Effect of Monochromatic Light on Expression of Estrogen Receptor (ER) and Progesterone Receptor (PR) in Ovarian Follicles of Chicken

<div><p>Artificial illumination is widely used in modern poultry houses and different wavelengths of light affect poultry production and behaviour. In this study, we measure mRNA and protein abundance of estrogen receptors (ERs) and progesterone receptors (PRs) in order to investigate the effect of monochromatic light on egg production traits and gonadal hormone function in chicken ovarian follicles. Five hundred and fifty-two 19-wk-old laying hens were exposed to three monochromatic lights: red (RL; 660 nm), green (GL; 560 nm), blue (BL; 480 nm) and control cool white (400–760 nm) light with an LED (light-emitting diode). There were 4 identical light-controlled rooms (n = 138) each containing 3 replicate pens (46 birds per pen). Water was supplied ad libitum and daily rations were determined according to the nutrient suggestions for poultry. Results showed that under BL conditions there was an increase in the total number of eggs at 300 days of age and egg-laying rate during the peak laying period. The BL and GL extended the duration of the peak laying period. Plasma melatonin was lowest in birds reared under BL. Plasma estradiol was elevated in the GL-exposed laying hens, and GL and BL increased progesterone at 28 wk of age. In the granulosa layers of the fifth largest preovulatory follicle (F5), the third largest preovulatory follicle (F3) and the largest preovulatory follicle (F1), ERα mRNA was increased by BL and GL. Treatment with BL increased ERβ mRNA in granulosa layers of F5, F3 and F1, while GL increased ERβ mRNA in F5 and F3. There was a corresponding increase in abundance of the proteins in the granulosa layers of F5, with an increase in PR-B, generated via an alternative splice site, relative to PR-A. Treatment with BL also increased expression of PR mRNA in all of the granulosa layers of follicles, while treatment with GL increased expression of PR mRNA in granulosa layers of SYF(small yellow follicle), F5 and F1. These results indicate that blue and green monochromatic lights promote egg production traits via stimulating gonadal hormone secretion and up-regulating expression of ERs and PRs. Changes in PR-B protein suggest that this form of the progesterone receptor is predominant for progesterone action in the granulosa layers of preovulatory follicles in chickens during light stimulation.</p></div

Effect of monochromatic light on plasma concentrations of melatonin, estradiol and progesterone.

<p>Results are expressed as mean ± SD (n = 5). BL = blue light, GL = green light, RL = red light, and CL = cool white light (control group). Least square means with different letters are significantly different (<i>P</i> < 0.05).</p

Effects of monochromatic light on ER isoforms and PR mRNA abundance in ovarian follicles of laying hens at 28 wk.

<p>(A) The abundance of ERα (B) ERβ, and (C) PR mRNA. Results are expressed as mean ± SD (n = 5). BL = blue light, GL = green light, RL = red light, and CL = cool white light (control group). ERα = estrogen receptor-α, ERβ = estrogen receptor-β, PR = progesterone receptor. SYF = small yellow follicle, F5 = the fifth largest preovulatory follicle, F3 = the third largest preovulatory follicle, and F1 = the largest preovulatory follicle. Least squares means with different letters are significantly different (<i>P</i> < 0.05).</p

Effects of monochromatic light on PR isoform protein content in the F5 of laying hens at 28 wk.

<p>(A) Protein abundance of PR-A and (C) PR-B. Proteins detected by western blot from the chicken F5 were quantified by densitometric analysis and normalized to β-actin protein content. (B) Representative blot of PR-A, PR-B, and β-actin. (D) PR-A or PR-B ratio to total protein abundance (PR-A+PR-B) in the four light groups. Results are expressed as mean ± SD (n = 5). BL = blue light, GL = green light, RL = red light, and CL = cool white light (control group). Least square means with different letters are significantly different (<i>P</i> < 0.05).</p

Effect of monochromatic light on EN300 and laying rate in PLP (%).

<p>EN300 = the total number of eggs at 300 days of age; egg-laying rate during PLP = egg-laying rate during the peak laying period. BL = blue light, GL = green light, RL = red light, and CL = cool white light (control group). Results are expressed as mean ± SD (n = 150). Least square means with different letters are significantly different (<i>P</i> < 0.05).</p

Effects of monochromatic light on ER isoform protein content in F5 of laying hens at 28 wk.

<p>(A) Protein abundance of ERα and (C) ERβ. Proteins detected by western blot from the chicken F5 were quantified by densitometric analysis and normalized to β-actin protein content. (B) Representative blots of ERα and β-actin, and (D) ERβ and β-actin. Results are expressed as mean ± SD (n = 5). BL = blue light, GL = green light, RL = red light, and CL = cool white light (control group). ERα = estrogen receptor-α, ERβ = estrogen receptor-β. Least squares means with different letters are significantly different (<i>P</i> < 0.05).</p

Muscle proteins were significantly enriched among the differentially expressed (DE) genes.

<p>(A) The significantly enriched GO categories (y-axis) in the DE genes with FDR <0.05. (B) The Log2 fold changes from both RNA-seq (dark) and arrays (grey) of the DE genes associated with muscle proteins and contractile fiber, cytoskeleton protein binding and heart development. (C) The ZBED6 peak at about 1 kb upstream of <i>Twist2</i> gene (grey box) displayed together with placental mammal conservation score. The peak maxima overlapped with a highly conserved region (grey box), which contained the consensus motif GCTCGC of ZBED6 only in the placental mammals. A 10-bp insertion (+10) was present within the consensus motif in the opossum genome, indicating lack of ZBED6 binding site in this region. (D) The relative expression levels of <i>Twist2</i> from RNA-seq and array in either <i>Zbed6</i>-silenced or control C2C12 cells. (E) The relative luciferase activity (the ratio of firefly to <i>Renilla</i>) of <i>Igf2</i>, <i>Twist2</i> and empty pGL3 basic constructs in <i>Zbed6</i>-silenced or control C2C12 cells. **, P<0.01; ***, P<0.001.</p

ZBED6 modulates the expression of Igf2 and Myogenin during differentiation of C2C12 cells.

<p>(A) Expression of <i>ZBED6</i>, <i>IGF2</i> and <i>Myogenin</i> mRNA monitored by qPCR before and after differentiation of control cells and transfected cells overexpressing ZBED6 from the pTRE-ZB vector. (B) Expression of <i>ZBED6</i>, <i>IGF2</i> and <i>Myogenin</i> mRNA monitored by qPCR before and after differentiation of control cells and cells in which ZBED6 has been silenced using siRNA. (C) Western blot analysis confirming altered expression of ZBED6 and Myogenin at the protein level. Actin was used as loading control.</p

RNA sequencing of <i>Zbed6</i>-silenced myoblast cells.

<p>(A) Quantitative PCR validation of <i>Zbed6</i>-silencing (light gray bar) and <i>Igf2</i> up-regulation (dark gray bar) two and four days post-<i>Zbed6</i> siRNA transfection. Biological triplicates were performed for both <i>Zbed6</i> and scrambled siRNA. Error bars, s.e.m. The asterisk indicates a significant difference (P<0.05) between control and <i>Zbed6</i>-silenced samples. (B) Western blot validation of <i>Zbed6</i>-silencing. The protein lysates from the pooled biological triplicates for each siRNA treatment were equally loaded to the protein gel after measuring the concentration. The specific band for ZBED6 protein (110 kDa) appeared above the 100-kDa band from the protein ladder and α-Tubulin was used as a reference protein. (C) Direct comparison of the read counts (y-axis) from <i>Zbed6</i>-silenced and negative control RNA-seq data across <i>Zbed6</i> and <i>Igf2</i> regions for both two and four days post siRNA transfection. The ChIP-seq data with ZBED6 antibody from Markljung et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094187#pone.0094187-Markljung1" target="_blank">[1]</a> are shown to pinpoint the binding sites of ZBED6.</p

Small nucleolar RNA genes significantly up-regulated according to RNA-seq both on day 2 and day 4 after <i>Zbed6</i> silencing and qPCR validation.

a<p>Student's t-test, P<0.05).</p