Chronic Toxicity and Reversibility of Antifertility Effect of Immunization against Gonadotropin-Releasing Hormone in Male Rats and Rabbits

Narender Kumar1, Toyin Savage, William DeJesus, Y. Y. Tsong, A. Didolkar and K. Sundaram

Center for Biomedical Research, Population Council, 1230 York Avenue, New York, New York 10021

Received June 3, 1998; accepted March 25, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chronic systemic toxicity of immunization with gonadotropin-releasing hormone, conjugated to tetanus toxoid (GnRH-TT), was investigated in male rats and rabbits in order to start Phase I clinical trials. Groups of rats and rabbits were immunized with GnRH-TT dissolved in aqueous adjuvant. The antigen was administered at weeks 0, 4, and 8, followed by boosters to maintain high antibody titers. At termination (8–9 months after first immunization), twenty rats and ten rabbits exhibiting the highest mean anti-GnRH titers and all the controls were selected for complete toxicological evaluation. In the rat study, a castrated control group was included for comparison with the immunized group. The hematological and serum chemistry parameters of immunized rats and rabbits were not affected in a significant manner. Most of the changes in serum chemistry of immunized rats were also found in castrated rats, indicating that the changes are most likely due to the withdrawal of androgenic support. The weights of the testes, epididymides, and sex accessory glands were lower in all immunized animals. There was significant atrophy of the germinal epithelium, which, however, sustained a population of Sertoli cells, spermatogonia, and pachytene spermatocytes. Other morphological changes in the prostate, seminal vesicles, pituitary, and mammary gland reflected the effect of androgen withdrawal. The decrease in the weight of liver, kidney, and heart seen in the immunized rats was also present in castrated rats and was not associated with any histopathological changes. The reversibility of immunization-induced infertility was investigated by mating the rats with normal females. Four months after the start of immunization, 9 out of 10 immunized rats were infertile whereas by nine months, all rats had regained fertility. Thus, it is concluded that immunization with GnRH-TT had no systemic toxicological effects in the adult male rats and rabbits for the period studied. The results also indicated that the GnRH-TT immunization had an antifertility effect in male rats. Fertility was restored following cessation of immunization and decline in anti-GnRH antibody titers.

Key Words: immunization; GnRH; toxicity; reversibility; pituitary response.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gonadotropin-releasing hormone (GnRH), a hypothalamic peptide hormone, is critical for the synthesis and secretion of gonadotropins from the pituitary, which in turn regulates gonadal function (Garner et al., 1990Go). Active immunization with GnRH conjugated to tetanus toxoid (GnRH-TT) has been shown to neutralize GnRH leading to inhibition of gonadal function in animals (Chappel et al., 1980Go; Fraser et al., 1974Go; Giri et al., 1990Go; Ladd et al., 1989Go, 1994Go; Upadhyay et al., 1989Go). A male contraceptive method based on this approach has been proposed (Awoniyi et al., 1992aGo; Ladd et al., 1988Go). The method involves immunization with GnRH-TT and administration of an androgen supplement to maintain sexual behavior and other androgen-supported functions (Awoniyi et al., 1992bGo; Ladd et al., 1988Go). Immunization against GnRH may also be useful for treating sex steroid-dependent abnormal growth of mammary glands and prostate (Jayashankar et al., 1989Go; Ravdin and Jordan, 1988Go). In order to initiate clinical trials, chronic systemic toxicity studies of GnRH-TT were carried out in male rats and rabbits. To be acceptable, a contraceptive method must also be reversible. In a separate study, the reversibility of GnRH-TT induced infertility was investigated in rats.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Test and Control Articles
Synthetic (Gln1)-GnRH was provided by the Salk Institute, La Jolla, CA. It was conjugated to tetanus toxoid (Wyeth Laboratories, Marietta, PA) at the N-terminal amino acid (Gln) using 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide and was designated as GnRH-TT. The test article was an off-white, lyophilized powder containing 0.73 mg GnRH-TT per mg of powder. The conjugate, when analyzed by high-performance-liquid chromatography on BioRad TSK-250 column showed one broad peak with a shoulder. Based on radioimmunoassay, there were 3.38 moles of (Gln1) GnRH per mole of (Gln1) GnRH-TT. GnRH-TT was dissolved in an aqueous adjuvant, PBS-PT (0.01 M phosphate buffered saline containing 2.5% Pluronic L 121 and 0.2% Tween 80). The adjuvant was used as control vehicle. GnRH-TT and the adjuvant were prepared as per GMP regulations.

Animals and Husbandry
Crl:CD® Br COBS® male (BW 300–325 g) and female rats (BW 200–225 g) were purchased from Charles River Laboratories (NY). New Zealand White rabbits (BW 3.0–3.5 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 (2–3 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 10–15 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 4–8 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 9Go. 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 12–15 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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TABLE 9 Antifertility Effect of GnRH-TT–Immunization and Its Recovery in Male Rats
 
Hormone Assays and Titer Determination
Serum T was determined with a Coat-a-Count Total T kit (Diagnostic Products Corp., Los Angeles, CA). The sensitivity of the assay was 0.001–0.002 nmol/L.

Anti-GnRH titers were measured in rat and rabbit serum samples by a radioimmunoassay method described earlier (Ladd et al., 1988Go). 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 {gamma}-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., 1985Go). 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, 1985Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic Toxicity in Rats
There was no treatment-related mortality in this study. Two rats belonging to an immunized group died during the study. One rat had a heat injury to the tail when the tail was immersed in warm water to obtain a blood sample. The injury led to infection and partial gangrene of the tail. The second rat died due to an overdose of anesthetic during blood sampling. There was no effect on the general behavior (aggressiveness or lethargy) in immunized versus control rats observed at monthly intervals. The immunized and castrated rats showed a trend towards decrease in body weight compared to intact control during the in-life phase of the study. However, at week 20, body weights of the castrated rats were significantly lower than the intact control.

The anti-GnRH antibody titers of rats are given in Table 1Go. 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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TABLE 1 GnRH Antibody Titers (nmol/L) in Rats and Rabbits Immunized with GnRH-TT
 
The rats in the immunized group showed significant differences in levels of some hematological and serum-chemistry parameters. Similar differences were also present in the castrated control group. (Tables 2, 3GoGo). The RBC count and hemoglobin concentrations were decreased whereas mean corpuscular volume increased in both immunized and castrated rats compared to intact controls. A significant decrease in the percentage of monocytes and increase in eosinophils were observed only in castrated rats (Table 2Go).


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TABLE 2 Hematological Parameters in GnRH-TT-immunized and Control Rats
 

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TABLE 3 Serum Chemistry Parameters in GnRH-TT-Immunized and Control Rats
 
The following serum chemistry measurements in both immunized and castrated rats showed significant changes in their concentrations compared to intact rats: glucose, albumin, and chloride were increased, while blood urea, potassium, phosphorus and globulin were decreased. In addition, creatinine and ALT levels were increased in the immunized rats whereas alkaline phosphate showed decreases only in the castrated rats when compared to intact controls (Table 3Go).

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 4Go). 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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TABLE 4 Relative Organ Weights (weight per 100-grams bw) and Terminal Body Weight in Immunized and Control Rats
 
Microscopic evaluation revealed treatment-associated changes in testes, epididymides, sex accessory glands, and the pituitary of immunized rats. There was significant atrophy of the germinal epithelium in the testes of immunized animals, an expected response to decrease in gonadotropins and testosterone. In 17 of 20 immunized rats, the germinal epithelium was markedly atrophied but sustained a population of Sertoli cells, spermatogonia, and pachytene spermatocytes. The remaining 3 immunized rats had mild to moderate atrophy of the germinal epithelium and reduced spermatogenesis. The interstitial cells were degenerated in 20/20 animals. The prostate and seminal vesicles in both immunized and castrated rats were atrophied and appeared to be prepubertal in structure. The pituitary glands of the immunized rats were morphologically similar to those of vehicle control males. Typical castration cells, however, were present in 10/10 castrated rats. The skin/mammary-gland histology slide was often devoid of mammary acinar tissue; however, when present it was markedly atrophied in both immunized and castrated rats, but of normal configuration in control rats. In spite of decrease in the weights of liver, heart, kidney, and adrenals in immunized rats, microscopic appearance showed no abnormalities.

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 5Go). 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 6Go).


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TABLE 5 Hematological Parameters in GnRH-TT–Immunized and Control Rabbits
 

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TABLE 6 Serum Chemistry Parameters in Immunized and Control Rabbits
 
The organ weights of the testes, epididymides, and sex accessory glands were significantly lower in 6 of the 10 immunized rabbits compared to the control rabbits. The remaining 4 immunized rabbits had normal weights of the testes, epididymides, and sex accessory glands in spite of high antibody titers. The comparison of the mean weights of the organs relative to body weight, however, reflected significantly lower weights of testes and epididymides in the immunized group (Table 7Go).


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TABLE 7 Relative Organ Weights and Terminal Body Weights in Control and Immunized Rabbits
 
The histopathological evaluation of different organs revealed no evidence of a toxicopathological effect associated with the vaccine. The degenerative changes in the testes, epididymides and sex accessory glands of immunized rabbits were related to the treatment. There was marked atrophy of the seminiferous tubule epithelium and interstitial (Leydig) cells of testes in 6 out of 10 immunized rabbits. The testes were prepubertal in appearance but maintained a population of Sertoli cells and spermatogonia. The histological appearance of the epididymides and sex accessory glands were also prepubertal in the above 6 immunized rabbits. The remaining 4 out of 10 immunized rabbits showed no histological alterations and were comparable to the organs of the control rabbits. All other organs in the immunized rabbits showed no histomorphological changes compared to controls.

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 8Go. 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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TABLE 8 GnRH Antibody Titers and Serum Testosterone Levels in Rats Used in Fertility Studies
 
Fertility tests of the males were started two weeks after the last GnRH-TT injection. Parallel studies were conducted with control males. The fertility data and the numbers of implantation sites in the pregnant rats are summarized in Table 9Go. All 12 males (100%) in the control group were fertile in all of the fertility tests. In the immunized group at the first mating; 10 weeks after first immunization, two of the 12 males (17%) were fertile. At the second mating (16 week), only one (8%) out of the 11 rats was fertile (Fig. 1Go). At the next mating (at week 26), 7 of the 11 (64%) immunized males were fertile while by week 37 all of the rats had regained fertility (Fig. 1Go). The male rats were necropsied after the last mating test. The weights of the testes, epididymides, and sex accessory glands of the immunized rats were similar to those of the control (Fig. 2Go).



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FIG. 1. The effect of immunization with GnRH-TT on the fertility of male rats at different time periods. At the time of the mating test, the immunized and control male rats were housed individually with 2 females for 4 days (Monday–Thursday). The females were separated and killed after 12–15 days, and the number of implantation sites were counted. The percentage of fertile males was calculated.

 


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FIG. 2. Organ weights of control and GnRH-TT-immunized rats after recovery following fertility tests. SV, seminal vesicles; VP, ventral prostate; Epid, epididymus.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was carried out to determine the chronic toxicity of immunization with GnRH-TT in male rats and rabbits. In addition, reversibility of the antifertility effect upon immunization in rats was investigated. Active immunization with synthetic GnRH in animals has been shown to lead to a decrease in serum levels of gonadotropins and testosterone, and thus spermatogenesis (Arimura et al, 1973Go; Chappel et al., 1980Go; Fraser et al., 1974Go; Ladd et al., 1989Go). Hence, GnRH immunization leads to a functional castration (Fraser et al., 1974Go), and thus, the effects of immunization against GnRH could be compared to that of surgical castration.

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., 1992Go), and follicle-stimulating hormone (Udupa and Sheth, 1987Go). 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., 1987Go). 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., 1986Go). 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., 1987Go, 1990Go). 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., 1994Go). 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., 1986Go). 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.


    ACKNOWLEDGMENTS
 
We thank Dr. Robert F. McConnell for the histopathological evaluations. This work was supported by NICHD grant NO1-HD-3–3180.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (212) 327-7678. E-mail: kumar{at}popcbr.rockefeller.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adams, T. E., and Adams, B. M. (1990). Reproductive function and feedlot performance of beef heifers actively immunized against GnRH. J. Anim. Sci. 68, 2793–2802.[Abstract/Free Full Text]

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