Implementation of meiosis prophase I programme requires a conserved retinoid-independent stabilizer of meiotic transcripts

Abstract

Sexual reproduction is crucially dependent on meiosis, a conserved, specialized cell division programme that is essential for the production of haploid gametes. Here we demonstrate that fertility and the implementation of the meiotic programme require a previously uncharacterized meiosis-specific protein, MEIOC. Meioc invalidation in mice induces early and pleiotropic meiotic defects in males and females. MEIOC prevents meiotic transcript degradation and interacts with an RNA helicase that binds numerous meiotic mRNAs. Our results indicate that proper engagement into meiosis necessitates the specific stabilization of meiotic transcripts, a previously little-appreciated feature in mammals. Remarkably, the upregulation of MEIOC at the onset of meiosis does not require retinoic acid and STRA8 signalling. Thus, we propose that the complete induction of the meiotic programme requires both retinoic acid-dependent and -independent mechanisms. The latter process involving post-transcriptional regulation likely represents an ancestral mechanism, given that MEIOC homologues are conserved throughout multicellular animals.

Figure 1. Meioc is a conserved and meiosis prophase I-specific gene.

(a) Schematic representation of MEIOC proteins in the indicated species (see also Supplementary Fig. 1). A single conserved domain of unknown function (DUF4582, grey boxes) is retrieved in the sequence of all proteins. It features a highly conserved coiled-coil domain composed of four helixes (black boxes). The length and percentage of identity of the complete sequence compared with that of humans are indicated on the right, and the identities of the coiled-coil domain are indicated in the grey boxes. Comparison of amino-acid sequences of helixes 1 and 2 are detailed for the indicated species. Blue indicates conservation (Clustal2.1). Represented species are as follows: worm, Caenorhabditis elegans; tunicate, Ciona intestinalis; wasp, Nasonia vitripennis; annelid, Capitella teleta; oyster, Crassostrea gigas; urchin, Strongylocentrotus purpuratus; zebrafish, Danio rerio; frog, Xenopus tropicalis; platypus, Ornithorhynchus anatinus; lizard, Anolis carolinensis; marsupial, Sarcophilus harrisii; mouse, Mus musculus; rat, Rattus norvegicus; human, Homo sapiens; and chick, Gallus gallus. (b) Meioc mRNA expression measured by RT–qPCR in whole fetal and postnatal mouse ovaries (black columns) and testes (white columns) at the indicated developmental stages. Arrows indicate meiosis initiation. Mean±s.e.m., n=3 pools of 3–10 gonads. (c) Western blot analysis of MEIOC in mouse testis protein extracts at the indicated postnatal ages (days post-partum). β-actin/ACTB was used as a control. (d,e) Immunofluorescence for MEIOC (green), SYCP3 (red) and DAPI (blue) in (d) adult wild-type testis sections (scale bar, 20 μm) and (e) fetal (15.5 d.p.c.) wild-type ovaries (scale bar, 20 μm).

Figure 2. MEIOC protein is expressed in germ cells from the onset of meiosis.

(a) Immunofluorescence staining for MEIOC (green), STRA8 (red) and DAPI (blue) in 8 days post-partum (d.p.p.) testis sections. Scale bar, 20 μm. (b) Confocal acquisitions of representative Meioc^+/+ and Meioc^−/− spermatocytes stained for SYCP3 (red), MEIOC (green) and DAPI (blue). Diplo, diplotene; Lepto, leptotene; Pachy, pachytene; Prelepto, preleptotene; Zygo, zygotene. Scale bar, 5 μm.

Figure 3. Meioc is required for fertility and meiosis completion.

(a) Testes and ovaries from adult Meioc^−/− mice (knockout straight) are significantly smaller in size than wild type and heterozygous gonads. Scale bars, 2 mm (left) and 5 mm (right). (b) Left: histological sections of adult Meioc^+/+, Meioc^−/−(knockout straight), Meioc^flox/flox and Meioc^flox/flox;Vasa Cre ovaries. Scale bar, 200 μm. Right: histological sections of Meioc^+/+, Meioc^−/−, Meioc^flox/flox and Meioc^flox/flox;Vasa Cre adult testes. Scale bar, 40 μm. (c) Representative magnifications of histological sections of adult Meioc^+/+, Meioc^−/− (left) ovaries and (right) testes. Scale bar, 40 and 10 μm. (d) Histological sections of 18.5-d.p.c. Meioc^+/+ and Meioc^−/− ovaries stained for cleaved-caspase 3 (C-CASP 3) and VASA/DDX4. Scale bar, 10 μm. (e) Histological sections of adult Meioc^+/+ and Meioc^−/− testes stained for TUNEL. Scale bar, 20 μm.

Figure 4. Early meiotic defects in Meioc^−/− ovaries.

(a) Histological sections of 14.5-d.p.c. Meioc^+/+ and Meioc^−/− ovaries stained for VASA/DDX4 and OCT4/POU5F1, STRA8 or BrdU. Scale bar, 10 μm. OCT4 identified oogonia, and STRA8 labelled pre-meiotic cells. BrdU was injected to pregnant females 3 h before harvesting fetal gonads. BrdU incorporation revealed replicating cells. (b) The percentage of cells positive for the indicated marker of interest and for VASA, a germ cell marker, was determined in 14.5- and 15.5-d.p.c. Meioc^+/+ (white columns) and Meioc^−/− (black columns) ovaries. Mean±s.e.m. At least 200 cells were counted per gonad analysed. n=3 embryos analysed; *P<0.05 (Student's t-test). (c) Histological sections of 15.5-d.p.c. Meioc^+/+ and Meioc^−/− ovaries stained for SYCP3 and γ-H2AX meiotic markers showing delayed meiosis entry and progression in Meioc-deficient ovaries. Scale bar, 40 μm. (d) Meiosis prophase I stages in Meioc^+/+ and Meioc^−/− 15.5-d.p.c. ovaries. e, early; l, late; L, leptotene; Og, oogonia; P, pachytene; PL, preleptotene; Sg, spermatogonia; Z, zygotene. Mean±s.e.m., n=3 mice analysed; *P<0.05; **P<0.01 (Student's t-test). Scale bar, 5 μm. (e) Histological sections of 18.5-d.p.c. Meioc^+/+ and Meioc^−/− ovaries stained for γ-H2AX and VASA, STRA8 and VASA or P63. Scale bar, 40 and 10 μm. (f) Histological sections of 3-d.p.p. Meioc^+/+ and Meioc^−/−ovaries stained for P63 or C-CASP 3. Scale bar, 40 and 10 μm.

Figure 5. Early meiotic defects in Meioc^−/− testes.

(a) Histological sections of Meioc^+/+ and Meioc^−/− testes at the indicated ages stained for VASA and SYCP3 or γ-H2AX or haematoxylin and eosin. d.p.c., days post conception; d.p.p., days post-partum. Scale bar, 10 μm. (b,c) Spermatogonial and meiosis prophase I stages in Meioc^+/+ (white columns) and ^−/− (black columns). (b) Eight-d.p.p. and (c) sixteen-d.p.p. testes. D, Diplotene; e, early; l, late; L, leptotene; P, pachytene; PL, preleptotene; Sg, spermatogonia; Z, zygotene. Mean±s.e.m., mice analysed n=3–7, *P<0.05; **P<0.01; ***P<0.001 (Student's t-test). (d) Immunofluorescence staining for SYCP3 (red), STRA8 (green) and DAPI (blue) in Meioc^+/+ and Meioc^−/− (left) 8-d.p.p. and (right) 16-d.p.p. testis sections. Scale bars, 20 μm.

Figure 6. Pleiotropic meiotic defects in Meioc-deficient spermatocytes.

(a,b) Meioc^+/+ and Meioc^−/− spermatocyte chromosome spreads at the indicated meiosis prophase I stages stained for (a) SYCP1 (green) and SYCP3 (red) or (b) γH2AX (red) and SYCP3 (green). Lepto, leptotene; Pachy, pachytene; Zygo, zygotene. (c) Representative Meioc^+/+ and Meioc^−/− spermatocyte chromosome spreads at pachytene- and zygotene-like stages stained with SYCP3 (red) and KASH5 (green; meiotic telomere marker). A graph shows the proportion of cells with clustered telomeres (bouquet) among zygotene cells in adult testes. This suggests an extensive duration of the bouquet stage, a transitory telomere clustering occurring during zygotene stage. Mean±s.e.m., mice analysed n=3, **P<0.01 (Student's t-test). (d) Quantification of meiosis prophase I most advanced stages per tubule in adult testes revealed that Spo11^−/− spermatocytes progress until the pachytene-like stage, whereas Meioc invalidation induces arrest at a zygotene-like stage and abnormal metaphases, regardless of the genotype. D, Diplotene; e, early; l, late; L, leptotene; M*, abnormal metaphases; P, pachytene; PL, preleptotene; Spg, spermatogonia; Z, zygotene. Mean±s.e.m.; n=3 mice analysed per genotype. For each cell type analysed, statistical comparison (analysis of variance, ANOVA) indicated no significant differences between Meioc^−/− and Meioc^−/−;Spo11^−/−. (e) Histological sections of adult testis from wild type (Meioc^+/+; Spo11^+/+), Spo11^−/−, Meioc^−/− and Meioc^−/−;Spo11^−/− mice. Scale bar, 40 μm. (f) Histological sections of 8-d.p.p. ovaries from wild type, Meioc^−/−, Spo11^−/− and Meioc^−/−; Spo11^−/− females. Scale bar, 40 μm.

Figure 7. Abnormal metaphase phenotype in Meioc-deficient gonads.

(a) Magnification of abnormal metaphases in 16-d.p.p. Meioc^−/− testis histological sections stained for haematoxylin and eosin (left) or γ-H2AX (right). Scale bar, 5 μm. (b) Magnification of chromosome spreads of meiotic metaphase I in Meioc^+/+ and abnormal metaphase in Meioc^−/− testes stained with DAPI, CREST and γ-H2AX. Scale bar, 5 μm. (c,d) Sixteen-d.p.p. Meioc^−/− and adult Meioc^+/+ testis sections were stained for (c) pH3 (green)/SYCP3 (red)/DAPI (blue) or for (d) α-TUBULIN (green) and DAPI (blue). Scale bar, 5 μm. M, metaphase; M*, abnormal metaphase. Similarly to meiotic metaphases, abnormal metaphases in Meioc mutants display SYCP3 staining but they are distinguished because of atypical rosette shape and hemispindle. (e) Representative histological sections of 10-d.p.p. Meioc^−/− and Meioc^+/+ testes showing preleptotene cells associated with abnormal metaphases in the mutant testes. Arrowheads indicate abnormal metaphases; arrows indicate preleptotene cells. Scale bar, 10 μm. (f) Magnification of abnormal metaphases and associated cells in a Meioc^−/− 16-d.p.p. seminiferious tubule. Adjacent sections were stained for BrdU or pH3 (green) and DAPI (blue). BrdU was injected to 15-d.p.p. Meioc^−/− mouse 36 h before harvesting the gonads. The presence of BrdU in the abnormal metaphases suggests that these cells were at the preleptotene stage before reaching a metaphase-like stage. Arrowheads indicate abnormal metaphases. Scale bar, 10 μm. (g) Graphs showing percentage of tubules presenting abnormal metaphases at 10- and 16-d.p.p. and in adult Meioc^+/+ (white columns) and Meioc^−/− (black columns) testes (left) and percentage of abnormal metaphases cells in 15.5-d.p.c. ovaries (right). Mean±s.e.m., mice analysed n=3–6, **P<0.01; ***P<0.001 (Student's t-test).

Figure 8. Meiosis prophase I genes are less stable in Meioc^−/− gonads.

(a,b) RT–qPCR measurements of MPI transcripts in whole Meioc^+/+ and Meioc^−/−(a) 14.5-d.p.c. ovaries and 8-d.p.p. testes; (b) Meioc^−/− adult testes were compared with Meiob^−/− adult testes as a reference. (c) Plot of RT–qPCR data for pre-mRNA and accumulated (mature) mRNA in Meioc^+/+ and Meioc^−/− fetal ovaries and postnatal testes. Dotted lines, projections of minimal and maximal values. (d) Fold loss of mRNA levels was measured after pulse chase ethynyl uridine incorporation performed in Meioc^−/− and Meiob^−/− adult testes. (e) Treatment of testes with actinomycin D to inhibit transcription revealed higher meiotic mRNA degradation levels in Meioc^−/− gonads. The relative expression of Rad21L after 12 and 24 h of actinomycin D treatment compared with mRNA expression levels without drug treatment is presented. Linear regressions were calculated, and slope coefficients representing degradation rates were compared between Meiob and Meioc mutant gonads. Data represent Rad21L expression following exposure to actinomycin D. For a,b,d, mean±s.e.m., n=3–4; *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).

Figure 9. MEIOC/YTHDC2 complex binds meiotic mRNA.

(a) Western blot analysis of co-IP of MEIOC and YTHDC2 in postnatal testes. (b) MEIOC coiled-coil domain interacts with YTHDC2. Upper panel: schematic representation of FLAG-tagged MEIOC complete protein and deletion mutants overexpressed in HEK-293 cells. Numbers in square brackets indicate the deleted amino acids, which are also represented with dotted lines. Blue boxes indicate the coiled-coil domain. Lower panel: co-IP was performed with anti-FLAG antibody, and samples were subjected to western blotting with anti-FLAG and anti-YTHDC2 antibodies. Deletion of the N terminus (ΔN1 and ΔN2) of MEIOC did not abolish the interaction with YTHDC2, whereas deletion of the C terminus of MEIOC (ΔC) or the second helix of the coiled-coil domain (ΔH2) abolished this interaction (c). Confocal acquisitions of MEIOC (red), YTHDC2 (green) and DAPI (blue) staining in spermatocytes. Scale bar, 10 μm. (d) Meioc^+/+ and Meioc^−/− 8- and 16-d.p.p. testis sections were stained for YTHDC2 (green), SYCP3 (red) and DAPI (blue). Scale bar, 40 μm. (e) Western blot analysis of YTHDC2 in mouse testis protein extracts at the indicated postnatal ages (days post-partum). β-actin/ACTB was used as a control. Ad, adult. (f) RT–qPCR analysis of mRNA bound after IP assays using anti-YTHDC2 antibody (YTH) or IgG in testicular protein extracts. Statistical analysis compares mRNA fold enrichment/input levels with that of Gapdh. Mean±s.e.m.; n=6. **P<0.01, ***P<0.001 (Student's t-test).

Figure 10. Meioc expression is independent of RA and Stra8 signalling.

(a) Gonads from 12.5-d.p.c. embryos were cultured and exposed to RA or DMSO. (b) Embryonic ovaries (12.5 d.p.c.) and male pups (10 d.p.p.) were exposed to RAR inverse agonist (BMS4693) or DMSO (CTR). For a,b, RA target genes Cyp26a1 and RARβ (normalized to β-actin) and Stra8 and Meioc (normalized to Vasa/Ddx4 as these are germ cell-specific) expression was measured using RT–qPCR. Mean±s.e.m.; n=3–5. For each gene analysed, different letters indicate significantly different data (multiple comparisons ANOVA). *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). (c) MEIOC (red), STRA8 (green) and DAPI (blue) staining in 10-d.p.p. postnatal testes treated with BMS493 as in b (contralateral gonads) or with DMSO (CTR). Scale bars, 20 μm. (d) MEIOC (red), STRA8 (green) and DAPI (blue) staining in Stra8^+/+ and Stra8^−/− testis sections. Scale bars, 20 μm. (e) Hypothetical model for RA-dependent and -independent regulation of the meiotic programme.