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

<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

Tumours observed in aged iso1 ko and iso2 ko animals.

<p>Tumours observed in aged iso1 ko and iso2 ko animals.</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

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

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

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

Natural History of Meningioma Development in Mice Reveals: A Synergy of and Mutations-5

<p><b>Copyright information:</b></p><p>Taken from "Natural History of Meningioma Development in Mice Reveals: A Synergy of and Mutations"</p><p></p><p>Brain Pathology (Zurich, Switzerland) 2008;18(1):62-70.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253711.</p><p>© 2007 The Authors; Journal compilation © 2007 International Society of Neuropathology</p>brainstem. B,C. Transitional meningioma covering the right trigeminal nerve. Some cells have prominent nucleoli, reminiscent of atypical features in human tumors (arrows). D. Ultrastructural study of a large transitional spinal meningioma, using paraffin-embedded material, demonstrated features characteristic of human meningioma. The arrow indicates tight junctions and asterisks show complex interdigitating cell processes. E. Detail (higher magnification) of the image in D. Immunostaining for Prostaglandin D2 synthase (PGDS) in murine meningiomas. Mouse meningiomas show two different PGDS staining patterns. F. Transitional meningioma showing multifocal process with both meningioma and discrete nodules. The “” part of the tumor shows multiple layers with strong cytoplasmic staining. G. Fibroblastic meningioma with regional positivity for PGDS. In some areas, the cytoplasmic staining is dispersed with few, abnormally distributed granules. H. Higher magnification reveals PGDS immunoreactive granules dispersed in the cytoplasm

Natural History of Meningioma Development in Mice Reveals: A Synergy of and Mutations-0

<p><b>Copyright information:</b></p><p>Taken from "Natural History of Meningioma Development in Mice Reveals: A Synergy of and Mutations"</p><p></p><p>Brain Pathology (Zurich, Switzerland) 2008;18(1):62-70.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253711.</p><p>© 2007 The Authors; Journal compilation © 2007 International Society of Neuropathology</p>brainstem. B,C. Transitional meningioma covering the right trigeminal nerve. Some cells have prominent nucleoli, reminiscent of atypical features in human tumors (arrows). D. Ultrastructural study of a large transitional spinal meningioma, using paraffin-embedded material, demonstrated features characteristic of human meningioma. The arrow indicates tight junctions and asterisks show complex interdigitating cell processes. E. Detail (higher magnification) of the image in D. Immunostaining for Prostaglandin D2 synthase (PGDS) in murine meningiomas. Mouse meningiomas show two different PGDS staining patterns. F. Transitional meningioma showing multifocal process with both meningioma and discrete nodules. The “” part of the tumor shows multiple layers with strong cytoplasmic staining. G. Fibroblastic meningioma with regional positivity for PGDS. In some areas, the cytoplasmic staining is dispersed with few, abnormally distributed granules. H. Higher magnification reveals PGDS immunoreactive granules dispersed in the cytoplasm

Arrowhead indicates lateral round Gd-DTPA enhancement corresponding to a fibroblastic meningioma associated with hydrocephalus characterized by enlarged ventricles (asterisks)

<p><b>Copyright information:</b></p><p>Taken from "Natural History of Meningioma Development in Mice Reveals: A Synergy of and Mutations"</p><p></p><p>Brain Pathology (Zurich, Switzerland) 2008;18(1):62-70.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253711.</p><p>© 2007 The Authors; Journal compilation © 2007 International Society of Neuropathology</p> Arrowhead indicates abnormal, thick Gd-DTPA enhancement corresponding to a histologically confirmed meningothelial meningioma that would be defined as olfactory meningioma in human pathology. A hypointense signal corresponding to an osteoma is indicated by an asterisk. Meningioma nested within areas of meningothelial proliferation revealed by lateral linear thick Gd-DTPA enhancement (arrowhead). Imaging of a false-positive case of meningioma. Coronal T1-weighted image after Gd-DTPA injection showing moderate gadolinium enhancement of enlarged trigeminal nerves (arrowhead). Histological analysis revealed osseous metaplasia developing within the trigeminal nerve ipsilateral to the transorbital injection site