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BOR - Papers in Press, published online ahead of print December 19, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.065938
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BIOLOGY OF REPRODUCTION 78, 786–793 (2008)
DOI: 10.1095/biolreprod.107.065938
© 2008 by the Society for the Study of Reproduction, Inc.

Immunology; 

Expression and Regulation of Functional Oxytocin Receptors in Bovine T Lymphocytes1

Kalidou Ndiaye , Daniel H. Poole , and Joy L. Pate  2

Department of Animal Sciences, The Ohio State University/Ohio Agricultural Research and Development Center, Wooster, Ohio 44691

ABSTRACT

The corpus luteum (CL) produces oxytocin (OXT), which has been proposed to regulate the pulsatile release of prostaglandin F2alpha during luteolysis in ruminants. This action of OXT is mediated via oxytocin receptors (OXTRs) present on uterine epithelial cells. It is hypothesized that luteal OXT acts as a paracrine regulator of resident immune cells. In the present study, OXTR mRNA expression in bovine lymphocytes was analyzed, as well as its regulation during the estrous cycle. OXTR transcripts were observed in freshly purified bovine peripheral blood mononuclear cells and T lymphocytes. OXTR mRNA in bovine lymphocytes on Day 3 was numerically greater than but not significantly different from that of Day 19 of the estrous cycle (P = 0.091). In cultured T cells, estradiol (E2) treatment significantly increased the steady-state concentrations of OXTR mRNA, but the stimulatory effect of E2 was inhibited by the addition of progesterone (P4). Each of the major T cell subsets (CD4+, CD8+, and gamma delta+) expressed OXTR mRNA, with no significant difference in expression among them. Western blot analyses demonstrated the presence of the bovine OXTR protein at about 45 kDa in lymphocytes, as well as expression of the 14-kDa precursor of OXT. When lymphocytes were treated with OXT, intracellular concentrations of calcium ([Ca2+]i) were rapidly and dramatically increased. This study demonstrated that bovine lymphocytes express OXTRs and that this expression can be regulated in a steroid-dependent manner. Furthermore, OXT elicited a functional [Ca2+]i response in T lymphocytes, supporting the possibility that OXT within the CL could act as a paracrine or autocrine regulator of resident T lymphocytes.

bovine, calcium, corpus luteum, lymphocytes, oxytocin, oxytocin receptor

INTRODUCTION

The nonapeptide oxytocin (OXT) is mainly produced by the hypothalamus and transported in neurosecretory vesicles to the posterior pituitary, where it is stored or released into the bloodstream after specific stimulation. The major endocrine functions of OXT include uterine contraction during parturition and the contraction of the mammary gland for milk ejection [1]. In addition, there is evidence that the corpus luteum (CL) of ruminants also synthesizes and secretes OXT [2, 3]. This discovery led to the proposal that OXT stimulates the pulsatile release of prostaglandin F2{alpha} (PGF2{alpha}) from the uterus, resulting in regression of the CL [4–7]. It has also been shown that OXT is involved in the regulation of the proliferation of various cell types, including mammary gland epithelial cells and endometrial epithelial cells under both physiological and neoplastic conditions [8–10].

The biological actions of OXT are exerted through specific, membrane-bound OXT receptors (OXTRs) [11–13]. The structure of human and bovine OXTR proteins has been elucidated [14, 15], and the predicted amino acid sequences indicate that the OXTR belongs to the large family of G-protein-coupled receptors with a typical seven-transmembrane domain structure [15]. Upon stimulation by OXT, OXTR initiates a signal cascade resulting in the hydrolysis of phosphatidylinositol 4, 5-bisphosphate by phospholipase C to diacylglycerol, which stimulates a protein kinase C, and to inositol triphosphate [16]. The inositol triphosphate pathway leads to [Ca2+]i release from intracellular stores and the diffusion of Ca2+ into the cytosol for induction of specific biological responses. The traditional role of OXT has been extended in light of several reports about its biological activities and in light of the presence of OXTR mRNA in tissues that have not previously been considered as conventional targets for OXT [10, 17, 18]. For instance, receptors for OXT have been recently identified in the thymus of embryonic mice [19] and in freshly isolated lymphocytes from human peripheral blood [20].

In the reproductive system, immune cells are involved in the progression of luteolysis once it has been initiated by PGF2{alpha} and likely contribute to both the continued decline in progesterone (P4) production and the structural demise of the CL [21–23]. The CL is composed of a heterogeneous mixture of cell types that consist of steroidogenic cells and nonsteroidogenic cells, including vascular endothelial cells, fibroblasts, and immune cells. In the cow, lymphocytes are present in the fully functional CL and increase at the time of luteal regression [24, 25]. In addition, cultured bovine luteal cells were shown to be potent stimulators of T lymphocyte proliferation [26]. It is proposed that communication between steroidogenic or endothelial cells and immune cells occurs throughout the life span of the CL, not only during luteolysis. The demonstration of OXTR mRNA expression in the CL of sheep [27] and pigs [28] and in cultured bovine luteal cells [29] suggests an autocrine or paracrine role for luteal OXT. It is plausible that OCT, acting as a paracrine factor, is involved in the regulation of the function of immune cells that reside in the CL.

The long-term goal of these studies is to identify factors involved in luteal cell regulation of T lymphocytes. The functional status of immune cells within the CL is likely regulated by a balance of paracrine factors, and alterations of this balance may influence whether immune cells regulate normal homeostasis of luteal tissue or exert proinflammatory function during luteolysis. Luteal OXT is proposed to be one of these factors. Therefore, the specific objectives of this study were to determine if bovine immune cells express OXTR mRNA, if temporal changes in expression occur during the estrous cycle, how such expression is regulated, and if OXTR proteins in lymphocytes are functional.

MATERIALS AND METHODS

Isolation of Peripheral Blood Mononuclear Cells and T Lymphocytes

Peripheral blood mononuclear cells (PBMC) were isolated from whole blood collected via jugular venipuncture on Days 3, 11, and 19 of the estrous cycle (day of estrus = Day 0; n = 4 cows on each day). The lymphocyte-rich white blood cell layer was obtained following centrifugation of whole blood, and PBMC were isolated by centrifugation over Ficoll-Paque Plus (Amersham Pharmacia Biotech). An aliquot of fresh PBMC was taken for total RNA extraction. The T lymphocytes were separated from the PBMC immunomagnetically by positive selection with anti-CD2 and anti-gamma-delta ({gamma}{delta}) antibodies using the AutoMACS Separator (Miltenyi Biotech, Germany) as recommended by the manufacturer. Briefly, PBMC (1 x 108 cells) were incubated with mouse anti-CD2 (MUC2A) and anti-{gamma}{delta} ({alpha}-TcR1-N24 GB21A; VMRD, Inc., Pullman, WA) antibodies (5 µg/ml for each antibody), washed as previously described [30], and labeled with rat anti-mouse IgG2a+b microbeads (Miltenyi Biotech). Anti-CD2 antibodies were used to selectively target the CD2+ T lymphocytes, including natural killer (NK) T cells, while anti-{gamma}{delta} antibodies targeted the {gamma}{delta} T lymphocytes. This separation procedure yielded a population of cells that was approximately 95% pure T lymphocytes, as determined by fluorescent labeling with anti-CD3 (MM1A; T cell receptor) and anti-CD335 (MCA2365; NK cells) antibodies (VMRD, Inc.). The negative fraction that contained the macrophages and B cells was collected to analyze OXTR mRNA expression for comparison with purified T cells. The number of viable T cells was determined using the Guava ViaCount Flex Reagent in the Guava EasyCyte system (Guava Technologies, Inc.). The T lymphocytes were subsequently divided for culture or for total RNA extraction. T cells were cultured in the presence or absence of Concanavalin A (Con A; 125 µg/ml; Calbiochem), 10 ng/ml of P4 (Sigma-Aldrich), and/or 15 pg/ml of 17β-estradiol (E2; Sigma-Aldrich). Cultures were carried out for 72 h at 37°C in RPMI-1640 medium containing 10% fetal bovine serum (Gibco; Invitrogen Corp.). At the end of the culture, T cells were collected for total RNA extraction and for OXTR mRNA quantification by reverse transcription followed by quantitative PCR (RT-qPCR). A portion of the freshly collected PBMC were further separated into different subpopulations using anti-CD4, anti-CD8, and anti-{gamma}{delta} antibodies (VMRD, Inc.), separately, in order to analyze OXTR mRNA expression in these different T cell subsets.

Collection and Dissociation of Corpora Lutea and Coculture Experiments

CL was collected transvaginally from cyclic cows (n = 4) during the midluteal phase (Day 11; Day 0 = day of estrus) of the estrous cycle. Immediately following CL removal, 500 ml of jugular venous blood was collected as described above. Handling of animals and surgical procedures were conducted according to protocols approved by the Institutional Laboratory Animal Care and Use Committee of the Ohio State University. Dissociation of luteal tissues was performed according to the procedure described previously [31]. Luteal tissues were minced and placed in 24 mM Hepes-buffered Hams F-12 culture medium (Gibco; Invitrogen Corp.) containing 0.5% BSA (Sigma-Aldrich), 20 µg/ml gentamycin (Gentamycin Reagent Solution; Invitrogen Corp.), and 2000 U/g tissue collagenase type I (Worthington Biochemical Corporation). Dissociation, dispersion, and centrifugations and washes were performed as described [31]. Following dissociation, luteal cells were resuspended in Hams F-12 culture medium, and the number of viable cells was determined using the Guava ViaCount program in the Guava EasyCyte system. The luteal cells (5 x 105 cells) were cocultured with T cells (2 x 106 cells) in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum and treated with P4 (0.5 mol/L), OXT (100 ng/ml; GenScript Corporation), and anti-OXT polyclonal antibody (50 ng/ml; Chemicon International). The concentration of anti-OXT that would effectively bind all of the OXT in the culture medium was calculated based on a preliminary experiment performed to determine OXT concentrations secreted by luteal cells over the 72-h culture. The assay for OXT was kindly performed in Dr. Joanne Fortune's laboratory, and the average concentration of OXT in luteal cell-conditioned medium was 1.75 ng/ml. Cocultures of luteal cells and T cells (TC-LC) were also carried out in the presence or absence of 50 µg/ml of aminoglutethimide (AG; Sigma-Aldrich), an inhibitor of the cytochrome P450 side-chain cleavage enzyme. This concentration of AG has been previously shown to inhibit endogenous P4 synthesis by cultured bovine luteal cells [32]. Additionally, exogenous P4 was added to the coculture in the presence of AG. All TC-LC cocultures were performed in a humidified atmosphere of 5% CO2 in air at 37°C. Total RNA was extracted from the T cells after 72 h of coculture to quantify OXTR mRNA by RT-qPCR. In a separate experiment, T cells were isolated from luteal tissues following the same procedure described above. These T cells, referred to as resident T cells, were collected for RNA extraction and for detection of OXTR mRNA.

Total RNA Extraction and RT-qPCR

Total RNA was extracted from freshly purified PBMC, T cells, and cultured cells using TRIzol reagent (Life Technologies; Invitrogen). The concentrations of total RNA were determined by measurement of optical density at 260 nm in the UV/Vis spectrophotometer (Beckmann). Total RNA was treated with RNase-free DNase I (Roche Molecular Biochemicals) to eliminate genomic DNA contamination. RT-qPCR was used to detect OXTR mRNA in total RNA from fresh PBMC, fresh T cells, cultured T cells treated with steroids (P4, E2), and T cells cocultured with luteal cells followed by treatment with P4, OXT, and anti-OXT. Oligonucleotide primers specific for OXTR transcript (forward: 5'-ATCCGCACGGTCAAGATG-3'; reverse: 5'-AGAGGAAGCGCTGCACAA-3') were designed to amplify a 226-bp OXTR cDNA fragment. These primers were designed from the bovine OXTR mRNA sequence [14] (GenBank accession number AF101724). Total RNA from bovine uterus was used as positive control in the present study. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment was amplified as a constitutively expressed gene with the following primers (forward: 5'-AAGATTGTCAGCAATGCC-3'; reverse: 5'-ACAGACACGTTGGGAG-3'). Total RNA (2 µg) was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories) according to the protocol of the manufacturer. Following the reverse transcription reaction, quantitative PCR was performed on the MJ Research Opticon 2 (Bio-Rad Laboratories) using the iQSYBR Green Supermix (Bio-Rad Laboratories) for a total of 35 cycles under the following conditions: denaturing, 94°C for 30 sec; annealing, 56°C for 45 sec; and extension, 72°C for 60 sec, followed by an extra elongation of 5 min at 72°C. Homologous standard curve prepared from purified OXTR cDNA PCR product was used to calculate the steady-state concentration of OXTR mRNA in triplicate wells for each sample. The PCR amplification products were electrophoretically separated on 1.5% agarose gels and visualized with ethidium bromide. For initial validation, the specific band corresponding to the size of the expected OXTR cDNA fragment was cut and purified using the QIAquick Gel Extraction Kit (Qiagen Sciences) for sequence confirmation. A control sample that was not reverse transcribed was used to confirm that the product obtained was not amplified from genomic DNA.

Protein Extraction and Immunoblotting

Fresh PBMC were isolated on Days 3, 11, and 19 of the estrous cycle from four different cows at each time. The proteins were extracted from lymphocytes using the CelLytic MT Cell Lysis Reagent (Sigma-Aldrich) in the presence of the protease inhibitor cocktail (Sigma-Aldrich) following the manufacturer's protocol and quantified according to the method of Bradford [33] (Bio-Rad Protein Assay; Bio-Rad Laboratories). Protein samples (100 µg) were subjected to electrophoresis on a 12% SDS-polyacrylamide gel and the separated proteins were blotted onto polyvinylidene difluoride membranes (Hybond-P; Amersham Pharmacia Biotech). Western analyses were performed as previously described [34] using a polyclonal rabbit anti-OXTR antibody (catalog #O4389; Sigma-Aldrich) raised against a synthetic peptide corresponding to the N-terminal extracellular domain of the OXTR. First, membranes were incubated with blocking buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 5% nonfat dry milk, and 0.05% Tween-20) for 2 h at room temperature followed by an overnight (12–16 h) incubation at 4°C with anti-OXTR antibody at a final concentration of 0.5 µg/ml. Membranes were washed twice with TBS-Tween (20 mM Tris [pH 7.4], 150 mM NaCl, 0.05% Tween-20) and incubated with the horseradish peroxidase-labeled anti-rabbit secondary antibody (Amersham Biosciences) at a dilution of 1:20 000. The antigen-antibody complex was visualized using the enhanced chemiluminescence system (ECL Western Blotting Analysis System; Amersham Biosciences) following the manufacturer's protocol. Membranes were exposed to Kodak Biomax light films (Kodak) and the films were developed in the SRX-101A Konica film processor (Konica Corporation, Japan). Membranes were also incubated with anti-OXT to determine if bovine lymphocytes express OXT along with its receptor. Beta actin was used as an internal control to verify the integrity of proteins in the samples.

Intracellular Calcium Measurements

To test OXT actions on [Ca2+]i concentrations, T lymphocytes were isolated as described above, washed with serum-free RPMI-1640, and loaded with 1 mol/L of the fluorescent Ca2+ indicator, Fura-2 AM (Molecular Probes, Eugene, OR), essentially according to the manufacturer's protocol and as described by Braileanu et al. [35]. Briefly, cells were incubated with Fura-2 at 37°C for 40 min in serum-free culture medium. The cells were washed for 30 min in calcium-free buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM Hepes, and 6 mM glucose; pH 7.4) to remove unincorporated Fura-2 prior to [Ca2+]i measurements. Fura-2-loaded cells were treated with OXT (100 ng/ml), and the measurements were performed using the BioTek FL600F Fluorescence Plate Reader (Bio-Tek Instruments, Inc.). [Ca2+]i concentrations were determined by the fluorescence measurements at 340 and 360 nm. The specificity of the response was evaluated by the treatment of cells with arginine vasopressin (Sigma-Aldrich). Results were obtained from separate experiments using T cells from four different animals. Each experiment was performed in triplicate, and the concentration in each replicate represents the average of 10 individual readings. Values were corrected for background fluorescence.

Statistical Analysis

Gene expression data were log-transformed and analyzed using covariate analysis within the mixed model of SAS (SAS Inst., Inc., Cary, NC) with GAPDH as the covariate. Data were presented as least-square means ± SEM, and differences were considered significant at P < 0.05. For calcium measurements, three-way analysis of variance was performed to determine whether differences existed between the different treatments. The Student-Newman-Keuls procedure was used to determine differences between specific means. Differences were considered significant at P < 0.05.

RESULTS

OXTR mRNA Expression in Bovine Lymphocytes

Real-time PCR was initially conducted to determine if OXTR mRNA are expressed in bovine lymphocytes. Experiments were performed on freshly collected PBMC, freshly purified T and B cells, and cultured T cells. After a maximal number of amplification cycles, OXTR cDNA was detected in all samples analyzed with a specific 226-bp amplicon (Fig. 1A). The same amplicon was observed in the uterine sample used as a positive control. Sequencing results confirmed that the observed amplicon was indeed OXTR transcript. This transcript was also detected in the resident T cells isolated from luteal tissues (Fig. 1B). Quantitative analyses showed that OXTR mRNA was greater in T cells than in PBMC (P < 0.05; Fig. 2A) or purified B cells/macrophages (P < 0.05; Fig. 2B), indicating that T cells are the main lymphocyte population that expresses OXTR mRNA. In addition, OXTR mRNA concentrations were greater in freshly isolated T cells compared with cultured T cells (P < 0.05; Fig. 2C). The expression of OXTR mRNA was also observed in peripheral blood T cells collected at three different stages of the estrous cycle. There was considerable variation among animals in expression of OXTR mRNA, such that there was no significant difference in OXTR mRNA concentrations between the early (Day 3), mid (Day 11), and late (Day 19) stages of the estrous cycle (Fig. 3). However, OXTR mRNA expression appeared numerically lower in cells collected on Day 19 of the estrous cycle compared to Day 3 (P = 0.091). Additionally, OXTR transcripts were found in each of the major T cell subsets, including CD4+, CD8+, and {gamma}{delta}+ T cells, with no significant differences in the steady-state concentrations of OXTR mRNA among T cell subsets (Fig. 4).


Figure 01
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  		FIG. 1. Detection of OXTR cDNA in bovine lymphocytes by PCR. A) Total RNA was extracted from fresh PBMC, fresh T and B lymphocytes, and cultured T cells (TC). B) Total RNA was extracted from PBMC, peripheral blood T cells (P-TC), and resident T cells (R-TC; T cells isolated from within luteal tissues). Uterus was used as a positive control sample. Specific amplification of the OXTR cDNA was observed in all samples. The bands represent PCR product obtained after 35 cycles.


Figure 02
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  		FIG. 2. Analysis of OXTR mRNA expression in bovine peripheral white blood cells. OXTR mRNA expression in fresh T cells was compared to fresh PBMC (A; n = 6 cows), B cells/macrophages (B; n = 4), and cultured T cells (C; n = 4). Data are presented as least-square means ± SEM, and different letters denote means that differed significantly (P < 0.05).


Figure 03
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  		FIG. 3. OXTR mRNA expression throughout the estrous cycle. Expression of OXTR mRNA was analyzed in freshly purified T cells from Day 3, Day 11, and Day 19 of the estrous cycle. Data are presented as least-square means ± SEM (n = 4 cows).


Figure 04
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  		FIG. 4. OXTR mRNA expression in different T cell subsets. Different T cell subsets were isolated using specific antibodies and analyzed for OXTR mRNA expression by RT-qPCR. Data are presented as least-square means ± SEM (n = 6 cows).

Western Blot Analyses

To investigate whether OXTR protein expression paralleled that of the mRNA, Western blot analyses were performed on lymphocyte protein samples from D3, D11, and D19 of the estrous cycle. A specific band of about 45 kDa corresponding to the bovine OXTR protein was observed in all protein extracts from lymphocytes collected at different times of the estrous cycle (Fig. 5A). Proteins extracted from the uterus were used as the positive control, and a single protein was recognized by the anti-OXTR antibody. Interestingly, one more band at about 31 kDa appeared in all lymphocyte samples (Fig. 5A). Both the 45-kDa and 31-kDa proteins appeared similar for the cells collected at each sampling time. Western blot analyses performed on the same samples also showed the presence of OXT in the lymphocytes at about 14 kDa, consistent with the size of OXT-neurophysin 1, the precursor protein to OXT (Fig. 5B). The presence of this precursor indicates production of OXT by bovine lymphocytes. The same size protein was observed in a pituitary sample used as a positive control for OXT. Beta actin protein used as an internal control was recognized in all samples by the monoclonal anti-beta actin antibody, and no changes were observed throughout the estrous cycle.


Figure 05
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  		FIG. 5. Representative Western blot analyses using antibodies against OXTR and OXT for protein expression in bovine lymphocytes. OXTR (A) and OXT-associated neurophysin 1 (B) proteins were observed in bovine lymphocytes throughout the estrous cycle, with corresponding β actin blots. The experiment was repeated using cells from four animals with similar results.

OXTR mRNA Regulation by Steroids

In order to better understand the regulation of OXTR mRNA expression in T cells, the effects of P4 and E2 were analyzed. T cells were cultured in the presence or absence of Con A and treated with P4 and E2 separately and in combination. E2 significantly increased OXTR mRNA expression (P < 0.05; Fig. 6, left panel). The effect of P4-only treatment on the steady-state concentrations of OXTR mRNA was not significant compared with the control cells (P = 0.092; Fig. 6, left panel), but P4 completely inhibited E2-stimulated OXTR mRNA expression. Surprisingly, the stimulatory effect of E2 on OXTR mRNA expression by T cells was completely abrogated when the cells were cultured in the presence of Con A (Fig. 6, right panel). In fact, expression of OXTR mRNA in Con A-stimulated T cells treated with E2 was lower than in control cells (P < 0.05; Fig. 6, right panel), and the steady-state concentration of OXTR mRNA in P4-treated cells was intermediate between controls and E2-treated cells (P = 0.077). In Con A-treated cells, OXTR mRNA expression was not different in P4 + E2-treated cells compared to either steroid administered alone (Fig. 6, right panel).


Figure 06
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  		FIG. 6. Regulation of OXTR mRNA expression in bovine T lymphocytes by P4 (10 ng/ml) and E2 (15 pg/ml). Left panel: Cells cultured without Con A. Right panel: Cells cultured with Con A. Bars represent relative concentrations of OXTR mRNA (mean ± SEM). Different letters denote significant differences within the Con A-treated groups (P < 0.05; n = 4 cows).

TC-LC Coculture

To further analyze P4 effects on OXTR mRNA expression by T cells, coculture experiments were conducted using T cells and luteal cells from Day 11 CL. T cells that were cultured in the presence of luteal cells tended (P = 0.0516) to have lower amounts of OXTR mRNA compared to T cells cultured alone, presumably due to the P4 produced by the cultured luteal cells (Fig. 7). Treatment of the TC-LC coculture with AG, which inhibits the production of endogenous P4, caused a significant increase in the steady-state concentrations of OXTR mRNA compared to the TC-LC with no AG (P < 0.05; Fig. 7). Addition of exogenous P4 to the coculture reversed the effect, causing a decrease in OXTR mRNA expression by T cells as compared to AG-treated cells (P < 0.05). Neither OXT nor anti-OXT affected OXTR mRNA expression compared to the TC-LC control.


Figure 07
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  		FIG. 7. Effects of P4, OXT, and anti-OXT on OXTR mRNA expression in TC-LC cocultures. Bars represent relative concentrations of OXTR mRNA (mean ± SEM), and different letters denote significant differences (P < 0.05; n = 3 cows).

Calcium Measurements

To determine if the OXTR protein found in the T cells is biologically active, [Ca2+]i concentrations were measured using the Fura-2 calcium indicator. When T cells were treated with OXT, concentrations of [Ca2+]i were rapidly and dramatically increased as compared with nontreated cells (P < 0.05; Fig. 8, A and B). The specificity of the Ca2+ response to OXT was demonstrated by lack of increase in [Ca2+]i after treatment with vasopressin (Fig. 8).


Figure 08
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  		FIG. 8. Oxytocin-stimulated [Ca2+]i mobilization in Fura-2-loaded T lymphocytes. Lymphocytes were stimulated with OXT (100 ng/ml) or with vasopressin (100 ng/ml). A) Time course of Ca2+ mobilization after OXT treatment in a representative experiment. B) Peak Ca2+ concentrations in response to OXT (mean ± SEM; n = 4 separate experiments). Blank = Fura 2-nonloaded T cells; Control = Fura 2-loaded T cells without treatments.

DISCUSSION

In the present study, OXT receptor mRNA and protein expression was demonstrated in bovine PBMC and T lymphocytes. The expression of OXTR mRNA in PBMC was lower than in purified T cells, indicating that T cells are the main white blood cell components that express OXTR mRNA; therefore, purification of T cells from macrophages and B lymphocytes enriched the OXTR message relative to GAPDH. This was confirmed by the observation that the negative fraction of PBMC, containing macrophages and B lymphocytes, had very little expression of OXTR mRNA compared with specifically purified T lymphocytes. Cultured T cells exhibited lower expression of OXTR mRNA compared with freshly purified T cells, perhaps because the culture medium did not contain factors that stimulate or sustain OXTR mRNA expression in T lymphocytes. It is also possible that OXTR mRNA is downregulated during the isolation procedure, and more than 72 h in culture is required for T cells to resume expression of OXTR mRNA in amounts comparable to the fresh cells. Alternatively, autocrine factors that downregulate OXTR mRNA may be produced during the culture. All the subsets of T cells (CD4+, CD8+, and {gamma}{delta}+) expressed OXTR mRNA, suggesting an important role for the OXT/OXTR system in the response of these immune cells.

OXTR transcripts were quantified by RT-qPCR in bovine lymphocytes and appeared differentially regulated by E2 and P4 in this in vitro model. Several studies have shown that OXTR mRNA from uterus and mammary gland tissues is upregulated by E2 and downregulated by P4 in vivo and in vitro [36–40]. In the rat uterus, OXTR mRNA was shown to be upregulated when concentrations of circulating estrogen are high and P4 concentrations are low [40, 41]. Similarly, in ewes, E2 treatment induced an increase in endometrial OXTR mRNA expression [42–44], but treatment with E2 after several days of continuous P4 treatment decreased OXTR mRNA expression [45, 46]. This observation may suggest that OXTR mRNA stimulation by E2 requires the absence or the decline of P4 in the cellular environment and/or an adequate balance of E2/P4 rather than only an increase in E2 concentrations. In the present experiment, E2 significantly increased OXTR mRNA expression in T cells cultured for 72 h. This stimulatory effect of E2 on OXTR mRNA lymphocytes agrees with previous studies examining OXTR mRNA in reproductive tissues. This result suggests that E2 may have an effect on immune cell functions by regulating OXTR mRNA expression from the T cells. However, the effect of E2 was completely abrogated when the T cells were stimulated with Con A. It is not clear if this surprising observation is the result of a direct inhibition of E2 action or its receptor by Con A, or if stimulated T cells produce autocrine/paracrine factors that antagonize the response to E2.

The steroid hormone P4 was also shown to regulate OXTR mRNA expression. One of the functions of P4 is the maintenance of uterine quiescence by decreasing uterine sensitivity to OXT. Previous studies showed that P4 had a negative effect on OXTR mRNA expression in endometrial epithelial cells [47–49]. In the endometrial cells, the OXTR gene was spontaneously upregulated when the cells were explanted away from the influence of circulating P4 [49]. Another in vitro study showed a decrease in OXTR mRNA numbers when bovine endometrial epithelial cells were exposed to P4 and treated with E2, suggesting that the combination of E2 with P4 leads to a low responsiveness of the endometrium to OXT by decreasing OXTR mRNA [48]. In bovine peripheral lymphocytes, OXTR mRNA expression was highest on Day 3 of the estrous cycle, began to decline by Day 11, and was further decreased by Day 19. The decrease in lymphocyte OXTR mRNA as the estrous cycle progressed is likely due to elevated P4 at midcycle compared to Day 3. E2 concentrations would be expected to increase around Day 19 of the estrous cycle, but P4 concentrations are still elevated at that time. Although E2 stimulated OXTR mRNA expression in T cells in vitro, its action was completely inhibited by simultaneous treatment with P4. Therefore, the temporal pattern of expression of OXTR mRNA in circulating lymphocytes is consistent with the observed steroid hormone effects on cultured lymphocyte OXTR mRNA expression. The decrease of the steady-state concentration of OXTR mRNA by the combination of P4 and E2 might suggest regulation of T cell responsiveness to OXT by these two steroids. This implies that resident T cells might be relatively unresponsive to OXT when P4 synthesis is maximal, although the effect of P4 was primarily to antagonize E2 stimulation of OXTR mRNA. Since the bovine CL does not contain appreciable amounts of E2, the local effect of P4 on lymphocyte OXTR mRNA may be less pronounced. Further, during times of tissue remodeling, such as during luteinization and luteolysis, P4 concentrations are lower, perhaps allowing for increased expression of OXTR mRNA. The importance of hormonal regulation of OXTR mRNA to the function of peripheral blood and resident T cells remains to be determined.

To determine if P4 synthesis by luteal cells could affect OXTR mRNA expression by T lymphocytes, T cells were cocultured with luteal cells. In the presence of AG, an inhibitor of endogenous P4 production by luteal cells, OXTR mRNA expression was greater compared to its expression in cells cultured in the absence of AG. When exogenous P4 was added to the cocultures in the presence of AG, a decrease in OXTR mRNA expression was observed. These data suggest that P4 produced by luteal cells may downregulate T lymphocyte expression of OXTR mRNA in this coculture system. In addition to P4, it is also possible that other factors, alone or in synergy with P4, may affect TC expression of OXTR mRNA. No effect of OXT or anti-OXT was observed on OXTR mRNA expression. This may indicate that OXT does not regulate its own receptor, although suppression of OXTR mRNA expression by luteal cell-produced P4 in those cultures may have masked any effects of OXT.

It is generally held that steroid hormones E2 and P4 act at a genomic level by binding to nuclear receptors and modulating the expression of specific target genes. Though there is no E2 response element on the bovine or ovine OXTR gene promoter regions [50], it has been reported that estrogen receptor can act through other sites on gene promoters [51]. Despite the well-described effects of P4, the molecular mechanisms that mediate its actions on lymphocytes are still mostly unknown. Although P4 appears to be responsible for the downregulation of OXTR mRNA expression, this effect is most likely an indirect nongenomic effect since there is no P4 response element reported on the promoter region of the OXTR gene [50, 52], and bovine T lymphocytes do not express nuclear P4 receptors [30]. Several reports suggest that P4 may act at the cell membrane through specific receptors [53–55], supporting the notion that P4 may act through membrane receptors to regulate OXTR mRNA expression by lymphocytes.

The OXTR protein was observed in lymphocyte samples at all stages of the estrous cycle. A specific band was recognized both in the lymphocytes and the uterus samples. The deduced amino acids from bovine OXTR protein [14] predicted a putative molecular size of 43.3 kDa, which is similar to the size observed in the present study at about 45 kDa. An additional protein reacting with the anti-OXTR antibody was observed at about 31 kDa in lymphocytes, but not in the uterus. It is not clear if the two bands observed in the lymphocytes represent two different, unrelated receptors from different splicing events of the OXTR gene, or if different posttranslational modifications occur as suggested by Kojro et al. [56]. Interestingly, in the pregnant rat uterus, a single OXTR band at 66 kDa was observed, whereas in the non-pregnant rat a second band of 30 kDa was also described [39]. The nature of the 30-kDa form was not identified, but could indicate possible modifications of the OXTR protein dependent on the physiological status of the tissue or the animal. The larger molecular mass of OXTR protein is likely the functional protein in correlation with what has been previously reported in the rabbit myometrium and amnion [57].

Oxytocin treatment induced an increase in [Ca2+]i in T cells, suggesting a functional response mediated by OXTR protein. Functional OXTR protein in T lymphocytes makes these immune cells potential targets for OXT. In addition, the present study also showed that bovine lymphocytes express OXT, as demonstrated by Western blot analysis. The presence of OXT and its receptor in the lymphocytes implies that OXT can have not only paracrine actions but also autocrine actions that could regulate the function of the lymphocytes. In summary, the present data provide evidence that OXTR mRNA expression in bovine lymphocytes is regulated in a steroid-dependent manner. Moreover, OXT elicited a functional [Ca2+]i response in T lymphocytes, supporting the hypothesis that OXT may act as a paracrine and/or autocrine regulator of resident immune cell functions within the CL throughout the estrous cycle. It is possible that, in addition to its well-known functions on reproductive tissues, OXT may exert an alternate role by regulating and maintaining a balance of anti-inflammatory and pro-inflammatory cytokines within the CL. Further studies will determine the nature of the T cell response to OXT.

ACKNOWLEDGMENTS

The authors wish to thank Mr. Justin Fear for assistance with sample collection and T cell isolation, Dr. Joanne Fortune for the assay of oxytocin, and Dr. William Weiss for assistance with the statistical analyses.

FOOTNOTES

1Supported by National Research Initiative Competitive Grant 2004-35203-14789 from the USDA Cooperative State Research, Education, and Extension Service Animal Reproduction Program. Salaries and research support also provided by State and Federal funds appropriated. Back

Correspondence: 2Joy L. Pate, Department of Animal Sciences, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Avenue, Wooster, OH 44691. FAX: 330 263 3949; e-mail: pate.1{at}osu.edu

Received: 5 October 2007.

First decision: 24 October 2007.

Accepted: 19 December 2007.

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