Recipient Organization
WEST VIRGINIA UNIVERSITY
886 CHESTNUT RIDGE RD RM 202
MORGANTOWN,WV 26505-2742
Performing Department
Physiology and Pharmacology
Non Technical Summary
Within the body, a part of the brain called the hypothalamus releases the hormone, gonadotropin releasing hormone (GnRH). This acts on the pituitary to release luteinizing hormone (LH), which acts on the ovaries to release estradiol (E2). E2then feeds back in a negative manner and inhibits the release of GnRH and LH. In many mammal species, as they reachpuberty, there is a decrease in the inhibition by E2, leading to an increase of GnRH and LH, which eventually leads to puberty onset. It is currently unknown what causes this change in E2-negative feedback, and therefore what controls the timing of puberty onset. Adding to the complexity, E2cannot act directly tosuppress GnRH, since the neurons that release GnRH lack receptors for estradiol, ERa. This means that there must be other neurons that haveERa and which acton GnRH neurons.Delayedpuberty onset in domestic animals leads to a decrease in lifetime productivity of the animal, therefore decreasing profitability for producers. In addition, delayed puberty in humans has a number of negative effects on health. Determining what mechanisms control the timing of puberty onset may allow for more options to address these issues.The first goal of this project is to determine where in the hypothalamus estradiol is acting. The first experiment involves placingE2in specific areas of the hypothalamus, namely either the preoptic area (POA) or the arcuate nucleus (ARC), of ewesafter removing the ovaries, and therefore any circulating estradiol. Blood samples will then be taken to determine if the E2implants reduce LH, indicating that E2is acting in either the POA or ARC (or both), or if there is no response. The second experiment will involve placing ICI, a compound that blocks the receptor for E2, in either the POA or ARC of ewes. The ovaries of the ewes will be removed, but E2will be givenso that there is a constant amount of estradiol in circulation. Blood samples will be taken to determine if the results supportthe first experiment, i.e., if E2is acting in that area, the ICI compound should block it from working and the LH levels will be increased.The second goal of the proposal is to try to determine the neurons in the hypothalamusthat act onGnRH neurons and thatmay be responsible for the change in E2-negative feedback that leads to puberty. We have identified a few populations of cells that we know act on GnRH in some way: ARC propiomelantocortin (POMC) neurons, ARC agouti-related peptide (AgRP) neurons, and POA NK3R-containing neurons. POMC and AgRP neurons are known to have ERa receptors. Approaches will be used that will enable us to assess changes in the number ofPOMC, AgRP and NK3R neurons with pubertal development as well as whether ERa expression in each of these different sets of neurons changes.If ERa expression one or more of these sets of neurons changes, then it might suggest that they are involved in the change in E2-negative feedback that leads to puberty onset.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The normal timing of puberty onset in domestic livestock is critical for maximal productivity and, therefore, profitabilityfor the producer. Puberty onset is caused by an increase in gonadotropin releasing hormone (GnRH) secretion, and, in turn, luteinizing hormone (LH),as a result of decreased estradiol (E2) negative feedback. GnRH neurons, located in the preoptic area of the hypothalamus, lack the receptors for estradiol, ERa. This indicates that the shift in E2negative feedback must happenin neurons afferent to GnRH neurons. It is currently unknown in what neurons or where this occurs. Aim 1 of this project is to determine if E2 acts within the preoptic area (POA) or the arcuate nucleus (ARC) of the hypothalamusto inhibit(LH)secretion in prepubertal female lambs. Thiswill be done by applying E2or blocking E2in those areas. Aim 2 of thisproposal focuses on defining changes in neuronal populations that contain ERaover development. Within the ARC, the neuronal populations targeted will be propiomelanocortin (POMC) neurons and agouti-related peptide (AgRP) neurons. Within the POA, the focus will be on neurons containing NK3R, the receptor for neurokinin B.
Project Methods
The first experiment conducted will involve ovariectomizing a group of ewes and placing guide cannulas in either the POA or the ARC. After a two week recovery, blood samples will be collected every 12 minutes for four hours, then assessed for LH using a radioimmunoassay. After this, the ewes will receive either a blank implant or an estradiol implant within the cannula for each area. After a week, blood samples will be collected using the same method. Treatments will then be reversed in a crossover design, allowing each animal to serve as its owncontrol.One week later, blood samples will be collected again, after which hypothalamic tissue will be collected. Blood samples will be analyzed for LH pulses using the following criteria:(1) a peak within two samples of the previous nadir; (2) amplitude greater than sensitivity of the LH assay; and (3) three standard deviations larger than the nadir directly preceding and following it. The hypothalamic tissue will be visualized with H&E staining to determine if the guide cannulas were placed in the proper region of the hypothalamus.The second experiment will also involveovariectomizing a group of ewes and placing guide cannulas in either the POA or the ARC, however E2implants will be placed subcutaneously in the ewes at the time of ovariectomy. After atwo week recovery, blood samples will be collected every 12 minutes for four hours, then run for LH using a radioimmunoassay. After this, the ewes will receive either a blank implant or an ICI implant, an ERa antagonist, within each targeted area. A week later, blood samples will be collected, and treatments will be reversed in a crossover design. One week later, blood samples will be collected again and the blank, ICI, and E2implants will be removed. One week later, a final blood collection will be performed and hypothalamic tissue will be collected. The same analyses forpulsatile LH secretion and guide cannula placement will be performed as for experiment 1.In the third experiment, hypothalamic tissue from ewes aged 5 months (prepubertal), 8 months (peripubertal), and 10 months (postpubertal) will be assessed using immunohistochemistry. ARC tissue will be stained for POMC and ERa, and AgRP and ERa. POA tissue will be stained for NK3R and ERa. The number of cell bodies containing each neuropeptide and the percentage colocalization with ERa at each stage of pubertal development will be analyzed using an Olympus Slide Scanner to visualizecells andOlyvia to perform quantitative analyses.