Evidence for a role of the alternatively spliced ED-I sequence of fibronectin during ovarian follicular development

This study was aimed at testing the hypothesis that different forms of fibr onectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovar ian follicle. In particular, we were interested in determining the effect o f the ED-I (also termed ED-A) type III repeat, which is absent in the plasm a form. Analysis of FN levels in follicular fluids corresponding to differe nt stages of development of bovine follicles revealed marked changes in the concentrations of ED-I+FN, whereas total FN levels remained relatively con stant. ED-I+FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological ro le for ED-I+FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 1 0-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-beta elicited a 2-fold stimulation of overall F N synthesis and a 4-fold increase in the synthesis of ED-I containing FN. T his effect was evident at the protein (Western blots) and messenger RNA (No rthern blots) levels. Although a negative correlation (P < 0.001) was detec ted between ED-I+FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I+FN was suggested by the observation that a recombi nant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I+FN as a growth regulatory factor was further strengthened by the fact that depletio n of FN from BGC-1-conditioned medium, which contained ED-I+FN, abrogated i ts mitogenic activity, whereas plasma FN was without effect. We propose tha t changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.