miR-301a-5p Regulates TGFB2 during Chicken Spermatogenesis

The process of spermatogenesis is complex and systemic, requiring the cooperation of many regulators. However, little is known about how micro RNAs (miRNAs) regulate spermatogenesis in poultry. In this study, we investigated key miRNAs and their target genes that are involved in spermatogenesis in chickens. Next-generation sequencing was conducted to determine miRNA expression profiles in five cell types: primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia (Spa), and chicken sperm. Next, we analyzed and identified several key miRNAs that regulate spermatogenesis in the four germline cell miRNA profiles. Among the enriched miRNAs, miRNA-301a-5p was the key miRNA in PGCs, SSCs, and Spa. Through reverse transcription quantitative PCR (RT-qPCR), dual-luciferase, and miRNA salience, we confirmed that miR-301a-5p binds to transforming growth factor-beta 2 (TGFβ2) and is involved in the transforming growth factor-beta (TGF-β) signaling pathway and germ cell development. To the best of our knowledge, this is the first demonstration of miR-301a-5p involvement in spermatogenesis by direct binding to TGFβ2, a key gene in the TGF-β signaling pathway. This finding contributes to the insights into the molecular mechanism through which miRNAs regulate germline cell differentiation and spermatogenesis in chickens.

Transcriptomic and Epigenetic Preservation of Genetic Sex Identity in Estrogen-feminized Male Chicken Embryonic Gonads

Whereas in ovo exposure of genetically male (ZZ) chicken embryos to exogenous estrogens temporarily feminizes gonads at the time of hatching, the morphologically ovarian ZZ-gonads (FemZZs for feminized ZZ gonads) are masculinized back to testes within 1 year. To identify the feminization-resistant “memory” of genetic male sex, FemZZs showing varying degrees of feminization were subjected to transcriptomic, DNA methylome, and immunofluorescence analyses. Protein-coding genes were classified based on their relative mRNA expression across normal ZZ-testes, genetically female (ZW) ovaries, and FemZZs. We identified a group of 25 genes that were strongly expressed in both ZZ-testes and FemZZs but dramatically suppressed in ZW-ovaries. Interestingly, 84% (21/25) of these feminization-resistant testicular marker genes, including the DMRT1 master masculinizing gene, were located in chromosome Z. Expression of representative marker genes of germline cells (eg, DAZL or DDX4/VASA) was stronger in FemZZs than normal ZZ-testes or ZW-ovaries. We also identified 231 repetitive sequences (RSs) that were strongly expressed in both ZZ-testes and FemZZs, but these RSs were not enriched in chromosome Z. Although 94% (165/176) of RSs exclusively expressed in ZW-ovaries were located in chromosome W, no feminization-inducible RS was detected in FemZZs. DNA methylome analysis distinguished FemZZs from normal ZZ- and ZW-gonads. Immunofluorescence analysis of FemZZ gonads revealed expression of DMRT1 protein in medullary SOX9+ somatic cells and apparent germline cell populations in both medulla and cortex. Taken together, our study provides evidence that both somatic and germline cell populations in morphologically feminized FemZZs maintain significant transcriptomic and epigenetic memories of genetic sex.

Mutator Foci Are Regulated by Developmental Stage, RNA, and the Germline Cell Cycle in Caenorhabditis elegans

RNA interference is a crucial gene regulatory mechanism in Caenorhabditis elegans. Phase-separated perinuclear germline compartments called Mutator foci are a key element of RNAi, ensuring robust gene silencing and transgenerational epigenetic inheritance. Despite their importance, Mutator foci regulation is not well understood, and observations of Mutator foci have been largely limited to adult hermaphrodite germlines. Here we reveal that punctate Mutator foci arise in the progenitor germ cells of early embryos and persist throughout all larval stages. They are additionally present throughout the male germline and in the cytoplasm of post-meiotic spermatids, suggestive of a role in paternal epigenetic inheritance. In the adult germline, transcriptional inhibition results in a pachytene-specific loss of Mutator foci, indicating that Mutator foci are partially reliant on RNA for their stability. Finally, we demonstrate that Mutator foci intensity is modulated by the stage of the germline cell cycle and specifically, that Mutator foci are brightest and most robust in the mitotic cells, transition zone, and late pachytene of adult germlines. Thus, our data defines several new factors that modulate Mutator foci morphology which may ultimately have implications for efficacy of RNAi in certain cell stages or environments.

Mutator foci are regulated by developmental stage, RNA, and the germline cell cycle in Caenorhabditis elegans

ABSTRACTRNA interference is a crucial gene regulatory mechanism in Caenorhabditis elegans. Phase-separated perinuclear germline compartments called Mutator foci are a key element of RNAi, ensuring robust gene silencing and transgenerational epigenetic inheritance. Despite their importance, Mutator foci regulation is not well understood, and observations of Mutator foci have been largely limited to adult hermaphrodite germlines. Here we reveal that punctate Mutator foci arise in the progenitor germ cells of early embryos and persist throughout all larval stages. They are additionally present throughout the male germline and in the cytoplasm of post-meiotic spermatids, suggestive of a role in paternal epigenetic inheritance. In the adult germline, transcriptional inhibition results in a pachytene-specific loss of Mutator foci, indicating that Mutator foci are partially reliant on RNA for their stability. Finally, we demonstrate that Mutator foci intensity is modulated by the stage of the germline cell cycle and specifically, that Mutator foci are brightest and most robust in the mitotic cells, transition zone, and late pachytene of adult germlines. Thus, our data defines several new factors that modulate Mutator foci morphology which may ultimately have implications for efficacy of RNAi in certain cell stages or environments.

Rapid accumulation of mutations in growing mycelia of a hypervariable fungus Schizophyllum commune

The number of mutations that occur per nucleotide per generation varies between species by several orders of magnitude. In multicellular eukaryotes, the per generation mutation rate depends both on the per cell division mutation rate and on the number of germline cell divisions per generation. In a range of species, from fungi to humans, the number of germline cell divisions is lower than that of somatic cells, reducing the mutation burden on the offspring. The basidiomycete Schizophyllum commune has the highest level of genetic polymorphism known among eukaryotes. In a previous study, it was also found to have a high per generation mutation rate, probably contributing to its high polymorphism. However, this rate has been measured only in a breeding experiment on Petri dishes, and it is unclear how this result translates to natural populations. Here, we used an experimental design that measures the rate of accumulation of de novo mutations in a linearly growing mycelium. We show that S. commune accumulates mutations at a uniform rate of 1.4·10−7 substitutions per nucleotide per meter of growth, which is 3 orders of magnitude higher than the corresponding rates in the oak Quercus robur and the fungus Armillaria gallica. This figure is consistent with the estimate obtained before, and suggests the lack of a dedicated germline in this system. If so, even a low per cell division mutation rate can translate into a very high per generation mutation rate when the number of cell divisions between consecutive meioses is large.

Social insect colony size is correlated with rates of substitution and DNA repair gene evolution

Larger numbers of germline cell divisions can increase the number of mutations inherited by offspring. Therefore, in systems where the number of offspring is dependent on the number of germline cell divisions, a higher overall rate of molecular evolution may be expected. Here I examine whether colony size in social insects, which varies from tens to millions, influences molecular evolutionary rates by analyzing several recently collected datasets. First, I find that colony size is negatively correlated with GC-content across 115 ant genera, indicative of a positive relationship between substitution rate and colony size. Second, genome-wide rates of molecular evolution are positively correlated with colony size in three clades of social insects including eight species in the ant genusPseudomyrmex, seven fungus-growing ants, and 11 bee species. The additional germline cell divisions necessary to maintain large colony sizes might lead to mutation accumulation in the germlines of queens of these species, a process similar to that which occurs in aging human males. I also find intensified constraint on DNA repair genes in species with large colonies, suggesting that the additional mutations that occur in these taxa increase selective pressure for improved replication fidelity. Colony size, a fundamental facet of eusociality, plays a previously unappreciated role in genome evolution.