Anti-GnRH vaccination triggered a good immunocastration response in all immunized male rats
Compared to EM, neither ORC nor IM had obvious effects on the body weight profile of rats (P > 0.05; Fig.1A). After two doses of GnRH vaccine injection, all the 9 immunized rats shown a substantial decrease (P < 0.001; Fig.1B and C) in testes weight. At decapitation, the average testes weight of immunized rats was reduced by 80% compared to that of EM (Fig.1C). Resultantly, serum concentrations of testosterone were reduced (P < 0.001; Fig.1D) to undectable levels, indicating a very good immunocastration efficacy in all the immunized rats. Compared to EM, both serum LH and FSH concentrations were substantially increased in ORC (P < 0.001), but reduced (P < 0.001) in IM (Fig.1E and F).
Differential efficacy of ORC versus IM in causing degeneration of SMG in male rats
Compared to EM, ORC caused a substantial degeneration of SMG. Both the weight and organ index of SMG were markedly decreased (P < 0.01) in rats following ORC (Fig. 2A-B). But, there was no obvious difference in either SMG weight or organ index between EM and IM (P > 0.05). And both the weight and organ index of SMG in IM were higher (P < 0.001) than in ORC.
Histological analysis shown that the SMG exhibited predominantly serous acini and a lower quantity of mucous acini, and an obvious degeneration of SMG acini and ducts in ORC (Fig. 3A and B). Statistical analysis indicated that ORC substantially decreased (P < 0.001) the percentage of both SMG acini and ducts (Fig. 4A and B), indicating a substantial degenarative alteration in the histological structure of SMG. Likewise, IM also moderately reduced the percentage of SMG acini (P = 0.0659) and ducts (P < 0.05), but both of which were still much higher (P < 0.001) than those in ORC (Fig. 3B and C; Fig. 4A and B).
Compared to EM, there was a substantial increase (P < 0.001) in lipid vacuoles in SMG in rats following ORC (Fig. 3E and Fig. 4C). Although, lipid depostion was also increased (P < 0.05) in SMG of rats following IM, it was markedly lower than that in ORC (P < 0.001; Fig. 3F and Fig. 4C).
Transcriptomic changes of SMG in response to ORC versus IM
To gain a better understanding of the mechanisms of testicular steroids deficiency on SMG physiology, we assessed the transcriptomic changes of SMG in response to ORC versus IM by RNA-seq. Hierarchical clustering comparing patterns of gene expression indicated a striking separation in SMG among groups (Fig. 5A), revealing a distinct functional dissociation in SMG between ORC/IM and EM, and between IM and ORC. Even though, SMG samples from IM were still clustered more closer to those from ORC than from EM, suggesting there exist some common causes to SMG degeneration between ORC and IM.
Pairwise comparison among groups revealed 851 differentially expressed genes (DEGs, Fig. 5B; see Methods for criteria). Of those, 647 DEGs were identifed between ORC and EM, of which 356 (55%) were upregulated and 291 (45%) were downregulated in ORC (Fig. 5C;Table S2). And, 369 DEGs were identified between IM and EM, of which 160 (44.6%) were upregulated and 209 (55.4%) were downregulated in IM (Fig. 5D;Table S2). There were 209 common DEGs between ORC and EM, and between IM and EM (Fig. 5B, yellow circle; Table S3). Except Brms1 and LOC100912599, all other 207 common DEGs showed same direction of changes (up- or downregulation) comparing EM (Fig. 5E). Of those common DEGs, 126 (61%) were common downregulated and 83 (39%) were common upreglated in ORC/IM (Table S3).
Functional enrichment analysis using DAVID (V6.8) shown that the common downregulated DEGs in ORC/IM were predominantly annotated into biological process (BP) of epithelial cell development/differentiation/morphogenesis, angiogenesis, ion transport, antomical structure/tube/tissue morphogenesis, secretion, gland/tube development (Fig. 5F). Dysfunction of these biological processes, especially dysfunction of epithelial cell development, angiogenesis and anatomical structure morphogenesis should cause SMG degeneration. Molecular function (MF) analysis showed that these common downregulated DEGs were mainly annotated into growth factor binding and fibroblast growth factor binding(Fgfr3, Fgfrl1 and Thbs1) (Fig. 5F), revealing that growth hormones and fibroblast growth factors might play important roles in SMG degeneration following ORC/IM treatments. They were mainly annotated into cellular component (CC) of membrane-bouded vesicle, extracellular vesicle/exosome, golgi apparatus, ect (Fig. 5F), suggesting a declined saliva secretion of SMG after androgen deprivation. And, KEGG pathway analysis highlighted PI3K-Akt signaling pathway, focal adhesion and ECM-receptor interaction (Fig. 5F) may play great roles in mediating androgen deprivation-induced SMG degeneration. While, the common upregulated DEGs were predominantly annotated into BP of oxoacid metabolic process, amide biosynthetic process, translation, programmed cell death, apoptotic process/signaling pathway, regulation of programmed cell death ect, into MF of aminoacyl-tRNA ligase activity, structural constituent of ribosome, ATP binding, ect, into CC of extracellular exosome, cytosolic ribosome, mitochondrion, into KEGG pathways of antibotics and amino acids biosynthesis, carbon metabolism, ect (Fig. 5G). Obviously, increased cell apoptosis/death also should be another important cause of SMG degeneration in androgen deprivation.
Identification of key candiate genes that mediate SMG degeneration in response to androgen deprivation
To identify candidate genes that mediate SMG degeneration in response to androgen deprivation, we checked the expression of reproductive hormone receptors in SMG across groups. Using FPKM ≥1 in at least one of the sample replicates as a stringent cutoff for gene expression , we found that SMG expresses androgen receptor (Ar) but not expresses receptors for estrogens, gonadotropins (LH and FSH) or gonadotropin-releasing hormone (GnRH) (Fig. 6A), suggesting androgen deprivation is of high possibility to be the main or even the only cause of SMG degeneration following surgical or immunological castration. Given disrupted epithelial cell development, angiogenesis, anatomical structure morphogenesis and enhanced cell apoptosis are directly relevant to SMG tissue structural and morphological remodeling, we selected the common regulated genes by both ORC and IM which are involved in these four functional terms to conduct futher analysis. De novo motif analysis of these selected genes revealed a high number of putative AR binding sites within their promoter regions (Fig. 6B), suggesting these genes might be the direct targets of androgens in regulating SMG structure and morphology remodeling. Of those important candidate genes, B4galt1, Angpt14, Ace, Klf4, Egf, Tgfb2, Wnt4 and Sox10 are simutaneously involved epithelial cell development, angiogenesis, anatomical structure morphogenesis and/or cell apoptosis and their expressions were all significantly downregulated by androgen deprivation (Fig. 6B), thus they might be the key candidate genes that mediate androgen deprivation-induced SMG degeneration. Besides, Dnase2b (deoxyribonuclease II beta), an enzyme responsible for nuclear degradation, palys a major role in cellular apoptosis . And, Kcnip3 (also called Dream) enhances cell apoptosis by altering endoplasmic reticulum calcium signaling . Both of the two genes were commonly upregulated to 7~10 fold by androgen deprivation, thus they appear to play key roles in SMG degeneration in rats following ORC/IM through enhancing cell apopotosis. Using RT-qPCR, we validated the expression changes of all these key candiate genes (Fig. 6C).
Functional enrichment analysis reveals the causes of differential degeneration response of SMG between ORC and IM
Functional enrichment analysis of the DEGs between IM and ORC indicated that the upregulated DEGs in IM were mainly annotated into CC of coated vesicle membrane, golgi apparatus and endoplasmic reticulum, into BP of golgi vesicle transport, protein transport, autophagosome assemby, gluconeogenesis, into MF of ion binding, and into KEGG pathways of protein processing in endoplasmic reticulum (Fig.7A; Table S4), suggesting a stronger capacity of SMG to secrete saliva and generate glucose in IM than in ORC. While, the downregulated DEGs in IM were mianly annotated into CC of ribosome, mitochondrion, into BP of oxidation-reduction process, amide biosynthetic process, gene expression, translation, into MF of structural constituent of ribosome, and into KEGG pathways of ribosome and oxidative phosphorylation (Fig.7B), highlighting a hyperfunction of ribosome and mitochondrion of SMG in ORC than in IM.
Gene set enrichment analysis (GSEA) using the whole expressed genes were performed. Interestingly, compared to either EM or IM, ORC rats consistently shown a positive enrichment score for protein translation (Fig. 8A and B) and oxidative phosphorylation (Fig. 8C and D), and negative score for vesicle transport (Fig. 8E and F), reaffirming hyperfunction of ribosome and mitochondrion, and lower salivary secretion of SMG in ORC rats than in IM/EM rats.
To further gain insights into the differential SMG degeneration response between ORC and IM, we isolated the genes whose expressions were differentially different between ORC and EM (adj < 0.05), but remained unchanged between IM and EM. We refered them to as the maintained DEGs (mDEGs). In other words, these mDEGs were selectively affected by ORC but not by IM. Thus, they are directly relevant to the differential SMG degeneration respone between IM and ORC. There were 647 DEGs between ORC and EM, of which 427 (66%) were the mDEGs (Fig. 9A; Table S2). Functional enrichment analysis showed that these mDEGs were predominantly annotated into CC of ribosome, mitochondrion, focal adhesion, cell-substrate junction, extracellular vesicle, into MF of structural constituent of ribosome, RNA binding, into BP of translation, ribosome biogenesis, gene expression, cellular component biogenesis, cellular lipid metabolic process, into KEGG pathways of ribosome, oxidative phosphorylation, protein processing in endoplasmic reticulum and metabolic pathways (Fig.9B). Of 427 mDEGs, 62 mDEGs are associated with ribosome (Fig.9C) and 37 associated with mitochondrion (Fig.9D). Almost, all of these mDEGs involved in ribosome and mitochondrion were expressed higher in ORC than in EM/IM (Fig.9 C and D), emphasizing hyperfunction of ribosome and mitochondrion of SMG in ORC again.
Integrated network analysis revealed key candidate genes that mediate the differential SMG degeneration response between ORC and IM
Oxidative stress and apoptosis have been considered as the mechnisms of dysfunction of salivary gland in women after menopause . Functional enrichment analysis of our SMG transcriptomic data highlighted that epithelial cell development/proliferation/morphogenesis, cell death, neuron development, anion transport, cell junction, extracellular vesicle, ect, might be associated with the degeneration response of SMG in deprivation of adrogens (Fig. 5F and G;Table S4). Besides, histology analysis shown that lipid depostion was also an important reason to cause SMG degeneration in deprivation of androgens. Comprehensive analysis suggests that hyperfunction of ribosome and mitochondrion might be associated with the differential degeneration response of SMG between IM and ORC (Fig. 7B and C). We isolated the mDEGs involved in those above terms to construct a network using cytoscape softeware (v3.9) (Fig. 10A). We further extracted a subnetwork (Fig. 10B) derived from 1) the mDEGs that were differentially expressed between IM and ORC, and 2) the mDGEs that are simutaneously involved in three or more functional terms. The mDEGs in Fig. 10B thus should play important roles in mediating the differential degeneration response of SMG between IM and ORC. Particularly, 27 genes (Fig. 10C) in this subnetwork are mDEGs and also DEGs between IM and ORC. We consider these 27 genes are the key candidate genes that mediate the differential degeneration response of SMG between IM and ORC. Of these 27 genes, Naa38 and Fam129b is involved in cell death; Cox5b, Ndufa1, LOC679739 (Ndufs6) and Tomm7 are all involved in mitochondrion; Rps14, Rpl23, Rpl13, Rpl18, Rps15a, Rps23 and Rps28 are all involved in ribosome; Scap and St6galnac2 are both associated with lipid metabolism. Expression of these genes was all upregulated in ORC but kept unchanged or downregulated in IM, in accordance with more severe degeneration of SMG in ORC than in IM (Fig. 10C). While, Hyou1 is involved in cell junction and extracellular vesicle; Gdf5 is involved in epithelial cell proliferation; C2cd4b is involved in both cell junction and cell-substrate junction; Tfg and Slc30a7 are both involved in extracellular vesicle. These genes were all downregulated in ORC but kept unchanged in IM (Fig. 10C), also in accordance with more severe degeneration of SMG in ORC than in IM.