Evaluating the impact of the hexosamine biosynthesis pathway and O-GlcNAcylation on glucose metabolism in bovine granulosa cells.

Granulosa cells (GCs) of ovarian follicles prefer glucose as a metabolic substrate for growth and maturation. Disruption of glucose utilization via the hexosamine biosynthesis pathway (HBP) impairs O-linked N-acetylglucosaminylation (O-GlcNAcylation) and inhibits proliferation of bovine GCs of both small (3-5 mm) and large (>8.5 mm) antral follicles. Knowing that 2-5% of all glucose in cells is utilized via the HBP, the aim of this study was to characterize glucose metabolism in bovine GCs and determine the impact of the HBP and O-GlcNAcylation on metabolic activity. The GCs were initially cultured in serum-containing medium to confluency and then sub-cultured in serum-free medium in 96 well plates (n = 10 ovary pairs). The cells were exposed to vehicle and inhibitors of the HBP and O-GlcNAcylation for 24 h. Extracellular acidification rate (ECAR; an indicator of glycolysis) and oxygen consumption rate (OCR; an indicator of oxidative phosphorylation) of the GCs were measured using a Seahorse xFe96 Analyzer, including the implementation of glycolytic and mitochondrial stress tests. GCs from small antral follicles exhibited overall greater metabolic activity than GCs from large antral follicles as evidenced by increased ECAR and OCR. Inhibition of the HBP and O-GlcNAcylation had no effect on the metabolic activity of GCs from either type of follicle. The glycolytic stress test indicated that GCs from both types of follicles possessed additional glycolytic capacity; but again, inhibition of the HBP and O-GlcNAcylation did not affect this. Interestingly, inhibition of cellular respiration by 2-Deoxy-D-glucose impaired OCR only in GCs from small antral follicles, but exposure to the mitochondrial stress test had no effect. Conversely, in GCs from large antral follicles, oxidative metabolism was impaired by the mitochondrial stress test and was accompanied by a concomitant increase in glycolytic metabolism. Immunodetection of glycolytic enzymes revealed that phosphofructokinase expression is increased in GCs of small antral follicles compared to large follicles. Inhibition of O-GlcNAcylation impaired the expression of hexokinase only in GCs of small antral follicles. Inhibition of O-GlcNAcylation also impaired the expression of phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase in GCs of both types of follicles, but had no effect on the expression of lactate dehydrogenase. The results indicate that GCs of small antral follicles possess greater aerobic glycolytic capacity than GCs from large antral follicles; but disruption of the HBP and O-GlcNAcylation has little to no impact on metabolic activity.

Stimulatory effects of TGFα in granulosa cells of bovine small antral follicles.

Intraovarian growth factors play a vital role in influencing the fate of ovarian follicles. They affect proliferation and apoptosis of granulosa cells (GC) and can influence whether small antral follicles continue their growth or undergo atresia. Transforming growth factor-alpha (TGFα), an oocyte-derived growth factor, is thought to regulate granulosa cell function; yet its investigation has been largely overshadowed by emerging interest in TGF-beta superfamily members, such as bone morphogenetic proteins (BMP) and anti-Mullerian hormone (AMH). Here, effects of TGFα on bovine GC proliferation, intracellular signaling, and cytokine-induced apoptosis were evaluated. Briefly, all small antral follicles (3-5 mm) from slaughterhouse specimens of bovine ovary pairs were aspirated and the cells were plated in T25 flasks containing DMEM/F12 medium, 10% FBS, and antibiotic-antimycotic, and incubated at 37 °C in 5% CO2 for 3 to 4 d. Once confluent, the cells were sub-cultured for experiments (in 96-, 12-, or 6-well plates) in serum-free conditions (DMEM/F12 medium with ITS). Exposure of the bGC to TGFα (10 or 100 ng/mL) for 24 h stimulated cell proliferation compared to control (P < 0.05; n = 7 ovary pairs). Proliferation was accompanied by a concomitant increase in mitogen-activated protein kinase (MAPK) signaling within 2 h of treatment, as evidenced by phosphorylated ERK1/2 expression (P < 0.05, n = 3 ovary pairs). These effects were entirely negated, however, by the MAPK inhibitor, U0126 (10uM, P < 0.05). Additionally, prior exposure of the bGC to TGFα (100 ng/mL) failed to prevent Fas Ligand (100 ng/mL)-induced apoptosis, as measured by caspase 3/7 activity (P < 0.05, n = 7 ovary pairs). Collectively, the results indicate TGFα stimulates proliferation of bGC from small antral follicles via a MAPK/ERK-mediated mechanism, but this action alone fails to prevent apoptosis, suggesting that TGFα may be incapable of promoting their persistence in follicles during the process of follicular selection/dominance.

A variety of hormones regulate ovarian function in the cow, thus influencing fertility. One such hormone, transforming growth factor-alpha, TGFα, is expressed by the oocyte (egg) of the bovine ovary; yet little other information about the actions of this molecule on ovarian cells is available. In this study, we determined that although TGFα directly stimulates growth and proliferation of cells of the bovine ovary, and does so via specific signaling mechanisms, it fails to prevent immune-mediated programmed cell death. The latter observation diminishes the importance of TGFα relative to other oocyte-derived hormones in terms of ovarian function and overall animal fertility.

History, insights, and future perspectives on studies into luteal function in cattle.

The corpus luteum (CL) forms following ovulation from the remnant of the Graafian follicle. This transient tissue produces critical hormones to maintain pregnancy, including the steroid progesterone. In cattle and other ruminants, the presence of an embryo determines if the lifespan of the CL will be prolonged to ensure successful implantation and gestation, or if the tissue will undergo destruction in the process known as luteolysis. Infertility and subfertility in dairy and beef cattle results in substantial economic loss to producers each year. In addition, this has the potential to exacerbate climate change because more animals are needed to produce high-quality protein to feed the growing world population. Successful pregnancies require coordinated regulation of uterine and ovarian function by the developing embryo. These processes are often collectively termed "maternal recognition of pregnancy." Research into the formation, function, and destruction of the bovine CL by the Northeast Multistate Project, one of the oldest continuously funded Hatch projects by the USDA, has produced a large body of evidence increasing our knowledge of the contribution of ovarian processes to fertility in ruminants. This review presents some of the seminal research into the regulation of the ruminant CL, as well as identifying mechanisms that remain to be completely validated in the bovine CL. This review also contains a broad discussion of the roles of prostaglandins, immune cells, as well as mechanisms contributing to steroidogenesis in the ruminant CL. A triadic model of luteolysis is discussed wherein the interactions among immune cells, endothelial cells, and luteal cells dictate the ability of the ruminant CL to respond to a luteolytic stimulus, along with other novel hypotheses for future research.

The corpus luteum (CL) forms on the ovary from the cellular remnants of the follicle following ovulation. The function of the CL is to produce progesterone that is required for successful pregnancy. In the absence of an embryo or sufficient embryonic signaling, the uterus will release a prostaglandin that kills the CL in a process called luteolysis. Therefore, the CL and the embryo share a symbiotic relationship, each requiring the other to be healthy and functional for survival. The Northeast Multistate Project, one of the oldest in the nation, has produced a large body of evidence that has enhanced our understanding of how the CL functions, its regulation, and the impact of ovarian activity on fertility of cattle. This review highlights some of the important advances made in the understanding of the ruminant CL.

Evidence and manipulation of O-GlcNAcylation in granulosa cells of bovine antral follicles†.

Glucose is a preferred energy substrate for metabolism by bovine granulosa cells (GCs). O-linked N-acetylglucosaminylation (O-GlcNAcylation), is a product of glucose metabolism that occurs as the hexosamine biosynthesis pathway (HBP) shunts O-GlcNAc sugars to serine and threonine residues of proteins. O-GlcNAcylation through the HBP is considered a nutrient sensing mechanism that regulates many cellular processes. Yet little is known of its importance in GCs. Here, O-GlcNAcylation in GCs and its effects on GC proliferation were determined. Bovine ovaries from a slaughterhouse, staged to the mid-to-late estrous period were used. Follicular fluid and GCs were aspirated from small (3-5 mm) and large (>10 mm) antral follicles. Freshly isolated GCs of small follicles exhibited greater expression of O-GlcNAcylation and O-GlcNAc transferase (OGT) than large follicles. Less glucose and more lactate was detectable in the follicular fluid of small versus large follicles. Culture of GCs revealed that inhibition of the HBP via the glutamine fructose-6-phosphate aminotransferase inhibitor, DON (50 µM), impaired O-GlcNAcylation and GC proliferation, regardless of follicle size. Direct inhibition of O-GlcNAcylation via the OGT inhibitor, OSMI-1 (50 µM), also prevented proliferation, but only in GCs of small follicles. Augmentation of O-GlcNAcylation via the O-GlcNAcase inhibitor, Thiamet-G (2.5 µM), had no effect on GC proliferation, regardless of follicle size. The results indicate GCs of bovine antral follicles undergo O-GlcNAcylation, and O-GlcNAcylation is associated with alterations of glucose and lactate in follicular fluid. Disruption of O-GlcNAcylation impairs GC proliferation. Thus, the HBP via O-GlcNAcylation constitutes a plausible nutrient-sensing pathway influencing bovine GC function and follicular growth.

Hyper-O-GlcNAcylation promotes epithelial-mesenchymal transition in endometrial cancer cells.

Diabetic women have a 2-3 fold increased risk of developing endometrial cancer, however, the molecular aspects of this risk are not fully understood. This study investigated the alteration of cellular O-GlcNAcylation of proteins as the potential mechanistic connection between these two conditions. The endometrial cancer cell line (Ishikawa) was utilized to study the effect of dysregulation of O-GlcNAcylation on epithelial mesenchymal transition (EMT). Hyper-O-GlcNAcylation (via 1 µM Thiamet-G/ThmG or 25 mM Glucose) enhanced the expression of EMT-associated genes (WNT5B and FOXC2), and protein expression of the EMT adhesion molecule, N-Cadherin. Reorganization of stress filaments (actin filaments), consistent with EMT, was also noted in ThmG-treated cells. Interestingly, Hypo-O-GlcNAcylation (via 50 µM OSMI-1) also upregulated WNT5B, inferring that any disruption to O-GlcNAc cycling impacts EMT. However, Hypo-O-GlcNAcylation reduced overall cellular proliferation/migration and the expression of pro-EMT genes (AHNAK, TGFB2, FGFBP1, CALD1, TFPI2). In summary, disruption of O-GlcNAc cycling (i.e., Hyper- or Hypo-O-GlcNAcylation) promoted EMT at both the molecular and cellular levels, but only Hyper-O-GlcNAcylation provoked cellular proliferation/migration, and cytoskeletal reorganization.

Changes in Myeloid Lineage Cells in the Uterus and Peripheral Blood of Dairy Heifers During Early Pregnancy.

Establishment of pregnancy requires interaction between the developing conceptus and the uterine mucosal immune system. Myeloid lineage cells (macrophages and dendritic cells) are key mediators of pregnancy in rodents and humans but relatively little is known regarding their role and distribution during early pregnancy in ruminants. We tested the hypothesis that myeloid lineage cell number, distribution, and function are altered during early pregnancy in dairy heifers. Dairy heifers were inseminated using sperm from a single bull (Day 0), and uteri and blood were collected at slaughter on Days 17 and 20 of pregnancy to investigate the response of myeloid lineage cells to the presence of a conceptus. Responses were compared to noninseminated heifers on Day 17 of the estrous cycle. Peripheral blood and uterine-derived immune cells were isolated magnetically and examined using flow cytometry. Immunohistochemical analysis was used to evaluate the spatial distribution of myeloid lineage cells in the endometrium and quantitative polymerase chain reaction was conducted to quantify abundance of mRNA transcripts associated with myeloid lineage cell function. Transcripts for major histocompatibility complex (MHC) II, cluster of differentiation (CD) 80, CD86, CD163, and indoleamine 2,3-dioxygenase (IDO) 1 were greater in endometrium of pregnant compared to cyclic heifers. Immunofluorescence analysis revealed increased labeling for MHCII and SIRPA in pregnant compared to cyclic heifers. There were approximately 50% more CD14+CD11c+ cells in the peripheral circulation of pregnant compared to cyclic heifers. A greater number of myeloid lineage cells were observed during early pregnancy, and this increase was most pronounced in and around the shallow glands. Furthermore, expression of molecules associated with a tolerogenic or alternatively activated phenotype of these cells also increased in pregnant heifers. The results support the hypothesis that myeloid lineage cells with a tolerogenic phenotype are involved in establishment of pregnancy in dairy heifers.

Molecular manipulation of keratin 8/18 intermediate filaments: modulators of FAS-mediated death signaling in human ovarian granulosa tumor cells.

BACKGROUND: Granulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence. Keratin intermediate filaments expressed in these tumors are a diagnostic marker, yet paradoxically, may also constitute a target for therapeutic intervention. In the current study, we evaluated keratin 8/18 (K8/18) filament expression as a mechanism of resistance to apoptosis in GCT, specifically focusing on regulation of the cell surface death receptor, Fas (FAS). METHODS: The GCT cell line, KGN, was transiently transfected with siRNA to KRT8 and KRT18 to reduce K8/18 filament expression. Expression of K8/18, FAS, and apoptotic proteins (PARP, cleaved PARP) were evaluated by fluorescence microscopy, flow cytometric analysis, and immunoblotting, respectively. The incidence of FAS-mediated apoptosis in KGN cells was measured by caspase 3/7 activity. All experiments were performed independently three to six times, using a fresh aliquot of KGN cells for each experiment. Quantitative data were analyzed by one- or two-way analysis of variance (ANOVA), followed by a Tukey's post-test for multiple comparisons; differences among means were considered statistically significant at P < 0.05. RESULTS: Control cultures of KGN cells exhibited abundant K8/18 filament expression (~90 % of cells), and minimal expression of FAS (<25 % of cells). These cells were resistant to FAS-activating antibody (FasAb)-induced apoptosis, as determined by detection of cleaved PARP and measurement of caspase 3/7 activity. Conversely, siRNA-mediated knock-down of K8/18 filament expression enhanced FAS expression (> 70 % of cells) and facilitated FasAb-induced apoptosis, evident by increased caspase 3/7 activity (P < 0.05). Additional experiments revealed that inhibition of protein synthesis, but not MEK1/2 or PI3K signaling, also prompted FasAb-induced apoptosis. CONCLUSIONS: The results demonstrated that K8/18 filaments provide resistance to apoptosis in GCT by impairing FAS expression. The abundance of keratin filaments in these cells and their role in apoptotic resistance provides a greater mechanistic understanding of ovarian tumorgenicity, specifically GCT, as well as a clinically-relevant target for potential therapeutic intervention.

Effect of cryotherapy on muscle recovery and inflammation following a bout of damaging exercise.

The purpose of this study was to determine the effect of cryotherapy on the inflammatory response to muscle-damaging exercise using a randomized trial. Twenty recreationally active males completed a 40-min run at a -10 % grade to induce muscle damage. Ten of the subjects were immersed in a 5 °C ice bath for 20 min and the other ten served as controls. Knee extensor peak torque, soreness rating, and thigh circumference were obtained pre- and post-run, and 1, 6, 24, 48, and 72 h post-run. Blood samples were obtained pre- and post-run, and 1, 6 and 24 h post-run for assay of plasma chemokine ligand 2 (CCL2). Peak torque decreased from 270 ± 57 Nm at baseline to 253 ± 65 Nm post-run and increased to 295 ± 68 Nm by 72 h post-run with no differences between groups (p = 0.491). Soreness rating increased from 3.6 ± 6.0 mm out of 100 mm at baseline to 47.4 ± 28.2 mm post-run and remained elevated at all time points with no differences between groups (p = 0.696). CCL2 concentrations increased from 116 ± 31 pg mL(-1) at baseline to 293 ± 109 pg mL(-1) at 6 h post-run (control) and from 100 ± 27 pg mL(-1) at baseline to 208 ± 71 pg mL(-1) at 6 h post-run (cryotherapy). The difference between groups was not significant (p = 0.116), but there was a trend for lower CCL2 in the cryotherapy group at 6 h (p = 0.102), though this measure was highly variable. In conclusion, 20 min of cryotherapy was ineffective in attenuating the strength decrement and soreness seen after muscle-damaging exercise, but may have mitigated the rise in plasma CCL2 concentration. These results do not support the use of cryotherapy during recovery.

Cytokeratin 18 expression inhibits cytokine-induced death of cervical cancer cells.

OBJECTIVES: In cervical cancer, increased cytokeratin 18 (CK18) filament expression is associated with disease progression. However, it may also provide resistance to cytokine-induced apoptosis. The present study tested whether CK18 expression influences susceptibility to cytokine-induced apoptosis. METHODS: The cervical cancer cell lines C-4II (high CK18 expression), ME-180 (low CK18 expression), and 2 subtypes of HeLa cells containing or lacking CK18 expression (CK18+ and CK18- cells, respectively) were exposed to vehicle (control), Fas ligand (FasL) (50 ng/mL), or tumor necrosis factor α (TNF-α; 10 ng/mL) without/with cycloheximide (CHX; 2.5 µg/mL) to test the hypothesis that diminished CK18 expression increases susceptibility to cytokine-induced apoptosis. RESULTS: Flow cytometric analysis of cell death via TUNEL staining revealed that cytokine-induced apoptosis was 2-fold greater in ME-180 cells than C-4II cells in response to FasL+CHX or TNF-α+CHX (P < 0.05). Similarly, there was a higher incidence of FasL-induced apoptosis in CK18- HeLa cells (23% and 91% apoptotic for FasL and FasL+CHX, respectively) than CK18+ HeLa cells (1% and 11%, respectively; P < 0.05). Surprisingly, TNF-α had no effect on either CK18+ or CK18- HeLa cells (P > 0.05). Caspase 3 activity was greater in CK18- HeLa cells than in CK18+ HeLa cells at 8 and 18 hours after FasL treatment (P < 0.05), an effect abrogated by the caspase 8 inhibitor IETD-fmk (P < 0.05). CONCLUSIONS: Cervical cancer cells with diminished CK18 expression are more susceptible to cytokine-induced apoptosis, particularly in response to FasL treatment. These observations suggest that relative CK18 expression is an important factor when considering therapeutic strategies to enhance immune cell-mediated death of cervical cancer cells.

Expression and distribution of cytokeratin 8/18 intermediate filaments in bovine antral follicles and corpus luteum: an intrinsic mechanism of resistance to apoptosis?

Apoptosis is a mechanism of cell elimination during follicular atresia and luteal regression. Recent evidence suggests sensitivity to apoptosis in some cell types is partly dependent upon cytokeratin-containing intermediate filaments. Specifically, cytokeratin 8/18 (CK8/18) filaments are thought to impart resistance to apoptosis. Here, cytokeratin filament expression within bovine ovarian follicles and corpora lutea (CL) was characterized and the potential relationship between cell-specific CK8/18 expression and apoptosis explored. Immunoprecipitation and western blot analysis confirmed CK8 associates with CK18 to form CK8/18 heterodimeric filaments within bovine ovarian cells. Immunostaining revealed populations of CK18-positive (CK18+) cells in healthy growing follicles that increased in postovulatory follicles. Atretic follicles at all stages of atresia also contained some CK18+ cells. However, no CK18+ cells were detected in primordial or primary follicles. In CL, developing CL contained a higher proportion of CK18+ cells (approximately 35%, range 30-70%) than mature CL (approximately 16%) and regressing CL (approximately 5%; P<0.05, n = 3-5 CL/stage), suggesting CK8/18 filament expression diminishes over time, as luteal cells become more susceptible to apoptosis. Dual-fluorescence labeling for CK18 and a cell death marker (TUNEL labeling) confirmed this view, demonstrating less death of CK18+ than CK18- luteal cells throughout the estrous cycle (P<0.05). The results indicate differential expression of CK8/18 filaments occurs in cells of bovine ovarian follicles and CL throughout the estrous cycle. The prevalence and cell-specific pattern of cytokeratin expression in these structures is consistent with the concept these filaments might impart resistance to apoptosis in ovarian cells as is seen in other cell types.

Immune cell-endothelial cell interactions in the bovine corpus luteum.

Early embryonic mortality accounts for a substantial portion of reproductive failure in agriculturally important livestock, including the dairy cow. The maintenance of early pregnancy requires a fully functional corpus luteum (CL) that is not susceptible to regression following fertilization, yet the cellular mechanisms of luteal regression are not clearly understood. Immune-cell accumulation within the CL at the time of regression is a well-documented phenomenon in a variety of species. In the dairy cow, immune-cell accumulation precedes luteal regression by several days and coincides with an increase in expression of the chemokine monocyte chemoattractant protein 1 (CCL2), suggesting that immune-mediated events promote tissue destruction. Recent studies indicate that endothelial cells comprising the CL are a primary source of CCL2 secretion. Moreover, although uterine-derived prostaglandin F(2α) (PGF) initiates luteal regression in the cow, PGF does not directly provoke CCL2 secretion by luteal endothelial cells. Instead, PGF-induced luteal regression is thought to require cooperative interaction among immune cells, endothelial cells, and steroidogenic cells of the CL to further promote CCL2 secretion, enhance immune-cell recruitment, and eliminate luteal tissue. This brief review focuses on putative interactions between immune cells and endothelial cells derived from the bovine CL that result in enhanced CCL2 expression and the elaboration of other inflammatory mediators (for example, cytokines), which perpetuate luteal regression. Fundamental knowledge of immune-endocrine interactions within the reproductive system of cows has relevance to other CL-bearing mammals, including humans and endangered animals, particularly in the development of methods to control and/or improve fertility. Thus, it is a timely topic for this symposium concerning ecological immunology and public health.

Cooperative expression of monocyte chemoattractant protein 1 within the bovine corpus luteum: evidence of immune cell-endothelial cell interactions in a coculture system.

Endothelial cells (EC) of the bovine corpus luteum (CL) are a known source of proinflammatory mediators, including monocyte chemoattractant protein 1 (CCL2) and endothelin 1 (EDN1). Here, a coculture system was devised to determine if immune cells and PGF 2alpha together affect CCL2 and EDN1 secretion by EC. Luteal EC were cultured either alone or together with peripheral blood mononuclear cells (PBMC), and treated without or with PGF 2alpha for 48 h (n = 6 experiments). Coculture of EC with PBMC increased CCL2 secretion an average of 5-fold higher compared with either cell type alone (P < 0.05). Basal secretion of EDN1 by EC was substantial (approximately 2 ng/ml), but was not affected by coculture with PBMC (P > 0.05). EC cocultured with concanavalin A-activated PBMC (ActPBMC) increased CCL2 secretion an average of 12-fold higher compared with controls (P < 0.05), but again, EDN1 secretion was unchanged (P > 0.05). Interestingly, PGF 2alpha did not alter either CCL2 or EDN1 secretion, regardless of culture conditions (P > 0.05). In a second series of experiments (n = 3 experiments), mixed luteal cells (MLC) were cultured alone or with PBMC as described above. Secretion of CCL2 and EDN1 was not affected by coculture or by PGF 2alpha (P > 0.05), but MLC produced less progesterone in the presence of ActPBMC (P < 0.05). Collectively, these results suggest that immune cells and EC can interact cooperatively to increase CCL2 secretion in the CL, but this interaction does not affect EDN1 secretion nor is it influenced by PGF 2alpha. Additionally, activated immune cells appear to produce a factor that impairs progesterone production by luteal steroidogenic cells.

Chemokines in the corpus luteum: implications of leukocyte chemotaxis.

Chemokines are small molecular weight peptides responsible for adhesion, activation, and recruitment of leukocytes into tissues. Leukocytes are thought to influence follicular atresia, ovulation, and luteal function. Many studies in recent years have focused attention on the characterization of leukocyte populations within the ovary, the importance of leukocyte-ovarian cell interactions, and more recently, the mechanisms of ovarian leukocyte recruitment. Information about the role of chemokines and leukocyte trafficking (chemotaxis) during ovarian function is important to understanding paracrine-autocrine relationships shared between reproductive and immune systems. Recent advances regarding chemokine expression and leukocyte accumulation within the ovulatory follicle and the corpus luteum are the subject of this mini-review.

Secretion of monocyte chemoattractant protein-1 by endothelial cells of the bovine corpus luteum: regulation by cytokines but not prostaglandin F2alpha.

Information regarding the regulation of monocyte chemoattractant protein-1 (MCP-1) in regression of the corpus luteum (CL) is limited. This study tested the hypothesis that endothelial cells derived from bovine CL are a source of MCP-1, and that proinflammatory cytokines, prostaglandin F2alpha (PGF2alpha), and progesterone regulate MCP-1 expression. Endothelial cells were treated without (Control) or with PGF2alpha (1 micro M), TNFalpha (100 ng/ml), interferon-gamma (IFNgamma, 200 IU/ml), and TNFalpha + IFNgamma for 24 and 48 h in the absence or presence of progesterone (P4, 250 ng/ml). Increases in MCP-1 mRNA and protein were observed in response to TNFalpha within 24 and 48 h of culture, respectively (P < 0.05). Interferon-gamma stimulated (P < 0.05) both MCP-1 mRNA and protein after 24 h of culture, and this effect was also sustained through 48 h of culture (P < 0.05). Cotreatment of cultures with TNFalpha + IFNgamma lead to further increases (P < 0.05) in MCP-1 in both 24- and 48-h cultures. Surprisingly, neither PGF2alpha nor P4 affected MCP-1 production. Subsequent experiments revealed that the endothelial cells lacked prostaglandin F2alpha receptor mRNA, and the MAPK pathway, although present and responsive to growth factor stimulation, was unresponsive to PGF2alpha stimulation. In summary, endothelial cells derived from bovine CL respond to TNFalpha and IFNgamma stimulation with an increase in MCP-1 secretion. In contrast, neither PGF2alpha nor P4 directly influenced endothelial expression of MCP-1. These results suggest that cytokines stimulate the synthesis of MCP-1 observed during PGF2alpha-induced luteal regression.

Expression of monocyte chemoattractant protein-1 and distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle.

This study characterizes the expression of monocyte chemoattractant protein-1 (MCP-1) and the relative distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle. Immunodetectable MCP-1 was evident in corpora lutea of cows at Days 6, 12, and 18 postovulation (Day 0 = ovulation, n = 4 cows/stage). Day 6 corpora lutea contained minimal MCP-1 that was confined primarily to blood vessels. In contrast, relatively intense staining for MCP-1 was observed in corpora lutea from Days 12 and 18 postovulation. MCP-1 was again most evident in the cells of the vasculature, but it was also observed surrounding individual luteal cells, particularly by Day 18. An increase in immunohistochemical expression of MCP-1 on Days 12 and 18 postovulation corresponded with increases in MCP-1 mRNA and protein in corpora lutea as determined by Northern blot analysis and ELISA. Monocytes and macrophages were the most abundant immune cells detected in the bovine corpus luteum, followed by CD8+ and CD4+ T lymphocytes. In all instances, Day 6 corpora lutea contained fewer immune cells than corpora lutea from Days 12 and 18. In conclusion, increased expression of MCP-1 was accompanied by the accumulation of immune cells in the corpora lutea of cows during the latter half of the estrous cycle (Days 12-18 postovulation). These results support the hypothesis that MCP-1 promotes immune cell recruitment into the corpus luteum to facilitate luteal regression. These results also raise a provocative issue, however, concerning the recruitment of immune cells several days in advance of the onset of luteal regression.