Mouse HORMAD1 Is a Meiosis I Checkpoint Protein That Modulates DNA Double-Strand Break Repair During Female Meiosis

ABSTRACT

Oocytes in embryonic ovaries enter meiosis I and arrest in the diplonema stage. Perturbations in meiosis I, such as abnormal double-strand break (DSB) formation and repair, adversely affect oocyte survival. We previously discovered that HORMAD1 is a critical component of the synaptonemal complex but not essential for oocyte survival. No significant differences were observed in the number of primordial, primary, secondary, and developing follicles between wild-type and Hormad1^−/− newborn, 8-day, and 80-day ovaries. Meiosis I progression in Hormad1^−/− embryonic ovaries was normal through the zygotene stage and in oocytes arrested in diplonema; however, we did not visualize oocytes with completely synapsed chromosomes. We investigated effects of HORMAD1 deficiency on the kinetics of DNA DSB formation and repair in the mouse ovary. We irradiated Embryonic Day 16.5 wild-type and Hormad1^−/− ovaries and monitored DSB repair using gammaH2AX, RAD51, and DMC1 immunofluorescence. Our results showed a significant drop in unrepaired DSBs in the irradiated Hormad1^−/− zygotene oocytes as compared to the wild-type oocytes. Moreover, Hormad1 deficiency rescued Dmc1^−/− oocytes. These results indicate that Hormad1 deficiency promotes DMC1-independent DSB repairs, which in turn helps asynaptic Hormad1^−/− oocytes resist perinatal loss.

HORMAD1, a synaptonemal complex protein, is a critical component of the meiosis I surveillance and monitors DNA double-strand break repair.

INTRODUCTION

Oocytes in mice enter meiosis circa Embryonic Day (E) 13.5 and arrest in diplonema of meiosis I. The oocytes in the embryonic mouse gonad are physically located in germ cell clusters (also known as cysts), where they are connected via intercellular bridges. Oocyte clusters break down shortly after birth to form primordial follicles, where individual oocytes become enveloped with a single layer of flat granulosa cells. Thus formed, primordial follicles are recruited regularly to grow and form a mature egg. More than half of oocytes are naturally lost during the primordial follicle formation [1]. Abnormal meiosis can accelerate oocyte loss and diminish the primordial follicle pool [2–7]. Synaptonemal complex proteins (Sycp1, Sycp2, and Sycp3) are important prosurvival factors, and their deficiency accelerates germ cell loss [5–11]. DMC1, a meiosis-specific recombinase, is important in oocyte survival, and its deficiency leads to the accumulation of the extensively resected double-strand breaks (DSBs) and to massive oocyte loss [3]. ATM is a kinase that controls DSB formation [4, 12], and its deficiency also leads to massive oocyte loss. Unrepaired DSBs are associated with genomic instability and trigger apoptosis with subsequent oocyte death [13]. SPO11 is a trans-esterase that initiates DSB formation [14]. Mice deficient in Spo11, and therefore unable to generate DSBs, have been used to determine the importance of DSBs in oocyte loss. Oocyte loss in Atm and Dmc1 mutants is partially rescued in Spo11-deficient mice, presumably because DSBs do not form [14, 15]. However, DSBs are not the only determinants of oocyte loss, because Spo11 mutants themselves show accelerated loss of oocytes [16]. These experiments suggest that DSB-dependent as well as DSB-independent mechanisms trigger oocyte loss when recombination defects occur.

Our group as well as others have recently discovered that meiosis-specific HORMA domain-containing 1 (HORMAD1) likely is the mammalian counterpart of yeast Hop1 and that Hormad1 deficiency disrupts mammalian synaptonemal complex formation, meiotic recombination, and chromosome segregation [9, 17–21]. Proteins with HORMA domain are critical components of the axial elements [22], and in nonmammalian organisms, several meiosis-specific HORMA proteins, such as Hop1 [23] and Red1 [24] in yeast, Him-3 [25, 26] in nematodes, and Asy1 [27] in plants, are critical for meiosis. In yeast, plants, and nematodes, HORMA domain proteins are critical components of the synaptonemal complex and essential for meiosis I [9, 19, 21]. Mouse and yeast HORMA domains in HORMAD1 and Hop1 share 28% amino acid identity, and HORMAD1 likely is the mammalian homologue of Hop1. Hop1 in yeast appears to bind near or at the sites of DSB formation and modulates the initial DSB cleavage [28]. Hop1 mutants in yeast have a reduced number of DSBs [20], and Hop1 may participate in recruiting DMC1, RAD51, and other proteins that are required for DNA repair during meiotic synapsis and recombination [19, 20]. Phosphorylation of Hop1 by Mec1/Tel1 yeast kinases is important for interhomologue recombination and prevents DMC1-independent repair of meiotic DSBs [21]. In mammals, HORMAD1 deficiency disrupts synaptonemal complex formation; however, folliculogenesis is apparently normal, with no gross evidence of accelerated oocyte loss [9, 19, 21]. These results indicate that HORMAD1 is an important checkpoint protein in female meiosis. Moreover, Hormad1 deficiency rescued accelerated loss of oocytes in Spo11 mutants, indicating that HORMAD1 is an important contributor to DSB-independent mechanisms of asynaptic surveillance [19, 21]. It is unknown whether HORMAD1 regulates DSB formation and how DSB formation and repair in Hormad1-deficient ovaries contributes to oocyte survival. To further understand how Hormad1 deficiency prevents an excessive loss of oocytes, we carefully assessed meiosis I in female meiocytes by studying the kinetics of DSB repair in Hormad1-deficient animals and the effects of Hormad1 deficiency on Dmc1 mutants. Our results are consistent with the interpretation that HORMAD1 regulates DSB repair by inhibiting DMC1-independent DSB repair mechanisms and that Hormad1 deficiency promotes DSB repair. HORMAD1 therefore acts as a pachytene-stage checkpoint protein in part by modulating DSB formation in female meiocytes.

MATERIALS AND METHODS

Animal Breeding

All mouse experiments were carried out on the 129S7/SvEvBrd × C57BL/6 hybrid background. All experimental and surgical procedures complied with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Hormad1^+/− mice were as previously described [9]. Atm^+/− and Dmc1^+/− mice were purchased from The Jackson Laboratory [2, 29].

Histology, Immunostaining, and Quantification

Ovaries were fixed in 10% buffered formalin (Sigma-Aldrich). Fixed tissues were embedded in paraffin, serially sectioned (section thickness, 5 μm), and stained with hematoxylin (Sigma-Aldrich) and periodic acid-Schiff. At least five pairs of testes and ovaries from each genotype were subjected to gross and microscopic analyses at each time point. Germ cell cysts and primordial, primary, and secondary follicles were defined as described previously [10]. Wild-type and mutant oocytes were stained concurrently with the same mixture of antibodies. Anti-NOBOX and anti-LHX8 antibody was used to identify oocytes [8, 10].

Quantification of the Immunofluorescence Signal

Wild-type and mutant oocytes were stained concurrently with the same mixture of antibodies. For multiphoton excitation laser-scanning microscopy (Olympus Fluoview FV1000MPE, Olympus), ovaries were fixed in 4% paraformaldehyde and incubated with the Scale A2 reagent for 2 wk [30]. Oocytes were labeled with anti-LHX8 antibody, and multiphoton excitation laser-scanning microscopy was used to detect immunofluorescence. Images were taken at 2-μm steps. In each experiment, when comparing wild-type and mutants, imaging of the ovaries was performed on the same day with the same microscope and camera settings. PerkinElmer Volocity 6.1 software was used to control for changes in illumination during the course of imaging and measurement of the immunofluorescence. We measured total fluorescence of γH2AX in identically sized rectangles that were placed over the cell boundaries as previously described [9]. Fifty individual oocytes were subjected to immunofluorescence analysis.

Ovary In Vitro Culture and Irradiation

Intact ovaries from E15.5 mice were cultured for 1 day as previously described [31]. Ovaries were isolated from E15.5 mice, and surrounding tissue, including the ovarian bursa, was removed. The ovaries were placed on Millicell-PC membrane inserts (pore size, 3.0 μm; diameter, 30 mm; Millipore Corp.) with media filling only the lower chamber. Each ovary was placed on the membrane with a single drop of medium, and eight ovaries were placed on each membrane. The medium was removed from the lower chamber until only a thin film covered the ovaries. The medium for organ culture was Waymouth MB752 (Life Technologies) supplemented with 0.23 mM pyruvic acid (Sigma-Aldrich), 10 μg/ml of streptomycin sulfate (Sigma-Aldrich), 75 μg/ml of penicillin G (Sigma-Aldrich), and 10% fetal bovine serum (Thermo Fisher). Organ cultures were maintained for 1 day in a 37°C incubator thoroughly infused with a gas mixture of 5% CO₂ and balanced air. The next day, select wild-type and Hormad1^−/− ovaries were subjected to irradiation via a Nordion Gamma Cell 1000 Irradiator. Ovaries received 2 Gy (1 Gy = 100 rads) at a dose of 120 rads/min. The irradiated ovaries were collected at 1 and 8 h after treatment and immediately processed to prepare chromosome spreads as previously described [9].

RESULTS

Hormad1 Deficiency Does Not Accelerate Ovarian Follicle Loss

Our previous gross assessment of the ovaries found no significant differences between the number of various follicles in wild-type and Hormad1-deficient animals [9]. We carefully reassessed these numbers by the well-accepted histomorphometric analysis [8] as well as by fluorescence imaging of the whole ovary [30]. Histomorphometric assessment of the newborn wild-type and Hormad1^−/− ovaries identified 1333.4 ± 238.0 (mean ± SEM throughout) and 1730.3 ± 100.3 primordial follicles, respectively, and the counts did not differ significantly (P > 0.1) (Supplemental Fig. S1, A–C, available online at www.biolreprod.org). At 8 days of age, 1128 ± 139 (n = 5) and 1383 ± 155.2 (n = 5) primordial follicles, 71.5 ± 2.4 and 73.8 ± 3.3 primary follicles, and 95.3 ± 4.7 and 115 ± 4.4 secondary follicles were counted in wild-type and Hormad1^−/− ovaries, respectively, and these counts did not differ significantly (P > 0.1) (Supplemental Fig. S1, D–F). Furthermore, in 80-day-old ovaries, we counted 619 ± 238.5 and 704 ± 147.0 primordial follicles, 45.7 ± 5.5 and 44.3 ± 7.2 primary follicles, 61.5 ± 9.2 and 48.8 ± 6.6 secondary follicles, 27.0 ± 10.2 and 26.6 ± 6.5 preantral follicles, and 10.3 ± 3.9 and 11.5 ± 4.1 antral follicles in wild-type (n = 5) and Hormad1^−/− (n = 5) ovaries, respectively, and these counts did not differ significantly (P > 0.1) (Supplemental Fig. S1, G–I).

Serial sectioning and histomorphometric oocyte counting is prone to errors due to many confounders [1, 32]. We therefore used multiphoton excitation laser-scanning microscopy on whole-mount ovaries that were treated with ScaleA2 reagent [30] as an alternative way to compare germ cell numbers in Hormad1^−/− and wild-type ovaries. We quantitated immunofluorescence of LHX8, an oocyte-specific marker that labels oocyte nuclei. We did not find significant difference in the LHX8 immunofluorescence between wild-type and Hormad1^−/− ovaries (Supplemental Fig. S1, J–L). The results of our analyses indicate that Hormad1 deficiency had no effect on the naturally occurring perinatal oocyte loss. Interestingly, a trend toward a greater number of primordial oocytes was observed in Hormad1-deficient ovaries, but this trend was not statistically significant.

Meiosis I in Hormad1-Deficient Oocytes

We also investigated the molecular anatomy of Hormad1^−/− female meiocytes to better understand events preceding massive perinatal oocyte loss. Meiosis in mouse ovaries commences circa E13.5 and arrests in the diplotene stage before birth. By E16.5, most oocytes show zygotene and pachytene stages of meiosis, whereas at E18.5, pachytene and diplotene stages predominate. The substages of meiosis I prophase are defined by chromosome configurations and structures: pairing, which occurs during the leptotene and zygotene stages; synapsis, which is completed at the onset of the pachytene stage; and desynapsis, which occurs during the diplotene stage [33]. Recombination is initiated by DSBs, and these breaks are repaired by either crossover or noncrossover events [16, 34, 35].

We compared the E16.5 and E18.5 wild-type and Hormad1^−/− ovaries for meiosis-stage progression using the centromere specific anti-CREST antibody and anti-SYCP2 antibody [18] (Fig. 1). In the wild type, zygonema is characterized by unsynapsed axes and 40 CREST foci, whereas pachynema is characterized by synapsed chromosomes with 20 CREST foci and diplonema is characterized by desynapsed axes with a D-loop structure. We did not find a significant difference in the number of zygotene-stage cells between the wild-type and Hormad1^−/− ovaries (Fig. 1, A and E). At E16.5, 35% fewer zygopachytene oocytes and 18% more early diplotene oocytes that had the D-loop were observed in the Hormad1^−/− ovaries as compared to the wild-type ovaries (P < 0.01). At E18.5, 19% fewer zygopachytene-like stage and 12% more early diplotene oocytes were observed in the Hormad1^−/− ovaries (Fig. 1, B and F) (P < 0.01). We did not identify oocytes with completely synapsed chromosomes in E16.5 or E18.5 Hormad1^−/− ovaries, versus 59% and 35% pachynema oocytes observed in E16.5 and E18.5 wild-type ovaries, respectively (Fig. 1, B, C, F, and G). Amazingly, the number of diplonema oocytes did not significantly differ between the wild-type and Hormad1^−/− ovaries (Fig. 1, D and F). Despite the lack of synapsed chromosomes, asynaptic oocytes are not eliminated in Hormad1-deficient ovaries, consistent with designating HORMAD1 as a pachytene checkpoint protein [19].

FIG. 1.

Meiosis I progression in Hormad1^−/− ovaries. Chromosomal spreads were performed on meiocytes from E16.5, E18.5, and newborn (NB) wild-type (+/+) and Hormad1^−/− (−/−) ovaries. Representative spreads are shown in A–D for wild-type ovaries and E–H for Hormad1^−/− ovaries. Immunofluorescence staining with anti-SYCP2 (green) and CREST (white) was used to label axial lateral elements and centromeres, respectively. The proportion of various meiotic stages observed at various ages in either wild-type (WT) or Hormad1^−/− (KO) ovaries is shown in a table below the figure panels. We failed to find pachynema oocytes in Hormad1^−/− embryonic ovaries. Incompletely synapsed zygotene had 40 CREST foci (in wild type ovaries; 40-30 foci in Hormad1^−/− ovaries). Hormad1^−/− zygopachynema show incompletely synapsed chromosomes with 20–30 CREST foci, whereas wild-type pachynema are synapsed (by SYCP2 staining) with 20 CREST foci. Wild-type and Hormad1^−/− diplotene contain D-loop structures. DNA was stained with 4′,6-diamidino-2-phenylindole (blue). Original magnification ×100.

Hormad1 Deficiency Accelerates DSB Repair

We previously showed that zygotene-stage Hormad1^−/− oocytes exhibit a drastically reduced number of γH2AX foci, a marker for DSB formation [9, 19]. DMC1, RAD51, and RPA, all homologous recombination DSB repair proteins, were also reduced in Hormad1^−/− ovaries [9]. We hypothesized that HORMAD1 modulates DSB formation, because unrepaired DSBs have been associated with accelerated oocyte loss [2–4]. We irradiated E16.5 wild-type and Hormad1^−/− ovaries with 2 Gy of gamma radiation (Fig. 2A) and used γH2AX as a marker for DSB foci formation at various intervals following irradiation [36, 37]. Both wild-type and Hormad1^−/− zygotene oocytes showed a greater than 2-fold increase in the γH2AX signal in ovaries exposed to 2 Gy for 1 h as compared to the nonirradiated ovaries (Fig. 2, Ba, Bb, Bd, Be, and C). Eight hours after radiation, wild-type zygotene oocytes retained almost 80% of the γH2AX signal, whereas only 46% of the γH2AX signal remained in the Hormad1^−/− zygotene oocytes. The drop in γH2AX signal in the Hormad1^−/− zygotene oocytes was statistically significant (P < 0.01), and the intensity of γH2AX signal in irradiated Hormad1^−/− zygotene oocytes did not significantly differ from nonirradiated Hormad1^−/− zygotene oocytes 8 h after irradiation or sham treatment (Fig. 2, Bc, Bf, and C). The rapid drop in γH2AX signal 8 h after irradiation in Hormad1^−/− oocytes is consistent with activation of DSB repair pathways.

FIG. 2.

Hormad1 deficiency accelerates DSB repair. A) Diagram of the experimental design. The E15.5 wild-type and Hormad1^−/− ovaries were harvested and cultured in Waymouth MB752 medium overnight while genotyping was performed. Cultured ovaries were exposed to 2 Gy of gamma rays. Chromosome spread assays were performed on irradiated wild-type and Hormad1^−/− ovaries either 1 or 8 h after the exposure to gamma rays. Nonirradiated ovaries (0 Gy) were collected and analyzed after the same culture period as irradiated ovaries. B) Immunofluorescence with anti-SYCP2 (green) and anti-γH2AX (red) antibodies in wild-type (a–c) and Hormad1^−/− zygotene oocytes (d–f) at various time points following irradiation. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Original magnification ×100. C) Relative γH2AX intensity, normalized against DAPI signal, for indicated genotype and postexposure time points. Error bars represent the SEM. Student t-test was used to calculate P-values (*P < 0.01).

Hormad1 Deficiency Accelerates DMC1-Independent DSB Repair Pathways

We investigated DMC1 and RAD51 foci formation in irradiated E16.5 wild-type and Hormad1^−/− ovaries to determine which DSB repair pathway was activated in Hormad1^−/− ovaries. DMC1 is a meiosis-specific recombinase important for DSB repair and interhomologue recombination. In the wild-type zygotene oocytes, the meiosis-specific recombinase DMC1 foci formation increased by 46% at 1 h after irradiation, and the increase persisted after 8 h (Fig. 3, Ac, Bc, Cc, and G). However, irradiation of Hormad1^−/− zygotene oocytes did not significantly affect DMC1 foci formation (Fig. 3, Dc, Ec, Fc, and G). We also assessed RAD51 foci formation. Unlike DMC1, that is germ cell specific, RAD51 is ubiquitously expressed, involved in mitosis and meiosis, and is also important in DSB repair and inter-homologue recombination. The irradiated wild-type zygotene oocytes showed a 42.8% increase in the number of RAD51 foci (Fig. 3, Ad, Bd, and H). The irradiated Hormad1^−/− zygotene oocytes also showed a 44% increase in the number of RAD51 foci (Fig. 3, Dd, Ed, and H). Eight hours after irradiation or sham treatment, the number of RAD51 foci was reduced to the level of nonirradiated wild-type and Hormad1^−/− zygotene oocytes (Fig. 3, Cd, Fd, and H). These data are consistent with the interpretation that Hormad1 deficiency interferes with DMC1 binding to DSB sites and that DSB repair may be accomplished in part by DMC1-independent DSB repair pathways.

FIG. 3.

Hormad1 deficiency leads to DSB repair by DMC1-independent manner. The E15.5 wild-type and Hormad1^−/− ovaries were cultured and either exposed to 2 Gy of gamma rays or not exposed to gamma rays (0 Gy). Chromosome spread assays were performed on irradiated ovaries after 1 and 8 h exposure time periods. Aa–Fe) Immunofluorescence with SYCP2 (green), RAD51 (red), and DMC1 (red) antibodies in wild-type (Aa–Ce) and Hormad1^−/− zygotene oocytes (Da–Fe). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Original magnification ×100. G) Graphic representation of DMC1 foci from 50 meiocytes at different exposure time points for specific genotypes. H) Graphic representation of RAD51 foci from 50 meiocytes at different exposure time points for specific genotypes. Error bars represent the SEM. Student t-test was used to calculate P-values (*P < 0.05).

Hormad1 Deficiency Rescues Oocyte Loss in Dmc1^−/− Ovaries

The above-described experiments are consistent with the interpretation that HORMAD1 inhibits DMC1-independent DSB repair pathways and that HORMAD1 deficiency allows DMC1-independent DSB repair to proceed. We therefore examined whether Hormad1 deficiency can rescue accelerated loss of oocytes observed in the Dmc1^−/− ovaries. DMC1-dependent interhomologue recombination is important in oocyte survival [2, 3, 13, 16, 38]. Dmc1 deficiency leads to the accumulation of the extensively resected DSBs and massive oocyte loss shortly after birth [2]. We hypothesized that Hormad1 deficiency would rescue Dmc1^−/− oocyte loss, in part, by derepressing DMC1-independent DSB repair pathways. We crossed Hormad1^−/− mice with Dmc1^−/− mice to generate double knockouts. We examined the ovarian histology in wild-type, Dmc1^−/−, Hormad1^−/−, and Dmc1^−/−Hormad1^−/− double-knockout mice. Three-day-old wild-type postnatal ovaries contain oocytes in germ cell clusters as well as primordial and some primary follicles. Histomorphometric assessment of the wild-type and Hormad1^−/− 3-day-old postnatal ovaries showed 1147.4 ± 108.6 primordial and 68.5 ± 25.4 primary follicles in the wild-type mice (n = 5), 1184.6 ± 236.1 primary and 78.0 ± 33.4 primordial in the Hormad1^−/− mice (n = 5), and 1317.8 ± 97.8 primary and 68.4 ± 19.2 primordial follicles in the Dmc1^−/−Hormad1^−/− double-knockout mice (n = 3) (Fig. 4, A, B, D, and E). The numbers of primordial and primary follicles in wild-type, Hormad1^−/−, and Dmc1^−/−Hormad1^−/− double-knockout ovaries at Postnatal Day 3 was not statistically significant. In contrast, very few primordial follicles were present in Dmc1^−/− single-knockout ovaries (Fig. 4, C and E). At Postnatal Day 20, 677.0 ± 96.4, 677.1 ± 122.9, and 878.4 ± 300.7 primordial follicles; 60.8 ± 14.7, 40.6 ± 8.3, and 68.2 ± 8.1 primary follicles; 66.8 ± 17.1, 37.6 ± 10.6, and 88.6 ± 7.3 secondary follicles; and 54.2 ± 9.2, 44.0 ± 20.1, and 77.0 ± 20.3 preantral follicles were counted in wild-type (n = 5), Hormad1^−/− (n = 5), and Dmc1^−/−Hormad1^−/− (n = 3) ovaries, respectively (Fig. 4, F and H–J). The numbers of primordial and primary follicles in wild-type, Hormad1^−/−, and Dmc1^−/−Hormad1^−/− ovaries at Postnatal Day 20 did not differ significantly (Fig. 4J). In contrast, almost no follicles remained in Dmc1^−/− ovaries, as previously reported [2, 3] (Fig. 4G). Moreover, the 6-mo-old Dmc1^−/−Hormad1^−/− ovaries were indistinguishable from the wild-type and Hormad1^−/− ovaries, whereas no oocytes were detectable in Dmc1^−/− ovaries (data not shown). Our data show that Hormad1 deficiency rescued oocyte loss observed in DMC1-deficient mice. We also checked DSB formation in wild-type, Dmc1^−/−, Hormad1^−/−, and Dmc1^−/−Hormad1^−/− zygotene oocytes using γH2AX antibody (Fig. 5, A–D). As expected, no significant difference was found in Dmc1^−/− as compared to wild-type oocytes, but Hormad1^−/− and Dmc1^−/− Hormad1^−/− oocytes showed a 50% reduced γH2AX signal compared to the wild-type zygonema (Fig. 5E). These results are consistent with our interpretation that HORMAD1 represses Dmc1-independent repair pathways and that HORMAD1 deficiency allows DMC1-independent pathways to operate and repair DSBs.

FIG. 4.

Hormad1 deficiency rescues oocyte survival in Dmc1^−/− ovaries. NOBOX (postnatal oocyte-specific marker [8]) was used to detect oocytes by immunohistochemistry of ovaries from 3- and 20-day-old mice. Oocytes in primordial follicle (PF) and primary follicle (PrF) stages are shown in 3-day-old wild-type (A), Hormad1^−/− (C), and Dmc1^−/−Hormad1^−/− (D) ovaries. No PF follicles are visible in 3-day-old Dmc1^−/−Hormad1^+/− ovaries (B). Oocytes in PF, PrF, secondary follicle (SF), and antral follicle (AF) stages are shown in 20-day-old wild-type (F), Hormad1^−/− (H), and Dmc1^−/−Hormad1^−/− (I) ovaries. No follicles were visible in 20-day old Dmc1^−/− ovaries (G). There were no statistically significant differences between the wild-type, Hormad1^−/−, and Dmc^−/−Hormad1^−/− double-knockout oocyte numbers at various follicle stages (E and J). Error bars represent the SEM. Bar = 50 μm.

FIG. 5.

Hormad1 deficiency activates DMC1-independent DSB repair. DSB formation in Dmc1^−/−, Hormad1^−/−, and Dmc1^−/−Hormad1^−/− zygonema as compared to the wild-type. Immunofluorescence with γH2AX (red; A′–D′) and SYCP2 (green, A″–D″) antibodies in wild-type (A), Dmc1^−/− (B), Hormad1^−/− (C), and Dmc1^−/−Hormad1^−/− (D) zygonema. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Original magnification ×100. E) Relative intensity of γH2AX was determined on 50 oocytes for various genotypes (A–D). The intensity of γH2AX was normalized against DAPI. Error bars represent the SEM. Student t-test was used to calculate P-values (*P < 0.01).

Hormad1 Deficiency Does Not Rescue Oocyte Loss in Atm^−/− Ovaries

We also examined the effects of Hormad1 deficiency in Atm knockout mice. ATM is a serine/threonine-specific protein kinase that has been associated with cell-cycle regulation, apoptosis, and response to DNA damage repair [15, 29, 39, 40]. Recent data support a role for ATM in DSB formation during meiosis I [12]. ATM appears to control SPO11 activity via a negative-feedback loop in which ATM activation by DSBs suppress further DSB formation [12]. We showed previously that ATM kinase activation lies downstream of HORMAD1 [9]. We therefore hypothesized that ATM deficiency would stimulate a dramatic increase in SPO11 activity and a concomitant increase in DSB formation, which Hormad1 deficiency will not rescue. Histomorphometric assessment at Postnatal Day 3 revealed only 10.6 ± 10.8 primordial follicles in the Atm^−/− and 24.8 ± 17.1 (n = 3) in Atm^−/−Hormad1^−/− mice as compared to more than 1,000 primordial follicles in the corresponding wild-type or Hormad1^−/− mice (Figs. 4, A and C, and 6, A–C). We examined synaptonemal complex formation and DSBs in E16.5 wild-type, Atm^−/−, Hormad1^−/−, and Atm^−/−Hormad1^−/− double-knockout oocytes using SYCP2 and γH2AX antibodies. Atm^−/− zygonema showed a 37.4% increase in γH2AX intensity as compared with wild-type zygonema, whereas Hormad1^−/− and Atm^−/−Hormad1^−/− zygonema showed a 57.8% and 22.1% decrease, respectively, compared to wild-type zygonema (Fig. 6, D–G and L). However, Atm^−/− Hormad1^−/− zygonema oocytes had a significantly greater number of DSBs than Hormad1^−/− single-knockout oocytes (Fig, 6, F and G). We also examined the zygopachynema in E16.5 wild-type, Atm^−/−, Hormad1^−/−, and Atm^−/−Hormad1^−/− mice. Atm^−/− and Atm^−/−Hormad1^−/− zygopachynema showed a 102.7% and 76.5% increase, respectively, in γH2AX intensity (P < 0.01), whereas Hormad1^−/− zygopachynema showed a 26.6% decrease, compared to wild-type zygonema (P < 0.05) (Fig. 6, H–L). Our data as well as those of others [9, 17, 19] are consistent with Atm deficiency causing an increase in the number of meiotic DSBs. Moreover, these results suggest that ATM is in part necessary for the DMC1-independent sister chromatid repair operating in Hormad1-deficient oocytes, because Hormad1 deficiency was unable to decrease the formation of γH2AX foci.

FIG. 6.

Hormad1 deficiency cannot rescue Atm^−/− mice. We generated Atm^−/−Hormad1^−/− double-knockout mice to assess effects of double deficiency on ovarian development and DSB formation. NOBOX was used to detect oocytes by immunohistochemistry on ovaries from 3-day-old mice. Only a few primordial follicles (PF) were visible in 3-day-old Atm^−/− and Atm^−/−Hormad1^−/− ovaries (P > 0.05) (A–C). These results indicate that Hormad1 deficiency cannot rescue Atm^−/− ovarian phenotype. Bar = 50 μm (A and B). D–K) Immunofluorescence with γH2AX (red) and SYCP2 (green) antibodies in wild-type (D and H), Atm^−/− (E and I), Hormad1^−/− (F and J), and Atm1^−/−Hormad1^−/− (G and K) ovaries. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). L) Relative intensity of γH2AX normalized against DAPI was calculated and plotted in a graph form for indicated genotypes and meiotic stages (D–K). Original magnification ×100. Error bars represent the SEM. Student t-test was used to calculate P-values (* P < 0.05, **P < 0.01).

DISCUSSION

Many meiosis-specific components of the synaptonemal complex are critical in early folliculogenesis. For example, mutations in Atm1, Dmc1, Sycp1, Sycp2, and Sycp3 accelerate oocyte loss [2–7, 29]. Perinatal germ cell loss in Hormad1-deficient mice does not differ significantly from that in wild-type mice [9, 19, 21], despite formation of asynaptic oocytes. These findings as well as the absence of pachynema in Hormad1^−/− meiocytes strongly support the argument that HORMAD1 is an important checkpoint protein essential for pachynema formation and elimination of asynaptic oocytes. HORMAD1 does this surveillance in part via DSB-independent pathways, because Hormad1 deficiency can rescue Spo11 mutants [19, 21]. Spo11 mutants do not form DSBs, hence other mechanisms that trigger oocyte death must be involved [14].

The role of Hormad1 in DSB repair and its role in oocyte survival have not been examined to date. Based on its HORMA domain and knockout phenotype in mice, HORMAD1 is the mammalian homologue to yeast Hop1. Hop1 in yeast has been implicated in facilitating interhomologue DSB repair and in repressing DMC1-independent intersister chromatid repair pathways [41, 42]. We therefore examined whether HORMAD1 actions in the mammalian system overlap what has been extensively studied with Hop1 in yeast. Our experimental findings are consistent with HORMAD1 playing a role similar to that of Hop1. First, our irradiation experiments show that gamma ray-induced DSBs persist over time in wild-type meiocytes but are significantly reduced in Hormad1^−/− meiocytes. Moreover, Hormad1 deficiency rescues Dmc1 knockouts, and these double knockouts show normal folliculogenesis and ovarian development. DSBs in the Dmc1^−/−Hormad1^−/− mice are also significantly reduced as compared to the Dmc1^−/− mice. MLH1, a protein that forms foci in later stages of recombination, is required for the formation of most of the crossovers (chiasmata) observed in mice and therefore is an excellent marker for meiotic crossovers [33, 43, 44]. The number of MLH1 foci in the Hormad1^−/− oocytes was significantly reduced as compared to the wild-type oocytes [9, 19]. The lack of MLH1 foci (crossover markers) in Hormad1^−/− oocytes also supports the interpretation that Hormad1 deficiency leads to DMC1-independent intersister DSB repair and noncrossover. The above-mentioned observations are all consistent with the interpretation that HORMAD1 blocks DMC1-independent DSB repair pathways and that in the absence of HORMAD1, DMC1 independent mechanisms activate intersister chromatid repair.

Our data also show that Hormad1 deficiency cannot rescue the Atm ovarian phenotype. Atm^−/− embryonic ovaries enter leptonema, zygonema, and pachynema, but oocytes begin to degenerate late in embryogenesis before dictyate arrest so that by 11 days, few oocytes remain [2–4]. The loss of oocytes in Atm^−/− ovaries is likely due to the meiosis arrest at the zygotene/pachytene stage of prophase I, as a result of abnormal chromosomal synapsis and subsequent chromosome fragmentation [4]. ATM does not appear to affect HORMAD1, because HORMAD1 phosphorylation and localization is normal in Atm^−/− gonads [9]. Moreover, HORMAD1 deficiency does not abrogate ATM autophosphorylation (phospho-S1981) in the ovary (data not shown). Why is it that Hormad1 deficiency cannot rescue Atm^−/− whereas Spo11 deficiency can? ATM inactivation leads to a great increase in DSBs, with 10-fold elevation in steady-state levels of SPO11-oligonucleotide complexes [12]. Whereas in Spo11 mutants DSBs do not form, in Atm mutants DSBs are produced at a higher rate [12]. Our data as well as previously reported Hormad1/Spo11 double-knockout rescue results [19, 21] are consistent with HORMAD1 modulating both DNA damage-dependent and DNA damage-independent responses. HORMAD1 clearly is a major checkpoint protein in oocyte meiosis and coordinates both checkpoint signaling and chromosome behavior (Fig. 7).

FIG. 7.

Summary of HORMAD1 functions as a major checkpoint in female meiosis. In early prophase, axial element formation causes DSBs by SPO11 and phosphorylated HORMAD1 localizes on unsynapsed chromosomes (leptonema and zygonema). HORMAD1 stimulates synaptonemal complex formation and DMC1-dependent interhomologue DSB repair [9, 17, 19, 21]. Sequential HORMAD1 and HORMAD2 binding to unsynapsed chromosome lead to meiotic silencing of unsynapsed chromosomes [46, 47] and synaptonemal complex formation. Synaptonemal complex formation leads to HORMAD1 depletion from the synapsed chromosomes (pachynema) [9, 17, 19, 21]. HORMAD1 and HORMAD2 were localized at the desynapsed axes in diplonema. After that, oocytes arrest in dictyate stage of meiosis I until ovulation in puberty. HORMAD1 blocks the DMC1-independent intersister DSB repair and consequently activates DMC1-dependent interhomologue DSB repair. Unrepaired DSBs associate with germ cell death, and HORMAD1 deficiency activates DMC1-independent intersister DSB repair, bypassing the DSB check point and ensuring oocyte survival [2–4, 13]. Original magnification ×100.

Phosphorylation of HORMAD1 and HORMAD2 correlates with their localization to unsynapsed chromosome axes but not to synapsed regions [20]. Phosphorylation is reduced in the absence of initiation of meiotic recombination, and these studies suggest that modifications of chromosome axis components signal recombination, checkpoint control, transcription, and synapsis regulation [20]. These findings are in line with recent experiments that show partial rescue of the Dmc1^−/− phenotype by the synaptonemal complex protein SYCP3 [45].

Another germ cell-specific HORMA domain protein, HORMAD2, was recently identified in mammals [17]. HORMAD2 localizes to the synaptonemal complex after HORMAD1 binding and is involved in the meiotic silencing mechanism at the unsynapsed chromatin [46, 47]. HORMAD1 and HORMAD2 do not appear to interact physically [20]. Interestingly, HORMAD2 has no effect on female fertility, oocyte numbers, and RAD51, DMC1, and MLH1 foci in the knockout [46]. HORMAD2 cannot rescue oocyte loss in Dmc1^−/− ovaries; however, Hormad2 deficiency can rescue oocyte loss in Spo11^−/− ovaries [46, 47]. HORMAD1, on the other hand, can rescue both Spo11^−/− [19] and Dmc1^−/− phenotypes (Fig. 4). Therefore, HORMAD1 has a dominant function in the female DSB repair pathway (Fig. 7) as well as in the female asynapsis surveillance. It has been proposed that HORMAD2 is important for the asynapsis surveillance in females based on its rescue of Spo11 as well as the barely significant increase in univalent-containing metaphase I oocytes [46]. This is indeed surprising, because HORMAD2 is functionally neutral in the female, at least based on the reported normal fertility and ovarian development phenotype. It would be interesting to assess whether Hormad2^−/− ovaries are more susceptible to environmental stress, such as chemotherapy and radiation.

Although HORMAD1 plays a critical role in meiotic surveillance, HORMAD1 deficiency does not decelerate the natural loss of oocytes that occurs as the germ cell clusters break down to form primordial follicles. The loss of oocytes at that stage is enormous—more than 50% are lost [1]—and likely determines individual endowment and reproductive life span. If HORMAD1 plays a major role in meiotic surveillance and asynapsis as well as unrepaired DNA damage are the cause of perinatal oocyte atresia, we would expect a higher number of primordial follicles in Hormad1^−/− animals. Although a trend to a greater number of primordial follicles was observed in Hormad1^−/− ovaries compared to wild-type ovaries (Supplemental Fig. S1), the trend was not statistically significant. These results suggest that meiotic errors per se may not be the major determinant of oocyte loss during formation of the primordial follicle and that other pathways are involved.