Specific localization of transcription factors in the chromatin of mouse mature spermatozoa.

We previously characterized a nuclease-hypersensitive fraction of mouse sperm chromatin, which is organized in a typical nucleosomal structure. A partial genomic library was constructed with the DNA from the nuclease-hypersensitive chromatin, which revealed a high content in retroposon/retroviral DNA sequences. Here we report that the cloned nuclease-hypersensitive DNA also contains clusters of potential sites for transcription factors: among those, binding sites for Oct-1, Oct-4, TBP, Ets-1, and C/EBP are most abundant. This observation prompted us to ask whether mature spermatozoa contain the corresponding protein factors. Indirect immunofluorescence experiments show that all analyzed factors are indeed present in the sperm heads. Moreover, transcription factors are associated with the nuclease-hypersensitive chromatin of spermatozoa, as endogenous nucleases that degrade the hypersensitive fraction also cause the concomitant release of transcription factors from sperm cells into the medium. Band-shift assays with proteins extracted from the supernatant, and immunofluorescence analysis of sperm pellets, indicate that transcription factors are largely recovered in the supernatant while being absent or poorly retained in spermatozoa. The possible involvement of these factors in early embryogenesis is discussed.

Nucleosomal domains of mouse spermatozoa chromatin as potential sites for retroposition and foreign DNA integration.

Exogenous DNA molecules are spontaneously taken up by sperm cells, internalized in nuclei, and eventually integrated in the sperm genome. The actual occurrence of the integration suggests that the sperm chromosomal DNA is not uniformly and tightly packed with protamines, implying the existence of genomic sites where the chromosomal DNA is accessible to foreign molecules. We have characterized a hypersensitive, nucleosomal subfraction of mouse sperm chromatin that is highly enriched in unmethylated retroposon DNA from a variety of families. Here we propose that both the integration of exogenous DNA molecules, and the endogenous retroposition activity, occur in the same site(s) of sperm chromatin.

Reverse transcriptase activity in mature spermatozoa of mouse.

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.

A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA.

We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of 'active' chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.

Deregulated expression of the RanBP1 gene alters cell cycle progression in murine fibroblasts.

RanBP1 is a molecular partner of the Ran GTPase, which is implicated in the control of several processes, including DNA replication, mitotic entry and exit, cell cycle progression, nuclear structure, protein import and RNA export. While most genes encoding Ran-interacting partners are constitutively active, transcription of the RanBP1 mRNA is repressed in non proliferating cells, is activated at the G1/S transition in cycling cells and peaks during S phase. We report here that forced expression of the RanBP1 gene disrupts the orderly execution of the cell division cycle at several stages, causing inhibition of DNA replication, defective mitotic exit and failure of chromatin decondensation during the telophase-to-interphase transition in cells that achieve nuclear duplication and chromosome segregation. These results suggest that deregulated RanBP1 activity interferes with the Ran GTPase cycle and prevents the functioning of the Ran signalling system during the cell cycle.

Activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA.

Mature sperm cells, either of epididymal origin or ejaculated and depleted of seminal fluid, are spontaneously able to bind exogenous DNA molecules which are subsequently internalized into sperm nuclei. Southern blot analysis showed that the internalized DNA was specifically cleaved by sperm endonucleases and showed typical fragmentation patterns of localized hypersensitivity. Nucleases were activated in response to the internalization of exogenous DNA by sperm cells and their activity increased with the DNA concentration. Nuclease activation was efficient in epididymal sperm cells, while being drastically reduced in ejaculated washed spermatozoa. Nucleases were Ca++ dependent, and were, respectively, inhibited and activated by preincubating sperm cells with Aurintricarboxylic Acid (ATA) and Ca++ Ionophore A23187, which are known to, respectively, inhibit and activate apoptosis in somatic cells. Moreover, nuclease activation also caused a partial degradation of the sperm endogenous chromosomal DNA; cleaved DNA fragments were released from the sperm cells to the medium. Taken together, these results suggest that a metabolically active process similar to apoptosis is triggered in the nuclei of mature sperm cells upon interaction with exogenous DNA.

Expression of the murine RanBP1 and Htf9-c genes is regulated from a shared bidirectional promoter during cell cycle progression.

The murine Htf9-a/RanBP1 and Htf9-c genes are divergently transcribed from a bidirectional promoter. The Htf9-a gene encodes the RanBP1 protein, a major partner of the Ran GTPase. The divergently transcribed Htf9-c gene encodes a protein sharing similarity with yeast and bacterial nucleic acid-modifying enzymes. We report here that both mRNA species produced by the Htf9-associated genes are regulated during the cell cycle progression, peak in S phase and decrease during mitosis. Transient expression experiments with reporter constructs showed that cell cycle expression is controlled at the transcriptional level, because the bidirectional Htf9 promoter is down-regulated in growth-arrested cells, is activated at the G1/S transition and reaches maximal activity in S phase, though with a different efficiency for each orientation. We have delimited specific promoter regions controlling S phase activity in one or both orientations: identified elements contain recognition sites for members belonging to both the E2F and Sp1 families of transcription factors. Together, the results suggest that the sharing of the regulatory region supports co-regulation of the Htf9-a/RanBP1 and Htf9-c genes in a common window of the cell cycle.