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.].

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.

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.

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.

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.

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.

Muscle dimensions of the foot in the orangutan and the chimpanzee.

The hindlimbs of two orangutans and four chimpanzees were dissected, and muscle parameters (mass, fascicle length, and physiological cross-sectional area: PCSA) were determined to explore possible interspecies variation in muscle dimensions. Muscle mass and PCSA were divided by the total mass and total PCSA of the entire foot muscles for normalization. The results indicate that the pedal interosseous and the intrinsic pedal digital extensor muscles in the orangutans probably have higher capacity for force production due to their relatively larger PCSAs than in chimpanzees. Moreover, the medial components of the intrinsic muscles exhibited relatively larger mass and PCSA ratios in orangutans. The mass and PCSA ratios of the hallucal muscles were larger in chimpanzees. These differences in foot muscle dimensions of the two species suggest that the orangutan is more specialized for hook-like digital gripping without involvement of the rudimentary hallux, while the chimpanzee is adapted to hallux-assisted power gripping in arboreal locomotion.

Computed tomography and radiographic lymphography of the thoracic duct by subcutaneous or submucosal injection.

A simple method of lymphography of the thoracic duct was investigated. Using three female beagles, contrast media were administered rectally, vaginally and into the perianal tissue. The administration sites were gently massaged, and imaging was carried out at constant intervals using computed tomography and radiograph. Moreover, Indian ink was administered into the rectum mucous membrane in dogs for proof of this method of lymphography, and the lymph drainage routes were observed. The investigation showed that clear computed tomography and radiographic contrast images of the thoracic duct were obtained by subcutaneous and submucosa injection of angiography contrast medium and 3D processing of these images revealed the three-dimensional positions and course of the thoracic duct and cisterna chyli.

Immunohistochemical localization and gene expression of carbonic anhydrase isoenzymes CA-II and CA-VI in canine lower airways and lung.

The immunohistolocalization and gene expression of carbonic anhydrase (CA) isoenzymes CA-II and CA-VI in the canine lower airways and lung were examined using specific canine CA-II and CA-VI antisera and the RT-PCR method. Laryngeal, tracheal and bronchial epithelia, serous acinar and bronchiolar secretory cells and pulmonary great alveolar cells showed immunopositive reactions to anti-CA-II and anti-CA-VI antisera. However, all mucous cells showed immunonegative reactions. The physiological roles of CA-II and CA-VI in the lower airways and lung may involve the maintenance of pH balance and the protection of mucosal surfaces against the acidic milieu.

Determination of full-length cDNA nucleotide sequence of equine carbonic anhydrase VI and its expression in various tissues.

A full-length cDNA clone of an equine carbonic anhydrase (CA)-VI was obtained from the equine parotid gland. The cDNA sequence was 1338 bp long and was predicted to encode a 319 amino acid polypeptide with a putative signal peptide of 18 amino acids. The deduced amino acid sequence of mature CA-VI showed the similarity of 70% to those of other mammalians reported. Westernblot analysis using anti-horse CA-VI peptide detected the single band in parotid gland, and the band reduced its size by treatment with N-glycosidase F. Additionally, CA-VI protein expression was confirmed in submandicular gland and weakly in liver. In contrast, RT-PCR analysis revealed signals in the digestive tract including duodenum, jejunum, ileum, cecum and colon as well as the salivary glands. In addition, certain signals were detected in testis, thyroid gland and liver, but not in nerve tissue, skeletal muscle, spleen or lymph node.

Dimensions of forelimb muscles in orangutans and chimpanzees.

Eight forelimbs of three orangutans and four chimpanzees were dissected and the muscle mass, fascicle length and physiological cross-sectional area (PCSA) of all forelimb muscles were systematically recorded to explore possible interspecies variation in muscle dimensions. Muscle mass and PCSA were divided by the total mass and total PCSA of the entire forelimb muscles for normalization. The results indicate that the mass and PCSA ratios of the monoarticular elbow flexors (M. brachialis and M. brachioradialis) are significantly larger in orangutans. In contrast, the mass ratios of the biarticular muscles in the upper arm (the short head of M. biceps brachii and the long head of M. triceps brachii) are significantly larger in chimpanzees. For the rotator cuff muscles, the force-generating capacity of M. subscapularis is significantly larger in orangutans, whereas the opposite rotator cuff muscle, M. infraspinatus, is larger in chimpanzees. These differences in forelimb muscle dimensions of the two species may reflect functional specialization for their different positional and locomotor behaviors.

Dimensions of the foot muscles in the lowland gorilla.

We dissected the hindlimb of a female western lowland gorilla and determined the muscle dimensions (mass, fascicle length, and physiological cross-sectional area: PCSA). Comparisons of the muscle parameters of the measured gorilla with corresponding reported human data demonstrated that the triceps surae muscles were larger and had more capacity to generate force than the other muscle groups in both species, but this tendency was more prominent in the human, probably as an adaptation to strong toe-off during bipedal walking. On the other hand, PCSAs of the extrinsic pedal digital flexors and digiti minimi muscles were larger in the western lowland gorilla, suggesting that the foot, particularly the fifth toe, has a relatively high grasping capability in the lowland gorilla.

Pathomechanism of cellular infiltration in the perivascular region of several organs in SAMP1/Yit mouse.

We investigated the histological changes of extra-intestinal organs, such as the liver, kidney, lung and pancreas in SAMP1/Yit mice, a human Crohn's disease model, using immunohistochemical techniques. The perivascular cellular infiltration was detected around the small vessels after 30 weeks. These infiltrating cells consisted of many CD4-positive T-lymphocytes, and small numbers of CD8- positive T-lymphocytes and IgG-positive B-lymphocytes. MAdCAM-1 and VCAM-1 were detected in vascular endothelial cells in non-affected regions of 13 and 20 week-old, as well as in the affected regions showing perivascular cellular infiltration after 30 weeks. In addition, integrin alpha4beta7 was detected on these infiltrating cells in the perivascular regions after 30 week-old. LT-beta and IL-12, cytokines of the Th-1-type immune response, were not observed in these affected regions. However, IL-4, one of the cytokines of the Th-2-type immune response, was detected on the perivascular infiltrating cells after 30 week-old. These results revealed that the changes in extra-intestinal organs were mainly caused by infiltration of CD4-positive T-lymphocytes into the perivascular regions in SAMP1/Yit mice. These cellular infiltrations were thought to be initiated by adhesion of CD4-positive T-lymphocytes to the endothelial cells mediated by MAdCAM-1 and integrin beta7. Immunohistochemistry for Th related cytokines indicated that the perivascular cellular infiltration was developed by the Th-2-type immune response in the extra-intestinal organs of SAMP1/Yit mouse.

Immunohistolocalization and gene expression of secretory carbonic anhydrase isoenzyme CA-VI in canine nasal cavity.

Immunolocalization of the secretory form of carbonic anhydrase isoenzyme, CA-VI were studied using a specific canine CA-VI antiserum, and CA-VI mRNA signals were also investigated using the reverse-transcriptase polymerase chain reaction (RT-PCR) in canine nasal mucosal epithelia and glands. Immunoreactivity to CA-VI was positive throughout the mucosal epithelial cells and in the cytoplasm of serous acinar and ductal epithelial cells of the nasal mucosa and glands, including the vestibule of the nose, but the mucous acinar cells of the glands were immunonegative. We detected CA-VI gene transcripts in the same regions as the CA-VI immunoreactivity. The physiological roles of CA-VI in the nasal mucosal epithelium and glands might maintain bicarbonate levels in nasal secretions and protect the mucosa against acid.

A macroscopic examination of M. biceps femoris and M. gluteus maximus in the orangutan.

The musculature of the hip and thigh in the orangutan has been described previously. Anatomically, there are various descriptions among primates in those structures, in particular, the relationship between M. biceps femoris and M. gluteus maximus, their derivatives, and the muscle segment. However, a detailed innervation system to this ischiofemoral part has not been described, thus there is still uncertainty as to with which muscle it is associated. In this analysis, we examined the gross anatomy of the hip and thigh muscles of the orangutan and chimpanzee, including their innervation. Also, a comparison was made with documented data of other primates. As a result of these observations, it was found that the ischiofemoral part in the orangutan is innervated by the same sciatic nerve branch (the common peroneal nerve) as the long head of M. biceps femoris, but not by the same nerve as M. gluteus maximus. Therefore, the ischiofemoral part is appropriately considered as a part of the long head of M. biceps femoris. It appears that this morphologic feature is an adjustment to the arboreal life of the orangutan. The development of the flexor complex of the thigh is necessary for this arboreal adaptation, resulting in a unique musculature of M. biceps femoris in the orangutan.

Muscle architecture of the upper limb in the orangutan.

We dissected the left upper limb of a female orangutan and systematically recorded muscle mass, fascicle length, and physiological cross-sectional area (PCSA), in order to quantitatively clarify the unique muscle architecture of the upper limb of the orangutan. Comparisons of the musculature of the dissected orangutan with corresponding published chimpanzee data demonstrated that in the orangutan, the elbow flexors, notably M. brachioradialis, tend to exhibit greater PCSAs. Moreover, the digital II-V flexors in the forearm, such as M. flexor digitorum superficialis and M. flexor digitorum profundus, tend to have smaller PCSA as a result of their relatively longer fascicles. Thus, in the orangutan, the elbow flexors demonstrate a higher potential for force production, whereas the forearm muscles allow a greater range of wrist joint mobility. The differences in the force-generating capacity in the upper limb muscles of the two species might reflect functional specialization of muscle architecture in the upper limb of the orangutan for living in arboreal environments.

Comparative observation of skeletal-dental abnormalities in wild, domestic, and laboratory rabbits.

Dietary habits must be considered as one of the major potential factors resulting in acquired malocclusions in rabbits. Although the dentition of the wild rabbit and the domesticated laboratory rabbit are basically identical, dietary habits are noticeably different. Therefore, the prevalence of tooth problems between these lagomorph species were investigated anatomically and radiographically. Mean measurements of the skull and dental arches suggested that wild rabbits have slightly shorter and wider skulls and dental arches compared with domestic laboratory rabbits. Root elongation of incisors and check teeth, and periodontal disease were more frequently observed in domestic laboratory rabbits. Diagnostic radiographs from domestic pet rabbits showed relatively higher crowns, severe root elongation, and advanced periodontitis. These results do not provide definitive evidence that dietary habits cause malocclusions, however they suggest that diet is a major factor in the initiation of malocclusions in rabbits.