In mammals, meiotic silencing of unsynapsed chromatin (MSUC) is initiated by the DNA damage response (DDR) pathway, as marked by γH2AX. During normal male meiosis, MSUC is restricted to the unsynapsed sex chromosomes, a process known as meiotic sex chromosome inactivation (MSCI). While the initiation of MSCI has been well studied, its full silencing dynamics and underlying structural mechanisms remain unclear. In contrast to MSCI, broader MSUC can occur on autosomes in response to synapsis failure, but its cell-to-cell variability obscures its quantification. To address these challenges, we introduce "digital-chromosome-banding", a single-cell-based approach that allows quantitative analysis of MSCI and MSUC at chromosomal resolution. Using this approach, we identified two distinct silencing transitions during MSCI, occurring from zygonema to early pachynema and from early to mid-pachynema. The latter step coincides with mature sex body formation and involves a gel-like diffusion barrier to enforce transcriptional repression. Applying this approach to synapsis-defective mouse models (Spo11-/- , Tardbp cKO, and Nelfb cKO), we observed divergent MSUC patterns that correlate with the severity of asynapsis. Comparative analysis between sexes also uncovered notable sexual dimorphisms in meiotic silencing. Together, our data provide a quantitative framework to dissect the spatiotemporal dynamics and sexual differences of meiotic silencing.

Preclinical Alzheimer’s disease (AD) trials can involve multiple years of follow-up and burdensome procedures for older individuals. Optimizing the design and conduct of these trials requires input from participants and their families. Since 2020, the Alzheimer’s Clinical Trials Consortium (ACTC) Research Participant Advisory Board has provided input on study attributes including: participant and study partner compensation, consent language, and result communication tools. A key recommendation from the advisory board is to design studies with the option to learn individual research results. Participants find results personally meaningful, even when the clinical relevance of results has not been established. After release of topline study results, A4 research sites were provided reports that included treatment arm assignment and individual research results (cognitive scores, amyloid PET tertile, and Clinical Dementia Rating scale results). Research sites were provided guidance on communicating research results in a training webinar. A survey was subsequently sent to all A4 sites to understand what methods were used to communicate results and support participants. The 42 sites that completed the survey reported sharing results with 445 participants, or 65% of participants active at the end of the study. Participants expressed interest in taking part in other studies and, where eligible, were referred. Those with worsening cognition and function were referred for clinical evaluation and care. Some A4 participants told sites that they expected “something tangible” and “more than a one-hour meeting” after the many years of participation. The A4 sites that hosted ‘thank you’ events to discuss study-level results, with separate one-on-one sessions to discuss individual results shared these were appreciated by participants. The active involvement of the diverse ACTC Participant Advisory Board has benefited the design of preclinical AD studies. The A4 experience demonstrates that it is feasible to share individual research results, and that a majority of participants will opt to learn. Greater involvement of the Advisory Board at the earliest stages of study design and planning will maximize impact of their feedback, and help researchers move toward the goal of optimized, inclusive trials.

Meiotic recombination generates crossovers (COs), reciprocal exchanges between homologous chromosomes critical for accurate chromosome segregation. Inappropriate CO frequency and distribution drive aneuploidy in human oocytes, with error rates up to 10-fold higher than in sperm despite females exhibiting higher CO frequencies. COs form in the context of the proteinaceous synaptonemal complex (SC) that tethers homologs during prophase I. SC length strongly correlates with CO number, and sexual dimorphism in recombination has long been attributed to longer SCs in females. However, this model is challenged by wild-derived PWD mice in which males consistently generate more COs despite having shorter SCs. Here, we exploit natural genetic variation among inbred mouse strains to dissect the structural and regulatory basis of sexually dimorphic CO regulation. Using cytological markers of SC assembly (SYCP3), recombination progression (RAD51, MSH4), class I CO designation (HEI10, MLH1/MLH3), and chiasmata, we show that SC length is not the sole predictor of CO number. PWD males exhibit stronger CO interference and higher CO number than females, despite reduced SC length. Notably, females show reduced efficiency in designating recombination intermediate to become COs, whereas PWD males display exceptional proficiency. Unexpectedly, although class II COs are rare, they play a disproportionate role in ensuring that every chromosome pair receives at least one CO, thereby safeguarding against aneuploidy. Together, these findings challenge the prevailing view that SC length is the primary determinant of sexually dimorphic CO rates and instead highlight sex-specific regulation of CO designation and pathway usage as key drivers of recombination outcomes.

Meiosis is a common feature in all sexually reproducing eukaryotic organisms. The goal of this unique cell cycle is to halve the genome in order to generate haploid gametes for fertilization. To achieve this, germ cells undergo a prolonged G2 phase that encompasses a prolonged prophase I. During this stage, homologous chromosomes must pair and become physically tethered in order to then segregate at the first meiotic division. The second meiotic division then segregates the paired sister chromatids, without a preceding DNA replication event, resulting in haploid gametes. Thus, meiosis involves two division events preceded by only one round of DNA replication. Importantly, though the process of meiosis is conserved amongst eukaryotes, there are distinct differences in the regulation of these events across species and between the sexes.

In meiotic prophase I, hundreds of DNA double-strand breaks are formed and subsequently repaired as noncrossovers or crossovers (COs). COs are essential for accurate chromosome segregation during the first meiotic division, and errors in this process result in aneuploidy, birth defects, or infertility. Such errors are more pronounced in females compared with males, indicating that CO regulation and surveillance are sexually dimorphic. We demonstrate here dual roles of cyclin N-terminal domain containing 1 (CNTD1) in ensuring appropriate CO between homologous chromosomes in oocytes and in establishing the pool of follicles in the postnatal ovary. CNTD1-deficient oocytes fail to form COs and exhibit a severely depleted follicle pool shortly after birth, which is temporally distinct from previously reported CO mutants. Further investigation indicates that follicle loss is CHK2-dependent, resulting from inappropriate retention of HORMAD1 and the absence of SKP1. These findings indicate that CNTD1 plays novel roles in CO designation and establishment of the follicular reserve in female mammals.

Alterations to the retina manifest in patients diagnosed with neurodegenerative diseases such as Alzheimer’s disease (AD). Retinal imaging techniques open the possibility for non-invasive evaluation of AD pathology. Clinically AD diagnosed patients exhibit retinal amyloid deposits. Few studies monitoring preclinical individuals exist, limiting the assessment of the feasibility of retinal imaging as a biomarker for early-stage AD risk detection. We compared retinal and cerebral amyloid in clinically normal individuals who screened positive for amyloid through positron emission tomography (PET) from the Anti-Amyloid Treatment in Asymptomatic Alzheimer Disease (A4) as well as a companion cohort of individuals who were negative on amyloid by amyloid PET in the Longitudinal Evaluation of Amyloid Risk and Neurodegeneration (LEARN) study. We quantified the number of curcumin-positive fluorescent retinal spots from a small subset of participants from both studies to determine retinal amyloid deposition at baseline. Participants from the A4 trial exhibited a greater number of retinal spots compared to those from the LEARN study. We report a positive correlation between retinal spots and brain amyloid, as measured by the standardized uptake value ratio (SUVr). The results of this small pilot study support the use of retinal fundus imaging for detecting amyloid deposition that is correlated with brain amyloid PET SUVr. A larger sample set is under analysis currently to fully ascertain the relationship between amyloid PET and retinal amyloid both cross-sectionally and longitudinally.

Argonaute proteins are best known for their role in microRNA-mediated post-transcriptional gene silencing. Here, we show that AGO3 and AGO4, but not AGO2, localize to the sex chromatin of pachytene spermatocytes where they are required for transcriptional silencing of XY-linked genes, known as Meiotic Sex Chromosome Inactivation (MSCI). Using an Ago413 -/- mouse, we show that AGO3 and AGO4 are key regulators of spermatogenesis, orchestrating expression of meiosis-related genes during prophase I while maintaining silencing of spermiogenesis genes. Premature overexpression of spermiogenesis genes during prophase I in Ago413 -/- mice results in subfertility, altered sperm morphology and reduced fertilization capability. We also identify BRG1, a BAF complex subunit, as an AGO3 interactor. Loss of AGO3 and AGO4 results in increased BRG1 in spermatocytes, suggesting that AGO3 aids in removing BRG1 from the XY chromatin to achieve MSCI and demonstrating a meiotic role for AGO3 in transcriptional control through the chromatin remodeling machinery.

Long-acting non-hormonal male contraceptives are urgently needed but developing strategies that are both effective and reversible presents significant challenges. Here, we investigated the potential of meiotic prophase I blockade as a promising and potentially reversible approach to male contraception. To do this, we utilized (+)-JQ1, a small-molecule inhibitor of the testis-specific protein, BRDT. Daily injections of (+)-JQ1 for three weeks resulted in disrupted spermatogenesis resulting in loss of spermatozoa and an inability to sire pups. While spermatogenic cells repopulated the testis within six weeks post drug cessation, full fertility restoration required a longer recovery period. We attribute this delay in full recovery to persistent issues with the pachytene transcriptional program, which is crucial for meiotic progression and spermatid development. These findings underscore the potential of pharmacological approaches to disrupt meiotic prophase I as a targeted, reversible male contraceptive strategy, providing new insights into developing effective non-hormonal contraceptive approaches. ### Competing Interest Statement The authors have declared no competing interest. Bill & Melinda Gates Foundation, https://ror.org/0456r8d26, INV-00371, INV-038185

Ovulation results from the cyclical recruitment of non-renewing, quiescent oocytes for growth. Therefore, the primordial follicles that are established during development from an oocyte encapsulated by granulosa cells are thought to comprise the lifelong ovarian reserve 1-4. However, using oocyte lineage tracing in mice, we observed that a subset of oocytes recruited for growth in the first juvenile wave remain paused for many months before continuing growth, ovulation, fertilization and development into healthy offspring. This small subset of genetically-labeled fetal oocytes, labeled with Sycp3-CreERT2, is distinguished by earlier entry and slower dynamics of meiotic prophase I. While labeled oocytes were initially found in both primordial follicles and growing follicles of the first wave, they disappeared from primordial follicles by puberty. Unexpectedly, these first-wave labeled growing oocytes persisted throughout reproductive lifespan and contributed to offspring at a steady rate beyond 12 months of age, suggesting that follicles can pause mid-growth for extended periods then successfully resume. These results challenge the conclusion from lineage tracing of granulosa cells that first-wave follicles make a limited contribution to fertility5 and furthermore suggest that growth-paused oocytes comprise a second and previously unrecognized ovarian reserve.

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.

Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis, and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. Topbp1 B5/B5 males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted, including phosphorylation and localization of the RNA:DNA helicase Senataxin. Topbp1 B5/B5 spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.

Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4 , Hei10, and Cntd1 , while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1 . Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.

During prophase I of meiosis, DNA double-strand breaks form throughout the genome, with a subset repairing as crossover events, enabling the accurate segregation of homologous chromosomes during the first meiotic division. The mechanism by which DSBs become selected to repair as crossovers is unknown, although the crossover positioning and levels in each cell indicate it is a highly regulated process. One of the proteins that localises to crossover sites is the serine/threonine cyclin-dependent kinase CDK2. Regulation of CDK2 occurs via phosphorylation at tyrosine 15 (Y15) and threonine 160 (T160) inhibiting and activating the kinase, respectively. In this study we use a combination of immunofluorescence staining on spread spermatocytes and fixed testis sections, and STA-PUT gravitational sedimentation to isolate cells at different developmental stages to further investigate the temporal phospho regulation of CDK2 during prophase I. Western blotting reveals differential levels of the two CDK2 isoforms (CDK233kDa and CDK239kDa) throughout prophase I, with inhibitory phosphorylation of CDK2 at Y15 occurring early in prophase I, localising to telomeres and diminishing as cells enter pachynema. Conversely, the activatory phosphorylation on T160 occurs later, specifically the CDK233kDa isoform, and T160 signal is detected in spermatogonia and pachytene spermatocytes, where it co-localises with the Class I crossover protein MLH3. Taken together, our data reveals intricate control of CDK2 both with regards to levels of the two CDK2 isoforms, and differential regulation via inhibitory and activatory phosphorylation.

Meiosis is characterized by highly regulated transitions in gene expression that require diverse mechanisms of gene regulation. For example, in male mammals, transcription undergoes a global shut-down in early prophase I of meiosis, followed by increasing transcriptional activity into pachynema. Later, as spermiogenesis proceeds, the histones bound to DNA are replaced with transition proteins, which are themselves replaced with protamines, resulting in a highly condensed nucleus with repressed transcriptional activity. In addition, two specialized gene silencing events take place during prophase I: meiotic silencing of unsynapsed chromatin (MSUC), and the sex chromatin specific mechanism, meiotic sex chromosome inactivation (MSCI). Notably, conserved roles for the RNA binding protein (RBP) machinery that functions with small non-coding RNAs have been described as participating in these meiosis-specific mechanisms, suggesting that RNA-mediated gene regulation is critical for fertility in many species. Here, we review roles of small RNAs and their associated RBPs in meiosis-related processes such as centromere function, silencing of unpaired chromatin and meiotic recombination. We will discuss the emerging evidence of non-canonical functions of these components in meiosis.

The A4 Study is a Phase 3 clinical trial investigating solanezumab in preclinical Alzheimer’s disease. The COVID-19 pandemic impacted the delivery of elective health services worldwide and social distancing restrictions created challenges for clinical trial participants to continue monthly infusions. In an effort to retain A4 participants, decentralized clinical trial methods were implemented, including deployment of mobile research nurses to conduct home visits. The A4 Study is a 4.5 year double-blind trial, followed by an optional 4 year open-label treatment period. The study launched in 2014 and 1163 males and females between the ages of 65 and 85 years old have been dosed. Home visits were not originally included in the study protocol, but were implemented in 2020 to mitigate the COVID-19 pandemic impact on study conduct. PCM Trials offered home visits as an option to allow participants to continue to receive investigational product. Mobile research nurses traveled to participant homes and administered the 30- to 60-minute intravenous infusions every 4 weeks. To date, more than 1,400 home infusions visits have been conducted in the A4 Study. 124 participants were enrolled in home infusions (99 in the United States; 1 in Canada and 24 in Australia) from 18 sites. Implementation of home visits led to several positive outcomes, including reduced participant burden (particularly for participants living in rural/remote locations), study continuity and maintenance of treatment regimen, and retention of participants. Considering the length of the study, the infusion frequency, and the age of the participants, home visits have been a valuable option to help study completion and compliance. Data integrity and participant safety were ensured and maintained with the use of home visits in the A4 study.

In the long-lived naked mole-rat (NMR), the entire process of oogenesis occurs postnatally. Germ cell numbers increase significantly in NMRs between postnatal days 5 (P5) and P8, and germs cells positive for proliferation markers (Ki-67, pHH3) are present at least until P90. Using pluripotency markers (SOX2 and OCT4) and the primordial germ cell (PGC) marker BLIMP1, we show that PGCs persist up to P90 alongside germ cells in all stages of female differentiation and undergo mitosis both in vivo and in vitro. We identified VASA+ SOX2+ cells at 6 months and at 3-years in subordinate and reproductively activated females. Reproductive activation was associated with proliferation of VASA+ SOX2+ cells. Collectively, our results suggest that highly desynchronized germ cell development and the maintenance of a small population of PGCs that can expand upon reproductive activation are unique strategies that could help to maintain the NMR's ovarian reserve for its 30-year reproductive lifespan.

Alterations to the retina manifest in patients diagnosed with neurodegenerative diseases such as Alzheimer’s disease (AD). Retinal imaging techniques open the possibility for non-invasive evaluation of AD pathology. Clinically AD diagnosed patients exhibit retinal spots that may be amyloid deposits. Few studies monitoring preclinical individuals exist, limiting the assessment of the feasibility of retinal imaging as a biomarker for early-stage AD risk detection. We compared extent and distribution of retinal fluorescent spots and cerebral amyloid in clinically normal individuals who screened positive for high amyloid levels through positron emission tomography (PET) from the Anti-Amyloid Treatment in Asymptomatic Alzheimer Disease (A4) as well as a companion cohort of individuals who exhibited low levels of amyloid PET in the Longitudinal Evaluation of Amyloid Risk and Neurodegeneration (LEARN) study. We quantified the number of curcumin-positive fluorescent retinal spots from participants from both studies to determine retinal amyloid deposition at baseline. Participants from the A4 trial exhibited a greater number of retinal spots compared to those from the LEARN study. We report a positive correlation between retinal spots and brain amyloid, as measured by the standardized uptake value ratio (SUVr). The results of this study support the use of retinal fundus imaging for and assessing relationships with brain amyloid PET SUVr. Further work is needed to determine the nature of retinal spots and their relationship with actual amyloid plaques cross-sectionally and longitudinally.

During meiotic prophase I, spermatocytes must balance transcriptional activation with homologous recombination and chromosome synapsis, biological processes requiring extensive changes to chromatin state. We explored the interplay between chromatin accessibility and transcription through prophase I of mammalian meiosis by measuring genome-wide patterns of chromatin accessibility, nascent transcription, and processed mRNA. We find that Pol II is loaded on chromatin and maintained in a paused state early during prophase I. In later stages, paused Pol II is released in a coordinated transcriptional burst mediated by the transcription factors A-MYB and BRDT, resulting in ~3-fold increase in transcription. Transcriptional activity is temporally and spatially segregated from key steps of meiotic recombination: double strand breaks show evidence of chromatin accessibility earlier during prophase I and at distinct loci from those undergoing transcriptional activation, despite shared chromatin marks. Our findings reveal mechanisms underlying chromatin specialization in either transcription or recombination in meiotic cells.

In meiotic prophase I, hundreds of double-strand breaks (DSB) are induced to initiate homologous recombination, resulting in the formation of non-crossovers (NCO) and crossovers (CO). Of the 250-300 DSBs that form in mice, only ∼10% are resolved as COs, with all remaining DSB events resolving as NCO. Thus, both the frequency and distribution of COs must be tightly regulated to ensure their appropriate localization across the genome. Errors in crossover designation and maturation can result in nondisjunction, leading to aneuploidy, germ cell death, birth defects, or infertility. That these errors are more evident in female meiosis compared to males suggests that the events of meiotic prophase I are sexually dimorphic with respect to DSB processing, resolution, and/or surveillance. Therefore, it is essential to investigate how COs are designated and regulated during prophase I in the female germline. Previous studies in our lab showed that Cyclin N-Terminal Domain Containing 1 (CNTD1) protein plays a critical role in CO designation during male meiosis, co-localizing with other essential CO proteins such as MutLγ (MLH1/MLH3) and crossover site-associated cyclin-dependent kinase-2 (CDK2) during pachynema. In the current study, we investigated whether analogous roles for CNTD1 existed in female meiosis. Similar to Cntd1 -/- males, Cntd1 -/- females are sterile, with no gross morphological ovarian defects in adults albeit with a depletion in ovarian follicle numbers. During prophase I, early DSB induction and repair occur normally, but oocytes from Cntd1 -/- females fail to accumulate MutLγ in pachynema. Phenotypically, meiotic prophase I disruption in Cntd1 -/- females is similar to that seen in Cntd1 -/- males. Importantly, whereas the Caernohabditis elegans ortholog of CNTD1, COSA-1, appears to interact with CDK-2 during pachynema in oocytes, CNTD1 in mouse oocytes lacks the CDK-interacting motif, suggesting that its function in oocytes does not involve a cyclin-like induction of CDK2 phosphorylation activity. In contradiction to the timing of prophase I disruption in Cntd1 -/- females at pachynema, we observe a dramatic depletion of primordial follicles, beginning shortly after birth and increasing dramatically through to adult ages. Those primordial follicles that persist, however, are able to develop through dictyate arrest into primary and larger follicular structures in pre-pubertal females, and only by adulthood were significant declines observed in all follicles. The early loss of oocytes in Cntd1 -/- females shortly after birth, considerably earlier than that observed for mice lacking MLH1 or MLH3, suggests a novel early role for CNTD1 in CO designation and/or in ensuring progression of oocytes into dictyate arrest at late pachynema.### Competing Interest StatementThe authors have declared no competing interest.