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a Division of Endocrinology, Department of Internal Medicine,
b Department of Pathology, and
c Department of Obstetrics and Gynecology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Regulation of PTH-rp protein production was assessed in serum-free monolayer cultures of porcine granulosa cells. Transforming growth factor (TGF)-ß1 (100 ng/ml) increased PTH-rp concentrations (assayed by two-site immunoradiometric assay of culture media) as well as corresponding PTH-rp mRNA accumulation (assessed by RT-PCR) in a time-dependent manner, with maximal responses of 3- to 5-fold at 96 h. TGF-ß1 dose-response studies revealed an ED50 of 0.24–0.38 ng/ml with a maximal effect at 30 ng/ml. Other growth factors and hormones, including insulin, insulin-like growth factor (type I), epidermal growth factor, FSH, estradiol, and interleukin-1, failed to alter PTH-rp secretion.
Biological effects of PTH-rp were evident in purified porcine theca cells. Using the Ca2+-sensitive fluorescent indicator dye, fura-2, and digital imaging videomicroscopy, we found that PTH-rp (1 µM) stimulated intracellular free calcium ion concentrations ([Ca2+]i) in single porcine theca cells. The [Ca2+]i elevation was characterized by a slow and prolonged rise. After PTH-rp stimulation, theca cells maintained responsiveness to hormone stimulation by LH, which elicited a typical theca cell [Ca2+]i response.
Our results allow a hypothesis of a paracrine intrafollicular signaling system involving interaction between theca cell-derived TGF-ß and granulosa cell-derived PTH-rp, with feedback by PTH-rp on theca cells. Alternatively, expression of mRNAs encoding PTH-rp and its receptor in corpora lutea suggests that this peptide may play a role in luteal cell function. The precise role of this intraovarian PTH-rp system will require further study.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many reproductive tissues express PTH-rp and its receptor, including the uterus, placenta, mammary gland, testis, and ovary. Histochemical studies have demonstrated the presence of immunoreactive PTH-rp in the human ovary [4], and there are reports of agents that modulate PTH-rp production in vitro [5,6]. Here, we evaluate the expression of gene transcripts for PTH-rp and its receptor in porcine ovary, and appraise whether gonadotropins or locally produced factors regulate production of PTH-rp by cultured granulosa cells. In addition, we assess the impact of PTH-rp on the cAMP or Ca2+ signaling pathways utilized by porcine theca and granulosa cells. Our data suggest the presence of a paracrine signaling system in the developing porcine follicle, which may involve an interaction between theca cell-derived transforming growth factor (TGF)-ß and granulosa cell-derived PTH-rp.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovine FSH (NIDDK oFSH-19) and LH (NIDDK oLH-26) were provided by the National Hormone and Pituitary Program, NIH (Bethesda, MD). The acetoxymethyl ester form of fura-2 was purchased from Calbiochem (San Diego, CA). Estradiol-17ß, TGF-ß1, interleukin-1 (IL-1), epidermal growth factor (EGF), porcine insulin, ionomycin, collagenase (type IV), and deoxyribonuclease I (DNase) were purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant insulin-like growth factor I (IGF-I) and PTH-rp (1–86) were purchased from Bachem (Torrance, CA). Fetal bovine serum, Eagle's minimal Essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM)/F12 (1:1), and penicillin-streptomycin were purchased from Gibco (Grand Island, NY).
Cell Culture
Follicular fluid and granulosa cells were collected from small (1–5 mm)-, medium (5–8 mm)-, and large (8–10 mm)-sized follicles of immature swine by fine-needle aspiration. Cells were removed by centrifugation at 3000 rpm for 20 min. Granulosa cells from small- and medium-sized follicles were washed three times by low-speed centrifugation (3000 rpm) in MEM, and approximately 2 x 106 viable granulosa cells were plated in 24-well dishes containing bicarbonate-buffered MEM and 3% fetal calf serum to permit cell attachment. Cells were allowed to attach for 16–24 h at 37°C and 5% CO2 and subsequently maintained in serum-free MEM with the indicated treatments for the designated time intervals [7]. Theca cells were isolated from small follicles (3–6 mm) by collagenase/DNase digestion of follicle linings after removal of granulosa cells [8,9]. For calcium experiments, cells were allowed to attach to 10-cm plastic culture dishes for 2 h to facilitate differential plating of cells. Theca cells were then dislodged from culture plates by manual agitation and were allowed to attach to glass chamber slides (Nalgene-Nunc International Naperville, IL) at a density of 105 cells/ml for 36 or 60 h in the presence of MEM, 3% fetal calf serum, and porcine insulin (3 µg/ml). Subsequently, medium was changed to serum-free Hepes-buffered MEM with 0.1% BSA and 3 µg/ml insulin, and incubation continued for an additional 12 h.
Measurement of Intracellular Free Calcium [Ca2+]i
A Cunningham chamber was built on each slide, and medium was replaced with a defined phenol red-free medium, which also served as the vehicle for all treatments (S-medium: 127 mM NaCl, 5 mM KCl, 2 mM MgCl, 1.8 mM CaCl2, 0.5 mM KH2PO4, 5 mM NaHCO3, 10 mM Hepes, 10 mM glucose, and 0.1% BSA, pH 7.4). Theca cells were incubated with the fluorescent Ca2+-sensitive indicator fura-2/AM (3 µM) at 37°C for 20 min. Cells were rinsed with S-medium to remove excess dye and incubated for 20 min to allow de-esterification of the fura-2/AM.
Cells were monitored at room temperature using a Zeiss Axioplan (Carl Zeiss, Thornwood, NY) epifluorescent microscope equipped with a Fluor x20 objective as previously described [10]. The excitation wavelength was selected by narrow band pass filters (360 or 380 nm; Corion, Holliston, MA). Emission fluorescence was evaluated over a broad band. Video images were recorded continuously at 380 nm excitation wave length from a field containing theca cells using a silicon-intensified target camera (SIT-68 DAGE-MTI, Michigan City, IN) and stored on 3/4-inch broadcast-quality videotape. Cells were treated with vehicle alone and then with various concentrations of PTH-rp for 1–5 min (86% were exposed for at least 4 min), followed by a vehicle wash, 5 µg/ml oLH to verify LH-responsive theca cells, and a final delivery of the Ca2+ ionophore ionomycin (10 µM) to verify cellular fura-2 responsiveness. To monitor nonspecific changes in fluorescence due to photobleaching or leakage, a short sequence of images was recorded at 360 nm excitation wave length (Ca2+ insensitive) at the beginning, during, and at the end of each experiment. Each PTH-rp concentration was tested on at least 5 slides in at least three different experiments using different batches of theca cells.
Image Analysis
The recorded video signal was captured and digitized using software run on a QX-7 image analysis system (Quantex, Sunnyvale, CA). Fluorescence intensity values were converted to relative values using the equation R = Fo/Fi (Fo: initial emission intensity; Fi: fluorescence at time i). Cells were classified as responsive when Fo/Fi exceeded baseline plus 3 standard deviations.
Reverse Transcription-Polymerase Chain Reaction(RT-PCR)
Total RNA was prepared from pools of freshly isolated granulosa and theca cells isolated from immature swine ovaries as described above and from corpora lutea collected 12 days after eCG/hCG induction of the estrous cycle, by solubilizing in TriReagent (Molecular Research Center, Cincinatti, OH). RNA was extracted with chloroform, ethanol precipitated, and resuspended in sterile water for quantitation by absorbance at 260/280 nm. Human amnion RNA, used as a control sample, was isolated by the guanidinium isothiocyanate extraction procedure. Partial porcine cDNAs were cloned from granulosa cells, theca cells, and corpus luteum by RT-PCR using the Access RT-PCR system (Promega, Madison, WI) and a Perkin-Elmer (Irvine, CA) DNA thermal cycler Model 480. For the PTH-rp receptor, a 167-base pair (bp) cDNA was amplified that corresponds to porcine sequence bases 563–730 [11] using 0.5 µM each of forward (5'-ATCATAAAGGCCACGCCTAC-3') and reverse primer (3'-GGAGTAGCCCACGGTGTAGA-5'). For the PTH-rp hormone, a 419-bp cDNA was amplified corresponding to human sequence bases 32–451 [12] using 1.2 µM each of forward (5'-AGACTGGTTCAGCAGTGGAG-3') and reverse primer (3'-GTTCGCCGTTTTTTCTTTTCC-5'). In both cases, 250 ng of total RNA was reverse transcribed at 48°C for 45 min, followed by 2 min at 94°C to inactivate enzyme. Second-strand synthesis and PCR amplification were carried out for 35 cycles; the profile for PTH-rp hormone was 30 sec at 94°C for denaturation, 30 sec at 59°C for annealing, 30 sec at 62°C for polymerization, with a final extension step at 62°C for 7 min. For the PTH-rp receptor, samples were incubated for 30 sec at 94°C for denaturation, 30 sec at 58°C for annealing, and 30 sec at 72°C for polymerization, followed by a final extension step at 72°C for 7 min. Single bands of expected size were purified on low-melt agarose gels and extracted on Wizard PCR prep minicolumns (Promega). Purified DNA was subcloned into the TA 2.1 PCR cloning vector (Invitrogen, San Diego, CA), and inserts were sequenced on an ABI Prism (Ramsey, NJ) automated sequencer model 377 (Biomolecular Research Facility, University of Virginia, Charlottesville, VA). Multiple samples from three different batches of ovaries were analyzed by PCR and independently sequenced in both directions.
Other Assays
PTH-rp concentrations in spent medium were quantified using a two-site immunoradiometric assay for the human peptide (Nichols Institute Diagnostics, San Juan Capistrano, CA). The assay has a sensitivity of 0.3 pM and shows no cross-reactivity with other hormones, including PTH. Dilution studies demonstrated that parallelism and recovery of PTH-rp (1–86) was 80–91% in porcine follicular fluid. Androstenedione was measured by RIA, using antibody-coated tubes manufactured by ICN Biomedicals (Costa Mesa, CA). The RIA has a sensitivity of 0.1 ng/ml and exhibits low cross-reactivity with dehydroepiandrosterone (2.7%), testosterone (0.6%), and estrone (0.2%). Progesterone, prostaglandin (PG) E2, cAMP, and total cellular DNA were measured as described previously [13].
Statistical Methods
Data are presented as the mean ± SEM of three or more independent experiments using separate batches of ovaries to confirm reproducibility of results. Untransformed data were submitted to one-way ANOVA and Duncan's multiple comparison test to determine significant (P < 0.05) treatment effects [14]. Nonlinear least-squares curve fitting of the dose-response curves was used to estimate the mean and 95% confidence intervals for the values of the half-maximally effective stimulatory concentrations (ED50). For Ca2+ experiments, only LH-responsive cells were included in the analysis, and treatment effects were evaluated using a 3 x 2 chi-square table.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify which ovarian cell types are responsible for production of PTH-rp and to determine whether these cells express receptors for PTH-rp, we performed RT-PCR on total RNA isolated from freshly purified granulosa or theca cells collected from small- and medium-sized follicles and from corpora lutea collected 12 days after eCG/hCG induction of the estrous cycle. We found cDNAs encoding a 167-bp segment of the PTH-rp receptor in all three tissues, corresponding to position 563–730 of the full-length porcine receptor [11]. This partial cDNA was 94% and 91% homologous to the human and rat PTH-rp receptors, respectively. In addition, a cDNA encoding a 419-bp portion of PTH-rp peptide was isolated from corpora lutea. This sequence corresponds to position 32–451 of the human coding sequence [12] and was 97% and 87% similar to the human and rat PTH-rp. A representative ethidium bromide-stained gel of cDNAs encoding PTH-rp and its receptor is presented in Figure 1.
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To assess the regulation of PTH-rp production, immature granulosa cells were cultured in serum-free medium for 24 or 48 h. Treatments consisted of FSH (100 ng/ml), estradiol (1 µg/ml), insulin (3 µg/ml), FSH+insulin, IL-1 (10 ng/ml), EGF (10 ng/ml), IGF-I (300 ng/ml), or IGF-I+EGF, each of which had no effect on PTH-rp production (Table 2. However, treatment with TGF-ß1 (10 ng/ml) for 48 h increased PTH-rp production threefold (P < 0.05). The effect of TGF-ß was dose and time dependent. Three- to fivefold increases above basal levels were observed at 48 and 96 h, respectively (Fig. 2). The ED50 values for TGF-ß were 0.38 ± 0.06 ng/ml at 48 h and 0.24 ± 0.02 ng/ml at 96 h. Increases in mRNA encoding PTH-rp were also observed in granulosa cells after 48 and 96 h of incubation with TGF-ß (30 ng/ml) (Fig. 2 inset).
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To evaluate the functional role of PTH-rp, we established separate serum-free cultures of purified granulosa and theca cells in the presence of human PTH-rp at concentrations ranging from 1 nM to 1 µM for various time intervals. We observed no effect on cAMP accumulation when theca cells were treated with PTH-rp for 1, 2, 5, 30, or 60 min or for 4 or 24 h. Moreover, PTH-rp had no effect on basal accumulation of androstenedione or PGE2 after 24 or 48 h of treatment. Similarly, production of progesterone, cAMP, or PGE2 by granulosa cells was unaffected by PTH-rp after 24 or 48 h of treatment.
Because activation of PTH-rp receptors by PTH-rp in other tissues is associated with increased soluble inositol phosphate formation and increases in [Ca2+]i [15,16], we determined whether PTH-rp evokes calcium transients in single theca cells. A total of 435 theca cells were imaged at 48 (n = 66) or 72 h (n = 369) after collection. As seen in Figure 3, PTH-rp at concentrations of 1 µM evoked significant (P < 0.001) [Ca2+]i responses in 58% of theca cells that were also LH-responsive. Although it was not statistically significant, lower concentrations of PTH-rp (10 nM) elicited a calcium rise in 28% of LH-positive cells versus 20% in controls. The response was characterized by a slow rise in [Ca2+]i that plateaued after a wash with vehicle (Figure 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present studies used RT-PCR to demonstrate the expression and localization of PTH-rp and its receptor in the porcine ovary. Specifically, the corpus luteum expressed mRNA for both PTH-rp protein and receptor. In contrast, within the developing follicle, both theca and granulosa cells expressed mRNA encoding receptors for PTH-rp, but no measurable peptide or mRNA for PTH-rp was detectable in untreated cell cultures. The latter presumably reflects very low transcript abundance in the absence of stimulation in vivo, since TGF-ß-stimulated granulosa cells produce PTH-rp mRNA and immunoreactive protein in a time- and dose-dependent manner in vitro. Similar TGF-ß effects were reported in human myometrial smooth muscle cells, endometrial stromal cells, and breast carcinoma cell lines [18,19]. In each of these cell types, the level of PTH-rp mRNA and the accumulation of immunoreactive PTH-rp in culture medium increased in response to TGF-ß. Given that theca cells are the major secretory source of TGF-ß1 in the porcine ovary [20,21] and that granulosa cells can synthesize and secrete PTH-rp when treated with TGF-ß (present studies), we speculate that local regulation of PTH-rp may occur in vivo via a similar paracrine mechanism.
Little is known about the functional role of PTH-rp in ovarian tissue. The present studies demonstrate that PTH-rp can stimulate increased [Ca2+]i in single theca cells. Fluorescence videomicroscopy further revealed that PTH-rp activates a [Ca2+]i signal of low amplitude with slow kinetics, characteristic of extracellular influx [22,23]. This [Ca2+]i response mode is not typical of the biphasic spike and plateau [Ca2+]i increases that occur when receptor-activated PLC hydrolyzes phosphatidylinositol-4,5-bisphosphate to produce soluble mediators (inositol 1,4,5-triphosphate and diacylglycerol) that mobilize Ca2+ from internal stores. Agonists known to increase [Ca2+]i through the inositol phosphate pathway typically evoke rapid Ca2+ transients that are sustained briefly [24]. This spike phase may be followed by a delayed plateau-like [Ca2+]i elevation. In contrast, the sustained slow-onset [Ca2+]i rise induced by PTH-rp resembles that observed in granulosa or Sertoli cells stimulated with cAMP or FSH [10,25]. Moreover, the failure of PTH-rp to stimulate cAMP accumulation in cultured theca cells suggests that PTH-rp-mediated increases in [Ca2+]i may occur by a non-cAMP-dependent second messenger pathway or possibly via direct activation of ion channels. Whether the PTH-rp receptor can act as an ion channel like the nicotinic acetylcholine [26], gamma-amminobutyric acid [27], and glycine [28] receptors is not known. Indeed, to our knowledge, there are no electrophysiologic studies that have characterized the putative ion channels mediating PTH-rp action on [Ca2+]i in ovarian cells.
The role of PTH-rp-mediated [Ca2+]i increases in ovarian theca cells is not currently known. One possibility is that the [Ca2+]i signal, independently of PLC activation or protein kinase C translocation, activates calmodulin. Activated Ca2+-calmodulin may mediate increased transport of cholesterol to mitochondria by a mechanism that involves the actin cytoskeleton [29]. Another possible role for the Ca2+ signal generated by PTH-rp is activation of a Ca2+-sensitive adenylyl cyclase or cAMP-phosphodiesterase isoform, thereby linking [Ca2+]i and cyclic nucleotide metabolism. Alternatively, increases in [Ca2+]i could facilitate the translocation of cytoplasmic protein kinase C isoforms to cellular or nuclear membranes to phosphorylate target substrates. Thus, ovarian cell signaling mechanisms induced by PTH-rp will require further elucidation.
In summary, the present studies demonstrate the presence of transcripts encoding PTH-rp and its receptor in the (swine) ovary, and PTH-rp protein in follicular fluid and granulosa cell spent medium. Expression of PTH-rp mRNA is greatest in the corpus luteum and least, but detectable, in granulosa cells differentiated in vitro with TGF-ß. PTH-rp receptor message is expressed at high levels in the corpus luteum and theca cells, and in smaller amounts in granulosa cells. In single theca cells, PTH-rp initiates sustained increases in cytoplasmic [Ca2+]i in the majority of cells exposed to this peptide hormone. These findings allow the speculation that PTH-rp may play a role in modulating ovarian function by increasing [Ca2+]i-dependent second messenger signaling and thereby contribute to the local control of follicular and/or luteal growth and development.
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FOOTNOTES |
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1 Supported in part by NIH Grants HD-16806 (to J.D.V.), HD-12335 (to M.E.B.), and U54-HD96008 (Specialized Cooperative Centers in Reproductive Research). 
2 Correspondence: James C. Garmey, Department of Internal Medicine, Box 202, University of Virginia Health Sciences Center, Charlottesville, VA 22908. Fax: 804 982 3923; jcg8p{at}virginia.edu
Accepted: September 22, 1999.
Received: June 28, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-): pleiotropic effects of TNF- on differentiated functions of untransformed swine granulosa cells. Endocrinology 1991; 129:641–648.This article has been cited by other articles:
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  I. Karpati, I. Seres, J. Matyus, T. Ben, G. Paragh, Z. Varga, and G. K. Which parameters affect cytosolic free calcium in polymorphonuclear leukocytes of haemodialysis patients? Nephrol. Dial. Transplant., July 1, 2001; 16(7): 1409 - 1415. [Abstract] [Full Text] [PDF]
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