ABSTRACT
The Plasmodium falciparum life cycle includes obligate transition between a human and mosquito host. Gametocytes are responsible for transmission from the human to the mosquito vector where gamete fusion followed by meiosis occurs. To elucidate how male and female gametocytes differentiate in the absence of sex chromosomes, we perform FACS-based cell enrichment of a P. falciparum gametocyte reporter line followed by single-cell RNA-seq. In our analyses we define the transcriptional programs and predict candidate driver genes underlying male and female development, including genes from the ApiAP2 family of transcription factors. A motif-driven, gene regulatory network analysis indicates that AP2-G5 specifically modulates male development. Additionally, genes linked to the inner membrane complex, involved in morphological changes, are uniquely expressed in the female lineage. The transcriptional programs of male and female development detailed herein allow for further exploration of the evolution of sex in eukaryotes and provide targets for future development of transmission blocking therapies.
Subject(s)
Gametogenesis , Plasmodium falciparum , Single-Cell Analysis , Transcriptome , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Gametogenesis/genetics , Female , Humans , Male , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Gene Regulatory Networks , RNA-Seq , Animals , Transcription Factors/metabolism , Transcription Factors/genetics , Germ Cells/metabolism , Cell Differentiation/geneticsABSTRACT
The Caenorhabditis elegans germline is arranged spatiotemporally and is therefore a powerful model system for the interrogation of meiotic molecular dynamics. Coupling this property with the temporal control that the auxin-inducible degron (AID) system allows can unveil new/unappreciated roles for critical meiotic factors in specific germline regions. Here we describe a widely used approach for the introduction of degron tags to specific targets and provide a procedure for applying the AID system to C. elegans meiotic DSB repair dynamics in the germline.
Subject(s)
Caenorhabditis elegans , DNA Breaks, Double-Stranded , Meiosis , Caenorhabditis elegans/genetics , Animals , DNA Repair , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Germ Cells/metabolismABSTRACT
Fertility requires the faithful proliferation of germ cells and their differentiation into gametes. Controlling these cellular states demands precise timing and expression of gene networks. Nucleic acid binding proteins (NBPs) play critical roles in gene expression networks that influence germ cell development. There has, however, been no functional analysis of the entire NBP repertoire in controlling in vivo germ cell development. Here, we analyzed germ cell states and germline architecture to systematically investigate the function of 364 germline-expressed NBPs in the Caenorhabditis elegans germ line. Using germline-specific knockdown, automated germ cell counting, and high-content analysis of germ cell nuclei and plasma membrane organization, we identify 156 NBPs with discrete autonomous germline functions. By identifying NBPs that control the germ cell cycle, proliferation, differentiation, germline structure and fertility, we have created an atlas for mechanistic dissection of germ cell behavior and gamete production.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Germ Cells , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Germ Cells/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Gene Expression Regulation, Developmental , Cell Differentiation/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Fertility/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cell Proliferation/genetics , Cell Cycle/genetics , Male , Gene Regulatory NetworksABSTRACT
In animals, stem cell populations of varying potency facilitate regeneration and tissue homeostasis. Notably, germline stem cells in both vertebrates and invertebrates express highly conserved RNA binding proteins, such as nanos, vasa, and piwi. In highly regenerative animals, these genes are also expressed in somatic stem cells, which led to the proposal that they had an ancestral role in all stem cells. In cnidarians, multi- and pluripotent interstitial stem cells have only been identified in hydrozoans. Therefore, it is currently unclear if cnidarian stem cell systems share a common evolutionary origin. We, therefore, aimed to characterize conserved stem cell marker genes in the sea anemone Nematostella vectensis. Through transgenic reporter genes and single-cell transcriptomics, we identify cell populations expressing the germline-associated markers piwi1 and nanos2 in the soma and germline, and gene knockout shows that Nanos2 is indispensable for germline formation. This suggests that nanos and piwi genes have a conserved role in somatic and germline stem cells in cnidarians.
Subject(s)
Germ Cells , RNA-Binding Proteins , Sea Anemones , Animals , Sea Anemones/genetics , Sea Anemones/metabolism , Germ Cells/metabolism , Germ Cells/cytology , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Cell Lineage/genetics , Stem Cells/metabolism , Stem Cells/cytology , Argonaute Proteins/metabolism , Argonaute Proteins/geneticsABSTRACT
Recently, we reported that, in the naked mole-rat (Heterocephalus glaber) ovary, there is mitotic expansion of the primordial germ cells (PGCs), and the initiation of the meiotic program occurs postnatally. This is opposite to almost all other mammals, including humans and mice, whose reproductive cycle begins very early in development. In both mouse and human, the ovaries become populated with PGCs in utero; these PGCs will later generate the oogonia. After mitotic proliferation, these cells will trigger the meiotic program and initiate meiotic prophase I. Given that all these processes happen in utero, their analysis has been very challenging; so the ability to study them postnatally and to manipulate them with inhibitors or other substances, in the naked mole-rat, opens new possibilities in the field. In this chapter, we present a comprehensive collection of protocols that permit the culture of whole naked mole-rat ovaries, followed by analysis of germ cells, from PGCs to oocytes, in meiotic prophase I, as well the obtention of single-cell suspension or single-nuclei suspension for RNASeq.
Subject(s)
Meiotic Prophase I , Mole Rats , Ovary , Animals , Female , Germ Cells/cytology , Germ Cells/metabolism , Meiosis , Oocytes/cytology , Oocytes/metabolism , Ovary/cytology , Sequence Analysis, RNA/methods , Single-Cell Gene Expression AnalysisABSTRACT
BACKGROUND: Exploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases. METHODS: Diaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development. RESULTS: Based on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3ß-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated. CONCLUSIONS: Overall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.
Subject(s)
Cell Proliferation , Formins , Germ Cells , Gonads , Mice, Knockout , Animals , Mice , Female , Male , Formins/genetics , Formins/metabolism , Cell Proliferation/genetics , Gonads/metabolism , Germ Cells/metabolism , Apoptosis/genetics , Testis/metabolism , Testis/growth & development , Testis/cytology , Cell Movement/genetics , Ovary/metabolism , Ovary/growth & development , Mice, Inbred C57BLABSTRACT
BACKGROUND: The piRNA pathway in animal gonads functions as an 'RNA-based immune system', serving to silence transposable elements and prevent inheritance of novel invaders. In Drosophila, this pathway relies on three gonad-specific Argonaute proteins (Argonaute-3, Aubergine and Piwi) that associate with 23-28 nucleotide piRNAs, directing the silencing of transposon-derived transcripts. Transposons constitute a primary driver of genome evolution, yet the evolution of piRNA pathway factors has not received in-depth exploration. Specifically, channel nuclear pore proteins, which impact piRNA processing, exhibit regions of rapid evolution in their promoters. Consequently, the question arises whether such a mode of evolution is a general feature of transposon silencing pathways. RESULTS: By employing genomic analysis of coding and promoter regions within genes that function in transposon silencing in Drosophila, we demonstrate that the promoters of germ cell-specific piRNA factors are undergoing rapid evolution. Our findings indicate that rapid promoter evolution is a common trait among piRNA factors engaged in germline silencing across insect species, potentially contributing to gene expression divergence in closely related taxa. Furthermore, we observe that the promoters of genes exclusively expressed in germ cells generally exhibit rapid evolution, with some divergence in gene expression. CONCLUSION: Our results suggest that increased germline promoter evolution, in partnership with other factors, could contribute to transposon silencing and evolution of species through differential expression of genes driven by invading transposons.
Subject(s)
DNA Transposable Elements , Evolution, Molecular , Gene Silencing , Germ Cells , Promoter Regions, Genetic , RNA, Small Interfering , Animals , DNA Transposable Elements/genetics , Germ Cells/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Drosophila Proteins/genetics , Drosophila/genetics , Argonaute Proteins/geneticsABSTRACT
BACKGROUND: Malaria, a global health concern, is caused by parasites of the Plasmodium genus, which undergo gametogenesis in the midgut of mosquitoes after ingestion of an infected blood meal. The resulting male and female gametes fuse to form a zygote, which differentiates into a motile ookinete. After traversing the midgut epithelium, the ookinete differentiates into an oocyst on the basal side of the epithelium. METHODS: Membrane proteins with increased gene expression levels from the gamete to oocyst stages in P. berghei were investigated utilizing PlasmoDB, the functional genomic database for Plasmodium spp. Based on this analysis, we selected the 184-kDa membrane protein, Pb184, for further study. The expression of Pb184 was further confirmed through immunofluorescence staining, following which we examined whether Pb184 is involved in fertilization using antibodies targeting the C-terminal region of Pb184 and biotin-labeled C-terminal region peptides of Pb184. RESULTS: Pb184 is expressed on the surface of male and female gametes. The antibody inhibited zygote and ookinete formation in vitro. When mosquitoes were fed on parasite-infected blood containing the antibody, oocyst formation decreased on the second day after feeding. Synthesized biotin-labeled peptides matching the C-terminal region of Pb184 bound to the female gamete and the residual body of male gametes, and inhibited differentiation into ookinetes in the in vitro culture system. CONCLUSIONS: These results may be useful for the further studying the fertilization mechanism of Plasmodium protozoa. There is also the potential for their application as future tools to prevent malaria transmission.
Subject(s)
Fertilization , Plasmodium berghei , Protozoan Proteins , Plasmodium berghei/genetics , Plasmodium berghei/metabolism , Animals , Female , Male , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Mice , Germ Cells/metabolism , Malaria/parasitology , Membrane Proteins/metabolism , Membrane Proteins/genetics , Zygote/metabolism , Anopheles/parasitology , Anopheles/metabolism , Oocysts/metabolism , Gametogenesis/geneticsABSTRACT
In animals with germ plasm, embryonic germline precursors inherit germ granules, condensates proposed to regulate mRNAs coding for germ cell fate determinants. In Caenorhabditis elegans, mRNAs are recruited to germ granules by MEG-3, a sequence non-specific RNA-binding protein that forms stabilizing interfacial clusters on germ granules. Using fluorescence in situ hybridization, we confirmed that 441 MEG-3-bound transcripts are distributed in a pattern consistent with enrichment in germ granules. Thirteen are related to transcripts reported in germ granules in Drosophila or Nasonia. The majority, however, are low-translation maternal transcripts required for embryogenesis that are not maintained preferentially in the nascent germline. Granule enrichment raises the concentration of certain transcripts in germ plasm but is not essential to regulate mRNA translation or stability. Our findings suggest that only a minority of germ granule-associated transcripts contribute to germ cell fate in C. elegans and that the vast majority function as non-specific scaffolds for MEG-3.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Germ Cells , Protein Biosynthesis , RNA, Messenger , RNA-Binding Proteins , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Germ Cells/metabolism , Germ Cells/cytology , RNA, Messenger/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Cytoplasmic Granules/metabolism , Gene Expression Regulation, Developmental , In Situ Hybridization, FluorescenceABSTRACT
In studying the molecular underpinning of spermatogenesis, we expect to understand the fundamental biological processes better and potentially identify genes that may lead to novel diagnostic and therapeutic strategies toward precision medicine in male infertility. In this review, we emphasized our perspective that the path forward necessitates integrative studies that rely on complementary approaches and types of data. To comprehensively analyze spermatogenesis, this review proposes four axes of integration. First, spanning the analysis of spermatogenesis in the healthy state alongside pathologies. Second, the experimental analysis of model systems (in which we can deploy treatments and perturbations) alongside human data. Third, the phenotype is measured alongside its underlying molecular profiles using known markers augmented with unbiased profiles. Finally, the testicular cells are studied as ecosystems, analyzing the germ cells alongside the states observed in the supporting somatic cells. Recently, the study of spermatogenesis has been advancing using single-cell RNA sequencing, where scientists have uncovered the unique stages of germ cell development in mice, revealing new regulators of spermatogenesis and previously unknown cell subtypes in the testis. An in-depth analysis of meiotic and postmeiotic stages led to the discovery of marker genes for spermatogonia, Sertoli and Leydig cells and further elucidated all the other germline and somatic cells in the testis microenvironment in normal and pathogenic conditions. The outcome of an integrative analysis of spermatogenesis using advanced molecular profiling technologies such as scRNA-seq has already propelled our biological understanding, with additional studies expected to have clinical implications for the study of male fertility. By uncovering new genes and pathways involved in abnormal spermatogenesis, we may gain insights into subfertility or sterility.
Subject(s)
RNA-Seq , Single-Cell Analysis , Spermatogenesis , Spermatogenesis/genetics , Humans , Male , Animals , Single-Cell Analysis/methods , Mice , RNA-Seq/methods , Germ Cells/metabolism , Testis/metabolism , Infertility, Male/genetics , Single-Cell Gene Expression AnalysisABSTRACT
As an RNA binding protein (RBP), DDX5 is widely involved in the regulation of various biological activities. While recent studies have confirmed that DDX5 can act as a transcriptional cofactor that is involved in the formation of gametes, few studies have investigated whether DDX5 can be used as a transcription factor to regulate the formation of primordial germ cells (PGCs). In this study, we found that DDX5 was significantly up-regulated during chicken PGC formation. Under different PGC induction models, the overexpression of DDX5 not only up-regulates PGC markers but also significantly improves the formation efficiency of primordial germ cell-like cells (PGCLC). Conversely, the inhibition of DDX5 expression can significantly inhibit both the expression of PGC markers and PGCLC formation efficiency. The effect of DDX5 on PGC formation in vivo was consistent with that seen in vitro. Interestingly, DDX5 not only participates in the formation of PGCs but also positively regulates their migration and proliferation. In the process of studying the mechanism by which DDX5 regulates PGC formation, we found that DDX5 acts as a transcription factor to bind to the promoter region of BMP4-a key gene for PGC formation-and activates the expression of BMP4. In summary, we confirm that DDX5 can act as a positive transcription factor to regulate the formation of PGCs in chickens. The obtained results not only enhance our understanding of the way in which DDX5 regulates the development of germ cells but also provide a new target for systematically optimizing the culture and induction system of PGCs in chickens in vitro.
Subject(s)
Bone Morphogenetic Protein 4 , Chickens , DEAD-box RNA Helicases , Germ Cells , Animals , Bone Morphogenetic Protein 4/metabolism , Bone Morphogenetic Protein 4/genetics , Germ Cells/metabolism , Chickens/genetics , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Chick Embryo , Gene Expression Regulation, Developmental , Transcription Factors/genetics , Transcription Factors/metabolism , Cell Proliferation , Cell Movement/genetics , Promoter Regions, GeneticABSTRACT
The fungicide tebuconazole (TEB) poses risks to human and animal health via various exposure routes. It induces toxicity in multiple organs and disrupts reproductive health by affecting steroid hormone synthesis and fetal development. In this study, we investigated the impact of TEB on fetal testes using in vitro models, focusing on germ, Sertoli, and Leydig cells, and explored the mechanisms underlying cellular damage. The results revealed significant damage to germ cells and disruption of Leydig cell development. TEB exposure led to a decrease in germ cell numbers, as indicated by histological and immunostaining analyses. TEB induced the up- and down-regulation of the expression of fetal and adult Leydig cell markers, respectively. Additionally, TEB-treated fetal testes exhibited increased expression of oxidative-stress-related genes and proteins. However, co-treatment with the antioxidant N-acetylcysteine mitigated TEB-induced germ cell damage and prevented abnormal Leydig cell development. These findings suggest that administration of antioxidants can prevent the intratesticular damage typically caused by TEB exposure.
Subject(s)
Leydig Cells , Organ Culture Techniques , Oxidative Stress , Reactive Oxygen Species , Testis , Triazoles , Male , Animals , Testis/drug effects , Testis/metabolism , Triazoles/pharmacology , Mice , Reactive Oxygen Species/metabolism , Leydig Cells/drug effects , Leydig Cells/metabolism , Oxidative Stress/drug effects , Organ Culture Techniques/methods , Sertoli Cells/drug effects , Sertoli Cells/metabolism , Antioxidants/pharmacology , Fetus/drug effects , Fungicides, Industrial/toxicity , Germ Cells/drug effects , Germ Cells/metabolismABSTRACT
Germ granules are biomolecular condensates present in most animal germ cells. One function of germ granules is to help maintain germ cell totipotency by organizing mRNA regulatory machinery, including small RNA-based gene regulatory pathways. The C. elegans germ granule is compartmentalized into multiple subcompartments whose biological functions are largely unknown. Here, we identify an uncharted subcompartment of the C. elegans germ granule, which we term the E granule. The E granule is nonrandomly positioned within the germ granule. We identify five proteins that localize to the E granule, including the RNA-dependent RNA polymerase (RdRP) EGO-1, the Dicer-related helicase DRH-3, the Tudor domain-containing protein EKL-1, and two intrinsically disordered proteins, EGC-1 and ELLI-1. Localization of EGO-1 to the E granule enables synthesis of a specialized class of 22G RNAs, which derive exclusively from 5' regions of a subset of germline-expressed mRNAs. Defects in E granule assembly elicit disordered production of endogenous siRNAs, which disturbs fertility and the RNAi response. Our results define a distinct subcompartment of the C. elegans germ granule and suggest that one function of germ granule compartmentalization is to facilitate the localized production of specialized classes of small regulatory RNAs.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Cytoplasmic Granules , Germ Cells , Caenorhabditis elegans/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Animals , Germ Cells/metabolism , Cytoplasmic Granules/metabolism , RNA, Messenger/metabolism , RNA, Messenger/genetics , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/genetics , Intrinsically Disordered Proteins/metabolism , Intrinsically Disordered Proteins/geneticsABSTRACT
The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Germ Cells , Nucleosomes , Promoter Regions, Genetic , RNA, Small Interfering , Animals , Caenorhabditis elegans/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Nucleosomes/genetics , Nucleosomes/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Germ Cells/metabolism , Chromatin Assembly and Disassembly/genetics , Chromatin/genetics , Chromatin/metabolism , Transcription, Genetic , Gene Expression Regulation/genetics , Heterochromatin/genetics , Heterochromatin/metabolism , Piwi-Interacting RNAABSTRACT
Cancer cells undergo major epigenetic alterations and transcriptomic changes, including ectopic expression of tissue- and cell-type-specific genes. Here, we show that the germline-specific RNA helicase DDX4 forms germ-granule-like cytoplasmic ribonucleoprotein granules in various human tumors, but not in cultured cancer cells. These cancerous DDX4 complexes contain RNA-binding proteins and splicing regulators, including many known germ granule components. The deletion of DDX4 in cancer cells induces transcriptomic changes and affects the alternative splicing landscape of a number of genes involved in cancer growth and invasiveness, leading to compromised capability of DDX4-null cancer cells to form xenograft tumors in immunocompromised mice. Importantly, the occurrence of DDX4 granules is associated with poor survival in patients with head and neck squamous cell carcinoma and higher histological grade of prostate cancer. Taken together, these results show that the germ-granule-resembling cancerous DDX4 granules control gene expression and promote malignant and invasive properties of cancer cells.
Subject(s)
Cytoplasmic Granules , DEAD-box RNA Helicases , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Humans , Animals , Mice , Cytoplasmic Granules/metabolism , Male , Cell Line, Tumor , Cell Proliferation , Alternative Splicing/genetics , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Germ Cells/metabolismABSTRACT
Germ granules have been hypothesized to deliver mRNAs of germ cell fate determinants to primordial germ cells. Now, a new study in Development finds that many mRNAs enriched in germ granules are not involved in germline development in Caenorhabditis elegans. To find out more about the story behind the paper, we caught up with first author Alyshia Scholl, second author Yihong Liu and corresponding author Geraldine Seydoux, Professor at Johns Hopkins University School of Medicine.
Subject(s)
Caenorhabditis elegans , Animals , Caenorhabditis elegans/genetics , Germ Cells/metabolism , Developmental Biology/history , History, 21st Century , History, 20th Century , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/geneticsABSTRACT
Embryonic germ cells develop rapidly to establish the foundation for future developmental trajectories, and in this process, they make critical lineage choices including the configuration of their unique identity and a decision on sex. Here, we use single-cell genomics patterns for the entire embryonic germline in Drosophila melanogaster along with the somatic gonadal precursors after embryonic gonad coalescence to investigate molecular mechanisms involved in the setting up and regulation of the germline program. Profiling of the early germline chromatin landscape revealed sex- and stage-specific features. In the male germline immediately after zygotic activation, the chromatin structure underwent a brief remodeling phase during which nucleosome density was lower and deconcentrated from promoter regions. These findings echoed enrichment analysis results of our genomics data in which top candidates were factors with the ability to mediate large-scale chromatin reorganization. Together, they point to the importance of chromatin regulation in the early germline and raise the possibility of a conserved epigenetic reprogramming-like process required for proper initiation of germline development.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin , Drosophila melanogaster , Embryonic Development , Animals , Male , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Chromatin/metabolism , Chromatin/genetics , Chromatin Assembly and Disassembly/genetics , Embryonic Development/genetics , Gene Expression Regulation, Developmental/genetics , Embryonic Germ Cells/metabolism , Embryonic Germ Cells/cytology , Germ Cells/metabolism , Epigenesis, Genetic , Female , Nucleosomes/metabolism , Nucleosomes/genetics , Single-Cell Analysis/methodsABSTRACT
This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.
Subject(s)
Cell Survival , Mice, Inbred C57BL , Organoids , Spermatogenesis , Testis , Testosterone , Male , Animals , Organoids/cytology , Organoids/metabolism , Organoids/drug effects , Testis/cytology , Testis/drug effects , Testis/metabolism , Testosterone/pharmacology , Spermatogenesis/drug effects , Cell Survival/drug effects , Mice , Cell Differentiation/drug effects , Germ Cells/cytology , Germ Cells/drug effects , Germ Cells/metabolism , Dexamethasone/pharmacologyABSTRACT
Stem cell loss in aging and disease is associated with nuclear deformation. Yet, how nuclear shape influences stem cell homeostasis is poorly understood. We investigated this connection using Drosophila germline stem cells, as survival of these stem cells is compromised by dysfunction of the nuclear lamina, the extensive protein network that lines the inner nuclear membrane and gives shape to the nucleus. To induce nuclear distortion in germline stem cells, we used the GAL4-UAS system to increase expression of the permanently farnesylated nuclear lamina protein, Kugelkern, a rate limiting factor for nuclear growth. We show that elevated Kugelkern levels cause severe nuclear distortion in germline stem cells, including extensive thickening and lobulation of the nuclear envelope and nuclear lamina, as well as alteration of internal nuclear compartments. Despite these changes, germline stem cell number, proliferation, and female fertility are preserved, even as females age. Collectively, these data demonstrate that disruption of nuclear architecture does not cause a failure of germline stem cell survival or homeostasis, revealing that nuclear deformation does not invariably promote stem cell loss.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Germ Cells , Homeostasis , Nuclear Lamina , Stem Cells , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Female , Germ Cells/metabolism , Drosophila melanogaster/metabolism , Stem Cells/metabolism , Nuclear Lamina/metabolism , Cell Nucleus/metabolism , Cell Proliferation , Drosophila/metabolism , Nuclear Envelope/metabolismABSTRACT
Atrazine is currently one of the most commonly used agrochemicals in the United States and elsewhere. Here, we studied the immunoexpression of molecular markers of mammalian testicular functions: androgen receptor (AR), promyelocytic leukemia zinc finger (PLZF), GDNF family receptor alpha-1 (GFRA1), VASA/DDX4 (DEAD-Box Helicase 4) as well as the levels of intratesticular and intra-epididymal estradiol (E2) and dihydrotestosterone (DHT), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukins (IL-1ß and IL-6, IL-10) and testicular chemokines (CXCL-1, CCL-2 and CCL3) in BalB/c mice after a sub-acute gavage treatment with a gonado-toxin, atrazine (50 mg/kg body wt.) for three days. We found high numbers of AR immunopositive Sertoli cells and low numbers of GFRA1, PLZF and VASA/DDX4-positive germ cells in the seminiferous tubule regions of the testes. While TNF-α level in the testes fell and remained unchanged in the epididymides, IFN-γ levels in the testes remained constant but increased in the epididymides. E2 and DHT concentrations remained unaltered in the testes but were changed in the epididymides. There were no significant changes in the levels of interleukins in the testis and epididymis. Intratesticular chemokines were also not significantly altered, except for CCL-4, which was increased in the testis. Light microscopy of the epididymis showed detached epithelium and some detached cells in the lumen. It is concluded that atrazine changed the inflammatory status of the gonads and highlighted Sertoli and undifferentiated spermatogonia as important targets for atrazine's toxic effects in the testis of mice. Concerning the epididymis, atrazine altered the epididymal hormonal concentrations and promoted histopathological modifications in its parenchyma.