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Dynamic changes in the expression of apoptosis-related genes in differentiating gonocytes and in seminomas.

ABSTRACT: Apoptosis is an integral part of the spermatogenic process, necessary to maintain a proper ratio of Sertoli to germ cell numbers and provide an adequate microenvironment to germ cells. Apoptosis may also represent a protective mechanism mediating the elimination of abnormal germ cells. Extensive apoptosis occurs between the first and second postnatal weeks, at the point when gonocytes, precursors of spermatogonial stem cells, should have migrated toward the basement membrane of the tubules and differentiated into spermatogonia. The mechanisms regulating this process are not well-understood. Gonocytes undergo phases of proliferation, migration, and differentiation which occur in a timely and closely regulated manner. Gonocytes failing to migrate and differentiate properly undergo apoptosis. Inadequate gonocyte differentiation has been suggested to lead to testicular germ cell tumor (TGCT) formation. Here, we examined the expression levels of apoptosis-related genes during gonocyte differentiation by quantitative real-time polymerase chain reaction, identifying 48 pro- and anti-apoptotic genes increased by at least two-fold in rat gonocytes induced to differentiate by retinoic acid, when compared to untreated gonocytes. Further analysis of the most highly expressed genes identified the pro-apoptotic genes Gadd45a and Cycs as upregulated in differentiating gonocytes and in spermatogonia compared with gonocytes. These genes were also significantly downregulated in seminomas, the most common type of TGCT, compared with normal human testicular tissues. These results indicate that apoptosis-related genes are actively regulated during gonocyte differentiation. Moreover, the down-regulation of pro-apoptotic genes in seminomas suggests that they could represent new therapeutic targets in the treatment of TGCTs.

SUBMITTER: Manku G

PROVIDER: S-EPMC4430938 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chromatin modifier Swi-independent 3a (SIN3A), together with associated histone deacetylases, influences gene expression during development and differentiation through a variety of transcription factors in a cell-specific manner. Sin3a is essential for the maintenance of inner cell mass cells of mouse blastocysts, embryonic fibroblasts, and myoblasts, but is not required for the survival of trophectoderm or Sertoli cells. To better understand how this transcriptional regulator modulates cells at different developmental stages within a single lineage, we used conditional gene targeting in mice to ablate Sin3a from perinatal quiescent male gonocytes and from postnatal differentiating spermatogonia. Mitotic germ cells expressing stimulated by retinoic acid gene 8 (Stra8) that lacked Sin3a exhibited increased DNA damage and apoptosis, yet collectively progressed through meiosis and spermiogenesis and generated epididymal sperm at approximately 50% of control levels, sufficient for normal fertility. In contrast, perinatal gonocytes lacking Sin3a underwent rapid depletion that coincided with cell cycle reentry, exhibiting 2.5-fold increased histone H3 phosphorylation upon cycling that suggested a prophase/metaphase block; germ cells were almost entirely absent two weeks after birth, resulting in sterility. Gene expression profiling of neonatal testes containing Sin3a-deleted gonocytes identified upregulated transcripts highly associated with developmental processes and pattern formation, and downregulated transcripts involved in nuclear receptor activity, including Nr4a1 (Nur77). Interestingly, Nr4a1 levels were elevated in testes containing Stra8-expressing, Sin3a-deleted spermatogonia. SIN3A directly binds to the Nr4a1 promoter, and Nr4a1 expression is diminished upon spermatogonial differentiation in vitro. We conclude that within the male germline, Sin3a is required for the mitotic reentry of gonocytes, but is dispensable for the maintenance of differentiating spermatogonia and subsequent spermatogenic processes.

Project description:The spermatogenic lineage is established after birth when gonocytes migrate to the basement membrane of seminiferous tubules and give rise to spermatogonial stem cells (SSC). In adults, SSCs reside within the population of undifferentiated spermatogonia (A(undiff)) that expands clonally from single cells (A(single)) to form pairs (A(paired)) and chains of 4, 8 and 16 A(aligned) spermatogonia. Although stem cell activity is thought to reside in the population of A(single) spermatogonia, new research suggests that clone size alone does not define the stem cell pool. The mechanisms that regulate self-renewal and differentiation fate decisions are poorly understood due to limited availability of experimental tools that distinguish the products of those fate decisions. The pluripotency factor SALL4 (sal-like protein 4) is implicated in stem cell maintenance and patterning in many organs during embryonic development, but expression becomes restricted to the gonads after birth. We analyzed the expression of SALL4 in the mouse testis during the first weeks after birth and in adult seminiferous tubules. In newborn mice, the isoform SALL4B is expressed in quiescent gonocytes at postnatal day 0 (PND0) and SALL4A is upregulated at PND7 when gonocytes have colonized the basement membrane and given rise to spermatogonia. During steady-state spermatogenesis in adult testes, SALL4 expression overlapped substantially with PLZF and LIN28 in A(single), A(paired) and A(aligned) spermatogonia and therefore appears to be a marker of undifferentiated spermatogonia in mice. In contrast, co-expression of SALL4 with GFRα1 and cKIT identified distinct subpopulations of A(undiff) in all clone sizes that might provide clues about SSC regulation. Collectively, these results indicate that 1) SALL4 isoforms are differentially expressed at the initiation of spermatogenesis, 2) SALL4 is expressed in undifferentiated spermatogonia in adult testes and 3) SALL4 co-staining with GFRα1 and cKIT reveals distinct subpopulations of A(undiff) spermatogonia that merit further investigation.

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Dynamic changes in the expression of apoptosis-related genes in differentiating gonocytes and in seminomas.

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