Representative 3D images and mammograms from two different patients.

<p>Left column: sample number 6 (fibrosis, sclerosing adenosis, normal ductal hyperplasia, intraductal papilloma), only spherical MC classified as no suspicious MC; right column: sample number 25 (DCIS high grade), fine linear MC. CC indicates craniocranialcaudal view and MLO indicates mediolateral oblique view.</p

Representative 3D images from different biopsies.

<p>Benign (group A: A–C) lesions, unclear biological potential lesions (group B: D–F) and malignant (group C: G–I) lesions: A (sample number 1, fine pleomorphic), B (sample number 6, no suspicious MC), C (sample number 7, no suspicious MC), D (sample number 15, coarse heterogeneous), E (sample number 16, fine pleomorphic), F (sample number 20, fine linear), G (sample number 22, fine linear), H (sample number 25, fine linear) and I (sample number 24, fine pleomorphic). For more details concerning the different samples, compare sample numbers in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169349#pone.0169349.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169349#pone.0169349.t002" target="_blank">2</a>.</p

Morphological parameters including object volume (μm³x10⁵) and the structure model index (±SD) of every specimen.

<p>Morphology: fl: fine linear, fp: fine pleomorphic, ch: coarse heterogeneous, ns: no suspicious MC, 1 indicating presence and 0 indicating absence of the respective type of microcalcification.</p

Overview of different morphological parameters including mean number, volume (μm³) and SMI of MC for all different groups (benign: including B-classification B1 and B2; unclear malignant potential: including B-classification B3 and B4; malignant lesions: including B-classification B5).

<p>Error bars indicate the standard error of the mean (SEM).</p

Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation

<div><p>The tumour suppressor Merlin, encoded by the gene <i>NF2</i>, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, <i>NF2</i> is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, <i>NF2</i> encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used <i>Nf2</i> isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new <i>Nf2</i>-dependent process. Additionally, we provide for the first time <i>in vivo</i> evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2.</p></div

Expression of <i>Nf2</i> in major organs of adult mice.

<p><b>(A)</b> RT-PCR using primers spanning exon 14 to exon 17 of <i>Nf2</i>. Isoform 1 (iso1) mRNA lacks exon 16, making it 45 bp smaller than isoform 2 (iso2). Cyclophilin D levels were used as an mRNA loading control. Arrows denote organs primarily expressing one of the two isoforms (testis, heart, muscle, spleen, liver). <b>(B)</b> Determination of mRNA expression levels in male organs using qPCR with primers specific for exon 2 (total <i>Nf2</i> mRNA); normalized to lung levels. <b>(C)</b> Determination of mRNA expression levels using qPCR with specific primers for isoform 1 mRNA or isoform 2 mRNA; normalized to lung levels. Relative isoform levels were consistent between the two methods (epi., epididymis; sem. ves., seminal vesicles; ant., anterior; wat, white adipose tissue).</p

<i>Nf2</i> is expressed in the seminiferous and epididymal epithelium.

<p><b>(A)</b><i>In situ</i> hybridisation analysis using radioactively labelled probes targeting exons 1–4 of <i>Nf2</i>. <i>Nf2</i> specific signals were present in the seminiferous tubules but not the interstitium of the testis (scale bar = 200 μm) and in all tubules of the epididymis (scale bar = 1000 μm). <b>(B)</b> IHC staining shows Merlin to be abundantly expressed in residual bodies in the testis and at the apical site of the epididymal epithelium. <b>(C)</b> Western blot analysis of Merlin protein levels in testicular lysates at different post-natal stages showing elevated expression of Merlin from P30 onwards.</p

Classical merlin regulated pathways are unaltered in ko testes.

<p>Average littersize of different breeding combinations over a period of 6 months, starting at 2 months of age, on a C57Bl/6 <b>(A)</b> and a mixed C57Bl/6-FVB/NJ <b>(B)</b> genetic background revealed a background dependent impact of the isoform 2 knockout on fertility. <b>(C)</b> Testis and seminal vesicle weight of both knockout lines was unchanged at 3 months (n = 5). <b>(D)</b> and <b>(E)</b> Western blot analysis of whole testis lysates indicated unaltered Hippo (phospho Yap) and MAPK (phospho Erk) signalling in both knockouts. Knockout of isoform 1 led to altered merlin protein levels in iso1 ko testis <b>(E)</b>.</p

Epididymal vesicle formation is altered in ko animals.

<p>H&E stained sections showed normal histology of iso1 ko and iso2 ko epidiymises. (scale bars = 200 μm (overview)/ 100μm (lower panels)).</p

Knockout of <i>Nf2</i> isoform 2 causes testicular atrophy in aged mice.

<p><b>(A)</b> Organization of the <i>Nf2</i> gene locus, splicing of isoform 1 and gene knockout strategy. Note that deletion of one isoform causes expression of the other. <b>(B)</b> Body weight of iso1 ko and iso2 ko animals. Development of mice was normal (n = 3 for each genotype and time point). <b>(C)</b> Percentage of aged (22–26 months old) animals displaying testicular atrophy in more than 30% of tubules. #: significant on an a α-level of <0.1 (Fisher’s exact test). <b>(D)</b> H&E-stained tissue sections showing testicular atrophy and empty epididymis of iso2 ko animals. Scale bar = 200 μm (testis)/ 50 μm (epididymis).</p