International Journal of

     Introduction
Gonadotropin releasing hormone (GnRH) is produced and secreted from the hypothalamus. This hypothalamic peptide hormone controls the synthesis and secretion of Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) from anterior pituitary, which in turn regulates the synthesis and secretion of sex steroid hormones (testosterone in males and estradiol and progestrone in females) and thus controls the whole reproductive function (1).
     A technique has been developed to cause a mammal to generate antibodies against its own hormone i.e., GnRH. In general the technique involves the injection of a synthetic GnRH conjugated to a non-native protein (Bovine Serum Albumin, Human Serum Albumin, Tetanus toxoid etc.).The conjugated GnRH when enters the circulation, raises the antibodies against it. Antibodies to the hypothalamic hormone reduce the circulating level of biologically active GnRH, thereby reducing the level of subsequent reproductive hormones and inducing the infertility. Active immunization of various mammals to GnRH, has been shown to lead in the case of males to testicular regression, reduction of testosterone secretion and cessation of spermatogenesis and in the case of females to loss of cycling and ovarian regression (2-5). A novel GnRH analogue conjugated to mycobacterium hsp70 has been reported to elicit a relatively higher level of antibodies that neutralize the activity of native GnRH (6).  Immunization against GnRH may also be useful for treating sex steroid-dependent abnormal growth of mammary glands and prostate (7). Recently a recombinant anti-GnRH vaccine has been designed with relatively higher immunogenicity for controlling the fertility (8). GnRH vaccine has been employed with good results for control of wild animal populations (9- 10). In connection to this, present experimental investigation is aimed to evaluate the infertility effects induced through GnRH immunization in male mice.
 
Materials and methods
     For the present study, forty adult male mice, Mus musculus (P) weighing 30±5 gms were used. The animals were divided into two groups of twenty each. Group-1 served as control, while group 2, were immunized with 50 µg GnRH-BSA conjugate (Sigma Aldrich) dissolved in 100 µl phosphate buffered solution (0.01N) and emulsified with an equal volume i.e., 100 µl Freund´s adjuvant for 30, 60, 90, and 120 days respectively. After above durations, five animals were weighed and then sacrificed and their organs testes and epididymides were dissected out quickly. Testes were weighed for observing gonadosomatic indices, while epididymides were cut to release sperms in normal saline (100mg tissue/ 2ml) for sperm suspension. For sperm motility, a drop of sperm suspension was placed on the Neubar’s chamber which was focused on the four squares (WBC squares) and the motile spermatozoa were counted along with the total number of spermatozoa in each small sub square. A total of minimum 10-12 separate fields were scored and the percent motility was calculated. For sperm count (millions/ml), sperm suspension was sucked upto 0.5 mark in the WBC pipette and then diluted   upto   the   11   mark   with    5%   sodium
bicarbonate solution. A drop of this fluid was then
placed on the Neubar’s chamber and the total number of spermatozoa was counted in 64 small WBC squares with the help of microscope. While, sperm count (millions/ml) was done by adding sodium bicarbonate (spermicide) to a part of sperm suspension and counted on Neubar`s chamber under microscope. For sperm morphology, one to two drops of sperm suspension were put on a glass slide, spread with other slide to form a smear, air dried and finally stained with Leishman`s stain and observed under microscope at a magnification of 400X   for observing normal and abnormal sperm morphological forms (11). Results of the experiments were expressed as mean and standard error of different groups.
 
Statistical analysis
     The statistical analysis in between control Vs experimental groups were done by Student′s ‘t’ test. The values for p<0.01 were considered significant while the values p<0.001 were considered as highly significant.
 
Results
     The body weight and GSI levels were lowered significantly in the later part of the experiment after GnRH-BSA immunizations when compared to control group (Table I). Besides this, the sperm motility, sperm count and sperm morphology were also significantly decreased in the immunized animals throughout the experiment and the effects were more prominent in later part of the experiment i.e., 90 and 120 days (Table II). However, morphological studies denote that more than 70 percent of the sperms were abnormal in shape characterized by pin head, large head, tapering head, oval head, small head, headless double head, bend neck, looped mid piece, coiled tail and double tail sperms in GnRH - BSA immunized animals of all groups i.e., 30, 60, 90 and 120 days when we compared with control groups (figures 1-5). The percentage of morphological abnormalities was more prominent and highly significant in the later part of the experiment i.e., 90 and 120 days.














Discussion
 
     In the pituitary, GnRH binds to the GnRH receptors on the gonadotropic cells to stimulate the release of follicle stimulating hormone (FSH) and luteinizing hormone (LH) to the circulation. The pulsatile secretion pattern of GnRH induces the cyclic release of LH and to a lesser extent of FSH. In male mammals, LH stimulates the secretion of androgens (e.g. testosterone) from the leydig cells in the testis. Production of antiserum to gonadotropin releasing hormone was found to be associated with gonadal atrophy in mammals after GnRH immunization (12-14). Antifertility effects of immunization against GnRH in male rats and rabbits were reported by Kumar el al (2000) (15). Malmgren et al (2001) also reported the changes in sexual behavior, androgen deprivation and testicular atrophy in male Stallions after GnRH immunization (16). Immunization with a GnRH analogue conjugated to mycobacterium hsp70 has induced changes in the reproductive system of male mice (17). Talwar et al (2004) reported the ability of a multimer recombinant anti-LHRH vaccine to cause decline of testosterone to castration level and atrophy of prostate of rats (18).  In addition Turkstra (2005) revealed active immunization against GnRH as an active tool to block the fertility axis in mammals (19). More recent reports on contraception have pointed out that the immunization against GnRH could be an alternative to castration of male and female animals (20-21). Our results also denote that after immunization with GnRH-BSA conjugate at different intervals significantly lowered the body weight, GSI, sperm motility, sperm count and modulated sperm morphology in male mice. These significant effects were more prominent in later part of the experiment. These results suggested that active immunization against GnRH produced antibodies which reduced the level of biologically active GnRH by hypothalamohypophysial gonadal axis which might inhibit androgen level in circulation resulting inhibition of spermatogenesis in mice. Alterations in GnRH level induced changes in reproductive function of male mice, which are duration dependent.
 
Conclusion
 
     We conclude that the immunization with GnRH-BSA at different intervals induced antifertility effects in male mice. Thus the hormonal conjugate may be used as a hormonal immunocontraceptive  to control fertility.
 
Acknowledgement
    
     This work was supported by Department of Biosciences, Barkatullah University, Bhopal, India.
     The author wishes to acknowledge Prof. Meenakshi Benerjee (Prof  & H.O.D.), Department of Biosciences, for experimental support.