A rabbit pAb was generated against peptide 395
of mouse ovastacin attached N terminal to a cysteine residue and conjugated to keyhole limpet hemocyanin (Sigma-Aldrich). mAbs IE-3 and M2c.2 that bind to the N- and C-terminal regions of ZP2, respectively, were previously described (East and Dean, 1984
; Rankin et al., 2003
), and the following antibodies and lectins were obtained commercially: LCA-FITC (Sigma-Aldrich), donkey anti–rabbit conjugated with Alexa Fluor 555 (Invitrogen), goat anti–rabbit DyLight 649 (Thermo Fisher Scientific), donkey anti–rabbit conjugated with HRP (Jackson ImmunoResearch Laboratories, Inc.), and goat anti–rat conjugated with HRP (Jackson ImmunoResearch Laboratories, Inc.).
Egg and embryo collection and culture
4–5-wk-old female mice were injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin followed by 5 IU of human chorionic gonadotropin 48 h later. Ovulated eggs and embryos were collected before and after mating, respectively, in M2 medium (Millipore) containing protease inhibitors (Roche). Embryos were subsequently cultured in potassium simplex optimized medium (Millipore) at 37°C in 5% CO2. All experiments were conducted in compliance with the guidelines of the Animal Care and Use Committee of the National Institutes of Health under the Division of Intramural Research, National Institute of Diabetes and Digestive and Kidney Diseases approved animal study protocols.
Immunofluorescence and confocal microscopy
Oocytes and embryos were fixed in 2% PFA for 30 min at 37°C, washed in PBS containing 0.3% polyvinylpyrrolidone (PVP), and then blocked in 0.3% BSA/0.1 M glycine (three times for 10 min) followed by permeabilization in 0.2% Triton X-100 for 15 min (Baibakov et al., 2007
). Oocytes and embryos were then incubated (1 h) with primary antibody (1:50), washed with 0.3% PVP/0.1% Tween (three times for 10 min), and incubated (45 min) with secondary antibody (1:100) followed by staining with Hoechst 33342 (Invitrogen) before imaging. Alternatively, eggs and embryos were stained with LCA-FITC (1:100). Samples were mounted in PBS, and confocal laser-scanning images were obtained on a confocal microscope (LSM 510; Carl Zeiss) with a 63× 1/2 W objective using the manufacture’s software. LSM images were exported as full-resolution TIF files and processed in Photoshop (Adobe) to adjust brightness and contrast.
Eggs and two-cell embryos were lysed in 2 or 4× Tris-glycine SDS loading buffer with DTT, separated on 4–20% Tris-glycine gels by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Invitrogen), blocked in 3 or 5% nonfat milk in PBS, and probed with primary antibodies followed by secondary antibodies conjugated to HRP (Gahlay et al., 2010
). Chemiluminescence was performed with ECL Plus (GE Healthcare), and signals were acquired by the Luminescent Image Analyzer LAS-3000 (Fujifilm) or with BioMax XAR film (Kodak).
For detection of ovastacin, blots were incubated with a 1:1,000 dilution of peptide-purified, rabbit anti–mouse ovastacin antibody (1.7 mg/ml) in 5% nonfat milk in TBS with 0.1% Tween 20 (TBST) at 4°C overnight. On the following day, blots were incubated with a 1:10,000 dilution of goat anti–rabbit HRP in TBST for 1 h at room temperature. For staining with LCA, blots were incubated with 10 µg/ml of the biotinylated LCA (US Biological) in 5% nonfat milk in TBST at 4°C overnight. Blots were then incubated with a 1:10,000 dilution of HRP-streptavidin (Thermo Fisher Scientific) in TBST and incubated for 1 h at room temperature.
Establishment of the Astl-null mouse line
is a single-copy gene that encodes ovastacin. Mouse lines lacking ovastacin protein were established using DNA recombineering (Liu et al., 2003b
) and targeted ablation in ES cells (Zheng and Dean, 2009
). The targeting construct contained positive (neomycin resistance) and negative (herpes simplex virus thymidine kinase) selectable markers and replaced exons 2 and 3 of Astl
and deleted the transcriptional and translational start sites. Correctly targeted ES cells were identified by Southern hybridization of SspI-digested genomic DNA using 32
P-labeled probes 5′ (−6,766 to −6,234 bp of the transcriptional start site) and 3′ (2,688–3,056 bp) to the targeting vector. Heterozygous null ES cells were injected into mouse blastocysts to establish chimeric founder lines. Germline transmission of the null allele and subsequent genotyping were determined by allele-specific PCR products of tail DNA. Primers P1 (5′-AGGCCTTGTCACCAGGTATG-3′) and P2 (5′-CCAGAGAATGAAGGGAGCAG-3′) were used to detect the normal allele (797 bp), and primers P2 and P3 (forward 5′-GGGAGGATTGGGAAGACAAT-3′) were used to detect the null allele (399 bp) in PCR genotyping of tail DNA. The PCR condition consisted of one cycle at 94°C for 5 min; 30 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min and 30 s; and a full extension cycle at 72°C for 10 min.
AstlHet and AstlNull females were cocaged with FVB males of proven fertility to determine the number and size of litters for a period of 8–10 mo.
Oocytes and embryos from AstlHet
females were fixed in 1.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and incubated at 4°C for 2 h. After extensive washing in the cacodylate buffer, the oocytes and embryos were embedded in 2% agarose. The samples were then dehydrated through a graded series of ethanol and processed for embedding in London Resin white. Ultrathin sections were obtained with an ultramicrotome (MICROM International GmbH) and mounted on Formvar-coated nickel grids. For lectin cytochemistry (Jiménez-Movilla et al., 2004
), grids were preincubated (for 10 min at room temperature) in PBS (1% BSA) and transferred to a drop of WGA-HRP lectin (Sigma-Aldrich) in PBS for 1 h. After rinsing in PBS, grids were floated on a drop of rabbit anti-HRP pAb (Sigma-Aldrich) diluted 1:500 in PBS for 1 h. Grids were then washed in PBS and floated on a drop of Protein A gold (15 nm)–conjugated antibody (1:60; Utrecht University) for 1 h. After washing in twice-distilled water, ultrathin sections were counterstained with uranyl acetate followed by lead citrate and imaged in a transmission electron microscope (Philips Tecnai 12; FEI). WGA-positive cortical granules were observed in five nonserial ultrathin sections (total; mean ± SEM/section) from three AstlNull
oocytes (180; 12.0 ± 0.7). AstlHet
(337; 22.5 ± 0.7) oocytes and two-cell embryos (no cortical granules) served as positive and negative controls, respectively.
Ovaries from 8-wk-old FVB mice were homogenized in cold lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol) with one tablet of protease inhibitor cocktail (Roche) added to every 10 ml of buffer. The sample was centrifuged (13,200 rpm at 4°C for 20 min), and supernatants were collected. 10 µg rabbit anti–mouse ovastacin antibody was added to the supernatant in a final volume of 800 µl and rotated overnight at 4°C. 30 µl protein G Sepharose beads (GE Healthcare) was equilibrated with lysis buffer and added to the protein–antibody mixture. This was rotated for an additional 2 h at 4°C. After a brief centrifugation, the supernatant was removed, and the beads were washed three times with 50 mM Tris, pH 8.0, 150 mM NaCl, and 1% NP-40 buffer. Two 30-µl aliquots of elution buffer (10 mM Tris, pH 8.0, 1 mM EDTA, and 1% SDS) were added to the beads for 10 min at 30°C. Eluates were separated on 12% SDS-PAGE gels before immunoblotting.
Sperm binding assay
Caudal epididymal sperm were isolated from wild-type FVB mice and placed under oil (Irvine Scientific) in human tubal fluid medium (Millipore) previously equilibrated with 90% N2
, 5% O2
, and 5% CO2
and capacitated by an additional 30–60 min of incubation at 37°C (Baibakov et al., 2007
). Sperm binding to 10–20 ovulated eggs and two-cell embryos isolated from normal and AstlNull
mice was quantified using capacitated sperm and normal two-cell embryos as wash controls. Samples were fixed in 2% PFA for 30 min, stained with Hoechst 33342, and imaged by confocal microscopy.
pDonr253 is a Gateway Donor vector modified from pDonr201 (Life Technologies). pDonr253 replaces the kanamycin resistance gene with a gene encoding spectinomycin resistance and contains several sequencing primer sites to aid in sequence verification of Entry clones. The following oligonucleotides (Eurofins MWG Operon) were used: 4771, 5′-GGGGACAACTTTGTACAAGAAAGTTGATTAGCCTTTTTCGAATTGCGGATGGCTCC-3′; 6088, 5′-GGGGACAACTTTGTACAAAAAAGTTGGCACCATGAAATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTC-3′; 11154, 5′-CGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCGGAGCGCCCCTGGCCTCCAGCTGCG-3′; 11155, 5′-CGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATGCGGCCGGAGCACCCTCAGCATCCAGATGTTC-3′; 11156, 5′-GCCTTTTTCGAATTGCGGATGGCTCCAGGATCCATCTTCGGACATCCCCTTGAAATGATT-3′; and 11157, 5′-GCCTTTTTCGAATTGCGGATGGCTCCAGGATCCGTCTCTGGGCACCTCTCTAATGTGAC-3′.
Mouse ovastacin was cloned using PCR from cDNA templates for baculovirus expression. A honeybee melittin signal peptide leader sequence was added to the 5′ end of each construct to enhance secretion of the proteins in insect cells, and a C-terminal noncleavable Strep2 tag (GSWSHPQFEKG) was added for purification purposes. Initial PCR was performed using Phusion DNA polymerase (New England Biolabs, Inc.) under standard conditions using a 40-s extension time and 200 nM of flanking primers. After five cycles of amplification, 200 nM of each adapter primer was added, and amplification was continued for 20 additional cycles. The final PCR products are flanked by Gateway recombination signal sequences attB1 at the 5′ end and attB2 at the 3′ end. The PCR products were cleaned using the QIAquick PCR purification kit (QIAGEN) and recombined into pDonr253 using the Gateway BP recombination reaction (Life Technologies) and the manufacturer’s protocols. BP reactions were transformed into Escherichia coli DH10B cells, and colonies were isolated on lysogeny broth plates containing 50 µg/ml spectinomycin. Plasmid DNA was prepared and sequenced using a variety of internal and external sequencing primers to verify the sequence.
The sequence-verified Entry clones were subcloned by Gateway LR recombination (Life Technologies) into pDest-8 for insect cell expression. Final expression clones were verified by size and restriction digest pattern. The expression clones were then transformed into E. coli DH10Bac (Life Technologies) and plated on selective media containing gentamycin, kanamycin, tetracycline, IPTG, and X-gal as per the manufacturer’s protocols. White colonies were selected from these plates, and bacmid DNA was generated by alkaline lysis plasmid preparation and verified by PCR amplification across the bacmid junctions.
The bacmid DNAs were complexed with XpressNOW transfection reagent (Lonza) and transfected into 100 ml of Sf-9 insect cells at 1.5 × 106 ml−1 in SFX-Insect medium (HyClone; Thermo Fisher Scientific). At 5 d after transfection, the cultures were centrifuged at 1,100 g, and the virus-containing supernatant was collected. For expression, one liter of High Five cells was set in SFX medium in a three-liter Erlenmeyer (Corning) at a cell concentration of ~1.5 × 106 ml−1 and infected (multiplicity of infection of three) with 40 ml of the recombinant baculovirus. The culture was grown in a shaker incubator at 21°C for 3 d, and the supernatant was collected after centrifugation at 1,100 g.
The conductivity of the supernatant was adjusted to 12.61 mS/cm with 20 mM Hepes, pH 7.3. The supernatant (41 ml) was applied to a 5-mL Q Sepharose column (GE Healthcare) equilibrated with 20 mM Hepes, pH 7.3, and 75 mM NaCl. A flow-through sample was collected, and then a 5-ml S Sepharose column (previously equilibrated in 20 mM Hepes, pH 7.3, and 75 mM NaCl) was added for subsequent purification on both columns. After completion of the load, the columns were washed to baseline in 20 mM Hepes, pH 7.3, and 75 mM NaCl. A 10-column volume elution from 75 mM to 1 M NaCl was completed by collecting 2.5-ml fractions across the gradient. Analysis was performed via SDS-PAGE/Coomassie staining and immunoblotting. The rabbit anti-ovastacin antibody was incubated overnight at 4°C, washed at room temperature with 1× TBS with Tween 20, incubated 1 h in 1:5,000 anti–rabbit secondary antibody, and washed five times in 1× TBS with Tween 20. SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) was used to develop the signal. The fractions with the highest concentration of ovastacin were used in subsequent assays.
In vitro cleavage assay
Zonae pellucidae were isolated from 150 oocytes by freeze thawing four times in 100 µl PBS, pH 7.4, 0.1% IGEPAL CA-630 (Sigma-Aldrich), and 0.5 M NaCl. Isolated zonae were solubilized in 30 µl PBS, 0.4% PVP, and 0.1% SDS by heating at 60°C for 30 min. Solubilized zona samples were incubated with recombinant ovastacin at 37°C over time (0–8 h), and cleavage was analyzed by immunoblotting with mAb M2c.2.