Previously, we found oviductal eggs to be significantly more penetrable and fertilizable in vitro than ovulated eggs collected from the ovarian bursa, while bursal eggs were comparable to mature (unovulated) follicular eggs. Incubation of follicular eggs with a soluble eluate of oviductal egg cumulus complexes (COF) increased sperm penetration: the activity was macromolecular, was destroyed at 56 degrees C, and was produced in the oviduct. We now report purification of this oviductal factor that enhances penetration of follicular eggs and have identified it as oviductin (OVN). Oviducts, 1-1.5 h post-LH from eCG-primed females, were homogenized and the cytosolic fraction was chromatographed on a Helix pomatia lectin affinity column; specific proteins were eluted with 0.2 M N-acetyl-D-galactosamine. Fractions were monitored by dot-blot assay using as the primary antibody monoclonal antibody (mAb) 1C4 against OVN. Proteins were resolved by one-dimensional SDS-gel electrophoresis, followed by electrotransfer and immunostaining of Western blots. OVN fractions were indexed to COF by quantitative dot-blot assay, and activity was bioassayed by penetration of follicular eggs within 1 h of coincubation with precapacitated sperm +/- factors: COF and BSA (high and low controls, respectively) and fractions from the lectin-isolated peak. The mean penetration rates for three isolations were 17 +/- 4.0a, 51.7 +/- 5.0b, and 49 +/- 2.7b% for BSA, COF, and column fractions, respectively (p < or = 0.05). Purified OVN bound to follicular zonae during culture. Acrosome-intact sperm heads bound OVN during 30 min of incubation both before (t = 0 h) and after capacitation (t = 5.5 h) (visualized by indirect immunofluorescence).(ABSTRACT TRUNCATED AT 250 WORDS)

Three sources of hamster periovulatory fluids (+/- heat inactivation at 56 degrees C), with bovine serum albumin (BSA) as control, were tested for effects on penetration of three classes of eggs by hamster sperm precapacitated in BSA. These fluids were a soluble extract of cumulus oophorus fluid (COF) from the ovulated hamster egg-cumulus complex, serum, and follicular fluid. Egg types were ovulated, salt-stored (ovulated), and follicular. In both COF and serum, there were significant differences among egg types in mean penetration, and significant effects of fluid addition. In contrast, there was no effect of follicular fluid and no differences between follicular and stored eggs. For the follicular eggs (combined data, normalized, ranked), patterns of response to the three factors (+/- heating) were different: only unheated COF and heated serum increased penetration significantly above BSA control levels (average rank 20.2, 41.4, 38, for BSA, COF (unheated), serum (heated), respectively). This indicated that the active component in COF was heat labile, not present in either serum or follicular fluid, and, therefore, of oviductal origin. Oviduct and/or COF exposure of eggs and sperm was tested for effects as an acrosome reaction inducing factor (ARIF) for acrosome reactions (AR; zona-bound and free-swimming sperm) and on sperm:zona binding and penetration. The COF ARIF for free-swimming sperm AR was heat stable. Penetration of follicular eggs increased after incubation in COF prior to sperm addition, but a greater response occurred when COF was added to eggs with sperm. In kinetic experiments, 25 min following sperm attachment, follicular eggs had lost 41% of initially bound sperm, vs. 23% for ovulated eggs, and had only 16 AR sperm/egg, vs. 26 for ovulated. Follicular eggs incubated in COF (then washed three times) had the same number of bound AR sperm as ovulated eggs. Acid solubilized zona pellucida (ASZP) from ovulated eggs was more effective as an ARIF per zona than ASZP from follicular eggs. Zonae of follicular eggs, as evidenced by dissolution times in beta-mercaptoethanol (beta-MEOH), were not "harder" than those of ovulated eggs. There were differences in lectin binding antigens on zonae of both fresh and stored, follicular and ovulated, eggs. We conclude that multiple biological factors orchestrate sperm:egg interactions in the ampulla. Our data are consistent with the presence of at least three effective components: 1) the oviductal lectin-binding antigen (ZPO or oviductin), 2) an additional heat-labile component, and 3) the heat-stable ARIF for free-swimming sperm.