The E3 ubiquitin ligase Cullin 4A regulates meiotic progression in mouse spermatogenesis

The Cullin-RING ubiquitin-ligase CRL4 controls cell cycle and DNA damage checkpoint response and ensures genomic integrity. Inactivation of the Cul4 component of the CRL4 E3 ligase complex in Caenorhabditis elegans by RNA interference results in massive mitotic DNA re-replication in the blast cells, largely due to failed degradation of the DNA licensing protein, CDT-1, and premature spermatogenesis. Here we show that inactivation of Cul4a by gene-targeting in mice only affected male but not female fertility. This male infertility phenotype resulted from a combination of decreased spermatozoa number, reduced sperm motility and defective acrosome formation. Agenesis of the mutant germ cells was accompanied by increased cell death in pachytene/diplotene cells with markedly elevated levels of phospho-p53 and CDT-1. Despite apparent normal assembly of synaptonemal complexes and DNA double strand break repair, dissociation of MLH1, a component of the late recombination nodule, was delayed in Cul4a−/− diplotene spermatocytes, which potentially led to subsequent disruptions in meiosis II and spermiogenesis. Together, our study revealed an indispensable role for Cul4a during male germ cell meiosis.     

Essential role of the CUL4B ubiquitin ligase in extra-embryonic tissue development during mouse embryogenesis

Mutations of the CUL4B ubiquitin ligase gene are causally linked to syndromic X-linked mental retardation (XLMR). However, the pathogenic role of CUL4B mutations in neuronal and developmental defects is not understood. We have generated mice with targeted disruption of Cul4b, and observed embryonic lethality with pronounced growth inhibition and increased apoptosis in extra-embryonic tissues. Cul4b, but not its paralog Cul4a, is expressed at high levels in extra-embryonic tissues post implantation. Silencing of CUL4B expression in an extra-embryonic cell line resulted in the robust accumulation of the CUL4 substrate p21^Cip1/WAF and G2/M cell cycle arrest, which could be partially rescued by silencing of p21^Cip1/WAF. Epiblast-specific deletion of Cul4b prevented embryonic lethality and gave rise to viable Cul4b null mice. Therefore, while dispensable in the embryo proper, Cul4b performs an essential developmental role in the extra-embryonic tissues. Our study offers a strategy to generate viable Cul4b-deficient mice to model the potential neuronal and behavioral deficiencies of human CUL4B XLMR patients.     

ZIP4H (TEX11) deficiency in the mouse impairs meiotic double strand break repair and the regulation of crossing over

We have recently shown that hypomorphic Mre11 complex mouse mutants exhibit defects in the repair of meiotic double strand breaks (DSBs). This is associated with perturbation of synaptonemal complex morphogenesis, repair and regulation of crossover formation. To further assess the Mre11 complex's role in meiotic progression, we identified testis-specific NBS1-interacting proteins via two-hybrid screening in yeast. In this screen, Zip4h (Tex11), a male germ cell specific X-linked gene was isolated. Based on sequence and predicted structural similarity to the S. cerevisiae and A. thaliana Zip4 orthologs, ZIP4H appears to be the mammalian ortholog. In S. cerevisiae and A. thaliana, Zip4 is a meiosis-specific protein that regulates the level of meiotic crossovers, thus influencing homologous chromosome segregation in these organisms. As is true for hypomorphic Nbs1 (Nbs1^ΔB/ΔB) mice, Zip4h^−/Y mutant mice were fertile. Analysis of spermatocytes revealed a delay in meiotic double strand break repair and decreased crossover formation as inferred from DMC1 and MLH1 staining patterns, respectively. Achiasmate chromosomes at the first meiotic division were also observed in Zip4h^−/Y mutants, consistent with the observed reduction in MLH1 focus formation. These results indicate that meiotic functions of Zip4 family members are conserved and support the view that the Mre11 complex and ZIP4H interact functionally during the execution of the meiotic program in mammals.     

Mouse strains with an active H2-Ea meiotic recombination hot spot exhibit increased levels of H2-Ea-specific DNA breaks in testicular germ cells

We devised a sensitive method for the site-specific detection of rare meiotic DNA strand breaks in germ cell-enriched testicular cell populations from mice that possess or lack an active recombination hot spot at the H2-Ea gene. Using germ cells from adult animals, we found an excellent correlation between the frequency of DNA breaks in the 418-bp H2-Ea hot spot and crossover activity. The temporal appearance of DNA breaks was also studied in 7- to 18-day-old mice with an active hot spot during the first waves of spermatogenesis. The number of DNA breaks detected rose as leptotene and zygotene spermatocytes populate the testis with a peak at day 14 postpartum, when leptotene, zygotene, and early pachytene spermatocytes are the most common meiotic prophase I cell types. The number of DNA breaks drops precipitously 1 day later, when middle to late pachytene spermatocytes become the dominant subtype. The recombination-related breaks in the hot spot likely reflect SPO11-induced double-strand breaks and/or recombination intermediates containing free 3' hydroxyl groups.     

Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination.     

PLK1 depletion alters homologous recombination and synaptonemal complex disassembly events during mammalian spermatogenesis

Homologous recombination (HR) is an essential meiotic process that contributes to the genetic variation of offspring and ensures accurate chromosome segregation. Recombination is facilitated by the formation and repair of programmed DNA double-strand breaks. These DNA breaks are repaired via recombination between maternal and paternal homologous chromosomes and a subset result in the formation of crossovers. HR and crossover formation is facilitated by synapsis of homologous chromosomes by a proteinaceous scaffold structure known as the synaptonemal complex (SC). Recent studies in yeast and worms have indicated that polo-like kinases (PLKs) regulate several events during meiosis, including DNA recombination and SC dynamics. Mammals express four active PLKs (PLK1–4), and our previous work assessing localization and kinase function in mouse spermatocytes suggested that PLK1 coordinates nuclear events during meiotic prophase. Therefore, we conditionally mutated Plk1 in early prophase spermatocytes and assessed stages of HR, crossover formation, and SC processes. Plk1 mutation resulted in increased RPA foci and reduced RAD51/DMC1 foci during zygonema, and an increase of both class I and class II crossover events. Furthermore, the disassembly of SC lateral elements was aberrant. Our results highlight the importance of PLK1 in regulating HR and SC disassembly during spermatogenesis.     

Mammalian meiosis involves DNA double-strand breaks with 3′ overhangs

Meiotic recombination in yeast is initiated at DNA double-strand breaks (DSBs), processed into 3′ single-strand overhangs that are active in homology search, repair and formation of recombinant molecules. Are 3′ overhangs recombination intermediaries in mouse germ cells too? To answer this question we developed a novel approach based on the properties of the Klenow enzyme. We carried out two different, successive in situ Klenow enzyme-based reactions on sectioned preparations of testicular tubules. Signals showing 3′ overhangs were observed during wild-type mouse spermatogenesis, but not in Spo11 ^?/? males, which lack meiotic DSBs. In Atm ^?/? mice, abundant positively stained spermatocytes were present, indicating an accumulation of non-repaired DSBs, suggesting the involvement of ATM in repair of meiotic DSBs. Thus the processing of DSBs into 3′ overhangs is common to meiotic cells in mammals and yeast, and probably in all eukaryotes.     

X-linked intellectual disability gene CUL4B targets Jab1/CSN5 for degradation and regulates bone morphogenetic protein signaling

Cullin 4B (CUL4B) is a scaffold protein involved in the assembly of cullin-RING ubiquitin ligase (E3) complexes. Contemporary reports have identified multiple mutations of CUL4B gene as being causally associated with X-linked intellectual disability (XLID). Identifying the specific protein substrates will help to better understand the physiological functions of CUL4B. The current study identified Jun activation domain-binding protein (Jab1/CSN5) in the COP9 signalosome (CSN) complex as a novel proteolytic target for the CUL4B ubiquitin ligase complex. The impaired degradation of Jab1 was observed in cells after RNAi-mediated CUL4B depletion. Integrity of DDB1-CUL4B-ROC1 was further demonstrated to be indispensable for the degradation of Jab1. In addition, the degradation of Jab1 is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of Jab1. Interestingly, CUL4B-silenced cells were shown to exhibit abnormal upregulation of bone morphogenetic protein (BMP) signaling. Furthermore, in vivo studies of embryonic fibroblasts in Cul4b-deficient mice demonstrated Jab1 accumulation and increased activation of the BMP signaling pathway. Together, the current findings demonstrate the CUL4B E3 ubiquitin ligase plays a key role in targeting Jab1 for degradation, potentially revealing a previously undocumented mechanism for regulation of the BMP signaling pathway involved with the CUL4B-based E3 complex. This observation may provide novel insights into the molecular mechanisms underlying CUL4B-associated XLID pathogenesis.     

CUL4B ubiquitin ligase in mouse development: A model for human X-linked mental retardation syndrome?

CUL4B, a member of the cullin-RING ubiquitin ligase family, is frequently mutated in X-linked mental retardation (XLMR) patients. The study by Liu et al. showed that Cul4b plays an essential developmental role in the extra-embryonic tissues, while it is dispensable in the embryo proper during mouse embryogenesis. Viable Cul4b-null mice provide the first animal model to study neuronal and behavioral deficiencies seen in human CUL4B XLMR patients.CUL4 is a member of the cullin-RING ubiquitin ligase family, the largest E3 ligase family, which appears to account for ∼20% of total protein degradation by the ubiquitin-proteasome system^1,2,3. CUL4 is conserved during evolution from yeast to human. In yeast, CUL4 encodes a single gene, but mammalian cells express two closely related paralogs, CUL4A and CUL4B with about 82% sequence identity. CUL4A and CUL4B assemble structurally similar E3 complexes through binding to an adaptor protein (DDB1) and a substrate receptor protein (DCAF) at the N-terminus, and a RING protein RBX1 at the C-terminus (Figure 1), and share functional redundancy in targeting substrates such as p21 and Cdt1 for ubiquitination and degradation^1,2. The Cul4a-null mice are viable and display no abnormal development and growth phenotypes, likely due to functional compensation from Cul4b^4,5. The only phenotype associated with Cul4a abrogation is the reproductive defects seen with male but not female mice, resulting from differential non-overlapping expression patterns of the two Cul4 genes during male meiosis⁶. On the other hand, germline deletion of Cul4b resulted in embryonic lethality around E9.5⁷, indicating a unique function of Cul4b that cannot be compensated by Cul4a during embryogenesis.Open in a separate windowFigure 1Differential expression of Cul4a and Cul4b in the embryo proper and extra embryonic tissues determines their fate. Before implantation, both Cul4a and Cul4b are expressed in the blastocyst. Following implantation, Cul4a is expressed in the embryo proper, but not in extra-embryonic tissues. Upon Cul4b deletion, p21 accumulates in extra-embryonic tissues to induce G2/M arrest and eventually embryonic death due to degeneration of extra-embryonic tissues. Expression of Cul4a in embryo prevents p21 accumulation and subsequent embryonic death.Mental retardation (MR) affects approximately 1%-3% of the population and is about 30% more common in males than in females⁸, suggesting a causal relationship with gene mutations on the X chromosome. To date, mutations in about 100 genes have been identified in X-linked MR (XLMR), much more than those found on autosomes⁹. In 2007, two independent groups reported that mutations of CUL4B (Xq24) ubiquitin ligase gene are associated with XLMR^10,11. CUL4B-deficient patients display a syndrome of delayed puberty, moderate short stature, hypogonadism, relative macrocephaly, central obesity, fine intention tremor, brachydactyly, and large tongue^10,11. Similarly, the neuronal and developmental deficiencies found in XLMR patients with CUL4B mutations are not compensated by CUL4A. The studies of the molecular pathogenesis of human XLMR are lagging partly due to the lack of an animal model for the disease.In the most recent study published in Cell Research, Zhou and coworkers¹² attempted to generate conditional Cul4b knockout mice with targeted deletion of Cul4b at exons 4 and 5, giving rise to a non-functional Cul4b fragment lacking both the DDB1-binding domain and the cullin homology domain for RBX1 recruitment. The chicken-actin (CAG)-Cre was used, which drives Cre-mediated recombination at the early zygote stage, leading to Cul4b deletion in both the embryo proper and extra-embryonic tissues. Like human CUL4B, the mouse Cul4b is also located on the X-chromosome. Intercrossing of male CAG-Cre with female Cul4b^fl/+ revealed that hemizygous deletion of Cul4b causes embryonic lethality. No embryos with the genotype of Cul4b^−/y survived beyond E9.5. Interestingly, the heterozygous Cul4b^+/− embryos also die in the uterus before E13.5, suggesting that the paternal X chromosome undergoes imprinted inactivation with only trace amount, if any, of Cul4b expression remaining in extra-embryonic tissues. Detailed analysis of dissected embryos revealed that dying Cul4b^+/− embryos (E12.5) lack blood supply from the yolk sacs, whereas the Cul4b^−/y embryos (E8.5) showed remarkable reduction in proliferation with growth arrest at G2/M and enhanced apoptosis. The authors went on and investigated why Cul4a failed to compensate the loss of Cul4b, and found a dynamic expression pattern, differing between two forms, during early embryonic development. Prior to implantation, both Cul4 proteins are detectable in the blastocysts. Shortly after implantation, while both forms are expressed in the embryo proper, only Cul4b is expressed in the extra-embryonic tissues. Thus, upon Cul4b deletion, extra-embryonic tissues without Cul4a compensation degenerate, eventually leading to embryonic death. Consistently, when the authors deleted Cul4b in the epiblast using the Sox2-Cre (targeted Cul4b deletion in embryos proper only), viable Cul4b-null mice are produced likely due to Cul4a compensation. Thus, Cul4b is essential for the development of extra-embryonic tissues, but is dispensable for embryogenesis itself.To study the potential underlying mechanism(s) of embryonic lethality upon Cul4b deletion in extra-embryonic tissues, the authors used an extra-embryonic cell line (XEN). Cul4b knockdown induced a remarkable cell cycle arrest at the G2/M phase, consistent with observation made in Cul4b-null embryos, and robust accumulation of p21, a universal inhibitor of cyclin dependent kinase and a known substrate of Cul4¹. To determine whether accumulated p21 is responsible for the G2/M arrest, the authors simultaneously knocked down both Cul4b and p21 in XEN cells and observed a partial abrogation of growth arrest, suggesting that p21 plays a causal role, at least in part. Unfortunately, due to unavailability of anti-mouse p21 antibody specific for immunohistochemical staining, the authors were not able to show if p21 is indeed accumulated in extra-embryonic tissues upon Cul4b deletion. However, whether p21 indeed plays a causal role in embryonic death upon Cul4b deletion can be unequivocally determined by a rescuing experiment in which simultaneous deletion of p21 should abrogate or at least delay embryonic lethality, if it is causal. Nevertheless, the study by Zhou''s group can be summarized as follows. Before implantation, both Cul4a and Cul4b ubiquitin ligases are expressed in the blastocyst (inner cell mass and trophoblast cells). Following embryo implantation, while Cul4b is expressed in both the embryo proper and extra embryonic tissues, Cul4a is only expressed in the embryo proper. The CAG-Cre-driven Cul4b deletion (in both the embryo proper and extra-embryonic tissues) causes significant p21 accumulation in Cul4a non-expressing extra-embryonic tissues, resulting in G2/M arrest, followed by embryonic death due to degeneration of extra-embryonic tissues. On the embryo side, Cul4b deletion has no detrimental consequence, benefiting from the compensatory effect of Cul4a for p21 targeting. The same holds true when Cul4b is deleted driven by embryonic specific Sox2-Cre (Figure 1).It is noteworthy that the studies by Zhou''s group revealed two distinct differences between Cul4b KO mice and CUL4B-associated XLMR patients. First, Cul4b deletion at the zygote stage causes embryonic lethality, whereas XLMR patients with CUL4B mutations live to adulthood. Second, the Cul4b-null allele cannot be transmitted from the mother to the offspring, whereas human XLMR patients inherit X-linked CUL4B mutations from their mothers. Nevertheless, viable Cul4b-null mice (upon epiblast ablation by Sox2-Cre) provide the first mouse model for mechanistic study of human XLMR diseases associated with CUL4B mutations in the following three aspects:First, as noted earlier, human CUL4B XLMR patients have multiple neuronal and developmental defects. An obvious follow-up study will be to use this mouse model for neurological and behavioral analyses to determine whether Cul4b-null mice indeed present some of human XLMR symptoms.Second, this model can also be used to validate whether accumulation of Cul4b substrates during various stages of brain development indeed plays a pathogenic role and contributes to the clinical symptoms of XLMR patients. For instance, WDR5, a recently identified gene affecting general cognitive ability¹³, was found to be a novel nuclear substrate of CUL4B, but not CUL4A¹⁴. Investigation into whether WDR5 is abnormally accumulated upon Cul4b deletion in vivo would rule in or rule out its potential association with human XLMR, although it was not the case in this study using an extra-embryonic cell line in vitro.Third, the viability of Cul4b-null mice upon epiblast-specific deletion provides opportunities to study neuronal specific ablation of Cul4b in association with the pathogenesis of CUL4B-associated XLMR. For example, Cul4b is expressed at high levels in the hippocampus and cerebrum of mouse brains; both regions are affected in MR patients¹⁵. Thus, the use of Cre mouse lines that target the deletion of Cul4b in the entire brain, selected brain areas, or specific neuronal cells in both spatial and temporal manners¹⁶ would reveal potential contributions of particular regions and cell types to the development and symptoms of CUL4B-associated XLMR.A number of questions that warrant future investigation remain unanswered. First, in addition to p21, what are the other Cul4B substrates, which also contribute to degeneration of extra-embryonic tissues upon Cul4b deletion, since simultaneous deletion of p21 only partially rescues the growth defects? Second, besides the difference in tissue/cell specific expression seen in this study, are Cul4a and Cul4b targeting a unique set of substrates non-redundantly, thus differentiating their physiological functions? A related question will be why CUL4A cannot compensate for the loss of CUL4B in CUL4B-associated XLMR patients? Third, what is the pathogenic mechanism for CUL4B-associated XLMR? Is it mainly due to pathological accumulation of many CUL4B substrates? Answers to these questions may offer insights into potential therapeutic strategies for the treatment of CUL4B-associated XLMR patients.In summary, the findings reported by Zhou''s group provide the first convincing evidence that demonstrates an essential role of Cul4b in the development of extra-embryonic tissues during mouse embryogenesis. The viable Cul4b conditional knockout mice, generated in this study, may serve as the first mouse model for future mechanistic studies of neuronal and behavioral deficiencies of human XLMR associated with CUL4B mutations. We look forward to more exciting discoveries of how Cul4b deficiency leads to the development of XLMR in years to come.     

Homology‐dependent repair is involved in 45S rDNA loss in plant CAF‐1 mutants

Arabidopsis thaliana mutants in FAS1 and FAS2 subunits of chromatin assembly factor 1 (CAF1) show progressive loss of 45S rDNA copies and telomeres. We hypothesized that homology‐dependent DNA damage repair (HDR) may contribute to the loss of these repeats in fas mutants. To test this, we generated double mutants by crossing fas mutants with knock‐out mutants in RAD51B, one of the Rad51 paralogs of A. thaliana. Our results show that the absence of RAD51B decreases the rate of rDNA loss, confirming the implication of RAD51B‐dependent recombination in rDNA loss in the CAF1 mutants. Interestingly, this effect is not observed for telomeric repeat loss, which thus differs from that acting in rDNA loss. Involvement of DNA damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double‐stranded breaks (measured as γ‐H2AX foci) in 45S rDNA. Occurrence of the foci is not specific for S‐phase, and is ATM‐independent. While the foci in fas mutants occur both in the transcribed (intranucleolar) and non‐transcribed (nucleoplasmic) fraction of rDNA, double fas rad51b mutants show a specific increase in the number of the intranucleolar foci. These results suggest that the repair of double‐stranded breaks present in the transcribed rDNA region is RAD51B dependent and that this contributes to rDNA repeat loss in fas mutants, presumably via the single‐stranded annealing recombination pathway. Our results also highlight the importance of proper chromatin assembly in the maintenance of genome stability.     

asunder Is a Critical Regulator of Dynein–Dynactin Localization during Drosophila Spermatogenesis

Spermatogenesis uses mitotic and meiotic cell cycles coordinated with growth and differentiation programs to generate functional sperm. Our analysis of a Drosophila mutant has revealed that asunder (asun), which encodes a conserved protein, is an essential regulator of spermatogenesis. asun spermatocytes arrest during prophase of meiosis I. Strikingly, arrested spermatocytes contain free centrosomes that fail to stably associate with the nucleus. Spermatocytes that overcome arrest exhibit severe defects in meiotic spindle assembly, chromosome segregation, and cytokinesis. Furthermore, the centriole-derived basal body is detached from the nucleus in asun postmeiotic spermatids, resulting in abnormalities later in spermatogenesis. We find that asun spermatocytes and spermatids exhibit drastic reduction of perinuclear dynein–dynactin, a microtubule motor complex. We propose a model in which asun coordinates spermatogenesis by promoting dynein–dynactin recruitment to the nuclear surface, a poorly understood process required for nucleus–centrosome coupling at M phase entry and fidelity of meiotic divisions.     

Synaptonemal complex assembly and H3K4Me3 demethylation determine DIDO3 localization in meiosis

Synapsis of homologous chromosomes is a key meiotic event, mediated by a large proteinaceous structure termed the synaptonemal complex. Here, we describe a role in meiosis for the murine death-inducer obliterator (Dido) gene. The Dido gene codes for three proteins that recognize trimethylated histone H3 lysine 4 through their amino-terminal plant homeodomain domain. DIDO3, the largest of the three isoforms, localizes to the central region of the synaptonemal complex in germ cells. DIDO3 follows the distribution of the central region protein SYCP1 in Sycp3−/− spermatocytes, which lack the axial elements of the synaptonemal complex. This indicates that synapsis is a requirement for DIDO3 incorporation. Interestingly, DIDO3 is missing from the synaptonemal complex in Atm mutant spermatocytes, which form synapses but show persistent trimethylation of histone H3 lysine 4. In order to further address a role of epigenetic modifications in DIDO3 localization, we made a mutant of the Dido gene that produces a truncated DIDO3 protein. This truncated protein, which lacks the histone-binding domain, is incorporated in the synaptonemal complex irrespective of histone trimethylation status. DIDO3 protein truncation in Dido mutant mice causes mild meiotic defects, visible as gaps in the synaptonemal complex, but allows for normal meiotic progression. Our results indicate that histone H3 lysine 4 demethylation modulates DIDO3 localization in meiosis and suggest epigenetic regulation of the synaptonemal complex.     

Inactivation of Nxf2 causes defects in male meiosis and age-dependent depletion of spermatogonia

In eukaryotes, mRNA is actively transported from nucleus to cytoplasm by a family of nuclear RNA export factors (NXF). While yeast harbors only one such factor (Mex67p), higher eukaryotes encode multiple NXFs. In mouse, four Nxf genes have been identified: Nxf1, Nxf2, Nxf3, and Nxf7. To date, the function of mouse Nxf genes has not been studied by targeted gene deletion in vivo. Here we report the generation of Nxf2 null mutant mice by homologous recombination in embryonic stem cells. Nxf2-deficient male mice exhibit fertility defects that differ between mouse strains. One third of Nxf2-deficient males on a mixed (C57BL/6 × 129) genetic background exhibit meiotic arrest and thus are sterile, whereas the remaining males are fertile. Disruption of Nxf2 in inbred (C57BL/6J) males impairs spermatogenesis, resulting in male subfertility, but causes no meiotic arrest. Testis weight and sperm output in C57BL/6J Nxf2^−/Y mice are sharply reduced. Mutant epididymal sperm exhibit diminished motility. Importantly, proliferation of spermatogonia in Nxf2^−/Y mice is significantly decreased. As a result, inactivation of Nxf2 causes depletion of germ cells in a substantial fraction of seminiferous tubules in aged mice. These studies demonstrate that Nxf2 plays a dual function in spermatogenesis: regulation of meiosis and maintenance of spermatogonial stem cells.     

DNA double-strand breaks and gamma-H2AX signaling in the testis

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, gamma-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit gamma-H2AX foci but show homogeneous nuclear gamma-H2AX staining, whereas in pachytene spermatocytes gamma-H2AX is only present in the sex vesicle. In response to ionizing radiation, gamma-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, gamma-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear gamma-H2AX staining in leptotene spermatocytes demonstrates a function for gamma-H2AX during meiosis. gamma-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA double-strand breaks.     

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 ^−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 ^−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 ^−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 ^−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2''s function and as a result leads to infertility and genomic instability in mice.     

Targeted inactivation of testicular nuclear orphan receptor 4 delays and disrupts late meiotic prophase and subsequent meiotic divisions of spermatogenesis

Testicular orphan nuclear receptor 4 (TR4) is specifically and stage-dependently expressed in late-stage pachytene spermatocytes and round spermatids. In the developing mouse testis, the highest expression of TR4 can be detected at postnatal days 16 to 21 when the first wave of spermatogenesis progresses to late meiotic prophase. Using a knockout strategy to delete TR4 in mice, we found that sperm production in TR4(-/-) mice is reduced. The comparison of testes from developing TR4(+/+) and TR4(-/-) mice shows that spermatogenesis in TR4(-/-) mice is delayed. Analysis of the first wave of spermatogenesis shows that the delay can be due to delay and disruption of spermatogenesis at the end of late meiotic prophase and subsequent meiotic divisions. Seminiferous tubule staging shows that stages X to XII, where late meiotic prophase and meiotic divisions take place, are delayed and disrupted in TR4(-/-) mice. Histological examination of testis sections from TR4(-/-) mice shows degenerated primary spermatocytes and some necrotic tubules. Testis-specific gene analyses show that the expression of sperm 1 and cyclin A1, which are genes expressed at the end of meiotic prophase, was delayed and decreased in TR4(-/-) mouse testes. Taken together, results from TR4(+/+) and TR4(-/-) mice indicate that TR4 is essential for normal spermatogenesis in mice.     

Persistence of histone H2AX phosphorylation after meiotic chromosome synapsis and abnormal centromere cohesion in poly (ADP-ribose) polymerase (Parp-1) null oocytes

In spite of the impact of aneuploidy on human health little is known concerning the molecular mechanisms involved in the formation of structural or numerical chromosome abnormalities during meiosis. Here, we provide novel evidence indicating that lack of PARP-1 function during oogenesis predisposes the female gamete to genome instability. During prophase I of meiosis, a high proportion of Parp-1^(−/−) mouse oocytes exhibit a spectrum of meiotic defects including incomplete homologous chromosome synapsis or persistent histone H2AX phosphorylation in fully synapsed chromosomes at the late pachytene stage. Moreover, the X chromosome bivalent is also prone to exhibit persistent double strand DNA breaks (DSBs). In striking contrast, such defects were not detected in mutant pachytene spermatocytes. In fully-grown wild type oocytes at the germinal vesicle stage, PARP-1 protein associates with nuclear speckles and upon meiotic resumption, undergoes a striking re-localization towards spindle poles as well as pericentric heterochromatin domains at the metaphase II stage. Notably, a high proportion of in vivo matured Parp-1^(−/−) oocytes show lack of recruitment of the kinetochore-associated protein BUB3 to centromeric domains and fail to maintain metaphase II arrest. Defects in chromatin modifications in the form of persistent histone H2AX phosphorylation during prophase I of meiosis and deficient sister chromatid cohesion during metaphase II predispose mutant oocytes to premature anaphase II onset upon removal from the oviductal environment. Our results indicate that PARP-1 plays a critical role in the maintenance of chromosome stability at key stages of meiosis in the female germ line. Moreover, in the metaphase II stage oocyte PARP-1 is required for the regulation of centromere structure and function through a mechanism that involves the recruitment of BUB3 protein to centromeric domains.