Conditional Deletion of Bmal1 in Ovarian Theca Cells Disrupts Ovulation in Female Mice

Abstract

Rhythmic events in female reproductive physiology, including ovulation, are tightly controlled by the circadian timing system. The molecular clock, a feedback loop oscillator of clock gene transcription factors, dictates rhythms of gene expression in the hypothalamo-pituitary-ovarian axis. Circadian disruption due to environmental factors (eg, shift work) or genetic manipulation of the clock has negative impacts on fertility. Although the central pacemaker in the suprachiasmatic nucleus classically regulates the timing of ovulation, we have shown that this rhythm also depends on phasic sensitivity to LH. We hypothesized that this rhythm relies on clock function in a specific cellular compartment of the ovarian follicle. To test this hypothesis we generated mice with deletion of the Bmal1 locus in ovarian granulosa cells (GCs) (Granulosa Cell Bmal1 KO; GCKO) or theca cells (TCs) (Theca Cell Bmal1 KO; TCKO). Reproductive cycles, preovulatory LH secretion, ovarian morphology and behavior were not grossly altered in GCKO or TCKO mice. We detected phasic sensitivity to LH in wild-type littermate control (LC) and GCKO mice but not TCKO mice. This decline in sensitivity to LH is coincident with impaired fertility and altered patterns of LH receptor (Lhcgr) mRNA abundance in the ovary of TCKO mice. These data suggest that the TC is a pacemaker that contributes to the timing and amplitude of ovulation by modulating phasic sensitivity to LH. The TC clock may play a critical role in circadian disruption-mediated reproductive pathology and could be a target for chronobiotic management of infertility due to environmental circadian disruption and/or hormone-dependent reprogramming in women.

Figure 1.. Circadian rhythms of ovarian sensitivity to phasic eLH treatment in adult and juvenile eCG-primed C57BL6/J mice.

A, Serum levels of LH at ZT12 on proestrus were suppressed by treatment with CET (n = 4; *, P < .05 vs saline [n = 5]). B, Dose-response curve for eLH treatment. All 3 doses (50, 200, or 800 IU; n = 3–4 per treatment) stimulated more oocyte release at ZT18 (*, P < .001). C, Rhythm of ovarian sensitivity to eLH in adult C57BL6/J mice (n = 3–6 per treatment time; *, P < .05 vs ZT3, ZT6, and ZT9). D, Free-running circadian rhythm of sensitivity to LH in C57BL6/J mice (*, P < .05 vs ZT6; n = 3–6 per treatment time). E, A diurnal rhythm of sensitivity to eLH in juvenile eCG-primed mice (**, P < .001 vs ZT0–ZT9 on estrus, n = 3–6 per treatment time). F, A circadian rhythm of responsiveness to eLH in juvenile-eCG-primed mice housed in DD (*, P < .05 vs the nadir at CT6 on estrus, n = 3–4 per treatment time). In F, the time of CET injection (CT5) is not shown due to scaling of the x-axis. In C–F, saline failed to stimulate ovulation. Data are presented as mean ± SEM and fit to a nonlinear regression (see Materials and Methods). The shaded areas in C–F indicate the dark phase.

Figure 2.. Targeted deletion of Bmal1 in specific cellular compartments in the ovary of GCKO and TCKO mice.

Targeted deletion of Bmal1 in granulosa (GCKO) and theca/stromal (TCKO) cells of the ovary using the CRE:LOX system. Transgenic GCKO (Cyp19-Cre;Bmal1^flx/flx) and TCKO (Cyp17-Cre;Bmal1^flx/flx) mice were crossed with TOM-GFP mice to produce TCKO-TOM-GFP and GCKO-TOM-GFP transgenic mice (see Supplemental Methods). As shown in A, CRE-driven eGFP expression in GCKO-TOM-GFP is nearly completely limited to the mural and cumulus GC layer with little to any GFP expression detected in the TC/SC compartment or the ovarian surface epithelium (OSE). B, In TCKO-TOM-GFP mice, CRE-driven GFP is more dispersed and spotty in the interstitial cells and limited the TC layer of larger antral follicles with no signal detected in the GC compartment or surface epithelium. Representative images of whole mount (C) pituitary gland and (D) liver from a GCKO mouse. A small number of cells in these tissues from both transgenic strains were positive for GFP (<5%). Scale bars: 0.25 μm shown in A (A and B), 0.05 mm shown in C (C and D), and 0.25 mm shown in E (E and F). Images are representative of 3–4 mice per genotype (LC, GCKO, and TCKO).

Figure 3.. Reproductive cycles, serum LH, and serum P4 levels on proestrus are not altered by conditional Bmal1 deletion in the ovary of GCKO and TCKO mice.

A, Representative reproductive cycle graphs from LC, GCKO, and TCKO transgenic mice. B, Analysis of (top) cycle length and (bottom) percentage of time spent in each day of the cycle reveals no significant difference between LC, GCKO, and TCKO transgenic mice (n = 9 per group). C, Serum LH measured on proestrus from ZT3–ZT18 reveals that both GCKO and TCKO transgenic mice had appreciable surges of serum LH with both rhythms peaking at ZT12 (*, P < .05, n = 3–4 samples per time point). D, Levels of serum P4 at ZT6 and ZT18 on proestrus were not significantly affected by targeted deletion of Bmal1 in either GCKO or TCKO transgenic mice (n = 3 samples per time point except for ZT6 in LC mice [n = 2]). Data are labeled according to genotype and time of serum collection. All data are presented as mean ± SEM.

Figure 4.. Phasic sensitivity to LH is disrupted in adult and juvenile eCG-primed conditional Bmal1 TCKO mice.

A, Rhythm of sensitivity to eLH in cycling adult LC (n = 3; both ZT18 and ZT6), GCKO (ZT18 n = 5, ZT6 n = 4), and TCKO (ZT18 n = 5, ZT6 n = 6) mice. Both LC and GCKO transgenic mice displayed strong diurnal rhythms of eLH sensitivity with peak responses to eLH treatment at ZT18 (*, P < .05 vs ZT6) for both. In contrast, adult cycling TCKO mice did not maintain a rhythm of sensitivity to eLH (P > .05 ZT18 vs ZT6). B, Diurnal rhythms of sensitivity to LH in juvenile-eCG-primed LC (n = 4–8 per time of treatment), GCKO (n = 5–6 per time of treatment), and TCKO (n = 4–6 per time of treatment) mice. Both LC (open circles) and GCKO (gray squares) mice displayed diurnal rhythms of responsiveness with significant peaks of responsiveness to eLH treatment at ZT9–ZT12 (*, P < .05 vs troughs at ZT0, ZT3, and ZT18). TCKO mice (black triangles) did not show a significant rhythm in response to phasic eLH. Data are presented as mean ± SEM and in B data are fit to a nonlinear regression (see Materials and Methods). In B, # indicates difference between TCKO and GCKO, and + indicates a significant difference between TCKO and LC mice.

Figure 5.. Rhythms of clock gene mRNA abundance in whole ovary are disrupted in both GCKO and TCKO transgenic mice.

Abundance of clock gene mRNA in ovaries recovered from LC (n = 3), GCKO (n = 3), and TCKO (n = 3) mice on proestrus. LC mice displayed diurnal rhythms of Bmal1, Per1, and Rev-erbα mRNA abundance (P < .05 indicated in top right of each panel). Rhythms of Bmal1 and Rev-erbα mRNA abundance were disrupted in both GCKO and TCKO mice. Although still rhythmic, the absolute level of Per1 mRNA was suppressed in both GCKO and TCKO mice. Although not significant by CircWave analysis, Cry1 mRNA abundance oscillated at low amplitude with a peak near midday in LC mice and was altered in both GCKO and TCKO mice. Clock mRNA displayed a low amplitude rhythm in TCKO mice that was not detected in LC or GCKO mice. Data are presented as mean ± SEM; n = 3 samples per time point except for ZT24 in TCKO mice (n = 2). (#, P < .05 GCKO vs TCKO; *, P < .05 LC vs GCKO or TCKO). Values for target mRNA abundance were calculated using the ΔΔCT method as described in Materials and Methods with β-actin as the reference gene. Data were fit to a nonlinear regression (see Materials and Methods).

Figure 6.. Conditional KO of Bmal1 in TCs alters rhythms of LHR mRNA abundance in the ovary.

Rhythms of Lhcgr and Fshr expression in whole ovary tissue recovered from LC (n = 3), GCKO (n = 3), and TCKO (n = 3) transgenic mice. In both LC and GCKO mice we detected a significant diurnal rhythm of Lhcgr mRNA abundance (P < .05 for both) that was abolished in TCKO transgenic mice. There was a marked increase in Lhcgr expression at ZT24 in TCKO mice (#, P < .05) when compared with the same time point in both LC and GCKO mice. A low amplitude oscillation of Fshr mRNA expression was detected in GCKO transgenic mice with a peak in the morning (ZT1.99). Daily variation of Fshr mRNA abundance was not significant in either LC or TCKO transgenic mice. Data are presented as mean ± SEM. n = 3 ovaries from 3 different mice per time point. #, P < .05 GCKO vs TCKO; *, P < .05 LC vs GCKO or TCKO. Only those rhythms labeled directly with a P value were considered significant by CircWave analyses. Values for target mRNA abundance were calculated using the ΔΔCT method as described in Materials and Methods, with β-actin as the reference gene. Data were fit to a nonlinear regression (see Materials and Methods).