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This study was designed to evaluate the effect of active immunization using recombinant GnRH-I protein on reproductive function in dogs. Six male and six female dogs were randomly assigned to either a control group or an immunization group (n = 3 males or 3 females/group). Dogs (aged 16 weeks) were immunized against GnRH-I with a maltose-binding protein-gonadotropin-releasing hormone I hexamer generated by recombinant DNA technology. Blood samples were taken at 4-week intervals after immunization. The serum concentrations of testosterone and estradiol and anti-GnRH-I antibodies were determined by RIA and ELISA, respectively. The results showed that active immunization with recombinant GnRH-I increased the serum levels of anti-GnRH antibodies (P < 0.05) and reduced the serum concentrations of testosterone (P < 0.05) and estradiol (P < 0.05) as compared with the controls. At 28 weeks of age, testes and ovaries were taken surgically for morphologic evaluation. Histologic studies performed on testicular and ovarian tissues revealed clear signs of atrophy in the recombinant GnRH-I-immunized dogs and a significant reduction (P < 0.05) in the weights and sizes of paired testes and ovaries in the treated dogs. Microscopically, spermatogonia were visible, but no spermatids and spermatozoa were detected in the seminiferous tubules. Neither early antral nor antral follicles were found in the immunized group. These results demonstrate that recombinant GnRH-I is an effective immunogen in dogs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Gonadotropin cell is the main responsible for the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and immunocastration reduces the concentrations of serum FSH and LH. A few studies have reported the histological structure of gonadotropin cells obtained from immunocastration animals at the light microscopy level. However, the ultrastructure of gonadotropin cells remains largely unexplored. The aim of this study was to evaluate and to compare ultrastructure of gonadotropin cell in gonadally intact boars and immunologically castrated male animals., Findings: In this study, serum and adenohypophysis tissue were collected from nine gonadally intact boars and nine male pigs treated with recombinant gonadotropin releasing hormone I (GnRH-I). Anti-GnRH-I antibodies in serum and the ultrastructure of gonadotropin cell in adenohypophysis were determined by enzymelinked immunosorbent assay and electron microscopy, respectively. The results demonstrated that active immunization against recombinant GnRH-I increased serum GnRH-I antibody levels (P<0.05). Ultramicroscopic analysis of gonadotropin cell revealed a decrease (P<0.05) in the number and size of the large granules and small granules in the recombinant GnRH-I immunized animals., Conclusions: We conclude that immunization against recombinant GnRH-I induces severe atrophy of granules in gonadotropin cell of boars, possibly reflecting GnRH-I regulation of gonadotropin cell.

This study was designed to explore the effect of active immunization against maltose binding protein-gonadotropin releasing hormone I hexamer ( MBP- GnRH-I6) on the reproductive function in cats. Each immunized cat was administered twice intramuscularly in the neck at 16 and 20 weeks old. The concentrations of the testosterone and estradiol and the level of anti- GnRH- I antibody in the serum were measured by radioimmunoassay and ELISA, respectively. The results showed that the weight and size of testicles and ovaries, and the concentrations of serum testosterone and estradiol in the immunized animals were lower than those of the control cats ( P < 0.05), but that the levels of anti- GnRH- I antibody were significant higher compared to control animals ( P < 0.05). Testicular tissues from the immunized male cats showed that seminiferous tubules were depauperate with the lumen relatively empty and that the differentiation of spermatogonia was not obvious. Tissues from the immunized female cats showed that the ovaries had many primordial follicles and primary follicles, but no secondary follicle was observed. These results showed active immunization against MBP- GnRH-I6 could make the gonads atrophy and reduce the concentrations of gonadal hormones, which suggested that MBP- GnRH-I6 was a very effective immunogen in the cat. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate and to compare testicular tissue in immunized and control boars. Eighteen male piglets, aged 12 weeks, were vaccinated twice intramuscularly with a maltose-binding protein–gonadotropin-releasing hormone I hexamer peptide (MBP-GnRH-I6). Blood samples were taken at 12, 18, 21 and 24 weeks of age. Serum concentrations of testosterone and GnRH-I antibodies were determined by radioimmunoassay. The pigs were sacrificed 6 weeks after the second immunization. Testicular weight and size were recorded and tissue samples were collected for histological examination. The results demonstrated that active immunization against MBP-GnRH-I6 increased serum GnRH-I antibody levels (P < 0.05) and reduced serum concentrations of testosterone (P < 0.05) when compared with controls. Histological studies performed on testicular tissue revealed clear signs of atrophy in the MBP-GnRH-I6 immunized pigs, and a significant reduction (P < 0.05) in paired testes weight and size were seen in the treated boars. Microscopically, the mean diameter of the seminiferous tubules was markedly reduced (P < 0.01). Spermatogonia were visible, as well as few spermatocytes, but no spermatozoa were detected in the seminiferous tubules. Ultramicroscopic analysis of testicular tissue revealed an increase in the thickness of the basement membrane and extensive damage in the cell organelles of the treated animals, including small spermatogonial size, decreased number of mitochondria and endoplasmic reticulum in the primary spermatocyte and spermatid, a shallow hollow for nuclear membranes in Sertoli cells and mitochondrial vacuolation in Leydig cells. We conclude that MBP-GnRH-I6 induces severe atrophy in the testes of immunized boars. [ABSTRACT FROM PUBLISHER]

The aim of this study was to investigate the efficacy of active immunisation against recombinant maltose binding protein-gonadotropin releasing hormone I (MBP-GnRH-I6) in preventing boar taint. The concentrations of testosterone and anti-GnRH-I antibodies in serum were determined by radioimmunoassay and enzyme linked immunosorbent assay, respectively. The mRNA levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in testes were analysed by real time quantitative PCR. The concentrations of androstenone and 3-methylindole in backfat were assayed by HPLC. Active immunisation against MBP-GnRH-I6 increased the serum concentration of anti-GnRH-I antibodies (P<0.05) and the levels of 3β-HSD and 17β-HSD mRNA in testis. However, it reduced the serum concentrations of testosterone (P<0.05) and androstenone. The concentration of 3-methylindole in backfat (P>0.05) was not changed. Thus, we conclude that vaccination against MBPGnRH- I6 is a practical and effective method to suppress the synthesis of androstenone. [ABSTRACT FROM AUTHOR]

Abstract: To determine whether hormone-receptor signaling pathways in the thymus are altered by active immunization against gonadotrophin-releasing hormone I (GnRH), 3-week-old Sprague–Dawley male rats received GnRH-tandem-OVA peptides (200μg/ml), and the effects were compared to a control group. Serum testosterone, LH and FSH concentrations were markedly reduced, with severe testicular atrophy, compared to controls, demonstrating effective blockade of the pituitary–gonadal axis. The reduction in LH and FSH concentrations in the thymus of immunized animals was lower than that observed in the serum, where a significant difference (P <0.001) in concentration was observed between both groups. Concentrations of GnRH were increased in the thymus of immunized rats. In thymic tissue, GnRHR, FSHR and LHR demonstrated stronger immunostaining, and AR weaker staining, in the immunized group compared to controls. Reproductive hormone receptor mRNA expression was consistent with protein variations in the immunized thymus. Compared to controls, GnRHR gene levels were significantly increased (P <0.05), however, AR mRNA expression were greatly decreased with immune week-age (P <0.05). Both FSHR and LHR mRNA expression levels were significantly higher in the treated group than in controls in the first three samples (P <0.05). When GnRHR was blocked by an antagonist in thymocytes, all reproductive hormone receptor gene expressions were significantly increased (P <0.001). In summary, these findings suggest that active immunization against GnRH can up-regulate GnRH receptor and gonadotropin receptor signaling, by stimulating thymic autocrine and paracrine function, whereas the androgen receptor is down-regulated due to a lack of testosterone secretion in the thymus. [Copyright &y& Elsevier]

A recombinant fusion protein, maltose-binding protein (MBP) - gonadotropin releasing hormone I (GnRH-I), was produced by cloning a gene fragment encoding a tandem repeated GnRH-I hexmer peptide (GnRH-I6) into the pMAL-c4x vector which was subsequently expressed in E. coli TB1. MBP-GnRH-I6 was affinity purified. MBP-GnRH-I6 was verified by SDS-PAGE and Western blot and immunogenicity tested in boars. Injection of the recombinant fusion protein into boars yielded a high-titer antibody specific for GnRH-I. This was followed by serum testosterone and the degeneration of spermatogenesis. These results showed that MBP-GnRH-I6 acted as a strong immunogen and could be a candidate for an immune antifertility vaccine. [ABSTRACT FROM AUTHOR]

Abstract: Immunization using recombinant maltose binding protein–gonadotropin releasing hormone (MBP–GnRH6) altered both testicular development and transcription of the pituitary GnRH receptor (GnRHR) gene in boars. Scrotal measurement and blood samples were taken at 4-week interval after immunization at 9 weeks of age. The concentrations of testosterone and anti-GnRH antibodies in serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. The results showed that active immunization with MBP–GnRH6 increased the serum concentration of anti-GnRH antibodies (P <0.05) and reduced the serum concentration of testosterone (P <0.05) as compared with MBP controls. At 25 weeks of age, boars were sacrificed and testes were evaluated histologically. Testicular development was suppressed in the MBP–GnRH6 immunized animals as compared with MBP immunized boars. MBP–GnRH6 immunized pigs exhibited mounting behavior 4 weeks later than MBP immunized boars. No mature spermatozoa were observed from MBP–GnRH6 immunized animals. By real-time quantitative PCR analysis, the amount of GnRHR mRNA in the pituitary tissue was found to be significantly lower in MBP–GnRH6 immunized animals than in controls (P <0.05). These data demonstrate that recombinant MBP–GnRH6 was effective in immunological castration in boars. [Copyright &y& Elsevier]

To investigate the effect of gonadotropin-releasing hormone 2-multiple antigen peptide (GnRH2-MAP) on reproductive function. In our study, 20-day-old male rats (n = 90) were randomly allocated to one of three treatment groups: GnRH2-MAP immunization, GnRH2 immunization, and non-immunized control groups. The immunized animals were administered three doses of GnRH2-MAP or GnRH2 vaccines from 0 to 6 weeks at 2-week intervals. The control group only received oil adjuvant. Blood and right testis samples were collected, and the left testis was weighed and its volume was measured at 0, 2, 4, 6, 8, 10 and 12 weeks after the first immunization. The serum antibody titer and testosterone concentration were determined by ELISA, and the right testis samples were collected for histological analysis. The results revealed that the serum of vaccinated rats elicited a significantly higher antibody titer and a lower T concentration compared with the control group two weeks after the first immunization ( P  < 0.05), but the highest antibody titer and lowest T concentration were found in animals treated with GnRH2-MAP ( P  < 0.05). The second immunization resulted in a significant decrease in testicular weight and volume ( P  < 0.05) in both immunized groups compared to the control, but these values were significantly lower in the GnRH2-MAP group than in the GnRH2 group. Furthermore, seminiferous tubules revealed more significant atrophy in the GnRH2-MAP group than in the GnRH2 group, and no sperm were observed in rats of the GnRH2-MAP group. Thus, GnRH2-MAP may be an effective antigen and a potential immunocastration vaccine with higher effectiveness. [ABSTRACT FROM AUTHOR]