Extrahepatic Expression of Fibrinogen by Granulosa Cells ... · estradiol and progesterone. The pituitary gonadotropin FSH regulates granulosa cell function during the follicular

0013.7227/93/1334-1645$03.00/O Endocrinology Copyright 0 1993 by The Endocrine Society

Vol. 133, No. 4 Printed in U.S.A.

Extrahepatic Expression of Fibrinogen by Granulosa Cells: Potential Role in Ovulation*

JEFF A. PARROTT, PATRICIA D. WHALEY, AND MICHAEL K. SKINNER?

Reproductive Endocrinology Center, University of California, San Francisco, California 94143-0556

ABSTRACT Granulosa cells from ovarian follicles were shown to express and

secrete fibrinogen under the control of FSH. Conditioned medium was collected from granulosa cell cultures and found to contain FSH- dependent 50-kilodalton (kDa) and 93- to 95-kDa proteins. N-Terminal microsequence analysis identified these proteins as fibrinogen /3- and y-chains, respectively. Proteins migrating at 93 and 95 kDa contain identical y-chain sequences at the N-terminal, suggesting differential processing of fibrinogen. These fibrinogen chains were specifically detected with antifibrinogen antibodies in immunoblot and immuno- precipitation analysis. Fibrinogen y-chain mRNA was detected in granulosa cells by polymerase chain reaction analysis, confirming fi- brinogen gene expression by these cells. Fibrinogen secretion by gran-

ulosa cells was measured by a competitive enzyme-linked immunosor- bent assay. Granulosa cells treated with FSH (100 rig/ml) secreted 2- 3 times more fibrinogen than untreated cells. These data show that fibrinogen, a major product of the liver, is also a secretory product of granulosa cells. This provides a novel extrahepatic site of fibrinogen expression. As hepatic parenchymal cells normally maintain high cir- culating levels of fibrinogen, the local production of fibrinogen in the ovary is anticipated to have specialized functions. Locally produced fibrinogen may be important in the clotting process following tissue rupture at ovulation. In addition, fibrinogen fragments may be involved in the mechanism of ovulation by increasing the activity of tissue-type plasminogen activator to control the proteolytic activity required for ovulation. (Endocrinology 133: 1645-1649, 1993)

G RANULOSA cells have an integral role in the mainte- nance and control of ovarian function. This epithelial cell helps form the follicle and provides the proper cytoar- chitectural support and microenvironment for the developing oocyte. During the process of folliculogenesis, granulosa cells proliferate and actively differentiate from a primordial stage of development through ovulation to a luteal stage. A major functional parameter of granulosa cells is the biosynthesis of estradiol and progesterone. The pituitary gonadotropin FSH regulates granulosa cell function during the follicular phase of development (1). Granulosa cell function can also be regulated by local cell-cell interactions involving neighboring thecal cells (2-5). A complete understanding of the physio- logical importance of granulosa cells requires further eluci- dation of specific cellular functions, in particular the identi- fication of FSH-dependent secretory products (6).

fibrinogen synthesis have not been reported. Due to high circulating levels of fibrinogen, any extrahepatic synthesis will probably have specialized local functions.

The current study was designed to investigate granulosa cell function and identifies fibrinogen as a FSH-regulated secretory product. The potential role of fibrinogen in the ovulatory process is discussed.

Materials and Methods

Tissue isolation and serum-free cell culture

Fibrinogen is primarily synthesized in the liver and re- leased into circulating blood, where it has an essential role in the final stage of blood clotting throughout the body (reviewed in Ref. 7). Fibrinogen is produced as a 340-kilo- dalton (kDa) multimeric protein composed of two sets of cy-, p-, and y-chains that are processed into proleolytic fragments by thrombin to produce fibrin. In addition to their role in blood clotting, fibrinogen fragments can also enhance the proteolytic activity of tissue-type plasminogen activator (tPA) (8). This activity is important for the fibrinolytic system, because tPA specifically converts plasminogen to plasmin, which is involved in the processing of fibrinogen (9). There- fore, fibrinogen has a dual function in its ability to alter tPA activity and facilitate blood clotting. Extrahepatic sites of

Bovine ovaries were obtained from young nonpregnant cycling heifers less than 10 min after death. Ovaries were delivered fresh on ice or immediately frozen at -70 C and delivered on dry ice by Bova-Max, Inc. (Fresno, CA). Granulosa cells were isolated by microdissection from fresh tissue and cultured as previously described (6). Cells were plated with an initial density of approximately lo6 cells/2 cm’ and maintained for 1-3 days at 37 C in a 5% CO* atmosphere in the absence of serum. The indicated cells were treated with FSH at 100 rig/ml (National Pituitary Agency, Baltimore, MD).

Gel electrophoresis

Conditioned medium from granulosa cell cultures was collected for l-3 days. When required, the medium was concentrated by ultrafiltra- tion using a lo-kDa exclusion limit membrane. Electrophoresis was performed on 7.5-15% gradient polyacrylamide gels using reducing conditions and the Laemmli sodium dodecyl sulfate buffer system (10). For fluorography and immunoprecipitation, cells were maintained in glycine- and methionine-free medium containing 5 rCi/ml [‘5S]methio- nine and 5 &/ml [3H]glycine. Gels were fluorographed with diphen- yloxazole in acetic acid, as previously described (11).

Received April 12, 1993. Peptide microsequencing

l This work was supported by NIH Grant HD-20922 (to M.K.S.). Concentrated granulosa cell conditioned medium (loo-fold concen- t To whom all correspondence should be addressed. trated) was electrophoresed and transferred to polyvinylidine membrane

1645

1646 FIBRINOGEN IN THE OVARY Endo. 1993 Voll33. No 4

(Bio-Rad, Richmond, CA) in 10 rnM 3-cyclohexylamino-propane-sulfonic acid buffer, pH 11.0, and 10% methanol. Transferred proteins were stained in 0.1% Coomassie blue R-250 in 40% methanol and destained in 40-50% methanol. Bands of interest were cut out and microsequenced on an Applied Biosystems 475A/900A peptide sequencer (Foster City, CA) using the Edman degradation procedure (12). Sequences were searched for homology in FIR/Swiss-Prot protein data banks.

Immunoprecipitation

Fibrinogen was immunoprecipitated using a double antibody precip- itation technique. Concentrated radiolabeled granulosa cell-conditioned medium (GCM) (30-fold concentrated) was preincubated with goat antihuman fibrinogen (F-2506, Sigma Chemical Co., St. Louis, MO) or goat serum in equal volume buffer (0.2 M Tris, pH 7.5; 50 PM phenol- methylsulfonylfluoride; 1 mM benzamadine; 0.5% Triton X-100; and 0.15 M NaCl). Samples were then incubated with rabbit antigoat im- munoglobulin G (Sigma) and centrifuged at 12,500 x g for 30 min. The pellet was electrophoresed, and the gel was fluorographed. All incuba- tions were performed at 4 C overnight with mixing.

Immunoblotting

Concentrated GCM (30.fold concentrated) was electrophoresed and transferred to nitrocellulose membrane (Bio-Rad) using 25 mM Tris-192 rnM glycine buffer in 20% methanol. Membranes were blocked in 2% BSA (Sigma) or 2% bovine hemoglobin for 2 h at room temperature. Primarv antibody (goat antihuman fibrinogen, 1:lOO; Sigma) was added for 1 h, followed’-by an alkaline phosphatase-conjugated secondary antibodv (rabbit antieoat immunoelobulin G, 1:lOOO; Sizrna) for 0.5-l h. Membranes were “washed in T&saline buffer with 6.1% Triton X- 100. Alkaline phosphatase was detected calorimetrically using nitro blue tetrayoleum and 5-bromo-4-chloro-3-indolyl phosphate substrates in 20 mM Na2C03 buffer, pH 9.6 (carbonate buffer).

Enzyme-linked immunosorbent assay (ELISA) of fibrinogen

Fibrinogen was measured by a competitive ELISA using Immulon 2 microtiter plates (Dynatech Laboratories, Alexandria, VA) coated with 0.1 fig bovine fibrinogen in 200 ~1 carbonate buffer for 4 h at room temperature. Samples were purified using a Cl8 Sep-Pak minicolumn (Millipore, Milford, MA) and neutralized with 10 M NaOH. Standards containing 10 ng to 1 rg bovine fibrinogen in 100 ~1 PBS were prepared fresh. Peroxidase-conjugated goat antihuman fibrinogen (Cappel, War- rington, PA) was also prepared fresh (l:lO,OOO dilution) in PBS-Tween (O.i% Tween-20). After initial coating, the plate was washed once with PBS-Tween. Quickly, 100 ~1 antibody were added for 10 min, followed bv 100 ~1 sample/standard, and the plate was rotated for a total of 1 h (25 C). After f&washes, peroxidasewas detected calorimetrically using o-phenylenediamine dihydrochloride (Zymed, South San Francisco, CA) substrate in 48 mM citric acid-102 mM NaHP04 containing HZ02. Plates were read on a micro-ELISA densitometer (Titertek, Flow Laboratories, Inc., Toronto, Ontario, Canada) using a 450.nm filter. Fibrinogen pro- duction was normalized to DNA content, as previously described (13, 14).

Polymerase chain reaction (PCR) of y-fibrinogen

Total RNA was extracted from -70 C frozen bovine ovaries using a guanidinium thiocyanate procedure and further purified by centrifuga- tion through a cesium chloride gradient (15). Total RNA was reverse transcribed to obtain cDNA using avian myoblastosis virus reverse transcriptase @omega, Madison, WI). The cDNA template was ampli- fied by PCR using 25-basepair (bp) primers whose sequences were derived from published human and bovine y-fibrinogen sequence (hu- man genomic positions 2995 and 5846) (16-19). The PCR primers correspond to the coding region of exons VI and VIII (separated by two introns), such that a 394-bp PCR product derived from bovine cDNA can be distinguished from PCR products derived from genomic DNA. The sequences of the primers were 5’-GAT GGG TCT GGA AAT GGA TGG ACT G-3’ (5’-primer) and 5’-GTA CTG AAC TGC ATG CCA

TTA TGG G-3’ (3’-primer). These primers correspond to identical sequences in bovine and human y-fibrinogen. Amplification was per- formed under stringent conditions for 45 cycles. The 394-bp fragment was visualized by UV illumination on 5% polyacrylamide gels stained with 0.5 rg/ml ethidium bromide.

Results

Granulosa cells were cultured in the presence of [35S] methionine and [3H]glycine to study specific secretory pro- teins. Radiolabeled GCM was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluo- rography (Fig. 1, lane 1). FSH treatment clearly stimulated certain secretory proteins while suppressing the secretion of others. Bands with apparent molecular masses of 50 and 93- 95 kDa were identified and investigated. Concentrated GCM (loo-fold) was electrophoresed in several lanes, and sepa- rated proteins were transferred to a polyvinylidine mem- brane in 3-cyclohexylamino-propane-sulfonic acid buffer. Transferred proteins were stained for 1 min in 0.1% Coom- assie blue. Although some bands were indistinguishable due to high protein concentration, the bands of interest were identified and excised. Similar bands from different lanes were combined for N-terminal microsequence analysis on an ABI 475A liquid phase peptide sequencer using the Edman degredation method. It was possible to sequence 5-20 amino acids in each run, after which background from contaminat- ing sequences was too high. These sequences were loaded into the PIR/Swiss Prot protein data banks to search for homologies. The bands of interest migrating at 50 and 93- 95 kDa matched the sequences of bovine fibrinogen /3- and y-chains, respectively (Fig. 2). In addition, each matching sequence immediately followed a lysine (k) residue, corre- sponding to an expected tryptic cleavage site of the protein.

The immunoreactivity of these bands was investigated using a primary antibody to human fibrinogen. Figure 1 shows the results of both immunoprecipitation and immu- noblot analysis with this antibody. In both experiments, the bands of interest clearly react with fibrinogen antibody. The immunoprecipitation was performed on radiolabeled GCM using a double antibody precipitation technique. Radiola- beled 50-kDa and 93- to 95-kDa proteins were specifically precipitated, demonstrating active synthesis and secretion of these proteins by granulosa cells. Immunoblot analysis also detected the 50-kDa and 93- to 95-kDa proteins. Periodically, a more than 200-kDa protein was detected (Fig. 1, lanes 4, 5, and 7) that presumably is the large mol wt precurser of fibrinogen. Additional bands of 65- to 70-kDa and 120- to 150-kDa were detected in lanes 2-4, which appear in the nonimmune serum and no primary antibody lanes, suggest- ing nonspecific interactions with these proteins and the sec- ondary antibody. These results demonstrate that the 50-kDa and 93- to 95-kDa proteins are immunologically similar to fibrinogen and are actively secreted by bovine granulosa cells in vitro.

To examine the production of fibrinogen at the transcrip- tional level, fibrinogen gene expression was studied in gran- ulosa cells. Fibrinogen is transcribed from three related genes encoding the (Y-, /3-, and y-subunits of the whole molecule.

FIBRINOGEN IN THE OVARY 1647

2 3 4 5 6 7 8

A NH2-- : , i. c.#

/L--1

WKGRQNQVQDNENWNEY FIBRINOGEN-B

GRQNQVQDNENWNEY 50K

B NH*~~~~~.~~~~~~~

+-l VKAlQlSYNPDQ FIBRINOGEN-Y

AIQISYNP 93K

AIQISYNPD 95K

FIG. 2. Schematic of fibrinogen fi- and y-protein chains (A and B, respectively). The N-terminal amino acid sequences of the granulosa cell-secreted 50-, 93-, and 95-kDa proteins are shown beneath the published bovine fibrinogen sequence. These sequences are a consensus of three experiments.

In several species each of the fibrinogen genes have been shown to be independently regulated in the liver (20). RNA was extracted from bovine granulosa cells and whole folli-

cles. Total RNA was reverse transcribed, and cDNA was used as template for PCR analysis (RT-PCR). Two primers were synthesized encoding regions from exons VI and VIII of the fibrinogen y-chain gene. Figure 3 shows the results of RT- PCR from ovarian follicle and liver RNA using the fibrinogen y-chain primers. The expected 394-bp fragment was gener- ated in bovine liver (positive control) and ovarian follicle cells, but not in rat liver. Similar results were obtained with purified granulosa cells (data not shown). Therefore, the fibrinogen y-chain gene is expressed in these cells and pro- vides additional support that fibrinogen is synthesized and secreted by granulosa cells.

Because FSH promotes granulosa cell differentiation, the effects of FSH treatment on fibrinogen secretion were ex- amined. A competitive ELISA was developed using a primary antibody to human fibrinogen that was conjugated to per- oxidase. This assay was useful in measuring fibrinogen in GCM and in standard solutions of purified bovine fibrinogen. As shown in Fig. 4B, FSH-treated cells secreted 2-3 times more immunoreactive fibrinogen than control cells. The as-

1648 FIBRINOGEN IN THE OVARY Endo l 1993 Vol133 l No 4

M C RL BL F

E 396-

IV

z aa : n

154-

FIG. 3. Expression of bovine fibrinogen mRNA. RT-PCR analysis was performed with 20 rg total RNA. Primers (indicated by arrows in the y-fibrinogen gene schematic) were designed to generate a 394-bp frag- ment from bovine cDNA template. M, Standard DNA ladder; C, no template, RL, rat liver; BL, bovine liver; F, bovine follicle. Data are representative of five experiments.

say is validated in Fig. 4A by demonstrating parallel curves of GCM and standard fibrinogen,

Discussion

The current study was designed to further investigate granulosa cell function through the identification of secreted proteins. A previous study with bovine granulosa cell cul- tures identified major secreted proteins of 200, 65, 25, and 15 kDa (6). In the current study granulosa cells were treated with FSH and found to secrete a number of FSH-stimulated

A 0.14- 0 STD

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A C 0.13 n FSH

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A 0.12

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6 0.11

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si 0.09

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FIBRINOGEN (ug) or GCM (ul)

products, including 50-kDa and 93- to 95-kDa radiolabeled secreted proteins. These proteins were isolated, and an N- terminal sequence was determined to potentially identify these granulosa cell products. The 50-kDa protein had a sequence identical to bovine fibrinogen ,&chain. A set of at least three proteins around 92 kDa was consistently detected, and the 93- and 95-kDa proteins were both found to have an identical sequence with bovine fibrinogen y-chain. As both the 93- and 95-kDa proteins had the same N-terminal sequence, these proteins are apparently processed differ- ently. These results suggest that granulosa cells may secrete fibrinogen in vitro.

To confirm the production of fibrinogen by granulosa cells, an antibody to fibrinogen was obtained. An immunoprecip- itation of radiolabeled secreted proteins specifically precipi- tated the 50-kDa and 93- to 95-kDa proteins. The precipita- tion of radiolabeled proteins indicates that these proteins are actively synthesized and secreted by granulosa cells. An immunoblot procedure also detected these same three pro- teins. Therefore, granulosa cells appear to actively secrete 50-kDa and 93- to 95-kDa proteins that have sequence similarity to fibrinogen and are immunologically similar to fibrinogen. The sizes of these proteins correspond to the anticipated sizes of processed forms of fibrinogen. Altema- tively, the y-dimer of fibrin corresponds to 93-95 kDa. Quantitation of fibrinogen production by granulosa cells indicated that FSH stimulated fibrinogen secretion. These data provide additional support that granulosa cells produce fibrinogen and suggest that fibrinogen may provide a useful model of granulosa cell cytodifferentiation.

The gene expression of fibrinogen y-chain was investi- gated with RT-PCR. The PCR primers used corresponded to exon regions that spanned two introns. Therefore, the PCR product was distinguished from the possible amplification of genomic DNA. Granulosa cells were found to contain fibrin- ogen y mRNA, confirming the expression of the gene by this cell type. Further studies are now needed to assess the gene expression of fibrinogen P- and a-chains. In addition, ex- amination of the fibrinogen subunit gene expression should

CONTROL FSH

TREATMENT

FIG. 4. Fibrinogen production by granulosa cells. Fibrinogen levels in GCM was determined using a competitive ELISA. A, Line graph of a typical experiment, validating the assay. Parallel curves of standard bovine fibrinogen (0), granulosa cell control medium (A), and FSH-treated granulosa cell media (m) are shown. B, Effects of FSH on fibrinogen production, represented as the mean k SEM percentage of the control value (n = 12). **, Statistical difference from control with analysis of variance (P c 0.01). All data were normalized for granulosa cell DNA levels (micrograms of fibrinogen per pg granulosa DNA), and the fibrinogen levels in conditioned medium ranged from 9-42 rg/ml.

FIBRINOGEN IN THE OVARY 1649

include a Northern analysis to detect potential spliced var- ients. The results of the current study provide one of the first examples of extrahepatic expression of fibrinogen. Because high concentrations of fibrinogen are present in the circula- tion, local production of fibrinogen will probably have spe- cialized functions.

7. Davie EW, Fujikawa K, Kisiel W 1991 The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30:10363- 10370

8.

9.

Fibrinogen secretion by granulosa cells is speculated to function during ovulation. As fibrinogen is essential for blood clotting and fibrinolysis during tissue repair (7, 8), fibrinogen secretion by granulosa cells may be important to ensure the integrity of the ovarian wall after ovulation. In addition, fibrinogen may be involved in the mechanism of ovulation because fibrinogen fragments enhance tPA activity. Previ- ously, tPA has been postulated to be involved in ovulation (21-24). Therefore, fibrinogen may regulate the proteolytic activity of tPA in follicular fluid and indirectly influence ovulation. Fibrinogen has also been shown to associate with hyaluronic acid, which is an abundant component of follic- ular fluid and may influence ovulation and subsequent fibri- nolysis and wound healing (25-27). Analysis of the potential role of fibrinogen in the ovary will involve the determination of fibrinogen levels in follicular fluid to assess serum US. granulosa cell contribution. Further analysis of fibrinogen secretion and fibrinogen gene expression during follicle de- velopment will provide insight into the physiological func- tion of granulosa cell-secreted fibrinogen.

10.

11.

12.

Robbins KC 1981 The regulation and control of the blood fibrion- olytic system. Prog Fibrin01 5:3-13 Petersen TE, Martzen MR, Ichnose A, Davie EW 1990 Character- ization of the gene for human plasminogen a key proenzyme in the fibrinolvtic svstem. 1 Biol Chem 265:6104-6111 Laemmii UK 1970 Cleavage of structural proteins during the assem- bly of the head of bacteriophage T4. Nature 227:680 Skinner MK, Griswold MD 1983 Fluorographic detection of radio- activity in polyacrylamide gels with 2,5diphenyl oxazole in acetic acid and its comparison with existing procedures. Biochem J 209:281 Matsudaira P 1987 Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035-10038

13.

14.

15.

16

17

Karsten U, Wollenberger A 1977 Improvements in the ethidium bromide method for direct fluorometric estimation of DNA and RNA in cell and tissue homogenates. Anal Biochem 77:464 Roberts AJ, Skinner MK 1990 Hormonal regulation of thecal cell function during antral follicle development in bovine ovaries. En- docrinology 127:2907 Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299 Brown WM, Dziegielewska KM, Foreman RC, Saunders NR 1989 Nucleotide and deduced amino acid sequence of a y subunit of bovine fibrinogen. Nucleic Acids Res 17:8387-6397 Chune DW. Harris IE. Davie EW 1990 Nucleotide seauences of the thyee genes coding’ for human fibrinogen. Adv Exp’ Med Biol 281:39-48

18.

Acknowledgments 19.

We thank Byron Glenn, Lisa Halbumt, and Susan Schlitz for technical assistance, and Linda Jaqua and Jennifer Simenc for assistance in the preparation of the manuscript, We also acknowledge Brian Mullaney, Andy Roberts, and L. N. Schaefer for support.

Chung DW, Chan W, Davie EW 1983 Characterization of a com- plementary deoxyribonucleic acid coding for the gamma chain of human fibrinogen. Biochemistry 22:3250-3256 Rixon MW, Chung DW, Davie EW 1985 Nucleotide sequence of the gene for the gamma chain of human fibrinogen. Biochemistry 24:2077-2086

20. Hirose S, Oda K, Ikehara Y 1988 Biosynthesis, assembly and secretion of fibrinogen in cultured rat hepatocytes. Biochem J 251:373-377

21. Cajander SB 1989 Periovulatory changes in the ovary morphology and expression of tissue type plasminogen activator. Prog Clin Biol Res 296:91-101

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