Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline.

Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita, frequently resulting in spontaneous bone marrow failure. A dyskeratosis congenita mutation in TPP1 (K170∆) that specifically compromises telomerase recruitment to telomeres is a valuable tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mouse knocked in for the equivalent of the TPP1 K170∆ mutation (TPP1 K82∆) and investigated both its hematopoietic and germline compartments in unprecedented detail. TPP1 K82∆ caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82∆ caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of dysfunctional seminiferous tubules, and a decrease in germ cells. Intriguingly, both TPP1 K82∆ mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to infertility at steady-state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice.

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice.

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Regulation of meiotic progression by Sertoli-cell androgen signaling.

Androgen receptor (AR) signaling in Sertoli cells is known to be important for germ-cell progression through meiosis, but the extent to which androgens indirectly regulate specific meiotic stages is not known. Here, we combine synchronization of spermatogenesis, cytological analyses and single-cell RNAseq (scRNAseq) in the Sertoli-cell androgen receptor knockout (SCARKO) mutant and control mice, and demonstrate that SCARKO mutant spermatocytes exhibited normal expression and localization of key protein markers of meiotic prophase events, indicating that initiation of meiotic prophase is not androgen dependent. However, spermatocytes from SCARKO testes failed to acquire competence for the meiotic division phase. ScRNAseq analysis of wild-type and SCARKO mutant testes revealed a molecular transcriptomic block in an early meiotic prophase state (leptotene/zygotene) in mutant germ cells, and identified several misregulated genes in SCARKO Sertoli cells, many of which have been previously implicated in male infertility. Together, our coordinated cytological and scRNAseq analyses identified germ-cell intrinsic and extrinsic genes responsive to Sertoli-cell androgen signaling that promotes cellular states permissive for the meiotic division phase.

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.

Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells.

Telomerase replicates chromosome ends in germ and somatic stem cells to facilitate their continued proliferation. Telomerase action depends on the telomeric protein TPP1, which recruits telomerase to telomeres and facilitates processive DNA synthesis. Here, we identify separation-of-function long (TPP1-L) and short (TPP1-S) isoforms of TPP1 that appear to be generated from separate transcripts and differ only in 86 amino acids at their N terminus. Although both isoforms retain the ability to recruit telomerase, only TPP1-S facilitates efficient telomere synthesis. We find that TPP1-S is the predominant isoform in somatic cells, and strikingly, TPP1-L is the major isoform in differentiated male germ cells. We observed that TERT expression persists in these germ cells, suggesting that TPP1-L could restrain telomerase in this context. We show how differential expression of TPP1 isoforms determines telomerase function and demonstrate how alternative transcription start sites allow one gene to perform distinct functions in different biological contexts.

Gametogenesis: A journey from inception to conception.

Gametogenesis, the process of forming mature germ cells, is an integral part of both an individual's and a species' health and well-being. This chapter focuses on critical male and female genetic and epigenetic processes underlying normal gamete formation through their differentiation to fertilization. Finally, we explore how knowledge gained from this field has contributed to progress in areas with great clinical promise, such as in vitro gametogenesis.

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

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.