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Ubiquitin is encoded in mice by two polyubiquitin genes, Ubb and Ubc, that are considered to be stress inducible and two constitutively expressed monoubiquitin (Uba) genes. Here we report that targeted disruption of Ubb results in male and female infertility due to failure of germ cells to progress through meiosis I and hypogonadism. In the absence of Ubb, spermatocytes and oocytes arrest during meiotic prophase, before metaphase of the first meiotic division. Although cellular ubiquitin levels are believed to be maintained by a combination of functional redundancy among the four ubiquitin genes, stress inducibility of the two polyubiquitin genes, and ubiquitin recycling by proteasome-associated isopeptidases, our results indicate that ubiquitin is required for and consumed during meiotic progression. The striking similarity of the meiotic phenotype in Ubb−/− germ cells to the sporulation defect in fission yeast (Schizosaccharomyces pombe) lacking a polyubiquitin gene suggests that a meiotic role of the polyubiquitin gene has been conserved throughout eukaryotic evolution.
Precisely timed destruction of key cell cycle regulators by the ubiquitin (Ub)-proteasome system orchestrates the complex biochemical events that control the eukaryotic cell division cycle, ensuring its fidelity and unidirectionality (26). The central event in this process is the covalent conjugation of Ub, a versatile, small protein that, in addition to its well-established role in directing protein destruction by the proteasome, serves a multitude of signaling functions in all eukaryotic cells, ranging from membrane trafficking to transcriptional regulation (14, 15). This critical dependence on Ub-dependent processes imposes a stringent requirement on cells to maintain adequate supplies of available Ub.
Cellular Ub exists in a dynamic equilibrium between Ub conjugates and free Ub monomer, maintained by the opposing activities of E3 Ub ligases and Ub isopeptidases (9). Ub supplies are ultimately maintained, however, by de novo synthesis of Ub from Ub gene transcripts. In budding yeast (Saccharomyces cerevisiae), Ub is encoded by four genes, three of which (UBI1, UBI2, and UBI3) encode fusion proteins between Ub and small protein components of the large and small ribosomal subunits and one of which (UBI4) encodes a Ub polyprotein composed of five Ub moieties arranged in a spacerless, tandem, head-to-tail array (23). UBI1, UBI2, and UBI3 supply all of the Ub required for vegetative growth and mitotic cell division, but the polyubiquitin gene, UBI4, is essential only during stress (12). Mammalian genomes have four functional Ub genes: two Ub-ribosomal fusion genes, Uba52 (accession no. MGI:98887) and Uba80 (accession no. MGI:1925544; also known as Rps27a); and two polyubiquitin genes, Ubb (accession no. MGI:98888) and Ubc (accession no. MGI:98889), consisting of four and nine tandem Ub coding units, respectively (2, 3, 11, 16, 25, 32). The relative contribution of each of these Ub genes to basal Ub levels varies widely among different tissues and cell types (29).
To assess the requirement for maintenance of adequate Ub supplies in cellular function and development, we have generated mice harboring a targeted disruption of the polyubiquitin B (Ubb) gene. Mice lacking one or both copies of Ubb develop normally and are viable at birth, but Ubb−/− mice of both genders are infertile. Ubb−/− germ cells progress to the pachytene stage and form apparently normal synaptonemal complexes in both males and females. Ubb−/− spermatocytes form XY bodies containing ubiquitinated histones but ultimately undergo cell death during pachytene, and males exhibit complete testicular degeneration by 2 years of age. Ubb−/− oocytes exhibit a heteromorphic meiotic arrest but do not proceed beyond metaphase I. These findings underscore a critical role for Ubb in germ cell development and meiosis.
Details of targeting vector construction and generation of Ubb−/− mice are described in the supplemental material.
Testes were isolated and fixed in 10% neutral buffered formalin and kept at 4°C until embedded in 2-hydroxyethyl methacrylate (Technovit 7100, Kulzer, Germany). Three-micrometer sections were stained with periodic acid Schiff and hematoxylin (PAS/H). Photomicrographs were taken on a Zeiss Axiovert 35 microscope using a SPOT RT camera and software from Diagnostics Instruments.
Ovaries were fixed in Bouin's fixative at room temperature for 4.5 h, dehydrated in ethanol, and placed in n-butanol overnight before being embedded in paraffin. Five-micrometer ovarian sections were prepared using a microtome and stained with hematoxylin and eosin by standard protocols.
Four females of each genotype (Ubb+/+ and Ubb−/−) were superovulated with an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG; Sigma) followed 48 h later by 5 IU of human chorionic gonadotropin (Sigma). Mice were euthanized 16 h later by CO2 inhalation, and the oviducts were dissected out and removed. The cumulus-oophorus-complex was removed and dissolved with hyaluronidase to facilitate oocyte counting.
Two to four females of each genotype (Ubb+/+, Ubb+/−, and Ubb−/−) were primed with an intraperitoneal injection of 5 IU PMSG (Sigma). Mice were euthanized 44 h later by CO2 inhalation, and antral follicles were punctured with a needle to expel germinal vesicle (GV) oocytes. Progression of meiotic maturation of denuded mouse oocytes was scored by monitoring the breakdown of GV (GVBD) with an inverted microscope (Olympus, Melville, NY) fitted with a Hoffman contrast lens. Oocytes showing clear nuclear membranes (GV) and nucleoli were classified as GV stage, those without a visible nuclear structure (i.e., exhibiting GVBD) were classified as GVBD stage, and those metaphase II arrested with a polar body were classified as MII. Time zero was the time of follicle puncture.
Meiotic spreads were prepared from spermatocytes and immunolabeled as previously described (27). The antibodies used were anti-synaptonemal complex protein 1 (SYCP1) rabbit immunoglobulin G (IgG) (ab15090, 1:500; Abcam); anti-synaptonemal complex protein 3 (SYCP3) mouse monoclonal IgG1 (clone Cor 10G11/7; Novus); anti-SYCP3 rabbit IgG (1:250; Abcam); anti-phosphorylated histone H2A.X (γH2AX) rabbit IgG (1:800; Upstate); anti-ubiquityl histone H2A (uH2A) mouse IgM (clone E6C5, 1:500; Upstate); and Alexa Fluor 488- or 594-conjugated anti-rabbit IgG, Alexa Fluor 594-conjugated anti-mouse IgG1, and Alexa Flour 594-conjugated anti-mouse IgM (Molecular Probes). (All Alexa-conjugated secondary antibodies were used at 1:1,000.) Meiotic spreads were stained with 4′,6′-diamidino-2-phenylindole (DAPI), mounted in SlowFade antifade solution (Molecular Probes), and imaged using a Leica DMRXE upright fluorescent microscope, a Micromax cooled charge-coupled device camera, and Metamorph image acquisition software.
Fetal ovaries were collected from 18.5-day-postcoitum (dpc) embryos. From each embryo, one ovary was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at room temperature and then washed in PBS overnight at 4°C for subsequent embedding and immunolabeling. The other ovary was used to prepare microspread oocyte nuclei. The ovaries were each placed in one well of a multiwell dish containing hypoextraction buffer (15 mM Tris, 50 mM sucrose, 20 mM citrate, 5 mM EDTA, 0.5 mM dithiothreitol [DTT], 0.09 mg/ml phenylmethylsulfonyl fluoride [PMSF], and 0.5 mg/ml collagenase added just prior to use). After 30 min of incubation at room temperature, each ovary was moved to one 10-μl drop of 0.1 M sucrose on a siliconized glass slide. Each ovary was macerated, an additional 10 μl of 0.1 M sucrose was added, and a suspension was made by gentle pipetting. The 20-μl suspension was then pipetted onto a glass slide coated with freshly prepared 1% paraformaldehyde containing 0.1% Triton X-100 (pH 9.2). The slides were incubated in a humid chamber for 4 h at room temperature, moved to the bench top for brief air drying, and then washed three times for 5 min each in 1:250 Photo-Flo (Kodak, Electron Microscopy Sciences). Slides were air dried and immunolabeled according to standard protocols (27). The antibodies used were anti-SYCP1 rabbit IgG (see above), anti-SYCP3 mouse monoclonal IgG1 (see above), Alexa Fluor 594-conjugated anti-rabbit IgG (1:1,000; Molecular Probes), and Alexa Fluor 488-conjugated anti-mouse IgG1 (1:1,000; Molecular Probes).
Embedded fetal ovaries were serially sectioned (5 μm thick), deparaffinized, and treated twice for 5 min each in 50 mM NH4Cl-PBS. Each section was incubated for 1 h at room temperature in blocking solution (10% normal goat serum, 3% bovine serum albumin [BSA], 0.1% Triton X-100 in PBS). Diluted primary antibody (in blocking solution) was applied to each section and incubated for 2 h at room temperature (or at 4°C overnight). Slides were washed three times for 10 min each in 10% blocking solution (in PBS), and diluted secondary antibody was applied to each section. Slides were incubated at room temperature for 2 h and then washed three times for 10 min each in blocking solution. Slides were then stained with DAPI and mounted in SlowFade antifade solution (Molecular Probes). The antibodies used were anti-germ cell nuclear antigen (GCNA) rat IgM (undiluted) (10) and Alexa Fluor 594-conjugated anti-rat IgM (1:1,000; Molecular Probes). Immunolabeled sections were imaged using a Leica DMRXE upright fluorescent microscope, a Micromax cooled charge-coupled device camera, and Metamorph image acquisition software. Immunolabeled slides were then postfixed in 4% paraformaldehyde for 10 min at room temperature to preserve immunolabeling and washed in PBS, and a terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay was performed according to the manufacturer's (Roche) protocol for in situ cell death detection with fluorescein. TUNEL-labeled sections were imaged a second time as described above.
Oocytes were collected from antral follicles after PMSG priming of wild-type (+/+; n = 1), heterozygous (+/−; n = 3), and knockout (−/−; n = 4) females. Fifteen hours past puncture, oocytes were treated with Tyrode's acid for 2 h at room temperature to remove the zona pellucida. They were fixed in a solution containing 0.1% Triton X-100, 4% paraformaldehyde, and 1 μM Taxol for 30 min at 37°C, washed, and blocked with 2% BSA in PBS overnight at 4°C. Oocytes were placed in anti-α-tubulin monoclonal antibody (DMC1, 1:500; Sigma) overnight at 4°C, washed, and then incubated with Alexa Fluor 488-conjugated anti-mouse IgG (1:1,700; Molecular Probes) and DAPI for 2 h at room temperature. Oocytes were mounted using Vectashield.
Tissues were homogenized with Potter-Elvehjem tissue grinder with polytetrafluoroethylene pestle (Thomas Scientific) in hypotonic buffer (10 mM sodium phosphate [pH 7.4] with protease inhibitor cocktail; Roche) with 0.1% digitonin and incubated on ice for 20 to 30 min. Tissue lysates were centrifuged at 13,000 rpm for 10 min at 4°C, and the supernatant was removed to measure protein concentration by bicinchoninic acid protein assay (Pierce). To prepare samples for enzyme-linked immunosorbent assay (ELISA), tissue lysates (24 μg) were treated with 2.4 μg Usp2-cc (catalytic core of ubiquitin-specific protease 2) in the presence of 1.4 mM β-mercaptoethanol and 140 mM NaCl in a 20-μl reaction volume for 30 min at 37°C (4, 8). Usp2-cc-treated tissue lysates were further diluted with 1% BSA-PBS containing 0.1% digitonin, and total Ub levels were measured by ELISA as described previously (28). For oocyte ELISA, oocytes at GV and GVBD stages were counted and collected in 3 μl hypotonic buffer containing 0.1% digitonin during maturation in vitro. Oocytes were lysed by repeated freeze-thaw cycle, treated with Usp2-cc, and subjected to ELISA measurement.
Total RNA was isolated from RNAlater-stabilized tissues using the RNeasy Plus kit (Qiagen). The RNA concentration was determined, and 200 ng of total RNA was subsequently treated with DNase I (Invitrogen) and reverse transcribed using iScript select cDNA synthesis kit (Bio-Rad) with oligo(dT) as a primer. For real-time PCR, 1/20 of cDNA was used as a template. Reverse transcription-PCR (RT-PCR) was accomplished using the iQ SYBR green supermix (Bio-Rad) and iCycler system with iCycler iQ software version 3.1 (Bio-Rad), with the following cycle profile: 95°C for 3 min; 45 cycles of 95°C for 10 s, 60°C for 30 s, 68°C for 30 s; 95°C for 1 min; and 55°C for 1 min followed by 80 cycles of 55°C for 10 s with an increase of 0.5°C per cycle for melt curve analysis. The mRNA expression levels of Ubc, Ubb, Uba52, and Uba80 were normalized to the level of β-actin or 18S rRNA (5). Primers used for real-time RT-PCR were as follows: Ubc-F (5′-GTTACCACCAAGAAGGTC-3′), Ubc-R (5′-GGGAATGCAAGAACTTTATTC-3′), Ubb-F (5′-TCTGAGGGGTGGCTATTAA-3′), Ubb-R (5′-TGCTTACCATGCAACAAAAC-3′), Uba52-F (5′-GTCAGCTTGCCCAGAAGTAC-3′), Uba52-R (5′-ACTTCTTCTTGCGGCAGTTG-3′), Uba80-F (5′-TGGCAAAATTAGCCGACTTCG-3′), Uba80-R (5′-AACACTTGCCACAGTAATGCC-3′), 18S rRNA-F (5′-CGGCTACCACATCCAAGGAA-3′), and 18S rRNA-R (5′-GCTGGAATTACCGCGGCT-3′). Control plasmid DNA was generated by subcloning each cDNA fragment into the pCR2.1 vector (Invitrogen), and 108 to 103 copies of plasmid DNA were used as a standard.
For direct visualization of green fluorescent protein (GFP) fluorescence in testicular and ovarian sections, tissues were fixed in 4% paraformaldehyde for 2 to 4 h at 4°C, cryoprotected with 30% sucrose, and sectioned with a cryostat. Thaw-mounted sections (5 μm thick) were washed with Tris-buffered saline (TBS) and incubated with TO-PRO-3 iodide (1:1,000; Molecular Probes, excitation/emission λ = 642/661 nm) in 0.3% Triton X-100-TBS (TBST) for 30 min at room temperature for DNA staining. Sections were washed with TBST followed by TBS only and mounted with ProLong Gold antifade reagent (Molecular Probes). To monitor Ubb or Ubc transcriptional activity, fluorescence from GFP was directly visualized. Confocal images were collected with Leica TCS SP2 laser-scanning system with sequential image recording. The GFP image was collected using a 488-nm excitation light from an Ar laser with the emission window at 500 to 535 nm, and the TO-PRO-3 iodide image was collected using a 633-nm excitation light from an He-Ne laser with the emission window at 650 to 750 nm.
A two-tailed unpaired Student's t test was used to compare the data between two groups. P < 0.05 was considered to be statistically significant.
Although testes of 7-day-old Ubb−/− mice were indistinguishable from those of wild-type littermates, they failed to increase in size with age, suggesting that Ubb is required for postnatal testicular development (Fig. (Fig.1A).1A). At 5 months of age, testes of Ubb−/− mice were substantially smaller, although seminal vesicle weights were very similar to those of littermate controls (Ubb+/+ and Ubb+/−), suggesting normal Leydig cell function (Fig. (Fig.1B;1B; also see Fig. S2 in the supplemental material). Testosterone levels in 5-month-old male mice were not significantly different among different genotypes, supporting the conclusion that Leydig cell function is intact in Ubb−/− mice (data not shown). Ovarian size and weight were also reduced significantly in 5-month-old Ubb−/− females compared to littermate controls (Fig. (Fig.1B;1B; also see Fig. S2 in the supplemental material). Interbreeding of homozygous (Ubb−/−) males and females did not yield any progeny, although vaginal plugs were observed (n = 2). Mating of homozygous males with heterozygous females (n = 14) or heterozygous males with homozygous females (n = 12) also did not yield any progeny over a 4-month period despite observation of vaginal plugs. These results demonstrate a strict requirement of Ubb for male and female fertility in the mouse.
At 7 days of age (d7), Ubb−/− testes display normal histology (Fig. (Fig.2A,2A, d7). Seminiferous tubules containing numerous Sertoli cells with radially oriented nuclei are interspersed among abundant spermatogonia. By 19 days, however, marked differences are seen between Ubb+/+ and Ubb−/− testes. Ubb+/+ testes contain seminiferous tubules with multiple layers of germ cells at different stages of maturation, including those that have completed meiosis to become round spermatids (Fig. (Fig.2A,2A, d19, +/+). Some seminiferous tubules have formed lumens, and all Sertoli cells have their nuclei located adjacent to the basement membrane. In contrast, seminiferous tubules from Ubb−/− testes are smaller and contain variable numbers of germ cells (Fig. (Fig.2A,2A, d19, −/−). Some of the seminiferous tubules contain abundant Sertoli cells but few spermatogonia (tubule 1), while others contain numerous germ cells that have not matured beyond the early pachytene spermatocyte stage of meiosis I (tubule 2). Furthermore, Ubb−/− testes at 19 days contain large clusters of heteropyknotic germ cells possibly undergoing cell death. Finally, many Sertoli cell nuclei are abnormally separated from the basement membrane and no seminiferous tubule lumens have formed. By 28 days, Ubb+/+ testes contain the full complement of germ cell types with multiple generations of germ cells present, whereas the most advanced germ cells in Ubb−/− testes remain in pachytene or are undergoing cell death (data not shown). By 40 days, the first wave of spermatogenesis is complete in Ubb+/+ testes; elongated and condensed spermatids are clearly visible (Fig. (Fig.2A,2A, d40, +/+). Seminiferous tubule lumens are well formed, and Sertoli cell nuclei are adjacent to the basement membrane. In contrast, Ubb−/− testes contain a paucity of germ cells; these have not matured beyond the pachytene stage (Fig. (Fig.2A,2A, d40, −/−). Many of these pachytene spermatocytes are undergoing cell death, as indicated by nuclear condensation and fragmentation. In addition, the testis architecture is severely disrupted, with absent seminiferous tubule lumens, focal clusters of detached Sertoli cells, and prominent interstitial Leydig cells. The last feature could be the result of Leydig cell hyperplasia but most likely reflects the failure of normal seminiferous epithelium growth, resulting in reduced size and mass of the seminiferous tubules. Finally, at 8.5 months, Ubb−/− testes are severely atrophic and completely abnormal; no germ cells are present, and the testes contain only sparse and degenerating Sertoli cells and prominent masses of Leydig cells (data not shown). At 2 years of age, the testes could not be identified due to complete organ degeneration (data not shown). This severe degenerative phenotype, which is not typical of other meiotic arrest mutants, suggests defects beyond those of the germ cells. Thus, germ cells from Ubb−/− mice fail to progress beyond the early pachytene spermatocyte stage of meiosis and undergo cell death; additionally testis development is impaired, with ultimate degeneration.
To further characterize the pachytene arrest, meiotic spreads were prepared from the spermatocytes of 21-day-old Ubb−/− and littermate control (Ubb+/−) mice. Immunolabeling with antibodies to the synaptonemal complex proteins SYCP1 and SYCP3 demonstrate that synaptonemal complexes form normally along homologous chromosomes in Ubb−/− pachytene spermatocytes (Fig. (Fig.2B).2B). Consistent with the histology data, diplotene nuclei could not be identified in meiotic spread preparations from Ubb−/− spermatocytes, whereas roughly half of Ubb+/− spermatocytes were found to be in diplotene at 21 days. The XY body, comprised of the partially synapsed and largely nonhomologous X and Y chromosomes, also forms in Ubb−/− pachytene spermatocytes, as indicated by immunolabeling with an antibody against phosphorylated histone H2A.X (γH2AX) (Fig. (Fig.2C).2C). This histone modification is associated with the transcriptionally silenced XY body and is normally a discrete focus in the mid-pachytene spermatocytes. In Ubb−/− spermatocytes, however, the γH2AX antibody does not label a discrete XY body in all pachytene spermatocytes (Fig. (Fig.2C).2C). This could reflect a delay in the progression of pachynema in Ubb−/− spermatocytes or a delay in the formation of the XY body. Although Ub content is decreased in Ubb−/− testes (see below), the XY body becomes associated with ubiquitinated H2A (uH2A) (Fig. (Fig.2D),2D), a modification which is thought to maintain silencing of unpaired chromosomal regions (1). Thus, data from both histology and meiotic chromatin analysis demonstrate a spermatogenetic arrest during pachytene and indicate that Ubb is required, either directly or indirectly, for exit from prophase of meiosis I.
At 2 and 5 months of age, both Ubb+/+ and Ubb+/− females display normal ovarian architecture, with numerous follicles present in different stages of development (primary, secondary, and antral or Graafian follicles), as well as prominent corpora lutea indicative of recent ovulation (Fig. (Fig.3A,3A, +/+, and data not shown for Ubb+/−). In contrast, ovaries from Ubb−/− females are much smaller (see Fig. Fig.1B)1B) and have few mature follicles and few corpora lutea (Fig. (Fig.3A,3A, −/−). Five-month-old Ubb−/− ovaries weigh on average 2 to 3 times less than Ubb+/+ and Ubb+/− ovaries (see Fig. S2 in the supplemental material). Because Ubb−/− spermatocytes fail to progress to diplonema, meiotic spread preparations were made from fetal Ubb−/− ovaries to assess whether knockout oocytes could progress to diplonema. Immunolabeling with antibodies against SYCP1 and SYCP3 revealed that, as in spermatocytes, synaptonemal complex proteins assemble normally along synapsed homologous chromosomes during female meiosis. In addition, while some normal diplotene oocyte nuclei were observed, there were also some abnormal diplotene-like nuclei with fragmented and elongated synaptonemal complexes (Fig. (Fig.3B).3B). Immunolabeling with an antibody against GCNA and the TUNEL assay revealed that Ubb−/− fetal ovaries have fewer oocytes and elevated apoptotic nuclei (Fig. (Fig.3C).3C). Therefore, some Ubb−/− oocytes do not progress normally to diplonema and are likely lost via apoptosis during fetal development. The reduction in ovary size and follicle number in adult Ubb−/− females is likely the result of oocyte loss during fetal development. Despite these abnormalities, the surviving oocytes in immature follicles from adult ovaries appear histologically to be in the normal dictyate (resting) stage of meiosis I. (Mammalian oocytes are arrested in embryonic or early postnatal life after completing the diplotene stage of meiosis I [7, 24] and resume meiosis only at the antral follicle stage .)
Following treatment with PMSG, Ubb−/− females formed morphologically normal antral follicles in which oocytes contained a normal-appearing GV (Fig. (Fig.3D);3D); however, the size of these antral follicles was smaller than those of Ubb+/+ females and never reached more than 100 to 150 μm (data not shown). To determine whether Ubb−/− females are capable of ovulation, they were superovulated with exogenous sources of follicle-stimulating hormone and luteinizing hormone (Fig. (Fig.3E).3E). Only rarely did Ubb−/− females ovulate any oocytes, and even these tended to have an abnormal morphology with a thin zona pellucida tightly adherent to the plasma membrane and no evidence of polar bodies (data not shown). These data suggest that Ubb is required for meiotic maturation.
To address the nature of the meiotic maturation defect, we assessed the ability of Ubb−/− oocytes to undergo spontaneous maturation in vitro. Whereas over 90% of Ubb+/+ (data not shown) and Ubb+/− oocytes underwent nuclear envelope dissolution (GVBD) within 90 min after isolation, GVBD was markedly delayed and ultimately observed in fewer than 70% of Ubb−/− oocytes (Fig. (Fig.4A,4A, left). Strikingly, while nearly 80% of Ubb+/+ (data not shown) and Ubb+/− oocytes extruded polar bodies after 24 h in vitro, signifying completion of meiosis I, Ubb−/− oocytes were never observed to complete this step (Fig. (Fig.4A,4A, right). These results suggest that two sequential defects occur in adult Ubb−/− oocytes. First, many of these oocytes cannot exit meiotic prophase since they cannot undergo GVBD, which occurs at the prophase-to-metaphase transition. Second, those that do exit prophase cannot complete meiosis I and form the first polar body. Oocytes from Ubb+/− females aligned their chromosomes on a well-formed spindle (Fig. (Fig.4B,4B, +/−) and also displayed chromosomes and spindle remnants in the polar body. In contrast, chromosomes from Ubb−/− oocytes only rarely aligned on a normal-looking spindle (2/19 oocytes; 10.5%); often, the spindle was malformed (7/19 oocytes; 36.8%) or was not present at all (10/19 oocytes; 52.6%) (Fig. (Fig.4B,4B, −/−). Thus, Ubb−/− oocytes cannot successfully negotiate the prophase-to-metaphase transition. The polyubiquitin gene Ubb therefore is necessary for proper progression through and exit from meiotic prophase, as well as for pre- and postnatal oocyte development.
The dramatic meiotic phenotypes of male and female Ubb−/− mice suggest that the Ubb gene contributes a specific, nonredundant role in Ub homeostasis during germ cell development. To assess the contribution of Ubb to the maintenance of Ub pools, we used indirect competitive ELISA (28) to measure total Ub abundance in various organs of 5-month-old mice of different Ubb genotypes (Fig. (Fig.5A).5A). Of the tissues examined, the Ub concentration (as a fraction of total protein) was highest in testes—nearly double that of brain, the tissue with the next highest Ub concentration—and more than 4 times higher than spleen. The Ub concentration in testes was reduced by homozygous loss of Ubb by ~70% (Fig. (Fig.5A)5A) in 5-month-old mice and by ~50% (data not shown) in 1-month-old mice. In contrast, Ub levels were largely unaffected by homozygous loss of Ubb in all other organs examined. Furthermore, Ubb mRNA is the most abundant Ub transcript in testes, constituting ~60% of the total Ub transcripts and ~75% of the total Ub coding “potential” (i.e., when normalized for the number of Ub coding units per transcript) (Fig. (Fig.5B5B and +/+ in Fig. Fig.5C,5C, right). Considering that germ cells comprise ~90% of the cells in the adult testes (20) and that postnatal spermatogenesis is accompanied by an approximate doubling of testicular Ub levels (data not shown), it is probable that the uniquely high levels of Ub in testes are due to the contribution of Ubb expression in germ cells.
In contrast, Ub levels in the ovary are comparable to those in other tissues and reduced only ~35% (P = 0.055) by homozygous disruption of Ubb (Fig. (Fig.5A),5A), consistent with the observation that Ubb mRNA represents only ~20% of the total Ub transcript in this organ (Fig. (Fig.5B)5B) and is predicted to contribute only ~30% of the total Ub coding potential (Fig. (Fig.5D,5D, right). We propose that the dramatic phenotype observed in Ubb−/− ovaries, despite the relatively minor contribution of Ubb to total Ub pools in this tissue, reflects the fact that oocytes constitute only a small fraction of the mass of this tissue. In fact, the Ub concentration in isolated GV oocytes from wild-type mice is 0.24 ± 0.01 ng/oocyte (Fig. (Fig.5E),5E), corresponding to approximately 10 μg/mg protein (19), and is directly comparable to the level in whole testes. Total Ub levels in freshly isolated GV oocytes from Ubb−/− mice were about 70% lower than in the wild type, confirming the overwhelming contribution of Ubb to maintenance of oocyte Ub levels (Fig. (Fig.5E).5E). Moreover, Ub levels fell significantly—by nearly 25%—upon GVBD in wild-type oocytes, suggesting that Ub is consumed during meiotic prophase. These data unequivocally establish that Ubb is the principal source of Ub in oocytes and that, in its absence, Ub levels decline during prophase I to less than 25% of wild-type levels, even in the absence of such dramatic Ub deficiency in whole ovary.
Although Ubc, like Ubb, is known to be stress inducible and contains heat shock elements (6, 13, 29), changes in Ubc transcript levels in Ubb−/− testes (Fig. (Fig.5C)5C) and ovaries (Fig. (Fig.5D)5D) are insufficient to compensate for the reduced Ub levels resulting from the loss of Ubb, even after correcting for the greater number of Ub coding units in Ubc mRNA. Surprisingly, we observed substantially increased levels of Uba transcripts in both testes and ovaries of Ubb−/− mice (Fig. 5C and D), reflecting a limited capacity of Uba genes to respond to Ub deficiency or perhaps an increased relative mass of cells in which the Uba genes contribute a higher proportion of total Ub.
In order to understand the consequences of loss of the Ubb and Ubc genes on Ub levels and germ cell maturation, it is necessary to identify the cell types in which these two genes are normally expressed. However, because the final protein products of all four Ub genes are chemically identical, it is not feasible to use immunohistochemistry to assess the spatial expression of these genes. We therefore, took advantage of the GFP-puro cassette that precisely replaces the polyubiquitin coding exons in the target construct to monitor polyubiquitin gene promoter activity (29). Since the promoter and all other regulatory elements of the Ubb and Ubc genes remain intact following recombinatorial disruption of the coding exon, the spatial expression of Ubb and Ubc genes can be assessed by GFP expression analysis (Fig. (Fig.6).6). Control experiments confirmed that the relative levels of GFP mRNA in tissues from Ubb+/− and Ubc+/− mice were correlated with the levels of Ubb and Ubc mRNA in wild-type mice (see Fig. S3A and S3B in the supplemental material). Moreover, GFP expression in tissues from Ubb+/− and Ubc+/− mice, measured by indirect competitive ELISA, also correlated with expression of the corresponding genes assessed by measuring Ubb and Ubc mRNA levels in wild-type mice (see Fig. S3A and S3C in the supplemental material).
Comparison of Ubb- and Ubc- driven GFP-puro expression by direct visualization of GFP fluorescence in testes of 40-day-old mice heterozygous for the marked allele reveals that, although both genes are prominently expressed in Leydig cells, they exhibit minimally overlapping and largely complementary patterns of expression in seminiferous tubules (Fig. (Fig.6A).6A). Although GFP expression in Ubb+/− testes is evident throughout the seminiferous epithelium including Sertoli cells, the band of fluorescence in a circumferential layer just above the basal layer of the epithelium, together with the absence of the classic “spokes on a wheel” pattern, typical of Sertoli cells, is most consistent with predominant pattern of expression in spermatocytes. In sharp contrast, GFP expression in Ubc+/− testes is restricted to Leydig cells and elongating spermatids. GFP fluorescence in Ubb−/− testes is prominent in spermatogonia and arrested pachytene spermatocytes, as well as in Leydig cells (data not shown). These differences in GFP expression patterns within the seminiferous epithelium of Ubb+/− and Ubc+/− testes point to differences in endogenous promoter activity and reflect the preferential expression of the Ubb gene in developing germ cells.
In ovaries, the predominant role of Ubb over Ubc is most striking in oocytes and granulosa cells, as demonstrated by direct visualization of GFP fluorescence (Fig. (Fig.6B);6B); GFP fluorescence is strong in follicles of Ubb+/− ovaries but much reduced in these cells in Ubc+/− ovaries. Interestingly, Ubc+/− ovaries exhibit strong GFP fluorescence in luteal cells, as in Ubb+/− ovaries. The high GFP expression in oocytes and granulosa cells from Ubb+/− ovaries, combined with the low expression of Ubc in these cells, strongly suggests that insufficient Ub in oocytes and their surrounding granulosa cells may be the principal cause of ovarian failure in Ubb−/− mice.
Ub is an abundant protein that plays essential roles in multiple cellular processes in all eukaryotic cells. Yet disruption of Ubb, one of four ubiquitously expressed, seemingly redundant genes, leads to an arrest of meiotic progression in male and female germ cells, as well as degenerative changes to the testes. These findings suggest that Ubb plays roles in both meiotic progression and gonadal physiology. The similarity between the meiotic phenotypes in Ubb−/− germ cells and the sporulation defects observed in ubi4 yeast signifies that this unique requirement for polyubiquitin gene expression in meiotic progression is remarkably conserved over more than a billion years of evolution.
The single polyubiquitin gene in budding (UBI4) or fission (ubi4+; Schizosaccharomyces pombe) yeast is essential for meiosis and for survival under stress (12, 22). Our finding that Ubc−/− (29) but not Ubb−/− (data not shown) mouse embryonic fibroblasts exhibit increased stress sensitivity suggests that these dual functions of UBI4/ubi4+ have been segregated into the two paralogous polyubiquitin genes in mammals. The finding that other organs in Ubb−/− mice develop normally suggests that Ubb is not required for mitotic progression, consistent with the lack of growth or mitotic phenotypes in ubi4 yeast (12, 22).
The Ubb−/− testes at 7 days of age are morphologically normal and do not exhibit greatly reduced numbers of germ cells. However, spermatocytes lacking Ubb are blocked in pachytene; they fail to enter the diplotene stage or progress to the first meiotic division phase to separate correctly synapsed chromosomes and eventually undergo cell death. This meiotic arrest suggests that the principal defect in Ubb−/− spermatogenesis occurs at or before the transition to the diplotene stage of prophase I. In contrast, the meiotic phenotype in female Ubb−/− mice is more heterogeneous, with some oocytes progressing to diplotene in vivo. The majority of oocytes isolated from Ubb−/− mice are competent to progress to GVBD, albeit more slowly, and a few are even able to form a metaphase spindle signifying the completion of prophase I. Like Ubb−/− oocytes, ubi4 zygotes in fission yeast exhibit abnormal spindle morphology and impaired chromosome separation in meiosis I (22). The occasional ubi4 zygotes observed to progress through the first meiotic division become arrested in the second (22). We propose that the heterogeneity in meiotic arrest in Ubb−/− oocytes and ubi4 zygotes is the result of cell-to-cell variation in the amount of available Ub, underscoring the paramount and conserved role for polyubiquitin gene expression in meiotic progression. ubi4+ transcription is highly induced during sporulation in fission yeast, yet cellular Ub levels do not appear to increase, leading to the surprising conclusion that Ub may be consumed during meiosis (22). (Ub is normally recycled by isopeptidases associated with the 26S proteasome.) Likewise, total Ub levels decrease between GV and GVBD in mouse oocytes in vitro (see Fig. Fig.5E),5E), suggesting that, as in yeast, significant amounts of Ub are consumed during meiotic progression. These parallels between mice and yeast support the conclusion that meiotic progression depends on expression of Ubb, most likely autonomously, in germ cells.
Although our data support the conclusion that Ubb constitutes the predominant source of Ub in germ cells, it is also clear that Ubb is expressed in somatic cells in the gonads, particularly those that, like Sertoli and granulosa cells, interact intimately with and support germ cells. Effects of Ubb loss are not limited to arrest of germ cell development during meiotic prophase; they include deficiencies in numbers of germ cells from early developmental stages as well as possible defects in supporting cells of the gonad (e.g., Sertoli cells display abnormal separation from the basement membrane) and ultimate atrophy of the gonads. The latter defect is not typical of other meiotic arrest or germ cell depletion phenotypes and thus cannot be explained solely by loss of meiotic germ cells. In testes, Ubb is expressed in somatic supporting cells, including Leydig cells and also Sertoli cells, and lack of Ub in these cells could account for detrimental gonadal degeneration phenotypes. Age-related gonadal atrophy may also be due to hypothalamic defects in the Ubb−/− mouse (K. Ryu et al., submitted for publication). Many, but not all, of the gonadal phenotypes exhibited by Ubb−/− mice are similar to phenotypes of mice with a deletion in the Gnrh1 gene (the hpg, or hypogonadal, mouse) (17, 18). Male mice (and humans) with Gnrh1 mutations exhibit small testes and arrest of spermatogenesis before the diplotene stage. However, female mice with Gnrh1 mutations are much more severely impaired than Ubb−/− females and have only streak gonads at birth. These interesting possibilities could be tested experimentally by gonad and germ cell transplantation analyses and by the generation of cell-type-specific “conditional” Ubb knockouts to determine the cell autonomy of the various germ cell defects due to Ubb gene disruption.
Although the phenotype of the Ubb−/− ovaries may be complicated by the expression of Ubb in both the somatic and germ cell compartments, several scenarios may account for the markedly reduced size of the Ubb−/− ovaries. It is possible that Ubb−/− oogonia do not efficiently complete fetal meiotic prophase and a large number of them degenerate in a manner reminiscent of the pachytene demise observed during spermatogenesis. Thus, the pool of oocytes available to form follicles is probably already decreased at birth. However, the observation that follicle growth is also impaired may indicate that oocyte signals instructive for follicle formation and early growth are defective in Ubb−/− oocytes, causing inefficient folliculogenesis during postnatal life.
In spite of additional phenotypes with incomplete penetrance, Ubb−/− oocytes display a complete blockade of meiosis I progression and are unable to extrude a polar body. This is true even for the rare oocytes with an apparently normal spindle. This failure to progress to anaphase is most likely with the result of impaired function of APC/C (anaphase-promoting complex/cyclosome). As in mitosis, progression through the two meiotic divisions requires APC/C-mediated destruction of securin and cyclins (21, 30), underscoring an essential role for APC/C-mediated Ub conjugation in meiotic progression. How APC/C activity contributes to the separation of homologous chromosomes in meiosis I and the identity of meiosis-specific APC/C substrates remain unknown. Our data demonstrate that Ubb plays an essential role in maintaining the Ub supplies required for APC/C function and predict that undegraded meiosis-specific APC/C substrates should accumulate in Ubb−/− germ cells.
Although Ub is universally required for nearly all cellular processes, our data suggest that loss of Ubb gives rise to profound defects in germ cell maturation that are surprising in view of the fact that mitotic cell cycle is not obviously affected. Given the established roles of Ub-dependent proteolysis in regulating progression through the meiotic and mitotic cell cycles and given that the single polyubiquitin gene in yeast is also essential for meiosis and its absence cannot be compensated for by any of the other yeast ubiquitin genes, it is perhaps not surprising that a Ub gene knockout results in meiotic arrest. Therefore, the genes which encode Ub serve nonredundant functions and suggest the possibility of unanticipated meiotic roles that are conserved throughout eukaryotic evolution.
We thank our colleagues in the Kopito lab for helpful discussions and comments, and we are especially grateful to Catherine Gilchrist for help in screening the mouse genomic DNA library and Gregory Barsh for helpful discussion. We also thank the Transgenic Research Center for DNA microinjection, embryonic stem cell manipulation, and generation of chimeric mice.
This work was funded in part by a grant from the National Institute of Aging (to R.R.K.) and by grants from the National Institute of Child Health and Human Development (to M.A.H. and M.C.).
Published ahead of print on 10 December 2007.
†Supplemental material for this article may be found at http://mcb.asm.org/.