A major function of granulosa cells of the ovarian follicle is to produce estradiol that is triggering the luteinizing hormone (LH) surge which in turn initiates ovulation. This cycle (among others) seems to be disturbed in high-yielding dairy cows ; however, the molecular mechanisms remain largely unclear. One line of evidence pointed toward an increased estradiol clearance from the periphery due to increased metabolic activity of the liver in those animals. However, approaches to increase estradiol concentrations by exogenous application of this hormone largely failed . Another explanation pointed toward a disturbance of granulosa cell function (specifically decreased estradiol production) probably mediated by harmful metabolic byproducts such as reactive oxygen species (ROS). Some ROS escape from endogenous defense systems. Overproduction of oxidants that overwhelms the cellular antioxidant capacity is called oxidative stress. The concentration of these reactive metabolites inevitably increases in high-performing dairy cows due to their increased metabolic activity by concurrently limited defense capacities [3, 4]. In previous work, we and others described oxidized or peroxidized lipids as potentially harmful ROS interfering with follicular functions [5, 6]. The production of ROS (in particular lipohydroperoxides) almost linearly paralleled oxidative metabolic rates and milk production in dairy cows . Another oxidatively modified lipid is oxidized low-density lipoprotein (ox-LDL). Ox-LDL is synthesized from native low-density lipoprotein by lipid peroxidation in a nonenzymatic reaction and reaches via the blood stream potentially every target tissue.
Ox-LDL binds to several cell surface scavenger receptors, such as lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). LOX-1 is a multiligand receptor which serves as sensor and integrator for different danger signals . The critical role of the ox-LDL—LOX-1 system in macrophages and endothelial cells and its implication in inflammation and cardiovascular diseases has intensively been studied; for review see for example . LOX-1 has also been described to be expressed from human preovulatory granulosa cells [10, 11]; however, a functional link between LOX-1 and follicular dysfunctions has not been substantiated so far.
We previously confirmed the expression of LOX-1 also from bovine granulosa cells . We demonstrated that inhibition of the LOX-1 signal pathway via an inhibiting antibody leads to an activation of estradiol production. These data pointed toward a critical role of the LOX-1 receptor system for proper estrogenesis in granulosa cells. An increased estrogenesis (possibly mediated via inhibiting LOX-1) might defend against decreased estradiol concentrations which are evident in high-yielding dairy cows and, therefore, defend against follicular dysfunctions in these animals. The aim of this study was to elucidate the signal transduction pathway of the LOX-1—estradiol connection.
Material and Methods
Preparation/Culture of Mural Granulosa Cells
Periovulatory follicles were produced by heat induction with a prostaglandin F2α analog (cloprostenol) followed by an ovulation induction of the developed dominant follicle with a GnRH analog (depherelin) as described [13, 14]. In brief, mural granulosa cells (MGCs) were isolated from preovulatory ovaries sampled by ovariectomy using Holstein heifers (420 ± 30 kg in body weight, 16 ± 2 month old) after cycle synchronization and from follicle > 15 mm in diameter. Morphological assessment and 17β-estradiol concentration of the follicle fluid (at least 20 ng/ml), exceeding the progesterone level, indicated that the follicle were healthy (vascularized, estrogenic) and maturing into the final preovulatory stage . Sixteen hours post-GnRH administration, the ovary bearing the periovulatory follicle was removed by ovariectomy and MGCs were prepared. Follicle was dissected from the ovary, the follicle fluid was aspirated, MGCs scraped from the theca interna, and collected by centrifugation (60 g, 5 min, 4°C) as described . The cells were resuspended in ice-cold water for 10 s to lyse erythrocytes. Lysis was terminated by addition of NaCl (to 150 mM) and BSA (to 1%), cells were collected and resuspended in cell culture medium (MegaCell, Sigma, Taufkirchen, Germany) supplemented with BSA (1%), insulin (100 ng/ml), l-glutamine (2.5 mM), and an antibiotic mixture (Sigma). Cell density was determined by cell counter (Multisizer II, Beckman-Coulter), cell viability by exclusion of trypan blue or 40 µM propidium iodide . Propidium iodide uptake was checked by flow cytometry; the viability of the cells was > 95%. The prepared MGCs represent a status 14 h after the LH peak and 12 ± 4 h before the expected ovulation. The procedures with animals were approved by a governmental Animal Care and Use Committee.
The procedure followed a described technique with fura-2 as indicator . Cells in Ca2+ free DPBS medium (Sigma) were loaded with the calcium-sensitive dye fura-2 AM (to 2 μM) in the dark (30 min with gentle shaking). After washing, the calcium signal (fura-2 ratio, excitation at 340 nm and 380 nm, emission at 510 nm) was recorded using a high-resolution residual-light fluorescence imaging system (Till Photonics, Gräfelfing, Germany). Cells were viewed continuously under the microscope (IX70, Olympus) in an open dish incubation chamber (ΔTC3, Bioptech, Butler, PA) at RT. For LOX-1 inhibition experiments we used an anti-LOX-1 antibody (BioVision, Milpitas, CA, #3659R-100) in a final concentration of 1.5 µg/ml. As positive control, we used 1 μM thapsigargin which mobilizes calcium ions from the endoplasmic reticulum.
The 17β-estradiol concentration was determined by a direct 3H-RIA in house assay in 10 µl duplicates as previously described . Estradiol production rates after application of anti-LOX-1 or unspecific antibodies have been expressed relative to unstimulated production rates.
qPCR and Western Blot
Aromatase gene expression has been measured using a quantitative real-time PCR assay on a i-cycler (Bio-Rad, München, Germany) as described . Aromatase expression levels have been normalized to a standard curve and have been expressed relative to the input of total RNA. For western blot analysis, an anti-aromatase antibody from BioVision (Milpitas, #3599-100) has been used according to standard protocols.
JC-1 and dihydrorhodamine 123 (DHR) assays have been used as described . JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) was used in a final concentration of 1.5 µM. JC-1 fluorescence was measured after an excitation at 480 nm and recorded at 590 and 530 nm after 15 min reaction time in a cytoplate reader (Millipore). The fluorescence produced per mg protein was calculated by the ratio (fluorescence (sample) − fluorescence (blank)) × dilution factor/volume of mitochondrial sample (ml) × mg protein/ml and expressed relatively to the basal fluorescence. DHR was measured in a final concentration of 0.7 µM by an argon laser-equipped flow cytometer (Gallios, Beckman-Coulter). Fluorescence was excited at 488 nm and emission was recorded at 525 ± 25 nm.
Effects were assessed by student's t-test. Values of P <. 0.05 were considered statistically significant. Results are presented as means ± SD when not otherwise stated. Test were performed by SAS statistical package (SAS Institute Inc. Cary, NC) and Sigma Stat of the Jandel Scientific Software (Erkrath, Germany). Graphic presentations were accomplished by Sigma Plot of the Jandel Scientific program package.
A major function of granulosa cells is the production of estradiol to support ovulation and later to sustain the growing embryo. However, the mechanism of this activation is incomplete understood. In a previous work, we demonstrated that inhibition of the LOX-1 signal pathway via an inhibiting antibody leads to an activation of estradiol production. This activation is partially reversed by additional application of fumonisin, an inhibitor of sphingosine metabolism .
Because sphingosines interfere with the endoplasmic reticulum leading to altered calcium concentrations , we asked whether calcium signals might also be involved in LOX-1 signal pathway. To test this hypothesis, we applied an inhibiting anti-LOX-1 antibody (BioVision, final concentration 1.5 µg/ml) to periovulatory MGCs and measured the Ca2+ signal via the calcium sensitive fluorophor fura-2 . As shown in Figure 1A, we received an oscillating Ca2+ signal upon anti-LOX-1 antibody treatment but not by using an unspecific control antibody. To test from which particular calcium pool this calcium signal might arise, we tested the combined activation of thapsigargin and the LOX-1 antibody. After treatment with thapsigargin, we observed an increased calcium release from MGCs. Because thapsigargin depletes specifically ER calcium pools, this signal is derived from ER pools. After that activation, we received an additional calcium release using the anti-LOX-1 antibody (Fig. 1B). This indicates that the LOX-1 pathway addresses (at least in part) alternative calcium pools independent from the ER (e.g., mitochondria). In the reciprocal experiment, we first applied the LOX-1 antibody to MGCs and received a calcium signal as expected. However, no further calcium release could be obtained after a subsequent thapsigargin application, indicating that anti-LOX-1 antibody additionally depleted ER pools (Fig. 1C). Taken together, these data indicate that the LOX-1 pathway addresses both ER-dependent and ER-independent calcium pools.
To substantiate an involvement of calcium signals with estradiol production, we tested whether inhibition of the Ca2+ signal interferes with the estradiol production in MGCs. Unstimulated MGCs produce 1.19 ± 0.35 ng/[ml × 106 cells × h] estradiol. Again, treatment with the inhibiting anti-LOX-1 antibody leads to calcium release as described above (Fig. 1). However, preincubation with 10 µM BAPTA, a membrane permeable calcium chelator , impeded this activation (Fig. 2). These data indicate that calcium signals are critical in LOX-1 receptor-mediated modulation of estradiol production in MGCs.
We next asked which downstream targets might be modulated by altered calcium concentrations. Because a cross-talk between calcium and aromatase activity has been described , we first investigated aromatase concentrations both on transcript and protein levels in MGCs. In unstimulated MGCs, we detected 134 ± 74 fg/µg total RNA of aromatase transcripts which are not significantly different to those of stimulated cells (105 ± 56 fg/µg total RNA). We also did not observe different protein concentrations in western blot analysis (data not shown). Since MGCs properly produce estradiol upon offering testosterone as substrate, we also have no indication of altered aromatase enzyme activities in those cells (12 + data not shown). Since we demonstrated above that the LOX-1 signal pathway addressed ER but also ER-independent calcium pools (Fig. 1), we next addressed the ER-independent calcium pool mitochondrion. As shown in Figure 3, application of the inhibiting anti-LOX-1 antibody leads to increased membrane potential ΔΨ as measured by the JC-1 assay. Incubation with a nonspecific antibody did not change ΔΨ. Application of testosterone did also not show any alteration, again indicating proper steroidogenesis in these cells. In contrast, application of MnTBAP (final concentration 2 µM), a cell-permeable superoxide dismutase mimetic, decreased the membrane potential, both in the presence and absences of LOX-1 antibody.
Concomitantly, application of anti-LOX-1 antibody (but not an unspecific antibody) leads to a decreased fluorescence in a dihydrorhodamine 123 (DHR) flow cytometric assay (Fig. 4). Because DHR is a mitochondrial-specific indicator of free radicals, these data indicate that mitochondrial-derived oxidative stress is decreased after incubation with the inhibiting anti-LOX-1 antibody.
Taken together, these data indicate that the LOX-1 pathway is important for proper estradiol production from MGCs. This activation appears to be mediated via altered calcium concentrations. Furthermore, activation of the LOX-1 pathway might lead to increased oxidative stress at least in part due to altered mitochondrial functions. Our data indicate a functional link between mitochondrial dysfunctions and follicular dysfunctions in dairy cows.
Reduced fecundity in dairy cows is a major problem leading to economic losses of several million Euro worldwide. The reasons for inappropriate fertility in high-yielding dairy cows but also in metabolically highly active woman such as endurance athletes are multifactorial in nature including disturbed female cycle and failure of ovulation which in turn prevents fertilization in these females and animals. The reason of follicular dysfunctions are at least in part ascribed to inappropriate delivery of estradiol and progesterone from preovulatory granulosa cells and the developing corpus luteum, respectively .
In a previous study, we suggested the multiligand receptor LOX-1 to play a serious role in estradiol production from MGCs . In this study, we demonstrate that calcium signals are critical for this pathway (Figs. 1 and 2). Calcium release per se is not sufficient for regulating estrogenesis in MGCs. Activation of the PAF receptor pathway (another pro-inflammatory signal transduction pathway which is active in granulosa cells) releases calcium signals ; however, this calcium release does not lead to altered estradiol concentrations (unpublished data). Probably, multiple signal transduction pathways need to be addressed, e.g., calcium release from ER-dependent and ER-independent calcium pools as demonstrated for LOX-1 (Figs. 1B and 1C). One critical compartment appears to be the mitochondrion, pointing to an involvement of ROS. Inhibiting the LOX-1 pathway leads to increased mitochondrial membrane potential ΔΨ by simultaneously decreased production of ROS (Figs. 3 and 4). A reciprocal regulation of ΔΨ and ROS production has frequently been observed in mitochondrial physiology . In turn, endogenous activation of the LOX-1 by the natural ligand ox-LDL is expected to decrease ΔΨ by concomitantly increased production of ROS.
Production of ROS in dairy cows and female endurance athletes is a double-edged sword. On the one side, moderate amounts of ROS are desired since, e.g., prostaglandin production from the corpus luteum mandatory needs this substrate. On the other hand overwhelming ROS production leads to harmful oxidatively modified molecules, such as lipohydroperoxides or ox-LDL. A very similar scenario has been developed in nutritional intervention studies in human. Intensive gifts of antioxidant vitamins (which decrease ROS production) have been shown to prevent the beneficial effects of physical exercise training . Thus, reasonable ROS production is mandatory for the training effect. In the dairy cow, the concentration of ROS inevitably increases due to their raised metabolic activity [5, 6]. Again, this high metabolic activity is desired to ensure high milk production. One constraint of the increased metabolic rates is an increased turn-over rate of steroids. It has been calculated that the metabolism of estrogen and progesterone in lactating cows is ∼ 2.3-fold higher compared to nonlactating cows . Thus, dairy cows might suffer from inappropriate estrogen and progesterone concentrations which in turn interfere with its reproductive functions.
In this study, we draw a line of evidence from modulation of LOX-1 receptor (endogenously probably activated by ox-LDL) via altered calcium release and mitochondrial alterations finally leading to disturbed estradiol production from follicular granulosa cells. The endogenous ligand triggering this harmful cycle might be ox-LDL, which concentration increases with increased metabolic rate and increasing ROS production. In future experiment, we will test whether this mechanism also holds true in vivo. Blocking the LOX-1 receptor signal pathway might be a promising way to improve steroid hormone concentrations in the dairy cow and defending against reproductive dysfunctions in this species.