Single-cell RNA-seq unravels alterations of the human spermatogonial stem cell compartment in patients with impaired spermatogenesis.
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ABSTRACT:
SUBMITTER: Di Persio S
PROVIDER: S-EPMC8484693 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Male infertility is a major health problem affecting about 8-12% of couples worldwide. Spermatogenesis starts in the early fetus and completes after puberty, passing through different stages. Male infertility can result from primary or congenital, acquired, or idiopathic causes. The absence of sperm in semen, or azoospermia, results from non-obstructive causes (pretesticular and testicular), and post-testicular obstructive causes. Several medications such as antihypertensive drugs, antidepressants, chemotherapy, and radiotherapy could lead to impaired spermatogenesis and lead to a non-obstructive azoospermia. Spermatogonial stem cells (SSCs) are the basis for spermatogenesis and fertility in men. SSCs are characterized by their capacity to maintain the self-renewal process and differentiation into spermatozoa throughout the male reproductive life and transmit genetic information to the next generation. SSCs originate from gonocytes in the postnatal testis, which originate from long-lived primordial germ cells during embryonic development. The treatment of infertility in males has a poor prognosis. However, SSCs are viewed as a promising alternative for the regeneration of the impaired or damaged spermatogenesis. SSC transplantation is a promising technique for male infertility treatment and restoration of spermatogenesis in the case of degenerative diseases such as cancer, radiotherapy, and chemotherapy. The process involves isolation of SSCs and cryopreservation from a testicular biopsy before starting cancer treatment, followed by intra-testicular stem cell transplantation. In general, treatment for male infertility, even with SSC transplantation, still has several obstacles. The efficiency of cryopreservation, exclusion of malignant cells contamination in cancer patients, and socio-cultural attitudes remain major challenges to the wider application of SSCs as alternatives. Furthermore, there are limitations in experience and knowledge regarding cryopreservation of SSCs. However, the level of infrastructure or availability of regulatory approval to process and preserve testicular tissue makes them tangible and accurate therapy options for male infertility caused by non-obstructive azoospermia, though in their infancy, at least to date.
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Project description:Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a man's life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from four-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation.
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Project description: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.
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Project description:We characterized spermatogenesis in the large yellow croaker (Larimichthys crocea), a marine fish of significant commercial value, based on a high-resolution single-cell RNA-seq atlas of testicular cells from three distinct developmental stages- juvenile, adult differentiating and regressed testes. We detailed continuous developmental trajectory of spermatogenic cells, from spermatogonia to spermatids, elucidating the molecular events involved in spermatogenesis. We uncovered dynamic heterogeneity in cellular compositions throughout the annual reproductive cycle, accompanied by strong molecular signatures within specific testicular cells. Notably, we identified a distinct population of SSCs and observed a critical metabolic transition from glycolysis to oxidative phosphorylation, enhancing our understanding of the biochemical and molecular characteristics of SSCs. Additionally, we elucidated the interactions between somatic cells and spermatogonia, illuminating the mechanisms that regulate SSCs development. Overall, this work enhances our understanding of spermatogenesis in seasonal breeding teleost and provides essential insights for the further conservation and culture of SSCs.
2024-11-15 | GSE269124 | GEO
Project description:Prepubertal cancer treatment leads to irreversible infertility in half of the male patients. Current in vitro spermatogenesis protocols and cryopreservation techniques are inadequate to expand spermatogonial stem/progenitor cells (SSPC) from testicles. Bone marrow derived mesenchymal stem cells (BM-MSC) bearing a close resemblance to Sertoli cells, improved spermatogenesis in animal models. We asked if a co-culture setup supported by syngeneic BM-MSC that contributes to the air-liquid interphase (ALI) could lead to survival, expansion and differentiation of SSPCs in vitro. We generated an ALI platform able to provide a real-time cellular paracrine contribution consisting of syngeneic BM-MSCs to neonatal C57BL/6 mice testes. We aimed to evaluate the efficacy of this culture system on SSPC pool expansion and spermatogenesis throughout a complete spermatogenic cycle by measuring the number of total germ cells (GC), the undifferentiated and differentiating spermatogonia, the spermatocytes and the spermatids. Furthermore, we evaluated the testicular cell cycle phases, the tubular and luminal areas using histochemical, immunohistochemical and flow cytometric techniques. Cultures in present of BM-MSCs displayed survival of ID4(+) spermatogonial stem cells (SSC), expansion of SALL4(+) and OCT4(+) SSPCs, VASA(+) total GCs and Ki67(+) proliferative cells at 42 days and an increased number of SCP3(+) spermatocytes and Acrosin(+) spermatids at 28 days. BM-MSCs increased the percentage of mitotic cells within the G2-M phase of the total testicular cell cycle increased for 7 days, preserved the cell viability for 42 days and induced testicular maturation by enlargement of the tubular and luminal area for 42 days in comparison to the control. The percentage of PLZF(+) SSPCs increased within the first 28 days of culture, after which the pool started to get smaller while the number of spermatocytes and spermatids increased simultaneously. Our findings established the efficacy of syngeneic BM-MSCs on the survival and expansion of the SSPC pool and differentiation of spermatogonia to round spermatids during in vitro culture of prepubertal mice testes for 42 days. This method may be helpful in providing alternative cures for male fertility by supporting in vitro differentiated spermatids that can be used for round spermatid injection (ROSI) to female oocyte in animal models. These findings can be further exploited for personalized cellular therapy strategies to cure male infertility of prepubertal cancer survivors in clinics.
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Project description:Human adult spermatogonial stem cells (hSSCs) must balance self-renewal and differentiation. To understand how this is achieved, we profiled DNA methylation and open chromatin (ATAC-seq) in SSEA4+ hSSCs, analyzed bulk and single-cell RNA transcriptomes (RNA-seq) in SSEA4+ hSSCs and differentiating c-KIT+ spermatogonia, and performed validation studies via immunofluorescence. First, DNA hypomethylation at embryonic developmental genes supports their epigenetic "poising" in hSSCs for future/embryonic expression, while core pluripotency genes (OCT4 and NANOG) were transcriptionally and epigenetically repressed. Interestingly, open chromatin in hSSCs was strikingly enriched in binding sites for pioneer factors (NFYA/B, DMRT1, and hormone receptors). Remarkably, single-cell RNA-seq clustering analysis identified four cellular/developmental states during hSSC differentiation, involving major transitions in cell-cycle and transcriptional regulators, splicing and signaling factors, and glucose/mitochondria regulators. Overall, our results outline the dynamic chromatin/transcription landscape operating in hSSCs and identify crucial molecular pathways that accompany the transition from quiescence to proliferation and differentiation.
| S-EPMC5832720 | biostudies-literature
Project description:Mammalian spermatogenesis is a complex developmental program that transforms mitotic testicular germ cells (spermatogonia) into mature male gametes (sperm) for production of offspring. For decades, it has been known that this several-weeks-long process involves a series of highly ordered and morphologically recognizable cellular changes as spermatogonia proliferate, spermatocytes undertake meiosis, and spermatids develop condensed nuclei, acrosomes, and flagella. Yet, much of the underlying molecular logic driving these processes has remained opaque because conventional characterization strategies often aggregated groups of cells to meet technical requirements or due to limited capability for cell selection. Recently, a cornucopia of single-cell transcriptome studies has begun to lift the veil on the full compendium of gene expression phenotypes and changes underlying spermatogenic development. These datasets have revealed the previously obscured molecular heterogeneity among and between varied spermatogenic cell types and are reinvigorating investigation of testicular biology. This review describes the extent of available single-cell RNA-seq profiles of spermatogenic and testicular somatic cells, how those data were produced and evaluated, their present value for advancing knowledge of spermatogenesis, and their potential future utility at both the benchtop and bedside.
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Project description:BACKGROUND: Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and reside within a specific microenvironment in the testes called "niche" which regulates stem cell properties, such as, self-renewal, pluripotency, quiescence and their ability to differentiate. METHODOLOGY/PRINCIPAL FINDINGS: Here, we introduce zebrafish as a new model for the study of SSCs in vertebrates. Using 5'-bromo-2'-deoxyuridine (BrdU), we identified long term BrdU-retaining germ cells, type A undifferentiated spermatogonia as putative stem cells in zebrafish testes. Similar to rodents, these cells were preferentially located near the interstitium, suggesting that the SSC niche is related to interstitial elements and might be conserved across vertebrates. This localization was also confirmed by analyzing the topographical distribution of type A undifferentiated spermatogonia in normal, vasa::egfp and fli::egfp zebrafish testes. In the latter one, the topographical arrangement suggested that the vasculature is important for the SSC niche, perhaps as a supplier of nutrients, oxygen and/or signaling molecules. We also developed an SSC transplantation technique for both male and female recipients as an assay to evaluate the presence, biological activity, and plasticity of the SSC candidates in zebrafish. CONCLUSIONS/SIGNIFICANCE: We demonstrated donor-derived spermato- and oogenesis in male and female recipients, respectively, indicating the stemness of type A undifferentiated spermatogonia and their plasticity when placed into an environment different from their original niche. Similar to other vertebrates, the transplantation efficiency was low. This might be attributed to the testicular microenvironment created after busulfan depletion in the recipients, which may have caused an imbalance between factors regulating self-renewal or differentiation of the transplanted SSCs.
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Project description: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.
| S-EPMC6713459 | biostudies-literature
Project description: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.
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