Mid-cycle Corpus Luteum Research Articles

12 Impact of progesterone signaling on tumor necrosis factor-alpha synthesis by luteal macrophages

Abstract Understanding the mechanism in which luteolysis occurs is an important step in improving the reproduction of livestock in the industry. The corpus luteum (CL) is a transient endocrine organ found on the ovary responsible for progesterone secretion. It is well known that prostaglandin F2-alpha (PGF) is responsible for luteal regression, however, the process by which luteal cells (LC) become sensitive to the luteolytic effects of PGF remain unclear. Therefore, the objective of this study was to determine if suppression of progesterone (P4) synthesis or blocking P4 signaling activates tumor necrosis factor (TNF)-alpha production by the luteal macrophages. Ovaries were collected from an abattoir and transported to the laboratory on ice. Ovary status (i.e. approximate days post ovulation), was determined by phenotypic description of ovarian structures. Only ovaries (n = 5) containing midcycle (d 12/13 post ovulation) CL were used in these experiments. Luteal tissue was isolated and dissociated to collect LC and and luteal macrophages (MAC). Isolated cells were incubated independently at 37°C at 5% CO2 for 24 h in 24-well plates. Luteal cells and MACs were then harvested and placed in the following conditions: 1) LC + MAC + DMSO (control), 2) LC + MAC + trilostane (TRIL 1.0mg/mL), 3) LC + MAC + TRIL 1.5 mg/mL, 4) MACS only, 5) MACS +LPS. Supernatant was harvested at 24 h whereas both cells and supernatant were harvested after 48 h, in vitro. Supernatant was immediately frozen for subsequent P4 analysis, via RIA. Data results were analyzed using a MIXED procedure of SAS and evaluated the effects of cell-type, treatment, and culture time. Statistical significance was determined at P < 0.05 and a tendency at 0.05 < P < 0.10. Overall, TRIL treatment reduced supernatant P4 concentration (8.3 and 6.8 ng/mL to 1.0 and 1.5 mg/mL TRIL, respectively) compared with controls (22.8 ng/mL; P = 0.0012); however there was not a difference when comparing the TRIL treated cultures. Progesterone concentrations tended to decrease over time (P = 0.0661). At 24 h in vitro, P4 concentrations were suppressed in 1.0 mg/mL TRIL and 1.5 mg/mL TRIL treated wells (5.3 and 4.2 ng/mL, respectively), compared with controls (16.4 ng/mL; P = 0.0014). Whereas P4 concentrations were reduced in the 1.5 mg/mL TRIL treated wells (1.6 ng/mL) compared with controls (9.95 ng/mL; P = 0.0419) and tended to differ in the1.0 mg/mL TRIL treated wells (2.4 ng/mL; P = 0.0634), at 48 h in vitro. Progesterone concentrations were undetectable in wells containing MAC only. Collectively, TRIL treatment effectively reduced P4 synthesis in LC cultures. Therefore, the greater concentration of TRIL is needed for P4 suppression in longer term culture systems. Additional analyses will be conducted to determine the effect of P4 suppression on macrophage function.

APN Expression in Serum and Corpus Luteum: Regulation of Luteal Steroidogenesis Is Possibly Dependent on the AdipoR2/AMPK Pathway in Goats

Adiponectin (APN) is an essential adipokine for a variety of reproductive processes. To investigate the role of APN in goat corpora lutea (CLs), CLs and sera from different luteal phases were collected for analysis. The results showed that the APN structure and content had no significant divergence in different luteal phases both in CLs and sera; however, high molecular weight APN was dominant in serum, while low molecular weight APN was more present in CLs. The luteal expression of both AdipoR1/2 and T-cadherin (T-Ca) increased on D11 and 17. APN and its receptors (AdipoR1/2 and T-Ca) were mainly expressed in goat luteal steroidogenic cells. The steroidogenesis and APN structure in pregnant CLs had a similar model as in the mid-cycle CLs. To further explore the effects and mechanisms of APN in CLs, steroidogenic cells from pregnant CLs were isolated to detect the AMPK-mediated pathway by the activation of APN (AdipoRon) and knockdown of APN receptors. The results revealed that P-AMPK in goat luteal cells increased after incubation with APN (1 μg/mL) or AdipoRon (25 μM) for 1 h, and progesterone (P4) and steroidogenic proteins levels (STAR/CYP11A1/HSD3B) decreased after 24 h. APN did not affect the steroidogenic protein expression when cells were pretreated with Compound C or SiAMPK. APN increased P-AMPK and reduced the CYP11A1 expression and P4 levels when cells were pretreated with SiAdipoR1 or SiT-Ca, while APN failed to affect P-AMPK, the CYP11A1 expression or the P4 levels when pretreated with SiAdipoR2. Therefore, the different structural forms of APN in CLs and sera may possess distinct functions; APN might regulate luteal steroidogenesis through AdipoR2 which is most likely dependent on AMPK.

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries.

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 μg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 μg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.

Effects of mancozeb, metalaxyl and tebuconazole on steroid production by bovine luteal cells in vitro

Mancozeb, metalaxyl and tebucanazole are widely used pesticides in agriculture and industry to treat plant pathogenic fungi. Livestock may be exposed to such substances by consuming contaminated plants. The present study was designed to evaluate the effects of these three fungicides on bovine luteal cell steroidogenesis. Luteal slices from mid-cycle corpus luteum were dissociated into single cell suspension in aerated (O2) culture media (DMEM/F12) by enzymatic digestion. The cells were incubated in newborn calf serum (10%) for 18 h and then with serum-free media containing mancozeb (0.01 μM, 0.1 μM, 1 μM), tebuconazole (1 μM, 10 μM, 100 μM) or metalaxyl (100 μM, 500 μM, 2500 μM) for additional 96 h. The medium was replaced on day 1 and 3; and the retrieved medium was stored at −20 °C until progesterone assay. Treatment of cells with three different fungicides induced dose dependent variable decrease in steroid synthesis during incubation periods. Incubation of cells with 1 μM mancozeb exhibited a 33% decline in steroid synthesis on day 3 and 48% decline on day 5 compared with controls. Treatment of cells with 100 μM tebuconazole and 500 μM metalaxyl resulted in a 65% and 31% decrease, respectively, in progesterone accumulation on day 5 of incubation. Fungicide induced suppressive effects on luteal steroidogenesis were as metalaxyl < tebuconazole < mancozeb. Results of the present study suggest that designated concentrations of all three fungicides studied might have varying degrees of adverse effects on luteal steroidogenesis.

Fibroblast growth factor-2 and transforming growth factor-beta1 oppositely regulate miR-221 that targets thrombospondin-1 in bovine luteal endothelial cells.

Thrombospondin-1 (THBS1) affects corpus luteum (CL) regression. Highly induced during luteolysis, it acts as a natural anti-angiogenic, proapoptotic compound. THBS1 expression is regulated in bovine luteal endothelial cells (LECs) by fibroblast growth factor-2 (FGF2) and transforming growth factor-beta1 (TGFB1) acting in an opposite manner. Here we sought to identify specific microRNAs (miRNAs) targeting THBS1 and investigate their possible involvement in FGF2 and TGFB1-mediated THBS1 expression. Several miRNAs predicted to target THBS1 mRNA (miR-1, miR-18a, miR-144, miR-194, and miR-221) were experimentally tested. Of these, miR-221 was shown to efficiently target THBS1 expression and function in LECs. We found that this miRNA is highly expressed in luteal cells and in mid-cycle CL. Consistent with the inhibition of THBS1 function, miR-221 also reduced Serpin Family E Member 1 [SERPINE1] in LECs and promoted angiogenic characteristics of LECs. Plasminogen activator inhibitor-1 (PAI-1), the gene product of SERPINE1, inhibited cell adhesion, suggesting that PAI-1, like THBS1, has anti-angiogenic properties. Importantly, FGF2, which negatively regulates THBS1, elevates miR-221. Conversely, TGFB1 that stimulates THBS1, significantly reduces miR-221. Furthermore, FGF2 enhances the suppression of THBS1 caused by miR-221 mimic, and prevents the increase in THBS1 induced by miR-221 inhibitor. In contrast, TGFB1 reverses the inhibitory effect of miR-221 mimic on THBS1, and enhances the upregulation of THBS1 induced by miR-221 inhibitor. These data support the contention that FGF2 and TGFB1 modulate THBS1 via miR-221. These in vitro data propose that dynamic regulation of miR-221 throughout the cycle, affecting THBS1 and SERPINE1, can modulate vascular function in the CL.

Early transcriptome responses of the bovine midcycle corpus luteum to prostaglandin F2α includes cytokine signaling

In ruminants, prostaglandin F2alpha (PGF2α)-mediated luteolysis is essential prior to estrous cycle resumption, and is a target for improving fertility. To deduce early PGF2α-provoked changes in the corpus luteum a short time-course (0.5–4 h) was performed on cows at midcycle. A microarray-determined transcriptome was established and examined by bioinformatic pathway analysis. Classic PGF2α effects were evident by changes in early response genes (FOS, JUN, ATF3) and prediction of active pathways (PKC, MAPK). Several cytokine transcripts were elevated and NF-κB and STAT activation were predicted by pathway analysis. Self-organizing map analysis grouped differentially expressed transcripts into ten mRNA expression patterns indicative of temporal signaling cascades. Comparison with two analogous datasets revealed a conserved group of 124 transcripts similarly altered by PGF2α treatment, which both, directly and indirectly, indicated cytokine activation. Elevated levels of cytokine transcripts after PGF2α and predicted activation of cytokine pathways implicate inflammatory reactions early in PGF2α-mediated luteolysis.

Lipopolysaccharide enhances apoptosis of corpus luteum in isolated perfused bovine ovaries in vitro.

Lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, has detrimental effects on the structure and function of bovine corpus luteum (CL) in vivo. The objective was to investigate whether these effects were mediated directly by LPS or via LPS-induced release of PGF2α. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and isolated perfused for 240 min. After 60 min of equilibration, LPS (0.5 μg/ml) was added to the medium of five ovaries, whereas an additional six ovaries were not treated with LPS (control). After 210 min of perfusion, all ovaries were treated with 500 iu of hCG. In the effluent perfusate, concentrations of progesterone (P4) and PGF2α were measured every 10 and 30 min, respectively. Punch biopsies of the CL were collected every 60 min and used for RT-qPCR to evaluate mRNA expression of receptors for LPS (TLR2, -4) and LH (LHCGR); the cytokine TNFA; steroidogenic (STAR, HSD3B), angiogenic (VEGFA121, FGF2), and vasoactive (EDN1) factors; and factors of prostaglandin synthesis (PGES, PGFS, PTGFR) and apoptosis (CASP3, -8, -9). Treatment with LPS abolished the hCG-induced increase in P4 (P≤0.05); however, there was a tendency (P=0.10) for increased release of PGF2α at 70 min after LPS challenge. Furthermore, mRNA abundance of TLR2, TNFA, CASP3, CASP8, PGES, PGFS, and VEGFA121 increased (P≤0.05) after LPS treatment, whereas all other factors remained unchanged (P>0.05). In conclusion, reduced P4 responsiveness to hCG in LPS-treated ovaries in vitro was not due to reduced steroidogenesis, but was attributed to enhanced apoptosis. However, an impact of luteal PGF2α could not be excluded.

Influence of controlled breeding techniques on fertility and plasma progesterone, protein and cholesterol profile in true anestrus and repeat breeding buffaloes

Aim: The aim was to evaluate the estrus response, conception rate and plasma profile of progesterone, protein and cholesterol following use of different hormonal protocols in anestrus and repeat breeding buffaloes. Materials and Methods: This study was carried out on 20 true anestrus, 20 repeat breeders, and 10 normal cyclic buffaloes. Ten anestrus buffaloes each were treated with standard controlled internal drug releasing (CIDR) i/vg device and Ovsynch (GPG) protocols with fix timed artificial insemination (FTAI), and blood samples were obtained on day 0, 7, 9/10 (AI) of treatment and day 21 post-AI. Ten repeat breeding buffaloes with mature mid-cycle palpable corpus luteum (CL) were treated with i/m injection of 25 mg prostaglandin F2α (PGF 2 α) with FTAI twice at 72 and 96 h later, whereas other ten repeat breeding buffaloes in standing estrus were inseminated with simultaneous i/m injection of buserelin acetate-gonadotropinreleasing hormone (GnRH) 20 μg. 10 buffaloes exhibiting first estrus within 90 days postpartum and inseminated without any treatment served as normal cyclic control. Blood samples were obtained on day of PG injection, day of AI and day 21 post-AI for estimation of plasma progesterone, protein, and cholesterol. Results: CIDR and Ovsynch protocols resulted in 100 and 80% induction of estrus with conception rates of 40 and 30% at induced estrus, respectively, in anestrus buffaloes. Mid-cycle PGF 2 α treatment resulted in 90% estrus induction and 40% conception rate at induced estrus, while Buserelin acetate-GnRH 20 μg injection at AI resulted in 30% conception rate in repeat breeders. In normal cyclic control group also, the first service conception rate was 30%. The mean plasma progesterone concentrations on day 0, 7, 9/10 (AI) of treatment and on day 21 post-AI were found to be significantly (p<0.05) different in both CIDR and Ovsynch protocols, being higher on day 7 (day of PG injection) and on day 21 post-AI than on day 0 and 9/10 (FTAI), which were near basal levels. The mean plasma progesterone level was significantly (p<0.01) higher on the day of initiation of mid-cycle PGF 2 α treatment (3.81±0.67 ng/ml) in a repeat breeding buffaloes suggesting luteal phase. The mean plasma P 4 levels on day 21 post-AI were significantly (p<0.01) higher than on the day of estrus in both repeat breeders and in normal cyclic controls. The plasma P 4 value on day 21 post-AI was significantly (p<0.01) higher in conceived than non-conceived buffaloes in all five groups. The mean plasma total cholesterol and total protein concentrations in anestrus and repeat breeding buffaloes under different treatments did not vary significantly between sampling days. However the cholesterol content was significantly (p<0.05) lower (79.96±2.17 vs. 92.27±6.04 mg/dl) and protein higher (8.14±0.73 vs. 7.69±0.59 g/dl) in conceived than non-conceived animals. In both anestrus and repeat breeding buffaloes, the values of cholesterol and protein were significantly lower than in normal cyclic control group (138.04±11.98 mg/dl and 7.82±0.11 g/dl, respectively). Conclusion: The results showed that CIDR was better than Ovsynch protocol in inducing fertile estrus in anestrus buffaloes, while mid-cycle PG treatment was superior over AI + GnRH in repeat breeders, and all four treatment protocols significantly influenced plasma P4 profile, but not the protein or cholesterol.

Luteal cells from functional and regressing bovine corpora lutea differentially alter the function of gamma delta T cells.

The luteal microenvironment is thought to direct the function of resident immune cells to facilitate either luteal function or regression. To determine if luteal cells from functional (Days 10-12) and regressing (8 h after administration of prostaglandin F2alpha) corpora lutea (CL) induce different responses in γδ T cells, luteal cells were cocultured with autologous γδ T cells isolated from peripheral blood. Proliferation, functional phenotypes, and cytokine synthesis were analyzed by flow cytometry. To determine if the luteal cells from functional CL induce hyporesponsiveness in γδ T cells, γδ(+) cells were cocultured with midcycle luteal cells and further stimulated with concanavalin A. Coculture of γδ(+) cells with midcycle luteal cells did not inhibit concanavalin A-induced proliferation. In a proliferation assay, luteal cells from midcycle CL predominantly induced proliferation of γδ(+) WC1(-) cells (P < 0.05), while luteal cells from regressing CL predominantly induced proliferation of γδ(+)WC1(+) cells (P < 0.05). Analysis of intracellular cytokines indicated that midcycle luteal cells increased the proportion of γδ(+) cells containing interleukin 10 (P < 0.05), but reduced the proportion of γδ(+) cells containing interferon gamma (IFNG; P < 0.05). There were no changes in the proportions of γδ(+) cells synthesizing interleukin 4 or tumor necrosis factor. Unexpectedly, coculture of γδ(+) cells with luteal cells from regressing CL had no effect on any of the cytokines analyzed. These data support the hypothesis that the function of resident T cells is differentially modulated depending on the status of the CL.

Accumulation of eosinophils in the infundibulum of the bovine oviduct just after ovulation.

ABSTRACTThis study was to investigate the localization and distribution of eosinophils (EOS) in the bovine oviduct throughout the estrous cycle. Histological studies revealed more abundant EOS in the infundibula of the oviducts ipsilateral to the preovulatory dominant follicle and the ovulated ovary. The number of EOS was higher in the infundibula of the oviducts ipsilateral to the ovulated ovary than those of the oviducts contralateral to the ovulated ovary. The infundibula of the oviducts ipsilateral to the preovulatory dominant follicle had higher number of EOS than those of the oviducts ipsilateral to the mid-cycle corpus luteum. The number of EOS in the isthmus, but not in the ampulla, was higher in the outer layers (tunica muscularis and tunica serosa) than in the inner layers (tunica mucosa and tunica submucosa) during the estrous cycle. Thus, the EOS number varied with the region of the bovine oviduct, with greater number in the infundibula of the oviduct ipsilateral to the ovulated ovary, suggesting the impact of ovulation.

Life or Death Decisions in the Corpus Luteum

The corpus luteum (CL) is an ephemeral endocrine organ. During its lifespan, it undergoes a period of extremely rapid growth that involves hypertrophy, proliferation and differentiation of the steroidogenic cells, as well as extensive angiogenesis. The growth phase is followed by a period in which remodelling of the tissue ceases, but it engages in unparalleled production of steroids, resulting in extraordinarily high metabolic activity within the tissue. It is during this stage that a critical juncture occurs. In the non-fertile cycle, uterine release of prostaglandin (PG)F(2α) initiates a cascade of events that result in rapid loss of steroidogenesis and destruction of the luteal tissue. Alternatively, if a viable embryo is present, signals are produced that result in rescue of the CL. This review article summarizes the major concepts related to the fate of the CL, with particular focus on recent insights into the mechanisms associated with the ability of PGF(2α) to bring about complete luteolysis. It has become clear that the achievement of luteolysis depends on repeated exposure to PGF(2α) and involves coordinated actions of heterogeneous cell types within the CL. Together, these components of the process bring about not only the loss in progesterone production, but also the rapid demise of the structure itself.

Prostaglandin F2α Induces Pro-Inflammatory T Lymphocyte Phenotypes in the Bovine Corpus Luteum.

Lymphocytes are present in the fully functional corpus luteum (CL) and have been shown to proliferate during luteal regression due to migration into, as well as proliferation within, the tissue. Previous work in our laboratory has demonstrated that functional interactions exist between peripheral T lymphocytes (PTC) and luteal cells in culture. Functionally distinct classes of lymphocytes express distinctive cell surface proteins. Thus, we hypothesize that the function, based on cell surface proteins, of resident T lymphocytes (RTC) in the CL are dependent upon luteal status, either functional or regressing. We previously demonstrated that gamma-delta positive (γδ+) lymphocytes responded to luteal cell stimulation in vitro and were capable of producing both pro-and anti-inflammatory cytokines. Based on those results, the objectives of the present study were to: 1) isolate and characterize functional classes of RTC based on cell surface proteins; 2) compare RTC phenotypes from midcycle or 8 hour post PGF2α CL to PTC phenotypes, and 3) determine if lymphocyte populations isolated from CL or peripheral blood change as a result of prostaglandin (PG) F2α injection. Peripheral and resident T lymphocytes were isolated from whole blood and from dissociated luteal cell suspensions immunomagnetically by positive selection for cell surface proteins (CD2 and γδ). This procedure has been validated in our laboratory and yielded a population of cells that were ≥95% pure T cells. Lymphocytes were labeled with specific antibodies and analyzed to evaluate functional phenotypes. These analyses revealed the presence of pro-inflammatory (CD4+,γδ+ WC1+) and anti-inflammatory (γδ+ CD8+) lymphocytes in both RTC and PTC. Analysis of RTC from midcycle CL (n=4) and 8 hr post PGF2α CL (n=5) revealed a notable decrease in γδ- CD8+ lymphocytes after a luteolytic injection of PGF2α. In contrast, after a luteolytic injection of PGF2α RTC displayed an increase in γδ- WC1+ lymphocytes (p<0.01) and a numerical increase of γδ+ WC1+ lymphocytes. Additionally, there was a small increase in CD4+ lymphocytes after a luteolytic injection of PGF2α. In peripheral blood, a greater proportion of γδ+ CD8+ lymphocytes was observed in midcycle (n=4) compared to after PGF2α injection (n=5). Moreover, an increase in γδ+ WC1+ lymphocytes was observed in PTC after a luteolytic injection of PGF2α. Overall, comparison of RTC to PTC demonstrated a smaller percentage of positively labeled γδ+ (p<0.05), CD4+ (p<0.05), and WC1+ (p<0.05) lymphocytes isolated from the CL than from peripheral blood. However, the percentage of positively labeled CD8+ lymphocytes was greater in RTC than in PTC (p<0.0001). Based on these data, it is concluded that when the CL is fully functional, such as at midcycle, the RTC are predominately anti-inflammatory, however upon the induction of luteolysis with PGF2α, the proportion of pro-inflammatory lymphocytes is increased. These are the first data collected in any species to demonstrate a shift in functional phenotypes of T lymphocyte subsets isolated from the corpus luteum. Further, differences in T lymphocyte subsets within the CL compared to those in the peripheral circulation implies tissue-specific regulation of lymphocyte trafficking and/or proliferation. This project was supported by National Research Initiative Competitive Grant no. 2004-35203-14789 from the USDA Cooperative State Research, Education, and Extension Service.

PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells

The hypotheses that PKCepsilon is necessary for: 1) PGF2alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKCepsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKCepsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.PKCepsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKCepsilon expression was ablated by 75%, the inhibitory effect of PGF2alpha on LH-stimulated P4 secretion was only 29% lower than in the LH-stimulated group. In contrast, it was reduced by 75% in the group where PKCepsilon expression had not been reduced (P < 0.05). Real time PCR analysis indicated that there were no differences in the expression of cyclooxygenase-2 (COX-2), aldoketoreductase 1B5 (AKR1B5), prostaglandin E synthase (PGES), hydroxyprostaglandin-15 dehydrogenase (PGDH) and PGE2 -9-reductase as a function of PKCepsilon down-regulation. Finally, LH stimulated secretion of P4 at each luteal stage (Day -4 and -10), and PGF2alpha inhibited this only in Day -10 cells (P < 0.05). When A23187 was used at concentrations greater than 0.1 μmol, the induced elevation in [Ca(2+)]i inhibited the effect of LH on secretion of P4 in Day -4 and -10 cells (P < 0.05, Fig. 5). The inhibitory effect of PGF2alpha on LH-stimulated P4 in Day -10 cells was reduced if an increase in [Ca(2+)]i was prevented with Bapta-AM. These results support the hypothesis that differential expression of PKCepsilon and an elevation of [Ca(2+)]i are important for acquisition of luteolytic response to PGF2alpha.

EXPRESSION OF mRNA FOR MEMBRANE PROGESTERONE RECEPTORS BY BOVINE T LYMPHOCYTES

It has been previously demonstrated in this laboratory 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 (mPRs), 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 mPR (mPR alpha, mPR beta, mPR gamma) and for progesterone receptor membrane component 1 (PGRMC1) mRNA expression by RT-qPCR. The mRNA of mPR alpha, mPR beta, mPR gamma, and PGRMC1 were observed in bovine T lymphocytes throughout the estrous cycle. Steady-state concentrations of mPR beta, mPR gamma, and PGRMC1 mRNA were not different (p>0.05) among D3, D11, and D19 of the estrous cycle. In contrast, expression of the mPR alpha subunit was significantly less on D19 TC as compared to D3 TC (p<0.05). When the mRNAs of the three receptors and PGRMC1 in lymphocytes were compared, mPR gamma mRNA was greater compared to PGRMC1 (p<0.05) while the mRNA concentrations of mPR alpha, mPR beta, and mPR gamma were all similar. Treatment with P4 or E2 did not affect mPRs 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 gamma-delta+) expressed mPR alpha, mPR beta, and mPR gamma mRNA. No significant differences were observed for all mPRs subunits and PGRMC1 transcripts between CD4+, CD8+, and gamma-delta+ T cells. However, the steady-state concentrations of mRNA were greater in purified T cell subsets as compared to PBMC. In conclusion, we found that bovine lymphocytes express membrane progestin receptors during the estrous cycle with a down-regulation in mPR alpha at the end of the cycle. These results suggest that P4 could modulate the activity and function of T lymphocytes through membrane receptors. Therefore, the high intracellular concentrations of P4 present in midcycle CL may exert immunosuppressive actions, preventing the activation and proliferation of T lymphocytes. This may explain why lymphocytes present within the fully functional CL do not elicit a proinflammatory response. This project was supported by National Research Initiative Competitive Grant no. 2004–35203–14789 from the USDA Cooperative State Research, Education, and Extension Service. (poster)

The class II major histocompatibility complex molecule BoLA-DR is expressed by endothelial cells of the bovine corpus luteum

Cells expressing class II major histocompatibility complex (MHC) molecules are found within the corpus luteum (CL) of several species. Expression and localization of class II MHC molecules in the bovine CL were examined in the present study. Immunohistochemical evaluation revealed class II MHC molecules on single cells in early CL (days 4 and 5 post-estrus). Two class II MHC-expressing cell types were observed in midcycle CL (days 10-12 post-estrus), single cells similar to those observed in the early CL, and endothelial cells. Not all endothelial cells expressed class II MHC, and further investigation revealed expression of only one type of class II MHC molecule, DR, on endothelial cells. Class II MHC was also localized to endothelial cells in late CL (day 18 post-estrus). Steroidogenic luteal cells were negative for class II MHC throughout the estrous cycle. Quantitative RT-PCR revealed higher (P < 0.05) concentrations of mRNA encoding the alpha-subunit of DR (DRA) in late CL when compared with those in the early CL. DRA mRNA abundance was also measured in cultures of mixed luteal and luteal endothelial (CLENDO) cells, in the presence or absence of tumor necrosis factor-alpha (TNF). No differences were found in the DRA mRNA concentration between mixed luteal and CLENDO cell cultures, and TNF had no effect on DRA mRNA concentration in both cell types. Expression of DR by endothelial cells of the midcycle CL may induce anergy of T lymphocytes, or stimulate them to secrete products that enhance normal luteal function.

Oxygen Concentration Is an Important Factor for Modulating Progesterone Synthesis in Bovine Corpus Luteum

Oxygen deficiency caused by a decrease in the blood supply is known to induce various responses of cells. Because luteal blood flow has been shown to decrease during luteolysis, a low-oxygen condition seems to be an integral part of the environment during luteolysis. To determine whether a low-oxygen condition is associated with functional luteolysis, we examined the influence of reduced oxygen tension on the luteal progesterone (P4) generating system in cultured bovine midluteal cells. Luteal cells obtained from midcycle corpus luteum (d 8-12) were incubated under different O2 concentrations (20, 10, 5, 3% O2) with or without LH for 24 h. P4 production decreased with decreasing O2 concentration but was significantly stimulated by LH regardless of O2 concentration. After 8 h of culture, both basal and LH-stimulated P4 production was significantly lower under 3% O2 than under 20% O2. Low-oxygen condition also inhibited pregnenolone production. Cytochrome P450 side-chain cleavage enzyme (P450scc) mRNA expression, measured by quantitative PCR, decreased under low-oxygen condition in both non-LH-treated and LH-treated cells. Low-oxygen condition did not affect the expressions of steroidogenic acute regulatory protein mRNA or protein, whereas steroidogenic acute regulatory protein mRNA expression was stimulated by LH during 4 h of culture. Low-oxygen condition also did not affect 3 beta-hydroxysteroid dehydrogenase/Delta 5-Delta 4 isomerase mRNA expression or the activity of the enzyme in the cells, regardless of the incubation period. The overall results indicate that a low-oxygen condition decreases P4 synthesis by attenuating P450scc production and P450scc activity in bovine luteal cells and suggest that oxygen deficiency is an essential condition for the progression of luteolysis in cattle.

Endothelin-converting Enzyme-1, Abundance of Isoforms a-d and Identification of a Novel Alternatively Spliced Variant Lacking a Transmembrane Domain

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH(2) terminus but share the catalytic domain located in the COOH-terminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternatively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3', which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, non-endothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH(2)-terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.

Differential effects of lysolipids on steroid synthesis in cells expressing endogenous LPA2 receptor

Incubation of ovarian luteal cells with the bioactive lipid mediator lysophosphatidic acid (LPA) for 180 min abolishes gonadotropin-induced steroid production with no attenuation of the cyclic AMP accumulation. Treatment with the lysolipid also diminishes [14C]steroid production in cells preloaded with either [14C]cholesterol or [14C]acetate. Neither the expression of steroidogenic acute regulatory (StAR) protein nor in vitro steroid synthesis is affected in isolated mitochondrial fractions. The LPA-induced attenuation of steroid production occurs only in the mid-cycle corpus luteum and is associated with a transient endogenous expression of mRNA for the lysophosphatidic acid A2 (LPA2) receptor (with no concomitant changes in the expression of LPA1 receptor). Expression of LPA2 is accompanied by LPA-induced sphingosine-1-phosphate (S1P) production. Because luteal cells, in the presence of the sphingosine kinase inhibitor dihydrosphingosine, can overcome the inhibitory effects of LPA on steroid synthesis, we suggest the possible requirement of intracellular S1P production. Interestingly, no LPA-induced inhibition of 8Br-cAMP-stimulated progesterone synthesis can be detected in Leydig tumor cell line MA10 cells expressing only LPA2 receptor. Surprisingly, however, exogenous S1P inhibits agonist-stimulated progesterone in both cell types by inhibiting cyclic AMP accumulation, suggesting different mechanisms of action.

Temporal and spatial expression of tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and -2) in the bovine corpus luteum

The matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), may mediate the dramatic structural and functional changes in the corpus luteum (CL) over the course of its life span. In addition to regulating MMP activity, TIMPs are also involved in a variety of cellular processes, including cell proliferation and steroidogenesis. In a series of initial studies, we determined that matrix metalloproteinase inhibitory activity was present in protein extracts from early (4 days old, estrus = day 0), mid (10–12 days old) and late (16 days old) CL (n = 3 for each stage). Reverse zymography revealed four metalloproteinase inhibitory protein bands with relative molecular masses that are consistent with those reported for TIMP-1 to -4. In order to gain a better understanding of TIMPs and their role in luteal function, we further characterized this inhibitory activity with a particular focus on the temporal and spatial expression of TIMP-1 and TIMP-2 in the bovine CL. Northern blotting revealed that the TIMP-1 transcript (0.9 kb) was expressed at a higher (p < 0.05) level in early and mid cycle CL than in the late stage. In contrast, two TIMP-2 mRNA species, one major 1 kb species and one minor 3.5 kb species, were significantly (p < 0.05) increased in the mid and late cycle CL than in the early. Western blotting analyses demonstrated no differences in TIMP-1 (29 kDa) protein levels between early and mid stages, while its levels decreased (p < 0.05) from the mid to late stage CL. Conversely, TIMP-2 (22 kDa) protein was detected at a low level in the early CL, but significantly (p < 0.05) increased in the mid and late stages. Immunohistochemistry revealed that both TIMP-1 and -2 were localized to large luteal cells from all three ages of CL. TIMP-1 was also localized in capillary smooth muscle cells, while TIMP-2 was restricted to the endothelial cells in the capillary compartment. In conclusion, the different temporal expression patterns of TIMP-1 and TIMP-2 suggest that TIMP-1 may be important for luteal formation and development, while TIMP-2 may play significant roles during luteal development and maintenance. Furthermore, the distinct localization of these two inhibitors in the vascular compartment indicates that they may serve diverse physiological functions during different stages of luteal angiogenesis.

Expression and regulation of interferon gamma-inducible proteasomal subunits LMP7 and LMP10 in the bovine corpus luteum.

The proteasome is a large, polymeric protease complex responsible for intracellular protein degradation and generation of peptides that bind to class I major histocompatibility complex (MHC) molecules. Interferon gamma (INFgamma) induces expression of alternative proteasomal subunits that affect intracellular protein degradation, thereby changing the types of peptides that bind to class I MHC molecules. These alterations in class I MHC peptides can influence whether cells and tissues are tolerated by the immune system. Expression of two INFgamma-inducible proteasomal subunits, LMP7 and LMP10, in bovine luteal tissue was examined in this study. Northern analysis revealed the presence of mRNA encoding LMP7 and LMP10 in luteal tissue. Steady-state amounts of LMP7 mRNA did not change during the estrous cycle, but LMP10 mRNA was low in early corpus luteum (CL) and elevated in midcycle and late CL. Tumor necrosis factor alpha alone and in the presence of LH and/or prostaglandin F2alpha elevated steady-state amounts of LMP10 mRNA but did not affect LMP7 mRNA in cultured luteal cells. Immunohistochemistry revealed the presence of LMP10 primarily in small luteal cells. Numbers of LMP10-positive cells were lower in early CL than in midcycle and late CL. The finding that INFgamma-inducible proteasomal subunits are expressed in luteal tissue when the CL is fully functional was unexpected and suggests that proteasomes in luteal cells may generate peptides capable of stimulating a class I MHC-dependent inflammatory response.