Evaluating the Efficacy of Dietary Intervention with Flaxseed in Chicken Oviductal Polyps: A Model for Human Leiomyoma

Author

Emily Isgur, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2015

Degree Name

Master of Science

Department

Molecular Cellular and Systemic Physiology

First Advisor

Hales, Dale

Abstract

Uterine fibroids or leiomyomas are benign smooth muscle tumors in the uterus that affect 70% of reproductive age women. Most women are asymptomatic, but 40% of those affected are plagued with intense pelvic pain, menorrhagia, and infertility. Leiomyomas are characterized by an overgrowth of extracellular matrix and fibrous tissue. What separates leiomyomas from other gynecological abnormalities is the need for both progesterone (P) and estrogen (E) for growth and maintenance. Laying hens are the only animals to spontaneously develop smooth muscle tumors (leiomyomas) in their reproductive tract, similar to women. Hen leiomyomas occur as polyps on the oviduct with increasing abundance as hens age, and have the same histological and molecular markers as human uterine fibroids. Flaxseed has two major biologically active components; oil in the germ, and phytoestrogen lignans in the hull. Flaxseed oil is very high in omega-3 fatty acids (alpha-linolenic acid) and these are known to act as inhibitors of prostaglandin pathways and as potent anti-inflammatories. Secoisolariciresinol diglucoside (SGD) in the hull is converted to the anti-estrogenic compounds, enterolactone and enterodiol. Our lab has previously shown that dietary intervention with flaxseed reduces incidence and severity of ovarian cancer. Similarly, flaxseed has also been shown to reduce the expression of steroidogenic enzymes in the ovary. The objective of this study is to analyze oviductal polyps from flax-fed and control-fed hens, to investigate the potential for flax-based dietary intervention for the treatment of uterine fibroids, as well as further characterize hen oviductal polyps. Three studies were conducted to assess this objective. First, 1200 6-month-old White leghorn hens were fed a diet supplemented with 10% whole flaxseed over a period of 5 years. Every 6 months, 20 hens were sacrificed and oviductal polyps were collected to determine if the flax diet had any effect on polyp size, number, and location. Normal oviductal, muscularis tissue and polyps were collected for histological analysis and for RNA. The second study consisted of six treatment groups 15% whole flax seed, 10% defatted flax meal (DFM), 5% flax oil (FXO), 5% corn oil, 5% Stearidonic Acid (SDA) oil, and 5% soy oil with 30 1.5 year old hens in each group, fed for 4 months. Hens were then sacrificed, and polyps were collected and fixed for histological analysis. A long-term component study where 3 year old hen were fed diets supplemented with 15% whole flaxseed, 10% defatted flax meal, 5% flax oil, 5% fish oil, and 5% corn oil for a one year period. Power analysis was conducted regarding ovarian cancer to determine the number of hens per group. It was determined 161 hens in control, 161 hens in DFM, 161 hens in flax oil, 161 hens in whole flax, 175 hens in fish oil, and 175 hens in the corn oil group. After one year, polyps were counted and collected for various tissue analyses. In the 5 year study, overall polyp incidence increased between the 3 and 3.5 year time points, with no difference between groups. Average polyp area per bird and largest polyp per bird significantly increased from 2.5 year old birds versus to 3.5 and 4 year old birds with no difference in diet groups. Average polyp number per bird had no significant difference between the time points. Incidence, average polyp number per bird, largest polyp per bird, and average polyp volume per bird in the one-year long-term component study showed no difference between diets. Uterine fibroid estrogen receptor α (ERα), progesterone receptor (PR), B-cell lymphoma 2 (BCL-2), transforming growth factor β3 (TGF-β3), collagen type 1 (COL1A1) and collagen type 3 (COL3A1) were then analyzed by qPCR and immunohistochemistry in both oviductal polyps and muscularis oviduct. In the 5 year long-term study, ERα mRNA expression decreased, while PR, BCL-2, COL1A1, and COL3A1 mRNA expression were significantly increased in hen oviductal polyps. In the one year long-term study ERα mRNA in oviductal polyps was decreased in all groups (DFM, FXO, fish oil, corn oil) except whole flax fed hens. Progesterone receptor mRNA expression was increased in the muscularis oviduct of only corn fed hens, while no change was seen in oviductal polyp. BCL-2 mRNA expression was decreased in all treatment groups in muscularis oviduct and increased in oviductal polyps from DFM supplemented birds. TGF-β3 mRNA expression was significantly increased in whole flax fed and fish oil birds in muscularis oviduct. In oviductal polyps, TGF-β3 was increased in whole flax, flax oil, fish oil, and corn oil but none were significant. COL1A1 and COL3A1 demonstrated a similar pattern and showed minimal change between diet groups as well as muscularis oviduct and oviductal polyps. Inflammatory pathway enzymes COX-1 and COX-2 were then analyzed by qPCR and immunohistochemistry. In the 5 year long-term study, no change was seen in COX-1, but COX-2 mRNA increased in whole flax fed oviductal polyps. Immunohistochemical sections of COX-1 had singular punctate staining in DFM and flax oil compared to other diets. COX-2 expression showed decreased immunohistochemical staining intensity in the flax oil fed hens, but not in any other group. In the one year long-term component study, COX-1 mRNA expression had no change in muscularis oviduct and oviductal polyps. COX-2 mRNA decreased in muscularis muscle tissue of flax oil and fish oil treated hens, but not in oviductal polyps. The steroidogenic capability of oviductal polyps was then analyzed by examining mRNA and protein expression. In the 5 year study steroidogenic pathway components (StAR, 3βHSD, CYP17, and CYP19 showed no mRNA expression in smooth muscle oviduct. In oviductal polyps StAR and CYP17 mRNA expression increased, but 3βHSD and CYP19 expression decreased with the flaxseed diet. 3βHSD protein expression was then shown immunohistochemically to shift its expression to a more punctate, cytoplasmic form in defatted flax meal fed hens. In the one year long-term component study, steroidogenic pathway components were found in the muscularis oviduct. mRNA expression of StAR decreased in flax oil and fish oil of muscularis oviduct, but increased in oviductal polyps from flax oil hens. 3βHSD mRNA expression increased in muscularis oviduct of corn oil fed hens, however expression was decreased in flax oil and fish oil groups. In oviductal polyps 3βHSD mRNA was decreased in all groups. CYP17 mRNA was decreased in muscularis oviduct of flax oil and fish oil fed birds, but increased in DFM hens. Oviductal polyp CYP17 mRNA expression increased in flax oil, flax oil and corn oil diets but expression was ameliorated in whole flax hens. CYP19 mRNA expression in muscularis oviduct significantly decreased in flax oil and fish oil fed hens. In oviductal polyps CYP19 mRNA expression was decreased in DFM, whole flax, and fish oil hens. CYP19 expression was increased in corn fed birds in muscularis oviduct and oviductal polyps. Immunohistochemical protein expression of 3βHSD and aromatase (CYP19) did not change between diet groups. This is the first demonstration of the steroidogenic capability of oviductal polyps. This study proposes supplementation of whole flaxseed as a mechanism of dietary intervention for uterine fibroids. As stated above, flaxseed may be working through the estrogen or inflammatory pathways to reduce overall incidence of oviductal polyps. The data suggests that flaxseed may not act on the growth of leiomyomas, but perhaps their initiation. Progesterone has been shown to upregulate important leiomyoma factors, such as BCL-2 and TGF-β3, while COX-2 upregulation has been previously shown to increase collagen deposition and TGF-β3 expression. The increases in the proinflammatory environment of the oviduct could initiate the upregulation of downstream targets of progesterone and COX-2. Similarly, an increase in PGE2 synthesis has been shown to induce transcription of steroidogenic pathways and could explain expression in oviductal polyps. Further studies need to be conducted to elucidate the role of PGE2 in oviductal polyps. The mechanisms of flaxseed and its components must also be further teased apart to elucidate their true impact and determine the exact action in oviductal polyps.