All animal care procedures and experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No.85-23, revised 1996) and were approved by the Local Animal Experimentation Ethics Committee and the French Ministry of Research (APAFIS#1215-2015090115013347 v5) and the Haut Conseil des Biotechnologies (HCB n°6461). Mice were housed under controlled photoperiod conditions (lights on from 08:00 to 20:00) and supplied with commercial food and tap water ad libitum. Embryos were collected from dated matings. Males were caged with females overnight, and the presence of a vaginal plug was examined the following morning. The following midday was defined as 0.5 day post conception (dpc). Mice were sacrificed via cervical dislocation, and fetuses were removed from the uterine horns before gonad isolation under a binocular microscope.

Cep250 invalidation was performed by using the transcription activator-like effector nuclease (TALEN) genome editing technology. The end of exon 5 of mouse Cep250 gene was targeted at the site of the original mutation associated with Caprine-like Generalized Hypoplasia Syndrome (SHGC) phenotype in cattle (chromosome: GRCm38:2:155 958 456-155 998 900 (forward strand)). The target sequences were selected using the ZiFiT Targeter program (http://zifit.partners.org). A potential TALEN target sequence identified via this program was selected empirically, with a preference for an 18-16-18 combination (16 bases for the spacer). The chosen sequences were TAGTGAGGAAGGAGAGCC for the left TALEN and GGAGGTAGGAGAACAAGA for the right (localization 2:165538094-165538145). No homology with the entire targeted sequence was identified (BLAST NCBI) at any other location in the genome, which might represent a potential off-target site. The TALEN kit used for TALE assembly was a gift from the Keith Joung Laboratory (Addgene kit # 1000000017).

TALENs were constructed according to the restriction enzyme and ligation (REAL) assembly method, as described previously (Reyon et al., 2012). The left TALEN was constructed by assembling units of the kit in the following order: 6-14-20-24-26-14-19-21-26-14-19-21-29-11-19-22 (by groups of four units). The entire insert was subcloned into the final JDS71 plasmid, which was cleaved at the bsmb1 site. Similarly, the right TALEN was constructed by assembling units of the kit in the following order: 7-15-20-24-30-15-17-25-27-12-20-21-27-12-20-22. The entire insert was subcloned into the final JDS71 plasmid. All inserts of the final plasmids were sequenced with primers 2978 (TTG​AGG​CGC​TGC​TGA​CTG) and 2980 (TTA​ATT​CAA​TAT​ATT​CAT​GAG​GCA​C). To prepare RNA from each plasmid for microinjection, 5 μg of the TALEN plasmid was linearized with 20 U of Pme1 enzyme (New England Biolabs) for 8 h at 37°C in a 100 μL reaction volume. The linearized fragment was electrophoresed on an agarose gel and purified using the Qiagen Gel Extraction Column Kit (Qiagen, Hilden, Germany). Messenger RNA (mRNA) was prepared from 1 μg of purified linearized plasmid, using the ARCA T7 capRNA pol kit (Cellscript, TEBUbio, France), and polyadenylated with the polyA polymerase tailing kit (Epicentre) according to the manufacturer’s instructions. The synthesized mRNA was purified using the RNEasy mini kit (Qiagen) and resuspended in distilled water; then, mRNA concentration was estimated using a Nanodrop spectrophotometer (ND-1000, Thermo Scientific). The mRNA was then diluted to 100 ng/μL with injection buffer (Millipore, France) and stored at -80°C until further use.

TALEN mRNAs (2 ng/µL) were microinjected into the pronuclei of fertilized eggs derived from superovulated C57BL/6 female mice crossed with males of the same strain. Embryos were transferred into the oviducts of pseudo-pregnant C57BL/6 female mice. The resulting offspring were genotyped by DNA analysis of tail biopsies, as described in the DNA sequencing subsection. Transgenic mice were crossed with C57BL/6 mice to derive F1 offspring, which were used to produce the Cep250 transgenic line.

The tail tips of mice were lysed in 100 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.2% SDS, and 200 mg/ml proteinase K overnight at 37°C and centrifuged for 10 min at 13,000 g. Genomic DNA was precipitated with an equal volume of isopropanol and resuspended in 10 mM Tris-HCl (pH 7.5) and 0.1 mM EDTA. DNA concentration was measured using a NanoDrop ND-1000 spectrophotometer.

Primers were designed, using Primer3 software, in a manner so that they annealed ∼150 bp upstream and downstream from the expected TALEN-target site. PCR was carried out in a final volume of 60 µL using 200 ng of DNA and primers, which amplified a 333 pb fragment of Cep250 surrounding the target site in exon 5 (for primer sequences, Supplementary Table S1), with standard GoTaq PCR reagents (Promega) on a Master Thermal Cycler (Eppendorf). The cycling conditions were as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 20 s, 58°C for 30 s, 72°C for 30 s. Sanger sequencing was performed using standard methods (Eurofins) to identify null mutations.

Transgenic mice were genotyped using the same pair of primers against Cep250 surrounding the TALEN restriction site and the same PCR protocol, as described above. The PCR products were precipitated and then digested with the FokI restriction enzyme (New England Biolabs). This enzyme recognizes the GGATG DNA sequences and thus only cleaves wild-type PCR products into two fragments. PCR products were electrophoresed on a 2% agarose gel.

At 5–21 weeks of age, the wild-type and Cep250^-/- mice were euthanized, and various tissues were dissected, weighed, and either frozen at −80°C for RNA/protein extraction, or rinsed in phosphate-buffered saline (PBS), fixed overnight in 4% paraformaldehyde (PFA)/PBS, and embedded in paraffin (EG 1160 Embedding Center; Leica).

Total RNA was extracted from the kidneys of wild-type and transgenic animals using the RNA NOW reagent (Ozyme). After DNase I treatment, the RNA (200 ng) was reverse transcribed into complementary DNA (cDNA) using the Superscript II kit (Invitrogen) with oligo (dT) according to the manufacturer’s instructions. The cDNA was then amplified via PCR with primers for the Cep250 and Gapdh genes (Supplementary Table S1). Primers were designed, using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/), from separate exons to produce 100–300 bp amplicons. The Gapdh gene was used as a reference to normalize the relative expression of the target genes.

Fetal and postnatal gonads were harvested and frozen in RLT lysis buffer for RNA extraction. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Valencia, CA, United States). cDNA was obtained via reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, United States) according to the manufacturer’s instructions. The 7900HT fast real-time PCR system (Applied Biosystems) and SYBR Green chemistry were used for RT–qPCR. The comparative Ct method was used to determine the relative quantities of mRNA, using β-actin or DEAD-Box Helicase 4 (Ddx4) mRNA as the endogenous reporter. The results are presented as a percentage of the control (i.e., expression of the control is defined as 1). Each RNA sample was analyzed in duplicate. All primers were used at a final concentration of 400 nM. The sequences of oligonucleotides used in this study are listed in Supplementary Table S1.

To isolate the centrosomal fraction from the testis, we followed the protocol described by Moudjou and Bornens (Moudjou and Bornens, 1998). Briefly, we crushed the testes using ultra-turrax in 2.5 ml of a solution containing 10 mM Tris-HCl (pH 7.2), 2 mM MgCl₂, 0.5% NP-40, and 1 mM DTT with EDTA-free Halt Protease Inhibitor Cocktail (Thermo), at 4°C. All the subsequent steps were performed at 4°C. Chromatin and nuclei were pelleted via centrifugation at 2,500 × g for 10 min. The supernatant was deposited on 5 ml of a 50% sucrose cushion (w/w) dissolved in 10 mM Tris-HCl, pH (7.2), 0.1% Triton X-100, and 1 mM DTT. The centrosomes were concentrated onto the sucrose cushion via centrifugation at 22,500 × g for 20 min. The supernatant was removed, and the sucrose cushions were pooled, including the interface containing the centrosomes. We then transferred the solution onto a discontinuous sucrose gradient comprising 10 ml of 70% sucrose (w/w), 6 ml of 40% sucrose (w/w), and 6 ml of 30% sucrose (w/w), in 10 mM Tris-HCl (pH 7.2) and 1 mM DTT, and centrifuged it at 116,000 × g for 75 min. Aliquots were collected from each fraction, in Eppendorf tubes, for analysis to identify the centrosome-containing fractions. The 2-D Quant kit (GE Healthcare) was used to determine protein concentration. Protein samples (50–100 µg) were mixed with 2× Laemmli and separated on a 7.5% polyacrylamide home-cast SDS-PAGE at 80 V for 20 min and then at 150 V until the front reached the bottom of the gel; a pink pre-stained protein ladder (Nippon Genetics) was used a reference. The resolved proteins were transferred onto a nitrocellulose membrane using Trans-Blot Turbo (Bio-Rad) at 2.5 A and 25 V maximum for 10 min. The proteins were then stained with a 5× Red Ponceau solution and destained in TBS-Tween 20 and 0.1% (TBST) for 15 min. Next, the membrane was blocked with 5% non-fat lyophilized milk in TBST for 1 h at room temperature, following which primary hybridization was carried out with the monoclonal antibody anti-CEP250 C-Nap1 (Santa Cruz; Sc390540), dissolved in TBST, for 1 h at room temperature. After three washes in TBST, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody. Signals were detected using the ECL prime enhanced chemiluminescence system (GE Healthcare) and a ChemiDoc Touch Imager system (Bio-Rad).

For histological analysis, the gonads were fixed in 4% formaldehyde. The fixed gonads were dehydrated, embedded in paraffin, and cut into 5 μm thick sections. The sections were mounted on slides, dewaxed, and rehydrated, following which they were boiled for 20 min in citrate buffer.

For immunohistochemical staining, the endogenous peroxidase activity was blocked by incubating the sections for 15 min with 3% hydrogen peroxide. The sections were then blocked with 2% horse serum for 30 min and incubated with primary antibodies overnight at 4°C (Supplementary Table S2 for antibodies). After three washes with PBS, the slides were incubated with peroxidase-conjugated secondary antibodies (ImmPRESSTM reagent kit, Vector Laboratories) for 30 min at room temperature. Antibodies were detected using DAB (DAB substrate reagent kit, Vector Laboratories) or the VIP Vector (VIP substrate reagent kit, Vector Laboratories). All counts were carried out blind and were done using Histolab analysis software (Microvision Instruments, Evry, France).

For immunofluorescence staining, sections were blocked for 1 h in a gelatin-blocking solution, containing 0.2% BSA, 0.2% gelatin, and 0.05% Tween in 1× PBS, and incubated with an appropriate primary antibody, diluted in the blocking solution, for 1.5 h at 37°C. After three washes with PBS containing 0.05% Tween, the slides were incubated with secondary antibodies, diluted in the blocking solution, at 37°C for 1 h. The slides were then incubated with DAPI and mounted in the ProLong Gold antifade mounting medium (Thermo Fisher). Imaging was performed using a Leica DM5500 B epifluorescence microscope (Leica Microsystems) equipped with a CoolSNAP HQ2 camera (Photometrics) and Leica MMAF software (Metamorph) or a confocal microscope (Spinning disc). Images were processed using the ImageJ software.

Pericentrin immunostaining was performed with dissociated testicular cells. After removal of tunica albuginea, testes were incubated in 1 mg/ml PBS-collagenase solution for 20 min at 32°C with pipeting up and down regularly. Dissociated cells were washed twice in PBS. Cells were counted using Malassez chamber with Trypan staining and placed in Cytospin device to reach 25,000 cell per slide. Samples were then fixed with 4% PFA solution. Cells were permeabilized by a 5 min incubation in 0.2% Triton X-100 solution and washed in PBS. Subsequent steps were performed as described above.

The testes used for spermatocyte spread preparation were collected from postnatal mice at 12 days post-partum (dpp). Cells were isolated via enzymatic digestion. Briefly, the albuginea was removed; then, the seminiferous tubules were isolated and incubated at 32°C for 1 h in the TrypLE Express reagent (Thermo Fisher) by pipetting regularly. Fetal bovine serum was used to terminate the TrypLe reaction. The isolated cells were resuspended in 0.1 M sucrose. The sucrose suspension was distributed on slides in humid chambers and fixed with 1% PFA (in H₂O, pH 9.2) and 0.001% Triton X-100 for 1.5 h. After fixation, the slides were rinsed with H₂O and 0.4% Photo-Flo 200 (Kodak).

After washing the spermatocyte chromosome spreads, the slides were air-dried and incubated with a gelatin-blocking solution (containing 0.2% BSA, 0.2% gelatin, and 0.05% Tween in 1× PBS) for 1 h at room temperature and then with primary antibodies, dissolved in blocking solution, overnight at room temperature (Supplementary Table S2 for the list of antibodies). Next, the slides were incubated with secondary antibodies at 37°C for 1.5 h, stained with DAPI, and mounted on the Prolongold medium. Imaging was performed using a Leica DM5500 B epifluorescence microscope (Leica Microsystems) equipped with a CoolSNAP HQ2camera (Photometrics) and Leica MMAF software (Metamorph). Images were processed using the ImageJ software.

In order to assess Cep250 abundance in various tissues, we first analyzed the publicly available RNA-seq datasets for human adult tissues. The testis displayed the highest Cep250 expression compared to the somatic tissues (Figure 1A). To refine Cep250 expression profile in the gonads, Cep250 expression was quantified via RT–qPCR at various stages in mouse fetal and postnatal gonads. Cep250 expression levels increased in post-natal testes from 5 dpp and onwards, indicating the possible roles of CEP250 in spermatogenesis and meiosis (Figure 1B). Next, we investigated the localization of CEP250 protein in post-natal testis sections. Consistent with its known centrosomal role, CEP250 colocalized with γ-tubulin in the spermatogonia and spermatocytes (Figures 1C,D; Supplementary Figure S1). Upon rotating the γ-tubulin image 90° clockwise relative to the CEP250 image, the existence of a specific signal for CEP250 at the centrosome was confirmed. CEP250 was localized between the two γ-tubulin foci which supported its possible role in centrosome cohesion during spermatogenesis (Figure 1D). Owing to the poor specificity of the signal, we could not exclude the possibility of the presence of CEP250 in other cellular compartments.

To further investigate the biological function of CEP250, we generated Cep250 mutant mice in a C57BL/6 background. Cep250 invalidation was performed by targeting exon 5 using the TALEN genome editing technology (Supplementary Figure S2A). The Cep250 mutant founder allele showed a 6-bp deletion and a 1-bp insertion, which resulted in the disruption of the open reading frame, thus inducing a premature stop codon (Supplementary Figures S2B,C). The heterozygous animals were healthy and were used to generate homozygous mice. Homozygous mutant mice developed normally, were viable, and were obtained at the expected Mendelian ratio. RT-PCR analysis revealed a considerable reduction in Cep250 expression (testis and kidney), while western blotting revealed a complete absence of CEP250 (testis) in homozygous mice (Supplementary Figures S3A–C). We confirmed the absence of CEP250 protein in mutant testes via the immunofluorescence staining of seminiferous tubule sections (Figure 2A). Thus, these animals could be considered as null mutants (Cep250 ^-/- ). Cep250 ^-/- male mice were fully infertile (Supplementary Table S3). Mutant testes were smaller than wild-type testes starting from 12 dpp (Figure 2B; Supplementary Figure S3D). The size difference was maximal during adulthood, with mutant testes being approximately 80% smaller than wild-type testes (Figures 2B,C; Supplementary Figure S3E). Notably, no difference was observed between the wild-type and Cep250 heterozygous testes. Thus, we used heterozygous mice as controls in subsequent experiments. The histological analysis of adult Cep250 ^-/- testis sections revealed complete absence of spermatids and spermatozoa in the seminiferous tubules (Figure 2D). Spermatocytes were exceedingly rare in mutant testes, and the few observed were arrested early during the meiotic prophase I (Supplementary Figure S4A). To better define the ontogenesis of testicular defects, postnatal testes were analyzed at 0, 5, and 10 dpp. At 0 and 5 dpp, no defects were observed in mutant testes (Figure 2E). At 10 dpp, when the first germ cells reached the early stages of meiotic prophase I, the density of leptotene and zygotene cells was substantially reduced in mutant testes (Figure 2E). These findings suggest that Cep250 invalidation impairs spermatogenesis, both by reducing the number of germ cells reaching meiosis and preventing the completion of meiotic divisions.

To determine the spermatogenic stages that were altered in Cep250 ^-/- mice, we analyzed DDX4 (VASA) expression at 10 and 15 dpp, when germ cells were exclusively spermatogonia or early spermatocytes. DDX4 is used as a germ cell marker, as this RNA helicase is found in the germline of all animals, and in embryonic germ cells, up to the level of gametes, in mice. Ddx4 mRNA levels were significantly reduced by 42 and 79%, respectively, in the 10 and 15 dpp mutant testes compared to those in heterozygous testes (Figure 3A). DDX4 immunostaining revealed fewer positive cells in both 10 and 15 dpp mutant testes (Figures 3B,C). In addition, many apoptotic cells that stained for cleaved caspase 3 were detected in the tubules of Cep250 ^-/- testes (Figures 3D,E), explaining the reduction in germ cell population detected in the mutant testes. Germ cell apoptosis was also observed in the adult Cep250 ^-/- testis (Supplementary Figure S4B). As germ cell survival is partly dependent upon the somatic niche, somatic cells in the mutant testes were analyzed using the Sertoli cell marker GATA1 and the common Sertoli and Leydig cell marker GATA4. Both GATA1 and GATA4 were unaffected in the post-natal mutant testes (Supplementary Figure S5A). Similarly, mRNA levels of the somatic cell-specific transcripts Sox9 (Sertoli cells), Gata4, Hsd3β (Leydig cells), and αSMA (peritubular myoid cells) were unaltered in the mutant compared to that in the control testes (Supplementary Figure S5B).

Altogether, these results indicate that CEP250 is essential for post-natal germ cell survival and development without affecting somatic cell development, suggesting an intrinsic role of Cep250 in germ cells.

To further characterize the stages of spermatogenesis affected by Cep250 invalidation, we performed RT-qPCR and immunostaining of different spermatogonia cell markers. mRNA levels of the undifferentiated spermatogonial marker Sall4 were unaltered, while c-Kit, expressed in differentiated spermatogonia, was significantly downregulated in the 10 and 15 dpp testes (Figure 4A). The population of undifferentiated and differentiating type A spermatogonia, stained for ZBTB16, was equivalent in the control and mutant postnatal testes (Figure 4B). STRA8 is a marker found both at low levels in differentiating type A spermatogonia, and at high levels in pre-leptotene cells (Zhou et al., 2008; Endo et al., 2015). As expected, STRA8 was expressed in type A spermatogonia and strongly expressed in preleptotene spermatocytes in the mutants. Quantification of STRA8-low spermatogonia cells revealed no significant difference in Cep250^-/- testes (Figure 4C). Spermatogonia can be divided into three different populations according to Ki67 and ZBTB16 staining. The Ki67-negative and ZBTB16-positive cells correspond to quiescent or slow-proliferating spermatogonial stem cells. The Ki67- and ZBTB16-positive cells correspond to the proliferating, undifferentiated spermatogonia or the differentiating type A spermatogonia, in which ZBTB16 staining progressively declines. The Ki67-positive and ZBTB16-negative cells represent a more differentiated population of spermatogonia. Interestingly, we observed a 24-fold reduction in the population of Ki67-positive and ZBTB16-negative differentiating spermatogonia in the mutants (Figure 4D; Supplementary Figure S6A), with no alteration in the number of the earlier spermatogonium populations. Additionally, in the control testes, we observed clusters of ZBTB16-negative pH3-stained mitotic metaphase cells, corresponding to dividing late-differentiating spermatogonia (Supplementary Figure S7). However, this was hardly observed in the Cep250 ^-/- testes, in which the pH3-positive spermatogonia were always ZBTB16-positive. Lastly, immunohistochemical detection of c-KIT confirmed the loss of differentiated spermatogonia (Figure 4E). Altogether, these findings indicate that the first spermatogonial divisions usually take place in the mutant testes, and that the differentiated spermatogonia are mostly altered (Figure 4F).

Surprisingly, we found that the differentiating spermatogonia with faint ZBTB16 staining were frequently positive for γH2AX, a DNA double-strand break (DSB) marker, specifically in the mutant testes (Supplementary Figure S8A). Most ZBTB16-positive metaphases were also γH2AX-positive in the mutants (Supplementary Figures S8A,B). This supports the hypothesis that spermatogonia undergo abnormal mitotic divisions, which could result in genome instability and abnormal γH2AX in late spermatogonia. Overall, these results allowed us to identify the stages of spermatogonia divisions affected by Cep250 invalidation. We found that a reduction in the population of late differentiating spermatogonia and the defects in dividing spermatogonia, likely induced the death of most germ cells before completing spermatogonia differentiation.

Despite a remarkable reduction in the population of differentiating spermatogonia, we still observed germ cells reaching the meiotic prophase I. However, we did not detect any meiotic metaphasic cells or post-meiotic germ cells, which suggested a second role of Cep250 in the regulation of meiotic prophase I progression. Accordingly, we next analyzed the mitosis-to-meiosis transition in Cep250 ^-/- testes using STRA8 immunostaining (Figure 5A). At 10 dpp, we observed Cep250 ^-/- tubules containing high STRA8-positive preleptotene spermatocytes, showing that spermatogonia that achieved differentiation entered meiosis. At 15 dpp we observed fewer preleptotene spermatocytes in the mutant testes than in the control testes. Stra8 mRNA levels were significantly reduced in both 10 and 15 dpp mutant testes when compared to those in the controls (Figure 5B). As Stra8 is specifically expressed in germ cells, we also normalized its expression with that of Ddx4 in order to consider the variation in the global germ cell number and obtain an estimation of the proportion of germ cells that initiate meiosis. When Stra8 expression was reported to that of Ddx4, it remained steady in mutants, indicating that a similar proportion of germ cells initiate meiosis at 10 and 15 dpp (Supplementary Figure S9).

As some germ cells were able to initiate meiosis in Cep250 ^-/- testes, we monitored the progression of spermatocytes through prophase I. To this end, we stained the lateral element of the synaptonemal complex SYCP3, a marker of prophase I spermatocytes, which allowed us to recognize the different stages of prophase I (Figure 6). In the control 10 dpp testes, most tubules contained early meiotic prophase I spermatocytes at the leptotene and zygotene stages (Griswold, 2015). At 15 dpp, all tubules contained SYCP3-positive, and all the stages of prophase I could be visualized. In accordance with the reduction in germ cell population observed in the meiotic prophase I, we observed rare SYCP3-positive cells in Cep250 ^-/- postnatal testes, both at 10 and 15 dpp (Figure 6A; Supplementary Figure S10). Consistently, mRNA levels of the meiotic markers Sycp3 and Dmc1 were significantly reduced in mutant testes compared to those in the controls (Figure 6B; Supplementary Figure S9). Interestingly, Cep250 ^-/- testes fully lacked diplotene spermatocytes, indicating an arrest of prophase I at the pachytene stage (Figure 6C).

Based on these observations, we investigated meiotic prophase I events to identify defects that prevent prophase I progression (Figure 6D). Two major events occur during prophase I: Homologous chromosome synapsis and DSB formation and repair. Immunostaining of the synaptonemal complex and DSBs on the spermatocyte chromosome spreads allowed us to monitor these events. In wild-type testes, synaptonemal complex began forming during the leptotene stage, with lateral elements stained by SYCP3 beginning to assemble along the chromosome axes. At zygotene, synaptonemal central elements, stained by SYCP1, formed a zipper-like structure between the lateral elements of homologous chromosomes. Except for the sex chromosomes, other chromosomes were fully synapsed at the pachytene stage. In parallel, DSBs formed at the leptotene and zygotene stages were monitored using γH2AX staining. At the pachytene stage, the DSBs were completely repaired and γH2AX remains exclusively on the sex chromosomes. In Cep250 ^-/- spermatocytes, DSBs formed normally at the leptotene and zygotene stages, and the homologous chromosomes initiated pairing (Figure 6D). At the pachytene-like stage, synapsing defects were observed, with some autosomal chromosomes stained for SYCP3 but not SYCP1, indicating a failure to complete synapsis. In addition, we observed the persistence of γH2AX, potentially indicating defects in DSB repair.

Altogether, we observed an arrest of meiotic prophase I at the pachytene stage, likely due to synapsis defects and γH2AX persistence in pachytene-like Cep250 ^-/- spermatocytes.

Considering the centromeric localization of CEP250, we investigated the centrosome structure during prophase I. We immunolocalized γ-tubulin in the postnatal testes. At the beginning of prophase I, the two centrioles were linked together, appearing as two overlapping γ-tubulin spots in the control mice. In contrast, we detected two separated or fragmented γ-tubulin spots in the Cep250 ^-/- early prophase I spermatocytes (Figure 7A). Pericentrin (PCNT) is another component of the pericentriolar material that behaved as γ-tubulin in control spermatocytes, forming two closely associated spots (Figure 7B). In Cep250 ^-/- meiocytes, PCNT staining was found both as foci colocalizing with the distant γ-tubulin spots and in other filament-like structures. These observations indicate that CEP250 is mandatory for centrosome cohesion in male meiocytes, which is consistent with previous studies in other cell types (Mayor et al., 2000; Bahe et al., 2005; Floriot et al., 2015).

Cep250 mRNA was steadily expressed in foetal and adult ovaries (Figure 1B). We detected CEP250 protein at the centrosomes of mitotic and meiotic germ cells in fetal wild-type ovaries but not in Cep250 ^-/- ovaries (Figure 8A). In contrast to males, Cep250 ^-/- females were fully fertile (Supplementary Table S4). Adult mutant ovaries had a normal size, and histological analysis showed a density of follicles equivalent to that of wild-type adult ovaries (Figure 8B). Accordingly, we observed a normal density of post-natal oocytes between 5 and 10 dpp (Figure 8C) and of SYCP3-positive germ cells in the mutant fetal ovaries (Figure 8D).

To assess the difference observed between male and female meiocytes in response to Cep250 invalidation, we examined centrosomes in wild-type meiotic oocytes at the beginning of prophase I. Interestingly, we could distinguish two different cell populations of germ cells at early stages of prophase I in the ovaries. Some early prophase I oocytes possessed two paired γ-tubulin foci, with CEP250 located between them (Figures 9A,B) as in spermatocytes (Figure 7A). In other oocytes, γ-tubulin foci were separated at the leptotene stage; in this case, CEP250 was not observed (Figures 9A,B). Both the populations of oocytes, with paired or separated foci, were also observed in Cep250 ^-/- ovaries.

These results suggest a sexual dimorphism in the control of the meiotic centrosome structure in mice. A precocious separation of centrosomes in males leads to spermatogenesis defects and germ cell death, whereas in females, germ cells normally progress through oogenesis even if the centrosomes split up.