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Gonadotropin-releasing hormone (GnRH) vaccines have been successfully used for the inhibition of gonadal development and function, but current GnRH-based vaccines often present variability in the response. Cross-reactive material 197 (CRM197) has been used as carrier molecules to enhance an immune response to associated antigens. So, the synthetic mammalian tandem-repeated GnRH hexamer (GnRH6) gene was integrated into the expression plasmid pET-21a. Recombinant GnRH6-CRM197 protein was subsequently overexpressed in Escherichia coli strain BL21 and purified through Nickel column affinity chromatography and the antigenicity and biological effects of GnRH6-CRM197 were evaluated in rats. Sixteen 4-month-old adult male rats were randomly divided into two groups: the GnRH6-CRM197 group ( n  = 8) and the control group ( n  = 8). The GnRH6-CRM197 group rats were subcutaneously immunized with 100 μg of GnRH6-CRM197, administered thrice at 2-week intervals with GnRH6-CRM197.The control group received only a white oil adjuvant. Following the initial immunization, the weights of animals were recorded, and blood samples were collected from the orbital sinus at 4, 4.5, 5, 5.5, 6, 6.5, and 7 months. Serum antibody titers and testosterone concentrations were quantified using ELISA and CLIA, respectively. Additionally, testicular tissues were collected for morphological examination. The results revealed a significant increase in serum GnRH antibody titers ( p  < 0.05), but a significant decrease in serum testosterone concentrations ( p  < 0.05), and the weight, length, width, and girth of the testis, and the number of spermatogonia cells, spermatocytes, and sperm cells in the immunized rats. Furthermore, seminiferous tubules revealed significant atrophy and no sperm were observed in the immunized animals. Thus, GnRH6-CRM197 may be an effective antigen and a potential immunocastration vaccine.

The study aimed to investigate the effect of Grid1, encoding the glutamate ionotropic receptor delta type subunit 1 (GluD1), on puberty onset in female rats. Grid1 mRNA and protein expression was detected in the hypothalamus of female rats at prepuberty and puberty. The levels of Grid1 mRNA in the hypothalamus, the fluorescence intensity in the arcuate nucleus and paraventricular nucleus of the prepubertal rats was significantly lower than pubertal. Additionally, the expression of Grid1 was suppressed in primary hypothalamus cells and prepubertal rat. Finally, investigated the effect of Grid1 knockdown on puberty onset and reproductive performance. Treatment of hypothalamic neurons with LV-Grid1 decreased the level of Grid1 and Rfrp-3 (encoding RFamide-related peptide 3) mRNA expression, but increased the Gnrh (encoding gonadotropin-releasing hormone) mRNA levels. After an ICV injection, the time for the rat vaginal opening occurred earlier. Moreover, Gnrh mRNA expression was increased, whereas Rfrp-3 mRNA expression was decreased in the hypothalamus. The concentration of progesterone (P 4 ) in the serum was significantly decreased compare with control group. Ovary hematoxylin-eosin staining revealed that the LV-Grid1 group mainly contained primary and secondary follicles. The reproductive performance of the rats was not affected by the Grid1 knockdown. Therefore, Grid1 may affect the onset of puberty in female rats by regulating the levels of Gnrh, and Rfrp-3 in the hypothalamus, as well as the concentrations of P 4 , but not reproduction performance.

The present study aimed to reveal the effects of immunocastration on the development of the immune system in rats. Seventy rats were randomly assigned into two groups: Control (n = 35) and immunized (n = 35). Twenty-day-old rats were immunized with gonadotropin-releasing hormone (GnRH) and booster immunization was administered every two weeks (three immunizations in total). From 20-day-old rats, we collected samples every two weeks, including five immunized rats and five control rats (seven collections in total). We collected blood samples, testicles, thymuses, and spleens. The results showed that GnRH immunization increased the GnRH antibody titers and reduced the testosterone concentration (both P < 0.05 ). Compared with the control group, the number of CD4+CD8- cells, CD4-CD8+ cells, and CD4+CD8+ cells increased ( P < 0.05 ) whereas the number of CD4-CD8- cells and CD4+CD25+ cells reduced in the immunized group ( P < 0.05 ) over time. GnRH immunization also increased the relative weights of thymus and spleen ( P < 0.05 ), serum concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17 and Interferon-γ (IFN-γ) over time ( P < 0.05 ), and changed the mRNA levels of IL-2, IL-4, IL-6. IL-10, IL-17, IFN-γ, CD4, D8, CD19 GnRH, and GnRH receptor ( GnRH-R ) in thymus and spleen. Thus, GnRH immunization enhanced the immune markers in thymus, spleen, and blood immune cytokines in rats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pan, Du, Tian, Xie, Zhang, Fu, Li, Ling, Zhang, Fang and Liu.)

In the present study, we evaluated how Ptprn-2 (encoding tyrosine phosphatase, receptor type, N2 polypeptide protein) affects the onset of puberty in female rats. We evaluated the expression of Ptprn-2 mRNA and protein in the hypothalamus-pituitary-ovary axis of female rats using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence at infancy, prepuberty, puberty, peripuberty, and adulthood. We evaluated the effects of Ptprn-2 gene knockdown on different aspects of reproduction-related biology in female rats, including the expression levels of puberty-related genes in vivo and in vitro, the time to onset of puberty, the concentration of serum reproductive hormones, the morphology of ovaries, and the ultrastructure of pituitary gonadotropin cells. Our results demonstrated that PTPRN-2 was primarily distributed in the arcuate nucleus (ARC), periventricular nucleus (PeN), adenohypophysis, and the ovarian follicular theca, stroma, and granulosa cells of female rats at various stages. Ptprn-2 mRNA levels significantly varied between peripuberty and puberty (P < 0.05) in the hypothalamus and pituitary gland. In hypothalamic cells, Ptprn-2 knockdown decreased the expression of Ptprn-2 and Rfrp-3 mRNA (P < 0.05) and increased the levels of Gnrh and Kiss-1 mRNA (P < 0.05). Ptprn-2 knockdown in the hypothalamus resulted in delayed vaginal opening compared to the control group (n = 12, P < 0.01), and Ptprn-2, Gnrh, and Kiss-1 mRNA levels (P < 0.05) all decreased, while the expression of Igf-1 (P < 0.05) and Rfrp-3 mRNA (P < 0.01) increased. The concentrations of FSH and P 4 in the serum of Ptprn-2 knockdown rats were lower than in control animals (P < 0.05). Large transverse perimeters and longitudinal perimeters (P < 0.05) were found in the ovaries of Ptprn-2 knockdown rats. There were fewer large secretory particles from gonadotropin cells in adenohypophysis tissue of the Ptprn-2 knockdown group compared to the control group. This indicates that Ptprn-2 knockdown can regulate levels of Gnrh, Kiss-1, and Rfrp-3 mRNA in the hypothalamus, regulate the concentration of serum FSH and P 4 , and alter the morphology of ovarian and gonadotropin cells, delaying the onset of puberty in female rats., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The aim of this study was to assess whether changes in kisspeptin and GnRH levels could be attributed to sex steroids at puberty onset. We used the ovariectomy (OVX) model in rats treated with 17β-estradiol (E2; OVX + E2), or oil (OVX + oil), and in intact rats treated with E2 (intact + E2) or oil only (intact + oil) to determine gene expression changes of Kiss1 and Gnrh1 in the hypothalamus and protein expression of kisspeptin and GnRH in the different areas of the hypothalamus. In the intact + E2 and OVX + E2 rats on the day of the onset of puberty, GnRH-immunoreactive (ir) cell numbers decreased (P < 0.05) in the arcuate nucleus but were increased in the preoptic area; Kisspeptin-ir cells increased (P < 0.05) in the arcuate nucleus, periventricular nucleus, and preoptic area; no difference (P > 0.05) was found in the paraventricularis nucleus for GnRH-ir or kisspeptin-ir cells. Additionally, levels of Kiss1 and Gnrh1 messenger RNA in the hypothalamus were significantly higher (P < 0.05) in the OVX + E2 or intact + E2 rats than in the OVX + oil or intact + oil animals, respectively. In the OVX + oil rats, OVX significantly increased (P < 0.05) levels of Gnrh1 and Kiss1 messenger RNA and the expression of GnRH and kisspeptin in the hypothalamus compared to intact + oil animals. These results suggest that kisspeptin and GnRH play major roles in modulating the activity of estrogen circuits at the onset of puberty., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (© 2015 Japanese Society of Animal Science.)

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.

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.

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 (200 μg/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 © 2013 Elsevier Inc. All rights reserved.)

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 hexamer 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.

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 (c) 2010 Elsevier B.V. All rights reserved.)