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In non-clinical animal studies for drug discovery, histopathological evaluation is the most powerful tool to assess testicular toxicity. However, histological analysis is extremely invasive; many experimental animals are needed to evaluate changes in the pathology and anatomy of the testes over time. As an alternative, small animal magnetic resonance imaging (MRI) offers a non-invasive methodology to examine testicular toxicity without radiation. The present study demonstrated the suitability of a new, ready-to-use compact MRI platform using a high-field permanent magnet to assist with the evaluation of testicular toxicity. To validate the utility of the MRI platform, male mice were treated with busulfan (40 mg/kg, intraperitoneal injection). Tenty-eight days after treatment, both testes in busulfan-treated and control mice (n = 3/group) were non-invasively scanned in situ by MRI at 1 tesla. On a T1-weighted, 3D gradient-echo MRI sequences (voxel size: 0.23 × 0.23 × 0.50 mm), the total testicular volume in busulfan-treated mice was significantly smaller than in controls. On T1-weighted images, the signal intensity of the testes was significantly higher in busulfan-treated mice than in controls. The mice were sacrificed, and the testes were isolated for histopathological analysis. The weight of the testes in busulfan-treated mice significantly decreased, similar to the results of the non-invasive analysis. Additionally, periodic acid-Schiff stain–positive effusions were observed in the interstitium of the busulfan-treated mouse testes, potentially explaining T1 shortening due to a high concentration of glycoproteinaceous content. The present data demonstrated a rapid evaluation of testicular toxicity in vivo by compact MRI.

BackgroundReactive blue 2 (RB2) dye specifically binds to the nuclei of human spermatozoa under weakly alkaline conditions, thus providing a new method to assess sperm quality. However, this technique has not yet been applied to other mammalian species, such as well-established rodent models, which could enable evaluation of the male reproductive toxicity of drug candidates in non-clinical studies.ObjectivesWe aimed to evaluate the usefulness of RB2 staining in assessing testicular and epididymal sperm toxicity in mice using a busulfan-induced infertility model.MethodsMale C57BL/6J mice were intraperitoneally administered 40 mg/kg of busulfan. After 28 days, the testes and epididymis were collected and stained with RB2 at pH 10. In vitro evaluations were conducted on uncoated glass slides with RB2 mixed with either protamines extracted from the spermatozoa or intracellular protein components from somatic cells without protamines.ResultsFollowing peanut agglutinin (PNA) lectin histochemistry, RB2-positive cells were observed in elongating and elongated spermatids at all stages except for stages IX–XI of the seminiferous epithelium. After busulfan administration, the proportion of RB2-positive germ cells in the seminiferous tubules decreased significantly, and no RB2-positive spermatozoa were found in the caput epididymis of treated mice. Aggregates were observed in the mixture of RB2 dye (pH 10) with protamines but not in the mixture of intracellular protein components without protamines, and this specificity was lost at neutral pH.Discussion and ConclusionOur study demonstrates that RB2 specifically stains steps 12–16 spermatids, indicating specific binding to protamine expressed in these spermatids. The RB2 staining technique has potential as a biomarker for male reproductive toxicity, allowing for the rapid visualization of protamination.

The male reproductive system consists of testes, a series of ducts connecting the testes to the external urethral orifice, accessory sex glands, and the penis. Spermatogonial stem cells differentiate and mature in testes and epididymides, and spermatozoa are ejaculated with exocrine fluids secreted by accessory sex glands. Many studies have clarified the detailed structure and function of the male reproductive system, and have shown that various biologic controls, including genomics, epigenetics, and the neuroendocrine-immune system regulate proliferation, differentiation, and maturation of germ cells. In other words (1) genetic deletion or abnormalities, (2) aberration of DNA methylation and histone modifications, as well as small RNA dysfunction, and (3) neuroendocrine-immune disorders are involved in functional failure of the male reproductive system. In this article, we review these three factors for germ cell microcircumstance, especially focused on the immunoendocrine environment. In particular, the relation between factors protecting germ cells with strong auto-immunogenicity and opposite factors compromising this protection are discussed. Reductions in sperm count, concentration, and semen quality are serious problems in developed countries, although the causes are complex and remain unclear. The accumulation of basic knowledge regarding the structure, function, and regulation of the male reproductive system under various experimental conditions will be important to resolve these problems.

It is difficult to correlate the direction of mandibular canal branches (MCBs) with altered sensation in dental treatments. In contrast, calcitonin gene–related peptide (CGRP) is related to vasodilation, bone formation, and the interaction with the peripheral nervous system. Therefore, we investigated the detailed morphological characteristics of MCBs using cone-beam computed tomography (CBCT) and observation of the CGRP distribution around the MCB. The MCB measurements were evaluated using principal component analysis (PCA) to identify morphological correlations. A total of 168 sides of mandibles from 84 cadavers were analyzed in this study. Most of the MCBs were primarily in the direction of the clock model from X to XI in sagittal sections and XII to I in coronal sections of the mandible. The structure of the MCB was divided into the fine canal branch (60.4%, 223/369), partial branch (24.4%, 90/369), and no canal branch (15.2%, 56/369). PCA indicated that the measurement element with the MCB and its structures were correlated in contrast to tooth factors. Positive CGRP reactions were clearly observed in the no-canal branch group compared to other groups. These data provide useful suggestions for MCB dynamics and information for clinical dental treatment.

Objectives Injury to the mandibular nerve (MN) branches may cause pain and irregular occlusal movement during mastication after mandibular dental treatments. Growing evidence indicates that the calcitonin gene‐related peptide (CGRP) plays a key role in the development of peripheral sensitization and the associated enhanced pain, suggesting it may be a sign to ensure a safe and reliable dental implant treatment. Our focus was on the distribution of the MN branches and their communication with the lingual nerve (LN), the localized expression of CGRP, and the identification of a pain area related to the mylohyoid muscle (MM) fascia in the mandibular floor. Material and Methods In this study, MM samples from 440 sides of 303 human cadavers aged 61–103 years were examined microscopically and immunohistochemically. These data were further evaluated by the use of principal component analysis. Results A complex but weak attachment site was identified for the fascia of the MM. CGRP expression was mainly located in small vessels and was scattered throughout the whole fascia of the MM. Communication between the MN and LN was found in 62.5% (275/440) of the samples. The results from the principal component analysis showed that the positive contributions were from the descending branch in the premolar region (correlation coefficient value R = 0.665), the ascending branch in the molar region (R = 0.709) and the intermediate branch of the digastric branch (R = 0.720) in component 1. In the fascia off the MM, strongly labeled CGRP‐positive cells were also found around the blood vessels and the nerve. Conclusions The findings reported in this study indicate that there is a risk of damage when pulling the fascia off the MM at the border of the molar and premolar regions during dental implant surgery.

Purpose We morphometrically analyzed human facial muscles, and evaluated the Yanagihara facial nerve grading system using our data. Methods We used 15 types of human facial muscle, 2 types of masticatory muscle and 2 types of skeletal muscle. The materials were obtained from 11 Japanese male cadavers aged 43–86 years. We counted the muscle fibers and measured the transverse area of the muscle fibers (TAMF), and then calculated the number of muscle fibers (NMF) per mm2 and the average TAMF. Results We found a significant correlation between average TAMF and NMF (r = − 0.70; p

Formaldehyde (FA) is frequently used to embalm human cadavers that are employed to teach gross anatomy to medical and dental students. However, exposure to FA is harmful to both students and educators. The aim of this study was to reduce the FA levels in the anatomy dissection hall by spraying an FA scavenger solution. We measured the changes in FA levels after administering FA scavenger solutions to liquid, wet paper towels, organs, and cadavers containing FA. Among l-cysteine, N-ethyl urea, and urea, the latter was found to have the strongest scavenging power towards the FA in the liquid. The molar concentration of urea that most efficiently reduced the levels of volatilized FA from the wet paper towels was the same as that of the FA. After spraying the urea solution, the volatilized FA levels immediately decreased, reaching their minimum at 60 min, and remained low even after 240 min. Spraying the urea solution onto the organs reduced the levels of FA volatilized from the surfaces of organs but not those from the insides of the organs. In the dissection hall used for the gross anatomy course at Tokyo Medical University, the FA levels were significantly decreased after spraying the urea solution onto the cadavers. Moreover, dissection could be performed without the cadavers putrefying during the 4-month course. These results indicate that various institutes could use urea solution spray to effectively reduce the FA levels in the dissection hall and thus ensure the safety of students and educators.

Aim Senior surgeons in Japan who participated in "cadaver-based educational seminar for trauma surgery (CESTS)" subsequently stated their interest in seminars for more difficult procedures. Therefore, we held a 1-day advanced-CESTS with saturated salt solution (SSS)-embalmed cadavers and assessed its effectiveness for surgical skills training (SST). Methods Data were collected from three seminars carried out from September 2015 to January 2018, including a 10-point self-assessment of confidence levels (SACL) questionnaire on nine advanced surgical skills, and evaluation of seminar content before, just after, and half a year after the seminar. Participants assessed the suitability of the two embalming methods (formalin solution [FAS] and SSS) for SST, just after the seminar. Statistical analysis resulted in P

Neonatal maternal separation (NMS) is an experimental model for early life stress, which affects the growth and development of various organs, resulting in adverse health effects in humans and animals. In our previous study, we demonstrated that NMS [(0.5-, 1-, 2-h/day NMS, from postnatal day (PND) 1-10] induced morphological changes to the male reproductive system, including decreased Sertoli cell numbers in mouse testes at PND 70. To clarify the mechanism by which NMS decreases Sertoli cell numbers, we evaluated the effects of NMS on mouse testes at PNDs 10 and 16. At PND 10, the Sertoli cell number was not significantly different among experimental groups; however, it decreased in 0.5- and 2-h/day NMS mice at PND 16. The termination of Sertoli cell proliferation in prepuberty can be induced by p27, a cyclin-dependent kinase inhibitor. At PND 10, we observed an increase in the number of p27-positive Sertoli cells in 2-h/day NMS mice. The seminiferous tubule diameters decreased significantly in 1- and 2-h/day NMS mice, and the relative interstitial area increased in 2-h/day NMS mice. Serum corticosterone level significantly increased, and serum testosterone level significantly decreased in the 2-h/day NMS mice. At PND 16, the tubule diameters and height of seminiferous epithelium were significantly higher in 0.5- and 2-h/day NMS mice. Our results suggest that NMS disturbs serum corticosterone and testosterone levels and increases the number of p27-positive Sertoli cells at PND 10, resulting in a decrease in the number of Sertoli cells at PND 16.

This study aimed to identify human body areas in which paramedical students are particularly eager to observe and that are of interest in relation to the type of healthcare profession by means of a questionnaire after anatomical observation. A total of 840 students from 27 schools who visited Chiba University School of Medicine for anatomical tours, were examined after responding to the open questionnaire along with their consent. Terms of the human body structure that were of high interest were abstracted from the responses, and the frequency was analyzed according to individual specialized field. As a result, 1746 words were abstracted overall, and a high level of interest was identified in the nervous system (482 words), digestive system (425 words), cardiovascular system (305 words), musculoskeletal system (254 words), and respiratory system (183 words) sequentially. When considering individual specialized fields, the highest interest was found in the nervous system by nurses, acupuncturists, and emergency medical technicians, the digestive system by pharmacists and nutritionists, and the musculoskeletal system by rehabilitationists. When providing instruction during the observation, explanation should focus on areas of higher interest, as clarified in this study. At the same time, instruction in less interesting but are still important areas for the students in their specialized field must be provided with ingenuity to cultivate interest.Mini-abstract: Highly interesting human body structures were the nervous system, digestive system, and musculoskeletal system, for nurses, acupuncturists, and emergency medical technicians; pharmacists and nutritionists; and the rehabilitation field, respectively.

Clavicle fracture is known to be one of the injuries frequently occurring in the elderly. The purpose of this study was to characterise the internal structures that might correlate with the higher incidence of lateral clavicle fracture in the elderly. Twenty clavicles were collected from ten Japanese cadavers ranging from 70 to 99 years (83.6 ± 7.6), scanned, and three-dimensional computed tomography (3D CT) images reconstructed. The clavicle lengths were divided into five equal segments. The four demarcation lines from the acromial end of the clavicle were defined as the observation points A, B, C, and D. The clavicles were then measured and analysed. It was shown that along the clavicles observation point A was the widest and points B and C the narrowest. Regarding the thickness, point D was the thickest among all four points, and there was no significant difference among the points A, B, and C. No male-female difference was found in either the cortical or cancellous bone ratio at all four points. Interestingly, the highest cortical bone ratio was observed at point B and the ratio was significantly decreased toward either end. The cancellous bone ratio was highest at point C and decreased toward both ends. Further observations showed that there were rays of trabeculae around point A, spreading from the superior-posterior edge or anterior edge toward each other and toward the lateral end and point B. Characteristics in the cortical and cancellous bone ratios and cancellous bone patterns might shed light on understanding the fractures in the lateral portion of the clavicle in the elderly.

Experimental autoimmune orchitis (EAO) may be used as a model to investigate immunological infertility in men. Murine EAO is induced via immunization with auto-immunogenic antigens (AIAgs) from testicular germ cells (TGCs). CD4 + T cells play a crucial role in EAO induction. However, whether AIAgs induce an immune response remains unclear. We aimed to identify self-antigens that induce EAO by screening a phage display library of random TGC peptides using IgG from EAO-induced A/J mice. Twenty TGC-specific AIAgs were detected, and G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) and heat shock protein A4L (HSPA4L) were identified as candidate AIAgs that induce EAO. Immunization with GIT1 or HSPA4L, emulsified in complete Freund's adjuvant, resulted in 66 % or 100 % incidence of EAO, respectively, indicating that HSPA4L is a most potent AIAg that induces EAO in mice. These findings may expectedly help improve the diagnostic procedures and treatment of immunological infertility in men.

Several intrauterine environmental factors can increase the future risk of type 2 diabetes. The microbiome can influence the balance between health and disease. However, the influence of the maternal gut microbiome on the future risk of diabetes in the fetus is unknown. The present study investigated the associations between maternal gut microbiome and differentially methylated regions of diabetes‐associated genes in umbilical cord samples. The present study included 10 pregnant participants from a birth cohort study. 16S ribosomal ribonucleic acid metagenome analysis of maternal stool samples and deoxyribonucleic acid methylation assays of umbilical cord samples were carried out. The present study found that changes in the UBE2E2 and KCNQ1 methylation rates in umbilical cord samples were associated with the proportion of Firmicutes in the maternal gut, albeit with marginal correlations after adjustment for age and body mass index. These findings suggest a link between the methylation of diabetes‐associated genes in fetuses and maternal microbiota components during pregnancy.

The musculocutaneous nerve innervates the brachial flexors; i.e., the coracobrachialis, biceps brachii, and brachialis. The musculocutaneous and median nerve sometimes share a communicating branch and also muscular branches to brachial flexors are sometimes transposed to median nerve. Because these variations constitute a potentially important clinical and surgical issue, we evaluated 130 upper limbs of 65 cadavers and 184 cases of musculocutaneous and median nerve variations in the literature and devised a novel system of classification that covers each pattern of variation. Our proposed classification was applicable in all of our cases and those previously reported. In addition, transposed innervation of the brachial flexors from the musculocutaneous nerve to the communicating branches was observed in one limb in our case series. In this case and all previously reported cases of transposed innervation of the brachial flexors, communicating branches between the musculocutaneous and median nerve were found and no brachial flexor branch arose from the musculocutaneous nerve distal to the communicating branches. Consequently, we established that, as per our novel classification, all patterns of communicating branches could be classified into five types and the patterns of transposed innervation of the brachial flexors into three types.

H19 is a tumor-suppressor gene, and changes in the methylation of the H19-differential methylation region (H19-DMR) are related to human health. However, little is known about the factors that regulate the methylation levels of H19-DMR. Several recent studies have shown that maternal environmental factors during pregnancy, such as smoking, drinking, chemical exposure, and nutrient intake, can alter the methylation levels of several genes in fetal tissues. In this study, we examined the effects of maternal factors on changes in the methylation levels of H19-DMR in the human umbilical cord (UC), an extra-embryonic tissue. Participants from the Chiba study of Mother and Children's Health (C-MACH) were enrolled in this study. Genomic DNA was extracted from UC samples, and the methylation level of H19-DMR was evaluated by methylation-sensitive high resolution melting analysis. Individual maternal and paternal factors and clinical information for newborns at birth were examined using questionnaires prepared in the C-MACH study, a brief-type self-administered diet history questionnaire (BDHQ) during early pregnancy (gestational age of 12 weeks), and medical records. Univariate and multivariate logistic regression analyses indicated that reduced H19-DMR methylation (

It is known that the activities of estrogen receptor α (ERα) can be modulated by epidermal growth factor (EGF) through the phosphatidylinostitol 3-kinase-alpha serine/threonine protein kinase (PI3K-AKT) pathway by phosphorylation. To clarify how ERα functions are regulated in endometrial cells during menstrual cycle, molecules related to phosphorylation of ERα (pERα) were examined. It was found that the expression of phosphorylated AKT on serine 473 (pAKT-Ser473) was increased during the proliferative phase, but decreased in the secretory phase. Although the expression of pAKT on threonine 308 in the proliferative phase was only identified in the wall of arterioles, it was strongly expressed in the cytoplasm of endometrial glandular cells after entering the secretory phase. Further observations revealed that while the expression of pERα-Ser104 was constant, pERα-Ser118 was expressed following a cyclic pattern similar to that of the pAKT-Ser473. Following treatment with specific inhibitors for EGFR-PI3K-AKT pathway, it was found that while the expression of pERα-Ser118 and pERα-Ser167 was inhibited, the induced apoptosis could be antagonized by the addition of estrogen, indicating that a mitochondrial pathway is involved. Therefore, pAKT and pERα or ERα could act cooperatively on coiled arterioles and endometrial cells in order to control menstrual cycle.

Maternal exposure to polychlorinated biphenyls (PCBs) results in abnormal fetal development, possibly because of epigenetic alterations. However, the association between PCB levels in cord serum with fetal DNA methylation status in cord tissue is unclear. This study aims to identify alterations in DNA methylation in cord tissue potentially associated with PCB levels in cord serum from a birth cohort in Chiba, Japan (male neonates = 32, female neonates = 43). Methylation array analysis identified five sites for female neonates (cg09878117, cg06154002, cg06289566, cg12838902, cg01083397) and one site for male neonates (cg13368805) that demonstrated a change in the methylation degree. This result was validated by pyrosequencing analysis, showing that cg06154002 (tudor domain containing 9: TDRD9) in cord tissue from female neonates is significantly correlated with total PCB levels in cord serum. These results indicate that exposure to PCBs may alter TDRD9 methylation levels, although this hypothesis requires further validation using data obtained from female neonates. However, since the present cohort is small, further studies with larger cohorts are required to obtain more data on the effects of PCB exposure and to identify corresponding biomarkers.

The toxicity of decabromodiphenyl ether (BDE-209) has been reported in several studies. However, there is not much known about the toxicological biomarkers that characterize BDE-209 exposure. In this study, we subcutaneously exposed mice to 0.025 mg/kg/day BDE-209 on postnatal days 1‑5 and sacrificed the animals at 12 weeks of age (day 84). Flow injection analysis and hydrophilic interaction chromatography-triple quadrupole mass spectrometry were used to determine the serum metabolomes of these mice in order to characterize the effects of BDE-209 exposure. Data analysis showed a good separation between control and exposed mice (R(2) = 0.953, Q(2) = 0.728, and ANOVA of the cross‑validated residuals (CV‑ANOVA): P‑value = 0.0317) and 54 metabolites were identified as altered in the exposed animals. These were selected using variable importance (VIP) and loadings scaled by a correlation coefficient criteria and orthogonal partial least squares discriminant analysis (OPLS‑DA). BDE‑209‑exposed mice showed lower levels of long-chain acylcarnitines and citrate cycle-related metabolites, and higher levels of some amino acids, long-chain phospholipids, and short-chain acylcarnitines. The disruption of fatty acid, carbohydrate, and amino acid metabolism observed in the serum metabolome might be related to the previously observed impaired spermatogenesis in mice with early postnatal exposure to a low dose of BDE-209.

Surface plasmon resonance (SPR) has been extensively used to characterize the interactions between molecules in terms of their binding specificity, affinity, and kinetics. Practical procedures, however, for measurement of the protein-DNA association in crude nuclear extract are yet to be defined.Crude nuclear extract was obtained from MCF-7 cells or recombinant estrogen receptor alpha (ERα) was used for analysis. To suppress signal from non-specific bindings in SPR assay using Biacore, DNA fragments with minimal protein binding activity were identified in a database for transcription factors and included in the study.It is known that when analytes were purified transcription factors, the dissociation curves in Biacore sensorgrams exhibit exponential tendency. Based on statistical analysis, the dissociation phase between the ERα complex from crude nuclear extract and DNA oligonucleotides could be fitted exponentially. Following extrapolation of the dissociation phase, theoretical amount of bound antibodies could be estimated and compared for significant difference.Our procedures made SPR technique such as Biacore a practical technique for measurement of protein-DNA associations in crude nuclear extract with reproducible and reliable results.

Biacore is a system that is extensively used to characterize the interactions between molecules in terms of their binding specificity, affinity, and kinetics. The practical procedures, however, for measurement of protein-DNA association in crude nuclear extract are yet to be defined. In the present study, DNA fragments with the least protein binding activity were identified in database for transcription factors and included in Biacore assay as control, so that the signals from non-specific binding were markedly suppressed. It was known that when analytes were purified transcription factors, the dissociation curves in Biacore sensorgrams showed exponential tendency. Further analysis showed that the interaction between ERα complex from crude nuclear extract and DNA oligos could be fitted to mono- or bi-exponential functions. Discrimination between orders of exponential function was based on the results of several statistical analyses with an average score of more than 95%. As exponential characteristics allow extrapolation of the dissociation, theoretical amount of bound anti-ERα antibodies could thus be evaluated statistically. Our procedures made Biacore a practical technique like Supershift Assay to measure protein-DNA association in crude nuclear extract with reproducible and reliable results.

Clavicle fracture is known to be one of the injuries frequently occurring in the elderly. The purpose of this study was to characterise the internal structures that might correlate with the higher incidence of lateral clavicle fracture in the elderly. Twenty clavicles were collected from ten Japanese cadavers ranging from 70 to 99 years (83.6 ± 7.6), scanned, and three-dimensional computed tomography (3D CT) images reconstructed. The clavicle lengths were divided into five equal segments. The four demarcation lines from the acromial end of the clavicle were defined as the observation points A, B, C, and D. The clavicles were then measured and analysed. It was shown that along the clavicles observation point A was the widest and points B and C the narrowest. Regarding the thickness, point D was the thickest among all four points, and there was no significant difference among the points A, B, and C. No male-female difference was found in either the cortical or cancellous bone ratio at all four points. Interestingly, the highest cortical bone ratio was observed at point B and the ratio was significantly decreased toward either end. The cancellous bone ratio was highest at point C and decreased toward both ends. Further observations showed that there were rays of trabeculae around point A, spreading from the superior-posterior edge or anterior edge toward each other and toward the lateral end and point B. Characteristics in the cortical and cancellous bone ratios and cancellous bone patterns might shed light on understanding the fractures in the lateral portion of the clavicle in the elderly.

Decabromodiphenyl ether (decaBDE), one of the polybrominated diphenyl ethers (PBDEs), is the most well‐known flame retardant and is used worldwide. In a previous study, we identified adverse effects of neonatal decaBDE exposure on mouse epididymides, such as decreased epididymal weight. On the other hand, neonatal exposure to diethylstilbestrol (DES), an artificial estrogenic compound, also causes several adverse effects on epididymides. DES exposure results in decreased epididymal weight, morphological abnormalities, and permanent alterations in the expression levels of several genes. The molecular mechanisms underlying the harmful effects of decaBDE exposure remain unclear. Many studies have reported that PBDEs have estrogenic activity, which may contribute to the induction of the adverse effects of decaBDE exposure. We aimed to examine the effects of neonatal decaBDE exposure on epididymides. Our data showed that (1) no histological change was observed on epididymal tissues from neonatal decaBDE exposure, unlike the effect of DES, (2) decaBDE exposure did not induce the alterations in gene expression observed with DES exposure; instead alterations in gene expression of certain oxidative stress‐related genes were observed, and (3) the expression of ubiquitin C increased in decaBDE‐exposed mouse epididymides. Our present data suggest the possibility that increased oxidative stress plays a role in the harmful effects observed in mouse epididymides after decaBDE‐exposure.

Exposure to di-(2-ethylhexyl) phthalate (DEHP) has been reported to induce spermatogenic disturbance through oxidant stress and affect the immune system as an adjuvant. However, the effect of DEHP on the testicular immune microenvironment has not yet been investigated. In the present study, we examined the testicular immune microenvironment after exposure to doses of DEHP, previously identified as no-observed-adverse-effect levels. Adult male mice were administered food containing 0%, 0.01% or 0.1% DEHP and then testes were analyzed. The results showed that a slight but significant spermatogenic disturbance appeared in the 0.1% DEHP group but not in the 0.01% DEHP group at 8 weeks. It was also demonstrated that lymphocytes and F4/80- and MHC class II- positive cells were significantly increased with the elevation of IL-10 and IFN-γ mRNA expressions in the testes of not only the 0.1% DEHP group but also the 0.01% DEHP group at 8 weeks. Histochemical analyses involving horseradish peroxidase (HRP) as a tracer showed that a little blood-borne HRP had infiltrated into the lumen of a few seminiferous tubules beyond the blood-testis-barrier in both the 0.1% and 0.01% DEHP groups at 8 weeks. This indicates that a dose of DEHP that has little effects on spermatogenesis can change the testicular immune microenvironment with functional damage of the blood-testis barrier.

Purpose Recent epidemiological studies have shown that environmental factors during the fetal period to early childhood might affect the risk of non-communicable diseases in adulthood. This is referred to as the developmental origins of health and disease (DOHaD) concept. The Chiba study of Mother and Children's Health (C-MACH) is a birth cohort study based on the DOHaD hypothesis and involves multiomics analysis. This study aims to explore the effects of genetic and environmental factors—particularly the fetal environment and postbirth living environment—on children's health, and to identify potential biomarkers for these effects. Participants The C-MACH consists of three hospital-based cohorts. The study participants are pregnant women at

The Graduate School of Medicine at Chiba University is planning to introduce computed tomography (CT) images of donated cadavers to the gross anatomy laboratory. Here we describe an anomaly of the right subclavian artery that was detected by interpretation of CT images prior to dissection. The anomaly was verified to be the right subclavian artery, as the last branch of the aortic arch, by subsequent dissection of the cadaver. We also identified an anomalous origin of the right vertebral artery by dissection. This anomaly was also visible on CT images, although it had not been recognized in the first interpretation of the CT images. Our results suggest that branching anomalies of arteries with a diameter of >1 cm are detectable on CT images even without the injection of contrast medium. We also discuss the utility of interpreting CT images prior to dissection as a means by which medical students can gain a better understanding of human body during the gross anatomy laboratory.

Cadmium, one of various environmental toxicants, is known to suppress systemic immunity and to injure the testicular capillary endothelia with resultant necrosis of testicular tissues in mice and rats treated with high doses. Recently, it also became evident that cadmium can affect the integrity of the blood-testis barrier (BTB), the endocrine function of Leydig cells, apoptosis of germ cells and systemic immunity, even on treatment with a low dose that does not induce spermatogenic disturbance. Experimental autoimmune orchitis (EAO), i.e., an organ-specific autoimmunity of the testis, can be induced by repeated immunization with testicular antigens, and its pathology is characterized by lymphocytic inflammation and spermatogenic disturbance. In the present study, we investigated the morphological and functional changes of testes in mice treated with a low dose of cadmium chloride (CdCl2 ) and also examined its toxicity as to susceptibility to EAO. The results showed that exposure to 3 mg CdCl2 kg(-1) body weight did not affect the spermatogenic state. However, the BTB at the tubuli recti and the rete testis, but not the seminiferous tubules, was slightly weakened, and intra-testicular mRNA expression of interleukin (IL)-6, tumor necrosis factor-α and IL-1β was significantly increased by the CdCl2 treatment. Furthermore, immunization with testicular antigens after the CdCl2 exposure significantly augmented the EAO severity. Therefore, exposure to a low dose of CdCl2 induces no significant disturbance of spermatogenesis, however, it does change the immunological microcircumstances in the testis, resulting in increased susceptibility to testicular autoimmunity.

Decabromodiphenyl ether (decaBDE) is a brominated flame retardant used in many commercial products such as televisions, computers, and textiles. Recent reports indicate that decaBDE adversely affects male reproductive organs in mice, but the underlying molecular mechanisms remain unknown. We hypothesized that decaBDE affects mouse testes by altering the expression and phosphorylation level of cortactin (CTTN), an F-actin-binding protein that is similar to flutamide, and we performed western blot analyses on testicular samples from mice subcutaneously injected with decaBDE (0.025, 0.25, and 2.5 mg/kg body weight/day) on postnatal days 1 to 5. Mice treated with low-dose decaBDE (0.025 mg/kg) showed reduced testicular weight, sperm count, elongated spermatid and Sertoli cell numbers, as well as induced Tyr phosphorylation of CTTN and reduced the expression level of p60 Src tyrosine kinase (SRC). Further, 0.25 and 2.5 mg/kg decaBDE-exposed groups produced an decrease the expression level of CTTN. High-dose decaBDE (2.5 mg/kg) showed increased abnormal germ cells, as well as induced Ser phosphorylation of CTTN and activated extracellular signal-regulated kinase (ERK1/2); however, high-dose decaBDE did not affect testicular weight and sperm count. These findings suggest that postnatal exposure to low-dose decaBDE inhibits mouse testicular development by increasing Tyr phosphorylation of CTTN, although different mechanisms may be involved depending on the dose of decaBDE.

Formaldehyde (FA) is an aldehyde used in antiseptics and adhesives. The World Health Organization (WHO) and other institutes have linked FA to sick building syndrome and allergic diseases. Recent studies have reported that cadavers embalmed using formalin and ethanol-based preservative solutions release FA vapor during dissection and that FA vapor may adversely affect students and lecturers in gross anatomy laboratories. However, few details have been reported correlating dissection stage with increased FA vapor release. In this study, we evaluated the vapor level of FA released in each dissection stage. Six cadavers for which consent was given for use in anatomy research and education were examined in this study. Using an active sampling method, FA vapor was collected above the thoracoabdominal region of each dissected cadaver. FA was eluted from each sampler using acetonitrile and analyzed by high-performance liquid chromatography. Our data show that FA levels significantly increase after skin incision and that the vapor level of FA released differs between male and female cadavers. We also found that subcutaneous adipose tissues of the thoracoabdominal-region release FA vapor and that female cadavers release significantly higher levels of FA per kilogram of subcutaneous adipose tissue than do male cadavers. Based on these data, we propose the methods be developed to prevent exposure to FA vapors released from cadavers. Electronic supplementary material The online version of this article (doi:10.1007/s11356-016-6744-8) contains supplementary material, which is available to authorized users.

Cortactin—an F-actin–binding protein—regulates actin polymerization and plays an important role in cytoskeleton actin dynamics. Cortactin functions are reportedly regulated by changes in its isoform (p80/85) profiles and phosphorylation status. Although essential for spermatogenesis, the exact role of cortactin in the testis has not been well elucidated. Herein, we examined its dynamics in isoform profiles and tyrosine phosphorylation levels during spermatogenesis in mice. Western blot analysis showed that the amount of the p85 isoform of cortactin particularly decreased during stages VI–VIII. In the p80 isoform, cortactin phosphorylations at both tyrosine 421 and 466 were detected during stages XII–V and VI–VIII. For the p85 isoform, tyrosine 466 phosphorylation of cortactin, not 421, was detected during stages XII–V and VI–VIII, and this phosphorylation particularly increased during stages VI–VIII. The tyrosine 466 phosphorylation level of the p85 isoform of cortactin relative to the total amount of the p85 isoform notably increased during stages VI–VIII. Additionally, immunohistochemical analysis showed localization of the tyrosine 466–phosphorylated cortactin around the heads of elongating and elongated spermatids. Finally, we examined the effect of flutamide—an antiandrogen—on cortactin isoform profiles and phosphorylation status. The amount of tyrosine 466–phosphorylated p85 isoform of cortactin relative to the total amount of the p85 isoform of cortactin decreased after flutamide injection, whereas no change was detected with regard to the total amount of cortactin p85 isoform. These data suggest the possibility that localization of the more tyrosine 466–phosphorylated p85 isoform of cortactin around the heads of elongated spermatids is important for stages VII–VIII processes and that its phosphorylation site of the p85 isoform might be a target for creating a novel contraceptive and treating infertility in the future.

Heat shock protein 90 (Hsp90) regulates diverse signaling pathways. Emerging evidence suggests that Hsp90 inhibitors, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), enhance DNA damage-induced cell death, suggesting that Hsp90 may regulate cellular responses to genotoxic stress. However, the underlying mechanisms are poorly understood. Here, we show that the Fanconi anemia (FA) pathway is involved in the Hsp90-mediated regulation of genotoxic stress response. In the FA pathway, assembly of 8 FA proteins including FANCA into a nuclear multiprotein complex, and the complex-dependent activation of FANCD2 are critical events for cellular tolerance against DNA cross-linkers. Hsp90 associates with FANCA, in vivo and in vitro, in a 17-AAG–sensitive manner. Disruption of the FANCA/Hsp90 association by cellular treatment with 17-AAG induces rapid proteasomal degradation and cytoplasmic relocalization of FANCA, leading to impaired activation of FANCD2. Furthermore, 17-AAG promotes DNA cross-linker–induced cytotoxicity, but this effect is much less pronounced in FA pathway-defective cells. Notably, 17-AAG enhances DNA cross-linker–induced chromosome aberrations. In conclusion, our results identify FANCA as a novel client of Hsp90, suggesting that Hsp90 promotes activation of the FA pathway through regulation of intracellular turnover and trafficking of FANCA, which is critical for cellular tolerance against genotoxic stress.

In the gross anatomy laboratory, which is one of the compulsory subjects in most medical and dental schools, participants cannot avoid exposure to formaldehyde (FA), which is emitted from cadavers during dissection. FA has been recognized as a harmful chemical and we have previously reported that symptoms felt by participants in a gross anatomy laboratory are similar to those of allergic diseases. Although immunoglobulin E (IgE)-mediated sensitization to FA is a matter of controversy, it is possible that IgE production is evoked during a gross anatomy laboratory and is responsible for the reported symptoms. In order to test this hypothesis, we examined the relationships between the personal FA exposure levels and plasma IgE levels in a gross anatomy laboratory. In the laboratory, the personal FA exposure levels ranged from 0.33 to 1.47 ppm. Total blood IgE levels did not increase significantly and specific IgE to FA was negative during the laboratory sessions. Thus, from this study, we cannot support the hypothesis that the exposure to FA triggers an IgE-mediated reaction in this study. In conclusion, exposure to FA does not induce IgE production during gross anatomy laboratories at our school.

Spinal muscular atrophy results from the loss of functional survival motor neuron (SMN1) alleles. Two nearly identical copies of SMN exist and differ only by a single non-polymorphic C to T transition in exon 7. This transition leads to alteration of exon 7 splicing; that is, SMN1 produces a full-length transcript, whereas SMN2 expresses a low level of full-length transcript and predominantly an isoform lacking exon 7. The truncated transcript of SMN encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. In this paper, we identified a cis-acting element (element 1), which is composed of 45 bp in intron 6 responsible for the regulation of SMN exon 7 splicing. Mutations in element 1 or treatment with antisense oligonucleotides directed toward element 1 caused an increase in exon 7 inclusion. An approximately 33-kDa protein was demonstrated to associate with a pre-mRNA sequence containing both element 1 and the C to T transition in SMN exon 7 but not with the sequence containing mutated element 1, suggesting that the binding of the approximately 33-kDa protein plays crucial roles in the skipping of SMN exon 7 containing the C to T transition.

Diethylstilbestrol (DES), an endocrine-disrupting chemical, is an infamous artificial estrogenic compound. Although neonatal exposure to DES has been shown to result in inflammation of the male reproductive system, it has not, to our knowledge, been reported to induce testicular inflammation. Here we report that neonatal exposure to DES caused granulomatous orchitis with spermatogenic disturbance in 4 of 17 ICR male mice at 12 weeks of age. In the animals with spermatogenic disturbance, we observed either seminiferous tubules containing only cells with Sertoli cell features (likely Sertoli cell syndrome), or tubule cells in maturation arrest that contained only spermatogonia and/or spermatocytes. Following neonatal DES exposure, 5-week-old mice exhibited inflammation in cauda epididymis; by 8 weeks, the inflammation had spread to all segments of epididymis but not the testis; by 12 weeks, inflammation of the epididymis was observed in all mice. These data indicated that cauda epididymis has increased sensitivity to neonatal DES exposure compared to other segments of epididymis and testis. The data also implied that neonatal DES exposure-induced inflammation in cauda epididymis extended gradually to the testis via corpus and caput during development.

Cadmium, one of various environmental toxicants, is known to suppress systemic immunity and to injure the testicular capillary endothelia with resultant necrosis of testicular tissues in mice and rats treated with high doses. Recently, it also became evident that cadmium can affect the integrity of the blood-testis barrier (BTB), the endocrine function of Leydig cells, apoptosis of germ cells and systemic immunity, even on treatment with a low dose that does not induce spermatogenic disturbance. Experimental autoimmune orchitis (EAO), i.e., an organ-specific autoimmunity of the testis, can be induced by repeated immunization with testicular antigens, and its pathology is characterized by lymphocytic inflammation and spermatogenic disturbance. In the present study, we investigated the morphological and functional changes of testes in mice treated with a low dose of cadmium chloride (CdCl2 ) and also examined its toxicity as to susceptibility to EAO. The results showed that exposure to 3 mg CdCl2 kg(-1) body weight did not affect the spermatogenic state. However, the BTB at the tubuli recti and the rete testis, but not the seminiferous tubules, was slightly weakened, and intra-testicular mRNA expression of interleukin (IL)-6, tumor necrosis factor-α and IL-1β was significantly increased by the CdCl2 treatment. Furthermore, immunization with testicular antigens after the CdCl2 exposure significantly augmented the EAO severity. Therefore, exposure to a low dose of CdCl2 induces no significant disturbance of spermatogenesis, however, it does change the immunological microcircumstances in the testis, resulting in increased susceptibility to testicular autoimmunity.

At the Medical School of Chiba University, educational dissection tours have been conducted for intra- and extramural students in other programs, such as students of nursing. In the 2006 school year there were more than 1,500 students. As presented in a previous report, we tested an educational program in which our medical students teach other students parts of splanchnology, neurology, and myology to promote student understanding of human physiology through their own teaching. Since this system, termed the "teaching assistant system," was fairly laborious for many medical students, we attempted to improve it by decreasing the students' load and reducing the frequency of teaching from several times to once during the one-term dissection practice. We assessed the improved method with questionnaires for medical students who had studied at the school in 2006 and 2007 (n = 206) before and after teaching other students. The response rate for the questionnaires was 91.3% (n = 188). The results were as follows. (1) Most medical students (69.7%) realized that the task of teaching had stimulating effects on their own learning motivation. (2) According to most of their evaluations (80.4%), the duties of teaching involved in the previous assistant system were laborious. In contrast, the ratio of medical students who considered teaching to be laborious decreased by about half (55.3%) in the present improved system. (3) Most students (79.8%) were satisfied with the teaching assistant system. We concluded that the improved teaching assistant system was effective for the dissection practice.

Spinal muscular atrophy is caused by the homozygous loss of survival motor neuron 1 (SMN1). SMN2, a nearly identical copy gene, differs from SMN1 only by a single nonpolymorphic C to T transition in exon 7, which leads to alteration of exon 7 splicing; SMN2 leads to exon 7 skipping and expression of a nonfunctional gene product and fails to compensate for the loss of SMN1. The exclusion of SMN exon 7 is critical for the onset of this disease. Regulation of SMN exon 7 splicing was determined by analyzing the roles of the cis-acting element in intron 7 (element 2), which we previously identified as a splicing enhancer element of SMN exon 7 containing the C to T transition. The minimum sequence essential for activation of the splicing was determined to be 24 nucleotides, and RNA structural analyses showed a stem-loop structure. Deletion of this element or disruption of the stem-loop structure resulted in a decrease in exon 7 inclusion. A gel shift assay using element 2 revealed formation of RNA-protein complexes, suggesting that the binding of the trans-acting proteins to element 2 plays a crucial role in the splicing of SMN exon 7 containing the C to T transition.