The Molecular Chaperone Hsp90α Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse

et al. (2010) The Molecular Chaperone Hsp90a Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse. PLoS ONE 5(12): e15770. doi:10.1371/journal.pone.0015770 The Molecular Chaperone Hsp90a Is Required for Meiotic Progression of Spermatocytes beyond Pachytene in the Mouse Iwona Grad 0 Christopher R. Cederroth 0 Joe l Walicki 0 Corinne Grey 0 Sofia Barluenga 0 Nicolas 0 Winssinger 0 Bernard De Massy 0 Serge Nef 0 Didier Picard 0 Suzannah Rutherford, Fred Hutchinson Cancer Research Center, United States of America 0 1 De partement de Biologie Cellulaire, Universite de Gene`ve, Sciences III, Gene`ve, Switzerland, 2 De partement de Me decine Ge ne tique et De veloppement, Universite de Gen e`ve, Centre Me dical Universitaire, Gene`ve, Switzerland, 3 Institut de Ge ne tique Humaine, IGH - CNRS, Montpellier, France, 4 Institut de Science et d'Inge nierie Supramole culaires, Universite de Strasbourg , Strasbourg , France The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90a and Hsp90b. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90b die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90a gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90a are apparently normal, with one major exception. Mutant male mice, whose Hsp90b levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90a may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects. - Funding: Work in the DP and SN laboratories was supported by the Swiss National Science Foundation and the Canton de Gene`ve. CG and BdM were supported by the Centre National de la Recherche Scientifique and the Agence Nationale de la Recherche (06-BLAN-0160-01 and 09-BLAN-0269-01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Hsp90 is an ubiquitous, highly conserved protein, comprising up to 2% of total cell proteins even under non-stressed conditions. It facilitates the folding and activity of a large number and variety of client proteins. The list of Hsp90 interacting proteins has already grown to almost 300 proteins and it keeps growing. The activity of Hsp90 is modulated by the interaction with a variety of cochaperones, which can act as regulators of the ATPase activity, influence the choice of clients, target client proteins for degradation, recruit other co-chaperones, or affect the cellular localization of the clients [14] (for a comprehensive and updated summary of Hsp90 facts, see http://www.picard.ch/downloads/Hsp90facts.pdf]). In humans and mice, there are two cytosolic Hsp90 isoforms, encoded by two separate genes, Hsp90a (gene Hsp90aa1) and Hsp90b (gene Hsp90ab1). With 85.8% sequence identity and 93.4% similarity, the two isoforms are highly homologous. Whereas Hsp90b is more or less constitutively and ubiquitously expressed, the expression of Hsp90a is heat-inducible and more tissue-specific [5]. So far, there is only limited evidence for isoform-specific functions. Provided the two isoforms are expressed at all, they are thought to be largely redundant. The known exceptions include the specific participation of Hsp90a in antigen processing [6] and in blocking caspase-2 activation [7]. It has also been demonstrated that Hsp90a can be secreted to promote the maturation of matrix metalloprotease 2 and cell invasiveness in metastasis [8], and cell migration in wound healing [9]. Recently, it was suggested that Hsp90a might play a role in female meiosis in the mouse, notably in the G2/M transition [10]. A role for Hsp90 in spermatogenesis had first been described in Drosophila melanogaster, where males with certain transheterozygous combinations of mutant hsp90 alleles are sterile and display a disrupted meiosis, possibly due to a defect in microtubule dynamics [11]. A study on testis in newt showed a role for Hsp90b in prolactin-induced apoptosis of spermatogonia [12]. The in vivo role of the Hsp90 machinery has been mainly investigated by genetic studies in yeast and pharmacologically with mammalian tissue culture cells. In contrast, the genetic analysis of the Hsp90 chaperone machine in the mouse is still in its infancy. Although an Hsp90b gene disruption was found to be early embryonic lethal ten years ago [13], this finding was not further investigated. Mutational analyses of some of the co-chaperones such as FKBP51, FKBP52 and p23 have highlighted the complexity of the Hsp90 machinery and the fact that interesting insights can be gained from genetically ablating components of this important housekeeping chaperone machine [1416]. The aim of this study was to investigate genetically the in vivo role of Hsp90a, the other core component of this molecular machine, in the mouse. Generation of Hsp90a gene disruption mutants in the mouse In order to assess the role of Hsp90a in vivo, a mutant mouse line was established from an embryonal stem cell clone carrying a gene trap insertion in the last intron of the Hsp90aa1 gene (Fig. 1A). This particular insertion of a gene trap vector into intron 10 could potentially encode an in-frame fusion protein consisting of a truncated Hsp90a lacking the C-terminal 36 amino acids and a bgalactosidase-neomycine resistance gene (bGeo). The very Cterminal domain of Hsp90a comprises the conserved sequence motif MEEVD, which is essential for the interaction with the tetratricopeptide repeats present in some Hsp90 co-chaperones. This is notably the case for the large immunophilins Cyp-40, FKBP51 and FKBP52, the serinethreonine protein phosphatase 5 and CHIP [17,18]. A more extensive portion of the C-terminal domain is required for dimerization and for viability in the budding yeast [19]. Whereas a truncation mut (...truncated)