•Hsa-miR-513b-5p expression was up-regulated in testicular specimens with maturation arrest.•Hsa-miR-513b-5p suppressed NT2 cell proliferation and induced apoptosis in vitro.•IRF2 was a direct target of hsa-miR-513b-5p.•Silencing of IRF2 enhanced hsa-miR-513b-5p-mediated effects on cell proliferation.•Hsa-miR-513b-5p or si.IRF2 could promote the expression of P53.Previous studies have reported the miR-513b is located on the X chromosome and is preferentially expressed in testis. However, the underlying mechanisms of miR-513b involved in spermatogenesis remains unknown. In this study, we found that hsa-miR-513b-5p was highly expressed in the testes of infertile males with maturation arrest compared with normal controls. Overexpression of hsa-miR-513b-5p suppressed testicular embryonal carcinoma (NT2) cell proliferation and induced apoptosis in vitro, whereas silencing of hsa-miR-513b-5p reversed these effects. In addition, we found that interferon regulatory transcription factor 2 (IRF2) was a direct and functional target of hsa-miR-513b-5p. Silencing of endogenous IRF2 enhanced hsa-miR-513b-5p-mediated effects on cell proliferation in NT2 cells, whereas overexpression of IRF2 reversed these effects. Moreover, immunoblotting showed that overexpression of hsa-miR-513b-5p or silencing of endogenous IRF2 could promote the expression of P53. Moreover, overexpression of hsa-miR-513b-5p in the absence of p53 could also induce cell apoptosis. Together, our results suggest that hsa-miR-513b-5p suppresses NT2 cell proliferation and promotes P53 protein expression by targeting IRF2, and abnormal testicular hsa-miR-513b-5p expression may contribute to maturation arrest.

During the process of human civilization, owning household pets has become increasingly popular. However, dogs and cats may be reservoirs or vectors of transmissible diseases to humans. Confronted with the overpopulation of pets, traditional contraception methods, surgical methods of sterilization, for animals are used, namely, ovariohysterectomy and orchidectomy. Therefore, a simple, nonsurgical, controllable, more effective and less expensive contraception method is highly desirable. In this study, we show that in situ testicular injection of methoxy poly(ethylene glycol)-modified gold nanorods with near-infrared irradiation in male mice can achieve short-lived or permanent male infertility. In a lower hyperthermia treatment, the morphology of testes and seminiferous tubules is only partly injured, and fertility indices are decreased to ∼10% at day 7, then recovered to 50% at day 60. In a higher hyperthermia treatment, the morphology of testes and seminiferous tubules are totally destroyed, and fertility indices are decreased to 0 at day 7. Overall, our results indicate a potential application of plasmonic nanomaterials for male contraception.

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumors. However, the mechanisms underlying the targeting and functions of miR-383 during spermatogenesis remain unknown. In this study, we found that fragile X mental retardation protein (FMRP) was associated with 88 miRNAs in mouse testis including miR-383. Knockdown of FMRP in NTERA-2 (NT2) (testicular embryonal carcinoma) cells enhanced miR-383-induced suppression of cell proliferation by decreasing the interaction between FMRP and miR-383, and then affecting miR-383 binding to the 3′-untranslated region of its target genes, including interferon regulatory factor-1 (IRF1) and Cyclin D1 both in vivo and in vitro. On the other hand, FMRP levels were also downregulated by overexpression of miR-383 in NT2 cells and GC1 (spermatogonia germ cell line). miR-383 targeted to Cyclin D1 directly, and then inhibited its downstream effectors, including phosphorylated pRb and E2F1, which ultimately resulted in decreased FMRP expression. Reduced miR-383 expression, dysregulated cyclin-dependent kinase 4 expression (one of the downstream genes of miR-383) and increased DNA damage were also observed in the testes of Fmr1 knockout mice and of MA patients with a downregulation of FMRP. A potential feedback loop between FMRP and miR-383 during spermatogenesis is proposed, and FMRP acts as a negative regulator of miR-383 functions. Our data also indicate that dysregulation of the FMRP–miR-383 pathway may partially contribute to human spermatogenic failure with MA.

While the immunogenicity and cytotoxicity of gold nanoparticles (AuNPs) are noted by many researchers, the mechanisms by which AuNPs exert these effects are poorly understood. In this study, we investigated the effects of polyethylene glycolylated AuNPs (PEG@AuNPs) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and interleukin-6 (IL-6) production and the associated molecular mechanism in RAW264.7 cells. The results showed that PEG@AuNPs were internalized more quickly by LPS-activated RAW264.7 cells than unstimulated cells, and they reached saturation within 24 hours. PEG@AuNPs enhanced LPS-induced production of NO and IL-6 and inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells, partially by activating p38 mitogen-activated protein kinases (p38 MAPK) and nuclear factor-kappaB pathways. In addition, the p38 MAPK inhibitor SB203580 attenuated PEG@AuNP-enhanced LPS-induced NO production and iNOS expression. Overproduction of NO and IL-6 is known to be closely correlated with the pathology of many diseases and inflammations. Thus, it is speculated that the highly biocompatible gold nanoparticles can induce immunotoxicity due to their potency to stimulate macrophages to release aberrant or excessive pro-inflammatory mediators.

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). However, the underlying mechanisms of miR-383 involved in the pathogenesis of MA remain unknown. In this study, we showed that downregulation of miR-383 was associated with hyperactive proliferation of germ cells in patients with mixed patterns of MA. Overexpression of miR-383 in NT2 (testicular embryonal carcinoma) cells resulted in suppression of proliferation, G1-phase arrest and induction of apoptosis, whereas silencing of miR-383 reversed these effects. The effects of miR-383 were mediated through targeting a tumor suppressor, interferon regulatory factor-1 (IRF1), and miR-383 was negatively correlated with IRF1 protein expression in vivo. miR-383 inhibited IRF1 by affecting its mRNA stability, which subsequently reduced the levels of the targets of IRF1, namely cyclin D1, CDK2 and p21. Downregulation of IRF1 or cyclin D1, but not that of CDK2, enhanced miR-383-mediated effects, whereas silencing of p21 partially inhibited the effects of miR-383. Moreover, miR-383 downregulated CDK4 by increasing proteasome-dependent degradation of CDK4, which in turn resulted in an inhibition of phosphorylated retinoblastoma protein (pRb) phosphorylation. These results suggest that miR-383 functions as a negative regulator of proliferation by targeting IRF1, in part, through inactivation of the pRb pathway. Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumor.

MicroRNAs (miRNAs) have been indicated to play key roles in ovarian follicular development. However, little is known about how the miRNA gene expression itself is regulated in the mammalian ovary. We previously reported that miR-224 is involved in TGF-β1-mediated follicular granulosa cell (GC) growth and estradiol (E2) production by targeting Smad4. Here, the transcriptional regulation of miR-224 expression in GCs was further investigated. Our results showed that both the tumor suppressor gene p53 and NF-κB p65 subunit suppressed the TGF-β1-induced increase in pri-miR-224 expression in GCs. ChIP assays demonstrated that TGF-β1 enhanced the binding of p53 and p65 to the proximal promoter region of GABAA receptor ε subunit (miR-224 host gene). p53 and p65 transcriptionally cooperated to inactivate the GABAA receptor ε subunit promoter. In addition, p53/p65 could up-regulate Smad4 expression by inhibiting its target miR-224 in GCs which contributed, at least partially, to the effects of miR-224 and Smad4 on GC proliferation and E2 release. Our results provide new data about the interplay between transcription factors involved in GC proliferation and function by cooperatively regulating miRNA expression.Highlights► GABAA receptor ε subunit (GABRE) is miR-224 host gene. ► TGF-β1 enhanced the binding of p53/p65 to the proximal promoter of GABRE in GCs. ► p53/p65 transcriptionally cooperated to inactivate the GABRE promoter in GCs. ► p53/p65 suppressed the TGF-β1-induced increase in pri-miR-224 expression in GCs. ► p53/p65 influenced GC proliferation and E2 release through miR-224-Smad4 pathway.