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• W1995043995 abstract "To study the role of Hsp90b1, an endoplasmic chaperone, we have built a conditional knockout by crossing Hsp90b1flox/flox with the Vasa-Cre transgenic line. Spermatozoa deficient in Hsp90b1 could not naturally fertilize oocytes and exhibited large and globular heads with abnormal intermediate pieces, a phenotype reminiscent of human globozoospermia. To study the role of Hsp90b1, an endoplasmic chaperone, we have built a conditional knockout by crossing Hsp90b1flox/flox with the Vasa-Cre transgenic line. Spermatozoa deficient in Hsp90b1 could not naturally fertilize oocytes and exhibited large and globular heads with abnormal intermediate pieces, a phenotype reminiscent of human globozoospermia. Hsp90b1 (gp96; glucose-related protein 94 [Grp94]) is an endoplasmic chaperone member of the heat shock protein 90 (Hsp90) family. It is involved in protein folding and in the targeting of malfolded proteins to endoplasmic reticulum (ER)–associated degradation (ERAD), in addition to participating in calcium storage. Beyond those basic and fundamental cellular functions, Hsp90b1 is well known for its role in immunity, cancer, neurologic disorders, and, more recently, development (1Eletto D. Dersh D. Argon Y. GRP94 in ER quality control and stress responses.Semin Cell Dev Biol. 2010; 21: 479-485Crossref PubMed Scopus (156) Google Scholar, 2Ni M. Lee A.S. ER chaperones in mammalian development and human diseases.FEBS Lett. 2007; 581: 3641-3651Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 3Yang Y. Li Z. Roles of heat shock protein gp96 in the ER quality control: redundant or unique function?.Mol Cells. 2005; 20: 173-182Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Hsp90b1 is a gene essential during embryonic development, because Hsp90b1−/− embryos die around day 7 of gestation owing to inability to generate mesoderm (4Wanderling S. Simen B.B. Ostrovsky O. Ahmed N.T. Vogen S.M. Gidalevitz T. et al.GRP94 is essential for mesoderm induction and muscle development because it regulates insulin-like growth factor secretion.Mol Biol Cell. 2007; 18: 3764-3775Crossref PubMed Scopus (112) Google Scholar).Knowing that other members of the Hsp90 family were expected to exert important roles during final oogenesis (5Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (34) Google Scholar) or during spermatogenesis (6Lee S.J. Expression of HSP86 in male germ cells.Mol Cell Biol. 1990; 10: 3239-3242PubMed Google Scholar), we wanted to determine whether Hsp90b1 was required for gametogenesis. Therefore, to circumvent the embryonic lethality observed in a Hsp90b1−/− line, we took advantage of the floxed Hsp90b1 allele created by Yang et al. (7Yang Y. Liu B. Dai J. Srivastava P.K. Zammit D.J. Lefrancois L. et al.Heat shock protein gp96 is a master chaperone for toll-like receptors and is important in the innate function of macrophages.Immunity. 2007; 26: 215-226Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). We used the transgenic line expressing Cre recombinase driven by the Vasa promoter to establish a new mouse model with the deletion of Hsp90b1 targeted in the embryonic male and female germlines (8Gallardo T. Shirley L. John G.B. Castrillon D.H. Generation of a germ cell–specific mouse transgenic Cre line, Vasa-Cre.Genesis. 2007; 45: 413-417Crossref PubMed Scopus (58) Google Scholar). Animal breeding and experiments were approved by the Departmental Veterinary Office (Haute-Garonne) according to French legislation.Hsp90b1flox/flox female mice were crossed with Hsp90b1flox/+ male mice which were either homozygous or heterozygous for the transgene Vasa-Cre (see Supplemental Materials and Methods, available online). The number of pups per litter produced by Vasa-Cre Hsp90b1flox/+ males was 6.1 ± 0.26 compared with 9.5 ± 0.47 pups per control litter generated by wild-type (WT) or Hsp90b1flox/flox males (≥17 litters were analyzed for each type of breeding). This suggested that some embryos died during gestation, probably owing to nonspecific activation of the transgenic Vasa promoter. This was consistent with the description made by Gallardo et al. in testing this transgene on the reporter line Gt(Rosa)26Sor LacZ (8Gallardo T. Shirley L. John G.B. Castrillon D.H. Generation of a germ cell–specific mouse transgenic Cre line, Vasa-Cre.Genesis. 2007; 45: 413-417Crossref PubMed Scopus (58) Google Scholar). In addition, we observed an abnormally high level of Hsp90b1+ allele transmission (94.3% instead of 50%; Supplemental Tables 1 and 2, available online) among the viable pups. When heterozygous Vasa-Cre/+ males were used, they transmitted the transgene to 25% of their offspring instead of 50% (Supplemental Table 2). Consequently, it was difficult to obtain experimental animals with the appropriate genotype leading to a complete loss of expression in the germline.Four experimental or mutant (MT) males (Vasa-Cre Hsp90b1flox/del) were obtained. One was used for preliminary observations, and three were included in the present study. They were overall normal, and at 3 months they were mated with WT females during a 4-week period. They did not produce any offspring and exhibited normal mating behavior and testis size (Supplemental Fig. 1, available online). This suggested that Hsp90b1 was required for normal spermatozoa functions, and further analyses were performed on mutant testes and epididymal sperm.Figure 1A (panels I–IV) shows that testis histology was grossly normal in MT males compared with WT. Hsp90b1 which was homogeneously expressed in WT seminiferous tubules (Fig. 1A, panel V) was completely absent in the germ cell population but still visible in the cytoplasm of Sertoli cells of MT males, as shown in Figure 1A (panel VI). Epididymis sections contained abundant spermatozoa, but closer examination revealed that the normal hook-shaped sperm head was identified only in WT male samples and large globular heads were seen in MT (Fig. 1A, panels VII and VIII, respectively and Supplemental Fig. 2, available online). To define the severity of the head distortion, we calculated the percentage of normal heads versus three categories of abnormal heads: 1) with a rather elongated head; 2) with a rounded-globular head; and 3) with an enlarged-deformed head. MT males (n = 200 spermatozoa) exhibited 86% of abnormal sperm of types 2 and 3 and no normal sperm (Fig. 1B). In addition, spermatozoa collected in the cauda epididymis of MT males were nearly immobile (data not shown).To better characterize the anomalies of the MT sperm head, we used three markers to stain the spermatozoa. Fluorescent Lens culinaris agglutinin–fluorescein isothiocyanate (FITC) complex, which interacts with glycoconjugates, exhibited a defined labeling of the acrosome in WT sperm (Fig. 1C, panel I) in contrast to diffuse and variable staining in MT sperm (Fig. 1C, panels II and III). Streptavidin-FITC complex, which binds endogenously biotinylated proteins present almost exclusively in mitochondria, showed a weak labeling of the intermediate piece in WT sperm (Fig. 1D, panels I and I′) in contrast to stronger staining concentrated at the abnormal sperm head in MT sperm (Fig. 1D, panels II and III). Fluorescent immunodetection of HSPA5 (BIP, Grp78), an ER chaperone, was visible mainly at the level of the intermediate piece in WT sperm (Fig. 1E, panel I) but strongly localized within the abnormal sperm head of MT sperm (Fig. 1E, panels II and III).This phenotype resembles human globozoospermia, which is characterized by round-headed spermatozoa, absent or highly abnormal acrosomic compartment, disorganization of mitochondria, and excessive cytoplasm around the nucleus (9Lin Y.N. Roy A. Yan W. Burns K.H. Matzuk M.M. Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis.Mol Cell Biol. 2007; 27: 6794-6805Crossref PubMed Scopus (161) Google Scholar). Globozoospermia causes infertility and even compromises the use of IVF owing to severe chromatin-nuclear anomalies (10Dam A.H. Koscinski I. Kremer J.A. Moutou C. Jaeger A.S. Oudakker A.R. et al.Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia.Am J Hum Genet. 2007; 81: 813-820Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 11Dam A.H. Feenstra I. Westphal J.R. Ramos L. van Golde R.J. Kremer J.A. Globozoospermia revisited.Hum Reprod Update. 2007; 13: 63-75Crossref PubMed Scopus (212) Google Scholar). A better molecular understanding of this rare human disease relies on basic studies exploiting genetically modified mouse models. For the time being, the Mouse Genome Informatics database (http://www.informatics.jax.org) provides a list of 43 genotypes with a phenotype description containing terms related to “globozoospermia.” This includes 21 identified genes and 18 chemically induced mutations awaiting further genomic identification. Within this list, several genes encode ER proteins or vesicle trafficking-related polypeptides such as Spata16 (a Golgi protein) (10Dam A.H. Koscinski I. Kremer J.A. Moutou C. Jaeger A.S. Oudakker A.R. et al.Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia.Am J Hum Genet. 2007; 81: 813-820Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), Gba2 (beta-glucosidase 2, a resident enzyme of the ER (12Yildiz Y. Matern H. Thompson B. Allegood J.C. Warren R.L. Ramirez D.M. et al.Mutation of beta-glucosidase 2 causes glycolipid storage disease and impaired male fertility.J Clin Invest. 2006; 116: 2985-2994Crossref PubMed Scopus (179) Google Scholar), Tisp40 (testes-specific bZIP transcription factor) (13Nagamori I. Yomogida K. Ikawa M. Okabe M. Yabuta N. Nojima H. The testes-specific bZip type transcription factor Tisp40 plays a role in ER stress responses and chromatin packaging during spermiogenesis.Genes Cells. 2006; 11: 1161-1171Crossref PubMed Scopus (34) Google Scholar), Gopc (Golgi-associated PDZ and coiled-coil motif–containing protein) (14Yao R. Ito C. Natsume Y. Sugitani Y. Yamanaka H. Kuretake S. et al.Lack of acrosome formation in mice lacking a Golgi protein, Gopc.Proc Natl Acad Sci U S A. 2002; 99: 11211-11216Crossref PubMed Scopus (214) Google Scholar), Pick1 (protein interacting with C kinase 1), which would cooperate with other proteins such as GOPC and CK2alpha in acrosome biogenesis and sperm head organization (15Xiao N. Kam C. Shen C. Jin W. Wang J. Lee K.M. et al.Pick1 deficiency causes male infertility in mice by disrupting acrosome formation.J Clin Invest. 2009; 119: 802-812Crossref PubMed Scopus (135) Google Scholar, 16Xu X. Toselli P.A. Russell L.D. Seldin D.C. Globozoospermia in mice lacking the casein kinase II alpha′ catalytic subunit.Nat Genet. 1999; 23: 118-121Crossref PubMed Scopus (325) Google Scholar), and Zpbp (protein binding the zona pellucida) (9Lin Y.N. Roy A. Yan W. Burns K.H. Matzuk M.M. Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis.Mol Cell Biol. 2007; 27: 6794-6805Crossref PubMed Scopus (161) Google Scholar). Because Hsp90b1 is an endoplasmic chaperone, it is expected to interact with other ER proteins to contribute to their folding and activity or to trigger their degradation (1Eletto D. Dersh D. Argon Y. GRP94 in ER quality control and stress responses.Semin Cell Dev Biol. 2010; 21: 479-485Crossref PubMed Scopus (156) Google Scholar, 2Ni M. Lee A.S. ER chaperones in mammalian development and human diseases.FEBS Lett. 2007; 581: 3641-3651Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 3Yang Y. Li Z. Roles of heat shock protein gp96 in the ER quality control: redundant or unique function?.Mol Cells. 2005; 20: 173-182Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). It could be speculated that Hsp90b1 is required to be the testis-specific chaperone of some of these ER proteins. Furthermore, as suggested by Asquith et al. (17Asquith K.L. Harman A.J. McLaughlin E.A. Nixon B. Aitken R.J. Localization and significance of molecular chaperones, heat shock protein 1, and tumor rejection antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction.Biol Reprod. 2005; 72: 328-337Crossref PubMed Scopus (88) Google Scholar), this chaperone could be involved “in the mechanisms by which mammalian spermatozoa both acquire and express their ability to recognize the zona pellucida.” A link between this function and globozoospermia was shown in the case of Zpbp1, which was identified as a zona pellucida–binding protein and loss of function of which provoked a globozoospermia phenotype (9Lin Y.N. Roy A. Yan W. Burns K.H. Matzuk M.M. Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis.Mol Cell Biol. 2007; 27: 6794-6805Crossref PubMed Scopus (161) Google Scholar).Taken together, the present data suggest that Hsp90b1, which was already known to be implicated in cancer (1Eletto D. Dersh D. Argon Y. GRP94 in ER quality control and stress responses.Semin Cell Dev Biol. 2010; 21: 479-485Crossref PubMed Scopus (156) Google Scholar, 2Ni M. Lee A.S. ER chaperones in mammalian development and human diseases.FEBS Lett. 2007; 581: 3641-3651Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar) and bipolar disorder (18Kakiuchi C. Ishiwata M. Nanko S. Kunugi H. Minabe Y. Nakamura K. et al.Association analysis of HSP90B1 with bipolar disorder.J Hum Genet. 2007; 52: 794-803Crossref PubMed Scopus (22) Google Scholar), should be studied also as a potential candidate in genetic screening of human cases of globozoospermia. In addition, it is worth mentioning that members of the Hsp90 family share an adenosine triphosphatase domain that can be blocked by anticancer drugs such as the derivatives of geldanamycin (1Eletto D. Dersh D. Argon Y. GRP94 in ER quality control and stress responses.Semin Cell Dev Biol. 2010; 21: 479-485Crossref PubMed Scopus (156) Google Scholar). Consequently, the use of those treatments could affect functions of the endoplasmic Hsp90b1, causing spermatogenesis defects. In conclusion, Hsp90b1 is a gene critically involved in reproduction, with biomedical implications in human health. Hsp90b1 (gp96; glucose-related protein 94 [Grp94]) is an endoplasmic chaperone member of the heat shock protein 90 (Hsp90) family. It is involved in protein folding and in the targeting of malfolded proteins to endoplasmic reticulum (ER)–associated degradation (ERAD), in addition to participating in calcium storage. Beyond those basic and fundamental cellular functions, Hsp90b1 is well known for its role in immunity, cancer, neurologic disorders, and, more recently, development (1Eletto D. Dersh D. Argon Y. GRP94 in ER quality control and stress responses.Semin Cell Dev Biol. 2010; 21: 479-485Crossref PubMed Scopus (156) Google Scholar, 2Ni M. Lee A.S. ER chaperones in mammalian development and human diseases.FEBS Lett. 2007; 581: 3641-3651Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 3Yang Y. Li Z. Roles of heat shock protein gp96 in the ER quality control: redundant or unique function?.Mol Cells. 2005; 20: 173-182Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Hsp90b1 is a gene essential during embryonic development, because Hsp90b1−/− embryos die around day 7 of gestation owing to inability to generate mesoderm (4Wanderling S. Simen B.B. Ostrovsky O. Ahmed N.T. Vogen S.M. Gidalevitz T. et al.GRP94 is essential for mesoderm induction and muscle development because it regulates insulin-like growth factor secretion.Mol Biol Cell. 2007; 18: 3764-3775Crossref PubMed Scopus (112) Google Scholar). Knowing that other members of the Hsp90 family were expected to exert important roles during final oogenesis (5Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (34) Google Scholar) or during spermatogenesis (6Lee S.J. Expression of HSP86 in male germ cells.Mol Cell Biol. 1990; 10: 3239-3242PubMed Google Scholar), we wanted to determine whether Hsp90b1 was required for gametogenesis. Therefore, to circumvent the embryonic lethality observed in a Hsp90b1−/− line, we took advantage of the floxed Hsp90b1 allele created by Yang et al. (7Yang Y. Liu B. Dai J. Srivastava P.K. Zammit D.J. Lefrancois L. et al.Heat shock protein gp96 is a master chaperone for toll-like receptors and is important in the innate function of macrophages.Immunity. 2007; 26: 215-226Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). We used the transgenic line expressing Cre recombinase driven by the Vasa promoter to establish a new mouse model with the deletion of Hsp90b1 targeted in the embryonic male and female germlines (8Gallardo T. Shirley L. John G.B. Castrillon D.H. Generation of a germ cell–specific mouse transgenic Cre line, Vasa-Cre.Genesis. 2007; 45: 413-417Crossref PubMed Scopus (58) Google Scholar). Animal breeding and experiments were approved by the Departmental Veterinary Office (Haute-Garonne) according to French legislation. Hsp90b1flox/flox female mice were crossed with Hsp90b1flox/+ male mice which were either homozygous or heterozygous for the transgene Vasa-Cre (see Supplemental Materials and Methods, available online). The number of pups per litter produced by Vasa-Cre Hsp90b1flox/+ males was 6.1 ± 0.26 compared with 9.5 ± 0.47 pups per control litter generated by wild-type (WT) or Hsp90b1flox/flox males (≥17 litters were analyzed for each type of breeding). This suggested that some embryos died during gestation, probably owing to nonspecific activation of the transgenic Vasa promoter. This was consistent with the description made by Gallardo et al. in testing this transgene on the reporter line Gt(Rosa)26Sor LacZ (8Gallardo T. Shirley L. John G.B. Castrillon D.H. Generation of a germ cell–specific mouse transgenic Cre line, Vasa-Cre.Genesis. 2007; 45: 413-417Crossref PubMed Scopus (58) Google Scholar). In addition, we observed an abnormally high level of Hsp90b1+ allele transmission (94.3% instead of 50%; Supplemental Tables 1 and 2, available online) among the viable pups. When heterozygous Vasa-Cre/+ males were used, they transmitted the transgene to 25% of their offspring instead of 50% (Supplemental Table 2). Consequently, it was difficult to obtain experimental animals with the appropriate genotype leading to a complete loss of expression in the germline. Four experimental or mutant (MT) males (Vasa-Cre Hsp90b1flox/del) were obtained. One was used for preliminary observations, and three were included in the present study. They were overall normal, and at 3 months they were mated with WT females during a 4-week period. They did not produce any offspring and exhibited normal mating behavior and testis size (Supplemental Fig. 1, available online). This suggested that Hsp90b1 was required for normal spermatozoa functions, and further analyses were performed on mutant testes and epididymal sperm. Figure 1A (panels I–IV) shows that testis histology was grossly normal in MT males compared with WT. Hsp90b1 which was homogeneously expressed in WT seminiferous tubules (Fig. 1A, panel V) was completely absent in the germ cell population but still visible in the cytoplasm of Sertoli cells of MT males, as shown in Figure 1A (panel VI). Epididymis sections contained abundant spermatozoa, but closer examination revealed that the normal hook-shaped sperm head was identified only in WT male samples and large globular heads were seen in MT (Fig. 1A, panels VII and VIII, respectively and Supplemental Fig. 2, available online). To define the severity of the head distortion, we calculated the percentage of normal heads versus three categories of abnormal heads: 1) with a rather elongated head; 2) with a rounded-globular head; and 3) with an enlarged-deformed head. MT males (n = 200 spermatozoa) exhibited 86% of abnormal sperm of types 2 and 3 and no normal sperm (Fig. 1B). In addition, spermatozoa collected in the cauda epididymis of MT males were nearly immobile (data not shown). To better characterize the anomalies of the MT sperm head, we used three markers to stain the spermatozoa. Fluorescent Lens culinaris agglutinin–fluorescein isothiocyanate (FITC) complex, which interacts with glycoconjugates, exhibited a defined labeling of the acrosome in WT sperm (Fig. 1C, panel I) in contrast to diffuse and variable staining in MT sperm (Fig. 1C, panels II and III). Streptavidin-FITC complex, which binds endogenously biotinylated proteins present almost exclusively in mitochondria, showed a weak labeling of the intermediate piece in WT sperm (Fig. 1D, panels I and I′) in contrast to stronger staining concentrated at the abnormal sperm head in MT sperm (Fig. 1D, panels II and III). Fluorescent immunodetection of HSPA5 (BIP, Grp78), an ER chaperone, was visible mainly at the level of the intermediate piece in WT sperm (Fig. 1E, panel I) but strongly localized within the abnormal sperm head of MT sperm (Fig. 1E, panels II and III). This phenotype resembles human globozoospermia, which is characterized by round-headed spermatozoa, absent or highly abnormal acrosomic compartment, disorganization of mitochondria, and excessive cytoplasm around the nucleus (9Lin Y.N. Roy A. Yan W. Burns K.H. Matzuk M.M. Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis.Mol Cell Biol. 2007; 27: 6794-6805Crossref PubMed Scopus (161) Google Scholar). Globozoospermia causes infertility and even compromises the use of IVF owing to severe chromatin-nuclear anomalies (10Dam A.H. Koscinski I. Kremer J.A. Moutou C. Jaeger A.S. Oudakker A.R. et al.Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia.Am J Hum Genet. 2007; 81: 813-820Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 11Dam A.H. Feenstra I. Westphal J.R. Ramos L. van Golde R.J. Kremer J.A. Globozoospermia revisited.Hum Reprod Update. 2007; 13: 63-75Crossref PubMed Scopus (212) Google Scholar). A better molecular understanding of this rare human disease relies on basic studies exploiting genetically modified mouse models. For the time being, the Mouse Genome Informatics database (http://www.informatics.jax.org) provides a list of 43 genotypes with a phenotype description containing terms related to “globozoospermia.” This includes 21 identified genes and 18 chemically induced mutations awaiting further genomic identification. Within this list, several genes encode ER proteins or vesicle trafficking-related polypeptides such as Spata16 (a Golgi protein) (10Dam A.H. Koscinski I. Kremer J.A. Moutou C. Jaeger A.S. Oudakker A.R. et al.Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia.Am J Hum Genet. 2007; 81: 813-820Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), Gba2 (beta-glucosidase 2, a resident enzyme of the ER (12Yildiz Y. Matern H. Thompson B. Allegood J.C. Warren R.L. Ramirez D.M. et al.Mutation of beta-glucosidase 2 causes glycolipid storage disease and impaired male fertility.J Clin Invest. 2006; 116: 2985-2994Crossref PubMed Scopus (179) Google Scholar), Tisp40 (testes-specific bZIP transcription factor) (13Nagamori I. Yomogida K. Ikawa M. Okabe M. Yabuta N. Nojima H. The testes-specific bZip type transcription factor Tisp40 plays a role in ER stress responses and chromatin packaging during spermiogenesis.Genes Cells. 2006; 11: 1161-1171Crossref PubMed Scopus (34) Google Scholar), Gopc (Golgi-associated PDZ and coiled-coil motif–containing protein) (14Yao R. Ito C. Natsume Y. Sugitani Y. Yamanaka H. Kuretake S. et al.Lack of acrosome formation in mice lacking a Golgi protein, Gopc.Proc Natl Acad Sci U S A. 2002; 99: 11211-11216Crossref PubMed Scopus (214) Google Scholar), Pick1 (protein interacting with C kinase 1), which would cooperate with other proteins such as GOPC and CK2alpha in acrosome biogenesis and sperm head organization (15Xiao N. Kam C. Shen C. Jin W. Wang J. Lee K.M. et al.Pick1 deficiency causes male infertility in mice by disrupting acrosome formation.J Clin Invest. 2009; 119: 802-812Crossref PubMed Scopus (135) Google Scholar, 16Xu X. Toselli P.A. Russell L.D. Seldin D.C. Globozoospermia in mice lacking the casein kinase II alpha′ catalytic subunit.Nat Genet. 1999; 23: 118-121Crossref PubMed Scopus (325) Google Scholar), and Zpbp (protein binding the zona pellucida) (9Lin Y.N. Roy A. Yan W. Burns K.H. Matzuk M.M. Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis.Mol Cell Biol. 2007; 27: 6794-6805Crossref PubMed Scopus (161) Google Scholar). Because Hsp90b1 is an endoplasmic chaperone, it is expected to interact with other ER proteins to contribute to their folding and activity or to trigger their degradation (1Eletto D. Dersh D. Argon Y. GRP94 in ER quality control and stress responses.Semin Cell Dev Biol. 2010; 21: 479-485Crossref PubMed Scopus (156) Google Scholar, 2Ni M. Lee A.S. ER chaperones in mammalian development and human diseases.FEBS Lett. 2007; 581: 3641-3651Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 3Yang Y. Li Z. Roles of heat shock protein gp96 in the ER quality control: redundant or unique function?.Mol Cells. 2005; 20: 173-182Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). It could be speculated that Hsp90b1 is required to be the testis-specific chaperone of some of these ER proteins. Furthermore, as suggested by Asquith et al. (17Asquith K.L. Harman A.J. McLaughlin E.A. Nixon B. Aitken R.J. Localization and significance of molecular chaperones, heat shock protein 1, and tumor rejection antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction.Biol Reprod. 2005; 72: 328-337Crossref PubMed Scopus (88) Google Scholar), this chaperone could be involved “in the mechanisms by which mammalian spermatozoa both acquire and express their ability to recognize the zona pellucida.” A link between this function and globozoospermia was shown in the case of Zpbp1, which was identified as a zona pellucida–binding protein and loss of function of which provoked a globozoospermia phenotype (9Lin Y.N. Roy A. Yan W. Burns K.H. Matzuk M.M. Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis.Mol Cell Biol. 2007; 27: 6794-6805Crossref PubMed Scopus (161) Google Scholar). Taken together, the present data suggest that Hsp90b1, which was already known to be implicated in cancer (1Eletto D. Dersh D. Argon Y. GRP94 in ER quality control and stress responses.Semin Cell Dev Biol. 2010; 21: 479-485Crossref PubMed Scopus (156) Google Scholar, 2Ni M. Lee A.S. ER chaperones in mammalian development and human diseases.FEBS Lett. 2007; 581: 3641-3651Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar) and bipolar disorder (18Kakiuchi C. Ishiwata M. Nanko S. Kunugi H. Minabe Y. Nakamura K. et al.Association analysis of HSP90B1 with bipolar disorder.J Hum Genet. 2007; 52: 794-803Crossref PubMed Scopus (22) Google Scholar), should be studied also as a potential candidate in genetic screening of human cases of globozoospermia. In addition, it is worth mentioning that members of the Hsp90 family share an adenosine triphosphatase domain that can be blocked by anticancer drugs such as the derivatives of geldanamycin (1Eletto D. Dersh D. Argon Y. GRP94 in ER quality control and stress responses.Semin Cell Dev Biol. 2010; 21: 479-485Crossref PubMed Scopus (156) Google Scholar). Consequently, the use of those treatments could affect functions of the endoplasmic Hsp90b1, causing spermatogenesis defects. In conclusion, Hsp90b1 is a gene critically involved in reproduction, with biomedical implications in human health. Supplemental materials and methodsAnimalsThe Hsp90b1flox/flox mice were generated previously and were kindly provided by Dr. Z. Li. They were described elsewhere (1Yang Y. Liu B. Dai J. Srivastava P.K. Zammit D.J. Lefrançois L. et al.Heat shock protein gp96 is a master chaperone for Toll-like receptors and is important in the innate function of macrophages.Immunity. 2007; 26: 215-226Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). They were maintained in a mixed genetic background. To delete Hsp90b1 in the male germline, Hsp90b1flox/flox mice were crossed with transgenic mice carrying Vasa promoter–mediated Cre recombinase. Vasa-Cre transgene is expressed in the male germline (2Gallardo T. Shirley L. John G.B. Castrillon D.H. Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre.Genesis. 2007; 45: 413-417Crossref PubMed Scopus (244) Google Scholar).All animal work was conducted according to relevant national and international guidelines. In particular, protocols for animal breeding and experiments were approved by the Departmental Veterinary Office (Haute-Garonne) according to French legislation (approval ID: 31-09-555-39).Sperm PreparationSpermatozoa were collected from the cauda epididymis as decribed elsewhere (3Salmand P.A. Jungas T. Fernandez M. Conter A. Christians E.S. Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.Biol Reprod. 2008; 79: 1092-1101Crossref PubMed Scopus (34) Google Scholar).Histology, Immunohistochemistry, and ImmunofluorescenceFor histology and immunohistochemistry, mouse testes were fixed with Bouin, and histologic preparations were performed according to classic procedures (Plateau Technique d’Histopathologie Expérimentale de l’IFR30, Plateforme d’Exploration Fonctionnelle/Génopole, Toulouse Midi-Pyrénées). Immunohistochemistry was performed as previously described (3Salmand P.A. Jungas T. Fernandez M. Conter A. Christians E.S. Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.Biol Reprod. 2008; 79: 1092-1101Crossref PubMed Scopus (34) Google Scholar, 4Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (73) Google Scholar).According to the experiment, histologic sections or spermatozoa were stained with the following fluorescent dyes: DNA (TO-PRO-3, dilution 1:500 in mounting medium; Invitrogen, Carlsbad, CA) and endoplasmic reticulum glycoconjugates (Lens culinaris agglutinin–fluorescein isothiocyanate complex [LCA-FITC], solution 100 μg/mL, dilution 1:10 in phosphate-buffered saline solution). Fluorescent staining and immunofluorescence protocols were adapted from previous work (3Salmand P.A. Jungas T. Fernandez M. Conter A. Christians E.S. Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.Biol Reprod. 2008; 79: 1092-1101Crossref PubMed Scopus (34) Google Scholar, 4Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (73) Google Scholar, 5Bierkamp C. Luxey M. Metchat A. Audouard C. Dumollard R. Christians E. Lack of maternal heat shock factor 1 results in multiple cellular and developmental defects, including mitochondrial damage and altered redox homeostasis, and leads to reduced survival of mammalian oocytes and embryos.Dev Biol. 2010; 339: 338-353Crossref PubMed Scopus (37) Google Scholar).Primary antibodies used in immunohistochemistry or immunofluorescence were as follow: Hspa5 (rabbit polyclonal, NB100-91794; Novus Biologicals, Littleton, CO), dilution 1:50; and Hsp90b1 (rabbit polyclonal, Ab13509; Abcam, Cambridge, MA), dilution 1:500, or (rat monoclonal, 9G10; Abcam), dilution 1:50.Confocal microscopy and imaging analysis was performed as described previously (4Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (73) Google Scholar, 5Bierkamp C. Luxey M. Metchat A. Audouard C. Dumollard R. Christians E. Lack of maternal heat shock factor 1 results in multiple cellular and developmental defects, including mitochondrial damage and altered redox homeostasis, and leads to reduced survival of mammalian oocytes and embryos.Dev Biol. 2010; 339: 338-353Crossref PubMed Scopus (37) Google Scholar). AnimalsThe Hsp90b1flox/flox mice were generated previously and were kindly provided by Dr. Z. Li. They were described elsewhere (1Yang Y. Liu B. Dai J. Srivastava P.K. Zammit D.J. Lefrançois L. et al.Heat shock protein gp96 is a master chaperone for Toll-like receptors and is important in the innate function of macrophages.Immunity. 2007; 26: 215-226Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). They were maintained in a mixed genetic background. To delete Hsp90b1 in the male germline, Hsp90b1flox/flox mice were crossed with transgenic mice carrying Vasa promoter–mediated Cre recombinase. Vasa-Cre transgene is expressed in the male germline (2Gallardo T. Shirley L. John G.B. Castrillon D.H. Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre.Genesis. 2007; 45: 413-417Crossref PubMed Scopus (244) Google Scholar).All animal work was conducted according to relevant national and international guidelines. In particular, protocols for animal breeding and experiments were approved by the Departmental Veterinary Office (Haute-Garonne) according to French legislation (approval ID: 31-09-555-39). The Hsp90b1flox/flox mice were generated previously and were kindly provided by Dr. Z. Li. They were described elsewhere (1Yang Y. Liu B. Dai J. Srivastava P.K. Zammit D.J. Lefrançois L. et al.Heat shock protein gp96 is a master chaperone for Toll-like receptors and is important in the innate function of macrophages.Immunity. 2007; 26: 215-226Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). They were maintained in a mixed genetic background. To delete Hsp90b1 in the male germline, Hsp90b1flox/flox mice were crossed with transgenic mice carrying Vasa promoter–mediated Cre recombinase. Vasa-Cre transgene is expressed in the male germline (2Gallardo T. Shirley L. John G.B. Castrillon D.H. Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre.Genesis. 2007; 45: 413-417Crossref PubMed Scopus (244) Google Scholar). All animal work was conducted according to relevant national and international guidelines. In particular, protocols for animal breeding and experiments were approved by the Departmental Veterinary Office (Haute-Garonne) according to French legislation (approval ID: 31-09-555-39). Sperm PreparationSpermatozoa were collected from the cauda epididymis as decribed elsewhere (3Salmand P.A. Jungas T. Fernandez M. Conter A. Christians E.S. Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.Biol Reprod. 2008; 79: 1092-1101Crossref PubMed Scopus (34) Google Scholar). Spermatozoa were collected from the cauda epididymis as decribed elsewhere (3Salmand P.A. Jungas T. Fernandez M. Conter A. Christians E.S. Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.Biol Reprod. 2008; 79: 1092-1101Crossref PubMed Scopus (34) Google Scholar). Histology, Immunohistochemistry, and ImmunofluorescenceFor histology and immunohistochemistry, mouse testes were fixed with Bouin, and histologic preparations were performed according to classic procedures (Plateau Technique d’Histopathologie Expérimentale de l’IFR30, Plateforme d’Exploration Fonctionnelle/Génopole, Toulouse Midi-Pyrénées). Immunohistochemistry was performed as previously described (3Salmand P.A. Jungas T. Fernandez M. Conter A. Christians E.S. Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.Biol Reprod. 2008; 79: 1092-1101Crossref PubMed Scopus (34) Google Scholar, 4Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (73) Google Scholar).According to the experiment, histologic sections or spermatozoa were stained with the following fluorescent dyes: DNA (TO-PRO-3, dilution 1:500 in mounting medium; Invitrogen, Carlsbad, CA) and endoplasmic reticulum glycoconjugates (Lens culinaris agglutinin–fluorescein isothiocyanate complex [LCA-FITC], solution 100 μg/mL, dilution 1:10 in phosphate-buffered saline solution). Fluorescent staining and immunofluorescence protocols were adapted from previous work (3Salmand P.A. Jungas T. Fernandez M. Conter A. Christians E.S. Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.Biol Reprod. 2008; 79: 1092-1101Crossref PubMed Scopus (34) Google Scholar, 4Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (73) Google Scholar, 5Bierkamp C. Luxey M. Metchat A. Audouard C. Dumollard R. Christians E. Lack of maternal heat shock factor 1 results in multiple cellular and developmental defects, including mitochondrial damage and altered redox homeostasis, and leads to reduced survival of mammalian oocytes and embryos.Dev Biol. 2010; 339: 338-353Crossref PubMed Scopus (37) Google Scholar).Primary antibodies used in immunohistochemistry or immunofluorescence were as follow: Hspa5 (rabbit polyclonal, NB100-91794; Novus Biologicals, Littleton, CO), dilution 1:50; and Hsp90b1 (rabbit polyclonal, Ab13509; Abcam, Cambridge, MA), dilution 1:500, or (rat monoclonal, 9G10; Abcam), dilution 1:50.Confocal microscopy and imaging analysis was performed as described previously (4Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (73) Google Scholar, 5Bierkamp C. Luxey M. Metchat A. Audouard C. Dumollard R. Christians E. Lack of maternal heat shock factor 1 results in multiple cellular and developmental defects, including mitochondrial damage and altered redox homeostasis, and leads to reduced survival of mammalian oocytes and embryos.Dev Biol. 2010; 339: 338-353Crossref PubMed Scopus (37) Google Scholar). For histology and immunohistochemistry, mouse testes were fixed with Bouin, and histologic preparations were performed according to classic procedures (Plateau Technique d’Histopathologie Expérimentale de l’IFR30, Plateforme d’Exploration Fonctionnelle/Génopole, Toulouse Midi-Pyrénées). Immunohistochemistry was performed as previously described (3Salmand P.A. Jungas T. Fernandez M. Conter A. Christians E.S. Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.Biol Reprod. 2008; 79: 1092-1101Crossref PubMed Scopus (34) Google Scholar, 4Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (73) Google Scholar). According to the experiment, histologic sections or spermatozoa were stained with the following fluorescent dyes: DNA (TO-PRO-3, dilution 1:500 in mounting medium; Invitrogen, Carlsbad, CA) and endoplasmic reticulum glycoconjugates (Lens culinaris agglutinin–fluorescein isothiocyanate complex [LCA-FITC], solution 100 μg/mL, dilution 1:10 in phosphate-buffered saline solution). Fluorescent staining and immunofluorescence protocols were adapted from previous work (3Salmand P.A. Jungas T. Fernandez M. Conter A. Christians E.S. Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin.Biol Reprod. 2008; 79: 1092-1101Crossref PubMed Scopus (34) Google Scholar, 4Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (73) Google Scholar, 5Bierkamp C. Luxey M. Metchat A. Audouard C. Dumollard R. Christians E. Lack of maternal heat shock factor 1 results in multiple cellular and developmental defects, including mitochondrial damage and altered redox homeostasis, and leads to reduced survival of mammalian oocytes and embryos.Dev Biol. 2010; 339: 338-353Crossref PubMed Scopus (37) Google Scholar). Primary antibodies used in immunohistochemistry or immunofluorescence were as follow: Hspa5 (rabbit polyclonal, NB100-91794; Novus Biologicals, Littleton, CO), dilution 1:50; and Hsp90b1 (rabbit polyclonal, Ab13509; Abcam, Cambridge, MA), dilution 1:500, or (rat monoclonal, 9G10; Abcam), dilution 1:50. Confocal microscopy and imaging analysis was performed as described previously (4Metchat A. Akerfelt M. Bierkamp C. Delsinne V. Sistonen L. Alexandre H. et al.Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.J Biol Chem. 2009; 284: 9521-9528Crossref PubMed Scopus (73) Google Scholar, 5Bierkamp C. Luxey M. Metchat A. Audouard C. Dumollard R. Christians E. Lack of maternal heat shock factor 1 results in multiple cellular and developmental defects, including mitochondrial damage and altered redox homeostasis, and leads to reduced survival of mammalian oocytes and embryos.Dev Biol. 2010; 339: 338-353Crossref PubMed Scopus (37) Google Scholar). Supplemental Figure 1. Supplemental Figure 2. Supplementary Table 1. Tabled 1Mating and expected production of Hsp90b1 germline deleted animals using the conditional knockout Hsp90b1flox and the Vasa-Cre transgene (Tg).MalesFemalesParent genotypeTg/Tg; Hsp90b1flox/++/+; Hsp90b1flox/floxTransmitted alleles (%)Tg (100%)Hsp90b1+ (50%)Hsp90b1del (50%)aThe floxed allele (flox) is recombined by the Cre recombinase expressed in the male germline, producing a deleted allele (del).+ (100%)Hsp90b1flox (100%)Offspring genotype (%)Tg/+; Hsp90b1+/flox (50%)Tg/+; Hsp90b1del/flox (50%) = experimental animalsbBased on this percentage and the number of pups generated (n = 105) with this type of mating, the number of experimental animals should have been 52, or ∼25 males instead of four as observed in the present study.(a) The floxed allele (flox) is recombined by the Cre recombinase expressed in the male germline, producing a deleted allele (del).(b) Based on this percentage and the number of pups generated (n = 105) with this type of mating, the number of experimental animals should have been 52, or ∼25 males instead of four as observed in the present study. Open table in a new tab Supplementary Table 2. Tabled 1Mating and expected transmission of conditional allele Hsp90b1flox 1Yang Y. Liu B. Dai J. Srivastava P.K. Zammit D.J. Lefrançois L. et al.Heat shock protein gp96 is a master chaperone for Toll-like receptors and is important in the innate function of macrophages.Immunity. 2007; 26: 215-226Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar and the transgene (Tg) Vasa-Cre 2Gallardo T. Shirley L. John G.B. Castrillon D.H. Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre.Genesis. 2007; 45: 413-417Crossref PubMed Scopus (244) Google Scholar.MalesFemalesMating 1: parent genotypeaThe expected results of mating 1 is described in Supplemental Table 1.Tg/Tg; Hsp90b1flox/+ (n = 3 males)+/+;Hsp90b1flox/flox Transmitted alleles(n = 105 pups)TgHsp90b1+ bThe percentage (94.3%) obtained for the paternal Hsp90b1+ allele implicated that the other paternal allele (Hsp90b1del) could be transmitted to only 5.7% of the offspring.P<0.001 Chi square for actual genotype segregation versus expected.Hsp90b1del(c)The floxed allele (flox) is recombined by the Cre recombinase expressed in the male germline, producing a deleted allele (del)., (d)The percentage (60%) obtained for the transmission of the Hsp90b1del allele is not compatible with the deduced/calculated paternal germline transmission as indicated in (b).+Hsp90b1floxeThe percentage (38%) obtained for the maternal Hsp90b1flox allele is lower than expected (100%; Supplemental Table 1). From (d) and (e), it can be suggested that the maternal Hsp90b1flox is recombined in somatic cells by unspecific expression of the Vasa-Cre transgene in 62% of the pups.P<0.001 Chi square for actual genotype segregation versus expected. n105996310538 Observed %10094.36010036.2Mating 2: parent genotypeTg/+; Hsp90b1flox/+ (n = 4 males)+/+; Hsp90b1flox/flox Transmitted alleles(n = 87 pups)Tg∗P<0.01 Chi square for actual genotype segregation versus expected.Hsp90b1+∗P<0.01 Chi square for actual genotype segregation versus expected.Hsp90b1del+Hsp90b1flox n2560NDNDND Observed %28.768.9NDNDND(a) The expected results of mating 1 is described in Supplemental Table 1.(b) The percentage (94.3%) obtained for the paternal Hsp90b1+ allele implicated that the other paternal allele (Hsp90b1del) could be transmitted to only 5.7% of the offspring.(c) The floxed allele (flox) is recombined by the Cre recombinase expressed in the male germline, producing a deleted allele (del).(d) The percentage (60%) obtained for the transmission of the Hsp90b1del allele is not compatible with the deduced/calculated paternal germline transmission as indicated in (b).(e) The percentage (38%) obtained for the maternal Hsp90b1flox allele is lower than expected (100%; Supplemental Table 1). From (d) and (e), it can be suggested that the maternal Hsp90b1flox is recombined in somatic cells by unspecific expression of the Vasa-Cre transgene in 62% of the pups.∗ P<0.01 Chi square for actual genotype segregation versus expected.∗∗ P<0.001 Chi square for actual genotype segregation versus expected. Open table in a new tab" @default.
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• W1995043995 title "Hsp90b1 knockout targeted to male germline: a mouse model for globozoospermia" @default.
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