Retraction: Nuclear Morphometric Analysis of Leydig Cells of Male Pubertal Rats Exposed In Utero to Di(n-butyl) Phthalate.

[This retracts the article on p. 439 in vol. 26, PMID: 24526819.].

In Utero Exposure to Di( n-butyl)phthalate Induces Morphological and Biochemical Changes in Rats Postpuberty.

Pregnant Sprague-Dawley rats were orally administered di( n-butyl)phthalate (DBP; 100 mg/kg/day) on gestation days (GD) 12 to 21. We investigated the male offspring and probed morphological alterations in Sertoli cells at 7, 9, 14, and 17 weeks of age. Parameters assessed in this study included offspring number, sex ratios, body weights, testis weights, seminiferous tubule (ST) profile numbers and diameters, number of vimentin-labeled Sertoli cells, and both testosterone and follicle-stimulating hormone (FSH) levels. Testicular weight/body weight ratios and the numbers and diameters of ST in maximum transverse testicular sections were statistically similar at weeks 7 and 9; however, at weeks 14 and 17, they were statistically different and displayed higher BrdU-positive Sertoli cells/Sertoli cell ratios in the DBP treatment group. Noteworthily, the serum FSH levels were higher and testicular testosterone levels were lower in the DBP treatment group. To our knowledge, the present study is the first to report that in utero DBP exposure significantly increased Sertoli cell numbers and their cellular proliferation from postpuberty to adulthood, with a significant decrease in testicular testosterone and an increase in FSH.

Three-dimensional reconstruction of deferent ducts papillae in urogenital duct system of the male rat.

BACKGROUND: The rodent ejaculatory ducts penetrate the male accessory sex gland complex and open into the urethra, anatomically similar to humans. Although the deferent ducts papillae in rodents have been described at the distal end of deferent ducts, they are absent in humans, and their detailed morphology has been unclear. METHODS: The detailed anatomical structures of the distal end of the deferent ducts of rats were investigated by the computer assisted three-dimensional reconstruction analysis using serial sections of the male accessory sex gland complexes in rats. RESULTS: The present study revealed that a pair of deferent ducts enters the ventral side of the male accessory sex gland complex, runs caudally parallel to the urethra, and then exits at about midsection of the dorso-lateral lobe of prostate. They are composed of mammilliform papillae, called the deferent duct papillae, which dorso-laterally protrude into the duct lumen from intra-ventral portion of the main duct of ampullary gland. The internal surface of the deferent ducts papillae is composed of ciliated columnar epithelium continuous from the deferent ducts, while their external surface is composed of the columnar secretory epithelium of the ampullary glands. Sphincter muscles were not observed in the deferent ducts papillae, while their lamina propria were occupied by many arterial or venous capillaries. CONCLUSIONS: The deferent ducts of rat terminated at the deferent ducts papillae that located at the main duct of ampullary glands that drained into the urethra. The deferent ducts papillae might be controlled by the expansion/contraction of well-developed papillary mucosal capillary vessels.

Adenocarcinoma of the ampullary glands of the ductus deferens in a Sprague-Dawley rat.

Spontaneously occurring proliferative lesions of the male accessory sex glands are infrequent in various strains of rats. In rodents, the ampullary glands are embedded in the prostate. Although 2 spontaneous cases of atypical hyperplastic lesions at the ampullary gland were previously described in Wistar rats, adenocarcinoma and/or adenoma in this gland have not been reported. This study describes adenocarcinomas in the bilateral ampullary glands in a 52-week-old intact male Sprague-Dawley rat housed as part of a control group in a toxicological experiment. At necropsy, the body weight (644.4 g) and the weight of the prostate with ampullary gland (2.75 g) were similar to others of the same control group, and it had a normal gross appearance. Histopathologically, both ampullary glands revealed microinvasive adenocarcinoma without vascular invasion. The morphological characteristics of the neoplasm varied in different regions of the gland. Other parts of the male accessory sex glands did not show proliferative lesions.

Vascular endothelial growth factor mRNA levels as a biomarker for short-term N-butyl-N-(4-hydroxybutyl) nitrosamine-induced rat bladder carcinogenesis bioassay.

Generically, carcinogenic effects of chemicals in bladder carcinogenesis are judged by induction of papillary or nodular (PN) hyperplasia in rats given N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) for 4 weeks and the test chemical for 22-28 weeks. However, upregulation of vascular endothelial growth factor (VEGF) begins early in rat BBN bladder carcinogenesis. To establish a short-term rat bladder carcinogenic bioassay, we analyzed the correlations between VEGF, VEGF mRNA and bladder lesions inductions at 10 and 26 weeks after BBN treatment. Six-week-old male Wistar (slc) rats were given 0.05% BBN for 4, 10 or 26 weeks. To avoid individual rat bias, the bladders were investigated by partial cystectomy at 10 weeks and total cystectomy at 26 weeks. After induction, PN hyperplasia and carcinoma in rats increased with the length of BBN treatment and immunohistochemical VEGF expression also increased following carcinogenesis, but the immunoreactivity of individual lesions was quite variable. Moreover, induction of PN hyperplasia at 10 weeks' BBN treatment was not significantly correlated with that at 26 weeks' treatment; thus, it was not possible to predict the carcinogenic effect due to the induction of PN hyperplasia at 26 weeks' BBN treatment by that at 10 weeks' treatment. However, VEGF mRNA levels of rat bladders at 10 weeks' BBN treatment revealed a strong significant correlation with the incidence of bladder lesions at 26 weeks' treatment. Here, we suggest that quantitative VEGF mRNA levels are a good biomarker for a short-term BBN-induced bioassay for rat bladder carcinogenesis.

Effects of in utero exposure to di(n-butyl) phthalate for estrogen receptors α, ß, and androgen receptor of Leydig cell on rats.

Estrogens and androgens affect male and female reproductive systems. Recently, we reported that prenatal di(n-butyl) phthalate (DBP) exposure induced atypical Leydig cells (LCs) hyperplasia during adulthood. The present study investigated the expression of estrogen receptor α (ERα), estrogen receptor ß (ERß), and androgen receptor (AR) in LCs of 5-, 7-, 9-, 14-, and 17-week-old Sprague-Dawley (srl) rats whose dams had been administered DBP intragastrically at 100 mg/kg/day or the vehicle (corn oil) from days 12 to 21 postconception. Immunohistochemical, Western blotting, and reverse transcription polymerase chain reaction analyses revealed that the expressions of ERα, ERß, and AR proteins and mRNAs in the DBP group were similar to those of the vehicle group at 5 and 7 weeks, but significantly higher ERα and lower ERß and AR levels were observed in the DBP group at 9 to 17 weeks. The rats prenatally exposed to DBP had seminiferous tubule degeneration and atypical hyperplasia of LCs during adulthood, which was associated with an increase in expression of ERα and a decrease of ERß and AR in the testis.

Atypical Leydig cell hyperplasia in adult rats with low T and high LH induced by prenatal Di(n-butyl) phthalate exposure.

The present study describes atypical Leydig cell (LC) hyperplasia in 20-week-old Sprague-Dawley rats with low testosterone and high luteinizing hormone levels after prenatal administration of 100 mg/kg/day di(n-butyl) phthalate on days 12 to 21 postconception. Light microscopy revealed LC hyperplasia surrounded by severely degenerated seminiferous tubules. Aggregated LCs had large ovoid nuclei with nucleoli and abundant eosinophilic cytoplasm. Immunohistochemical analysis showed expression of proliferating cell nuclear antigen and vimentin in many hyperplastic LCs. Electron microscopy revealed atypical nuclei, abundant free ribosomes, stripped rough endoplasmic reticulum, intermediate-size filaments, elongated cytoplasmic filopodia, atypical tight junctions, and cilia formations, but smooth endoplasmic reticulum was scarcely observed.

Male Sprague-Dawley rats exposed to in utero di(n-butyl) phthalate: dose dependent and age-related morphological changes in Leydig cell smooth endoplasmic reticulum.

When 100 mg/kg/day of di(n-butyl) phthalate (DBP) was intragastrically administered to pregnant Sprague-Dawley rats throughout gestation days 12 to 21, the male pups had similar body weights with no apparent physical differences (e.g., litter size, sex ratio) compared to that of the vehicle group. However, prominent age-related morphological alterations in the smooth endoplasmic reticulum (sER) of testicular Leydig cells (LCs) were observed once these animals reached puberty. At weeks 5 to 7, the abundant sER with non-dilated cisternae was distributed in LCs. Subsequently, although the number of LCs significantly increased, the amount of sER was significantly decreased at 9 to 14 weeks of age and had disappeared at 17 weeks. In contrast, the number of LCs and the amount of sER in LCs of the lower dose groups (10, 30, and 50 mg/kg/day) were similar to those of the vehicle group. Further, serum testosterone levels in the 100 mg/kg dose group were significantly lower during 5 to 17 weeks of age. While their luteinizing hormone (LH) level was significantly lower at 5 to 7 weeks of age, it became significantly higher during 9 to 17 weeks. The amount of sER in LCs decreased with age with the increase in LCs proliferation and serum LH levels in rat exposed in utero to DBP in a dose-dependent manner.

Nuclear Morphometric Analysis of Leydig Cells of Male Pubertal Rats Exposed In Utero to Di(n-butyl) Phthalate.

We recently reported that prenatal rat exposure to di(n-butyl) phthalate (DBP) induced Leydig cell (LC) hyperplasia after nine weeks (wks) of age, yet the number of LCs was similar to that of the vehicle group until seven weeks. Nuclear pleomorphism of hyperplastic LCs is common and is considered to be continuous progressive degeneration. Thus, computer-assisted image cell nuclear analysis of LCs was performed on 5- and 7-wk-old Sprague-Dawley (SD) rats whose dams had been administered DBP (i.g.) at 100 mg/kg/day or vehicle (corn oil) on gestation day 12 to 21. The results of the 5-wk-old DBP group were similar to those of the vehicle group; LC nuclei of the 7-wk-old DBP group showed normal ploidy and similar amounts of DNA. However, the size, elongation and peripheral chromatin aggregation parameters were significantly higher, and the reticular chromatin distribution and isolated chromatin aggregation parameters were significantly lower compared with the vehicle group. The present study quantitatively demonstrated nuclear morphological alterations in rat LCs at 7 wks old (puberty) due to the prenatal DBP administration before apparent LC hyperplasia developed.

Retraction: In utero-exposed di(n-butyl) phthalate induce dose dependent, age-related changes of morphology and testosterone-biosynthesis enzymes/associated proteins of Leydig cell mitochondria in rats.

Editor's AnnouncementIn utero-exposed di(n-butyl) phthalate induce dose dependent, age-related changes of morphology and testosterone-biosynthesis enzymes/associated proteins of Leydig cell mitochondria in ratsMasaya Motohashi, Michael F. Wempe, Tomoko Mutou, Yuya Okayama, Norio Kansaku, Hiroyuki Takahashi, Masahiro Ikegami, Masao Asari, Shin Wakui(The Journal of Toxicological Sciences, 41, 195-206, 2016) I have retracted the above paper as Editor-in-Chief of The Journal of Toxicological Sciences since I have serious concerns about it, primarily due to inappropriate authorship on a non-negligible scale.When it was brought to my attention that there was inappropriate authorship in this paper, I contacted the co-authors to confirm this point. I found out that the majority of them considered their listing as co-authors to be inappropriate. In addition, the majority agreed to the retraction of this paper.These facts raise concerns about the paper. From the standpoint of maintaining the integrity of the research community, I felt that such a paper should be retracted at once.Accordingly, I sent a summary of my concerns about the paper to the corresponding author, Dr. Shin Wakui. I also had an online interview with him to discuss this matter. I told Dr. Wakui that inappropriate authorship on a non-negligible scale is a serious problem that raises concerns about the paper.I prepared a draft of this Editor's Announcement and sent it to Dr. Wakui for review prior to revision and release. Although he did not agree to the retraction, I have decided to take this action from the standpoint of maintaining the integrity of the research community.I coordinated my response to this issue with Dr. Akira Naganuma, Editor-in-Chief of Fundamental Toxicological Sciences, a sister journal of The Journal of Toxicological Sciences. Toshiyuki Kaji, Ph.D.Editor-in-ChiefThe Journal of Toxicological Sciences.

Ultrastructural immunohistochemical study of L-type amino acid transporter 1-4F2 heavy chain in tumor microvasculatures of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced rat bladder carcinoma.

Angiogenesis is essential for tumor growth, and an enhanced vasculature supplying nutrients and oxygen might reflect malignant potential. L-type amino acid transporter 1 (LAT1/4F2hc) comprises a major nutrient transport system responsible for the Na+-independent transport of large neutral amino acids. Seventy five to seventy eight percent N-butyl-N-(4-hydroxybutyl) nitrosamine-induced rat bladder carcinoma cells showed high LAT1/4F2hc expression. While the intracarcinoma microvasculatures of fenestrated endothelial cells highly expressing LAT1/4F2hc might progressively transport essential amino acids from the microvasculatures to the extracellular matrix, non-fenestrated endothelial cells and pericytes did not. The present study revealed that the tumor angiogenesis is one of target anti-L-type amino acid transporter 1 drug.

In utero-exposed di(n-butyl) phthalate induce dose dependent, age-related changes of morphology and testosterone-biosynthesis enzymes/associated proteins of Leydig cell mitochondria in rats.

Female pregnant Sprague-Dawley rats were intragastrically (ig) administered di(n-butyl) phthalate (DBP) at four doses (0, 10, 50 and 100 mg/kg) during gestation days (GD) 12-21 (n = 5 per group). The age-related morphological changes of Leydig cell mitochondrion (LC-Mt) and testosterone biosynthesis enzymes/associated genes/proteins expression levels were investigated. As compared to the control (no DBP), the 10 mg, and 50 mg DBP dose groups, the 100 mg DBP dose group at weeks 5 and 7 showed a significant amount of small LC-Mt. Thereafter, from weeks 9 to 17, the LC-Mt size and quantity in the 100 mg DBP dose group increased and became statistically similar to the other dose groups; hence, dose and time-dependent LC-Mt changes were observed. Throughout the study, the 100 mg DBP dose group had significantly lower testosterone levels. In addition, the 100 mg DBP dose group displayed lower StAR (StAR, steroidogenic acute regulatory protein) and P450scc (CYP11a1, cholesterol side-chain cleavage enzyme) levels at weeks 5 and 7, but they became statistically similar to all other dose groups at weeks 9 to 17; in contrast, the SR-B1 (Sarb1, scavenger receptor class B member 1) levels were similar for all DBP dose groups. The rats in utero 100 mg DBP /kg/day (GD 12-21) exposure results from this study indicate a dose-dependent, age-related morphological change in LC-Mt which are linked to reductions in testosterone biosynthesis genes / proteins expression, specifically StAR and P450scc.

Male rats exposed in utero to di(n-butyl) phthalate: Age-related changes in Leydig cell smooth endoplasmic reticulum and testicular testosterone-biosynthesis enzymes/proteins.

This study investigated the age-related (i.e., weeks 5, 7, 9, 14 and 17) morphological changes of Leydig cell smooth endoplasmic reticulum (LCs-ER) and testicular testosterone biosynthesis/protein expression in rats in utero exposed to di(n-butyl) phthalate (DBP) (intragastrically; 100mg/kg/day) on days 12-21 post-conception. Ultrastructural observations revealed the LCs-ER of the DBP group were non-dilated until peri-puberty, and thereafter decreased and disappeared. RT-PCR and Western blotting analyses revealed that StAR and P450scc levels in the DBP group were significantly lower at 5 and 7 weeks compared with the vehicle group but became similar during weeks 9-17. Although 3ß-HSD, P450c17, and 17ß-HSD levels of mRNA and protein in the DBP group were similar to the vehicle control group at 5 and 7 weeks of age, they were significantly lower during weeks 9-17. In utero DBP exposure results in age-related LCs-ER changes corresponding to reduction of testicular testosterone biosynthesis enzymes/associated proteins.