The luteinizing hormone surge regulates circadian clock gene expression in the chicken ovary

Abstract

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors' previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary.

Fig. 1.. Ovulation records and experimental design.

Animals were housed in a 16:8 LD cycle with lights on at ZT0 and off at ZT16. A) Representative oviposition record for hens used in experiments 1 and 2. Ovipositions were monitored for 30 days prior to sacrifice. Hens were placed on a 16:8 light:dark schedule. The time of oviposition is indicated by the black dot. Arrows indicates the time of sacrifice on the day of the experiment. Data are double-plotted. Shaded area indicates the time of lights-off. B) The chicken ovary showing the hierarchy of follicles and a schematic of the experimental design for experiment 2. To account for time-of-day dependent change in clock gene transcript expression, all animals were sacrificed at ZT6. Oviposition was monitored at 30-min intervals from ZT3 to ZT12, and again at ZT14, to time late ovipositions. The oviposition cycle in this group of hens was approximately 24.25 h. The time of the previous oviposition was used to predict the time of the LH surge, which reliably occurs ~4 h prior to oviposition (Tischkau et al., 1996). Animals in Group 1 were sacrificed at ZT6, but prior to the LH surge based on predicted oviposition at ZT11 on the day of the experiment. Animals in Group 2 were sacrificed at ZT6, but after the LH surge, based on a predicted oviposition at ZT8. Also depicted is the chicken ovary, with hierarchical follicles (F1-F5), small yellow follicles (SYF), large white follicles (LWF), small white follicles (SWF) and a postovulatory follicle (POF). Only F1 follicles were used for this experiment.

Fig. 2.. Daily mRNA levels of circadian clock-related genes in the chicken.

Samples were collected every 6 hours under a light/darkness (L16:D8) schedule (n=3-4 per timepoint). A) Polyacrylamide gel electrophoresis of real-time quantitative PCR products demonstrates amplification of single bands corresponding to the predicted size for chicken Bmal1, Cry1 and Per2. Each sample was run on a separate gel with a 100 bp marker. Quantitative RT-PCR was used to measure Bmal1 (▲), Cry1 (♦) and Per2 (■) transcripts in chicken pineal gland (B), medio-basal hypothalamus (C), liver (D), F1 theca (E) and F1 granulosa (F) tissues. Results are shown as mean fold-change to lowest (trough) level ± SEM (top to bottom). Results of statistical analysis (ANOVA) are described in the results section. White and black bars at the top of the figure indicates the time of lights on and off, respectively.

Fig. 3.. Effects of the endogenous LH surge on circadian clock gene transcripts in the chicken.

Animals were sacrificed either before or after an endogenous LH surge, predicted from the time of the previous oviposition (see Fig. 1). All tissues were obtained at ZT6 to control for time-of day dependent changes in gene expression. Transcript levels for Bmal1, Per2 and Cry1 were determined by quantitative PCR. Data represent mean ± SEM with n=4 for each group. Statistical significance was determined using ANOVA with Bonferroni’s post-hoc analysis (*, ** and **** represent p < 0.05, p < 0.01 and p < 0.0001, respectively).

Fig. 4.. Effects of LH on Bmal1 and Per1 rhythms in cultured granulosa cells.

Per1 (A and C) and Bmal1 (B and D) transcripts were assessed after LH treatment of unsynchronized (A and B) or serum shocked (C and D) SIGC. Data means ± SEM of three independent experiments. Statistical significance was determined using ANOVA with Bonferroni’s post-hoc analysis but is not indicated on the graphs. Peaks for Per1 transcript levels occurred at 1h, 16h and 40h after LH treatment or serum shock. Peaks in Bmal1 transcripts occurred at 8h and 32 h after LH treatment or serum shock. LH treatment caused a significant 4-8 h phase delay in transcript expression peaks for both Per1 and Bmal1.

Fig. 5.. Acute effects of LH on Egr-1 and Per1 transcripts in cultured granulosa cells.

Transcript levels for the LH target gene, Egr-1 (A) Per1 (B) were determined by quantitative PCR in SIGC treated with either forskolin, LH, SQ22536 or LH/SQ22536. Data represent mean ± SEM with n=4-6 for each group. Statistical significance was determined using ANOVA with Bonferroni’s post-hoc analysis (* and ** represent p < 0.05 and p < 0.01, respectively).