A bilateral third head of the gastrocnemius which is morphologically similar to the plantaris.

PURPOSE: An extra muscle was observed on both sides of the popliteal fossa in the cadaver of a 78-year-old Japanese male during dissection. The aim of this case report was to identify whether this variant is a double plantaris or a third head of the gastrocnemius according to its morphological characteristics and innervation. METHODS: The muscles were displayed by careful dissection and delineation of surrounding structures. The size of each of the muscle bellies and tendons of those extra muscles were measured manually by the vernier caliper. RESULTS: The origin of each extra muscle was lateral to the tibial nerve and superior to the plantaris, and each extra muscle which transitioned to a descending tendon parallel to the plantaris had a cone-shaped belly. However, the tendon of the extra muscles was fused into the investing fascia of the gastrocnemius with a tendon length of 4.5 cm on the left and 4.6 cm on the right. The extra muscles were innervated by the branch of the tibial nerve to the medial head of the gastrocnemius on both sides. CONCLUSION: Although they had an origin and shape similar to that of the plantaris, we identified the extra muscles in this case as a third head of the gastrocnemius, because of innervation to the plantaris arises directly from the tibial nerve. This case highlighted that the innervation is essential to understanding the myogenesis of extra muscles, especially in cases which are difficult to categorize based on the morphological features of the muscle.

Changes in expression levels of oxidative stress-related genes in mouse epididymides by neonatal exposure to low-dose decabromodiphenyl ether.

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.

Persistent median artery: paedogenesis of the antebrachial arterial system in the human body.

The persistence of the median artery in adult life, a remnant of the early brachial artery in the embryonic stage, has been reported in many anatomical and clinical studies. Herein, we aimed to investigate the prevalence and origin of the median artery in cadavers. We examined 53 adult Japanese cadavers and carefully dissected 106 upper limbs, and the arterial systems in the forearms and hands were observed macroscopically. We found the palmar type of the median artery on 106 sides in 53 cadavers, and found that it passes through the carpal tunnel and forms the superficial palmar arch in the hand in only two cadavers. The antebrachial type, ending in the forearm before reaching the wrist joint, was detected on 63 sides in 44 cadavers. The proportions of the origins of the median artery examined in this study were as follows: originating from the common interosseous artery (CIA) on 15 sides (23%), anterior interosseous artery (AIA) on 9 sides (14%), ulnar artery (UA) on 16 sides (24%), and CIA-UA trunk on 26 sides (39%). None arose from other arteries in the upper limbs. Based on our results and the current theory on vascular development, we conclude that the term 'persistent median artery' must be strictly used for the one that arises from the arteries in the forearm except for the radial artery, and the presence of this ontogenetic remnant can be interpreted as paedogenesis in the human body. We also describe that the increasing trend in the incidence of the median artery since the nineteenth century, as pointed out by a few researchers, may represent 'nearly neutral evolution' at the phenotypic level in human populations.

Effects on the local immunity in the testis by exposure to di-(2-ethylhexyl) phthalate (DEHP) in mice.

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.

Cadmium exposure increases susceptibility to testicular autoimmunity in mice.

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-1ß 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.

A novel categorization of the muscular branches of the tibial nerve within the popliteal fossa.

BACKGROUND: The muscular branches of the tibial nerve within the popliteal fossa innervate the gastrocnemius, soleus, plantaris, and popliteus muscles. Various branching patterns have been described in textbooks; however, the underlying fundamental rules explaining the patterns remain unclear. Understanding the fundamental rule explaining the branching pattern of the innervating nerves is essential for understanding the ontogeny of skeletal muscles. Therefore, this study aimed at establishing a theory to explain the branching pattern of the muscular branches of the tibial nerve within the popliteal fossa. METHODS: The branching patterns of the muscular branches of the tibial nerve within the popliteal fossa were examined macroscopically in 62 lower limbs derived from 31 adult cadavers (22 males and 9 females, aged 49-95 years). RESULTS: The branch to the medial head of the gastrocnemius muscle invariably arose from the posteromedial side of the tibial nerve. The branches to the soleus muscle and lateral head of the gastrocnemius muscle had a common trunk in all the lower limbs and invariably arose from the posterolateral side. The branches to the plantaris and popliteus muscles arose anteriorly from the tibial nerve in this order (plantaris branch first, followed by the popliteus branch). These branches invariably arose more distally than the branch to both the heads of the gastrocnemius and soleus muscles. CONCLUSIONS: Based on these fundamental branching patterns, we suggest a novel branching categorization. The branches could be categorized into a posterior group and an anterior group, which has independent branches to the plantaris and popliteus muscles. This fundamental branching pattern and novel categorization contribute to the understanding of the ontogeny of the skeletal muscles around the flexor compartment of the leg.

Malformation spectrum induced by ketoconazole after single administration to pregnant rats during the critical period - comparison with vitamin A-induced malformation spectrum.

Azole derivatives have teratogenic effects in rodents. In the present study, malformations and their sensitive windows induced by high-dose ketoconazole (KCZ), an azole derivative, without maternal toxicity were investigated. In addition, the malformation spectrum determined was compared with that induced by vitamin A palmitate (VAP). Pregnant rats were administered a single dose of KCZ by oral gavage on specific individual days from gestational days 8 to 15 (GDs 8-15). Maternal animals were subjected to necropsy on GD 20, and the obtained fetuses were examined for external, visceral and skeletal malformations. The malformation spectrum of VAP was identified from available published data (Noda, Sato, and Udaka, 1982) and a complementary study (single administration of VAP at 1 200 000 IU kg(-1) ). Embryonic lethality was observed in dams given KCZ on GDs 9-12 with peak incidence on GDs 10 and 11 with complete resorption. KCZ induced major malformations included cleft palate, digital anomalies, misshapen limbs and unique discontinuous ribs, and the sensitive window for each was identified. Compared with the malformations induced by VAP, unique malformations (e.g. discontinuous ribs by KCZ, neural tube defects by VAP), similar malformations with similar sensitive windows (e.g. digital and limb malformations) and similar malformations with different sensitive windows (e.g. embryonic lethality and cleft palate) were distinguished, suggesting that the mechanisms of several of the types of KCZ-induced malformation are related to excessive vitamin A.

Neonatal corticosterone administration increases p27-positive Sertoli cell number and decreases Sertoli cell number in the testes of mice at prepuberty.

Cortisol and corticosterone (CORT) are steroid, antistress hormones and one of the glucocorticoids in humans and animals, respectively. This study evaluated the effects of CORT administration on the male reproductive system in early life stages. CORT was subcutaneously injected at 0.36 (low-), 3.6 (middle-), and 36 (high-dosed) mg/kg body weight from postnatal day (PND) 1 to 10 in ICR mice. We observed a dose-dependent increase in serum CORT levels on PND 10, and serum testosterone levels were significantly increased only in high-dosed-CORT mice. Triiodothyronine levels were significantly higher in the low-dosed mice but lower in the middle- and high-dosed mice. However, testicular weights did not change significantly among the mice. Sertoli cell numbers were significantly reduced in low- and middle-dosed mice, whereas p27-positive Sertoli cell numbers increased in low- and middle-dosed mice. On PND 16, significant increases in testicular and relative testicular weights were observed in all-dosed-CORT mice. On PND 70, a significant decrease in testicular weight, Sertoli cell number, and spermatozoa count was observed. These results revealed that increased serum CORT levels in early life stages could induce p27 expression in Sertoli cells and terminate Sertoli cell proliferation, leading to decreased Sertoli cell number in mouse testes.

The influence of environmental exposure to formaldehyde in nasal mucosa of medical students during cadaver dissection.

BACKGROUND: Environmental exposure to formaldehyde is commonly associated with clinical symptoms such as mucosal irritation and olfactory disorders. However, the impact of such exposure on the development of mucosal inflammation and its outcome has not been carefully evaluated. METHODS: The observational non-comparative study was planned. The study population consisted of group of 41 medical students who had signed up for a cadaver dissection course as part of their gross anatomy teaching at the school of medicine Chiba University in Japan. During such dissection course, the students are exposed to variable levels of environmental formaldehyde routinely employed for the preservation the cadavers. The subjects were evaluated by a detailed medical examination. We measured their serum IgE levels. In addition, an olfaction test and nasal mucosal sensitivity to histamine was serially determined, immediately before and after the course and 6 months after the completion of the course. RESULTS: Olfactory abnormalities were observed in 13/41 (32%) subjects and increased nasal mucosal hypersensitivity to histamine was observed in 17/41 (41%) during and immediately after completion of the course. These subjects had evidence of preexisting allergic rhinitis. 6/41 (15%) other students with no prior evidence of allergic rhinitis also exhibited formaldehyde associated clinical symptoms during the dissecting course. However, the symptoms disappeared upon completion of the course in all subjects studied. CONCLUSIONS: Temporary abnormalities in the olfaction test and increased nasal mucosal hypersensitivity to histamine were observed in a few students with preexisting allergic rhinitis after environmental exposure of high concentrations of formaldehyde. These effects appeared to be transient.

[Trial to stimulate learning motivation of medical students in the dissection practice "teaching assistant system"].

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.

[The physical therapy undergraduate students' responses to the gross human anatomy subjects].

Instruction in gross human anatomy is one of the important items in the subject for co-medical students of the physical therapist course. The physical therapy undergraduate students are required to have a solid understanding of the structure and formation of the human body. Therefore, their good-understanding of the course on the gross human anatomy and their experience of the gross human anatomy laboratory (observation practice) are acquired to improve their knowledge of the human body. To clarify the student responses to the gross human anatomy course including the gross human anatomy laboratory, several questionnaires were administered to the freshman physical therapy undergraduate student for two years. We found that more than 80% of the students, who felt a negative attitude for gross human anatomy before the course started, had a positive attitude about the gross human anatomy after going through the course. The experience of the gross human anatomy laboratory increased the students' activity of learning and they thought more about the dignity of being human after the course than before viewing. In addition, the results suggested that the multiple experiences of the gross human anatomy course are useful for the physical therapy undergraduate students to improve the quality of their understanding of the human body.

Maternal-fetal transfer rates of PCBs, OCPs, PBDEs, and dioxin-like compounds predicted through quantitative structure-activity relationship modeling.

The present study aims to predict the maternal-fetal transfer rates of the polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and polybrominated diphenyl ethers (PBDEs), and dioxin-like compounds using a quantitative structure-activity relationship model. The relation between the maternal-fetal transfer rate and the contaminants' physicochemical properties was investigated by multiple linear regression (MLR), partial least square regression (PLS), and random forest regression (RF). The 10-fold cross-validation technique estimated low predictive performances for both MLR and PLS models (R 2CV = 0.425 ± 0.0964 for MLR and R 2CV = 0.492 ± 0.115 for PLS) and is in agreement with an external test (R 2pred = 0.129 for MLR and R 2pred = 0.123 for PLS). In contrast, the RF model exhibits good predictive performance, estimated through 10-fold cross-validation (R 2CV = 0.566 ± 0.0885) and an external test set (R 2pred = 0.519). Molecular weight and polarity were selected in all models as important parameters that may predict the ability of a molecule to cross the placenta to the fetus.

Early changes in sperm motility, acrosome reaction, and gene expression of reproductive organs in rats treated with sulfasalazine.

Previously, we reported that decreased epididymal expression of CD59 and decay accelerating factor (DAF) genes may affect sperm motility and the acrosome reaction in rats treated long-term (28 days) with sulfasalazine. To investigate the early effects of sulfasalazine on the male reproductive tract, we presently examined sperm motility, the acrosome reaction, and gene expression in the testes and epididymides of rats treated with sulfasalazine for 1, 7 or 14 days. Reduced sperm motility and acrosome reactions were noted on day 7, however, there were no remarkable changes in testicular gene expression. On the other hand, attenuated epididymal gene expression of CD59 and DAF was observed as early as day 1. As CD59 and DAF are secreted from the epididymis and play a role in sperm maturation, we hypothesize that sulfasalazine affects sperm maturation as an early effect and that CD59 and DAF genes are related to the negative effect.

Reduction of dioxins and polychlorinated biphenyls (PCBs) in human body.

The accumulation of persistent lipophilic organic pollutants like dioxins and PCBs in human body is of great concern since many of these compounds may elicit adverse health effects on humans. To reduce dioxins and PCBs with long half-lives that are absorbed into the human body, we need to work actively to minimize accumulation of dioxins and PCBs taken. Lot of manner has been tested such as foods containing dietary fibers and chlorophyll, lipids (squalane etc) and anion exchange resins. Cholestyramine, a cholesterol lowering agent, was no efficacy in humans. Authors have conducted a pilot study to demonstrate the effect lowering dioxin in human bodies using colestimide. Nine patients on cdestimide for 6 months, showed mean 20% decrease respectively in both dioxin and PCB levels, and the maximum percentage decrease was approximately 40%. From a standpoint to avoid the influence on high-risk group and high-risk life stage other than next generation, the world-wide cooperation for reducing environmental chemicals is greatly appealed.

Assessment of questionnaire-based PCB exposure focused on food frequency in birth cohorts in Japan.

We investigated the relationship between food frequency questionnaire (FFQ) responses and serum polychlorinated biphenyl (PCB) levels of mothers and fathers recruited from the Chiba Regional Center, which is one of the 15 regional centers of the Japan Environment and Children's Study (mothers: n = 1477, fathers: n = 219). The expected PCB values were estimated from the participants' FFQ answers and medical records (age, body mass index and number of deliveries). Based on the stepwise forward selection results of Bayesian regression models, age and fish and egg consumption were positively associated with PCB concentrations and a number of deliveries were negatively associated with PCB concentrations in mothers, whereas only age was positively associated with PCB concentrations in fathers.These findings indicated that the estimation of daily dietary intake may be useful for the prediction of PCB concentration for mothers.

Formaldehyde exposure in a gross anatomy laboratory--personal exposure level is higher than indoor concentration.

GOAL, SCOPE AND BACKGROUND: Cadavers for gross anatomy laboratories are usually prepared by using embalming fluid which contains formaldehyde (FA) as a principal component. During the process of dissection, FA vapors are emitted from the cadavers, resulting in the exposure of medical students and their instructors to elevated levels of FA in the laboratory. The American Conference of Governmental Industrial Hygienists (ACGIH) has set a ceiling limit for FA at 0.3 ppm. In Japan, the Ministry of Health, Labour and Welfare has set an air quality guideline defining two limit values for environmental exposure to FA: 0.08 ppm as an average for general workplaces and 0.25 ppm for specific workplaces such as an FA factory. Although there are many reports on indoor FA concentrations in gross anatomy laboratories, only a few reports have described personal FA exposure levels. The purpose of the present study was to clarify personal exposure levels as well as indoor FA concentrations in our laboratory in order to investigate the relationship between them. METHODS: The gross anatomy laboratory was evaluated in the 4th, 10th and 18th sessions of 20 laboratory sessions in total over a period of 10 weeks. Air samples were collected using a diffusive sampling device for organic carbonyl compounds. Area samples were taken in the center and four corners of the laboratory during the entire time of each session (4-6 hours). Personal samples were collected from instructors and students using a sampling device pinned on each person's lapel, and they were 1.1 to 6 hours in duration. Analysis was carried out using high performance liquid chromatography. RESULTS AND DISCUSSION: Room averages of FA concentrations were 0.45, 0.38 and 0.68 ppm for the 4th, 10th and 18th sessions, respectively, ranging from 0.23 to 1.03 ppm. These levels were comparable to or relatively lower than the levels reported previously, but were still higher than the guideline limit for specific workplaces in Japan and the ACGIH ceiling limit. The indoor FA concentrations varied depending on the contents of laboratory sessions and seemed to increase when body cavity or deep structures were being dissected. In all sessions but the 4th, FA levels at the center of the room were higher than those in the corners. This might be related to the arrangement of air supply diffusers and return grills. However, it cannot be ruled out that FA levels in the corners were lowered by leakage of FA through the doors and windows. Average personal exposure levels were 0.80, 0.45 and 0.51 ppm for instructors and 1.02, 1.08 and 0.89 ppm for students for the 4th, 10th and 18th session, respectively. The exposure levels of students were significantly higher than the mean indoor FA concentrations in the 4th and 10th sessions, and the same tendency was also observed in the 18th session. The personal exposure level of instructors was also significantly higher than the indoor FA level in the 4th session, while they were almost the same in the 10th and 18th sessions. Differences in behavior during the sessions might reflect the differential personal exposure levels between students and instructors. CONCLUSION: The present study revealed that, if a person is close to the cadavers during the gross anatomy laboratory, his/her personal exposure level is possibly 2 to 3-fold higher than the mean indoor FA concentration. This should be considered in the risk assessment of FA in gross anatomy laboratories. RECOMMENDATION AND OUTLOOK: If the risk of FA in gross anatomy laboratories is assessed based on the indoor FA levels, the possibility that personal exposure levels are 2 to 3-fold higher than the mean indoor FA level should be taken into account. Otherwise, the risk should be assessed based on the personal exposure levels. However, it is hard to measure everyone's exposure level. Therefore, further studies are necessary to develop a method of personal exposure assessment from the indoor FA concentration.

Levels of formaldehyde vapor released from embalmed cadavers in each dissection stage.

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.

Chiba study of Mother and Children's Health (C-MACH): cohort study with omics analyses.

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 <13 weeks gestation. Women who underwent an examination in one of the three hospitals received an explanation of the study. The participants consented to completing questionnaire surveys and the collection and storage of biological and house/environmental samples. Participants were provided unique study numbers. All of the data and biological specimens will be stored in the Chiba University Center for Preventive Medical Sciences and Chiba University Center for Preventive Medical Sciences BioBank, respectively. FINDINGS TO DATE: Consent to participate was obtained from 433 women. Of these women, 376 women completed questionnaires in the early gestational period. The mean age was 32.5 (4.4) years. The mean body mass index (BMI) was 21.1 (3.0) kg/m(2). Before pregnancy, 72.3% of the women had a BMI of 18.5-24.9 kg/m(2). During early pregnancy, 5.0% of the participants smoked. FUTURE PLANS: Primary outcomes are allergy, obesity, endocrine and metabolic disorders, and developmental disorders. Genome-level, metabolome-level, umbilical cord DNA methylation (epigenome), gut microbiota and environmental chemical exposure variables will be evaluated. We will analyse the relationships between the outcomes and analytical variables.

Long-term alteration of gene expression without morphological change in testis after neonatal exposure to genistein in mice: toxicogenomic analysis using cDNA microarray.

In this study, we carried out toxicogenomic analysis using in-house cDNA microarray to ascertain the long-term effects of neonatal exposure to genistein, also known as phytoestrogen, on testicular gene expression in mice. Male ICR mice, 1 day after birth, were exposed for 5 days to genistein (1000 microg/mouse/day) or diethylstilbestrol (DES) (50 microg/mouse/day), used as an example of a potent estrogen, and their testes were used when they were 12 weeks old. Since exposure to DES was been reported to induce morphological changes and alteration of gene expression in reproductive organs, DES was used as a positive control. Genistein-treated mice did not show any histological abnormalities or increased apoptotic cells in testes, but these abnormalities and increases were found in DES-treated mice. On the other hand, mRNA expression analysis using in-house cDNA microarray revealed that 2 down-regulated genes (GeneBank accession No. W49392 and AI430907) were detected in genistein-treated mouse testes. Moreover, real-time PCR analysis revealed that mRNAs of the W49392 gene, estrogen receptor alpha (ERalpha) and androgen receptor (AR), were down-regulated in the testes of both genistein-treated and DES-treated mice. In our present study using toxicogenomic analysis, long-term alteration in testicular mRNA expression, without morphological change in testes, was detected after neonatal treatment with genistein, indicating that the W49392 gene, in addition to ERalpha and AR, might be useful as a biological marker for predicting the effects of neonatal exposure to DES and genistein.

Association of testicular undescent induced by prenatal flutamide treatment with thickening of the cremaster muscle in rats.

Background and Aims: Previously, in cryptorchid rats, which were induced by prenatal exposure to flutamide, we found a thickening of the cremaster muscle. This study was undertaken to quantify the increase of the cremaster muscle thickness in the cryptorchid rats, and to examine its possible relationship with the proliferation of muscle cells. Methods: To obtain cryptorchid rats, pregnant Wistar rats were subcutaneously injected with flutamide (100 mg/kg per day) during gestational days 16-17. Serial sections of the scrotum, containing the testis and cremaster muscle, were prepared from the control and cryptorchid rats that were 2-6 weeks of age, and stained with hematoxylin-eosin for morphometry, or stained with antibody against the proliferating cell nuclear antigen (PCNA) to analyze the cell proliferation ability. Results: The thickened cremaster muscle was always associated with cryptorchid testis and, in the case of unilateral cryptorchidism, the cremaster muscle of the contralateral (descended testis) side exhibited normal thickness. The average thickness of the affected cremaster muscle was 0.80 and 1.89 mm at 4 and 6 weeks of age, respectively, although that of the normal muscle was 0.28 and 0.33 mm at the same time period, respectively. Conclusion: Our results showed that the cremaster muscle of the cryptorchid rats was significantly thicker than that of the control rats. The immunohistochemical analysis revealed that a thickened cremaster muscle contained many PCNA-positive nuclei even at 4 weeks of age, in contrast to the control, which had only a few positive nuclei. Our present study indicates that continuous proliferation of the muscle cells associated with cryptorchid testis increases the thickness of cremaster cells in rats exposed to flutamide prenatally. (Reprod Med Biol 2003; 2: 109-113).