In addition to genetic factors, external environmental factors also play important roles in the sex determination and differentiation of fish. Therefore, fish can be useful subjects to study sexual differentiation. With the continuous progress and development of transcriptomic and proteomic technologies, transcriptomics and proteomics have been widely used in the study of reproductive and sexual differentiation-related mechanisms in fish, including tilapia, especially in the identification of DEGs, the screening of functional genes, and the discovery and verification of important regulatory pathways [14–17]. After treatment with progesterone receptor inhibitor (RU486), 7148 genes were differentially expressed in gonad of female tilapia; the results revealed that fshr and lhr were significantly downregulated and ars was significantly upregulated after RU486 treatment, which might account for the masculinization and infertility of female fish . Transcriptomic analysis of the gonads of female and male tilapia at different developmental stages revealed that estrogen may play an important role in female sex determination and maintenance of phenotypic sex, which lays the foundation for future studies into the molecular mechanisms of sex determination and maintenance of phenotypic sex in non-model teleosts . Analysis of the transcriptome of the gonads of control female, high-temperature-treated female, and high-temperature-induced neomale tilapia identified a number of genes that may be involved in GSD + TE (genotypic sex determination + temperature effects), which should be useful for investigating the molecular mechanisms of GSD + TE in fish . Study of the gonadal proteome in fish during sex reversal or gonadal differentiation, and the screening of important functional proteins related to reverse acquisition and gonadal differentiation are of great significance for the study of vertebrate sex determination and differentiation [21, 22]. GnRH immunization of mammals, such as mice, sheep, cattle, and pigs, can reduce the levels of FSH and LH, and inhibit the development of the gonads, achieving artificial intervention in animal sex differentiation . Intervention in fish sex differentiation by GnRH immunization, investigation of the mechanism of action of GnRH in regulating sex differentiation, and screening of sex determination factors are of great significance for uncovering the sex-differentiation and sex-controlling mechanisms. However, no transcriptomic or proteomic studies of tilapia gonad after GnRH immunization have been reported.
In this study, the gonadal transcripts of tilapia immunized with GnRHa were sequenced, and an average of 88.28% clean reads were mapped to the reference genome. Sun et al. sequenced the gonadal transcripts and mapped an average of 80.09% clean reads to the reference genome . The reference genome of tilapia used by Sun et al. was Orenil1.1 (GCA_000188235.2) , while the reference genome used in this study was O_niloticus_UMD_NMBU (GCA_001858045.3). Orenil1.1 is a genome assembled by next-generation sequencing (Illumina), with a full length of 927,679,487 bp, while O_niloticus_UMD_NMBU was assembled by third-generation sequencing (PacBio), with a full length of 1,005,681,550 bp. Combined with other genome parameters, the integrity of the O_niloticus_UMD_NMBU genome is better than that of the Orenil1.1 genome, resulting in better alignment in this study. The failure in aligning 11.72% clean reads to the reference genome in this study might have been due to factors such as wrongly sequenced reads remaining after quality control, interference of alternatively spliced genes, contamination by other genomes, and poor integrity and assembly of the reference genome.
In this study, 644 DEGs were identified by transcriptomic analysis, including 80 upregulated genes and 564 downregulated ones. Moreover, 1150 DEPs were identified by proteomic analysis, including 351 upregulated proteins and 799 downregulated ones. Overall, 209 genes showed consistent differential expression patterns at mRNA and protein levels, of which 9 were upregulated and 200 were downregulated. The results of the three kinds of analysis showed that the expression of gonadal genes was inhibited by GnRHa immunization in tilapia, and the functions of downregulated genes were mainly focused on single-organism process, binding, cellular process, metabolic process, and catalytic activity, while the main involved pathways include ECM–receptor interaction, focal adhesion, cardiac muscle contraction, and oxidative phosphorylation. It is notable that six DEGs are involved in the GnRH signaling pathway, and the expression of these six genes is downregulated, indicating that GnRHa immunization can inhibit the expression of genes related to the gonadotropin releasing hormone signal transduction pathway.
Forty-seven downregulated genes were obtained by combined analysis of the transcriptomic and proteomic data, among which the differential expression of zona pellucida sperm-binding protein 3 (ZPBP3), apolipoprotein A-I (apo A-I), and cytoplasmic 1 was most significant. Zona pellucida sperm binding protein (ZPBP) is closely related to sperm motility, capacitation, acrosome reaction, and sperm–egg binding. Studies have shown that ZPBP1 and ZPBP2 were mainly expressed in testicular tissue of mammals, while ZPBP3 was found in tilapia [23, 24]. In this study, it was found that ZPBP3 could be expressed in the gonad of female tilapia, which may be related to the specific sex differentiation mechanism of fish. Meanwhile, the expression of ZPBP3 in gonad of tilapia was significantly downregulated after GnRHa immunization, indicating that ZPBP3 plays an important role in the gonad development of tilapia. Apo A-I is the major component of high-density lipoprotein, which plays an important role in reverse cholesterol transport and lipid metabolism, and is also a very important innate immune molecule, providing a platform for the assembly of several immune complexes [25, 26]. Previous studies showed that apo A-I was present in multiple tissues of Branchiostoma belcheri, and the expression of apo A-I mRNA was highest in the gonads; the expression of apo A-I mRNA increased significantly in the process of infection, suggesting that apo A-I may be involved in immune stress response and play an important role in the innate defense immune system of Amphioxus . The apo A-I expression profile in Monopterus albus immunized with Aeromonas hydrophila showed that infection could downregulate the expression of apo A-I in the small intestine, spleen, and liver, indicating that apo A-I might be involved in the innate immune system of this species . In this study, the expression of apo A-I in gonad of tilapia immunized with GnRHa was significantly downregulated, which also indicated that apo A-I plays an important role in the immune response of tilapia. Actin is one of the most highly conserved proteins in eukaryotic cells. It is mainly present in the cytoplasm, is the main component of cytoskeleton microfilaments, and is necessary for a variety of cell functions, such as the division, movement, and growth of cells . Actin (cytoplasmic 1) can be expressed in several tissues of tilapia, but its expression level in gonad is much higher than that in other tissues, indicating that this gene may play an important role in gonad development and gonad functions of tilapia . In this study, the expression of actin (cytoplasmic 1) in gonad of tilapia immunized with GnRHa was significantly downregulated, which indicated that GnRHa immunization could inhibit the expression of important genes involved in gonad development and provide a reference for revealing the mechanism of GnRH immunization.