Prolactin action on luteal protein expression in the corpus luteum.

0013-7227/91/1294-1821$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 4 Printed in U.S.A.

Prolactin Action on Luteal Protein Expression in the Corpus Luteum* CONSTANCE T. ALBARRACIN AND GEULA GIBORIf Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612

ABSTRACT. Although it is well established that PRL is essential for luteal cell steroidogenesis and growth in the rat, its exact mechanism of action remains unknown. Whereas PRL stimulates growth and induces the content of specific proteins in several target tissues, its effect on proteins in the corpus luteum has not been examined. To determine whether PRL affects the synthesis of specific luteal protein(s), corpora lutea were obtained from rats hypophysectomized on day 3 of pregnancy and then treated with or without PRL (125 ng x 2/day) for 4 days. In this rat model, corpora lutea are exquisitely responsive to PRL and produce high levels of progesterone in response to PRL stimulation. Analysis of cytosolic proteins on one- and two-dimensional gel electrophoresis revealed a dramatic inhibitory effect of PRL on a 37,000 mol wt (MW) protein. This PRL-regulated protein (PRP) resolved into five separate isoelectric variants (pis 5.5, 5.6, 6.15, 6.6, and 6.85). Since differences in phosphorylation state can alter the isoelectric point of proteins, we examined the possibility that the 37,000 MW PRP is a substrate for phosphorylation. Luteal homogenates were incubated with [32P]ATP in the presence or absence of Ca2+, Ca2+/calmodulin, Ca2+/phospholipid, or cAMP. Phosphorylation of PRP was not affected by PRL treatment or the addition of specific cofactors. The least abundant isoelectric species (pi 6.85) was identified by Western blot analysis as annexin I, a known regulator of phospholipase A2 and prostaglandin synthesis. To determine whether the other four isoelectric species represent variants of the same protein, we developed

P

RL, a polypeptide hormone secreted by the anterior pituitary, has a diverse array of actions. In mammals, it acts on both the mammary gland and corpus luteum. In the mammary gland, PRL stimulates the expression of milk proteins, increasing both casein gene transcription and messenger RNA half-life (1, 2). In the rat corpus luteum, PRL is essential for progesterone biosynthesis and luteal cell growth (3, 4). Progesterone production by the corpus luteum is totally inhibited if Received May 14,1991. Address all correspondence and reprint requests to: Dr. Geula Gibori, Department of Physiology and Biophysics, University of Illinois, College of Medicine (M/C 901), Box 6998, Chicago, Illinois 60680. * Presented in part at the 22nd Annual Meeting of the Society for the Study of Reproduction, Columbia, MO, and at the Eighth Ovarian Workshop, Maryville, TN. This study was supported by NIH Grant HD-11119 (to G.G.) and by a Sigma Xi grant-in-aid (to C.T.A.). t NIH Merit Awardee.

a polyclonal antibody to the 6.15 isoelectric species which is remarkably regulated by PRL. In the absence of PRL, the 6.15 isoelectric species (37K) is the major protein of the PRP band. Conversely, PRL treatment resulted in the virtual disappearance of this luteal protein. The antibody recognized the 37K alone, indicating that the other 37,000 MW isoelectric species were distinct proteins. To examine the time course of PRL action on the expression of the 37K, luteal cells from day 3 pregnant rats were cultured with different doses of PRL from 6 h to 5 days. Western blot analysis of luteal cellular proteins indicated that PRL caused a decrease in the expression of the 37K within 6 h of treatment. Although it is well known that estradiol together with PRL is required for optimal growth of the rat corpus luteum, estradiol alone had no inhibitory action on the 37K nor did it affect the inhibitory action of PRL. Developmental studies performed on corpora lutea obtained from rats at different stages of pregnancy have revealed that the 37K is absent from highly active steroidogenic corpora lutea and appears abruptly before parturition during luteolysis. In summary, the results of this investigation demonstrate that PRL substantially down-regulates the expression of the abundant 37,000 MW protein(s) in the corpus luteum. The least abundant of these proteins has been identified as annexin I. The major protein (37K), to which we developed a specific antibody, is highly tissue-specific and is expressed only in the corpus luteum undergoing luteolysis. This 37K protein, which is remarkably regulated by PRL, can serve as a marker for both PRL action in the corpus luteum as well as luteal regression. {Endocrinology 129: 1821-1830,1991)

PRL is removed from the circulation (5) or if P R L binding to its receptor is prevented by antibodies against the P R L receptor (6). The rat corpus luteum thus provides a model which is highly and specifically responsive to PRL, allowing the systematic study of PRL action. In addition, progesterone levels provide a measurable focal endpoint of P R L action in the corpus luteum. Although the physiological roles of P R L have been well established, our knowledge on the cellular and molecular action of P R L in general and on the corpus luteum in particular is very limited, and the precise nature of the cellular events leading to the biological effects of P R L remains highly speculative. The corpus luteum is unique in that it produces both progesterone as well as substances that prevent progesterone secretion, such as prostaglandin F 2 a (PGF 2a ) (7,

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PRL EFFECT ON LUTEAL PROTEINS

8) and 20 a-hydroxysteroid dehydrogenase (20 a-HSD) (9). 20 a-HSD is the enzyme responsible for the conversion of progesterone to 20 a-hydroxyprogesterone, a weak progestagen, which cannot sustain pregnancy. PRL is known to dramatically inhibit the activity of 20 a-HSD, (9) but whether PRL has any effect on the amount of this enzyme has not been demonstrated. PRL has also been postulated to inhibit PG formation in the corpus luteum, thus preventing the PGF2«-induced decrease in progesterone biosynthesis (10). Recently, PRL has been shown to affect annexin expression in the pigeon crop sac (11). Annexins, also known as lipocortins, are a family of proteins which inhibit phospholipase A2, a key enzyme in PG biosynthesis (12-14). Thus, PRL may ultimately regulate progesterone production in the corpus luteum by affecting annexin expression. Cloning and expression studies have shown that the PRL receptor is a 40K protein with a large extracellular region involved in hormone binding, a single transmembrane segment, and a very short cytoplasmic domain (15). The PRL receptor is not a tyrosine kinase, and although various intracellular mediators have been proposed to be involved in PRL action, there are no clear effects of PRL on either cAMP, cGMP, or inositol phospholipids in the mammary gland (2,16-19). Despite these observations, PRL has been shown to enhance protein kinase-induced phosphorylation of cellular proteins in this tissue (20, 21). Phosphorylation/dephosphorylation of proteins is a major mechanism for controlling the activities of enzymes. Whether PRL induces alterations in the phosphorylation state of key proteins in the corpus luteum is still unknown. In this study, we have examined the changes in protein synthesis and phosphorylation that accompanies PRL stimulation of luteal steroidogenesis and growth.

Materials and Methods Materials

Endo • 1991 Vol 129 • No 4

(Boston, MA); silastic medical grade tubing was purchased from Dow Corning Corp. (Midland, MI); collagenase and dispase were obtained from Worthington Diagnostic Inc. (Freehold, NJ); bovine testis calmodulin (CaM) and 1% Nutridoma were purchased from Pharmacia Fine Chemicals (Piscataway, NJ) and Boehringer Mannheim Biochemicals (Indianapolis, IN), respectively. Animal model and tissue preparation Pregnant Sprague-Dawley rats were obtained from Holtzman (Madison, WI) on day 2 of pregnancy and were maintained at 24-26 C on a daily 14-h light, 10-h dark cycle with free access to Purina rat chow and water. Rats were hypophysectomized using a transauricular approach on day 3 of pregnancy. Surgery was performed under ether anesthesia with minimal stress to the animals. Completeness of hypophysectomy was evaluated by examination of the pituitary removed at the time of operation, visualization of the pituitary fossa at autopsy and serum progesterone levels of less than 2 ng/ml for vehicletreated control or more than 40 ng/ml for PRL-treated animals. Hypophysectomized rats were injected sc with PRL (NIDDK ovine PRL-18; 30 IU/mg) in 50% polyvinylpyrrolidone, pH 9.0, twice daily (125 ng in 0.25 ml/injection). Control rats were treated with vehicle. An additional group of intact pregnant rats was included in the study. For the estradiol studies, rats were treated with estradiol-filled silastic implants either alone or with PRL. Blood samples were obtained by cardiac puncture under light ether anesthesia. Animals were killed 2-4 days after surgery, and corpora lutea were dissected from the ovaries, weighed, and rapidly frozen at -80 C until processed. Various tissues were also obtained from the same animals. Tissues were suspended in 2 ml cold homogenization buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol at pH 7.4, and 250 mM sucrose followed by homogenization in a PotterElvejehm homogenizer (Wheaton, Millville, NJ). The luteal homogenate was centrifuged at 1000 x g for 10 min to remove cell debris followed by centrifugation at 100,000 X g for 60 min to obtain particulate and cytosolic fractions. The fractions were then stored at -80 C and aliquots assayed for protein content using the Bradford assay (22). One and two dimensional analysis

Tris, EDTA, phenylmethylsulfonyl fluoride, dithiothreitol, j8-mercaptoethanol, polyvinylpyrrolidone (MW 40,000), Coomassie brilliant blue R250, bromophenol blue, Ponceau S, cAMP, 3-isobutyl-l-methylxanthine (IBMX), 1,2-diolein, dimethylsulfoxide, Freund's adjuvant, deoxyribonuclease (DNase), Ham's F12, and McCoy's 5a were obtained from Sigma Chemical Co. (St. Louis, MO); acrylamide and bis-acrylamide were products of Accurate Chemical Inc. (Westbury, NY) and Eastman Kodak (Rochester, NY), respectively; ultrapure urea was obtained from ICN Biomedical, Inc. (Cleveland, OH); ampholines were products of Serva (Westbury, NY), LKB (Piscataway, NJ), and Sigma; [7-32P]ATP was purchased from the Amersham Corporation (Arlington Heights, IL) or from NEN Research Products (Wilmington, DE); estradiol 17/3 was a product of Steraloids Co. (Wilton, NH); [l,2,6,7-3H]progesterone (94.1 Ci/mmol) was a product of New England Nuclear Co.

Luteal proteins were solubilized and separated by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5-18% gradient gels as described by Laemmli (23). Isoelectric focusing of cytosolic proteins (100 jig/tube) was performed with a pH range of 4.5-8 using the system of O'Farrell (24). Separation in the second dimension was carried out on 7.5-18% gradient gels and the proteins stained with Coomassie blue. Development of polyclonal antibody Cytosolic luteal proteins (200 /ig/tube) from vehicle-treated rats were separated by two-dimensional electrophoresis. Proteins were transferred onto nitrocellulose and the abundant 6.15 isoelectric species (37K) of the 37,000 MW band was


PRL EFFECT ON LUTEAL PROTEINS excised for injection into rabbits. The excised protein was dissolved in 0.5 ml dimethylsulfoxide (25), emulsified with an equal vol of Freund's complete adjuvant, and injected intradermally on multiple sites on the back of a New Zealand white male rabbit (Hazelton, Denver, PA). A booster injection (prepared exactly like the initial injection except that Freund's incomplete adjuvant was used) was given every 2 weeks thereafter. Rabbits were bled 4 weeks after the initial injection and then every 2 weeks after that. The antisera was screened for the presence of antibodies specific for the 37K using Western blot analysis. After the second boost, we detected rising titers of an antibody specific for the 37K. All Western blot analyses were conducted using antisera from the fourth bleeding obtained after the third booster injection. This antisera recognized the 37K at dilutions ranging from 1:1000-1:8000. Western blot analysis The gel was washed twice for 15 min in 20 mM Tris base, 500 mM NaCl, pH 7.5 (TBS) followed by the electrophoretic transfer of proteins onto nitrocellulose paper in 25 mM Tris, 192 mM glycine, and 20% methanol buffer at 250 mA for 17 h at 4 C. The nitrocellulose was stained with either fast green or Ponceau S to visualize the proteins, whereas the gel was stained with Coomassie blue to assess efficiency of protein transfer. The blot was washed twice with TBS for 15 min each, blocked with 1-3% gelatin in TBS for 1 h followed by two 15-min washes with TBS. The nitrocellulose was then incubated for 2 h with a 1:2000 dilution of primary antibody. This concentration was shown to be optimal after immunoblotting with serial dilutions ranging from 1:1000-1:8000 of either annexin I antisera or the antibody generated to the 37K. Annexin I antisera was kindly provided by Dr. R. Blake Pepinsky of Biogen Research Corporation (Cambridge, MA) (26). The blots were then washed in TBS for 2 X 15 min and immunoreactive proteins detected using either 125I-labeled protein A (2 X 105 cpm/ml) (ICN, Irvine, CA) or alkaline phosphatase-labeled secondary antibody (Stratagene, La Jolla, CA). All washes and incubations were carried out at room temperature. Blots incubated with 125I-labeled protein A were exposed to Kodak XAR5 film with or without a DuPont Cronex intensifying screen (DuPont, Wilmington, DE) for 17 h at -80 C. Phosphorylation assay Ca2+- and cAMP-dependent phosphorylation were studied in a cell-free system using the appropriate cofactors and equal amounts of proteins. Previous experiments demonstrated optimal conditions for phosphorylation using 25-75 ng protein per reaction after an incubation period of 1 min at 30 C. The phosphorylation assay was carried out using equal amounts of proteins in a final vol of 100 fA 25 mM Tris (pH 7.4), 6 mM MgSO4,1 mM EDTA, 1 mM dithiothreitol with or without Ca2+ (1 mM), CaM (1 jug/ml), phospholipids (L-a-phosphatidyl-Lserine, 40 Mg/ml; diolein, 4 Mg/ml), or cAMP (1 fiM) plus 1 mM IBMX. The mixture was preequilibrated for at least 5 min at 30 C, phosphorylation was initiated by the addition of 1-4 /*Ci [7-32P]ATP (0.1-0.2 mM), incubated for 1 min, and terminated with the addition of 40 ^1 stop solution containing 20% glycerol, 10% mercaptoethanol, 9% SDS, 0.125 M Tris, and 0.02% bromophenol blue followed by boiling for 3 min. Proteins were

1823

then separated by SDS-PAGE with the appropriate mol wt (MW) markers included in all gels. Gels were subsequently stained in 20% methanol, 7% acetic acid, and 0.05% Coomassie blue overnight and destained in 10% methanol and 7% acetic acid for 4-6 h. The gels were then dried and exposed to Kodak XAR-5 film with or without an intensifying screen. Luteal cell culture Luteal cells were dispersed according to the method of Nelson et al. (27). Briefly, whole corpora lutea were obtained from day 3 pregnant rats and dispersed in collagenase (50 U/ml), dispase (2.4 U/ml), and DNase (200 U/ml) at 37 C at 30-min intervals for 2 h followed by treatment with 0.02% EDTA in PBS (pH 7.5) containing 2% BSA and 25 mM HEPES. Cells were cultured in Ham's F12:McCoy's 5a with 10% fetal calf serum for the first 24 h and with 1% Nutridoma thereafter. RIA Serum progesterone was assayed by RIA (28) after hexane extraction using a highly specific antiserum kindly provided by Dr. G. D. Niswender (Colorado State University, Fort Collins, CO) (GDN-337).

Results Effect of PRL on luteal proteins To examine the effect of PRL on luteal protein content, rats were hypophysectomized on day 3 of pregnancy and treated with PRL for 4 days. Luteal proteins obtained 4 days after hypophysectomy were analyzed by SDS-PAGE, and serum progesterone was measured by RIA. As shown in Fig. 1 (left panel), PRL treatment induced a remarkable decrease in the expression of a 37,000 MW protein. This PRL-regulated protein (PRP) was localized primarily in the cytosol and was barely detectable in the particulate fraction. The down-regulatory effect of PRL was accompanied by a dramatic increase in progesterone production (Fig. 1, right panel), suggesting an inverse relationship between PRP and steroidogenesis. On two-dimensional gel electrophoresis, the 37,000 MW PRP resolved into five different isoelectric species with the most abundant species virtually disappearing after PRL treatment (Fig. 2). Phosphorylation of 37,000 MW PRP Since transfer of phosphate groups during protein phosphorylation can result in the addition of negative charges which can alter the isoelectric point of proteins, we wanted to determine whether PRP is phosphorylatable. Cytosolic proteins obtained from rats treated with or without PRL were phosphorylated in the presence of cofactors to Ca2+-CaM, Ca2+/phospholipid, and cAMPdependent kinases. Basal protein phosphorylation was monitored in the absence of appropriate cofactors. Figure 3 shows the luteal phosphoprotein profile of PRL- (right



1824

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PROTEIN PROFILE

Endo • 1991 Vol 129 • No 4

-PROLACTIN PROTEIN PROFILE

kD

O

O

2

Q.

Q.

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205 116

7.97

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1 16 97

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97 66

HYPOX

HYPOX*PRL

NORMAL

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B

+PROLACTIN PROTEIN PROFILE

4.5

FlG. 1. Effect of PRL on luteal proteins. Rats were hypophysectomized on day 3 of pregnancy and treated with 125 ng PRL twice daily (HYPOX + PRL) or vehicle (HYPOX) for 4 days. Normal day 7 pregnant rats (NORMAL) were also included as control. Corpora lutea were isolated and homogenized in buffer. The homogenate was centrifuged at 1000 X g for 10 min to remove cell debris, and a supernatant cell extract was obtained. Protein samples were separated by SDSPAGE on 7.5-18% gradient gels (leftpanel). Each treatment group has at least four rats. The arrow represents the PRP whose content is increased by PRL. The right panel represents serum progesterone levels.

7.97

kD 205 1 16 97

36 29

panel) and vehicle (leftpanel)-treated rats. No phosphate transfer could be observed in the PRP. Phosphorylation of this protein was not induced by cofactors nor was it affected by PRL treatment, suggesting that the different variants of PRP seen in two-dimensional gels are not due to differences in their phosphorylation states using the above conditions.

24

20

L

Identification of annexin I by western blot analysis

FlG. 2. Two-dimensional analysis of PRP. Isoelectric focusing was done using a pH range of 4.5-8 using cytosolic proteins (100 jig/tube) from corpora lutea taken from non-PRL (-PROLACTIN) and PRL (+PROLACTIN) treated rats. Proteins were separated on the second dimension by SDS-PAGE and then transferred onto nitrocellulose and stained with fast green. The large arrow on the left indicates PRP. Small arrows 1-5 represent the isoelectric species of PRP.

Since PRP is within the MW range of annexin I (26), a protein found to be regulated by PRL in pigeon crop sac (11), it became of interest to examine whether the luteal PRP is annexin. Cytosolic and particulate fractions were obtained from corpora lutea of hypophysectomized rats treated with or without PRL and from intact pregnant rats. Equal amounts of proteins were separated by one-dimensional SDS-PAGE, electroblotted, and probed with a highly specific antibody to annexin (26). As shown in Fig. 4, the antibody reacted with the cytosolic PRP. PRL treatment caused a marked decrease in

the content of this protein to levels equivalent to those of the normal nonhypophysectomized pregnant group. However, Western blot analysis of the two-dimensional gels (Fig. 5) demonstrated that not all of the five isoelectric species were recognized by the annexin I antibody. The antibody recognized only the least abundant isoelectric species (pi 6.85) with an isoelectric point similar to annexin I (29) (Fig. 5, right panels). The content of this protein also decreased after PRL treatment. These results indicate that PRP is made up of several proteins, one of which was immunoreactive with the annexin I antibody.


PRL EFFECT ON LUTEAL PROTEINS - PROLACTIN

1825

+ PROLACTIN

kD COFACTOR

o

o

o 116 97 66 45

36 29 24

20 14

Protein (ug/lane)

50 50

50 100 100 100 CYTOSOLIC

50

50

50

100

100 100

PARTICULATE

FIG. 3. Phosphorylation of luteal proteins. Equal amounts of luteal cytosolic proteins (50 jig) were incubated with [Y- 3 2 P]ATP in the presence or absence of Ca2+ (1 mM), Ca2+/CaM (1 /ug/ml), Ca2+ with phospholipids (phosphatidylserine, 40 Mg/ml; diolein, 4 ^g/ml), or cAMP (1 nM) with IBMX (1 mM). Phosphorylated proteins were visualized by autoradiography. Arrows indicate the 37K protein. The autoradiographs above represent samples taken from hypophysectomized rats treated with (right) or without (left) PRL.

FIG. 4. Identification of annexin I by Western blot. Cytosolic and particulate fractions were obtained by differential centrifugation. Luteal proteins (50 and 100 jig/lane) of rats hypophysectomized and treated for 4 days with (+) or without (—) PRL or of intact pregnant rats (NORMAL) were separated by SDS-PAGE and electroblotted onto a nitrocellulose membrane. The blots were probed with a 1:2000 dilution of a polyclonal antibody to annexin I and [125I]protein A (2 x 105 cpm/ml).

Development of a specific polyclonal antibody to the 37K

with PRL, PRL + estradiol, or estradiol alone. Luteal proteins obtained from these different treatment groups were resolved by one-dimensional gel electrophoresis (Fig. 8). Estradiol had no inhibitory effect when administered alone, nor did it affect the inhibitory action of PRL on PRP (Fig. 8). It is also evident, as shown in Fig. 8 {lower panel), that the presence of PRP has a striking correlation with low serum levels of progesterone. To further characterize PRL regulation of the 37K, cells were dispersed from corpora lutea of day 3 pregnant rats and cultured with different doses of PRL (1-1000 ng/ml). Luteal cells were harvested at different times thereafter. Western blot analyses indicated that within 6 h the cellular content of the 37K is decreased by the highest dose of PRL (Fig. 9). This inhibition can be demonstrated even after 5 days of culture in serum-free media (data not shown). The inhibitory effect of PRL appears to be an all or none effect and is not observed at lower concentrations of the hormone.

To further establish that these different species were not isoforms of the same protein, we developed a polyclonal antibody highly specific to the major isoelectric variant (pi 6.15). Western blot analysis revealed that the antibody recognizes only the 6.15 isoelectric species (37K) and none of the other 37,000 MW proteins (Fig. 6), indicating that these proteins are not variants of the 37K. In addition, the 37K antisera did not immunoreact with a purified sample of annexin obtained from Dr. R. B. Pepinsky (data not shown). To examine the tissue specificity of the major 37K, both steroidogenic and nonsteroidogenic tissues were obtained from normal rats and from hypophysectomized rats treated with or without PRL (Fig. 7). In addition, proteins were isolated from the postpartum corpus luteum, mammary gland, and from placental tissue. Figure 7 shows that the 37K is present in the corpus luteum alone and could not be demonstrated in any of the other tissues examined. These experiments indicate that the 37K protein is expressed specifically in the corpus luteum and is immunologically distinct from annexin I, a ubiquitous protein present in various tissues (26). Hormonal regulation of the 37,000

MWprotein(s)

To examine estradiol effect on PRL inhibition of PRP, day 3 pregnant rats were hypophysectomized and treated

Ontogeny of the 37K during pregnancy

Since the 37K was observed in corpora lutea undergoing luteolysis after hypophysectomy, we examined the developmental expression of the PRL-regulated 37K during pregnancy. Luteal proteins were obtained from normal rats from days 5-21 of pregnancy. Equal amounts of



1826 A

Endo • 1991 Vol 129 • No 4

-PROLACTIN IMMUNOBLOT

PROTEIN PROFILE

kD 205 116 97 66 45 •

292420

+ PROLACTIN

B

+ PROLACTIN PROTEIN PROFILE

IMMUNOBLOT

4.5

2 0L

2QL

FIG. 5. Identification of annexin I by Western blot analysis of two-dimensional gels. Equal amounts of cytosolic luteal proteins taken from nonPRL and PRL-treated rats were resolved by two-dimensional electrophoresis. Arrows indicate isoelectric species of PRP. Annexin I in non-PRL (A) and PRL (B)-treated luteal tissues were compared by immunoblot analyses using the antibody to annexin I. The arrows in the right panel represent specific recognition of the 6.85 species of PRP by the annexin I antibody. kD

4.5

66 45 -

29 24 -

FiG. 6. Western blot analysis of two-dimensional gel using the 37K antisera. Cytosolic luteal proteins from hypophysectomized were resolved by one-dimensional (left) and two-dimensional (right) electrophoresis. Proteins were transferred onto nitrocellulose paper and the blots probed with a 1:2000 dilution of a polyclonal antibody to 37K protein and [125I]protein A (2 x 105 cpm/ml). Immunoreactive proteins were detected by autoradiography. Arrow indicates the 37K protein and the specific recognition of the 6.15 species of PRP.

cytosolic proteins were loaded on SDS polyacrylamide gels and immunoblotted with the 37K antisera. The results shown in Fig. 10 indicate that the 37K is abundantly expressed at the end of pregnancy when progesterone secretion by the corpus luteum is markedly de-

creased. This finding combined with those observed in Fig. 7, which indicate that this protein is also abundantly expressed in the involuting postpartum corpus luteum, suggests that the 37K is either the result or the cause of luteolysis. Discussion The present study demonstrates, for the first time, that PRL markedly down-regulates the expression of major 37,000 MW proteins (PRP) in the corpus luteum. This inhibitory effect is temporally related to PRL stimulation of progesterone synthesis and suggests an important role for PRP as a mediator of PRL action on luteal steroidogenesis. PRP resolves into several isoelectric variants, two of which (pi 6.15 and 6.85) are considerably reduced by PRL. However, the different isoelectric forms do not share common antigenic sites as evidenced by the absence of cross-reaction among the different species. A highly specific antibody to annexin I recognized only one variant (pi 6.85), whereas the antibody developed against the major 6.15 isoelectric variant (37K) immunoreacted



1827 PROTEIN

a SKELETAL > MUSCLE PANCREAS

I 4

_j

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ADRENAL

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I i £I si I

TREATMENT

kD 205

116 97 66

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FIG. 7. Tissue-specific expression of the 37K protein. Proteins were obtained from postpartum corpus luteum, mammary gland, and from placental tissue. In addition, proteins were isolated from skeletal muscle, pancreas, spleen, adrenal, liver, lung, thymus, brain, kidney, oviduct, and uterus of hypophysectomized rats treated with vehicle (—PRL) and PRL (+PRL) for 3 days and from normal day 6 pregnant rats (normal). Hypox CL represents luteal tissue from hypohysectomized rats. Western blot analysis was done using the 37K antisera.

only with this species. The 37K antisera did not crossreact with recombinant annexin I (obtained from Dr. R. B. Pepinsky), further indicating that the different isoelectric forms are separate proteins, immunologically distinct from each other. Recent studies in the pigeon crop sac have shown that PRL affects annexin gene expression (11). Annexins were initially characterized as mediators of the antiinflammatory action of glucocorticoids (30, 31). Although their induction by glucocorticoids is now questionable (32, 33), there is sufficient evidence to indicate that annexins are immunologically active proteins capable of regulating cellular inflammatory responses in the same manner as glucocorticoids (31, 34). Studies have shown that purified annexin decreases the activity of natural killer cells and the antibody-dependent cellular cytotoxicity of lymphocytes in a dose-dependent manner (35). In addition, T suppressor function induced by purified annexin is selectively inhibited by antibodies against annexin (36). The functional role of annexin in the corpus luteum and its down-regulation by PRL remains to be defined. It is possible that annexin may provide the missing link between PRL and the immune system. It has become increasingly evident that PRL can regulate the immune system affecting both humoral and cellular immunity. Reduction of circulating levels of PRL, either by hypophysectomy or bromocriptine, suppresses im-

|

45

•S

3015

FIG. 8. Effect of PRL and estradiol on the 37K protein. Proteins were obtained from rats hypophysectomized on day 3 of pregnancy and treated with vehicle (-PRL); PRL (+PRL); estradiol (sc implant; E2); PRL with estradiol (PRL + E2) for 3 days and from normal day 6 pregnant rats (normal). The bottom panel represents serum progesterone levels of the respective treatment group.

PROLACTIN (ng/ml)

o •o

x o a

37K

FIG. 9. Time course and dose response of the 37K protein to PRL treatment. Luteal cells were obtained from day 3 pregnant rats and cultured with or without PRL (0-1000 ng/ml) for 6 h of culture. Hypox CL indicates luteal cytosolic proteins obtained from hypophysectomized rats. The 37K was detected using Western blot analysis.



1828 DAYS OF PREGNANCY 9

10

11

14

1 5 1 7 1 9

1 I

21

-37K

PROTEIN

FlG. 10. Ontogeny of the 37K protein during pregnancy. Equal amounts of luteal cytosolic proteins were obtained from pregnant rats on days 5-21 of pregnancy. Proteins were resolved on 10% gels and immunoblotted with 37K antisera. Immunoreactive proteins were visualized using alkaline phosphatase-labeled secondary Ab. Hypox CL are luteal cytosolic proteins obtained from hypophysectomized rats not treated with PRL.

mune responses (37, 38). PRL replacement restores immune competence, enhancing antibody production and T lymphocyte function (37-39). More recently, PRL has been shown to induce lymphocyte proliferation (39) and IL-2 receptor expression (40). Whether PRL regulation of luteal steroidogenesis and function may involve the immune system with annexin serving a pivotal role is not known. It is certainly possible considering the downregulation of annexin in the corpus luteum by PRL and the close negative association of this protein with steroidogenesis. Regulation of annexin expression may also represent a mechanism by which PRL may regulate PG synthesis in the corpus luteum since annexins are known to inhibit phospholipase A2 activity (12, 31, 32). Our results indicate a predominantly cytosolic localization of this protein in the corpus luteum. Annexin I has a calcium-dependent association with membranes and has been demonstrated in association with particulate fractions. It is possible that chelation of free calcium by EGTA contained in our homogenization buffer results in dissociation of annexin I from the membranes resulting in very minimal amounts of the protein in the particulate fraction. This ability of EGTA to cause dissociation of annexin from membrane has been amply demonstrated in various tissues (41-43). Changes in the phosphorylation state has been a well established mode of regulation of enzyme or protein activity. In the corpus luteum, PRL has been shown to alter the Ca/CaM-dependent phosphorylation of elongation factor 2, a protein crucial for the proper translation of proteins (44). However, the phosphorylation of

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PRP was not affected by any of the cofactors involved. Although annexin has been shown to be a substrate for phosphorylation and is an integral part of PRP, no phosphate transfer onto this protein could be demonstrated. It is possible that phosphorylation sites have been filled in vivo, preventing further phosphate transfer onto the molecule during the assay. Furthermore, studies involving the phosphorylation of annexin have been done on purified samples using concentrations several orders of magnitude greater than what is found in vivo. Another possibility is that annexin is differentially regulated in different tissues. Indeed, PRL appears to have tissuespecific effects on annexin, decreasing annexin content in the corpus luteum and stimulating annexin expression in the pigeon crop sac (11). Studies in lymphocytes indicate that in vivo and in vitro phosphorylation of annexin is accompanied by a decrease or complete loss of its phospholipase A2 inhibitory activity (45), whereas phosphorylation of annexin I in human placenta enhances its Ca2+-dependent phospholipid binding (46, 47). Bearing in mind that phosphorylation has different effects on the activity of annexin obtained from these two different tissues, it is more than likely that regulation of annexin activity by phosphorylation occurs in a tissuespecific manner. The identity of the major 6.15 species (37K) of the 37,000 MW PRP has not yet been determined. The 37K is the predominant species in corpora lutea undergoing luteolysis. PRL treatment causes its virtual disappearance from the corpus luteum and a concomitant increase in progesterone production. The relative abundance and tissue-specific expression of this protein in the corpus luteum, its marked regulation by PRL, and its close reverse association with progesterone levels in the corpus luteum strongly suggests that the 37K plays an important role in the tropic action of PRL in the corpus luteum. A possible candidate for this protein is 20 a-HSD, a 36,000 MW cytosolic protein (48) responsible for the conversion of progesterone to 20 a-hydroxyprogesterone, a weak progestagen, which cannot sustain pregnancy. PRL is known to inhibit the activity of 20 a-HSD (9) but whether it has any effect on the amount of this enzyme is not known. At present, 20 a-HSD has not been totally purified, and there are no available antibodies to this protein. Although the expression of the 37K throughout pregnancy closely parallels the ontogeny of 20 aHSD activity in the rat ovary (49), we have failed to detect 20 aHSD activity in this protein. It is interesting to note the absence of the 37K during the development of pregnancy and its abrupt appearance at the end of pregnancy when the ability of the corpus luteum to produce progesterone is markedly reduced. This protein could signal the termination of pregnancy and the onset of parturition. The cloning of the 37K, presently in progress in our


PRL EFFECT ON LUTEAL PROTEINS laboratory, should provide more definitive answers regarding its identity and function in the corpus luteum. It is well known that PRL and estradiol are required for optimal luteal growth (50-52). PRL maintains estrogen receptor numbers, thus exerting a permissive effect on the action of this hormone (51, 52). Similarly, estradiol induces PRL receptors (53), thereby enhancing PRL action. However, these two hormones do not appear to have a synergistic effect on the 37K. Estradiol alone does not affect the luteal content of the 37K protein by itself, nor does it affect PRL inhibition of the 37K and PRL stimulation of progesterone synthesis. This indicates that the inhibitory action of PRL on the luteal 37K is independent from estradiol stimulation of the tropic effect of PRL. In summary, we have shown, by both in vivo and in vitro methods, that PRL causes a remarkable downregulation of the expression of several 37,000 MW proteins in the rat corpus luteum. One of these proteins was immunologically identified as annexin I. We have developed a specific antibody to the major PRL-regulated 37K (pi 6.15) and demonstrated that this protein is abundantly and specifically expressed in corpora lutea undergoing luteolysis. Whether the down-regulation of this protein is necessary for the normal process of steroidogenesis and whether PRL stimulation of progesterone biosynthesis occurs as a result of a decrease in the luteal content of this protein remains to be investigated. However, it is clearly evident from the results of this investigation that the 37K protein is consistently and profoundly affected by PRL treatment and can serve as an excellent marker for both PRL action in the corpus luteum as well as luteal regression.

Acknowledgments We wish to express our sincere appreciation to Dr. R. B. Pepinsky for his gifts of recombinant lipocortin and lipocortin antisera and for critical reading of this manuscript. We wish to thank Dr. G. D. Niswender for kindly providing the progesterone antiserum, Linda Alaniz for photographs and Rosemary Clepper for animal care.

References 1. Guyette WA, Matusik RJ, Rosen JM 1979 Prolactin-mediated transcriptional and posttranscriptional control of casein gene expression. Cell 17:1013-1023 2. Matusik RJ, Rosen JM 1980 Prolactin regulation of casein gene expression: possible mediators. Endocrinology 106:252-259 3. Rothchild 11981 The regulation of the mammalian corpus luteum. Recent Prog Horm Res 37:183-298 4. Armstrong DT, Miller LS, Knudsen KA 1969 Regulation of lipid metabolism and progesterone production in rat corpora lutea and ovary interstitial elements by prolactin and luteinizing hormone. Endocrinology 85:393-401 5. Gibori G, Richards JS 1978 Dissociation of two distinct luteotropic effects of prolactin: regulation of luteinizing hormone-receptor content and progesterone secretion during pregnancy. Endocrinology 102:767-774

1829

6. Shiu RPC, Friesen HG 1976 Blockade of prolactin action by an antiserum to its receptors. Science 192:259-261 7. Phariss BB & Wyngarden LJ 1969 The effect of prostaglandin F2a on the progestagen content of ovaries from pseudopregnant rats. Proc Soc Exp Biol Med 130:92-94 8. Bartmann W, Beck G, Lerch U, Teufel H, Scholkens B 1979 Luteolytic prostaglandins. Synthesis and biological activity. Prostaglandins 17:301-311 9. Lamprecht SA, Lindner HR, Strauss III SF 1969 Induction of 20 a hydroxysteroid dehydrogenase in rat corpora lutea by pharmacological blockade of pituitary prolactin secretion. Biochim Biophys Acta 187:133-143 10. Ueda K, Ochiai K, Rothchild 11985 A luteotropic action of prolactin suppression of intraluteal prostaglandin product or effect? Endocrinology 116:772-778 11. Horseman ND 1988 A prolactin-inducible gene product which is a member of the calpactin/lipocortin family. Mol Endocrinol 3:773779 12. Flower RJ, Blackwell GJ 1976 The importance of phospholipaseA2 in prostaglandin biosynthesis. Biochem Pharmacol 25:285-291 13. Pepinsky RB, Tizard R, Mattaliano RJ, Sinclair LK, Miller GT, Browning JL, Chow EP, Burne C, Huang K-S, Pratt D, Wachter L, Hession C, Frey AZ, Wallner BP 1988 Five distinct calcium and phospholipid binding proteins share homology with lipocortin I. J Biol Chem 263:10799-10811 14. Crumpton MJ, Dedman JR 1990 Protein terminology tangle. Nature 345:212 15. Boutin JM, Jolicoeur C, Okamura H, Gagnon J, Edery M, Shirota M, Benville D, Dusanter-Fourt I, Djiane J, Kelly PA 1988 Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene. Cell 53:69-77 16. Bolander Jr FF 1985 Possible roles of calcium and calmodulin in mammary gland differentiation in vitro. J Endocrinol 104:29-34 17. Houdebine LM, Djiane J, Dusanter-Fourt I, Martel P, Kelly PA, Devinoy E, Servely JL 1985 Hormonal action controlling mammary activity. J Dairy Sci 68:489-500 18. Devinoy E, Hubert C, Jolivet G, Thepot D, Clergue N, Desaleux M, Dion M, Servely JL, Houdebine LM 1988 Recent data on the structure of rabbit milk protein genes and on the mechanism of the hormonal control of their expression. Reprod Nutr Dev 28:1145-1151 19. Etindi RN, Rillema JA 1988 Prolactin induces the formation of inositol triphosphate in cultured mouse mammary gland explants. Biochim Biophys Acta 968:385-391 20. Turkington RW, Riddle M 1969 Hormone-dependent phosphorylation of nuclear proteins during mammary gland differentiation in vitro. J Biol Chem 244:6040-6046 21. Rillema JA 1985 Actions of prolactin on [32P] -phosphate uptake and incorporation into proteins and phospholipids in mouse mammary gland explants. In: MacLeod RM, Turner MO, Scapagnini U (eds) Prolactin, Basic and Clinical Correlates. Liviana Press, Padova, vol 1:335-341 22. Bradford MM 1976 A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of dye-binding. Anal Biochem 72:248-254 23. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage, T4. Nature 227:680-685 24. O'Farrell PH 1975 High resolution two-dimensional electrophoresis of proteins. J Biol Chem 205:4007-4021 25. Knudsen KA 1985 Proteins transferred to nitrocellulose for use as immunogens. Anal Biochem 147:285-288 26. Pepinsky RB, Sinclair LK, Browning JL, Mattaliano RJ, Smart JE, Chow EP, Falbel T, Ribolini A, Garwin JL, Wallner BP 1986 Purification and partial sequence analysis of a 37-kDa protein that inhibits phospholipase A2 activity from rat peritoneal exudates. J Biol Chem 261:4239-4246 27. Nelson S, Jayatilak PG, Hunzicker-Dunn M, Gibori G Characterization of two luteal cell types from the pregnant rat corpus luteum. Program of the 20th Annual Meeting of the Society for the Study of Reproduction, Urbana IL, 1987, p 135 (Abstract) 28. Gibori G, Antzak E, Rothchild I 1977 The role of estrogen in the regulation of luteal progesterone secretion in the rat after day 12


1830


of pregnancy. Endocrinology 100:1483-1495 29. Creutz CE, Zaks WJ, Hamman HC, Crane S, Martin WH, Gould KL, Oddie KM, Parsons SJ 1987 Identification of chromaffin granule-binding proteins. Relationship of the chromobindins to calelectrin, synhibin, and the tyrosine kinase substrates p35 and p36. J Biol Chem 262:1860-1868 30. Blackwell GJ, Carnuccio R, DiRosa M, Flower RJ 1980 Macrocortin: a polypeptide causing the anti-phospholipase effect of glucocorticoids. Nature 287:147-149 31. Hirata F, Schiffmann E, Venkatasubramanian K, Salomon D, Axelrod J 1980 A phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids. Proc Natl Acad Sci USA 77:2533-2536 32. Bronnegard M, Anderson O, Edwall D, Lund J, Norstedt G, Carstedt-Duke J 1988 Human calpactin II (lipocortin I) messenger ribonucleic acid is not induced by glucocorticoids. Mol Endocrinol 2:732-739 33. Bienkowski MJ, Petro MA, Robinson LJ 1989 Inhibition of thromboxane A synthesis in U937 cells by glucocorticoids. Lack of evidence for lipocortin I as the second messenger. J Biol Chem 264:6536-6544 34. Cirino G, Flower RJ, Browning JL, Sinclair LK, Pepinsky RB 1987 Recombinant human lipocortin 1 inhibits thromboxane release from guinea-pig isolated perfused lung. Nature 328:270-272 35. Hattori T, Hirata F, Hoffman T, Hizuta A, Herberman RB 1983 Inhibition of human natural killer (NK) activity and antibody dependent cellular cytotoxicity (ADCC) by lipomodulin, a phospholipase inhibitory protein. J Immunol 131:662-665 36. Hirata F, Iwata M 1983 Role of lipomodulin, a phospholipase inhibitory protein, in immunoregulation by thymocytes. J Immunol 130:1930-1936 37. Nagy E, Berczi I, Wren GE, Asa SL, Kovacs K 1983 Immunomodulation by bromocriptine. Immunopharmacology 6:231-243 38. Bernton EW, Meltzer MS, Holaday JW 1988 Suppression of macrophage activation and T-lymphocyte function in hypoprolactinemic mice. Science 239:401-404 39. Viselli SM, Mastro AM, Prolactin stimulation of lymphocyte proliferation. Program of the 30th Annual Meeting of the American Society of Cell Biology, San Diego CA, 1990, p 230a (Abstract) 40. Mukherjee P, Mastro AM, Hymer WC 1990 Prolactin induction of interleukin-2 receptors on rat splenic lymphocytes. Endocrinology 126:88-94

Endo • 1991 Vol 129 • No 4

41. Edwards HC, Booth AG 1987 Calcium-sensitive, lipid-binding cytoskeletal proteins of the human placental microvillar region. J Cell Biol 105:303-311 42. Glenney J 1986 Phospholipid-dependent Ca+2 binding by the 36kDa tyrosine kinase substrate (calpactin) and its 33-kDa core. J Biol Chem 261:7247-7252 43. Turgeon TL, Cooper RH, Waring DW 1991 Membrane-specific association of annexin I and annexin II in anterior pituitary cells. Endocrinology 128:96-102 44. Albarracin CT, Palfrey HC, Rao MC, Gibori G Effect of prolactin on the lOOkD/elongation factor 2 in the corpus luteum. Program of the 72nd Annual Meeting of The Endocrine Society, Atlanta GA, 1990, p 194 (Abstract) 45. Hirata F 1981 The regulation of lipomodulin, a phospholipase inhibitory protein in rabbit neutrophils by phosphorylation. J Biol Chem 256:7730-7733 46. Schlaepfer D, Haigler H 1987 Characterization of calcium-dependent phospholipid binding and phosphorylation of lipocortin I. J Biol Chem 262:6931-6937 47. Ando Y, Imamura S, Hong Y-M, Owada MK, Kakunaga T, Kannagi R 1989 Enhancement of calcium sensitivity of lipocortin I in phospholipid binding induced by limited proteolysis and phosphorylation at the amino terminus as analyzed by phospholipid affinity column chromatography. J Biol Chem 264:6948-6955 48. Pongsawasdi P, Anderson B 1984 Kinetic studies of rat ovarian 20 a-hydroxysteroid dehydrogenase. Biochim Biophys Acta 799:51-58 49. Wiest WG, Kidwell WR, Balogh Jr K 1968 Progesterone catabolism in the rat ovary: a regulatory mechanism for progestational potency during pregnancy. Endocrinology 82:844-859 50. Rodway RG, Garris DR 1982 Potentiation by prolactin of the luteotrophic effect of oestradiol in the pregnant rat. Acta Endocrinol (Copenh) 101:287-292 51. Gibori G, Richards JS, Keyes PL 1979 Synergistic effects of prolactin and estradiol on the luteotropic process in the pregnant rat: regulation of estradiol receptors by prolactin. Biol Reprod 21:419423 52. Basuray R, Jaffe R, Gibori G 1983 Role of decidual luteotropin and prolactin in the control of luteal cell receptors for estradiol. Biol Reprod 28:551-556 53. Kelly PA, Posner BI, Friesen HG 1975 Effect of hypophysectomy, ovariectomy, and cycloheximide on specific binding sites for lactogenic hormones in rat liver. Endocrinology 97:1408-1415


Prolactin secretion and corpus luteum function in women with luteal phase deficiency.

ATF3 expression in the corpus luteum: possible role in luteal regression.

Expression of aldo-keto reductase 1C23 in the equine corpus luteum in different luteal phases.

Ever-changing cell interactions during the life span of the corpus luteum: relevance to luteal regression.

Transition of the rabbit corpus luteum to estrogen dependence during early luteal development.

Hormonal, luteal, and follicular changes during initiation of persistent corpus luteum in mares.

Effect of tokishakuyakusan on corpus luteum endothelin.

Luteal regression vs. prepartum luteolysis: regulatory mechanisms governing canine corpus luteum function.

Ruptured corpus luteum with hemoperitoneum.

A new perspective on the mechanism of corpus luteum regression.

A role for intraluteal estrogen in the mediation of luteinizing hormone action on the rat corpus luteum during pregnancy.

Corpus luteum function in ageing inbred mice.

[The corpus luteum reactivation test (CLR-test): a simple method for the characterization of the corpus luteum function (proceedings)].

Expression, action, and steroidal regulation of insulin-like growth factor-I (IGF-I) and IGF-I receptor in the rat corpus luteum: their differential role in the two cell populations forming the corpus luteum.

Relaxin gene expression in the sow corpus luteum during the cycle, pregnancy, and lactation.

Effect of post-ovulatory administered oestrogens on corpus luteum function.

[Proceedings: Influence of exogenous on corpus luteum function].

Production a corpus luteum angiogenic factor responsible for proliferation of capillaries and neovascularization of the corpus luteum.

The role of GPR1 signaling in mice corpus luteum.

Distribution of leukocyte subpopulations in the human corpus luteum.

Cytological observations of dissociated rat corpus luteum.

Experimental aspects of corpus luteum function.

Combined pregnancy and ruptured corpus luteum cyst.

The corpus luteum of the guinea pig. II. Cytochemical studies on the Golgi complex, GERL, and lysosomes in luteal cells during maximal progesterone secretion.