9913-7227/92/1304-1951$03.99/O Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 130, No. 4 Printed

in U.S.A.

Basic Fibroblast Growth Factor Inhibits Basal and Stimulated Relaxin Secretion by Cultured Porcine Luteal Cells: Analysis by Reverse Hemolytic Plaque Assay* MICHAEL

J TAYLOR

AND CHERYL

L CLARK

Department of Veterinary Physiology and Pharmacology,Iowa State University, Ames, Iowa 50010

ABSTRACT. The role of the basic fibroblast growth factor (basic FGF) in the control of secretion of the ovarian protein hormone relaxin (RLX) by porcine large luteal cells (LLCs) was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with proteinA-coupled sheep erythrocytes. In the presence of complement and porcine relaxin antiserum, a zone of hemolysis (a plaque) developed around a RLX-releasing LLC. The rate of plaque development in time-course experiments was used as an index of the rate of RLX secretion. Monolayers were bathed in medium containing graded concentrations of basic FGF in the presence or absence of a stimulatory secretagogue [O.Ol PM prostaglandin

Et (PGWI.

Exposure of luteal cell-containing monolayers to basic FGF resulted in a significant reduction (P < 0.05) in the rate of RLXinduced plaque formation, evidence of an inhibitory effect of basic FGF on the rate of basal RLX secretion. This suppressive effect was variable in onset (l-3 h of incubation) and dose related. Minimally and maximalls effective doses were about 10 and 30 rig/ml basic FGF, respectively. Higher doses of basic FGF

B

(20 and 30 rig/ml) entirely suppressed RLX from a substantial subset of LLCs (lO-20% of all LLCs), an indication of a differentially sensitive subpopulation. Addition of basic FGF (30 ng/ ml) to monolayers also treated with PGEs resulted in a significant (P < 0.05) attenuation of the stimulatory effect of PGEz on RLX secretion, evidence that these agents functionally interact in the modulation of RLX. We conclude that these results taken in association with the prior demonstration of the presence of basic FGF in luteal tissue suggest that basic FGF serves as a local inhibitory mechanism that regulates RLX secretion. Furthermore, the ability of basic FGF to counteract the effect of PGEl implies that intraluteal stimulatory/inhibitory agents may act in concert to achieve tine control of RLX secretion. The observation of a preferentially responsive subpopulation is consistent with the possibility that basic FGF is implicated in heteroaenous RLX secretion. Nevertheless, the phy‘siological role(s) if basic FGF in the control of RLX secretion and the interrelationships of basic FGF with other local and systemic secretagogues remain to be clearly defined. (Endocrinology 130: 1951-1956,1992)

shifts in endocrine capabilities. One of the best characterized luteal hormones is the insulin-like polypeptide hormone relaxin (RLX), a hormone identified in human, rat, and porcine luteal tissue during pregnancy (5). In swine, RLX is present in minimal amounts in granulosa and thecal cells, but the subsequent luteinization of these cell types results in an abrupt initiation of RLX synthesis and secretion. Moreover, RLX secretion by the CL increases progressively as gestation advances (5). The factors that regulate the timing and/or magnitude of this major switch in endocrine ability are unknown, but the presence of intraluteal basic FGF together with its documented effects on ovarian steroidogenesis (3,4,6) raise the interesting possibility that basic FGF might also influence RLX secretion. The recent demonstration that basic FGF directly modulates the secretion of another polypeptide hormone, PRL, by rat lactotropes (7) is consistent with this possibility. The specific objectives of this study were to determine whether basic FGF could influence basal or stimulated rates of RLX secretion and if all large luteal cells (LLCs)

ASIC fibroblast growth factor (basic FGF) is a 146amino acid peptide that influences the proliferation, differentiation, and function of a wide range of mesoderm and neuroectoderm cells (1). This growth factor is potentially well suited, therefore, to influencing the complex morphogenic and functional changes that occur during the follicular and luteal phases of ovarian cycles, and an increasing body of evidence supports this role (2, 3). In this context, basic FGF has been specifically implicated in the development and function of the corpus luteum (CL). Luteal tissue contains basic FGF, which is present as a truncated (missing the first 15 residues), but biologically active, form (4). The differentiation of follicle cell types (granulosa and thecal) into mature luteal cells is accompanied not only by morphological changes, but also by equally remarkable Received October 3,1QQl. Address all correspondence and requests for reprints to: Dr. M. J. Taylor, Department of Veterinary Physiology and Pharmacology, Iowa State University College of Veterinary Medicine, Ames, Iowa 50010. * This work was supported by NIH Grant HD-22786. 1951

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1952

BASIC

FGF AND

RELAXIN

respond in the same manner. To achieve these goals, we employed a standard RLX reverse hemolytic plaque assay (RLX-RHPA) to monitor relaxin release. The unique strength of this technique is that it detects hormone release by individual cells maintained in monolayer culture (reviewed in Ref. 8). Moreover, time-course studies with the RLX-RHPA that track the percentage of RLXreleasing LLCs permit the identification of RLX stimulatory/inhibitory secretagogues as well as differentially responsive subsets of cells (9, 10). Materials

and Methods

Full details of the collection of tissue, cell dispersion, and plaque assay methodology were provided previously (ll), and only a brief description is provided here. Animals Ovaries from pregnant pigs (days 30-40 of gestation) were collected within 30 min of death at the Meat Laboratory, Iowa State University. Gestational age was determined by the day of mating with an intact boar and was confirmed by fetal crown-rump length (12). Both ovaries were placed in medium and transported back to the laboratory within lo-60 min. The medium used in this step (and all other subsequent steps in cell dispersion and assays) was Dulbecco’s Modified Eagle’s Medium (DMEM) obtained from Gibco (Grand Island, NY) and supplemented with antibiotics (100 U/ml penicillin G and 100 lg/ml streptomycin sulfate; Gibco) and 0.1% BSA (fraction V; Sigma, St. Louis, MO). This medium will be referred to hereafter as DMEM-0.1% BSA. Cell dispersion Three or four individual CL were incised, and the luteal tissue was gently enucleated from the fibrous capsule and then washed two or three times with fresh DMEM-0.1% BSA. Approximately one quarter of each CL was then placed in a sterile 65-mm petri dish containing 3 ml DMEM-0.1% BSA, minced finely with sterile scalpel blades in a laminar flow hood, and washed repeatedly with fresh medium. The luteal fragments were then placed in a Spinner Suspension Flask (Bellco, Vineland, NJ) containing 15 ml DMEM-0.1% BSA, 0.12% collagenase (type III, Cooper Biomedical, Freehold, NJ). The tissue was incubated for 60 min, after which time the fragments were drawn in and out of a sterile lo-ml pipette, incubated for a further 10 min, and then centrifuged at 1500 x g for 10 min. The cells were washed twice in Spinner’s Minimum Essential Medium and passed through a 75-pm mesh (Tetco, Inc., Elmsford, NY) to remove cell clumps. An aliquot of the mixed cell suspension was taken for LLC counts (using a hemocytometer) and cell viability (trypan blue exclusion). Yields were normally about l-2 x lo6 LLCs, and cell viability was 80-95%. The mixed luteal cell suspension was finally resuspended in DMEM-0.1% BSA at a final concentration of 0.25 X lo6 LLCs/ ml.

SECRETION

Endo I’01130

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1992 No 4

RHPA Incubation chambers (Cunningham chambers) were constructed by attaching a glass coverslip to a poly+lysine-treated glass microscope slide using double stick tape. The mixed luteal cell suspension was then mixed 1:l with a 12% suspension of sheep erythrocytes to which protein-A (Sigma) had been chemically attached (13). This mixture was then infused into each chamber by capillary action and incubated for 45 min (5% CO,95% air, humidified atmosphere) to allow cell attachment. Each chamber was flushed with fresh medium to form a monolayer one cell deep on the chamber floor and then incubated overnight in the presence of DMEM-0.1% BSA. The monolayers were washed again with fresh medium to remove cell products that had accumulated overnight and immediately filled with fresh medium containing porcine relaxin antiserum (1:80; no. 280, obtained from Dr. 0. D. Sherwood) containing stated concentrations of growth factor and/or secretagogue. Basic FGF was obtained from Peninsula Laboratories [brain-derived basic FGF-( l-24); bovine; no. 80471, stored as a stock solution of 120 pg/ml in sterile double distilled water at -20 C and diluted appropriately in medium immediately before experimentation. Monolayers exposed to medium containing porcine relaxin antiserum (1:80) alone acted as controls. After infusion of growth factor, prostaglandin E, (PGE,), or control medium, the monolayers were incubated for 1-12 h. At the end of each incubation period, each chamber was filled with medium containing guinea pig serum as a source of complement (1:40; Gibco), incubated for 50 min to complete plaque formation, fixed with 8% glutaraldehyde in normal saline, and stored at 4 C immersed in the same fixative in air-tight containers. Each monolayer was examined microscopically to score the percentage of LLCs that formed plaques; at least 200 LLCs/ monolayer were counted, and triplicate chambers were prepared at each time point. Percentages from the triplicate monolayers were averaged. Small luteal cells (i.e. cells

Basic fibroblast growth factor inhibits basal and stimulated relaxin secretion by cultured porcine luteal cells: analysis by reverse hemolytic plaque assay.

The role of the basic fibroblast growth factor (basic FGF) in the control of secretion of the ovarian protein hormone relaxin (RLX) by porcine large l...
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