Sexual reproduction of male mammals requires genes involved in meiosis, sperm formation and maturation as well as fertilization many of which are controlled by developmental stage-specific transcription factors 12. Induction of post-meiotic gene expression is in part dependent upon the τ activator isoform of CREM (Cyclic AMP-Responsive Element Modulator) essential for spermatogenesis (for reviews, see 345). Using a yeast two-hybrid assay CREM was shown to bind Fhl5 which is specifically expressed in meiotic spermatocytes and highly induced in post-meiotic round spermatids. Fhl5 is a protein containing four and a half LIM domains which are protein-protein interaction motifs found in many factors required for processes such as transcription, cell structure and motility as well as signal transduction 6789. The protein was shown to mediate strong reporter gene expression in transfection assays using heterologous promoter constructs in yeast and mammalian cells and its dynamic pattern of nuclear and cytoplasmic localization during early and late stages of spermiogenesis is mediated via direct interaction with the Kif17b kinesin motor protein 10111213. Fhl5 is a member of a family of five related LIM domain proteins one of which, Fhl4, is also transcribed in testis showing peak induction in spermatids 1114. Fhl5 expression in pachytene spermatocytes and round spermatids was proposed to be dependent upon CREM not only in rodents but also in human; weak transcription of Fhl5 was found in three out of four infertile patients whose testes contained meiotic germ cells normally expressing the gene, suggesting a link between impaired Fhl5 function and spermatogenic arrest in a subgroup of individuals 15161718.

While CREM is essential for spermatogenesis, Fhl5 is not because mutant male mice were found to be fertile. However, they displayed partially abnormal sperm maturation and morphology which was suggested to be a consequence of impaired CREM/Fhl5-dependent post-meiotic gene expression 19. Three testicular genes reported to be directly controlled by CREM continued to be expressed normally in the absence of Fhl5 but since numerous testicular transcripts were shown to depend upon CREM many candidate genes remained to be investigated 1920.

To identify Fhl5 target genes, a whole-genome expression profiling experiment using total testicular samples and high-density oligonucleotide microarrays (GeneChips) containing probes for all known mouse protein-coding genes was carried out. We report that deletion of Fhl5 does not have a measurable effect on testicular gene expression at the level of sensitivity reached by microarray analysis when wild-type and Fhl5 mutant mice are examined in an isogenic background.

Fhl5 has initially been identified as a CREM-binding factor required for gene activation in the male germline. It was our aim to verify the hypothesis that abnormal sperm maturation and morphology observed in mutant mice lacking Fhl5 was due to impaired CREM-dependent gene expression. To this end we carried out two independent profiling experiments using total testicular samples and MG430 2.0 GeneChips covering all known protein-coding genes of the mouse genome.

The first experiment was based on total testicular samples from adult nine week-old Fhl5-/- mice produced in a mixed genetic background (C57BL/6 and 129/SvPas) 19 that were compared to data previously obtained with wild-type samples isolated at the same age but from another strain (B6129SF2/J) 14. We considered this cost-effective and time-saving approach acceptable in spite of the influence of genetic backgrounds on gene expression which is especially pertinent in the case of human samples 25 because Fhl5 was a potent activator in transfection experiments 1026. However, among the genes we found in our initial experiment using Fhl5+/+ and -/- samples from different backgrounds none played a role in spermiogenesis, sperm maturation or fertility and not a single one fell into the group of CREM target genes previously identified by microarray analysis 20. A detailed second expression analysis of litter mates failed to yield any differentially expressed genes in 12 samples from adult mice including five wild-type and seven mutant animals. It should be emphasized that these mice stemmed from a back-crossed strain background that displayed a stronger phenotype than the Fhl5 deletion in the initial mixed background (P. Sassone-Corsi, unpublished observation).

The most obvious explanation of these results is that the loci identified in the first experiment showed distinct expression patterns because we compared samples from different genetic backgrounds while the second experiment based on isogenic litter mates revealed that Fhl5 is not essential for testicular gene expression at the level of sensitivity achieved with microarrays and whole gonad samples. This result is in keeping with a previous report that the absence of Fhl5 has no effect on the expression of known CREM targets such as TNP1, PRN1 or PRN2 as assayed by PCR 19. It also concurs with the fact that point mutations in the human ortholog of Fhl5 occur with the same frequency in fertile individuals and azoo- or oligozoospermic patients which argues against an essential role for the protein in human spermatogenesis 1827. It is possible that Fhl5's in vivo role as a transcriptional regulator is too subtle to be detected by whole-genome profiling using total testicular samples and that highly enriched populations of round spermatids would have to be used instead. We consider this unlikely, however, since Fhl5 was reported to be a strong activator and approximately 60% of the adult testicular cell mass consists of spermatids 10.

An alternative explanation is that a related factor to a large degree compensates the deletion of Fhl5 such that transcriptional effects are masked while sperm maturation and morphology remain partially abnormal. One likely candidate is the LIM-domain protein Fhl4 which was initially shown to be expressed in testis 1128 and later found to be absent in spermatogonia and highly induced in spermatocytes and spermatids by microarray analysis 14. Fhl4 weakly activates a heterologous reporter construct in yeast and mammalian cells but it cannot, as opposed to Fhl5, bind to CREM in a yeast two-hybrid assay 11. However, this does not exclude the native Fhl4 protein from being able to partially take over roles of Fhl5 in the physiological context of adult testicular tissue, especially in the absence of functional Fhl5.

The corollary of our work is that deletion of Fhl5 does not cause any significant changes in testicular expression at the genome-wide level at a level detectable by high-density oligonucleotide microarray expression profiling when compared to an isogenic wild-type sample. We speculate that this is the case because Fhl5 is redundant with Fhl4, a structurally related factor showing a similar testicular expression pattern. If this idea were correct, mice lacking Fhl4 should display a phenotype reminiscent of the one reported for Fhl5 mutants while deletion of both genes should disrupt spermatogenesis at the post-meiotic stage because of impaired CREM-dependent gene expression.

The authors declare that they have no competing interests.

AL processed and interpreted the data and contributed to the manuscript. FC contributed to data analysis. PD processed the samples and produced the raw microarray data. PSC and MP designed research and MP interpreted the data and wrote the paper. All authors have read and approved the final manuscript.