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

a, Schematic representation of the simulated annual rhythm in photoperiod. Quail were collected in 16hr light, Shr dark photoperiod and then every 2 weeks the photoperiod was decreased by 2hrs to 14hr, 12hr, 10hr and then a SL short photoperiod. Photoperiod was then increased to mimic the vernal transition and birds were collected at 1Ohr, 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 t he 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 ofMBH punches identifies distinct waves of transcripts as quail transition across photoperiodic conditions. f-g, qPCR assays for proopiomelanocortin (POMC) and deiodinase type-3 (DI03) confirmed restricted activation during 10v-SL and SL-I 0v phases, respectively. h-i, vimentin immunoreactivity in the median eminence (ME) shows tanycyte morphology growth is limited to 10a, SL, and 10v photoperiods. j, Heat-map illustrating photoperiodic transitions in pitutiary transcripts. k-m, confirmed that FSH/J is elevated under non-stimulatory photoperiods followed by increased prolactin (PRL) in 14v and thyrotropin stimulating hormone-(TSH/J) in 16v. n, diagram summarizing that long photoperiods increase GNRH synthesis and release into the pituitary gland to stimulate FSH/J and induce testis growth. Transition to autumnal equinox phases results in reduced FSH/J expression and regressed testis. Prolonged exposure to short photoperiods inhibits GNRH expression, triggers tanycyte extension, maintains low FSH/J and regressed testis. Vernal transitions in photoperiod to the equinox results in resumption of GNRH and elevated FSH/J 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.

FSHP 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, FSH/3 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, FSH/3 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).

Endogenous and light-induced FSHP expression.

a, FSH/J expression increased during the photoinduced transition from 8L to 10v, and 12v. FSH/J 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 FSH/J 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 FSH/J expression. c, D/03 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