Center for Biomedical Research, Population Council, 1230 York Avenue, New York, New York 10021
Received June 3, 1998; accepted March 25, 1999
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ABSTRACT |
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Key Words: immunization; GnRH; toxicity; reversibility; pituitary response.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Animals and Husbandry
Crl:CD® Br COBS® male (BW 300325 g) and female rats (BW 200225 g) were purchased from Charles River Laboratories (NY). New Zealand White rabbits (BW 3.03.5 kg) were purchased from Hazelton Dutchland Laboratories, Inc. (NY). The animals were housed at the Laboratory Animal Research Center (LARC) of the Rockefeller University in New York City according to the NIH guidelines outlined in the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Rats were given food and water ad libitum and housed under standard conditions with a photo period of 12L:12D. The rats were housed 2 per cage for the chronic toxicity study. Rats for fertility testing were housed singly in hanging wire-bottom cages. Rabbits were housed singly and given a restricted diet of approximately 125 g per day, in order to prevent an excessive increase in body weight. Water was allowed ad libitum.
Experimental Design
Toxicity study in rats.
A total of 90 male rats were used for this study. After an acclimatization period, rats were randomly allocated: 60 to the vehicle control, Group 1; the treatment group (Group 2, 60 rats) or the castrated group (Group 3, 10 rats). The rats in Group 3 were bilaterally castrated on the day of initiation of the treatment, to compare with immunized rats. The test article (GnRH-TT in adjuvant), and vehicle (adjuvant) were administered by intramuscular injection in the lateral part of the thigh. A total of 200 µg GnRH-TT suspended in 0.5 ml of the adjuvant was administered to rats in the treatment group. For the primary immunization phase, rats were injected 3 times at 4-week intervals. A booster injection was given to all rats in Group 2, 12 weeks after the last injection, in order to maintain high antibody titers. Control rats received a similar number of injections with the vehicle. The total duration of the study was 8 months.
All signs of ill health and/or behavior changes (aggressiveness or lethargy) in response to treatment were observed and recorded for individual animals at the time of treatment and at monthly intervals. The rats were weighed prior to initiation of treatment, weekly for the first 8 weeks and at monthly intervals thereafter. For the purpose of determining antibody titers, blood (23 ml) was collected from the caudal artery of each rat, at the base of the tail, in Groups 1 and 2, beginning 8 weeks after the start of the treatment and at monthly intervals thereafter. To facilitate blood collection, the tails were immersed in warm water (50°C) for 1015 s. Serum was prepared and stored at 20°C until assayed.
During the terminal phase, all of the control rats, 20 intact and 10 castrated, and 20 immunized rats were used for the hematology, serum chemistry, and histopathology evaluations. Twenty out of 60 immunized rats were selected, based on the highest mean antibody titers determined in the blood samples of the previous 4 months. The remaining forty immunized rats were used in other mechanistic studies such as pituitary responsiveness to GnRH and changes in GnRH-receptor (data not included). Food was removed overnight from the rats selected for necropsy. On the day of necropsy, rats were anaesthetized with methoxyflurane and exsanguinated through the abdominal aorta.
An aliquot of the blood was used for hematological examination. The rest of the blood was allowed to clot and the serum collected. The following parameters were determined: white blood cell count (WBC), red blood cell count (RBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), differential blood count, alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, chloride, phosphorus, calcium, sodium, potassium, urea nitrogen (BUN), creatinine, total proteins, albumin, globulin, triglycerides, and cholesterol. At necropsy, the weights of testes, epididymides, sex accessory glands, kidneys, liver, heart, adrenals, pituitary, brain, spleen, and thyroid were determined. The following tissues were submitted for histopathological evaluation; heart, lung, trachea, liver, spleen, kidney, urinary bladder, adrenal, salivary gland, thymus, pituitary, thyroid, brain, eye, esophagus, stomach, duodenum, jejunum, ileum, mesenteric lymph node, caecum, colon, rectum, femur, sternum (bone and marrow), pancreas, skeletal muscle, mammary gland, skin, aorta, sciatic nerve, testis, epididymidis, and sex accessory gland. The histopathological evaluation was performed by a certified pathologist. The changes in the tissues were marked as normal, mild, moderate, or severe.
Toxicity study in rabbits.
Forty male rabbits were randomly allocated to the treatment group (30) or to the control group (10). The controls received 0.5 ml of vehicle (adjuvant) and treatment group rabbits received 500 µg GnRH-TT in 0.5-ml vehicle. Rabbits were injected (intramuscularly) in the lateral part of the thigh at weeks 0, 4, and 8 for primary immunization. A booster was given 12 weeks after the last injection. Two months after the primary immunization and at monthly intervals thereafter, blood was collected from the ear vein of each rabbit for antibody-titer determination.
At termination, 10 immunized rabbits exhibiting the highest mean antibody titers during the previous 4 months and the 10 control rabbits were used for toxicological evaluation. Food was removed overnight from all the rabbits selected for necropsy. On the day of necropsy, blood samples were obtained from the median artery or marginal vein of the ear. Blood samples were subjected to hematological and biochemical analyses as described earlier. Rabbits were sacrificed by carbon dioxide asphyxiation and subjected to autopsy procedures. Postmortem examination was performed by a certified pathologist. The condition of thoracic viscera, and abdominal viscera was examined. The urinary bladder, gastrointestinal tract, lungs, liver, and kidney were examined whole and after incision at several points. The organs/tissues for weighing and histopathological analysis were selected as described for rats.
Reversal of fertility in GnRH-TT immunized male rats.
Active immunization against GnRH results in loss of support to the testicular spermatogenesis. To assure that the antifertility effects were not permanent, fertility tests were performed in immunized rats at 48 week-intervals after primary immunization. A total of 24 male rats were divided into control and immunized groups (12 rats per group). Group 1: intact control rats treated with the vehicle alone, and Group 2: those immunized with GnRH-TT. Immunization was by 3 intramuscular injections of 200 µg GnRH-TT in vehicle given on day 1 and at the end of weeks 4 and 8. Two months after the first injection and at monthly intervals thereafter, antibody titers and serum testosterone levels were measured.
The fertility of the male rats in the control and immunized group was determined at specific time intervals shown in Table 9. Males were housed singly in wire-bottom steel hanging cages. For each fertility test, two normal female rats were placed with a male for 4 days. The females were examined daily for the presence of sperm in the vaginal lavage and the presence of vaginal plugs on the paper under the cage. The presence of plug or sperm in the vagina was taken as evidence of positive mating behavior. At the end of the mating period, female rats were removed and housed in plastic cages, and were killed 1215 days after separation from the male. The number of implantations and resorbing sites were recorded. The presence of one or more implantation sites was taken as indicative of male fertility. At the end of the study, male rats were killed and the testes, epididymides, seminal vesicles, and ventral prostate were removed and weighed.
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Anti-GnRH titers were measured in rat and rabbit serum samples by a radioimmunoassay method described earlier (Ladd et al., 1988). Briefly, sera from immunized rats or rabbits were diluted 1:100 in 1% BSA. One hundred-µl aliquots of diluted sera were incubated overnight at room temperature in 1% BSA pre-coated polystyrene tubes with 100 µl of [125 I]-GnRH (~15,000 cpm). Antibody-bound [125I]-GnRH was precipitated with 400 µl of polyethylene glycol 8000 (25% solution) and 200 µl of bovine
-globulin (5 mg/ml). Bound and free fractions were separated by centrifugation at 1000 x g for 25 min. The supernatant was aspirated and the pellet-counted in an automatic gamma counter. Antibody titers were expressed as nmol of [125I]-GnRH bound per liter of serum. Non specific binding was determined by measuring antibody titers in non-immunized animals and did not exceed 0.6 nmol/L.
In the antifertility experiment, the titers were also determined by titrating the individual sera by a dilution method (Tsong et al., 1985). Briefly, [125I]-GnRH (15,000 cpm) was added to serial dilutions of serum to a final volume of 0.3 ml and incubated overnight at room temperature. Bound and unbound radioactivity was separated as described above. Antibody titers were expressed as the reciprocal of the dilution of serum at which 30% of the added, labeled GnRH was bound.
Statistical Analysis
Analysis of variance was employed to detect significant treatment effects between groups. The level of significance was determined by Fisher's LSD test using BMDP Software (Dixon, 1985).
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RESULTS |
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The anti-GnRH antibody titers of rats are given in Table 1. The titers increased after the primary immunization but started to decline by week 25. A single booster administered at week 27 restored the high titers by week 30, and the titers remained high until the end of the study. The twenty rats with the highest mean antibody titers from a group of sixty immunized rats were selected for necropsy.
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The relative organ weights of testes, epididymides (in immunized group), ventral prostate, seminal vesicles, liver, heart, and kidneys were significantly lower in both immunized and castrated rats compared to intact controls (Table 4). In addition, in the immunized rats the weights of the pituitary and adrenal glands were significantly lower than those of intact and castrated rats.
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Chronic Toxicity in Rabbits
There was no mortality in this study. The mean body weights of the immunized rabbits during the study and at termination were not significantly different from the control group. The anti-GnRH-TT antibody titers of immunized rabbits after the primary immunization remained high up to week 17 and thereafter started to decline. A booster given at week 27, however, restored the titers to a high level until the end of the study. The mean antibody titers from the last 4 blood samples of immunized rabbits were calculated and 10 rabbits with mean highest antibody titers were selected for the necropsy.
No significant differences in the hematological parameters estimated in the blood of immunized compared to control rabbits were observed (Table 5). The serum chemistry evaluation showed that the concentrations of total protein, albumin and alanine aminotransferase were decreased while cholesterol was increased in the immunized rabbits compared to controls (Table 6
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Reversibility of Antifertility in Rat
The mean antibody titers and serum testosterone levels in the control and immunized rats used in the fertility studies are shown in Table 8. The antibody titers in the immunized group declined gradually after the last immunization. The serum levels of testosterone in the immunized group were low between 10 and 15 weeks after the first immunization. Then, starting from week 19 the levels of testosterone started to rise gradually, although, by the end of the study, the levels were still significantly lower than in the control group.
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DISCUSSION |
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In both rats and rabbits, immunization against GnRH led to significant atrophy of androgen target organs, i.e., testes, epididymides, prostate, and seminal vesicles. In this regard, rabbits were less sensitive than rats. The testes in immunized rats (20/20) and rabbits (6/10) were atrophied and showed marked regression of the interstitial (Leydig) cells and germinal epithelium but sustained a population of Sertoli cells, spermatogonia, and pachytene spermatocytes. The epididymides had no spermatozoa in the ducts. The prostate and seminal vesicles in all immunized rats and in 60% of rabbits were prepubertal in appearance. Four of the 10 immunized rabbits had reproductive organs that appeared normal despite high anti-GnRH titers. Serum levels of testosterone were also normal in these 4 rabbits. The reasons for this are not clear. This could be due to the production of anti-idiotypic antibodies, which can mimic the biological functions of their original antigen as described earlier for insulin (Schechter et al., 1984), luteinizing hormone (Sairam, et al., 1992), and follicle-stimulating hormone (Udupa and Sheth, 1987
). The relative weights of liver, heart, and kidney were lower in immunized rats but not in rabbits, However, no histopathological changes were observed in any of these organs.
While androgens play an important physiological role in the development and maintenance of the male reproductive system, they also exert an anabolic effect on other organs such as liver, kidney, heart, and skeletal muscle (Mooradian et al., 1987). The body weights of the immunized rats at termination were significantly lower than weights of intact rats. Similar effects of GnRH immunization and castration have been shown on body weight and body composition (Fletcher et al., 1986
). The pituitary and adrenal glands in the immunized rats were significantly smaller than those in the intact and castrated rats, though morphologically they were similar to that of intact rats. The smaller pituitary gland in the immunized rats may reflect a decreased secretory activity. GnRH has been shown to increase the number of gonadotrophs and the synthesis and release of gonadotropins in the rat (Garner et al., 1987
, 1990
). Castrated rats showed the presence of castration cells (vacuoles) in the pituitary. The reason for the decrease in weights of adrenal glands in the immunized rats is not clear. It is possible that GnRH may play a role in adrenal stimulation, since the expression of the GnRH receptor has been demonstrated in rat adrenals (Kakar et al., 1994
). Compared to rabbits, the rats showed a greater response to GnRH-TT immunization based on its effectiveness in terms of regression of androgen target organs and changes in the hematological and serum chemistry parameters. Rats were also more sensitive to androgen deprivation.
The safety of the vaccine was also evaluated by determining whether or not its effects on the pituitary-testicular axis were reversible. Immunization against GnRH led to the suppression of testosterone levels and testicular regression in the rats. The contraceptive effect of immunization was indicated by the observation that 92% of immunized rats became infertile 16 weeks after the primary immunization, corresponding to their high anti-GnRH titers. Fertility was restored in all immunized rats following a decline in anti-GnRH titers and increase in serum testosterone levels by week 37 of the study. The presence of early spermatogenic stages allowed the reversibility of spermatogenic arrest with declining anti-GnRH titers. These results indicate that immunization against GnRH had been effective in blocking spermatogenesis, and that the effects of immunization on fertility and tissue weights were reversible.
These results also showed that active immunization of male animals against GnRH leads to suppression of the pituitary-testicular axis, which results in the regression of primary and secondary sex glands. The weights of adrenals and pituitary and some of the hematological and serum chemistry parameters were altered in the immunized rats. However, most of these changes were also encountered in castrated rats, indicating that the effects are due to androgen withdrawal. The changes in the serum chemistry parameters specific to immunized animals were within the normal range of the rats and rabbits of this age group (Wolford et al., 1986). In addition, no histopathological changes were observed in the immunized animals as a result of changes in serum chemistry or organ/tissue weights. Immunization of male rats with GnRH-TT resulted in reduced fertility, 10 weeks after the last immunization. Fertility was fully restored by week 37 in all the immunized male rats. In conclusion, active immunization against GnRH-TT showed no systemic toxic effects in male rats and rabbits.
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ACKNOWLEDGMENTS |
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NOTES |
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