Theriogenology xxx (2015) 1–7
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Serum antibody immunoreactivity to equine zona protein after SpayVac vaccination Tracy A. Mask a, b, Kathryn A. Schoenecker a, Albert J. Kane c, Jason I. Ransom d, Jason E. Bruemmer b, * a
Fort Collins Science Center, U.S. Geological Survey, Fort Collins, Colorado, USA Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado, USA Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Fort Collins, Colorado, USA d Department of Ecosystem Science and Sustainability, Colorado State University, Fort Collins, Colorado, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 December 2014 Received in revised form 13 March 2015 Accepted 18 March 2015
Immunocontraception with porcine ZP (pZP) can be an effective means of fertility control in feral horses. Previous studies suggest that antibodies produced after pZP vaccination may both inhibit fertilization and cause follicular dysgenesis. Zonastat-H, PZP-22, and SpayVac are three pZP vaccines proposed for use in horses. Although all these vaccines contain the pZP antigen, variations in antigen preparation and vaccine formulation lead to differences in antigenic properties among them. Likewise, despite numerous efficacy and safety studies of Zonastat-H and PZP-22, the contraceptive mechanisms of SpayVac remain unclear. The preparation of pZP for SpayVac is thought to include more nonzona proteins, making it less pure than the other two vaccines. This may result in increased antigenicity of the vaccine. We therefore investigated the immunoreactivity of serum antibodies from SpayVac-vaccinated mares to equine zona protein. Western blot analyses revealed an immunoreactivity of these antibodies to protein isolated from mature equine oocytes, ZP, follicular tissues, and ovarian tissues. Immunohistochemical analyses were used to locate the binding of serum antibodies to the ZP of immature oocytes in ovarian stromal tissue. We also found serum antibodies from SpayVac-treated mares to be predominantly specific for zona protein 3. Collectively, our results suggest a model where serum antibodies produced in response to SpayVac vaccination are immunoreactive to equine zona protein in vitro. Our study lends insight into the contraceptive mechanisms underlying the infertility observed after SpayVac vaccination. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Contraception Equus caballus Fertility control Immunocontraception Porcine ZP
1. Introduction Feral horse (Equus caballus) populations residing on lands managed by the Bureau of Land Management and United States Forest Service are federally protected under the Wild Free-Roaming Horses and Burros Act (Public Law 92–195). Provided this protection, in combination with a lack of natural predators, many horse populations can grow
* Corresponding author. Tel.: þ1 970 491 8409; fax: 970-491-3557. E-mail address: [email protected] (J.E. Bruemmer). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.03.012
10% to 25% annually [1]. Because their habitats remain constant in size and limited in forage availability, feral horse populations often overwhelm available resources. Researchers have investigated the use of immunocontraceptive vaccines as an approach to control feral horse population growth [2]. Immunocontraceptive vaccines stimulate an animal’s own immune system to interfere with a critical reproductive event [2]. When an antigen is combined with an immunologic adjuvant and injected into an animal, antigen-specific antibodies are produced [3]. These antibodies are thought to bind not only to the antigen for which they are specific but also to structurally similar endogenous
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proteins in the target animal. Immunocontraceptive vaccines can therefore target specific hormones or proteins within the animal [4]. Zonastat-H (Humane Society of the United States, Washington, DC, USA), PZP-22 [5], and SpayVac (Immunovaccine Inc., Halifax, Nova Scotia, Canada) are immunocontraceptive vaccines that incorporate the porcine ZP (pZP) antigen. Vaccination with the pZP antigen is reported to stimulate an immune response that can be measured by serum antibody titers [6,7]. Infertility is thought to be achieved by the binding of these serum antibodies to ZP proteins on mature oocytes, thereby inhibiting sperm binding and penetration [8–10]. Follicular dysgenesis characterized by diminished ovulatory ability, absence of mature oocytes and follicles, decreased ovarian weights, and a decline in ovarian estrogen secretion has also been reported after pZP vaccination [11–13]. These studies suggest that serum antibodies produced in response to pZP vaccination may act both on ZP of ovulated oocytes in the oviduct and during folliculogenesis in the ovary. Despite these valuable pathologic studies in multiple species, the molecular mechanisms by which antibodies produced in response to SpayVac vaccination result in infertility remain unclear. Although serum antibodies may not accurately predict the contraceptive efficacy of a vaccine, understanding whether these antibodies are capable of binding key proteins involved in folliculogenesis and fertilization is a central part of understanding how the vaccine may be acting to inhibit fertility. Our objectives were to (1) determine whether vaccination of feral horse mares with SpayVac results in the production of antibodies immunoreactive to equine zona protein and ovarian tissues and (2) identify the location of binding of these antibodies to equine oocytes and ovarian tissue. 2. Materials and methods 2.1. Animal use No live animals were used exclusively for this study. All tissue and serum samples from nonvaccinated mares were collected as part of other ongoing research under protocols approved by the Institutional Animal Care and Use Committee at Colorado State University. Serum from SpayVacvaccinated mares was obtained from samples previously collected under protocols approved by the Fort Collins Science Center’s Institutional Animal Care and Use Committee as part of a study on SpayVac immunocontraception (Roelle, U.S. Geological Survey, Pauls Valley, OK, USA). 2.2. Samples Equine ovaries and renal tissues were harvested from necropsy samples collected at the Animal Reproduction and Biotechnology Laboratory, Colorado State University (Fort Collins, CO, USA). Oocytes were collected by aspirating follicular fluid from follicles on the ovaries. Oocytes were stored in PBS at 4 C. Tissue samples were either fixed in 4% paraformaldehyde and sent to the Colorado State University’s Veterinary Diagnostics Laboratory to be embedded in paraffin or frozen and stored at 80 C.
Serum from vaccinated mares of known titer levels was obtained from a study on SpayVac immunocontraception. Mares were vaccinated with 200-mg pZP (Immunovaccine Inc.). Blood samples were collected and processed 3 months after vaccination, and serum was stored at 80 C. Serum antibody titers from these samples were determined using ELISA by Immunovaccine Inc. Titers were read as average optical density across three readings of the same sample and then reported as a percent of standard, where the standard was a serum sample containing a large number of antibodies specific to the vaccine. Percent of standard values, or titers, ranged from less than 50.00 to as high as 178.33, with 100.00 representing a sample that had an antibody profile similar to that of the standard. Mares with higher titer levels, specifically values greater than 100.00, were considered to have an increased number of vaccine-specific antibodies in their serum. Because antibodies against the vaccine are thought to be responsible for the contraceptive effect, higher titer levels correlate to increased vaccination efficacy. Serum samples from eight mares of high titer levels ranging from 97.83% to 178.33% of standard were therefore used interchangeably throughout the study. Serum from two nonvaccinated mares was obtained from other ongoing research at the Animal Reproduction and Biotechnology Laboratory. All serum samples were stored at 80 C. 2.3. Protein isolation and quantification Isolation of protein was performed using radioimmunoprecipitation assay (RIPA) lysis buffer (150-mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50-mM Tris, pH 8.0, 1-mM EDTA). Samples were suspended in RIPA, homogenized and sonicated twice, and subjected to centrifugation for 10 minutes at 10,000 revolutions per minute (Centrifuge Model #5417R; Eppendorf, Germany). The supernatant containing protein was recovered, and the DNA-containing pellet was discarded. All protein was isolated just before use and never frozen. To isolate protein from equine oocytes, cumulus cells were mechanically stripped from four oocytes by a series of passages through a 10-mL embryo pipette (Bioniche, Pullman, WA, USA), while the oocytes were suspended in PBS. Oocytes were suspended in 40-mL RIPA lysis buffer and processed as detailed previously. The final supernatant volume was divided into two 20-mL samples. To isolate protein from the ZP of equine oocytes, the cumulus cells were mechanically stripped from four equine oocytes and ultrasharp splitting blades (Bioniche) were used to make an incision in each ZP. Turbulence was created to free each ZP from the ooplasm, and the ZP of the four oocytes were suspended in 40-mL RIPA lysis buffer. Protein was isolated as previously described, and the final volume of the supernatant was divided into two 20-mL samples. To isolate protein from ovarian and renal tissues, a small section of each tissue was excised from the sample, suspended in 300-mL RIPA lysis buffer, and processed as detailed previously. Protein content was quantified following the manufacturer’s protocol for the Pierce BCA
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Protein Assay Kit (Catalog #23227; Thermo Scientific, Rockford, IL, USA). An aliquot containing 40 mg of each protein sample was transferred to individual 1.7-mL conical tubes and brought up to 20 mL in volume with PBS. 2.4. Electrophoresis and Western blot analysis Protein samples were incubated with 4-mL 6X Laemmli sample buffer (375-mM Tris-HCL, pH 6.8, 48% glycerol, 6% sodium dodecyl sulfate, 0.01% bromophenol blue, 20% dithiothreitol), loaded into 10% Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad, Hercules, CA, USA) in duplicate, separated by electrophoresis, and transferred to Protran nitrocellulose membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Membranes were blocked in 5% blocking buffer (5-g nonfat dried milk in 100-mL 1X TBSTd50-mM Tris-HCL, pH 7.4, 150-mM NaCl, 0.1% Tween 20) for 1 hour and then cut down the middle such that each half contained one set of the duplicate protein samples. One half of each membrane was incubated in a dilution (1:50 in 1% blocking bufferd1-g nonfat dried milk in 100mL 1X TBST) of either serum from a SpayVac-vaccinated mare or serum from a nonvaccinated mare overnight. Both membranes were then incubated with horseradish peroxidase (HRP) –conjugated goat polyclonal secondary antibody to horse immunoglobulin G (IgG; 1:10,000 in 1% blocking buffer; ab102396; Abcam, San Francisco, CA, USA) for 1 hour. Membranes were exposed to SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) for 5 seconds and imaged immediately on a Molecular Imager ChemiDoc XRSþ System (Bio-Rad). Membranes were washed between each step with 1X TBST for 15 minutes. A mouse anti–b actin monoclonal primary antibody (1:100 in 1% BSA; ab8226; Abcam) followed by an HRPconjugated donkey antimouse IgG polyclonal secondary antibody (1:1000 in 1% BSA; ab97030; Abcam) was used as a loading control for protein isolated from follicular and ovarian tissues. A rabbit anti-ZP3 polyclonal primary antibody (1:100 in 1% blocking buffer; ap17235b; Abgent) followed by HRPconjugated goat antirabbit IgG polyclonal secondary antibody (1:1000 in 1% blocking buffer; #170-6515; Bio-Rad) was used for protein isolated from oocytes and ZP as a loading control. Binding specificity was determined by preincubation of the Western blot membranes with either the rabbit antiZP3 polyclonal antibody (ap17235b; Abgent) or rabbit antiZP4 polyclonal antibody (ap12724b; Abgent) overnight before incubation with serum from a SpayVac-vaccinated mare overnight and use of the HRP-conjugated goat polyclonal secondary antibody to horse IgG (1:10,000 in 1% blocking buffer; ab102396; Abcam). 2.5. Immunohistochemistry Three consecutive 5-mm sections were sliced from tissue embedded in paraffin blocks and placed on two glass slides. The slides were incubated at 55 C for 30 minutes. Citrisolv (Thermo Scientific) along with an alcohol gradient was used to dewax and rehydrate the tissue slices. After rehydration, one of the three slices of tissues was subjected to a hematoxylin and eosin staining protocol. The slide was incubated in hematoxylin for 1 minute, rinsed
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under running tap water for 5 minutes, incubated in eosin for 5 minutes, rinsed once more under running tap water, and dehydrated through an alcohol and citrisolv gradient. The slide was then left to air dry before being mounted with a coverslip using Cytoseal (Thermo Scientific). The other slide, containing two tissue slices, was boiled in 10-mM sodium citrate (pH 6.0) for 20 minutes and blocked in 5% natural goat serum in 1% BSA (0.1-g BSA in 10-mL 1X PBS) for 2 hours. The two tissue slices were then incubated with a dilution (1:50 in 1% BSA) of serum from either a SpayVac-vaccinated mare or nonvaccinated mare overnight. The following day, both tissue slices were incubated with the HRP-conjugated goat polyclonal secondary antibody to horse IgG (1:1000 in 1% BSA; ab102396; Abcam) for 1 hour. Impact DAB Peroxidase Substrate Solution (Vector Laboratories, Burlingame, CA, USA) was then applied for 10 minutes. The slide contents were dehydrated through an alcohol and citrisolv gradient, allowed to dry, and mounted with a coverslip using cytoseal. The slide was washed between each step with 1X PBS for 10 minutes, and each incubation step took place in a moist chamber. All samples were imaged using light microscopy. Binding specificity of serum antibodies from SpayVacvaccinated mares was determined by presaturating a sample of serum from a SpayVac-vaccinated mare with ZP3 and ZP4 synthetic blocking peptides (bp17235b and bp12724b, respectively; Abgent). An aliquot of 30 mg of each peptide was added to 10 mL of serum in 500-mL 1% BSA before use as the primary antibody for immunohistochemical analysis. The HRP-conjugated goat antihorse IgG polyclonal antibody was used as the secondary antibody (1:1000 in 1% BSA; ab102396; Abcam). 3. Results 3.1. Immunoreactivity of serum antibodies from SpayVacvaccinated mares to equine zona protein Western blot analysis revealed the immunoreactivity of serum from SpayVac-vaccinated mares to equine zona protein. Antibodies in the serum of SpayVac-vaccinated mares bound to protein isolated from whole equine oocytes and equine ZP (Fig. 1A), as well as to protein isolated from equine ovarian and follicular tissues (Fig. 1C). These antibodies are not endogenously found in serum from nonvaccinated mares (Fig. 1B, D) and did not bind protein isolated from equine renal tissue (Fig. 1C). Western blot analyses of equine oocytes and ZP were repeated twice and analyses of equine tissues were repeated four times with different protein and serum samples to verify consistency. 3.2. Location of binding of serum antibodies from SpayVacvaccinated mares to equine ovarian tissue Immunohistochemical staining of equine ovarian tissue located the binding of serum antibodies from SpayVacvaccinated mares to the ZP of immature oocytes of primary follicles (Fig. 2E). These serum antibodies did not bind oocytes in primordial follicles, where the ZP was not yet formed as part of folliculogenesis (Fig. 2B). They are also not found in serum from nonvaccinated mares (Fig. 2C, F).
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Fig. 1. Immunoreactivity of serum antibodies from SpayVac-vaccinated mares for equine zona protein. Western blot analysis of protein isolated from the equine ZP of two oocytes, two whole equine oocytes (OC), 40-mg equine ovarian tissue (OT), 40-mg equine follicular tissue (FT), and 40-mg equine renal tissue (RT) using serum from a SpayVac-vaccinated mare (A and C) or serum from a nonvaccinated mare (B and D). Protein quantities are expressed per lane. Anti-ZP3 (B) and anti– b actin (D) are used as loading controls. Renal tissue was used as a negative control. Molecular weight standards appear in the leftmost lane of each gel.
Immunohistochemical analyses were repeated twice with different serum and tissue samples. 3.3. Specificity of serum antibodies from SpayVac-vaccinated mares for equine ZP3 The addition of ZP3 and ZP4 blocking peptides to a sample of serum from a SpayVac-vaccinated mare successfully saturated antibodies in the serum and blocked their ability to bind zona proteins in the tissue (Fig. 3C). In
the absence of blocking peptides, serum antibodies from the SpayVac-vaccinated mare bound to an immature oocyte in the ovarian tissue (Fig. 3B). Preincubation of a Western blot membrane with antiZP3 polyclonal antibody before incubation with serum from a SpayVac-vaccinated mare blocked the ability of serum antibodies to bind to the protein sample (Fig. 4A). These serum antibodies maintained their ability to bind the protein samples after preincubation with anti-ZP4 polyclonal antibody (Fig. 4B).
Fig. 2. Localization of serum antibodies from SpayVac-vaccinated mares to the ZP of immature oocytes. Immunohistochemical staining of equine ovarian tissue using serum from a SpayVac-vaccinated mare (B and E) or serum from a nonvaccinated mare (C and F). Serum antibodies bind the ZP of oocytes in primary follicles (E) but did not bind primordial follicles (B). Immunohistochemical staining using serum from nonvaccinated mares is not observed (C and F). H&E staining of ovarian tissue containing a primordial follicle (A) and primary follicle (D). Bar ¼ 20 mm. GC, granulosa cells.
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Fig. 3. Specificity of serum antibodies from SpayVac-vaccinated mares for zona protein. H&E staining of of ovarian tissue containing a primary follicle (A). Immunohistochemical staining of equine ovarian tissue using serum from a SpayVac-vaccinated mare (B) or serum from the same SpayVac-vaccinated mare after presaturation of the serum with ZP3 and ZP4 blocking peptides (C). Bar ¼ 20 mm.
4. Discussion 4.1. Immunoreactivity of serum antibodies from SpayVacvaccinated mares to equine zona protein Through Western blot analyses of mature preovulatory equine oocytes and ZP, we revealed the presence of antibodies in serum from SpayVac-vaccinated mares that are immunoreactive to equine zona protein. The use of serum from nonvaccinated mares as a control verified that these antibodies are not endogenous to the mare. Western blot analyses also confirmed the immunoreactivity of serum antibodies from SpayVac-vaccinated mares to protein isolated from equine ovarian and follicular tissues. 4.2. Location of binding of serum antibodies from SpayVacvaccinated mares to equine ovarian tissue The immunoreactivity of serum antibodies from SpayVacvaccinated mares to protein isolated from equine ovarian tissue is explained by the localization of antibodies to the ZP of immature oocytes in the tissue. We failed to locate the binding of these serum antibodies to follicular tissue. Because the synthesis of the ZP is an integrated process involving both the oocyte and granulosa cells [14,15], the immunoreactivity of serum antibodies from SpayVac-treated mares to equine follicular tissue is thought to be due to the presence of zona protein precursors in the granulosa cells. Although Western
blot analysis may have detected these zona protein precursors because of its high level of sensitivity and stringent cell lysis protocol to release intracellular proteins, high background staining observed in immunohistochemical analyses may have masked any localization to these cells [16]. High background staining is common when the tissue is of the same species as the secondary antibody used [16]. Conversely, the high background staining observed may also be indicative of an immunoreactivity between serum antibodies from SpayVac-vaccinated mares to other proteins located in follicular tissue. Because it is likely that the preparation of SpayVac results in nonzona proteins, such as ovarian and follicular proteins [17], it is possible that serum antibodies from SpayVac-vaccinated mares are immunoreactive to nonzona proteins as well. More research however is required to investigate the immunoreactivity of serum from SpayVacvaccinated mares to other ovarian and follicular proteins and how this immunoreactivity may relate to efficacy and reversibility of the vaccine. 4.3. Specificity of serum antibodies from SpayVac-vaccinated mares for equine ZP3 Our presaturation and preincubation experiments are indicative of both a specificity of serum antibodies from SpayVac-vaccinated mares for equine zona protein 3 and a lack of nonspecific binding. Because zona proteins are large glycoproteins with varying levels of posttranslational
Fig. 4. Specificity of serum antibodies from SpayVac-vaccinated mares for equine zona protein 3. Western blot analysis of protein isolated from equine ovarian tissue (OT), equine follicular (FT), and equine renal (RT) tissues using serum from a SpayVac-vaccinated mare after preincubation with rabbit anti-ZP3 polyclonal antibody (A) or rabbit anti-ZP4 polyclonal antibody (B). Anti–b actin antibody was used as a loading control. Renal tissue was used as a negative control. Molecular weight standards appear in the leftmost lanes of each gel. Forty micrograms of protein was loaded per lane.
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modifications, each protein can be observed in a range of molecular weights making the protein difficult to accurately identify by electrophoresis [18–20]. Our experiments lend clarity to the specificity of these serum antibodies. Likewise, this specificity for ZP3 is in agreement with the finding by Wassarman [18] that ZP3 is the major component of the porcine ZP, representing as much as 70% to 80% of total protein content. When the pZP antigen is isolated, the entire ZP is homogenized [21], resulting in a compilation of all zona protein families. It follows that 70% to 80% of the pZP antigen is likely accounted for by ZP3 and, when injected, the mare may produce substantially more antibodies against ZP3 than the other proteins. Furthermore, the pZP antigen is thought to work by eliciting the production of antibodies that bind to ZP proteins on mature oocytes, thereby interfering with sperm binding and penetration [8–10]. Because the zona protein families of ZP1, ZP2, and ZP4 are thought to only be involved in ZP structure, whereas ZP3 has a known role in sperm binding and penetration [20], this specificity for ZP3 coincides with the conclusions of prior studies. 4.4. Limitations of results There are a few limitations to our study. First, our study focused exclusively on SpayVac as it is less documented in published literature and potentially induces greater duration of infertility in field application. Although all three vaccines incorporate the pZP antigen, they are not a homogenous set of compounds. For instance, it is likely that the preparation of SpayVac results in more nonzona proteins, such as ovarian and follicular proteins, than the other two vaccines [17]. This variation in purity may account for the immunoreactivity of serum antibodies from SpayVacvaccinated mares to ovarian and follicular tissues seen in our experiments. Specifically, the high background staining observed during our immunohistochemical analysis of equine ovarian tissue may be due to the presence of antibodies immunoreactive to ovarian proteins produced as a result of SpayVac vaccination. Second, serum samples from vaccinated mares were selectively chosen for their reported high antibody titer levels. Differences in antibody titer and quality depend on the animal immunized and contribute to variability in efficacy among individuals [22]. Accordingly, although serum samples were taken from mares immunized with the same dose of SpayVac, one mare was still able to conceive despite vaccination. Nonetheless, our experiments showed that all eight mares produced antibodies immunoreactive to equine zona protein, regardless of whether the antibodies successfully suppressed the mare’s fertility. It is likely that although each mare had sufficient serum antibodies to successfully bind the protein in our experimental samples, overall availability of these antibodies in vivo may not have been enough to saturate all possible protein-binding sites and thus result in infertility. 4.5. Conclusions Our results suggest a model where serum antibodies produced in response to SpayVac vaccination are
immunoreactive to equine zona proteins in vitro. When zona proteins are isolated from equine tissues and made available to serum samples from SpayVac-vaccinated mares, antibodies in the serum are consistently able to recognize the protein. These serum antibodies appear immunoreactive not only to the ZP of mature preovulatory oocytes isolated from Graafian follicles but also to immature oocytes embedded in ovarian tissue. Although we illustrate a pathway by which serum antibodies from SpayVac-vaccinated mares could act if they are biologically available to these tissues under the correct conditions, it has yet to be investigated whether these antibodies are present in the follicular fluid of follicles or able to permeate ovarian tissue after SpayVac vaccination. We simply showed a model by which antibodies produced in response to SpayVac vaccination may bind zona proteins in vivo, thereby interfering with a critical reproductive component, and result in infertility. Acknowledgments This research was funded by the United States Geological Survey Wildlife Program and conducted through a cooperative effort between the USGS Fort Collins Science center, Bureau of Land Management, United States Department of Agriculture’s Animal and Plant Health Inspection Service, and Colorado State University. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the United States Government or Colorado State University. This research could not have been conducted without the support, cooperation, laboratory use, and sample contribution of the U.S. Geological Survey, Bureau of Land Management, and Colorado State University. References [1] National Research Council. Using science to improve the BLM wild horse and burro program: a way forward. Washington, DC: National Academies Press; 2013. [2] Kirkpatrick JF, Lyda RO, Frank KM. Contraceptive vaccines for wild: a review. Am J Reprod Immunol 2011;66:40–50. [3] Pashine A, Valiante NM, Ulmer JB. Targeting the innate immune response with improved vaccine adjuvants. Nat Med Suppl 2005;11: 563–8. [4] Barber MR, Fayrer-Hosken RA. Evaluation of somatic and reproductive immunotoxic effects of the porcine zona pellucida vaccination. J Exp Zool 2000;286:641–6. [5] Turner JW, Liu IKM, Flanagan DR, Rutberg AT, Kirkpatrick JF. Immunocontraception in wild horses: one inoculation provides two years of infertility. J Wildl Manage 2007;71:662–7. [6] Willis P, Heusner GL, Warren RJ, Kessler D, Fayrer-Hosken RA. Equine immunocontraception using porcine zona pellucida: a new method for remote delivery and characterization of the immune response. J Equine Vet Sci 1994;14:364–70. [7] Turner JW, Kirkpatrick JF, Liu IKM. Effectiveness, reversibility, and serum antibody titers associated with immunocontraception in captive white-tailed deer. J Wildl Manage 1996;60:45–51. [8] Sacco AG. Antigenic cross-reactivity between human and pig zona pellucida. Biol Reprod 1977;16:164–73. [9] Liu IKM, Bernoco M, Feldman M. Contraception in mares heteroimmunized with pig zonae pellucidae. J Reprod Fertil 1989;85: 19–29. [10] Kirkpatrick JF, Liu IKM, Turner Jr JW. Remotely-delivered immunocontraception in feral horses. Wildl Soc Bull 1990;18:326–30. [11] Skinner SM, Mills T, Kirchick HJ, Dunbar BS. Immunization with zona pellucida proteins results in abnormal ovarian follicular
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techniques in veterinary diagnostic laboratories. J Vet Diagn Invest 2008;4:393–413. Miller LA, Fagerstone KA, Wagner DC, Killian GJ. Factors contributing to the success of a single-shot, multiyear PZP immunocontraceptive vaccine for white-tailed deer. Hum Wildl Confl 2009;3:103–15. Wassarman PM. Zona pellucida glycoproteins. Annu Rev Biochem 1988;57:415–42. Dunbar BS, Avery S, Lee V, Prasad S, Schwahn D, Schwoebel E, et al. The mammalian zona pellucida: its biochemistry, immunochemistry, molecular biology, and developmental expression. Reprod Fertil Dev 1994;6:331–47. Prasad SV, Skinner SM, Carino C, Wang N, Cartwright J, Dunbar BS. Structure and function of the proteins of the mammalian zona pellucida. Cells Tissues Organs 2000;166:148–64. Yurewicz EC, Sacco AG, Subramanian MG. Isolation and preliminary characterization of a purified pig zona antigen (PPZA) from porcine oocytes. Biol Reprod 1983;29:511–23. Ramos-Vara JA. Technical aspects of immunohistochemistry. Vet Pathol 2005;42:405–26.
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Theriogenology journal homepage: www.theriojournal.com
Serum antibody immunoreactivity to equine zona protein after SpayVac vaccination Tracy A. Mask a, b, Kathryn A. Schoenecker a, Albert J. Kane c, Jason I. Ransom d, Jason E. Bruemmer b, * a
Fort Collins Science Center, U.S. Geological Survey, Fort Collins, Colorado, USA Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado, USA Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Fort Collins, Colorado, USA d Department of Ecosystem Science and Sustainability, Colorado State University, Fort Collins, Colorado, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 December 2014 Received in revised form 13 March 2015 Accepted 18 March 2015
Immunocontraception with porcine ZP (pZP) can be an effective means of fertility control in feral horses. Previous studies suggest that antibodies produced after pZP vaccination may both inhibit fertilization and cause follicular dysgenesis. Zonastat-H, PZP-22, and SpayVac are three pZP vaccines proposed for use in horses. Although all these vaccines contain the pZP antigen, variations in antigen preparation and vaccine formulation lead to differences in antigenic properties among them. Likewise, despite numerous efficacy and safety studies of Zonastat-H and PZP-22, the contraceptive mechanisms of SpayVac remain unclear. The preparation of pZP for SpayVac is thought to include more nonzona proteins, making it less pure than the other two vaccines. This may result in increased antigenicity of the vaccine. We therefore investigated the immunoreactivity of serum antibodies from SpayVac-vaccinated mares to equine zona protein. Western blot analyses revealed an immunoreactivity of these antibodies to protein isolated from mature equine oocytes, ZP, follicular tissues, and ovarian tissues. Immunohistochemical analyses were used to locate the binding of serum antibodies to the ZP of immature oocytes in ovarian stromal tissue. We also found serum antibodies from SpayVac-treated mares to be predominantly specific for zona protein 3. Collectively, our results suggest a model where serum antibodies produced in response to SpayVac vaccination are immunoreactive to equine zona protein in vitro. Our study lends insight into the contraceptive mechanisms underlying the infertility observed after SpayVac vaccination. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Contraception Equus caballus Fertility control Immunocontraception Porcine ZP
1. Introduction Feral horse (Equus caballus) populations residing on lands managed by the Bureau of Land Management and United States Forest Service are federally protected under the Wild Free-Roaming Horses and Burros Act (Public Law 92–195). Provided this protection, in combination with a lack of natural predators, many horse populations can grow
* Corresponding author. Tel.: þ1 970 491 8409; fax: 970-491-3557. E-mail address: [email protected] (J.E. Bruemmer). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.03.012
10% to 25% annually [1]. Because their habitats remain constant in size and limited in forage availability, feral horse populations often overwhelm available resources. Researchers have investigated the use of immunocontraceptive vaccines as an approach to control feral horse population growth [2]. Immunocontraceptive vaccines stimulate an animal’s own immune system to interfere with a critical reproductive event [2]. When an antigen is combined with an immunologic adjuvant and injected into an animal, antigen-specific antibodies are produced [3]. These antibodies are thought to bind not only to the antigen for which they are specific but also to structurally similar endogenous
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proteins in the target animal. Immunocontraceptive vaccines can therefore target specific hormones or proteins within the animal [4]. Zonastat-H (Humane Society of the United States, Washington, DC, USA), PZP-22 [5], and SpayVac (Immunovaccine Inc., Halifax, Nova Scotia, Canada) are immunocontraceptive vaccines that incorporate the porcine ZP (pZP) antigen. Vaccination with the pZP antigen is reported to stimulate an immune response that can be measured by serum antibody titers [6,7]. Infertility is thought to be achieved by the binding of these serum antibodies to ZP proteins on mature oocytes, thereby inhibiting sperm binding and penetration [8–10]. Follicular dysgenesis characterized by diminished ovulatory ability, absence of mature oocytes and follicles, decreased ovarian weights, and a decline in ovarian estrogen secretion has also been reported after pZP vaccination [11–13]. These studies suggest that serum antibodies produced in response to pZP vaccination may act both on ZP of ovulated oocytes in the oviduct and during folliculogenesis in the ovary. Despite these valuable pathologic studies in multiple species, the molecular mechanisms by which antibodies produced in response to SpayVac vaccination result in infertility remain unclear. Although serum antibodies may not accurately predict the contraceptive efficacy of a vaccine, understanding whether these antibodies are capable of binding key proteins involved in folliculogenesis and fertilization is a central part of understanding how the vaccine may be acting to inhibit fertility. Our objectives were to (1) determine whether vaccination of feral horse mares with SpayVac results in the production of antibodies immunoreactive to equine zona protein and ovarian tissues and (2) identify the location of binding of these antibodies to equine oocytes and ovarian tissue. 2. Materials and methods 2.1. Animal use No live animals were used exclusively for this study. All tissue and serum samples from nonvaccinated mares were collected as part of other ongoing research under protocols approved by the Institutional Animal Care and Use Committee at Colorado State University. Serum from SpayVacvaccinated mares was obtained from samples previously collected under protocols approved by the Fort Collins Science Center’s Institutional Animal Care and Use Committee as part of a study on SpayVac immunocontraception (Roelle, U.S. Geological Survey, Pauls Valley, OK, USA). 2.2. Samples Equine ovaries and renal tissues were harvested from necropsy samples collected at the Animal Reproduction and Biotechnology Laboratory, Colorado State University (Fort Collins, CO, USA). Oocytes were collected by aspirating follicular fluid from follicles on the ovaries. Oocytes were stored in PBS at 4 C. Tissue samples were either fixed in 4% paraformaldehyde and sent to the Colorado State University’s Veterinary Diagnostics Laboratory to be embedded in paraffin or frozen and stored at 80 C.
Serum from vaccinated mares of known titer levels was obtained from a study on SpayVac immunocontraception. Mares were vaccinated with 200-mg pZP (Immunovaccine Inc.). Blood samples were collected and processed 3 months after vaccination, and serum was stored at 80 C. Serum antibody titers from these samples were determined using ELISA by Immunovaccine Inc. Titers were read as average optical density across three readings of the same sample and then reported as a percent of standard, where the standard was a serum sample containing a large number of antibodies specific to the vaccine. Percent of standard values, or titers, ranged from less than 50.00 to as high as 178.33, with 100.00 representing a sample that had an antibody profile similar to that of the standard. Mares with higher titer levels, specifically values greater than 100.00, were considered to have an increased number of vaccine-specific antibodies in their serum. Because antibodies against the vaccine are thought to be responsible for the contraceptive effect, higher titer levels correlate to increased vaccination efficacy. Serum samples from eight mares of high titer levels ranging from 97.83% to 178.33% of standard were therefore used interchangeably throughout the study. Serum from two nonvaccinated mares was obtained from other ongoing research at the Animal Reproduction and Biotechnology Laboratory. All serum samples were stored at 80 C. 2.3. Protein isolation and quantification Isolation of protein was performed using radioimmunoprecipitation assay (RIPA) lysis buffer (150-mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50-mM Tris, pH 8.0, 1-mM EDTA). Samples were suspended in RIPA, homogenized and sonicated twice, and subjected to centrifugation for 10 minutes at 10,000 revolutions per minute (Centrifuge Model #5417R; Eppendorf, Germany). The supernatant containing protein was recovered, and the DNA-containing pellet was discarded. All protein was isolated just before use and never frozen. To isolate protein from equine oocytes, cumulus cells were mechanically stripped from four oocytes by a series of passages through a 10-mL embryo pipette (Bioniche, Pullman, WA, USA), while the oocytes were suspended in PBS. Oocytes were suspended in 40-mL RIPA lysis buffer and processed as detailed previously. The final supernatant volume was divided into two 20-mL samples. To isolate protein from the ZP of equine oocytes, the cumulus cells were mechanically stripped from four equine oocytes and ultrasharp splitting blades (Bioniche) were used to make an incision in each ZP. Turbulence was created to free each ZP from the ooplasm, and the ZP of the four oocytes were suspended in 40-mL RIPA lysis buffer. Protein was isolated as previously described, and the final volume of the supernatant was divided into two 20-mL samples. To isolate protein from ovarian and renal tissues, a small section of each tissue was excised from the sample, suspended in 300-mL RIPA lysis buffer, and processed as detailed previously. Protein content was quantified following the manufacturer’s protocol for the Pierce BCA
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Protein Assay Kit (Catalog #23227; Thermo Scientific, Rockford, IL, USA). An aliquot containing 40 mg of each protein sample was transferred to individual 1.7-mL conical tubes and brought up to 20 mL in volume with PBS. 2.4. Electrophoresis and Western blot analysis Protein samples were incubated with 4-mL 6X Laemmli sample buffer (375-mM Tris-HCL, pH 6.8, 48% glycerol, 6% sodium dodecyl sulfate, 0.01% bromophenol blue, 20% dithiothreitol), loaded into 10% Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad, Hercules, CA, USA) in duplicate, separated by electrophoresis, and transferred to Protran nitrocellulose membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Membranes were blocked in 5% blocking buffer (5-g nonfat dried milk in 100-mL 1X TBSTd50-mM Tris-HCL, pH 7.4, 150-mM NaCl, 0.1% Tween 20) for 1 hour and then cut down the middle such that each half contained one set of the duplicate protein samples. One half of each membrane was incubated in a dilution (1:50 in 1% blocking bufferd1-g nonfat dried milk in 100mL 1X TBST) of either serum from a SpayVac-vaccinated mare or serum from a nonvaccinated mare overnight. Both membranes were then incubated with horseradish peroxidase (HRP) –conjugated goat polyclonal secondary antibody to horse immunoglobulin G (IgG; 1:10,000 in 1% blocking buffer; ab102396; Abcam, San Francisco, CA, USA) for 1 hour. Membranes were exposed to SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) for 5 seconds and imaged immediately on a Molecular Imager ChemiDoc XRSþ System (Bio-Rad). Membranes were washed between each step with 1X TBST for 15 minutes. A mouse anti–b actin monoclonal primary antibody (1:100 in 1% BSA; ab8226; Abcam) followed by an HRPconjugated donkey antimouse IgG polyclonal secondary antibody (1:1000 in 1% BSA; ab97030; Abcam) was used as a loading control for protein isolated from follicular and ovarian tissues. A rabbit anti-ZP3 polyclonal primary antibody (1:100 in 1% blocking buffer; ap17235b; Abgent) followed by HRPconjugated goat antirabbit IgG polyclonal secondary antibody (1:1000 in 1% blocking buffer; #170-6515; Bio-Rad) was used for protein isolated from oocytes and ZP as a loading control. Binding specificity was determined by preincubation of the Western blot membranes with either the rabbit antiZP3 polyclonal antibody (ap17235b; Abgent) or rabbit antiZP4 polyclonal antibody (ap12724b; Abgent) overnight before incubation with serum from a SpayVac-vaccinated mare overnight and use of the HRP-conjugated goat polyclonal secondary antibody to horse IgG (1:10,000 in 1% blocking buffer; ab102396; Abcam). 2.5. Immunohistochemistry Three consecutive 5-mm sections were sliced from tissue embedded in paraffin blocks and placed on two glass slides. The slides were incubated at 55 C for 30 minutes. Citrisolv (Thermo Scientific) along with an alcohol gradient was used to dewax and rehydrate the tissue slices. After rehydration, one of the three slices of tissues was subjected to a hematoxylin and eosin staining protocol. The slide was incubated in hematoxylin for 1 minute, rinsed
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under running tap water for 5 minutes, incubated in eosin for 5 minutes, rinsed once more under running tap water, and dehydrated through an alcohol and citrisolv gradient. The slide was then left to air dry before being mounted with a coverslip using Cytoseal (Thermo Scientific). The other slide, containing two tissue slices, was boiled in 10-mM sodium citrate (pH 6.0) for 20 minutes and blocked in 5% natural goat serum in 1% BSA (0.1-g BSA in 10-mL 1X PBS) for 2 hours. The two tissue slices were then incubated with a dilution (1:50 in 1% BSA) of serum from either a SpayVac-vaccinated mare or nonvaccinated mare overnight. The following day, both tissue slices were incubated with the HRP-conjugated goat polyclonal secondary antibody to horse IgG (1:1000 in 1% BSA; ab102396; Abcam) for 1 hour. Impact DAB Peroxidase Substrate Solution (Vector Laboratories, Burlingame, CA, USA) was then applied for 10 minutes. The slide contents were dehydrated through an alcohol and citrisolv gradient, allowed to dry, and mounted with a coverslip using cytoseal. The slide was washed between each step with 1X PBS for 10 minutes, and each incubation step took place in a moist chamber. All samples were imaged using light microscopy. Binding specificity of serum antibodies from SpayVacvaccinated mares was determined by presaturating a sample of serum from a SpayVac-vaccinated mare with ZP3 and ZP4 synthetic blocking peptides (bp17235b and bp12724b, respectively; Abgent). An aliquot of 30 mg of each peptide was added to 10 mL of serum in 500-mL 1% BSA before use as the primary antibody for immunohistochemical analysis. The HRP-conjugated goat antihorse IgG polyclonal antibody was used as the secondary antibody (1:1000 in 1% BSA; ab102396; Abcam). 3. Results 3.1. Immunoreactivity of serum antibodies from SpayVacvaccinated mares to equine zona protein Western blot analysis revealed the immunoreactivity of serum from SpayVac-vaccinated mares to equine zona protein. Antibodies in the serum of SpayVac-vaccinated mares bound to protein isolated from whole equine oocytes and equine ZP (Fig. 1A), as well as to protein isolated from equine ovarian and follicular tissues (Fig. 1C). These antibodies are not endogenously found in serum from nonvaccinated mares (Fig. 1B, D) and did not bind protein isolated from equine renal tissue (Fig. 1C). Western blot analyses of equine oocytes and ZP were repeated twice and analyses of equine tissues were repeated four times with different protein and serum samples to verify consistency. 3.2. Location of binding of serum antibodies from SpayVacvaccinated mares to equine ovarian tissue Immunohistochemical staining of equine ovarian tissue located the binding of serum antibodies from SpayVacvaccinated mares to the ZP of immature oocytes of primary follicles (Fig. 2E). These serum antibodies did not bind oocytes in primordial follicles, where the ZP was not yet formed as part of folliculogenesis (Fig. 2B). They are also not found in serum from nonvaccinated mares (Fig. 2C, F).
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Fig. 1. Immunoreactivity of serum antibodies from SpayVac-vaccinated mares for equine zona protein. Western blot analysis of protein isolated from the equine ZP of two oocytes, two whole equine oocytes (OC), 40-mg equine ovarian tissue (OT), 40-mg equine follicular tissue (FT), and 40-mg equine renal tissue (RT) using serum from a SpayVac-vaccinated mare (A and C) or serum from a nonvaccinated mare (B and D). Protein quantities are expressed per lane. Anti-ZP3 (B) and anti– b actin (D) are used as loading controls. Renal tissue was used as a negative control. Molecular weight standards appear in the leftmost lane of each gel.
Immunohistochemical analyses were repeated twice with different serum and tissue samples. 3.3. Specificity of serum antibodies from SpayVac-vaccinated mares for equine ZP3 The addition of ZP3 and ZP4 blocking peptides to a sample of serum from a SpayVac-vaccinated mare successfully saturated antibodies in the serum and blocked their ability to bind zona proteins in the tissue (Fig. 3C). In
the absence of blocking peptides, serum antibodies from the SpayVac-vaccinated mare bound to an immature oocyte in the ovarian tissue (Fig. 3B). Preincubation of a Western blot membrane with antiZP3 polyclonal antibody before incubation with serum from a SpayVac-vaccinated mare blocked the ability of serum antibodies to bind to the protein sample (Fig. 4A). These serum antibodies maintained their ability to bind the protein samples after preincubation with anti-ZP4 polyclonal antibody (Fig. 4B).
Fig. 2. Localization of serum antibodies from SpayVac-vaccinated mares to the ZP of immature oocytes. Immunohistochemical staining of equine ovarian tissue using serum from a SpayVac-vaccinated mare (B and E) or serum from a nonvaccinated mare (C and F). Serum antibodies bind the ZP of oocytes in primary follicles (E) but did not bind primordial follicles (B). Immunohistochemical staining using serum from nonvaccinated mares is not observed (C and F). H&E staining of ovarian tissue containing a primordial follicle (A) and primary follicle (D). Bar ¼ 20 mm. GC, granulosa cells.
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Fig. 3. Specificity of serum antibodies from SpayVac-vaccinated mares for zona protein. H&E staining of of ovarian tissue containing a primary follicle (A). Immunohistochemical staining of equine ovarian tissue using serum from a SpayVac-vaccinated mare (B) or serum from the same SpayVac-vaccinated mare after presaturation of the serum with ZP3 and ZP4 blocking peptides (C). Bar ¼ 20 mm.
4. Discussion 4.1. Immunoreactivity of serum antibodies from SpayVacvaccinated mares to equine zona protein Through Western blot analyses of mature preovulatory equine oocytes and ZP, we revealed the presence of antibodies in serum from SpayVac-vaccinated mares that are immunoreactive to equine zona protein. The use of serum from nonvaccinated mares as a control verified that these antibodies are not endogenous to the mare. Western blot analyses also confirmed the immunoreactivity of serum antibodies from SpayVac-vaccinated mares to protein isolated from equine ovarian and follicular tissues. 4.2. Location of binding of serum antibodies from SpayVacvaccinated mares to equine ovarian tissue The immunoreactivity of serum antibodies from SpayVacvaccinated mares to protein isolated from equine ovarian tissue is explained by the localization of antibodies to the ZP of immature oocytes in the tissue. We failed to locate the binding of these serum antibodies to follicular tissue. Because the synthesis of the ZP is an integrated process involving both the oocyte and granulosa cells [14,15], the immunoreactivity of serum antibodies from SpayVac-treated mares to equine follicular tissue is thought to be due to the presence of zona protein precursors in the granulosa cells. Although Western
blot analysis may have detected these zona protein precursors because of its high level of sensitivity and stringent cell lysis protocol to release intracellular proteins, high background staining observed in immunohistochemical analyses may have masked any localization to these cells [16]. High background staining is common when the tissue is of the same species as the secondary antibody used [16]. Conversely, the high background staining observed may also be indicative of an immunoreactivity between serum antibodies from SpayVac-vaccinated mares to other proteins located in follicular tissue. Because it is likely that the preparation of SpayVac results in nonzona proteins, such as ovarian and follicular proteins [17], it is possible that serum antibodies from SpayVac-vaccinated mares are immunoreactive to nonzona proteins as well. More research however is required to investigate the immunoreactivity of serum from SpayVacvaccinated mares to other ovarian and follicular proteins and how this immunoreactivity may relate to efficacy and reversibility of the vaccine. 4.3. Specificity of serum antibodies from SpayVac-vaccinated mares for equine ZP3 Our presaturation and preincubation experiments are indicative of both a specificity of serum antibodies from SpayVac-vaccinated mares for equine zona protein 3 and a lack of nonspecific binding. Because zona proteins are large glycoproteins with varying levels of posttranslational
Fig. 4. Specificity of serum antibodies from SpayVac-vaccinated mares for equine zona protein 3. Western blot analysis of protein isolated from equine ovarian tissue (OT), equine follicular (FT), and equine renal (RT) tissues using serum from a SpayVac-vaccinated mare after preincubation with rabbit anti-ZP3 polyclonal antibody (A) or rabbit anti-ZP4 polyclonal antibody (B). Anti–b actin antibody was used as a loading control. Renal tissue was used as a negative control. Molecular weight standards appear in the leftmost lanes of each gel. Forty micrograms of protein was loaded per lane.
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modifications, each protein can be observed in a range of molecular weights making the protein difficult to accurately identify by electrophoresis [18–20]. Our experiments lend clarity to the specificity of these serum antibodies. Likewise, this specificity for ZP3 is in agreement with the finding by Wassarman [18] that ZP3 is the major component of the porcine ZP, representing as much as 70% to 80% of total protein content. When the pZP antigen is isolated, the entire ZP is homogenized [21], resulting in a compilation of all zona protein families. It follows that 70% to 80% of the pZP antigen is likely accounted for by ZP3 and, when injected, the mare may produce substantially more antibodies against ZP3 than the other proteins. Furthermore, the pZP antigen is thought to work by eliciting the production of antibodies that bind to ZP proteins on mature oocytes, thereby interfering with sperm binding and penetration [8–10]. Because the zona protein families of ZP1, ZP2, and ZP4 are thought to only be involved in ZP structure, whereas ZP3 has a known role in sperm binding and penetration [20], this specificity for ZP3 coincides with the conclusions of prior studies. 4.4. Limitations of results There are a few limitations to our study. First, our study focused exclusively on SpayVac as it is less documented in published literature and potentially induces greater duration of infertility in field application. Although all three vaccines incorporate the pZP antigen, they are not a homogenous set of compounds. For instance, it is likely that the preparation of SpayVac results in more nonzona proteins, such as ovarian and follicular proteins, than the other two vaccines [17]. This variation in purity may account for the immunoreactivity of serum antibodies from SpayVacvaccinated mares to ovarian and follicular tissues seen in our experiments. Specifically, the high background staining observed during our immunohistochemical analysis of equine ovarian tissue may be due to the presence of antibodies immunoreactive to ovarian proteins produced as a result of SpayVac vaccination. Second, serum samples from vaccinated mares were selectively chosen for their reported high antibody titer levels. Differences in antibody titer and quality depend on the animal immunized and contribute to variability in efficacy among individuals [22]. Accordingly, although serum samples were taken from mares immunized with the same dose of SpayVac, one mare was still able to conceive despite vaccination. Nonetheless, our experiments showed that all eight mares produced antibodies immunoreactive to equine zona protein, regardless of whether the antibodies successfully suppressed the mare’s fertility. It is likely that although each mare had sufficient serum antibodies to successfully bind the protein in our experimental samples, overall availability of these antibodies in vivo may not have been enough to saturate all possible protein-binding sites and thus result in infertility. 4.5. Conclusions Our results suggest a model where serum antibodies produced in response to SpayVac vaccination are
immunoreactive to equine zona proteins in vitro. When zona proteins are isolated from equine tissues and made available to serum samples from SpayVac-vaccinated mares, antibodies in the serum are consistently able to recognize the protein. These serum antibodies appear immunoreactive not only to the ZP of mature preovulatory oocytes isolated from Graafian follicles but also to immature oocytes embedded in ovarian tissue. Although we illustrate a pathway by which serum antibodies from SpayVac-vaccinated mares could act if they are biologically available to these tissues under the correct conditions, it has yet to be investigated whether these antibodies are present in the follicular fluid of follicles or able to permeate ovarian tissue after SpayVac vaccination. We simply showed a model by which antibodies produced in response to SpayVac vaccination may bind zona proteins in vivo, thereby interfering with a critical reproductive component, and result in infertility. Acknowledgments This research was funded by the United States Geological Survey Wildlife Program and conducted through a cooperative effort between the USGS Fort Collins Science center, Bureau of Land Management, United States Department of Agriculture’s Animal and Plant Health Inspection Service, and Colorado State University. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the United States Government or Colorado State University. This research could not have been conducted without the support, cooperation, laboratory use, and sample contribution of the U.S. Geological Survey, Bureau of Land Management, and Colorado State University. References [1] National Research Council. Using science to improve the BLM wild horse and burro program: a way forward. Washington, DC: National Academies Press; 2013. [2] Kirkpatrick JF, Lyda RO, Frank KM. Contraceptive vaccines for wild: a review. Am J Reprod Immunol 2011;66:40–50. [3] Pashine A, Valiante NM, Ulmer JB. Targeting the innate immune response with improved vaccine adjuvants. Nat Med Suppl 2005;11: 563–8. [4] Barber MR, Fayrer-Hosken RA. Evaluation of somatic and reproductive immunotoxic effects of the porcine zona pellucida vaccination. J Exp Zool 2000;286:641–6. [5] Turner JW, Liu IKM, Flanagan DR, Rutberg AT, Kirkpatrick JF. Immunocontraception in wild horses: one inoculation provides two years of infertility. J Wildl Manage 2007;71:662–7. [6] Willis P, Heusner GL, Warren RJ, Kessler D, Fayrer-Hosken RA. Equine immunocontraception using porcine zona pellucida: a new method for remote delivery and characterization of the immune response. J Equine Vet Sci 1994;14:364–70. [7] Turner JW, Kirkpatrick JF, Liu IKM. Effectiveness, reversibility, and serum antibody titers associated with immunocontraception in captive white-tailed deer. J Wildl Manage 1996;60:45–51. [8] Sacco AG. Antigenic cross-reactivity between human and pig zona pellucida. Biol Reprod 1977;16:164–73. [9] Liu IKM, Bernoco M, Feldman M. Contraception in mares heteroimmunized with pig zonae pellucidae. J Reprod Fertil 1989;85: 19–29. [10] Kirkpatrick JF, Liu IKM, Turner Jr JW. Remotely-delivered immunocontraception in feral horses. Wildl Soc Bull 1990;18:326–30. [11] Skinner SM, Mills T, Kirchick HJ, Dunbar BS. Immunization with zona pellucida proteins results in abnormal ovarian follicular
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