Temporally desynchronizing the circadian clock results in arrested spermatogonial differentiation and reduced fertilization in zebrafish and mice. (a and b) Confocal images of the testes from Tg(RARE-gata2a : NLS-EYFP; hsp70l : clock1a; CG2) and sibling males after heat shock at ZT0 (a) or ZT12 (b) for 7 consecutive days (n = 9–15). (c) FISH images of Tg(hsp70l : clock1a; CG2) and sibling male testes with kita or sycp3 probe, and confocal images of Tg(hsp70l : clock1a; CG2; piwil1 : mCherry) and sibling male testes, after heat shock at ZT0 or ZT12 for 7 consecutive days (n = 3 × 5). (d) Quantification of the images in (c) (n = 6 −18). (e–g) Fertilization rates by pairwise crosses of WT females and Tg(hsp70l : clock1a; CG2) males or sibling males following heat shock just once (e) or for 7 consecutive days (f) starting at ZT0 or ZT12 or 7 days after removing the treatment from heat shock starting at ZT0 or ZT12 for 7 consecutive days (g) (n = 3 × 10). (h) Fertilization rates of desynchronization and control groups’ sperms with WT oocytes by IVF (n = 3). (i) Heatmaps of loss-of-rhythmicity genes in the desynchronization and control groups. (j) Top-20 enriched GO terms BP of the loss-of-rhythmicity genes in the desynchronization group. (k) Expression of Cry1, Smcp, Zbtb16 and Cyp26b1 in the control (black) and desynchronization (red) groups. (a–c), Arrows indicate spermatogonia. Sg, spermatogonia; Sc, spermatocyte; Sz, spermatozoon. Scale bar, 100 μm. **P < 0.01; ***P < 0.001 (Figs S8 and S9).
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