Active immunization with recombinant GnRH6-CRM197 inhibits reproductive function of male rats

Abstract

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.

Keywords:

• Active immunization
• gonadotropin-releasing hormone hexamer-cross reacting material 197 (GnRH6-CRM197;) reproductive function
• rat

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors’ contributions

Assisted with study design and analyzing and interpreted data, drafted and completed manuscript: XBG, XY; collected the samples and developed vaccine and immunized rats: MXL, MYD, JTL; Expressed and identified the vaccine and raised rats: SSX, ZHP, YYZ; assisted with hormone analyses and interpreted data: ZYW, WZ, PQ; prepared the tissue section and observation: YL, YSL; oversaw overall study design and interpreted data: FGF.

Additional information

Funding

This research received financial support from various sources, including the National Natural Science Foundation of China (grant number 31972629), and the Innovation Fund Designated for Graduate Students of Anhui Agricultural University (grant numbers X202310364037, X202210364181, X202210364145).