Loss of BMAL1 in ovarian steroidogenic cells results in implantation failure in female mice

Abstract

The circadian clock plays a significant role in many aspects of female reproductive biology, including estrous cycling, ovulation, embryonic implantation, onset of puberty, and parturition. In an effort to link cell-specific circadian clocks to their specific roles in female reproduction, we used the promoter that controls expression of Steroidogenic Factor-1 (SF1) to drive Cre-recombinase-mediated deletion of the brain muscle arnt-like 1 (Bmal1) gene, known to encode an essential component of the circadian clock (SF1-Bmal1(-/-)). The resultant SF1-Bmal1(-/-) females display embryonic implantation failure, which is rescued by progesterone supplementation, or bilateral or unilateral transplantation of wild-type ovaries into SF1-Bmal1(-/-) dams. The observation that the central clock, and many other peripheral clocks, are fully functional in this model allows the assignment of the implantation phenotype to the clock in ovarian steroidogenic cells and distinguishes it from more general circadian related systemic pathology (e.g., early onset arthropathy, premature aging, ovulation, late onset of puberty, and abnormal estrous cycle). Our ovarian transcriptome analysis reveals that deletion of ovarian Bmal1 disrupts expression of transcripts associated with the circadian machinery and also genes critical for regulation of progesterone production, such as steroidogenic acute regulatory factor (Star). Overall, these data provide a powerful model to probe the interlocking and synergistic network of the circadian clock and reproductive systems.

Keywords: circadian rhythm; fertility; ovary; steroidogenesis.

Fig. 1.

Effect of Bmal1 deletion on wheel running activity. (A) Representative activity records of individual Bmal1^+/+, Bmal1^−/−, Bmal1^fx/fx, and SF1-Bmal1^−/− females are presented in double-plotted format. The bar above shows the light-dark cycle. DD, constant darkness; LD, light-dark. Gray shading indicates constant darkness in the actograms. (B–E) Effects of genotype on free running period (B), amplitude of circadian rhythm (C), and activity level in LD (D) and DD (E). *P < 0.05, by one-way ANOVA. Genotypes are indicated at the bottom of B–E.

Fig. 2.

Effect of Bmal1 deletion on the onset of vaginal opening, estrous cycle, and ovary histology. (A) Age at vaginal opening. Each point represents an individual animal. P values (Wilcoxon rank sum test) for comparison of SF1-Bmal1^−/− (n = 10) vs. Bmal1^fx/fx (n = 12) and Bmal1^−/− females (n = 6) vs. Bmal1^+/+ (n = 5) are shown. (B) Estrous cycle length (mean ± SEM) of SF1-control (four Bmal1^fx/fx and one Bmal1^fx/+Cre^sf1) and SF1-Bmal1^−/− females (n = 6). (C) Proportion of time (mean ± SEM) spent in each estrous stage in control (n = 5) and SF1-Bmal1^−/− females (n = 6). (D) Representative histology sections of ovaries from control and SF1-Bmal1^−/− females at 3.5 dpc. L, corpus luteum. (Scale bars: 0.5 mm.)

Fig. 3.

Expression of Dbp, Bmal1, Nr1d2, Per1, Per2, Rora, Lhcgr, and Star mRNAs in ovaries. Values are expressed as mean ± SEM, setting mean expression value of Bmal1^+/+ at ZT12 on 3.5 dpc to 1. Ovaries were harvested every 4 h from 2.5 to 3.5 dpc as shown on the timeline at the top. (A) Gene expression assay on C57BL/6J females obtained from JAX (n = 3 per time point) by microarray (∆, right axis) and qPCR (♦, left axis). ★, pMMC-β<0.05 in COSOPT. (B) qPCR gene expression assay on mice of the indicated genotype bred in our mouse facility (n ≥ 4 per time point⋅genotype⁻¹). (C) Microarray gene expression assay on C57/B6 females (same animals as A), Bmal1^−/−(n = 3 per time point), Bmal1^fx/fx and SF1-Bmal1^−/− (n = 4 per time point⋅genotype⁻¹).*P < 0.05 (two-way ANOVA between Bmal1^fx/fx and SF1-Bmal1^−/−).

Fig. 4.

Effects of Bmal1 deletion on serum progesterone and PRL levels. Each point represents a single animal. Progesterone levels in Bmal1^−/− and SF1-Bmal1^−/− females at 3.5 dpc (A) and 6.5 dpc (B) and PRL levels in SF1-Bmal1^−/− females at 3.5 dpc (C) and 6.5 dpc (D) are shown. SF1-control includes Bmal1^fx/fx and Bmal1^fx/+Cre^sf1 females. P values, by Wilcoxon sum test, are indicated.

Fig. 5.

Effects of progesterone or PRL treatment on implantation at 6.5 dpc or 10.5 dpc. Representative whole uteri (1) and H&E-stained sections (2) are shown. SF1-Bmal1^−/− females (B–D and F–H) and wild-type controls (A and E) received corn oil (A, B, E, and F), progesterone (C and G), or PRL (D and H) until 6.5 dpc (A–D) or 10.5 dpc (E–H). (Scale bars: 0.5 mm.)