Breeding efficiency in 129.MOLF-L1 congenic mice with or without <i>in utero</i> flutamide, DES, or radiation treatment.

a<p>Values given as absolute number and percentage of total plug-positive females.</p>b<p>Mean ± SEM.</p

Morphology and histology of the testes in 4-week-old 129.MOLF-L1 male mice irradiated <i>in utero</i> with two doses of 0.8 Gy and controls.

<p>Testicular morphology showing bilateral (<b>A</b>) and unilateral (<b>B</b>) TGCTs from irradiated mice. Note that the testis without TGCT after <i>in utero</i> radiation exposure (B) is smaller than the normal untreated testis from a 4-week-old mouse (insert in B). Testis sections showing teratoma containing tissues apparently from multiple dermal origins identified by morphology (<b>C</b>), and TGCT containing only neuroepithelial cells (NE) (<b>D</b>). CA: cartilage; BM: bone marrow; MS: muscle, EEP: endodermal epithelium. ST: seminiferous tubule; BV: blood vessel. Sections from testes of 4-week old irradiated (<b>E</b>) and contol (<b>F</b>) mice without TGCT showing qualitatively normal spermatogenesis. Bar: 0.5 cm in A & B, and 50 µm in C–F.</p

Testicular germ cell tumor (TGCT) incidence and related characterization in 129.MOLF-L1 congenic mice with or without <i>in utero</i> flutamide, DES, or radiation treatment.

a<p>Mean ± SEM.</p>b<p>Values given as absolute number and percentage of mice, testes, or tumors analyzed.</p>c<p>Since the incidence of tumors in the DES-treated mice was not increased from historical controls or the concurrent flutamide controls, we did not complete this arm of the study, and cannot rigorously conclude that there is no increase in tumor incidence in DES-treated mice compared to a sham-treated control group.</p>d<p>Two cryptorchid testes (weights, 19 and 34 mg) were excluded from this average.</p>e<p>Significantly different between treated and control mice, Fisher's exact Chi square test: <i>P</i><0.01. Other differences were not significant (<i>P</i>≥0.05).</p>f<p>Significantly different between treated and control mice, <i>t</i> test: <i>P</i><0.01. Other differences were not significant (<i>P</i>≥0.05).</p

Increased testicular germ cell tumor incidence (TGCT) in 129 and L1 mice exposed <i>in utero</i> to cyclophosphamide (CP) or radiation.

a<p>Values given as absolute number and percentage of mice, testes, or tumors analyzed.</p>b<p>Data from 129S5 and 129S1/SvImJ mice were pooled.</p>c<p>Significantly different between treated and control mice (<i>P</i><0.05; Fisher's exact test).</p>d<p>Significantly different between CP-exposed and irradiated mice of the same strain (<i>P</i><0.05; Fisher's exact test).</p>e<p>The odd number is due to the presence of only one testis in one of the mice analyzed, which was not considered for bilateral TGCT analysis.</p>f<p>Significantly different between treated and control mice (<i>P</i><0.05; <i>t</i> test).</p>g<p>Data from a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093311#pone.0093311-Shetty1" target="_blank">[5]</a> were included for comparison.</p

Reduction of testis weights and sperm counts in 8-week-old 129S1/SvImJ mice exposed to cyclophosphamide (CP) <i>in utero</i>.

a<p>Significantly different between treated and control mice (<i>P</i><0.01; <i>t</i>-test).</p

Histology of testes and ovaries from 129 mice exposed to cyclophosphamide at 7.5 mg/kg/day on E10.5 and 11.5.

<p>(A-D) Testes from 4-week-old mice. (A) TGCT characterized as a teratoma originating from multiple dermal layers; (B) TGCT containing only neuroepithelial cells. Abbreviations: BM: bone marrow; CA: cartilage; EEP: endodermal epithelium; NE: neuroepithelial cells; MS: muscle; ST: seminiferous tubule. (C) non-TGCT-bearing testis showing active and atrophic tubules. (D) High magnification of atrophic tubules containing only Sertoli cells. (E-H) Ovaries from 7-day-old mice. (E, G) From a mouse treated on E10.5 and 11.5 with 7.5 mg CP/kg/day. (F, H) Control ovary of the same age. G and H are the magnified views from portions of E and F respectively, showing primordial (PO), primary-transitional (PT) and primary (PR) follicles. The bar represents 100 μm in A, B, C, E & F; 30 μm in D; 10 μm in G and H.</p

Effect of cyclophosphamide (CP) or radiation on days 10.5 and 11.5 after mating on breeding efficiency of 129 and L1 mice.

<p>Abbreviation: ND, no data.</p>a<p>Values given as absolute number and percentage of total vaginal plug-positive females.</p>b<p>The number of male offspring per group ranged from 19 to 51.</p>c<p>Data from 129S5 and 129S1/SvImJ mice were pooled.</p>d<p>Significantly different between treated and control mice (<i>P</i><0.05; Fisher's exact test).</p>e<p>Significantly different between treated and control mice (<i>P</i><0.05; <i>t</i> test).</p>f<p>Historical data from previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093311#pone.0093311-Shetty1" target="_blank">[5]</a> were used for comparison.</p

Androgen-Responsive MicroRNAs in Mouse Sertoli Cells

<div><p>Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs – small, non-coding RNAs – are one class of androgen-regulated <em>trans</em>-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.</p> </div

Expression pattern of testosterone-responsive miRNAs.

<p>Real-time RT-PCR analysis of selected miRNA expression in total cellular RNA prepared from the adult mouse tissues. All values are normalized against RNU19 levels. Bar graphs represent the mean fold increase ± SEM of miRNA expression over background for at least two RT reactions assayed in duplicate from three separate mice. Several miRNAs negatively regulated by the androgen were specifically expressed in the testis.</p

MiR-471 regulates expression of Foxs1 in Sertoli cells.

<p>(<b>A</b>) Putative miR-471 binding sequence in the <i>Foxd1</i> 3′ UTR. (<b>B</b>) We co-transfected 15P1 Sertoli cells with <i>Renilla</i> luciferase expression construct pRL-CMV and firefly luciferase construct containing pMIR-<i>Foxd1</i> 3′ UTR in the absence and presence of miR-471 mimic. We normalized firefly luciferase activity of each sample to <i>Renilla</i> luciferase activity. Graphs show mean ± SEM of three independent experiments (performed in duplicate for each experiment). * <i>p</i><0.01; *** <i>p</i><0.001. (<b>C</b>) Real-time RT-PCR analysis of miR-471-overexpressing cells by using <i>Foxd1</i>-specific primers. (<b>D</b>) Western blot analysis of 15P1 cells transfected with miR-471 mimic by using anti-Foxd1 antibody (1∶1000). Tubulin was used as a loading control. Gel photographs represent three independent experiments. Values below the gel were quantified using Image J software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>). Foxd1 protein level for the control was set to 1.</p