Synaptonemal Complex in Human Biology and Disease.

The synaptonemal complex (SC) is a meiosis-specific multiprotein complex that forms between homologous chromosomes during prophase of meiosis I. Upon assembly, the SC mediates the synapses of the homologous chromosomes, leading to the formation of bivalents, and physically supports the formation of programmed double-strand breaks (DSBs) and their subsequent repair and maturation into crossovers (COs), which are essential for genome haploidization. Defects in the assembly of the SC or in the function of the associated meiotic recombination machinery can lead to meiotic arrest and human infertility. The majority of proteins and complexes involved in these processes are exclusively expressed during meiosis or harbor meiosis-specific subunits, although some have dual functions in somatic DNA repair and meiosis. Consistent with their functions, aberrant expression and malfunctioning of these genes have been associated with cancer development. In this review, we focus on the significance of the SC and their meiotic-associated proteins in human fertility, as well as how human genetic variants encoding for these proteins affect the meiotic process and contribute to infertility and cancer development.

The Oncogenic FOXL2 C134W Mutation Is a Key Driver of Granulosa Cell Tumors.

Adult-type granulosa cell tumors (AGCT) are the most common type of malignant ovarian sex cord-stromal tumors. Most AGCTs carry the somatic variant c.402C>G (p.C134W) affecting the transcription factor FOXL2. Germline dominant variants in FOXL2 are responsible for blepharophimosis syndrome, which is characterized by underdevelopment of the eyelid. In this work, we generated a mouse model harboring the C134W variant of FOXL2 to evaluate in vivo the poorly understood oncogenic role of FOXL2. The mutation was dominant regarding eyelid hypoplasia, reminiscent of blepharophimosis syndrome. Interestingly, Foxl2+/C134W female mice had reduced fertility and developed AGCTs through a progression from abnormal ovaries with aberrant granulosa cells to ovaries with stromal hyperplasia and atypia and on to tumors in adut mice. The genes dysregulated in mouse AGCTs exhibited the hallmarks of cancer and were consistent with a gain-of-function of the mutated allele affecting TGFß signaling. A comparison of these data with previous results on human AGCTs indicated similar deregulated pathways. Finally, a mutational analysis of mouse AGCT transcriptomic data suggested the absence of additional driver mutations apart from FOXL2-C134W. These results provide a clear in vivo example in which a single mutational hit triggers tumor development associated with profound transcriptomic alterations. SIGNIFICANCE: A newly generated mouse model carrying a FOXL2 mutation characteristic of adult-type granulosa cell tumors shows that FOXL2 C134W shifts the transcriptome towards a signature of granulosa cell cancer and drives tumorigenesis.

The Oocyte-Specific Linker Histone H1FOO Is Not Essential for Mouse Oogenesis and Fertility.

Meiosis is a highly conserved specialized cell division process that generates haploid gametes. Many of its events are associated with dynamically regulated chromosomal structures and chromatin remodeling, which are mainly modulated by histone modifications. Histone H1 is a linker histone essential for packing the nucleosome into higher-order structures, and H1FOO (H1 histone family, member O, oocyte-specific) is a H1 variant whose expression pattern is restricted to growing oocytes and zygotes. To further explore the function of H1FOO, we generated mice lacking the H1foo gene by the CRISPR/Cas9 technique. Herein, we combine mouse genetics and cellular studies to show that H1foo-null mutants have no overt phenotype, with both males and females being fertile and presenting no gross defects in meiosis progression nor in synapsis dynamics. Accordingly, the histological sections show a normal development of gametes in both male and female mice. Considering the important role of oocyte constituents in enhancing mammalian somatic cell reprogramming, we analyzed iPSCs generation in H1foo mutant MEFs and observed no differences in the absence of H1FOO. Taken all together, in this work we present the first in vivo evidence of H1FOO dispensability for mouse fertility, clarifying the debate in the field surrounding its essentiality in meiosis.

A truncating variant of RAD51B associated with primary ovarian insufficiency provides insights into its meiotic and somatic functions.

Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B -(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability.

Meiotic chromosome synapsis depends on multivalent SYCE1-SIX6OS1 interactions that are disrupted in cases of human infertility.

Meiotic reductional division depends on the synaptonemal complex (SC), a supramolecular protein assembly that mediates homologous chromosomes synapsis and promotes crossover formation. The mammalian SC has eight structural components, including SYCE1, the only central element protein with known causative mutations in human infertility. We combine mouse genetics, cellular, and biochemical studies to reveal that SYCE1 undergoes multivalent interactions with SC component SIX6OS1. The N terminus of SIX6OS1 binds and disrupts SYCE1's core dimeric structure to form a 1:1 complex, while their downstream sequences provide a distinct second interface. These interfaces are separately disrupted by SYCE1 mutations associated with nonobstructive azoospermia and premature ovarian failure (POF), respectively. Mice harboring SYCE1's POF mutation and a targeted deletion within SIX6OS1's N terminus are infertile with failure of chromosome synapsis. We conclude that both SYCE1-SIX6OS1 binding interfaces are essential for SC assembly, thus explaining how SYCE1's reported clinical mutations give rise to human infertility.

A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1.

Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with three cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse showed that it behaves as a hypomorphic allele compared to a new loss-of-function (knock-out) mouse model. Hsf2bpS167L/S167L females show reduced fertility with smaller litter sizes. To obtain mechanistic insights, we identified C19ORF57/BRME1 as a strong interactor and stabilizer of HSF2BP and showed that the BRME1/HSF2BP protein complex co-immunoprecipitates with BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L variant showed a strongly decreased staining of both HSF2BP and BRME1 at the recombination nodules and a reduced number of the foci formed by the recombinases RAD51/DMC1, thus leading to a lower frequency of crossovers. Our results provide insights into the molecular mechanism of HSF2BP-S167L in human ovarian insufficiency and sub(in)fertility.

The PSMA8 subunit of the spermatoproteasome is essential for proper meiotic exit and mouse fertility.

The ubiquitin proteasome system regulates meiotic recombination in yeast through its association with the synaptonemal complex, a 'zipper'-like structure that holds homologous chromosome pairs in synapsis during meiotic prophase I. In mammals, the proteasome activator subunit PA200 targets acetylated histones for degradation during somatic DNA double strand break repair and during histone replacement during spermiogenesis. We investigated the role of the testis-specific proteasomal subunit α4s (PSMA8) during spermatogenesis, and found that PSMA8 was localized to and dependent on the central region of the synaptonemal complex. Accordingly, synapsis-deficient mice show delocalization of PSMA8. Moreover, though Psma8-deficient mice are proficient in meiotic homologous recombination, there are alterations in the proteostasis of several key meiotic players that, in addition to the known substrate acetylated histones, have been shown by a proteomic approach to interact with PSMA8, such as SYCP3, SYCP1, CDK1 and TRIP13. These alterations lead to an accumulation of spermatocytes in metaphase I and II which either enter massively into apoptosis or give rise to a low number of aberrant round spermatids that apoptose before histone replacement takes place.

Shugoshin protects centromere pairing and promotes segregation of nonexchange partner chromosomes in meiosis.

Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.

Ubiquitin-specific protease 26 (USP26) is not essential for mouse gametogenesis and fertility.

Ubiquitin-specific protease 26 (USP26) is a deubiquitylating enzyme belonging to the USPs family with a transcription pattern restricted to the male germline. Since protein ubiquitination is an essential regulatory mechanism during meiosis, many efforts have been focused on elucidating the function of USP26 and its relationship with fertility. During the last decade, several studies have reported the presence of different polymorphisms in USP26 in patients with non-obstructive azoospermia (NOA) or severe oligozoospermia suggesting that this gene may be associated with human infertility. However, other studies have revealed the presence of these and novel polymorphisms, including nonsense mutations, in men with normal spermatogenesis as well. Thus, the results remain controversial and its function is unknown. In the present study, we describe the in vivo functional analysis of mice lacking USP26. The phenotypic analysis of two different Usp26-null mutants showed no overt-phenotype with both males and females being fertile. Cytological analysis of spermatocytes showed no defects in synapsis, chromosome dynamics, DNA repair, or recombination. Histopathological analysis revealed a normal distribution and number of the different cell types in both male and female mice. Finally, normal counts were observed in fertility assessments. These results represent the first in vivo evidence showing that USP26 is not essential for mouse gametogenesis.

Local activation of mammalian separase in interphase promotes double-strand break repair and prevents oncogenic transformation.

Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double-strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology-directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1, and Mms21 represent the corresponding kinase, methyltransferase, and SUMO ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation.

The Post-anaphase SUMO Pathway Ensures the Maintenance of Centromeric Cohesion through Meiosis I-II Transition in Mammalian Oocytes.

The production of haploid gametes requires the maintenance of centromeric cohesion between sister chromatids through the transition between two successive meiotic divisions, meiosis I and meiosis II. One mechanism for the cohesion maintenance is shugoshin-dependent protection of centromeric cohesin at anaphase I onset [1-3]. However, how centromeric cohesion is maintained during late anaphase I and telophase I, when centromeric shugoshin is undetectable [1-3], remains largely unexplored. Here we show that the centromeric small ubiquitin-related modifier (SUMO) pathway is critical for the maintenance of centromeric cohesion during post-anaphase-I periods in mouse oocytes. SUMO2/3 and E3 ligase PIAS are enriched near centromeres during late anaphase I and telophase I. Specific perturbation of the centromeric SUMO pathway results in precocious loss of centromeric cohesin at telophase I, although shugoshin-dependent centromeric protection at anaphase I onset remains largely intact. Prevention of the SUMO perturbation during post-anaphase-I periods restores the maintenance of centromeric cohesion through the meiosis I-II transition. Thus, the post-anaphase-I centromeric SUMO pathway ensures continuous maintenance of centromeric cohesion through the meiosis I-II transition.

piRNA-associated proteins and retrotransposons are differentially expressed in murine testis and ovary of aryl hydrocarbon receptor deficient mice.

Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR+/+ and AhR-/- mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in AhR-/- than in AhR+/+ testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Unexpectedly, AhR-/- males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR+/+ mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR-/- ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR+/+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements.

C14ORF39/SIX6OS1 is a constituent of the synaptonemal complex and is essential for mouse fertility.

Meiotic recombination generates crossovers between homologous chromosomes that are essential for genome haploidization. The synaptonemal complex is a 'zipper'-like protein assembly that synapses homologue pairs together and provides the structural framework for processing recombination sites into crossovers. Humans show individual differences in the number of crossovers generated across the genome. Recently, an anonymous gene variant in C14ORF39/SIX6OS1 was identified that influences the recombination rate in humans. Here we show that C14ORF39/SIX6OS1 encodes a component of the central element of the synaptonemal complex. Yeast two-hybrid analysis reveals that SIX6OS1 interacts with the well-established protein synaptonemal complex central element 1 (SYCE1). Mice lacking SIX6OS1 are defective in chromosome synapsis at meiotic prophase I, which provokes an arrest at the pachytene-like stage and results in infertility. In accordance with its role as a modifier of the human recombination rate, SIX6OS1 is essential for the appropriate processing of intermediate recombination nodules before crossover formation.

Distinct Roles of Meiosis-Specific Cohesin Complexes in Mammalian Spermatogenesis.

Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1ß, we generated Smc1ß-/-Rec8-/- and Smc1ß-/-Rad21L-/- mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1ß complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1ß and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in Smc1ß-/-Rad21L-/- cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion.

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.

Mutant cohesin in premature ovarian failure.

Premature ovarian failure is a major cause of female infertility. The genetic causes of this disorder remain unknown in most patients. Using whole-exome sequence analysis of a large consanguineous family with inherited premature ovarian failure, we identified a homozygous 1-bp deletion inducing a frameshift mutation in STAG3 on chromosome 7. STAG3 encodes a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion. Female mice devoid of Stag3 are sterile, and their fetal oocytes are arrested at early prophase I, leading to oocyte depletion at 1 week of age.

STAG3 is a strong candidate gene for male infertility.

Oligo- and azoospermia are severe forms of male infertility. However, known genetic factors account only for a small fraction of the cases. Recently, whole-exome sequencing in a large consanguineous family with inherited premature ovarian failure (POF) identified a homozygous frameshift mutation in the STAG3 gene leading to a premature stop codon. STAG3 encodes a meiosis-specific subunit of the cohesin complex, a large proteinaceous ring with DNA-entrapping ability that ensures sister chromatid cohesion and enables correct synapsis and segregation of homologous chromosomes during meiosis. The pathogenicity of the STAG3 mutations was functionally validated with a loss-of-function mouse model for STAG3 in oogenesis. However, and since none of the male members of this family was homozygous for the mutant allele, we only could hypothesized its putative involvement in male infertility. In this report, we show that male mice devoid of Stag3 display a severe meiotic phenotype that includes a meiotic arrest at zygonema-like shortening of their chromosome axial elements/lateral elements, partial loss of centromeric cohesion at early prophase and maintenance of the ability to initiate but not complete RAD51- and DMC1-mediated double-strand break repair, demonstrating that STAG3 is a crucial cohesin subunit in mammalian gametogenesis and supporting our proposal that STAG3 is a strong candidate gene for human male infertility.

Cohesin removal precedes topoisomerase IIα-dependent decatenation at centromeres in male mammalian meiosis II.

Sister chromatid cohesion is regulated by cohesin complexes and topoisomerase IIα. Although relevant studies have shed some light on the relationship between these two mechanisms of cohesion during mammalian mitosis, their interplay during mammalian meiosis remains unknown. In the present study, we have studied the dynamics of topoisomerase IIα in relation to that of the cohesin subunits RAD21 and REC8, the shugoshin-like 2 (Schizosaccharomyces pombe) (SGOL2) and the polo-like kinase 1-interacting checkpoint helicase (PICH), during both male mouse meiotic divisions. Our results strikingly show that topoisomerase IIα appears at stretched strands connecting the sister kinetochores of segregating early anaphase II chromatids, once the cohesin complexes have been removed from the centromeres. Moreover, the number and length of these topoisomerase IIα-connecting strands increase between lagging chromatids at anaphase II after the chemical inhibition of the enzymatic activity of topoisomerase IIα by etoposide. Our results also show that the etoposide-induced inhibition of topoisomerase IIα is not able to rescue the loss of centromere cohesion promoted by the absence of the shugoshin SGOL2 during anaphase I. Taking into account our results, we propose a two-step model for the sequential release of centromeric cohesion during male mammalian meiosis II. We suggest that the cohesin removal is a prerequisite for the posterior topoisomerase IIα-mediated resolution of persisting catenations between segregating chromatids during anaphase II.

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.