Article Figures & Data
Figures
- Figure 1.
Long photoperiods induce physiological and hypothalamic neuroendocrine change associated with reproduction in adult and juvenile Japanese quail. a, Measurements of testis length showed a significant interaction between photoperiod and age. b, Fat score ratings, as established by Wingfield and Farner, were overall higher in adults and in 16L photoperiod conditions. c, qRT-PCR analyses show a significant photoperiod by age interaction on DIO2 expression in the MBH. d, A significant photoperiod by age interaction was also found in MBH DIO3 expression. Physiological and transcriptomic data from qRT-PCR were analyzed by two-way ANOVA and Tukey's HSD with an α value of p = 0.05. The letters above each group indicate pairwise comparisons by Tukey's HSD where appropriate.
- Figure 2.
Volcano plots comparing age and photoperiod revealed a high number of differentially expressed transcripts. Upregulated transcripts (logFC > 1) are colored red, and downregulated transcripts (logFC < −1) are colored blue. a, Significant differentially expressed transcripts across photoperiod treatments in adults (n = 206) included FSHß, PRL, and NTS. b, Significant differentially expressed transcripts across photoperiod treatments in juveniles (n = 184) included HAPLN1. c, Significant differentially expressed transcripts across 16L age groups (n = 685) included GH. d, Significant differentially expressed transcripts across 8L age groups (n = 678) also included GH. A p-value of <0.01 was deemed significant for all plots.
- Figure 3.
a, A heatmap of the top 50 most significant transcripts in the pituitary gland. Among the most significant transcripts were FSHß and GH. b–f, Targeted qRT-PCR analyses were performed in order to investigate the transcription of genes of interest influenced by photoperiod and age. b–d, There were significant main effects of both photoperiod and age on FSHß, LHß, and PRL expression, as well as an interaction effect between photoperiod and age on LHß expression. e, There was a significant photoperiod by age interaction on GH expression. f, There was a significant main effect of photoperiod and age on OPN5 expression. For the pituitary sequencing heatmap analysis, a p-value of 0.01 was deemed significant. Targeted qRT-PCR analyses were performed using two-way ANOVA and Tukey's HSD with an α value of p = 0.05. The letters above each group indicate pairwise comparisons by Tukey's HSD where appropriate. Additional data relating to these analyses are provided in Extended Data Figures 3-1 and 3-2.
Tables
- Table 1.
Targeted qRT-PCR primer sequences and specifications, including forward and reverse primer sequences, annealing temperature, melting temperature, and expected product sizes for DIO2, DIO3, OPN5, LHβ, FSHβ, PRL, and GH
Gene Primer Annealing temperature (°C) Melting temperature (°C) Length (bp) DIO2 CGCCTACAAGCAGGTCAAAC 60 82 242 CACACTTGCCACCAACACTCTT DIO3 AGGCTCTCTTCCTTCGGGAT 60 83 180 TAGCACTTGCTAGGCAGCAC OPN5 ATGGCATCAGACTGCAACTCC 60 84 499 AAGGAACAGTAGCCCAGAACG LHΒ TTTACCGCAGCCCTTTGGGT 60 87 125 AGAGCCACGGGTAGGATGACTTT FSHΒ CTGCGGTGACCATCCTGAATCTTT 62 85 396 GCTTCCATTGTGACTGAAGGAGCA PRL AATGAAACCCCGACCCTGAG 60 79 630 CCCCTAGTGCAACTTGAGACC GH GCTGCCGAGACATACAAAGAG 60 81 109 GAGCTGGGATGGTTTCTGAG ß-actin AATCAAGATCATTGCCCCAC 60 84 114 TAAGACTGCTGCTGACACC - Table 2.
Summary of two-way ANOVA analyses associated with Figures 1 and 3, including F- and P-statistics associated with the main effects of photoperiod and age, and their interaction, on testis length, fat score, and the expression of DIO2, DIO3, FSHβ, LHβ, PRL, GH, and OPN5E
Photoperiod Age Photoperiod:age Testis length F(1,15) = 198.46, p = 4.69 × 10−10*** F(1,15) = 112.75, p = 2.26 × 10−8*** F(1,15) = 81.17, p = 1.94 × 10−7*** Fat score F(1,16) = 9.23, p = 7.83 × 10−3** F(1,16) = 6.15, p = 2.46 × 10−2* F(1,16) = 0.00, p = 1.00 n.s. DIO2 F(1,16) = 0.77, p = 3.94 × 10−1 n.s. F(1,16) = 6.94, p = 1.81 × 10−2* F(1,16) = 22.12, p = 2.4 × 10−4*** DIO3 F(1,17) = 10.78, p = 4.38 × 10−3** F(1,17) = 13.40, p = 1.94 × 10−3** F(1,17) = 15.30, p = 1.12 × 10−3** FSHβ F(1,17) = 17.69, p = 5.95 × 10−4*** F(1,17) = 29.54, p = 4.45 × 10−5*** F(1,17) = 0.83, p = 3.75 × 10−1 n.s. LHβ F(1,17) = 9.47, p = 6.83 × 10−3** F(1,17) = 74.37, p = 1.29 × 10−7*** F(1,17) = 4.68, p = 4.51 × 10−2* PRL F(1,17) = 9.27, p = 7.34 × 10−3** F(1,17) = 14.64, p = 1.35 × 10−3** F(1,17) = 1.92, p = 1.83 × 10−1 n.s. GH F(1,16) = 3.46, p = 8.14 × 10−2 n.s. F(1,16) = 21.48, p = 2.75 × 10−4*** F(1,16) = 5.67, p = 3.00 × 10−2* OPN5 F(1,16) = 17.01, p = 7.96 × 10−4*** F(1,16) = 24.79, p = 1.36 × 10−4*** F(1,16) = 0.05, p = 8.21 × 10−1 n.s. Additional data relating to these analyses are provided in Extended Data Tables 2-1–2-3.
Nonsignificant p-values are indicated by “n.s.,” p-values <0.05 are indicated by “*,” <0.01 by “**,” and <0.001 by “***.”
Table 2-1
Raw data including physiology measurements, MBH and pituitary qPCR fold changes in gene expression, and pituitary RNA-sequencing differential expression data generated using edgeR. Differential expression analyses include those relating to simultaneous comparison of all groups, age comparisons, and photoperiod comparisons. Download Table 2-1, XLS file.
Table 2-2
Functional pathway analyses comparing 8L birds with 16L birds using the Database for Annotation, Visualization and Integrated Discovery (DAVID, Sherman et al 2022). Categories and functional analysis terms have been reported alongside associated gene count, significance levels, and a total list of significantly differentially expressed genes within each category. Download Table 2-2, XLS file.
Table 2-3
Functional pathway analyses comparing juvenile birds with adult birds using the Database for Annotation, Visualization and Integrated Discovery (DAVID, Sherman et al 2022). Categories and functional analysis terms have been reported alongside associated gene count, significance levels, and a total list of significantly differentially expressed genes within each category. Download Table 2-3, XLS file.
Figure 3-1
Plots comparing transcript count data from Oxford Nanopore RNA-sequencing. A-C. PRL, FSHß, and GH were significantly differentially expressed across all groups. D. OPN5 was not found to be differentially expressed, contrasting qPCR data. E. NTS was significantly differentially expressed, whereas F-G. MEF2A and MEF2D were not significantly differentially expressed across treatment groups. For all analyses, a P-value of P<0.01 was deemed significant. Download Figure 3-1, TIF file.
Figure 3-2
Schematic diagram representing photoperiod and developmental changes in pituitary cell type transcript expression. Expression of FSHß, LHß, PRL, TSHß and GH in pituitary cells is dependent on both the age of the quail and the experienced photoperiod conditions. Blank cells are included to indicate that transcriptomic changes are occurring in existing cells, rather than e.g., as a result of the production of new cells. Download Figure 3-2, TIF file.
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