Immunoneutralization of native GnRH-I
A N-terminal modified GnRH-I peptide was conjugated to tetanus toxoid (TT), TT-CHWSYGLRPGNH2 (TT-GnRH-I), as described previously and used to immunize male animals (NZB X BALB/c F1) mice, Sprague–Dawley rats, Beagles and Scottish Suffolk-crossbred rams). The mice and rats (aged 6 and 7 weeks old, respectively, n ¼ 5) were randomized and immunized at fortnightly intervals with 50 mg equivalent of GnRH-I, intraperitoneally (i.p.). An equivalent sized group for each species was not immunized (untreated controls). Adult dogs (aged 3 years old, n ¼ 4) were immunized subcutaneously with either 50 mg (low dose) or 200 mg (high dose) equivalent of GnRH-I at fortnightly intervals (study weeks 0, 2, 4 and 6). Adult post-pubertal rams (aged 18 months old) were immunized intramuscularly (i.m.) with 200 mg equivalent of GnRH-I in study weeks 0, 3 and 6. In general, the subcutaneous route of administration is preferred for this type of vaccine in larger animals, but better responses can be obtained with i.m injections in the sheep. The immunogen was made up to 100 ml (mice and rats) or 1 ml (dogs) with PBS, pH 7.4 and adsorbed with an equal volume of Imject alum (Perbio Science Ltd., UK) for 30 min before use. The sheep work was carried out at an off-site facility, therefore the formulation was made up sterile, consisting of equal volumes (1 ml) immunogen and Imject alum, with additional 0.01% (w/v) sodium azide. The formulation was stored at 4 8C until used. In order to check the effect of the addition of sodium azide on immunogenicity, a group of mice, were immunized with this variation in formulation but found to show no significant differences (data not shown). Immunized animals were monitored to assess the effect of the vaccine over a 3-month-period. During the study, weekly body weights, endocrine, antibody and gonadal measurements (testicle width in the mice and rats, testicle diameter in the rams and scrotal diameter in the dogs) were taken. All the following measurements were carried out blind, by randomly coding the samples and the treatments were not revealed until the end of the study. The observers and the analysts were unaware of the treatments given to the animals.
Histological evaluation of the reproductive organs following GnRH-I ablation
Following sacrifice of the animals, the testes, epididymides, seminal vesicles (excluding the dog) and prostate from the untreated control and treated animals were weighed and prepared for histology. In the case of the dog and sheep, sections were prepared from untreated animals of equivalent age to the experimental groups. The organs were fixed for 24 h in PBS, containing 10% (v/v) formalin and stained with haematoxylin and eosin, or Goldner’s trichrome as described before. Morphological changes in the accessory sex glands were evaluated under a microscope at low (100X) and high (400X) magnification. Fifty randomly selected tubular crosssections were studied with respect to the presence of spermatozoa, round and elongated spermatids, spermatogonia, testicular macrophages and Leydig cells. Scoring system was developed to quantify human spermatogenesis, a modification to account for the difference in seminiferous tubular epithelium between humans and dogs. This modified score system was used in this study. A score of 10 was assigned when the germinal epithelium was organized with a regular thickness and an open lumen, with 50–80% of the lumen containing spermatozoa. An average score of 7 was considered the lowest standard for a normal testes, while an average score <7 was considered a positive anti-fertility effect on spermatogenesis. However, all other scores of testicular spermatogenesis were evaluated based on the criteria described in Table 1.
Measurement of GnRH-I and tetanus toxoid specific antibody levels
For the rodents, 3 days after each immunization tail bleeds were carried out into heparinized capillary tubes (Hawsley and Sons Ltd., UK) and the blood centrifuged at 1000 X g for 20 min to obtain plasma for measurement of antibody levels. In the rams, 10 ml jugular vein blood was collected into 8 ml lithium heparin plasma separation vacuettes, at fortnightly intervals prior to immunisation and the blood was centrifuged at 1000 X g for 20 min to obtain plasma. The plasma was decanted and stored at -20 8C until analysed. In the dogs, blood sampling was carried out by jugular venepuncture (4 ml) into heparinized tubes prior to immunisation, at fortnightly intervals and plasma separated following centrifugation at 1000 X g for 20 min. The antibody responses were determined using BSA-CHWSYGLRPG-NH2-coated 96 well tissue culture plates (TPP, Finland). The peptide conjugate was diluted in PBS, pH 7.4 and incubated (1 mg/ 100 ml per well) at 37°C for 1 h. The plates were washed three times with wash buffer (PBS, pH 7.4 and 0.05% Tween-20) and blocked with 150 ml per well 3% (w/v) non-fat milk protein solution (Marvel, Premier Brands,UK) in wash buffer for 1 h at 37 8C. The rodent plasma samples were diluted 1:1000 with PBS and 100 ml per well incubated at 37 8C for 1 h. The sheep and dog plasma were diluted in blocking solution and left standing for 30 min at room temperature to decrease high background levels and non-specific binding, prior to incubating 100 ml per well at 37°C for 1 h. All samples were carried out in triplicate. The plates were washed three times with wash buffer and replaced with 100 ml per well horse-radish peroxidase labelled H þ L specific IgG diluted with PBS (1:5000 dilution goat anti-mouse or goat anti-rat or rabbit anti-sheep from Perbio Science Ltd., UK or 1:1000 dilution rabbit antidog from Sigma–Aldrich Ltd.). The plates were incubated for 45 min at 37°C, washed three times with PBS (without Tween) and developed with TMB substrate 100 ml per well (250 ml of stock 6mg/ml 3,30,5,50-tetramethyl benzidine in dimethylsulphoxide, added to 25 ml 0.1 M sodium acetate buffer, pH 5.5 with 4 ml 30% (v/v) hydrogen peroxide). The reaction was stopped with 50 ml per well 10% (v/v) sulphuric acid after 15 min and the A450 read. The means of the triplicate readings were calculated and plotted on a graph against the study week. In order to examine the antibody reponse to tetanus toxoid in the sheep and dogs, the plates were coated with tetanus toxoid, instead of the BSA conjugate and the method described above followed.
Endocrine analysis
Testosterone concentrations in the plasma were determined using a direct competitive radioimmunoassay (Coat-a-Count Total Testosterone; Euro/ DPC Ltd., UK), according to the manufacturers’ instructions. For the rodents, it was only feasible to take single measurements at the end of the study. However, it was possible to sample the larger animals more frequently; the sheep were sampled six times, at hourly intervals and the dogs 17 times every 30 min, at the beginning and end of the study.
Statistical analysis
A Student’s t-test (unpaired) was carried out using Statview Version 5.1 software.