MORC2B is required for chromosomal synapsis in meiosis in both sexes.

<p>(A) Surface spread nuclei of spermatocytes from the testes of juvenile wild type and <i>Morc2b</i>^-/- males were immunostained for synaptonemal complex proteins (SYCP1 and SYCP2). The sex chromosomes in wild type pachytene spermatocyte (left) are labelled. Three paired chromosomes in <i>Morc2b</i>^-/- spermatocytes are indicated by arrowheads. (B) Surface spread nuclei of oocytes from wild type and <i>Morc2b</i>^-/- embryonic day 17.5 (E17.5) embryos were immunostained for SYCP1 and SYCP2. Note the apparent pairing and alignment of presumably homologous chromosomes judged by the equal length of SC axial elements in the <i>Morc2b-</i>deficient oocyte. (C) Surface spread nuclei of spermatocytes from wild type and <i>Morc2b</i>^-/- postnatal day 18 testes were immunostained for SYCP3 and HORMAD1. Arrows indicate synapsed regions. Scale bars, 10 μm.</p

MORC2B is essential for meiotic progression and fertility

<div><p>The microrchidia (MORC) family proteins are chromatin-remodelling factors and function in diverse biological processes such as DNA damage response and transposon silencing. Here, we report that mouse <i>Morc2b</i> encodes a functional germ cell-specific member of the MORC protein family. <i>Morc2b</i> arose specifically in the rodent lineage through retrotransposition of <i>Morc2a</i> during evolution. Inactivation of <i>Morc2b</i> leads to meiotic arrest and sterility in both sexes. <i>Morc2b</i>-deficient spermatocytes and oocytes exhibit failures in chromosomal synapsis, blockades in meiotic recombination, and increased apoptosis. Loss of MORC2B causes mis-regulated expression of meiosis-specific genes. Furthermore, we find that MORC2B interacts with MORC2A, its sequence paralogue. Our results demonstrate that <i>Morc2b</i>, a relatively recent gene, has evolved an essential role in meiosis and fertility.</p></div

MORC2B is essential for male meiosis.

<p>(A) Targeted inactivation of the <i>Morc2b</i> gene. Deletion of exon 2 removes the entire coding region and thus results in a null allele. The neomycin selection marker PGKNeo is flanked by loxP sites. HyTK provides negative selection by ganciclovir. (B) Dramatic reduction in testis size in 3-month-old <i>Morc2b</i>^-/- males. (C) Western blot analysis of postnatal day 20 (PND20) <i>Morc2b</i>^+/- and <i>Morc2b</i>^-/- testes. ACTB serves as a loading control. (D) Histological analysis of 8-week-old <i>Morc2b</i>^+/- and <i>Morc2b</i>^-/- testes. Abbreviations: Pa, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids. Scale bars, 25 μm. (E) TUNEL analysis of seminiferous tubules from the testes of 8-week-old wild-type and <i>Morc2b</i>^-/- males. TUNEL-positive cells are shown in green. DNA was counterstained with DAPI. Scale bars, 25 μm. The graph on the left shows quantification of TUNEL-positive cells. Only tubule cross-sections with at least one TUNEL-positive cell (3 wild type testes and 3 <i>Morc2b</i>^-/- testes) were analyzed. 18 to 24 tubules per testis were counted. Statistical analysis was performed with Student’s <i>t</i>-test.</p

Persistence of γH2AX in <i>Morc2b</i>^-/- spermatocytes.

<p>(A, B) Spread nuclei of spermatocytes from wild type (A) and <i>Morc2b</i>^-/- (B) males at PND25 were immunostained with anti-SYCP2 and anti-γH2AX antibodies. Representative images of wild type spermatocytes at the leptotene through diplotene stages are shown. In <i>Morc2b</i>^-/- males, zygotene-like spermatocytes formed lateral elements and contained a high level of γH2AX, whereas pachytene-like spermatocytes showed more prominent lateral elements, alignment of lateral elements, and a low level of γH2AX. (C) Percentage of spermatocytes at meiotic stages (leptotene through diplotene). The number of spermatocytes analysed: wild type, 460; <i>Morc2b</i>^-/-, 480. Scale bars, 10 μm.</p

MORC2B interacts with its paralogue MORC2A.

<p>(A) Identification of MORC2B-associated proteins in lysates from PND20 mouse testes by IP and mass spectrometry. The gel was stained with SYPRO Ruby. Gel slices corresponding to the two unique protein bands present in wild-type but not in <i>Morc2b</i>^-/- testes (*~110 kDa; ** ~65 kDa) were cut from both lanes and subject to mass spectrometry. Proteins with at least three unique peptides are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007175#pgen.1007175.s006" target="_blank">S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007175#pgen.1007175.s007" target="_blank">S3</a> Tables. (B) Co-IP analysis of MORC2A and MORC2B from PND20 testicular protein extracts. (C) Co-IP analysis of MORC2A and MORC2B in HEK 293T cells. Expression constructs for tagged MORC2A and MORC2B were transfected into HEK 293T cells alone or together as indicated, followed by co-IP with anti-V5 antibody and western blotting with antibodies as indicated. Input was 10% of the lysate used for IP.</p

MORC2B is essential for meiotic recombination.

<p>Immunofluorescence was performed on spread nuclei of spermatocytes from <i>Morc2b</i>^+/- and <i>Morc2b</i>^-/- testes at postnatal day 18. Based on the morphology of the synaptonemal complex (SYCP2 immunolabelling), spermatocytes were categorized into the following stages: leptotene (Le), zygotene (Zy), pachytene (Pa), zygotene-like (Zy-like), and pachytene-like (Pa-like). Each dot represents the number of DNA repair protein foci per cell. Solid lines show the average number of foci for each category of spermatocytes. (A) RPA2 foci. (B) MEIOB foci. (C) RAD51 foci. (D) DMC1 foci. Representative images of spermatocytes at pachytene or pachytene-like stages are shown (A-C). Spermatocytes at zygotene and zygotene-like stages are shown in D. Scale bars, 10 μm. Statistics was performed by Student’s <i>t</i> test.</p

<i>Morc2b</i> is a retrotransposed germ cell-specific derivative of <i>Morc2a</i>.

<p>(A) Gene structure of <i>Morc2a</i> and <i>Morc2b</i>. Coding regions are shown in black. Percent identities of nucleotide (nt) and amino acid (aa) sequences in the coding region are shown. 5’ and 3’ untranslated regions (UTRs) lack significant nt identity and are represented as open boxes. (B) Timing of the <i>Morc2b</i> retrotransposition. mya, million years ago. (C) Western blot analysis of MORC2A and MORC2B in lysates from adult mouse tissues. ACTB served as a loading control. (D, E) Expression and subcellular localization of MORC2B in adult testes. Immunofluorescence analysis of MORC2B was performed on frozen sections from wild type (D) and <i>Morc2b</i>^-/- (E) testes. DNA was stained with DAPI. Insets show enlarged view of boxed regions. Abbreviations: Pa, pachytene spermatocytes; Spc, spermatocytes; RS, round spermatids; ES, elongated spermatids; Sertoli, Sertoli cells. Scale bars, 25 μm. (F) RT-PCR expression analysis of <i>Morc2b</i> in juvenile testes. <i>Actb</i> serves as a control. (G) Quantification of <i>Morc2b</i> expression in juvenile testes by real-time PCR. The expression level of <i>Morc2b</i> was normalized to <i>Actb</i>. The relative expression level at postnatal day 20 was set at 1.0.</p

Transcript profiling analysis of wild type and <i>Morc2b</i>^-/- testes.

<p>(A) Volcano plot of transcript levels between wild type and <i>Morc2b</i>^-/- testes at postnatal day 12. The expression cut-off is at least 1 FPKM in either wild type or <i>Morc2b</i>^-/- testes. The differentially expressed genes (FDR < 0.05) are highlighted in red (upregulated in <i>Morc2b</i>^-/-) and green (downregulated in <i>Morc2b</i>^-/-). (B) Validation of differentially expressed genes by real-time PCR analysis. Statistics was performed with Student’s <i>t</i>-test: *, p<0.05; **, p<0.01; ***, p<0.001. (C) GO term enrichment in differentially expressed genes.</p

MORC2B is essential for oogenesis.

<p>(A) Histological analysis of the ovaries from 3-month-old <i>Morc2b</i>^+/- and <i>Morc2b</i>^-/- females. (B, C) Postnatal loss of oocytes in <i>Morc2b</i>^-/- ovaries. Frozen sections from postnatal day 0 (PND0) and 2 (PND2) ovaries were immunostained with anti-YBX2 antibodies. YBX2 is specifically expressed in oocytes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007175#pgen.1007175.ref032" target="_blank">32</a>]. (D) TUNEL analysis of PND0 ovaries. Scale bars: 100 μm (A), 50 μm (B-D).</p