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Gene Expression Profiling of the SSC-Enriched Thy1+ and SSC-Depleted Thy1- Fractions of Prepubertal Mouse Testes

ABSTRACT: Self-renewal and differentiation of spermatogonial stem cells (SSCs) provides the foundation for testis homeostasis, yet mechanisms that control their functions in mammals are poorly defined. We used microarray transcript profiling to identify specific genes whose expression are augmented in the SSC-enriched Thy1+ germ cell fraction of mouse pup testes. Comparisons of gene expression in the Thy1+ germ cell fraction to the Thy1-depeleted testis cell population identified 202 genes that are expressed 10-fold or higher in Thy1+ cells. This database provided a mining tool to investigate specific characteristics of SSCs and identify novel mechanisms that potentially influence their functions.

ORGANISM(S): Mus musculus

PROVIDER: GSE14222 | GEO | 2008/12/30

SECONDARY ACCESSION(S): PRJNA111255

REPOSITORIES: GEO

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Project description:In the adult mammalian testis, spermatogenic differentiation starts from a minute population of spermatogonial stem cells (SSCs). SSCs are generated after birth from the fetal gonocytes, themselves derived from the primordial germ cells (PGCs), which are specified during the first days after implantation. Transcriptome profiling of purified preparations evidenced the preferential accumulation in SSCs of transcripts of PU.1 (Sfpi1), a regulatory gene previously identified in hematopoietic progenitors. In situ immunolabeling and RNA determination showed a complex pattern of expression in the adult testis, first in SSCs and early spermatogonia followed by de novo expression in pachytene spermatocytes. Spermatogenesis in a null mutant (PU.1(G/G)) was arrested at the prenatal stage, with reduced numbers of gonocytes owing to a defect in proliferation already noticeable at E12.5. Transcripts of several germinal markers, including vasa (Mvh, Ddx4), Oct4 (Pou5f1), Dazl and Taf4b, were detected, whereas stella (PGC7, Dppa3) was not. Germ cells of PU.1(G/G) newborn testes grafted in nude mice did not initiate the postnatal replicative stage, whereas grafts of their wild-type littermates underwent complete spermatogenesis. During embryonic development, PU.1 transcription was initiated as early as the blastocyst stage, with a generalized expression at E6.5 in the embryonic ectoderm. PU.1 therefore appears to play a determinant role in at least two distinct lineages and, given its wide range of expression, possibly in other stem cells. Experiment Overall Design: Our starting point was the establishment of a transcriptome profile of purified preparations of SSCs, on the assumption that crucial genes would appear among those expressed at elevated levels in the stem cells. Work on adult SSCs is made difficult by their small number. We previously reported an efficient procedure, in which a truncated version of the Stra8 promoter is used to express in SSCs a neutral heterologous surface protein including two domains of the human CD4 antigen (huCD4) (Giuili et al., 2002). Once fractionated by immunomagnetic sorting, the huCD4-positive fraction is homogeneous for the expression of known SSC markers and highly efficient in the colonization of a sterile recipient.

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Gene Expression Profiling of the SSC-Enriched Thy1+ and SSC-Depleted Thy1- Fractions of Prepubertal Mouse Testes

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