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. 2023 Jan 13:13:1023104.
doi: 10.3389/fimmu.2022.1023104. eCollection 2022.

Effect of GnRH immunocastration on immune function in male rats

Fuqiang Pan  1 , Huiting Du  1 , Weiguo Tian  1 , Huihui Xie  1 , Bochao Zhang  1 , Wanzhen Fu  1 , Yunsheng Li  1   2   3 , Yinghui Ling  1   2   3 , Yunhai Zhang  1   2   3 , Fugui Fang  1   2   3 , Ya Liu  1   2   3
Affiliations

Effect of GnRH immunocastration on immune function in male rats

Fuqiang Pan et al. Front Immunol. .

Abstract

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.

Keywords: GnRH; immune function; immunocastration; male rats; spleen; thymus.

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Conflict of interest statement

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.

Figures

Figure 1
Figure 1
Mean (± SEM) of the serum antibody titers in the gonadotropin-releasing hormone (GnRH) immunization group (n = 35) and control group (n = 35). The ordinate is the OD of the serum GnRH antibody. The abscissa is the age of weeks after first immunization and Arrows indicate the primary vaccination and subsequent boosters. * Indicates a significant difference (P < 0.05).
Figure 2
Figure 2
(A) Images of testicles of the gonadotropin-releasing hormone (GnRH) immunization and control groups from 10 Week rats after first immunization. (B) Histopathological observation of testes samples from the control group (a, b, c), and the gonadotropin-releasing hormone (GnRH) immunization group (d, e, f) at 10 weeks after first immunizaton. (a-c) and (d-f) represents different magnifications (100, 200, and 400 times, respectively). S indicates seminiferous tubules; Sperm cells at various stages (arrows), and spermatozoa (square area) are indicated. (C) Mean ( ± SEM) the relative weight of the testicle in gonadotropin-releasing hormone (GnRH) immunization group (n=35) and control group (n=35). Arrows indicate the primary vaccination and subsequent boosters. * Indicates a significant difference (P < 0.05). (D) Mean (± SEM) of the serum testosterone concentration in the gonadotropin-releasing hormone (GnRH) immunization group (n =35) and control group (n = 35). The ordinate is the concentration of testosterone. The abscissa is the age of weeks after first immunization and arrows indicate the primary vaccination and subsequent boosters. The testosterone levels were detected using an enzyme-linked immunosorbent assay (ELISA). * Indicates a significant difference (P < 0.05).
Figure 3
Figure 3
Mean (± SEM) of the serum IFN-γ (A), TNF-α (B), IL-2 (C), IL-4 (D), IL-6 (E), IL-10 (F), and IL-17 (G) concentrations in the gonadotropin-releasing hormone (GnRH) immunization group (n = 35) and control group (n = 35). All were detected using enzyme-linked immunosorbent assays (ELISAs). The ordinate is the concentration of immue cytokine. The abscissa is the age of weeks after first immunization and arrows indicate the primary vaccination and subsequent boosters. * Indicates a significant difference (P<0.05).
Figure 4
Figure 4
(A) The proportions of lymphocytes in blood of rats were separated and were analyzed by flow cytometry compared to control groups.Up left is CD45,up right is CD4 and CD8(CD45 for gate), down left is CD3 and CD19(CD45 for gate),down right is CD4 and CD25(CD45 for gate), (B) Mean (± SEM) of the serum CD3+CD19-、CD3-CD19+、CD4+CD8-、CD4-CD8+、CD4+CD8+、CD4-CD8-、CD4+CD25+ and the ratio of CD4+ and CD8+ (all from the gate of CD45+ cells)concentrations in the gonadotropin-releasing hormone (GnRH) immunization group (n = 35) and control group (n = 35). Flow cytometry was used to detect the number of immune cells. The ordinate is the ratio of immue cytokine. The abscissa is the age of weeks after first immunization. * Indicates a significant difference (P<0.05).
Figure 5
Figure 5
(A) Mean (± SEM) of the relative weight of the thymus in the gonadotropin-releasing hormone (GnRH) immunization group (n = 35) and control group (n = 35). Arrows indicate the primary vaccination and subsequent boosters. * Indicates a significant difference (P < 0.05). (B) Histopathological observation of the thymus of the control group (a, b, e, g, i, k) and gonadotropin-releasing hormone (GnRH) immunization group (b, d, f, h, j, l). All observations were performed on samples taken from 4 week old rats after first immunization. Numbers 1, 2, 3, and 4 represent a magnification of 40, 100, 200, and 400 times, respectively.
Figure 6
Figure 6
Mean (± SEM) of the effects of gonadotropin-releasing hormone (GnRH) immunization on the mRNA expression of reproduction-related genes in the thymus of the control (n = 35) and GnRH immunization (n=35) groups. (A–F) indicate the detection time at different ages. * Indicates a significant difference (P < 0.05).
Figure 7
Figure 7
Mean (± SEM) of the effects of gonadotropin-releasing hormone (GnRH) immunization on the mRNA expression levels of immunity-related genes in the thymus of the control (n = 35) and GnRH immunization (n = 35) groups. (A-F) represent the detection time at different ages. * Indicates a significant difference (P < 0.05).
Figure 8
Figure 8
(A) Mean (± SEM) of the relative weight of the spleen in the gonadotropin-releasing hormone (GnRH) immunization group (n = 35) and the control group (n = 35). Arrows indicate primary vaccination and subsequent boosters. * Indicates a significant difference (P < 0.05). (B) Histopathological observation of the spleen of the control group (a, c, e, g, i, k) and the gonadotropin-releasing hormone (GnRH) immunization group (b, d, f, h, j, l). All observations were performed on samples taken from 4-week-old rats after first immunization. Numbers 1, 2, 3, and 4 represent a magnification of 40, 100, 200, and 400 times, respectively.
Figure 9
Figure 9
Mean ( ± SEM) effects of gonadotropin-releasing hormone (GnRH) immunization on the mRNA expression of reproduction-related genes in the spleen of the control (n = 35) and GnRH immunization groups (n = 35). (A–F) represent the detection time at different ages. * Indicates a significant difference (P < 0.05).
Figure 10
Figure 10
Mean (± SEM) of the effects of gonadotropin-releasing hormone (GnRH) immunization on mRNA expression of immunity-related genes in the spleen of the control (n = 35) and GnRH immunization groups (n = 35). (A-F) Indicate the detection time at different ages. * Indicates a significant difference (P < 0.05).
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