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Transforming growth factor-beta (TGF-beta), a multifunctional polypeptide growth factor, is produced by follicular cells in the ovary. However, there is little information indicating that TGF-beta is produced in the post-ovulatory follicle, i.e. the corpus luteum. In the present communication we present evidence that bovine luteal cells secrete large amounts of TGF-beta when maintained in moderate-term monolayer culture. Using TGF-beta 1 and TGF-beta 2 subtype-specific antibodies to neutralize the bioactivity it was found that 80-90% TGF-beta activity in luteal cell conditioned medium (LCCM) is due to TGF-beta 1, whereas < or = 10% TGF-beta activity in LCCM is due to TGF-beta 2. TGF-beta subtype nonspecific antibodies effectively and completely neutralized all TGF-beta activity in LCCM. The ratio of TGF-beta 1:TGF-beta 2 as estimated on the basis of neutralization studies was supported by visual observation of TGF-beta 1 and TGF-beta 2 protein bands on Western blotting. Using a modified and rapid mink lung epithelial cell bioassay and authentic TGF-beta to generate standard curves, the amount of TGF-beta secreted by luteal cells in vitro was quantitated. The concentration of luteal cell secreted proteins in the medium increased with time during 7 days of culture. Likewise, the TGF-beta concentration in LCCM increased during 7 days. To study the effect of duration of culture on the rate of TGF-beta secretion by luteal cells, conditioned medium was collected at 24 h intervals and replaced with fresh nutrient medium.(ABSTRACT TRUNCATED AT 250 WORDS)

An important but poorly understood aspect of mammalian follicle development involves the regulation of theca cell proliferation. To investigate the premise that growth factors regulate theca cell proliferation, porcine theca cells were prepared by collagenase/DN'ase digestion of follicle linings after the removal of the granulosa cells and allowed to attach for 24 h. This method provided a monolayer of theca cells that had little if any granulosa cell contamination and which secreted high levels of androstenedione relative to granulosa cells during moderate-term culture (33-fold difference, P less than 0.01). In medium containing fetal calf serum (10%), theca cells were significantly more responsive to platelet-derived growth factor (PDGF) than epidermal growth factor (EGF) in terms of proliferation (13.4 +/- 0.2-vs. 7.0 +/- 0.1-fold increases relative to the initial cell count, P less than 0.05). This is in contrast to granulosa cells which were significantly more responsive to EGF than PDGF (7.1 +/- 0.1 vs. 4.0 +/- 0.2 fold-increases, P less than 0.05). Since serum has been shown to contain both EGF and PDGF, proliferation studies were performed using plasma-derived serum (PDS) which is growth factor restricted to examine more closely the direct effects of growth factors. In medium containing 0.25% PDS and within experiments, PDGF (1-25 ng/ml) stimulated theca cell proliferation in a dose-dependent manner (2.3-fold increase relative to controls, P less than 0.05) whereas EGF did not. EGF, however, markedly enhanced the proliferative action of PDGF (6.4-fold increase relative to controls, P less than 0.05). Insulin-like growth factor I and low density lipoprotein, factors which enhance markedly the proliferative effects of EGF and PDGF in terms of granulosa cell proliferation, exhibited only a modest synergistic effect with respect to EGF and PDGF upon theca cells (9.5-fold increase vs. a 6.4-fold increase above controls, P less than 0.05). Temporal studies in vitro indicate that theca cell proliferation is low during the first 3-day exposure to growth factors irrespective of treatment (a 2-fold increase over the seeding density). During the second 3-day exposure, however, theca cell proliferation increases 4- to 5-fold. The temporal pattern of theca cell proliferation stimulated by fetal calf serum supplemented with EGF or PDGF and PDS-containing medium supplemented with PDGF, EGF, insulin-like growth factor I, and low density lipoprotein is similar. These results suggest that PDGF is a major mitogen toward porcine theca cells and that EGF greatly enhances its activity.(ABSTRACT TRUNCATED AT 400 WORDS)