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BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.010249
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BIOLOGY OF REPRODUCTION 68, 1447–1454 (2003)
DOI: 10.1095/biolreprod.102.010249
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Expression and Regulation of Interferon {gamma}-Inducible Proteasomal Subunits LMP7 and LMP10 in the Bovine Corpus Luteum1

Matthew J. Cannona, and Joy L. Pate2,a

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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} (INF{gamma}) 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 INF{gamma}-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 F2{alpha} 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 INF{gamma}-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.

corpus luteum, corpus luteum function, immunology, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of T lymphocytes by major histocompatibility complex (MHC) molecules on cells of the corpus luteum (CL) may serve to stimulate an immune response that facilitates luteal regression. In the cow, T lymphocytes are present in luteal tissue throughout the estrous cycle, with greater numbers present in CL collected during midcycle and near the time of luteal regression [1, 2]. Messenger RNAs encoding the cytokines interferon {gamma} (IFN{gamma}), tumor necrosis factor {alpha} (TNF{alpha}), and interleukin 1ß (IL-1ß) are present within the bovine CL [1, 3]. TNF{alpha} protein has been detected in the bovine CL immunohistochemically and in media from an in vivo luteal microdialysis system [4, 5]. T lymphocytes, activated as a result of interactions with cells expressing MHC molecules, are one possible source of these cytokines. Bovine luteal cells express class I and class II MHC molecules [6] and stimulate the proliferation of T lymphocytes in vitro [7]. Expression of class II MHC molecules in luteal tissue is upregulated near the end of the estrous cycle and in response to prostaglandin (PG) F2{alpha} but is downregulated in the presence of a developing embryo [6]. This finding suggests that MHC-mediated interactions between luteal cells and T lymphocytes may facilitate the process of luteal regression.

Of the two classes of MHC molecules, class I MHC molecules are found on nearly all nucleated cells. Class I MHC molecules are cell surface proteins that allow cells to present peptides derived from proteins synthesized within the cell to T lymphocytes. This process provides the means by which the immune system surveys somatic cells for expression of virus-derived proteins and aberrantly synthesized endogenous proteins [8]. Peptides presented to T cells via class I MHC molecules are generated intracellularly by a multicatalytic protease complex called the proteasome, which is responsible for intracellular protein degradation via the ubiquitin-dependent proteasome pathway [9, 10]. Under normal circumstances, the proteolytic core of the proteasome is composed of three types of constitutively expressed subunits [11, 12]. Under inflammatory conditions, the constitutively expressed subunits are replaced by three IFN{gamma}-inducible subunits, referred to as LMP2, LMP7, and LMP10 [13–15]. Replacement of the constitutive proteasomal subunits with IFN{gamma}-inducible subunits alters the proteolytic cleavage patterns of the proteasome [16–19] thereby altering the peptides presented to T lymphocytes by class I MHC molecules [9, 10].

Bovine luteal cells potently stimulate T lymphocyte proliferation in vitro, and they do so to a greater degree when isolated from CL induced to regress in vivo by administration of PGF2{alpha} [7]. This effect is due to changes in the luteal cells rather than T lymphocytes in response to PGF2{alpha} in vivo [20], but the mechanism responsible for enhancing the ability of luteal cells to stimulate T lymphocyte proliferation is unknown. One possibility is that proteasomal processing may be altered within luteal cells near the time of luteal regression. Changes in proteasomal processing may result in the presentation of peptides, in the context of class I MHC molecules, capable of inducing a breakdown in immune system tolerance and stimulation of an autoimmune inflammatory response during luteal regression. The experiments described herein were conducted to examine the hypothesis that expression of IFN{gamma}-inducible proteasomal subunits increases in the CL near the time of luteal regression. The objective of the present study was to examine the expression of two of the IFN{gamma}-inducible proteasomal subunits, LMP7 and LMP10, in luteal tissue throughout the estrous cycle and following induction of luteal regression with PGF2{alpha}. Previous studies have shown that TNF{alpha} regulates expression of both LMP7 and LMP10 in other cell types [21, 22]. Therefore, a second objective of the study was to determine the effects of LH and PGF2{alpha}, the primary hormonal regulators of luteal function, and the cytokine TNF{alpha}, which has been implicated as a mediator of luteal function, on expression of LMP7 and LMP10 by luteal cells in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Powdered Ham F-12 culture medium, gentamicin, fetal bovine serum, Escherichia coli DH5{alpha} chemically competent cells, and TRIzol reagent were all purchased from Life Technologies (Grand Island, NY). Recombinant murine TNF{alpha} was purchased from Life Technologies and R & D Systems (Minneapolis, MN). The National Hormone and Pituitary Program (Baltimore, MD) provided bovine LH (NIAMMD-bLH-4). Insulin-transferrin-selenium premix was obtained from BD Biosciences (Bedford, MA). BSA (fraction V), PGF2{alpha}, Hepes buffer, and Bouin fixative were purchased from Sigma Chemical Co. (St. Louis, MO). Type I collagenase was acquired from Worthington Biochemical Corp. (Freehold, NJ). SDS and 3-(N-morpholino)propanesulfonic acid (MOPS) buffer were acquired from Amresco (Solon, OH). Digoxigenin-labeled rNTP mix (10x), alkaline-phosphatase-conjugated anti-digoxigenin Fab fragments, CDP-Star chemiluminescent substrate, blocking reagent, and ampicillin were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Restriction enzymes, RNA polymerases, recombinant RNase inhibitor, Moloney murine leukemia virus reverse transcriptase and TaqBead Hot Start Polymerase were purchased from Promega (Madison, WI). Hybond N+ membranes were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Tissue culture flasks were from Corning (Acton, MA). All other chemicals were purchased from Sigma Chemical Co. or Fisher Scientific (Fairlawn, NJ).

Tissue Collection

Multiparous lactating Holstein and Jersey cows between 3 and 6 yr of age were used in the present study. Animals were housed indoors, had complete freedom of movement, and were fed a total mixed ration ad libitum. CL were removed transvaginally from regularly cycling dairy cows. Each CL was cut into four equal pieces, three of which were immediately snap frozen in liquid nitrogen, transported to the lab, and stored at -80°C until RNA extraction was performed. The fourth piece was cut into 4-mm cubes that were fixed in Bouin fixative and subsequently processed for immunohistochemistry. CL (n = 4–6 for each time point) were removed early (Day 5) in the estrous cycle, during midcycle (Days 9–12), or late (Day 18) in the estrous cycle. Luteal regression was induced by i.m. administration of 25 mg of PGF2{alpha} (Lutalyse; Upjohn Co., Kalamazoo, MI) to cows in the midluteal phase of the estrous cycle (Days 9–12 postestrus), and samples were collected 0, 0.5, 1, 4, 12, or 24 h later. For cell culture experiments, CL were removed during midcycle, immediately placed in Ham F-12 culture medium on ice, and transported to the laboratory for dissociation. Handling of animals and surgical procedures were carried out according to protocols approved by the Institutional Laboratory Animal Care and Use Committee of Ohio State University (Animal Use Protocol 99-AG002).

Luteal Cell Dissociation and Culture

Dissociation of luteal cells was carried out according to procedures described previously [23]. Following dissociation, cells were resuspended in F-12 medium containing insulin (5 µg/ml), transferrin (5 µg/ml), selenium (5 ng/ml), gentamicin (20 µg/ml), and LH (1 ng/ml). Cell viability was determined via trypan blue exclusion, and the number of viable cells was counted using a hemacytometer.

Cell culture was performed in a humidified atmosphere of 5% CO2 in air at 37°C. Dispersed luteal cells (4 x 106 cells/flask) were cultured in serum-coated 25-cm2 flasks in a total of 4 ml of culture medium. Cells were allowed to adhere overnight, medium was replaced, and cultures were treated with LH (10 ng/ml), PGF2{alpha} (10 ng/ml), or TNF{alpha} (50 ng/ml). These concentrations exert significant effects on bovine luteal cell function in vitro [23–27]. Treatments were also combined in all possible combinations to examine interactions between these regulators of luteal function. Media and treatments were replaced after 24 h. In a previous study [26], an effect of TNF{alpha} on expression of MHC molecules in cultured bovine luteal cells was observed by Day 4 of culture. Because cell surface proteins were measured in that study, whereas steady state mRNA concentrations were measured in the present study, an earlier time point was chosen for collection of samples. Total RNA was extracted from cultured cells 48 h after initiation of treatments.

Cloning of Bovine LMP7 and LMP10 Partial cDNAs

Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify partial LMP7 and LMP10 cDNA sequences from total cellular RNA extracted from bovine luteal tissue. Primers were designed based on regions of highest homology between published human and murine sequences. Forward and reverse primers for LMP7 were 5'-CTCGCCTTCAAGTTCCAGCA-3' and 5'-TGCAGCAGGTCACTGACATC-3', respectively. Amplification using these primers resulted in a single 581-base pair (bp) cDNA fragment, which was the size expected for the amplified LMP7 cDNA product. Forward and reverse primers for LMP10 were 5'-ACGCGAGCCACTAACGATTC-3' and 5'-TCCACCTCCATAGCCTGCAC-3', respectively. It was expected that these primers would yield a cDNA fragment 650 bp in length, and RT-PCR using these primers resulted in amplification of a single 650-bp LMP10 cDNA fragment. Amplification products were cloned and sequenced as described previously [28] to determine the identity of the cDNA inserts. The resulting partial bovine LMP7 cDNA sequence shares 91%, 90%, and 85% cDNA sequence identity with homologous regions of porcine, human, and murine LMP7 cDNAs, respectively, and has been assigned GenBank accession number AF214525. The partial bovine LMP10 cDNA sequence generated in this study shares 86% and 82% sequence identity with homologous regions of the human and murine LMP10 cDNA sequences, respectively, and has been assigned GenBank accession number AF473832.

RNA Isolation and Northern Analysis

Frozen tissue was homogenized in TRIzol reagent using a Polytron tissue homogenizer (Brinkman Instruments, Westbury, NY). RNA was isolated from cultured cells by adding TRIzol reagent directly to culture flasks. Following tissue homogenization or cell lysis, total cellular RNA was isolated according to the manufacturer's instructions. The final RNA precipitate was resuspended in diethyl pyrocarbonate (DEPC)-treated double-distilled water, and RNA concentration was determined spectrophotometrically.

Total cellular RNA (15 µg) was electrophoretically separated on 1.5% agarose denaturing gels containing 20 mM MOPS buffer, 5 mM sodium acetate, 1 mM EDTA, and 0.66 M formaldehyde. Following electrophoresis, gels were stained for 15 min with 1 µg/ml ethidium bromide in DEPC-treated water, destained for 1 h in DEPC-treated water, and photographed using type 55 positive/negative film (Polaroid Corp., Cambridge, MA). RNA was transferred to Hybond-N+ membranes using 10x saline-sodium citrate. Following transfer, membranes were baked at 80°C for 2 h to crosslink RNA to the membranes. Gels were restained with ethidium bromide following transfer and examined under ultraviolet light to confirm complete transfer of RNA.

Northern analysis was performed using digoxigenin-labeled riboprobes. Antisense digoxigenin-labeled riboprobes were synthesized using linearized plasmids containing the partial cDNA sequences of bovine LMP7 or LMP10. Northern analysis procedures were carried out according to specifications in the DIG Nonradioactive Nucleic Acid Labeling and Detection System manual (Roche Molecular Biochemicals). Corresponding sense riboprobes were also synthesized and were used as controls to confirm riboprobe specificity (data not shown). All prehybridization and hybridization procedures were performed at 68°C in a Hybaid Micro-4 hybridization oven (ThermoHybaid, Franklin, MA). Following hybridization and extensive washing, bound riboprobes were detected using the CDPStar chemiluminescent detection system (Roche Molecular Biochemicals). Membranes were exposed to Biomax ML film (Eastman Kodak Co., Rochester, NY) to detect chemiluminescence.

To quantify steady state concentrations of RNA for each message, densitometry was performed using a PDI 420oe scanning densitometer. The densitometric values (in arbitrary densitometric units) of each band were normalized for the densitometric values of the corresponding 18S rRNA.

Semiquantitative RT-PCR

Semiquantitative RT-PCR was performed using a GeneAmp PCR System 9700 thermal cycler (Perkin Elmer, Boston, MA). LMP7 and LMP10 primers were the same as those used to generate 581-bp and 650-bp partial bovine LMP7 and LMP10 cDNA fragments for cloning, respectively. An 854-bp glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA fragment was amplified in parallel reactions carried out in separate tubes using ovine-specific primers described previously [29] and was used as an external control. The number of cycles in which the reactions entered the logarithmic phase of amplification was determined individually for each primer set. The number of cycles to be used for each primer set was determined by performing serial reactions with a single control sample, incrementally increasing the number of cycles and determining empirically the cycle number at which amplification was in the logarithmic phase. In these experiments, G3PDH amplification was in the logarithmic phase at 25 cycles, LMP7 amplification was logarithmic at 26 cycles, and LMP10 amplification was logarithmic at 30 cycles. Reverse transcription was performed on 200 ng of total RNA, followed by 25 cycles (G3PDH), 26 cycles (LMP7), or 30 cycles (LMP10) of PCR using the following conditions: denaturation at 95°C for 30 sec; annealing at 58°C for 30 sec; extension at 72°C for 60 sec. Following amplification, PCR products were separated on 1.5% agarose gels and visualized with ethidium bromide. Densitometric values (in arbitrary densitometric units) of LMP7 or LMP10 bands were normalized to values of corresponding G3PDH bands.

Immunohistochemistry

Immunohistochemistry was performed on paraffin-embedded bovine luteal tissue according to previously described procedures [28]. LMP10 was detected in luteal tissue sections (5 µm) using rabbit polyclonal anti-human LMP10 antiserum (Afiniti Research Products Ltd., Exeter, U.K.) at a dilution of 1:2500. Nonimmune rabbit serum was used as a control to demonstrate antibody specificity, and ovarian stromal tissue devoid of LMP10 mRNA (as determined by RT-PCR; data not shown) was used as a negative control. Cells positively stained for LMP10 were quantified by two independent observers. The total number of LMP10-positive cells was counted in three random fields per slide, at 400x, using coded slides.

Statistical Analysis

A randomized complete block design was used to test the effect of time of the estrous cycle and after PGF2{alpha} administration on steady state LMP7 and LMP10 mRNA concentrations. Treatments were blocked by time and by Northern blot to account for variability due to differences in blots. The statistical model was Yij = µ + Ti + Bj + {epsilon}ij, where T = time, B = block, and {epsilon} is the interaction term. Blocks and time were considered fixed effects [30]. All statistical analyses were performed using Sigma Stat software (Jandel Corporation, San Rafael, CA). A two-way ANOVA was performed to determine whether differences existed among mean densitometric values of LMP7 and LMP10 mRNA at different stages of the estrous cycle and at time points following in vivo administration of PGF2{alpha}. In the instance of a significant F value, the Student-Newman-Keuls test was used to determine differences between specific means.

To determine whether differences existed in steady state concentrations of LMP7 or LMP10 mRNA in cultured cells, a one-way ANOVA followed by the Student-Newman-Keuls test was performed on densitometric values of LMP7 and LMP10 bands (normalized to corresponding G3PDH bands) to determine differences between specific treatment means. One-way ANOVAs and Student-Newman-Keuls procedures were also performed to determine whether differences existed between numbers of cells immunohistochemically stained for LMP10 in luteal tissue sections. All differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To determine patterns of LMP7 and LMP10 expression in the CL, steady state concentrations of mRNA were examined in luteal tissue using Northern analysis. The results of Northern analysis of LMP7 mRNA are displayed in Figure 1. A single 1.2-kilobase (kb) LMP7 transcript, similar in size to that characterized in human placental tissue [21, 31], was observed in luteal tissue RNA (Fig. 1A). No band was present in RNA extracted from ovarian stromal tissue (negative control). There were no differences in the relative steady state concentrations of LMP7 mRNA in luteal tissue collected throughout the estrous cycle or at any time following induction of luteal regression with PGF2{alpha} (P > 0.10; Fig. 1B).



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  		FIG. 1. A) Representative Northern blot of LMP7 in luteal tissue samples collected early (n = 6), during midcycle (n = 6), or late (n = 6) in the estrous cycle and at 0 (n = 6), 0.5 (n = 5), 1 (n = 5), 4 (n = 5), 12 (n = 4), or 24 (n = 5) hours after administration of a luteolytic dose of PGF2{alpha}. Ovarian stromal tissue RNA was used as a negative control. Corresponding 18S rRNA from each sample is also shown. B) Steady state concentrations of LMP7 mRNA in luteal tissue. Bars represent densitometric values of LMP7 mRNA normalized to 18s rRNA. No significant differences were detected (P > 0.10)

Northern analysis revealed the presence of a transcript of approximately 1.2 kb, identical in size to the human LMP10 transcript [32], corresponding to LMP10 mRNA in all luteal tissue samples (Fig. 2A). Ovarian stromal tissue RNA (negative control) was devoid of any corresponding band. In contrast to LMP7, steady state concentrations of LMP10 mRNA were lower (P < 0.05) in luteal tissue collected early in the estrous cycle than in that collected during midcycle or late in the estrous cycle (Fig. 2B). As for LMP7 mRNA, no changes were observed in steady state concentrations of LMP10 mRNA during PGF2{alpha}-induced luteal regression (P > 0.10; Fig. 2B).



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  		FIG. 2. A) Representative northern blot of LMP10 in luteal tissue samples collected early (n = 6), during midcycle (n = 6), or late (n = 6) in the estrous cycle and at 0 (n = 6), 0.5 (n = 5), 1 (n = 5), 4 (n = 5), 12 (n = 4), or 24 (n = 5) hours after administration of a luteolytic dose of PGF2{alpha}. Ovarian stromal tissue RNA was used as a negative control. Corresponding 18S rRNA from each sample is also shown. B) Steady state concentrations of LMP10 mRNA in luteal tissue. Bars represent densitometric values of LMP10 mRNA normalized to 18s rRNA. Different letters denote significant differences (P < 0.05)

Immunohistochemical localization revealed the presence of LMP10 in both small and large luteal cells (Fig. 3). The number of cells positively stained for LMP10 in early luteal tissue was lower than that in midcycle or late luteal tissue (Figs. 3, a–c, and 4; P < 0.05). This increase in the number of LMP10-positive cells present in midcycle CL is similar to the increase in steady state LMP10 mRNA concentrations found in midcycle CL. There was no difference in the number of positively stained cells between midcycle CL and CL collected late in the estrous cycle. Although small cells devoid of LMP10 immunoreactivity were observed occasionally (Fig. 3d, arrows) the majority of small and large luteal cells contained immunoreactive LMP10. LMP10 staining was more intense in small luteal cells than in large luteal cells. No staining was observed in luteal tissue when nonimmune rabbit serum was used in place of rabbit anti-LMP10 primary antiserum (Fig. 3e). Ovarian stromal tissue, chosen as a negative control because of the lack of detectable LMP10 mRNA, exhibited only negligible background staining (Fig. 3f).



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  		FIG. 3. Immunohistochemical localization of LMP10 in bovine luteal tissue. a–d) Luteal tissue stained with anti-LMP10 antisera. Positively stained cells are sparse in early luteal tissue (a) compared with midcycle (b) or late (c) luteal tissue. x100. Higher magnification (d, x400) shows more intense LMP10 immunoreactivity localized to small luteal cells. Arrows indicate small luteal cells lacking LMP10 immunoreactivity. e) Nonimmune rabbit antiserum substituted for anti-LMP10 antiserum. f) Ovarian stroma (negative control).



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  		FIG. 4. Number of cells stained positively for LMP10 in bovine luteal tissue sections (n = 4 CL/time point). Bars represent numbers of LMP10-positive cells per high-power field (x400) in sections of luteal tissue collected at different stages of the estrous cycle. Different letters denote significant differences (P < 0.05)

Treatment of cultured luteal cells with LH, PGF2{alpha}, and/or TNF{alpha} had no effect on steady state concentrations of LMP7 mRNA as determined by semiquantitative RT-PCR (Fig. 5). In contrast, steady state LMP10 mRNA concentrations in cultured luteal cells increased in response to treatment with TNF{alpha} (Fig. 6). A representative example of results obtained using semiquantitative RT-PCR to measure LMP10 mRNA in cultured luteal cells is shown in Figure 6A. Apparent increases in amounts of the 650-bp amplicon corresponding to LMP10 mRNA were evident in samples from cultures treated with TNF{alpha} (Fig. 6A). Treatment of cultures with TNF{alpha} resulted in increases in the steady state concentrations of LMP10 mRNA, regardless of the presence or absence of LH and/or PGF2{alpha} (P < 0.05; Fig. 6B).



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  		FIG. 5. A) Representative gel showing products of LMP7 RT-PCR amplification of RNA from untreated luteal cell cultures (control) and from cultures of luteal cells treated with LH (10 ng/ml), PGF2{alpha} (10 ng/ml), and/or TNF{alpha} (50 ng/ml). Corresponding G3PDH amplification products are also shown. B) Steady state concentrations of LMP7 mRNA in cultured bovine luteal cells treated with LH, PGF2{alpha}, and/or TNF{alpha}. Bars represent densitometric values of LMP7 amplification products normalized to G3PDH (n = 4 CL). No significant differences were detected (P > 0.10)



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  		FIG. 6. A) Representative gel showing products of LMP10 RT-PCR amplification of RNA from untreated luteal cell cultures (control) and from cultures of luteal cells treated with LH (10 ng/ml), PGF2{alpha} (10 ng/ml), and/or TNF{alpha} (50 ng/ml). Corresponding G3PDH amplification products are also shown. B) Steady state concentrations of LMP10 mRNA in cultured bovine luteal cells treated with LH, PGF2{alpha}, and/or TNF{alpha}. Bars represent densitometric values of LMP10 amplification products normalized to G3PDH (n = 4 CL). Different letters denote significant differences (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The proteasome is a large cytosolic protease complex composed of multiple subunits. Proteasomal degradation is responsible for the majority of intracellular protein turnover. Proteasomal processing of intracellular proteins also generates peptides that are presented to T cells via class I MHC molecules [9, 33]. The catalytic core of the proteasome, referred to as the 20S proteasome, is composed of two heterologous families of subunits, {alpha} and ß, the latter of which contains the proteolytically active sites [11, 12]. INF{gamma} induces expression of three inducible subunits, LMP2, LMP7, and LMP10, which replace the constitutively expressed ß subunits [13, 14, 34–36], thereby altering the peptide cleavage patterns of the proteasome [16–19, 37, 38]. Using mice deficient in LMP2 or LMP7 expression, Fehling and coworkers [39] and Van Kaer and coworkers [40] demonstrated that the IFN{gamma}-inducible subunits are necessary for antigen processing. In these mutant mice, expression of cell surface class I MHC is reduced, and processing of specific antigenic peptides is impaired. Although these mice display no overt phenotypic signs of reproductive dysfunction, no studies were carried out to determine whether ovarian physiology, and specifically luteal regression, was abnormal in these animals.

This study is the first to describe the expression of INF{gamma}-inducible subunits of the proteasome in the CL of any species. In this study, mRNAs encoding LMP7 and LMP10 were detected in CL collected at all stages of the estrous cycle. Steady state concentrations of LMP7 mRNA did not change throughout the estrous cycle. In contrast, LMP10 mRNA concentrations were greater in midcycle and late CL than in early CL, and this increase in steady state LMP10 mRNA concentration was similar to the increase in the number of cells containing LMP10 protein. The gene encoding LMP7 is located within the class II MHC gene region [41–43], whereas the gene encoding LMP10 is located elsewhere [13, 14]. Expression of both genes is potently induced by IFN{gamma} [13, 34–37], and IFN{gamma} mRNA has been detected within bovine luteal tissue [1, 3]. Steady state concentrations of IFN{gamma} mRNA are highest in midcycle CL and decline to a nadir in late CL [3], but it is unknown whether concentrations of IFN{gamma} protein correspond directly to steady state mRNA concentrations in the CL. Thus, the presence of IFN{gamma} in the CL may account for the unexpected expression of LMP7 and LMP10 observed in the CL throughout the estrous cycle.

Previous studies have shown that TNF{alpha} can regulate LMP7 and LMP10 expression in other cell types [21, 22]. In the present study, steady state concentrations of LMP10 mRNA were elevated in cultured luteal cells in response to TNF{alpha}, suggesting that this cytokine may regulate expression of LMP10 within the CL. However, the finding in the present study that LMP10 mRNA concentrations did not change following in vivo administration of PGF2{alpha} seems incongruous with the results of a study by Shaw and Britt [5], in which TNF{alpha} was detectable in luteal dialysate only after initiation of luteal regression, whether spontaneous or PGF2{alpha} induced. However, because of the inefficiency of the in vivo luteal microdialysis system used in that study, it may not have been possible to detect low concentrations of TNF{alpha} in the CL prior to initiation of luteal regression. Thus, the absence of TNF{alpha} during the estrous cycle cannot be confirmed from that study nor can conclusions be drawn about changes in the concentrations of TNF{alpha} in the CL throughout the estrous cycle. Concentrations of LMP10 mRNA may be maximal in the CL by the midluteal phase, and any further increases in TNF{alpha}, such as those observed after initiation of luteal regression, then would not affect steady state LMP10 concentrations. This possibility could explain the lack of effect of in vivo PGF2{alpha} administration on LMP10 mRNA concentrations observed in the present study.

The lack of effect of TNF{alpha} on steady state LMP7 mRNA concentrations in the present study is enigmatic and remains unexplained. However, the increase in the steady state concentration of LMP10 mRNA in the CL is similar to increases in numbers of TNF{alpha}-producing cells shown in other studies. Hehnke-Vagnoni and coworkers [44], using porcine CL, demonstrated that endothelial cells are a source of TNF{alpha}. The increase in the number of LMP10-positive cells demonstrated in the present study is similar to the increase in the number of endothelial cells observed from early cycle to midcycle in previous studies using bovine and ovine CL [45, 46]. Macrophages within the CL are also a likely source of TNF{alpha} [47, 48], and in a study conducted by Townson and coworkers [2] the number of macrophages in the bovine CL was greater during midcycle and near the time of luteal regression than in the early part of the cycle. Because the results of the present study demonstrate that TNF{alpha} increases steady state LMP10 mRNA concentrations in bovine luteal cells and because increases in LMP10 expression are similar to increases in numbers of TNF{alpha}-producing cells within the CL, we conclude that TNF{alpha} is a primary regulator of LMP10 expression in the bovine CL.

Luteal cells from regressing CL are more potent stimulators of T lymphocyte proliferation than are luteal cells from fully functional CL [7]. One possible explanation for this difference is that the array of self-peptides presented to T cells in the context of class I MHC molecule changes during luteal regression. Changes in the expression of the IFN{gamma}-inducible proteasomal subunits could affect the repertoire of antigenic peptides generated by proteasomes in luteal cells, thus altering the repertoire of self-peptides presented to T cells. Such a change could result in a breakdown of peripheral immune tolerance of the CL. It was therefore hypothesized that a change in the array of self-peptides presented to T cells in the context of class I MHC molecules could explain the enhancement of T cell proliferation in response to PGF2{alpha}.

At the outset of these studies the initial hypothesis was that IFN{gamma}-inducible proteasome subunits are upregulated in luteal tissue near the time of luteal regression. Benyo and coworkers [6] previously demonstrated that expression of class II MHC molecules on the surface of luteal cells increases near the time of luteal regression and also in response to PGF2{alpha}. The class II transactivating factor (CIITA) coordinately regulates expression of class II MHC genes [49, 50]. Because LMP7 is located within the class II gene region, steady state concentrations of LMP7 mRNA within luteal tissue were expected to increase near the time of luteal regression and in response to PGF2{alpha}, similar to the increases observed in cell surface class II MHC molecules [6]. The presence of LMP7 mRNA in luteal tissue at all time points examined in the present study was therefore unexpected. Further, the lack of any change in LMP7 mRNA concentrations in response to PGF2{alpha} was surprising, given the increases in cell surface class II MHC molecules in response to PGF2{alpha}. One caveat is that in the present study we measured steady state concentrations of mRNA following PGF2{alpha} administration, whereas Benyo and coworkers [6] measured cell-surface proteins. Therefore, the possibility that PGF2{alpha} enhances the translation of mRNA encoding class II MHC gene products without affecting steady state concentrations of mRNA must be taken into consideration.

In conclusion, this study demonstrates the expression of LMP7 and LMP10, two IFN{gamma}-inducible subunits of the proteasome, in the bovine CL. Though it was expected that LMP7 and LMP10 would be expressed during regression of the CL, the finding that LMP7 mRNA and LMP10 mRNA are present throughout the estrous cycle was unexpected. Steady state concentrations of LMP7 mRNA are unaffected by TNF{alpha}, but LMP10 mRNA concentrations were elevated in response to TNF{alpha}. As a result of LMP7 and LMP10 expression in the CL, proteasomes within luteal cells may generate a repertoire of peptides presented to T cells in the context of class I MHC molecules that is different from that found in other tissues. The generation and presentation of altered arrays of self-peptides in the context of class I MHC by luteal cells could predispose the CL to a breakdown in immune tolerance. Such a breakdown in tolerance could promote a transient autoimmune-type response in luteal tissue that would facilitate the process of luteal regression.


   ACKNOWLEDGMENTS
 
The authors thank Jodi Winkler for excellent technical assistance and Dr. Michele McGuinness for assistance with Northern blotting procedures.


   FOOTNOTES
 
1 This work was supported by NIH grant HD37550 to J.L.P. Salaries and research support were also provided by state and federal funds. Back

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

Received: 14 August 2002.

First decision: 30 August 2002.

Accepted: 1 November 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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