Meiotic telomere distribution and Sertoli cell nuclear architecture are altered in Atm- and Atm-p53-deficient mice

The ataxia telangiectasia mutant (ATM) protein is an intrinsic part of the cell cycle machinery that surveys genomic integrity and responses to genotoxic insult. Individuals with ataxia telangiectasia as well as Atm(-/-) mice are predisposed to cancer and are infertile due to spermatogenesis disruption during first meiotic prophase. Atm(-/-) spermatocytes frequently display aberrant synapsis and clustered telomeres (bouquet topology). Here, we used telomere fluorescent in situ hybridization and immunofluorescence (IF) staining of SCP3 and testes-specific histone H1 (H1t) to spermatocytes of Atm- and Atm-p53-deficient mice and investigated whether gonadal atrophy in Atm-null mice is associated with stalling of telomere motility in meiotic prophase. SCP3-H1t IF revealed that most Atm(-/-) p53(-/-) spermatocytes degenerated during late zygotene, while a few progressed to pachytene and diplotene and some even beyond metaphase II, as indicated by the presence of a few round spermatids. In Atm(-/-) p53(-/-) meiosis, the frequency of spermatocytes I with bouquet topology was elevated 72-fold. Bouquet spermatocytes with clustered telomeres were generally void of H1t signals, while mid-late pachytene and diplotene Atm(-/-) p53(-/-) spermatocytes displayed expression of H1t and showed telomeres dispersed over the nuclear periphery. Thus, it appears that meiotic telomere movements occur independently of ATM signaling. Atm inactivation more likely leads to accumulation of spermatocytes I with bouquet topology by slowing progression through initial stages of first meiotic prophase and an ensuing arrest and demise of spermatocytes I. Sertoli cells (SECs), which contribute to faithful spermatogenesis, in the Atm mutants were found to frequently display numerous heterochromatin and telomere clusters-a nuclear topology which resembles that of immature SECs. However, Atm(-/-) SECs exhibited a mature vimentin and cytokeratin 8 intermediate filament expression signature. Upon IF with ATM antibodies, we observed ATM signals throughout the nuclei of human and mouse SECs, spermatocytes I, and haploid round spermatids. ATM but not H1t was absent from elongating spermatid nuclei. Thus, ATM appears to be removed from spermatid nuclei prior to the occurrence of DNA nicks which emanate as a consequence of nucleoprotamine formation.     

The temporal and spatial distribution of the proliferation associated Ki-67 protein during female and male meiosis

We used immunolocalization in tissue sections and cytogenetic preparations of female and male gonads to study the distribution of the proliferation marker pKi-67 during meiotic cell cycles of the house mouse, Mus musculus. During male meiosis, pKi-67 was continuously present in nuclei of all stages from the spermatogonium through spermatocytes I and II up to the earliest spermatid stage (early round spermatids) when it appeared to fade out. It was not detected in later spermatid stages or sperm. During female meiosis, pKi-67 was present in prophase I oocytes of fetal ovaries. It was absent in oocytes from newborn mice and most oocytes of primordial follicles from adults. The Ki-67 protein reappeared in oocytes of growing follicles and was continuously present up to metaphase II. Thus, pKi-67 was present in all stages of cell growth and cell division while it was absent from resting oocytes and during the main stages of spermiocytogenesis. Progression through the meiotic cell cycle was associated with extensive intranuclear relocation of pKi-67. In the zygotene and pachytene stages, most of the pKi-67 colocalized with centromeric (centric and pericentric) heterochromatin and adjacent nucleoli; the heterochromatic XY body in male pachytene, however, was free of pKi-67. At early diplotene, pKi-67 was mainly associated with nucleoli. At late diplotene, diakinesis, metaphase I and metaphase II of meiosis, pKi-67 preferentially bound to the perichromosomal layer and was almost absent from the heterochromatic centromeric regions of the chromosomes. After the second division of male meiosis, the protein reappeared at the centromeric heterochromatin and an adjacent region in the earliest spermatid stage and then faded out. The general patterns of pKi-67 distribution were comparable to those in mitotic cell cycles. With respect to the timing, it is interesting to note that relocation from the nucleolus to the perichromosomal layer takes place at the G2/M-phase transition in the mitotic cell cycle but at late diplotene of prophase I in meiosis, suggesting physiological similarity of these stages.     

Novel phosphorylation of histone H3 at threonine 11 that temporally correlates with condensation of mitotic and meiotic chromosomes in plant cells

A novel mitosis-specific phosphorylation site in histone H3 at threonine 11 has been described for mammalian cells. This modification is restricted to the centromeric region while phosphorylation at the classical H3 sites, Ser10 and Ser28 occurs along the entire chromosomal arms. Using phosphorylation state-specific antibodies we found that phosphorylation at threonine 11 occurs also in plant cells, during mitosis as well as meiosis. However, in contrast to animal cells, ph(Thr11)H3 was distributed along the entire length of condensed chromosomes, whereas H3 phosphorylated at Ser10 and Ser28 appeared to be restricted to centromeric/pericentromeric chromatin. Phosphorylation at Thr11 started in prophase and ended in telophase, it correlated with the condensation of mitotic and meiotic chromosomes and was independent of the distribution of late replicating heterochromatin and Giemsa-banding positive regions. Interestingly, treatment of cells with the phosphatase inhibitor cantharidin revealed a high level of Thr11 phosphorylation in interphase cells, in this case particularly in pericentromeric regions. These data show that histone modifications are highly dynamic. Moreover, animal and plant organisms may have evolved individual histone codes.     

Cathepsin L stabilizes the histone modification landscape on the Y chromosome and pericentromeric heterochromatin

Posttranslational histone modifications and histone variants form a unique epigenetic landscape on mammalian chromosomes where the principal epigenetic heterochromatin markers, trimethylated histone H3(K9) and the histone H2A.Z, are inversely localized in relation to each other. Trimethylated H3(K9) marks pericentromeric constitutive heterochromatin and the male Y chromosome, while H2A.Z is dramatically reduced at these chromosomal locations. Inactivation of a lysosomal and nuclear protease, cathepsin L, causes a global redistribution of epigenetic markers. In cathepsin L knockout cells, the levels of trimethylated H3(K9) decrease dramatically, concomitant with its relocation away from heterochromatin, and H2A.Z becomes enriched at pericentromeric heterochromatin and the Y chromosome. This change is also associated with global relocation of heterochromatin protein HP1 and histone H3 methyltransferase Suv39h1 away from constitutive heterochromatin; however, it does not affect DNA methylation or chromosome segregation, phenotypes commonly associated with impaired histone H3(K9) methylation. Therefore, the key constitutive heterochromatin determinants can dynamically redistribute depending on physiological context but still maintain the essential function(s) of chromosomes. Thus, our data show that cathepsin L stabilizes epigenetic heterochromatin markers on pericentromeric heterochromatin and the Y chromosome through a novel mechanism that does not involve DNA methylation or affect heterochromatin structure and operates on both somatic and sex chromosomes.     

Regulation of meiosis during mammalian spermatogenesis: the A-type cyclins and their associated cyclin-dependent kinases are differentially expressed in the germ-cell lineage

To begin to examine the function of the A-type cyclins during meiosis in the male, we have examined the developmental and cellular distribution of the cyclin A1 and cyclin A2 proteins, as well as their candidate cyclin-dependent kinase partners, Cdk1 and Cdk2, in the spermatogenic lineage. Immunohistochemical localization revealed that cyclin A1 is present only in male germ cells just prior to or during the first, but not the second, meiotic division. By contrast, cyclin A2 was expressed in spermatogonia and was most abundant in preleptotene spermatocytes, cells which will enter the meiotic pathway. Immunohistochemical detection of Cdk1 was most apparent in early pachytene spermatocytes, while staining intensity diminished in diplotene and meiotically dividing spermatocytes, the cells in which cyclin A1 expression was strongest. Cdk2 was highly expressed in all spermatocytes. Notably, in cells undergoing the meiotic reduction divisions, Cdk2 appeared to localize specifically to the chromatin. This was not the case for spermatogonia undergoing mitotic divisions. In the testis, cyclin A1 has been shown to bind both Cdk1 and Cdk2 but we show here that cyclin A2 binds only Cdk2. These results indicate that the A-type cyclins and their associated kinases have different functions in the initiation and passage of male germ cells through meiosis.     

Expression and secretion of human apolipoprotein A-I in the heart

Nuclear envelope-peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino-terminal sequencing show that one of these activities co-isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post-translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio-temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.     

Mammalian E-type Cyclins Control Chromosome Pairing,Telomere Stability and CDK2 Localization in Male Meiosis

Loss of function of cyclin E1 or E2, important regulators of the mitotic cell cycle, yields viable mice, but E2-deficient males display reduced fertility. To elucidate the role of E-type cyclins during spermatogenesis, we characterized their expression patterns and produced additional deletions of Ccne1 and Ccne2 alleles in the germline, revealing unexpected meiotic functions. While Ccne2 mRNA and protein are abundantly expressed in spermatocytes, Ccne1 mRNA is present but its protein is detected only at low levels. However, abundant levels of cyclin E1 protein are detected in spermatocytes deficient in cyclin E2 protein. Additional depletion of E-type cyclins in the germline resulted in increasingly enhanced spermatogenic abnormalities and corresponding decreased fertility and loss of germ cells by apoptosis. Profound meiotic defects were observed in spermatocytes, including abnormal pairing and synapsis of homologous chromosomes, heterologous chromosome associations, unrepaired double-strand DNA breaks, disruptions in telomeric structure and defects in cyclin-dependent-kinase 2 localization. These results highlight a new role for E-type cyclins as important regulators of male meiosis.     

HP1γ links histone methylation marks to meiotic synapsis in mice

During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri- and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9a- and Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2-mediated H3K9me3 and its recognition by the Cbx3 gene product HP1γ. We further found that centromere clustering and synapsis were commonly affected in G9a- and Cbx3-deficient spermatocytes. These genetic observations suggest that HP1γ/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1γ/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.     

Maturation promoting factor destabilization mediates human chorionic gonadotropin induced meiotic resumption in rat oocytes

Human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone (LH) and triggers meiotic maturation and ovulation in mammals. The mechanism by which hCG triggers meiotic resumption in mammalian oocytes remains poorly understood. We aimed to find out the impact of hCG surge on morphological changes, adenosine 3′,5′‐cyclic monophosphate (cAMP), guanosine 3′,5′‐cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), Wee1, early mitotic inhibitor 2 (Emi2), anaphase‐promoting complex/cyclosome (APC/C), meiotic arrest deficient protein 2 (MAD2), phosphorylation status of cyclin‐dependent kinase 1 (Cdk1), its activity and cyclin B1 expression levels during meiotic resumption from diplotene as well as metaphase‐II (M‐II) arrest in cumulus oocyte complexes (COCs). Our data suggest that hCG surge increased cyclic nucleotides level in encircling granulosa cells but decreased their level in oocyte. The reduced intraoocyte cyclic nucleotides level is associated with the decrease of Cdc25B, Thr161 phosphorylated Cdk1 and Emi2 expression levels. On the other hand, hCG surge increased Wee1, Thr14/Tyr15 phosphorylated Cdk1, APC/C as well as MAD2 expression levels. The elevated APC/C activity reduced cyclin B1 level. The changes in phosphorylation status of Cdk1 and reduced cyclin B1 level might have resulted in maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggered resumption of meiosis from diplotene as well as M‐II arrest in rat oocytes.     

Abnormal sperm development in pcd 3J-/- mice: the importance of Agtpbp1 in spermatogenesis

Homozygous Purkinje cell degeneration (pcd) mutant males exhibit abnormal sperm development. Microscopic examination of the testes from pcd(3J)-/- mice at postnatal days 12, 15, 18 and 60 revealed histological differences, in comparison to wild-type mice, which were evident by day 18. Greatly reduced numbers of spermatocytes and spermatids were found in the adult testes, and apoptotic cells were identified among the differentiating germ cells after day 15. Our immunohistological analysis using an antihuman AGTPBP1 antibody showed that AGTPBP1 was expressed in spermatogenic cells between late stage primary spermatocytes and round spermatids. A global gene expression analysis from the testes of pcd(3J)-/- mice showed that expression of cyclin B3 and de-ubiquitinating enzymes USP2 and USP9y was altered by >1.5-fold compared to the expression levels in the wild-type. Our results suggest that the pcd mutant mice have defects in spermatogenesis that begin with the pachytene spermatocyte stage and continue through subsequent stages. Thus, Agtpbp1, the gene responsible for the pcd phenotype, plays an important role in spermatogenesis and is important for survival of germ cells at spermatocytes stage onward.     

Histone Phosphorylation and Pericentromeric Histone Modifications in Oocyte Meiosis

Epigenetic regulation of pericentromeric heterochromatin is crucial for proper interactions between kinetochores and spindle microtubules governing accurate chromosome segregation. Here, we first examined the dynamic distribution of phosphorylated serine 10 and 28 on H3 during mouse oocyte maturation and early embryo development using immunofluorescent staining and confocal microscopy. Our results revealed strong signals of phosphorylated H3/ser10 and 28 in the pericentromeric heterochromatin area and continuous persistent staining of the chromosome periphery, respectively. A panel of specific antibodies against various acetylated lysine, dimethylated lysine or phosphorylated serine residues on histone H3 or H4 were used to investigate the effects of Trichostatin A (TSA), a general inhibitor of histone deacetylases (HDACs), on histone modifications of pericentromeric heterochromatin. Unexpectedly, TSA treatment was unable to alter the acetylation and methylation status of pericentromeric heterochromatin, however, it resulted in significant dephosphorylation of H3/ser10 at this site during mouse oocyte meiosis, which is likely to play a role in the TSA-induced defective chromosome segregation. Furthermore, by using ZM447439, an inhibitor of Aurora kinases, we revealed that Aurora kinases may participate in the regulation of histone phosphorylation during mouse oocyte maturation.     

Testicular germ cell apoptosis in Bcl6-deficient mice

Bcl6 protein has been detected in testicular germ cells, mainly spermatocytes, of normal mice, but its physiological role is largely unknown. The number of spermatozoa in the cauda epididymis of adult Bcl6-deficient (Bcl6-/-) mice is lower than that of Bcl6+/+ mice. We have found numerous apoptotic spermatocytes at the metaphase I stage with induction of Bax protein in adult Bcl6-/- testes. Developmentally, the incidence of germ cell apoptosis of Bcl6-/- mice was similar to that of Bcl6+/+ mice until six weeks of age and increased after eight weeks of age. The incidence of apoptosis in heterozygous Bcl6+/- mice was also higher than that of Bcl6+/+ mice. Since the activated form of p38 MAP kinase was detected in spermatocytes of adult Bcl6-/- mice, the germ cell apoptosis may be induced by stressors. Treatment of testes of adult Bcl6+/+ mice with a mild hyperthermia resulted in germ cell apoptosis predominantly in metaphase I spermatocytes with induction of Bax protein and activation of p38 MAP kinase and this apoptosis mimics that in adult Bcl6-/- mice. Thus, Bcl6 may play a role as a stabilizer in protecting spermatocytes from apoptosis induced by stressors.     

Reinterpreting pericentromeric heterochromatin

In fission yeast, pericentromeric heterochromatin is directly responsible for the sister chromatid cohesion that assures accurate chromosome segregation. In plants, however, heterochromatin and chromosome segregation appear to be largely unrelated: chromosome transmission is impaired by mutations in cohesion but not by mutations that affect heterochromatin formation. We argue that the formation of pericentromeric heterochromatin is primarily a response to constraints on chromosome mechanics that disfavor the transmission of recombination events in pericentromeric regions. This effect allows pericentromeres to expand to enormous sizes by the accumulation of transposons and through large-scale insertions and inversions. Although sister chromatid cohesion is spatially limited to pericentromeric regions at mitosis and meiosis II, the cohesive domains appear to be defined independently of heterochromatin. The available data from plants suggest that sister chromatid cohesion is marked by histone phosphorylation and mediated by Aurora kinases.     

Molecular cloning and characterization of SRG-L, a novel mouse gene developmentally expressed in spermatogenic cells

Full-length cDNA of a novel mouse gene upregulated in late stages of spermatogenic cells was cloned from mouse testis using overlapping RT-PCR and RACE. The mRNA of the gene was expressed mainly in diplotene/pachytene spermatocytes, round and elongating spermatids. We named this gene as SRG-L (Spermatogenesis Related Gene expressed in late stages of spermatogenic cells, GenBank Accession No. AY352586). The tissue-specific analysis showed a higher expression level in testis and spleen. The gene is mapped on chromosome 8q33.1 and contains 18 exons. The full-length of cDNA is 2,843 bp with an open reading frame (ORF) of 2,625 bp that encodes a 104 kDa protein (874 amino acids) with a putative transmembrane region. The bioinformatics analysis revealed that the SRG-L has two conserved regions, transglutaminase-like homologues domain and D-serine dehydratase domain, rich phosphorylation sites and methylation sites. The SRG-L protein was detected in diplotene/pachytene spermatocytes and spermatids by immunohistochemical staining and Western blot. The results suggest that SRG-L may play definite roles regulating differentiation of germ cells during spermatogenesis, particularly during meiosis and spermiogenesis.     

Elimination of male germ cells in transgenic mice by the diphtheria toxin A chain gene directed by the histone H1t promoter

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.     

Pericentromeric heterochromatin domains are maintained without accumulation of HP1

The heterochromatin protein 1 (HP1) family is thought to be an important structural component of heterochromatin. HP1 proteins bind via their chromodomain to nucleosomes methylated at lysine 9 of histone H3 (H3K9me). To investigate the role of HP1 in maintaining heterochromatin structure, we used a dominant negative approach by expressing truncated HP1alpha or HP1beta proteins lacking a functional chromodomain. Expression of these truncated HP1 proteins individually or in combination resulted in a strong reduction of the accumulation of HP1alpha, HP1beta, and HP1gamma in pericentromeric heterochromatin domains in mouse 3T3 fibroblasts. The expression levels of HP1 did not change. The apparent displacement of HP1alpha, HP1beta, and HP1gamma from pericentromeric heterochromatin did not result in visible changes in the structure of pericentromeric heterochromatin domains, as visualized by DAPI staining and immunofluorescent labeling of H3K9me. Our results show that the accumulation of HP1alpha, HP1beta, and HP1gamma at pericentromeric heterochromatin domains is not required to maintain DAPI-stained pericentromeric heterochromatin domains and the methylated state of histone H3 at lysine 9 in such heterochromatin domains.