Biology subjects

Roig, I.

Publications and source records attributed to Roig, I..

10 recordsLinked to original sources

bioRxiv2025

TRIP13 fosters both transcriptional silencing and DSB repair during meiosis

During meiosis, the coordinated suppression of global gene transcription and induction of double-strand breaks (DSBs) genome wide is crucial for successful gamete formation. The AAA+ ATPase TRIP13 is a master meiotic regulator involved in numerous cellular processes, including DSB repair, organization of the meiotic chromosome axis, and meiotic silencing of unsynapsed axes. Here, we examine the involvement of TRIP13 in transcription suppression and DSB repair during meiotic prophase I in mice. Because TRIP13 is required for silencing of sex chromosomes at pachynema, we hypothesized that it may also be involved in the global transcription suppression that begins at the onset of meiotic prophase. Indeed, as predicted, we observed increased incorporation of 5-ethynyl uridine in early stages of meiotic prophase I and a higher presence of the active form of RNA polymerase II in Trip13 mutant spermatocytes. RNA sequencing data revealed that more than two-thirds of upregulated transcripts came from the sex chromosomes. Given this more general role in transcription silencing, we also tested whether TRIP13 promotes the transcription block that causes meiotic arrest in synapsis-deficient oocytes. However, Trip13 ablation did not rescue Spo11-/- oocytes, which experience asynapsis without DSB repair defects. Instead, Trip13 mutation did increase the number of oocytes in Dmc1-/- Chk2-/- mice, in which DSBs persist unrepaired. This rescue seems attributable to a previously unknown function of TRIP13 in preventing DSB repair by non-canonical pathways, as we observed fewer homologous recombination markers along with an increased presence of non-homologous end joining markers in the triple mutant cells. These findings underscore TRIP13s multifaceted role in transcription and DSB repair regulation during meiosis, highlighting its importance and potential for further meiotic regulation and fertility research.

cell biology↗

bioRxiv2025

The dynamics of ciliogenesis in prepubertal mouse meiosis reveal new clues about testicular maturation during puberty

The primary cilium, a solitary and non-motile extension of the plasma membrane, has recently been identified in adult male mouse spermatocytes. However, very little is known about when these cilia emerge during testicular maturation and what their function is. In the context of fertility establishment during puberty, this study investigates the dynamics of ciliogenesis in prepubertal mouse spermatocytes. Our findings reveal that primary cilia are not an intrinsic feature of spermatocytes during the first wave of meiosis, which initiates at 8 days post-partum (dpp). Instead, cilia begin polymerizing at 20 dpp, after first meiotic wave has been completed, and are present in spermatocytes across all stages of prophase I. Thus, no direct correlation between cilia polymerization and the initiation of synapsis or desynapsis was found, although chemical ablation of cilia may delay DNA repair during prophase I. Typical adult cilia features, which are shorter and restricted to zygotene spermatocytes, are settled upon acquisition of sexual maturity. This study also highlights that the emergence of ciliated spermatocytes in prepuberal mice coincides with the onset of flagellogenesis, hinting at a potential link between the regulation of the formation of both types of axonemes within the tissue developing environment. Proteomic analyses further identify temporal regulators of axoneme assembly, providing valuable targets for future research to unravel the molecular pathways underlying ciliogenesis, flagellogenesis, and their roles in spermatogenesis. We explored distinct regulatory mechanisms of ciliogenesis during the first meiotic wave and found that Aurora kinase A (AURKA) is a critical regulator of cilia disassembly during late diplotene, with evidence suggesting that centrosome migration and cilia depolymerization are mutually exclusive events during meiosis. In summary, this study provides the first detailed characterization of primary cilia dynamics during early testicular maturation in mice, revealing their spatiotemporal regulation, candidate molecular mediators, and potential roles during meiosis. These findings lay the groundwork for understanding the physiological relevance of meiotic cilia in spermatogenesis and testicular development.

cell biology↗

bioRxiv2025

SIRT7 links H3K36ac epigenetic regulation with genome maintenance in the aging mouse testis

ABSTRACT/SUMMARYReproductive aging is an increasing health concern affecting family planning and overall well-being. While extensively studied in females, the mechanisms driving male reproductive aging remain largely unexamined. Here we found that mammalian Sirtuin 7 (SIRT7) sustains spermatogenesis in an age-dependent manner through the control of histone 3 lysine 36 acetylation (H3K36ac). SIRT7 deficiency in mice resulted in increased levels of H3K36ac in spermatogonia and spermatocytes. In a germ cell line, SIRT7 deficiency disrupted nucleosome stability and increased vulnerability to genotoxic stress. Importantly, undifferentiated spermatogonia, which are required for continuous sperm production, decreased prematurely in Sirt7-/- mice and showed genome damage accumulation. These changes were concurrent with age-dependent defects in homologous chromosome synapsis and partial meiotic arrest. Taken together, our results indicate that SIRT7 connects H3K36ac epigenetic regulation to long-term genome stability in male germ cells, ensuring steady-state spermatogenesis during the lengthy male reproductive lifespan.

cell biology↗

bioRxiv2024

BEND2 is a crucial player in oogenesis and reproductive aging

Infertility affects up to 12% of couples globally, with genetic factors contributing to nearly half of the cases. Advances in genomic technologies have led to the discovery of genes like Bend2, which plays a crucial role in gametogenesis. In the testis, Bend2 expresses two protein isoforms: full-length and a smaller one. Ablation of both proteins results in an arrested spermatogenesis. Because the Bend2 locus is on the X chromosome, and the Bend2-/y mutants are sterile, BEND2s role in oogenesis remained elusive. In this study, we employed a novel Bend2 mutation that blocks the expression of the full-length BEND2 protein but allows the expression of the smaller BEND2 isoform. Interestingly, this mutation does not confer male sterility and mildly affects spermatogenesis. Thus, it allowed us to study the role of BEND2 in oogenesis. Our findings demonstrate that full-length BEND2 is dispensable for male fertility, and its ablation leads to a reduced establishment of the ovarian reserve. These results reveal a critical role for full-length BEND2 in oogenesis and provide insights into the mechanisms underlying the establishment of the ovarian reserve. Furthermore, these findings hold relevance for the diagnostic landscape of human infertility.

cell biology↗

bioRxiv2019

The DNA damage response regulates the oocyte pool in mammals

Mammalian oogonia proliferate without completing cytokinesis producing germ cell cysts. Within these cysts, oocytes differentiate and enter meiosis, promote genome-wide double-strand break (DSBs) formation which repair by homologous recombination leads to synapsis of the homologous chromosomes. Errors in homologous recombination or synapsis trigger the activation of surveillance mechanisms, traditionally called pachytene checkpoint, to either repair them or send the cells to programmed death. Contrary to what is found in spermatocytes, most oocytes present a remarkable persistence of unrepaired DSBs at pachynema. Simultaneously, there is a massive oocyte death accompanying the oocyte cyst breakdown. This oocyte elimination is thought to be required to properly form the follicles, which constitute the pool of germ cells females will use during their adult life. Based on all the above mentioned, we hypothesized that the apparently inefficient meiotic recombination occurring in mouse oocytes may be required to eliminate most of the oocytes in order to regulate the oocyte number, promote cyst breakdown and follicle formation in mammalian females. To test this idea, we analyzed perinatal ovaries to evaluate the oocyte population, cyst breakdown and follicle formation in control and mutant mice for the effector kinase of the DNA damage response, CHK2. Our results confirm the involvement of CHK2 in the elimination of oocytes that accumulate unrepaired DSBs and show that CHK2 regulates the number of oocytes in fetal ovaries. We also show that CHK2 is required to eliminate oocytes as a result of LINE-1 activation, which was previously shown to be responsible for fetal oocyte loss. Nonetheless, the number of oocytes found in Chk2 mutant ovaries three days after birth was similar to that of control ovaries, suggesting the existence of CHK2-independent mechanisms capable of eliminating oocytes. In vitro inhibition of CHK1 rescued the oocyte number in Chk2 mutant ovaries suggesting that CHK1 regulates postnatal oocyte death. Moreover, both CHK1 and CHK2 functions are required to timely breakdown cyst and form follicles. Altogether, we propose the DNA damage response controls the number of oocytes present perinatally and is required to properly break down oocyte cysts and form follicles, highlighting the importance of the DNA damage response in setting the reserve of oocytes each female will use during their entire lifespan.

developmental biology↗

bioRxiv2018

ATR function is indispensable to allow proper mammalian follicle development.

Mammalian female fertility relies on the proper development of follicles. Right after birth in mouse, oocytes associate with somatic ovarian cells to form follicles. These follicles grow during adult lifetime to produce viable gametes. In this study, we analyzed the role of the ATM and rad3-related (ATR) kinase in mouse oogenesis and folliculogenesis using a hypomorphic mutation of the Atr gene (Murga et al., 2009). Female mice homozygote for this allele have been reported to be sterile. Our data show that female meiotic prophase is not grossly altered when ATR levels are reduced. However, follicle development is majorly compromised since Atr mutant ovaries present a decrease of growing follicles. Comprehensive analysis of follicular cell death and proliferation suggest that wild-type levels of ATR are required to achieve optimal follicular development. Altogether, these findings suggest that reduced ATR expression causes sterility due to defects in follicular progression rather than in meiotic recombination. We discuss the implication of these findings for the use of ATR inhibitors as anti-cancer drugs and its possible side effects on female fertility.

cell biology↗

bioRxiv2018

Spermatoproteasome-deficient mice are proficient in meiotic DNA repair but defective in meiotic exit

Meiotic recombination generates crossovers which are essential to ensure genome haploidization. The ubiquitin proteasome system regulates meiotic recombination through its association to the synaptonemal complex, a zipper-like structure that holds homologs and provides the structural framework for meiotic recombination. Here we show that the testis-specific 4s subunit (PSMA8) of the spermatoproteasome is located at the synaptonemal complex and is essential for the assembly of its activator PA200. Accordingly, synapsis-deficient mice show delocalization of PSMA8 from the synaptonemal complex. Genetic analysis of Psma8-deficient mice shows normal meiotic DNA repair, crossing over formation and an increase of spermatocytes at metaphase I and metaphase II which either enter into apoptosis or slip to give rise to an early spermatid arrest and infertility. Thus, spermatoproteasome-dependent histone degradation is dispensable for meiotic recombination. We show that PSMA8 deficiency alters the proteostasis of several key meiotic players such as acetylated histones, SYCP3, SYCP1, CDK1 and TRIP13 which in turn leads to an aberrant meiotic exit and early spermatid arrest prior to the histone displacement process that take place subsequently.

cell biology↗

bioRxiv2017

Shu complex SWS1-SWSAP1 is required for mouse meiotic recombination in concert with the BRCA2 C terminus

Homology recognition and DNA-strand invasion ensure faithful homolog pairing and segregation during the first meiotic division1. RAD51 and DMC1 recombinases catalyze these steps2, with BRCA2 promoting their assembly into nuclear foci3. The recently identified human SWS1-SWSAP1 complex, related to the Shu complex in yeast, promotes RAD51 focus formation in cell lines4,5. We show here that mouse SWS1-SWSAP1 is critical for meiotic homologous recombination (HR) by promoting the assembly of RAD51 and DMC1 on early recombination intermediates. Absence of the complex perturbs meiotic progression in males and females and both sexes are sterile, although a fraction of meiocytes form crossovers. Remarkably, loss of the DNA damage checkpoint kinase CHK2 rescues fertility specifically in females without rescuing crossover numbers. Unlike the Shu complex, the BRCA2 C terminus (known to be required for RAD51 stabilization6,7) is dispensible for RAD51 and DMC1 focus formation. However, concomitant loss of the BRCA2 C terminus aggravates the meiotic defects in Shu mutant spermatocytes. These results point to a complex interplay of factors that ensure recombinase function and hence meiotic progression in the mouse.

genetics↗

bioRxiv2017

H2AFX AND MDC1 PROTECT GENOMIC INTEGRITY IN MALE GERM CELLS BY PROMOTING RECOMBINATION AND ACTIVATION OF THE RECOMBINATION-DEPENDENT CHECKPOINT

In somatic cells, H2afx and Mdc1 are close functional partners in DNA repair and damage response. However, it is not known whether they are also involved in the maintenance of genome integrity in meiosis. By analyzing chromosome dynamics in H2afx-/- spermatocytes, we found that synapsis of the autosomes and X-Y chromosomes were impaired in a relevant fraction of cells. Such defect correlated with an abnormal recombination profile. Conversely, Mdc1 was dispensable for the synapsis of the autosomes, and only played a minor role in X-Y synapsis, relatively to H2afx. This suggested that those genes have non-overlapping functions in chromosome synapsis. However, we observed that both genes play a similar role in the assembly of MLH3 onto chromosomes, a key step in crossover formation. Moreover, we showed that H2afx and Mdc1 cooperate in promoting the activation of the recombination-dependent checkpoint, a mechanism that restrains the differentiation of cells with unrepaired DSBs. This occurs by a mechanism that involves P53. Overall, our data showed that, in male germ cells, H2afx and Mdc1 promote the maintenance of genome integrity.

developmental biology↗

bioRxiv2017

ATR Is Required To Complete Meiotic Recombination In Mice

Precise execution of recombination during meiosis is essential for forming chromosomally balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Binding of replication protein A (RPA) at resected DSBs fosters association of RAD51 and DMC1, the primary effectors of homology search. It is well appreciated that cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these responses remain poorly understood, particularly in mammals. Here we provide evidence that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

cell biology↗