SUN4 is a spermatid type II inner nuclear membrane protein that forms heteromeric assemblies with SUN3 and interacts with lamin B3.

SUN domain proteins are conserved proteins of the nuclear envelope and key components of the LINC complexes (for 'linkers of the nucleoskeleton and the cytoskeleton'). Previous studies have demonstrated that the testis-specific SUN domain protein SUN4 (also known as SPAG4) is a vital player in the directed shaping of the spermatid nucleus. However, its molecular properties relating to this crucial function have remained largely unknown, and controversial data for the organization and orientation of SUN4 within the spermatid nuclear envelope have been presented so far. Here, we have re-evaluated this issue in detail and show robust evidence that SUN4 is integral to the inner nuclear membrane, sharing a classical SUN domain protein topology. The C-terminal SUN domain of SUN4 localizes to the perinuclear space, whereas the N-terminus is directed to the nucleoplasm, interacting with the spermiogenesis-specific lamin B3. We found that SUN4 forms heteromeric assemblies with SUN3 in vivo and regulates SUN3 expression. Together, our results contribute to a better understanding of the specific function of SUN4 at the spermatid nucleo-cytoplasmic junction and the process of sperm-head formation.

Achiasmatic meiosis in the unisexual Amazon molly, Poecilia formosa.

Unisexual reproduction, which generates clonal offspring, is an alternative strategy to sexual breeding and occurs even in vertebrates. A wide range of non-sexual reproductive modes have been described, and one of the least understood questions is how such pathways emerged and how they mechanistically proceed. The Amazon molly, Poecilia formosa, needs sperm from males of related species to trigger the parthenogenetic development of diploid eggs. However, the mechanism, of how the unreduced female gametes are produced, remains unclear. Cytological analyses revealed that the chromosomes of primary oocytes initiate pachytene but do not proceed to bivalent formation and meiotic crossovers. Comparing ovary transcriptomes of P. formosa and its sexual parental species revealed expression levels of meiosis-specific genes deviating from P. mexicana but not from P. latipinna. Furthermore, several meiosis genes show biased expression towards one of the two alleles from the parental genomes. We infer from our data that in the Amazon molly diploid oocytes are generated by apomixis due to a failure in the synapsis of homologous chromosomes. The fact that this failure is not reflected in the differential expression of known meiosis genes suggests the underlying molecular mechanism may be dysregulation on the protein level or misexpression of a so far unknown meiosis gene, and/or hybrid dysgenesis because of compromised interaction of proteins from diverged genomes.

The testis-specific LINC component SUN3 is essential for sperm head shaping during mouse spermiogenesis.

Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome-manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain-containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype.

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 SMC1ß. 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.

Evolutionary history of the mammalian synaptonemal complex.

The synaptonemal complex (SC), a key structure of meiosis that assembles during prophase I, has been initially described 60 years ago. Since then, the structure has been described in many sexually reproducing organisms. However, the SC protein components were characterized in only few model organisms. Surprisingly, they lacked an apparent evolutionary relationship despite the conserved structural organization of the SC. For better understanding of this obvious discrepancy, the evolutionary history of the SC and its individual components has been investigated in Metazoa in detail. The results are consistent with the notion of a single origin of the metazoan SC and provide evidence for a dynamic evolutionary history of the SC components. In this mini review, we recapitulate and discuss new insights into metazoan SC evolution.

CDK2 regulates nuclear envelope protein dynamics and telomere attachment in mouse meiotic prophase.

In most organisms, telomeres attach to the nuclear envelope at the onset of meiosis to promote the crucial processes of pairing, recombination and synapsis during prophase I. This attachment of meiotic telomeres is mediated by the specific distribution of several nuclear envelope components that interact with the attachment plates of the synaptonemal complex. We have determined by immunofluorescence and electron microscopy that the ablation of the kinase CDK2 alters the nuclear envelope in mouse spermatocytes, and that the proteins SUN1, KASH5 (also known as CCDC155) and lamin C2 show an abnormal cap-like distribution facing the centrosome. Strikingly, some telomeres are not attached to the nuclear envelope but remain at the nuclear interior where they are associated with SUN1 and with nuclear-envelope-detached vesicles. We also demonstrate that mouse testis CDK2 phosphorylates SUN1 in vitro. We propose that during mammalian prophase I the kinase CDK2 is a key factor governing the structure of the nuclear envelope and the telomere-led chromosome movements essential for homolog pairing.

Analysis of meiosis in SUN1 deficient mice reveals a distinct role of SUN2 in mammalian meiotic LINC complex formation and function.

LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun1(-/-) meiocytes attached telomeres retained the capacity to form bouquet-like clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun1(-/-) mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional.

The meiotic nuclear lamina regulates chromosome dynamics and promotes efficient homologous recombination in the mouse.

The nuclear lamina is the structural scaffold of the nuclear envelope and is well known for its central role in nuclear organization and maintaining nuclear stability and shape. In the past, a number of severe human disorders have been identified to be associated with mutations in lamins. Extensive research on this topic has provided novel important clues about nuclear lamina function. These studies have contributed to the knowledge that the lamina constitutes a complex multifunctional platform combining both structural and regulatory functions. Here, we report that, in addition to the previously demonstrated significance for somatic cell differentiation and maintenance, the nuclear lamina is also an essential determinant for germ cell development. Both male and female mice lacking the short meiosis-specific A-type lamin C2 have a severely defective meiosis, which at least in the male results in infertility. Detailed analysis revealed that lamin C2 is required for telomere-driven dynamic repositioning of meiotic chromosomes. Loss of lamin C2 affects precise synapsis of the homologs and interferes with meiotic double-strand break repair. Taken together, our data explain how the nuclear lamina contributes to meiotic chromosome behaviour and accurate genome haploidization on a mechanistic level.

Dynamic localization of SMC5/6 complex proteins during mammalian meiosis and mitosis suggests functions in distinct chromosome processes.

Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase IIα, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIα colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase IIα colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase IIα, in regulating centromere cohesion during meiosis II.

Hydra meiosis reveals unexpected conservation of structural synaptonemal complex proteins across metazoans.

The synaptonemal complex (SC) is a key structure of meiosis, mediating the stable pairing (synapsis) of homologous chromosomes during prophase I. Its remarkable tripartite structure is evolutionarily well conserved and can be found in almost all sexually reproducing organisms. However, comparison of the different SC protein components in the common meiosis model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Mus musculus revealed no sequence homology. This discrepancy challenged the hypothesis that the SC arose only once in evolution. To pursue this matter we focused on the evolution of SYCP1 and SYCP3, the two major structural SC proteins of mammals. Remarkably, our comparative bioinformatic and expression studies revealed that SYCP1 and SYCP3 are also components of the SC in the basal metazoan Hydra. In contrast to previous assumptions, we therefore conclude that SYCP1 and SYCP3 form monophyletic groups of orthologous proteins across metazoans.

Mouse CCDC79 (TERB1) is a meiosis-specific telomere associated protein.

BACKGROUND: Telomeres have crucial meiosis-specific roles in the orderly reduction of chromosome numbers and in ensuring the integrity of the genome during meiosis. One such role is the attachment of telomeres to trans-nuclear envelope protein complexes that connect telomeres to motor proteins in the cytoplasm. These trans-nuclear envelope connections between telomeres and cytoplasmic motor proteins permit the active movement of telomeres and chromosomes during the first meiotic prophase. Movements of chromosomes/telomeres facilitate the meiotic recombination process, and allow high fidelity pairing of homologous chromosomes. Pairing of homologous chromosomes is a prerequisite for their correct segregation during the first meiotic division. Although inner-nuclear envelope proteins, such as SUN1 and potentially SUN2, are known to bind and recruit meiotic telomeres, these proteins are not meiosis-specific, therefore cannot solely account for telomere-nuclear envelope attachment and/or for other meiosis-specific characteristics of telomeres in mammals. RESULTS: We identify CCDC79, alternatively named TERB1, as a meiosis-specific protein that localizes to telomeres from leptotene to diplotene stages of the first meiotic prophase. CCDC79 and SUN1 associate with telomeres almost concurrently at the onset of prophase, indicating a possible role for CCDC79 in telomere-nuclear envelope interactions and/or telomere movements. Consistent with this scenario, CCDC79 is missing from most telomeres that fail to connect to SUN1 protein in spermatocytes lacking the meiosis-specific cohesin SMC1B. SMC1B-deficient spermatocytes display both reduced efficiency in telomere-nuclear envelope attachment and reduced stability of telomeres specifically during meiotic prophase. Importantly, CCDC79 associates with telomeres in SUN1-deficient spermatocytes, which strongly indicates that localization of CCDC79 to telomeres does not require telomere-nuclear envelope attachment. CONCLUSION: CCDC79 is a meiosis-specific telomere associated protein. Based on our findings we propose that CCDC79 plays a role in meiosis-specific telomere functions. In particular, we favour the possibility that CCDC79 is involved in telomere-nuclear envelope attachment and/or the stabilization of meiotic telomeres. These conclusions are consistent with the findings of an independently initiated study that analysed CCDC79/TERB1 functions.

Protein SYCP2 is an ancient component of the metazoan synaptonemal complex.

During the first meiotic prophase, chromosome synapsis is mediated by the synaptonemal complex (SC), an evolutionarily conserved meiosis-specific structure. In mammals, 7 SC protein components have been identified so far. Despite some controversy in the past, we have shown that SC proteins are ancient in metazoans and very likely formed an ancestral SC structure in the ancestor of metazoans. Protein components SYCP1, SYCP3, SYCE2, and TEX12 were identified in basal-branching metazoans, while other components (SYCE1 and SYCE3) are more recent elements. However, the evolutionary history of mammalian SYCP2 is not known. Here, we investigated this aspect with the aid of bioinformatic tools as well as with RNA and protein expression analysis. We conclude that SYCP2 belongs to the group of ancient SC proteins that was already present in the common ancestor of metazoans more than 500 million years ago.

A novel mouse synaptonemal complex protein is essential for loading of central element proteins, recombination, and fertility.

The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE-specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE-specific proteins, which in turn would promote synapsis between homologous chromosomes.

Regulation of meiotic telomere dynamics through membrane fluidity promoted by AdipoR2-ELOVL2.

The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a conserved membrane fluidity sensor, AdipoR2, regulates the meiosis-specific lipidome in mouse testes by promoting the synthesis of sphingolipids containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs). AdipoR2 upregulates the expression of a fatty acid elongase, ELOVL2, both transcriptionally and post-transcriptionally, to synthesize VLC-PUFA. The depletion of VLC-PUFAs and subsequent accumulation of palmitic acid in AdipoR2 knockout testes stiffens the cellular membrane and causes the invagination of the nuclear envelope. This condition impairs the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Further, the stiffened membrane impairs the formation of intercellular bridges and the germ cell syncytium, which disrupts the orderly arrangement of cell types within the seminiferous tubules. According to our findings we propose a framework in which the highly-fluid membrane microenvironment shaped by AdipoR2-ELOVL2 underpins meiosis-specific chromosome dynamics in testes.

The mammalian synaptonemal complex: protein components, assembly and role in meiotic recombination.

The synaptonemal complex (SC) is a proteinaceous structure of chromosome bivalents whose assembly is indispensable for the successful progression of the first meiotic division of sexually reproducing organisms. In this mini-review we will focus on recent progress dealing with the composition and assembly of the mammalian SC. These advances mainly resulted from the systematic use of knockout mice for all known mammalian SC proteins as well as from protein polymerization studies performed in heterologous systems.

Evolution of the Degenerated Y-Chromosome of the Swamp Guppy, Micropoecilia picta.

The conspicuous colour sexual dimorphism of guppies has made them paradigmatic study objects for sex-linked traits and sex chromosome evolution. Both the X- and Y-chromosomes of the common guppy (Poecilia reticulata) are genetically active and homomorphic, with a large homologous part and a small sex specific region. This feature is considered to emulate the initial stage of sex chromosome evolution. A similar situation has been documented in the related Endler's and Oropuche guppies (P. wingei, P. obscura) indicating a common origin of the Y in this group. A recent molecular study in the swamp guppy (Micropoecilia. picta) reported a low SNP density on the Y, indicating Y-chromosome deterioration. We performed a series of cytological studies on M. picta to show that the Y-chromosome is quite small compared to the X and has accumulated a high content of heterochromatin. Furthermore, the Y-chromosome stands out in displaying CpG clusters around the centromeric region. These cytological findings evidently illustrate that the Y-chromosome in M. picta is indeed highly degenerated. Immunostaining for SYCP3 and MLH1 in pachytene meiocytes revealed that a substantial part of the Y remains associated with the X. A specific MLH1 hotspot site was persistently marked at the distal end of the associated XY structure. These results unveil a landmark of a recombining pseudoautosomal region on the otherwise strongly degenerated Y chromosome of M. picta. Hormone treatments of females revealed that, unexpectedly, no sexually antagonistic color gene is Y-linked in M. picta. All these differences to the Poecilia group of guppies indicate that the trajectories associated with the evolution of sex chromosomes are not in parallel.

Protein SYCP2 provides a link between transverse filaments and lateral elements of mammalian synaptonemal complexes.

Synaptonemal complexes (SCs) are evolutionarily conserved meiosis-specific nuclear structures critically involved in synapsis, recombination, and segregation of homologous chromosomes. SCs are proteinaceous structures composed of (a) two lateral elements (LEs), to which the chromatin of the homologs is attached, (b) numerous transverse filaments (TFs) that link the LEs, and (c) a central element (CE). Major protein components of mammalian SCs are the TF protein SYCP1 and the LE proteins SYCP2 and SCYP3. How SCs become assembled is presently poorly understood, in particular, it is not known how TFs assemble at the plane of LEs to interconnect the homologous chromosomes. Therefore, we have investigated possible interactions between SYCP1 and other SC proteins. In immunoprecipitation experiments we could find that SYCP1 and SYCP2 interact in extracts of meiotic cells. Using the yeast two-hybrid system, we were able to demonstrate that the C-terminus of SYCP1 directly interacts with SYCP2. These results were confirmed by different interaction traps. Furthermore, we could narrow down the interacting domain of the SYCP2 molecule to its C-terminal region. We propose that SYCP2 acts as a linker between SYCP1 and SYCP3 and therefore would be the missing connecting link between LEs and TFs essential for proper chromosome synapsis.

Expression of lamin C2 in mammalian oocytes.

Lamin C2 (LMN C2) is a short product of the lamin a gene. It is a germ cell-specific lamin and has been extensively studied in male germ cells. In this study, we focussed on the expression and localization of LMN C2 in fully-grown germinal vesicle (GV) oocytes. We detected LMN C2 in the fully-grown germinal vesicle oocytes of various mammalian species with confirmation done by immunoblotting the wild type and Lmnc2 gene deleted testes. Expression of LMN C2 tagged with GFP showed localization of LMN C2 to the nuclear membrane of the oocyte. Moreover, the LMN C2 protein notably disappeared after nuclear envelope breakdown (NEBD) and the expression of LMN C2 was significantly reduced in the oocytes from aged females and ceased altogether during meiotic maturation. These results provide new insights regarding LMN C2 expression in the oocytes of various mammalian species.

Synaptonemal complex protein SYCP3: Conserved polymerization properties among vertebrates.

Synaptonemal complexes (SCs) are meiosis-specific, nuclear structures that are critically involved in synapsis, recombination and segregation of homologous chromosomes. Although the SC structure is conserved in evolution this is not the case for its protein components. To provide information on SC proteins which would be important for our understanding of the conserved SC structure and function, here we compared ortholog SYCP3 proteins of two evolutionary distant vertebrate species, namely rat and medaka fish. To this end we have investigated the polymerization properties of both proteins by immunocytochemistry, electron microscopy and cell fractionation. We found that despite of the sequence differences that have accumulated over the last 450 million years mammalian and fish SYCP3 have similar properties that allow them to co-assemble higher order structures under experimental conditions. We also provide a likely explanation as to how heterozygous mutations in the SYCP3 gene can lead to a defective meiosis.

Mammalian protein SCP1 forms synaptonemal complex-like structures in the absence of meiotic chromosomes.

Synaptonemal complexes (SCs) are evolutionary conserved, meiosis-specific structures that play a central role in synapsis of homologous chromosomes, chiasmata distribution, and chromosome segregation. However, it is still for the most part unclear how SCs do assemble during meiotic prophase. Major components of mammalian SCs are the meiosis-specific proteins SCP1, 2, and 3. To investigate the role of SCP1 in SC assembly, we expressed SCP1 in a heterologous system, i.e., in COS-7 cells that normally do not express SC proteins. Notably, under these experimental conditions SCP1 is able to form structures that closely resemble SCs (i.e., polycomplexes). Moreover, we show that mutations that modify the length of the central alpha-helical domain of SCP1 influence the width of polycomplexes. Finally, we demonstrate that deletions of the nonhelical N- or C-termini both affect polycomplex assembly, although in a different manner. We conclude that SCP1 is a primary determinant of SC assembly that plays a key role in synapsis of homologous chromosomes.