Diet influences community dynamics following vaginal group B streptococcus colonization.

The vaginal microbiota plays a pivotal role in reproductive, sexual, and perinatal health and disease. Unlike the well-established connections between diet, metabolism, and the intestinal microbiota, parallel mechanisms influencing the vaginal microbiota and pathogen colonization remain overlooked. In this study, we combine a mouse model of Streptococcus agalactiae strain COH1 [group B Streptococcus (GBS)] vaginal colonization with a mouse model of pubertal-onset obesity to assess diet as a determinant of vaginal microbiota composition and its role in colonization resistance. We leveraged culture-dependent assessment of GBS clearance and culture-independent, sequencing-based reconstruction of the vaginal microbiota in relation to diet, obesity, glucose tolerance, and microbial dynamics across time scales. Our findings demonstrate that excessive body weight gain and glucose intolerance are not associated with vaginal GBS density or timing of clearance. Diets high in fat and low in soluble fiber are associated with vaginal GBS persistence, and changes in vaginal microbiota structure and composition due to diet contribute to GBS clearance patterns in nonpregnant mice. These findings underscore a critical need for studies on diet as a key determinant of vaginal microbiota composition and its relevance to reproductive and perinatal outcomes.IMPORTANCEThis work sheds light on diet as a key determinant influencing the composition of vaginal microbiota and its involvement in group B Streptococcus (GBS) colonization in a mouse model. This study shows that mice fed diets with different nutritional composition display differences in GBS density and timing of clearance in the female reproductive tract. These findings are particularly significant given clear links between GBS and adverse reproductive and neonatal outcomes, advancing our understanding by identifying critical connections between dietary components, factors originating from the intestinal tract, vaginal microbiota, and reproductive outcomes.

Intestinal microbial circadian rhythms drive sex differences in host immunity and metabolism.

Circadian rhythms dynamically regulate sex differences in metabolism and immunity, and circadian disruption increases the risk of metabolic disorders. We investigated the role of sex-specific intestinal microbial circadian rhythms in host metabolism using germ-free and conventionalized mice and manipulation of dietary-derived fat, fiber, and microbiota-accessible carbohydrates. Our findings demonstrate that sex differences in circadian rhythms of genes involved in immunity and metabolism depend on oscillations in microbiota, microbial metabolic functions, and microbial metabolites. Further, we show that consuming an obesogenic, high-fat, low-fiber diet produced sex-specific changes in circadian rhythms in microbiota, metabolites, and host gene expression, which were linked to sex differences in the severity of metabolic dysfunction. Our results reveal that microbial circadian rhythms contribute to sex differences in immunity and metabolism and that dietary factors can entrain new circadian rhythms and modify the magnitude of sex differences in host-microbe circadian dynamics.

Perspectives: Methods for Evaluating Primate Spermatogonial Stem Cells.

Mammalian spermatogenesis is a complex, highly productive process generating millions of sperm per day. Spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis and can either self-renew, producing more SSCs, or differentiate to initiate spermatogenesis and produce sperm. The biological potential of SSCs to produce and maintain spermatogenesis makes them a promising tool for the treatment of male infertility. However, translating knowledge from rodents to higher primates (monkeys and humans) is challenged by different vocabularies that are used to describe stem cells and spermatogenic lineage development in those species. Furthermore, while rodent SSCs are defined by their biological potential to produce and maintain spermatogenesis in a transplant assay, there is no equivalent routine and accessible bioassay to test monkey and human SSCs or replicate their functions in vitro. This chapter describes progress characterizing, isolating, culturing, and transplanting SSCs in higher primates.

Genomic study of TEX15 variants: prevalence and allelic heterogeneity in men with spermatogenic failure.

Introduction: Human spermatogenesis is a highly intricate process that requires the input of thousands of testis-specific genes. Defects in any of them at any stage of the process can have detrimental effects on sperm production and/or viability. In particular, the function of many meiotic proteins encoded by germ cell specific genes is critical for maturation of haploid spermatids and viable spermatozoa, necessary for fertilization, and is also extremely sensitive to even the slightest change in coding DNA. Methods: Here, using whole exome and genome approaches, we identified and reported novel, clinically significant variants in testis-expressed gene 15 (TEX15), in unrelated men with spermatogenic failure (SPGF). Results: TEX15 mediates double strand break repair during meiosis. Recessive loss-of-function (LOF) TEX15 mutations are associated with SPGF in humans and knockout male mice are infertile. We expand earlier reports documenting heterogeneous allelic pathogenic TEX15 variants that cause a range of SPGF phenotypes from oligozoospermia (low sperm) to nonobstructive azoospermia (no sperm) with meiotic arrest and report the prevalence of 0.6% of TEX15 variants in our patient cohort. Among identified possible LOF variants, one homozygous missense substitution c.6835G>A (p.Ala2279Thr) co-segregated with cryptozoospermia in a family with SPGF. Additionally, we observed numerous cases of inferred in trans compound heterozygous variants in TEX15 among unrelated individuals with varying degrees of SPGF. Variants included splice site, insertions/deletions (indels), and missense substitutions, many of which resulted in LOF effects (i.e., frameshift, premature stop, alternative splicing, or potentially altered posttranslational modification sites). Conclusion: In conclusion, we performed an extensive genomic study of familial and sporadic SPGF and identified potentially damaging TEX15 variants in 7 of 1097 individuals of our combined cohorts. We hypothesize that SPGF phenotype severity is dictated by individual TEX15 variant's impact on structure and function. Resultant LOFs likely have deleterious effects on crossover/recombination in meiosis. Our findings support the notion of increased gene variant frequency in SPGF and its genetic and allelic heterogeneity as it relates to complex disease such as male infertility.

Single-cell analysis of human testis aging and correlation with elevated body mass index.

Aging men display reduced reproductive health; however, testis aging is poorly understood at the molecular and genomic levels. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from both young and older men and examined age-related changes in germline development and in the testicular somatic cells. Age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and somatic cells ranged from moderate to severe. Altered pathways included signaling and inflammation in multiple cell types, metabolic signaling in Sertoli cells, hedgehog signaling and testosterone production in Leydig cells, cell death and growth in testicular peritubular cells, and possible developmental regression in both Leydig and peritubular cells. Remarkably, the extent of dysregulation correlated with body mass index in older but not in younger men. Collectively, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging and their possible exacerbation by concurrent chronic conditions such as obesity.

Variants in GCNA, X-linked germ-cell genome integrity gene, identified in men with primary spermatogenic failure.

Male infertility impacts millions of couples yet, the etiology of primary infertility remains largely unknown. A critical element of successful spermatogenesis is maintenance of genome integrity. Here, we present a genomic study of spermatogenic failure (SPGF). Our initial analysis (n = 176) did not reveal known gene-candidates but identified a potentially significant single-nucleotide variant (SNV) in X-linked germ-cell nuclear antigen (GCNA). Together with a larger follow-up study (n = 2049), 7 likely clinically relevant GCNA variants were identified. GCNA is critical for genome integrity in male meiosis and knockout models exhibit impaired spermatogenesis and infertility. Single-cell RNA-seq and immunohistochemistry confirm human GCNA expression from spermatogonia to elongated spermatids. Five identified SNVs were located in key functional regions, including N-terminal SUMO-interacting motif and C-terminal Spartan-like protease domain. Notably, variant p.Ala115ProfsTer7 results in an early frameshift, while Spartan-like domain missense variants p.Ser659Trp and p.Arg664Cys change conserved residues, likely affecting 3D structure. For variants within GCNA's intrinsically disordered region, we performed computational modeling for consensus motifs. Two SNVs were predicted to impact the structure of these consensus motifs. All identified variants have an extremely low minor allele frequency in the general population and 6 of 7 were not detected in > 5000 biological fathers. Considering evidence from animal models, germ-cell-specific expression, 3D modeling, and computational predictions for SNVs, we propose that identified GCNA variants disrupt structure and function of the respective protein domains, ultimately arresting germ-cell division. To our knowledge, this is the first study implicating GCNA, a key genome integrity factor, in human male infertility.

Transcriptome profiling reveals signaling conditions dictating human spermatogonia fate in vitro.

Spermatogonial stem cells (SSCs) are essential for the generation of sperm and have potential therapeutic value for treating male infertility, which afflicts >100 million men world-wide. While much has been learned about rodent SSCs, human SSCs remain poorly understood. Here, we molecularly characterize human SSCs and define conditions favoring their culture. To achieve this, we first identified a cell-surface protein, PLPPR3, that allowed purification of human primitive undifferentiated spermatogonia (uSPG) highly enriched for SSCs. Comparative RNA-sequencing analysis of these enriched SSCs with differentiating SPG (KIT+ cells) revealed the full complement of genes that shift expression during this developmental transition, including genes encoding key components in the TGF-ß, GDNF, AKT, and JAK-STAT signaling pathways. We examined the effect of manipulating these signaling pathways on cultured human SPG using both conventional approaches and single-cell RNA-sequencing analysis. This revealed that GDNF and BMP8B broadly support human SPG culture, while activin A selectively supports more advanced human SPG. One condition-AKT pathway inhibition-had the unique ability to selectively support the culture of primitive human uSPG. This raises the possibility that supplementation with an AKT inhibitor could be used to culture human SSCs in vitro for therapeutic applications.

Single-Cell RNA Sequencing of Human, Macaque, and Mouse Testes Uncovers Conserved and Divergent Features of Mammalian Spermatogenesis.

Spermatogenesis is a highly regulated process that produces sperm to transmit genetic information to the next generation. Although extensively studied in mice, our current understanding of primate spermatogenesis is limited to populations defined by state-specific markers from rodent data. As between-species differences have been reported in the duration and differentiation hierarchy of this process, it remains unclear how molecular markers and cell states are conserved or have diverged from mice to man. To address this challenge, we employ single-cell RNA sequencing to identify transcriptional signatures of major germ and somatic cell types of the testes in human, macaque, and mice. This approach reveals similarities and differences in expression throughout spermatogenesis, including the stem/progenitor pool of spermatogonia, markers of differentiation, potential regulators of meiosis, RNA turnover during spermatid differentiation, and germ cell-soma communication. These datasets provide a rich foundation for future targeted mechanistic studies of primate germ cell development and in vitro gametogenesis.

An isolated CLASP TOG domain suppresses microtubule catastrophe and promotes rescue.

Microtubules are heavily regulated dynamic polymers of αß-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the cytoplasmic linker associated protein (CLASP) family that reduce catastrophe and/or increase rescue also contain arrayed TOGs, but how CLASP TOGs contribute to activity is poorly understood. Here, using Saccharomyces cerevisiae Stu1 as a model CLASP, we report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. The two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. The structure of Stu1-TOG2 reveals a CLASP-specific residue that likely confers distinctive tubulin-binding properties. The isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro, contradicting the expectation that regulatory activity requires an array of TOGs. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function.

A tethered delivery mechanism explains the catalytic action of a microtubule polymerase.

Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple αß-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast αß-tubulin. Like TOG1, TOG2 binds selectively to a fully 'curved' conformation of αß-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two αß-tubulins. Preferential interactions between TOGs and fully curved αß-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized αß-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end.