Plasma testosterone levels in adult TRα^AMI-ARO males, in basal conditions and after hCG stimulation, compared with control mice and mRNA levels of Cyp11a1/P450ssc, Cyp17a1/P450c17 and StAR in TRα^AMI-ARO testes.

<p><b>(a)</b> In both TRα^AMI-ARO (11 males) and controls (9 males), hCG induced a significant increase (***<i>P</i> < 0.001) in testosterone secretion, as expected. Plasma testosterone levels were unchanged in TRα^AMI-ARO compared with the control. Data are shown as mean ± SEM. Two-way ANOVA followed by Bonferroni’s <i>post-hoc</i> test. Black bars, basal levels; white bars, hCG stimulated. <b>(b)</b> The amount of cDNA of 3 steroidogenic genes (Cyp11a1/P450ssc, Cyp17a1/P450c17 and StAR) was compared in TRα^AMI-ARO (black bars) and control males (white bars). For each genotype, 6 mice were tested in triplicate using TaqMan quantitative real-time PCR, as described in materials and methods. Normalization was performed with two housekeeping genes, β-actin and GAPDH, that both exhibited similar expression among samples. mRNA expression is presented as percent of mean control level. A non-parametric test (Mann-Whitney) was used for statistical analysis. NS: not significant.</p

Histological sections of adult TRα^AMI-ARO testes at low (a) and high (b) magnification.

<p>Seminiferous tubules were fully developed in TRα^AMI-ARO. Their epithelium exhibited normal structure and organization. Hematoxylin staining. Bar represents 100 μm.</p

Primers for PCR genotyping of TRα^AMI-ARO line and for RT-PCR of TRα1 [27] and actin genes.

<p>Primers for PCR genotyping of TRα^AMI-ARO line and for RT-PCR of TRα1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119392#pone.0119392.ref027" target="_blank">27</a>] and actin genes.</p

Whole testicular sperm reserve and testis weight in adult TRα^AMI-ARO, and percentage of proliferating SC in TRα^AMI-ARO at P3 <i>in vivo</i> and in testicular explants using organotypic <i>in vitro</i> cultures with or without exogenous T3.

<p>We observed a significant increase in whole testicular sperm reserve <b>(a)</b> and in testis weight <b>(b)</b> (***<i>P</i> < 0.001; n = 15 for controls and n = 16 for TRα^AMI-ARO group). Data are shown as mean ± SEM. Statistical analyses were performed using Student’s <i>t</i>-test. White bars, control; black bars, TRα^AMI-ARO (a, b). After BrdU immunohistochemical labeling, BrdU negative and BrdU positive SC were counted and the SC proliferation index was calculated. <b>(c)</b> When BrdU was injected <i>in vivo</i> 3 h before sacrifice, proliferation of Sertoli cells increased in P3 testes of TRα^AMI-ARO mice in comparison with the control (***<i>P</i> < 0.001; n = 5 animals for each genotype). <b>(d)</b> When BrdU was added <i>in vitro</i> 3 h before the end of the organotypic cultures of P3 testicular explants, the SC proliferation index significantly decreased in the control in presence of T3 compared with vehicle (***<i>P</i> < 0.001, n = 6). In contrast, T3 had no effect on SC proliferative index in TRα^AMI-ARO mice (<i>P</i> > 0.05, n = 5). Data are shown as mean ± SEM. Statistical analyses: two-way ANOVA followed by Bonferroni’s <i>post-hoc</i> test. White bars, control; black bars, TRα^AMI-ARO.</p

Production and characterization of ARO-iCre transgenic mouse line.

<p><b>(a)</b> Schematic diagram of the DNA construct used for producing ARO-iCre mice. The mammalian codon-improved Cre recombinase (iCre, 1051 bp; white box) followed by an SV40 poly(A) signal cassette (right grey box) is controlled by 304 bp of the human Cyp19/aromatase(IIa)_-278 promoter (left grey box). Arrows indicate position and direction of primers. Oligonucleotide sequences are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119392#pone.0119392.t001" target="_blank">Table 1</a>. <b>(b)</b> Cre excision is restricted to ovaries (Ov) and testes (Te) of iCre^+/0;IGF1R^flox/WT mice. The excision was only observed in gonads, not in other tissues (He, heart; Mu, muscle; Lu, lung; Sp, spleen; Br, brain; Li, liver; Pa, pancreas) from males (M) and females (F). RT-, negative control (testis cDNA without RT). PCR detected wild-type (W, 256 bp) and floxed (F, 312 bp) alleles in all tissues, and the excised allele (E, 204 bp) exclusively in gonads. <b>(c)</b> Cre recombinase activity detected in somatic cells of testes in adult ARO-iCre males. After crossing the ARO-iCre mice with a ROSA26 Cre reporter mouse, β-galactosidase activity was detected in both SC and LC. No activity was present in germ cells. Right micrograph, low magnification; left micrographs, details in higher magnification. ST, seminiferous tubules. In, interstitium. Arrow head points to SC. Bar represents 50 μm.</p

Physiological, endocrine and cellular characteristics in TRα^AMI-ARO (present study), TRα^AMI-SC and TRα^Null/Null transgenic lines.

<p>**<i>P</i> < 0.01;</p><p>***<i>P</i> < 0.001;</p><p>ND, not determined.</p><p>Physiological, endocrine and cellular characteristics in TRα^AMI-ARO (present study), TRα^AMI-SC and TRα^Null/Null transgenic lines.</p

The mitochondrial morphology is unaffected in germ cells of p43−/− mice.

<p>Mitochondria observed in germ cells in the adult testis of wild type mice (WT) and in the adult testis of p43−/− mice at 5 months of age (p43−/−). Mitochondria present in germ cells of p43−/− mice were morphological identical at those presenting in germ cells of WT mice. Data are shown as the mean+/− SEM; statistical analyses; two-way ANOVA followed by Bonferroni’s post-test (n = 60). Arrowheads: Mitochondria.</p

CDK4 expression is increased in P43−/− testis at P3.

<p>Western blot analyses were performed with p43−/− and WT proteins extract testes at P3 (n = 5 for P43−/−, n = 4 for WT) using a specific CDK4 and ß-actin antibodies. Normalization was achieved using ß-actin levels. Data are shown as the mean+/− SEM and statistical analyses were performed using the student t test. CDK4 level was increased (P<0,001).</p

Percentage of <i>in vivo</i> proliferating Sertoli cells in p43−/− testis at P3 and P10.

<p>A) Immunohistochemical labelling of proliferating cells revealed by BrdU incorporation injected three hours before sacrifice in P3 and P10 testis of p43−/− mice. Arrowheads: Sertoli cells; arrows: germ cells; star: myoid peritubular cells; black: BrdU-positive; white: BrdU-negative. Scale bar 10 µm. B) BrdU negative and positive Sertoli cells were counted and proliferation index was calculated. In p43−/− testis, SC proliferation was increased at P3 in comparison to WT mice (P<0.001). At P10 there is no difference. (n = 3/genotype/age). Data are shown as the mean+/− SEM; statistical analyses; two-way ANOVA followed by Bonferroni’s post-test.</p

Mitochondrial gene expression is altered in p43−/− testis at P3.

<p>The volcano plot is an arbitrary representation (proposed by the web-based RT² prolifer PCR Array Data Analysis program) of the fold change (FC) for each of the 84 genes in the array. It represents the log2C FC of each gene expression between the p43−/− group (n = 6) and the control group (n = 4) versus the negative Log10 P values from the t-test. The red vertical line indicates that the gene expression fold change threshold is 2. The blue horizontal line indicates that the P value of the T-test threshold is 0.05. Genes, which were significantly up-regulated in p43−/− mice in comparison with controls, were indicated.</p