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Histological alterations in ovaries of pubertal female rats induced by triphenyltin
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Experimental and Toxicologic Pathology 60 (2008) 313–321 www.elsevier.de/etp
Histological alterations in ovaries of pubertal female rats induced by triphenyltin B. Watermanna,, K. Groteb, K. Gnassa, H. Kolodzeya, A. Thomsena, K.E. Appeld, D. Candia-Carnevalie, U. Schulte-Oehlmannc a
LimnoMar, Hamburg, Bei der Neuen Muenze 11, D-22145 Hamburg, Germany Institute of Clinical Pharmacology and Toxicology, Charite´ University Medical School Berlin, Garystr, 5, 14195 Berlin, Germany c Department of Ecology and Evolution-Ecotoxicology, Johann Wolfgang Goethe University Frankfurt, Siesmayerstr. 70, D-60054 Frankfurt a.M, Germany d Center for Experimental Toxicology, Federal Institute for Risk Assessment, Thielallee 88-92, 14195 Berlin, Germany e Dipartimento di Biologia Luigi Gorini, Universita’ degli Studi di Milano (University of Milano), Via Celoria 26, 20133 Milano, Italy b
Received 6 August 2007; accepted 31 March 2008
Abstract Triphenyltin is an organotin compound that has been used extensively as an antifouling biocide and as an agricultural pesticide. Certain organotin compounds act as endocrine-active agents and have been reported to affect reproduction in mollusks and mammals. Here we studied the histopathological effects of 2 or 6 mg triphenyltin chloride (TPTCl)/kg b.w. on the reproductive tissue and the thymus of female pubertal rats as part of a comprehensive pubertal assay. Beginning at postnatal day (PND) 23 female Wistar rats were treated daily per gavage until their first estrus after PND 53. Reproductive organs were removed and histologically evaluated. While no histological changes were observed in oviduct, uterus, vagina and mamma, an increase in the number of all follicle stages occurred at both dose levels. Furthermore, exposure to 2 mg TPTCl/kg b.w. led to a significant reduction in the diameter of tertiary follicles. A significant increase in the number of atretic follicles was observed in tertiary and preovulatory follicles after exposure to 6 mg TPTCl. The thymus displayed a decreased number of apoptotic cells in both dose groups. We conclude that peripubertal administration of 2 and 6 mg TPTCl/kg b.w. caused effects on ovarian follicles of female rats. r 2008 Elsevier GmbH. All rights reserved. Keywords: Triphenyltin; Puberty; Female rat; Reproduction; Ovary; Endocrine-active compounds
Introduction Organotin compounds (OTC) are chemicals widely used for numerous purposes such as industrial catalysts and stabilizers and technical or agricultural biocides. Corresponding author. Tel.: +49 40 678 99 11; fax: +49 40 679 92 04. E-mail address: [email protected] (B. Watermann).
0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.03.009
Some trisubstituted organotins have biocidal properties and have been used extensively as antifoulants in ship paints as algaecides and molluscicides. They are leaching into the aquatic systems, resulting in bioconcentration and subsequent exposure of humans via fish and seafood. Certain OTCs are endocrine-active compounds and have shown to induce the so-called imposex phenomenon that is characterized by the development of male sex characteristics in female snails (Gibbs et al.,
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1991a, b; Oehlmann et al., 1996, 1999; Matthiessen and Gibbs, 1998; Schulte-Oehlmann et al., 2000). The suggested underlying mechanism of action is the inhibition of the conversion of androgens to estrogens mediated by the aromatase cytochrome P450 enzyme. There is an evidence that exposure to these OTCs can also affect female and male reproduction in mammals. Exposure of male and female pubertal rats to trisubstituted OTCs resulted in effects on reproductive organ weights and sexual hormone concentrations (Grote et al., 2004, 2006). Furthermore, in a recent study a shift in the day of parturition was observed in rat dams that were exposed to TPT during gestation and lactation (Grote et al., 2007). Administration of phenyltins during early pregnancy caused implantation failure as well as increased resorption and postimplantation loss in rats (Noda et al., 1992; Ema and Miyawaki, 2002; Adeeko et al., 2003). Changes in ovarian weight as well as uterine weight and serum progesterone concentrations were observed after exposure of rats to TBT and TPT, respectively (Wester et al., 1990; Ema et al., 1999; Ogata et al., 2001). Although the reproductive toxicity of TPT has been studied extensively during its registration process as a pesticide, there are several unclear aspects regarding its mechanism of action. The results presented here, are part of a comprehensive study that was carried out with the aim to investigate possible hormone-like effects of TPT on the neuro-endocrine axis during the sensitive period of puberty with possible influence on female reproductive development of rats. For this purpose a modified protocol of the rodent 20-day thyroid/pubertal female assay was applied, which is recommended by the US EPA for the evaluation of socalled endocrine disruptors (Goldman et al., 2000). This assay was designed by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) for the detection of chemicals that are agonists or antagonists to the estrogen or androgen receptors, that can alter steroidogenesis, disturb the hypothalamic–pituitary– gonadal axis or alter thyroid hormone function (EDSTAC, 1998). In this paper, the effects of peripubertal treatment with two doses of TPT on the tissues of ovaries, uterus, oviduct, vagina and mammary gland as well as on the thymus of female rats are evaluated by histological sections.
cology and Toxicology, Department of Toxicology, Benjamin Franklin Medical Center under specificpathogen-free (SPF) conditions in climate-controlled rooms. Twenty animals for each dosage group were kept together in Type IV Macrolons cages. Softwood granulate 8–15s (econ. Altromin, Lage, FRG) was used as bedding for the animals. The rats were housed at a constant light cycle (12 h of light, 12 h of darkness). Relative humidity was 5075% at a room temperature of 2171 1 C. Animals received autoclaved commercial diet (Altromin s1324, Fa. Altromin Lage, FRG) and tap water ad libitum.
Materials and methods
Quantification and measurement of follicles
Animals and animal husbandry
For the quantification and measurement of follicles the ovaries were completely cut in multiple sections with intervals of 50 mm. The number of follicles was determined in 7–16 serial sections (the variation in section number was due to large differences in ovary
Female weanling Wistar rats (HsdCpb:WU) were purchased from Harlan Winkelmann, Borchen, FRG. Animals were kept at the Institute of Clinical Pharma-
Animal dosing and treatment period Animals were weighed 2 days after weaning (23 days of age) and randomized into two treatment groups and one control group with approximately equal mean body weights and variances. Animals were treated daily from 23 days of age at dose levels of 2 or 6 mg TPT/kg b.w./ day that were chosen to avoid general toxicity. Triphenyltin chloride (purity ¼ 97.0%) was purchased from Sigma-Aldrich Chemie GmbH, Steinheim, FRG. The substance was dissolved in pharmaceutical peanut oil and administered per gavage at a volume of 5 ml/kg. The control group received pharmaceutical peanut oil only. The animals were treated until their first estrous cycle after PND 53 and subsequently sacrificed in estrus determined through vaginal smear. Animals were killed by decapitation and trunk blood was collected. Reproductive organs and thymus of nine animals per dose group were removed, placed into Bouin’s fixative and subsequently evaluated by light microscopy.
Histology, histometry and immunohistochemistry One ovary per animal, oviduct, uterus, vagina, mammary gland and thymus were fixed in Bouin’s solution for 24 h and then transferred to 80% ethanol. The organs were trimmed according to the procedure outlined for the Registry of Industrial Toxicology Animal-data (Bahnemann et al. 1995). After dehydration the fixed tissue samples were embedded in paraffin and cut in sections of 2–3 mm. For routine histology sections were stained with haematoxylin–eosin (HE).
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size) of all animals in each group. The number of follicles was counted in sections where the oocyte nucleus was present. The diameter was measured in follicles of four randomly selected animals, by using an image analysis system (Kontron, Carl Zeiss). The maximum diameter at right angles to it was used to obtain a mean diameter for each such follicle (Hirshfield and Midgley, 1978).
Quantification of mitotic granulosa cells and of atresia in ovaries and thymus The number of mitotic granulosa cells was determined in one proliferating cell nuclear antigen (PCNA)immunolabelled and hematoxylin-counterstained section per animal of all follicles irrespective of the stage. These sections used as the mitotic stages could be easily counted. The atretic index of the ovary was determined in two ways (1) Normal and atretic follicles were counted in the HEstained serial sections for all follicle stages except the primordial stage. The determination of atresia in primordial follicles was disregarded due to incertainty of error-free determination. Atretic follicles were diagnosed based on the following cellular criteria: shrinking of nucleus, blebbing of nucleus, pyknotic nuclei of granulosa cells, infiltration of blood cells into the follicle. (2) In a subsample of 2–4 rats per group the atretic index was determined in all follicle stages by counting the atretic follicles in two ss-apostainlabelled sections per animal. The atretic index in the ovary was calculated dividing the number of atretic follicles by the total number of follicles. In addition, the number of apoptotic cells was calculated in apostain-immunolabelled sections of the thymus of three randomly selected rats per group, counting the apoptotic thymocytes in five powerfields per animal at a magnification of 400.
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41:200 in PBS. One hundred microlitres of EnVision+( Peroxidase, Mouse, Ready-to-use; DAKO no. K4000) was added followed by incubation for 30 min in a humidity chamber and subsequently rinsed with PBS. Afterwards, application of chromagen solution NovaRED and incubation in a humidity chamber for 15 min followed by washing in aqua dest for 5 min (NovaRED Subtrate Kit—for Peroxidase—Vector no. SK-4800) was carried out. Counterstaining was performed with haematoxylin. (2) Monoclonal antibodies to single-stranded DNA are specific and sensitive cellular markers of apoptosis which detect cells even in the early stage of apoptosis (Frankfurt et al., 1996) 100 ml of monoclonal antibody F7-26 (Alexis no. APO-20A-078-L001) was added to deparaffinized 3–4 mm sections mounted on slides, incubated at room temperature in a humidity chamber for 30 min and rinsed in PBS. Afterwards, 100 ml of peroxidase-conjugated antimouse IgM (Zytomed no. 04 6820) was applied, again incubated for 30 min in a humidity chamber and finally rinsed with PBS. The working concentration of the second antibody, peroxidase-conjugated rat monoclonal antimouse IgM was 1:100 in PBS. Slides were stained with chromagen solution NovaRED and incubated in a humidity chamber for 15 min, followed by washing in aqua dest for 5 min. Counterstaining was done with haematoxylin.
Statistical analysis The statistical evaluation was performed using GraphPad Prism version 4 for Windows, GraphPad Software, San Diego, California, USA. For the statistical evaluation of the measured follicle diameter the ANOVA test followed by Dunett’s multiple comparison test was applied. For the statistical evaluation of the number of follicles per ovary, the numbers of mitotic granulosa cells in follicles, the atretic index and the number of apoptotic cells in thymus the Kruskal–Wallis test with Dunn’s multiple comparison test was applied. For statistical confirmation a probability error of 5% (po0.05) was defined.
Immunohistochemical methods (1) PCNA is an auxiliary protein of DNA polymerases and enzymes necessary for DNA synthesis and used as a marker of proliferation (Oktay et al., 1995). In addition PCNA is involved in DNA repair. One hundred microlitres of the monoclonal mouse antiPCNA (PCNA clone PC 10; DAKO no. M 0879) was applied to deparaffinized 3–4 mm sections mounted on slides, and incubated at room temperature in a humidity chamber for 30 min and then rinsed in PBS. Working concentration (PCNA) ¼
Results No differences have been observed among control and treated animals by histological evaluation of oviduct, uterus, vagina and mamma (data not shown). In contrast, several alterations in the ovary of exposed rats were observed and semi-quantified. The number of follicles per ovary counted in serial sections of all animals, revealed significant differences between control
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and rats exposed to 2 and 6 mg TPT in primary follicles and in preovulatory follicles for the 6 mg TPT dose group (Fig. 1). In secondary and tertiary follicles an increase in number was encountered as well, but was not found to be statistically significant. The diameter of primary and secondary follicles was not altered compared with the control (Fig. 2). A reduction in the diameter of tertiary follicles in the exposure groups was observed with statistical significance for the 2 mg/kg TPT group. The diameter of preovulatory follicles was decreased in the 2 mg/kg TPT group, but not in a statistically significant manner.
The mitotic activity in granulosa cells, expressed as the number of PCNA-positive cells per follicle, exhibited a marked but not statistically significant decrease in the 2 mg/kg group compared to the control, whereas after administration of 6 mg TPT/kg b.w. only a minor decrease was observed (Fig. 3). In sections immuno-labelled for the demonstration of PCNA, no difference in intensity or the number of labelled granulosa cells could be stated between the control and the exposure groups. The nuclei of intact oocytes were labelled in all groups in a comparable intensity. In contrast, granulosa cells with pyknotic
Fig. 1. Number of follicles per ovary of rats after treatment with TPT; median and interquartile range; number of animals in each group: N ¼ 9, *po0.05 and **po0.01.
Fig. 2. Diameter of follicles in mm, mean & SEM, measured in four animals per group, *po0.01.
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nuclei in atretic follicles were not labelled irrespective of exposure (data not shown). Atretic follicles of rats of the control group (Fig. 4) and of the exposure groups did not show structural differences. In some of the animals of the 2 mg/kg group a more irregular pattern of atresia and more residual bodies could be observed (Fig. 5). The atretic index evaluated in HE-stained sections revealed an increase in both treatment groups compared to the control in all follicle stages evaluated. The
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increase was statistically significant in tertiary and preovulatory follicles in the 6 mg TPT group (Fig. 6). The atretic index established on a subsample of sections immunolabelled for single-stranded DNA corroborated the findings yielded on HE sections. The thymus of all rats displayed no pathological alterations, but differences in the rate of apoptosis between control and exposed animals were observed. The number of apoptotic cells per thymus seems to decrease in the rats treated with both doses (Fig. 7).
Discussion
Fig. 3. Number of mitotic granulosa cells per follicle, median and interquartile range, N ¼ 9, no significant differences.
During female reproductive life, most ovarian follicles undergo a degenerative process called atresia at some stage of their development, and only few follicles reach the ovulatory stage. Several atretogenic (enhancing apoptosis) and antiatretogenic (inhibiting apoptosis) factors have been identified (Durlinger et al., 2000) with estrogens suppressing follicular atresia and androgens enhancing atresia. Under physiological conditions the majority of atresia occurs in secondary and tertiary follicles, while preovulatory follicles undergo a low rate of atresia (Kaipia and Hsueh, 1997). This situation was present in the control group whereas in the exposure group the atretic index was significantly elevated. Along with this elevation a more irregular segmentation of the ooplasm was observed in atretic follicles of the exposure
Fig. 4. Atretic tertiary follicle of female rat of the control group with segmented ooplasm of oocyte (soc) surrounded by granulosa cells (gr), bar ¼ 10 mm, HE.
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Fig. 5. Atretic tertiary follicle of female rat treated with 2 mg TPT/kg with segmented ooplasm of oocyte (soc) and residual bodies (arrows), surrounded by granulosa cells (gc), an ¼ antrum, bar ¼ 10 mm, HE.
Fig. 6. Atretic index of follicles ¼ number of atretic follicles/total number of follicles; mean and SEM; number of animals in each group: N ¼ 9, po0.05 and **po0.01.
groups compared to the controls. The significance of this observation deserves a more detailed investigation. Effects of chemicals on reproduction may be due to direct impact on reproductive organs and/or due to indirect effects on homeostasis of sexual hormone concentrations and their regulation. In the ovarian
follicles of rats, androgens are utilized as substrates for estrogen synthesis, act via androgen receptor (AR) as an enhancer of follicular differentiation and as an inhibitor of folliculogenesis. Androgens produced in increasing amounts by thecal cells under LH stimulation as follicular development progresses, serve as an obligatory
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Apoptotic cells in thymus
number of apoptotic cells
10 8 6 4 2 0 ctrl
2 mg/kg
6 mg/kg
Fig. 7. Apoptotic cells in thymus per power field at magnification of 400, five microscopic fields per animal, N ¼ 3.
substrate for cytochrome P450 aromatase-catalyzed estrogen synthesis in granulosa cells. It is assumed that the final stage of follicular development is accompanied with a loss of AR receptors in granulosa cells involving a shift in androgen utilization from direct action via AR to increase metabolism via cytochrome P450 aromatase to estrogens (Tetsuka and Hillier, 1997). It is assumed that for healthy follicular development, a smooth transition of androgen utilization from action via AR to metabolism and production of estrogens via cytochrome P450 aromatase is necessary. Disturbance in folliculogenesis and maturation can be induced by withdrawal of estrogen or administration of androgens (Billig et al., 1993a, b, 1996). As mentioned above, PCNA labelling in oocytes was not used as marker of proliferation due to the fact that oocytes are meiotically arrested. Instead PCNA labelling was used as an indicator of DNA repair or preparation of the oocytes for multiple mitoses after fertilization (Muskhelishvili et al., 2005). No difference in the intensity of labelling in relation to the control group could be stated. In contrast, labelling of granulosa cells with PCNA was used as proliferation marker, and also revealed no differences in labelling intensity of exposed animals compared to the control. Peripubertal treatment of female rats with TPT in this study resulted in a statistically significant increase in the number of primary and preovulatory follicles in both exposure groups. In parallel, a statistically significant decrease in follicle size in tertiary follicles after a dose of 2 mg TPT corresponded to a reduction of the mitotic activity of granulosa cells in relation to the control. TPT exposure probably exerted a stimulating effect on follicle development, but follicle progression and maturation was impaired. This would explain the significant increase in the atretic index in both exposure groups in relation to the control.
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As the number of primordial follicles is defined prenatally and no further multiplication takes place after birth, the increase in the number of primary to preovulatory follicle stages might be due to the mentioned enhanced follicle development. These findings correspond with the statistically significant increase in ovarian weight at PND 53 (control: 9079; 2 mg TPTCl: 106711; 6 mg TPTCl: 111712 mg, mean7SD) previously described by Grote et al. (2006). In addition, the increase of atretic follicles in the ovary may correspond to the measured inhibition of aromatase activity in the ovary (Grote et al., 2006). The observed alterations are in agreement with results reported by Newton and Hays (1968), who observed a decreased number of mature follicles and an increased incidence of atresia in early follicle growth in the ovaries of female rats after administration of 20 mg/kg b.w. of triphenyltin acetate and triphenyltin chloride. On the other hand, the lacking inhibition of aromatase activity in the brain and the increase in estrogen levels with increasing TPT concentrations in serum reported earlier (Grote et al., 2006) correspond with the decrease of apoptotic cells in the thymus. It is well known that circulating estrogens act as inhibitors of apoptosis (Billig et al., 1993a, b). However, this observation is in contrast to the reports that TBTO can cause an increase of apoptosis in the thymus of rats (Raffray et al., 1993). In this study the rate of atresia expressed as apoptotic index increased with increasing dose of TPT and predominantly affected tertiary and preovulatory follicles. Atresia of follicles occurred as apoptotic cell death displaying the special features typical for oocytes (Devine et al., 2000). The fragmentation/segmentation of apoptotic oocytes in the ovary as well as in the fallopian tubes is regarded as an organ-specific phenomenon which occurs during ovulation and can be induced by androgens like testosterone (Shinohara, 1981; Shinohara and Matsuda, 1982). There is a substantial evidence that fragmentation and death of ovulated murine oocytes is due to apoptosis (Perez et al., 1999). Apoptosis in follicles is slightly different to the classical apoptotic cascade in other tissues. Atretic follicles from primary through antral follicles display pyknotic, darkly staining granulosa cells losing the contact to the oocyte. At the same time the proliferation activity of granulosa cells decreases during follicular atresia, (Durlinger et al., 2000) a process that could be confirmed in this study. We conclude that daily administration of 2 or 6 mg TPT/kg b.w. resulted in effects on different cell and tissue levels with predominant effects in the ovary of peripubertal female rats, which might have been caused by impact of TPT on the sexual hormone homeostasis. The histological evaluation of ovaries and thymus of female rats exposed daily to 2 and 6 mg TPT revealed a variety of effects on the cellular and tissue level mainly
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indicating an endocrine action of TPT, leading to a hormonal imbalance, evident as a disturbance in ovarian follicular development. In relation to the target organ and the concentration of TPT, the action was directed as well into an androgenic as an antiandrogenic effect. The increase in the number of all follicle stages at both dose levels may reflect an antiandrogenic effect. Whereas the significant increase in the number, and the reduction of the diameter of tertiary and preovulatory follicles may reflect an androgenic effect. Compared with the results regarding sexual development, (Grote et al., 2006) complex effects on the ovaries can be stated as well.
Acknowledgments This work was supported by the Federal Institute for Risk Assessment, Berlin, Germany and the EU Commission in the framework of the Research Project COMPRENDO. The authors would like to thank Heike Marburger for her technical assistance in this study.
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Histological alterations in ovaries of pubertal female rats induced by triphenyltin B. Watermanna,, K. Groteb, K. Gnassa, H. Kolodzeya, A. Thomsena, K.E. Appeld, D. Candia-Carnevalie, U. Schulte-Oehlmannc a
LimnoMar, Hamburg, Bei der Neuen Muenze 11, D-22145 Hamburg, Germany Institute of Clinical Pharmacology and Toxicology, Charite´ University Medical School Berlin, Garystr, 5, 14195 Berlin, Germany c Department of Ecology and Evolution-Ecotoxicology, Johann Wolfgang Goethe University Frankfurt, Siesmayerstr. 70, D-60054 Frankfurt a.M, Germany d Center for Experimental Toxicology, Federal Institute for Risk Assessment, Thielallee 88-92, 14195 Berlin, Germany e Dipartimento di Biologia Luigi Gorini, Universita’ degli Studi di Milano (University of Milano), Via Celoria 26, 20133 Milano, Italy b
Received 6 August 2007; accepted 31 March 2008
Abstract Triphenyltin is an organotin compound that has been used extensively as an antifouling biocide and as an agricultural pesticide. Certain organotin compounds act as endocrine-active agents and have been reported to affect reproduction in mollusks and mammals. Here we studied the histopathological effects of 2 or 6 mg triphenyltin chloride (TPTCl)/kg b.w. on the reproductive tissue and the thymus of female pubertal rats as part of a comprehensive pubertal assay. Beginning at postnatal day (PND) 23 female Wistar rats were treated daily per gavage until their first estrus after PND 53. Reproductive organs were removed and histologically evaluated. While no histological changes were observed in oviduct, uterus, vagina and mamma, an increase in the number of all follicle stages occurred at both dose levels. Furthermore, exposure to 2 mg TPTCl/kg b.w. led to a significant reduction in the diameter of tertiary follicles. A significant increase in the number of atretic follicles was observed in tertiary and preovulatory follicles after exposure to 6 mg TPTCl. The thymus displayed a decreased number of apoptotic cells in both dose groups. We conclude that peripubertal administration of 2 and 6 mg TPTCl/kg b.w. caused effects on ovarian follicles of female rats. r 2008 Elsevier GmbH. All rights reserved. Keywords: Triphenyltin; Puberty; Female rat; Reproduction; Ovary; Endocrine-active compounds
Introduction Organotin compounds (OTC) are chemicals widely used for numerous purposes such as industrial catalysts and stabilizers and technical or agricultural biocides. Corresponding author. Tel.: +49 40 678 99 11; fax: +49 40 679 92 04. E-mail address: [email protected] (B. Watermann).
0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.03.009
Some trisubstituted organotins have biocidal properties and have been used extensively as antifoulants in ship paints as algaecides and molluscicides. They are leaching into the aquatic systems, resulting in bioconcentration and subsequent exposure of humans via fish and seafood. Certain OTCs are endocrine-active compounds and have shown to induce the so-called imposex phenomenon that is characterized by the development of male sex characteristics in female snails (Gibbs et al.,
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1991a, b; Oehlmann et al., 1996, 1999; Matthiessen and Gibbs, 1998; Schulte-Oehlmann et al., 2000). The suggested underlying mechanism of action is the inhibition of the conversion of androgens to estrogens mediated by the aromatase cytochrome P450 enzyme. There is an evidence that exposure to these OTCs can also affect female and male reproduction in mammals. Exposure of male and female pubertal rats to trisubstituted OTCs resulted in effects on reproductive organ weights and sexual hormone concentrations (Grote et al., 2004, 2006). Furthermore, in a recent study a shift in the day of parturition was observed in rat dams that were exposed to TPT during gestation and lactation (Grote et al., 2007). Administration of phenyltins during early pregnancy caused implantation failure as well as increased resorption and postimplantation loss in rats (Noda et al., 1992; Ema and Miyawaki, 2002; Adeeko et al., 2003). Changes in ovarian weight as well as uterine weight and serum progesterone concentrations were observed after exposure of rats to TBT and TPT, respectively (Wester et al., 1990; Ema et al., 1999; Ogata et al., 2001). Although the reproductive toxicity of TPT has been studied extensively during its registration process as a pesticide, there are several unclear aspects regarding its mechanism of action. The results presented here, are part of a comprehensive study that was carried out with the aim to investigate possible hormone-like effects of TPT on the neuro-endocrine axis during the sensitive period of puberty with possible influence on female reproductive development of rats. For this purpose a modified protocol of the rodent 20-day thyroid/pubertal female assay was applied, which is recommended by the US EPA for the evaluation of socalled endocrine disruptors (Goldman et al., 2000). This assay was designed by the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) for the detection of chemicals that are agonists or antagonists to the estrogen or androgen receptors, that can alter steroidogenesis, disturb the hypothalamic–pituitary– gonadal axis or alter thyroid hormone function (EDSTAC, 1998). In this paper, the effects of peripubertal treatment with two doses of TPT on the tissues of ovaries, uterus, oviduct, vagina and mammary gland as well as on the thymus of female rats are evaluated by histological sections.
cology and Toxicology, Department of Toxicology, Benjamin Franklin Medical Center under specificpathogen-free (SPF) conditions in climate-controlled rooms. Twenty animals for each dosage group were kept together in Type IV Macrolons cages. Softwood granulate 8–15s (econ. Altromin, Lage, FRG) was used as bedding for the animals. The rats were housed at a constant light cycle (12 h of light, 12 h of darkness). Relative humidity was 5075% at a room temperature of 2171 1 C. Animals received autoclaved commercial diet (Altromin s1324, Fa. Altromin Lage, FRG) and tap water ad libitum.
Materials and methods
Quantification and measurement of follicles
Animals and animal husbandry
For the quantification and measurement of follicles the ovaries were completely cut in multiple sections with intervals of 50 mm. The number of follicles was determined in 7–16 serial sections (the variation in section number was due to large differences in ovary
Female weanling Wistar rats (HsdCpb:WU) were purchased from Harlan Winkelmann, Borchen, FRG. Animals were kept at the Institute of Clinical Pharma-
Animal dosing and treatment period Animals were weighed 2 days after weaning (23 days of age) and randomized into two treatment groups and one control group with approximately equal mean body weights and variances. Animals were treated daily from 23 days of age at dose levels of 2 or 6 mg TPT/kg b.w./ day that were chosen to avoid general toxicity. Triphenyltin chloride (purity ¼ 97.0%) was purchased from Sigma-Aldrich Chemie GmbH, Steinheim, FRG. The substance was dissolved in pharmaceutical peanut oil and administered per gavage at a volume of 5 ml/kg. The control group received pharmaceutical peanut oil only. The animals were treated until their first estrous cycle after PND 53 and subsequently sacrificed in estrus determined through vaginal smear. Animals were killed by decapitation and trunk blood was collected. Reproductive organs and thymus of nine animals per dose group were removed, placed into Bouin’s fixative and subsequently evaluated by light microscopy.
Histology, histometry and immunohistochemistry One ovary per animal, oviduct, uterus, vagina, mammary gland and thymus were fixed in Bouin’s solution for 24 h and then transferred to 80% ethanol. The organs were trimmed according to the procedure outlined for the Registry of Industrial Toxicology Animal-data (Bahnemann et al. 1995). After dehydration the fixed tissue samples were embedded in paraffin and cut in sections of 2–3 mm. For routine histology sections were stained with haematoxylin–eosin (HE).
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size) of all animals in each group. The number of follicles was counted in sections where the oocyte nucleus was present. The diameter was measured in follicles of four randomly selected animals, by using an image analysis system (Kontron, Carl Zeiss). The maximum diameter at right angles to it was used to obtain a mean diameter for each such follicle (Hirshfield and Midgley, 1978).
Quantification of mitotic granulosa cells and of atresia in ovaries and thymus The number of mitotic granulosa cells was determined in one proliferating cell nuclear antigen (PCNA)immunolabelled and hematoxylin-counterstained section per animal of all follicles irrespective of the stage. These sections used as the mitotic stages could be easily counted. The atretic index of the ovary was determined in two ways (1) Normal and atretic follicles were counted in the HEstained serial sections for all follicle stages except the primordial stage. The determination of atresia in primordial follicles was disregarded due to incertainty of error-free determination. Atretic follicles were diagnosed based on the following cellular criteria: shrinking of nucleus, blebbing of nucleus, pyknotic nuclei of granulosa cells, infiltration of blood cells into the follicle. (2) In a subsample of 2–4 rats per group the atretic index was determined in all follicle stages by counting the atretic follicles in two ss-apostainlabelled sections per animal. The atretic index in the ovary was calculated dividing the number of atretic follicles by the total number of follicles. In addition, the number of apoptotic cells was calculated in apostain-immunolabelled sections of the thymus of three randomly selected rats per group, counting the apoptotic thymocytes in five powerfields per animal at a magnification of 400.
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41:200 in PBS. One hundred microlitres of EnVision+( Peroxidase, Mouse, Ready-to-use; DAKO no. K4000) was added followed by incubation for 30 min in a humidity chamber and subsequently rinsed with PBS. Afterwards, application of chromagen solution NovaRED and incubation in a humidity chamber for 15 min followed by washing in aqua dest for 5 min (NovaRED Subtrate Kit—for Peroxidase—Vector no. SK-4800) was carried out. Counterstaining was performed with haematoxylin. (2) Monoclonal antibodies to single-stranded DNA are specific and sensitive cellular markers of apoptosis which detect cells even in the early stage of apoptosis (Frankfurt et al., 1996) 100 ml of monoclonal antibody F7-26 (Alexis no. APO-20A-078-L001) was added to deparaffinized 3–4 mm sections mounted on slides, incubated at room temperature in a humidity chamber for 30 min and rinsed in PBS. Afterwards, 100 ml of peroxidase-conjugated antimouse IgM (Zytomed no. 04 6820) was applied, again incubated for 30 min in a humidity chamber and finally rinsed with PBS. The working concentration of the second antibody, peroxidase-conjugated rat monoclonal antimouse IgM was 1:100 in PBS. Slides were stained with chromagen solution NovaRED and incubated in a humidity chamber for 15 min, followed by washing in aqua dest for 5 min. Counterstaining was done with haematoxylin.
Statistical analysis The statistical evaluation was performed using GraphPad Prism version 4 for Windows, GraphPad Software, San Diego, California, USA. For the statistical evaluation of the measured follicle diameter the ANOVA test followed by Dunett’s multiple comparison test was applied. For the statistical evaluation of the number of follicles per ovary, the numbers of mitotic granulosa cells in follicles, the atretic index and the number of apoptotic cells in thymus the Kruskal–Wallis test with Dunn’s multiple comparison test was applied. For statistical confirmation a probability error of 5% (po0.05) was defined.
Immunohistochemical methods (1) PCNA is an auxiliary protein of DNA polymerases and enzymes necessary for DNA synthesis and used as a marker of proliferation (Oktay et al., 1995). In addition PCNA is involved in DNA repair. One hundred microlitres of the monoclonal mouse antiPCNA (PCNA clone PC 10; DAKO no. M 0879) was applied to deparaffinized 3–4 mm sections mounted on slides, and incubated at room temperature in a humidity chamber for 30 min and then rinsed in PBS. Working concentration (PCNA) ¼
Results No differences have been observed among control and treated animals by histological evaluation of oviduct, uterus, vagina and mamma (data not shown). In contrast, several alterations in the ovary of exposed rats were observed and semi-quantified. The number of follicles per ovary counted in serial sections of all animals, revealed significant differences between control
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and rats exposed to 2 and 6 mg TPT in primary follicles and in preovulatory follicles for the 6 mg TPT dose group (Fig. 1). In secondary and tertiary follicles an increase in number was encountered as well, but was not found to be statistically significant. The diameter of primary and secondary follicles was not altered compared with the control (Fig. 2). A reduction in the diameter of tertiary follicles in the exposure groups was observed with statistical significance for the 2 mg/kg TPT group. The diameter of preovulatory follicles was decreased in the 2 mg/kg TPT group, but not in a statistically significant manner.
The mitotic activity in granulosa cells, expressed as the number of PCNA-positive cells per follicle, exhibited a marked but not statistically significant decrease in the 2 mg/kg group compared to the control, whereas after administration of 6 mg TPT/kg b.w. only a minor decrease was observed (Fig. 3). In sections immuno-labelled for the demonstration of PCNA, no difference in intensity or the number of labelled granulosa cells could be stated between the control and the exposure groups. The nuclei of intact oocytes were labelled in all groups in a comparable intensity. In contrast, granulosa cells with pyknotic
Fig. 1. Number of follicles per ovary of rats after treatment with TPT; median and interquartile range; number of animals in each group: N ¼ 9, *po0.05 and **po0.01.
Fig. 2. Diameter of follicles in mm, mean & SEM, measured in four animals per group, *po0.01.
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nuclei in atretic follicles were not labelled irrespective of exposure (data not shown). Atretic follicles of rats of the control group (Fig. 4) and of the exposure groups did not show structural differences. In some of the animals of the 2 mg/kg group a more irregular pattern of atresia and more residual bodies could be observed (Fig. 5). The atretic index evaluated in HE-stained sections revealed an increase in both treatment groups compared to the control in all follicle stages evaluated. The
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increase was statistically significant in tertiary and preovulatory follicles in the 6 mg TPT group (Fig. 6). The atretic index established on a subsample of sections immunolabelled for single-stranded DNA corroborated the findings yielded on HE sections. The thymus of all rats displayed no pathological alterations, but differences in the rate of apoptosis between control and exposed animals were observed. The number of apoptotic cells per thymus seems to decrease in the rats treated with both doses (Fig. 7).
Discussion
Fig. 3. Number of mitotic granulosa cells per follicle, median and interquartile range, N ¼ 9, no significant differences.
During female reproductive life, most ovarian follicles undergo a degenerative process called atresia at some stage of their development, and only few follicles reach the ovulatory stage. Several atretogenic (enhancing apoptosis) and antiatretogenic (inhibiting apoptosis) factors have been identified (Durlinger et al., 2000) with estrogens suppressing follicular atresia and androgens enhancing atresia. Under physiological conditions the majority of atresia occurs in secondary and tertiary follicles, while preovulatory follicles undergo a low rate of atresia (Kaipia and Hsueh, 1997). This situation was present in the control group whereas in the exposure group the atretic index was significantly elevated. Along with this elevation a more irregular segmentation of the ooplasm was observed in atretic follicles of the exposure
Fig. 4. Atretic tertiary follicle of female rat of the control group with segmented ooplasm of oocyte (soc) surrounded by granulosa cells (gr), bar ¼ 10 mm, HE.
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Fig. 5. Atretic tertiary follicle of female rat treated with 2 mg TPT/kg with segmented ooplasm of oocyte (soc) and residual bodies (arrows), surrounded by granulosa cells (gc), an ¼ antrum, bar ¼ 10 mm, HE.
Fig. 6. Atretic index of follicles ¼ number of atretic follicles/total number of follicles; mean and SEM; number of animals in each group: N ¼ 9, po0.05 and **po0.01.
groups compared to the controls. The significance of this observation deserves a more detailed investigation. Effects of chemicals on reproduction may be due to direct impact on reproductive organs and/or due to indirect effects on homeostasis of sexual hormone concentrations and their regulation. In the ovarian
follicles of rats, androgens are utilized as substrates for estrogen synthesis, act via androgen receptor (AR) as an enhancer of follicular differentiation and as an inhibitor of folliculogenesis. Androgens produced in increasing amounts by thecal cells under LH stimulation as follicular development progresses, serve as an obligatory
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Apoptotic cells in thymus
number of apoptotic cells
10 8 6 4 2 0 ctrl
2 mg/kg
6 mg/kg
Fig. 7. Apoptotic cells in thymus per power field at magnification of 400, five microscopic fields per animal, N ¼ 3.
substrate for cytochrome P450 aromatase-catalyzed estrogen synthesis in granulosa cells. It is assumed that the final stage of follicular development is accompanied with a loss of AR receptors in granulosa cells involving a shift in androgen utilization from direct action via AR to increase metabolism via cytochrome P450 aromatase to estrogens (Tetsuka and Hillier, 1997). It is assumed that for healthy follicular development, a smooth transition of androgen utilization from action via AR to metabolism and production of estrogens via cytochrome P450 aromatase is necessary. Disturbance in folliculogenesis and maturation can be induced by withdrawal of estrogen or administration of androgens (Billig et al., 1993a, b, 1996). As mentioned above, PCNA labelling in oocytes was not used as marker of proliferation due to the fact that oocytes are meiotically arrested. Instead PCNA labelling was used as an indicator of DNA repair or preparation of the oocytes for multiple mitoses after fertilization (Muskhelishvili et al., 2005). No difference in the intensity of labelling in relation to the control group could be stated. In contrast, labelling of granulosa cells with PCNA was used as proliferation marker, and also revealed no differences in labelling intensity of exposed animals compared to the control. Peripubertal treatment of female rats with TPT in this study resulted in a statistically significant increase in the number of primary and preovulatory follicles in both exposure groups. In parallel, a statistically significant decrease in follicle size in tertiary follicles after a dose of 2 mg TPT corresponded to a reduction of the mitotic activity of granulosa cells in relation to the control. TPT exposure probably exerted a stimulating effect on follicle development, but follicle progression and maturation was impaired. This would explain the significant increase in the atretic index in both exposure groups in relation to the control.
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As the number of primordial follicles is defined prenatally and no further multiplication takes place after birth, the increase in the number of primary to preovulatory follicle stages might be due to the mentioned enhanced follicle development. These findings correspond with the statistically significant increase in ovarian weight at PND 53 (control: 9079; 2 mg TPTCl: 106711; 6 mg TPTCl: 111712 mg, mean7SD) previously described by Grote et al. (2006). In addition, the increase of atretic follicles in the ovary may correspond to the measured inhibition of aromatase activity in the ovary (Grote et al., 2006). The observed alterations are in agreement with results reported by Newton and Hays (1968), who observed a decreased number of mature follicles and an increased incidence of atresia in early follicle growth in the ovaries of female rats after administration of 20 mg/kg b.w. of triphenyltin acetate and triphenyltin chloride. On the other hand, the lacking inhibition of aromatase activity in the brain and the increase in estrogen levels with increasing TPT concentrations in serum reported earlier (Grote et al., 2006) correspond with the decrease of apoptotic cells in the thymus. It is well known that circulating estrogens act as inhibitors of apoptosis (Billig et al., 1993a, b). However, this observation is in contrast to the reports that TBTO can cause an increase of apoptosis in the thymus of rats (Raffray et al., 1993). In this study the rate of atresia expressed as apoptotic index increased with increasing dose of TPT and predominantly affected tertiary and preovulatory follicles. Atresia of follicles occurred as apoptotic cell death displaying the special features typical for oocytes (Devine et al., 2000). The fragmentation/segmentation of apoptotic oocytes in the ovary as well as in the fallopian tubes is regarded as an organ-specific phenomenon which occurs during ovulation and can be induced by androgens like testosterone (Shinohara, 1981; Shinohara and Matsuda, 1982). There is a substantial evidence that fragmentation and death of ovulated murine oocytes is due to apoptosis (Perez et al., 1999). Apoptosis in follicles is slightly different to the classical apoptotic cascade in other tissues. Atretic follicles from primary through antral follicles display pyknotic, darkly staining granulosa cells losing the contact to the oocyte. At the same time the proliferation activity of granulosa cells decreases during follicular atresia, (Durlinger et al., 2000) a process that could be confirmed in this study. We conclude that daily administration of 2 or 6 mg TPT/kg b.w. resulted in effects on different cell and tissue levels with predominant effects in the ovary of peripubertal female rats, which might have been caused by impact of TPT on the sexual hormone homeostasis. The histological evaluation of ovaries and thymus of female rats exposed daily to 2 and 6 mg TPT revealed a variety of effects on the cellular and tissue level mainly
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indicating an endocrine action of TPT, leading to a hormonal imbalance, evident as a disturbance in ovarian follicular development. In relation to the target organ and the concentration of TPT, the action was directed as well into an androgenic as an antiandrogenic effect. The increase in the number of all follicle stages at both dose levels may reflect an antiandrogenic effect. Whereas the significant increase in the number, and the reduction of the diameter of tertiary and preovulatory follicles may reflect an androgenic effect. Compared with the results regarding sexual development, (Grote et al., 2006) complex effects on the ovaries can be stated as well.
Acknowledgments This work was supported by the Federal Institute for Risk Assessment, Berlin, Germany and the EU Commission in the framework of the Research Project COMPRENDO. The authors would like to thank Heike Marburger for her technical assistance in this study.
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