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Departments of Animal Science and Pharmacology and Therapeutics (S.K.), McGill University, Montreal, Canada H3G 1Y6; Fondazione Santa Lucia Istituto e Cura a Carattere Scientifico (C.C.), 00179 Rome, Italy; Department of Physiology, Institute of Biomedicine (N.K.), University of Turku, FI-20520 Turku, Finland; Department of Pharmacology (J.H., P.S.-C.), School of Medicine, University of California, Irvine, Irvine, California 92697; Center for Genomics and Bioinformatics (C.H.), Karolinska Institutet, S-171 77 Stockholm, Sweden; Department of Human Physiology and Pharmacology (L.M.), University of Rome "La Sapienza," 00185 Rome, Italy; and Department of Target Discovery (J.A.G., M.v.D.), NV Organon, 5340 BH Oss, The Netherlands
Address all correspondence and requests for reprints to: Paolo Sassone-Corsi, Department of Pharmacology, School of Medicine, University of California, Irvine, Irvine, California 92697. E-mail: psc{at}uci.edu.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Segregation of chromosomes is a highly complex process that can succumb to errors due to a failure in the molecular machinery that drives cell division. Aneuploidy arising in the germline is associated with the majority of human early embryonic loss (5) and is a characteristic of cancerous cells. The conserved serine/threonine kinases that constitute the Aurora family are essential for cell division and chromosome dynamics (6). The subcellular localization of Aurora kinases is dynamically and tightly coupled to their biochemical and morphological functions during mitosis. Aurora-B and Aurora-C are chromosomal passenger proteins, localized on centromeres from prophase to the metaphase-anaphase transition. At anaphase they transfer to the spindle midzone, and at cytokinesis they associate with the midbody (6, 7, 8). Both Aurora-B and Aurora-C phosphorylate histone H3 at Ser10 and Ser28 and form a complex with INCENP (inner centromere protein) and survivin (7, 8, 9). Blocking Aurora-B activity by RNA interference, mutation, or through chemical interference elicits abnormal mitosis with failure in chromosome alignment, segregation, and cytokinesis (10, 11). The mitotic role of Aurora-C is less well established, although in vitro evidence in HeLa cells indicates that it may serve a similar role to Aurora-B in the regulation of centrosome function, chromosome segregation, and cytokinesis (12).
The role of Aurora kinases in the differentiation program of male germ cells is not well established. We undertook to examine the expression profile of Aurora-B and -C kinases in mouse spermatogenesis. Given the specialized localization of Aurora-B and Aurora-C in the testis, we concentrated our analysis on their cell-specific and developmentally regulated expression. To establish the respective role of Aurora-B and Aurora-C, we generated mouse mutants designed to specifically target these kinases. A transgenic mouse model was developed to ectopically express normal or mutant forms of the Aurora-B kinase, whereas the gene encoding for the Aurora-C kinase was mutated by homologous recombination. The study of these mutant mice provides evidence for a differential function of Aurora kinases in spermatogenesis and delineates future strategies to the understanding of their role in pathological situations.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), and Aurora-C appears to be preferentially expressed in the testis (Fig. 1A and Ref. 13). As a first step in determining the expression patterns of Aurora-B and -C kinases in the testis, we performed in situ hybridization. The results indicated high expression in the germ cells and stage-specific expression because positive reactivity to Aurora antisense probes was restricted to a subset of tubules (Fig. 1, B and C). Because of the essential role demonstrated for Aurora-B in mitosis (14, 15, 16), we further delineated the developmental regulation of Aurora-B expression by immunohistochemistry in testes collected at different times during the first wave of spermatogenesis and assessed according to the appearance of spermatogenic cell types (17). Aurora-B is present at postnatal d 5 (type A spermatogonia), 8 (type A and B spermatogonia), 14 (zygotene), and in pachytene spermatocytes in adult testis through stages IX–XII (Fig. 2A). Although Aurora-B expression during spermatogenesis has been described (18, 19), the exact cell type and colocalization with phosphorylated histone H3 (P-H3) at Ser10 has not been established. To unequivocally address this question, we generated two independent, highly specific antibodies raised in both rabbit (20) and rat, targeting the N-terminus of Aurora-B. In combination with P-H3, we examined Aurora-B immunolocalization using a highly accurate staging method that allows for determination of the exact spermatogenic stage and cell type (21). Histone H3 phosphorylation at Ser-10 by Aurora-B is a hallmark of chromatin condensation in mitosis (22). Aurora-B and P-H3 colocalize in heterochromatic regions in the nucleus of mitotic spermatogonia and in late meiotic cells from stages IX–XII (Fig. 2B). During metaphase, Aurora-B accumulates at the inner centromeres (Fig. 2B). The distribution of Aurora-C in spermatogenesis has recently been described (23) and has been shown to overlap the localization of Aurora-B reported here. The subcellular localization of Aurora-B (Fig. 2) and Aurora-C (8, 23, 24) and their similar catalytic activity (7, 8, 12) prompted us to further explore their functions in spermatogenesis by developing transgenic and mutant mouse models.
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), under the control of the pachytene stage-specific ß-4-galactosyltransferase promoter (25). This promoter becomes active in meiotic cells at 21 d of age (25), corresponding to the appearance of late pachytene cells (17). Two transgenic lines were produced, one encoding the wild-type Aurora-B (TG-wt), and a second expressing an inactive dominant negative kinase (TG-m) (16). Southern (Fig. 3B) and ribonuclease (RNase) protection analyses (Fig. 3C) confirmed integration of transgenes and tissue-specific expression restricted to the testis. Expression of the transgene was confirmed by immunofluorescence on spread spermatocytes using an antibody against the Myc-tag (data not shown) and by Western analysis of protein extracts from the seminiferous tubules (Fig. 3D). Transgenic founders were identified, and those with copy numbers determined to be between five and 10 by Southern blot were selected for intensive breeding.
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). Reduced Aurora-B activity in TG-m mice was confirmed by scoring for P-H3 in spermatogenic cell counts and Western analysis (Fig. 3, F–H).
Disrupted Spermatogenesis in Aurora-B Transgenic Mice Expressing the Kinase Inactive Aurora-B
All Aurora-B transgenic mice were analyzed at reproductive maturity (>8 wk of age), after performance assessment in breeding trials, which lasted a minimum of 2 months. As the transgene was not activated until postnatal d 21, analysis of Aurora-B transgenics during the first wave of spermatogenesis was not warranted. The Aurora-B TG-wt animals are healthy and mate normally, whereas the TG-m mice are subfertile with 48% (n = 25) failing to produce a litter in comparison with the TG-wt (n = 17) and nontransgenic (NT) (n = 12) littermate controls (Table 1). Sperm counts from the TG-m mice are reduced in comparison with TG-wt and NT mice (Fig. 4A). Fertility, testis size, and histology of the TG-wt are not different from NT littermates (P > 0.05). Morphological analysis reveals that infertile TG-m males have reduced testis size (Fig. 4B and Table 1), and examination of the seminiferous tubules shows disorganization of the spermatogenic stages as evidenced by the lack of a transition from a strong spot, to a dark zone, to a pale zone (22) (Fig. 4C). Histological analysis of seminiferous epithelium from infertile TG-m mice demonstrated severely impaired spermatogenesis (Fig. 4D). There is a greatly reduced number of round and elongating spermatids and abnormal cell associations within the tubules (Figs. 4D and 5).
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). Abnormal cell types identified by this method included degenerating metaphase I and II (MI and MII) cells and binucleate cells (Fig. 5, B–D). We also observed so-called "false anaphase" cells because they lacked a cleavage furrow, failed to undergo cytokinesis, and gave rise to binucleate cells (Fig. 5D).
Spermatogenic Cell Abnormalities in Transgenic Mice Expressing the Kinase Inactive Aurora-B
Close examination of histological preparations from TG-m mice revealed various abnormalities, including apoptotic cells, binucleate spermatocytes, multinucleated cells, and pyknotic cells; the latter two defects characteristic of degenerating round spermatids (26) (Figs. 4 and 5). There is an increase in the levels of apoptosis in comparison with NT mice, as shown by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) immunohistochemical staining (P 
0.05) (Fig. 4, E–F). On average, two apoptotic cells are expected in any given tubule in normal mice (27). In TG-m animals (n = 3), 16% of apoptotic cells were at metaphase vs. 3% (n = 3) in NT mice or TG-wt (data not shown), suggesting that there is a partial arrest in meiosis. In support of this scenario, activation of a testis-specific checkpoint as has been demonstrated in previous studies (28).
Inactivation of Mouse Aurora-C by Homologous Recombination
The high expression levels of Aurora-C in the testis relative to other tissues suggests that it may have a specific function in male germ-cell development. Furthermore, given the emerging data suggesting that Aurora-B and Aurora-C share overlapping functions in vitro (8, 12, 23), it was pertinent to examine the possible contribution of Aurora-C to the phenotype observed in the Aurora-B transgenic mutant mice and to solve the question of the contribution of the Aurora-C kinase to spermatogenesis. To address this issue the gene encoding the Aurora-C kinase was targeted for mutation by homologous recombination in the germline. Murine Aurora-C is encoded by seven exons. The gene was disrupted by insertion of a Lac Z neomycin resistance cassette into exons II–IV (Fig. 6). The targeting vector was electroporated into ES cells and homologous recombination was detected by Southern analysis. After injection of blastocysts and backcrossing of chimeras, aurora-C+/– mice were obtained. Genotyping of progeny by Southern analysis or PCR revealed the existence of progeny where only the modified allele could be detected (Fig. 6, B and C). At 9 wk of age, six male and six female homozygous-null mice were killed, and all organs and tissues were isolated for histopathological analysis. No relevant changes were observed in gross anatomy, organ weight, hematology, or physiology of these animals (data not shown). Therefore, Aurora-C is not required for mouse viability. Tissues from heterozygotes were analyzed for ß-galactosidase expression and confirmed tissue-specific expression patterns observed by RNase protection analysis. As expected, ß-galactosidase expression was greatest in the testis and ovary (data not shown).
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). In comparison with the wild type, aurora-C–/– mice had smaller (31% of wild type) litters (P 0.05), and some (40%) of the males failed to produce pups (n = 4). Sperm counts and testis weight for the aurora-C–/– mice were normal (data not shown).
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). Leydig and Sertoli cells were normal in size, shape, and location within the seminiferous epithelium and appeared to be functionally normal based on the unaltered size and weight of the testis and seminal vesicles. The levels of apoptosis in the seminiferous epithelium of mutant mice were normal (Fig. 7, B and C). Indeed, on average two apoptotic cells are expected in any given tubule in normal mice (29). The percentage of apoptotic cells was similar between the mutant and wild-type mice (P 
0.05) (Fig. 7, B and C).
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, C and D). The most common abnormal head morphology observed in aurora-C-null sperm was a shortened apex (Fig. 8C). Analysis by electron microscopy revealed significant (P 0.05) defects in the sperm cells from aurora-C–/– mice (Fig. 8, A and B). Interestingly, these included defective nuclear chromatin condensation and acrosome detachment and malformation (Fig. 8, A and B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The severe disruption of spermatogenesis in the transgenic mice expressing a mutated form of the Aurora-B kinase indicates that Aurora-B is a critical enzyme controlling key signaling events in spermatogenesis. In TG-m animals we observed cells arrested at MI and MII. These cellular defects highlight the multifunctional roles played by this kinase in postmitotic spermatogenic events. During spermatogenesis there are two checkpoints that govern apoptosis: the first occurs at stage IV and corresponds to the detection of abnormalities associated with synapsis, and the second is activated at stage XII and is associated with the detection of errors in the spindle or the orientation of bivalents on the metaphase plate (30). In the TG-m mice spermatogenesis is interrupted at stage XII, consistent with errors in chromosome alignment and segregation. It has been shown that the Aurora-B complex has several conserved functions from yeast to mammals, including the regulation of chromosome movements, chromosome congression through influence on microtubules dynamics, and histone H3 phosphorylation (15, 16, 20). These functions are in keeping with the reduced levels of histone H3 phosphorylation and the high level of apoptosis in M-phase spermatocytes from TG-m mice (Figs. 3 and 4).
The arrest of cells at MI in TG-m mice supports a role for Aurora-B in the cell division phase (6, 10). The presence of binucleated cells in TG-m animals is likely to occur when Aurora-B function is completely ablated at the division phase, and both MI and MII checkpoints are overridden. We observe what we term as a failing anaphase (Figs. 4 and 5), in which cells fail to undergo cytokinesis. Importantly, a role for Aurora-B in cytokinesis has been demonstrated in mitotic cells (10, 11), a function that our findings indicate being conserved in meiosis. Interestingly, binucleate cells have been reported for T-Rex-U2OS cells expressing a kinase-dead Aurora-B mutant (31) and in cyclin A1-null mice (29).
Recently Aurora-C has been described as a chromosomal passenger in male meiosis (23), suggesting a potential contribution in the aforementioned events during spermatogenesis. The similar localization of Aurora-C and Aurora-B in meiotic cells (23) could suggest overlapping functions. Aurora-C localizes to chromocenters in diplotene spermatocytes and at centromeres in MI and MII and persists to a lesser extent in round spermatids (23). Thereby, we have considered the possibility that Aurora-C may operate by physiologically compensating for Aurora-B dysfunctions in the Aurora-B TG-m mice. Although the severe disruption in spermatogenesis that we observe in the TG-m argues against this scenario, it is still conceivable that the dominant negative Aurora-B protein produced in the TG-m mice may also interfere with Aurora-C function. It is noteworthy that in HeLa cells transfected with a catalytically dead Aurora-C, Aurora-B was correctly localized in 100% of the cells, suggesting that a dominant negative Aurora-B may not alter Aurora-C function (8). In any case, the severe phenotype of the Aurora-B TG-m mice, considered together with the normal meiosis in the Aurora-C null males, suggests that Aurora-B is a principal operator of chromosomal dynamics and cell division in spermatogenesis. This possibility is highlighted by the observation that in cells coexpressing Aurora-B and Aurora-C kinases, Aurora-B was preferentially associated with INCENP, whereas little Aurora-C was associated with INCENP (8).
In contrast to the Aurora-B TG-m mice, Aurora-C-null males have normal testis weights, sperm counts, levels of apoptotic cells, and progression of meiotic events. However, Aurora-C nulls did display a subfertility phenotype because they had reduced litter sizes or failed to produce a litter. Our analyses by light and electron microscopy revealed that the defect is in late spermiogenesis, with defects in chromatin condensation, acrosome formation, and head shape. These abnormalities suggest that Aurora-C has critical functions in the reorganization of the chromatin during spermiogenesis that may be related to its kinase activity. In vitro kinase assays revealed that Aurora-C does not phosphorylate transition proteins or protamines (Monaco, L., and P. Sassone-Corsi, unpublished observations); however, a role in chromatin condensation is highly probable based on its localization in the spermatid nucleus (23).
Despite reports that in vitro Aurora-C functions as a chromosomal passenger protein (8, 12, 23), we did not observe defects in spermatocytes in the Aurora-C null males. Recent in vitro genetic and RNA interference studies, designed to inhibit the function of the Aurora-kinases (9, 12, 31), suggest a degree of cooperation between the Aurora kinases. At least in vitro, knockdown of one kinase can be compensated for by enhanced activity due to overexpression of the compensating Aurora kinase (8). Our in vivo studies do not rule out that Aurora-B may compensate for Aurora-C and vice versa.
Spermatogenesis is a highly complex cell differentiation process that involves massive alterations in the epigenetic program (32). Sperm contain more histone variants than any somatic cell and undergo a drastic alteration in chromatin composition with the replacement of the majority of the histones by transition proteins followed by the protamines. Given the incidence of heterogenous chromatin condensation in the Aurora-C null males, a critical role for Aurora-C in the epigenetic program of the developing male germ cell is indicated. Idiopathic male infertility is associated with genetic and epigenetic abnormalities. Such abnormalities include chromosome translocations and aneuploidies, Y chromosome microdeletions, and abnormal packaging of the DNA (33). Our findings reveal essential functions of the Aurora-B and Aurora-C kinases in mammalian spermatogenesis, placing them into a privileged position as candidate genes that may be mutated and serve in the diagnosis of men presenting with idiopathic infertility or even as a future target for novel contraceptives.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-32UTP] antisense riboprobes specific to either endogenous Aurora-B or Aurora-C, or the transgenes were generated using an in vitro transcription kit (Promega). A mouse ß-actin riboprobe was used as internal control to monitor RNA quality and amounts.
In Situ Hybridizations
In situ hybridizations were performed as previously described (35). The Aurora-B and -C cDNA were used to generate 35S-labeled antisense riboprobes by in vitro transcription (Promega, Madison, WI). As control for nonspecific signal, consecutive sections were hybridized with sense probes.
Immunohistochemical and Immunoblotting Analysis of Germ Cells
Distribution of Aurora-B and P-H3 in mouse germ cells was analyzed using a transillumination-assisted microdissection method for stage-specific isolation of spermatogenic cells (21). Antisera used were: polyclonal rat or rabbit anti-Aurora-B generated in our laboratory (20) and anti-P-H3 (Upstate Cell Signaling Solutions, Lake Placid, NY). Spermatocytes positively labeled for Ser10/P-H3 were counted from 10 tubule cross-sections from five wild type, five TG-m mice, or five TG-wt mice. Immunoblots were performed by standard techniques and revealed by enhanced chemiluminescence (Pierce, Rockford, IL).
Histology and TUNEL Analysis
Testis sections were subjected to TUNEL analysis using Apoptag Peroxidase in situ apoptosis detection kit (Chemicon International, Temecula, CA). Tubule cross-sections were scored as apoptotic when three or more positively stained cells were observed per tubule cross-section as described (28). Percent M-phase arrested cells were determined by counting 100 apototic cells in tubules from three TG-m and three NT mice. A cell was determined to be in M phase by the presence of chromosomes aligned on the metaphase plate. The mean number of apoptotic cells was determined by counting TUNEL positive cells in 10 tubule cross-sections from TG-m (n = 3) and NT (n = 3) mice.
In Vitro Aurora-B Kinase Assays
Kinase assays were performed as previously described (20). The level of kinase activity was measured by scanning densitometry of autoradiograms that were normalized to levels of substrate protein determined from densitometry of Coomassie stained gels. Data from in vitro kinase assays are representative of multiple independent studies.
Western Blotting
Total testis extracts were resolved by standard SDS-PAGE, then electroblotted onto Protran nitrocellulose (Schleicher & Schuell, Keene, NH). The membranes were incubated overnight at 4 C in PBS with 5% low-fat milk and 0.05% Tween 20 and rabbit anti-P-H3 (Upstate) or anti-Myc. Donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody (Jackson Laboratories, Bar Harbor, ME) was diluted in 5% milk in PBS with 0.05% Tween 20, and labeling was detected using enhanced chemiluminescence (Pierce). Membranes were exposed to Kodak autoradiography BioMax film (Kodak, Rochester, NY).
Generation of Aurora-B Transgenic Mice
For generation of ß-4-galactosyltransferase-Aurora-B lines, mouse Aurora-B cDNA was obtained by RT-PCR from a 129/sv mouse (GenBank accession no. BC003261), then cloned into pCS2-MTK, in frame with the ß-4-galactosyltransferase promoter (26). The resulting 2.13-kb targeting fragment was microinjected into fertilized (B57Bl/6J*SJL x B57Bl/6J*SJL) oocytes to obtain transgenic founders, which were crossed with (129/sv) wild-type mice to create the transgenic lines. Southern blot and PCR analyses were used to verify incorporation of the transgene. Several lines for both TG-wt (n = 3) and TG-m (n = 6) were obtained, with a comparable number of transgene copies integrated in the genome. Genomic DNA was extracted from tail biopsies and subjected to either PCR or Southern blot analysis. For expression analysis and characterization of the phenotype, mice were killed at reproductive maturity after identified performance in breeding trials that commenced at 6 wk of age. Mice were watered and fed standard mouse chow ad libitum and housed under conditions of controlled light (12-h light, 12-h dark cycle) at 27 C. Breeding trials were conducted for a period of 2 months, and female mice were checked for the presence of vaginal plugs. All animal procedures were approved by the Animal Care and Use Committee of the Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France.
Generation and Genotyping of Aurora-C Mutant Mice
The Aurora-C gene was disrupted by replacing exons II–IV with an internal ribosome entry site Lac/Z/MC1 neomycin resistance cassette using homologous recombination. Embryonic stem cell clones from 129/SVpas mice containing the targeted mutation were screened by Southern blot. Positive clones were injected into C57BL/6 blastocysts following standard procedures. Male chimeras were bred with C57BL/6 mice to identify mice where germline transmission of the mutant allele had taken place. Heterozygous mice were backcrossed for a minimum of five generations with C57BL/6 mice before they were used in these studies. Genotyping was performed using DNA extracted from mice tails and performed by PCR or Southern blot. Mice were killed at reproductive maturity after identified performance in breeding trials that commenced at 8 wk of age. Mice were watered and fed standard mouse chow ad libitum and housed under conditions of controlled light (12-h light, 12-h dark cycle) at 27 C. Breeding trials were conducted for a period of 2 months, and female mice were checked for the presence of vaginal plugs. All animal procedures were approved by the Animal Care and Use Committee of the Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasbourg, France.
Phase Contrast and Electron Microscopy Analysis of Aurora-C Null and Wild-Type Sperm
For phase-contrast microscopy, mature sperm were collected from the cauda epididymis of Aurora-C-null (n = 3) and wild-type mice (n = 3) and spread on Superfrost plus slides (Fisher Scientific, Pittsburgh, PA). Samples were air-dried, fixed with 4% paraformaldehyde, and washed in PBS. Slides were stained with toluidine blue and analyzed by phase-contrast microscopy. In each sperm sample 100 sperm were examined for morphological appearance. For electron microscopy, mature spermatozoa from cauda epididymis of the Aurora-C +/+ (n = 1) and Aurora-C –/– (n = 1) mice were fixed in 2.5% glutaraldehyde and prepared according to standard procedures. For electron microscope analysis of sperm, 159 sperm from an Aurora-C –/– mouse and 100 from a wild type were counted and classified as either normal or showing acrosome defects or areas of heterogenous chromatin condensation.
Spermatid Number
Sperm was counted from homogenized cauda epidiymydes from Aurora-B TG-wt (n = 25), Aurora-B TG-m (n = 17), Aurora-C null (n = 6), and wild-type (n = 6) mice using standard procedures.
Statistical Analysis
Significant differences between two groups of data were evaluated using Student’s t tests. To test for differences among more than two groups of data, data were first tested for normality, then analyzed using the ANOVA-GLM procedure in Statistical Analysis Systems (SAS Institute, Cary, NC).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online December 27, 2006
Abbreviations: INCENP, Inner centromere protein; M phase, mitotic phase; MI, metaphase I; MII, metaphase II; NT, nontransgenic; P-H3, phosphorylated histone H3; RNase, ribonuclease; TG-wt, wild-type transgene; TG-m, mutant transgene; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.
Received for publication August 11, 2006. Accepted for publication December 19, 2006.
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