Pregnancy-Associated Plasma Protein-A Is the Insulin-Like Growth Factor Binding Protein-4 Protease Secreted by Human Ovarian Granulosa Cells and Is a Marker of Dominant Follicle Selection and the Corpus Luteum

Abstract

Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases are important in ovarian function. IGFs stimulate granulosa steroidogenesis, an effect that is inhibited by IGFBP-4 and augmented by IGFBP-4 proteolysis. We have recently identified the IGFBP-4 protease in human ovarian follicular fluid (FF) as pregnancy-associated plasma protein-A (PAPP-A). In the current study, we identify the IGFBP-4 protease secreted by cultured human ovarian granulosa cells as PAPP-A, based on specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with PAPP-A polyclonal antibodies and immunorecognition by PAPP-A monoclonal antibodies in ELISA. PAPP-A was barely detectable in conditioned media (CM) from granulosa derived from </=9 mm androgen-dominant follicles, but was secreted by cultured granulosa from estrogen-dominant follicles >/=9 mm, coincident with dominant follicle selection, and by luteinizing granulosa. PAPP-A levels in CM from the latter did not change in response to IGF-II or hCG (100 ng/mL). A naturally occurring inhibitor of PAPP-A, proform of eosinophil major basic protein (proMBP), was detected by ELISA in estrogen-dominant follicular fluid FF, but not in CM from granulosa or luteinizing granulosa cells treated with IGF-II (0-200 ng/mL), FSH (0-100 ng/mL) or hCG (0-100 ng/mL), suggesting an alternative source (other than granulosa) for proMBP, compared to PAPP-A. The data demonstrate granulosa cells as a source of PAPP-A in human ovary and suggest that PAPP-A is a marker of ovarian follicle selection and corpus luteum formation. In addition the data suggest complex regulation of this system in human ovary.

Pregnancy‐associated plasma protein‐A (PAPP‐A) expression and insulin‐like growth factor binding protein‐4 protease activity in normal and malignant ovarian surface epithelial cells

“…Previous studies have described the expression of PAPP-A in healthy follicles and corpora lutea in human 11,38 and murine 39 ovaries, consistent with elevated PAPP-A expression (and decreased IGFBP-4 protein) being associated with IGF-induced estrogen production and selection of dominant follicles. 10 -12 Our results extend these analyses and show that normal, unmodified OSE cell cultures express abundant PAPP-A mRNA and protein.…”

“…6 By neutralizing the IGF-sequestering activity of IGFBP-4 by proteolysis to fragments with reduced affinity for IGF, activation of PAPP-A results in acute local increases in the levels of IGF available for interaction with IGF receptors in vitro. [7][8][9] Beyond its role in pregnancy, PAPP-A is expressed by osteoblasts 6,7 and human granulosa cells 10,11 and plays an important role in regulating IGF bioavailability in developing ovarian follicles. 12,13 In addition, PAPP-A activation is implicated in injury-repair responses in the vasculature through local increases in bioavailable IGF.…”

Cell Surface Detachment of Pregnancy-associated Plasma Protein-A Requires the Formation of Intermolecular Proteinase-Inhibitor Disulfide Bonds and Glycosaminoglycan Covalently Bound to the Inhibitor

“…The initial recognition is followed by the formation of intermolecular PAPP- (17). Covalent complex formation allows the proMBP GAG to compete with the PAPP-A receptor for PAPP-A surface binding, leading to detachment of the PAPP-A⅐proMBP complex (4). Importantly, the formation of intermolecular disulfide bonds between PAPP-A and proMBP results in the inhibition of PAPP-A proteolytic activity (15), and therefore, the release of bound IGF cannot occur.…”

“…Monoclonal antibodies against PAPP-A (PA-1A) or proMBP (PM-5A) 4 followed by peroxidase-conjugated anti-(mouse IgG) (P0260, DAKO) were used for detection. The wells were blocked by incubation with PBS containing 2% bovine serum albumin.…”

Role of apoptosis-related factors in follicular atresia

“…Dominant and subordinate follicles differ in their IGFBP contents (Monget et al, 1996). Their presence is controlled at the level of synthesis (Armstrong et al, 1998) by the gonadotropins and by the presence of proteases which breakdown low molecular weight IGFBP in the healthy antral follicles of cows Fortune, 2001, 2003a, b), humans (Conover et al, 2001), and mice (Conover et al, 2002).…”