Progress 08/01/04 to 01/01/08
Outputs
OUTPUTS: Terminated Position with the University, no report given. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Terminated Position with the University, no report given.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We have previously demonstrated that progesterone (P4) inhibits luteal cell-induced T lymphocyte proliferation in a dose-dependent manner, yet freshly purified lymphocytes did not express the nuclear P4 receptor. The effects of P4 on immune cell functions within the corpus luteum (CL), and the exact mechanisms by which P4 might exert such effects are not fully understood. However, recent reports described the presence of membrane receptors for P4 in different tissues. The objectives of the present study were to determine whether bovine lymphocytes express membrane progestin receptors (mP4Rs), to study the regulation of these receptors, and to analyze their expression in the different T lymphocyte subpopulations. Bovine peripheral blood mononuclear cells (PBMC) were isolated from whole blood of four animals at different stages (days 3, 11, and 19) of the estrous cycle. The T cells (TC) were separated from PBMC, and frozen for RNA or cultured for 72 hours in RPMI-1640
containing 10 % heat-inactivated fetal bovine serum, and treated with progesterone and/or 17beta-estradiol (E2). Total RNA was extracted from fresh TC along with cultured TC, and examined for the three distinct forms of mP4R and for progesterone receptor membrane component 1 (PGRMC1) mRNA expression by RT-qPCR. The mRNA of mP4Ra, mP4Rb, mP4Rg, and PGRMC1 were observed in bovine T lymphocytes throughout the estrous cycle. Steady-state concentrations of mP4Ra, mP4Rb, mP4Rg, and PGRMC1 mRNA were not significantly different among D3, D11, and D19 of the estrous cycle. Nevertheless, for all mP4R subunits, expressions in T cells were lower at D19 compared with D3. Treatment with P4 or E2 did not affect mP4Rs or PGRMC1 mRNA expression although a slight decrease was observed when T lymphocytes were treated with the combination of P4 and E2. We found that all T lymphocyte subsets (CD4+, CD8+, and gd+) expressed mP4Ra, mP4Rb, mP4Rg, and PGRMC1 mRNA. No significant differences were observed for
all mP4Rs subunits and PGRMC1 transcripts between CD4+, CD8+, and gd+ T cells, whereas the steady-state concentrations of mRNA were greater in purified T cell subsets as compared with whole PBMC.
PARTICIPANTS: Joy L Pate, PD Kalidou Ndiaye, Postdoc Daniel Poole, PhD student
TARGET AUDIENCES: Scientists
PROJECT MODIFICATIONS: None
Impacts
We found that bovine lymphocytes express membrane progestin receptors during the estrous cycle with a down-regulation at the end of the cycle. These results suggest that P4 could modulate the activity and function of T lymphocytes through membrane receptors. Therefore, P4 from the CL might exert immunosuppressive actions preventing the activation and proliferation of T lymphocytes in a fully functional CL, thus allowing different reproductive events to successfully take place.
Publications
- Davis, TL and Pate, JL. 2007. Proliferation of major histocompatibility nonrestricted gamma delta T cells is stimulated by bovine luteal cells. Biol. Reprod. 77,914-922
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Progress 01/01/06 to 12/31/06
Outputs
Oxytocin is a posterior pituitary hormone whose primary action is to stimulate uterine contraction or milk ejection via oxytocin receptors (OTR). The corpus luteum (CL) also produces oxytocin which is involved in the regulation of pulsatile release of prostaglandin F2α during luteolysis. Another role of oxytocin is proposed to be immunosuppressive to prevent full activation of proinflammatory responses during early and midcycle corpus luteum development. To verify the hypothesis that oxytocin produced in the CL of the cow may serve as a paracrine factor to regulate function of resident immune cells, it was first necessary to determine if bovine immune cells express OTR. Receptors for oxytocin have been identified and characterized in some tissues involved in immune function in the rat as well as in freshly prepared lymphocytes from human peripheral blood. Here we report the expression of OTR message in bovine peripheral blood mononuclear cells (PBMCs) and T
lymphocytes (TC). In the present study, bovine PBMCs were isolated from whole blood of four different cows at different stages (day 3, 11, and 19) of the estrous cycle. TC were isolated and cultured in RPMI-1640 containing 10 % heat-inactivated fetal bovine serum, in the presence or absence of Concanavalin A (Con A, 1mg/ml) for 72 hours. Total RNA was extracted from fresh PBMCs and fresh TC along with cultured TC, and examined (2μg of total RNA) for OTR expression by real-time reverse transcription-polymerase chain reaction (RT-qPCR). The presence of OTR was observed in fresh PBMCs and TC at day 3, 11, and 19 of the estrous cycle. No difference was observed in OTR expression between total PBMCs and TC. However, OTR mRNA was greater in fresh cells compared with cultured cells with or without Con A. From these data, we conclude that OTR is expressed in bovine PBMCs and TC during the estrous cycle but OTR expression was dramatically reduced when cells were placed in culture.
Furthermore, the expression of OTR in lymphocytes suggest that oxytocin could serve as a signaling molecule between luteal cells and immune cells.
Impacts
This is the first report of oxytocin receptor expression in lymphocytes of cattle. These findings could lead to new approaches to regulation of fertility and/or disease resistance in ruminants.
Publications
- Ndiaye, K and Pate, JL. 2006. Evidence for Oxytocin Receptor Expression in Bovine Peripheral Blood Mononuclear Cells and T Lymphocytes. Biol. Reprod. Abstracts, p. 74
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Progress 01/01/05 to 12/31/05
Outputs
Osteopontin, aka Eta-1, is produced in a variety of tissues and is thought to participate in tissue remodeling events and cell-mediated immunity. The objective of this study was to examine the temporal expression of Eta-1 in the CL throughout the estrous cycle and luteolysis. CL were collected from cyclic dairy cows on days 4, 5 , 10 and 18 of the estrous cycle and at 0.5, 1, 4 and 8 hours after a luteolytic injection of PGF administered on day 10 (n= 4 cows at each time of collection). One-half of each CL was processed for immunohistochemistry and RNA was isolated from the remaining half. Steady state concentrations of specific mRNAs were quantified by RT-qPCR. Eta-1 mRNA was expressed in all luteal tissues, with lowest concentrations observed in CL from day 4 of the estrous cycle. Concentration of Eta-1 tended to increase by day 5 with a further significant elevation on day 18. Of particular interest was the finding that Eta-1 mRNA increased in concentration by 1
hour after PGF. Immunohistochemical analysis confirmed the presence of Eta-1 protein within the CL. The proinflammatory actions of Eta-1 may be partly achieved by an increase in the interleukin (IL)12 to IL10 ratio. The effects of Eta-1 on IL12 and IL10 depend on interaction with integrin and CD44, respectively. Concentrations of CD44 mRNA paralleled those of Eta-1 during the estrous cycle, with significantly increased concentrations observed in day 10 and day 18 CL compared to those in day 4 CL. Changes in integrin mRNA were less dramatic, although an increase in concentration was observed between days 5 and 10 of the estrous cycle. Although CD44 mRNA did not change during luteolysis, integrin mRNA declined by 4 hours post PGF. Concentrations of both IL12 and IL10 mRNA increased markedly between day 5 and day 10 of the estrous cycle. Although IL10:IL12 was always greater than 1.0, the ratio tended to be less when Eta-1 concentrations were high, supporting a role for Eta-1 as a
proinflammatory mediator. In conclusion, Eta-1 is produced within the CL, with increased expression observed during luteolysis. Temporal changes in CD44 and integrin, which interact directly with Eta-1, as well as alterations in IL12:IL10 mRNAs, further reflect a proinflammatory role for Eta-1 during luteolysis.
Impacts
Changes in Eta-1 in the CL could signal immune cell responses, which would regulate ovarian activity. This research could lead to new methods to enhance fertility in dairy cattle.
Publications
- Winkler, JL and Pate JL. 2005. Expression of Early T Cell Activation Factor (Eta-1), also known as osteopontin, in the bovine corpus luteum (CL). Biol. Reprod. abstracts, p. 204
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Progress 01/01/04 to 12/31/04
Outputs
Experiments have been initiated, but it is too early to have conclusive results.
Impacts
This research could lead to new methods to enhance fertility in dairy cattle.
Publications
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