Male Mouse Meiosis Research Articles

The DNA helicase FANCJ (BRIP1) functions in double strand break repair processing, but not crossover formation during prophase I of meiosis in male mice.

Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events.

Both 20S and 19S components of proteasome are essential for the meiosis in male mice

Both 20S and 19S components of proteasome are essential for the meiosis in male mice

The Organotypic Culture of Mouse Seminiferous Tubules as a Reliable Methodology for the Study of Meiosis In Vitro.

Male mouse meiosis has been traditionally studied using descriptive methods like histological sections and spreading or squashing techniques, which allow the observation of fixed meiocytes in either wildtype or genetically modified mice. For these studies, the sacrifice of the males and the extraction of the testicles are required to obtain the material of study. Other functional in vivo studies include the administration of intravenous or intraperitoneal drugs, or the exposure to mutagenic agents or generators of DNA damage, in order to study their impact on meiosis progression. However, in these studies, the exposure times or drug concentration are important limitations to consider when acknowledging animal welfare. Recently, several approaches have been proposed to offer alternative methodologies that allow the in vitro study of spermatocytes with a considerable reduction in the use of animals. Here we revisit and validate an optimal technique of organotypic culture of fragments of seminiferous tubules for meiotic studies. This technique is a trustable methodology to develop functional studies that preserve the histological configuration of the seminiferous tubule, aim homogeneity of the procedures (the use of the same animal for different study conditions), and allow procedures that would compromise the animal welfare. Therefore, this methodology is highly recommendable for the study of meiosis and spermatogenesis, while it supports the principle of 3R's for animal research.

Kinase PLK1 regulates the disassembly of the lateral elements and the assembly of the inner centromere during the diakinesis/metaphase I transition in male mouse meiosis.

PLK1 is a serine/threonine kinase with crucial roles during mitosis. However, its involvement during mammalian male meiosis remains largely unexplored. By inhibiting the kinase activity of PLK1 using BI 2536 on organotypic cultures of seminiferous tubules, we found that the disassembly of SYCP3 and HORMAD1 from the lateral elements of the synaptonemal complex during diakinesis is impeded. We also found that the normal recruitment of SYCP3 and HORMAD1 to the inner centromere in prometaphase I spermatocytes did not occur. Additionally, we analyzed the participation of PLK1 in the assembly of the inner centromere by studying its implication in the Bub1-H2AT120ph-dependent recruitment of shugoshin SGO2, and the Haspin-H3T3ph-dependent recruitment of Aurora B/C and Borealin. Our results indicated that both pathways are regulated by PLK1. Altogether, our results demonstrate that PLK1 is a master regulator of the late prophase I/metaphase I transition in mouse spermatocytes.

The Male Mouse Meiotic Cilium Emanates from the Mother Centriole at Zygotene Prior to Centrosome Duplication

Cilia are hair-like projections of the plasma membrane with an inner microtubule skeleton known as axoneme. Motile cilia and flagella beat to displace extracellular fluids, playing important roles in the airways and reproductive system. On the contrary, primary cilia function as cell-type-dependent sensory organelles, detecting chemical, mechanical, or optical signals from the extracellular environment. Cilia dysfunction is associated with genetic diseases called ciliopathies and with some types of cancer. Cilia have been recently identified in zebrafish gametogenesis as an important regulator of bouquet conformation and recombination. However, there is little information about the structure and functions of cilia in mammalian meiosis. Here we describe the presence of cilia in male mouse meiotic cells. These solitary cilia formed transiently in 20% of zygotene spermatocytes and reached considerable lengths (up to 15-23 µm). CEP164 and CETN3 localization studies indicated that these cilia emanate from the mother centriole prior to centrosome duplication. In addition, the study of telomeric TFR2 suggested that cilia are not directly related to the bouquet conformation during early male mouse meiosis. Instead, based on TEX14 labeling of intercellular bridges in spermatocyte cysts, we suggest that mouse meiotic cilia may have sensory roles affecting cyst function during prophase I.

CLPP Depletion Causes Diplotene Arrest; Underlying Testis Mitochondrial Dysfunction Occurs with Accumulation of Perrault Proteins ERAL1, PEO1, and HARS2.

Human Perrault syndrome (PRLTS) is autosomal, recessively inherited, and characterized by ovarian insufficiency with hearing loss. Among the genetic causes are mutations of matrix peptidase CLPP, which trigger additional azoospermia. Here, we analyzed the impact of CLPP deficiency on male mouse meiosis stages. Histology, immunocytology, different OMICS and biochemical approaches, and RT-qPCR were employed in CLPP-null mouse testis. Meiotic chromosome pairing and synapsis proceeded normally. However, the foci number of the crossover marker MLH1 was slightly reduced, and foci persisted in diplotene, most likely due to premature desynapsis, associated with an accumulation of the DNA damage marker γH2AX. No meiotic M-phase cells were detected. Proteome profiles identified strong deficits of proteins involved in male meiotic prophase (HSPA2, SHCBP1L, DMRT7, and HSF5), versus an accumulation of AURKAIP1. Histone H3 cleavage, mtDNA extrusion, and cGAMP increase suggested innate immunity activation. However, the deletion of downstream STING/IFNAR failed to alleviate pathology. As markers of underlying mitochondrial pathology, we observed an accumulation of PRLTS proteins ERAL1, PEO1, and HARS2. We propose that the loss of CLPP leads to the extrusion of mitochondrial nucleotide-binding proteins to cytosol and nucleus, affecting late meiotic prophase progression, and causing cell death prior to M-phase entry. This phenotype is more severe than in mito-mice or mutator-mice.

Global phosphoproteomic analysis identified key kinases regulating male meiosis in mouse.

Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation, there is a lack of systemic characterization of phosphorylation regulation of meiosis in mammals. Using the phosphoproteomic approach, we profiled large-scale phosphoproteome of purified primary spermatocytes undergoing meiosis I, and identified 14,660 phosphorylation sites in 4419 phosphoproteins. Kinase-substrate phosphorylation network analysis followed by in vitro meiosis study showed that CDK9 was essential for meiosis progression to metaphase I and had enriched substrate phosphorylation sites in proteins involved in meiotic cell cycle. In addition, histones and epigenetic factors were found to be widely phosphorylated. Among those, HASPIN was found to be essential for male fertility. Haspin knockout led to misalignment of chromosomes, apoptosis of metaphase spermatocytes and a decreased number of sperm by deregulation of H3T3ph, chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC). The complicated protein phosphorylation and its important regulatory functions in meiosis indicated that in-depth studies of phosphorylation-mediated signaling could help us elucidate the mechanisms of meiosis.

Haspin participates in AURKB recruitment to centromeres and contributes to chromosome congression in male mouse meiosis.

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This essential step regulates the accuracy of cell division and must therefore be precisely regulated. One of the main centromeric regulatory signaling pathways is the haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. During mitosis, the haspin kinase phosphorylates histone H3 at threonine 3 (H3T3ph), an essential epigenetic mark that recruits the CPC, in which the catalytic component is Aurora B kinase (AURKB). However, the centromeric haspin-H3T3ph-CPC pathway remains largely uncharacterized in mammalian male meiosis. We have analyzed haspin functions by either its chemical inhibition with LDN-192960 in cultured spermatocytes, or the ablation of the Haspin gene in Haspin-/- mice. Our studies suggest that haspin kinase activity is required for proper chromosome congression both during meiotic divisions and for the recruitment of Aurora B and kinesin MCAK (also known as KIF2C) to meiotic centromeres. However, the absence of H3T3ph histone mark does not alter borealin (or CDCA8) and SGO2 centromeric localization. These results add new and relevant information regarding the regulation of the haspin-H3T3ph-CPC pathway and centromere function during meiosis.

Trim41 is required to regulate chromosome axis protein dynamics and meiosis in male mice.

Meiosis is a hallmark event in germ cell development that accompanies sequential events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 knockout (KO) spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome. Furthermore, mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 (ΔRING-TRIM41) and found that ΔRING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result suggested that TRIM41 exerts its function on the chromosome axes. Our study revealed that Trim41 is essential for preventing SYCP3 overloading, suggesting a TRIM41-mediated mechanism for regulating chromosome axis protein dynamics during male meiotic progression.

KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.

PLK1 regulates centrosome migration and spindle dynamics in male mouse meiosis

Cell division requires the regulation of karyokinesis and cytokinesis, which includes an essential role of the achromatic spindle. Although the functions of centrosomes are well characterised in somatic cells, their role during vertebrate spermatogenesis remains elusive. We have studied the dynamics of the meiotic centrosomes in male mouse during both meiotic divisions. Results show that meiotic centrosomes duplicate twice: first duplication occurs in the leptotene/zygotene transition, while the second occurs in interkinesis. The maturation of duplicated centrosomes during the early stages of prophase I and II are followed by their separation and migration to opposite poles to form bipolar spindles I and II. The study of the genetic mouse model Plk1(Δ/Δ) indicates a central role of Polo-like kinase 1 in pericentriolar matrix assembly, in centrosome maturation and migration, and in the formation of the bipolar spindles during spermatogenesis. In addition, in vitro inhibition of Polo-like kinase 1 and Aurora A in organotypic cultures of seminiferous tubules points out to a prominent role of both kinases in the regulation of the formation of meiotic bipolar spindles.

PDS 5 proteins regulate the length of axial elements and telomere integrity during male mouse meiosis

Cohesin cofactors regulate the loading, maintenance, and release of cohesin complexes from chromosomes during mitosis but little is known on their role during vertebrate meiosis. One such cofactor is PDS5, which exists as two paralogs in somatic and germline cells, PDS5A and PDS5B, with unclear functions. Here, we have analyzed their distribution and functions in mouse spermatocytes. We show that simultaneous excision of Pds5A and Pds5B results in severe defects during early prophase I while their individual depletion does not, suggesting their functional redundancy. Shortened axial/lateral elements and a reduction of early recombination nodules are observed after the strong depletion of PDS5A/B proteins. Moreover, telomere integrity and their association to the nuclear envelope are severely compromised. As these defects occur without detectable reduction in chromosome-bound cohesin, we propose that the dynamic behavior of the complex, mediated by PDS5 proteins, is key for successful completion of meiotic prophase I.

Histone methyltransferase PRDM9 is not essential for meiosis in male mice.

A hallmark of meiosis is the rearrangement of parental alleles to ensure genetic diversity in the gametes. These chromosome rearrangements are mediated by the repair of programmed DNA double-strand breaks (DSBs) as genetic crossovers between parental homologs. In mice, humans, and many other mammals, meiotic DSBs occur primarily at hotspots, determined by sequence-specific binding of the PRDM9 protein. Without PRDM9, meiotic DSBs occur near gene promoters and other functional sites. Studies in a limited number of mouse strains showed that functional PRDM9 is required to complete meiosis, but despite its apparent importance, Prdm9 has been repeatedly lost across many animal lineages. Both the reason for mouse sterility in the absence of PRDM9 and the mechanism by which Prdm9 can be lost remain unclear. Here, we explore whether mice can tolerate the loss of Prdm9. By generating Prdm9 functional knockouts in an array of genetic backgrounds, we observe a wide range of fertility phenotypes and ultimately demonstrate that PRDM9 is not required for completion of male meiosis. Although DSBs still form at a common subset of functional sites in all mice lacking PRDM9, meiotic outcomes differ substantially. We speculate that DSBs at functional sites are difficult to repair as a crossover and that by increasing the efficiency of crossover formation at these sites, genetic modifiers of recombination rates can allow for meiotic progression. This model implies that species with a sufficiently high recombination rate may lose Prdm9 yet remain fertile.

Transition from a meiotic to a somatic-like DNA damage response during the pachytene stage in mouse meiosis.

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms can also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of γH2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid-pachytene to diplotene, γH2AX appeared as large discrete foci. This late repair pattern was mediated initially by NHEJ, involving Ku70 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early “meiotic” repair pathway that acts by default at the beginning of meiosis is replaced from mid-pachytene onwards by a “somatic-like” repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, changes in the gene expression profile and acquisition of competence to proceed to metaphase.

ATR is a multifunctional regulator of male mouse meiosis

Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis.

Transient reduction of DNA methylation at the onset of meiosis in male mice

BackgroundMeiosis is a specialized germ cell cycle that generates haploid gametes. In the initial stage of meiosis, meiotic prophase I (MPI), homologous chromosomes pair and recombine. Extensive changes in chromatin in MPI raise an important question concerning the contribution of epigenetic mechanisms such as DNA methylation to meiosis. Interestingly, previous studies concluded that in male mice, genome-wide DNA methylation patters are set in place prior to meiosis and remain constant subsequently. However, no prior studies examined DNA methylation during MPI in a systematic manner necessitating its further investigation.ResultsIn this study, we used genome-wide bisulfite sequencing to determine DNA methylation of adult mouse spermatocytes at all MPI substages, spermatogonia and haploid sperm. This analysis uncovered transient reduction of DNA methylation (TRDM) of spermatocyte genomes. The genome-wide scope of TRDM, its onset in the meiotic S phase and presence of hemimethylated DNA in MPI are all consistent with a DNA replication-dependent DNA demethylation. Following DNA replication, spermatocytes regain DNA methylation gradually but unevenly, suggesting that key MPI events occur in the context of hemimethylated genome. TRDM also uncovers the prior deficit of DNA methylation of LINE-1 retrotransposons in spermatogonia resulting in their full demethylation during TRDM and likely contributing to the observed mRNA and protein expression of some LINE-1 elements in early MPI.ConclusionsOur results suggest that contrary to the prevailing view, chromosomes exhibit dynamic changes in DNA methylation in MPI. We propose that TRDM facilitates meiotic prophase processes and gamete quality control.

Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells

The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3+/− germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse.

Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes.

The distribution and regulation of the cohesin complexes have been extensively studied during mitosis. However, the dynamics of their different regulators in vertebrate meiosis is largely unknown. In this work, we have analyzed the distribution of the regulatory factor Sororin during male mouse meiosis. Sororin is detected at the central region of the synaptonemal complex during prophase I, in contrast with the previously reported localization of other cohesin components in the lateral elements. This localization of Sororin depends on the transverse filaments protein SYCP1, but not on meiosis-specific cohesin subunits REC8 and SMC1β. By late prophase I, Sororin accumulates at centromeres and remains there up to anaphase II The phosphatase activity of PP2A seems to be required for this accumulation. We hypothesize that Sororin function at the central region of the synaptonemal complex could be independent on meiotic cohesin complexes. In addition, we suggest that Sororin participates in the regulation of centromeric cohesion during meiosis in collaboration with SGO2-PP2A.

Male idiopathic infertility and the TP53 polymorphism in codon 72.

Many environmental agents affect the development of male germ cells at different stages. Apoptosis is common during normal spermatogenesis; it plays an important role in controlling the number of germ cells and the disposal of defective stem cells to produce functional sperm. The presence of p53 in primary spermatocytes suggests that it plays a role in the prophase of meiosis. p53 is expressed in the testis in both spermatocytes and spermatogonia. This suggests that the p53 gene (TP53) is important for apoptosis regulation during spermatogenesis, and may be associated with male infertility. The main causes of male infertility are genetic, physical, and pathological abnormalities, intense and prolonged exercise, aging, drug use, and long periods of sexual abstinence. Approximately 20% of male infertility is idiopathic. The Trp53 gene is involved in meiosis in male rats and mice suggesting that the p53 plays a critical role in spermatogenesis. We investigated the association between the TP53 polymorphism in codon 72 and idiopathic male infertility in 208 semen samples: 106 showed abnormal semen analysis results and were from infertile men, and 102 were from fertile individuals (the control group). Changes in Trp53 expression are associated with the main phase regulating meiotic progression with a peak in the pachytene stage, and Trp53-deficient mice exhibit degenerative syndrome (giant cells). The genotypic and allelic frequencies were not significantly different among the groups in this study; the results suggest that the TP53 polymorphism in codon 72 is not associated with the pathogenesis of idiopathic male infertility or failure of spermatogenesis.

The epigenetic processes of meiosis in male mice are broadly affected by the widely used herbicide atrazine

BackgroundEnvironmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. We studied the effects of the widely used herbicide atrazine (ATZ) on meiosis, a key step of gametogenesis, in male mice.MethodsGene expression pattern was analysed by Gene–Chip array. Genome-wide mapping of H3K4me3 marks distribution was done by ChIP-sequencing of testis tissue using Illumina technologies. RT-qPCR was used to validate differentially expressed genes or differential peaks.ResultsWe demonstrate that exposure to ATZ reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. Using Gene-Chip and ChIP-Seq analysis of H3K4me3 marks, we found that a broad range of cellular functions, including GTPase activity, mitochondrial function and steroid-hormone metabolism, are affected by ATZ. Furthermore, treated mice display enriched histone H3K4me3 marks in regions of strong recombination (double-strand break sites), within very large genes and reduced marks in the pseudoautosomal region of X chromosome.ConclusionsOur data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2095-y) contains supplementary material, which is available to authorized users.