Melatonin improves the ability of spermatozoa to bind with oocytes in the mouse.

CONTEXT AND AIMS: Melatonin is a powerful antioxidant regulating various biological functions, including alleviating male reproductive damage under pathological conditions. Here, we aim to analyse the effect of melatonin on normal male reproduction in mice. METHODS: Male mice received an intraperitoneal injection of melatonin (10mg/kg body weight) for 35 consecutive days. The testis and epididymis morphology, and epididymal sperm parameters were examined. PCNA, HSPA2, SYCP3, ZO-1 and CYP11A1 expressions in epididymis or testis were detected by immunohistochemistry or Western blotting. Male fertility was determined by in vivo and in vitro fertilisation (IVF) experiments. The differentially expressed sperm proteins were identified by proteomics. KEY RESULTS: No visible structural changes and oxidative damage in the testis and epididymis, and no significant side effects on testis weight, testosterone levels, sperm motility, and sperm morphology were observed in the melatonin-treatment group compared with the control group. Spermatogenesis-related molecules of PCNA, SYCP3, ZO-1, and CYP11A1 showed no significant differences in melatonin-treated testis. However, PCNA and HSPA2 increased their expressions in the epididymal initial segments in the melatonin-treatment group. Normal sperm fertilisation, two-cell and blastocyst development were observed in the melatonin-treated group, but melatonin significantly enhanced the sperm binding ability characterised as more sperm binding to one oocyte (control 7.2±1.3 versus melatonin 11.8±1.5). Sperm proteomics demonstrated that melatonin treatment enhanced the biological process of cell adhesion in sperm. CONCLUSIONS AND IMPLICATIONS: This study suggests that melatonin can promote sperm maturation and sperm function, providing important information for further research on the physiological function and protective effect of melatonin in male reproduction.

Bisphenol A exposure induces testicular oxidative damage via FTO/m6A/Nrf2 axis during postnatal development in mice.

Bisphenol A (BPA), a commonly used plasticizer in the production of polycarbonate plastics and epoxy resins, has been shown to induce male reproductive toxicity. However, the effects of BPA exposure on early testicular development have not been thoroughly studied, and the underlying mechanism is yet to be elucidated. In the current study, neonatal male mice were exposed to BPA at 0, 0.1, and 5 mg/kg, respectively, by daily subcutaneous injection during postnatal day (PND) 1-35 to explore its effects on testicular development at PND 36 (the end of the first round of spermatogenesis). Morphological analyses showed that BPA exposure significantly induced apoptosis of testicular cells (p < 0.01 and p < 0.001) and reduced the thickness of seminiferous epithelium (p < 0.01). In addition, BPA exposure significantly decreased the total antioxidant capacity of testes and levels of transcription factor Nrf2 as well as its downstream antioxidant molecules of NQO1 and GPx-1 (p < 0.05 and p < 0.01). Furthermore, global m6A modifications of mRNAs were upregulated accompanied by declined m6A demethylase (FTO) in the testes of BPA groups (p < 0.05 and p < 0.01). MeRIP-quantitative real-time polymerase chain reaction (qPCR) demonstrated that BPA exposure markedly increased the m6A modification of Nrf2 mRNA (p < 0.05 and p < 0.01). These findings suggest that upregulation of m6A induced by inhibited FTO may be involved in BPA-induced testicular oxidative stress and developmental injury during postnatal development, which provides a new idea to reveal the mechanism underlying BPA interfering with testicular development.

GSK1904529A, a Potent IGF-IR Inhibitor, Reverses MRP1-Mediated Multidrug Resistance.

Overexpression of multidrug-resistant efflux transporters is one of the major causes of chemotherapy failure. MRP1, a 190 kDa efflux transporter, confers resistance to a wide of range of chemotherapeutic drugs. Here we study the cellular effects of GSK1904529A in reversing MRP1-mediated drug resistance. Cytotoxicity of GSK1904529A was determined by MTT assay. Reversal effects of GSK1904529A in combination with MRP1 substrates were determined. The intracellular accumulation and efflux of MRP1 substrate was measured by scintillation counter and protein expression was determined by Western blotting analysis. Cell cycle effects of GSK1904529A in combination with MRP1 substrates were determined by flow cytometric analysis. GSK1904529A, at non-toxic concentrations, enhanced the cytotoxicity of MRP1 substrates in HEK293/MRP1 cells. Furthermore, GSK1904529A increased the intracellular accumulation of [3 H]-vinblastine by inhibiting the efflux function of MRP1. GSK1904529A did not alter the expression level of MRP1, induced a G0/G1 phase cell cycle arrest. Our results indicated that GSK1904529A significantly increased the sensitivity of MRP1 overexpressing cells to chemotherapeutic agents. Furthermore, GSK1904529A enhanced the efficacy of chemotherapeutic drugs that are substrates of MRP1. J. Cell. Biochem. 118: 3260-3267, 2017. © 2017 Wiley Periodicals, Inc.

[Long-term effects of neonatal exposure to bisphenol A on testes structure and the expression of Boule in testes of male mice].

OBJECTIVE: To investigate the long-term effects and the mechanism of neonatal bisphenol A( BPA) exposure on mouse testicular structure and Boule expression. METHODS: A total of 12 pregnant ICR mice were randomly divided into three groups:blank control group, negative control group( corn oil) and BPA 100 µg/kg group. After delivery, BPA was given daily by neck subcutaneous injection to the offspring male mice from postnatal day( PND 1) to PND 21. The offspring male mice were sacrificed on PND35 and PND 70. Morphological changes of testes were detected with hematoxylin-eosin staining, the level of Boule mRNA expression was determined by RT-PCR, the expression of Boule protein was detected by immunofluorescence and Western blotting. RESULTS: Compared with blank control group and negative control group, the diameter and the epithelium thickness of seminiferous epithelium in the same period( PND 35, PND 70)were significantly decreased and the lumen was significantly increased( P < 0. 05) in the testes of BPA( 100 µg/kg) group. In addition, the expressions of Boule mRNA and protein were decreased remarkably( P < 0. 05, P < 0. 01) in testes of BPA 100 µg/kg group. CONCLUSION: Neonatal BPA exposure has a long-term effect on mouse testicular development and may affect testicular development by decreasing the expression of Boule mRNA and protein in testes.

[Effects of bisphenol A exposure during lactation on testicular mitochondria in male mouse offspring].

OBJECTIVE: To study whether maternal bisphenol A (BPA) exposure during lactation stage will induce oxidative stress and apoptosis in male offspring's testis by mitochondrial pathway. METHODS: After delivery, maternal mice were randomly divided into four groups, including high dose group (50 mg/kg), medium dose group (5 mg/kg), low dose group (0.01 mg/kg) and solvent control group. BPA were given daily by gavage to maternal mice during lactation stage. The pups were raised and sacrificed at PND 75. The effect of BPA exposure on the activity of testicular mitochondrial succinate dehydrogenase (SDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in male offspring were detected. The expressions of cytochrome C (Cyt C), caspase 3 and apoptosis-inducing factor (AIF) in testes of male offspring were detected by Western blot. Changes of mitochondrial substructure in testicular cells were observed by transmission electron microscopy. RESULTS: The exposed mice had a significant decrease in the activities of testicular mitochondrial SDH (P < 0.05) in all groups and with notable decrease in the activities of GSH-Px and SOD in the high and medium-dose groups (P < 0.05). BPA also caused testicular mitochondrial MDA increased markedly (P < 0.05). Western blot showed that the expression levels of Cyt C, caspase 3 and AIF increased significantly in the testes of BPA-treated groups (P < 0.05). Under transmission electron microscopy, mitochondria swell, crests obscure and disappear were founded in the high and medium-dose groups. CONCLUSION: During lactation stage, maternal exposed to BPA induce oxidative stress and apoptosis of testicular cells by possible mechanisms of mitochondrial pathway,which would cause adverse effects on the early development of testis.

Design of Nanodrug Delivery Systems for Tumor Bone Metastasis.

Tumor metastasis is a complex process that is controlled at the molecular level by numerous cytokines. Primary breast and prostate tumors most commonly metastasize to bone, and the development of increasingly accurate targeted nanocarrier systems has become a research focus for more effective anti-bone metastasis therapy. This review summarizes the molecular mechanisms of bone metastasis and the principles and methods for designing bone-targeted nanocarriers and then provides an in-depth review of bone-targeted nanocarriers for the treatment of bone metastasis in the context of chemotherapy, photothermal therapy, gene therapy, and combination therapy. Furthermore, this review also discusses the treatment of metastatic and primary bone tumors, providing directions for the design of nanodelivery systems and future research.

Fabrication of Enhanced Mechanical Properties and Intrinsic Flame-Retardant Polyurethane Elastomer Containing 4-(Phenylethynyl) Di(Ethylene Glycol) Phthalate.

In this study, the aromatic acetylene compound 4-(phenylethynyl) di(ethylene glycol) phthalate (PEPE) was used as a chain extender, partially replacing 1,4-butanediol. To synthesize an intrinsic flame-retardant thermoplastic polyurethane elastomer (TPU) with an aromatic acetylene structure, PEPE was synthesized by a two-step polymerization. The flame retardancy, thermal stability, and mechanical properties of TPU were studied. The microstructure of TPU char was investigated by scanning electron microscopy to analyze the flame-retardant mechanism. The tensile strength of TPU containing 1.35 wt% PEPE was 39.2 MPa, which was almost twice as much as neat TPU, showed a dramatic decrease in the peak heat release rate and total heat release, and declined by 46.2% and 24.5%, respectively. After the flame-retardant TPU burned, a cross-linked network foaming char structure was formed. The results showed that PEPE improved the mechanical properties of TPU and conferred good stability that promoted the formation of charcoal and reduced heat release during the combustion of TPU.

Neonatal bisphenol A exposure induces meiotic arrest and apoptosis of spermatogenic cells.

Bisphenol A (BPA) is a widely used industrial plasticizer, which is ubiquitously present in the environment and organisms. As an endocrine disruptor, BPA has caused significant concerns regarding its interference with reproductive function. However, little is known about the impact of BPA exposure on early testicular development. The aim of the present study was to investigate the influence of neonatal BPA exposure on the first wave of spermatogenesis. Newborn male mice were subcutaneously injected with BPA (0.01, 0.1 and 5 mg/kg body weight) daily from postnatal day (PND) 1 to 21. Histological analysis of testes at PND 22 revealed that BPA-treated testes contained mostly spermatogonia and spermatocytes with markedly less round spermatids, indicating signs of meiotic arrest. Terminal dUTP nick-end labeling (TUNEL) assay showed that BPA treatment significantly increased the number of apoptotic germ cells per tubule, which corroborated the observation of meiotic arrest. In addition, BPA caused abnormal proliferation of germ cells as revealed by Proliferating Cell Nuclear Antigen (PCNA) immunohistochemical staining. Mechanistically, BPA-treated testes displayed a complete lack of BOULE expression, which is a conserved key regulator for spermatogenesis. Moreover, BPA significantly increased the expression of estrogen receptor (ER) α and ß in the developing testis. The present study demonstrated that neonatal BPA exposure disrupted meiosis progression during the first wave of spermatogenesis, which may be, at least in part, due to inhibition of BOULE expression and/or up-regulation of ERα/ß expression in BPA-exposed developing testis.

Tea nanoparticle, a safe and biocompatible nanocarrier, greatly potentiates the anticancer activity of doxorubicin.

An infusion-dialysis based procedure has been developed as an approach to isolate organic nanoparticles from green tea. Tea nanoparticle (TNP) can effectively load doxorubicin (DOX) via electrostatic and hydrophobic interactions. We established an ABCB1 overexpressing tumor xenograft mouse model to investigate whether TNP can effectively deliver DOX into tumors and bypass the efflux function of the ABCB1 transporter, thereby increasing the intratumoral accumulation of DOX and potentiating the anticancer activity of DOX. MTT assays suggested that DOX-TNP showed higher cytotoxicity toward CCD-18Co, SW620 and SW620/Ad300 cells than DOX. Animal study revealed that DOX-TNP resulted in greater inhibitory effects on the growth of SW620 and SW620/Ad300 tumors than DOX. In pharmacokinetics study, DOX-TNP greatly increased the SW620 and SW620/Ad300 intratumoral concentrations of DOX. But DOX-TNP had no effect on the plasma concentrations of DOX. Furthermore, TNP is a safe nanocarrier with excellent biocompatibility and minimal toxicity. Ex vivo IHC analysis of SW620 and SW620/Ad300 tumor sections revealed evidence of prominent antitumor activity of DOX-TNP. In conclusion, our findings suggested that natural nanomaterials could be useful in combating multidrug resistance (MDR) in cancer cells and potentiating the anticancer activity of chemotherapeutic agents in cancer treatment.

[Expression and biology identification of the human epididymis-specific gene ESC42 in E. coli].

OBJECTIVE: To provide materials for the study of the function of ESC42 protein specifically expressed in the human epididymis. METHODS: The ESC42 gene was amplified from the human epididymis cDNA library by PCR and then cloned into prokaryotic expression vector pGEX-4T-1, expressed and purified by recombinant DNA techniques. The specificity of ESC42 protein was identified by Western blot and MALDI-TOF-MS. The database was searched by Ms-Fit. RESULTS: The recombinant plasmid expressed a Mr 38 x 10(3) fusion protein in E. coli at a level of 30% of the total protein, and the purity was as high as 99%. The ESC42 protein was identified by ESC42 monoclonal antibody and its molecular weight was 11 978.12, tested by MALDI-TOF-MS. The peptide mass fingerprint analysis showed that the coverage rate of the sequence reached 48% with 100% matching. The motif scan in Prosite database reveal that ESC42 belonged to the beta-defensin family and had antibacterial activity. CONCLUSION: Obtaining high purity of rhESC42 protein may lay a foundation for the study of its functions.

Estrogenic and toxic effects of polychlorinated biphenyls on cultured ovarian germ cells of embryonic chickens.

Polychlorinated biphenyls (PCBs) are man-made ubiquitous pollutants that have detrimental effects on reproduction and endocrine functions in a variety of species. In the present study, estrogenic and toxic effects of PCBs on embryonic chicken ovarian development were evaluated by a germ-somatic cell co-culture system. Ovarian cells were cultured in serum-free medium and challenged with a mixture of PCBs (Aroclor 1254). Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release. Results showed that lower PCBs (0.1-1 microg/ml) manifested mainly estrogenic effect to stimulate germ cell proliferation, while higher PCBs (10 microg/ml) imposed severe toxicity on germ and somatic cells. The toxic effect of PCBs could be attenuated by an antioxidant tocopherol. PCBs induced condensed nuclear chromosome in ovarian cells and caused cell exfoliation and breakdown within initial hours of treatment. After 24 h, the estrogenic effect of PCBs began to exhibit and the survived germ cells manifested proliferation. Inhibition of the estrogenic effect of PCBs by tamoxifen led to increased toxicity on germ cells and somatic cells. These results indicate that PCBs exposure may interfere with ovarian germ cell proliferation and cause reproductive disorder via both toxic and estrogenic actions in embryonic chickens.

Effects of follicle-stimulating hormone and 17beta-estradiol on proliferation of chicken embryonic ovarian germ cells in culture.

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.

Effects of follicle-stimulating hormone and androgen on proliferation of cultured testicular germ cells of embryonic chickens.

A germ-Sertoli cell coculture model was established to study effects of follicle-stimulating hormone (FSH) and testosterone (T) on testicular germ cell proliferation of the embryonic chickens. Germ and somatic cells were dispersed from 18-day-old embryonic testes and cultured in 96-well plates. Germ cells were characterized by expression of stem cell factor receptor c-kit. Germ cell proliferation was assessed by an increase in cell number and expression of proliferating cell nuclear antigen (PCNA). Results showed that the germ and Sertoli cells kept alive in serum-free McCoy's 5A medium supplemented with insulin, transferrin, and selenite (ITS medium). Germ cells adhered to the free surface of Sertoli cells that spread the filopodia and formed a monolayer in ITS medium. In the serum-containing medium, Sertoli cells displayed an increment with a flat squamous form and only a few very large germ cell masses were found in the free surface of Sertoli cells. Many germ cells showed apoptosis in the McCoy's 5A medium without ITS or serum. Only germ cells showed positive staining for c-kit in the coculture. Ovine FSH (0.25-1.0 IU/ml) significantly increased the number of germ cells, and PCNA-labeling index (P < 0.05). FSH also induced stronger c-kit expression compared with the control. In the FSH-treated groups, germ cells were manifested distinct knob-like form. Similar stimulating effect was found in the germ cell number by T treatments (10(-7)-10(-6)M). Furthermore, FSH (0.5 IU/ml) combined with T significantly promoted higher testicular germ cell proliferation (P < 0.05) compared with either FSH or T alone, which indicated that interaction of FSH and T might be additive. The above results showed that the serum-free germ-Sertoli cell coculture model allowed evaluating hormonal regulation of testicular germ cell proliferation. FSH and T promoted testicular germ cell proliferation probably through indirect effects on Sertoli cells.