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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
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.
Parathyroid hormone-related peptide (PTH-rp) and the PTH-rp receptor are expressed in certain cancers as well as in many normal tissues. To evaluate the expression of this Ca2+-regulating hormone and its receptor in porcine ovary, we isolated partial cDNAs encoding homologous PTH-rp and PTH-rp receptor using reverse transcription-polymerase chain reaction (RT-PCR). The cDNA encoding PTH-rp (419 base pairs [bp]) was 92% and 87% homologous to human and rat sequences, respectively, while the PTH-rp receptor clone (167 bp) was 94% and 91% identical to the human and rat genes. Qualitative estimates of PTH-rp mRNA by RT-PCR indicated that the PTH-rp gene is expressed at high levels in the corpus luteum but is undetectable in granulosa and theca cells isolated from small (1–5 mm) and medium-sized (5–8 mm) antral follicles. In contrast, PTH-rp receptor transcripts were most abundant in corpora lutea and theca cells, and least abundant (albeit detectable) in granulosa cells.
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
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