LUTEINIZING HORMONE REGULATION OF CIRCADIAN CLOCK GENES IN THE AVIAN OVARY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0205709
• Project Start Date: Oct 1, 2005
• Project End Date: Sep 30, 2006
• Program Code: [(N/A)]- (N/A)

Project Director
Tischkau, S.

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
VETERINARY RES AND EXTENSION

Non Technical Summary
This proposal investigates how endogenous timing mechanisms generated within the brain are used to coordinate ovarian function with the environmental cycle of light and darkness. The major objective is to examine how pituitary hormones can influence the efficiency of egg production in an avian model system. 1) Development of a simplified model system to study regulation of organismic timing. 2) Discovery of mechanisms by which timing information is coordinated within a physiological context. 3) Discovery of the regulation and function of circadian clock genes in the ovary. 4) Development of alternative contraceptive strategies. 5) Development of new therapies for infertility. 6) Increase efficiency of egg production in the poultry industry.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3299	1020	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1020 - Physiology;

Keywords

circadian rhythms

avian ovary

luteinizing hormone

Goals / Objectives
Within an individual, physiological function is similarly organized into near 24 hour patterns to assure coordinated function among organ systems. Organismic rhythmicity relies on successful generation and transmission of temporal information by central clocks, or pacemakers, to peripheral targets. Although the molecular structure that underlies generation of circadian rhythmicity is well established, routes that convey timing information from central pacemakers to the periphery are poorly understood. Nowhere is harmonious timing performance more important than in the hypothamalic-pituitary-ovarian axis. This proposal examines central regulation of circadian clock genes within the avian ovary by the gonadotropin, luteinizing hormone. The proposal relies heavily on development of the avian system as an important model system for use in determining mechanisms that underlie dissemination of timing information from central pacemakers to peripheral clocks.

Project Methods
The near 24 hour periodicity of the chicken reproductive cycle affords an ideal model system for these studies. The proposal tests the hypothesis that central pacemakers act through the neuroendocrine axis to regulate expression of circadian clock genes in the ovary. Specifically, this proposal utilizes state-of-the-art molecular (real-time PCR and immunoblots) and behavioral (on-line activity assessment) technologies to assess whether 1) the chicken ovary contains components of a functional molecular circadian clock, and 2) output from the hypothalamic-pituitary axis regulates timing of ovarian circadian clock gene expression. These studies will provide critical new understanding of how circadian timing directs neuroendocrine control of reproductive function, specifically, and will establish a defined structure for global investigation of physiologic regulation of circadian timing. Importantly, our studies are broadly applicable to discernment of mechanisms that link gene expression to physiologic function and behavior. Practical implications of understanding timing mechanisms include increased success in assisted reproductive technologies and minimally, increased efficiency of egg production in the poultry industry.

Progress 10/01/05 to 09/30/06

Outputs
This project examined expression of circadian clock gene transcripts in the ovary of the domestic hen. Because the avian ovary essentially functions as a semi-independent circadian clock, it was pertinent to examine whether the basic molecular mechanisms that govern function of other vertebrate clocks was present and functional in the avian ovary. Because studies of the avian circadian clock genes using quantitative real-time PCR had never been reported, we first designed unique primer sets for three canonical clock genes, Bmal1, Period (Per) and Cryptochrome 1 (Cry1). At the initiation of these studies, the genome of the domestic hen was not yet published, so primers were designed from conserved regions of the genes by comparison of their human, mouse, rat and Drosophila counterparts. We were successful in producing primer sets to identify transcripts for each of the genes. Samples were collected from adult cycling hens at 6 h intervals over the course of a 24 h period. As controls, we collected samples from the primary clock in the suprachiasmatic nucleus, as well as from the pineal and liver. 24-h oscillations in Per, Bmal1 and Cry1 were demonstrated in all tissues sampled. Importantly, Per and Bmal1 ocillations were out-of-phase with each other, suggesting that a functional clockwork may be present in the ovary. As predicted from the mammalian literature, the phase of the clockwork oscillatory behavior shifted several hours in the peripheral tissues (ovary, liver, pineal) compared to the brain. We next examined whether luteinizing hormone (LH) might act as a factor to set the phase of the ovarian clock. LH did alter expression of both Per and Bmal1, suggesting that endocrine regulatory elements are important in regulating the synchrony of the ovarian clock with the primary clock in the brain. These finding are currently being prepared for publication. These findings demonstrate for the first time that an ovarian clock is present and may be an important component in the endocrine regulation of the ovary. Alterations of the clockwork mechanism, through light therapy may provide a means to increase reproductive efficiency in the hen.

Impacts
The impact of the circadian timing system on organismic health is only beginning to be appreciated. Our studies demonstrate that luteinizing hormone regulation of an ovarian clock provides timing cues that are essential for the efficient production of fertilizable ova.

Publications

• Howell, R.E., Bahr, J.M. and Tischkau, S.A. 2006. Characterization of the avian circadian system using Bmal1, Cry1, Per, and the effect of luteinizing hormone. Annual Meeting of the Society for the Study of Reproduction, Omaha, NE.