Isolation of THY1+ Undifferentiated Spermatogonia from Mouse Postnatal  Testes Using Magnetic-activated Cell Sorting (MACS)

Liao, Hung-Fu; Kuo, Joyce; Lin, Hsien-Hen; Lin, Shau-Ping

doi:10.21769/bioprotoc.2072

Cited by 3 publications

(3 citation statements)

References 18 publications

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“…Since TRIM71 is usually expressed in undifferentiated cells, we hypothesized that its expression in mice testes is present in undifferentiated spermatogonia, also known as spermatogonial stem cells (SSCs). To validate this hypothesis, we used wild type testes cell suspensions to enrich SSCs (THY1.2 +) via magnetic-activated cell sorting (MACS) ( Liao et al, 2016 ). We found an increase of Trim71 mRNA as well as the mRNAs of the known murine SSC-specific markers Sall4 ( Gassei and Orwig, 2013 ) and Lin28a ( Zheng et al, 2009 ) – but not the differentiating spermatogonia marker Lin28b ( Gaytan et al, 2013 ) – in the THY1.2 + enriched cell population ( Figures 1D,E ).…”

Section: Resultsmentioning

confidence: 99%

TRIM71 Deficiency Causes Germ Cell Loss During Mouse Embryogenesis and Is Associated With Human Male Infertility

Fernández

Emich

Port

et al. 2021

Front. Cell Dev. Biol.

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Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis.

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Section: Resultsmentioning

confidence: 99%

TRIM71 Deficiency Causes Germ Cell Loss During Mouse Embryogenesis and Is Associated With Human Male Infertility

Fernández

Emich

Port

et al. 2021

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

show abstract

“…Since TRIM71 is a stem cell-specific protein, we hypothesized that its expression in mice testes is present in undifferentiated spermatogonia, also known as spermatogonial stem cells (SSCs). To validate this hypothesis, we used wild type testes cell suspensions to enrich SSCs (THY1.2+) via magnetic-activated cell sorting (MACS) 35 . We found an increase of Trim71 mRNA as well as the mRNAs of the known SSC-specific markers Sall4 36 and Lin28a 37 – but not the differentiating spermatogonia marker Lin28b 38 – in the THY1.2+ enriched cell population (Fig.…”

Section: Resultsmentioning

confidence: 99%

TRIM71deficiency causes germ cell loss during mouse embryogenesis and promotes human male infertility

Emich

Port

et al. 2021

Preprint

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Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling the complex molecular network orchestrating germ cell development. TRIM71 is a stem cell-specific factor essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype, which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of developing germ cells in the testes’ seminiferous tubules. Infertility originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, in vitro growth competition assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells, a human GCT-derived cell line which we used as a surrogate model for proliferating PGCs, showed that TRIM71 promotes NCCIT cell proliferation and survival. Our data collectively suggest that germ cell loss in cKO mice results from combined defects during the specification and maintenance of PGCs prior to their sex determination in the genital ridges. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with SCO phenotype. Our work reveals for the first time an association of TRIM71 variants with human male infertility, and uncovers further developmental roles for TRIM71 in the generation and maintenance of germ cells during mouse embryogenesis.

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“…For the isolation of spermatogonial RNA, C57BL/6J and Cast/EiJ mice were obtained from The Jackson Laboratory and kept in the SPF animal facility of Max Planck Institute of Immunobiology and Epigenetics until sacrifice. In order to isolate spermatogonia from mice, testes were dissected and digested according to the protocol by Liao et al ((H.-F. Liao et al, 2016), with minor modifications. Briefly, we euthanized 6 weeks old mice with CO 2 and quickly dissected testes, removed the tunica albuginea and loosened the seminiferous tubules.…”

Section: Mouse Tissue Isolation and Rna Extractionmentioning

confidence: 99%

Genetic polymorphisms lead to major, locus-specific, variation in piRNA production in mouse

Casas

Sierra

Moreta-Moraleda

et al. 2022

Preprint

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PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that silence transposons in the animal germline. PiRNAs are produced from long single-stranded non-coding transcripts, from protein-coding transcripts, as well as from transposons. While some sites that produce piRNAs are in deeply conserved syntenic regions, in general, piRNAs and piRNA-producing loci turnover faster than other functional parts of the genome. To learn about the sequence changes that contribute to the fast evolution of piRNAs, we set out to analyse piRNA expression between genetically different mice. Here we report the sequencing and analysis of small RNAs from the mouse male germline of four classical inbred strains, one inbred wild-derived strain and one outbred strain. We find that genetic differences between individuals underlie variation in piRNA expression. We report significant differences in piRNA production at loci with endogenous retrovirus insertions. Strain-specific piRNA-producing loci include protein-coding genes. Our findings provide evidence that transposable elements contribute to inter-individual differences in expression, and potentially to the fast evolution of piRNA-producing loci in mammals.

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Isolation of THY1+ Undifferentiated Spermatogonia from Mouse Postnatal Testes Using Magnetic-activated Cell Sorting (MACS)

Cited by 3 publications

References 18 publications

TRIM71 Deficiency Causes Germ Cell Loss During Mouse Embryogenesis and Is Associated With Human Male Infertility

TRIM71 Deficiency Causes Germ Cell Loss During Mouse Embryogenesis and Is Associated With Human Male Infertility

TRIM71deficiency causes germ cell loss during mouse embryogenesis and promotes human male infertility

Genetic polymorphisms lead to major, locus-specific, variation in piRNA production in mouse

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