Human follicular fluid contains pro- and C-terminal immunoreactive alpha-inhibin precursor proteins

Abstract

The majority of immunoactive inhibin in human follicular fluid (hFF) is devoid of pituitary cell bioactivity and is hypothesized to contain alpha-inhibin monomeric proteins known to cross-react with an inhibin antiserum (Monash 1989). The aim of this study was to define more precisely the nature of these inhibin-immunoreactive proteins using alpha-inhibin sequence-specific antisera. First, a polyclonal antiserum was raised to precursor amino acids 21-35 (PIN-1) and was used in a RIA to measure pro-alpha-inhibin-immunoreactive proteins. Western blotting was used to confirm these findings. Secondly, the binding epitope of the Monash antiserum was defined by peptide analysis to be located within the C-terminus (precursor amino acids 326-341) of alpha-inhibin, and this assay was then used to monitor the presence of C-terminal sequences. Similar levels of pro-alpha-inhibin immunoreactivity (PIN-1 RIA; N-terminus alpha-inhibin precursor) were detected in hFF collected from women with normal menstrual cycles during the follicular phase and from multiple follicles from a woman undergoing ovarian hyperstimulation during an in vitro fertilization (IVF) protocol. Western blotting with the PIN-1 antibody confirmed the presence of immunoreactive proteins of 57,000 and 29,000 mol wt in the follicular fluids of both normal cycle and IVF follicles. However, ovarian hyperstimulation elevated intrafollicular C-terminal immunoreactivity (Monash RIA) compared to that in normal cycle hFF. Furthermore, intrafollicular estradiol and progesterone were significantly correlated to C-terminal activity in follicles from IVF, but not in normal cycles. These data show that 1) both pro- and C-terminal alpha-inhibin proteins are secreted into follicular fluids from normal and IVF cycles, suggesting that alpha-inhibin precursor proteins may be physiologically relevant in the process of folliculogenesis; and 2) IVF and normal cycle follicular fluids differ in their production and processing of inhibin.