Guanghui Wei

Characterization of constitutive promoters for piggyBac transposon-mediated stable transgene expression in mesenchymal stem cells (MSCs).

Multipotent mesenchymal stem cells (MSCs) can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells. The analyzed promoters include three viral promoters (CMV, CMV-IVS, and SV40), one housekeeping gene promoter (UbC), and two composite promoters of viral and housekeeping gene promoters (hEFH and CAG-hEFH). CMV-derived promoters are shown to drive the highest transgene expression in HEK-293 cells, which is however significantly reduced in MSCs. Conversely, the composite promoter hEFH exhibits the highest transgene expression in MSCs whereas its promoter activity is modest in HEK-293 cells. The reduced transgene expression driven by CMV promoters in MSCs may be at least in part caused by DNA methylation, or to a lesser extent histone deacetlyation. However, the hEFH promoter is not significantly affected by these epigenetic modifications. Taken together, our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term and high level transgene expression using the piggyBac transposon vector in progenitor cells such as MSCs.

The Mechanism of Environmental Endocrine Disruptors (DEHP) Induces Epigenetic Transgenerational Inheritance of Cryptorchidism

Discussion on the role of DEHP in the critical period of gonadal development in pregnant rats (F0), studied the evolution of F1-F4 generation of inter-generational inheritance of cryptorchidism and the alteration of DNA methylation levels in testis. Pregnant SD rats were randomly divided into two groups: normal control group and DEHP experimental group. From pregnancy 7d to 19d, experimental group was sustained to gavage DEHP 750mg/kg bw/day, observed the incidence of cryptorchidism in offspring and examined the pregnancy rate of female rats through mating experiments. Continuous recording the rat’s weight and AGD value, after maturation (PND80) recording testis and epididymis’ size and weight, detected the sperm number and quality. Subsequently, we examined the evolution morphological changes of testicular tissue for 4 generation rats by HE staining and Western Blot. Completed the MeDIP-sequencing analysis of 6 samples (F1 generation, F4 generation and Control). DEHP successfully induced cryptorchidism occurrence in offspring during pregnancy. The incidence of cryptorchidism in F1 was 30%, in F2 was 12.5%, and there was no cryptorchidism coming up in F3 and F4. Mating experiment shows conception rate 50% in F1, F2 generation was 75%, the F3 and F4 generation were 100%. HE staining showed that the seminiferous epithelium of F1 generation was atrophy and with a few spermatogenic cell, F2 generation had improved, F3 and F4 generation were tend to be normal. The DNA methyltransferase expression was up-regulated with the increase of generations by Real Time-PCR, immunohistochemistry and Western Blot. MeDIP-seq Data Analysis Results show many differentially methylated DNA sequences between F1 and F4. DEHP damage male reproductive function in rats, affect expression of DNA methyltransferase enzyme, which in turn leads to genomic imprinting methylation pattern changes and passed on to the next generation, so that the offspring of male reproductive system critical role in the development of imprinted genes imbalances, and eventually lead to producing offspring cryptorchidism. This may be an important mechanism of reproductive system damage.

Effects of diethylhexyl phthalate (DEHP) on INSL3 mRNA expression by Leydig cells derived from mouse embryos and in newborn mice.

Insulin-like factor 3 (INSL3) regulates testicular descent during fetal life, and Insl3 gene inactivation results in cryptorchidism. Little is known, however, about whether the plasticizer diethylhexyl phthalate (DEHP), a contaminant found widely in the environment, influences INSL3 expression. In this study, primary cultures of Leydig cells from mouse embryos were treated in vitro with DEHP. We also treated pregnant mice with DEHP from gestation day 12 to postnatal day 3 in order to study the effect of DEHP in vivo. INSL3 mRNA expression levels in primary Leydig cell cultures and in the testes of newborn mice were significantly lower following DEHP treatment. DEHP also caused detrimental morphological changes in both primary cultures of Leydig cells and the testes of newborn mice. These results suggest that the downregulation of INSL3 mRNA by DEHP might cause abnormalities of gubernacular development, which might be one of the mechanisms for development of cryptorchidism.

Urban fine particulate matter exposure causes male reproductive injury through destroying blood-testis barrier (BTB) integrity.

Blood-testis barrier (BTB) provides a suitable microenvironment for germ cells that is required for spermatogenesis. Exposure to particulate matter (PM) is recognized to occasion male reproductive impairment, but the mechanism of which remains unclear. Male Sprague-Dawley (SD) rats were used to establish animal models with PM2.5 exposure concentration of 0, 10, and 20mg/kg.b.w. once a day for four weeks. Success rate of mating, sperm quality, epididymal morphology, expressions of spermatogenesis markers, superoxide dismutases (SOD) activity and expression in testicular tissues, and expressions of BTB junction proteins were detected. In addition, in vitro experiments were also performed. After PM2.5 treatment, reactive oxygen species (ROS) production and apoptosis of Sertoli cells were analyzed. Our results indicated that after PM2.5 exposure male rats presented inferior uberty and sperm quality, with decreased expressions of spermatogenesis markers, escalated SOD activity and expression levels, and reduced expressions of tight junction, adherens junction, and gap junction proteins in testicular tissues. Meantime, PM2.5-treated Sertoli cells displayed increased SOD production and apoptosis. PM2.5 exposure engenders male reproductive function injury through breaking BTB integrity.

Protective Effects of Vitamin E against reproductive toxicity induced by di(2-ethylhexyl) phthalate via PPAR-Dependent Mechanisms

Abstract Objective: To investigate the protective/ameliorative effects of vitamin E on di-2-(ethylhexyl) phthalate (DEHP)-induced reproductive toxicity, particularly in testicular toxicity in male rats, emphasizing peroxisome proliferator-activated receptor (PPAR)-dependent mechanism. Methods: Sprague-Dawley females were exposed by oral route to DEHP alone or associated with vitamin E from gestation day (GD) 12.5 to postnatal day (PND) 3 according to the following treatment regimens: vehicle control (corn oil), vitamin E (200u2009mg/kg)+corn oil, DEHP (500u2009mg/kg)+corn oil, and DEHP (500u2009mg/kg)+vitamin E (200u2009mg/kg)+corn oil. Variables including litter size, sex ratio, pup weight, post-implantation losses, and the number of viable pups were also assessed. Three male pups per litter were randomly selected and necropsied to measure paired testes weight, apoptosis, and gene expression on PND 3. To evaluate the long-term protective effects of vitamin E, three randomly selected males were necropsied to measure testis histology on PND 70. Results: Supplementation of vitamin E (200u2009mg/kg) reduced malformations, increased testes weight and prevented the maternal bodyweight loss induced by DEHP. Litter size, sex ratio, and number of viable pups were unaffected, but vitamin E co-administration declined testicular cell apoptosis, decreased the PPARs expression, and protected testis histology. Conclusions: Vitamin E cotreatment showed protective effects against DEHP-induced testicular toxicity, including reproductive malformations, testicular weight, apoptosis and histology, and the mechanisms maybe associated with PPARs.

Effect of KnockOut serum replacement on germ cell development of immature testis tissue culture

To compare KnockOut serum replacement (KSR) and fetal bovine serum (FBS) for the development of germ cells. Testicular tissues from Sprague-Dawley rats were cultured for 4xa0weeks in culture media supplemented with FBS or KSR. Tissue area was measured at the beginning and end of the culturing period. Testicular histology, development of the germ cells, and the diameter of seminiferous tubules were analyzed by hematoxylin and eosin staining. After 4xa0weeks in culture, apoptosis and expression of the stage-specific spermatogenesis marker genes Kit, Sycp3, and Crisp1 were assayed. Tissues cultured in KSR-supplemented media were able to sustain growth and gradually increase seminiferous tubule diameter during the culture period. In addition, spermatogonia, primary spermatocytes, secondary spermatocytes, and round spermatids were observed after 4xa0weeks in culture, and reverse transcription-PCR confirmed expression of the marker genes. In comparison, tissues cultured in FBS-supplemented media showed dwindling testicular organization, necrotic seminiferous tubules, and expression of Kit, but inconsistent expression of Sycp3 and Crisp1 KnockOut serum replacement outperforms FBS as a growth media supplements for culturing immature spermatogonial tissue culture.

New Technique for the Treatment of Buried Penis in Children.

OBJECTIVEnTo present our treatment experience of buried penis, which has no consensus therapeutic technique for all cases of buried penis, by using a new technique for the repair of this condition, in which the approach is through the ventral penile root.nnnMATERIALS AND METHODSnWe performed a retrospective review of 153 patients (median age: 6.5 years) who underwent repair of a buried penis between March 2005 and March 2013. The technique involves the creation of a wedge-shaped cut of the ventral penile skin, followed by fixation of the subcutaneous penile skin at the base of the degloved penis to the Buck fascia at the 2- and 10-oclock positions. The ventral outer preputial skin is split down the midline, and the dorsal inner preputial skin is cut with oblique incision.nnnRESULTSnAll patients were followed for an average of 12 months after repair. Other than 2 cases (1.3%) of trapped penis with a ring of scar tissue, which required subsequent excision, there were no complications and the cosmetic appearance was satisfactory.nnnCONCLUSIONnThe described ventral penile approach is a simple and effective procedure with good cosmetic outcomes and few complications.

Urban fine particulate matter (PM2.5) exposure destroys blood–testis barrier (BTB) integrity through excessive ROS-mediated autophagy

Abstract Context: Blood–testis barrier (BTB), constituted by tight junctions (TJs), adherens junctions and gap junctions, is important for spermatogenesis. PM2.5 is known to impair testicular functions and reproduction. However, its effects on BTB and the underlying mechanisms remain obscure. Objective: To investigate the roles of autophagy in BTB toxicity induced by PM2.5. Materials and methods: Sprague–Dawley rats were developmentally exposed to normal saline (NS) or PM2.5 with the doses of 9u2009mg/kg b.w. and 24u2009mg/kg b.w. via intratracheal instillation for seven weeks. Success rate of mating, sperm quality, testicular morphology, expressions of BTB junction proteins and autophagy-related proteins were detected. In addition, expressions of oxidative stress markers were also analyzed. Results: Our results demonstrated that developmental PM2.5 exposure induced noticeable decreased fertility, significantly reduced sperm count, increased sperm abnormality rate and severe testicular damage in histomorphology. The expressions of TJ (such as ZO-1 and occludin), gap junction (such as connexin43) were down-regulated significantly after PM2.5 treatment. Intriguingly, PM2.5 simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II and p62, suggesting that the accumulated autophagosomes resulted from impaired autophagy degradation. Moreover, the expressions of HO-1 levels remarkably increased and expression levels of Gpx and SOD were significantly decreased after PM2.5 exposure. Vitamins E and C could alleviate the PM2.5-induced oxidative stress, reverse the autophagy defect and restore the BTB impairment. Conclusions: Taken together, the results suggest that PM2.5 exposure destroys BTB integrity through excessive ROS-mediated autophagy. Our finding could contribute to a better understanding of PM2.5-induced male reproductive toxicity.

Testicular developmental impairment caused by flutamide-induced and DEHP-induced cryptorchid rat models is mediated by excessive apoptosis and deficient autophagy

Abstract Background: Cryptorchidism is a common condition of childhood, and it is known to impair fertility potential. However, the underlying mechanisms remain unclear. Methods: This study constructed two cryptorchid rat models to investigate the roles of apoptosis and autophagy in testicular impairment induced by cryptorchidism. Pregnant rats were randomly divided into three groups. Group I: non-treated rats were used as controls. Group II: injected with drug Flutamide (Flu) 25u2009mg/kg/bw/d from gestation day (GD) 11–19. Group III: daily intragastric administration of 750u2009mg/kg/bw/d di-2-ethylhexylphosphate (DEHP) from GD 7–19. The cubs were feed normally and the testes were excised on postnatal day (PND) 30. Results: Our results demonstrated cryptorchidism models induced noticeable decreased fertility, significantly reduced sperm count, increased sperm abnormality rate, decreased testosterone and severe testicular damage in histomorphology. Intriguingly, the level of apoptosis marker FAS, Cytochrome C and caspase-3 increased in Flu-induced and DEHP-induced groups. DEHP-induced treatment simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II and p62. Significant decrease of autophagy gene (LC3-II and p62) expression is found in Flu-induced rats testes. Conclusion: Taken together, deficient autophagy is involved in testicular spermatogenesis damage of cryptorchidism rats. And this autophagy defect is caused by deficient degradation.

Histological and Biochemical Evaluation of Urethral Scar following Three Different Hypospadias Repairs: An Experimental Study in Rabbits

Introduction Several urethroplasties have been employed in the surgical treatment of hypospadias. Neourethral strictures are among the most common postoperative complications that often require reoperation. Materials and Methods We created a hypospadias model in New Zealand white male rabbits through a hypospadias‐like defect and acute repair. A total of 24 animals were randomly allocated into three groups: tubularized incised‐plate urethroplasty (TIPU) group (8), perimeatal‐based flap urethroplasty (Mathieu) group (8), onlay island flap urethroplasty (onlay) group (8), and corresponding surgical procedures were immediately performed to reconstruct neourethra. The rabbits were killed postoperatively at 5 days, 2 weeks, 6 weeks, and 3 months, respectively. The penile tissue was harvested for histological and biochemical investigations to evaluate the expressions of transforming growth factor &bgr;1 (TGF‐&bgr;1) and &agr;‐smooth muscle actin (&agr;‐SMactin) in all groups. Results All rabbits were operated on uneventfully. The amount of collagen content was increased in the Mathieu and onlay groups than in the TIPU group (p < 0.05). Biochemical analysis showed that the expression of TGF‐&bgr;1 in the TIPU group was decreased compared with the two other groups at 2 or 6 weeks postoperatively (p < 0.01). The expression pattern regarding &agr;‐SMactin was similar at 6 weeks or 3 months postoperatively (p < 0.01). Conclusion The neourethra repaired by TIPU was practically resumed to normal anatomy and scarring was less apparent than the two other groups. Therefore, TIPU is considered as a relatively rational approach for hypospadias repair. The activity of fibroblasts has been increased in the long term, which may be the pathogenesis of neourethral stricture following hypospadias repair.