Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Hermann BP

PROVIDER: S-EPMC6384825 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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

Hermann Brian P BP Cheng Keren K Singh Anukriti A Roa-De La Cruz Lorena L Mutoji Kazadi N KN Chen I-Chung IC Gildersleeve Heidi H Lehle Jake D JD Mayo Max M Westernströer Birgit B Law Nathan C NC Oatley Melissa J MJ Velte Ellen K EK Niedenberger Bryan A BA Fritze Danielle D Silber Sherman S Geyer Christopher B CB Oatley Jon M JM McCarrey John R JR

Cell reports 20181101 6

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 da ...[more]

PMID: 30404016

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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.

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.

Project description:Graphical abstract Highlights • Background: The risk of infertility after chemotherapy is unpredictable in children with cancer.• Our in vitro study demonstrated short-term damage of chemotherapy on immature mouse testis.• Cisplatin and doxorubicin induce acute spermatogonial stem cell loss that does not persist two months later.• High concentrations of cisplatin impair later phases of spermatogenesis in the long-term. Chemotherapy can affect testis development of young boys with cancer, reducing the chances of fatherhood in adulthood. Studies using experimental models are needed to determine the damage caused by individual chemotherapy drugs in order to predict the risk of infertility and direct patients towards appropriate fertility preservation options. Here, we investigated the individual role of two drugs, cisplatin and doxorubicin, using an in vitro culture model of prepubertal (postnatal day 5) mouse testis that supports induction and maintenance of full spermatogenesis. Twenty-four hour exposure with either drug at clinically-relevant doses (0.25, 0.5 or 0.75 ?g/mL for cisplatin, or 0.01, 0.03 or 0.05 ?g/mL for doxorubicin), induced an acute significant loss of spermatogonial stem cells (SSCs; PLZF+), proliferating SSCs (PLZF+BrdU+), total germ cells (MVH+), and spermatocytes (SCP3+) one week after chemotherapy exposure. By the time of the first (Week 4) and second (Week 8) waves of spermatogenesis, there was no longer any effect on SSC or proliferating SSC numbers in drug-exposed testis compared to untreated tissue: however, the populations of total germ cells and spermatocytes were still lower in the higher-dose cisplatin treated groups, along with a reduced frequency of round and elongated spermatids in both cisplatin- and doxorubicin-treated testis fragments. Overall, this study details a direct impairment of germ cell development following acute chemotherapy-induced damage during the prepubertal phase, most likely due to an effect on SSCs, using an in vitro culture system that successfully recapitulates key events of mouse spermatogenesis.

Dataset Information

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

Publications

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

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