Title : RUNX1 maintains the identity of the fetal ovary through an interplay with FOXL2.

Publication date : 11. 2019

Authors : B Nicol, SA Grimm, F Chalmel, E Lecluze, M Pannetier, E Pailhoux, E Dupin-De-Beyssat, Y Guiguen, B Capel, HH Yao

Sex determination of the gonads begins with fate specification of gonadal supporting cells into either ovarian pre-granulosa cells or testicular Sertoli cells. This fate specification hinges on a balance of transcriptional control. Here we report that expression of the transcription factor RUNX1 is enriched in the fetal ovary in rainbow trout, turtle, mouse, goat, and human. In the mouse, RUNX1 marks the supporting cell lineage and becomes pre-granulosa cell-specific as the gonads differentiate. RUNX1 plays complementary/redundant roles with FOXL2 to maintain fetal granulosa cell identity and combined loss of RUNX1 and FOXL2 results in masculinization of fetal ovaries. At the chromatin level, RUNX1 occupancy overlaps partially with FOXL2 occupancy in the fetal ovary, suggesting that RUNX1 and FOXL2 target common sets of genes. These findings identify RUNX1, with an ovary-biased expression pattern conserved across species, as a regulator in securing the identity of ovarian-supporting cells and the ovary.

Title : Dissecting Cell Lineage Specification and Sex Fate Determination in Gonadal Somatic Cells Using Single-Cell Transcriptomics.

Publication date : 03. 2019

Authors : I Stévant, F Kühne, A Greenfield, MC Chaboissier, ET Dermitzakis, S Nef

Sex determination is a unique process that allows the study of multipotent progenitors and their acquisition of sex-specific fates during differentiation of the gonad into a testis or an ovary. Using time series single-cell RNA sequencing (scRNA-seq) on ovarian Nr5a1-GFP

Title : The Mammalian Spermatogenesis Single-Cell Transcriptome, from Spermatogonial Stem Cells to Spermatids.

Publication date : 11. 2018

Authors : BP Hermann, K Cheng, A Singh, L Roa-De La Cruz, KN Mutoji, IC Chen, H Gildersleeve, JD Lehle, M Mayo, B Westernströer, NC Law, MJ Oatley, EK Velte, BA Niedenberger, D Fritze, S Silber, CB Geyer, JM Oatley, JR McCarrey

Spermatogenesis is a complex and dynamic cellular differentiation process critical to male reproduction and sustained by spermatogonial stem cells (SSCs). Although patterns of gene expression have been described for aggregates of certain spermatogenic cell types, the full continuum of gene expression patterns underlying ongoing spermatogenesis in steady state was previously unclear. Here, we catalog single-cell transcriptomes for >62,000 individual spermatogenic cells from immature (postnatal day 6) and adult male mice and adult men. This allowed us to resolve SSC and progenitor spermatogonia, elucidate the full range of gene expression changes during male meiosis and spermiogenesis, and derive unique gene expression signatures for multiple mouse and human spermatogenic cell types and/or subtypes. These transcriptome datasets provide an information-rich resource for studies of SSCs, male meiosis, testicular cancer, male infertility, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve spermatogenesis in vitro.

Title : The Mammalian Spermatogenesis Single-Cell Transcriptome, from Spermatogonial Stem Cells to Spermatids.

Publication date : 11. 2018

Authors : BP Hermann, K Cheng, A Singh, L Roa-De La Cruz, KN Mutoji, IC Chen, H Gildersleeve, JD Lehle, M Mayo, B Westernströer, NC Law, MJ Oatley, EK Velte, BA Niedenberger, D Fritze, S Silber, CB Geyer, JM Oatley, JR McCarrey

Spermatogenesis is a complex and dynamic cellular differentiation process critical to male reproduction and sustained by spermatogonial stem cells (SSCs). Although patterns of gene expression have been described for aggregates of certain spermatogenic cell types, the full continuum of gene expression patterns underlying ongoing spermatogenesis in steady state was previously unclear. Here, we catalog single-cell transcriptomes for >62,000 individual spermatogenic cells from immature (postnatal day 6) and adult male mice and adult men. This allowed us to resolve SSC and progenitor spermatogonia, elucidate the full range of gene expression changes during male meiosis and spermiogenesis, and derive unique gene expression signatures for multiple mouse and human spermatogenic cell types and/or subtypes. These transcriptome datasets provide an information-rich resource for studies of SSCs, male meiosis, testicular cancer, male infertility, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve spermatogenesis in vitro.

Title : Genome-wide identification of FOXL2 binding and characterization of FOXL2 feminizing action in the fetal gonads.

Publication date : 12. 2018

Authors : B Nicol, SA Grimm, A Gruzdev, GJ Scott, MK Ray, HH Yao

The identity of the gonads is determined by which fate, ovarian granulosa cell or testicular Sertoli cell, the bipotential somatic cell precursors choose to follow. In most vertebrates, the conserved transcription factor FOXL2 contributes to the fate of granulosa cells. To understand FOXL2 functions during gonad differentiation, we performed genome-wide analysis of FOXL2 chromatin occupancy in fetal ovaries and established a genetic mouse model that forces Foxl2 expression in the fetal testis. When FOXL2 was ectopically expressed in the somatic cell precursors in the fetal testis, FOXL2 was sufficient to repress Sertoli cell differentiation, ultimately resulting in partial testis-to-ovary sex-reversal. Combining genome-wide analysis of FOXL2 binding in the fetal ovary with transcriptomic analyses of our Foxl2 gain-of-function and previously published Foxl2 loss-of-function models, we identified potential pathways responsible for the feminizing action of FOXL2. Finally, comparison of FOXL2 genome-wide occupancy in the fetal ovary with testis-determining factor SOX9 genome-wide occupancy in the fetal testis revealed extensive overlaps, implying that antagonistic signals between FOXL2 and SOX9 occur at the chromatin level.

Title : Single-Cell RNA Sequencing Analysis Reveals Sequential Cell Fate Transition during Human Spermatogenesis.

Publication date : 10. 2018

Authors : M Wang, X Liu, G Chang, Y Chen, G An, L Yan, S Gao, Y Xu, Y Cui, J Dong, Y Chen, X Fan, Y Hu, K Song, X Zhu, Y Gao, Z Yao, S Bian, Y Hou, J Lu, R Wang, Y Fan, Y Lian, W Tang, Y Wang, J Liu, L Zhao, L Wang, Z Liu, R Yuan, Y Shi, B Hu, X Ren, F Tang, XY Zhao, J Qiao

Spermatogenesis generates mature male gametes and is critical for the proper transmission of genetic information between generations. However, the developmental landscapes of human spermatogenesis remain unknown. Here, we performed single-cell RNA sequencing (scRNA-seq) analysis for 2,854 testicular cells from donors with normal spermatogenesis and 174 testicular cells from one nonobstructive azoospermia (NOA) donor. A hierarchical model was established, which was characterized by the sequential and stepwise development of three spermatogonia subtypes, seven spermatocyte subtypes, and four spermatid subtypes. Further analysis identified several stage-specific marker genes of human germ cells, such as HMGA1, PIWIL4, TEX29, SCML1, and CCDC112. Moreover, we identified altered gene expression patterns in the testicular somatic cells of one NOA patient via scRNA-seq analysis, paving the way for further diagnosis of male infertility. Our work allows for the reconstruction of transcriptional programs inherent to sequential cell fate transition during human spermatogenesis and has implications for deciphering male-related reproductive disorders.

Title : A Comprehensive Roadmap of Murine Spermatogenesis Defined by Single-Cell RNA-Seq.

Publication date : 09. 2018

Authors : CD Green, Q Ma, GL Manske, AN Shami, X Zheng, S Marini, L Moritz, C Sultan, SJ Gurczynski, BB Moore, MD Tallquist, JZ Li, SS Hammoud

Spermatogenesis requires intricate interactions between the germline and somatic cells. Within a given cross section of a seminiferous tubule, multiple germ and somatic cell types co-occur. This cellular heterogeneity has made it difficult to profile distinct cell types at different stages of development. To address this challenge, we collected single-cell RNA sequencing data from ∼35,000 cells from the adult mouse testis and identified all known germ and somatic cells, as well as two unexpected somatic cell types. Our analysis revealed a continuous developmental trajectory of germ cells from spermatogonia to spermatids and identified candidate transcriptional regulators at several transition points during differentiation. Focused analyses delineated four subtypes of spermatogonia and nine subtypes of Sertoli cells; the latter linked to histologically defined developmental stages over the seminiferous epithelial cycle. Overall, this high-resolution cellular atlas represents a community resource and foundation of knowledge to study germ cell development and in vivo gametogenesis.

Title : Single-cell RNA-seq uncovers dynamic processes and critical regulators in mouse spermatogenesis.

Publication date : 09. 2018

Authors : Y Chen, Y Zheng, Y Gao, Z Lin, S Yang, T Wang, Q Wang, N Xie, R Hua, M Liu, J Sha, MD Griswold, J Li, F Tang, MH Tong

A systematic interrogation of male germ cells is key to complete understanding of molecular mechanisms governing spermatogenesis and the development of new strategies for infertility therapies and male contraception. Here we develop an approach to purify all types of homogeneous spermatogenic cells by combining transgenic labeling and synchronization of the cycle of the seminiferous epithelium, and subsequent single-cell RNA-sequencing. We reveal extensive and previously uncharacterized dynamic processes and molecular signatures in gene expression, as well as specific patterns of alternative splicing, and novel regulators for specific stages of male germ cell development. Our transcriptomics analyses led us to discover discriminative markers for isolating round spermatids at specific stages, and different embryo developmental potentials between early and late stage spermatids, providing evidence that maturation of round spermatids impacts on embryo development. This work provides valuable insights into mammalian spermatogenesis, and a comprehensive resource for future studies towards the complete elucidation of gametogenesis.

Title : Single-cell RNA-seq uncovers dynamic processes and critical regulators in mouse spermatogenesis.

Publication date : 09. 2018

Authors : Y Chen, Y Zheng, Y Gao, Z Lin, S Yang, T Wang, Q Wang, N Xie, R Hua, M Liu, J Sha, MD Griswold, J Li, F Tang, MH Tong

A systematic interrogation of male germ cells is key to complete understanding of molecular mechanisms governing spermatogenesis and the development of new strategies for infertility therapies and male contraception. Here we develop an approach to purify all types of homogeneous spermatogenic cells by combining transgenic labeling and synchronization of the cycle of the seminiferous epithelium, and subsequent single-cell RNA-sequencing. We reveal extensive and previously uncharacterized dynamic processes and molecular signatures in gene expression, as well as specific patterns of alternative splicing, and novel regulators for specific stages of male germ cell development. Our transcriptomics analyses led us to discover discriminative markers for isolating round spermatids at specific stages, and different embryo developmental potentials between early and late stage spermatids, providing evidence that maturation of round spermatids impacts on embryo development. This work provides valuable insights into mammalian spermatogenesis, and a comprehensive resource for future studies towards the complete elucidation of gametogenesis.

Title : Transcriptomic analysis of mRNA expression and alternative splicing during mouse sex determination.

Publication date : 12. 2018

Authors : L Zhao, C Wang, ML Lehman, M He, J An, T Svingen, CM Spiller, ET Ng, CC Nelson, P Koopman

Mammalian sex determination hinges on sexually dimorphic transcriptional programs in developing fetal gonads. A comprehensive view of these programs is crucial for understanding the normal development of fetal testes and ovaries and the etiology of human disorders of sex development (DSDs), many of which remain unexplained. Using strand-specific RNA-sequencing, we characterized the mouse fetal gonadal transcriptome from 10.5 to 13.5 days post coitum, a key time window in sex determination and gonad development. Our dataset benefits from a greater sensitivity, accuracy and dynamic range compared to microarray studies, allows global dynamics and sex-specificity of gene expression to be assessed, and provides a window to non-transcriptional events such as alternative splicing. Spliceomic analysis uncovered female-specific regulation of Lef1 splicing, which may contribute to the enhanced WNT signaling activity in XX gonads. We provide a user-friendly visualization tool for the complete transcriptomic and spliceomic dataset as a resource for the field.