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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: Biochim Biophys Acta. 2017 Sep 11;1859(12):2413–2419. doi: 10.1016/j.bbamem.2017.09.012

Influence of omega-3 fatty acids on bovine luteal cell plasma membrane dynamics.1

Michele R Plewes 2, Patrick D Burns 2,3, Richard M Hyslop 4, B George Barisas 5
PMCID: PMC5659747  NIHMSID: NIHMS910082  PMID: 28912100

Abstract

Fish oil is a rich source of omega-3 fatty acids which disrupt lipid microdomain structure and affect mobility of the prostaglandin F2α (FP) receptor in bovine luteal cells. The objectives of this study were to determine the effects of individual omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on 1) membrane fatty acid composition, 2) lipid microdomain structure, and 3) lateral mobility of the FP receptor in bovine luteal cells. Ovaries were collected from a local abattoir (n = 5/experiment). The corpus luteum was resected and enzymatically digested using collagenase to generate a mixed luteal cell population. In all experiments, luteal cells were treated with 0, 1, 10 or 100 μM EPA or DHA for 72 h to allow incorporation of fatty acids into membrane lipids. Results from experiment 1 show that culturing luteal cells in the presence of EPA or DHA increased these luteal fatty acids. In experiment 2, both EPA and DHA increased spatial distribution of lipid microdomains in a dose-dependent manner. Single particle tracking results from experiment 3 show that increasing both EPA and DHA concentrations increased micro- and macro-diffusion coefficients, increased domain size, and decreased residence time of FP receptors. Collectively, results from this study demonstrate similar effects of EPA and DHA on lipid microdomain structure and lateral mobility of FP receptors in cultured bovine luteal cells. Moreover, only 10 μM of either fatty acid was needed to mimic the effects of fish oil.

Keywords: bovine, luteal membrane, omega-3 fatty acids, lipid microdomains, FP receptor

Graphical abstract

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1. Introduction

The corpus luteum (CL) is a transient endocrine gland formed from the ovulatory follicle following ovulation. Progesterone is a steroid hormone synthesized and secreted by the gland that is vital for the establishment and maintenance of pregnancy in the cow [1]. In the absence of an embryo, the uterus releases prostaglandin (PG) F2α in a series of 6 - 8 pulses at approximately days 15 to 18 following ovulation [2, 3]. Luteal cells of the CL express FP receptors [4, 5], a G-protein coupled, membrane-bound receptor, and binding of PGF2α triggers an intracellular signaling cascade that causes regression of the gland [6, 7], allowing for another estrous cycle to occur, and with that, another opportunity to become pregnant. In the pregnant cow, the embryo must inhibit uterine PGF2α secretion to maintain the structure and function of the CL. Embryonic mortality often occurs when a viable embryo fails to effectively control maternal PGF2α secretion, which results in the regression of the CL and termination of the pregnancy [8–10]. Therefore, diminishing or altering sensitivity of luteal cells to PGF2α during early pregnancy may prevent regression of the CL.

The plasma membrane of cells is a complex and highly dynamic structure composed of lipids, carbohydrates, and proteins [11, 12]. The lipids form a bilayer that serves as a selective barrier separating the external from the internal environment of the cell. There are three major lipid classes found within mammalian biological membranes, namely glycerophospholipids, cholesterol, and sphingolipids. Glycerophospholipids are the predominant lipid of the plasma membrane that forms the lipid bilayer. Cholesterol tightly packs and interacts with sphingolipids forming distinct patches 10 to 200 nm in diameter referred to as lipid microdomains [13]. The interaction between cholesterol and sphingolipids limits incorporation into the glycerophospholipid bilayer, causing it to form detergent-resistant microdomains [14]. This unique environment between cholesterol and sphingolipids plays a role in recruitment of membrane-bound receptors and associated intracellular signaling molecules allowing for downstream signal transduction within the cell [15–17]. Therefore, altering lipid microdomain structure of luteal cells may affect sensitivity to the luteolytic actions of PGF2α.

Long-chain saturated, unsaturated, and polyunsaturated fatty acids are the basic constituents of glycerophospholipids and sphingolipids found within the plasma membrane. These fatty acids play a vital role in the structural integrity of the plasma membrane, membrane fluidity, and signal transduction. Omega-3 fatty acids are a class of long-chain polyunsaturated fatty acids with the first double bond at the third carbon atom from the omega terminal end. Alpha-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are common omega-3 fatty acids found within biological membranes, including the plasma membrane. The high degree of unsaturation of EPA and DHA, five and six double bonds, respectively, can hinder the quasi liquid-order of lipid microdomains [18]. Therefore, increasing incorporation of either EPA or DHA into membrane glycerophospholipids and sphingolipids may influence microdomain structure.

Fish oil obtained from cold water fishes is abundant in both EPA and DHA, and supplementing the diet with fish oil or fish meal has been reported to affect lipid microdomain structure [15, 19–22]. Furthermore, a recent study from this laboratory demonstrated increased lateral mobility of the FP receptor in bovine luteal cells treated with fish oil [22]. However, which omega-3 fatty acid in fish oil affects lipid microdomain structure is largely unknown. Most studies show that DHA may have greater influence on the disruption of lipid microdomains [15, 23–26], despite the fact that EPA alters microdomain structure and lipid composition as well [18]. The objectives of this study were to determine the effects of fish oil, EPA, and DHA on 1) luteal cell fatty acid composition, 2) lipid microdomain structure, and 3) lateral mobility of the FP receptor on the plasma membrane of bovine luteal cells. Understanding the influence that individual omega-3 fatty acids, EPA and DHA, have on membrane structure is critical for making the appropriate dietary recommendations for improving reproductive efficiency in mammalian females.

2. Methods

2.1. Tissue collection, cell preparation, and cell culture

Bovine ovaries containing a CL were collected at a local abattoir and transported to the laboratory at the University of Northern Colorado in 1× sterile PBS. Gross ovarian morphology was used to determine the age of the CL as previously described [27] and only mature midcycle CL (n = 5/experiment) were used in these studies. Ovaries with a mature CL were then submerged in 70% ethanol and 0.06% quaternary ammonium to destroy any microorganisms that may be present on the outside of the ovary from time of collection.

The CL was removed from the ovary using sterile techniques under a laminar flow hood, and placed into a sterile 60-mm2 Petri dish containing 10 mL of ice-cold Ca+2/Mg+2-free Hank’s balanced salt solution (HBSS, pH 7.34). Capsular connective tissue was carefully dissected away to expose luteal tissue. Luteal tissue was removed and cut into approximately 1 mm3 fragments and 1 g of tissue was placed into T-25 plugged culture flasks containing 5 mL of dissociation medium (HBSS containing 2000 units of collagenase type 1 per g tissue and 0.1% BSA). Tissue was incubated at 37 °C for 45 min in a shaking water bath. Following incubation, the supernatant was removed and transferred to a sterile 15-mL culture tube. Cells were then washed 3 × with sterile 1× PBS and re-suspended in 10 mL of culture medium (Ham’s F12 supplemented with 5% fetal bovine serum, insulin-transferrin-selenium-supplement (Invitrogen, Carlsbad, CA, USA), 1× antibiotic-antimycotic (Gibco, Waltham, Massachusetts, USA), pH 7.34), until cell concentration and viability was determined.

Trypan blue was used to determine the viability of cells, and a hemocytometer was used to estimate cell concentration. In the current study, only preparations with a cell population of greater than 85% viability were used for each experiment. Cell cultures were maintained at 37 °C in an atmosphere of 95% humidified air and 5% CO2.

2.2. Lipid preparation for in vitro culture

Eicosapentaenoic acid and DHA were purchased from Cayman Chemical (Ann Arbor, MI, USA) and added to culture medium containing 33 mg/mL fatty acid-free BSA at the appropriate concentration for each experiment as described by Mattos et al. [28]. Fatty acids from a commercial fish oil (Pharmavite, Mission Hills, CA) were also pre-bound to BSA prior to the addition to culture as previously described [22]. Control medium was prepared as stated above without the addition of lipids.

2.3 Experiment 1: Effects of fish oil, eicosapentaenoic, and docosahexaenoic acids on omega-3 polyunsaturated fatty acid composition of bovine luteal cells

Mixed luteal cells were plated in T-75 culture flasks at 2 × 106 cells/flask (n = 2 T-75 flasks/treatment/CL). Cells were maintained at 37 °C in an atmosphere of 95% humidified air and 5% CO2 until flasks reached 85 to 90% confluency. After reaching desired confluency, culture medium was removed and cells were treated with control medium, fish oil (0.3% vol/vol), or medium supplemented with EPA or DHA at 1, 10 or 100 μM. Cells were cultured an additional 72 h with supplemented medium to allow for incorporation of long-chain fatty acids into biological membranes. Following incubation, cells were removed from culture flasks and washed 6 × with 1× sterile PBS to remove excess free fatty acids. Cells were placed into a 16 × 100 mm reaction tube and freeze-dried for 24 h prior to methylation. Samples were maintained in the dark to minimize light-induced oxidation of fatty acids. Fatty acids were methylated using direct methylation as previously described [29].

An Agilent 7890A Series gas-liquid chromatograph (Wilmington, DE, USA) with a mass spectrometer (GC-MS) detector was used to determine long-chain fatty acid composition. The instrument was equipped with a 30-m × 0.20-mm (i.d.) fused silica capillary column (Supelcowax10; Supelco Inc., Bellefonte, PA, USA) and electron impact ionization was used. The injector temperature was set at 250 °C and 1 μL of fatty acid methyl ester samples was applied to the column using split-less mode. The carrier gas utilized was helium with a flow rate of 1 mL/min. Oven temperature was programmed from an initial temperature of 140 °C, which was held for 10 min, and then increased to a final temperature of 250 °C at the rate of 2.5 °C/min. The final temperature of 250 °C was held constant for 10 min for a total run time of 65 min. Standard fatty acid methyl ester mixtures were used to calibrate the instrument using reference standard GLC 68-D (Nu-Chek Prep, Inc. Waterville, MN, USA). Chromatograms were generated for each analysis using ChemStation Plus Chromatograph Manager (Agilent Technologies, Boulder, CO, USA). Identification of long-chain fatty acids obtained from cultured cells were determined by comparing the mass spectrometry analysis and relative retention times of fatty acid to the known set of standards. These peaks were then calculated as normalized area percentages of fatty acids.

2.4.1 Experiment 2: Effects of fish oil, eicosapentaenoic, and docosahexaenoic acids on spatial distribution of lipid microdomains

Mixed luteal cells were plated in four-chamber glass-bottom culture dishes (Cellvis, Mountain View, CA, USA) at 5 × 104 cells/dish (n = 2 chambers/treatment). Cells were incubated overnight at 37 °C in an atmosphere of 95% humidified air and 5% CO2 to allow cells to adhere to glass cover slips. Culture medium was removed and cells were treated with supplemented medium as described in experiment 1 for 72 h allowing for incorporation of fatty acids into cell membranes.

2.4.2 Lipid microdomain labeling and visualization

Lipid microdomains were labeled as previously described and validated in our laboratory [22, 30]. Following labeling of microdomains, cells were viewed using a Zeiss confocal microscope using a 40× water immersion objective (1.2 N.A). The appropriate laser was used to excite the Alexa-555 fluorophore. Approximately 10–15 cells were randomly selected from each dish and 1 μm slice images were generated from bottom to top of each cell. Whole cell fluorescence intensity was determined from three-dimensional images using ImageJ software. A total of 55, 69, 42, 55, 48, 32, 45, and 74 mixed luteal cells were analyzed from control, fish oil, 1, 10, 100 μM EPA and DHA treatments, respectively.

2.5 Experiment 3: Effects of fish oil, eicosapentaenoic, and docosahexaenoic acids on lateral mobility of FP receptors on the plasma membrane of bovine luteal cells

Mixed luteal cells were cultured in four-chamber glass bottom culture dishes at 5 × 104 cells/dish. Cells were cultured as described in experiment 2 for 72 h. Cells were then prepared using a validated protocol for single particle tracking as previously described [22]. In brief, polyclonal FP receptor antibody (Cayman Chemical, Ann Arbor, MI, USA) was conjugated to biotin per manufacturer’s instructions using a DSB-X biotin protein labeling kit (Life Technologies, Carlsbad, CA, USA). Receptors were prepared for single particle tracking experiments using biotinylated FP receptor antibody and Quantum dot® 605 Streptavidin Conjugate (Thermo-Fisher Scientific, Waltham MA, USA).

A Zeiss confocal microscope equipped with a high-speed camera (Hamamatsu Photonics, Japan) was used to capture individual receptor trajectories. Ultraviolet light was used to excite Quantum dots and emission of light was collected as 605 nm. Receptors were visualized using 100× oil objective and an acquisition image size of 512 × 512 pixel (33.3 μm × 33.3 μm). Receptors were recorded for 29 s (30 frames/s) and only receptors containing a minimum of 10 s of recordings were used for the final analysis in this study.

Video Spot Tracker v08.01 software (Computer Integrated Systems for Microcopy and Manipulation, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA) was used to track the motion of the receptor, generating individual X-Y coordinates. Trajectories were given as X-Y pixel and converted to μm. Mean square displacement of trajectories was calculated and plotted as a function of time to determine both micro (initial velocity of FP receptor) and macro (average velocity with confinement of FP receptor) diffusion coefficients according to the equations reported by Daumas et al [31]. Residence time and domain size were determined from each individual receptor as previously described [22]. A total of 22, 22, 22, 20, 28, 22, 24, and 25 receptor trajectories were analyzed from control, fish oil, 1, 10, 100 μM EPA and DHA supplemented luteal cells, respectively.

2.6 Statistical Analysis

All data are reported as least square means ± standard error of the mean and unless otherwise indicated, significance was declared at P < 0.05. Effects of EPA and DHA on luteal fatty acid composition, lipid microdomain staining intensity, lateral mobility of FP receptors (micro- and macro- diffusion coefficients), domain sizes, and residence time of receptors were analyzed using one-way analysis of variance (ANOVA). The model included concentration (0, 1, 10, or 100 μM) of fatty acid (EPA or DHA), fish oil, CL, concentration × fatty acid interaction, and residual error as sources of variation. Corpus luteum was considered a random variable in the model. Calculations were made using the mixed-model procedure of SAS (SAS Institute Inc. Cary, USA) and pairwise t-tests (PDIFF option) to separate means if ANOVA was significant. The effects of concentration of EPA and DHA on lipid microdomain staining intensity and lateral mobility of FP receptor were further analyzed using linear regression. Calculations were made using the procedure regression of SAS.

3. Results

3.1 Effects of fish oil, eicosapentaenoic, and docosahexaenoic acids on omega-3 polyunsaturated fatty acid composition of bovine luteal cells

In experiment 1, GC-MS was used to determine the effects of fish oil, EPA, and DHA on long-chain fatty acid composition in cultured bovine luteal cells. Fish oil treatment did not affect content of palmitic, palmitoleic, stearic, oleic, linoleic, and α-linolenic fatty acids when compared to control cells (data not shown). Luteal content of EPA and DHA was greater for cells treated with fish oil as compared to control cells (Fig 1). Regardless of concentration, there was no effect of culturing luteal cells in the presence of EPA on content of palmitic, palmitoleic, stearic, oleic, linoleic, α-linolenic (Fig. 1A), or DHA (Fig. 1C). Eicosapentaenoic acid concentration was greater for cells treated with either 10 or 100 μM EPA when compared to control-treated cells (Fig 1B). Additionally, there was no difference in luteal EPA composition for cells treated with either 10 or 100 μM EPA. However, cells treated with 100 μM EPA had decreased arachidonic acid when compared to control-treated cells (data not shown).

Figure 1. Effects of fish oil, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acid on relative composition (weight percent) of bovine luteal cell omega-3 long-chain polyunsaturated fatty acids.

Figure 1

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Panel A: Luteal α-linolenic acid. Bovine serum albumin (BSA) control (n = 5; solid grey bars), 0.03% fish oil (vol/vol; n = 5; hatched bars), EPA (n = 5; open bars), and DHA (n = 5; solid bars). Panel B: Luteal EPA. BSA control (n = 5; solid grey bars), fish oil (n = 5; hatched bars), EPA (n = 5; open bars), and DHA (n = 5; solid bars). Panel C: Luteal DHA. BSA control (n = 5; solid grey bars), fish oil (n = 5; hatched bars), EPA (n = 5; open bars), and DHA (n = 5; solid bars). *Significant difference within class of fatty acid compared to BSA control; P < 0.05. Non-detectable (N.D.).

Similar results were obtained in cells treated with DHA. Regardless of concentration, there was no effect of DHA on luteal fatty acid content of palmitic, palmitoleic, stearic, oleic, linoleic, α-linolenic, arachidonic acid, or EPA (Fig 1B). Similar to the effects of EPA on luteal fatty acid composition, DHA was greater for cells treated with either 10 or 100 μM DHA as compared to control-treated cells (Fig 1C). As observed with EPA, luteal DHA was less in cells treated with 1 μM DHA as compared to higher doses, but there was no difference between either 10 or 100 μM DHA-treated cells (Fig 1C). While 100 μM DHA decreased arachidonic acid by 57% when compared to control cells, this was not significant (data not shown).

3.2 Effects of fish oil, eicosapentaenoic, and docosahexaenoic acids on spatial distribution of lipid microdomains

Experiment 2 investigated the effects of fish oil and increasing concentrations of EPA and DHA on spatial distribution of lipid microdomains on the plasma membrane of bovine luteal cells. Fish oil decreased total cell fluorescent intensity of microdomains when compared to control-treated cells. Increasing both EPA and DHA concentrations from 1 to 100 μM resulted in a linear disruption of lipid microdomains (EPA y = −137.8× + 31221; DHA y = −117.8× + 31552) respectively, which was detected by decreased total cell florescent intensity (Fig 2). Both 10 and 100 μM EPA and DHA decreased total cell fluorescent intensity of microdomains when compared to control-treated cells, but was not different when compared to fish oil-treated cells.

Figure 2. Effects of fish oil, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acid on spatial distribution of lipid microdomains.

Figure 2

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Panel A: Representative micrographs obtained from (A) Bovine serum albumin (BSA) control, (B) 1 μM EPA, (C) 10 μM EPA, (D) 100 μM EPA, (E) 0.03% fish oil (vol/vol), (F) 1 μM DHA, (G) 10 μM DHA, and (H) 100 μM DHA. Panel B: Mean fluorescent intensity for luteal cells obtained from BSA control, (n = 55 cells), 1 μM EPA (n = 42 cells), 10 μM EPA (n = 55 cells), 100 μM EPA (n = 48 cells), fish oil (n = 69 cells), 1 μM DHA (n = 32 cells), 10 μM DHA (n = 45 cells), and 100 μM DHA (n = 74 cells); BSA control vs. fish oil, EPA or DHA; *P < 0.05. Micron bar represents 20 μm.

3.3 Effects of fish oil, eicosapentaenoic, and docosahexaenoic acids on lateral mobility of FP receptors on the plasma membrane of bovine luteal cells

In experiment 3, live-cell single molecule tracking was used to observe lateral mobility of the bovine FP receptors in real time. Eicosapentaenoic acid and DHA had a significant influence on lateral mobility of receptors in a dose dependent manner. There was a linear increase in micro- (Y = 0.0024x + 0.279; P < 0.05) and macro- (Y = 0.00006x + 0.0042; P < 0.05) diffusion of FP receptors for EPA-treated cells. Similar results were obtained for DHA-treated cells for micro- (Y = 0.003x + 0.315; P < 0.05) and macro- (Y = 0.00007x + 0.0040; P < 0.05) diffusion. Both micro- and macro-diffusion of FP receptors were increased in cells treated with either 10 or 100 μM EPA or DHA as compared to control cells. Within equivalent concentrations of EPA and DHA, there was no difference in micro- or macro- diffusion. Additionally, micro- and macro- diffusion of FP receptors increased in fish oil-treated cells when compared to control cells (P < 0.05; Fig 3A and 3B). However, there was no difference in either micro- or macro-diffusion of FP receptors in cells treated with 10 or 100 μM of either EPA or DHA as compared to fish oil-treated cells.

Figure 3. Effects of fish oil, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acid on lateral mobility of prostaglandin F2α receptors on bovine luteal cells.

Figure 3

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(A) micro- and (B) macro-diffusion coefficient of the prostaglandin F2α (FP) receptor on bovine luteal cells treated with Bovine serum albumin (BSA) control medium, (n = 22 FP receptors), 1 μM EPA (n = 22 FP receptors), 1 μM DHA (n = 22 FP receptors), 10 μM EPA (n = 20 FP receptors), 10 μM DHA (n = 24 FP receptors), 100 μM EPA (n = 28 FP receptors), 100 μM DHA (n = 25 FP receptors), and 0.03% fish oil (vol/vol; n = 22 FP receptors); BSA control vs. fish oil, EPA or DHA; mean ± standard error of the mean * P < 0.05.

Eicosapentaenoic acid and DHA had a significant influence on domain size and time a FP receptor resided within a domain, which is referred as residence time, in a dose dependent manner. There was a linear increase in domain size (Y = 0.0013x + 0.181; P < 0.05) and decrease in residence time (Y = − 0.008x + 4.56; P < 0.05) for EPA-treated cells. Similar increase and decrease, respectively, were obtained for DHA-treated cells for domain size (Y = 0.0016x + 0.201; P < 0.05) and residence time (Y = −0.009x + 4.65; P < 0.05). Both 10 and 100 μM EPA and DHA affected domain size and residence time when compared to control-treated cells. This is in agreement with spatial distribution data, indicating both EPA and DHA increased disruption of lipid microdomains. Furthermore, domain size was increased in fish oil-treated cells when compared to control cells (Fig 4A). Residence time of FP receptors was decreased in fish oil-treated cells when compared to control cells (Fig 4B). There was no difference in domain size or residence time for FP receptors in cells treated with 10 or 100 μM of either EPA or DHA as compared to fish oil-treated cells.

Figure 4. Effects of fish oil, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acid on domain size and residence time of prostaglandin F2α receptors on bovine luteal cells.

Figure 4

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(A) domain size and (B) residence time of the prostaglandin F2α (FP) receptors on bovine luteal cells treated with Bovine serum albumin (BSA) control medium, (n = 22 FP receptors), 1 μM EPA (n = 22 FP receptors), 1 μM DHA (n = 22 FP receptors), 10 μM EPA (n = 18 FP receptors), 10 μM DHA (n = 24 FP receptors), 100 μM EPA (n = 28 FP receptors), 100 μM DHA (n = 25 FP receptors), and 0.03% fish oil (vol/vol; n = 22 FP receptors); BSA control vs. fish oil, EPA or DHA; mean ± standard error of the mean * P < 0.05.

Discussion

Eicosapentaenoic acid and DHA are two common omega-3 fatty acids found in fish by-products which can be readily incorporated into glycerophospholipids and sphingolipids. Numerous studies have demonstrated that dietary supplementation with fish by-products increase the percentage of omega-3 fatty acids into the plasma membrane of cells [21, 22, 29, 32–34] and remodel lipid microdomains [19]. However, recent data using artificial membranes have shown a tendency for glycerophospholipids containing DHA to be incorporated into lipid microdomains twice as efficiently as those having EPA [35], indicating that DHA may be preferentially incorporated into lipid microdomains. Yet, it is still unclear which individual omega-3 fatty acid plays a greater role in disrupting organization of microdomains. Most studies have reported that DHA has greater influence on lipid microdomain structure [15, 23–26], despite the fact that DHA-containing glycerophospholipids are more ordered than those containing EPA [35, 36]. A recent study in the literature showed that EPA and DHA equally disrupt lipid microdomains in immune cells [37]. The present study investigated the effects of individual omega-3 fatty acids, EPA and DHA, on luteal cell fatty acid composition, spatial distribution of lipid microdomains, and lateral mobility of the FP receptor on the plasma membrane of cultured bovine luteal cells.

Results from experiment 1 show that luteal cells treated with fish oil had increased content of EPA and DHA as compared to cells cultured in control medium. The percentage of EPA and DHA in fish oil-treated cells was comparable to the percentage of EPA and DHA in cells of corpora lutea collected from cows supplemented with fish meal [29, 30]. Free fatty acids of EPA or DHA were both readily incorporated into biological membranes of luteal cells as well. Neither of these fatty acids had a significant influence on abundance of other major long-chain fatty acids commonly found in biological membranes with the exception of arachidonic acid. While both EPA and DHA decreased the percentage of arachidonic acid at the highest dose examined (100 μM), only EPA was shown to be significant from control cells. Inclusion of fish meal in the diet of cows has been reported to decrease arachidonic acid in both endometrial and luteal tissue [29, 32]. The decrease in arachidonic acid observed in whole animal studies was most likely due to the incorporation of EPA into reproductive tissues, which resulted in displacement of arachidonic acid.

Lipid microdomains consist of cholesterol that is tightly packed with sphingolipid, which does not integrate well into the disordered glycerophospholipid bilayer of the plasma membrane. This aggregation of cholesterol and sphingolipid causes the formation of distinct patches ranging in size from 10 to 200 nm [13]. Clustering of lipid microdomains can be visualized through binding of fluorescently labeled cholera toxin B to monosialotetrahexosylganglioside and antibody cross-linking, resulting in distinct patches on the plasma membrane [38]. It has been demonstrated that incorporation of fatty acids from fish by-product diminished microdomain clustering in bovine luteal cells in vitro [22] and in vivo [30]. However, it was not clear from these previous studies which omega-3 fatty acid in fish by-products contributed to the disruption of lipid microdomain structure.

Experiment 2 investigated the influence of EPA and DHA on lipid microdomain structure. Data from experiment 2 show that cells treated with fish oil had decreased total florescence intensity which is consistent with a previous study from this laboratory [22] When assessing an individual fatty acids, both EPA and DHA had a similar effect on microdomain structure in this cell type. Increasing the dose of both EPA and DHA from 1 to 100 μM resulted in a linear decline in total cell florescence intensity. These data are in agreement with several studies showing omega-3 fatty acids alter lipid microdomain structure [21, 35]. The decrease in florescent intensity was most likely due to an increase in lipid microdomain size, leading to a decreased ability for cross-linking of cholera toxin B subunit. However, unlike the current study wherein culturing luteal cells in presences of individual omega-3 fatty acids or fish oil resulted in a decrease in total fluorescent intensity, other studies using immune cells treated with fish oil observed an increased intensity. Discrepancies between our data and previously reported studies regarding fluorescent intensity may be due to experimental methodology. In this study, live cells adhered to cover slips were used to determine spatial distribution of lipid microdomains, while other studies determined fluorescent intensity in fixed non-adherent cells. The adhesion of cells to cover slips may influence the cytoskeleton in EPA-, DHA-, and fish oil-treated cells, thereby affecting cross-linking of antibody to cholera toxin B, resulting in reduced fluorescent intensity. Furthermore, fixing of cells may influence the cytoskeleton of cells [39], leading to an apparent increase in fluorescent intensity in cells treated with fish oil.

Cholesterol plays a significant role in the formation and integrity of lipid microdomains. Early work using single particle tracking showed that these domains are approximately 200 nm in diameter and “trap” membrane proteins for several seconds. The removal of plasma membrane cholesterol disrupts these domains as indicated by reduced number of domains and residence time of proteins within a domain [40]. Mobility and residence time of FP receptors of bovine luteal cells are affected by fish oil treatment [22] and tissue obtained from fish meal supplemented animals [30]. This suggests that in addition to cholesterol, omega-3 fatty acids may influence FP receptor membrane dynamics within the plasma membrane and microdomains of luteal cells.

Experiment 3 examined the influence of either EPA or DHA on lateral mobility of the FP receptor on the plasma membrane of luteal cells. Cells cultured in the presence of fish oil had increased micro- and macro-diffusion coefficients. In addition to increased diffusion rate of receptors, size of domains increased and residence time within domains decreased. These results on FP receptor dynamics for fish oil-treated cells are very similar to a previously reported study from this laboratory [22]. Furthermore, both EPA and DHA had a significant influence on lateral mobility of FP receptor in a dose-dependent manner. Micro- and macro-diffusion coefficients were increased in cells cultured in 10 or 100 μM of either EPA or DHA compared to control cells, yet were similar to fish oil-treated cells. Our results show that both EPA and DHA have similar influences on FP receptor dynamics with as little as 10 μM concentration.

To our knowledge, this is the first study investigating the effects of individual omega-3 fatty acids, EPA and DHA, on bovine luteal cells. Plewes et al. [22] demonstrated fish oil increases both spatial distribution of lipid microdomains, as well as lateral mobility of the FP receptor of bovine luteal cells. Moreover, in vivo studies demonstrated that dietary fish meal supplementation alters plasma fatty acid composition, luteal tissue fatty acid composition [29], and lipid microdomain integrity [30]. Much work has been done to determine the beneficial effects of fish by-products on a number of cell types; however, the mechanistic role of omega-3 fatty acids is still unknown. A number of studies have shown DHA has a greater impact on membrane dynamics. In bovine reproductive luteal cells, both individual omega-3 fatty acids, EPA and DHA, have similar effects on disruption of lipid microdomains and lateral mobility of FP receptors. Additionally, the effects 10 or 100 μM of either EPA or DHA exert on lipid microdomains and lateral mobility of the FP receptor are not different to fish oil.

Adequate secretion of progesterone by the CL is required for maintenance of pregnancy in all mammalian females [41, 42]. Prostaglandin F2α secreted from the uterus is the endogenous luteolysin in the bovine [2, 43, 44]. Early embryonic mortality occurs when the conceptus is unable to adequately attenuate uterine section of PGF2α, resulting in loss of the gland [8]. One way to improve fertility may be to decrease luteal sensitivity to PGF2α. There are several ways to reduce luteal sensitivity which include 1) altering PGF2α metabolism within the CL, 2) decreasing cell signaling, or 3) mitigating number of responsive FP receptors on the plasma membrane of luteal cells. The current study demonstrated that omega-3 fatty acids increase the lateral mobility of FP receptors. It is possible that this increase in lateral mobility may result in a decrease in number of responsive FP receptors on the plasma membrane, resulting in diminished cell signaling. In fact, in fish oil-treated cells, the mobility of the FP receptor is unaffected by the presence of PGF2α, while lateral mobility becomes confined in control luteal cells [22], which may be necessary for downstream signaling. Taken together, the inclusion of omega-3 polyunsaturated fatty acids in the diet may reduce luteal sensitivity to PGF2α by altering membrane dynamics, which may increase embryo survival. Lastly, results from this study demonstrate individual omega-3 polyunsaturated fatty acids, EPA and DHA, have the same effects as fish oil. This may allow for development of more economical feeding strategies, such as inclusion of DHA containing algae in the diet.

Summary Sentence.

Eicosapentaenoic and docosahexaenoic acids disrupt lipid microdomains and increase FP receptor mobility on plasma membranes of bovine luteal cells.

Highlights.

  • EPA and DHA readily incorporate into biological membranes of bovine luteal cells

  • EPA or DHA disrupt spatial distribution of lipid microdomain in bovine luteal cells

  • Both EPA or DHA increase the lateral mobility of FP receptor

  • Both EPA or DHA decreases residence time, and increases domain size

  • Minimal concentration of EPA or DHA was required to mimic effects of fish oil

Acknowledgments

The authors thank Kenneth Cochran, Chad Wangeline, and Casey Rogers at the University of Northern Colorado Instrumentation and Fabrication Services for their assistance with microscopy. Additionally, the authors would like to thank the Chemistry and Biochemistry Department at the University of Northern Colorado for the use of the GC-MS instrument.

Footnotes

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1

This project was supported by National Research Initiative Competitive Grant no. 2013-6715-20966 from the USDA National Institute of Food and Agriculture to P.D.B and National Institutes of Health, Award Number CA175937 to B.G.B.

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