Immunogen Preparation
The peptide sequences (2 µmol, 90% purity, synthesized by Immune Systems Ltd, Torquay, UK) were conjugated to TT (0.1 µmol) using sulpho-maleimido benzoic succinimide (S-MBS, Perbio Science Ltd, Cheshire, UK) as described previously.
Immunization Schedule
Male Sprague–Dawley rats were housed in a fully climatized room: room temperature 220C; relative humidity 55%; light and dark cycles of 12 hr each; illumination 60 Lux. In study week 0, at the age of 7 weeks, the rats were randomized, divided into groups of eight and ear-coded. The immunogen (50 µg equivalent of peptide) was mixed in a 1:1 ratio with an aluminium hydroxide based adjuvant, Imject alum (Perbio Science Ltd, Cheshire, UK). The mixture was left for 30 min before use. The animals were immunized intraperitoneally in study weeks 1, 3, 5, 7.
Antibody Estimations
Three days after each immunization, tail bleeds were carried out into capillary tubes (Hawksley and Sons Ltd, West Sussex, UK) and the blood centrifuged at 1000 g for 20 min to obtain serum for measurement of antibody levels. The sera were frozen until specific enzyme-linked immunosorbent assays (ELISAs) were carried out on peptide-bovine serum albumin (BSA) coated plates as described previously.
Measurement of Specific
Antibody Levels Peptide–bovine serum albumin conjugate [equivalent to 1 µg peptide ⁄ well in 100 µL phosphate buffered saline (PBS), pH 7.4] was coated onto tissue culture grade 96-well plates for 1 hr at 370C. The plates were washed twice with wash buffer (PBS, containing 0.01% Tween 20) and blocked with 3% (w ⁄ v) Marvel in PBS– Tween, for 1 hr at 370C. The plates were washed thrice with wash buffer. Rat sera (0.1 mL, diluted 1:1000 in PBS, prepared from tail bleeds) was incubated per well for 1 h at 37C (carried out in triplicate for each sample). The plates were washed thrice with PBS– Tween. Horse radish peroxidase-labelled goat-anti-rat IgG (H + L chain specific, Perbio Science Ltd, Cheshire, UK) was diluted 1:3000 in PBS and 100 µL ⁄ well incubated for 45 min at 37C. The plates were washed thrice with PBS (without Tween) and developed with 0.1 mL TMB substrate ⁄ well (250 µL of stock 6 mg ⁄mL 3,3´,5,5´-tetramethylbenzidine in dimethylsulphoxide, added to 25 mL 0.1 m sodium acetate buffer, pH 5.5, with 4 µL 30% (v ⁄ v) hydrogen peroxide). The reaction was stopped with 50 µL ⁄ well 10% (v ⁄ v) sulphuric acid after 15 min and the A450 read. The means of the triplicate results were calculated and plotted on a graph against the study week number.
Assessment of Antibody Subclasses
Peptide specific ELISAs were carried out as described above, but horse radish peroxidase-labelled goatanti- rat IgM, IgG1-IgG2c (manufactured by Bethyl Laboratories Inc., Texas, USA, distributed by Universal Biologicals Ltd, Gloucestershire, UK), diluted 1:5000 in PBS, were used in place of whole-labelled goat-anti-rat IgG. Pooled antisera from TTCHWSYGLRPG- NH2 or HWSYGLRPGC-TT immunized animals (according to which sera were being tested), taken at postmortem, was used as a positive control, with labelled goat-anti-rat whole IgG for the second antibody. The means of triplicate results were plotted on a graph against the study week number.
Competitive ELISA to Detect Antibody Binding to Native GnRH
Plates were coated with peptide–BSA conjugate and blocked as above. The plates were washed thrice with wash buffer. Ten serial dilutions (1:10) of native GnRH (Sigma–Aldrich, Dorset, UK), ranging from 2 mg ⁄mL down, were made up in PBS. Fifty microlitres per well of each dilution was incubated in triplicate together with 50 µL ⁄ well rat sera (prepared as above, diluted 1:1000), for 1 h at 370C. The plates were washed thrice with PBS–Tween. Horse radish peroxidase-labelled goat-anti-rat IgG (H + L chain specific, Perbio Science Ltd, Cheshire, UK) was diluted 1:3000 in PBS and 100 µL ⁄ well incubated for 45 min at 370C. The plates were washed thrice with PBS (without Tween), developed and read as above. The means of the triplicate results were calculated and plotted on a graph against concentration of GnRH.
Measurement of Reproductive Physiological Effects
Single measurements of combined testicular widths were carried out on a weekly basis throughout the studies. This was found to provide an appropriate observation of the effect of immunization on reproductive function. At the end of each study, the animals were exsanguinated by section of the abdominal aorta distal to the renal arteries; the blood was retained for further analyses. Brief autopsies were also performed on the animals to identify any adverse effects of immunization; the testes, seminal vesicles and epididymis were weighed. Organs to be investigated histologically were fixed in phosphate buffered saline, containing 10% formalin.
Testosterone Estimation
Testosterone concentrations were determined in the serum, prepared from blood obtained at autopsy. A direct competitive radioimmunoassay (Coat-a-Count Total Testosterone; Euro ⁄DPC Ltd, Gwynedd, UK) was used according to the manufacturers’ instructions. The assay has been validated by the manufacturer for use with rat serum and has a detection limit of 0.04 ng ⁄mL.
Gonadotrophin Estimation
The follicle-stimulating hormone (FSH) concentrations were measured using a homologous rat FSH assay, the components [FSH for iodination ¼ rFSH-I-9, reference luteinizing hormone (LH) ¼ rFSH-RP-2, antiserum ¼ anti-rFSH-S-11] for which were kindly supplied by Dr A. F. Parlow of National Hormone and Peptide Program (NHPP), Harbor-UCLA Medical Center, California. Approximately 10 µg rat FSH was iodinated with I using a standard Chloramine-T technique, aliquoted and stored at )200C until required. Prior to use, an aliquot was thawed and diluted in sample buffer to give approximately 20,000 cpm in 25 µL. Each sample was run in quadruplicate with 25 µL of rat serum, 25 µL I rat FSH and 50 L of 1:32,000 rat anti-oFSH being added to each tube. The antibody titre was modified to give the optimum sensitivity at 1:40,000, this being 40% of the recommended value. After 24 hr incubation at room temperature, normal rat serum and donkey antirabbit serum was added and incubated overnight at 40C in order to produce large complexes. The tubes were centrifuged at 40C for 30 min after which the supernatant was removed by aspiration to leave a pellet, the radioactive content of which was read for 1 min using a Packard Auto Gamma Counter and suitable software to calculate the FSH concentration. From the standard curves produced, the FSH concentrations were calculated. In each run, quality control samples were included, these being rat sera of known concentrations. The sensitivity was calculated to be 1.56 ng ⁄mL.
A similar assay was donated by NHPP and used to measure the LH plasma concentrations (LH for iodination ¼ rLH-I-10, reference LH ¼ rLH-RP-3, antiserum ¼ anti-rLH-S-11). In this assay, the antibody titre was used at the recommended 1:2,000,000. As with the FSH assay, known concentrations were included with each run. The sensitivity was 0.39 ng ⁄mL.
Histology and Staining of Reproductive Organs
The formalin fixed tissues were cut into 0.5 cm2 pieces, and embedded in Tissue Tek (Raymond A Lamb Ltd, East Sussex, UK), at ) -700C. The tissues were sectioned (thickness 6 lm) in a cryotome (Thermo Shandon Inc., Pensylvania, USA), at ) -150C. They were fixed for 10 min onto silanized superfrost slides (BDH Ltd, Leicestershire, UK) with Carnoy’s fluid (60% absolute alcohol, 30% chloroform and 10% glacial acetic acid) and stained with an adapted Goldener’s trichrome method. The slides were dipped in Weigert’s haematoxylin (3 min), rinsed in running tap water for 15 min, dipped in Ponceau acid fuchsin mixture (5 min), 1% acetic acid (three dips), 4% phosphomolybdic acid- Orange G (12 min), 1% Light green stain (5 min), 1% acetic acid (three dips), 96% alcohol (1 min), 100% alcohol (2 min), 100% alcohol (3 min), xylene (5 min) and mounted.