Expression of parathyroid hormone-related peptide (PTH-rp) and its receptorin the porcine ovary: regulation by transforming growth factor-beta and possible paracrine effects of granulosa cell PTH-rp secretion on theca cells.
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 Ca(2+)-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. Regulation of PTH-rp protein production was assessed in serum-free monolayer cultures of porcine granulosa cells. Transforming growth factor (TGF)-beta1 (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-beta1 dose-response studies revealed an ED(50) 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 Ca(2+)-sensitive fluorescent indicator dye, fura-2, and digital imaging videomicroscopy, we found that PTH-rp (1 microM) stimulated intracellular free calcium ion concentrations ([Ca(2+)](i)) in single porcine theca cells. The [Ca(2+)](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 [Ca(2+)](i) response. Our results allow a hypothesis of a paracrine intrafollicular signaling system involving interaction between theca cell-derived TGF-beta 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.
Garmey
J C
JC
Division of Endocrinology, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. jcg8p@virginia.edu
Schnorr
J A
JA
Bruns
M E
ME
Bruns
D E
DE
Seaner
R M
RM
Ferguson II
J E
JE
Luking Jayes
F C
FC
Aguirre
C
C
Veldhuis
J D
JD
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D006062:Gonadotropins / Q000494:pharmacology; D006107:Granulosa Cells / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D010053:Ovary / Q000378:metabolism*; D019899:Paracrine Communication / Q000502:physiology*; D010281:Parathyroid Hormone / Q000096:biosynthesis*; D044162:Parathyroid Hormone-Related Protein; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D018016:Receptors, Parathyroid Hormone / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D013552:Swine; D013799:Theca Cells / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000502:physiology*
D006062:Gonadotropins; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D012333:RNA, Messenger; D018016:Receptors, Parathyroid Hormone; D016212:Transforming Growth Factor beta; D002118:Calcium