THE SECRETION OF TRANSFORMING GROWTH-FACTOR-BETA BY BOVINE LUTEAL CELLS-INVITRO

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-beta1 and TGF-beta2 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 whereas less-than-or-equal-to 10% TGF-beta activity in LCCM is due to TGF-beta2. TGF-beta subtype nonspecific antibodies effectively and completely neutralized all TGF-beta activity in LCCM. The ratio of TGF-beta1:TGF-beta2 as estimated on the basis of neutralization studies was supported by visual observation of TGF-beta1 and TGF-beta2 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. The rate of TGF-beta secretion increased gradually on days 2, 3 and 4 of culture and declined slowly thereafter. Interestingly, measurement of the rate of total protein secretion into the conditioned medium at 24 h intervals showed a steady decline with the duration of culture. The significant decrease in protein secretion by luteal cells during the latter part of prolonged culture resulted in an increase in TGF-beta secretion per unit total protein secretion (i.e. ng TGF-beta/mug protein). These studies for the first time demonstrate that bovine luteal cells secrete high amounts of latent TGF-beta1 and relatively little TGF-beta2. It is suggested that luteal cell secreted TGF-beta may play a role as an autocrine or paracrine regulator of luteal function and may modify gonadotropin-dependent ovarian follicular functions.