Mei Hua Jin

Enhanced TGF-β1 is involved in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced oxidative stress in C57BL/6 mouse testis

2,3,7,8-Tedtrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic endocrine disruptors and has been reported to induce oxidative stress in the reproductive organs. However, the mechanism by which TCDD induces oxidative stress is unclear. The aim of this study is to examine the role of the general cytokine, TGF-beta1, in TCDD-induced oxidative stress in the male reproductive system. To examine the effect of TCDD on antioxidant enzyme activity, we administered TCDD orally to C57BL/6 mice at 1 microgkg/day for 4 days. Using Smad2-siRNA, we examined the involvement of Smad and non-Smad pathways in TCDD-induced oxidative stress. We also measured the mRNA levels of typical antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and analyzed the activation of TGF-beta1, and the downstream signals, Smad2, Smad4, transcription factors (c-Jun, ATF3), and three major MAPKs (JNK, ERK, p38). After TCDD treatment, the mRNA levels of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) were significantly decreased. In addition, TGF-beta1 activity increased and the receptor-activated protein, Smad2, was activated while Smad4 was not. The levels of major transcription factors, c-Jun and ATF3, and the regulator of these transcription factors, MAPK, were also increased by TCDD administration. The mRNA levels of the 3 antioxidant enzymes in the Smad2-siRNA and TCDD co-treated group were higher than that of the TCDD-only treated group but still decreased when compared to control. C-Jun and ATF3 levels were also increased in Smad2-siRNA and TCDD co-treated testes compared to control. However, the levels of c-Jun and ATF3 were lower than those in the group treated with TCDD only. Of the three MAPKs which showed increase in expression after TCDD treatment, p38 was the only one that showed a decrease with Smad2 inhibition, while both ERK and JNK expression were unaffected. In conclusion, we found that the activated TGF-beta1-Smad pathway is involved in TCDD-induced oxidative stress. Furthermore, the effects of TCDD on the testes are caused by the coordinated action of both Smad and non-Smad pathways.

Toxic effects of lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on development of male reproductive system: involvement of antioxidants, oxidants, and p53 protein.

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD) is a potent endocrine disruptor compound and induces multiple organ dysfunctions. The effect of TCDD exposure both in adults and in utero has been well established. However, little is known about the effects of TCDD acquired through mothers milk on the development of the male reproductive system. The aim of this study was to investigate the effects and mechanisms of TCDD from lactational exposure. TCDD (1 μg/kg) was administered to C57BL/6 mouse mothers for 4 days from the day of delivery. On postnatal day 30 (PND 30) and postnatal day 60 (PND 60), body weight, body length, and anogenital distance (AGD) of male offspring were measured. The weights of the testes and epididymides were also measured. Epididymides were used for sperm counts, and testes were used to measure the activity of antioxidant enzymes (SOD, CAT, GPX, GR), the parameters of oxidative stress (hydrogen peroxide, MDA), and testosterone. In addition, expression of p53 and the proapoptotic protein, Bax, were analyzed by Western blot. TCDD exposure decreased body weight, body length, and AGD in both PND 30 and PND 60 groups compared with the control group. The activity of all antioxidant enzymes at PND 60 was decreased after TCDD treatment. TCDD treatment decreased testicular testosterone levels in both the PND 30 and PND 60 groups. The expression of p53 and Bax were also upregulated by TCDD and did not return to normal levels by PND 60. These data suggest that TCDD affects development of male offspring when the mother is exposed to TCDD during lactation. In addition, oxidative stress is a major mediator of TCDD‐induced adverse effects, and p53 may play an important role in this mechanism.

Decreased Interstitial Cells of Cajal-like Cells, Possible Cause of Congenital Refluxing Megaureters: Histopathologic Differences in Refluxing and Obstructive Megaureters

OBJECTIVES To evaluate the location of interstitial cells of Cajal-like cells and the ureteral aspect using histopathologic studies of obstructive and refluxing megaureters to reveal the different pathogenesis of megaureters in the human urinary tract. The underlying pathophysiology of obstructive megaureter and refluxing megaureter is poorly understood. METHODS The data from 14 patients with obstructive megaureter (7 boys and 7 girls), with a mean age of 12 months (range 2-84), and 9 patients with refluxing megaureter (7 boys and 2 girls), with a mean age of 11 months (range 4-24), were compared. We investigated the difference in the histopathologic aspects using Massons trichrome, terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (for apoptosis), and human c-kit antibody (CD117 for interstitial cells of Cajal-like cells) between the obstructive and refluxing megaureters. RESULTS The proportion of smooth muscle was significantly lower in segments of refluxing megaureter (32.04% +/- 4.96%) than in the segments of obstructive megaureter (52.48% +/- 3.46%; P < .01). The number of apoptotic cells was significantly increased in the obstructive megaureter (mean 1661 +/- 135.1 cells) compared with the refluxing megaureter (mean 375.2 +/- 65.14 cells; P < .0001). The number of c-kit positive cells was significantly lower in the refluxing megaureter (mean 83.60 +/- 48.84 cells) than in the obstructive megaureter (mean 463.6 +/- 100.4 cells; P < .05). CONCLUSIONS The differences in the histopathologic aspects can provide information on the possible pathophysiology of obstructive and refluxing megaureters. Ureteral peristalsis can be affected by the increased myocyte apoptosis in the obstructive megaureter and by the decreased number of interstitial cells of Cajal-like cells and smooth muscle content in refluxing megaureters. Additional research is warranted to elucidate the exact pathophysiology.

Function of the Cold Receptor (TRPM8) Associated with Voiding Dysfunction in Bladder Outlet Obstruction in Rats

Purpose Bladder outlet obstruction (BOO) causes storage and voiding dysfunction in the lower urinary tract. We investigated the expression of transient receptor potential cation channel subfamily M member 8 (TRPM8) to evaluate the relationship between TRPM8 expression and overactive bladder (OAB) in a rat model of BOO. Methods Fifty female Sprague-Dawley rats were divided into 4 groups; normal (n=10), normal-menthol (n=10), BOO (n=15), BOO-menthol (n=15). After 3 weeks, cystometry was performed by infusing physiological saline and menthol (3 mM) into the bladder at a slow infusion rate. The histological changes and expression of TRPM8 in the bladder were investigated by Massons trichrome staining, immunofluorescence and reverse transcription-polymerase chain reaction. Results Cystometry showed that the intercontraction interval (ICI; 428.2±23.4 vs. 880.4±51.2, P<0.001), micturition pressure (MP; 25.7±1.01 vs. 71.80±3.01, P<0.001), and threshold pressure (2.9±0.25 vs. 9.2±1.58, P<0.01) were significantly increased in BOO rats. The bladder wall was significantly dilated compared with the control. Detrusor muscle hypertrophy and a thick mucosa layer were observed in BOO bladder. After menthol treatment, ICIs were decreased and MPs were increased in the menthol treatment groups. TRPM8-positive cells and mRNA were predominantly increased in the bladder and dorsal root ganglia of all groups compared with the normal group. Conclusions Increased bladder wall thickness and proportion of collagen probably affect voiding dysfunction. Furthermore, an increase of TRPM8 expression in BOO may induce entry of Ca2+ from the extracellular space or stores. The increase of Ca2+ probably causes contraction of smooth muscle in BOO. However, OAB symptoms were not observed after menthol treatment although the expression of TRPM8 was abundant in the bladder epithelium after menthol treatment. Although OAB in BOO models may be caused by complex pathways, regulation of TRPM8 presents possibilities for OAB treatment.

In utero exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin affects the development of reproductive system in mouse.

Purpose Exposure of male reproductive organs to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) has been reported to cause developmental changes. In this study, we evaluated the effects of in utero TCDD exposure on male reproductive development. Materials and Methods Pregnant C57BL/6 mice were administered a single intraperitoneal injection of TCDD (1 µg/kg) on gestation day (GD) 15. The offspring were examined in the immature stage on postnatal day (PND) 30 and in the mature stage on PND 60. The testes were examined for histological changes, androgen receptor (AR), proliferating cell nuclear antigen (PCNA) and apoptosis following the measurement of morphological changes. Results Anogenital distance (AGD) and testis weights were reduced by TCDD exposure both on PND 30 and PND 60 while body weights and length of male offspring were not affected by TCDD. The regular sperm developmental stage was impaired with TCDD treatment on PND 30. However, no difference was found between the control group and TCDD groups on PND 60. Simultaneously, the expression of AR was also reduced on PND 30, while it was increased on PND 60 compared with the control group. The expression of PCNA was decreased whereas apoptosis was not affected by TCDD both on PND 30 and PND 60. Conclusion These results suggest that in utero exposure to TCDD influences the development of testes by inhibiting the expression of AR and PCNA. Moreover, the adverse effects of TCDD on male offspring reduced over time.

The effect of intravesical electrical stimulation on bladder function and synaptic neurotransmission in the rat spinal cord after spinal cord injury.

To investigate the effects of intravesical electrical stimulation (IVES) on bladder function and synaptic neurotransmission in the lumbosacral spinal cord in the spinalized rat, as the clinical benefits of IVES in patients with increased residual urine or reduced bladder capacity have been reported but studies on the mechanism of IVES have mainly focused on bladder Aδ afferents in central nervous system‐intact rats.

c-fos Expression in Bladder-Specific Spinal Neurons after Spinal Cord Injury Using Pseudorabies Virus

Purpose c-fos expression in spinal neurons that are activated by lower urinary tract stimulation are not organ specific. In this experiment, we demonstrated changes of c-fos expression in bladder-specific preganglionic neurons (PGNs) and interneurons using pseudorabies virus (PRV). Materials and Methods Forty Sprague-Dawley rats were used. We identified the neuronal pathway associated with the bladder by injecting PRV into the detrusor. An immunohistochemical method was used to stain Fos-protein encoded by the c-fos gene. Immunofluorescent staining for PRV was performed to evaluate changes in bladder-specific spinal neurons. Results Immunofluorescent staining with choline acetyltransferase (ChAT) revealed that the sacral parasympathetic nucleus (SPN) regions contained 9.8 PGNs/section. In rats with chronic spinal cord injury by intravesical saline instillation, 82.4 ± 10.3% of PGNs in SPN exhibited Fos-immunoreactive (IR). Two and a half days after PRV infection, PRV-IR PGNs were observed at 5.4 PGNs/ section, and 2.7 ± 1.6% of them exhibited Fos-IR. Unlike ChAT-IR PGNs, PRV-IR PGNs are bladder-specific neurons and PRV-IR and Fos-IR cells found in the back of PRV-IR PGNs are bladder-specific interneurons. Three days after PRV infection, we observed many PRV-IR and Fos-IR cells in the dorsal commissure. These neurons are interneurons distributed in the bladder. Conclusion We confirmed that in chronic spinal cord injury, the patterns of c-fos expression in bladder-specific spinal neurons were similar to those in voiding-reflex related spinal neurons, which had already been demonstrated earlier. We believe that our methodology can be applied to study interactions between voiding and other organs as well, such as the urethra and prostate.

Timing of Prepubertal Androgen Administration May Have Different Effects on Future Fertility as Well as Penile Size in Normal Male Rats

OBJECTIVES To investigate the effects of androgen administration at different periods of prepubertal life on penile size and potential fertility in hormonally normal rats. METHODS Eighty Sprague-Dawley rats were divided into 4 groups; group 1 received weekly injections of testosterone enanthate (8 mg/100 g) at 1-3 weeks group 2 at 3-5 weeks, group 3 at 1-5 weeks, and group 4 was control (n = 20 each). Animals were killed at age 60 days. Penile length, penile, testicular, and epididymal weight, testicular fertility index, serum follicular stimulating hormone, luteinizing hormone, and testosterone levels were measured and quantitative real time polymerase chain reaction of penile androgen receptor messenger ribonucleic acids was performed. RESULTS Penile length in the 3-5-week injection group was significantly longer than controls (41.18 vs 39.13 mm, P = .011), and it was smaller than controls in the 1-3 week injection group (37.24 vs 39.13 mm, P = .020). The penile wet weight showed a similar pattern to stretched penile length; however, statistical significance was not found. Testicular weight was significantly less in the testosterone injection group than controls (1.79, 2.16, 2.24, and 2.78 g in groups 1-4, respectively) as well as the epididymal weight (0.48, 0.42, 0.59, and 0.69 in groups 1-4, respectively). The mean tubular diameter and germ cell count were significantly reduced in the testosterone treated group. Total penile androgen receptor messenger ribonucleic acid level was significantly lower in group 3 as compared with controls. CONCLUSIONS Testosterone administered during early prepubertal life resulted in smaller penis than controls, as well as having an adverse effect on testicular development which can jeopardize potential fertility.

Neurologic Mechanisms Underlying Voiding Dysfunction due to Prostatitis in a Rat Model of Nonbacterial Prostatic Inflammation

Purpose The neurological molecular mechanisms underlying the voiding dysfunction associated with nonbacterial chronic prostatitis/chronic pelvic pain syndrome remain poorly understood. In this study, we assessed whether prostate inflammation activated bladder afferent neurons, leading to bladder dysfunction, and sought to elucidate the underlying mechanisms. Methods Thirty male Sprague-Dawley rats were divided into 3 groups: sham-saline, formalin-injected, and capsaicin-pretreated and formalin-injected. Chemical prostatitis was induced by 0.1 mL of 10% buffered formalin injected into the ventral prostate. Capsaicin was injected subcutaneously to desensitize capsaicin-sensitive nerves. In each group, conscious cystometry was performed, and c-fos expression within the spinal cord was determined immunocytochemically. Double immunofluorescent staining with c-fos and choline acetyltransferase (ChAT) was performed. On the third day after pseudorabies virus (PRV) infection, c-fos and PRV double-staining was performed. Results Intraprostatic formalin significantly increased the maximal voiding pressure and decreased the intercontraction interval, compared with controls. Pretreatment with capsaicin significantly reversed these effects. More c-fos-positive cells were observed in the sacral parasympathetic nucleus (SPN) and dorsal gray commissure (DCM) in the prostatitis group than in the sham group. c-fos-positive cells decreased in the capsaicin-pretreated group. Preganglionic neurons labeled by c-fos and ChAT were observed in the SPN in rats with prostatitis. Interneurons labeled by c-fos and PRV were identified in the DCM after PRV infection. Conclusions Our results suggest that prostate inflammation activates afferent nerve fibers projecting to the lumbosacral spinal cord, producing reflex activation of spinal neurons innervating the bladder and bladder hyperreflexia. This is mediated by capsaicin-sensitive prostate afferent neurons.

Neonatal Bladder Irritation Is Associated With Vanilloid Receptor TRPV1 Expression in Adult Rats

Purpose To evaluate whether mild chemical irritation of the bladder in neonatal rats is associated with persistent vanilloid receptor transient receptor potential vanilloid subfamily 1 (TRPV1) activity in adult rats. Methods Female Sprague-Dawley rats were used. Ten-day-old rat pups underwent bladder sensitization via intravesical infusion of 0.2% acetic acid in saline with or without prior bladder desensitization with capsaicin. After 8 weeks, 3 groups of rats (control [group 1], bladder sensitization [group 2], and bladder desensitization [group 3]) underwent cystometry. Inflammation of bladder tissue and the expression of TRPV1 in bladder tissue and dorsal root ganglia (DRG) were also evaluated. Results The bladder sensitization group showed more frequent voiding contractions. TRPV1 expression in adult bladder tissue was elevated in group 2. TRPV1 mRNA levels in the bladder and DRG were significantly higher in group 2 than in group 1. Moreover, group 2 had significantly more DRG neurons (identified by uptake of the retrograde label Fast Blue) that exhibited TRPV1 immunoreactivity. Conclusions We found a significant association between neonatal bladder sensitization and persistent TRPV1 activity in adult rats. This is the first study to focus on the underlying pathogenesis of bladder overactivity from childhood to adulthood. Our findings could lead to the development of new strategies for the treatment and prevention of adult urinary symptoms arising from childhood urinary tract dysfunction.