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Immunocontraception Is Induced in BALB/c Mice Inoculated With Murine Cytomegalovirus Expressing Mouse Zona Pellucida 3¹

Microbiology, School of Biomedical and Chemical Sciences,³ University of Western Australia, QEII Medical Centre, Nedlands, Western Australia, 6009 Australia Pathology, School of Surgery and Pathology,⁴ University of Western Australia, Nedlands, Western Australia, 6009 Australia

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Immunocontraception, the prevention of oocyte fertilization through immunological means, could potentially be used to control plaguing mouse populations in Australia. This paper describes the construction of a mouse-specific betaherpesvirus, murine cytomegalovirus, which has been engineered to express the murine zona pellucida 3 (ZP3) gene. A single inoculation of this recombinant virus resulted in almost complete infertility, persistent anti-ZP3 antibody production, and profound changes to ovarian morphology in BALB/c mice in the absence of significant virus replication during the acute phase of infection. Murine cytomegalovirus may prove to be useful as a vector for the delivery of a mouse-specific immunocontraceptive agent to target populations of wild mice in the field.

female reproductive tract, fertilization, follicle, immunology, ovary

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Immunocontraception is the prevention or interruption of oocyte fertilization through immunological means [1]. Successful immunocontraception induced by the inoculation of whole porcine zona pellucida (ZP) in adjuvant has been reported in feral horses [2, 3] and has been proposed as a useful tool in controlling other large animal species such as elks and elephants [3, 4]. Inoculation of whole murine but not whole porcine ZP has produced infertility in mice [5].

Murine ZP is made up of three sulphated glycoproteins, designated ZP1, ZP2, and ZP3, and forms a matrix that surrounds the oocyte of the growing follicle and ovulated egg. The ZP matrix induces the acrosome reaction in acrosome-intact spermatozoa that have penetrated the cumulus oophorus mass surrounding the ovulated egg, and modification of the ZP after fusion protects against polyspermy [6]. ZP3 is the primary sperm receptor and as such is a potential target for immunocontraception [6]. ZP3 peptides have been used to induce infertility in certain mouse strains, however, the strain-specific nature of the response reduces the usefulness of ZP3 peptides when an outbred mouse population is targeted [7, 8]. Mice in which the mZP3 gene has been disrupted by targeted mutagenesis have smaller ovaries than do mice with a functional mZP3 gene and they exhibit normal follicular development and oocyte growth in the absence of the ZP matrix, but they are completely infertile [9].

Although direct inoculation of an immunocontraceptive agent may be appropriate for the management and control of larger mammals, a freely disseminating virally vectored immunocontraceptive agent may be a more practical approach for the control of an erupting murine population that has spread over a large area.

Murine cytomegalovirus (MCMV) has been proposed as a candidate virus for virally vectored immunocontraception in Australia, predominantly for its species-specificity and its capacity to infect mice with more than one strain of virus simultaneously [10, 11]. This coinfection of different strains of MCMV in one host infers that a recombinant MCMV expressing a fertility antigen should be able to infect a population that is already naturally exposed to the virus. MCMV has been found present in every population of wild mice tested in Australia to date [12–14]. The persistence of the virus in an isolated and fluctuating Mus domesticus population [12] suggests that a fertility antigen expressed by a recombinant MCMV has the potential to be expressed over a long interval. MCMV establishes in mice a persistent or latent acute infection of major organs such as the spleen, liver, and lung, before establishing itself in the salivary gland, where it can persist for long periods of time [15]. Furthermore, MCMV has been shown to be amenable to engineering with the establishment of recombinant viruses that are stable and replication competent both in vitro and in vivo [16]. The MCMV ie2 gene has been reported to be dispensable for in vivo growth and has been shown to be a useful site for cloning in foreign genes [17].

In this study we explored the effect of a recombinant MCMV that expresses murine ZP3 at the ie2 region of the virus. This virus can elicit a long-term immunocontraceptive effect in BALB/c mice and induces profound pathological changes to the ovarian structure even though in vivo replication appears to be minimal.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals

Specific-pathogen-free BALB/c (ARC) mice were obtained from the Animal Resources Centre (Murdoch, Perth, Western Australia) and maintained under minimal disease conditions. Mouse care was based on the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes endorsed by the National Health and Medical Research Council, and was approved by the University of Western Australia's Animal Experimentation Ethics Committee. Sentinel mice are regularly screened by an independent institution for antibodies to a suite of murine pathogens, including MCMV.

Cells

Primary mouse embryo fibroblast (MEF) cell cultures were prepared by the trypsin dispersion of minced 15-day-old embryos from BALB/c mice as previously described [18]. After thawing, cells were grown in modified Eagle medium (MEM) + 10% newborn calf serum (NCS) and maintained in MEM + 2% NCS. M210B4 cells were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in RPMI 1640 medium.

Viruses

K181 (Smith) is the laboratory strain of MCMV, and its origins have been described elsewhere [11]. The parental MCMV used for transfection was the K181 (Smith) strain containing the lacZ gene under the control of the human cytomegalovirus IE1 promoter in the nonessential ie2 region of the MCMV genome, designated RM427+, and was obtained from Prof. E. Mocarski (Stanford University, Stanford, CA). RM427+ differs from the previously described RM427 in that it contains an intact sgg1 gene [19]. All viruses used were derived from tissue culture.

Plasmids

Plasmid DNA preparation and cloning were performed by standard cloning methods [20]. All restriction enzymes were obtained from Promega (Madison, WI) and used according to the manufacturer's instructions. The mouse ZP3 gene was engineered to contain flanking HindIII and EcoRI restriction sites and was provided by Dr. Ron Jackson of CSIRO Canberra, Australia [21]. This was cloned into a shuttle plasmid, pMV11, containing both the human cytomegalovirus IE1 promoter and a polyadenylation stop sequence. This expression cassette was excised with HindIII and blunt-ended by a fill-in reaction using Klenow DNA polymerase (Promega). This was cloned into the double HpaI site of the HindIII-L fragment of K181 (designated pK181-H3L), which had been dephosphorylated using calf intestinal alkaline phosphatase. We received pK181-H3L from Dr. A. Scalzo (University of Western Australia, Perth, Australia). By cloning into the HpaI site, the pK181-H3L plasmid has no TATA box, and the transcriptional start site of the ie2 gene is also removed. The resultant plasmid, designated pK181-H3L/MV11/ZP3, was linearized with HindIII and ethanol precipitated prior to cotransfection with RM427+ viral DNA. Prof. E. Mocarski (Stanford University, Stanford, CA) provided the plasmid pON427+, which contains the lacZ gene under the control of the human cytomegalovirus promoter for the manufacture of a revertant virus.

Generation of Recombinant Virus

MEF cell cultures infected with RM427+ and grown to 100% cytopathic effect were harvested to prepare viral DNA. Cells and media were centrifuged at 480 x g for 10 min, the resultant pellet was washed in Mouse Osmolarity Buffered Saline (MOBS), and the cells were lysed in 10% SDS and incubated with Proteinase K for 1 h at 37°C. DNA was precipitated in 5 M NaCl and ethanol, spooled onto sterile glass loops, and resuspended in nuclease-free water.

Transfection Procedure

MEF cell cultures were grown to 70% confluency. DNA from RM427+ 10–50µg and 2µg of linearized pK181-H3L/MV11/ZP3 DNA were coprecipitated onto the cells by calcium phosphate coprecipitation using the Cellphect procedure as detailed in the manufacturer's instructions (Amersham Pharmacia Biotechnology UK Ltd., Buckinghamshire, U.K.). At the completion of the incubation period the cells were osmotically shocked with 1% glycerol in MOBS for 1 min. Cell monolayers were allowed to grow until viral plaques formed. Primary viral plaques were individually assessed in triplicate on MEF cells and stained with either a viable or nonviable X-gal stain. Nonstaining plaques were chosen and purified three times before expanding them into a working stock. The resultant virus was designated rK181-ZP3. Similar methods were employed to produce a revertant virus using rK181-ZP3 viral DNA and the plasmid pON427+. This resulted in a revertant virus exhibiting a blue-staining phenotype when stained with 5-bromo-4-chloro-3-inodyl ß-D-galactopyranoside, which was designated rK181-ZP3-REV.

Polymerase Chain Reaction (PCR) and Reverse Transcriptase-PCR

Polymerase chain reaction was used to screen monolayers containing rK181-ZP3 plaques to confirm the phenotype of the white virus. Cell monolayers were dislodged, digested with proteinase K, phenol extracted, and the DNA was precipitated by ethanol precipitation. Internal ZP3 primers 5'-accctcgccctgtgagtggcc-3' and 5'-aattactacagttgccatggc-3' resulted in a fragment of the predicted size (662 base pairs; bp) and MCMV ie1 primers 5'-CATTAAAAACTATTGGTTCTA-3' and 5'-CCCATAGCCGAGCCCAATGCA-3' resulted in a fragment of the predicted size (641 bp). RNA was made using the Ultraspec RNA isolation system (Biotecx Laboratories, Inc., East Houston, TX) and complementary DNA was produced with Promega random primers. The ie2 primers 5'-TCTTGTTTCCATAGAGTCATCGCT-3' and 5'-GTCGAAGAGGCGGTGCAGCTCTAT-3' that result in a 527-bp fragment when the gene is correctly spliced were used to confirm the disruption of the ie2 gene. PCR was carried out using a Perkin Elmer Gene Amp PCR reagent kit (Roche, Nutley, NJ) and PCR was performed in a Perkin Elmer Gene Amp PCR System 2400.

Restriction Fragment-Length Polymorphism and Southern Blot

Viral DNA was purified as previously described and restriction fragment-length polymorphism (RFLP) was performed using the restriction enzyme HindIII. Fragments were resolved on a 0.9% agarose gel and compared with the parental viral RFLP profile. A Southern blot was carried out using a gel-purified ZP3 fragment that was labeled using the Boehringer-Mannheim digoxigenin (DIG) DNA labeling and detection kit (Boehringer-Mannheim, GmbH, Mannheim, Germany).

In Vitro Growth Curve

Recombinant and parental viruses were infected onto bone marrow stromal cell, M210B4, monolayers in 6-well trays at a multiplicity of infection of three. Cells and media were collected in triplicate at various intervals over a 44-h period. Samples were sonicated and plaque assayed in duplicate on the M210B4 cells.

In Vivo Characterization of rK181-ZP3

Groups of weanling (21 days old) and adult (8 wk old) mice were inoculated i.p. with 2 x 10⁴ plaque forming units (pfu) of either rK181-ZP3 or with the parental virus, RM427+, and the spleen, liver, salivary glands, and lungs were plaque assayed for the presence of infectious virus. Three mice were examined for each virus from each time point. DNA was extracted from salivary gland homogenates and was tested by PCR using the MCMV ie1 primers described previously.

Plaque Assay

Mouse organs were homogenized at 10% w/v in MEM + 2% NCS. Organ suspensions were clarified at 1900 x g for 30 min and dilutions of supernatant were plated onto confluent MEF cells in 24-well trays. After 1 h of incubation in 5% CO₂ the suspension was removed and a 0.5% methylcellulose/MEM + 2% NCS layer was applied. The plates were incubated for 4 days at 37°C in 5% CO₂, and stained with 1 ml of 0.05% methylene blue with 10% formaldehyde overnight. Plates were washed, allowed to dry, and the number of plaques was enumerated. The limit of detection of this assay for organ homogenates was 25 pfu per organ.

Hyperimmune Serum

All hyperimmune serum (HIS) was generated by three 100-µl i.p. inoculations at 2-wk intervals of either 2 x 10⁴ pfu of virus or 15 µg of ZP3 protein in TDM adjuvant (Sigma Chemical Company, St. Louis, MO). Serum was collected 2 wk after the final inoculation.

Immunofluorescence Microscopy

For direct immunofluorescence, frozen sections of ovaries from mice inoculated with rK181-ZP3 or the control virus rK181-ZP3-REV 35 days after inoculation were examined. The sections were fixed in methanol at -20°C for 5 min, washed in Tris-buffered saline (TBS; pH 7.4), and blocked with 10% normal goat serum diluted in TBS for 30 min at room temperature. The sections were then incubated with a Fab fragment of goat anti-mouse immunoglobulin antibody conjugated with fluorescein isothiocyanate (FITC) (Biosource International, Camarillo, CA) at 37°C for 30 min, mounted in 50% glycerol in TBS, and examined by fluorescent microscopy. For indirect immunofluorescence, frozen sections of uninfected mouse ovaries were fixed in methanol at -20°C for 5 min. Sections were blocked as before, washed in TBS, and incubated with a 1:10 dilution of ZP3 hyperimmune serum or control hyperimmune serum at 37°C for 30 min. Sections were washed again and then incubated with the goat anti-mouse immunoglobulin-FITC Fab fragment as previously described, mounted in 50% glycerol, and examined using a fluorescent microscope. The control hyperimmune serum was generated as previously described by three inoculations of a recombinant MCMV engineered in this laboratory to express the protein, ovalbumin.

Ovarian Histology

Ovaries from mice inoculated with rK181-ZP3 and rK181-ZP3-REV were fixed in Bouin fixative for 24 h and then transferred to 70% ethanol. Within 7 days of collection, organs were embedded in paraffin and 5-µm sections were cut and stained with hematoxylin and eosin, and periodic acid Schiff reagent.

MCMV Enzyme-Linked Immunosorbent Assay

To obtain MCMV antigen, the K181 laboratory strain of MCMV was grown on MEF until a 100% cytopathic effect was observed. Medium was collected and ultracentrifuged at 300 000 x g for 2 h at 4°C. The deposit was diluted in MOBS and sonicated twice for 15 sec to disrupt any remaining whole fibroblasts. The deposit was clarified by centrifugation for 5 min at 550 x g, and the resultant antigen was aliquoted and stored at -70°C. ELISA plates were coated with 7.5 µg of crude protein per well in Carb/Bicarb buffer (pH 7.5) and incubated overnight at 4°C. Plates were washed, and serum was diluted from 1:10 in 2-fold dilutions. Normal uninfected mouse serum was used as a negative control, and K181 hyperimmune serum was used as a positive control on all plates. Plates were blanked individually. MOBS with 0.05% v/v Tween-20 and 0.1% w/v BSA was used to wash plates, and MOBS with 0.05% Tween-20 and 1.0% BSA was used as the antibody diluent. Serum was incubated overnight at 4°C. Immunoglobulins G1 and G2a (IgG1, IgG2a) biotin-labeled conjugates were used as secondary antibodies (Southern Biotech. Association Inc., Birmingham, AL) and were incubated at 1:2000 at room temperature for 1 h. Streptavidin alkaline phosphatase (Amersham Pharmacia Biotechnology) was used as the tertiary antibody (room temperature, 1 h) and 5 mg of p-nitrophenyl phosphate substrate tablets (Sigma) dissolved in 5 ml of 10% diethanolamine buffer at pH 9.8 were used to develop the color reaction. ELISA plates were read at 405 nm after 15 min of incubation at room temperature. A positive serum was designated as one with an absorbance greater than the mean of the normal mouse serum values plus three times the standard deviation. This value was calculated for each ELISA group and is designed to eliminate false positive results.

ZP3 ELISA and Avidity Assay

Dr. Ron Jackson (CSIRO) supplied the ZP3 antigen. Briefly, Vero cells were infected with a recombinant myxoma virus expressing murine ZP3 and were grown until complete cell lysis was achieved. Medium was centrifuged at 550 x g for 5 min to remove cellular debris. Triton X-100 was added to 1% v/v, and the medium was passed twice through a wheat germ agglutinin column (Sigma) that had been equilibrated with Tris (pH 8.0). Bound ZP3 was released with a 100 mM N-acetylglucosamine/0.5% sodium deoxycholate/10 mM Tris (pH 8.0) wash. Eluted ZP3 was dialyzed in water, aliquoted, and stored at -20°C. ELISA plates were coated with 7.5 µg of crude protein extract overnight at room temperature. Plates were then blocked with MOBS + 5% skim milk powder at room temperature for 2 h before adding serum. The plates were washed after each step with MOBS + 0.05% Tween-20, and MOBS + 10% Superblock (Pierce, Rockford, IL) was used as the antibody diluent. Serum was incubated overnight at room temperature. The conjugates we used and incubation protocols we followed were the same as those described for the MCMV ELISA. Normal uninfected mouse serum used as a negative control and ZP3 hyperimmune serum was used as a positive control on all plates. Because normal mouse serum had little background activity, a graphical interpretation of results was required to determine the antibody titer. Absorbance values were graphed against serum dilutions and the point at which the linear regression of these values intersected the y-axis determined individual serum titers. The serum dilution immediately lower than the intersect was designated the ZP3 antibody titer. The avidity assay we used was described previously by Ross et al. [22]. In this experiment, ZP3 antigen was applied to ELISA plates (7.5 µg per well) and the avidity of antibody to ZP3 in rK181-ZP3 HIS was compared with that of ZP3 protein + adjuvant HIS. All samples were processed in triplicate at a 1:50 dilution and 0, 0.5, 1, 1.5, 2, and 3 M concentrations of a chaotrophic agent (sodium thiocyanate) were used to determine the relative affinity of the sera to the ZP3 antigen.

Estrous Cycle Determination

Two groups of 12 8-wk-old female mice were inoculated i.p. with either rK181-ZP3 or K181 using the same dose of virus described below. Mice were housed in groups of four in transparent cages next to cages of competent adult male mice. Three weeks after inoculation vaginal smears were taken daily from the female mice for at least five complete cycles using the method described by Allen [23]. Mice were housed in a 12L:12D, 0600–1800 h cycle, and smears were taken at midday (1200 h). The smears were heat fixed and stained with 0.1% methylene blue, and the stage of the cycle was determined by the presence and relative proportion of cornified epithelial cells, nucleated epithelial cells, and leukocytes.

In Vivo Breeding Experiments

Short term In all breeding experiments, 8-week-old female and male BALB/c mice were used. Groups of 10 female BALB/c mice were inoculated i.p. with 2 x 10⁴ pfu virus or MOBS + 0.5% fetal calf serum, which was used as a diluent in all virus inoculations, i.p. A male mouse was introduced to each female 21 days postinoculation and was removed once pregnancy was established. The presence of mating plugs was recorded. The number of live births and implantation scars were recorded immediately after birth of a litter, and after 35 days, any remaining mice were killed and examined for the presence of embryos and implantation scars.

Long-term breeding trios Three groups of nine female mice were inoculated i.p. with 2 x 10⁴ pfu of rK181-ZP3, RM427+, or diluent only. Twenty-one days after inoculation a male mouse was introduced to three female mice, and remained present for the duration of the experiment. The number of pups born was recorded on a daily basis, and cumulative 10-day data were graphed. Pups that were eaten or stillborn were not included in the total. The fertility of the male mice was confirmed by introducing a control female C57BL/6j mouse into each of the rK181-ZP3 trios. This mouse was removed after pregnancy was established. The experiment was terminated after 240 days.

Long-term breeding pairs Six female BALB/c mice were inoculated i.p. with 2 x 10⁴ pfu rK181-ZP3, RM427+, or diluent only, and one male was introduced to one female immediately, and the male remained in the box for the duration of the experiment. The breeding output (the number of pups born) over 100 days was recorded. Pups that were eaten or stillborn were not included. In this experiment the male was added immediately after inoculation. The experiment was terminated after 100 days.

Statistical Analysis

Statistical analysis was performed by paired t-test or by ANOVA where applicable.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Construction and Characterization of Recombinant MCMVs

Recombinant MCMVs expressing murine ZP3 were produced by homologous recombination of the ie1 and ie2 regions flanking the ZP3 expression cassette in pK181-H3L/MV11/ZP3 with the identical ie1 and ie2 regions flanking the lacZ gene in RM427+ DNA. This recombination event effectively replaces one gene cassette with another and produces a mixture of blue-staining and nonstaining (or white) viruses that are purified by choosing individual plaques three times. The white recombinant virus, which contained the murine ZP3 gene under the control of the human CMV IE1 promoter, was designated rK181-ZP3 (Fig. 1A).

View larger version (47K): [in this window] [in a new window]   FIG. 1. The construction and DNA characterization of recombinant murine cytomegaloviruses. A) The lacZ gene under the control of an HCMV immediate early promoter was cloned between the two HpaI restriction enzyme sites of the K181 HindIII-L fragment of the virus. From this parental recombinant virus, the murine ZP3 under the control of the HCMV immediate early 1 promoter was exchanged for the lacZ gene by homologous recombination. B) HindIII digestion of viral DNA: 1 kb ladder (lane 1), K181 (lane 2), RM427+ (lane 3), rK181-ZP3 (lane 4), rK181-ZP3-REV (lane 5), DIG-labeled kb ladder. C) Southern blot probed with ZP3 DIG-labeled probe (from previous gel: DIG-labeled ladder; lane 6), rK181-ZP3-REV (lane 5), rK181-ZP3 (lane 4). D) PCR of putative rK181-ZP3 DNA. One kb ladder (lane 1), MCMV ie1 (lane 2), MCMV ie2 RT-PCR (lane 3), mZP3 (lane 4), PCR negative control (lane 5)

A virus that had reverted to the blue-staining phenotype, rK181-ZP3-REV, was constructed using pON427+ to ensure that no unexpected mutations occurred as a result of the cotransfection and recombination process.

RFLP using HindIII, and a Southern blot of the transferred DNA fragments with a DIG-labeled L fragment containing the ZP3 gene, were used to verify the structure of recombinant and parental viruses. HindIII RFLP confirmed that the respective L fragment sizes were as expected (Fig. 1B: K181, 7238 bp; RM427+ and rK181-ZP3-REV, 10608 bp; rK181-ZP3, 9016 bp). The proper insertion of the gene cassette was confirmed by DIG labeling of the ZP3 9016 bp fragment (Fig. 1C). PCR was also used to confirm that rK181-ZP3 had been successfully engineered into the ie2 region of the genome. A 641-bp fragment was generated by MCMV ie1 PCR (Fig. 1D, lane 2), a 662-bp fragment was generated by PCR with the ZP3 internal primers (Fig. 1D, lane 4), and no fragment was detected by RT-PCR of the ie2 gene (Fig. 1D, lane 3).

In Vitro Characterization of rK181-ZP3

The in vitro growth in M210B4 cells for all recombinant viruses tested was similar to the laboratory strain of MCMV, K181 (Fig. 2), demonstrating that the insertion of a foreign gene into the ie2 region had no deleterious effect on virus replication. Differences observed in titers and growth kinetics at various time points are most likely attributable to slight differences in the input viral doses. There was no significant difference in peak viral titers.

View larger version (24K): [in this window] [in a new window]   FIG. 2. One-step growth curve of recombinant and parental viruses: K181, RM427+, rK181-ZP3, and rK181-ZP3-REV. Error bars shown are standard errors of the mean

Indirect Immunofluorescence of Serum Generated by rK181-ZP3

Hyperimmune serum from mice inoculated with rK181-ZP3 contained specific antibodies to ZP as shown in Figure 3A, where discrete and specific binding of antibody to follicular ZP can be seen. This pattern of binding also occurred with serum generated by a single inoculation of virus and has been seen with serum taken 100 days after inoculation (results not shown). Incubation of ovalbumin hyperimmune serum on ovarian sections from uninfected mice did not produce significant background immunofluorescence (Fig. 3B), demonstrating the specificity of the reaction. Normal mouse serum produced no background immunofluorescence (result not shown).

View larger version (82K): [in this window] [in a new window]   FIG. 3. Immunofluorescence of mouse ovaries. Indirect immunofluorescence: rK181-ZP3 HIS (A), rK181-OvaTfr HIS (B). Direct immunofluorescence: D35 rK181-ZP3 infected (C), rK181-ZP3-REV infected (D). Original magnification: A and B, x100; C and D, x200

Direct Immunofluorescence of Ovaries of Mice Infected With rK181-ZP3

Mice inoculated with rK181-ZP3 produced antibodies specific for ZP at Day 35 after inoculation, as shown by the detection of endogenous antibody binding to the follicular ZP of ovarian sections from these mice (Fig. 3C) and the absence of binding to other ovarian structures. There was no evidence of endogenous antibody binding to ovarian sections from mice inoculated with rK181-ZP3-REV from the same time point after inoculation (Fig. 3D), indicating that the antibody detected by this method is specific for the ZP.

In Vivo Characterization of rK181-ZP3

The in vivo growth of rK181-ZP3 and RM427+ was determined by the plaque assay of various organs collected at Days 3, 7, 21, and 35 postinoculation. The replication of rK181-ZP3 was severely impaired in both adult and weanling BALB/c mice when compared with the RM427+ parental virus (Table 1).

PCR to detect the MCMV ie1 gene was used to further examine salivary gland homogenates from both adult and weanling mice inoculated with rK181-ZP3. The salivary gland from one of three adult mice was PCR positive at 21 and 35 days postinoculation. One of three juvenile mice was PCR positive at Day 35, although all samples from Day 21 were negative (results not shown). There was no evidence of morbidity associated with infection with this recombinant virus.

ZP3 and MCMV ELISAs

Antibodies to ZP3 were detected in sera from mice at Days 21, 35, 58, and 108 postinoculation (Table 2). Antibodies of the IgG1 and IgG2a isotypes increased to comparable titers over this period. Sera from individual mice were also titrated on uninfected ovarian sections using indirect immunofluorescence. Titers peaked at Day 58 (1:160) and remained high at Day 108 (data not shown). Antibodies to MCMV were detected in sera from mice taken at Days 7, 21, 35, 58, and 108 postinoculation (Table 3). The IgG1 response peaked at Day 58, whereas the IgG2a response continued to increase up to 108 days.

Avidity Assay

In this assay, increasing concentrations of sodium thiocyanate were used to disrupt antigen-antibody binding. The rK181-ZP3 hyperimmune serum had a greater avidity for the ZP3 antigen than hyperimmune serum produced by immunization with ZP3 protein in adjuvant (Fig. 4). The absorbance of rK181-ZP3 HIS remained at a similar intensity regardless of the concentration of sodium thiocyanate, whereas the absorbance of ZP3 protein + adjuvant HIS antibody began to decline from a sodium thiocyanate concentration of 1.5 M.

View larger version (21K): [in this window] [in a new window]   FIG. 4. Avidity assay of ZP3 hyperimmune serum. Error bars shown are standard deviations

Effect of rK181-ZP3 on the Estrous Cycle

It was interesting to find was no difference in the mean duration of the estrous cycle over five complete cycles between mice inoculated with the laboratory strain of MCMV, K181, and those inoculated with rK181-ZP3. Mice inoculated with K181 had an average cycle length of 5.6 ± 1.9 days (SD), and those inoculated with rK181-ZP3 had an average cycle length of 5.7 ± 1.4 days. All mice exhibited predictable cycles, and the cellular content of the smears was generally that as described by Allen [23] and Bronson et al. [24]. No significant difference was observed in the duration of time spent at any stage of the estrous cycle (P < 0.05, data not shown).

Breeding Studies in Mice Infected With rK181-ZP3

Short term Mice inoculated with rK181-ZP3 showed an almost complete suppression of fertility over one breeding cycle with only one pup being born during the experiment, and only one uterine scar was observed at autopsy. In contrast, only one female mouse in the two control groups did not produce litters or they were not pregnant when the experiment was completed. Mice inoculated with diluent or the parental virus RM427+ had a similar output of pups, with an average litter size of 5.1 and 4.8 pups, respectively (Table 4). One mouse from the group inoculated with diluent was pregnant with 10 embryos, and one mouse inoculated with RM427+ was pregnant with 7 embryos at the completion of the experiment.

Long-term breeding trios No pups were born to mice that were inoculated with rK181-ZP3 for the duration of the experiment (250 days after the male mice were introduced). In contrast, the females inoculated with diluent produced 460 pups over this period. To verify the fertility of the male mice, one uninoculated female C57BL/6j mouse was included in each box. In all cases, this mouse subsequently became pregnant and produced a litter in an appropriate time. Litter output is shown in Figure 5A. The uterine horns of all mice inoculated with rK181-ZP3 were inspected at the completion of the experiment, and no uterine scars were observed. In comparison, mice inoculated with the diluent or with RM427+ produced offspring consistently over the course of the experiment. Mice inoculated with RM427+ were followed only for the first 100 days of the experiment. The cumulative breeding output from these mice was comparable to those inoculated with the diluent and the results are not shown.

View larger version (27K): [in this window] [in a new window]   FIG. 5. Long-term breeding experiments. A) Long-term breeding trios: diluent (MOBS + 0.5% FCS), rK181-ZP3. B) Long-term breeding pairs: diluent, K181, rK181-ZP3

Long-term breeding pairs To determine the fertility of individual mice, long-term breeding pairs were set up. Litter output over 100 days for mice inoculated with rK181-ZP3, RM427+, or diluent only is shown in Figure 5B, with male mice being introduced to the females on the day of virus inoculation. Three of the six female mice inoculated with rK181-ZP3 had one litter and no subsequent pregnancies. The remaining three mice had no litters, giving a mean number of litters for the duration of the experiment of 0.5 ± 0.5 (standard deviation). After 43 days, males known to be fertile were placed into the boxes with female mice that had not had a litter. These females did not litter subsequently, and the possibility that they had impaired fertility prior to inoculation with the recombinant virus cannot be excluded. However, all 6 females from each of the control groups littered throughout the experiment with an average of 5 litters ± 0.9 for the mice inoculated with diluent and 4.5 ± 1.2 for mice inoculated with RM427+. The differences between the ZP3-infected mice were highly significant (P < 0.01) when compared with either the K181-infected or diluent-inoculated control mouse groups.

Ovarian Histology of Mice Infected With rK181-ZP3

Generally, there was considerable variation in the numbers of follicles present in the ovaries from individual mice at different time points, but even when follicles appeared to be normal, there was morphological evidence of structural abnormality in most follicles that were examined.

BALB/c mice that were inoculated with rK181-ZP3 exhibited specific ovarian lesions. Ovaries taken from mice 7 days after viral inoculation had a normal concentration of follicles, but many of these follicles displayed degradation of the ZP, which often was poorly delineated.

At 21 days postinoculation with rK181-ZP3, significant changes to the structural morphology of the ovarian tissue were observed. The concentration of primary follicles was reduced when compared with sections from mice inoculated with rK181-ZP3-REV, and those present showed nuclear fragmentation and, occasionally, complete absence of the nucleus. Often, the ZP layer was poorly delineated. The population of secondary follicles was not reduced, but the morphology appeared abnormal. Nuclei appeared to be condensed or were completely absent, the ZP was fragmented, and vascularization of the granulosa was observed, indicating that these follicles were unlikely to progress to a tertiary stage. No tertiary follicles were observed in ovaries from this time point. Neither inflammatory cells nor other cellular infiltrate were seen. Ovaries from mice inoculated with rK181-ZP3 and taken 35 days postinoculation showed similar histopathological features to those taken 21 days postinoculation.

Ovaries from BALB/c mice inoculated with rK181-ZP3 and examined at either 58 or 108 days postinoculation showed a markedly reduced concentration of both primary and secondary follicles, and again, no tertiary follicles were observed (Fig. 6A). All follicles present were morphologically abnormal and often showed evidence of ZP fragmentation, nuclear condensation or dissolution, or both. Again, no inflammatory cells or other cellular infiltrate were observed. In addition, greater numbers of thecal lutein-like cells and interstitial cells became evident in the ovarian stroma. A control ovary from a mouse infected with rK181-ZP3-REV from the same time point is shown in Figure 6B for comparison.

View larger version (126K): [in this window] [in a new window]   FIG. 6. Hematoxylin and eosin-stained sections of ovaries from mice inoculated with rK181-ZP3 or rK181-ZP3-REV (control). A) rK181-ZP3 D58 postinoculation, x40. B) rK181-ZP3-REV (control) D58 postinoculation, x40. C) Day 150 rK181-ZP3, x100. D) Day 150 rK181-ZP3, x400. The thin arrow shows a remnant primordial follicle; the thick arrow shows interstitial cell aggregates

By 150 days after inoculation with rK181-ZP3, no secondary follicles could be detected in the ovaries of inoculated mice. A few primary follicles could still be found, but most did not possess ova (Fig. 6, C and D). Again, aggregates of interstitial cells (thecal lutein-like) were observed in large numbers in the stroma.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study we have demonstrated that an immunocontraceptive effect can be elicited in BALB/c mice by a single i.p. inoculation of MCMV that has been engineered to express the murine ZP3 gene. The in vivo effects of the recombinant virus, apart from inducing infertility, were benign and there was no evidence of morbidity associated with infection. The immunocontraceptive effect was present by 21 days postinoculation and was long-lasting, with a single vaccination inducing infertility that was sustained until the end of the experiment (250 days). Postmortem assessment of the infertile mice showed that there were no uterine scars, suggesting that an interruption of fertilization had occurred rather than a direct abortive effect being induced by the virus.

In other examples of immunocontraception using whole porcine ZP as the immunogen, the titer of antibody produced was directly associated with fertility success or failure. A decline in antibody to a defined critical level in either mares or elks resulted in conception [2, 3]. A paper describing a recombinant ectromelia (mousepox) virus expressing murine ZP3 showed that a single inoculation in BALB/c mice was able to produce 6 mo of infertility that was correlated with high serum antibody titers to ZP3. A reduction in titer could be linked to a resumption of fertility, and infertility could be restored at reinoculation [21].

In contrast, when using rK181-ZP3 to induce infertility in BALB/c mice, there was no clear relationship between ZP3 antibody titer and infertility. Infertility was manifest by 21 days postinoculation as demonstrated in both the long-term breeding trio experiment in which all mice inoculated with rK181-ZP3 were infertile until Day 240, and the short-term breeding experiment in which 1 mouse out of 10 mated 21 days postinoculation successfully produced a litter of one pup. Only minimal serum antibody titers to murine ZP3 were detectable by ELISA at this time point. Higher antibody titers were detected at later time points, and the serum response did not appear to wane significantly over time.

Although peripheral anti-ZP3 antibody titers produced by inoculation with rK181-ZP3 were low, the antibody produced was sufficient to coat the ZP of developing ovarian follicles in vivo. In the indirect immunofluorescence assay, hyperimmune serum from mice inoculated with rK181-ZP3 bound to the follicular ZP of an uninfected ovary. This implies that peripheral antibody with affinity for ovarian ZP3 was produced. The avidity assay performed also showed that ZP3 antibody from hyperimmune serum produced using MCMV as a delivery vector had a greater avidity for purified murine ZP3 antigen when compared with serum of a similar titer produced by inoculation of purified ZP3 protein and adjuvant. In addition, serum antibody was shown to be sufficient to delay the onset of live births in an experiment that involved the passive transfer of a single aliquot of ZP3 HIS to recipient female mice mated immediately with male mice. Fertility was delayed by an average of 16.3 days (unpublished data).

Histological analysis of the ovaries of mice infected with rK181-ZP3 showed a progressive reduction in the numbers of mature follicles present and increasing evidence of ZP injury with profound changes present by 21 days postinoculation. At 100 days postinoculation, no tertiary follicles and only small numbers of morphologically abnormal primary and secondary follicles were present, compared with ovaries from control mice. By 150 days, only a few primary follicles were present and most of these possessed damaged ova. Furthermore, there was an absence of cellular infiltrate in the ovary at all time points examined, indicating that an inflammatory response did not occur. The absence of inflammatory cells and other cellular infiltrates is a distinguishing feature of the viral effect elicited by rK181-ZP3.

Female mice appeared to be cycling normally, presumably due to the presence of some functioning follicles and of increased numbers of thecal lutein cells, however, we have no information on the hormonal profiles of these mice. The cycles of mice at 150 days postinoculation have not been examined and it is doubtful that the ovaries of mice with this degree of follicle depletion would be able to sustain an estrous cycle.

From all of these results, we speculate that the observed infertility is due to a failure in ovulation that is related to the continual depletion of functional ovarian follicles in female mice infected with rK181-ZP3, which by 150 days, is almost complete. This may be due to several mechanisms acting at different times after inoculation. At early time points, a cell-signaling failure in secondary follicles leading to increased atresia may occur, or an up-regulation in apoptosis in primary follicles may be significant, as evidenced by nuclear fragmentation being observed from an early time point. The effects of rK181-ZP3 at early time points are currently being further explored in this laboratory. It is interesting that CBA mice that are resistant to the immunocontraceptive effect of rK181-ZP3 show these early pathological manifestations (unpublished data). At later time points, the presence of peripheral antibody with a strong avidity for ZP3 may lead to the further destruction of primary follicles that would maintain the immunocontraceptive effect. It is interesting to speculate whether the ovaries would be able to recover from this depletion should the immunological pressure be removed.

For an immunocontraceptive virus to be successful in a field environment, a number of further issues must be addressed. MCMV is believed to be transmitted by saliva or by sexual contact [25], and it may be important that mating behavior is unaffected. The short-term breeding experiment provides preliminary evidence that mating behavior is unchanged, because all groups had the same number of breeding plugs when observed over several days after mating (results not shown). It is also imperative that a disseminating immunocontraceptive virus be both transmittable throughout the target population and species-specific to ensure that the virus does not affect nontarget species. Work is proceeding in our laboratory to address both of these matters.

MCMV is proving to be an extremely useful vector for the presentation of fertility antigens to the murine immune system.

	ACKNOWLEDGMENTS

 
We thank Nicole Harvey for technical assistance with PCR and Southern blotting, Slavica Pervan and Angela Coletti from the UWA Department of Pathology for preparation and staining of sections, and the Australian Neuromuscular Research Institute for use of their fluorescent microscope. Thanks also to Lyn Hinds and Alec Redwood for assistance with manuscript preparation.

	FOOTNOTES

 
¹ This work was funded by the Grains Research Development Corporation and the Pest Animal Control Cooperative Research Centre.

² Correspondence: Malcolm A. Lawson, Department of Microbiology, UWA, QEII Medical Centre, Nedlands, Western Australia, 6009 Australia. FAX: 61 08 93462912; mlawson{at}cyllene.uwa.edu.au

Received: 28 October 2002.

First decision: 19 November 2002.

Accepted: 3 January 2003.

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