Oocyte Collection and Maturation In Vitro

Ovaries were obtained from a local slaughterhouse and transported to the laboratory in physiological saline at 20°C. They were briefly washed in 70% ethanol and then in physiological saline. Oocytes were obtained by aspiration of large antral follicles (>4 mm). Only oocytes surrounded by compact cumuli (cumulus-oocyte complexes [COCs]) were used for culture. The COCs were cultured in maturation medium [29] supplemented with 15% estrus cow serum (ECS) and 5 IU/ml of Suigonan PG-600 (Intervet International B.V.) at 38.5°C in an atmosphere of 5% CO₂ [29]. Samples were collected at 0 h (germinal vesicle stage [GV]), 24 h (metaphase II [MII]), and 20 h (1-cell zygote) after fertilization. At the end of culture, the cumulus cells were removed from oocytes by vortexing. Denuded oocytes were washed in PBS and stored at −80°C for immunoblotting experiments. Some oocytes and zygotes were evaluated for meiotic progression and fertilization by staining with 0.1 μg/ml of Hoechst 33258 (Sigma) and observed under an Olympus IX70 epifluorescence microscope.

Inhibition of UCHL1 in the Oocyte

To investigate the role of UCHL1 in the oocyte, the following specific inhibitors of UCHL1 were added to the medium at 20 μM concentration (unless stated otherwise) at the beginning of culture: C30 [30] (alternative designation LDN-57444 [662086; Merck]) and C16 [31] (kindly provided by Dr. Gregory D. Cuny, Harvard Center for Neurodegeneration and Repair, Brigham & Women's Hospital and Harvard Medical School, Boston, MA). C30 is a reversible, competitive, active site-directed isatin oxime with consistent preference for UCHL1 over UCHL3 by 28-fold. C16 is an uncompetitive inhibitor of UCHL1 that binds only to the Michaelis complex and not to free enzyme. Stock solutions of C16 and C30 were prepared in dimethyl sulfoxide (DMSO) (D2438; Sigma) and kept frozen at −20°C. Controls were treated with an equivalent amount of DMSO. Final working solutions of C16 and C30 were diluted immediately before usage. Meiotic progression was evaluated at 24 h after isolation (extrusion of first polar body [PB]). The COCs were washed thoroughly in fertilization medium (FM) before IVF.

In Vitro Fertilization

Frozen ejaculate from the same fertile bull (provided by the Czech Breeders Association), stored in sperm pellet form, was used in all experiments. Basic FM was used for sperm thawing, washing, and swim up [29]. Sperm pellets were plunged into 2 ml of FM warmed up to 39°C, centrifuged in conic tubes for 10 min at 250 × g, and washed twice in FM. After the second wash, sperm pellets were incubated for 10 min at 39°C and divided into two equal parts; each part was layered under 1 ml of FM in 8-ml tubes and then incubated for 10 min at 39°C for the swim-up procedure. The supernatants (2 × 0.7 ml) with motile spermatozoa were pooled and centrifuged. Spermatozoa were placed into each well of four-well Nunclon dishes (Thermo Fisher Scientific, Denmark) with 500 μl of FM supplemented with 1.5 mg/ml of crystallized bovine serum albumin (BSA), 0.25 mg/ml of D-penicillamin (Sigma), 2% fetal calf serum, and 5 IU/ml of heparin (Sigma). The final sperm concentration was 0.5 × 10⁶ sperm per milliliter of FM. Washed COCs were coincubated with spermatozoa for 20 h at 38.5°C in an atmosphere of 5% CO₂. No inhibitors were added to FM during IVF. The number of pronuclei and CG status were assessed at 20 h after fertilization as already described.

Parthenogenetic Activation

Bovine oocyte maturation and in vitro embryo culture were performed as described previously [32]. C30 inhibitor was dissolved in DMSO and added to maturation medium at 20 μM concentration; DMSO alone was added to control maturation medium. After 24 h of maturation, oocytes were stripped of cumulus cells by vortexing. Oocytes were activated according to an established method [33] with slight modifications. Briefly, oocytes were incubated in 5 μM ionomycine (Sigma) in Tyrode lactate-Hepes supplemented with 3 mg/ml of BSA for 5 min and subsequently in 2 mM 6-DMAP (Sigma) in EmbryoAssist medium (Medicult) supplemented with 15% ECS for 5 h. After activation, oocytes were transferred into pure EmbryoAssist medium with ECS. Embryo development was examined at 32, 44, 56, 92, 120, 156, and 180 h after activation, and the numbers of 2-cell, 4-cell, early 8-cell, and late 8-cell embryos and morulae and blastocysts were counted at each time point.

In Vitro mRNA Production for Microinjection

The pCMVFL3 vector with cDNA clone (OVR010079H10) containing the full-length porcine UCHL1 sequence (GenBank accession number AK234541.1) was a gift from Professor Hirohide Uenishi (National Institute of Agrobiological Sciences, Tsukuba, Japan). The porcine-specific protein sequence (Q6SEG5) was aligned to the bovine protein sequence (ENSBTAT00000006692) using BLAST analysis, and identity between both sequences was 96%. To generate the template for transcription, full-length porcine cDNA of UCHL1 was cloned into the SpeI site (for N-terminal green fluorescent protein [GFP] tags) of the phageRNA (pRNA)-enhanced GFP (EGFP) or pRNA-empty vector containing a T3 promoter and Xenopus globin 5′ untranslated region (UTR), 3′ UTR, and Kozak sequences for high mRNA stability and efficient translation initiation [34]. EGFP-UCHL1 mRNA and wild-type UCHL1 mRNA for microinjection were produced by in vitro transcription of the linearized vector using mMESSAGE mMACHINE T3 kit (1348; Ambion). After in vitro transcription, mRNAs were immediately polyadenylated using Poly(A) Tailing kit (AM1350; Ambion) for stabilization. They were purified using RNeasy Mini kit (74104; Qiagen). Identity of products was confirmed by sequencing. EGFP mRNA for control microinjection was transcribed from an empty pRNA-EGFP vector. The mRNAs were aliquoted (5 μl) at 500 ng of RNA per microliter and stored at −80°C until used for microinjection.

Oocyte Microinjection

Freshly isolated GV-stage oocytes were partly denuded from cumulus cells and cultured for 1 h in manipulation medium (MM) without hormones. Oocytes were microinjected with 5 pl of the mRNA solution using an MIS-5000 micromanipulator (Burleigh; Exfo Life Sciences) and a PM 2000B4 microinjector (MicroData Instrument). Pipettes for microinjection were prepared using a P97 Pipette Puller (Sutter Instrument Company). The microinjection medium was the MM (110 mM NaCl, 10 mM glucose, 8 mM Hepes, 5 mM KCl, 2 mM CaCl₂.H₂O, 0.5 mM KH₂PO₄, 0.4 mM MgSO₄.7H₂O, 0.1% NaHCO₃, 1 mg/ml of polyvinyl alcohol, 0.2 mg/ml of sodium pyruvate, and antibiotics). Oocytes were microinjected with Uchl1 mRNA with dextran-fluorescein isothiocyanate (FITC) or with EGFP-UCHL1 mRNA. Control oocytes were injected with dextran-FITC (1 mg/ml, 70 kDa; Sigma) (Supplemental Fig. S1 available at  www.biolreprod.org). Following microinjection, oocytes were incubated in MM with PG-600. Only oocytes displaying EGFP fluorescence or dextran-FITC fluorescence were used for analysis. Oocytes were dry frozen at −80°C for immunoblotting or fixed in 4% paraformaldehyde (16005; Sigma) for immunofluorescence, in which case they were mounted in an antifade medium containing DNA stain 4′,6′-diamidino-2-phenylindole (DAPI) and analyzed by confocal microscopy.

Immunoblotting of Oocyte Extracts

Unless stated otherwise, all reagents were obtained from Sigma. Exact numbers of oocytes (20, 50, or 100 oocytes per extract) were lysed in 3× Blue Loading Buffer (7722; Cell Signaling) with or without dithiothreitol (DTT) and then subjected to SDS-PAGE gel (12% or 15% acrylamide, 0.75-mm thick). To limit the amount of disulfide-bonded oligomers formed in cells, the thiol-blocking agent N-ethylmaleimide (NEM) was added to the cell lysis buffer [35, 36]. Proteins were transferred to Immobilon P membrane (Millipore Corporation) using a semidry blotting system (Whatman Biometra GmbH) for 30 min at 5 mA/cm². Blocking of the membrane was performed in 5% nonfat milk in Tris-buffered saline (TBS)-Tween buffer (TBS-T) (20 mM Tris, pH 7.4, 137 mM NaCl, and 0.5% Tween 20) for 1 h. After three washes for 10 min in TBS-T, the membrane was incubated overnight with the first antibody. All antibodies were diluted in 5% skim milk-TBS-T at 1:1000 dilution. UCHL1 was detected by mouse monoclonal antibody (58593; Santa Cruz Biochemicals) or by goat polyclonal antibody (5937; Chemicon). Antiubiquitin antibodies included mouse monoclonal antimonoubiquitin (0508; Sigma), mouse antipolyubiquitin (K⁶³ linkage specific, PW0600; Biomol), and mouse antipolyubiquitin (58595; Santa Cruz Biochemicals). Washed membranes were incubated with appropriate horseradish peroxidase-conjugated secondary anti-IgG antibodies (1:10 000 dilution; Jackson Immunoresearch) in 5% nonfat milk-TBS-T for 1 h at room temperature. Protein bands were visualized by an ECL-PLUS detection system (GE Healthcare) according to the manufacturer's instructions. All Western blot experiments described in Results were performed at least two times, and representative images are shown in the figures. Band intensities were measured using ImageJ software (National Institutes of Health). Data were normalized to the number of oocytes (equal number of oocytes per line in each sample). The control was considered 100%.

Immunocytochemistry

For immunofluorescence microscopy, denuded and ZP-free oocytes (dezoned in 0.25% pronase) were washed in PBS, fixed for 60 min at 4°C in 4% paraformaldehyde in PBS, and again washed in PBS. Oocytes were permeabilized with 1% Triton X-100 for 20 min, washed and blocked for 2 h in 2% BSA in PBS, and incubated with primary antibody overnight at 4°C. All primary and secondary antibodies were diluted in 0.2% BSA in PBS. Anti-UCHL1 antibody (5937; Chemicon) was used at 1:500 dilution; antiubiquitin antibodies were used as already described. Oocytes were incubated with appropriate secondary antibodies (Alexa Fluor 488 and 543; Invitrogen) for 60 min at room temperature. As a control, oocytes were incubated with secondary antibody only. Lens culinaris agglutinin (LCA rhodamine, 1042; Vector Laboratories) was used as a marker of CGs [37]. F-actin was stained with 1 mg/ml of Phalloidin-Alexa Fluor 568 (12380; Molecular Probes). Oocytes and zygotes were mounted in mounting medium with DAPI (Vectashield; Vector Laboratories). To prevent deformities, 100-μm coverslip spacers (Zweckform) were used. Samples were examined under a Leica SP2 inverted confocal microscope equipped with an Acousto-Optical Beam Splitter (Leica Microsystems). Fluorescence intensities were measured using ImageJ software.

Statistical Analysis

Data were analyzed using SigmaStat 3.0 software (Jandel Scientific). z-Test was used for analysis of differences in proportions of characteristics of interest within groups. Student t-test or Mann-Whitney rank sum test was used for analysis of densitometric measurements. P < 0.05 was considered significant.

Protein Levels of UCHL1 Are Stable During Oocyte Maturation

An immunoblotting approach was used to detect expression of UCHL1 in bovine oocytes; the specific band of approximately 27 kDa (monomer form) was recognized and did not significantly change during oocyte maturation. We used two UCHL1-specific inhibitors, C16 and C30, to disrupt UCHL1 activity [30, 31]. Oocytes were treated with both inhibitors for 24 h at a concentration of 20 μM. We observed no difference in UCHL1 (27-kDa form) expression levels between GV- and MII-stage oocytes or any differences between control oocytes and oocytes treated with specific UCHL1 inhibitors (P > 0.05) (Fig. 1). Meiotic progression was not impaired in the presence of UCHL1 inhibitors (data not shown).

FIG. 1.

The level of the monomeric form of UCHL1 does not change during oocyte maturation. Bovine oocytes were analyzed by immunoblotting (50 oocytes per lane). Protein lysates were prepared from GV-stage and MII-stage oocytes. Individual groups of MII oocytes were treated with UCHL1 inhibitor C16 or C30 or DMSO (vehicle solvent for inhibitors). A representative image from three independent experiments is shown.

In summary, we observed stable levels of the monomeric form of UCHL1 during oocyte maturation. Inhibition of hydrolytic activity of UCHL1 did not block meiotic progression of bovine oocytes.

Subcellular Localization of UCHL1 in Bovine Oocytes

To study localization of UCHL1 in bovine oocytes, we used two different approaches. We microinjected oocytes with in vitro-synthesized UCHL1 mRNA fused with EGFP (EGFP-UCHL1). This UCHL1 mRNA was based on the porcine UCHL1 sequence, which is highly homologous to the bovine sequence (98% amino acid sequence homology [see Materials and Methods]). Two different concentrations (125 or 250 ng/μl) of in vitro-synthesized mRNA were used. At 1 h after microinjection of 125 ng/μl of EGFP-UCHL1 mRNA into GV-stage oocytes, the majority of EGFP signal was predominantly detected in cortical and subcortical regions (Fig. 2A). Low-level signal was visible throughout the cytoplasm and GV nucleoplasm (Fig. 2A). However, at the MII stage, EGFP-UCHL1 localized exclusively to the subcortical region (Fig. 2B). In contrast, oocytes injected with 250 ng/μl of EGFP-UCHL1 mRNA were unable to progress beyond the MI stage of meiosis. EGFP-UCHL1 fluorescent signal in such mRNA-injected oocytes was detected in the oocyte cortex, subcortically, and in the perivitelline space (PVS). The differential interference contrast (DIC) images showed colocalization of this PVS signal with small droplets or vesicles (Fig. 2, E and F, arrows).

FIG. 2.

Localization of GFP-tagged UCHL1 in bovine oocytes. A–D) Representative confocal images of bovine oocytes microinjected with 125 ng/μl of UCHL1 mRNA. A) At 1 h after microinjection of mRNA, EGFP-UCHL1 protein is localized predominantly in the subcortical region, with low signal in the cytoplasm and some accumulation in the oocyte nucleus and GV (10 of 11 oocytes examined had the described phenotype). B) At 24 h after microinjection, oocytes reached MII, and EGFP-UCHL1 fluorescence was predominantly visible in the oocyte cortex (14 of 14 examined). C) As a negative control for EGFP-UCHL1, EGFP mRNA (125 ng/μl) was used, resulting in even distribution of EGFP throughout the ooplasm and complete exclusion from the nucleus at 1 h after microinjection (23 of 23 examined). D) Microinjected control oocytes with EGFP mRNA (125 ng/μl) at 24 h after microinjection (18 of 18 examined). E) Oocytes microinjected with 250 ng/μl of EGFP-UCHL1 mRNA and arrested in MI; arrows indicate EGFP-UCHL1 fluorescence in the PVS. This fluorescence colocalizes with PVS membrane vesicles in the DIC image (10 of 10 examined). F) Detailed image of E. Green indicates EGFP-UCHL1 or EGFP; blue, DAPI used for DNA staining. Original magnification ×300 (A–E) and ×350 (F).

The specificity of localization of EGFP-UCHL1 was confirmed in control experiments using EGFP mRNA alone (125 or 250 ng/μl). EGFP signal was visible throughout the cytoplasm and was absent from the nucleus (Fig. 2, C and D).

These results were further confirmed by immunocytochemistry using specific anti-UCHL1 antibody (Fig. 3, A and B). Immunolocalization of UCHL1 agreed with EGFP-UCHL1 localization in microinjection experiments. The antibody labeled cortical and subcortical regions in GV-stage and MII-stage oocytes; in GV oocytes, fluorescent signal was also detected in the nucleus (Fig. 3A). Localization of native UCHL1 was unaffected in oocytes treated with C16 and C30 inhibitors (data not shown).

FIG. 3.

Localization of native UCHL1. Polyclonal antibody against UCHL1 detects subcortical and nuclear accumulation of endogenous UCHL1 in a GV-stage oocyte (A) (22 of 22 examined) and an MII-stage oocyte (B) (19 of 19 examined). Green indicates UCHL1; blue, DAPI for DNA staining. Original magnification ×300.

Inhibition of UCHL1 Causes Polyspermy During Bovine IVF

UCHL1 protein has been implicated in antipolyspermy defense in vitro in both mouse [27] and pig [28]. To explore the mechanistic basis for these observations, we studied the effects of downregulation of oocyte UCHL1 on bovine IVF. To investigate the role of UCHL1 during meiotic maturation of bovine oocytes, we treated oocytes with UCHL1-specific membrane-permeant inhibitors C16 and C30.

We did not use the RNA interference method, as UCHL1 expression is stable during oocyte maturation and its turnover is low (Supplemental Fig. S2). Because these inhibitors are highly specific [30, 31], we were able to use them at a relatively low concentration of 20 μM. Higher concentration of UCHL1-specific inhibitors (100 μM) did not block meiotic progression in bovine oocytes; however, 30% of MII-stage oocytes extruded all their chromosomes in the form of two PBs (Supplemental Fig. S3).

We then examined fertilization rates and occurrence of polyspermy after IVF in oocytes treated for the whole oocyte maturation period of 24 h with specific UCHL1 inhibitors (C16 and C30) and in control oocytes matured without inhibitors (with DMSO as a vehicle control). In the majority of zygotes that originated from oocytes treated with UCHL1 inhibitors, more than two pronuclei were found at 20 h after IVF (60% in the C16-treated group and 66% in the C30-treated group) (Fig. 4 and Table 1). When oocytes were stained for chromatin or DNA, the predominant pattern was the presence of three pronuclei in inhibitor-treated oocytes, suggestive of dispermic fertilization. In contrast, only 10% of dispermic or polyspermic zygotes were detected in vehicle control (DMSO [P < 0.001]), while the overall fertilization rate was not significantly different between treatments. These results suggest that, although inhibition of UCHL1 through meiosis does not impair nuclear maturation, the efficiency of antipolyspermy defense is reduced; thus, UCHL1 activity during in vitro maturation of bovine oocytes affects the rate of sperm penetration or incorporation during fertilization of MII oocytes.

FIG. 4.

Incidence of polyspermy in UCHL1 inhibitor-treated oocytes. Formation of supernumerary male pronuclei occurs in zygotes obtained by IVF of oocytes matured in the presence of UCHL1 inhibitor C16 or C30. Values from three independent replicates are shown. Different lowercase letters indicate statistical significance (P < 0.001).

TABLE 1.

Polyspermy incidence in one-cell bovine zygotes 20 h post IVF.*

Because developmental competence of embryos is impaired in polyspermy [38], we examined developmental competence of UCHL1 inhibitor-treated oocytes. We performed parthenogenic activation of oocytes treated with C30 (n = 59) and DMSO (n = 44). No significant difference was found in developmental competence of parthenotes produced from oocytes cultivated in 20 μM C30 compared with the control group (P > 0.05 for each developmental stage examined) (Fig. 5).

FIG. 5.

Developmental competence of embryos after parthenogenetic activation. The numbers of embryos reaching individual developmental stages are shown on the y-axis. The number of 2-cell-stage (2c) embryos is considered 100%. The proportion of oocytes that do not develop beyond the 1-cell stage was not significantly different in C30-treated (38%) vs. control (34%) oocytes. Developmental competence was examined in two independent experiments; bars represent the mean ± SEM.

Failure of CG Translocation During Meiotic Maturation in UCHL1-Inhibited Oocytes

The foregoing results suggested that inhibition of UCHL1 aggravates polyspermy in vitro. We next sought to determine whether downregulation of UCHL1 function inhibits the landmark mechanism necessary for blocking polyspermy (i.e., CG maturation and CG extrusion or exocytosis). We focused on distribution changes of CGs in MII oocytes. In GV-stage oocytes, CGs form clusters; as oocytes progress through meiosis, CG clusters dissociate, and CGs invade the cortical region, forming a thin layer [37, 39]. We stained CGs with rhodamine-labeled LCA; for classification of CG distribution, we adopted the previously established terms of oocyte-like pattern (typical of GV stage), intermediate pattern, and egg-like pattern (typical of MII stage) [37].

Migration of CGs was indeed altered in oocytes treated with UCHL1 inhibitors C16 and C30 (Fig. 6, C and D). A majority of MII oocytes or eggs treated with inhibitors during in vitro maturation displayed an oocyte-like CG localization pattern similar to that of GV-stage oocytes (Fig. 6A). Karyokinesis or meiotic progression was not altered (data not shown). In control groups treated with DMSO, CGs relocalized uniformly to the cortical area of the oocyte, displaying an egg-like pattern at MII (Fig. 6B). Data on CG translocation are summarized in Figure 7 and Supplemental Table S1.

FIG. 6.

Downregulation of UCHL1 affects migration or cortical translocation of CGs during oocyte maturation. A–D) Representative confocal images show GV-stage oocytes fixed immediately after isolation (A). B) Normal cortical migration of CGs (egg like) in an MII oocyte treated with DMSO. C and D) The CG distribution pattern in oocytes cultured in the presence of UCHL1 inhibitor C16 or C30 is similar to that of GV-stage (oocyte like) oocytes. E and F) Egg-like CG status in oocytes microinjected with 125 ng/μl of EGFP-UCHL1 mRNA (E) and oocyte-like CG distribution following treatment with 250 ng/μl of EGFP-UCHL1 mRNA concentration (F). Images are representative of three independent replicates. A summary of CG distribution for all groups is given in Supplemental Table S1. Original magnification ×175.

FIG. 7.

Proportion of oocytes with the oocyte-like pattern of CG distribution in control and UCHL1 inhibitor-treated groups. Bars represent the mean ± SEM. Values from three independent experiments are given. Different lowercase letters indi cate statistical significance (P < 0.001).

In GV-stage oocytes injected with 125 ng/μl of EGFP-UCHL1 mRNA, redistribution of CGs was not impaired (Fig. 6E), At the 250 ng/μl concentration, EGFP-UCHL1 mRNA blocked redistribution of CGs during oocyte maturation (Fig. 6F).

Inspired by these observations, we investigated if high polyspermy observed in inhibitor-treated zygotes coincided with impaired GC extrusion. Oocytes were matured in the presence of 20 μM C16 or C30, washed thoroughly, and fertilized using a standard IVF protocol without inhibitors. We observed high retention of CGs in inhibitor-matured zygotes (Fig. 8, B and C) but not in control zygotes at 20 h after fertilization (Fig. 8A). Data on CG extrusion are summarized in Figure 9 and Supplemental Table S2. Figure 10 shows the positive correlation between polyspermy ratio and CG extrusion at 20 h after IVF. Migration of CGs is a cytoskeleton-dependent process in which F-actin plays the major role [40, 41]. When we stained F-actin with Phalloidin-Alexa Fluor 568, we found greater abundance of transzonal projections (TZPs) in oocytes treated during in vitro maturation with C16 and C30 (Supplemental Fig. S4). The retraction or depolymerization of F-actin-rich TZPs is a hallmark of oocyte maturation, and their retention beyond the GV breakdown stage is indicative of cytoskeletal dysfunction.

FIG. 8.

Exocytosis of CGs in zygotes obtained by IVF of oocytes matured with UCHL1 inhibitor C16 or C30. Confocal images of zygotes at 20 h after IVF were acquired with identical settings for all treatments. A) Low fluorescence intensity of the LCA-rhodamine conjugate in a 1-cell zygote raised from oocytes matured in the presence of DMSO vehicle. B and C) Zygotes developed from oocytes matured with UCHL1 inhibitor C16 and C30, respectively. Images are representative of three independent experiments. The CG status in 1-cell zygotes for all groups is summarized in Supplemental Table S2. Original magnification ×230.

FIG. 9.

Absence of CG exocytosis in 1-cell zygotes obtained by IVF of oocytes matured with UCHL1 inhibitor C16 or C30. Oocytes were vehicle treated (DMSO) or exposed to 20 μM C16 or C30 for 24 h. Bars represent the mean ± SEM. Values are from three independent experiments. Different lowercase letters indicate statistical significance (P < 0.001). The CG status in 1-cell zygotes for all groups is summarized in Supplemental Table S2.

FIG. 10.

Coincidence of polyspermy and failed CG extrusion in bovine zygotes treated with inhibitors of UCHL1. Analysis of data was obtained from experiments monitoring the number of oocyte-incorporated spermatozoa (see Fig. 4) and CG exocytosis at 20 h after IVF (see Fig. 8). Coefficient of determination R² = 0.9275.

Treatment of Oocytes with Specific UCHL1 Inhibitors Reduces Monoubiquitin Levels and Increases Protein Ubiquitination

UCHL1 maintains the cytoplasmic pool of unconjugated monoubiquitin by recycling polyubiquitin chains. Consequently, we monitored free monoubiquitin levels and polyubiquitin in oocytes treated with C30 and C16. The average free monoubiquitin level decreased by 50% to 70% compared with controls (Fig. 11B). Conversely, formation of polyubiquitin chains and ubiquitination of proteins were increased 4 to 5 times in MII-stage oocytes treated with inhibitors compared with GV-stage oocytes and DMSO-treated MII oocytes (Fig. 11A). Overexpression of UCHL1 in oocytes injected with 125 ng/μl of EGFP-UCHL1 mRNA at the GV stage resulted in 2- to 4-fold increase of free ubiquitin (monoubiquitin) at 24 h after microinjection (Fig. 11C). In contrast, we detected no change in ubiquitination of proteins after microinjection (data not shown). Results were similar for both types of mRNA injected (EGFP-UCHL1 and nontagged UCHL1) (Supplemental Fig. S1).

FIG. 11.

Monoubiquitin and polyubiquitin levels in oocytes treated with UCHL1 inhibitors and after overexpression of UCHL1. UCHL1 was inhibited using 20 μM C16 or C30 during the in vitro maturation period (24 h). Control oocytes were cultured with inhibitor vehicle or solvent (DMSO) for the same period. A) In the presence of C16 or C30, chemiluminescence signal for ubiquitin is detected at a higher molecular mass in immunoblots; image is representative of two independent replicates. B) The monoubiquitin pool is decreased in C16- or C30-treated MII oocytes compared with control MII- and GV-stage oocytes; image is representative of three independent replicates. C) Microinjection of 125 ng/μl of UCHL1 or EGFP-UCHL1 mRNA increased the monoubiquitin pool in injected oocytes compared with control oocytes microinjected with dextran; image is representative of two indepenent replicates.

Taken together, these results demonstrate that UCHL1 promotes disassembly of polyubiquitin chains during meiotic maturation. Conversely, UCHL1 regulates formation of polyubiquitin chains in bovine oocytes.

Presumptive Oligomeric Forms of UCHL1 Increase in the Presence of UCHL1 Inhibitors

Increased formation of polyubiquitin chains and protein ubiquitination observed in the presence of UCHL1 inhibitors could account for increased ubiquitin ligase activity of dimeric and oligomeric forms of UCHL1 [15, 42]. By immunoblotting analysis, we identified several higher-mass UCHL1 bands at approximately 37 kDa (Supplemental Fig. S5) and in the range of 50–150 kDa (Fig. 12A). Expression of UCHL1 monomer did not change during meiotic maturation of bovine oocytes. Also, the 37-kDa UCHL1 band, presumably the monoubiquinated form of UCHL1 [17], did not change significantly during oocyte maturation (Supplemental Fig. S5). However, the content of presumptive UCHL1 oligomers (75–150 kDa) increased 6-fold in oocytes treated with C16 or C30 inhibitors (Fig. 12A).

FIG. 12.

Formation of presumptive UCHL1 oligomers in the presence of UCHL1 inhibitor C16 or C30. A) UCHL1 oligomers migrate predominantly at the 75-kDa level in the presence of the disulfide bond-reducing agent DTT. B) UCHL1 oligomers migrate at higher molecular mass when DTT is omitted from the oocyte lysis buffer. Images representative of two independent experiments are shown.

Treatment of control and inhibitor-exposed oocyte lysates with 40 mM DTT (disulfide bond-reducing agent) and 4 mM NEM (alkylating reagent) was used to determine whether the presumptive UCHL1 oligomers could be linked by disulfide bonds. In the presence of DTT, both 50- and 75-kDa UCHL1 species were visible (Fig. 12A), likely corresponding to the presumptive dimer and oligomer of UCHL1, respectively. Without DTT, UCHL1 bands were observed at 50, 75, 100, and 150 kDa (Fig. 12B). These masses could correspond to multimerization of 27-kDa UCHL1, forming dimers, tetramers, and hexamers. The lack of presumptive oligomers within the range of 100–150 kDa in the presence of DTT strengthens the hypothesis that presumptive oligomers are cross-linked by disulfide bonds. In contrast, 50- and 75-kDa oligomers were not sensitive to DTT treatment. Finally, data shown in Figure 12 suggest increased formation of presumptive UCHL1 oligomers in the presence of inhibitors C16 and C30 during the in vitro maturation period.

Inhibition of UCHL1 Increases Formation of K63-Linked Multiubiquitin Chains

Polyubiquitin chains are linked through the C-terminal G76 residue of one ubiquitin molecule bound covalently to one of seven internal lysine residues (K6, K11, K27, K29, K33, K48, or K63) of another ubiquitin molecule [43–46]. In most cases, all molecules within an isopeptide polyubiquitin chain are linked at the same internal lysine position. Consequently, polyubiquitin chains can be distinguished based on the internal linkage site (e.g., K48 or K63 chains). In dimeric or oligomeric form, UCHL1 functions as a ubiquitin ligase that promotes formation of K63-linked polyubiquitin chains [15, 47]. To determine if this is the case in bovine oocytes, we performed immunocytochemical analysis of matured oocytes with mouse monoclonal antibody specific to K63-linked polyubiquitin chains. We found increased fluorescent signal in the cytoplasm for K63-linked ubiquitin antibody in oocytes treated with UCHL1 inhibitor C30 (Fig. 13B) compared with control oocytes treated with DMSO (Fig. 13A). Densitometric evaluation of these oocyte groups clearly showed higher fluorescence intensity (6.96 and 6.19, respectively) in C16- and C30-treated oocytes compared with 0.56 in the control group. These data support our hypothesis that inhibition of hydrolase activity of UCHL1 stimulates ligase activity of this enzyme in bovine oocytes, resulting in increased protein stabilization or modification through K63-linked polyubiquitination.

FIG. 13.

Inhibition of UCHL1 stimulates formation of K63-linked polyubiquitin chains. A) Oocytes treated with DMSO only (0 of 10 examined). B) Immunocytochemical analysis shows higher fluorescence intensity of K63-linked ubiquitin chains in oocytes matured for 24 h in the presence of UCHL1 inhibitor (9 of 12 examined). Images were recorded with identical acquisition settings. Original magnification ×300.