Evaluation of infertility efficacy of the E. coli expressed STF2-GnRH vaccine in male cats.

Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus and anti-GnRH antibodies are not formed under normal conditions. However, administration an excess of recombinant GnRH protein results in the formation of anti-GnRH. We evaluated the efficacy of the recombinant Salmonella typhimurium flagellin fljB (STF2)-GnRH vaccine in inducing infertility in 17 intact male cats. The first vaccination and a boosting vaccine was injected for examination. Serum was obtained from blood collected at monthly intervals and anti-GnRH antibodies and testosterone concentrations were determined. Six months after the vaccination, testicular samples are obtained and used for histological examination. Compared with sham control group, the injection groups showed an increase in anti-GnRH antibody titers and testosterone concentrations tended to be reduced in the injection groups and increased in the control group. Histological evaluations and Johnsen's testicular biopsy scores revealed testicular hypoplasia in the 2 injection groups. Consequently, normal sexual maturation with sperm production was observed in the control group. In contrast, the cats that received the GnRH vaccine showed weak (2 of 7 cats) or moderate (4 out of 7 cats) dose-dependent infertility effects. On the basis of the results, the STF2-GnRH vaccine was identified to be effective in inducing infertility in male cats. The results of this study thus indicate the possibility of immunological castration targeting feral cats.

Rationalising drug delivery using nanoparticles: a combined simulation and immunology study of GnRH adsorbed to silica nanoparticles.

Silica nanoparticles (SiNPs) have been shown to have significant potential for drug delivery and as adjuvants for vaccines. We have simulated the adsorption of GnRH-I (gonadotrophin releasing hormone I) and a cysteine-tagged modification (cys-GnRH-I) to model silica surfaces, as well as its conjugation to the widely-used carrier protein bovine serum albumin (BSA). Our subsequent immunological studies revealed no significant antibody production was caused by the peptide-SiNP systems, indicating that the treatment was not effective. However, the testosterone response with the native peptide-SiNPs indicated a drug effect not found with cys-GnRH-I-SiNPs; this behaviour is explained by the specific orientation of the peptides at the silica surface found in the simulations. With the BSA systems, we found significant testosterone reduction, particularly for the BSA-native conjugates, and an antibody response that was notably higher with the SiNPs acting as an adjuvant; this behaviour again correlates well with the epitope presentation predicted by the simulations. The range of immunological and hormone response can therefore be interpreted and understood by the simulation results and the presentation of the peptides to solution, paving the way for the future rational design of drug delivery and vaccine systems guided by biomolecular simulation.

ZNF185-derived peptide induces fertility suppression in mice.

Zinc finger protein 185 (ZNF185) belongs to the ZNF family and is involved in male reproduction. However, it is unclear whether ZNF185 may be a target candidate for contraceptive vaccines. In this study, antigenic peptides derived from ZNF185 were prepared, and their immune contraceptive effects were investigated using mice. Results from enzyme-linked immunosorbent assay (ELISAs) showed that peptide immunization induced an antibody titre increase that reached a peak in week 12. Peptide-3 and peptide-4 were then chosen for subsequent experiments. The results of the fertility assays showed that peptide immunization inhibited the mating and fertility rates of the mice, whereas there were no obvious changes in the number of pups per litter. Subsequently, epididymal sperm was analysed. The results demonstrated that the sperm count and sperm motility were significantly decreased in the peptide group, while the amount of abnormal sperm was significantly increased in the peptide-3 group. The male reproductive organs were also evaluated. There were no obvious differences in testis or epididymal weights, in the diameters of the seminiferous tubules, or in the thicknesses of the seminiferous epithelium between the peptide group and the phosphate buffer saline (PBS) group. In addition, histological analysis indicated that there were no obvious pathologic changes in testis and epididymal histology in the peptide group; however, the number of spermatozoa present in the epididymal lumen of the peptide group was significantly decreased when compared with the PBS group. Our study demonstrates for the first time that peptides derived from ZNF185 may induce fertility suppression in mice without damaging reproductive organs. These peptides have the potential to be used as a male contraceptive vaccine.

Two B-cell epitope vaccines based on uPA effectively inhibit fertility in male mice.

uPA, a trypsin-like serine protease, was found to take active part in male reproduction. Our previous work had demonstrated the antifertility effects of its full length protein immunization, but with immune tolerance and other latent side effects. Here we discovered two effective B-cell epitopes of uPA for male contraception in growth factor-like domain and kringle domain respectively. Together with carrier protein, immunization of these two epitope peptides could induce high titers of specific antibodies in male mice. Significant reduction of fertility was observed in these two groups in mating trial without evident systemic illness or abnormal mating behavior. Epididymal sperms of immunized males exhibited impaired progressive motility and ability to fertilize eggs in vitro. The immunization of another predicted epitope in serine protease domain and the control groups showed no similar positive results. Importantly, T cells were not activated after the challenge of these B-cell epitopes itself, which suggests that these vaccines do not induce cell-mediated autoimmunity. Taken together, our study discovered two uPA B-cell epitopes as novel targets for male immunocontraception with minimum side effects. Considering their high identity with human uPA protein, these two epitope vaccines hold great promise to be developed for man use in the future.

Contraceptive efficacy of recombinant fusion protein comprising zona pellucida glycoprotein-3 fragment and gonadotropin releasing hormone.

Contraceptive vaccines have been used for the management of wildlife population. In the present study, we have examined the contraceptive potential of Escherichia coli-expressed recombinant fusion protein comprising of 'promiscuous' T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker (KK), dog ZP3 fragment (aa residues 307-346), triglycine spacer (GGG), T cell epitope of bovine RNase (bRNase; aa residues 94-104), GnRH, T cell epitope of circumsporozoite protein of Plasmodium falciparum (CSP; aa residues 362-383), and GnRH. SDS-PAGE analysis of the purified refolded protein revealed a dominant ∼12 kDa band, which in Western blot reacted with mouse polyclonal antibodies against dog ZP3 fragment and mouse monoclonal antibodies against GnRH. Immunization of female FvB/J mice following two booster schedule with the above recombinant protein supplemented with alum led to high antibody titres against the immunogen as well as ZP3 and GnRH as determined by ELISA. The immune sera reacted with zona pellucida of mouse oocyte and also inhibited in-vitro fertilization. The qRT-PCR studies showed decrease in the ovarian GnRH receptor in mice immunized with the recombinant fusion protein. Mating studies revealed high contraceptive efficacy of the recombinant protein as in two independent experiments, 90% of the immunized female mice failed to conceive. Following one booster immunization schedule, 50% of the immunized female mice failed to conceive. However, in adjuvanted controls, all the female mice became pregnant. To conclude, the recombinant protein described herein has a good potential to be developed as candidate contraceptive vaccine.

Complexes of trophoblastic peptides and heat shock protein 70 as a novel contraceptive vaccine in a mouse model.

The concept of contraceptive vaccines has interested reproductive biologists and immunologists for nearly 2 decades, but no approach has been approved. In this study, a new immunocontraceptive vaccine that targets placental trophoblasts was expored. We demonstrated that after in-vitro binding with heat shock protein 70, trophoblast-derived peptides can activate T cells both in vitro and in vivo. The activated T cells have a Th1 bias and specifically cause cytolysis of trophoblasts, leading to the termination of pregnancy. Such activated T cells seem to have an effect on early gestation, rather than influencing preimplantation. We did not observe side-effects of this vaccine in mice. In conclusion, a novel contraceptive strategy is described that uses heat shock protein 70-trophoblastic peptide complexes to generate a specific T-cell immune response against placental trophoblasts. This type of vaccine targeting the post-implantation phase does not generate a permanent effect but possibly raises an ethical issue.

Vaccination with an Epitope Peptide of IZUMO1 to Induce Contraception in Female Mice.

PROBLEM: The development of a new and suitable contraceptive methods, as well as in-depth and systematic research into underlying contraceptive mechanisms, is crucial. IZUMO1 plays an important role in the fusion of the sperm and ovum during fertilization. Izumo(-/-) mice are infertile. Therefore, IZUMO1 may be a potential target for the development of a contraceptive vaccine. METHOD OF STUDY: Linear B-cell epitopes (BCE) were identified in IZUMO using biosynthetic peptides and used to immunize female mice. RESULTS: Five IZUMO BCE were identified: DLVLDCL177-183, YSFYRV196-201 (named BCE-2), YLT217-219, SMVGPED221-227, and DAGNY228-232. Active immunization with the BCE-2 vaccine sharply decreased the fertility rate in female mice in a safe and reversible manner. In vitro fertilization showed that the BCE-2 vaccine interferes with and blocks the fusion of the sperm and the ovum. CONCLUSIONS: B-cell epitopes-2 may be a new candidate for the development of contraceptive vaccine due to its effectiveness, safety, and reversibility.

Ovarian and oocyte targets for development of female contraceptives.

INTRODUCTION: Steroid hormone-based contraceptives have been used by women for long time since their introduction. Efforts have been made to make steroidal contraceptives cost-effective, safe and improve their users' compliance. In addition, attempts have been made to develop nonsteroidal contraceptives. Contraceptive vaccines have been investigated as an alternate strategy for contraception. AREAS COVERED: The currently used steroidal contraceptives are reviewed. In addition, status of emerging nonsteroidal contraceptives that inhibit folliculogenesis, oocyte maturation, ovulation and endometrium receptivity targeting phosphodiesterase 3, angiopoietins, gonadotropin-releasing hormone, COX-2, progesterone/estrogen receptor and follicle-stimulating hormone receptor are presented. Various approaches to develop contraceptive vaccines aiming to inhibit ovarian follicle development, ovulation, fertilization and implantation including their current applications and limitations are discussed. EXPERT OPINION: Development of new nonsteroidal contraceptives, in addition to long-acting steroidal contraceptives, is pertinent for offering wider choice to women. It is imperative that basic research to discover new targets in the ovaries must be undertaken to facilitate development of novel contraceptives. Further, efforts on studying the feasibility and safety of contraceptive vaccines may be continued to bring these within the realm of application as contraceptives for humans.

Construction of a catsper1 DNA vaccine and its antifertility effect on male mice.

Cation channel of sperm 1 (CATSPER1) is a unique sperm cation channel protein, and essential for sperm function and male fertility. CATSPER1 exclusively expresses in meiotic and postmeiotic spermatogenic cells, thus belongs to the spermatogenesis-specific antigen that escape central tolerance. We have previously demonstrated the immunocontraceptive potential of its transmembrane domains and pore region, and reported the antifertility effects of its B-cell epitopes on male mice. Aiming to develop DNA vaccine targeting CATSPER1 for male contraception, here the whole open reading frame of mouse Catsper1 was cloned into the plasmid pEGFP-N1 to obtain a DNA vaccine pEGFP-N1-Catsper1. The vaccine was confirmed to be transcribed and translated in mouse N2a cell in vitro and mouse muscle tissue in vivo. Intramuscular injection with the vaccine on male mice induced specific immune reaction and caused significant inhibition on sperm hyperactivated motility and progressive motility (P<0.001 for both), and consequently reduced male fertility. The fertility rate of experimental group was 40.9%, which was significant lower (P=0.012) than control group (81.8%). No significant change in mating behavior, sperm production and histology of testis/epididymis was observed. Given that Catsper1 exhibits a high degree of homology among different species, Catsper1 DNA vaccine might be a good strategy for developing an immunocontraceptive vaccine for human and animal use.

Effect of anti-gonadotropin-releasing factor vaccine and band castration on indicators of welfare in beef cattle.

Angus crossbred bulls (n = 60; 257 ± 5.4 d of age; initial BW 358.8 ± 3.78 kg) were used to study the effect of a vaccine against gonadotropin-releasing factor (GnRF) and band castration on behavioral and physiological indicators of pain. Cattle were randomly assigned to 1 of 3 treatments: bulls, band-castrated calves without pain mitigation (castrated), and immune-vaccinated animals administered an anti-GnRF vaccine (vaccinated). All animals were fitted with a radio frequency ear tag so that individual animal feed intake and feeding behavior were recorded daily over the entire trial using an electronic feed bunk monitoring system. Two doses of anti-GnRF vaccine were administrated on d -35 and 0 and band castration was performed on d 0. Animal BW was recorded weekly starting on d -36 until d 56. Visual analog scores (VAS) were measured on d -36 -35, -1, and 0, and salivary cortisol concentration was measured at -30, 0, 30, 60, 120, and 270 min on d -35 and 0 after castration. Saliva and blood were obtained on d 1, 2, 5, and 7 and weekly until d 56 for determination of cortisol and complete blood cell count. Video data were collected for pain, sexual, and aggressive behavior daily the first week and once a week until d 56. Data were analyzed with a mixed-effect model with castration, time, and their interactions as main effects. Vaccinated calves had reduced ADG and intake (P < 0.05 and P < 0.001, respectively) during the first week after vaccination. Band-castrated calves had reduced ADG and intake (P < 0.001) until the end of the study. No differences in salivary cortisol and VAS were observed among groups at d -35 after the first vaccination and before band castration. However, on d 0, castrated cattle had greater cortisol concentrations and VAS (P < 0.05 and P < 0.01, respectively) than bulls and vaccinated animals. Complete blood cell count did not differ (P > 0.05) between treatments on d 0, 1, and 2. At d 56, vaccinated calves had greater (P < 0.05) final BW than band-castrated calves and both had less final BW than bulls. There was no indication that vaccination caused any physiological or behavioral changes indicative of pain. In contrast, band castration resulted in elevated cortisol scores and VAS indicative of a pain response and behavior related to pain (P < 0.001) until d 42 of the study. The present study demonstrates that anti-GnRF vaccine is a viable animal welfare-friendly alternative to traditional band castration in beef cattle under North American feedlot practices.

[Contraceptive vaccinogen - current state of development]. / Stav vývoje kontracepcního vakcinogenu.

OBJECTIVE: To present an overview of published results of research and practice in the field of searching for the contraceptie vaccinogen. DESIGN: Review article. SETTINGS: Department of Gynecology and Obstetrics, Medical Faculty in Pilsen, Charles University in Prague and University Hospital in Pilsen. OBJECTIVE OF STUDY: The issue of fertility is a growing problem especially in developed countries. The questions of contraception in terms of family planning are intensively discussed. Regardless of the unfavorable development of the number of the population in developed countries, various kinds of contraceptives are still in demand. The widespread availability and reliability confirms a dominant position of hormonal contraception. Due to the known and contemplated negative effects of hormonal contraceptives on the individual as well as on the environment, the immunological aproach offers to become an option with long lasting effect. Therefore it is not surprising that research on contraceptive vaccinogen is most supported in countries such as China or India. Experiences in research and treatment of otherwise unexplained infertility are also applicable in opposing intentions as immunologically induced down-regulation of fertility. CONCLUSION: Long term research of immunogenicproperties of human sperm indicates, that the possible induction of an immune response to this gametes currently appears to be the safest and also the most difficult way to achieve immunological contraception potentially in both, man and woman.

Effects of Improvac and Bopriva on the testicular function of boars ten weeks after immunization.

This study investigated the effects of Improvac and Bopriva, two anti-GnRF immunization products, on testicular function in boars. We predicted that both products would diminish testicular function; however, we specifically tested the hypothesis that the duration of efficacy for Bopriva would be longer than that of Improvac. Animals were immunized with either Improvac or Bopriva and then observed ten weeks after the second injection. Serum GnRF antibody titers rose after the second injection and peaked approximately two weeks later. At the same time testosterone concentrations decreased to undetectable levels and remained below assay detection for at least six weeks. At approximately eight weeks, testosterone began to increase in animals treated with Improvac though levels remained decreased in Bopriva treated animals throughout the ten weeks. Daily sperm production at 10 weeks was significantly reduced in both treatment groups; however, the reduction was greater in Bopriva treated boars. Examination of testes of both treatments revealed incomplete spermatogenesis with impaired spermatid production and reduced seminiferous tubule diameter. These findings were universal in Bopriva treated animals, but Improvac treated animals exhibited morphologies intermediate between Bopriva treated animals and control boars. Overall testicular function in Bopriva boars remained suppressed ten weeks post-immunization while Improvac boars appeared to be recovering.

Preparation, characterization, and determination of immunological activities of transfer factor specific to human sperm antigen.

OBJECTIVE. The objective of this study was to prepare, characterize, and determine immunological activities of specific transfer factor (STF) specific to human sperm antigen (HSA) for the preparation of antisperm contraceptive vaccine that can be used as an immunocontraceptive. METHODS. HSA-STF was prepared using the spleens of rabbits vaccinated with HSA. The specific immunological activities were examined by lymphocyte proliferation test (LPT), leukocyte adhesion inhibition test (LAIT), and by determining the concentrations of IL-4, γ -IFN, and IL-21. HSA-STF was a helveolous substance, having a pH value of 7.0 ± 0.4 and UV absorption maxima at 258 ± 6 nm. It contained seventeen amino acids; glycine and glutamic acids were the highest in terms of concentrations (38.8 µ g/mL and 36.3 µ g/mL, resp.). RESULTS. The concentration of polypeptide was 2.34 ± 0.31 mg/mL, and ribose was 0.717 ± 0.043 mg/mL. The stimulation index for lymphocyte proliferation test was 1.84, and the leukocyte adhesion inhibition rate was 37.7%. There was a statistically significant difference between the cultural lymphocytes with HSA-STF and non-HSA-STF for γ -IFN and IL-21 (P < 0.05), but there was no statistical significance for IL-4 (P > 0.05). CONCLUSION. HSA-STF was prepared and characterized successfully. It had immunological activity which could transfer the immune response specific to HSA and prove to be a potential candidate for the development of male immunocontraceptive agents.

The use of contraception as a disease management tool in wildlife.

Contraception offers potential as a tool for managing certain diseases in wildlife, most notably venereally transmitted diseases or diseases transmitted at parturition. Brucellosis is an excellent example of an infectious disease present in wild populations that could potentially be managed through immunocontraception. Previous studies in bison (Bison bison) suggest that a single injection of GonaCon (National Wildlife Research Center, U.S. Department of Agriculture/Animal and Plant Health Inspection Service/Wildlife Services, Fort Collins, Colorado 80521, USA) results in 3 or more yr of infertility. Ongoing studies will determine if the use of GonaCon in bison decreases shedding of Brucella abortus from infected animals and will better define the duration of infertility following a single injection

Long-term methods and effects of remotely treating wildlife with immunocontraception.

The development of sophisticated delivery equipment, as well as safer and more effective drugs, has made remote delivery of animal drugs a standard and readily available tool for wildlife professionals, veterinarians, ranchers, and animal control officers. In the 1980s, researchers began treating a wide variety of wildlife with injectable porcine zona pellucida immunocontraceptive vaccines. Remote delivery of immunocontraceptives has been proven effective at the individual and population level for wild horses and urban deer. However, it took only a short time at each study site to understand that each time an animal was treated with remotely delivered darts, it became more difficult to re-treat; researchers were required to adjust to the increased wariness of their targets. Multiyear vaccines will not reduce the need for researchers who can adapt to the many challenges of applying these nonlethal methods of population control in the field. Training, experience, and persistence are required for field personnel to adapt and develop new techniques for continued retreatment of previously treated free-ranging animals.

Steroid hormones, boar taint compounds, and reproductive organs in pigs according to the delay between immunocastration and slaughter.

The producer of vaccine against GnRH recommends that immunocastrated pigs are to be slaughtered within 4 to 6 weeks after the second vaccination (V2). The objective of the study was to examine the effect of shorter or longer delay on steroid hormones, boar taint compounds, and morphologic and histologic traits of reproductive organs. Forty male pigs (individually housed and fed a commercial diet) were assigned within litter to four treatment groups, 10 pigs were left entire (EM27) and the others were vaccinated against GnRH (Improvac, Pfizer Animal Health) at the age of 12 and 19 weeks. Pigs were slaughtered at 21 (IC21), 24 (IC24), and 27 (IC27 and EM27) weeks of age. Two EM27 pigs died during the experiment, one IC21 pig was excluded because of illness, one IC27 pig was a nonresponder, and two pigs (IC24 and IC27) were hermaphrodites. To assess the effect on steroid hormones, blood was taken at 12, 15, 19, 21, and 24 weeks of age. Subcutaneous fat and reproductive organs were sampled after slaughter for determination of androstenone, skatole, morphologic, and histologic measurements. Immmunocastration interrupted the rise of estrogen and caused a substantial fall of testosterone in IC21, IC24, and IC27 pigs. As a result, androstenone and skatole levels were successfully reduced regardless of the time elapsed from V2. The weight of the reproductive organs was also drastically reduced, the shrinkage being proportional to the length of the interval between V2 and slaughter and was the most evident for vesicular glands, followed by bulbourethral glands, and testes. Corresponding changes were observed also on a histologic level with a progressive decrease in the size and number of Leydig cells, a diminishing immunoreactivity of 3ß-hydroxysteroid dehydrogenase/Δ-5-4 isomerase, and luteinizing hormone receptor, along with a shrinkage of tubuli seminiferi, atrophy of seminiferous epithelium, and a loss of germ cells, indicating a disruption in testicular spermatogenetic function. Regression of the glandular tissue with a decreasing amount of secreta was also observed for bulbourethral and vesicular glands. The investigated physiologic, morphologic, and histologic traits were progressive with the increasing delay to slaughter (clearly seen already 2 weeks after V2), though no signs of functional or morphological restoration was observed within 8 weeks after V2.

Vaccination against gonadotropin-releasing factor (GnRF) with Bopriva significantly decreases testicular development, serum testosterone levels and physical activity in pubertal bulls.

The aim of this study was to evaluate the effects of vaccination against gonadotropin-releasing factor (GnRF) on testicular development, testosterone secretion, and physical activity in pubertal bulls. The experiment was performed using 44 bulls aged between 6 and 7 mo. Twenty-three animals were vaccinated twice 4 wk apart with 1 mL of Bopriva (Pfizer, Animal Health, Parkville, Australia) and 21 bulls served as matched controls. Serum GnRF antibody titer and testosterone concentration as well as body weight and scrotal circumference were determined in all bulls for 24 wk from the first vaccination. In addition, physical activity was analyzed in 11 vaccinated and in 10 control animals using the ALPRO DeLaval activity meter system (DeLaval AG, Sursee, Switzerland). The results show that vaccination significantly (P < 0.05) influenced all parameters evaluated except body weight. Antibody titers to GnRF began to rise 2 wk after the first vaccination and reached peak values 2 wk after the second injection. Significant group differences in anti-GnRF titer were present for 22 wk following the first vaccination. Testosterone concentrations were significantly lower between weeks 6 to 24 after first vaccination in bulls with Bopriva compared with control animals. In vaccinated bulls testicular development was impaired after the second injection and scrotal circumference was significantly smaller between weeks 8 to 24 after first vaccination. Physical activity of vaccinated bulls was reduced after the booster injection with significant group differences for a continuous period of 106 days. In conclusion, vaccination against GnRF with Bopriva in pubertal bulls decreased testosterone levels in peripheral blood, testicular development, and physical activity but did not affect weight gain.

Passive transfer of maternal GnRH antibodies does not affect reproductive development in elk (Cervus elaphus nelsoni) calves.

Gonadotropin-releasing hormone is intermittently released from the hypothalamus in consistent patterns from before birth to final maturation of the hypothalamic-pituitary-gonadal axis at puberty. Disruption of this signaling via GnRH vaccination during the neonatal period can alter reproduction at maturity. The objective of this study was to investigate the long-term effects of GnRH-antibody exposure on reproductive maturation and function in elk calves passively exposed to high concentrations of GnRH antibodies immediately after birth. Fifteen elk calves (eight males and seven females) born to females treated with GnRH vaccine or sham vaccine during midgestation were divided into two groups based on the concentration of serum GnRH antibodies measured during the neonatal period. Those with robust (>15 pmol (125)I-GnRH bound per mL of serum) titers (N = 10; four females and six males) were designated as the exposed group, whereas those with undetectable titers (N = 5; three females and two males) were the unexposed group. Onset of puberty, reproductive development, and endocrine function in antibody-exposed and unexposed male and female elk calves were compared. Neonatal exposure to high concentrations of GnRH antibodies had no effect on body weight (P = 0.968), endocrine profiles (P > 0.05), or gametogenesis in either sex. Likewise, there were no differences between groups in gross or histologic structure of the hypothalamus, pituitary, testes, or ovaries. Pituitary stimulation with a GnRH analog before the second potential reproductive season induced substantial LH secretion in all experimental elk. All females became pregnant during their second reproductive season and all males exhibited similar mature secondary sexual characteristics. There were no differences between exposure groups in hypothalamic GnRH content (P = 0.979), pituitary gonadotropin content (P > 0.05) or gonadal structure. We concluded that suppressing GnRH signaling through immunoneutralization during the neonatal period likely does not alter long-term reproductive function in this species.

Birth control vaccine targeting leukemia inhibitory factor.

The population explosion and unintended pregnancies resulting in elective abortions continue to impose major public health issues. This calls for a better method of contraception. Immunocontraception has been proposed as a valuable alternative that can fulfill most, if not all, of the properties of an ideal contraceptive. There are several targets that are being explored for contraceptive vaccine development. Leukemia inhibitory factor (LIF), a member of interleukin-6 family, is required for embryo development and successful blastocyst implantation in several mammalian species. The present study was conducted to examine if LIF can be a target for the development of a birth control vaccine. Three sequences from LIF and two sequences from LIF-receptor (LIF-R) that span the regions involved in ligand-receptor binding were delineated, and peptides were synthesized based upon these sequences. Antibodies raised against these five peptides reduced LIF bioactivity in an in vitro culture assay using BA/F3 mLIF-R-mpg130 cells. Vaccines were prepared by conjugating these peptides to various carrier proteins. Immunization of female mice with these peptide vaccines induced a long-lasting, circulating as well as local antibody response in various parts of the genital tract, and resulted in a significant (P ≤ 0.05) inhibition in fertility in all the three trials; the LIF-R peptide vaccines proved to be a better vaccine target. The data indicate that LIF/LIF-R is an excellent target for the development of a birth control vaccine. This is the first study, to our knowledge, that examined LIF/LIF-R as a target for immunocontraception. The findings of this study can be easily translated to humans since LIF/LIF-R is also important for implantation and pregnancy in women.

Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice.

The transforming growth factor ß (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.