Intrafollicular insulin-like growth factor-binding protein levels in equine ovarian follicles during preovulatory maturation and regression

The profiles of insulin-like growth factor-binding proteins (IGFBPs) in follicular fluid have been characterized in a number of mammals (rats, pigs, sheep, cattle, humans) and are good indicators of follicular status. We studied the IGFBP profiles of equine serum and ovarian follicular fluid recovered at various stages of the follicular phase. The levels of IGFBPs were related to the morphology and the steroidogenic activity of the follicles. Follicular fluids were recovered by ultrasound-guided follicular aspiration. In the first experiment, the dominant follicles of 10 mares were partly punctured (aspiration of 0.5-2.2 ml of fluid) once at the early dominant stage (22-25 mm in diameter) and a second time at the preovulatory stage (PO), 34 h after induction of ovulation. Among these 10 PO follicles, 5 were classified as healthy whereas the other 5 were classified as hemorrhagic, as assessed by ultrasonic morphology and subsequent ovulation or not. In another group of mares (n = 5), the largest follicle was punctured once at the late dominant stage (33-35 mm in diameter) and then at the PO stage, 34 h after induction of ovulation. Serum was prepared at each puncture session. In the second experiment, follicular fluid was recovered from the dominant and contemporary cohort subordinate follicles (n = 5 mares). Samples were individually assayed for estradiol-17 beta and progesterone content by RIA, and IGFBPs were studied by using Western ligand blotting and densitometry. Equine serum and follicular fluid displayed IGFBP at 42-44 kDa (likely corresponding to IGFBP-3), 28-32 kDa (likely corresponding to IGFBP-5), 24 kDa (likely corresponding to IGFBP-4), and 35 kDa, identified as IGFBP-2 by immunoblotting, plus one band at 120 kDa. IGFBP were clearly more abundant in serum than in fluid from healthy follicles. In the follicular fluid, 42-44-kDa IGFBP was the major binding protein, and its level was almost constant at the various physiological statuses studied. Follicular development of the dominant follicle in each mare was characterized by a decrease in intrafollicular IGFBP-2 and 28-32-kDa IGFBP levels before LH stimulation and by an increase in IGFBP-2 after LH stimulation. Follicular regression of large follicles, as well as subordinate ones, was characterized by a low level of intrafollicular estradiol-17 beta and was associated with an increase in IGFBP-2, 24-kDa IGFBP, and 28-32-kDa IGFBP intrafollicular levels. Taking these results together, we have demonstrated clear correlations between the intrafollicular levels of estradiol-17 beta and IGFBP-2 and 28-32-kDa IGFBP. Therefore, follicular growth and regression in the mare are associated with specific changes in IGFBP levels. These changes could be of crucial importance for follicular development in ovulation or atresia.