- previously uncovered sexual dimorphism in the basal and stress-responsive gene transcription of a biological system necessary for facilitating sexual behavior and reproduction, the hypothalamic-pituitary-gonadal (HPG) axis. - In this study, we delve further into understanding the mechanistic underpinnings of how changes in the environment can affect reproduction by testing the alternative splicing response of the HPG axis to an external stressor in both sexes.. - In both stress and control treatments, we identified a higher incidence of splicing activity in the pituitary in both sexes as compared to other tissues. - Of these splicing events, the core exon event is the most abundant form of splicing and more frequently occurs in the coding regions of the gene. - Overall, we observed less splicing activity in the 3 ’ UTR (untranslated region) end of transcripts than the 5 ’ UTR or coding regions.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - In the human gen- ome, approximately 80% of exons are >. - The inclusion of an exon in the final mRNA product is entirely driven by cis- and trans-acting elements/factors. - Previous research has revealed unique roles for alternative splicing in the immune response of chickens with avian pathogenic E.coli [25], mediation of abiotic stress response pathways of plants [26], and enhanced fear memory of mice [27]. - In vertebrates, a symphony of physiological events is required to regulate sexual behavior and reproduction, and these mechanisms are driven by an interconnected biological system made up of the hypothalamus in the brain, the pituitary gland, and the gonads (testes/ovaries . - Understanding how the alternative splicing land- scape of the reproductive axis changes in the face of stress will not only offer more insight into how stress can affect reproduction, but deepen our proximate knowledge of bio- logical processes and sexaully-biased behavioral responses in general.. - In this study, we traverse beyond the level of transcription to test for sex-biased alternative splicing patterns in the HPG axis of the rock dove in response to a restraint stress stimulus. - To our knowledge, this is the first report of sex-specific splicing events in the HPG axis in response to a stressor.. - Our first aim was to understand sex-typical splicing in the hypothalamus (hyp) and pituitary (pit) by assessing each tissue for alternative splicing events between males and females. - female splicing comparison: events by type In total, we identified 158 splicing events in the hypo- thalamus and 225 events in the pituitary. - When com- pared to the hypothalamus, the 42% increase of splicing event abundance seen in the pituitary is significant. - In both tissues, more events were identified in the control state compared to the stress condition (hyp: 99 control/59 stress, pit: 123 control/102 stress), but only the relationship in the hypothalamus was statistically significant (Fig. - Of these core exon splice events, there were al- most twice as many in the pituitary compared to hypothalamus (pit: 74 control/67 stress, hyp: 48 control/. - Retained intron events in the hypothalamus were the only event to differ significantly between treatments (ChiSq p = 0.012), with nearly 3 times (280% increase) more splicing events in the control state than the stressed. - Both core exon events in the hypothalamus and retained intron events in the pituitary reflected a similar increased abundance in splicing events of the control state, though these rela- tionships were not significant (ChiSq p: CE-Hyp = 0.053, RI-Pit = 0.052). - female splicing comparison: genes of interest Using our comparison of male to female splicing pat- terns, we were able to identify sex-specific alternatively spliced genes in the stress response. - a transcription factor that regu- lates transcription of gonadotropin-releasing hormone (GnRH is alternatively spliced in the male pituit- ary stress response. - Through alternative splicing of the POU2F1 gene in the pituitary, males may be altering sig- naling pathways within the HPG axis to optimize stress response.. - Alternatively spliced between sexes in the pituitary, P-selectin (SELP) is known to preserve im- mune function in mice [58]. - In the list of significant Table 1 Statistics for all Chi-Square Tests. - 2), splicing of organic and heterocyclic compound genes is underrepresented in the pituitary, while small molecular and drug binding is overrepresented in the hypothalamus regardless of treatment. - There was an interaction seen in the hypo- thalamus. - splicing of organic and heterocyclic compound genes was overrepresented in the stress treatment, though underrepresented in the control group.. - Alternatively spliced genes in the male vs female analysis revealed, in both tissues, more events in the control versus restraint stress condition. - In the control vs splicing comparison, more splicing occurred in the male hypothalamus. - while in the gonad, more splicing occurred in the female. - Red stars represent statistical significance between abundances in males and females, with more core exon splice events occurring in the female gonad than male (ChiSq p =3.01e-3), and more hypothalamic retained intron events found in males than females (ChiSq p =0.041). - This, perhaps, is not surprising given that the CDS regions are more abundant in the genome and alteration to these regions will ultimately result in changes to the protein sequence. - The abundance of al- ternatively spliced exons present in the 5′ &. - In the hypothalamus, more spliced exons occurred in the 5’UTR than genomic proportions would predict, with more dramatic shifts in the restraint stress treatment than the control (ChiSq, p = 0.015). - In the pituitary, the control group exhibited more spliced exons of the 5’UTR and both treatment groups presented more than a 6% decrease of splicing events in 3’UTR regions than predicted from genomic values (ChiSq p: 5 ’ UTR UTR = 2.60e-7).. - Splicing in the pituitary is more prevalent in heterocyclic and organic cyclic compound binding genes, while splicing in the hypothalamus affects small molecule and drug binding loci. - Parent ontology terms along the y-axis are in descending order from most frequent in the genome to less frequent. - stress splicing comparison: events by type Similar to our comparison of male-female alternative spli- cing, control-stress splicing reveals more splicing in the pituitary than other tissues (ChiSq p = 1.04e-6). - The male hypothalamus displays 59% more splicing events than the female hypo- thalamus, and we identified 78% more splicing events in the ovaries than the testes (Fig. - more core exon splice events occurred in the ovaries than in the testes (ChiSq p = 3.01e-3). - Estradiol 17- beta-dehydrogenase 11 (HSD17B11) was alternatively spliced between treatments in the male hypothalamus.. - 3 Distributions of core exon splicing event lengths for between-sex spliced loci in the control (light blue) and stress (orange) states as well as control-stress spliced loci in male (blue) and female (green) states. - In the pituitary of control vs stress comparison, lengths of spliced exons are significantly larger in the male pituitary than that of spliced exons in the female pituitary (Wilcoxon, p = 0.022). - if a particular type of gene or genes related to a specific biological process were more readily spliced in the stress response. - These terms were more abundant than genomic predictions in the male hypothalamus, female pituitary, and male gonad, while these same terms were less abundant than expected in the female hypothalamus and female gonad. - Similar to the male- female splicing analysis of splicing in protein motifs, we found no significant difference in the distribution of events occurring in any one motif (Figure S6).. - Similar to our male-female analysis, we also identified a significantly lower abun- dance of splicing events in the 3’UTR regions of all tis- sues in the control-stress analysis (ChiSq p: hyp = 0.042, pit = 2.87e-12, gon = 8.77e-4). - There were fewer events in the 5’UTR of the pituitary (ChiSq p = 2.04e-4), and more events occurred in the pituitary CDS than ex- pected (ChiSq p = 3.50e-4).. - Binding genes were more frequently spliced in the male hypothalamus and the female pituitary. - Parent ontology terms along the y axis are in descending order from most frequent in the genome to least frequent. - The only two instances that there appears to be a binary shift is in the case of alternate acceptors (AA), specifically in the hypo- thalamus of the male vs female analysis, and in the hypothalamus of the control vs stress analysis. - The additional 9 events this included for the male-female comparison were all found in the pituitary (3 control, 6 stress). - Sixty-one events in the control-stress comparison had deltaPSI values between − 0.1 and 0.1, 58. - of which were also in the pituitary (53 male, 5 female) and 3 in the female gonad. - Most significant splicing events have abs(deltaPSI) values greater than 0.1 in the male- female comparison. - The 53 events in the male pituitary with low abs(deltaPSI) may suggest the male pituitary responds to stress not only by splicing genic regions at higher PSI values, but also by numerous, low-level splicing events.. - There were significantly fewer splicing events in the 3 ’ UTR, agreeing with our previous findings, indicating that splicing machinery may selectively avoid this transcript region. - We discovered a sig- nificant sex difference in the splicing profile of hypothal- amic and pituitary tissues. - When subjects underwent a restraint stress treatment, we observed a decrease in the total number of significant splicing events between sexes as compared to controls (Fig. - If this pattern of convergence is not seen at other physiological levels, it is possible that the decrease in splicing differences between males and females in the restraint stress treatment may re- flect a stress response unique to the splicing level that does not exist in the proteomic or methylomic levels or. - Thus, an active sex-specific physiologically-driven alternative splicing landscape under stress may become reconciled in the HPG axis to shunt focus to other biological pro- cesses to support survival. - The remaining splicing differences may lie in loci generally implicated in the stress response, though fu- ture studies are needed to say this definitively. - This pat- tern was consistent with our previous report of increased differential gene expression (DGE) in the pituitary fol- lowing restraint stress [7], suggesting this gland may be a targeted site for mediating a stress response in the HPG axis through both gene expression and alternative splicing mechanisms. - Regarding overall splicing activity, males exhibited greater responsiveness to stress than fe- males in the hypothalamus and pituitary, with a signifi- cant increase in response of the male hypothalamus compared to that of the female. - Regarding the effects of stress treatment, more core exon events were present in the female gonads as compared to the male gonads. - This may then signify that the stress response of the female gonad is driven more heav- ily by core exon alterations to a transcript, while tran- scripts in the male gonad are more readily modified with all types of splicing events.. - The only other splicing event type that differed signifi- cantly between the stress response of males and females was the retention of introns, with more retained intron events occurring in the male hypothalamus. - terms in the pituitary spliced genes, it appears that males and females may differ in how cells in the hypothalamus bind various protein products. - While the function of genes in the male and female pituitary related to “binding” ex- hibit fewer alternative splicing events than expected by. - Specifically, we observed el- evated splicing of these genes in the male hypothalamus, female pituitary, and male gonad. - Though the alternative splicing detected in both sexes exhibited smaller than expected exon sizes, size distributions in the female pituitary were smaller than those of the male pituitary. - This may suggest that splicing in the female pituitary is targeted towards minute alteration, while genes in the male pituitary are modulated by movement/removal of larger sequence segments. - In all comparisons, we found that the vast majority of splicing occurs in the protein coding region, or coding sequence (CDS) of a gene. - When we examine the fre- quency of splicing location in relation to the genomic expectation (assuming an even distribution of splicing across all genic regions), we see that variation from expectation actually lies in the untranslated regions.. - 6% decrease in splicing of the 3′-UTR genes in the. - Splicing differences identified within the HPG axis of each sex in stressed birds as compared to con- trols displayed lower 3’UTR events in all tissues, and lower 5’UTR events in the pituitary. - The increased abun- dance of 5’UTR alternative splicing events in males as compared to female subjects suggests that differences in the 5’UTR region of transcripts exist between the sexes (regardless of control/stress state). - Males and females may differ in the 5’UTR, but both sexes seem to minimize splicing of this region in the pituitary in re- sponse to restraint stress. - The decrease in splicing events in the 3’UTR could signify that splicing events in the HPG axis do not favor the 3’UTR in response to re- straint stress. - Degradation of mRNA transcripts can increase due to environmental stress [103], and it may be that splicing events in the 3’UTR would result in undesired degradation of tran- scripts, or perhaps no degradation at all.. - We did not identify any differences in the frequency of protein motif splicing between the sexes or treatments.. - In both sexes and treatment groups, we identified more alternative splicing activity in the pituitary as compared to the hypothalamus and gonads. - We found less splicing in the 3’UTR and more splicing in the 5’UTR than expected in both sex and treatment comparisons, suggesting that the 3′ end of transcripts is more biologically constrained than the. - However, despite this reduction in sex- splicing differences, the male hypothalamus and female ovary experienced increased splicing activity in the face of stress as compared to the female hypothalamus and male testes, respectively. - Birds in the re- straint stress treatment group were captured in <. - We calculated differences of counts from expected values and did not include parent ontology terms of very low abundance in the genome (<. - Alternative splicing in the control of gene expression. - Distributions of exons and introns in the human genome. - Widespread sex differences in gene expression and splicing in the adult human brain. - Hypothalamic-pituitary-gonadal endocrine system in the hagfish.. - The physiological basis of parental feeding behavior in the ring dove (Streptopelia Risoria). - On the origin of species by means of natural selection, or the preservation of favoured races in the struggle for life. - Genomic diversity and evolution of the head crest in the rock pigeon. - Oct-1 is involved in the transcriptional repression of the gonadotropin-releasing hormone receptor gene. - Hypophysiotropic signal frequency and the functioning of the pituitary- ovarian system in the rhesus monkey. - Gonadal steroid hormone receptors and sex differences in the hypothalamo-pituitary-adrenal axis.. - A vertebrate-conserved cis-regulatory module for targeted expression in the main hypothalamic regulatory region for the stress response. - Sex differences in the molecular signature of the developing mouse hippocampus. - The role of alternative splicing in the control of immune homeostasis and cellular differentiation
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