Performing Department
Poultry Science
Non Technical Summary
Background: Over 70% of women develop uterine fibroid tumors by the time they reach 50 years old. Uterine fibroids have a significant effect on fertility and quality-of-life, costing the US $34.4 billion every year. To date, little progress has been made in determine causes of the disease, in part due to a lack of experimental models for study of the disease. In particular, virtually no study has examined how developmental environment, such as childhood diet, affects the disease risk. Egg-type chicken hens provide an excellent model for studying developmental origin and mechanisms of uterine fibroids as hens spontaneously develop fibroid tumors at a high rate. In addition, our team was the first to discover that hen oviductal fibroids share key characteristics with human uterine fibroids, such as molecular markers. Our goals of the proposed research are to: 1) determine the effect of low- and high-protein diet during postnatal period and timing of sexual maturity on risks of developing uterine fibroid, 2) use the obtained data to apply for PAR-13-204 by NIH and USDA, and 3) help establish guidelines for optimal nutrition during development to alleviate incidence and severity of fibroid tumors. Significance: Childhood diet as a potential origin of uterine fibroids has been practically untested. This proposal pioneers the field because not only does it examine the mechanistic link between postnatal diet and fibroid risk, but also disentangle diet from timing of sexual maturity in relation to fibroid risk. We hypothesize that high-protein postnatal nutrition and early onset of sexual maturity increase risks of uterine fibroids through alteration in the insulin-like growth factor (IGF) system and cell proliferation signals. Approach: To test the hypothesis, egg-type female chicks will be exposed to 1) various levels of protein in the diet and 2) alterations of photoperiod to manipulate onset of sexual maturity. Size and number of fibroid tumors will be determined biyearly along with plasma IGF and IGF binding protein (IGFBP) levels, as well as IGF receptor levels, cell proliferation signals including Bcl-2, PCNA, and an apoptosis signal, caspase-3 in fibroid tissues. The incidence of fibroid tumors is expected to rise with increasing proportion of protein during rearing and decreasing age at sexual maturity, accompanied by high levels of IGF, Bcl-2, PCNA, and low levels of IGFBP and caspase-3. The long-term goal of the proposed research is to lower prevalence of uterine fibroids to improve quality of life for millions of women. The results from this research will inform recommended consumption guidelines for protein during infancy and prepubertal period to prevent or lessen the severity of fibroid tumors.
Animal Health Component
100%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
1. Gather preliminary data to be included in a PAR-13-204 proposal (a NIH-USDA grant titled "Dual purpose with dual benefit: research in biomedicine and agriculture using agriculturally important domestic animal species (R01)")2. Understand the causal relationship among postnatal diet, timing of onset of sexual maturity and uterine fibroid3. Help establish a guideline for optimal nutrition during development to alleviate incidence and severity of fibroid tumors
Project Methods
Experimental Design 270 one-day old female chicks will be randomly allocated into 6 groups following a 3x2 full factorial design (n=45): three diets containing 10%, 18% or 25% crude protein, and two levels (early or late) of onset of sexual maturity. All diets will be isocaloric and fall within the range of protein content given to hens in a commercial setting (North and Bell, 1990). The hens will be raised on the assigned diet from 1 day of age to until euthanasia. All chickens will have ad lib access to food. Onset of sexual maturity will be controlled by photoperiod manipulation: day length will be switched from 8L:16D to 16L:8D, on 16 or 24 weeks. 10 Data collection and analyses Hens will be bled to obtain serum levels of IGF and IGFBP at 18 weeks (pre-sexual maturity), 26 weeks (post-sexual maturity), and every 6 months thereafter. Fifteen hens per treatment will be euthanized at 6 months, 1 year, and 1.5 years of age by CO2 narcosis. Size, number, and location of fibroid tumors will be recorded. Fibroid tissues will be collected from each hen and half will be snap-frozen using dry ice and the other half will be fixed in 10% formalin. Normal oviduct tissue from the same bird will serve as a control for comparison. Frozen tissue will be later homogenized, centrifuged, and supernatant will be used to quantify IGF-I, IGF-IR, and IGFBP-1 protein levels using a commercial ELISA kits (RayBiotech, Inc., Norcross, GA). The fixed tissue will be embedded in paraffin blocks and sliced into 5 μm sections for immunohistochemistry. Three immunohistochemical staining will be performed on 3 consecutive sections using primary antibodies against Bcl-2 (R&D Systems Inc., Minneapolis, MN), proliferating cell nuclear antigen (Lab Vision Corp., Fremont, CA), and caspase-3, a marker of cell apoptosis (Abcam, Cambridge, MA). Statistical analyses The effects of protein content of rearing diet and onset of sexual maturity on size, number of fibroid tumors will be analyzed using two-way ANCOVA with body weight as a covariate. The effects of protein content of rearing diet and onset of sexual maturity on IGF system, Bcl-2, PCNA, and caspase-3 staining will be analyzed using independent repeated measures ANCOVAs with respective levels in the normal tissues for each bird as a covariate. The alpha level will be set to 0.05.