultures were obtained from immature Shiba goats. Methods: The testes of 2-month-old Shiba goats were cut into smaller pieces and seeded in medium. At 1, 3, 6 and 9 h after administration of various concentrations of BPA, the specimens underwent light and transmission electron microscopic observations Results: At 1 h after BPA treatment, vacuolization and nuclear membrane rupture appeared within the nucleoplasm and cytoplasm of Sertoli cells. Such alterations tended to gradually increase in number in time- and dose-dependent manners. Thus, because of BPA treatment, apoptotic spermatogenic cells, necrotic spermatogenic cells, apoptotic Sertoli cells and necrotic Sertoli cells could be identified. Particularly in the Sertoli cell, ruptured vesicles could be found within the multivesicular nuclear body. Conclusion: The treatment with BPA at a low concentration tends to lead spermatogenic and Sertoli cells to apoptosis, whereas a higher concentration tends to lead spermatogenic and Sertoli cells to necrosis. Therefore, this study showed that testicular tissue culture is an advantageous avenue for screening the testicular toxicity of chemicals in ruminants. (Reprod Med Biol 2004; 3: 205-210).

Single administration of di(n-butyl) phthalate delays spermatogenesis in prepubertal rats.

Morphological alterations in seminiferous tubules caused by single administration of di(n-butyl) phthalate (DBP) in 3-week-old rats were investigated throughout the first wave of spermatogenesis. Single administration of DBP (500 mg/kg) showed progressive detachment and displacement of spermatogenic cells and disappearance of tubular lumen at 3h after treatment, and then showed thin seminiferous epithelia and wide tubular lumen at day 1 (D1). At D1, quite significant numbers of apoptotic spermatogenic cells were detected, and then they gradually decreased in accordance with the passage of time. In contrast, the testes revealed lower weight gain, even after completion of first wave of spermatogenesis in the DBP-treated group, compared to the control. In order to clarify whether spermatogenic cells differentiate into mature spermatids in the DBP-treated rats, immunohistochemical staining for Hsc 70t, a specific marker for elongate spermatids, was carried out. As a result, the decrease in mature spermatids in the DBP-treated testes, compared to the control, was demonstrated. For example, at D20 (41-day-old) after treatment, the most advanced spermatids in the tubules from rats in the DBP-treated groups were steps 2-4, while those of the control were steps 12-13. Moreover, in some tubules, pachytene spermatocytes were the most advanced spermatogenic cell. At D30 (51-day-old) after treatment, maturation of spermatogenic cells in the DBP-treated rats proceeded further, and the most advanced spermatids in tubules were steps 8-9, while those of the control were steps 15-19. These results lead us to the postulation that a single administration of DBP to prepubertal rats delays maturation of spermatogenic cells, even after completion of first wave of spermatogenesis.

Disappearance of vimentin in Sertoli cells: a mono(2-ethylhexyl) phthalate effect.

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.

Expression pattern of alphavbeta3 and alphavbeta5 integrin mRNA in mouse fetal gonads.

The alphavbeta3 and alphavbeta5 integrins are known as transmembrane receptors capable of binding to the RGD amino acid peptide sequence. In mouse early gonadogenesis, some proteins containing the RGD sequence are deposited into extracellular space and participate in morphogenesis. We analyzed the expression patterns of the alphavbeta3 and alphavbeta5 integrins in mouse developing gonads (10.5-13.5 days post coitum) using whole-mount in situ hybridization. The alphav integrin mRNA was homogenously expressed in developing gonadal regions. On the other hand, the beta3 integrin mRNA was found only in large and round cells (presumptive germ cells), whereas beta5 integrin was localized in gonadal somatic cells, with the exception of coelomic epithelial cells. The beta3 integrin-expressed cells were determined to be primordial germ cells because the number of these cells was drastically reduced in busulfan-treated gonads. In this study, we demonstrated that the alphavbeta3 and alphavbeta5 integrins are widely localized in the mouse developing gonads and discussed their presumptive functions on mouse gonadogenesis.