Changes in follicular fluid steroids, insulin-like growth factors (IGF) and IGF-binding protein concentration, and proteolytic activity during equine follicular development2

Abstract

The objective of the present study was to evaluate changes in equine follicular fluid insulin-like growth factor binding protein (IGFBP) proteolytic activity as well as steroid, IGF, and IGFBP concentrations during follicular development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6 to 15 mm; n = 54), medium (16 to 25 mm; n = 23), or large (> 25 mm; n = 15), and follicular fluid was collected. Estradiol and androstenedione levels in follicular fluid were greater (P < 0.05), and IGFBP-3 concentrations tended to be greater (P < 0.10) in large than in small or medium follicles, whereas IGFBP-2, -4, and -5 levels were less (P < 0.05) in large than in small or medium follicles. Estradiol and androstenedione concentrations were negatively correlated (P < 0.01) with IGFBP-2, -4, and -5 but not IGFBP-3 concentrations. To evaluate proteolysis of IGFBP, follicular fluid was incubated with human 125I-labeled IGFBP-2, -3, and -5 and protein separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of 125I-lableled IGFBP-2 or -3, and the small amount of proteolysis of IGFBP-2 and -3 did not differ (P > 0.10) among follicle classes. However, more 125I-labeled IGFBP-5 was cleaved (P < 0.05) by follicular fluid from large follicles collected during the follicular phase than large follicles during the luteal phase, and small or medium follicles from follicular and luteal phase mares indicating that a protease to IGFBP-5 exists in estrogen-dominant equine follicles. This IGFBP-5 protease was inhibited by kallikrein/serine protease and metalloprotease inhibitors. We conclude that the tendency of estrogen-dominant follicles of mares to have greater levels of IGFBP-3 and lesser levels of IGFBP-2 does not appear to be due to differences in proteolysis, whereas changes in IGFBP-5 levels are likely due to changes in activity of a serine protease or metalloprotease. Changes in IGFBP may alter levels of bioavailable IGF that stimulate steroidogenesis and mitogenesis in developing mare follicles.

Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles

“…In the mare, previous experiments have shown by WLB that serum contained higher levels of IGFBP-2 and IGFBP-4 than the corresponding preovulatory follicles (Gérard & Monget 1998, Bridges et al 2002. Immunoblotting experiments confirmed that the native 32 kDa form of IGFBP-2 was present at higher levels in serum (Fig.1A, lanes 1, 3 and 5) than in the corresponding dominant follicles (Fig.…”

“…Recently, Bridges et al (2002) claimed that equine preovulatory follicles contained proteolytic activity against IGFBP-4 but not IGFBP-2. The reason for the discrepancy with the present work is unknown.…”

“…Moreover, we have shown in the ewe, and more recently in the cow and the sow, that this decrease is also partly due to an increase in IGFBP-2 proteolytic degradation by PAPP-A (Besnard et al 1996b, Monget et al 2003, suggesting a well-conserved mechanism in mammalian species. In contrast, Bridges et al (2002) recently claimed that equine preovulatory follicles did not contain any proteolytic activity degrading IGFBP-2, suggesting a species-specific mechanism in the horse.…”

Follicle selection in cattle and horses: role of intrafollicular factors

“…In cattle, a greater proteolytic activity for BP-4 and -5 occurred in the LF than in the second LF apparently before the equivalent of beginning of deviation and was temporally associated with greater concentrations of free IGF-I in the LF and greater BP-4 and -5 in the second LF (Rivera & Fortune 2003). Proteolytic activity for BP-2 (Mazerbourg et al 2003), BP-4 (Mazerbourg et al 2000) and BP-5 (Bridges et al 2002) has also been reported in dominant follicles but well after the beginning of deviation in horses. In addition, a more recent report (Gerard et al 2004) indicated a greater BP-2 proteolytic activity in early dominant (25 mm) and late dominant (35 mm) follicles than in subordinate follicles; however, differential proteolytic activity of follicles in relation to deviation has not been reported in horses.…”

“…Thus, changes in intrafollicular IGFBPs lead to changes in IGF bioavailability and the up or down regulation of gonadotrophin actions on follicular cells. Four IGFBPs (BP-2, -3, -4 and -5) have been detected in follicular fluid of cattle (Echternkamp et al 1994, de la Sota et al 1996, Mihm et al 2000, Austin et al 2001 and at least four or five different IGFBPs (BP2, -3, -4, -5 and a high molecular weight complex of 90-135 kDa) in a follicular fluid of horses (Gerard & Monget 1998, Bridges et al 2002. However, only BP-2, -4 and -5 in cattle (Beg et al 2001, Rivera & Fortune 2003 and BP-2 in horses (Donadeu & Ginther 2002) have been studied in association with deviation.…”