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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Meiotic chromosome segregation requires homologous pairing, synapsis and crossover recombination during meiotic prophase. The checkpoint kinase ATR has been proposed to be involved in the quality surveillance of these processes, although the underlying mechanisms remain largely unknown. In our present study, we generated mice lacking HORMAD2, a protein that localizes to unsynapsed meiotic chromosomes. We show that this Hormad2 deficiency hampers the proper recruitment of ATR activity to unsynapsed chromosomes. Male Hormad2-deficient mice are infertile due to spermatocyte loss as a result of characteristic impairment of sex body formation; an ATR- and γH2AX-enriched repressive chromatin domain is formed, but is partially dissociated from the elongated sex chromosome axes. In contrast to males, Hormad2-deficient females are fertile. However, our analysis of Hormad2/Spo11 double-mutant females shows that the oocyte number is negatively correlated with the frequency of pseudo–sex body formation in a Hormad2 gene dosage-dependent manner. This result suggests that the elimination of Spo11-deficient asynaptic oocytes is associated with the HORMAD2-dependent pseudo–sex body formation that is likely initiated by local concentration of ATR activity in the absence of double-strand breaks. Our results thus show a HORMAD2-dependent quality control mechanism that recognizes unsynapsis and recruits ATR activity during mammalian meiosis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Meiosis is a specialized type of cell division that produces haploid gametes. Accurate chromosomal segregation at meiosis I requires a physical connection between homologous chromosomes (chiasma) to form during meiotic prophase I. Chiasma formation is accomplished by the proper progression of meiosis-specific chromosomal events including homologous pairing, synapsis and crossover recombination, the quality of which is monitored by a surveillance mechanism known as the pachytene checkpoint in many species (Roeder & Bailis 2000; de Rooij & de Boer 2003). Pachytene checkpoints are assumed to detect and respond to DNA damage, unrepaired recombination intermediates and chromosome asynapsis. In mammals, mutations of various meiotic genes cause defects in homologous recombination and synapsis leading to meiotic cell death due to the actions of the pachytene checkpoint (Burgoyne et al. 2009). To date, however, the molecular nature of the mammalian pachytene checkpoints has remained largely unknown (Burgoyne et al. 2009; Handel & Schimenti 2010) in spite of the critical importance of this mechanism in the protection against gamete aneuploidy.

The molecular mechanisms underlying the pachytene checkpoint are currently best understood in the budding yeast, Saccharomyces cerevisiae. The yeast pachytene checkpoints are triggered by recombination failures caused by mutations in various meiotic genes involved in homologous recombination and synapsis (Hochwagen & Amon 2006). They are also dependent on DNA double-strand break (DSB) formation by the Spo11 protein (Malone et al. 2004). A similar DNA damage–dependent checkpoint is also assumed to operate during mammalian meiosis. It has been shown that the Dmc1−/− oocytes (defective in the repair of SPO11-induced DSBs) are eliminated as a response to the presence of unrepaired DSBs (Di Giacomo et al. 2005). As an early response to meiotic programmed DSB formation, the chromatin-wide phosphorylation of histone H2AX on Ser-139, forming γH2AX, is observed in the leptotene stage and is dependent on the ATM checkpoint kinase (Bellani et al. 2005). This DSB-dependent γH2AX disappears as chromosomal synapsis and recombinational repair progress during the zygotene stage in the normal process of meiosis, whereas it persists beyond the zygotene stage if synapsis and recombination fail, and might be recognized as a sign of persistent DNA damage (Barchi et al. 2005). In somatic cells, γH2AX is rapidly formed in response to DSBs and is assumed to have a role in the DNA-damage checkpoint (Fernandez-Capetillo et al. 2002). It is suggested that mitotic DNA-damage checkpoint proteins, including TOPBP1 (Perera et al. 2004) and Rad1 (Freire et al. 1998), have a similar function in mammalian meiosis (Hochwagen & Amon 2006; Handel & Schimenti 2010) as they do in that of yeast (Lydall et al. 1996; Usui et al. 2001).

In mammalian meiotic prophase, there is another wave of γH2AX formation that is restricted on unsynapsed chromosomes, and initiates from the late zygotene stage (Mahadevaiah et al. 2001; Turner et al. 2005), indicating its association with persistent asynapsis. This phenomenon is markedly evident during male meiosis in the form of γH2AX-rich sex body formation, because the mammalian sex chromosomes X and Y are heteromorphic and thus are naturally asynaptic except for the pseudo-autosomal region. The sex body chromatin is characterized by chromosome-wide transcriptional inactivation accompanied by the accumulation of γH2AX and heterochromatin proteins (Handel 2004). This silencing mechanism is referred to as meiotic sex chromosome inactivation (MSCI), the failure of which results in the spermatocyte cell death (Royo et al. 2010). MSCI has been proposed to originate from a more fundamental mechanism, meiotic silencing of unsynapsed chromatin (MSUC), an evolutionarily conserved mechanism that suppresses gene transcription and genetic recombination of unsynapsed chromatin (Huynh & Lee 2005; Schimenti 2005). One proposed role of MSCI is to shield the unsynapsed axes or persistent DSBs on these axes from a putative surveillance mechanism that would otherwise sense the male sex chromosomes as aberrant. In contrast, particularly in female meiosis, MSUC is proposed to be involved in the elimination of asynaptic oocytes due to the silencing of crucial genes (Turner et al. 2005; Burgoyne et al. 2009). Although the consequences of MSCI in males and MSUC in females are the opposite in terms of cell survival, the chromosome-wide silencing mechanism associated with γH2AX formation is consistently engaged in the quality control of mammalian meiotic prophase. Recent studies have also showed that MSCI/MSUC is dependent on γH2AX formation (Fernandez-Capetillo et al. 2003) that is catalyzed by the checkpoint kinase ATR (Turner et al. 2004) with the assistance of other DNA-damage response (DDR) factors including BRCA1, TOPBP1 and MDC1 (Ichijima et al. 2011). This may suggest that MSCI/MSUC is dependent on persistent DSBs until the pachytene stage on unsynapsed chromosomes.

Meanwhile, and interestingly, a similar silencing mechanism has also been shown to function at unsynapsed chromosomes even in the absence of DSBs. Spo11−/− spermatocytes and oocytes, which harbor extensive asynapsis due to the lack of DSB formation, form a sex body-like structure termed ‘the pseudo–sex body’ (Barchi et al. 2005; Bellani et al. 2005) whose formation is also dependent on DDR factors (Ichijima et al. 2011) and occurs at the same timing with the true sex body. This suggests that unsynapsis itself can possibly activate ATR and other DDR factors leading to γH2AX formation in the absence of DSBs at the late zygotene/early pachytene stage. To date, however, it remains largely unknown how DDR factors are recruited to unsynapsed chromosomes and then activated to execute gene silencing, particularly at the late zygotene/early pachytene stage. Also unknown is the relative contribution of DSB-dependent and DSB-independent mechanisms in the activation of DDR factors in MSCI/MSUC.

Recently, we and others have generated and analyzed gene-targeted mice for Hormad1 (Shin et al. 2010; Daniel et al. 2011; Kogo et al. 2012), a mammalian ortholog of the evolutionarily conserved, meiosis-specific HORMA domain proteins including Hop1 in yeasts (Hollingsworth et al. 1990; Lorenz et al. 2004), ASY1 and PAIR2 in plants (Armstrong et al. 2002; Nonomura et al. 2006) and HIM-3 in the worm (Zetka et al. 1999). The Hormad1 deficiency in mice causes a failure in homologous pairing and synapsis, but allows asynaptic oocytes to survive and complete meiosis, suggesting that HORMAD1 is involved in the quality surveillance of synapsis in female meiosis. The Hormad1 deficiency in mice also affected the MSUC mechanism (Daniel et al. 2011), consistent with the proposed role of this pathway in the elimination of asynaptic oocytes (Burgoyne et al. 2009). However, HORMAD1 localizes to unsynapsed chromosome axes from the leptotene stage, consistent with its involvement in DSB formation and synapsis, suggesting that the onset of MSUC at the late zygotene stage might be dependent on additional factors that collaborate with HORMAD1.

In our present study, we examined the function of mouse Hormad2 by gene targeting. Although another group recently reported the results of Hormad2 gene targeting (Wojtasz et al. 2012), our findings in female meiosis further show that oocyte number and the frequency of pseudo–sex body formation are correlated with the gene dosage of Hormad2 in the Spo11-deficient background. These results strongly suggest that the HORMAD2-dependent MSUC is responsible for the elimination of asynaptic oocytes. In addition, a unique pattern of interaction failure between the sex chromosomes and the repressive chromatin domain in the Hormad2-deficient spermatocytes suggests that a HORMAD2-dependent mechanism is required for sufficient γH2AX formation to accomplish MSCI, in addition to DSB-dependent γH2AX formation. The further characterization of HORMAD2 function should accelerate our understanding of the molecular basis of MSUC/MSCI and its involvement in the pachytene checkpoint in mammalian meiosis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

HORMAD2 is required for male fertility

Our previous study and reports from other laboratories have shown that mouse HORMAD1 is indispensable for multiple functions in mammalian meiosis including DSB formation, synaptonemal complex formation and a meiotic prophase checkpoint mechanism (Shin et al. 2010; Daniel et al. 2011; Kogo et al. 2012). Consistent with its functions, mouse HORMAD1 predominantly localizes to the unsynapsed chromosome axes in leptotene and zygotene meiocytes (Wojtasz et al. 2009; Fukuda et al. 2010). In mammals, there is another meiosis-specific HORMA domain protein, HORMAD2. In contrast to the abundance of HORMAD1 at the early stages of meiosis, HORMAD2 is readily detectable only after the late zygotene or early pachytene stage in spermatocytes by immunohistochemical analysis of testis sections (Fig. 1A) using our antibodies (Fig. S1 in Supporting Information). Positive HORMAD2 staining was found to be prominent in a single fiber-like structure, presumably a pair of sex chromosomes at the pachytene stage (Fig. 1A, inset). This distribution of HORMAD2 was confirmed by immunofluorescent staining of meiotic chromosome spreads (Fig. 1B). Whereas others detected HORMAD2 on unsynapsed axes of leptotene and zygotene chromosomes (Wojtasz et al. 2009), only a small fraction of zygotene spermatocytes that appear to be at relatively late stages was positive for HORMAD2 using our antibodies (results not shown). This discrepancy is likely due to insufficient sensitivity of our antibodies, and thus, the HORMAD2 protein level at leptotene and zygotene stages is assumed to be fairly lower than that at pachytene stage. This suggests that HORMAD2 might have a negligible role, if any, at the early stages of meiosis unlike HORMAD1.

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Figure 1. Phenotypes of Hormad2−/− male mice. (A) Immunohistochemical analysis of HORMAD2 in adult mouse testis. HORMAD2 is barely detectable in leptotene (L) or early zygotene nuclei (results not shown) and is observed as a single fiber-like structure, presumably indicative of a pair of sex chromosomes, in late zygotene (lZ), pachytene (P) and early diplotene (eD) spermatocytes. The intensity of the HORMAD2 signal peaks at the pachytene stage and becomes faint at the late diplotene stage (lD). Roman numerals indicate the stages of the seminiferous tubules. Inset shows magnified view of pachytene spermatocytes. (B) Immunofluorescent analysis of meiotic chromosome spreads from adult mouse spermatocytes. The axial/lateral elements of the synaptonemal complex were labeled with SYCP3. HORMAD2 is predominantly localized on sex chromosome axes (XY) from early pachytene to diplotene spermatocytes. (C) Immunofluorescent staining of HORMAD2 for wild-type and Hormad2−/− pachytene spermatocytes. Monochrome images of HORMAD2 staining are shown on the right panel. (D) Litter sizes for wild-type, Hormad2+/− and Hormad2−/− males (black, mean ± SD) and females (gray, mean ± SD). Only the male Hormad2−/− mice are infertile. (E) Testicular weights of wild-type, Hormad2+/− and Hormad2−/− males were measured as the gonadosomatic index (GSI, mean ± SD). *P < 0.0001 compared to the other groups. (F) Ovarian weights of wild-type, Hormad2+/− and Hormad2−/− females were measured as the gonadosomatic index (GSI, mean ± SD). (G) Testes of 10-week-old wild-type, Hormad2+/− and Hormad2−/− male mice. (H) Hematoxylin and eosin staining of testis (top and middle panels) and epididymis (bottom panel) sections from 10-week-old wild-type and Hormad2−/− littermates. The middle panel shows a single cross-section of the seminiferous tubule of each genotype at a high magnification. (I) TUNEL staining of testis sections from 10-week-old wild-type and Hormad2−/− littermates. Asterisks indicate stage IV seminiferous tubules with extensive apoptosis. Magnified images of DAPI-stained stage IV seminiferous tubules, as identified by the presence of intermediate spermatogonia (In), mitotic intermediate spermatogonia (m) and spermatogonia B (SgB), were shown below. Spermatocytes (sc), spermatids (st) and sperm (sp) are present in the wild-type stage IV tubules, whereas apoptotic spermatocytes (asc) are found in the Hormad2−/− stage IV tubules. (J) Analysis of developmental stages of spermatocytes using chromosome spreads from adult wild-type and Hormad2−/− testis. The percentages of spermatocytes belonging to four different developmental stages including leptotene (Lepto), zygotene (Zygo), pachytene (Pachy) and diplotene (Diplo) stages, as judged by the SYCP3-stained axis morphology using photographs of random fields, are shown. The different fractions of spermatocyte stages between the two genotypes are consistent with the absence of mid-late pachytene and diplotene spermatocytes in the Hormad2−/− testis.

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To further examine the function of mammalian HORMAD2 in meiosis, we generated Hormad2 knockout mice via gene targeting (Fig. S2 in Supporting Information). The absence of HORMAD2 protein was confirmed by Western blotting of Hormad2−/− testis (Fig. S2D in Supporting Information) and by immunofluorescent staining of chromosome spreads from Hormad2−/− spermatocytes (Fig. 1C). The Hormad2-null mutant mice were infertile only in males, whereas the null mutant female mice and the heterozygous mice of both sexes were fully fertile (Fig. 1D). The weight of the Hormad2-deficient testis was <1/3 of the wild-type or heterozygous testis (Fig. 1E,G), whereas the weight and histology of the ovary were unaffected by the Hormad2 genotypes (Fig. 1F and results not shown). The histology of the testis and epididymis further showed the complete absence of sperm in the Hormad2-deficient males (Fig. 1H). TUNEL staining also showed that spermatocyte apoptosis was confined to the stage IV seminiferous tubules (Fig. 1I, asterisks), similar to other meiotic prophase-defect phenotypes (Handel & Schimenti 2010). This notion was supported by the absence of diplotene-stage spermatocytes in chromosome spreads of adult Hormad2−/− testes (Fig. 1J).

Synapsis and sex body formation in Hormad2−/− spermatocytes

To examine the cause of spermatocyte apoptosis as a result of the Hormad2 deficiency, we first observed meiotic chromosome synapsis in Hormad2−/− spermatocytes. In most wild-type pachytene spermatocytes, all of the autosomal homologous chromosomes were found to be fully synapsed (Fig. 2A). Similar findings were obtained for most Hormad2−/− pachytene spermatocytes, but the spermatocytes that contained one or a few chromosome pairs with incomplete synapsis were slightly increased in number compared with wild type (Fig. 2B,C). A similar increase in incomplete synapsis was also observed in the pachytene oocytes of fetal Hormad2−/− females (Fig. S3 in Supporting Information). A recent report failed to detect this synapsis defect in the Hormad2-deficient meiocytes (Wojtasz et al. 2012), probably because they analyzed synaptonemal complex formation grossly using all stages of meiocytes, whereas we specifically examined the partial synapsis failure in the pachytene meiocytes. These results suggest that HORMAD2 is dispensable for homologous pairing and synapsis but has a supporting role in completing synapsis. However, this small increase in the number of cells with incomplete synapsis is unlikely to be responsible for the massive spermatocyte apoptosis.

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Figure 2. Mild synapsis defects and aberrant sex body formation in the Hormad2−/− spermatocytes. (A and B) Representative immunofluorescent images of pachytene chromosomes from 10-week-old wild-type (A) and Hormad2−/− (B) littermates. Pachytene spermatocytes having one large γH2AX-rich domain, the sex body (XY), are shown. Representative images of ‘complete’ and ‘incomplete’ synapsis in the Hormad2−/− spermatocytes are shown in the left and right panels of (B), respectively. The axial/lateral elements of the synaptonemal complex are labeled with SYCP3. SYCP1 is a component of the central element of the synaptonemal complex and thus is a marker of synapsis. A chromosome pair showing incomplete synapsis is indicated with an arrow. (C) Quantification of incomplete synapsis in the wild-type (n = 37) and Hormad2−/− (n = 25) pachytene spermatocytes, as confirmed by the presence of one large γH2AX-rich domain. The percentage of cells with complete synapsis or with incomplete synapsis in one or more pairs of chromosomes is shown. (D and E) Various morphologies of the γH2AX-enriched domains. Representative immunofluorescent images of pachytene chromosomes from adult wild-type (D) and Hormad2−/− (E) spermatocytes with SYCP3 (chromosome axes) and γH2AX (the sex body) staining are shown. The sex body morphology was classified into four categories: normal [round-shaped γH2AX domain entirely covers the condensed sex chromosomes (XY)], partial [γH2AX domain is condensed, but is absent from a part of the stretched XY (arrow)], diffuse (diffuse γH2AX staining entirely covers the stretched XY) and axial (γH2AX staining is weakly detected on the XY axes). (F) Quantitative analysis of the sex body morphology in adult wild-type (n = 39) and Hormad2−/− (n = 40) pachytene spermatocytes. The percentages of cells with a sex body morphology determined as normal, partial, diffuse or axial are shown. (G) Immunohistochemical analysis of testes sections from the adult Hormad2−/− and wild-type male mice with HORMAD1 (unsynapsed chromosome axes) and ATR (the sex body) staining. Representative images of spermatocytes at the zygotene stage (Zygotene, many HORMAD1-positive chromosomal fibers), very early pachytene stage (Pachytene-1, only one HORMAD1-positive chromosomal fiber, that is a pair of sex chromosomes, and little nucleoplasmic HORMAD1 staining) or early pachytene stage (Pachytene-2, only one HORMAD1-positive chromosomal fiber and intense nucleoplasmic HORMAD1 staining) are shown. HORMAD1 (sex chromosomes) and the ATR-rich domain (sex body) are colocalized in some Hormad2−/− spermatocytes as in the wild-type spermatocytes (arrowheads). Other Hormad2−/− spermatocytes show a dissociation of ATR-rich domain from the sex chromosomes (white arrows), or weak/no ATR staining on the axes of the sex chromosomes (orange arrows). Magnified images of the HORMAD1-stained sex chromosomes with partially associated ATR-rich domain are shown in the middle panel.

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During our analysis of synapsis, we observed a morphological abnormality of the sex chromosome axes in the Hormad2−/− spermatocytes (Fig. 2B). We thus examined sex body formation in the Hormad2−/− spermatocytes in further detail, as the failure of MSCI is known to cause spermatocyte apoptosis (Royo et al. 2010). For the analysis of sex body formation in Hormad2−/− pachytene spermatocytes, γH2AX was immunostained on chromosome spreads. In wild-type pachytene spermatocytes, the γH2AX-enriched chromatin domain appeared rounded in shape and formed on the entire sex chromosome (Fig. 2D). Although a morphologically similar γH2AX-enriched domain was found in the Hormad2−/− pachytene spermatocytes, it often contained only a part of the sex chromosome (Fig. 2E, partial). In these spermatocytes, the sex chromosome axes were also often stretched long and thus likely failed to undergo the characteristic conformational changes that result in a compact folded form during normal sex body formation (Ichijima et al. 2011). As a result, a part of the sex chromosome was located outside of the repressive chromatin domain (Fig. 2E, arrow), presuming the failure of MSCI. In many other Hormad2−/− pachytene spermatocytes, γH2AX-positive chromatin was not rounded but appeared to be distributed on the entire sex chromosome in a diffuse manner (Fig. 2E, diffuse). This γH2AX staining profile is similar to that observed in the wild-type spermatocytes during the zygotene/pachytene transition (results not shown) (Page et al. 2012). In addition to this, a weak γH2AX signal was detectable only on the sex chromosome axis in a small fraction of Hormad2−/− pachytene spermatocytes (Fig. 2E, axial). A typical sex body was rarely observed in Hormad2−/− pachytene spermatocytes (Fig. 2F).

We also examined sex body formation by immunohistochemical analysis of Hormad2−/− testis sections using ATR kinase and HORMAD1 as a marker of the sex body and the unsynapsed chromosome axes, respectively. We found that round-shaped, ATR-positive sex bodies were formed in many Hormad2−/− early pachytene spermatocytes (Fig. 2G). The sex chromosomes marked by HORMAD1 and the ATR-rich domains were observed to have fully colocalized in only a small number of Hormad2−/− spermatocytes (Fig. 2G, arrowheads). Interestingly, many other Hormad2−/− spermatocytes showed the characteristic localization of the ATR-rich domain at one end of the sex chromosome (Fig. 2G, white arrows and magnified views in the middle panel). A subpopulation of the Hormad2−/− spermatocytes also showed weak or no ATR staining on the sex chromosome axes (Fig. 2G, orange arrows) and likely corresponds to the cells with ‘axial’ γH2AX staining in the chromosome spreads. Taken together, our findings show that a Hormad2 deficiency does not completely impair γH2AX formation on the sex chromosomes but causes a failure in the compaction of the sex chromosome axes resulting in the dissociation of a part of them from the ATR- and γH2AX-enriched repressive chromatin domain. The other group confirmed by RNA FISH analysis of three X-linked genes that this sex body abnormality corresponds with the failure of MSCI, particularly in the distal region of X chromosome from the pseudo-autosomal region (Wojtasz et al. 2012).

Distribution of MSCI factors in Hormad2−/− spermatocytes

We next investigated the distribution of MSCI factors including BRCA1, ATR, MDC1 and γH2AX in the Hormad2-deficient pachytene spermatocytes (Fig. 3). In wild-type spermatocytes, BRCA1, which has been reported as a MSUC initiator protein required for ATR activation (Turner et al. 2005), localizes on the axes of sex chromosomes (Fig. 3A). ATR, MDC1 and γH2AX distribute on the entire chromatin of the sex chromosome forming the sex body (Fig. 3B,C). In Hormad2−/− pachytene spermatocytes with ‘partial’ sex bodies, ATR, γH2AX and MDC1 colocalize and were found to be dissociated from part of the sex chromosome (Fig. 3D–F, arrows). In contrast, BRCA1 localized on the entire sex chromosome axes, as was observed in wild type (Fig. 3D), suggesting that this process is unaffected by the Hormad2 deficiency. These results together suggest that HORMAD2 functions after the recruitment of BRCA1 to the unsynapsed axes during normal MSCI. We conclude from our results that HORMAD2 is essential for the conformation changes in the sex chromosome axis coupled with the aggregation formation of ATR and other DDR factors in normal spermatocytes, whereas HORMAD2 is dispensable for the recruitment of BRCA1 to unsynapsed axes and subsequent spreading of DDR factors to chromatin loops, which are likely to be dependent on the presence of DSBs. Consistent with this notion, the ATR activation by unrepaired DSBs is shown to be HORMAD2 independent (Wojtasz et al. 2012). Our current understanding of the processes involved in the sex body formation is schematically shown in Fig. 3G, taking previous studies of Hormad1−/− and Mdc1−/− mice into account (Daniel et al. 2011; Ichijima et al. 2011; Kogo et al. 2012).

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Figure 3. Distribution of the components involved in meiotic sex chromosome inactivation (MSCI) in wild-type and Hormad2−/− spermatocytes. (A–C) Representative immunofluorescent images of pachytene chromosomes with a ‘normal’ sex body obtained from the wild-type testis. (D–F) Representative immunofluorescent images of pachytene chromosomes with a ‘partial’ sex body obtained from the Hormad2−/− testes. Distributions of BRCA1 (A, D), ATR (B, E), MDC1 (C, F) and γH2AX (A, B, D and E) are shown. The axial/lateral elements of the synaptonemal complex are labeled with SYCP3. HORMAD1 localizes to unsynapsed chromosome axes and thus is used as a marker of the sex chromosomes in the pachytene spermatocytes (C, F). Merged images (top), separate red (the second panel from the top) and green (the third panel from the top) fluorescent images, and monochrome images of SYCP3 staining (the bottom panel) are shown. Arrows indicate parts of the sex chromosome axes (XY) that are devoid of DDR factors. (G) Schema of our current understanding of the processes involved in sex body formation. Both double-strand breaks and HORMAD1 are likely to be involved in the axial localization of BRCA1 (Mahadevaiah et al. 2008; Daniel et al. 2011). MDC1 plays a role in the spreading of DDR factors to the chromatin (Ichijima et al. 2011). The results from the present study show that HORMAD2 is dispensable for the MDC1-dependent spreading of DDR factors but is required for the compaction of the sex chromosome axis that is essential for the silencing of entire chromosomes. Notably, the aggregation of the ATR- and γH2AX-enriched domain can occur in the absence of HORMAD2 (dashed arrow), resulting in the failure of MSCI with the ‘partial’ sex body.

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Combined Hormad2 deficiency abrogates oocyte death in Spo11−/− ovary

Although the Hormad2−/− female is fertile, Hormad2 mRNA is normally abundantly expressed in the fetal ovary (Fig. S4A in Supporting Information) and the HORMAD2 protein shows a specific localization on the unsynapsed region of the oocyte chromosomes in wild-type mice (Wojtasz et al. 2009), suggesting a possible function in female meiosis. As shown above, HORMAD2 is essential for MSCI and thus is also possibly involved in the mechanism of MSUC that has been proposed to be important in the meiotic prophase checkpoint during female meiosis (Burgoyne et al. 2009). We also observed an increased abundance of HORMAD2 in the Spo11−/− ovary (Fig. S4B in Supporting Information), consistent with its abundant localization on unsynapsed chromosome axes in the Spo11−/− oocytes (Fig. 4A). Furthermore, we found that the chromosomal localization of HORMAD2 was largely decreased in the Hormad1−/− oocytes and spermatocytes (Fig. 4A,B), suggesting that the recruitment of HORMAD2 to unsynapsed axes is dependent on HORMAD1. This observation was also supported by the results of Western blot analysis showing a prominent decrease in chromatin-bound HORMAD2 in the Hormad1−/− testis (Fig. S4C in Supporting Information). The possible direct interaction of HORMAD1 with HORMAD2 was further confirmed by immunoprecipitation and in vitro binding assays (Wojtasz et al. 2012). These results raise the possibility that HORMAD2 might be responsible for the HORMAD1-dependent elimination of asynaptic oocytes that has been previously proposed by us and other groups (Daniel et al. 2011; Kogo et al. 2012).

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Figure 4. Oocyte death and pseudo–sex body formation in the Spo11−/− ovary are abolished with a combined Hormad2 deficiency. (A, B) Immunofluorescent analysis of meiotic chromosome spreads obtained from a 16-dpc ovary (A) and adult testis (B) of wild-type, Spo11−/− and Hormad1−/− mice. Representative images of HORMAD2 staining in zygotene-like oocytes and spermatocytes of each genotype are shown. The chromosome axes were labeled with SYCP3. (C) Ovarian weights of 3-week-old Hormad2+/+ Spo11+/+, Hormad2+/+ Spo11−/−, Hormad2+/− Spo11−/− and Hormad2−/− Spo11−/− females were measured as the gonadosomatic index (GSI, mean ± SD). *P < 0.05 compared with the other groups. (D) c-KIT immunostaining of ovary sections from 3-week-old mice of each genotype. The anti-c-KIT antibody stains the cytoplasm of immature oocytes. (E) Total oocyte number per ovary in each genotype, as calculated from the numbers of c-KIT-positive oocytes and follicular oocytes in every tenth serial section of the entire ovary (mean ± SD). *P < 0.005 compared with the other groups. (F) Pseudo–sex body formation in the 18.5-dpc oocytes of each genotype. Representative images of chromosome spreads from the 18.5-dpc oocytes with γH2AX and SYCP3 staining are shown. (G) Quantification of pseudo–sex body formation in the 18.5-dpc oocytes. The frequency of pseudo–sex body-positive oocytes in Hormad2+/+ Spo11+/+ (n = 153), Hormad2+/+ Spo11−/− (n = 58), Hormad2+/− Spo11−/− (n = 141) or Hormad2−/− Spo11−/− (n = 100) ovaries is shown.

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To examine this possibility, we generated and characterized double-mutant females of Spo11 and Hormad2. In 3-week-old mice, the Spo11-deficient ovary was smaller than the wild-type ovary (Fig. 4C) and c-KIT-positive oocytes were found to be almost completely absent (Fig. 4D,E) as reported previously (Baudat et al. 2000; Di Giacomo et al. 2005). Interestingly, the decrease in the ovarian weight in the Spo11−/− background was recovered to wild-type levels by a combined heterozygous and homozygous deficiency of Hormad2 (Fig. 4C). In addition, the number of oocytes recovered partially in the Hormad2+/− Spo11−/− ovary and recovered fully to wild-type levels in the Hormad2−/− Spo11−/− ovary (Fig. 4D,E). These results clearly show that HORMAD2 is required for the elimination of asynaptic oocytes caused by the Spo11 deficiency. The testicular size and testicular histology of the Spo11−/− Hormad2−/− males were similar to those of each single mutant as expected from the common phenotype of spermatocyte death at the pachytene stage (results not shown).

Combined Hormad2 deficiency impairs pseudo–sex body formation in Spo11−/− oocytes

As a mechanism for the elimination of asynaptic oocytes, it is suggested that the silencing of crucial genes by MSUC mediated by the pseudo–sex body formation might be responsible (Burgoyne et al. 2009). To examine whether the Hormad2 deficiency also affects MSUC in the Spo11−/− oocytes, the frequency of pseudo–sex body formation was examined by γH2AX staining of the oocyte chromosome spreads obtained from 18.5-dpc fetal ovaries (Fig. 4F,G). The pseudo–sex body was sometimes observed on unsynapsed chromosome regions in wild-type oocytes (16.3%, n = 153). Most of the Spo11−/− oocytes contained the pseudo–sex body (74.1%, n = 58), consistent with previous observations (Daniel et al. 2011). In contrast, the pseudo–sex body did not form in the Hormad2−/− Spo11−/− oocytes (0%, n = 100) and was detected in approximately half of the Hormad2+/− Spo11−/− oocytes (45.4%, n = 141). Taking these results together, the oocyte number is negatively correlated with the frequency of the pseudo–sex body formation along with the Hormad2 genotype in the Spo11−/− background. We conclude from this that HORMAD2 is essential for MSUC mediated by the pseudo–sex body formation in the absence of DSBs. The possible role of HORMAD2 in pseudo–sex body formation was also evidenced from our examination of male meiosis. By immunofluorescence analysis of chromosome spreads of 15.5-dpp testes, the spermatocytes positive for the γH2AX-enriched domain (pseudo–sex body) are largely reduced, but not eliminated completely, by the Hormad2 deficiency in the Spo11−/− background (Fig. S5 in Supporting Information). This residual activity for the pseudo–sex body formation is likely to be male specific and was also observed in the Hormad1−/− Spo11−/− males (Kogo et al. 2012). Notably, the axial localization of HORMAD1 is not likely to be disrupted by the Hormad2 deficiency in the Spo11−/− spermatocytes (Fig. S5A in Supporting Information). This suggests that HORMAD1 alone is not sufficient, and that its recruitment of HORMAD2 to the unsynapsed axes is critical, for pseudo–sex body formation.

Distribution of BRCA1 in Hormad2−/− Spo11−/− oocytes

We next investigated whether the HORMAD2 deficiency affects the distribution of BRCA1 in a Spo11−/− background. In the wild-type pachytene oocytes, the BRCA1 signal on synapsed chromosome axes was below detectable levels (Fig. 5A), whereas that on unsynapsed chromosome axes was prominent and colocalized with a γH2AX-positive chromatin domain (Fig. 5B, arrow). In the Spo11−/− pachytene-like oocytes, BRCA1 was found to be weakly distributed on most unsynapsed chromosomes axes, but was accumulated in a limited region (Fig. 5C, arrow). Pseudo–sex body formation was found to be associated with the accumulation of BRCA1; some Hormad2+/− Spo11−/− oocytes that contained the pseudo–sex body always showed prominent BRCA1 at unsynapsed axes within the pseudo–sex body (Fig. 5D, arrow), whereas the remaining oocytes showed neither appreciable levels of BRCA1 accumulation nor pseudo–sex body formation (Fig. 5E). The Spo11−/− Hormad2−/− pachytene-like oocytes also showed neither pseudo–sex body formation nor BRCA1 accumulation; BRCA1 was weakly detectable on chromosome axes (Fig. 5F).However, BRCA1 was found to be recruited to the unsynapsed chromosome axes, and the γH2AX-enriched domain was observed in the Hormad2−/− oocytes (Fig. S6 in Supporting Information), suggesting that HORMAD2 is dispensable for the phosphorylation of H2AX on the unsynapsed chromosomes in the presence of DSBs. Taken together, we propose from these results that HORMAD2 is required for the local concentration of BRCA1 followed by ATR activation in the absence of DSBs, leading to pseudo–sex body formation and consequent oocyte apoptosis (Fig. 5G). The possible involvement of HORMAD2 phosphorylation in this process is discussed below.

image

Figure 5. Distribution of BRCA1 in the 18.5-dpc oocytes. Meiotic chromosome spreads were obtained from wild-type (A, B), Hormad2+/+ Spo11−/− (C), Hormad2+/− Spo11−/− (D, E), Hormad2−/− Spo11−/− (F) oocytes. Representative merged images of immunostaining results for γH2AX, SYCP3 and BRCA1 (A–F) and matched exposure images of BRCA1 staining (A′–F′) are shown. Arrows indicate the γH2AX-enriched domain (pseudo–sex body), where the BRCA1 signal is strong on the chromosome axes. Note that weak BRCA1 signals are detectable on the unsynapsed chromosome axes in oocytes of the Spo11−/− background. (G) Hypothetical schema for the mechanism of pseudo–sex body formation. HORMAD2 (and putatively its phosphorylation) may play a role in the induction of a localized concentration of BRCA1, leading to the activation of meiotic silencing of unsynapsed chromatin.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Putative function of HORMAD2 in MSCI

We observed a unique defect in sex body formation in the Hormad2−/− spermatocytes. The ATR- and γH2AX-enriched repressive domains can form the round-shaped sex body–like structure in the absence of HORMAD2, whereas the Hormad2 deficiency causes the dissociation of a portion of the sex chromosome axes from the repressive domain (Figs 2 and 3G, partial). A recent study of Mdc1−/− mice has implicated the chromosome-wide spreading of γH2AX formation with ATR activity to the entire chromatin in the compaction of sex chromosomes (Ichijima et al. 2011), although its molecular basis is not fully established. In addition, both DSB-dependent and DSB-independent mechanisms have been proposed to activate ATR and γH2AX formation on the sex chromosomes, but their relative contribution to MSCI remains largely unknown. Recently, Wojtasz et al. clearly showed that HORMAD2 is required for the accumulation of ATR along unsynapsed axes, but not at DSB or on DSB-associated chromatin loops (Wojtasz et al. 2012), leading to a failure in sex body formation. They suggest that the DSB-independent ATR recruitment is required to establish MSCI along the entire length of the sex chromosomes. In addition to that, our present results showed that many Hormad2-deficient spermatocytes are capable to form a sex body that has a ‘diffuse’ appearance on the entire sex chromosome (Fig. 2E,F). This is likely the normal first step of MSCI, as the similar sex body is formed on the extended (not condensed) X chromosome at early pachynema in wild-type mice (Page et al. 2012). We speculate that this γH2AX formation, which is likely DSB dependent, is not sufficient to complete MSCI forming a round-shaped sex body containing the entire chromosomes. We speculate from the phenotype of Hormad2-deficient spermatocytes that this completion of MSCI requires (i) further HORMAD2-dependent, DSB-independent γH2AX formation by ATR and (ii) a confinement of the sex chromosomes within the aggregation of γH2AX-enriched domain in the context of compaction of the chromosome axes. Given that the activated ATR has been suggested to aggregate autonomously at certain domains on chromosomes (Fukuda et al. 2012), our results suggest that HORMAD2 on sex chromosome axes might serve as a physical connection to ATR and any of other factors on the suppressive chromatin domain, as a mechanism enforcing the compaction of sex chromosomes. Interestingly, a similar inadequate targeting of ATR activity has been also observed on the sex chromosomes in Brca1Δ11/Δ11 spermatocytes (Turner et al. 2004), suggesting that BRCA1 and HORMAD2 function in the same pathway for the ATR activation leading to MSCI. Taken together, we conclude from our present results and previous results that HORMAD2 is required for the DSB-independent, BRCA1-dependent activation of ATR on the sex chromosomes that is essential for normal sex body formation.

Putative function of HORMAD2 in MSUC with extensive asynapsis

We also examined the possible involvement of HORMAD2 in the elimination of asynaptic oocytes and in the MSUC in Spo11−/− females. Although MSUC has been implicated in the elimination of asynaptic oocytes, it is still an incompletely understood process (Handel & Schimenti 2010; Kogo et al. 2012). In our present study, we observed that a Hormad2 deficiency abrogated both oocyte cell death and pseudo–sex body formation in the Spo11−/− background, supporting the proposed hypothesis that MSUC is responsible for the elimination of asynaptic oocytes. Interestingly, the heterozygous Hormad2 mutation also partially suppressed both of these phenomena, suggesting a critical importance of HORMAD2 in these processes. In terms of MSUC factors, BRCA1 is intensely accumulated in the region forming pseudo–sex body and is weakly distributed on the entire axes of unsynapsed chromosomes in the Spo11−/− background but the local accumulation of BRCA1 does not occur in the absence of HORMAD2. Although it remains an enigma as to why only a limited portion of unsynapsed chromosomes is subject to MSUC, the concentration of MSUC factors at a certain region might be triggered by the local concentration of BRCA1 and subsequent activation of ATR with other DDR factors and their amplification of the reaction at a certain part of unsynapsed chromosomes (Fig. 5G). Thus, the lack of pseudo–sex body formation in the Spo11−/− Hormad2−/− oocytes implicates the putative function of HORMAD2 in the initiation of MSUC activation (Fig. 5G). Interestingly, a recent study showed that the HORMAD2 phosphorylation, which is presumably dependent on the ATR kinase, is largely reduced in the Brca1Δ11/Δ11 testis (Fukuda et al. 2012). This suggests an interesting hypothesis to be examined that the HORMAD2 is a target of BRCA1-dependent ATR activation, and that in turn the phosphorylated HORMAD2 facilitates the concentration of BRCA1, generating a positive feedback loop to amplify MSUC response leading to pseudo–sex body formation (Fig. 5G).

Functional difference between HORMAD1 and HORMAD2

In mammals, there are two meiosis-specific HORMA domain proteins, HORMAD1 and HORMAD2, both of which have been shown to preferentially localize to unsynapsed chromosome axes (Wojtasz et al. 2009). In contrast to the prominent localization of HORMAD1 to the unsynapsed chromosome axes from leptotene to diplotene stages of meiotic prophase, we observed by immunohistochemistry that HORMAD2 was negligible at earlier stages (leptotene and zygotene stages), but was readily detectable from late zygotene and intensely detected at pachytene stage on the sex chromosome axis (Fig. 1A). Consistent with these spatio-temporal distributions, HORMAD1 is essential for the evolutionarily conserved earlier events such as DSB formation in leptotene stage and homologous pairing and synapsis in zygotene stage (Shin et al. 2010; Daniel et al. 2011; Kogo et al. 2012), whereas HORMAD2 is dispensable in these functions, although a small reduction in the number of recombination foci and a small increase in the number of incomplete synapsis were observed by others (Wojtasz et al. 2012) and in the present study (Fig. 2C and Fig. S3B in Supporting Information), respectively. Instead, HORMAD2 was found to be essential for MSCI in spermatocytes and for MSUC in the Spo11−/− oocytes at the pachytene stage. Previous studies of the Spo11−/− Hormad1−/− female mice have shown that the Hormad1 deficiency abrogates the pseudo–sex body formation by impairing the BRCA1 localization to unsynapsed chromosome axes (Daniel et al. 2011). In the present study, the Hormad2 deficiency affected the MSUC, but not the axial localization of HORMAD1 (Fig. S5A in Supporting Information), suggesting that the axial distribution of HORMAD1 alone is not sufficient for MSUC. HORMAD1 is rather likely required for the recruitment of HORMAD2 and BRCA1, both of which are essential for the pseudo–sex body formation. In addition, the heterozygous mutant of Hormad2 partially affected the MSUC in the Spo11−/− background (Fig. 4G), whereas that of Hormad1 did not (results not shown), suggesting that HORMAD2, rather than HORMAD1, plays a central role in MSUC and the consequent elimination of asynaptic oocytes.

Checkpoint function of HORMAD2 in female meiosis

The present results show that HORMAD2 is not essential for the normal process of female meiosis, particularly homologous pairing and synapsis, but is required for quality surveillance during this process. These characteristics of HORMAD2 meet the definition of a checkpoint protein, that is, a protein that only becomes essential in response to a defect in cell-cycle events. Although the same checkpoint function for synapsis failure could be attributed to HORMAD1 (Daniel et al. 2011; Kogo et al. 2012), HORMAD1 is a multivalent protein that is also essential for the homologous pairing and synapsis. In this context, HORMAD1 is not a typical checkpoint protein, but serves as a signature of unsynapsed chromosomes that have remained beyond the zygotene/pachytene transition. In addition, HORMAD1 is likely to have a role in facilitating synapsis in the absence of SPO11-dependent DSBs (Daniel et al. 2011; Kogo et al. 2012), which may help to ensure the completion of synapsis. The putative HORMAD1-dependent recruitment of HORMAD2 to unsynapsed chromosome axes at the end of the zygotene stage is a possible mechanism underlying the surveillance function of HORMAD2, that is, in detecting unsynapsed chromosomes. A recent paper has suggested that the phosphorylation of both HORMAD1 and HORMAD2, which we have also confirmed in our laboratory (results not shown), might be involved in the MSUC mechanism (Fukuda et al. 2012). Besides MSUC, however, there still remains another possibility that these phosphorylation events may serve as recognition signals for downstream signaling proteins of an as yet uncharacterized meiotic prophase checkpoint mechanism. Given that the phosphorylation of Hop1, an ortholog of the mammalian HORMADs, is essential for DSB-dependent pachytene checkpoints in yeast (Carballo et al. 2008), the phosphorylation of HORMADs is assumed to be important for putative checkpoint signaling in mammals. Although the molecular mechanism of pachytene checkpoint signaling has not yet been established as a part of mammalian meiosis, it would be worth examining in the future whether the meiotic HORMA domain proteins have a role as checkpoint proteins in an evolutionarily conserved mechanism mediated by phosphorylation signaling.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Preparation of antibodies

For the detection of HORMAD2, two different antisera were prepared (Fig. S1 in Supporting Information) via the immunization of rabbits with KLH-conjugated synthetic peptide KVSEPVTVFIPNRK (Thermo Electron) corresponding to the C-terminal 14 residues of mouse HORMAD2 (rabbit anti-HORMAD2_C), and the immunization of guinea-pigs (Shibayagi Co.) with KLH-conjugated synthetic peptide SHNTRTLKASKNTIFPSQ (Invitrogen) corresponding to residues 7–24 of mouse HORMAD2 (guinea-pig anti-HORMAD2_N). Rabbit polyclonal antibodies for HORMAD2 were affinity-purified with biotin-conjugated peptides (Invitrogen) and AffinityPak Streptavidin Columns (Pierce). Guinea-pig polyclonal antibody for HORMAD2 was affinity-purified using peptides (Invitrogen) and a Microlink peptide coupling kit (Pierce). The characterization of these purified antibodies is detailed in Fig. S1B (Supporting Information).

Mice

The Hormad2 mutants (Acc. No. CDB0576K: http://www.cdb.riken.jp/arg/mutant%20mice%20list.html) were generated as described at http://www.cdb.riken.jp/arg/Methods.html. Briefly, BAC clones containing the Hormad2 locus of C57BL/6J mice were purchased from the BACPAC Resources Center (http://bacpac.chori.org/) and used as templates to amplify the 3.9 kb 5′- and 10.4 kb 3′-homologous arms of the Hormad2 targeting vector with LA Taq polymerase (Takara) and the following primer sets: 5′-CCATTACTCGAGGGAGCATGTCAGCAGATAGGAAG-3′ and 5′-CATGCGCTAGCTACACTTGGGACGGGAAAATCGTG-3′ for the 5′-homologous arm, and 5′-GCGGCCGCTTCCCTCACTCACAGACTGTGTTAATC-3′ and 5′-GTCGACTCCAGAACCTGGTGTTGCCCTCAGTGCTG-3′ for the 3′-homologous arm. Gene targeting was performed in TT2 ES cells by homologous recombination. The targeted ES cells were screened by PCR using the primers ES_F 5′-CTTCACTAGCTGTGAAGAACCTGGAC-3′ and Neo 5′-CTGACCGCTTCCTCGTGCTTTACG-3′ (Fig. S2A in Supporting Information). Correct targeting of the PCR-positive ES cell clones was further verified by Southern blotting using the 5′- and 3′-external probes, 5′P-ES and 3′P-ES (Fig. S2B in Supporting Information). The Hormad2-targeted ES cells were injected into eight-cell-stage embryos of ICR mice, and chimeric offspring were bred with C57BL/6J mice. The Spo11-targeted mice were kindly provided by S. Keeney and M. Jasin. Mice were bred and maintained under specific pathogen-free conditions in accordance with the guidelines of the Animal Care and Use Committee at Fujita Health University. Experimental animals were compared with controls from the same litter (if available) or from other litters from the same matings. The described phenotypes were consistently observed irrespective of their ES cell clone of origin, number of successive brother–sister mating generations or backcrossing with C57BL/6J mice (more than eight generations).

PCR genotyping

PCR genotyping of Hormad2 alleles was performed using Ex Taq polymerase (Takara) and the following primers (Fig. S2A,C in Supporting Information): a common forward primer (CoF 5′-GAACACGATTTTCCCGTCCCAAG-3′), a neo-specific primer (neo 5′-CTGACCGCTTCCTCGTGCTTTACG-3′) and a Hormad2-specific reverse primer (H2R 5′-GCTAGCTTGAGGTTAATGGTTCC-3′). PCR genotyping of Spo11 alleles was performed as previously described (Baudat et al. 2000).

Analysis of reproductive phenotypes

To examine male fertility, four sets of littermates with three different Hormad2 genotypes were mated with wild-type C57BL/6J females. Each male was mated with more than two females, and the offspring were counted. To evaluate female fertility, seven sets of littermates with three different Hormad2 genotypes were mated with males of proven fertility. Each female was mated more than twice (confirmed by vaginal plug observations), and the offspring were counted.

Histology and immunofluorescent staining of meiotic chromosomes

Preparation of paraffin tissue sections, routine histology, immunohistochemistry and TUNEL staining of tissue sections were performed as previously described (Kogo et al. 2012). The stages of the seminiferous tubules were determined according to the classification developed by Russell et al. (Russell 1990) as previously described (Kogo et al. 2012). The number of total oocytes was analyzed based on immunostaining for c-KIT and nuclear counterstaining with DAPI as previously described (Kogo et al. 2012). Meiotic chromosome spreads were prepared using the previously described drying-down technique with some modifications (Peters et al. 1997; Kogo et al. 2012).

Antibodies for immunofluorescent staining

Immunohistochemical staining of paraffin tissue sections was performed using the following primary antibodies: affinity-purified rabbit anti-HORMAD2_C (0.5 μg/mL), affinity-purified rabbit anti-HORMAD1_C (0.05 μg/mL), goat anti-ATR (1:200, N-19; Santa Cruz Biotechnology) and goat anti-c-KIT (1:200, M-14; Santa Cruz Biotechnology). Immunofluorescent staining of meiotic chromosome spreads was performed using the following primary antibodies: affinity-purified rabbit anti-HORMAD2_C (2.0 μg/mL), guinea-pig anti-SYCP3 antiserum (1 : 30 000), mouse anti-γH2AX (1 : 5000, JBW301; Upstate), rabbit anti-SYCP1 (1 : 500; Novus Biologicals), goat anti-BRCA1 (1 : 30, M-20; Santa Cruz Biotechnology), goat anti-ATR (1 : 100, N-19; Santa Cruz Biotechnology) and affinity-purified rabbit anti-HORMAD1_C (2.0 μg/mL). The Alexa fluorochrome-conjugated secondary antibodies were used for detection as previously described (Kogo et al. 2012).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank M. Tong, T. Kato, S. Nishiyama, B. Hasbaira and A. Kogo for helpful discussions, T. Abe for help in generating the chimeric mice, R. Wang, M. Jasin and S. Keeney for providing Spo11-targeted mice and S. Nagao and K. Hikita for help with breeding the mice. This work was supported in part by a Grants-in-Aid for Young Scientists (B), Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (to H. Kogo); a Grants-in-Aid for Scientific Research (B), MEXT, Japan (to H. Kurahashi).

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
gtc12005-sup-0001-AppendixS1.docWord document60KAppendix S1 Experimental procedures.
gtc12005-sup-0002-FigureS1.pdfapplication/PDF180KFigure S1 Antibody preparation and detection of mouse HORMAD2.
gtc12005-sup-0003-FigureS2.pdfapplication/PDF302KFigure S2 Targeted disruption of the mouse Hormad2 gene.
gtc12005-sup-0004-FigureS3.pdfapplication/PDF214KFigure S3 Increased incomplete synapsis in Hormad2−/− oocytes.
gtc12005-sup-0005-FigureS4.pdfapplication/PDF259KFigure S4 Expression and localization of HORMAD2 in the fetal ovary and the HORMAD1-dependent chromatin association of HORMAD2.
gtc12005-sup-0006-FigureS5.pdfapplication/PDF293KFigure S5 Pseudo sex body formation in the pachytene spermatocytes obtained from the 15.5 dpp Hormad2+/+ Spo11−/− and Hormad2−/− Spo11−/− testes.
gtc12005-sup-0007-FigureS6.pdfapplication/PDF220KFigure S6 Distribution of BRCA1 in 0.5 dpp Hormad2−/− oocytes.

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