Vernal increase in pituitary FSHPβ precedes molecular switches in MBH and testes growth.

a, Schematic representation of the simulated annual rhythm in photoperiod. Quail were collected in 16hr light, 8hr dark photoperiod and then every 2 weeks the photoperiod was decreased by 2hrs to 14hr, 12hr, 10hr and then a 8L short photoperiod. Photoperiod was then increased to mimic the vernal transition and birds were collected at 10hr, 12hr, 14hr and 16hr light photoperiods. Testis volume confirmed critical day length (i.e.>12hr) induced growth. b, Body mass and c, abdomenal fat deposition increased until the autumnal equinox (12a), and then increased during the vernal photoperiod transitions. d, Diagram highlighting hypothalamic preoptic area (POA), mediobasal hypothalamus (MBH) and pitutiary gland. Tanycytes in the MBH gate GnRH release into the pituitary. e, Heat-map of RNA-seq of MBH punches identifies distinct waves of transcripts as quail transition across photoperiodic conditions. f-g, qPCR assays for proopiomelanocortin (POMC) and deiodinase type-3 (DIO3) confirmed restricted activation during 10v-8L and 8L-10v phases, respectively. h-i, vimentin immunoreactivity in the median eminence (ME) shows tanycyte morphology growth is limited to 10a, 8L, and 10v photoperiods. j, Heat-map illustrating photoperiodic transitions in pitutiary transcripts. k-m, confirmed that FSHβ is elevated under non-stimulatory photoperiods followed by increased prolactin (PRL) in 14v and thyrotropin stimulating hormone-β (TSHβ) in 16v. n, diagram summarizing that long photoperiods increase GNRH synthesis and release into the pituitary gland to stimulate FSHβ and induce testis growth. Transition to autumnal equinox phases results in reduced FSHβ expression and regressed testis. Prolonged exposure to short photoperiods inhibits GNRH expression, triggers tanycyte extension, maintains low FSHβ and regressed testis. Vernal transitions in photoperiod to the equinox results in resumption of GNRH and elevated FSHβ expression without testis growth. Data are mean +/- SEM, and residual dot plot. a-c, f,g,k,m One-way ANOVA with Bonferonni corrected Tukey’s test for multiple comparisons. h, I One way ANOVA with Tukey tests for significant pairwise comparison. Letters denote significant difference between photoperiod phase. Raw data available in Table S1.

FSHPβ is constitutively expressed during the vernal equinox.

a, schematic representation of four photoperiod treatment groups with arrows to indicate the daily sampling time. b, testes volume remained in a regressed non-functional state in autumnal equinox (12a), short photoperiod (8L) and vernal equinox (12v). Photoperiods that exceeded the critical day length (i.e.,> 12hr) induced testes growth. c, Pituitary circadian clock gene ARNTLI maintained daily rhythmic expression waveforms across all photoperiods (P<0.001), no significant difference between photoperiod treatment (P=0.42) d, PER3 displayed a daily waveform across 12a, 8L, 12v and 16L groups (P<0.001) and was anti-phase compared to ARNTLI. No significance difference between photoperiod treatments (P=0.31) e, FSHβ expression was significantly higher in 12v compared to long photoperiods (16L; P<0.001), autumnal equinox (12a; P<0.001) and short photoperiod (8L; P<0.001), but was not rhythmic (P=0.66). f, Similarly, PRL was high in 16L compared to 12a (P<0.001), and 8L (P<0.001), there was no significant daily rhythm (P=0.52). g, FSHβ promoter was devoid of circadian gene binding D-box and E-box motifs but contains a series of hormone and nutrient responsive motifs. b-f Two-way ANOVA followed by Tukey’s pairwise tests, rhythmic analyses were conducted using GraphPad Prism. ╪ indicates significant photoperiod treatment effect; # denotes significant time of day effect. Data are mean +/- SEM, and residual dot plot (a-d). Rhythmic analyses are presented in Table S7 and raw data are available in Table S1.

Endogenous and light-induced FSHP expression.

a, FSHβ expression increased during the photoinduced transition from 8L to 10v, and 12v. FSHβ also showned a smaller, yet significant increase in expression after prolonged exposure to 8L. Y-axis is presented in log-scale due to the significant increase in FSHβ expression in 10v and 12v. b, OPN5 was detected in the pituitary gland and showed a significant increase in expression in the transition to 10v and 12v similar to FSHβ expression. c, DIO3 was significantly reduced in 12v quail compared to all other treatment groups. d, GNRH expression remained constant during the transition from 8L to 12v. However, continued exposure to 8L was observed to increase GNRH expression. Data are mean +/- SEM, and residual dot plot (a-d). One-way ANOVA with Tukey’s test for multiple comparisons. Letters denote significant difference between photoperiod phase. Raw data available in Table S1. e, Schematic representation of the endogenous and light-dependent increase in pituitary cell types during the transition from 8L to stimulatory 16v light treatments. Increased color indicates increased transcript expression.