Regulation of Microcystin-LR-Induced Toxicity in Mouse Spermatogonia by miR-96

Microcystin (MC)-LR is a cyclic heptapeptide that acts as a potent reproductive system toxin, especially by decreasing sperm quality through affecting spermatogonia. However, the molecular mechanisms of MC-induced spermatogonial cytotoxicity still remain unclear. The present study was designed to investigate changes in microRNA (miRNA) profiles and their potential functions in spermatogonia (GC-1 cell line) following treatment with MC-LR. With microarray analysis, 101 miRNAs were identified to be significantly altered in GC-1 cells treated with MC-LR. Among the 25 miRNAs associated with spermatogenesis, miR-96 was down-regulated most dramatically and thus selected for further functional analysis. Deleted-in azoospermia-associated protein 2 (DAZAP2) was predicted to have a binding sequence for miR-96 within its 3′-untranslated region. Fluorescent reporter assay confirmed that DAZAP2 was the target gene of miR-96. The expression of DAZAP2 decreased significantly when miR-96 was up-regulated. Consistently, down-regulation of miR-96 significantly increased the level of DAZAP2. Up-regulation of miR-96 promoted cell viability in GC-1 cells as a result of exposure to MC-LR. Our study suggested a crucial role for miR-96 in the regulation of cytotoxic effects of MC-LR in spermatogonia, which provides new perspectives in the diagnosis and treatment strategies for MC-induced male infertility

Combined effects of NP and MBP on cell apoptosis and necrosis.

<p>a) Annexin V/PI binding assessment by flow cytometric analysis after 48 h of treatment. Viable cells are double negative and are seen in the lower left quadrant. Cells in the lower right quadrant that are Annexin V⁺/PI⁻ are apoptotic. The Annexin V⁺/PI⁺ cell population in the upper right quadrant has been described as necrotic or advanced apoptotic. The upper left quadrant or Annexin V⁻/PI⁻ may represent isolated nuclei from cells having been stripped of cytoplasmic membranes, cells in late necrosis, or cellular debris. b) Comparison of the mixture effects of NP and MBP on cell apoptosis and necrosis was obtained experimentally and by mathematical modeling. (mean ± SD) *: <i>P</i><0.05; **: <i>P</i><0.01 <i>vs.</i> control.</p

Combined effects of NP and MBP on Inhibin-B concentration.

<p>a) Effects of NP and MBP on concentration of Inhibin-B secreted by Sertoli cells following NP and MBP treatment for 24 and 48 h. b) Comparison of the mixture effects of NP and MBP on Inhibin-B concentration was obtained experimentally and by mathematical modeling. (mean ± SD) *: <i>P</i><0.05; **: <i>P</i><0.01 <i>vs.</i> control.</p

Combined effects of NP and MBP on cell viability.

<p>a) Effects of NP and MBP exposure on Sertoli cell viability were determined by measuring cytotoxicity <i>in vitro</i> 24 and 48 h after incubation. b) Comparison of the mixture effects of NP and MBP on cell viability was obtained experimentally and by mathematical modeling. (mean ± SD) *: <i>P</i><0.05; **: <i>P</i><0.01 <i>vs.</i> control.</p

Immunofluorescence photomicrographs of Sertoli cells stained with FSHR.

<p>Sertoli cells were positive FSHR staining (red), and nuclei were revealed by DAPI (blue). Figure 1A was shown as negative control. Purity of the Sertoli cells expressing FSHR was more than 95% in Figure 1B.</p

Additional file 1 of Human placental extract suppresses mast cell activation and induces mast cell apoptosis

Additional file 1: Fig S1. The gating strategies of apoptotic mast cells. An example is given to show the gating strategy and the apoptotic cells were revealed by the annexin V+ quadrants. (A) C57 cells. (B) HMC-1 cells

LxT1 mice display normal natural IgE levels.

<p>Similar serum IgE levels in LxT1 and B6 mice in the absence of active immunization. Each symbol represents an individual mouse. Black bar represents mean values. Representative of 2 experiments.</p

CD23^low surface levels in LxT1 mice due to a recessive mutation located on chromosome 8.

<p>(A) Male LxT1 mice were crossed to female BALB/c mice to generate F1 mice, from which females were backcrossed to the male LxT1 to generate offspring for analysis of CD23 phenotypes and DNA for genetic mapping. (B) The region of chromosome 8 (0 to 28.309.058) corresponds to the CD23^low phenotype observed in LxT1 mice based on the genetic linkage analysis.</p

Decreased intracellular CD23 level and normal CD23 and ADAM10 mRNA transcripts.

<p>The CD23 total (Total) protein pool in LxT1 B cells was lower than that of the control (A, left), with a similar reduction in CD23 cell surface only (Sur) (A, right) and intracellular (intra) levels (B). Representative of one experiment, n = 3/genotype. (C) Comparable CD23 mRNA levels in B cells from LxT1 and B6 mice. (D) Similar levels of ADAM10 mRNA expression in PLN B cells from LxT1 and B6 mice. RNA isolated from CD19⁺ splenic (SPL) and peripheral lymph node (PLN) B cells was analyzed by q-PCR. CD23 and ADAM10 transcripts were normalized to 18S rRNA. Representative of one experiment, n = 3/genotype.</p

Normal CD23 surface levels and hyper IgE in 129S4 and 129S1 mice.

<p>Flow cytometry analysis of spleen cells from nine months old B6, 129S4 and129S1 mice. (A) Gating on CD19⁺ splenic B cells, graph shows CD23^low surface levels are not observed on B cells in 129S4 and 129S1 mice compared to B6, with a different proportion of CD23^low/− among these three strains. (B) Gating on mature B cells (CD19⁺CD93⁻), dot plots show increased MZ (CD21^hiCD23^low/−) and CD21⁻CD23⁻ B cell populations and decreased FO B cells (CD21^hiCD23^hi) in 129S4 and 129S1 compared to B6. (C) Within each population gated in Fig. 4B, CD23 surface levels are expressed similarly in 129S4, 129S1 and B6 mice. (D) Serum IgE levels are higher in the two 129 substrains compared to LxT1 mice. LxT1 mice were three to nine months old, while 129S4 and 129S1 substrains were nine months old.</p