Evidence for two antigenically distinct molecular weight variants of prostaglandin H synthase in the rat ovary.

Evidence for Two Antigenically Distinct Molecular Weight Variants of Prostaglandin H Synthase in the Rat Ovary

Winona Y. L. Wong and JoAnne S. Richards Department of Cell Biology Baylor College of Medicine Houston, Texas 77030

Two affinity-purified polyclonal antibodies have been generated that differentially recognize two mol wt (Mr) variants of prostaglandin H synthase (PGS) in the rat ovary: antibody-2 recognized PGS of 72,000 Mr (PGS72), and antibody-3 recognized PGS of 69,000 Mr (PGS69). Immunoblot analyses showed that PGS72 was rapidly induced by LH in granulosa cells of preovulatory (PO) follicles and was associated with the increased production of prostaglandins (PGs) obligatory for ovulation. PGS72 was low (negligible) in other ovarian tissues, including PO follicles, corpora lutea, and interstitium. In contrast, PGS69 was constitutively present in small antral and PO follicles (primarily in thecal cells), was unaffected by LH, and was found at higher levels in corpora lutea throughout pregnancy and in the ovarian interstitium. PGS69 (but not PGS72) was also detected by immunoblots in rat adrenal glands, heart, uterus, and kidney. Immunofluorescent localization of PGS72 and PGS69 to ovarian tissue sections confirmed the cell-specific distribution of PGS observed by immunoblot analyses of cell extracts. Immunofluorescent detection of PGS72 required methanol fixation, whereas PGS69 was also observed with paraformaldehyde fixation and Triton X-100 permeabilization, further suggesting biochemical differences in these molecules. Immunoreactive PGS69 in PO follicles, thecal cells, and granulosa cells was associated with low amounts of indomethacin-sensitive production of PGs by these tissues in vitro, which was unaffected by inhibitors of transcription or translation. In contrast, stimulation of PGs in PO follicles by LH in vitro correlated with the marked induction of PGS72, but not PGS69, and was sensitive to both transcriptional and translational inhibitors. Collectively, these studies provide the first evidence that the rat ovary contains two immunologically distinct forms and Mr variants of PGS, each of which is selectively regulated by hormones, localized to specific cell types, differentially sensitive to 0888-8809/91/1269-1279$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society

inhibitors of transcription/translation, and differentially solubilized for immunocytochemical localization. (Molecular Endocrinology 5: 1269-1279, 1991)

INTRODUCTION

Prostaglandins (PGs) comprise a class of potent regulatory molecules that are produced by many tissues and are involved in a myriad of biological processes: injury, inflammation, vasodilation/constriction, and renal function (1, 2); mitosis and cell transformation (3-6); and implantation and ovulation (7, 8). The rate-limiting enzyme for prostanoid biosynthesis is PG endoperoxide synthase (PGS). PGS is found in situ as a dimer and functions as both a cyclooxygenase and a hydroperoxidase, converting arachadonic acid to prostaglandin H2 (PGH2) through formation of an unstable intermediate PGG2. Because of its biological significance, this enzyme has been studied extensively: it requires heme for functional activity (9), undergoes autoinactivation with a t1/2 of 10 min (10,11), is inhibited by aspirin and indomethacin (1), and is localized to the endoplasmic reticulum, plasma membrane, and nuclear envelope (12, 13). The activity and content of the enzyme have been shown to be regulated in a tissue-specific manner in vitro and in vivo by cytokines (14), peptides (15), and steroids (14-16). For example, interleukin-1 (IL-1) increases PGS activity and content in human dermal fibroblasts by mechanisms involving transcription and translation and is inhibited in these same cells by dexamethasone (14). In serum-starved 3T3 cells, plateletderived growth factor or phorbol ester increase PGE synthesis, which is blocked by transcription and translation inhibitors (17). Most recently, recombinant DNAs encoding sheep (18, 19), mouse (20), and human (21) PGS have been cloned and used to measure steady state levels of mRNA after various treatments in vitro (16, 17, 20, 22) and in vivo (23). Notably, changes in PG production and immunoreactive PGS have not always been associated with similar changes in PGS mRNA (17, 23).

1269

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 23:12 For personal use only. No other uses without permission. . All rights reserved.

RESULTS Characterization of Anti-PGS Antibodies Antibodies were generated in two rabbits against purified ovine seminal vesicle PGS (oPGS), obtained from Oxford Biochemical (Oxford, Ml). The antibodies were subjected to affinity purification, tested for recognition of oPGS (50 and 100 ng) by immunoblot analysis, and compared to the PGS antibody (antibody-1) used in previous studies (23,30). All three polyclonal antibodies detected a single oPGS-immunoreactive band (Mr = 70,000; Fig. 1). Taking into consideration the dilutions used for each antibody (antibody 1,1:50; antibodies 2 and 3, 1:15), the affinities of these antibodies for the oPGS antigen appear similar. Each of these antibodies was additionally character-

100

8

100

Our attention has been focused on the role of PGs in the mammalian ovary. In the rat (24) and rabbit (25,26), PG biosynthesis is markedly stimulated by the preovulatory surge of LH and is obligatory for ovulation, a process likened to an inflammatory response (27). After ovulation, PGs are subsequently involved in regulating the function of the corpus luteum. PGs such as PGE2 which support luteal cell function are distinguished from those such as PGF2« which terminate the functional lifespan of this structure. Immunofluorescent and immunoblot analyses conducted in several laboratories using either polyclonal or monoclonal antibodies have localized PGS to both folliclular and luteal ovarian compartments (28, 30). For example, an affinity-purified polyclonal antibody selectively localized PGS to granulosa cells of preovulatory (PO) follicles 5-7 h after exposure to an ovulatory dose of hCG in vivo or LH/ forskolin in vitro (23, 30). In contrast, specific monoclonal antibodies not only demonstrated PGS in PO follicles stimulated by hCG, but also showed significant immunostaining of PGS to thecal cells, corpora lutea, interstitial cells (28, 29), and kidney (28). Furthermore, although the 15- to 40-fold induction of PGS by LH in PO follicles is inhibited by transcriptional (a-amanitin) and translational (cycloheximide) antagonists, the increase in enzyme protein was not associated with any increase in PGS mRNA, as assessed by Northern blot analyses (23) using a full-length mouse cDNA probe (20). Rather, low amounts of PGS mRNA were observed in ovaries containing primarily PO follicles, and this was decreased by hCG (23). One explanation for these apparent divergent results concerning the amounts and tissue localization of PGS as well as the regulation of PGS mRNA in the rat ovary is that there is more than one PGS enzyme. The studies described herein provide the first evidence that the rat ovary contains two immunologically distinct forms and mol wt (Mr) variants of PGS, each of which is selectively regulated by hormones, localized to specific cell types, and differentially solubilized for immunocytochemical localization.

8

8

100

Vol 5 No. 9

MOL ENDO-1991 1270

10 *3 -

-

—

97.4 -

-

—

200

69

-

-

46

-

-

•

-

m•

—

AntiPGS 1 AntiPGS2 AntiPGS3 Fig. 1. Detection of Ovine PGS by Affinity-Purified Antibodies Purified ovine seminal vesicle PGS (50 and 100 ng) was subjected to immunoblot analysis using affinity-purified antiPGS antibodies: antibody 1 (diluted 1:50) (30), antibody 2 (1:15), and antibody 3 (1:15). All blots were processed simultaneously and exposed for equal lengths of time. Migration of Mr standards is indicated to the left of the figures.

ized for recognition of PGS in rat ovarian samples. Cell extracts from PO follicles, PO follicles collected 7 h after an ovulatory dose of hCG (PO + hCG, ovulatory follicles), and corpora lutea (luteal, CL) collected from rats on days 20 and 15 of pregnancy were run on triplicate blots and assessed for PGS content using antibody 1, 2, or 3. As expected from our previous studies using antibody 1 (23,30), this antibody did not detect demonstrable amounts of PGS in PO follicles unless they were exposed for 7 h in vivo to an ovulatory dose of hCG and detected only low amounts of PGS in corpora lutea (Fig. 2, left). As noted by the arrows in Fig. 2, the upper PGS band in luteal tissue appeared to migrate slightly faster (Mr = 69,000) than the PGS band in the ovulatory follicle (Mr = 72,000). In contrast, the lower PGS band (presumed to be a breakdown product) of PGS in the luteal samples migrated more slowly (Mr = 61,000) than that in the ovulatory follicle (Mr = 59,000). One of the new antibodies, antibody 2, detected PGS in the ovarian samples in a manner similar to that of antibody 1; PGS signals were absent in PO follicles, high in ovulatory follicles, and barely detectable in luteal tissue (Fig. 2, center). The intensity of the PGS signal in the ovulatory follicle was consistent with the strong signal of oPGS standard by antibody 2, observed above (Fig. 1)The third antibody, antibody 3, exhibited a pattern of immunoreactivity which differed greatly from that of antibodies 1 and 2. Notably, antibody 3 detected significant amounts of PGS in all samples, including PO follicles and corpora lutea, the immunopositive signal was of approximately equal intensity in all samples, and (as noted by the arrows in Fig. 2), the PGS band of the corpora lutea appeared to migrate slightly faster (Mr = 69,000) than the PGS detected in the ovulatory follicles by antibodies 1 and 2. It is important to note that antibody 3 recognized the 72,000 Mr PGS band in the ovulatory follicles, but relative to a similar dilution of


1271

Two Antigenic Forms of PGS

OV

o o

o o +

o

O O CM _ 3 Q . Q . Q

M r x 10 9 7.4-

+

10

O O 0_ Q.

*Q

O W

CM iQ Q

6 94 6-

AntiPGS- 1

AntiPGS-2

AntiPGS-3

Fig. 2. Detection of PGS in Rat Follicles and Corpora Lutea by Antibodies 1,2, and 3 Solubilized membrane extracts (300 ng) prepared from PO follicles collected before (PO) or 6 h after an ovulatory dose (10 IU, iv) of hCG (PO+hCG), and corpora lutea (CL) collected from rats on day 15 (D15) or 20 (D20) of pregnancy were analyzed by immunoblot with antibody 1 (1:50), antibody 2 (1:15), or antibody 3 (1:15).

antibody 2, the signal was far less intense. Thus, antibody 3 appears to have a lower affinity for the induced protein or contains a subpopulation of antibodies that cross-reacts with the induced protein but represents a small proportion of the polyclonal population. Antibody 3 also detected PGS in small antral (SA) follicles and in corpora lutea isolated on days 6,12,15, 20, and 23 of pregnancy (data not shown). The SA follicle content of PGS was unchanged by FSH, and corpora luteal levels did not change significantly over the days of pregnancy examined. Taken together, these results suggest that two antigenically distinct forms and Mr variants of PGS exist in the rat ovary; one form (detected preferentially with antibodies 1 and 2) is transiently induced in ovulatory follicles, and a second form (detected preferentially by antibody 3) is present in PO follicles, ovulating follicles, and corpora lutea and is not responsive to hCG. Resolution of PGS Variants by Lower Percentage Polyacrylamide Gel Electrophoresis (PAGE) To provide a direct comparison of relative Mr values, the antigenically distinct isoforms of PGS were visualized on a single Western blot by sequential blotting with antibodies 2 and 3. PGS (PGS72; Mr = 72,000) was visualized by antibody 2 in PO follicles treated in vitro with LH (PO + LH, ovulatory) along with an accompanying breakdown band (Mr = 59,000; Fig. 3A). Fteincubation of the blot with antibody 3 visualized a protein band at Mr 69,000 (PGS69) and an accompanying band at Mr 61,000 in the adjacent PO follicle and luteal samples (Fig. 3B). The differences in the mobility of both the upper bands and their associated lower breakdown bands could be clearly seen upon comparison of the induced ovulatory follicle with the PO follicle or luteal tissue. On a lower percent polyacrylamide gel

(7.5%), PGS72 in ovulatory follicles resolved as a doublet (Mr = 72,000 and 70,000; Fig. 3C, left). The biochemical basis for the doublet of PGS in the ovulatory sample is unclear, but may be related to glycosylation. The lower Mr band of the PGS72 doublet is unlikely to be PGS69, because detection of this band by antibody 2 is restricted to ovulatory samples. When these same samples were analyzed with antibody 3, PGS69 was observed in equal amounts in both PO and ovulatory samples (Fig. 3C, right). Comparison of Thecal and Granulosa Cell Contents of the PGS Variants To determine whether the two forms of PGS were expressed in a cell-specific manner, granulosa and thecal cells from PO follicles were assessed separately for PGS content, first with antibody 2 and then with antibody 3. As expected, antibody 2 detected high amounts of immunopositive PGS72 in the granulosa cells of PO follicles incubated with LH (Fig. 4A). Only a faint band could be detected in either granulosa or thecal cells of untreated PO follicles (Fig. 4A). When the untreated PO granulosa and thecal cell lanes were tested with antibody 3, PGS69 and its associated breakdown band (Mr = 61,000) were observed in abundance in thecal cells (Fig. 4B). These same bands were observed in the PO granulosa cells after much longer exposure. Excision and counting of the immunopostitive bands determined that the content of PGS72 was 40-fold higher in granulosa cells of PO follicles incubated with LH than in those incubated in medium alone. PGS69 in the thecal component was 4-fold higher than that in the granulosa cells. Thus, PGS69 is present primarily in thecal cells of PO follicles.


Vol 5 No. 9

MOL ENDO-1991 1272

GC

Mr x 10" 3

B

B

GC

Th

Fig. 4. Cell/Tissue-Specific Localization of PGS72 and PGS69 A, PO follicles were incubated for 6 h in medium alone (PO) or with LH (500 ng/ml; PO + LH; two experiments). After incubation, the granulosa cells (GC) were separated from the thecal cells (Th), and soluble cell extracts (300 fig) were analyzed by immunoblot with antibody 2 (1:25). The blot was overexposed to visualize PGS72 in PO Th samples. 1 2 5 ILabeled PGS bands cut from the nitrocellulose were counted (PO GC, 110 cpm; PO + LH GC, 3667 and 4411 cpm; PO Th, 134 cpm). B, Nitrocellulose strips containing the PO GC and Th lanes were reexamined using antibody 3 (1:10; GC, 189 cpm; Th, 720 cpm). Arrows at left of figures mark the position of oPGS standard.

Ab2

Ab3

Fig. 3. Comparison of Relative Mr of PGS72 and PGS69 A, Solublized cell extracts (300 nQ) from PO follicles (PO), PO follicles incubated for 6 h with LH (PO + LH), and day 15 corpora lutea (CL) were analyzed with antibody 2 (1:25). B, The blot was washed in buffer and reincubated with antibody 3 (1:10) and [125l]protein-A. C, PO and PO + LH samples were further resolved on 7.5% SDS-PAGE and analyzed with antibody 2 (Ab 2) or antibody 3 (Ab 3). The estimated Mr values of PGS bands are indicated to the right of each figure.

PGF2a and PGE2 Production by PO Follicles, Granulosa Cells, and Thecal Cells in Vitro To verify that PGS activity in rat ovarian tissues was correlated with immunopositive PGS, PG production by granulosa and thecal cells was determined by measuring the concentrations of PGF2« and PGE2 in medium samples by RIA (Table 1). To eliminate substrate limitations and allow PG production to more directly reflect the content of enzymatically active PGS in the cells, basal production of PGs by thecal and granulosa cells was measured in the presence or absence of mellitin, a known stimulator of arachadonic acid release (31). Granulosa cells of PO follicles produced low quantities of both PGF2a and PGE2 in the absence of mellitin, which were increased ~3-fold in the presence of mellitin. Thecal cells of PO follicles produced higher amounts of PGs than granulosa cells with or without mellitin. In

the presence of mellitin, thecal cell production of PGF2a and PGE2 was 2 and 1.7 times, respectively, that of mellitin-treated granulosa cells (Table 1). These relative differences in PG production by PO thecal and granulosa cells are consistent with the relative amount of PGS69 detected by antibody 3 in these tissues (Fig. 4B). Indomethacin inhibited PG production by PO thecal cells incubated with mellitin and reduced granulosa cell PG production under these same treatments. LH in the presence of mellitin stimulated PG production not only in granulosa cells (~9-fold), but also in thecal cells (~3fold) over mellitin alone. Although the overall production of PGs increased for both cell types with LH treatment, the ratio of PGE2 to PGF2a differed greatly between them. Granulosa cells produced PGE2 almost exclusively, whereas thecal cells produced both PGF2« as well as PGE2. Induction of PGS72 and PGS69 in Granulosa and Thecal Cells Incubated with Hormones in Vitro To verify that gonadotropins directly induced PGS72 in granulosa cells and to determine whether the LH stimulation of PG production in thecal cells in vitro was associated with changes in PGS69, PO granulosa cells, and thecal cells were incubated separately with either FSH (500 ng/ml) or LH (500 ng/ml), and cell extracts were examined with antibody 2 and antibody 3. PGS72 and its associated breakdown band (Mr = 59,000) were


1273


Table 1. Production of PGF2a and PGE2 by PO Granulosa Cells (GC) and PO Thecal Cells (TC) Incubated for 6 h in Vitro

GC Control + Mellitin + Mellitin + + Mellitin + TC + Mellitin + Mellitin + + Mellitin +

PGF2a (pg//ig Protein)

PGE2 (pg/ng Protein)

Total PG (pg/^g Protein)

0.5 0.9 0.04 1.70 1.0 1.7 0.6

1.3 4.3 0.7 39.0 3.8 7.3 2.4 26.0

1.8 5.2 1.1 40.7 4.8 9.0 3.0 31.3

indomethacin LH

indomethacin LH

5.3

LH = 500 ng/ml; mellitin = 3 x 10~7 M; indomethacin = 12.5 HM.

induced by FSH in granulosa cells, but not by LH in thecal cells (Fig. 5). Addition of mellitin to the incubations had no effect on FSH induction of PGS72. When the theca cell samples were subsequently examined using antibody 3, PGS69 was shown to be abundant in all samples and, per microgram loaded protein, did not change in response to LH or LH plus mellitin (Fig. 5). Production of PGF2» by the incubated tissues exhibited the relative amounts and changes of immunopositive PGS (Fig. 5, legend). Content of PGS69 and PGS72 in Other Rat Tissues To provide additional verification that antibody 3 was detecting authentic PGS, other tissues were examined with this antibody. Microsomal preparations of residual Granulosa 1 1

1

"3

Q)

s+ o PGS72

•

zCO

s

Z

u.

0

5

+

5

Mrx10"3 MrxiO

mmIff*

59 !

PGS69

Immunodetection of PGS in Primary Human Dermal Fibroblasts by Western Blot Analysis Using Antibody 3 To determine whether antibody 3 could detect PGS protein in another species, extracts from cultured pri-

Theca

1

ovarian tissue (ovaries from which PO follicles have been removed), adrenal gland, heart, uterus, and kidney all contained immunoreactive PGS69 as well as the accompanying 61,000 Mr breakdown band (Fig. 6). The highest amounts were observed in kidney and uterine preparations, tissues known to produce PGs and exhibit immunoreactive PGS (28, 32). Importantly, PGS72 was not detected in any of these tissues in either membrane (Fig. 6) (30) or cytosolic fractions (not shown). Solubilized membrane extracts of primary human dermal fibroblasts were also negative for PGS when antibody 2 was used (Fig. 6).

!

PGS69

j

vim*^

69

Fig. 5. PGS72 and PGS69 Contents in PO Granulosa and Thecal Cells Incubated Separately with Gonadotropins and Mellitin (Mel) PO granulosa and thecal cells were separated and incubated individually with FSH (500 ng/ml; granulosa cells) or LH (500 ng/ml; thecal cells) with or without [control (C)] mellitin (3 x 10~7 M) (31) for 6 h, and cell extracts (250 ^g except theca control; 125 ^g) were examined with antibody 2 (1:25) and antibody 3 (1:10). 125l-Labeled PGS bands were excised and counted: granulosa cells, 147 cpm; granulosa plus FSH, 803 cpm; granulosa cells plus FSH and mellitin, 547 cpm; thecal cells, 1319 cpm; thecal cells plus LH, 1904 cpm; and thecal cells plus LH and mellitin, 1925 cpm. PGF 2Q content in the media was measured by RIA, (pg/Mg protein): GC control 0.25, + FSH 3.4, FSH + mel 4.75; Theca control 3.8, +LH 10.65, LH + mel 11.42.

PGS72

Fig. 6. Immunodetection of PGS72 and PGS69 in Different Tissues Solubilized membrane extracts (300 ng) from rat residual ovaries (Res. Ovary; ovaries from which PO follicles had been removed), adrenal, heart, kidney, and uterus were examined for PGS69 content with antibody 3. Adrenal, heart, kidney, and uterus along with follicles exposed for 6 h to hCG in vivo (PO + hCG) and human dermal fibroblasts (HDF) were examined with antibody 2. Fifty nanograms of purified sheep PGS (oPGS) were included as a standard. Upper panel, Antibody 3; lower panel, antibody 2.


Vol 5 No. 9

MOL ENDO-1991 1274

mary human dermal fibroblasts were subjected to immunoblot analysis with this antibody. This tissue has been shown to contain immunoreactive PGS, which is increased (3- to 4-fold) by hormones (14). A single protein band migrating with the same apparent Mr as that reported for human PGS (Mr- 68,000) (21) was detected by antibody 3 in cell extracts of cultured human dermal fibroblasts (Fig. 7). Moreover, the content of this protein increased 3- to 4-fold after 12-h incubation with either 1 HIM 8-bromo-cAMP (1,986 cpm) or 2 U/ml IL-1 (1,263 cpm) compared to medium alone (479 cpm). These results are similar to those published by Raz et al. (14) using polyclonal antibodies and suggest that antibody 3 recognizes authentic PGS in this human tissue.

Table 2. PGF2a Production and PGS Content of PO Follicles Incubated for 6 h in Vitro

Effects of Transcriptional and Translational Inhibitors on PGF2a Production and PGS69 Content of Incubated PO Follicles

3-fold increase in immunopositive PGS in LH-treated follicles was due to the cross-reactivity of antibody 3 with PGS72. As observed in our previous studies (23), LH induced increases in PGS72 were blocked by inclusion of either a-amanitin or cycloheximide. In contrast, the content of PGS69 remained unchanged compared to that in control follicles (Table 2). These studies indicate that whereas PGS72 induction requires both transcriptional and translational events, the folliclular content and enzymatic activity of PGS69 are stable for up to 6 h in the absence of transcription or translation.

PO follicles were incubated for 6 h in the presence or absence of LH with or without cycloheximide or aamanitin. PGF2« production by these follicles was low, but detectable, in untreated follicles and increased 18fold with LH treatment. Inclusion of cycloheximide or «amanitin blocked the LH-induced rise in PGF2a production, but did not have a significant effect on the low basal production of PGF2a (Table 2). To determine whether the continued basal production of PGF2« in the presence of the inhibitors reflected the continued presence of PGS69 enzyme, antibody 3 was used to examine the content of PGS in these follicles; immunopositive bands were excised and quantitated on a 7counter. Antibody 3 detected significant amounts of immunopositive PGS69 in the PO follicle control. The

Mrx10

cpm

479 1986

1263

2146

Fig. 7. Immunodetection of PGS in Human Dermal Fibroblasts by Antibody-3 Primary human dermal fibroblasts cultured in 20% fetal bovine serum were incubated for 12 h in medium containing 5% fetal bovine serum alone or with 1 mM 8-bromo-cAMP or 2 U/ml human IL-1 a. Solubilized membrane extracts (300 ^g) were examined for immunopositive PGS with antibody 3 (1:10). Fifty nanograms of oPGS (Oxford Biochemicals) were included as a standard (PGS std).

PO Follicle

Control + LH + LH + Cycloheximide + LH + a-Amanitin

PGF2o (pg/Follicle)

PGS (cpm; antibody 3)'

6.3 116.6 7.2

116 370 127

6.2

102

LH = 500 ng/ml; cycloheximide = 10 ^g/ml; a-amanitin = 25 M g/ml. 8 Cell extracts (300 ng) were analyzed by Western blot analysis with antibody 3 (1:10); 125l-labeled PGS bands were excised and counted.

Immunocytochemical Localization of PGS72 and PGS69 with Antibodies 2 and 3, Respectively Antibodies 2 and 3 were used to immunolocalize PGS in sections from ovaries of hCG-primed immature rats (see Materials and Methods) collected 6 h after an ovulatory dose of hCG in vivo. Immunocytochemical localization of PGS with the two antibodies required selective fixation and solubilization procedures. Detection of PGS72 by antibody 1 required methanol fixation (30), and with this protocol, granulosa cells of ovulatory follicles imunostained intensely (Fig. 8A) in a manner identical to our previous studies (30). In contrast, detection of PGS69 by antibody 3 was observed with fixation procedures used by others (28,29,32), namely paraformaldehyde fixation and Triton X-100 permeabilization. In different prepared sections, PGS69 was localized to several compartments in the ovary (ovarian interstitium, germinal epithelium, thecal cells, and corpora lutea), but was most strikingly observed in cells in the thecal cell layer of staged follicles (Fig. 8B). This pattern of staining observed with antibody 3 is similar to that observed by Curry, Jr., et al. (28) using monoclonal antibodies initially selected for positive immunoreactivity with kidney tissue. Also note the vesicular type of staining in the thecal layer (observed with either fixation method) compared to the finer punctate staining in granulosa cells. No immunostaining was observed in the absence of the primary antibodies (Fig. 8C).

DISCUSSION Polyclonal antibodies against purified sheep seminal vesicle PGS have been generated, affinity purified, and



Fig. 8. Indirect Immunofluorescent Localization of PGS72 and PGS69 in Ovulatory Follicles Indirect immunofluorescent localization of PGS was performed in rat ovarian follicles isolated 6 h after the administration of 10ILJ hCG, iv. Shown are cryosections fixed in methanol and examined with antibody 2 (A), fixed with paraformaldehyde-Triton X-100 and examined with antibody 3 (B), or fixed with paraformaldehyde-Triton X-100 and examined without primary antibodies (C). Granulosa cells (G) exhibited intense immunostaining with antibody 2 (A), but not with antibody 3 (B; with either fixation protocol). No signal was detected in thecal cells (T) with antibody 2 (A), whereas the thecal cell layer (T) and selected areas of the interstitium (I) immunostained positively with antibody 3 (B). No signal was detected when sections were probed with secondary antibody (goat antirabbit immunoglobulin G) alone in G, T, or germinal epithelium (GE) C, Magnification: A-C, x147. Exposures and development times for each section were identical, thus allowing direct comparison. Representative figures from three different follicles are shown.

1275

shown to recognize nanogram amounts of purified oPGS standard. When the antibodies were used to immunolocalize and characterize PGS in rat ovarian and extraovarian tissues, two immunodistinct forms of the enzyme were identified. Based on several criteria, we propose that these two immunodistinct forms of PGS represent two authentic isoforms of the enzyme. First, each immunodistinct variant differs in its apparent Mr, as determined by Western blot analyses using 10% and 7.5% PAGE. One form, 72,000 Mr, recognized selectively by antibodies 1 and 2, has been designated PGS72. The other variant, 69,000 Mr, recognized selectively by antibody 3, has been designated PGS69. The breakdown products of these two variants also differ in their apparent Mr (59,000 and 61,000, respectively). Second, the antigenic and Mr differences of the PGS variants were associated with specific biochemical differences in localization and regulation of the enzymes. As described, PGS72 required methanol fixation/permeabilization for detection in cells by immunocytochemistry, whereas PGS69 was observed with paraformaldehyde/Triton X-100. Immunostaining of PGS72 was diffuse, whereas that for PGS69 appeared in large granules. These differences provide indirect evidence that the association of PGS72 to subcellular structures differs from that of PGS69. Furthermore, each variant was differentially sensitive to inhibitors of transcription and translation in short term incubations. Induction by LH of PGS72 and PG production were blocked by aamanitin or cycloheximide (Table 2) (23), whereas the content of PGS69 and basal production of PGs were not. Taken together, these observations indicate that PGS72 and PGS69 have distinct biochemical properties, the nature of which will require purification/cloning of each variant. Third, each immunodistinct variant of PGS was selectively regulated by hormones and localized to specific ovarian and extraovarian tissues. PGS72 was detected exclusively in granulosa cells of PO follicles incubated with ovulatory doses of LH or FSH for 6 h or exposed to hCG in vivo for 7 h. No immunopositive PGS72 was observed in rat heart, uterus, adrenal, or kidney, tissues known to produce PGs. In contrast, PGS69 was low in granulosa cells of PO follicles and was not induced by either FSH or LH. PGS69, however, was found in abundance in all of the extraovarian tissues examined and in ovarian thecal, interstitial, and luteal tissues. Within the PO follicle, the relative distribution of PGS72 and PGS69 was associated with mellitin- and hormoneinduced production of PGs, thus providing evidence that the enzyme content in these tissues was associated with enzyme activity. Specifically, basal production of PGs (PGE2 and PGF2l) was 3-fold greater in thecal cells than in granulosa cells, reflecting the relative abundance of PGS69 in the two tissues. However, in response to FSH/LH (in the presence of mellitin), granulosa cells exhibited a 10-fold increase in the production of PGs and PGS72 content, whereas thecal cells exhibited only a 3-fold increase in PGs, with no change in


MOL ENDO-1991 1276

PGS69 in the 6-h interval analyzed. Together, PGS69 and PGS72 provide a means for follicles to produce low and high amounts of PGs in response to specific hormonal/environmental cues. Furthermore, PGS69 appears to be predominantly localized to cells within the thecal layer of PO follicles and thereby may play a key role in mediating LH-induced increases in vascular permeability and vasodilation that occur in ovulating follicles. Specific induction of PGS72 by FSH and LH within the granulosa cell layer may provide a means to coordinate the effects of PGs on thecal tissue with the effects of PGs on granulosa cells, cumulus cells, and perhaps even the oocyte during the ovulatory period. Although specific effects of PGs on ovarian cell function during this period remain to be clearly defined, it is well known that indomethacin, an inhibitor of PGS activity, blocks ovulation (33-38). The presence of two immunodistinct forms of PGS in the rat ovary may explain dissimilar results observed among previous studies. For example, detection of PGS69 in rat kidney as well as several ovarian compartments (theca, interstitium, and corpora lutea) by antibody 3 mimics the localization of PGS in rat kidney and ovary by other investigators using monoclonal antibodies that have been specifically selected for recognition of renal PGS (29). In contrast, our previous studies in which ovarian PGS was localized exclusively to granulosa cells of PO follicles exposed to ovulatory doses of LH (30) were based on data obtained using antibody 1, which selectively recognizes PGS72, the form induced by LH. In addition, antibody 3 and the monoclonal antibody (29) appear to recognize not only PGS69 but also PGS72, the latter with less affinity. Thus, whereas antibodies 1 and 2 detect a 10- or greater fold increase in PGS72 in ovulatory follicles (23, 30), antibody 3 and the monoclonal antibody detect increases of ~3- to 5-fold (Table 2) (29, 39). Although no other study has yet reported the detection of more than one PGS protein in any tissue, other investigators have postulated the existence of more than one pool of the enzyme based on suggestive biochemical evidence. Needleman and colleages have noted differences in the ability of glucocoticoids to inhibit basal and increased levels of PGS activity in studies of IL-1 treated human dermal fibroblasts (14) and lipopolysaccharide-stimulated human monocytes (15). Whereas stimulation of PGS activity by IL-1 or lipopolysaccarides could be inhibited by dexamethasone (90% and 80%, respectively), basal PGS activity was inhibited much less (20% and 40%, respectively) (14,15). Tsai et al. (40) have also suggested that there are two pools of PGS based on kinetic studies. A biphasic pattern was observed for PGS synthesis and degradation in human umbilical venous endothelial cells; in the presence of phorbol ester or IL-1, the rate of rapid phase enzyme synthesis was enhanced, whereas the slow phase synthesis rate and the degradation rates remained unchanged. The molecular basis by which the oPGS antigen generated antibodies with different specificities for the

Vol 5 No. 9

rat PGS isoforms remains to be determined. One possibility is that the oPGS standard used conains two isoforms. Based on sodium dodecyl sulfate-PAGE (SDS-PAGE), polychromatic silver staining, and immunoblotting, we have no evidence for two Mr or antigenic variants. Equally plausible is the explanation that antibodies 1 and 2 comprise a large population of high affinity antibodies that selectively recognize epitopes shared by the oPGS antigen and rat PGS72. In contrast, the polyclonal population comprising antibody 3 preferentially recognizes epitopes shared by oPGS and rat PGS69. That antibody 3 also recognizes PGS72 suggests that a subpopulation cross-reacts with the induced protein, but either represents a small subclass of the polyclonal population or exhibits weak affinity to the PGS72 epitopes. Answers to these complex questions can only be resolved by monoclonal epitope mapping of PGS isoforms and cloning of the rat PGS cDNAs. Several cDNA clones encoding for PGS have been isolated from sheep (18, 19), mouse (20), and human (21) libraries. All of these clones show a high degree of amino acid and nucleotide sequence homology. These cDNAs have been used to quantitate PGS mRNA in several tissues, including human dermal fibroblasts (14), monocytes (15), and endothelial cells (11, 41); mouse 3T3 fibroblasts (17, 20) and myoblasts (42); and rat mesangial cells (43) and ovarian follicles and corpora lutea (23). Fold changes in mRNA, protein, and PG synthesis have averaged 3- to 4-fold in 3T3 cells (17, 20) and mesangial cells (43). No change was observed between ovaries containing PO follicles and corpora lutea, whereas hCG caused PGS mRNA in PO follicles to decline (23). This decline in PGS mRNA in ovaries at a time when the content of PGS72 increases 10-fold or greater has been difficult to reconcile. However, recent studies in which Northern blot hybridizations have been performed under conditions of low stringency have detected in sheep tracheal mucosa cells and rat mesangial cells not only the 2.8-kilobase (kb) mRNA observed in most tissues examined to date, but also a 4.0-kb mRNA, the nature of which has yet to be determined (43, 44). Even more recently, Xie et al. (45) have reported the isolation and characterization of a cDNA from chicken embryo fibroblasts transformed with Rous sarcoma virus (RSV), which has some degree of similarity in its nucleotide sequence (60%) and deduced amino acid sequence (59%) to the mouse, sheep, and human recombinant PGS clones and hybridizes to a 4.1-kb mRNA. These investigators propose that this RSV-induced chicken embryo fibroblast mRNA may encode a new form of PGS. These results provide strong, albeit indirect, evidence to support our immunological and biochemical data that at least two forms of authentic PGS may exist in the rat ovary. Whether the two immunodistinct forms and Mr variants of PGS that we have observed in the rat ovary are products of two separate genes remains to be determined. The tissue distribution of PGS immunoreactive with antibody 3 in human dermal fibroblasts and rat kidney and ovary, and the presence of a 2.8-kb PGS



transcript in these tissues suggest that this variant of PGS is encoded by the gene for which cDNAs now exist (18-21). In contrast, PGS72 appears to represent another form of the enzyme and may be encoded by a separate gene, similar to or distinct from the gene encoding the PGS induced by RSV in chicken embryo fibroblasts.

1277

EDTA) and centrifuged at 30,000 x g to obtain a particulate pellet and a soluble (cytosol) cell fraction. The crude membrane pellet was resuspended in PE buffer containing 10 mM 3-[(3-cholamidopropyl)dimethyl-ammonio]1-propansulfanat (CHAPS) and sonicated (three times; 3 sec each time). Sonicates were briefly centrifuged (microfuge; 5 min) to remove large particulate material, and supernatants (solubilized cell extracts) were saved for further protein and immunoblot analyses. Immunoblot Analyses

MATERIALS AND METHODS Animals Intact immature female rats and timed pregnant rats were obtained from Holtzman Co. (Madison, Wl). Immature hypophysectomized female rats were obtained from Johnson Laboratories (Chicago, IL) 1 day after surgery (day 26). Six-monthold female rabbits were obtained from JoJo Rabbitry (Bandera, TX) for antibody generation.

Solubilized membrane extract proteins (200-400 ^g) were resolved by one-dimensional SDS-PAGE (4.5% stacking gel and 10% separating unless otherwise specified) and electrophoretically transferred to nitrocellulose filters (0.45 HM; Schleicher and Schuell, Keene, NH), essentially as described previously (30). Antibodies and iodinated [125l]protein-A (1 x 106 cpm/ml) were diluted in 10 mM Tris-buffered isotonic saline, pH 7.0, containing 2% Carnation (nonfat) dry milk and 0.1% merthiolate and were used sequentially to visualize immunopositive proteins, as described previously (30). Apparent Mr values were determined using Rainbow Mr standards.

Tissues SA follicles were dissected manually from ovaries of immature 29-day-old rats. PO follicles were isolated from ovaries of immature 29-day-old rats after treatment with a low dose of hCG (0.15 IU twice daily for 2 days) (46). Ovarian tissue remaining after removal of the PO follicles was retained as residual tissue. Corpora lutea were isolated from ovaries of pregnant rats on days 15 and 20 of gestation. Kidneys, adrenals, and hearts were isolated from the immature rats; uteri were isolated from hypophysectomized immature 29-day-old rats treated with 17^-estadiol (1.5 mg/day for 3 days), followed by FSH (NIH ovine FSH-16; 1.0 M g twice daily for 2 days). Short Term Follicle and Cell Incubations PO follicles from intact rats were incubated in Dulbecco's Minimum Essential Medium and Ham's F-12 Nutrients (1:1) at 37 C and with 95% oxygen-5% CO2 for 5-7 h in the presence of hormones, inhibitors, and other compounds, as indicated for each experiment. After the incubations, follicles in each treatment (—60 follicles/group) were pooled, collected by centrifugation, frozen, and stored at - 7 0 C until appropriate cell extracts were prepared. In selected experiments, granulosa and thecal cells of PO follicles were isolated and incubated separately (in multiwell plates; 95% air-5% CO2) or isolated after whole follicle incubations. Granulosa and thecal cells were also frozen and stored before the preparation of cell extracts. Media were saved for RIAs. Fibroblast Cultures Primary human dermal fibroblasts (a kind gift from Dr. Fred Ledley, Baylor College of Medicine, Houston, TX) were obtained from a skin biopsy and grown to confluence in Minimum Essential Medium containing 20% fetal bovine serum and glutamine, (95% air-5% O2) on 150-mm dishes. As cells were approaching confluency, experiments were initiated by adding fresh medium containing 5% fetal bovine serum with no added hormones, with 1 mM 8-bromo-cAMP, or with 2 U/ml recombinant human IL-1a. Cells were incubated for 12 h, then collected by trypsin treatment and centrifugation. Cell pellets were washed with PBS buffer and were frozen and stored at - 7 0 C until cell extracts were prepared. Preparation of Cellular Extracts All tissues were homogenized, as described previously (30), in PE buffer (10 mM potassium phosphate, pH 6.8, and 1 mM

Antibodies Antibodies to PGS were generated in rabbits and affinity purified using ovine PGS as the antigen and ligand, respectively, as described previously (30). Antibody 1 has been used previously; the two new antibodies are designated antibody 2 and antibody 3. RIA PGF2a and PGE2 were quantified by RIA of unextracted samples of incubation medium. RIA of PGF2a was performed according to the method of Johnson ef a/. (47), with an intraassay coefficient of variation of 4.3% and an interassay coefficient of 10.8%. The limits of sensitivity for the PGF2o assay was 2.5 pg/ml. PGE2 RIAs were performed according to the method of Jacobs ef al. (48), with intra- and interassay coefficients of variation less than 10% and a limit of sensitivity of 8.2 pg/ml. Immunofluorescence Ovaries of immature 29-day-old rats treated with low doses of hCG were collected within 6 h after injection of an ovulatory dose of hCG (10 IU, iv), embedded in O.C.T. compound, and stored at - 7 0 C. Frozen sections of the ovaries (kept at - 2 0 C until stained) were prepared, as previously described (30), using 2% paraformaldehyde fixation and 1 % Triton X-100 solubilization (31) or a snap 30-sec methanol fixation (30), and incubated with 50 /J affinity-purified antibody 2 or 3 (straight) and fluorescein-conjugated goat antirabbit immunoglobulin G (1:50 in PBS). Sections were washed, mounted in 90% glycerol in PBS containing 1 % phenylenediamine, and observed with a Zeiss Photomicroscope III (Zeiss, New York, NY). Materials Ovine LH (NIH oLH-23) and ovine FSH (NIH oFSH-16) were obtained from the National Hormone and Pituitary Program (Baltimore, MD); hCG from Organon Special Chemicals (West Orange, NJ); recombinant human IL-1« from Genzyme (Boston, MA); indomethacin, a-amanitin, mellitin, and estradiol (for injections) from Sigma (St. Louis, MO); cycloheximide from Nutritional Biochemicals (Cleveland, OH); CHAPS from Boehringer-Mannheim (Indianapolis, IN); electrophoretic supplies from Bio-Rad (Richmond, CA); tissue culture reagents from Gibco (Grand Island, NY); purified PGS from Oxford Biomedical


MOL ENDO-1991 1278

Research, Inc. (Oxford, Ml); 125Mabeled protein-A from ICN Biomedicals, Inc. (Casa Mesa, CA); CNBr-activated Sepharose 4B from Pharmacia LKB Biotechnology, Inc. (Piscataway, NJ); fluorescein isothiocyanate-labeled goat antirabbit immunoglobulin G from Atlantic Antibodies (Scarborough, ME); Freund's Complete and Incomplete Adjuvents from Difco Laboratories (Detroit, Ml); and Rainbow Mr markers from Amersham (Arlington Heights, IL).

Acknowledgments We would like to thank Dr. Joy Pate and Sandy Jones for help with the PGF2a RIAs, Drs. Dan Carson and Andrew Jacobs for help with the PGE2 RIAs, and Dana Gaddy-Kurten for help with the immunofluorescent microscopy.

Received May 20, 1991. Revision received June 27, 1991. Accepted July 1,1991. Address requests for reprints to: Dr. JoAnne S. Richards, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. This work was supported in part by NIH Grant HD-16229.

REFERENCES 1. Lee JB, Katayama S 1985 Prostaglandins, thromboxanes and leukotrienes. In: Wilson JD, Foster DW (eds) Textbook of Endocrinology. Saunders, Philadelphia, pp 13451362 2. Smith WL 1989 The eicosanoids and their biochemical mechansisms of action. Biochem J 259:315-324 3. Birkenfeld S, Zaltsman YA, Krispin M, Zakut H, Zor U, Kohen F 1987 Antitumor effects of inhibitors of arachadonic acid cascade on experimentally induced intestinal tumors. Dis Colon Rectum 30:43-46 4. Balakrishnan A, Cramer S, Bandyopadhyay GK, Imagawa W, Yang J, Elias J, Beattie CW, Gupta TKD, Nandi S 1989 Differential proliferative response to linoleate in cultures of epithelial cells from normal human breast and fibroadenomas. Cancer Res 49:857-862 5. Young MR, Knies S 1984 Prostaglandin E production by Lewis lung carcinoma: mechanism for tumor establishment in vivo. J Natl Cancer Inst 72:919-922 6. Lala PK, Parhar RS 1988 Cure of B16F10 melanoma lung metastasis in mice by chronic indomethacin therapy combined with repeated rounds of interleukin 2: characteristics of killer cells generated in situ. Cancer Res 48:10721079 7. LeMaire WJ, Leidner R, Marsh JM 1975 Pre and post ovulatory changes in the concentration of prostaglandins in rat Graafian follicles. Prostaglandins 9:221-229 8. Behrman HR, Caldwell BV 1986 Prostaglandins, thromboxanes, and leukotrienes. In: Yen SSC, Jaffe RB (eds) Reproductive Endocrinology. Saunders, Philadelphia, pp 154-176 9. Smith WL, DeWitt DL, Kraemer SA, Andrews MJ, Hla T, Maciag T, Shimokawa T 1990 Structure-function relationships in sheep, mouse, and human prostaglandin endoperoxide G/H synthases. Adv Prostaglandin Tromboxane Leukotriene Res 20:14-21 10. Fagan JM, Goldberg AL 1986 Inhibitors of protein and RNA synthesis cause a rapid block in prostaglandin production at the prostaglandin synthase step. Proc Natl Acad Sci USA 83:2771-2775 11. Wu KK, Hatzakis H, Lo SS, Seong DC, Sanduja SK, Tain HH 1988 Stimulation of cte novo synthesis of prostaglandin G/H synthase in human endothelial cells by phorbol ester. J Biol Chem 263:19043-19047 12. DeWitt DL, Rollins TE, Day JS, Gauger JA, Smith WL

Vol 5 No. 9

1981 Orientation of the active site and antigenic determinants of prostaglandin endoperoxide synthase in the endoplasmic reticulum. J Biol Chem 256:10375-10382 13. Smith WL 1986 Prostaglandin biosynthesis and its compartmentalization in vascular smooth muscle and endothelial cells. Annu Rev Physiol 48:251-262 14. Raz A, Wyche A, Needleman P 1989 Temporal and pharmacological division of fibroblast cyclooxygenase expression into transcriptional and translational phases. Proc Natl Acad Sci USA 86:1657-1661 15. Fu JY, Masferrer JL, Seibert K, Raz A, Needleman P1990 The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes. J Biol Chem 265:16737-16740 16. Bailey MJ, Makheja AN, Pash J, Verma M 1988 Corticosteroids suppress cyclooxygenase mesenger RNA levels and prostanoid synthesis in cultured vascular cells. Biochem Biophys Res Commun 157:1159-1163 17. Lin A H , Bienkowski MJ, Gorman RR 1989 Regulation of prostaglandin H synthase mRNA levels and prostaglandin biosynthesis by platelet-derived growth factor. J Biol Chem 264:17379-17383 18. DeWitt DL, Smith WL 1988 Primary structure of prostaglandin G/H synthase from the sheep vesicular gland determined from the complementary DNA sequence. Proc Natl Acad Sci USA 85:1412-1416 19. Merlie JP, Fagan D, Mudd J, Needleman P 1988 Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J Biol Chem 263:3550-3553 20. Dewitt DL, Kraemer SA, Meade EA1990 Serum induction and superinduction of PGG/H synthase mRNA levels in 3T3 fibroblasts. Adv Prostaglandin Thromboxane Leukotriene Res 21:65-68 21. Yokoyama C, Tanabe T 1989 Cloning of human gene encoding prostaglandin endoperoxide synthase and primary structure of the enzyme. Biochem Biophys Res Commun 165:888-894 22. Simonson MS, Wolfe JA, Dunn MJ 1990 Regulation of prostaglandin synthesis by differential expression of the gene encoding prostaglandin endoperoxide synthase. Adv Prostaglandin Thromboxane Leukotriene Res 21:69-72 23. Wong WYL, DeWitt DL, Smith WL, Richards JS 1989 Rapid induction of prostaglandin endoperoxide synthase in rat preovulatory follicles by luteinizing hormone and cAMP is blocked by inhibitors of transcription and translation. Mol Endocrinol 3:1714-1723 24. Bauminger S, Lindner HR 1975 Periovulatory changes in ovarian prostaglandin formation and their hormonal control in the rat. Prostaglandins 9:737-751 25. Testart J, Thebault A, Lefevre B 1983 In-vitro ovulation of rabbit ovarian follicles isolated after the endogenous gonadotrophin surge. J Reprod Fertil 68:413-418 26. Espey LL 1986 Cycloheximide inhibition of ovulation, prostaglandin biosynthesis and steroidogenesis in rabbit ovarian follicles. J Reprod Fertil 78:679-683 27. Espey LL 1980 Ovulation as an inflammatory reaction-a hypothesis. Biol Reprod 22:73-106 28. Curry Jr TE, Malik A, Clark MR 1987 Ovarian prostaglandin synthase: immunohistochemical localization in the rat. Am J Obstet Gynecol 157:537-543 29. Curry Jr TE, Bryant C, Haddix AC, Clark MR 1990 Ovarian prostaglandin endoperoxide synthase: cellular localization during the rat estrous cycle. Biol Reprod 42:307-316 30. Hedin L, Gaddy-Kurten D, Kurten R, DeWitt DL, Smith WL, Richards JS 1987 Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and evidence for hormonal induction preceding ovulation. Endocrinology 121:722-731 31. Wang J, Leung PCK 1989 Synergistic stimulation of prostaglandin E2 production by calcium ionophore and protein kinase C activation in rat granulosa cells. Biol Reprod 40:1000-1006 32. Smith WL, Wilkin GP1977 Immunohistochemistry of pros-


1279


33.

34.

35. 36.

37.

38. 39.

40.

taglandin endoperoxide-forming cyclooxygenases: the detection of the cyclooxygenases in rat, rabbit, and guinea pig kidneys by immunofluorescence. Prostaglandins 13:873-892 Grinwich DL, Kennedy TG, Armstrong DT 1972 Dissociation of ovulatory and steroidogenic actions of luteinizing hormone in rabbits with indomethacin, an inhibitor of prostaglandin biosynthesis. Prostaglandins 1:89-96 Armstrong DT, Grinwich DL 1972 Blockade of spontaneous and LH-induced ovulation in rats by indomethacin, an inhibitor of prostaglandin biosynthesis. Prostaglandins 1:21-28 Tsafriri A, Koch Y, Lindner HR 1973 Ovulation rate and serum LH levels in rats treated with indomethacin or prostaglandin E2. Prostaglandins 3:461 -467 Sogn JH, Curry Jr TE, Brannstrom M, LeMaire WJ, Koos RD, Papkoff H, Hanson PO 1987 Inhibition of folliclestimulating hormone-induced ovulation by indomethacin in the perfused rat ovary. Biol Reprod 36:536-542 Munalulu BM, Hillier K, Peddie MJ 1987 Effect of human chorionic gonadotrophin and indomethacin on ovulation, steroidogenesis and prostaglandin synthesis in preovulatory follicles of PMSG-primed immature rats. J Reprod Fertil 80:229-234 Espey LL, Norris C, Saphire D 1986 Effect of time and dose of indomethacin on follicular prostaglandins and ovulation in the rabbit. Endocrinology 119:746-754 Huslig RL, Malik A, Clark MR 1987 Human chorionic gonadotropin stimulation of immunoreactive prostaglandin synthase in the rat ovary. Mol Cell Endocrinol 50:237246 Tsai Al, Sanduja R, Wu KK, Evidence for two pools of prostaglandin H synthase in human endothelial cells. 7th

41. 42.

43.

44.

45.

46.

47.

48.

International Conference on Prostaglandins and Related Compounds, Florence, Italy, 1990 (Abstract 41) Maier JA, Hla T, Maciag T 1990 Cyclooxygenase is an immediate early gene induced by interleukin-1 in human endothelial cells. J Biol Chem 265:10805-10808 SteinerSM, Keutzer JC, Hirschhorn RR 1991 Prostaglandin endoperoxide synthase (cyclooxygenase) mRNA and protein production in mouse myoblasts and a differentiation-defective variant. Exp Cell Res 192:643-646 Simonson MS, Wolfe JA, Konieczkowski M, Sedor JR, Dunn MJ 1991 Regulation of prostaglandin endoperoxide synthase gene expression in cultured rat mesangial cells: induction by serum via a protein kinase-C-dependent mechanism. Mol Endocrinol 5:441-451 Rosen GD, Birkenmeier TM, Raz A, Holtzman MJ 1989 Identification of a cyclooxygenase-related gene and its potential role in prostaglandin formation. Biochem Biophys Res Commun 164:A1358-A1365 Xie W, Chipman JG, Robertson DL, Erickson RL, Simmons DL 1991 Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 88:2692-2696 Richards JS, Bogovich K1982 Effects of human chorionic gonadotropin and progesterone on follicular development in the immature rat. Endocrinology 111:1429-1438 Johnson MS, Ottobre AC, Ottobre JS 1988 Prostaglandin production by corpora lutea of rhesus monkeys: characterization of incubation conditions and examination of putative regulators. Biol Reprod 39:839-846 Jacobs AL, Decker GL, Glasser SR, Julian J, Carson DD 1990 Vectorial secretion of prostaglandins by polarized rodent uterine epithelial cells. Endocrinology 126:21252136


Prostaglandin H synthase.

Two antigenically distinct species of human interferon.

Prostaglandin H synthase and xenobiotic oxidation.

Crystallization of prostaglandin-H synthase for X-ray structure analysis.

Xenobiotic metabolism by prostaglandin H synthase.

Spectroscopic Evidence for the Two C-H-Cleaving Intermediates of Aspergillus nidulans Isopenicillin N Synthase.

Induction of prostaglandin H synthase in rat preovulatory follicles by gonadotropin-releasing hormone.

Regulation of prostaglandin H synthase activity in dog urothelial cells.

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages.

Chemical modification of prostaglandin H synthase with diethyl pyrocarbonate.

Body weight, hyperinsulinemia, and gonadotropin levels in the polycystic ovarian syndrome: evidence of two distinct populations.

H synthase 2 expression in rat endometrial stromal cells.

Prostaglandin D2 production and identification of prostaglandin H synthase within canine mast cell granule.

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat.

Distinct properties of the two isoforms of CDP-diacylglycerol synthase.

The role of prostaglandin H synthase in 3-methylindole-induced pneumotoxicity in goat.

Molecular Taxonomic Evidence for Two Distinct Genotypes of Mycobacterium yongonense via Genome-Based Phylogenetic Analysis.

Inhibition of rat brain prostaglandin D synthase by inorganic selenocompounds.

Influenza vaccine strain selection: equivalence of two antigenically distinct haemagglutinin variants of 1989 H3N2 influenza A virus in protection of mice.

Evidence that two naturally occurring human insulin receptor alpha-subunit variants are immunologically distinct.

The molecular weight of, and evidence for two types of subunits in, the molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

A high molecular weight centrosomal protein of mammalian cells is antigenically related to myosin II.

Influence of prostaglandins and a prostaglandin H synthase inhibitor on cervical secretion of the guinea-pig.

Common Variants of the Prostaglandin-Endoperoxide Synthase 2 Gene and Hepatocellular Carcinoma Susceptibility.