RESEARCH ARTICLE Molecular Reproduction & Development 81:646–654 (2014)

Auto-Amplification System for Prostaglandin F2a in Bovine Corpus Luteum ASUKA KUMAGAI,1 SHIN YOSHIOKA,1 RYOSUKE SAKUMOTO2, 1 2

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KIYOSHI OKUDA1*

Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan Animal Physiology Research Unit, National Institute of Agrobiological Sciences, Ibaraki, Japan

SUMMARY The bovine corpus luteum (CL) is hypothesized to utilize a local auto-amplification system for prostaglandin (PG) F2a production. The objective of the present study was to determine if such a PGF2a auto-amplification system exists in the bovine CL, and if so, which factors regulate it. PGF2a significantly stimulated intra-luteal PGF2a production in all luteal phases, but did not affect PGE2 production. The stimulatory effect of exogenous PGF2a on CL PGF2a production was lower at the early luteal phase. Indomethacin, an inhibitor of prostaglandin-endoperoxide synthase (PTGS), significantly suppressed the PGF2a-stimulated PGF2a production by luteal tissue, indicating that the PGF2a in the medium was of luteal origin. Consistent with these secreted-PGF2a profiles, PGF2a receptor (PTGFR) protein expression was higher during the mid and late luteal phases than at early and developing luteal phases. Treatment of cultured bovine luteal cells obtained from the mid-luteal phase with PGF2a (1 mM) significantly increased the expressions of PTGS2, PGF synthase (PGFS), and carbonyl reductase1 (CBR1) at 24 hr post-treatment. Together, these results suggest the presence of a local auto-amplification system for PGF2a mediated by PTGS2, PGFS, and CBR1 in the bovine CL, which may play an important role in luteolysis.



Corresponding author: Laboratory of Reproductive Physiology Graduate School of Natural Science and Technology Okayama University Okayama 700-8530, Japan. E-mail: [email protected]

The authors have no conflicts of interest to declare. Asuka Kumagai and Shin Yoshioka contributed equally to this study.

Mol. Reprod. Dev. 81: 646654, 2014. ß 2014 Wiley Periodicals, Inc. Received 4 December 2013; Accepted 12 April 2014

INTRODUCTION The corpus luteum (CL) is indispensable for the establishment and maintenance of pregnancy because it secretes progesterone. When pregnancy does not occur, the CL undergoes functional luteolysis to decrease progesterone production, followed by structural luteolysis that results in luteal cell death (Knickerbocker et al., 1988; McCracken et al., 1999). Luteolysis in ruminants is induced by a positive-feedback loop between uterine prostaglandin (PG) F2a and luteal oxytocin (OXT) (Kotwica et al., 1999; McCracken

ß 2014 WILEY PERIODICALS, INC.

Grant sponsor: Japan Society for the Promotion of Science (JSPS); Grant number: 24380155 Published online 9 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22332

et al., 1999), wherein OXT stimulates the release of uterine PGF2a (Silvia and Taylor, 1989). On the other hand, administration of an OXT receptor antagonist in combination with a PGF2a analogue did not prevent increases of pulsatile PGF2a in the blood, raising the possibility that OXT is

Abbreviations: AA, arachidonic acid; CBR1, carbonyl reductase 1; CL, corpus luteum; IL, interleukin; OXT, oxytocin; PG, prostaglandin; PGFS, PGF synthase; PTGFR, PGF2a receptor; PTGS2, prostaglandin-endoperoxide synthase 2; TNF, tumor necrosis factor-alpha.

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not essential for initiating PGF2a release during luteolysis in cattle (Kotwica et al., 1999). Therefore, other mechanisms are though to stimulate PGF2a synthesis and secretion. Complex interactions between PGF2a and PGE2 signaling help regulate CL lifespan. In many mammals, PGE2 has been identified as an important luteoprotective factor that lengthens the lifespan of the CL and sustains progesterone production (Zelinski-Wooten et al., 1990; Ford and Christenson, 1991), whereas uterine PGF2a is the main luteolytic factor (McCracken et al., 1999). Despite these defined functions, CL lifespan is not prolonged by hysterectomy in rabbits, rhesus macaques, or humans, suggesting that uterine-produced PGF2a is not essential for luteolysis; instead PGF2a of luteal origin is thought to induce luteolysis in these species (Neill et al., 1969; Beling et al., 1970; Lytton and Poyser, 1982). The CL has the capacity to synthesize PGF2a. Injection of a PGF2a analogue increases the concentration of PGF2a in ovarian venous plasma in pigs, cattle, and rats (Diaz et al., 2000; Hayashi et al., 2003; Taniguchi et al., 2010), and increases the expression of prostaglandinendoperoxide synthase 2 (PTGS2) mRNA in sheep, pigs, cattle, rabbits, and rats (Tsai and Wiltbank, 1997; Diaz et al., 2000; Hayashi et al., 2003; Zerani et al., 2007; Taniguchi et al., 2010). These observations suggest that the CL of these species has an auto-amplification system in which PGF2a stimulates intra-luteal PGF2a production. PGF2a plays a physiological role in the initiation of luteal regression by binding to the PGF2a receptor (PTGFR), a seven transmembrane G-protein-coupled protein (Steele and Leung, 1993). PGF2a biosynthesis begins with the liberation of arachidonic acid from the membrane by the hormone-responsive enzyme cytosolic phospholipase A2 (Farooqui et al., 2000). Liberated arachidonic acid is metabolized to PGH2 by PTGS1 and PTGS2, which are both inhibited by indomethacin (Vane et al., 1998). PTGS1 is constitutively expressed in many tissues, whereas PTGS2 is induced by stimuli such as growth factors, cytokines, and hormones (Smith and Dewitt, 1996; Vane et al., 1998). PGH2 is converted to PGF2a by PGF synthase (PGFS) (Olofsson and Leung, 1994; Smith and Dewitt, 1996), which is regulated by cytokines such as tumor necrosis factor a (TNF) and interleukin (IL)-6 (Franczak et al., 2012). Carbonyl reductase1 (CBR1), a 9-keto reductase, converts PGE2 to PGF2a and is found in CL of rabbits, rats, and pig (Wintergalen et al., 1995; Inazu and Fujii, 1997; Wasielak et al., 2008). It has been reported that regulation of the CBR1 gene is induced through a xenobiotic response element (XRE) in the promoter region (Lakhman et al., 2007). The objective of the present study was to elucidate if such an auto-amplification system for PGF2a exists in the bovine CL during the luteal phase, and which factors regulate such a system. We examined PTGFR expression, the availability of enzymes involved in PGF2a biosynthesis, and the effects of PGF2a on PGF2a and PGE2 production in bovine luteal tissue during the luteal phase.

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RESULTS Effects of PGF2a on PGF2a and PGE2 Production in Bovine Luteal Tissue We first asked how much of the exogenous PGF2a was removed from the cultures after the washing step of our procedure to ensure minimal carryover during the 2-hr postexposure phase. PGF2a concentrations in the first through sixth washes revealed an average of 11.7  3.9 ng/ml (mean  standard error of the mean) (Table 1) in the final wash, indicating that the six washes were sufficient to remove the majority of the exogenous PGF2a. Thus, we were confident that we could measure a stimulatory effect of exogenous PGF2a on endogenous PGF2a production in conditioned media from the 2-hr post-treatment phase. Within the mid luteal phase, exogenous PGF2a directly stimulated the endogenous production of PGF2a in luteal tissue (Fig. 1). The PTGS inhibitor indomethacin significantly suppressed this PGF2a-stimulated PGF2a production (P < 0.05), indicating that the increased PGF2a in the medium was luteal in origin (Fig. 1). Basal PGF2a production tended to be higher at the early luteal phase (mean, 14.63 ng/ml/g tissue) than at the late luteal phase (mean, 5.47 ng/ml/g tissue; P ¼ 0.066). PGF2a significantly stimulated intra-luteal PGF2a production in all phases (P < 0.05; Fig. 2). There was no significant difference among the different luteal phases regarding the amount of intra-luteal PGF2a production after PGF2a treatment. Exogenous PGF2a did not affect PGE2 production (Fig. 3), although intra-luteal PGE2 production was higher at the early luteal than at the late luteal phase (Fig. 3). Overall, there was no difference in the PGE2/ PGF2a ratio between the early versus late luteal phases (Fig. 4a). On the other hand, the PGE2/PGF2a ratio was higher at the early luteal phase than at the other luteal phases after PGF2a supplementation (P < 0.05; Fig. 4b).

Effect of PGF2a on the PGF2a Production in Cultured Bovine Luteal Cells Compared with the control group, PGF2a increased PGF2a production at 4, 8, and 24 hr after treatment. Furthermore, PGF2a abundance significantly increased in a time-dependent manner after PGF2a treatment (P < 0.05; Fig. 5).

PTGFR Protein Expression in Bovine Luteal Tissue Throughout the Luteal Phase PTGFR was detected in the bovine luteal tissue throughout the luteal phase (n ¼ 45 cows per phase). PTGFR protein abundance was higher at the mid and late luteal phases than at the early and developing luteal phases (P < 0.05; Fig. 6).

Effects of PGF2a on the Protein Expression of PTGS, PGFS, and CBR1 in Cultured Bovine Luteal Cells To elucidate the direct effects of PGF2a on luteal PGF2a synthesis-related enzymes in bovine luteal cells, PGF2a

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TABLE 1. PGF2a Concentrations in the First Through Sixth Washes n 1 2 3 Average

Wash 1

Wash 2 (ng/ml)

Wash 3 (ng/ml)

Wash 4 (ng/ml)

Wash 5 (ng/ml)

Wash 6 (ng/ml)

þþþ þþþ þþþ 

þþþ þþþ 26.3  

þþþ 14.5 16.1 

40.1 12.7 11.7 21.5

31.5 12.7 9.1 17.8

19.0 9.3 6.8 11.7

þþþ PGF2a concentration too high to measure within the standard curve without dilution.

Figure 1. Effects of PGF2a (1 mM) and indomethacin (10 mM) on PGF2a production in cultured bovine luteal tissues at the mid luteal phase. All values are expressed as a percentage of control. Different letters indicate significant differences (P < 0.05), as determined by ANOVA followed by the Fisher’s PLSD as a multiple comparison test (mean  standard error of the mean, n ¼ 4 cows).

Figure 3. Stimulatory effects of PGF2a (1 mM) on PGE2 in cultured bovine luteal tissues during the different luteal phases (mean  standard error of the mean, n ¼ 4 cows/phase).

was applied to cultures for 4 or 24 hr. PGF2a significantly elevated the abundance of PTGS, PGFS, and CBR1 at 24 hr post-treatment; PGFS levels were elevated as early as 4 hr post-treatment (P < 0.05), but PTGS or CBR1 levels were not affected at this time point (Fig. 7).

DISCUSSION

Figure 2. The stimulatory effect of PGF2a (1 mM) on PGF2a production in cultured bovine luteal tissues during the different luteal phases. Asterisks indicate significant differences ( P < 0.01,  P < 0.001), as determined by two-way ANOVA followed by the Bonferroni test (mean  standard error of the mean, n ¼ 4 cows/phase).

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In many mammals, PGF2a acts as a luteolytic factor (Lytton and Poyser, 1982) while PGE2 acts as a luteoprotective factor (Zelinski-Wooten et al., 1990; Ford and Christenson, 1991; Weems et al., 2011). CL lifespan may be regulated, in part, by complex interactions between PGF2a and PGE2 (Arosh et al., 2004), where the ratio of intra-luteal luteotrophic and luteolytic PGs (PGE2 and PGF2a, respectively) is important for achieving luteolysis (Pate et al., 2012). In the present study, unstimulated luteal PGF2a production tended to be higher at the early luteal phase (14.63 ng/ml/g tissue) than at the late luteal phase (5.47 ng/ml/g tissue) (P ¼ 0.066) (Fig. 2), which is in agreement with previous reports (Milvae and Hansel, 1983; Rodgers et al., 1988). Luteal PGE2 production was also higher at the early luteal phase (4.27 ng/ml/g tissue) than at the late luteal phase (1.16 ng/ml/g tissue) (Fig. 3b). As a consequence, there was no difference in the PGE2/PGF2a ratio between the early versus late luteal phases throughout the estrous cycle (Fig. 4a). Following PGF2a treatment, however, luteal PGF2a production was stimulated during

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Figure 4. Effect of PGF2a (1 mM) on PGE2/PGF2a ratio in cultured bovine luteal tissues during the different luteal phases. Different superscript letters indicate significant differences (P < 0.05), as determined by ANOVA followed by the TukeyKramer multiple comparison test (mean  standard error of the mean, n ¼ 4 cows/phase).

all luteal phases, although the stimulatory effect of PGF2a was lower at the early luteal phase than at the other luteal phases (Fig. 2). Yet PGF2a did not affect PGE2 production (Fig. 3), resulting in a higher PGE2/PGF2a ratio at the early luteal phase than at the late luteal phase (Fig. 4b). The difference inPGE2/PGF2a ratio after PGF2a treatment among luteal phases suggests that the reactivity of luteal cells to PGF2a is one of the key factors underlying the acquisition of luteolytic capacity by the CL. The inhibitory effects of indomethacin on PGF2a secretion were not strong (Fig. 1). This may be a result of the methodology used: cells were treated for 2 hr with PGF2a in combination with indomethacin; this may not have been long enough to completely block the PTGS activity, particularly in the presence of exogenous PGF2a. In the present study, PGF2a stimulated PGF2a production in both bovine luteal tissue and luteal cells. The stimulatory effect of PGF2a on PGF2a secretion from mid-stage CL tissue was much stronger than from luteal cells obtained

Figure 5. Effect of PGF2a on the PGF2a production in cultured bovine luteal cells at different time points. Different letters and asterisks indicate significant differences (P < 0.05), as determined by ANOVA followed by the TukeyKramer multiple comparison test (mean  standard error of the mean, n ¼ 4 cows/phase).

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from the same stage (Figs. 1 and 5). These results suggest that a PGF2a auto-amplification system exists in luteal cells and in the CL, which also includes endothelial cells and immune cells. In fact, PTGFR is in bovine luteal cells (Sakamoto et al., 1995) as well as in luteal endothelial cells (Lee et al., 2009). Therefore, PGF2a production in the bovine may be a result of synergistic activity by these different CL cell types. PTGFR protein was present in bovine luteal tissue throughout the luteal phase, and was significantly higher at the mid and late luteal phases than at the early and

Figure 6. Changes in the abundance of PTGFR in cultured bovine luteal tissues during the different luteal phases. Lower panels are expressed as the relative ratio of PTGS2 to ACTB protein. Different letters indicate significant differences (P < 0.05), as determined by one-way ANOVA followed by the TukeyKramer test (mean  standard error of the mean, n ¼ 3 cows/phase).

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Figure 7. Effects of 4 hr (a) or 24 hr (b) of exogenous PGF2a stimulation on the accumulation of PTGS2, PGFS, or CBR1 in cultured bovine luteal cells at the mid luteal phase. Representative Western blot bands for PTGS2, PGFS, CBR1, and ACTB are shown in the upper panel. The lower panels are expressed as the relative ratio of PTGS2, PGFS, or CBR1 to ACTB protein. Different letters indicate significant differences (P < 0.05), as determined by ANOVA followed by a Fisher’s PLSD multiple comparison test (mean  standard error of the mean, n ¼ 35 cows).

developing luteal phases (Fig. 5). These data are consistent with receptor accumulation in rabbits, where PTGFR amounts increased four- to five-fold from the early to the mid and late luteal phases (Boiti et al., 2001). Furthermore, the binding capacity (Bmax) of PGF2a increased from the early (1.17  0.16 pmol mg1 protein) to the late (1.58  0.28 pmol mg1 protein) luteal phases, whereas the affinity (Kd) was almost the same from the early (18.3  2.12 nmol l1) to the late (22.4  1.26 nmol l1) luteal phases (Sakamoto et al., 1995). Together, these findings suggest that the difference in luteolytic capacity at the different stages is dependent on the amount of PTGFR in the CL. The number of immune cells, such as T lymphocytes and macrophages, also increases from the late to the regressed luteal phases (Penny et al., 1999). Immune cells produce a variety of cytokines, including IL-1b and TNF (Pate, 1995), which both increase PTGFR expression in human uterine mytocytes and follicular granulosa cells (Narko et al., 1997; Liang et al., 2008). In the present study, PTGFR protein expression was highest at the late luteal phase. Thus, based on similar expression profiles, bovine CL PTGFR

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could beat least partly regulated by cytokines such as IL-1b and TNF; since PTGFR expression was also high in the mid luteal phase, however, other PTGFR-regulating mechanisms may be responsible for the levels observed in the bovine CL. PTGS2, PGFS, and CBR1 abundance significantly increased in cultured bovine luteal cells at 24 hr after PGF2a treatment (Fig. 5), suggesting that the auto-amplification system of PGF2a is mediated by PTGS2, PGFS, and CBR1. These three enzymes are known to participate in PGF2a synthesis. PGFS levels significantly increased as early as 4 hr after PGF2a treatment, whereas PTGS2 and CBR1 abundance were not affected (Fig. 6). On the other hand, PGF2a-dependent PGF2a production by luteal tissues was observed at 4 hr (Fig. 6). This discrepancy may be due to differences between luteal tissue and the luteal cells: luteal tissues are composed of steroidogenic luteal cells, endothelial cells, fibroblasts, and immune cells such as lymphocytes and macrophages. Since IL-1b and TNF stimulate PGF2a production in cultured bovine luteal cells (Nothnick and Pate, 1990; Benyo and Pate, 1992; Townson and Pate, 1994), it is possible that intra-luteal PGF2a

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production increases due to interactions between steroidogenic luteal cells and the other stromal cells. Further studies are needed to clarify this possibility. Together, the present findings suggest that inter- and intra-cellular mechanisms are involved in PGF2a-stimulated PGF2a production (Fig. 8). In this model, a small amount of uterine or exogenous PGF2a binds to luteal PTGFR, thus activating an intra-cellular signaling pathway that may stimulate not only luteal OXT production but also luteal PGF2a production. Uterine PGF2a synthesis increases under a positive-feedback loop between uterine PGF2a and luteal OXT. Thereafter, an auto-amplification loop of intra-luteal PGF2a production mediated by PTGS2, PGFS, and CBR1 maybe activated by the steadily rise in uterine PGF2a concentration. This auto-amplification system for PGF2a production may aid in the progression towards CL luteolysis.

MATERIALS AND METHODS Collection of Bovine Corpora Lutea Ovaries with CL from Holstein-Friesian cows were collected at a local abattoir within 1020 min after exsanguination. Luteal phases were classified as early (Days 23), developing (Days 56), mid (Days 812), and late (Days 1516) phases after ovulation by macroscopic observation of the ovary as described previously (Miyamoto et al., 2000). After determination of the phases, the ovaries with CL were submerged in ice-cold physiological saline, and transported to the laboratory.

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Luteal Cell Isolation The mid-stage CL was collected for cell culture. Luteal tissue was enzymatically dissociated, and luteal cells were cultured as described previously (Okuda et al., 1992). Luteal cells were suspended in culture medium (Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 medium, 1:1 [v/v]; no. D8900, Sigma, St. Louis, MO) supplemented with 5% calf serum and 20 mg/ml gentamicin (no. 15750-011, Gibco BRL Life Technologies, Rockville, MD). Cell viability was higher than 80%, as assessed by trypan blue exclusion. The cell suspension contained very few endothelial cells or fibrocytes (05%), and no erythrocytes.

Experimental Procedures Effects of PGF2a on PGF2a and PGE2 production in bovine luteal tissues throughout the estrous cycle For tissue culture, luteal strips at the early, developing, mid, and late luteal phases were obtained from the center of the CL with 0.75 cm on a side. If there was a cavity in the CL, we excluded such CLs from samples. Luteal strips were washed three times in sterile saline solution containing penicillin (100 IU/ml) and streptomycin (100 mg/ml). The tissues were cut into small pieces with a scalpel, and subsequently washed another three times in Hank’s Balanced Salt Solution (HBSS) supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ml), and 0.1% bovine serum albumin (BSA) (Roche, Manheim, Germany; 10735078001). After hanging the tissues with steel needles (TOP, Tokyo, Japan; 8N01B), individual luteal tissues (triplicates per treatment) were placed into culture-glass tubes (12  75 mm; Kimble Chase Life Science and Research Products LLC, NJ; 73500-13100) containing 2 ml of culture medium. Luteal tissues were exposed to PGF2a (1 mM) (BIOMOL International Incorporated, PA; PG-008) or PGF2a (1 mM) in combination with the cyclooxyganase inhibitor indomethacin (10 mM) (Sigma Aldrich; I7378) for 2 hr in 38.08C with gentle shaking. After the tissues were washed six times with 38.08C-HBSS to remove PGF2a, the tissues were cultured for an additional 2 hr in fresh medium without PGF2a at 38.08C with gentle shaking. Each wash and/or 1 ml of conditioned media were collected into 1.5-ml tubes containing 10 ml of a stabilizer solution (0.3 M EDTA, 1% (w/v) acid acetyl salicylic, pH 7.3), and frozen at 308C until analysis. The concentrations of PGF2a and PGE2 in the culture medium were determined by enzyme immunoassay (EIA) as described below. The weight of each tissue was determined after incubation to normalize PGF2a and PGE2 concentration.

Effect of PGF2a on the PGF2a production in cultured bovine luteal cells The dispersed luteal cells were seed-

Figure 8. Schematic diagram of an auto-amplification system for PGF2a production in bovine luteal cells. AA, arachidonic acid; CBR1, carbonyl reductase-1; OXT, oxytocin; PGE2, prostaglandin E2; PGFS, PGF synthase; PGF2a, prostaglandin F2a; PGH2, prostaglandin H2; PTGS2, prostaglandin-endoperoxide synthase 2.

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ed at 2.0  105 viable cells/ml into 24-well plates (662160; Greiner Bio-One, Frickenhausen, Germany), and cultured in a humidified atmosphere of 5% CO2 in air at 38.08C. After 24 hr of culture, the medium was replaced with fresh medium containing 0.1% BSA (10 735 078 001; Roche Applied Science, Penzberg, Germany) with or without PGF2a

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PTGFR protein expression in bovine luteal tissues throughout the luteal phase Luteal tissues cultured in experiment 1 were frozen at 808C to determine PTGFR abundance by Western blotting. Luteal tissues were homogenized on ice in homogenization buffer (25 mM TrisHCl, 300 mM sucrose, 2 mM EDTA, complete protease inhibitor cocktail [11 697 498 001; Roche Diagnostics, Rotkreuz, Switzerland], pH 7.4) using a tissue homogenizer (Physcotron [NITI-ON, Inc., Chiba, Japan; NS-50]), followed by a filtration step using a metal wire mesh (150 mm). For protein analysis, nuclei were removed from the tissue homogenates by centrifugation at 600g for 30 min. The resultant supernatant, which contains cytosol and membrane fractions, was used for PTGFR protein analysis.

Effects of PGF2a on the protein expressions of PTGS2, PGFS, and CBR1 in cultured bovine luteal cells The dispersed luteal cells were seeded at 2.0  105

viable cells/ml in a 25-cm2 culture flask (Greiner Bio-One, Frickenhausen, Germany, no. 658175), and cultured in a humidified atmosphere of 5% CO2 in air at 38.08C. After 24 hr of culture, the medium was replaced with fresh medium containing 0.1% BSA (10 735 078 001; Roche Applied Science, Penzberg, Germany) with or without PGF2a (1 mM). After an additional 4 or 24 hr of culture, the cultured cells were scraped and suspended in buffer containing protease inhibitor (Protease Inhibitor Cocktail Tablets; Roche #11 697 498 001) for determination of protein abundance. Samples were frozen at 808C until analysis by Western blotting. The cultured cells were lysed in 200 ml of lysis buffer (20 mM TrisHCl, 150 mM NaCl, 1% TritonX100, 10% glycerol [Sigma, no. G7757], complete protease inhibitor, pH 7.4). The cell lysate was used for PTGS2, PGFS, CBR1 protein analyses.

PGF2a and PGE2 Determination The concentrations of PGF2a and PGE2 in the culture media were determined with an EIA test, as described previously (Uenoyama et al., 1997; Skarzynski et al., 2000). The PGF2a standard curves ranged from 15.6 to 4,000 pg/ml; intra- and inter-assay coefficients of variation averaged 8.6% and 12.1%, respectively. The PGE2 standard curve ranged from 0.07 to 20 ng/ml; intra- and inter-assay coefficients of variation averaged 6.9% and 9.7%, respectively.

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Chemical Industries, Ltd., Osaka, Japan; 137-06862], pH 6.8), and heated at 958C for 10 min. Samples (30 mg total protein) were electrophoresed on a 10% SDSPAGE for 1 hr at 200 V. The separated proteins were electrophoretically transferred to a 0.2-mm nitrocellulose membrane (Invitrogen; LC2000) at 100 V for 2 hr in transfer buffer (25 mM TrisHCl, 192 mM glycine, 20% methanol, pH 8.3). The membrane was washed in TBS-T (0.1% Tween 20 in TBS [25 mM TrisHCl, 137 mM NaCl, pH 7.5]), and then incubated in blocking buffer (5% fat-free dry milk powder in TBS-T) for 1 hr at room temperature. After the blocking step, the membrane was cut into pieces and separately incubated overnight at 48C with a primary antibody in blocking buffer specific to PTGFR (1:100, Cayman, MI; no. 101802), PTGS2 (1:1,000, ALPHA DIAGNOSTIC, San Antonio; no. 70209A), PGFS (1:400, kindly donated by Dr. Watanabe K, University of East Asia), CBR1 (1:400, abcam, Cambridge, UK; no. ab4148), or b-actin (ACTB) (1:4,000, Sigma, St. Louis; A2228). After incubation, the membrane pieces were washed three times for 10 min in TBS-T, and then incubated with secondary antibody in TBST (anti-rabbit IgG, HRP-linked whole antibody produced in donkey [Amersham Biosciences Corp., San Francisco, CA; no. NA931; 1:8,000, for PTGFR; 1:10,000, for PTGS2; 1:4,000, for PGFS]; anti-goat IgG, HRP-linked whole antibody produced in donkey [Santa Cruz Biotechnology; no. sc-2020; 1:5,000, for CBR1]; anti-mouse IgG, HRP-linked whole antibody produced in sheep [Amersham Biosciences Corp.; no. NA931; 1:80,000, for b-actin) for 1 hr at room temperature, and washed three times in TBS for 10 min at room temperature. Signal was detected using an ECL Western Blotting Detection System (Amersham Biosciences Corp.; RPN2109). ACTB protein expression was used as an internal control. The intensity of signal in each sample was estimated by measuring the optical density in the defined area by computerized densitometry using NIH Image (National Institutes of Health).

Statistical Analysis The experimental data are shown as the mean  standard error of the mean from 3 to 6 cows per phase. The statistical significance of differences in the protein expression was assessed by analysis of variance (ANOVA) followed by a Fisher protected least-significant difference procedure (PLSD) as a multiple-comparison test using Stat View (SAS Institute, Inc., NC). Comparisons among the luteal phase were performed by one-way ANOVA followed by the TukeyKramer test. Furthermore, to compare the effect of PGF2a on PGF2a and PGE2 secretion among the luteal phase, the statistical significance of differences was assessed by two-way ANOVA followed by the Bonferroni test.

Western Blotting Analysis Protein concentration was determined by the method of Osnes et al. (1993), using BSA as a standard. The proteins were then solubilized in SDS gel-loading buffer (50 mM TrisHCl, 2% SDS [Nacalai Tesque, Tokyo, Japan; 3160794], 10% glycerol, 1% b-mercaptoethanol [Wako Pure

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ACKNOWLEDGMENT This research was supported by Grant-in-Aid for Scientific Research (no. 24380155) of the Japan Society for the Promotion of Science (JSPS).

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Mol. Reprod. Dev. 81:646–654 (2014)

Auto-amplification system for prostaglandin F2α in bovine corpus luteum.

The bovine corpus luteum (CL) is hypothesized to utilize a local auto-amplification system for prostaglandin (PG) F2α production. The objective of the...
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