See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/269723786 Sexual maturation and fertility of mice exposed to triphenyltin during prepubertal and pubertal periods Article  in  Toxicology Reports · December 2014 DOI: 10.1016/j.toxrep.2014.12.006 CITATIONS 4 READS 49 7 authors, including: Some of the authors of this publication are also working on these related projects: STPs in Latin America View project Phytochemistry and Pharmacology of Solanum View project Ana Paula A Favareto Universidade do Oeste Paulista 23 PUBLICATIONS   224 CITATIONS    SEE PROFILE Camila Lopes Federal University of Rio de Janeiro 7 PUBLICATIONS   17 CITATIONS    SEE PROFILE Wilma De Grava Kempinas São Paulo State University 150 PUBLICATIONS   2,041 CITATIONS    SEE PROFILE Francisco Jose Roma Paumgartten Fundação Oswaldo Cruz 44 PUBLICATIONS   155 CITATIONS    SEE PROFILE All content following this page was uploaded by Camila Lopes on 30 January 2015. 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https://www.researchgate.net/institution/Fundacao_Oswaldo_Cruz?enrichId=rgreq-e450a2131caf680065fb70db233d059a-XXX&enrichSource=Y292ZXJQYWdlOzI2OTcyMzc4NjtBUzoxOTEzNDcwMDY3MzAyNDBAMTQyMjYzMjA4OTU3Ng%3D%3D&el=1_x_6&_esc=publicationCoverPdf https://www.researchgate.net/profile/Francisco_Jose_Roma_Paumgartten?enrichId=rgreq-e450a2131caf680065fb70db233d059a-XXX&enrichSource=Y292ZXJQYWdlOzI2OTcyMzc4NjtBUzoxOTEzNDcwMDY3MzAyNDBAMTQyMjYzMjA4OTU3Ng%3D%3D&el=1_x_7&_esc=publicationCoverPdf https://www.researchgate.net/profile/Camila_Lopes6?enrichId=rgreq-e450a2131caf680065fb70db233d059a-XXX&enrichSource=Y292ZXJQYWdlOzI2OTcyMzc4NjtBUzoxOTEzNDcwMDY3MzAyNDBAMTQyMjYzMjA4OTU3Ng%3D%3D&el=1_x_10&_esc=publicationCoverPdf Accepted Manuscript Title: Sexual maturation and fertility of mice exposed to triphenyltin during prepubertal and pubertal periods Author: Marcia S. Campos Mello Isabella F. Delgado Ana Paula A. Favareto Camila M.T. Lopes Marcelo M. Batista Wilma De-Grava Kempinas Francisco J.R. Paumgartten PII: S2214-7500(14)00160-7 DOI: http://dx.doi.org/doi:10.1016/j.toxrep.2014.12.006 Reference: TOXREP 148 To appear in: Received date: 6-9-2014 Revised date: 10-12-2014 Accepted date: 11-12-2014 Please cite this article as: M.S.C. Mello, I.F. Delgado, A.P.A. Favareto, C.M.T.L. ; Marcelo M. Batista, W.D.-G. Kempinas, F.J.R. Paumgartten, Sexual maturation and fertility of mice exposed to triphenyltin during prepubertal and pubertal periods., Toxicol. Rep. (2014), http://dx.doi.org/10.1016/j.toxrep.2014.12.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. http://dx.doi.org/doi:10.1016/j.toxrep.2014.12.006 http://dx.doi.org/10.1016/j.toxrep.2014.12.006 Page 1 of 35 Acc ep te d M an us cr ip t 1 1 2 Sexual maturation and fertility of mice exposed to 3 triphenyltin during prepubertal and pubertal periods. 4 5 6 7 Marcia S. Campos Mello1,5, Isabella F. Delgado2, Ana Paula A Favareto4, 8 Camila M.T. Lopes2; Marcelo M. Batista3, Wilma De-Grava Kempinas4, 9 and Francisco J.R. Paumgartten1 10 11 12 1Laboratory of Environmental Toxicology, Department of Biological Sciences, National 13 School of Public Health, 2Department of Immunology, National Institute of Health 14 Quality Control, 3Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ 21045-900, 15 Brazil 4Dept of Morphology, State University of São Paulo (UNESP), Botucatu, São 16 Paulo, Brasil. 5Dept of Biochemistry, Federal University of Rio de Janeiro State, 17 UNIRIO, Rio de Janeiro, Brazil. 18 19 20 Correspondence: Francisco J.R. Paumgartten, Laboratory of Environmental Toxicology, 21 National School of Public Health, FIOCRUZ, Av. Brasil 4036, rooms 101-104, Rio de Janeiro, 22 RJ21040-361, Brasil. Phone: +55.21.38829018, Fax: +55.21.31094664, Email: 23 paum@ensp.fiocruz.br. 24 *Manuscript http://ees.elsevier.com/toxrep/viewRCResults.aspx?pdf=1&docID=401&rev=2&fileID=16996&msid={3E1E1C3E-3F1C-4455-BC29-1D9B70BEE42D} Page 2 of 35 Acc ep te d M an us cr ip t 2 ABSTRACT 25 This study investigated the effects of pre- and peripubertal exposure 26 (PND15-45) to triphenyltin hydroxide (TPT: 0, 1.875, 3.75, 7.5 and 15 mg/kg 27 bw/d po) on mouse sexual maturation and fertility. Half of the mice were 28 euthanized on PND46 (males) or PND53 (females) and the remaining mice 29 were submitted to fertility tests on PND 65-75. TPT caused a transient decrease 30 of weight gain at 3.75 mg/kg bw/d, and deaths and body weight deficits at 31 higher doses. Delays of testes descent (TD), vaginal opening (VO) and first 32 estrus (FE) occurred at doses ≥3.75 (TD) and ≥7.5 mg/kg bw/d (VO, FE), 33 respectively. Body weight on the days of TD, VO and FE did not differ among 34 groups. TPT at doses ≥3.75 mg/kg decreased sperm and spematid counts at 35 the end of treatment (PND46) but no alteration was noted later on PND75. 36 Testicular histopathology (PND46) showed a dose-dependent reduction of 37 seminiferous tubules diameter, a greater degree of vacuolation in Sertoli cells 38 and germ cell degeneration and necrosis in TPT-treated mice. TPT did not 39 affect the outcome of fertility tests. Study-derived NOAEL was 1.875 mg TPT/kg 40 bw/d for males and 3.75 mg TPT/kg bw/d for females. The detrimental effects of 41 TPT on spermatogenesis were reversed after treatment discontinuation. 42 43 Key words: triphenyltin, TPTH, organotin compounds, puberty, postnatal exposure, 44 fertility. 45 46 Page 3 of 35 Acc ep te d M an us cr ip t 3 1. Introduction 47 Triphenyl (TPT) and tributyl (TBT) tins are powerful biocides (algicides, 48 acaricides, insecticides, molluscicides, miticides, fungicides, slimicides, 49 bactericides) that have a diversity of applications such as agricultural pesticides, 50 disinfectants, preservatives for wood and antifouling agents in paints for ship 51 hulls and fish farm nets and cages. The marked toxicity and putative endocrine 52 disrupting properties of organotin compounds have raised concerns about the 53 impact of their widespread use on the environment and human health. 54 Marine contamination by tri-substituted organotins has been associated 55 with increased occurrence of imposex, or the superimposition of male type 56 genital organs (penis and vas deferens), in female bivalve and gastropod 57 mollusks (Titley-O‟Neil et al, 2011). Since TBT and TPT act as competitive 58 inhibitors of a cytochrome P450-aromatase that converts androgen into 59 estrogen, it has been hypothesized that imposex might result from elevated 60 concentrations of unconverted androgens in organotin-exposed mollusks 61 (Horiguchi, 2006). Some authors, however, pointed out that while organotin 62 compounds are potent inducers of imposex, their effective concentrations as 63 aromatase inhibitors are high. Along this line, hypotheses about modes of 64 action other than inhibition of aromatase have been proposed for imposex 65 (Horiguchi, 2006). 66 A variety of potential deleterious effects of tri-organotins on mammals 67 have been described including a marked toxicity to mammalian development 68 and reproduction. Nonetheless, the critical molecular targets for organotin 69 induced reproductive toxicity remain unclear. Within this context, it has been 70 debated whether aromatase inhibition plays a role in mediating developmental 71 Page 4 of 35 Acc ep te d M an us cr ip t 4 toxic effects of tri-organotin compounds on mammals. Nakanishi (2008), for 72 instance, reported that in human choriocarcinoma cells, organotins instead of 73 inhibiting estradiol biosynthesis substantially enhanced it along with an increase 74 of both aromatase activity and 17-hydroxysteroid dehydrogenase type I 75 activity, an enzyme that converts estrone (a weak estrogen) into the biologically 76 active estrogen 17-estradiol at the same low concentrations. 77 Several studies including those by Kanayama et al (2005), Hiromori et al 78 (2009), and Le Maire et al (2009) demonstrated that TBT and TPT are 79 nanomolar agonists for retinoid X receptor alpha (RXR-) and peroxisome 80 proliferator-activated receptor (PPAR) gamma. Along this line, it has been 81 suggested that some toxic effects (and potential anticancer effects) of organotin 82 compounds are mediated via actions on the RXR and PPAR- nuclear 83 receptors (Nakanishi, 2008). 84 Although a number of studies have found detrimental effects of tri-85 organotins (TBT and TPT) on rodent reproductive organs and function, mostly 86 in rats, reports in the literature are far being consistent among compounds, 87 doses and routes of exposure, species and toxic responses. A study by Lang-88 Podratz et al (2012), for instance, described that TBT given orally to adult rats, 89 at a dose as low as 100ng/kg/d for two weeks, disrupted cycling regularity, 90 markedly reduced ovary weights and 17-estradiol concentrations in the serum, 91 increased progesterone concentrations and produced histological changes in 92 the ovary. Similar histological findings (increased number of atretic tertiary and 93 preovulatory follicles) were noted by Watermann (2008) in rats receiving TPTCl 94 at an oral dose as high as 6 mg/kg/d from PND 23 until 53, while only minor 95 Page 5 of 35 Acc ep te d M an us cr ip t 5 changes were found at a lower dose (2mg/kg/d). Moreover, Grote et al (2006) 96 reported that in female rats TPT (6 mg/kg/d po) administered from PND 23 to 53 97 retarded the puberty onset (day of vaginal opening) and increased 17-estradiol 98 blood serum concentrations. A previous study by the same authors (Grote et al, 99 2004) had found that oral treatment of male rats with TBT (15 mg/kg/d) or TPT 100 (6 and 12 mg/k/d) during the pre- and peripubertal periods (PND23-53) also 101 delayed puberty onset (preputial separation), and decreased testosterone and 102 luteinizing hormone (LH) serum concentrations. 103 The reproductive toxicity of tri-organotin compounds was less studied in 104 mice than in rats. Nonetheless, Si et al (2011) described that TBT (1, 10, 100 105 g/kg/d) given orally to female mice during gestation and lactation retarded 106 testes descent and acquisition of cliff-avoidance reflex in the exposed offspring 107 (100 g/kg/d). A subsequent study by the authors found that a similar treatment 108 with TBT (gestation + lactation) advanced the day of vaginal opening and the 109 day of the first estrus and that exposed female offspring (10 and 100g/kg/d) 110 also exhibited altered cycling regularity in adulthood (Si et al 2012). Data on the 111 toxicity of orally administered TPT (fentin) to mice during postnatal development 112 have been provided as well. Delgado et al (2009) treated pregnant mice (GD7-113 17) with TPT (7.5, 15 and 30 mg TPT/kg/d po) and noticed that the two highest 114 doses produced a marked perinatal mortality. Postnatal growth, days of vaginal 115 opening and testes descent, and other landmarks of somatic development, 116 however, remained unaffected in the offspring exposed in utero to TPT. In a 117 further study, the authors treated female mice with TPT (1.875, 3.75, or 7.5 118 mg/kg/d po) during pregnancy and lactation (GD6-PND21) and evaluated 119 offspring somatic development and fertility (Sarpa et al, 2010). Results showed 120 Page 6 of 35 Acc ep te d M an us cr ip t 6 no discernible effect of TPT at any tested dose on offspring development and 121 fertility. 122 This study was undertaken to investigate whether exposure to TPT 123 beginning before puberty and extending over pubertal onset period would 124 adversely affect sexual maturation and fertility in Swiss mice. Since half of the 125 treated mice were evaluated at the end of treatment and the remaining animals 126 one month later, data were also obtained on the reversibility of TPT induced 127 detrimental effects. 128 129 2. Materials and Methods 130 2.1. Animals 131 Female Swiss Webster mice, from the FIOCRUZ Central Animal House 132 (CECAL) breeding stock, were used. Mice were housed individually in standard 133 plastic cages with stainless-steel covers and autoclaved wood shavings as 134 bedding and kept under controlled temperature (232ºC), relative humidity 135 (approximately 70%) and day/night cycle (lights on from 8:00 a.m. to 8:00 p.m.). 136 A pelleted diet (Nuvital, for laboratory rats and mice, Nuvilab Ltd, Curitiba, PR, 137 Brazil) and filtered tap water were provided ad libitum. All procedures were 138 performed in accordance with Brazilian animal protection and welfare laws. The 139 study protocol was approved by the Ethics Committee on the Use of Animals of 140 Oswaldo Cruz Foundation (CEUA-FIOCRUZ, License Nr 0077-01). 141 2.2 Mating, pregnancy, parturition and culling. 142 Two females were placed into the cage of one male for 2 h at the end of 143 the dark period (6:00–8:00 a.m.) and copulation was confirmed by the presence 144 of a vaginal plug. The day on which mating was confirmed was designated as 145 Page 7 of 35 Acc ep te d M an us cr ip t 7 pregnancy day ´0`. From pregnancy day 18 onwards cages were inspected 146 twice a day (8:00 a.m. and 5:00 pm) for deliveries. The first 24 h after birth was 147 considered as postnatal day (PND) 1. On PND 5, litter size was adjusted by 148 culling to 10 pups (five females and five males, whenever possible). Natural 149 litters with 10 or fewer pups were not standardized. 150 2.3. Treatment, weaning and euthanasia 151 Triphenyltin hydroxide (TPT, purity ≥96.0%) was from Aldrich, Inc. On 152 PND 15, all pups were weighed and allocated at random (within their litters) to 153 one of five treatment groups (0, 1.875, 3.75, 7.5 and 15 mg TPT/kg bw/d). Male 154 and female mice (N = approximately 40 per gender and treatment group) from 155 20 litters were daily treated from PND 15 through to 45. After weaning on PND 156 21, dams were euthanized and up to 5 pups of a same gender and litter were 157 housed in one cage. TPT or its vehicle alone (pharmaceutical grade corn oil, 158 Sigma Chemical Co) was administered by gavage at a fixed volume of 10mL/kg 159 bw/d. A control group received the vehicle only. Males and females from 10 160 litters were killed on the day following that of last dose of TPT or its vehicle 161 (PND 45). Pups from the remaining litters received no further treatment until 162 euthanasia on PND 80-90. Fertility tests took place on PND 65-70. 163 2.4. General toxicity 164 During the period of treatment (PND 15-45) body weights were 165 determined on PNDs 15, 21, 25, 30, 35 and 45 while cages were inspected for 166 deaths and clinical signs of toxicity on a daily basis. Cage-side observations of 167 clinical signs of toxicity included, but were not limited to: behavioral 168 abnormalities, CNS depression symptoms, slow or irregular breathing, 169 Page 8 of 35 Acc ep te d M an us cr ip t 8 appearance of diarrhea, bleeding, changes in skin and fur, eyes and mucous 170 membranes, tumoral masses, edema and other abnormalities. 171 2.5. Vagina opening and testis descent 172 Occurrence of testes descent (by scrotum palpation) or vaginal opening 173 (visual inspection) was evaluated every morning (8:00-10:00 am) from PND 15 174 onwards. 175 2.6 Estrous cycle 176 From the day of VO onwards, for 20 consecutive days, vaginal smears 177 were prepared every morning (8:00–9:00 h) to determine the day on which the 178 first estrus took place and characterize the estrous cycle. Cytological findings 179 for staging the estrous cycle were as follows: pro-estrus, predominance of 180 round pro-nucleated epithelial cells; estrous, presence of cornified epithelial 181 cells; meta-estrous, presence of both cornified epithelial cells and leukocytes 182 and mucus, diestrus, predominance of leukocytes. 183 2.7 Euthanasia 184 On the day following the last dose of TPT or its vehicle, pups from 10 185 litters selected at random were killed by cervical dislocation. A blood sample 186 was collected by cardiac puncture. Liver, spleen, thymus, uterus, ovaries, testis, 187 epididymis and seminal vesicle with coagulating glands (without fluid) were 188 removed and weighed. Pups of the remaining 10 litters received no further 189 treatment. 190 2.8 Measurement of the number of spermatids in the testis and epididymal 191 sperm count 192 Following removal of the tunica albuginea, the right testis from each male 193 mouse was minced and homogenized in 1 ml of 0.9% NaCl containing 0.5% 194 Page 9 of 35 Acc ep te d M an us cr ip t 9 Triton X-100 for manual homogenization. After dilution in 0.9% NaCl, the 195 number of homogenization-resistant spermatids of each testis was counted with 196 a Neubauer chamber. Similarly, the cauda of epididymis was cut into small 197 pieces, minced and homogenized, and the sperm (spermatozoa) were counted 198 as described above (Amann et al. 1986). 199 2.9 Sperm morphology 200 To assess the percentage of morphologically abnormal sperm (detected 201 in the head or tail) the deferens ducts were rinsed with 0.3 ml 0.9% NaCl and a 202 sperm suspension was obtained. The smear on the slide (lamina) was prepared 203 with an aliquot of sperm suspension carefully stained with Congo red and 204 Gencian violet. Two hundred sperm per animal were examined at 1000x 205 magnification and morphologically normal and abnormal sperm were scored 206 according to the presence or absence of defects in the head (e.g., double-207 headed, amorphous head, reduced hook, banana head, no hook head, 208 detached head, dwarf head, giant head, pin head) or tail (crooked flagellum, 209 broken flagellum, coiled flagellum, tip coiled flagellum, bent flagellum tip) of the 210 sperm (Robb et al. 1978). 211 2.10 Histopathology and morphometry 212 The left testis from each male mouse was fixed with Bouin‟s solution that 213 was further replaced with Millonig‟s phosphate buffered formalin as modified by 214 Carson. After fixation, testes were embedded in paraffin, cut and mounted on 215 slides and subsequently stained with hematoxylin/eosin. One hundred 216 seminiferous tubules in randomly selected cross sections of the testis from each 217 mouse were identified regarding the stages of spermatogenesis. Twenty 218 essentially round seminiferous tubules per testis were examined to assess the 219 Page 10 of 35 Acc ep te d M an us cr ip t 10 mean tubule diameter. The height of the germinal epithelium was measured in 220 stages VII/VIII seminiferous tubules. 221 2.11 Testosterone level 222 Plasma testosterone concentrations were determined (samples in 223 duplicate) using a commercially available competitive immunoassay kit (Free 224 Testosterone ELISA, ARP, Inc™). 225 2.12. Fertility test 226 Fertility tests were carried out with half of mice from each treatment 227 group when they were approximately 65 day old. One male and 3 females from 228 the same treatment group and different litters (to avoid brother–sister mating), 229 chosen at random, were housed in the same cage for 15 d. Females were 230 examined every day in the morning for the presence of a vaginal plug. On the 231 day a vaginal plug was found (GD 0), females were transferred to individual 232 cages. Females that had not been impregnated by males during the first 233 cohabitation period were placed again into a male cage for a second mating 234 test. The second mating test was similar to the first, except that untreated males 235 of proven fertility were used. Males that did not impregnate at least 2 females 236 during the first mating test had a second 15-d cohabitation period with 3 237 untreated females. On day 16 of pregnancy females were killed by CO2 238 inhalation. The gravid uteri were weighed with their contents. The number of 239 dead and living fetuses and resorptions were recorded. The number of 240 implantation sites was determined by the Salewski‟s method (Salewski, 1964). 241 Liver, spleen, thymus, gravid uterus and ovaries were removed and weighed. 242 Male mice that took part in the fertility test were killed by cervical 243 dislocation on PND 75. Liver, spleen, thymus, testis, epididymis and seminal 244 Page 11 of 35 Acc ep te d M an us cr ip t 11 vesicle with coagulating glands (without fluid) were removed and weighed. 245 Collection of epididymal sperm and sperm counting was performed as 246 described in a previous section. 247 2.13. Statistical analysis 248 Data were analyzed by one-way analysis of variance (ANOVA) followed 249 by Bonferroni‟s post hoc test, or by the Kruskal–Wallis test followed by the 250 Mann–Whitney U test, whenever the data did not fit a normal distribution. 251 Proportions were evaluated by the chi-square test or by the Fisher exact test. 252 Statistical evaluation was performed using a SPSS® program and differences 253 were considered as statistically significant whenever P<0.05. 254 3. Results 255 3.1 Effects of TPT on pup survival and body weight gain. 256 No clinical sign of toxicity was noted at the two lowest doses (1.875 or 257 3.75 mg/kg bw/d) of TPT. A number of deaths, however, occurred among mice 258 treated with the second highest (7.5 mg/kg bw/d: 7.4% of males and 3.7% of 259 females) and the highest (15 mg/kg bw/d: 80.8% of males and 80.9% of 260 females) doses of TPT. All deaths of pups treated with TPT took place during 261 the second week of treatment within a few days of weaning (PND 21 to 25) 262 (Table 1). 263 The body weight changes of mouse pups treated with TPT are shown in 264 Figure 1. The treatment caused a dose-dependent reduction of body weight 265 gain in males (Figure 1A) at TPT doses 3.75 mg/kg bw/d while weight gain of 266 females (Figure 1B) was decreased only at the two highest doses (7.5 and 15 267 mg/kg bw/d). It is of note that the weight gain deficit during treatment with TPT 268 was transient. Except for the highest dose group (15 mg/kg bw/d) no differences 269 Page 12 of 35 Acc ep te d M an us cr ip t 12 in body weights between control and TPT treated mice were noted at the end of 270 treatment (PND45). Except for body weight gain deficit no other clinical signs of 271 toxicity were noted. 272 3.2 Effect of treatment with TPT on the onset of puberty 273 As shown in Table 2, the day on which landmarks of puberty onset 274 appear in female mice was not affected by treatment with the lowest doses of 275 TPT. At the two highest doses (7.5 and 15 mg/kg bw/d), however, TPT delayed 276 the day of vaginal opening and the day of the first estrus. The time (days) 277 between vaginal opening and the first estrus remained unaltered at all doses of 278 TPT (Table 2). The day of testes descent was delayed in male pups treated with 279 doses of TPT 3.75 mg/kg bw/d. At first sight, these findings seem to indicate 280 that TPT at the highest doses tested delayed puberty onset in both male and 281 female mice. Nonetheless, TPT-treated pups did not differ from controls 282 regarding pup body weight on the days of vaginal opening, first estrus and 283 testes descent (Table 2). Taken together, these results suggest that delays of 284 puberty onset reflect a general retardation of growth and somatic maturation 285 rather than specific effects on male and female endocrine-sensitive endpoints. 286 3.3 Effects of TPT on male reproductive organs, testosterone 287 concentrations, and sperm parameters at the end of treatment period 288 On the day following the last dose of TPT (PND 46) mice that had been 289 treated with doses of TPT 3.75 mg/kg bw/d exhibited dose-dependent 290 reductions of the number of spermatids (testes) and spermatozoa (epididymis) 291 compared to controls (Table 3). A small and statistically non-significant increase 292 in the proportion of sperm with an abnormal morphology was also noted at the 293 two highest doses of TPT. A decrease of male reproductive organs (testis, 294 Page 13 of 35 Acc ep te d M an us cr ip t 13 epididymis and seminal vesicle) weight, consistent with a nearly 10% body 295 weight reduction, was detected in male mice treated with the highest dose of 296 TPT (15 mg/kg bw/d), but not among those mice that received 7.5 mg/kg bw/d 297 or lower doses (Table 3). On PND 46, free testosterone concentrations in the 298 plasma of TPT-treated mice did not differ from the concentrations measured in 299 control group mice (Table 3). 300 The morphometrical analysis showed that mice that received doses of 301 TPT 3.75 mg/kg bw/d presented a smaller seminiferous tubule diameter and a 302 reduced germinal epithelium height (Table 3), a finding suggestive that 303 treatment with this tri-organotin compound impaired mouse spermatogenesis. 304 Histopathology of testicular tissue on PND46 revealed sparse alterations of 305 slight to moderate severity such as increased degree of vacuolation in Sertoli 306 cells (Figure 2, panels E and F), presence of immature germ cells (round 307 spermatids) and cellular debris in the tubule lumen and signs of degeneration 308 and necrosis of germ cells in the seminiferous tubules of TPT-treated mice 309 (Figure 2). No alteration of Leydig cell morphology was noted in the testis of 310 TPT-treated mice. 311 3.4. Effect of TPT on male liver and female liver, ovary and uterus (PND46). 312 Post-mortem examination after euthanasia on PND46 revealed no 313 abnormality and no alteration of reproductive organ (ovary and uteri) weight in 314 female mice treated with TPT. The relative weights of liver (%; [liver wt / body 315 wt] x100), however, were increased (Kruskal Wallis test and Mann-Whitney U 316 test, p<0.05) in males treated with TPT doses ≥7.5 mg/kg b wt/d (mg TPT /kg 317 bw/d; 0: 5.6±0.4, 1.785: 5.7±0.3, 3.75: 5.8±0.4, 7.5: 6.2±0.7, 15.0: 6.6±0.3) and 318 Page 14 of 35 Acc ep te d M an us cr ip t 14 females treated with doses ≥3.75 mg/kg b wt/d (mg TPT /kg bw/d; 0: 5.0±0.4, 319 1.785: 5.4±0.6, 3.75: 5.7±0.6, 7.5: 6.0±0.6, 15.0: 6.3±0.4). 320 3.5 Effects of TPT on fertility tests and sperm parameters 30 days after 321 treatment discontinuation 322 The outcome of fertility tests carried out approximately one month after 323 discontinuation of treatment indicated that pre/peripubertal exposure to TPT in 324 the dose range tested did not affect reproductive performance of male and 325 female mice. As shown in Table 4, ratios of pregnant females per mated 326 females and of males that copulated per males that took part in the fertility test 327 did not differ between controls and TPT-treated mice and, in all dose-groups 328 percentages of pregnant females were higher than 90% and in most cases 329 100%. One male that did not copulate, and two females that did not become 330 pregnant in the test were subsequently mated with an untreated female or male. 331 In this second mating, females were not successfully impregnated by their male 332 partners, a result that confirmed that the male and two females that failed in the 333 first test were in fact infertile. Moreover, data from C-section (performed on GD 334 16) of females that were impregnated in the fertility test revealed no difference 335 between control and TPT-treated groups regarding gravid uteri weight (with 336 their contents), and numbers of dead and living fetuses, resorptions and 337 implantations. Post-mortem examination at the C-section revealed no gross 338 pathology or weight change of maternal organs and no externally visible 339 abnormality in the recovered fetuses. 340 TPT-treated male mice euthanized after fertility test mating period 341 (approx PND 75) exhibited no discernible difference from controls regarding 342 body weight and reproductive organs (testes, epididymis and seminal vesicle) 343 Page 15 of 35 Acc ep te d M an us cr ip t 15 weight, spermatid count, sperm count, proportion of sperm with an abnormal 344 morphology, and plasma free testosterone concentrations (Table 5). 345 346 4. Discussion 347 Data from this study showed that treatment of mice with oral doses of 348 TPT ≥7.5 mg/kg/d from PND15 onwards resulted in a dose-dependent reduction 349 of pup weight gain and high mortality rates. All deaths occurred during the 350 second week of treatment within a few days of weaning (PND 21-25). No death 351 occurred at the two lowest doses of TPT tested in this study. A small and 352 transient reduction of weight gain was noted at 3.75mg/kg bw/d while no toxic 353 effect of TPT was detected among mice treated with 1.875 mg/kg bw/d (Table 354 1, Figure 1). Therefore, under the conditions of this study, the NOAELs for 355 general toxicity findings (weight gain reductions and mortality) were 1.875 356 mg/kg bw/d (males) and 3.75 mg/kg bw/d (females). The same NOAEL was set 357 for male reproductive endpoints (testes descent delay, sperm count and 358 seminiferous tubule diameter reduction) while 3.75 mg/kg bw/d was the study 359 derived NOAEL for female reproductive endpoints. 360 Delays in the day of vaginal opening completion (VO) and of day of the 361 first estrus (FE) indicated that TPT at doses ≥7.5 mg/kg bw/d retarded puberty 362 onset in females. TPT at doses ≥3.75 mg/kg bw/d also delayed the day on 363 which testes descent occurred. Since the body weight on the day on which 364 these landmark events occurred did not differ between control and TPT-treated 365 mice, it seems fair to conclude that delays in puberty onset in males and 366 females were associated with a general retardation of growth and somatic 367 maturation. Notwithstanding this fact, study results lend support to the 368 Page 16 of 35 Acc ep te d M an us cr ip t 16 interpretation that in rodents the testis is a target organ for TPT toxicity. Soon 369 after treatment discontinuation (PND46) both spermatid and sperm counts were 370 decreased in a dose-dependent manner in males treated with TPT at doses 371 ≥3.75 mg/kg bw/d. It is of note that spermatid and sperm count reductions were 372 consistent with histological examination findings that revealed shorter diameters 373 of seminiferous tubules and a number of signs of degeneration and necrosis of 374 germ cells (Figure 2) in the testis of males treated with TPT at doses ≥3.75 375 mg/kg bw/d (Table 3). Fertility tests undertaken nearly 30 days after 376 discontinuation of TPT administration revealed no decline of fertility in treated 377 animals (Table 4). Since fertility of males on PND 65-75 was unaffected by 378 doses of TPT that caused testicular injury and decreased sperm count on 379 PND46, either the reduction of sperm count was insufficient to decrease the 380 reproductive performance, or a recovery of TPT adverse effects on sperm 381 parameters was reversed during the 30 days that elapsed between 382 discontinuation of treatment and the fertility test. In rats and mice, the sperm 383 count of a normal ejaculate is far in excess of that number required for 100% of 384 impregnation success. Therefore, if sperm quality and motility are good, a 385 substantial decline of sperm count is required to affect the outcome of rodent 386 fertility tests. Since no decline in sperm count was detected in TPT-treated 387 males on PND75, it seems fair to conclude that adverse effects of TPT on 388 mouse spermatogenesis were reversed with treatment discontinuation. 389 Two previous studies from our laboratory had found no effect of TPT on 390 the puberty onset (VO and testes descent) of the offspring of mice treated 391 (same dose range) during pregnancy (Sarpa et al, 2010) or pregnancy and 392 lactation (Delgado et al 2009). Our previous negative results are not at odds 393 Page 17 of 35 Acc ep te d M an us cr ip t 17 with present the study findings. In this study, pups were directly exposed during 394 prepubertal and pubertal periods while in the other investigations offspring were 395 exposed via placenta (pregnancy) or via placenta and maternal milk (pregnancy 396 and lactation). Kinetic investigations showed that, albeit passing through the 397 placenta and accumulating in rodent embryos, only minimal amounts of 398 organotin compounds are transferred via maternal milk to suckling pups (Cooke 399 et al, 2009, Moser et al, 2009). Therefore, our previous findings consistently 400 demonstrated that prenatal exposure to TPT did not affect puberty onset in 401 mice. In this study pups were exposed directly (by gavage) to TPT and 402 exposure extended over post-weaning pre- and pubertal periods. 403 To the authors‟ knowledge, there is no other study on the effects of 404 similar pre- and pubertal exposures to TPT on sexual maturation of mice. 405 Reddy et al (2006) reported that spermatid and sperm counts were reduced in 406 adult (sexually mature) male mice that received three ip injections of 25μg/kg 407 bw of TPT. They also found decreased activities of testicular 3β- and 17β-408 hydroxysteroid dehydrogenases in treated mice and suggested that TPT 409 impaired spermatogenesis due to the inhibition of testosterone production. 410 As mentioned previously we found a disruption of seminiferous tubules 411 morphology along with unaltered serum concentrations of free testosterone in 412 mice treated orally with TPT during pre- and pubertal periods. 413 In male rats, Grote et al (2004, 2009) observed that orally administered 414 TPT (6 mg/kg bw/d) for 30 days (from PND 23 onwards) (Grote et al 2004), or 415 TPT (2 mg/kg bw/d) during pregnancy, lactation (maternal treatment), and post-416 weaning period (given directly to pups) until PND 53 (Grote et al 2009), 417 decreased testosterone serum concentrations. Grote et al (2009), however, 418 Page 18 of 35 Acc ep te d M an us cr ip t 18 detected no change in serum testosterone concentrations in mature male rats 419 (PND53) the treatment of which with TPT (2 mg/kg/bw/d, administered to 420 mothers) had been discontinued at weaning. Grote et al (2004) found that TPT 421 (6 mg/kg/d po PND23-53), albeit decreasing testosterone serum concentrations, 422 did not alter the day of preputial separation (PS). Nonetheless, a further study 423 by the same authors (Grote et al 2009) revealed that puberty onset (PS) was 424 delayed in rats treated with TPT (2mg/kg bw/d po) until weaning (PND 23) (no 425 effect on testosterone) and in those that continued to be treated until PND 53 426 (decrease of testosterone concentrations). Data by Grote et al (2004, 2009) 427 therefore suggested that, as far as male rats are concerned, the retardation of 428 puberty onset did not hinge on the depression of testosterone serum 429 concentrations caused by TPT. 430 Grote et al (2006, 2010) also described the effects of TPT on female rats. 431 A study by Grote et al (2006) found that TPT administered orally from PND 23 432 onwards had a biphasic effect on puberty onset; at a dose as high as 6 mg/kg 433 bw/d TPT delayed VO while at a lower dose (2 mg/kg bw/d) it advanced VO. A 434 subsequent study by the same authors (Grote et al, 2010), involving pre- and 435 postnatal (PND 21 or PND53) exposure to TPT (2 mg/kg bw/d), found an 436 advancement of puberty onset (VO) that was more pronounced in the group the 437 treatment of which was discontinued at weaning (PND21). Based on the 438 foregoing Grote et al proposed that the prenatal period would be a 439 developmental window of susceptibility to effects of TPT on puberty onset and 440 that males would be more susceptible than females to TPT-induced disruption 441 of sexual maturation. 442 Page 19 of 35 Acc ep te d M an us cr ip t 19 Contrasting to the foregoing results by Grote et al (2006) we did not 443 observe a treatment-related advancement of puberty onset (VO) at any dose 444 level. The retardation of VO and FE in mice treated with 7.5 mg/kg bw/d was 445 consistent with Grote et al„s (2006) results showing VO retardation in rats 446 treated with 6 mg/kg bw/d. No alteration of female puberty onset, however, was 447 noted in mice treated with the two lowest doses (1.875 and 3.75 mg/kg bw/d), 448 whereas Grote et al (2006) reported that VO was advanced in rats treated with 449 2 mg/kg bw/d. For a similar period of treatment (PND 23 onwards), Grote et al 450 (2004) found no effect of TPT on rat preputial separation (6 mg/kg bw/d) while 451 we detected a delay of testes descent and reduction of sperm count in mice 452 treated with 3.75 and 7.5 mg/kg bw/d. 453 The effects of TBT on sexual maturation were investigated by Si et al in 454 the offspring of female mice treated orally (0, 1, 10, 100 g/kg bw/d) GD6 and 455 throughout lactation until weaning (PND21). The authors reported results for 456 males (Si et al, 2011) and females (Si et al, 2012) in separate publications. Both 457 testes descent and acquisition of cliff-drop avoidance reflex were slightly, albeit 458 significantly retarded in males exposed to the highest dose (100 g/kg bw/d) 459 while a similar marked advancement of VO and FE, with a shortening of VO to 460 FE time interval was noted in females treated with all tested doses. Moreover, 461 Si et al (2012) also demonstrated that TBT-treated females exhibited body 462 weights lower than those of untreated controls on the day of VO and FE and 463 that in addition to an early puberty onset TBT also disrupted normal cycling in 464 mature female mice. Nonetheless, the effect of 1 g/kg bw/d was almost 465 identical to that of a 100-fold higher dose (100g/kg bw/d). The absence of any 466 Page 20 of 35 Acc ep te d M an us cr ip t 20 change of the magnitude of the toxic response over such a broad range of 467 doses tested by Si et al (2012) is an intriguing finding. 468 At any rate, a distinction should be made between “programming effects” 469 of exposure to organotin compounds during prenatal and or neonatal periods 470 affecting further sexual maturation, and effects on puberty onset somatic 471 landmarks arising from exposures that take place later (i.e., pre/pubertal 472 exposures) when the process is going on. Effects of TBT on female mouse 473 puberty onset estrous cyclicity and reported by Si et al (2012) possibly arise 474 from a epigenetic “programming” effect elicited during in utero and neonatal 475 periods and appear to be persistent. The effects reported in the present study, 476 on the other hand, were transient effects of doses of TPT that were also 477 associated to non-reproductive toxicity. 478 Recently, Mitra et al (2013) reported that, in in vitro germ-cell Sertoli cell 479 co-cultures, TBT (300-1000 nM concentration range) induced stress proteins 480 and mitochondrial depolarization leading to caspase-3-activation and apoptotic 481 (at lower concentrations) and necrotic (at higher concentrations) cell deaths. 482 They also noted that Sertoli cells were more susceptible than germ cells. 483 Moreover, the authors demonstrated that, in rats, an oral dose as high as 50 484 mg/kg bw disrupted the blood-testicular-barrier. Mitra et al (2013) suggested 485 that damage to Sertoli cells plays a pivotal role in tri-organotin compounds-486 mediated testicular toxicity. The histopathological changes in the seminiferous 487 tubules of TPT treated mice (vacuolation in Sertoli cells and degeneration and 488 necrosis of germ cells) seem to be consistent with Mitra et al„s hypothesis. 489 In conclusion, repeated exposure of mice to TPT by the oral route during 490 pre- and pubertal periods caused in addition to non-reproductive toxic effects, a 491 Page 21 of 35 Acc ep te d M an us cr ip t 21 high mortality and a delay of puberty onset in females (vaginal opening and first 492 estrus) and in males (testes descent), a decline of spermatid and sperm counts, 493 and slight to moderate damage to testicular Sertoli and germ cells. There was 494 an overlap between doses that cause general toxicity (deaths and weight gain 495 deficits) and reproductive toxicity in males (NOAEL 1.875 mg/kg bw/d) and 496 females (NOAEL 3.75 mg/kgbw/d). TPT-induced decline of spermatid/sperm 497 counts were reversed after treatment discontinuation. No effect of pre-498 pubertal/pubertal treatment with TPT was detected on the outcome of fertility 499 tests performed with mature mice. It is of note that NOAELs for reproductive 500 toxicity determined in this rodent study are orders of magnitude much higher 501 than estimated human exposures. Notwithstanding the fact that data on human 502 exposure to organotins (OTCs) are scanty, a Finnish study estimated that the 503 average daily intake of OTCs through fish consumption (a main dietary source 504 of organotins) was 3.2 ng/kg bw /d, which corresponds to 1.3% from tolerable 505 daily intake of 250 ng/kg bw/d set by the European Food Safety Authority 506 (Airaksinen et al, 2010). A study by Rantakokko et al (2008) determined blood 507 concentrations of TPTs in Finnish fishermen and their relatives and found that in 508 only 27 out of 300 samples analyzed TPT concentrations were higher than limit 509 of quantification of the method (0.04 ng/ml). The maximum TPT blood 510 concentration found by Rantakokko et al (2008) was 0.56 ng/ml. 511 512 Study highlights 513 - TPT administered by the oral route during pre-/pubertal period was 514 markedly toxic to mice and study-derived NOAELs for general toxicity 515 Page 22 of 35 Acc ep te d M an us cr ip t 22 (weight gain deficits and deaths) were as low as 1.875 mg/kg bw/d for males 516 and 3.75 mg/kg bw/d for females. 517 - Reproductive toxicity was noted at TPT doses at which signs of general 518 toxicity were also apparent. NOAEL for detrimental effects on male 519 reproductive parameters was 1.875 mg/kg bw/d (delay of testes descent, 520 reductions of sperm count and seminiferous tubules diameter on PND46, 521 and for effects on female reproductive parameters was 3.75 mg/kg bw/d 522 (delay of vaginal opening and day of first estrus). 523 - TPT induced reductions of spermatid and sperm counts were reversed 524 after treatment discontinuation. 525 - No decline of fertility was noted in mice treated with TPT during 526 pre/pubertal period tested 30 days after treatment discontinuation. 527 528 Acknowledgements 529 The research project was funded by the Brazilian National Research 530 Council (CNPq), FAPERJ, and FIOCRUZ (PAPES-III). This study is part of 531 MSCM doctoral thesis (INCQS-FIOCRUZ). FJRP, IFD and WGK are recipients 532 of research fellowships from CNPq. 533 534 Page 23 of 35 Acc ep te d M an us cr ip t 23 535 References 536 Airaksinen R, Rantakokko P, Turunen AW, Vartiainen T, Vuorinen PJ, 537 Lappalainen A, Vihervuori A, Mannio J, Hallikainen A (2010). Organotin 538 intake through fish consumption in Finland. Environ Res. 110(6):544-7. 539 Amann RP (1986). Detection of alterations in testicular and epididymal function 540 in laboratory animals. Environ Health Perspect. 70:149-58. 541 Cooke GM, Forsyth DS, Bondy GS, Tachon R, Tague B, Coady L (2008). 542 Organotin speciation and tissue distribution in rat dams, fetuses, and 543 neonates following oral administration of tributyltin chloride. J Toxicol 544 Environ Health A. 71(6):384-95. 545 Delgado IF, Viana VG, Sarpa M, Paumgartten FJ (2009). Postnatal 546 development and resistance to Plasmodium yoelii infection of mice 547 prenatally exposed to triphenyltin hydroxide. Environ Toxicol.;24(6):629-548 35. 549 Grote K, Andrade AJ, Grande SW, Kuriyama SN, Talsness CE, Appel KE, 550 Chahoud I (2006). Effects of peripubertal exposure to triphenyltin on 551 female sexual development of the rat. Toxicology. 222(1-2):17-24 552 Grote K, Stahlschmidt B, Talsness CE, Gericke C, Appel KE, Chahoud I (2004). 553 Effects of organotin compounds on pubertal male rats. Toxicology. 554 202(3):145-58 555 Hiromori Y, Nishikawa J, Yoshida I, Nagase H, Nakanishi T (2009). Structure-556 dependent activation of peroxisome proliferator-activated receptor 557 (PPAR) gamma by organotin compounds. Chem Biol Interact. 558 180(2):238-44. 559 Page 24 of 35 Acc ep te d M an us cr ip t 24 Horiguchi T (2006). Masculinization of female gastropod mollusks induced by 560 organotin compounds, focusing on mechanism of actions of tributyltin 561 and triphenyltin for development of imposex. Environ Sci. 13(2):77-87. 562 Kanayama T, Kobayashi N, Mamiya S, Nakanishi T, Nishikawa J (2005). 563 Organotin compounds promote adipocyte differentiation as agonists of 564 the peroxisome proliferator-activated receptor gamma/retinoid X receptor 565 pathway. Mol Pharmacol. 67(3):766-74 566 Lang-Podratz P, Delgado Filho VS, Lopes PF, Cavati Sena G, Matsumoto 567 ST,Samoto VY, Takiya CM, de Castro Miguel E, Silva IV, Graceli JB 568 (2012). Tributyltin impairs the reproductive cycle in female rats. J Toxicol 569 Environ Health A. 75(16-17):1035-46. 570 Le Maire A, Grimaldi M, Roecklin D, Dagnino S, Vivat-Hannah V, Balaguer P, 571 Bourguet W (2009). Activation of RXR-PPAR heterodimers by organotin 572 environmental endocrine disruptors. EMBO Rep. 10(4):367-73. 573 Mitra S, Srivastava A, Khandelwal S (2013). Tributyltin chloride induced 574 testicular toxicity by JNK and p38 activation, redox imbalance and cell 575 death in sertoli-germ cell co-culture. Toxicology. 314(1):39-50 576 Moser VC, McGee JK, Ehman KD (2009). Concentration and persistence of tin 577 in rat brain and blood following dibutyltin exposure during development. J 578 Toxicol Environ Health A. 72(1):47-52. 579 Nakanishi T (2008). Endocrine disruption induced by organotin compounds; 580 organotins function as a powerful agonist for nuclear receptors rather 581 than an aromatase inhibitor. J Toxicol Sci. 33(3):269-76. 582 Page 25 of 35 Acc ep te d M an us cr ip t 25 Rantakokko P, Turunen A, Verkasalo PK, Kiviranta H, Männistö S, Vartiainen T 583 (2008). Blood levels of organotin compounds and their relation to fish 584 consumption in Finland. Sci Total Environ. 399(1-3):90-5 585 Reddy PS, Pushpalatha T, Reddy PS (2006). Reduction of spermatogenesis 586 and steroidogenesis in mice after fentin and fenbutatin administration. 587 Toxicol Lett. 166(1):53-9. 588 Robb GW, Amann RP, Killian GJ (1978). Daily sperm production and 589 epididymal sperm reserves of pubertal and adult rats. J Reprod Fertil. 590 54(1): 103-7. 591 Salewski E (1964). Faerbemethoden zum Makroskopischen Nachweis von 592 Implantationsstellen am Uterus der Ratte. Naunyn-Schmiedebergs Archiv 593 fuer Experimentelle Pathologie und Pharmakologie 247: 367 594 Sarpa M, De-Carvalho RR, Delgado IF, Paumgartten FJ (2007). Developmental 595 toxicity of triphenyltin hydroxide in mice. Regul Toxicol Pharmacol 596 49(1):43-52. 597 Sarpa M, Tavares Lopes CM, Delgado IF, Paumgartten FJ (2010). Postnatal 598 development and fertility of offspring from mice exposed to triphenyltin 599 (fentin) hydroxide during pregnancy and lactation. J Toxicol Environ 600 Health A. 73(13-14):965-71. 601 Si J, Han X, Zhang F, Xin Q, An L, Li G, Li C (2012). Perinatal exposure to low 602 doses of tributyltin chloride advances puberty and affects patterns of 603 estrous cyclicity in female mice. Environ Toxicol. 27(11): 662-70. 604 Si J, Li P, Xin Q, Li X, An L, Li J (2013). Perinatal exposure to low doses of 605 tributyltin chloride reduces sperm count and quality in mice. Environ 606 Toxicol. doi: 10.1002/tox.21892. [Epub ahead of print] 607 Page 26 of 35 Acc ep te d M an us cr ip t 26 Si J, Li J, Zhang F, Li G, Xin Q, Dai B (2012). Effects of perinatal exposure to 608 low doses of tributyltin chloride on pregnancy outcome and postnatal 609 development in mouse offspring. Environ Toxicol. 27(10): 605-12. 610 Titley-O'Neal CP, Munkittrick KR, Macdonald BA (2011). The effects of 611 organotin on female gastropods. J Environ Monit. 13(9):2360-88. 612 Watermann B, Grote K, Gnass K, Kolodzey H, Thomsen A, Appel KE, Candia-613 Carnevali D, Schulte-Oehlmann U (2008). Histological alterations in 614 ovaries of pubertal female rats induced by triphenyltin. Exp Toxicol 615 Pathol. 60(4-5):313-21. 616 617 618 619 620 621 622 623 624 625 626 627 628 Page 27 of 35 Acc ep te d M an us cr ip t 27 LEGENDS TO FIGURES 629 630 Figure 1. Body weight gain (g) of male (A) and female (B) mice treated orally 631 (gavage) with triphenyltin hydroxide (0, 1.875, 3.75, 7.5 or 15 mg/kg bw/d) 632 suspended in corn oil from postnatal day 15 through to 45. The height of the 633 histogram bar corresponds to mean±SD. A star indicates that the mean body 634 weights differ (p<0.05, ANOVA followed by Bonferroni‟s post hoc test) from the 635 mean body weight of controls (0 mg/kg bw/d) at the same postnatal day. 636 637 638 Figure 2. Testicular histology changes in mice treated with triphenyltin 639 hydroxide (0, 1.875, 3.75, 7.5 or 15 mg/kg bw/d po) from PND 15 to 45. Mice 640 were euthanized at the end of treatment (PND 46). Testes were fixed in Bouin‟s 641 solution and Millonig‟s buffered formalin as modified by Carson and stained with 642 hematoxylin-eosin. Typical histology of a control mouse testis is shown in panel 643 A, while all remaining panels illustrate histological changes found in TPT-treated 644 animals (C,D,E: 1.875 mg/kg bw/d; G,I: 3.75 mg/kg bw/d; B,F,J: 7.5 mg/kg 645 bw/d), such as an increased degree of vacuolation of Sertoli cells (panels E,F, 646 asterisk *), vacuole apparently associated with germ cell deaths (panel D, 647 asterisk *), acidophilic cells with pyknotic nuclei and hypercondensation of 648 chromatin (panels I and J, arrows), multinucleated cell aggregates (panel C, 649 arrow head), immature germ cells and cell debris within the tubule lumen 650 (panels G and H); seminiferous tubule with vacuolation and few germ cell layers 651 (panel B, arrow head). Magnification: 400 x. 652 653 Page 28 of 35 Acc ep te d M an us cr ip t 28 Table 1. Survival of mice treated orally with TPT (0, 1.875, 3.75, 7.5, 15 mg/kg body wt/ day) from postnatal day (PND) 15 to 45. Pups alive TPT (mg/kg body wt /day po) 0 1.875 3.75 7.5 15 Females PND 15, N (%) 23 (100) 22 (100) 21 (100) 27 (100) 21 (100) PND 45, N (%) 23 (100) 22 (100) 21 (100) 26 (96.3) + 4 (19) + a Males PND 15, N (%) 26 (100) 26 (100) 24 (100) 27 (100) 26 (100) PND 45, N (%) 26 (100) 26 (100) 24 (100) 25 (92.6)+ 5 (19.2) + a + All deaths occurred between PND 21 (weaning) and PND 25. a differ (chi-square test, p<0.05) from other group survival rates. Page 29 of 35 Acc ep te d M an us cr ip t 29 Table 2. Landmarks of puberty onset in mice treated orally with TPT (0, 1.875, 3.75, 7.5, 15 mg/kg body wt/ day) from postnatal day (PND) 15 to 45. Landmarks TPT (mg/kg body wt /day po) 0 1.875 3.75 7.5 15 Female pups, N 23 22 21 26 4 Vaginal opening (VO) day 27 (22-30) 27.5 (23-32) 28 (24-38) 31 (24-44) a 39.5 (31-42) a Body weight on VO day (g) 17.0±2.4 17.0±2.6 16.7±3.0 17.6±2.6 17.9±0.9 First estrus (FE) day 40 (29-48) 38 (26-53) 41 (31-50) 48.5 (31-53) a, b 51.5 (51-52) a Body weight on FE day (g) 24.8±3.5 24.4±3.1 26.0±2.0 25.0±2.4 24.8±1.6 Time between VO and FE (days) 12.0±4.2 11.8±6.5 13.0±5.2 11.6±4.4 11.5±2.1 Male pups, N 26 26 24 25 5 Testes descent (TD) day 21 (19-25) 21 (20-25) 22.5 (20-28) a 23 (21-29) a 29 (27-30) a Body weight on TD day (g) 11.4±1.1 11.0±1.1 10.8±1.1 10.6±1.1 11.6±1.8 Kruskal-Wallis test and Mann Whitney U test: a  control (0 mg/kg bw/d) group; b  lowest dose (1.875 mg/kg bw/d) group. Page 30 of 35 Acc ep te d M an us cr ip t 30 Table 3. Reproductive organ weights, sperm parameters, and testosterone concentrations in male mice treated orally with TPT (0, 1.875, 3.75, 7.5, 15 mg/kg body wt/ day) from postnatal day (PND) 15 to 45 and killed at the end of treatment period (PND 46). Treatment on PND 15-45: Reproductive parameters on PND 46 TPT (mg/kg body wt /day po) 0 1.875 3.75 7.5 15 Male pups, N 14 14 12 13 4 Reproductive organ weights (g) Testis right 0.120.02 0.120.01 0.110.01 0.120.02 0.100.01 b left 0.110.01 0.120.01 0.110.01 0.110.02 0.100.01 Epididymis 0.070.01 0.070.01 0.070.01 0.060.01 0.050.01 a, b Seminal vesicle 0.160.03 0.160.03 0.150.02 0.130.01 a, b 0.080.03 a, b, c, d Spermatid count (x 106/testis), N 8.9±1.7 8.8±2.1 6.9±1.7 6.1±2.1 a, b 4.1±1.0 a, b Sperm count (x 106/ cauda epididymis), N 16.7±3.9 15.0±3.3 12.4±2.0 a 10.3±3.1 a, b 3.0±0.9 a, b, c, d Sperm with abnormal morphology, % 12.70 13.32 10.50 14.50 15.00 Seminiferous tubule diameter, µm 231.1±11.70 227.0±8.0 219.4±7.80 a 218.6±11.31 a 215.0±12.8♠ Germinal epithelium height#, µm 68.8±5.9 68.8±6.6 62.2±3.9 a, b 64.2±3.9 63.7±1.5 Plasma free testosterone level, pg/mL 16.7±21.8 34.2±32.8 14.4±22.9 18.2±20.7 0.12±0.09 Male pup body weight on PND46 (g) 40.83.1 41.92.3 41.93,6 41.64.2 35.53.2 a, b, c, d # Germinal epithelium height was measured in stage VII/VIII seminiferous tubules. ANOVA and Bonferroni‟s post hoc test: a  0 mg/kg bw/d; b  1.875 mg/kg bw/d, c  3.75 mg/kg bw/d, d 7.5mg/kg bw/d. ♠ Data from three mice were evaluated because no stage VII-VIII was found in the slide from one mouse testis. Page 31 of 35 Acc ep te d M an us cr ip t 31 Table 4. Outcome of fertility test carried out with male and female mice treated orally with TPT (0, 1.875, 3.75, 7.5, 15 mg/kg body wt/ day) from postnatal day (PND) 15 to 45 approximately 20 days after the end of treatment (PND 65) Treatment on PND 15-45: TPT (mg/kg body wt /day po) 0 1.875 3.75 7.5 15 First mating Copulating males/ Mated males (%) 12/12 (100%) 12/12 (100%) 12/12 (100%) 11/12 (91.6%) 2/2 (100%) Males impregnating a female/ Mated males 12/12+ (100%) 10/10 (100%) 9/10 (90%) 11/12 (91.6%) 2/2 (100%) Pregnant females / Mated females (%) 12/12+ (100%) 10/10 (100%) 9/10 (90%) 11/12 (91.6%) 2/2 (100%) Second mating Copulating males / Mated males (%) - - - 0/1 (0%) - Pregnant females / Mated females (%) - - 0/1(0%) 0/1 (0%) - First plus second mating Copulating males / Mated males (%) 12/12 (100%) 12/12 (100%) 12/12 (100%) 11/12 (91.6%) 2/2 (100%) Males impregnating a female/ Mated males 12/12+ (100%) 10/10 (100%) 9/10 (90%) 11/12 (91.6%) 2/2 (100%) Pregnant females / Mated females (%) 12/12+ (100%) 10/10 (100%) 9/10 (90%) 11/12 (91.6%) 2/2 (100%) Implantation sites (N, total) per litter (meanSD) 149 13.61.9 140 14.02.2 123 13.73.2 150 13.61.6 27 13.53.5 Live fetuses (N, total) per litter (meanSD) 143 13.02.0 135 13.52.0 110 12.23.4 145 13.21.5 23 11.50.7 + One pregnant female died during gestation. All other females were pregnant and survived to scheduled C-section (GD16). Statistical analysis revealed no difference between control and TPT-treated groups. Page 32 of 35 Acc ep te d M an us cr ip t 32 Table 5. Reproductive organ weights, sperm parameters, and testosterone concentrations in mice treated orally with TPT (0, 1.875, 3.75, 7.5, 15 mg/kg body wt/ day) from postnatal day (PND) 15 to 45 and evaluated nearly 30 days after the last administered dose (PND 76). Treatment on PND 15-45: Reproductive parameters on PND 76 TPT (mg/kg body wt /day po) 0 1.875 3.75 7.5 15 Male pups, N 12 12 12 12 1 Reproductive organ weights (g) Testis right 0.13±0.02 0.12±0.02 0.13±0.02 0.13±0.02 0.15 left 0.12±0.01 0.12±0.01 0.13±0.02 0.12±0.02 0.13 Epididymis 0.09±0.01 0.09±0.02 0.09±0.01 0.09±0.02 0.10 Seminal vesicle 0.19±0.03 0.19±0.04 0.20±0.03 0.20±0.03 0.25 Spermatid count (x 106/testis), N 10.0±3.8 8.3±3.7 8.2±3.0 6.4±2.8 10.0 Sperm count (x 106/ cauda epididymis), N 30.2±6.6 30.6±8.1 29.9±6.0 26.9±6.5 20.0 Sperm with abnormal morphology, % 17.8 19.4 18.6 19.4 19.0 Plasma free testosterone level, pg/mL 27.7±30.2 40.0±34.9 38.6±24.6 38.5±34.7 - Male pup body weight on PND 76 (g) 45.4±3.6 44.3±5.4 44.4±3.8 43.7±5.4 45.4 Statistical analysis (0, 1.875, 3.75 and 7.5 mg/kg bw/d) revealed no difference between dose groups. Page 33 of 35 Acc ep te d M an us cr ip t 33 * * * * *  control * * * * * * * * * * * * * * * * * (g ) (g ) Page 34 of 35 Acc ep te d M an us cr ip t 0 5 10 15 20 25 30 35 40 45 0 1.87 3.75 7.5 15 PND 15 PND 21 PND 25 PND 30 PND 35 PND 45 0 5 10 15 20 25 30 35 0 1.87 3.75 7.5 15 PND 15 PND 21 PND 25 PND 30 PND 35 PND 45 TPT (mg/kg body wt/ d) TPT (mg/kg body wt/ d) B o d y w e ig h t (g ) B o d y w e ig h t (g ) A B * Figure Page 35 of 35 Acc ep te d M an us cr ip t A B C D * * E * * F H G Figure2 V iew publication stats V iew publication stats https://www.researchgate.net/publication/269723786