Differential contribution of the MTOR and MNK pathways to the regulation of mRNA translation in meiotic and postmeiotic mouse male germ cells.

Translation of stored mRNAs accounts for protein synthesis during the transcriptionally inactive stages of spermatogenesis. A key step in mRNA translation is the assembly of the initiation complex EIF4F, which is regulated by the MTOR (mammalian target of rapamycin) and MNK1/2 (MAP kinase-interacting kinase 1 and 2) pathways. We investigated the expression and activity of regulatory proteins of these pathways in male germ cells at different stages of differentiation. All translation factors analyzed were expressed in germ cells throughout spermatogenesis. However, while EIF4G and PABP1 (poly[A]-binding protein 1) were more abundant in postmeiotic cells, MTOR and its target EIF4EBP1 (4E-BP1) decreased steadily during spermatogenesis. In vivo labeling showed that pachytene spermatocytes display higher rates of protein synthesis, which are partially dependent on MTOR and MNK activity. By contrast, haploid spermatids are characterized by lower levels of protein synthesis, which are independent of the activity of these pathways. Accordingly, MTOR and MNK activity enhanced formation of the EIF4F complex in pachytene spermatocytes but not in round spermatids. Moreover, external cues differentially modulated the activity of these pathways in meiotic and haploid cells. Heat shock decreased MTOR and MNK activity in pachytene spermatocytes, whereas round spermatids were much less sensitive. On the other hand, treatment with the phosphatase inhibitor okadaic acid activated MTOR and MNK in both cell types. These results indicate that translational regulation is differentially dependent on the MTOR and MNK pathways in mouse spermatocytes and spermatids and suggest that the late stages of germ cell differentiation display constitutive assembly of the translation initiation complex.

Transcriptome analysis of differentiating spermatogonia stimulated with kit ligand.

Kit ligand (KL) is a survival factor and a mitogenic stimulus for differentiating spermatogonia. However, it is not known whether KL also plays a role in the differentiative events that lead to meiotic entry of these cells. We performed a wide genome analysis of difference in gene expression induced by treatment with KL of spermatogonia from 7-day-old mice, using gene chips spanning the whole mouse genome. The analysis revealed that the pattern of RNA expression induced by KL is compatible with the qualitative changes of the cell cycle that occur during the subsequent cell divisions in type A and B spermatogonia, i.e. the progressive lengthening of the S phase and the shortening of the G2/M transition. Moreover, KL up-regulates in differentiating spermatogonia the expression of early meiotic genes (for instance: Lhx8, Nek1, Rnf141, Xrcc3, Tpo1, Tbca, Xrcc2, Mesp1, Phf7, Rtel1), whereas it down-regulates typical spermatogonial markers (for instance: Pole, Ptgs2, Zfpm2, Egr2, Egr3, Gsk3b, Hnrpa1, Fst, Ptch2). Since KL modifies the expression of several genes known to be up-regulated or down-regulated in spermatogonia during the transition from the mitotic to the meiotic cell cycle, these results are consistent with a role of the KL/kit interaction in the induction of their meiotic differentiation.

Dynamic regulation of histone H3 methylation at lysine 4 in mammalian spermatogenesis.

Spermatogenesis is a highly complex cell differentiation process that is governed by unique transcriptional regulation and massive chromatin alterations, which are required for meiosis and postmeiotic maturation. The underlying mechanisms involve alterations to the epigenetic layer, including histone modifications and incorporation of testis-specific nuclear proteins, such as histone variants and protamines. Histones can undergo methylation, acetylation, and phosphorylation among other modifications at their N-terminus, and these modifications can signal changes in chromatin structure. We have identified the temporal and spatial distributions of histone H3 mono-, di-, and trimethylation at lysine 4 (K4), and the lysine-specific histone demethylase AOF2 (amine oxidase flavin-containing domain 2, previously known as LSD1) during mammalian spermatogenesis. Our results reveal tightly regulated distributions of H3-K4 methylation and AOF2, and that H3-K4 methylation is very similar between the mouse and the marmoset. The AOF2 protein levels were found to be higher in the testes than in the somatic tissues. The distribution of AOF2 matched the cell- and stage-specific patterns of H3-K4 methylation. Interaction studies revealed unique epigenetic regulatory complexes associated with H3-K4 methylation in the testis, including the association of AOF2 and methyl-CpG-binding domain protein 2 (MBD2a/b) in a complex with histone deacetylase 1 (HDAC1). These studies enhance our understanding of epigenetic modifications and their roles in chromatin organization during male germ cell differentiation in both normal and pathologic states.

PARP-1 interaction with VP1 capsid protein regulates polyomavirus early gene expression.

Poly(ADP-ribose)polymerases are involved in fundamental cellular events as well as they seem to be associated to some viral infection process. In this work, the poly(ADP-ribose)polymerase-1 (PARP-1) role in the polyomavirus life cycle has been investigated. Early viral transcription was reduced by competitive inhibitors of PARPs in Swiss 3T3 cells and almost abolished in PARP-1 knockout fibroblasts and in wild-type fibroblasts when PARP-1 was silenced by RNA interference. In vivo chromatin immunoprecipitation assays showed that poly(ADP-ribosyl)ation (poly(ADP-ribose)) facilitates the release of the capsid protein viral protein 1 (VP1) from the chromatin of infecting virions. In vitro experiments demonstrated that VP1 stimulates the enzymatic activity of PARP-1 and binds non-covalently both protein-free and PARP-1-bound poly(ADP-ribose). Our studies suggest that PARP-1 promotes the complete VP1 displacement from viral DNA favouring the viral early transcription.