scholarly journals Dissecting the initiation of female meiosis in the mouse at single-cell resolution

2019 ◽  
Author(s):  
Wei Ge ◽  
Jun-Jie Wang ◽  
Rui-Qian Zhang ◽  
Shao-Jing Tan ◽  
Fa-Li Zhang ◽  
...  
Keyword(s):  
Germ Cell ◽  
Single Cell ◽  
Germ Cells ◽  
Molecular Mechanisms ◽  
Gene Expression Pattern ◽  
Cell Populations ◽  
Molecular Pathways ◽  
Cell Clusters ◽  
Prophase I ◽  
New Genes

ABSTRACTGerm cell meiosis is one of the most finely orchestrated events during gametogenesis with distinct developmental patterns in males and females. However, in mammals, the molecular mechanisms involved in this process remain not well known. Here, we report detailed transcriptome analyses of cell populations present in the mouse female gonadal ridges (E11.5) and the embryonic ovaries from E12.5 to E14.5 using single cell RNA sequencing (scRNA seq). These periods correspond with the initiation and progression of meiosis throughout the first stage of prophase I. We identified 13 transcriptionally distinct cell populations and 7 transcriptionally distinct germ cell subclusters that correspond to mitotic (3 clusters) and meiotic (4 clusters) germ cells. By comparing the signature gene expression pattern of 4 meiotic germ cell clusters, we found that the 4 cell clusters correspond to different cell status en route to meiosis progression, and therefore, our research here characterized detailed transcriptome dynamics during meiotic prophase I. Reconstructing the progression of meiosis along pseudotime, we identified several new genes and molecular pathways with potential critical roles in the mitosis/meiosis transition and early meiotic progression. Last, the heterogeneity within somatic cell populations was also discussed and different cellular states were identified. Our scRNA seq analysis here represents a new important resource for deciphering the molecular pathways driving meiosis initiation and progression in female germ cells and ovarian somatic cells.

scholarly journals Transcriptome Landscape Reveals Underlying Mechanisms of Ovarian Cell Fate Differentiation and Primordial Follicle Assembly

2019 ◽  
Author(s):  
Jun-Jie Wang ◽  
Wei Ge ◽  
Qiu-Yue Zhai ◽  
Jing-Cai Liu ◽  
Xiao-Wen Sun ◽  
...  
Keyword(s):  
Germ Cell ◽  
Single Cell ◽  
Cell Fate ◽  
Germ Cells ◽  
Granulosa Cells ◽  
Primordial Follicle ◽  
Gene Clusters ◽  
Bioinformatics Analyses ◽  
Primordial Follicles ◽  
New Genes

AbstractPrimordial follicle assembly in mammals occurs at perinatal ages and largely determines the ovarian reserve available to support the reproductive lifespan. The primordial follicle structure is generated by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time-series on mouse ovaries coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to PD3 are reported for the first time. The time frame of analysis comprises the breakdown of germ cell cysts and the assembly of primordial follicles. Confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified ten distinct gene clusters associated to germ cells and eight to granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. Building single-cell pseudo temporal trajectories five states and two branch points of fate transition for the germ cells, and three states and one branch point for the granulosa cells were revealed. Moreover, GO and ClueGO term enrichment enabled identifying biological processes, molecular functions and cellular components more represented in germ cells and granulosa cells or common to both cell types at each specific stage. Finally, by SCENIC algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell specific transcription programs throughout the investigated period.

scholarly journals A Novel Method to Identify the Differences Between Two Single Cell Groups at Single Gene, Gene Pair, and Gene Module Levels

2021 ◽  
Vol 12 ◽  
Author(s):  
Lingyu Cui ◽  
Bo Wang ◽  
Changjing Ren ◽  
Ailan Wang ◽  
Hong An ◽  
...  
Keyword(s):  
Single Cell ◽  
Molecular Mechanisms ◽  
Single Gene ◽  
Cell Types ◽  
Human Pancreas ◽  
Biological Difference ◽  
Cell Clusters ◽  
Cell Groups ◽  
Network Modules ◽  
Differential Gene

Single-cell sequencing technology can not only view the heterogeneity of cells from a molecular perspective, but also discover new cell types. Although there are many effective methods on dropout imputation, cell clustering, and lineage reconstruction based on single cell RNA sequencing (RNA-seq) data, there is no systemic pipeline on how to compare two single cell clusters at the molecular level. In the study, we present a novel pipeline on comparing two single cell clusters, including calling differential gene expression, coexpression network modules, and so on. The pipeline could reveal mechanisms behind the biological difference between cell clusters and cell types, and identify cell type specific molecular mechanisms. We applied the pipeline to two famous single-cell databases, Usoskin from mouse brain and Xin from human pancreas, which contained 622 and 1,600 cells, respectively, both of which were composed of four types of cells. As a result, we identified many significant differential genes, differential gene coexpression and network modules among the cell clusters, which confirmed that different cell clusters might perform different functions.

286 EXPRESSION OF PLURIPOTENT STEM CELL MARKERS IN DOMESTIC CAT SPERMATOGONIAL CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 290 ◽  
Author(s):  
R. H. Powell ◽  
M. N. Biancardi ◽  
J. Galiguis ◽  
Q. Qin ◽  
C. E. Pope ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Basement Membrane ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Cell Populations ◽  
Surface Markers ◽  
Cell Markers

Spermatogonial stem cells (SSC), progenitor cells capable of both self-renewal and producing daughter cells that will differentiate into sperm, can be manipulated for transplantation to propagate genetically important males. This application was demonstrated in felids by the successful xeno-transplantation of ocelot mixed germ cells into the testes of domestic cats, which resulted in the production of ocelot sperm (Silva et al. 2012 J. Androl. 33, 264–276). Spermatogonial stem cells are in low numbers in the testis, but have been identified and isolated in different mammalian species using SSC surface markers; however, their expression varies among species. Until recently, little was known about the expression of SSC surface markers in feline species. We previously demonstrated that many mixed germ cells collected from adult cat testes express the germ cell markers GFRα1, GPR125, and C-Kit, and a smaller population of cells expresses the pluripotent SSC-specific markers SSEA-1 and SSEA-4 (Powell et al. 2011 Reprod. Fertil. Dev. 24, 221–222). In the present study, our goal was to identify germ cell and SSC-specific markers in SSC from cat testes. Immunohistochemical (IHC) localization of germ cell markers GFRα1, GPR125, and C-Kit and pluripotent SSC-specific markers SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 was detected in testis tissue from both sexually mature and prepubertal males. Testes were fixed with modified Davidson’s fixative for 24 h before processing, embedding, and sectioning. The EXPOSE Mouse and Rabbit Specific HRP/DAB detection IHC kit (Abcam®, Cambridge, MA, USA) was used for antibody detection. Staining for SSEA-1, SSEA-4, TRA-1-60, TRA-1-81, and Oct-4 markers was expressed specifically at the basement membrane of the seminiferous tubules in both adult and prepubertal testes. The GFRα1 and GPR125 markers were detected at the basement membrane of the seminiferous tubules and across the seminiferous tubule section. However, C-Kit was not detected in any cell. Using flow cytometry from a pool of cells from seven adult testes, we detected 45% GFRα1, 50% GPR125, 59% C-Kit, 18% TRA-1-60, 16% TRA-1-81 positive cells, and a very small portion of SSEA-1 (7%) and SSEA-4 (3%) positive cells. Dual staining of germ cells pooled from 3 testes revealed 3 distinct cell populations that were positive for GFRα1 only (23%), positive for both GFRα1 and SSEA-4 (6%), and positive for SSEA-4 only (1%). Our IHC staining of cat testes indicated that cells along the basement membrane of seminiferous tubules were positive for SSC-specific markers, and flow cytometry analysis revealed that there were different cell populations expressing both germ cell and SSC-specific markers. Flow cytometry results show overlapping germ cell populations expressing SSEA-4 and GFRα1, and IHC results reveal that SSEA-4 positive cells are spermatogonia, whereas GFRα1 positive cells include other stages of germ cells, indicating that the small population of cells positive only for SSEA-4 is undifferentiated cat SSC.

scholarly journals The single-cell epigenetic regulatory landscape in mammalian perinatal testis development

2021 ◽  
Author(s):  
Jinyue Liao ◽  
Hoi Ching Suen ◽  
Shitao Rao ◽  
Alfred Chun Shui Luk ◽  
Ruoyu Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Fate ◽  
Germ Cells ◽  
Somatic Cells ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cellular Heterogeneity ◽  
Cell Populations ◽  
Cell Type

AbstractSpermatogenesis depends on an orchestrated series of developing events in germ cells and full maturation of the somatic microenvironment. To date, the majority of efforts to study cellular heterogeneity in testis has been focused on single-cell gene expression rather than the chromatin landscape shaping gene expression. To advance our understanding of the regulatory programs underlying testicular cell types, we analyzed single-cell chromatin accessibility profiles in more than 25,000 cells from mouse developing testis. We showed that scATAC-Seq allowed us to deconvolve distinct cell populations and identify cis-regulatory elements (CREs) underlying cell type specification. We identified sets of transcription factors associated with cell type-specific accessibility, revealing novel regulators of cell fate specification and maintenance. Pseudotime reconstruction revealed detailed regulatory dynamics coordinating the sequential developmental progressions of germ cells and somatic cells. This high-resolution data also revealed putative stem cells within the Sertoli and Leydig cell populations. Further, we defined candidate target cell types and genes of several GWAS signals, including those associated with testosterone levels and coronary artery disease. Collectively, our data provide a blueprint of the ‘regulon’ of the mouse male germline and supporting somatic cells.

scholarly journals A clusterability measure for single-cell transcriptomics reveals phenotypic subpopulations

2021 ◽  
Author(s):  
Maria Mircea ◽  
Mazene Hochane ◽  
Xueying Fan ◽  
Susana M. Chuva de Sousa Lopes ◽  
Diego Garlaschelli ◽  
...  
Keyword(s):  
Single Cell ◽  
Random Matrix Theory ◽  
Random Matrix ◽  
Matrix Theory ◽  
Unsupervised Clustering ◽  
Cell Populations ◽  
Cell Clusters ◽  
Transcriptomics Data

The ability to discover new cell populations by unsupervised clustering of single-cell transcriptomics data has revolutionized biology. Currently, there is no principled way to decide, whether a cluster of cells contains meaningful subpopulations that should be further resolved. Here we present SIGMA, a clusterability measure derived from random matrix theory, that can be used to identify cell clusters with non-random sub-structure, testably leading to the discovery of previously overlooked phenotypes.

scholarly journals Single-Cell RNA Sequencing Defines the Regulation of Spermatogenesis by Sertoli-Cell Androgen Signaling

2021 ◽  
Vol 9 ◽  
Author(s):  
Congcong Cao ◽  
Qian Ma ◽  
Shaomei Mo ◽  
Ge Shu ◽  
Qunlong Liu ◽  
...  
Keyword(s):  
Single Cell ◽  
Sertoli Cells ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Cell Populations ◽  
Wild Type ◽  
Cellular Processes ◽  
Transcriptomic Sequencing ◽  
Transcriptional Changes

Androgen receptor (AR) signaling is essential for maintaining spermatogenesis and male fertility. However, the molecular mechanisms by which AR acts between male germ cells and somatic cells during spermatogenesis have not begun to be revealed until recently. With the advances obtained from the use of transgenic mice lacking AR in Sertoli cells (SCARKO) and single-cell transcriptomic sequencing (scRNA-seq), the cell specific targets of AR action as well as the genes and signaling pathways that are regulated by AR are being identified. In this study, we collected scRNA-seq data from wild-type (WT) and SCARKO mice testes at p20 and identified four somatic cell populations and two male germ cell populations. Further analysis identified that the distribution of Sertoli cells was completely different and uncovered the cellular heterogeneity and transcriptional changes between WT and SCARKO Sertoli cells. In addition, several differentially expressed genes (DEGs) in SCARKO Sertoli cells, many of which have been previously implicated in cell cycle, apoptosis and male infertility, have also been identified. Together, our research explores a novel perspective on the changes in the transcription level of various cell types between WT and SCARKO mice testes, providing new insights for the investigations of the molecular and cellular processes regulated by AR signaling in Sertoli cells.

229 LONG-TERM PROPAGATION AND CRYOPRESERVATION OF CAT SPERMATOGONIAL STEM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 246
Author(s):  
L. M. Vansandt ◽  
M. Dickson ◽  
R. Zhou ◽  
L. Li ◽  
B. S. Pukazhenthi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Spermatogonial Stem Cells ◽  
Domestic Cat ◽  
Feeder Cell ◽  
Cell Clusters ◽  
Fetal Fibroblasts

Spermatogonial stem cells (SSC) are unique adult stem cells that reside within the seminiferous tubules of the testis. As stem cells, SSC maintain the ability to self-replicate, providing a potentially unlimited supply of cells and an alternate source for preservation of the male genome. While self-renewing, long-term SSC culture has been achieved in mice, there is virtually no information regarding culture requirements of felid SSC. Therefore, the objectives of this study were to (1) evaluate the ability of 3 feeder cell lines to support germ cell colony establishment in domestic cats (Felis catus), and (2) assess long-term culture using the best feeder(s). Cells isolated enzymatically from peripubertal cat testes (n = 4) and enriched by differential plating were cultured on mouse embryonic fibroblasts (STO line), mouse-derived C166 endothelial cells, and primary cat fetal fibroblasts (cFF). Colony morphology was assessed every other day and immunocytochemistry (ICC) was performed to investigate expression of SSC markers. At 5 days in vitro (DIV), a cluster forming activity assay was used to estimate the number of SSC supported by each feeder cell line. Differences among treatments were compared using Tukey-Kramer adjustment for pair-wise mean comparisons. Data were expressed as mean cluster number ± SE per 105 cells input. When cultured on STO feeders, cat germ cells were distributed as individual cells. On both C166 cells and cFF feeders, germ cell clumps (morphologically consistent with SSC colonies in other species) were observed. Immunocytochemistry revealed that the single germ cells present on STO feeders were positive for UCHL1 and weakly expressed PLZF and OCT4. Cells within the germ cell clumps on C166 cells and cFF co-expressed all 3 SSC markers. The C166 cells supported a higher number of germ cell clusters (77.4 ± 13.8) compared with STO (3.5 ± 1.1, P = 0.0003) or cFF (22.7 ± 1.0, P = 0.0024). Therefore, subsequent subculture experiments were performed exclusively with C166 feeder layers. Cultures from 2 donors were passaged at 12 DIV and periodically as needed thereafter. Germ cell clumps consistently reestablished following each subculture and immunocytochemistry analysis confirmed maintenance of all 3 SSC markers. Cells were also positive for alkaline phosphatase activity. Cells that had been cryopreserved in culture medium with 5% (vol/vol) dimethyl sulphoxide after144 DIV (7 passages) were thawed and cultured for an additional 18 days. These cells continued to express SSC markers and form germ cell clusters. Taken together, these data demonstrate that C166 feeder cells can facilitate colony establishment and in vitro propagation of germ cell clumps in the domestic cat. This represents an important first step towards attainment and optimization of a long-term SSC culture system in the cat. This system would provide a mechanism to explore regulation of spermatogenesis, test species-specific drugs, and produce transgenic biomedical models.

Single-cell RNA sequencing analysis of mouse follicular somatic cells

2021 ◽  
Author(s):  
Sen Li ◽  
Lei-Ning Chen ◽  
Hai-Jing Zhu ◽  
Xie Feng ◽  
Feng-Yun Xie ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Somatic Cells ◽  
Metabolic Reprogramming ◽  
Follicle Development ◽  
Sequencing Analysis ◽  
Cell Clusters ◽  
New Genes ◽  
Variable Genes ◽  
Cell Cell

Abstract Within the development of ovarian follicle, in addition to cell proliferation and differentiation, sophisticated cell–cell cross talks are established among follicular somatic cells such as granulosa cells (GCs) and theca cells. To systematically reveal the cell differentiation and signal transductions in follicular somatic cells, we collected the mouse follicular somatic cells from secondary to ovulatory stage, and analyzed the single cell transcriptomes. Having data filtered and screened, we found 6883 high variable genes in 4888 single cells. Then follicular somatic cells were clustered into 26 cell clusters, including 18 GC clusters, 4 theca endocrine cell (TEC) clusters, and 4 other somatic cell clusters, which include immune cells and Acta2 positive theca externa cells. From our data, we found there was metabolic reprogramming happened during GC differentiation. We also found both Cyp19a1 and Cyp11a1 could be expressed in TECs. We analyzed the expression patterns of genes associated with cell–cell interactions such as steroid hormone receptor genes, insulin signaling genes, and cytokine/transformation growth factor beta associated genes in all cell clusters. Lastly, we clustered the highly variable genes into 300 gene clusters, which could be used to search new genes involved in follicle development. These transcriptomes of follicular somatic cells provide us potential clues to reveal how mammals regulating follicle development and could help us find targets to improve oocyte quality for women with low fertility.

scholarly journals BMP4 promotes mouse iPS cell differentiation to male germ cells via Smad1/5, Gata4, Id1 and Id2

Reproduction ◽  
2017 ◽  
Vol 153 (2) ◽  
pp. 211-220 ◽  
Author(s):  
Shi Yang ◽  
Qingqing Yuan ◽  
Minghui Niu ◽  
Jingmei Hou ◽  
Zijue Zhu ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Germ Cell ◽  
Germ Cells ◽  
Real Time Pcr ◽  
Molecular Mechanisms ◽  
Ips Cells ◽  
Male Germ Cell ◽  
Western Blots ◽  
Male Germ Cells ◽  
Downstream Effectors

Generation of male germ cells from pluripotent cells could provide male gametes for treating male infertility and offer an ideal model for unveiling molecular mechanisms of spermatogenesis. However, the influence and exact molecular mechanisms, especially downstream effectors of BMP4 signaling pathways, in male germ cell differentiation of the induce pluripotent stem (iPS) cells, remain unknown. This study was designed to explore the role and mechanism of BMP4 signaling in the differentiation of mouse iPS cells to male germ cells. Embryoid body (EB) formation and recombinant BMP4 or Noggin were utilized to evaluate the effect of BMP4 on male germ cell generation from mouse iPS cells. Germ cell-specific genes and proteins as well as the downstream effectors of BMP4 signaling pathway were assessed using real-time PCR and Western blots. We found that BMP4 ligand and its multiple receptors, including BMPR1a, BMPR1b and BMPR2, were expressed in mouse iPS cells. Real-time PCR and Western blots revealed that BMP4 could upregulate the levels of genes and proteins for germ cell markers in iPS cells-derived EBs, whereas Noggin decreased their expression in these cells. Moreover, Smad1/5 phosphorylation, Gata4 transcription and the transcripts of Id1 and Id2 were enhanced by BMP4 but decreased when exposed to Noggin. Collectively, these results suggest that BMP4 promotes the generation of male germ cells from iPS cells via Smad1/5 pathway and the activation of Gata4, Id1 and Id2. This study thus offers novel insights into molecular mechanisms underlying male germ cell development.

scholarly journals Insights from the Applications of Single-Cell Transcriptomic Analysis in Germ Cell Development and Reproductive Medicine

2021 ◽  
Vol 22 (2) ◽  
pp. 823
Author(s):  
Hyeonwoo La ◽  
Hyunjin Yoo ◽  
Eun Joo Lee ◽  
Nguyen Xuan Thang ◽  
Hee Jin Choi ◽  
...  
Keyword(s):  
Germ Cell ◽  
Single Cell ◽  
Germ Cells ◽  
Cell Formation ◽  
Cell Development ◽  
Transcriptomic Analysis ◽  
Germ Cell Development ◽  
A Genome ◽  
Genome Scale ◽  
Germ Cell Formation

Mechanistic understanding of germ cell formation at a genome-scale level can aid in developing novel therapeutic strategies for infertility. Germ cell formation is a complex process that is regulated by various mechanisms, including epigenetic regulation, germ cell-specific gene transcription, and meiosis. Gonads contain a limited number of germ cells at various stages of differentiation. Hence, genome-scale analysis of germ cells at the single-cell level is challenging. Conventional genome-scale approaches cannot delineate the landscape of genomic, transcriptomic, and epigenomic diversity or heterogeneity in the differentiating germ cells of gonads. Recent advances in single-cell genomic techniques along with single-cell isolation methods, such as microfluidics and fluorescence-activated cell sorting, have helped elucidate the mechanisms underlying germ cell development and reproductive disorders in humans. In this review, the history of single-cell transcriptomic analysis and their technical advantages over the conventional methods have been discussed. Additionally, recent applications of single-cell transcriptomic analysis for analyzing germ cells have been summarized.

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