Selection of the Dominant Follicle and Insulin-Like Growth Factor(IGF)-Binding Proteins: Evidence that Pregnancy-Associated Plasma Protein A Contribu
Abstract., http://www.100md.com
Development of a dominant follicle is associated with decreased intrafollicular low molecular weight IGF-binding proteins (namely IGFBP-2, -4, and -5) and increased proteolysis of IGFBP-4 by pregnancy-associated plasma protein A (PAPP-A). In addition to IGFBP-4 proteolytic activity, bovine follicular fluid contains strong proteolytic activity for IGFBP-5, but not for IGFBP-2. Here we show that the IGFBP-5 protease present in bovine follicular fluid is a neutral/basic pH-favoring, Zn2+ metalloprotease very similar to the previously described IGFBP-4 protease. We hypothesized that immunoneutralization and immunoprecipitation with anti-PAPP-A antibodies would result in abrogation of the IGFBP-4, but not the IGFBP-5, proteolytic activity in follicular fluid. As expected, anti-PAPP-A antibodies were able to neutralize and precipitate the IGFBP-4, but not the IGFBP-5, proteolytic activity of human pregnancy serum, which was used as a positive control for PAPP-A. Surprisingly, immunoneutralization and immunoprecipitation of follicular fluid from bovine preovulatory follicles with anti-PAPP-A antibodies abrogated both IGFBP-4 and IGFBP-5 proteolysis. Quantitative results derived from phosphorimaging revealed a complete inhibition of both IGFBP-4 and -5 proteolysis by follicular fluid incubated for 2 or 5 h in the presence of anti-PAPP-A antibodies. After 18 h of incubation, anti-PAPP-A antibodies still inhibited IGFBP-5 degradation, although with an efficiency lower than that for IGFBP-4 degradation. Both proteolytic activities have identical electrophoretic mobility, and a single band (~ 400 kDa) was detected by Western immunoblotting of bovine follicular fluid with anti-PAPP-A antibodies. Proteolysis of IGFBP-5 was readily detectable in follicular fluid from dominant follicles and was negligible in subordinate follicles from the same cohort. These results suggest that an active intrafollicular IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is an important determinant of follicular fate.
Introductiondo43/x4, 百拇医药
A CRITICAL, but poorly understood, transition in follicular development is the selection of a dominant follicle capable of ovulating from a cohort of follicles recruited by a small increase in circulating FSH (1, 2, 3). In addition to the well documented increase in estradiol-synthesizing capacity (4, 5), dominant follicles are characterized by decreased levels of low molecular weight (mol wt) IGF-binding proteins (IGFBP-2, -4, and -5) (6, 7, 8, 9). Compelling evidence for an obligatory role of the IGF system in antral follicular development is provided by the infertile IGF-I-null mouse in which follicles are arrested at the preantral/early antral stage and fail to respond to gonadotropin administration (10). It is hypothesized that the decrease in the inhibitory, low mol wt IGFBPs in dominant follicles increases the amount of bioactive IGF available to synergize with FSH in promoting follicular growth and estradiol production (9, 11, 12, 13). Proteolytic activity against IGFBP-4 has been detected in the follicular fluid of human (14), bovine, ovine, and porcine (15) preovulatory follicles and is identical to pregnancy-associated plasma protein A (PAPP-A), first isolated from human pregnancy serum over 20 yr ago (16). Using cattle as the experimental model, we showed recently that there are lower levels of IGFBP-4 in dominant vs. subordinate follicles, very close to the time of follicular selection, due to an FSH-dependent metalloprotease activity against IGFBP-4 in the dominant follicle (17, 18).
Depending on the biological system, IGFBP-5 may exert either stimulatory (19) or inhibitory (20) effects on the IGF-receptor interaction; however, there seems to be consensus that IGFBP-5 inhibits IGF’s actions on ovarian cells (11, 12). The mechanisms that subserve the reduction in levels of the inhibitory IGFBP-5 in the dominant follicle are poorly understood. Recent reports indicate that follicular fluid from porcine (21), ovine (22), and equine (23) preovulatory follicles contains a metalloprotease or serine protease (equine) that degrades IGFBP-5. In addition, an uncharacterized proteolytic activity against IGFBP-5 has been reported recently in follicular fluid from bovine preovulatory follicles (24). A common theme emerging from those studies is that identification of the putative IGFBP-5 protease(s) remains elusive.%, http://www.100md.com
The goal of the current study was to characterize the putative IGFBP-5 protease activity in bovine follicular fluid and to investigate its potential association with selection of the dominant follicle. Cattle provide an excellent model for studies of the mechanisms of follicular selection for dominance because a follicular wave is recruited about every 7–8 d during the 21-d estrous cycle, the dominant follicle of any cohort has ovulatory capacity during its tenure of dominance if the corpus luteum is regressed naturally or experimentally, individual follicles of the cohort can be followed from day to day by ultrasound imaging, and the follicles are large enough to provide abundant follicular fluid for analysis of potential regulatory molecules.
Materials and Methodsj42'gd, 百拇医药
Animals and experimental protocolsj42'gd, 百拇医药
The main objective of this study was to characterize the putative IGFBP-5 protease present in follicular fluid from bovine preovulatory follicles. To that end, Holstein heifers with regular estrous cycles were injected with prostaglandin F2{alpha} (PGF2{alpha} ; 25 mg, im; Lutalyse, Pharmacia \|[amp ]\| Upjohn, Inc., Kalamazoo, MI) on d 7 of the estrous cycle (d 0 = day of estrus) to induce luteolysis. Luteolysis is followed by initiation of a follicular phase and differentiation of the dominant follicle of the first follicular wave into a preovulatory follicle. The ovaries of each heifer were examined daily by transrectal ultrasonography as described previously (5). Follicular fluid from the preovulatory follicle (n = 4 heifers) was obtained after ovariectomy performed 24 h after injection of PGF2{alpha} . In this experimental model (25, 26), estrus and the LH surge occur about 48–60 h after PGF2{alpha} ; thus, follicles were obtained about 24–36 h before the expected time of the LH surge.
To begin to explore the potential physiological role of intrafollicular IGFBP-5 proteolysis in the establishment of follicular dominance, follicular fluid samples were also obtained from the dominant follicle and the companion subordinate follicles on d 3 of the follicular wave (emergence of the wave = d 0; n = 4), close to the time of follicular selection. Luteolysis, a follicular phase, and ovulation were induced by injecting heifers with PGF2{alpha} during the midluteal phase. The ovaries of each heifer were examined daily by transrectal ultrasonography, as described above, to observe ovulation and the initiation of the first follicular wave of the next estrous cycle. Follicular fluid samples were obtained on d 3 of the follicular wave by ultrasound-guided follicular aspiration as previously described (17). Follicular fluid samples were centrifuged, and aliquots were stored at -80 C for later determinations. Animals were used in accordance with procedures approved by the Cornell University animal care and use committee (Protocol 86-214-99).
Analysis of IGFBP-2, -4, and -5 proteolytic activities-}:q(, http://www.100md.com
The ability of intrafollicular proteases to degrade IGFBP-2, IGFBP-4, or IGFBP-5 was assessed by incubating 2 or 5 µl follicular fluid plus substrate for 18 h at 37 C in a solution of 20 mM Tris (pH 7.5) containing 137 mM NaCl (TBS) and 0.1% BSA (final volume, 20 µl). In characterization experiments, 50 ng recombinant human (rh) IGFBP-2, -4, or -5 (Austral Biologicals, San Ramon, CA) were used as substrate. Protease assay samples were subjected to SDS-PAGE, followed by Western ligand blotting/phosphorimaging to quantify the percentage of substrate loss, as previously described (17). In addition, IGFBP-5 proteolytic activity in samples obtained from dominant and subordinate follicles of the first follicular wave was assessed by incubating 5 µl follicular fluid with approximately 50,000 cpm 125I-labeled rhIGFBP-5 for 18 h at 37 C. This assay was chosen to assess proteolytic activity against IGFBP-5 because the presence of abundant endogenous IGFBP-5 in subordinate follicles could interfere with determinations using cold rhIGFBP-5 substrate followed by Western ligand blotting. Iodination of rhIGFBP-5 was performed by the chloramine-T method as previously described (24).
Partial characterization of IGFBP-5 protease activity(w8jki, 百拇医药
To characterize the putative IGFBP-5 protease detected in follicular fluid from preovulatory follicles, rhIGFBP-5 was used as the substrate, followed by Western ligand blot analysis/phosphorimaging. The time and pH dependence of the IGFBP-5 degradation was assessed after incubation of 50 ng rhIGFBP-5 with follicular fluid from preovulatory follicles at 37 C. To provide initial mechanistic classification of the IGFBP-5 protease activity present in bovine follicular fluid, the following set of standard protease inhibitors (Sigma-Aldrich, St. Louis, MO), corresponding to the four protease classes recognized by the International Union of Biochemistry (27), was used: 1,10-phenanthroline (10 mM; metalloprotease inhibitor), trans-epoxysuccinyl- L-leucylamido-(4- guanidino)-butane (E-64; 10 µM; cysteine protease inhibitor), aprotinin (2 µg/µl; serine protease inhibitor), pepstatin (1 µM; aspartic protease inhibitor), phenylmethylsulfonyl fluoride (PMSF; 1 mM; serine protease inhibitor), chymostatin (100 µM; an inhibitor of chymotrypsin-like serine proteases and some cysteine proteases), and the nonspecific divalent cation chelator EDTA (5 mM). The inhibitors and doses used were chosen based on reported specificity and efficacy, respectively (27, 28, 29).
Immunoneutralization and immunoprecipitation of PAPP-A6^{'fjm, http://www.100md.com
Human pregnancy serum (hPS) was used as a positive control for PAPP-A in the experiments described below. Immunoneutralization was performed by preincubating an aliquot (5 µl) of hPS or follicular fluid from bovine preovulatory follicles with vehicle (PBS), nonimmune immunoglobulin G (IgG), or antihuman PAPP-A polyclonal antibody (both from DAKO Corp., Carpinteria, CA) for 1 h at room temperature. After preincubation, 50 ng rhIGFBP-4 or rhIGFBP-5 substrate were added, and the reaction mix was incubated at 37 C for various intervals as indicated. At the end of the incubation, samples were analyzed by Western ligand blotting.6^{'fjm, http://www.100md.com
Immunoprecipitation was performed as previously described (30). Briefly, 100 µl hPS or follicular fluid from bovine preovulatory follicles were preadsorbed by incubation with 100 µl Protein A/G Plus Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 30 min with frequent manual mixing, to reduce substances present in the samples that bind nonspecifically to Protein A/G Plus Agarose. After centrifugation, the supernatant was collected. This step was repeated three times. The supernatant was then divided into three aliquots and incubated overnight with vehicle (PBS), normal IgG (40 µg), or rabbit antihuman PAPP-A polyclonal antibody (40 µg) on a rocking platform. The samples were mixed with 100 µl Protein A/G Plus Agarose with frequent manual mixing for 1 h, followed by centrifugation and collection of the supernatant. This step was repeated twice. All incubations were performed at 4 C. The final supernatant was used in IGFBP protease assays.
Western ligand blot analysis\wae@9g, 百拇医药
Western ligand blot analysis was performed as previously described (17). Briefly, samples were subjected to electrophoresis in 12% SDS-PAGE gels under nonreducing conditions and transferred onto nitrocellulose membranes. The membranes were incubated with 1.2 x 106 cpm [125I]IGF-II (specific activity, 340–430 µCi/µg) overnight at 4 C, then washed, air-dried, and subjected to autoradiography and phosphorimaging. Mol wts of intact IGFBP species were estimated by running the samples in parallel with protein mol wt standards (2,850–43,000 mol wt range; Life Technologies, Inc., Grand Island, NY) on the same gel.\wae@9g, 百拇医药
Western immunoblot analysis of PAPP-A\wae@9g, 百拇医药
Follicular fluid samples (2.5 or 5 µl) were mixed with 2x loading buffer, boiled for 3 min, and resolved on a 6% SDS-PAGE gel under nonreducing conditions. Proteins were transferred to nitrocellulose membranes and subjected to immunoblot analysis using rabbit anti-PAPP-A polyclonal antibodies (DAKO Corp.). Briefly, membranes were washed twice in distilled water for 15 min each time and blocked with TBS (20 mM Tris and 138 mM NaCl, pH 7.4) containing 0.1% Tween 20 and 3% nonfat dry milk (blocking buffer) for 1.5 h. The membranes were then incubated with the primary antibody (1 µg/ml) diluted in blocking buffer for 1 h. Next, the membranes were washed once with distilled water and three times with TBS containing 0.1% Tween 20 (15 min/wash). The membranes were then incubated with antirabbit horseradish peroxidase-labeled antibody (dilution, 1:5000; Santa Cruz Biotechnology, Inc.) for 1 h. After five washes (15 min each) with TBS containing 0.1% Tween 20 and two washes with distilled water, the membranes were incubated with enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Inc.) for 1 min and exposed to x-ray films (Kodak, Rochester, NY). All incubations were performed at room temperature. Mol wts were estimated by running the samples in parallel with prestained protein standards (SeeBlue, Invitrogen, Carlsbad, CA).
Estimation of mol wt of the IGFBP-4 and IGFBP-5 protease activities present in bovine follicular fluid by native gel electrophoresisn, http://www.100md.com
To estimate the mol wt of the IGFBP-4 and IGFBP-5 protease activities present in bovine follicular fluid, we performed native gel electrophoresis as previously described (31). Briefly, two aliquots of 10 µl follicular fluid from preovulatory follicles were mixed with equivalent volumes of 2x native sample buffer [125 mM Tris-Cl (pH 6.8), 20% (v/v) glycerol, and 0.02% bromophenol blue]; samples were not heated. The samples were loaded onto a native 4% stacking and 5% separating Tris polyacrylamide mini-gel (Hoefer, San Francisco, CA) and electrophoresed in 25 mM Tris/192 mM glycine (pH 8.3) at 150 V at 4 C until the bromophenol blue reached the bottom of the gel. Prestained mol wt standards (SeeBlue, Invitrogen) were run in parallel with the samples.n, http://www.100md.com
After electrophoresis, the gel was removed from the glass plates, and the two sample lanes were separated. Individual lanes were cut into 2.5-mm slices, beginning at the bottom of the loading wells and progressing down the gel (gel length, 5 cm). Each slice was minced; placed in 25 µl protease assay buffer [20 mM Tris (pH 7.5), 137 mM NaCl, and 0.1% BSA] containing 100 ng IGF-II, 1 mM CaCl2, and either 50 ng rhIGFBP-4 (one lane) or 75 ng rhIGFBP-5 (the other lane); and incubated for 18 h at 37 C. After incubation, samples were subjected to 12% SDS-PAGE, followed by ligand blotting.
Phosphorimaging and autoradiography/|l, 百拇医药
Phosphor screen autoradiography of ligand blots or gels (72–96 h) involved scanning, digitalization, and processing of the image with a BAS1000 phosphorimager (Fuji Photo Film Co., Ltd., Tokyo, Japan). A Molecular Dynamics, Inc., software package (Bio-Imaging Analyzer, BAS1000 MacBas, Fuji Photo Film Co., Ltd.) was used to further process the data and obtain an image of the original radioactive sample in pixel values proportional to the amount of radioactivity. The background was subtracted from each sample. Additionally, x-ray films were exposed to nitrocellulose membranes or gels to obtain autoradiographs documenting the presence and size of bands in blots and gels. Quantitative results, shown in Fig. 1B, were obtained by densitometry of x-ray films./|l, 百拇医药
fig.ommitteed/|l, 百拇医药
Figure 1. Presence of IGFBP-4 and IGFBP-5, but not IGFBP-2, proteolytic activity in follicular fluid from bovine preovulatory follicles. A, Autoradiograph of a representative Western ligand blot of protease assay samples containing 2 µl follicular fluid from the same preovulatory follicle plus 50 ng rhIGFBP-4 (lanes 1 and 2), rhIGFBP-5 (lanes 3 and 4), or rhIGFBP-2 (lanes 5 and 6), incubated for 0 h (lanes 1, 3, and 5) or 18 h (lanes 2, 4, and 6) at 37 C. The approximate mol wt of intact IGFBPs is indicated to the right of the autoradiograph. B, Quantitative results (mean ± SEM) derived from densitometry of autoradiographs of Western ligand blots. The experiment was replicated with follicular fluid from four different preovulatory follicles. a vs. b, P < 0.01.
Statistical analysis7, 百拇医药
A t test was used for pairwise comparisons of densitometric data for 0 vs. 18 h of incubation (Fig. 1B). Data derived from phosphorimaging were analyzed by ANOVA, and individual means were compared by Tukey’s multiple comparison test. Values are presented as the mean ± SEM.7, 百拇医药
Results7, 百拇医药
IGFBP-4 and IGFBP-5, but not IGFBP-2, are degraded by follicular fluid from bovine preovulatory follicles7, 百拇医药
Previous work has shown that follicular fluid from bovine preovulatory follicles has a proteolytic activity that degrades IGFBP-4 (17). To determine whether the other low mol wt IGFBPs are also regulated by proteolytic degradation, we performed protease assays by incubating 2 µl follicular fluid from preovulatory follicles with 50 ng rhIGFBP-2, -4, or -5. Figure 1A shows a representative ligand blot of protease assays of follicular fluid frm the same preovulatory follicle using rhIGFBP-2, -4, or -5 as substrate, and quantitative results (n = 4) derived from densitometry are shown in Fig. 1B. Significant degradation of rhIGFBP-4 and -5 (P < 0.01), but not rhIGFBP-2 (P > 0.05), was observed after 18 h of incubation. As expected, no degradation of endogenous IGFBP-3 was seen after 18 h of incubation.
Partial characterization of the IGFBP-5 proteolytic activity present in follicular fluid from bovine preovulatory folliclesezw, 百拇医药
In a previous study we provided evidence that the IGFBP-4 proteolytic activity present in bovine follicular fluid is a neutral/basic pH-favoring, Zn2+ metalloprotease (17). Given the presence of strong IGFBP-5 degradation in follicular fluid from bovine preovulatory follicles (Fig. 1), we investigated the characteristics of this putative protease.ezw, 百拇医药
Time-course experiments revealed increasing IGFBP-5 proteolysis during the first 6 h of incubation (Fig. 2A), with approximately 30%, 40%, 60%, 70%, and 80% of the rhIGFBP-5 degraded after 1, 2, 4, 6, and 18 h of incubation, respectively (based on replication of the experiment with samples from four different preovulatory follicles; data not shown). To estimate the pH optimum of the IGFBP-5 protease, follicular fluid from preovulatory follicles was assessed for its ability to degrade rhIGFBP-5 over a broad range of pH values (pH 3–10). Proteolysis of rhIGFBP-5 was inhibited below pH 5, whereas weakly acidic, neutral, and weakly basic pHs promoted IGFBP-5 proteolysis (data not shown), as we showed previously for IGFBP-4 proteolysis (17).
fig.ommitteed&#:as;#, 百拇医药
Figure 2. Characterization of the IGFBP-5 proteolytic activity present in follicular fluid from bovine preovulatory follicles. A, Time course of rhIGFBP-5 degradation. Shown is a representative Western ligand blot showing degradation of rhIGFBP-5 (50 ng) by follicular fluid (5 µl) from preovulatory follicles as a function of time. B, Effect of class-selective protease inhibitors on IGFBP-5 proteolytic activity. Shown is a representative Western ligand blot showing degradation of rhIGFBP-5 (50 ng) by follicular fluid (5 µl) from preovulatory follicles. The samples were not incubated (-20 C) or were preincubated for 1 h at room temperature in the absence (None) or the presence of PMSF (1 mM), 1,10-phenanthroline (10 mM), EDTA (5 mM), E-64 (10 µM), pepstatin (1 µM), aprotinin (2 µg/ml), or chymostatin (100 µM). After preincubation, samples were assayed for their ability to degrade rhIGFBP-5 during an additional 18 h of incubation. Both experiments were replicated with follicular fluid from four different preovulatory follicles, and similar results were obtained.&#:as;#, 百拇医药
To provide initial mechanistic classification of the IGFBP-5 protease activity present in follicular fluid, we assessed its susceptibility to a set of standard protease inhibitors (27). Follicular fluid from preovulatory follicles was preincubated for 1 h in the absence or presence of the inhibitors at the concentrations detailed in Materials and Methods. Figure 2B shows a representative Western ligand blot of protease assay samples incubated in the absence or presence of different inhibitors. This experiment was replicated with follicular fluid samples from four different preovulatory follicles, and quantitative results derived from phosphorimaging of the gels were obtained and analyzed statistically (data not shown). Preincubation with the nonspecific divalent cation chelator EDTA (5 mM) completely inhibited proteolytic activity (P < 0.001). Although this observation suggests the possibility of a metalloprotease (cation dependence), it does not rule out the possibility of a cation-activated or stabilized nonmetalloprotease. However, the metalloprotease inhibitor 1,10-phenanthroline (10 mM) proved equally effective (P < 0.001). Given that 1,10-phenanthroline has a much higher stability constant for Zn2+ (2.5 x 10-6 M-1) than for Ca2+ (3.2 x 10-1 M-1), this observation is virtually diagnostic for a Zn2+ metalloprotease (27). Pretreatment with pepstatin A (1 µM), an acetylated pentapeptide established as a highly selective aspartic protease inhibitor, proved ineffective (P > 0.05). Similarly, E-64 (10 µM), a peptide epoxide recognized as a highly specific cysteine protease inhibitor, had no effect (P > 0.05) on IGFBP-5 proteolytic activity. Chymostatin (100 µM), an inhibitor of chymotrypsin-like serine proteases and of some cysteine proteases, did not inhibit IGFBP-5 degradation. Finally, aprotinin (2 µg/µl) and PMSF (1 mM), serine protease inhibitors, proved ineffective (P > 0.05). Taken together, these characterization studies suggest that follicular fluid from bovine preovulatory follicles contains an IGFBP-5, neutral/basic pH-favoring, Zn2+ metalloprotease. The previously described IGFBP-4 protease has the same characteristics (17).
Immunoneutralization and immunoprecipitation of IGFBP-4 and IGFBP-5 proteolytic activity with polyclonal antibodies against human PAPP-A3:s#$@&, http://www.100md.com
Recent studies have shown that the IGFBP-4 protease present in follicular fluid from human ovarian follicles (14, 32) and from several domestic species (15) is PAPP-A. The similarity between the previously described IGFBP-4 protease (17) and the IGFBP-5 proteolytic activity (present study) in bovine follicular fluid together with the recent finding that PAPP-A can also cleave IGFBP-5, at least in experiments in vitro with recombinant PAPP-A and substrate (33), raised the question of whether the IGFBP-5 proteolysis observed in bovine follicular fluid reflects the existence of an independent IGFBP-5 proteolytic system or simply the contribution of PAPP-A to IGFBP-5 proteolysis. We hypothesized that immunoneutralization and immunoprecipitation of bovine follicular fluid with polyclonal antibodies against human PAPP-A would result in inhibition and immunodepletion, respectively, of the IGFBP-4, but not the IGFBP-5, proteolytic activity. As hPS has a high concentration of PAPP-A, it was also incubated with antibodies against human PAPP-A as a positive control for the effects of the antiserum.
In an initial immunoneutralization experiment, we assessed proteolysis of IGFBP-4 or -5 by hPS or bovine follicular fluid incubated for 18 h with PBS, nonimmune IgG, or anti-PAPP-A antibodies. Interestingly, when the sample was hPS (the positive control for PAPP-A), polyclonal antibodies against human PAPP-A (1 or 5 µg), but not PBS or nonimmune IgG, were able to immunoneutralize IGFBP-4 (Fig. 3, top panel, lanes 1–6), but not the IGFBP-5 (Fig. 3, bottom panel, lanes 1–6), proteolytic activity. Surprisingly, incubation of follicular fluid from bovine preovulatory follicles with polyclonal antibodies against human PAPP-A, but not with PBS or nonimmune IgG, inhibited proteolysis of both IGFBP-4 (Fig. 3, top panel, lanes 7–12) and IGFBP-5 (Fig. 3, bottom panel, lanes 7–12). This initial experiment also suggested that, in contrast to the apparent complete neutralization of IGFBP-4 proteolysis, anti-PAPP-A antibodies substantially reduced, but did not completely block, IGFBP-5 degradation by bovine follicular fluid.
fig.ommitteedv]i8\, 百拇医药
Figure 3. Immunoneutralization of IGFBP-4 and IGFBP-5 degradation with polyclonal antibodies against human PAPP-A. Western ligand blots of protease assay samples containing 50 ng rhIGFBP-4 (top) or rhIGFBP-5 (bottom) and 5 µl hPS (lanes 1–6) or bovine follicular fluid (bFF; lanes 7–12) after incubation for 0 h (lanes 1 and 7) or 18 h (lanes 2–6 and 8–12) at room temperature in the presence of PBS (lanes 1–2 and 7–8), 1 or 5 µg nonimmune IgG (lanes 3 and 4 and 9 and 10), or 1 or 5 µg polyclonal antibodies against human PAPP-A (lanes 5 and 6 and 11 and 12). Note the presence of endogenous IGFBP-3 (43 kDa) in bovine follicular fluid, but not in hPS.v]i8\, 百拇医药
Results of the time-course experiment (Fig. 2A) suggest that after 18 h of incubation, IGFBP-5 could have been overdigested, thereby impairing our ability to assess the relative contributions of PAPP-A vs. other potential IGFBP-5 proteases. Therefore, in a follow-up experiment, we performed a kinetic analysis of the inhibition of IGFBP-4 and -5 degradation by anti-PAPP-A antibodies. Representative ligand blots of IGFBP-4 and -5 protease assays are shown in Fig. 4, and quantitative results derived from phosphorimaging are shown in Fig. 5 (left and right panels, respectively). These results show that, indeed, anti-PAPP-A antibodies, but not nonimmune IgG, completely inhibited (P < 0.01) both IGFBP-4 and -5 degradation by bovine follicular fluid when the incubation time was reduced to 2 or 5 h (Fig. 5, lanes 5, 7, 12, and 14). Interestingly, complete inhibition of IGFBP-4 proteolysis and partial (although significant, P < 0.05) inhibition of IGFBP-5 proteolysis by bovine follicular fluid were also observed after 18 h of incubation with anti-PAPP-A antibodies, but not with nonimmune IgG. In contrast, proteolysis of IGFBP-5 by hPS was not inhibited (P > 0.05) by anti-PAPP-A antibodies regardless of the time of incubation, whereas IGFBP-4 degradation by hPS was completely blocked after 2, 5, or 18 h of incubation in the presence of anti-PAPP-A antibodies, but not nonimmune IgG. The last observation argues for the specificity of the antiserum and the existence of an independent (not related to PAPP-A) IGFBP-5 proteolytic system in hPS, as previously suggested (34).
fig.ommitteedty\-r&c, http://www.100md.com
Figure 4. Kinetic analysis of the inhibition of IGFBP-4 and -5 degradation by anti-PAPP-A antibodies. Western ligand blots of 50 ng rhIGFBP-4 (left panels) or rhIGFBP-5 (right panels) incubated alone (lanes 1 and 8, respectively), with 5 µl hPS (lanes 2–3 and 9–10) or follicular fluid from different (b1, b2) bovine preovulatory follicles (lanes 4–7 and 11–14) for 0 h (lanes 1 and 8), 2 h (top panels), 5 h (middle panels), or 18 h (bottom panels) at room temperature in the presence of 5 µg nonimmune IgG (lanes 2, 4, 6, 9, 11, and 13) or 5 µg anti-PAPP-A antibodies (lanes 3, 5, 7, 10, 12, and 14). Note the presence of endogenous IGFBP-3 (43 kDa) in bovine follicular fluid, but not in hPS. This experiment was replicated with samples from four different preovulatory follicles.ty\-r&c, http://www.100md.com
fig.ommitteedty\-r&c, http://www.100md.com
Figure 5. Quantitative results (mean ± SEM) of kinetic analysis of the inhibition of IGFBP-4 and -5 degradation by anti-PAPP-A antibodies (n = 4 preovulatory follicles). Recombinant human IGFBP-4 (BP-4) or -5 (BP-5) was incubated alone, with hPS, or with follicular fluid from bovine preovulatory follicles (bFF) in the presence of nonimmune IgG (IgG) or anti-PAPP-A antibodies (PAPP-A) at room temperature for 2, 5, or 18 h. After incubation, protease assay samples were subjected to SDS-PAGE, followed by ligand blotting and phosphorimaging. *, P < 0.05; **, P < 0.01 [vs. control (BP-4 or BP-5)].
In the next experiment, polyclonal antibodies against human PAPP-A were mixed with hPS or bovine follicular fluid to immunoprecipitate PAPP-A before the protease assays. Immunoprecipitation with PAPP-A antibodies, but not nonimmune IgG, resulted in complete depletion of IGFBP-4 (Fig. 6A, top panel) and substantial (yet not complete) depletion of IGFBP-5 (Fig. 6A, bottom panel) proteolytic activities from bovine follicular fluid. Quantitative results derived from phosphorimaging (Fig. 6B) showed that anti-PAPP-A antibodies, but not nonimmune IgG, significantly (P < 0.05) depleted both IGFBP-4 and IGFBP-5 proteolytic activities from bovine follicular fluid; however, some IGFBP-5 degradation was still observed after immunoprecipitation with anti-PAPP-A antibodies. In contrast, immunoprecipitation with anti-PAPP-A antibodies (but not nonimmune IgG) resulted in full depletion of IGFBP-4 (P < 0.01), but not IGFBP-5 (P > 0.05), proteolytic activity from hPS (Fig. 6A, lanes 2 and 3, and Fig. 6B). Taken together, these experiments provide evidence that PAPP-A accounts for the IGFBP-4 proteolytic activity present in bovine follicular fluid and that it also contributes to substantial degradation of IGFBP-5.
fig.ommitteed.z1&, 百拇医药
Figure 6. Immunoprecipitation of IGFBP-4 and IGFBP-5 proteolytic activities with polyclonal antibodies against PAPP-A. A, Western ligand blots of protease assay samples containing 50 ng rhIGFBP-4 (top panel) or rhIGFBP-5 (bottom panel) incubated alone (lanes 1 and 10), in the presence of 30 µl supernatant of hPS (lanes 2 and 3) or follicular fluid from three different (b1, b2, and b3) preovulatory follicles (lanes 4–9) for 0 h (lanes 1) or 18 h (lanes 2–10) at room temperature. Samples of hPS and bovine follicular fluid were immunoprecipitated with nonimmune IgG (lanes 2, 4, 6, and 8) or anti-PAPP-A antibodies (lanes 3, 5, 7, and 9) before the protease assays. Note the presence of endogenous IGFBP-3 (43 kDa) in bovine follicular fluid, but not in hPS. B, Quantitative results (mean ± SEM) derived from phosphorimaging of ligand blots (n = 3). *, P < 0.05; **, P < 0.01 [vs. control (BP-4 or BP-5)]..z1&, 百拇医药
Immunodetection of PAPP-A in follicular fluid from bovine preovulatory follicles.z1&, 百拇医药
The antibody used in the immunoneutralization and immunoprecipitation experiments was raised against human PAPP-A/proform of eosinophil major basic protein (pro-MBP). Therefore, its specificity for PAPP-A in bovine follicular fluid (i.e. lack of cross-reactivity with other molecules) was tested by Western immunoblotting of bovine follicular fluid. As shown in Fig. 7, a specific band of about 400 kDa was detected after resolution of 2.5 or 5 µl follicular fluid from preovulatory follicles on a 6% SDS-PAGE gel under nonreducing conditions. Recombinant PAPP-A is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bonded subunits (35). Using the same polyclonal antibodies, PAPP-A/pro-MBP purified from human pregnancy serum has been shown to migrate as an approximately 200-kDa band under reducing conditions (35) and as a >400-kDa band after electrophoresis of human pregnancy serum under nonreducing conditions (30). Therefore, the detection by Western immunoblotting of a unique band of the expected size in bovine follicular fluid rules out the possibility of significant cross-reaction of the antibody with other related molecules (for example, PAPP-A2) and the presence of inactive proteolytic fragments (detectable by immunoblotting) previously reported to be generated by autocleavage (36).
fig.ommitteedau+'ep:, 百拇医药
Figure 7. Western immunoblot analysis of PAPP-A in follicular fluid from bovine preovulatory follicles. Follicular fluid (2.5 µl, lane 1; 5 µl, lane 2) was resolved on a 6% SDS-PAGE gel under nonreducing conditions and analyzed by Western immunoblotting using a polyclonal antibody against human PAPP-A/pro-MBP. The relative migration of molecular markers is indicated. This experiment was replicated three times.au+'ep:, 百拇医药
Estimation of the mol wt of the IGFBP-4 and IGFBP-5 protease activities present in bovine follicular fluid by native gel electrophoresisau+'ep:, 百拇医药
Follicular fluid was subjected to native gel electrophoresis. Gel lanes were cut into 2.5-mm slices (from the bottom of the wells and progressing down the gel), and slices from each individual lane were incubated with either rhIGFBP-4 or rhIGFBP-5. Proteolysis of rhIGFBP-4 and rhIGFBP-5 was observed for slices 1–3, indicating that the proteolytic activity in both cases had migrated 0–7.5 mm into the gel (data not shown). Given that the total length of the gel was 50 mm, both proteolytic activities had an Rf value of 0–0.15. The largest standard, myosin (~ 250 kDa), was located in slice 6, representing an Rf value of 0.3 and a migration faster than the protease activities. These data suggest that both proteolytic activities in bovine follicular fluid are large proteins (or complexes) with molecular masses greater than 250 kDa. Adding to the evidence that anti-PAPP-A antibodies immunoneutralized and immunodepleted both proteolytic activities (Figs. 3–6) and to the detection of a single band (~ 400 kDa) by Western immunoblotting (Fig. 6), the observation that both substrates (IGFBP-4 and IGFBP-5) were degraded by the same slices (no. 1–3) strongly suggests that the same molecular entity is responsible for both proteolytic activities. Interestingly, PAPP-A in bovine follicular fluid, shown in the present study to be the major contributor to IGFBP-5 degradation, is different from the complement component C1s, an 88-kDa IGFBP-5 serine protease secreted by human fibroblasts (37).
Potential role for IGFBP-5 proteolysis in the establishment of follicular dominance]6t, 百拇医药
To start investigating the role of IGFBP-5 proteolysis in the establishment of follicular dominance, we compared the IGFBP-5 protease activity in follicular fluid from companion dominant and subordinate follicles (n = 4) collected on d 3 after the emergence of the first follicular wave of the cycle (d 4/5 of the estrous cycle). Several characteristics of dominant and subordinate follicles used in this experiment were reported previously (17). Briefly, dominant follicles were larger (P < 0.05), and their follicular fluid had higher (P < 0.05) estradiol and similar (P > 0.05) protein concentrations compared with subordinate follicles. As shown in Fig. 8A, extensive proteolysis of the [125I]IGFBP-5 substrate and the appearance of proteolytic fragments of approximately 19 and 17 kDa were observed after 18 h of incubation with follicular fluid from the dominant follicle, but not with follicular fluid from the two largest subordinate follicles. Quantitative results (n = 4) derived from phosphorimaging (Fig. 8B) showed significant (P < 0.05) degradation of the [125I]IGFBP-5 substrate after 18 h of incubation with follicular fluid from dominant follicles, but not subordinate follicles. This evidence points to the intrafollicular IGFBP-5 proteolytic system as an integral component of the mechanisms leading to the establishment of follicular dominance.
fig.ommitteed}, 百拇医药
Figure 8. Evidence for a role for IGFBP-5 proteolysis in the establishment of ovarian follicular dominance. Follicular fluid obtained from a dominant follicle (DF) and the two largest companion subordinate follicles (SF1 and SF2) was incubated with [125I]IGFBP-5 substrate for 0 h (lane 1) or 18 h (lanes 2–4) at 37 C. After incubation, protease assay samples were subjected to SDS-PAGE and autoradiography (A). Quantitative results derived from phosphorimaging (mean ± SEM) were obtained from four sets of samples of dominant and companion subordinate follicles (B). *, P < 0.05 vs. DF incubated for 0 h.}, 百拇医药
Discussion}, 百拇医药
The results of the present study suggest that PAPP-A not only accounts for the IGFBP-4 proteolytic activity present in follicular fluid from bovine preovulatory follicles, but also makes a significant contribution to proteolysis of IGFBP-5. In addition, IGFBP-5 proteolysis was readily detectable in the dominant follicle and was negligible in subordinate follicles of the same cohort. The observation that proteolysis of IGFBP-5 is detected early during differentiation of a dominant follicle and persists through preovulatory development suggests a potential role for proteolysis of IGFBP-5 in determining follicular fate. Compelling evidence that human PAPP-A is also capable of cleaving IGFBP-5, in addition to IGFBP-4, at least in experiments in vitro, has been recently provided by Laursen et al. (33) using highly purified recombinant proteins. To our knowledge, the present study constitutes the first evidence of the existence of an IGFBP-4/-5 proteolytic system with physiological relevance in which PAPP-A is the dominant IGFBP protease involved.
We have previously shown that selection of a dominant follicle in cattle is associated with increased IGFBP-4 proteolysis by a neutral/basic pH-favoring, Zn2+ metalloprotease (17, 18). Herein, we report the presence of IGFBP-5 proteolytic activity in follicular fluid with striking similarities to the previously described IGFBP-4 protease. Several lines of evidence support the idea that both IGFBP-4 and -5 are, indeed, subjected to proteolysis by PAPP-A in bovine follicular fluid. 1) Characterization studies showed that IGFBP-5 is significantly degraded by follicular fluid from bovine preovulatory follicles within 4–6 h of incubation in vitro at neutral/basic pH, conditions very similar to those reported for IGFBP-4 proteolysis (17). 2) The IGFBP-5 proteolytic activity was sensitive only to EDTA and 1,10-phenanthroline, strongly suggesting, as in the case of IGFBP-4 degradation (17), that the IGFBP-5 proteolytic activity in bovine follicular fluid is a metalloprotease. 3) Anti-PAPP-A antibodies, but not nonimmune IgG, immunoneutralized and immunodepleted both IGFBP-4 and IGFBP-5 proteolytic activities in bovine follicular fluid. 4) PAPP-A was immunodetected as a single approximately 400-kDa migrating band after electrophoresis of bovine follicular fluid under nonreducing conditions, ruling out cross-reactivity of the polyclonal PAPP-A antibody with another protease(s) in follicular fluid. 5) Both proteolytic activities have identical electrophoretic mobility, as determined by native gel electrophoresis (data not shown). 6) Cleavage of IGFBP-5 by the putative IGFBP-5 protease present in bovine follicular fluid appears to occur at only one site, as suggested by the generation of radiolabeled proteolytic fragments of approximately 19 and 17 kDa after the incubation with [125I]IGFBP-5 substrate.
All of the above findings are in agreement with recently published reports showing that 1) PAPP-A is the IGFBP-4 protease produced by granulosa cells (32, 38) and found in follicular fluid of preovulatory follicles from humans (14) and from several domestic species, including cattle (15); 2) highly purified human recombinant PAPP-A cleaves both IGFBP-4 and -5, with similar kinetics (33); and 3) proteolysis of IGFBP-5 by PAPP-A occurs at one site, between Ser143 and Lys144 (33), thereby generating a pattern of proteolytic fragments similar to that observed in the present study. Interestingly, comparison of the reported site of cleavage of IGFBP-5 (R137KKLTQS{downarrow} KFVGGAE) by PAPP-A (33) with the site of cleavage of IGFBP-4 (S129TSGGKM{downarrow} KVNGAPR) by a fibroblast-derived IGFBP-4 protease (39) shown to be identical to PAPP-A (40) shows a Lys residue present in both P1' positions, hydrophobic residues in the P2' positions, and a Gly residue in both P4' positions (33). In addition, PAPP-A was found to undergo autocleavage at two major sites, showing similarities with, but also marked differences from, the cleavage site in IGFBP-4 (36). It has been proposed that the lack of unifying elements in sequences around the cleavage sites of PAPP-A implies that the specificity of PAPP-A is determined by steric restrictions more than by required interactions between PAPP-A and specific substrate side-chains (36).
Mutational analysis of the proteolytic domain of PAPP-A allowed its unequivocal classification as a member of the metzincin superfamily of metalloproteinases (36). All metzincins contain an elongated zinc-binding motif (HEXHXXGXXH) that coordinates the catalytic zinc ion of the active site and, as suggested by their common name, a Met residue believed to be important for the integrity of the catalytic site (36). Not surprisingly, the above-described catalytic site is absolutely conserved across species (15, 35, 41), and the full human and mouse PAPP-A amino acid sequences share 89% and 93% identity and similarity, respectively (41). PAPP-A exists in hPS as a covalent, heterotetrameric 2:2 complex with pro-MBP, PAPP-A/pro-MBP (42). In the present study immunoprecipitation and immunoneutralization experiments were performed with polyclonal antibodies raised against PAPP-A/pro-MBP purified from human pregnancy serum (35), which raised concerns about species specificity and cross-reactivity of the antibody with other molecules. The strictly conserved catalytic site and the overall high degree of homology of PAPP-A amino acid sequence across species argue against major species specificity concerns. Indeed, the same anti-PAPP-A/pro-MBP antibodies have been previously used to characterize PAPP-A in several domestic species (15), and recently, the successful use of a mouse antihuman PAPP-A monoclonal antibody in Western blot analysis of rat PAPP-A has been reported (43).
At present, only one protein with global homology to PAPP-A, a specific IGFBP-5 protease termed PAPP-A2 (44) or PAPP-E (45), has been described. Although some cross-reactivity of the polyclonal anti-PAPP-A/pro-MBP antibody with similar molecules, namely PAPP-A2, cannot be completely ruled out, this possibility seems unlikely considering that 1) Western immunoblot analysis of bovine follicular fluid samples resolved on SDS-PAGE under nonreducing conditions revealed the presence of a unique, specific signal migrating as an approximately 400-kDa band; 2) the electrophoretic mobility of the two proteolytic activities, as shown by native gel electrophoresis, was identical (data not shown); and 3) complete abrogation of the IGFBP-4, but not the IGFBP-5, protease activity in human pregnancy serum (used as a positive control for PAPP-A) was observed in immunoneutralization and immunoprecipitation experiments using polyclonal anti-PAPP-A antibodies. Unlike circulating PAPP-A, which is found in a 2:2 heterotetrameric PAPP-A/pro-MBP complex of about 500 kDa, PAPP-A2 is a monomer of approximately 220 kDa, and in addition, there is a limited degree of similarity between the two molecules (PAPP-A2 shares only 45% of its residues with PAPP-A). All of the above suggest that a high degree of cross-reactivity of polyclonal anti-PAPP-A with related (PAPP-A2/others) molecules is unlikely.
Of note is the observation that when we used hPS as a positive control for PAPP-A, immunoneutralization and immunoprecipitation resulted in inhibition of IGFBP-4, but not IGFBP-5, proteolysis after 2, 5, or 18 h of incubation. In a recent study (30) very little effect of PAPP-A antibodies on IGFBP-5 degradation was observed after incubation of PAPP-A antibodies with hPS for 5 h, whereas partial blockage was observed when the incubation time was reduced to 40 min. Similarly, polyclonal antibodies against human PAPP-A inhibited IGFBP-4, but not IGFBP-5, protease activity in follicular fluid from preovulatory follicles of women undergoing in vitro fertilization procedures (14). The failure of polyclonal antibodies against PAPP-A to inhibit IGFBP-5 degradation in hPS (present study) and human follicular fluid (14) argues for the existence of another major IGFBP-5 protease(s), presumably PAPP-A2, in those physiological fluids. At any rate, caution should be exercised in the interpretation of these apparently conflicting results; species differences, type of biological material under study, and/or methodological considerations (type of assay, time of incubation, or concentration of reagents) may well explain some of the apparently contradictory findings among different studies. Whether PAPP-A is the only intrafollicular protease responsible for proteolysis of IGFBP-5 or whether other minor IGFBP-5 proteases also contribute to the decrease in IGFBP-5 concentrations in follicular fluid of dominant, estrogen-active follicles in cattle awaits further investigation.
In this study we provide evidence that the IGFBP-5 proteolytic system is active in the dominant follicle, but not in the subordinate follicles of the same cohort. This is a novel piece of information considering that in previous reports of intrafollicular degradation of IGFBP-5 (21, 23, 24, 29) follicular fluid samples were obtained from preovulatory follicles well after selection had occurred. We have previously shown that, when dominant follicles are first detected as slightly larger than companion subordinate follicles, their capacity to secrete estradiol and levels of proteolytic activity against IGFBP-4 in follicular fluid are much greater than those of subordinate follicles, but they do not differ in other characteristics assessed (5, 17). In light of the present results, it is temping to speculate that an active IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is a key factor in the mechanisms leading to selection of a dominant follicle.1], 百拇医药
Acknowledgments
We thank D. Bianchi for care of experimental animals, and Dr. Mark S. Roberson for helpful discussions during the course of these experiments and critical reading of the manuscript.!h, 百拇医药
Received June 27, 2002.!h, 百拇医药
Accepted for publication October 16, 2002.!h, 百拇医药
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Development of a dominant follicle is associated with decreased intrafollicular low molecular weight IGF-binding proteins (namely IGFBP-2, -4, and -5) and increased proteolysis of IGFBP-4 by pregnancy-associated plasma protein A (PAPP-A). In addition to IGFBP-4 proteolytic activity, bovine follicular fluid contains strong proteolytic activity for IGFBP-5, but not for IGFBP-2. Here we show that the IGFBP-5 protease present in bovine follicular fluid is a neutral/basic pH-favoring, Zn2+ metalloprotease very similar to the previously described IGFBP-4 protease. We hypothesized that immunoneutralization and immunoprecipitation with anti-PAPP-A antibodies would result in abrogation of the IGFBP-4, but not the IGFBP-5, proteolytic activity in follicular fluid. As expected, anti-PAPP-A antibodies were able to neutralize and precipitate the IGFBP-4, but not the IGFBP-5, proteolytic activity of human pregnancy serum, which was used as a positive control for PAPP-A. Surprisingly, immunoneutralization and immunoprecipitation of follicular fluid from bovine preovulatory follicles with anti-PAPP-A antibodies abrogated both IGFBP-4 and IGFBP-5 proteolysis. Quantitative results derived from phosphorimaging revealed a complete inhibition of both IGFBP-4 and -5 proteolysis by follicular fluid incubated for 2 or 5 h in the presence of anti-PAPP-A antibodies. After 18 h of incubation, anti-PAPP-A antibodies still inhibited IGFBP-5 degradation, although with an efficiency lower than that for IGFBP-4 degradation. Both proteolytic activities have identical electrophoretic mobility, and a single band (~ 400 kDa) was detected by Western immunoblotting of bovine follicular fluid with anti-PAPP-A antibodies. Proteolysis of IGFBP-5 was readily detectable in follicular fluid from dominant follicles and was negligible in subordinate follicles from the same cohort. These results suggest that an active intrafollicular IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is an important determinant of follicular fate.
Introductiondo43/x4, 百拇医药
A CRITICAL, but poorly understood, transition in follicular development is the selection of a dominant follicle capable of ovulating from a cohort of follicles recruited by a small increase in circulating FSH (1, 2, 3). In addition to the well documented increase in estradiol-synthesizing capacity (4, 5), dominant follicles are characterized by decreased levels of low molecular weight (mol wt) IGF-binding proteins (IGFBP-2, -4, and -5) (6, 7, 8, 9). Compelling evidence for an obligatory role of the IGF system in antral follicular development is provided by the infertile IGF-I-null mouse in which follicles are arrested at the preantral/early antral stage and fail to respond to gonadotropin administration (10). It is hypothesized that the decrease in the inhibitory, low mol wt IGFBPs in dominant follicles increases the amount of bioactive IGF available to synergize with FSH in promoting follicular growth and estradiol production (9, 11, 12, 13). Proteolytic activity against IGFBP-4 has been detected in the follicular fluid of human (14), bovine, ovine, and porcine (15) preovulatory follicles and is identical to pregnancy-associated plasma protein A (PAPP-A), first isolated from human pregnancy serum over 20 yr ago (16). Using cattle as the experimental model, we showed recently that there are lower levels of IGFBP-4 in dominant vs. subordinate follicles, very close to the time of follicular selection, due to an FSH-dependent metalloprotease activity against IGFBP-4 in the dominant follicle (17, 18).
Depending on the biological system, IGFBP-5 may exert either stimulatory (19) or inhibitory (20) effects on the IGF-receptor interaction; however, there seems to be consensus that IGFBP-5 inhibits IGF’s actions on ovarian cells (11, 12). The mechanisms that subserve the reduction in levels of the inhibitory IGFBP-5 in the dominant follicle are poorly understood. Recent reports indicate that follicular fluid from porcine (21), ovine (22), and equine (23) preovulatory follicles contains a metalloprotease or serine protease (equine) that degrades IGFBP-5. In addition, an uncharacterized proteolytic activity against IGFBP-5 has been reported recently in follicular fluid from bovine preovulatory follicles (24). A common theme emerging from those studies is that identification of the putative IGFBP-5 protease(s) remains elusive.%, http://www.100md.com
The goal of the current study was to characterize the putative IGFBP-5 protease activity in bovine follicular fluid and to investigate its potential association with selection of the dominant follicle. Cattle provide an excellent model for studies of the mechanisms of follicular selection for dominance because a follicular wave is recruited about every 7–8 d during the 21-d estrous cycle, the dominant follicle of any cohort has ovulatory capacity during its tenure of dominance if the corpus luteum is regressed naturally or experimentally, individual follicles of the cohort can be followed from day to day by ultrasound imaging, and the follicles are large enough to provide abundant follicular fluid for analysis of potential regulatory molecules.
Materials and Methodsj42'gd, 百拇医药
Animals and experimental protocolsj42'gd, 百拇医药
The main objective of this study was to characterize the putative IGFBP-5 protease present in follicular fluid from bovine preovulatory follicles. To that end, Holstein heifers with regular estrous cycles were injected with prostaglandin F2{alpha} (PGF2{alpha} ; 25 mg, im; Lutalyse, Pharmacia \|[amp ]\| Upjohn, Inc., Kalamazoo, MI) on d 7 of the estrous cycle (d 0 = day of estrus) to induce luteolysis. Luteolysis is followed by initiation of a follicular phase and differentiation of the dominant follicle of the first follicular wave into a preovulatory follicle. The ovaries of each heifer were examined daily by transrectal ultrasonography as described previously (5). Follicular fluid from the preovulatory follicle (n = 4 heifers) was obtained after ovariectomy performed 24 h after injection of PGF2{alpha} . In this experimental model (25, 26), estrus and the LH surge occur about 48–60 h after PGF2{alpha} ; thus, follicles were obtained about 24–36 h before the expected time of the LH surge.
To begin to explore the potential physiological role of intrafollicular IGFBP-5 proteolysis in the establishment of follicular dominance, follicular fluid samples were also obtained from the dominant follicle and the companion subordinate follicles on d 3 of the follicular wave (emergence of the wave = d 0; n = 4), close to the time of follicular selection. Luteolysis, a follicular phase, and ovulation were induced by injecting heifers with PGF2{alpha} during the midluteal phase. The ovaries of each heifer were examined daily by transrectal ultrasonography, as described above, to observe ovulation and the initiation of the first follicular wave of the next estrous cycle. Follicular fluid samples were obtained on d 3 of the follicular wave by ultrasound-guided follicular aspiration as previously described (17). Follicular fluid samples were centrifuged, and aliquots were stored at -80 C for later determinations. Animals were used in accordance with procedures approved by the Cornell University animal care and use committee (Protocol 86-214-99).
Analysis of IGFBP-2, -4, and -5 proteolytic activities-}:q(, http://www.100md.com
The ability of intrafollicular proteases to degrade IGFBP-2, IGFBP-4, or IGFBP-5 was assessed by incubating 2 or 5 µl follicular fluid plus substrate for 18 h at 37 C in a solution of 20 mM Tris (pH 7.5) containing 137 mM NaCl (TBS) and 0.1% BSA (final volume, 20 µl). In characterization experiments, 50 ng recombinant human (rh) IGFBP-2, -4, or -5 (Austral Biologicals, San Ramon, CA) were used as substrate. Protease assay samples were subjected to SDS-PAGE, followed by Western ligand blotting/phosphorimaging to quantify the percentage of substrate loss, as previously described (17). In addition, IGFBP-5 proteolytic activity in samples obtained from dominant and subordinate follicles of the first follicular wave was assessed by incubating 5 µl follicular fluid with approximately 50,000 cpm 125I-labeled rhIGFBP-5 for 18 h at 37 C. This assay was chosen to assess proteolytic activity against IGFBP-5 because the presence of abundant endogenous IGFBP-5 in subordinate follicles could interfere with determinations using cold rhIGFBP-5 substrate followed by Western ligand blotting. Iodination of rhIGFBP-5 was performed by the chloramine-T method as previously described (24).
Partial characterization of IGFBP-5 protease activity(w8jki, 百拇医药
To characterize the putative IGFBP-5 protease detected in follicular fluid from preovulatory follicles, rhIGFBP-5 was used as the substrate, followed by Western ligand blot analysis/phosphorimaging. The time and pH dependence of the IGFBP-5 degradation was assessed after incubation of 50 ng rhIGFBP-5 with follicular fluid from preovulatory follicles at 37 C. To provide initial mechanistic classification of the IGFBP-5 protease activity present in bovine follicular fluid, the following set of standard protease inhibitors (Sigma-Aldrich, St. Louis, MO), corresponding to the four protease classes recognized by the International Union of Biochemistry (27), was used: 1,10-phenanthroline (10 mM; metalloprotease inhibitor), trans-epoxysuccinyl- L-leucylamido-(4- guanidino)-butane (E-64; 10 µM; cysteine protease inhibitor), aprotinin (2 µg/µl; serine protease inhibitor), pepstatin (1 µM; aspartic protease inhibitor), phenylmethylsulfonyl fluoride (PMSF; 1 mM; serine protease inhibitor), chymostatin (100 µM; an inhibitor of chymotrypsin-like serine proteases and some cysteine proteases), and the nonspecific divalent cation chelator EDTA (5 mM). The inhibitors and doses used were chosen based on reported specificity and efficacy, respectively (27, 28, 29).
Immunoneutralization and immunoprecipitation of PAPP-A6^{'fjm, http://www.100md.com
Human pregnancy serum (hPS) was used as a positive control for PAPP-A in the experiments described below. Immunoneutralization was performed by preincubating an aliquot (5 µl) of hPS or follicular fluid from bovine preovulatory follicles with vehicle (PBS), nonimmune immunoglobulin G (IgG), or antihuman PAPP-A polyclonal antibody (both from DAKO Corp., Carpinteria, CA) for 1 h at room temperature. After preincubation, 50 ng rhIGFBP-4 or rhIGFBP-5 substrate were added, and the reaction mix was incubated at 37 C for various intervals as indicated. At the end of the incubation, samples were analyzed by Western ligand blotting.6^{'fjm, http://www.100md.com
Immunoprecipitation was performed as previously described (30). Briefly, 100 µl hPS or follicular fluid from bovine preovulatory follicles were preadsorbed by incubation with 100 µl Protein A/G Plus Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 30 min with frequent manual mixing, to reduce substances present in the samples that bind nonspecifically to Protein A/G Plus Agarose. After centrifugation, the supernatant was collected. This step was repeated three times. The supernatant was then divided into three aliquots and incubated overnight with vehicle (PBS), normal IgG (40 µg), or rabbit antihuman PAPP-A polyclonal antibody (40 µg) on a rocking platform. The samples were mixed with 100 µl Protein A/G Plus Agarose with frequent manual mixing for 1 h, followed by centrifugation and collection of the supernatant. This step was repeated twice. All incubations were performed at 4 C. The final supernatant was used in IGFBP protease assays.
Western ligand blot analysis\wae@9g, 百拇医药
Western ligand blot analysis was performed as previously described (17). Briefly, samples were subjected to electrophoresis in 12% SDS-PAGE gels under nonreducing conditions and transferred onto nitrocellulose membranes. The membranes were incubated with 1.2 x 106 cpm [125I]IGF-II (specific activity, 340–430 µCi/µg) overnight at 4 C, then washed, air-dried, and subjected to autoradiography and phosphorimaging. Mol wts of intact IGFBP species were estimated by running the samples in parallel with protein mol wt standards (2,850–43,000 mol wt range; Life Technologies, Inc., Grand Island, NY) on the same gel.\wae@9g, 百拇医药
Western immunoblot analysis of PAPP-A\wae@9g, 百拇医药
Follicular fluid samples (2.5 or 5 µl) were mixed with 2x loading buffer, boiled for 3 min, and resolved on a 6% SDS-PAGE gel under nonreducing conditions. Proteins were transferred to nitrocellulose membranes and subjected to immunoblot analysis using rabbit anti-PAPP-A polyclonal antibodies (DAKO Corp.). Briefly, membranes were washed twice in distilled water for 15 min each time and blocked with TBS (20 mM Tris and 138 mM NaCl, pH 7.4) containing 0.1% Tween 20 and 3% nonfat dry milk (blocking buffer) for 1.5 h. The membranes were then incubated with the primary antibody (1 µg/ml) diluted in blocking buffer for 1 h. Next, the membranes were washed once with distilled water and three times with TBS containing 0.1% Tween 20 (15 min/wash). The membranes were then incubated with antirabbit horseradish peroxidase-labeled antibody (dilution, 1:5000; Santa Cruz Biotechnology, Inc.) for 1 h. After five washes (15 min each) with TBS containing 0.1% Tween 20 and two washes with distilled water, the membranes were incubated with enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Inc.) for 1 min and exposed to x-ray films (Kodak, Rochester, NY). All incubations were performed at room temperature. Mol wts were estimated by running the samples in parallel with prestained protein standards (SeeBlue, Invitrogen, Carlsbad, CA).
Estimation of mol wt of the IGFBP-4 and IGFBP-5 protease activities present in bovine follicular fluid by native gel electrophoresisn, http://www.100md.com
To estimate the mol wt of the IGFBP-4 and IGFBP-5 protease activities present in bovine follicular fluid, we performed native gel electrophoresis as previously described (31). Briefly, two aliquots of 10 µl follicular fluid from preovulatory follicles were mixed with equivalent volumes of 2x native sample buffer [125 mM Tris-Cl (pH 6.8), 20% (v/v) glycerol, and 0.02% bromophenol blue]; samples were not heated. The samples were loaded onto a native 4% stacking and 5% separating Tris polyacrylamide mini-gel (Hoefer, San Francisco, CA) and electrophoresed in 25 mM Tris/192 mM glycine (pH 8.3) at 150 V at 4 C until the bromophenol blue reached the bottom of the gel. Prestained mol wt standards (SeeBlue, Invitrogen) were run in parallel with the samples.n, http://www.100md.com
After electrophoresis, the gel was removed from the glass plates, and the two sample lanes were separated. Individual lanes were cut into 2.5-mm slices, beginning at the bottom of the loading wells and progressing down the gel (gel length, 5 cm). Each slice was minced; placed in 25 µl protease assay buffer [20 mM Tris (pH 7.5), 137 mM NaCl, and 0.1% BSA] containing 100 ng IGF-II, 1 mM CaCl2, and either 50 ng rhIGFBP-4 (one lane) or 75 ng rhIGFBP-5 (the other lane); and incubated for 18 h at 37 C. After incubation, samples were subjected to 12% SDS-PAGE, followed by ligand blotting.
Phosphorimaging and autoradiography/|l, 百拇医药
Phosphor screen autoradiography of ligand blots or gels (72–96 h) involved scanning, digitalization, and processing of the image with a BAS1000 phosphorimager (Fuji Photo Film Co., Ltd., Tokyo, Japan). A Molecular Dynamics, Inc., software package (Bio-Imaging Analyzer, BAS1000 MacBas, Fuji Photo Film Co., Ltd.) was used to further process the data and obtain an image of the original radioactive sample in pixel values proportional to the amount of radioactivity. The background was subtracted from each sample. Additionally, x-ray films were exposed to nitrocellulose membranes or gels to obtain autoradiographs documenting the presence and size of bands in blots and gels. Quantitative results, shown in Fig. 1B, were obtained by densitometry of x-ray films./|l, 百拇医药
fig.ommitteed/|l, 百拇医药
Figure 1. Presence of IGFBP-4 and IGFBP-5, but not IGFBP-2, proteolytic activity in follicular fluid from bovine preovulatory follicles. A, Autoradiograph of a representative Western ligand blot of protease assay samples containing 2 µl follicular fluid from the same preovulatory follicle plus 50 ng rhIGFBP-4 (lanes 1 and 2), rhIGFBP-5 (lanes 3 and 4), or rhIGFBP-2 (lanes 5 and 6), incubated for 0 h (lanes 1, 3, and 5) or 18 h (lanes 2, 4, and 6) at 37 C. The approximate mol wt of intact IGFBPs is indicated to the right of the autoradiograph. B, Quantitative results (mean ± SEM) derived from densitometry of autoradiographs of Western ligand blots. The experiment was replicated with follicular fluid from four different preovulatory follicles. a vs. b, P < 0.01.
Statistical analysis7, 百拇医药
A t test was used for pairwise comparisons of densitometric data for 0 vs. 18 h of incubation (Fig. 1B). Data derived from phosphorimaging were analyzed by ANOVA, and individual means were compared by Tukey’s multiple comparison test. Values are presented as the mean ± SEM.7, 百拇医药
Results7, 百拇医药
IGFBP-4 and IGFBP-5, but not IGFBP-2, are degraded by follicular fluid from bovine preovulatory follicles7, 百拇医药
Previous work has shown that follicular fluid from bovine preovulatory follicles has a proteolytic activity that degrades IGFBP-4 (17). To determine whether the other low mol wt IGFBPs are also regulated by proteolytic degradation, we performed protease assays by incubating 2 µl follicular fluid from preovulatory follicles with 50 ng rhIGFBP-2, -4, or -5. Figure 1A shows a representative ligand blot of protease assays of follicular fluid frm the same preovulatory follicle using rhIGFBP-2, -4, or -5 as substrate, and quantitative results (n = 4) derived from densitometry are shown in Fig. 1B. Significant degradation of rhIGFBP-4 and -5 (P < 0.01), but not rhIGFBP-2 (P > 0.05), was observed after 18 h of incubation. As expected, no degradation of endogenous IGFBP-3 was seen after 18 h of incubation.
Partial characterization of the IGFBP-5 proteolytic activity present in follicular fluid from bovine preovulatory folliclesezw, 百拇医药
In a previous study we provided evidence that the IGFBP-4 proteolytic activity present in bovine follicular fluid is a neutral/basic pH-favoring, Zn2+ metalloprotease (17). Given the presence of strong IGFBP-5 degradation in follicular fluid from bovine preovulatory follicles (Fig. 1), we investigated the characteristics of this putative protease.ezw, 百拇医药
Time-course experiments revealed increasing IGFBP-5 proteolysis during the first 6 h of incubation (Fig. 2A), with approximately 30%, 40%, 60%, 70%, and 80% of the rhIGFBP-5 degraded after 1, 2, 4, 6, and 18 h of incubation, respectively (based on replication of the experiment with samples from four different preovulatory follicles; data not shown). To estimate the pH optimum of the IGFBP-5 protease, follicular fluid from preovulatory follicles was assessed for its ability to degrade rhIGFBP-5 over a broad range of pH values (pH 3–10). Proteolysis of rhIGFBP-5 was inhibited below pH 5, whereas weakly acidic, neutral, and weakly basic pHs promoted IGFBP-5 proteolysis (data not shown), as we showed previously for IGFBP-4 proteolysis (17).
fig.ommitteed&#:as;#, 百拇医药
Figure 2. Characterization of the IGFBP-5 proteolytic activity present in follicular fluid from bovine preovulatory follicles. A, Time course of rhIGFBP-5 degradation. Shown is a representative Western ligand blot showing degradation of rhIGFBP-5 (50 ng) by follicular fluid (5 µl) from preovulatory follicles as a function of time. B, Effect of class-selective protease inhibitors on IGFBP-5 proteolytic activity. Shown is a representative Western ligand blot showing degradation of rhIGFBP-5 (50 ng) by follicular fluid (5 µl) from preovulatory follicles. The samples were not incubated (-20 C) or were preincubated for 1 h at room temperature in the absence (None) or the presence of PMSF (1 mM), 1,10-phenanthroline (10 mM), EDTA (5 mM), E-64 (10 µM), pepstatin (1 µM), aprotinin (2 µg/ml), or chymostatin (100 µM). After preincubation, samples were assayed for their ability to degrade rhIGFBP-5 during an additional 18 h of incubation. Both experiments were replicated with follicular fluid from four different preovulatory follicles, and similar results were obtained.&#:as;#, 百拇医药
To provide initial mechanistic classification of the IGFBP-5 protease activity present in follicular fluid, we assessed its susceptibility to a set of standard protease inhibitors (27). Follicular fluid from preovulatory follicles was preincubated for 1 h in the absence or presence of the inhibitors at the concentrations detailed in Materials and Methods. Figure 2B shows a representative Western ligand blot of protease assay samples incubated in the absence or presence of different inhibitors. This experiment was replicated with follicular fluid samples from four different preovulatory follicles, and quantitative results derived from phosphorimaging of the gels were obtained and analyzed statistically (data not shown). Preincubation with the nonspecific divalent cation chelator EDTA (5 mM) completely inhibited proteolytic activity (P < 0.001). Although this observation suggests the possibility of a metalloprotease (cation dependence), it does not rule out the possibility of a cation-activated or stabilized nonmetalloprotease. However, the metalloprotease inhibitor 1,10-phenanthroline (10 mM) proved equally effective (P < 0.001). Given that 1,10-phenanthroline has a much higher stability constant for Zn2+ (2.5 x 10-6 M-1) than for Ca2+ (3.2 x 10-1 M-1), this observation is virtually diagnostic for a Zn2+ metalloprotease (27). Pretreatment with pepstatin A (1 µM), an acetylated pentapeptide established as a highly selective aspartic protease inhibitor, proved ineffective (P > 0.05). Similarly, E-64 (10 µM), a peptide epoxide recognized as a highly specific cysteine protease inhibitor, had no effect (P > 0.05) on IGFBP-5 proteolytic activity. Chymostatin (100 µM), an inhibitor of chymotrypsin-like serine proteases and of some cysteine proteases, did not inhibit IGFBP-5 degradation. Finally, aprotinin (2 µg/µl) and PMSF (1 mM), serine protease inhibitors, proved ineffective (P > 0.05). Taken together, these characterization studies suggest that follicular fluid from bovine preovulatory follicles contains an IGFBP-5, neutral/basic pH-favoring, Zn2+ metalloprotease. The previously described IGFBP-4 protease has the same characteristics (17).
Immunoneutralization and immunoprecipitation of IGFBP-4 and IGFBP-5 proteolytic activity with polyclonal antibodies against human PAPP-A3:s#$@&, http://www.100md.com
Recent studies have shown that the IGFBP-4 protease present in follicular fluid from human ovarian follicles (14, 32) and from several domestic species (15) is PAPP-A. The similarity between the previously described IGFBP-4 protease (17) and the IGFBP-5 proteolytic activity (present study) in bovine follicular fluid together with the recent finding that PAPP-A can also cleave IGFBP-5, at least in experiments in vitro with recombinant PAPP-A and substrate (33), raised the question of whether the IGFBP-5 proteolysis observed in bovine follicular fluid reflects the existence of an independent IGFBP-5 proteolytic system or simply the contribution of PAPP-A to IGFBP-5 proteolysis. We hypothesized that immunoneutralization and immunoprecipitation of bovine follicular fluid with polyclonal antibodies against human PAPP-A would result in inhibition and immunodepletion, respectively, of the IGFBP-4, but not the IGFBP-5, proteolytic activity. As hPS has a high concentration of PAPP-A, it was also incubated with antibodies against human PAPP-A as a positive control for the effects of the antiserum.
In an initial immunoneutralization experiment, we assessed proteolysis of IGFBP-4 or -5 by hPS or bovine follicular fluid incubated for 18 h with PBS, nonimmune IgG, or anti-PAPP-A antibodies. Interestingly, when the sample was hPS (the positive control for PAPP-A), polyclonal antibodies against human PAPP-A (1 or 5 µg), but not PBS or nonimmune IgG, were able to immunoneutralize IGFBP-4 (Fig. 3, top panel, lanes 1–6), but not the IGFBP-5 (Fig. 3, bottom panel, lanes 1–6), proteolytic activity. Surprisingly, incubation of follicular fluid from bovine preovulatory follicles with polyclonal antibodies against human PAPP-A, but not with PBS or nonimmune IgG, inhibited proteolysis of both IGFBP-4 (Fig. 3, top panel, lanes 7–12) and IGFBP-5 (Fig. 3, bottom panel, lanes 7–12). This initial experiment also suggested that, in contrast to the apparent complete neutralization of IGFBP-4 proteolysis, anti-PAPP-A antibodies substantially reduced, but did not completely block, IGFBP-5 degradation by bovine follicular fluid.
fig.ommitteedv]i8\, 百拇医药
Figure 3. Immunoneutralization of IGFBP-4 and IGFBP-5 degradation with polyclonal antibodies against human PAPP-A. Western ligand blots of protease assay samples containing 50 ng rhIGFBP-4 (top) or rhIGFBP-5 (bottom) and 5 µl hPS (lanes 1–6) or bovine follicular fluid (bFF; lanes 7–12) after incubation for 0 h (lanes 1 and 7) or 18 h (lanes 2–6 and 8–12) at room temperature in the presence of PBS (lanes 1–2 and 7–8), 1 or 5 µg nonimmune IgG (lanes 3 and 4 and 9 and 10), or 1 or 5 µg polyclonal antibodies against human PAPP-A (lanes 5 and 6 and 11 and 12). Note the presence of endogenous IGFBP-3 (43 kDa) in bovine follicular fluid, but not in hPS.v]i8\, 百拇医药
Results of the time-course experiment (Fig. 2A) suggest that after 18 h of incubation, IGFBP-5 could have been overdigested, thereby impairing our ability to assess the relative contributions of PAPP-A vs. other potential IGFBP-5 proteases. Therefore, in a follow-up experiment, we performed a kinetic analysis of the inhibition of IGFBP-4 and -5 degradation by anti-PAPP-A antibodies. Representative ligand blots of IGFBP-4 and -5 protease assays are shown in Fig. 4, and quantitative results derived from phosphorimaging are shown in Fig. 5 (left and right panels, respectively). These results show that, indeed, anti-PAPP-A antibodies, but not nonimmune IgG, completely inhibited (P < 0.01) both IGFBP-4 and -5 degradation by bovine follicular fluid when the incubation time was reduced to 2 or 5 h (Fig. 5, lanes 5, 7, 12, and 14). Interestingly, complete inhibition of IGFBP-4 proteolysis and partial (although significant, P < 0.05) inhibition of IGFBP-5 proteolysis by bovine follicular fluid were also observed after 18 h of incubation with anti-PAPP-A antibodies, but not with nonimmune IgG. In contrast, proteolysis of IGFBP-5 by hPS was not inhibited (P > 0.05) by anti-PAPP-A antibodies regardless of the time of incubation, whereas IGFBP-4 degradation by hPS was completely blocked after 2, 5, or 18 h of incubation in the presence of anti-PAPP-A antibodies, but not nonimmune IgG. The last observation argues for the specificity of the antiserum and the existence of an independent (not related to PAPP-A) IGFBP-5 proteolytic system in hPS, as previously suggested (34).
fig.ommitteedty\-r&c, http://www.100md.com
Figure 4. Kinetic analysis of the inhibition of IGFBP-4 and -5 degradation by anti-PAPP-A antibodies. Western ligand blots of 50 ng rhIGFBP-4 (left panels) or rhIGFBP-5 (right panels) incubated alone (lanes 1 and 8, respectively), with 5 µl hPS (lanes 2–3 and 9–10) or follicular fluid from different (b1, b2) bovine preovulatory follicles (lanes 4–7 and 11–14) for 0 h (lanes 1 and 8), 2 h (top panels), 5 h (middle panels), or 18 h (bottom panels) at room temperature in the presence of 5 µg nonimmune IgG (lanes 2, 4, 6, 9, 11, and 13) or 5 µg anti-PAPP-A antibodies (lanes 3, 5, 7, 10, 12, and 14). Note the presence of endogenous IGFBP-3 (43 kDa) in bovine follicular fluid, but not in hPS. This experiment was replicated with samples from four different preovulatory follicles.ty\-r&c, http://www.100md.com
fig.ommitteedty\-r&c, http://www.100md.com
Figure 5. Quantitative results (mean ± SEM) of kinetic analysis of the inhibition of IGFBP-4 and -5 degradation by anti-PAPP-A antibodies (n = 4 preovulatory follicles). Recombinant human IGFBP-4 (BP-4) or -5 (BP-5) was incubated alone, with hPS, or with follicular fluid from bovine preovulatory follicles (bFF) in the presence of nonimmune IgG (IgG) or anti-PAPP-A antibodies (PAPP-A) at room temperature for 2, 5, or 18 h. After incubation, protease assay samples were subjected to SDS-PAGE, followed by ligand blotting and phosphorimaging. *, P < 0.05; **, P < 0.01 [vs. control (BP-4 or BP-5)].
In the next experiment, polyclonal antibodies against human PAPP-A were mixed with hPS or bovine follicular fluid to immunoprecipitate PAPP-A before the protease assays. Immunoprecipitation with PAPP-A antibodies, but not nonimmune IgG, resulted in complete depletion of IGFBP-4 (Fig. 6A, top panel) and substantial (yet not complete) depletion of IGFBP-5 (Fig. 6A, bottom panel) proteolytic activities from bovine follicular fluid. Quantitative results derived from phosphorimaging (Fig. 6B) showed that anti-PAPP-A antibodies, but not nonimmune IgG, significantly (P < 0.05) depleted both IGFBP-4 and IGFBP-5 proteolytic activities from bovine follicular fluid; however, some IGFBP-5 degradation was still observed after immunoprecipitation with anti-PAPP-A antibodies. In contrast, immunoprecipitation with anti-PAPP-A antibodies (but not nonimmune IgG) resulted in full depletion of IGFBP-4 (P < 0.01), but not IGFBP-5 (P > 0.05), proteolytic activity from hPS (Fig. 6A, lanes 2 and 3, and Fig. 6B). Taken together, these experiments provide evidence that PAPP-A accounts for the IGFBP-4 proteolytic activity present in bovine follicular fluid and that it also contributes to substantial degradation of IGFBP-5.
fig.ommitteed.z1&, 百拇医药
Figure 6. Immunoprecipitation of IGFBP-4 and IGFBP-5 proteolytic activities with polyclonal antibodies against PAPP-A. A, Western ligand blots of protease assay samples containing 50 ng rhIGFBP-4 (top panel) or rhIGFBP-5 (bottom panel) incubated alone (lanes 1 and 10), in the presence of 30 µl supernatant of hPS (lanes 2 and 3) or follicular fluid from three different (b1, b2, and b3) preovulatory follicles (lanes 4–9) for 0 h (lanes 1) or 18 h (lanes 2–10) at room temperature. Samples of hPS and bovine follicular fluid were immunoprecipitated with nonimmune IgG (lanes 2, 4, 6, and 8) or anti-PAPP-A antibodies (lanes 3, 5, 7, and 9) before the protease assays. Note the presence of endogenous IGFBP-3 (43 kDa) in bovine follicular fluid, but not in hPS. B, Quantitative results (mean ± SEM) derived from phosphorimaging of ligand blots (n = 3). *, P < 0.05; **, P < 0.01 [vs. control (BP-4 or BP-5)]..z1&, 百拇医药
Immunodetection of PAPP-A in follicular fluid from bovine preovulatory follicles.z1&, 百拇医药
The antibody used in the immunoneutralization and immunoprecipitation experiments was raised against human PAPP-A/proform of eosinophil major basic protein (pro-MBP). Therefore, its specificity for PAPP-A in bovine follicular fluid (i.e. lack of cross-reactivity with other molecules) was tested by Western immunoblotting of bovine follicular fluid. As shown in Fig. 7, a specific band of about 400 kDa was detected after resolution of 2.5 or 5 µl follicular fluid from preovulatory follicles on a 6% SDS-PAGE gel under nonreducing conditions. Recombinant PAPP-A is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bonded subunits (35). Using the same polyclonal antibodies, PAPP-A/pro-MBP purified from human pregnancy serum has been shown to migrate as an approximately 200-kDa band under reducing conditions (35) and as a >400-kDa band after electrophoresis of human pregnancy serum under nonreducing conditions (30). Therefore, the detection by Western immunoblotting of a unique band of the expected size in bovine follicular fluid rules out the possibility of significant cross-reaction of the antibody with other related molecules (for example, PAPP-A2) and the presence of inactive proteolytic fragments (detectable by immunoblotting) previously reported to be generated by autocleavage (36).
fig.ommitteedau+'ep:, 百拇医药
Figure 7. Western immunoblot analysis of PAPP-A in follicular fluid from bovine preovulatory follicles. Follicular fluid (2.5 µl, lane 1; 5 µl, lane 2) was resolved on a 6% SDS-PAGE gel under nonreducing conditions and analyzed by Western immunoblotting using a polyclonal antibody against human PAPP-A/pro-MBP. The relative migration of molecular markers is indicated. This experiment was replicated three times.au+'ep:, 百拇医药
Estimation of the mol wt of the IGFBP-4 and IGFBP-5 protease activities present in bovine follicular fluid by native gel electrophoresisau+'ep:, 百拇医药
Follicular fluid was subjected to native gel electrophoresis. Gel lanes were cut into 2.5-mm slices (from the bottom of the wells and progressing down the gel), and slices from each individual lane were incubated with either rhIGFBP-4 or rhIGFBP-5. Proteolysis of rhIGFBP-4 and rhIGFBP-5 was observed for slices 1–3, indicating that the proteolytic activity in both cases had migrated 0–7.5 mm into the gel (data not shown). Given that the total length of the gel was 50 mm, both proteolytic activities had an Rf value of 0–0.15. The largest standard, myosin (~ 250 kDa), was located in slice 6, representing an Rf value of 0.3 and a migration faster than the protease activities. These data suggest that both proteolytic activities in bovine follicular fluid are large proteins (or complexes) with molecular masses greater than 250 kDa. Adding to the evidence that anti-PAPP-A antibodies immunoneutralized and immunodepleted both proteolytic activities (Figs. 3–6) and to the detection of a single band (~ 400 kDa) by Western immunoblotting (Fig. 6), the observation that both substrates (IGFBP-4 and IGFBP-5) were degraded by the same slices (no. 1–3) strongly suggests that the same molecular entity is responsible for both proteolytic activities. Interestingly, PAPP-A in bovine follicular fluid, shown in the present study to be the major contributor to IGFBP-5 degradation, is different from the complement component C1s, an 88-kDa IGFBP-5 serine protease secreted by human fibroblasts (37).
Potential role for IGFBP-5 proteolysis in the establishment of follicular dominance]6t, 百拇医药
To start investigating the role of IGFBP-5 proteolysis in the establishment of follicular dominance, we compared the IGFBP-5 protease activity in follicular fluid from companion dominant and subordinate follicles (n = 4) collected on d 3 after the emergence of the first follicular wave of the cycle (d 4/5 of the estrous cycle). Several characteristics of dominant and subordinate follicles used in this experiment were reported previously (17). Briefly, dominant follicles were larger (P < 0.05), and their follicular fluid had higher (P < 0.05) estradiol and similar (P > 0.05) protein concentrations compared with subordinate follicles. As shown in Fig. 8A, extensive proteolysis of the [125I]IGFBP-5 substrate and the appearance of proteolytic fragments of approximately 19 and 17 kDa were observed after 18 h of incubation with follicular fluid from the dominant follicle, but not with follicular fluid from the two largest subordinate follicles. Quantitative results (n = 4) derived from phosphorimaging (Fig. 8B) showed significant (P < 0.05) degradation of the [125I]IGFBP-5 substrate after 18 h of incubation with follicular fluid from dominant follicles, but not subordinate follicles. This evidence points to the intrafollicular IGFBP-5 proteolytic system as an integral component of the mechanisms leading to the establishment of follicular dominance.
fig.ommitteed}, 百拇医药
Figure 8. Evidence for a role for IGFBP-5 proteolysis in the establishment of ovarian follicular dominance. Follicular fluid obtained from a dominant follicle (DF) and the two largest companion subordinate follicles (SF1 and SF2) was incubated with [125I]IGFBP-5 substrate for 0 h (lane 1) or 18 h (lanes 2–4) at 37 C. After incubation, protease assay samples were subjected to SDS-PAGE and autoradiography (A). Quantitative results derived from phosphorimaging (mean ± SEM) were obtained from four sets of samples of dominant and companion subordinate follicles (B). *, P < 0.05 vs. DF incubated for 0 h.}, 百拇医药
Discussion}, 百拇医药
The results of the present study suggest that PAPP-A not only accounts for the IGFBP-4 proteolytic activity present in follicular fluid from bovine preovulatory follicles, but also makes a significant contribution to proteolysis of IGFBP-5. In addition, IGFBP-5 proteolysis was readily detectable in the dominant follicle and was negligible in subordinate follicles of the same cohort. The observation that proteolysis of IGFBP-5 is detected early during differentiation of a dominant follicle and persists through preovulatory development suggests a potential role for proteolysis of IGFBP-5 in determining follicular fate. Compelling evidence that human PAPP-A is also capable of cleaving IGFBP-5, in addition to IGFBP-4, at least in experiments in vitro, has been recently provided by Laursen et al. (33) using highly purified recombinant proteins. To our knowledge, the present study constitutes the first evidence of the existence of an IGFBP-4/-5 proteolytic system with physiological relevance in which PAPP-A is the dominant IGFBP protease involved.
We have previously shown that selection of a dominant follicle in cattle is associated with increased IGFBP-4 proteolysis by a neutral/basic pH-favoring, Zn2+ metalloprotease (17, 18). Herein, we report the presence of IGFBP-5 proteolytic activity in follicular fluid with striking similarities to the previously described IGFBP-4 protease. Several lines of evidence support the idea that both IGFBP-4 and -5 are, indeed, subjected to proteolysis by PAPP-A in bovine follicular fluid. 1) Characterization studies showed that IGFBP-5 is significantly degraded by follicular fluid from bovine preovulatory follicles within 4–6 h of incubation in vitro at neutral/basic pH, conditions very similar to those reported for IGFBP-4 proteolysis (17). 2) The IGFBP-5 proteolytic activity was sensitive only to EDTA and 1,10-phenanthroline, strongly suggesting, as in the case of IGFBP-4 degradation (17), that the IGFBP-5 proteolytic activity in bovine follicular fluid is a metalloprotease. 3) Anti-PAPP-A antibodies, but not nonimmune IgG, immunoneutralized and immunodepleted both IGFBP-4 and IGFBP-5 proteolytic activities in bovine follicular fluid. 4) PAPP-A was immunodetected as a single approximately 400-kDa migrating band after electrophoresis of bovine follicular fluid under nonreducing conditions, ruling out cross-reactivity of the polyclonal PAPP-A antibody with another protease(s) in follicular fluid. 5) Both proteolytic activities have identical electrophoretic mobility, as determined by native gel electrophoresis (data not shown). 6) Cleavage of IGFBP-5 by the putative IGFBP-5 protease present in bovine follicular fluid appears to occur at only one site, as suggested by the generation of radiolabeled proteolytic fragments of approximately 19 and 17 kDa after the incubation with [125I]IGFBP-5 substrate.
All of the above findings are in agreement with recently published reports showing that 1) PAPP-A is the IGFBP-4 protease produced by granulosa cells (32, 38) and found in follicular fluid of preovulatory follicles from humans (14) and from several domestic species, including cattle (15); 2) highly purified human recombinant PAPP-A cleaves both IGFBP-4 and -5, with similar kinetics (33); and 3) proteolysis of IGFBP-5 by PAPP-A occurs at one site, between Ser143 and Lys144 (33), thereby generating a pattern of proteolytic fragments similar to that observed in the present study. Interestingly, comparison of the reported site of cleavage of IGFBP-5 (R137KKLTQS{downarrow} KFVGGAE) by PAPP-A (33) with the site of cleavage of IGFBP-4 (S129TSGGKM{downarrow} KVNGAPR) by a fibroblast-derived IGFBP-4 protease (39) shown to be identical to PAPP-A (40) shows a Lys residue present in both P1' positions, hydrophobic residues in the P2' positions, and a Gly residue in both P4' positions (33). In addition, PAPP-A was found to undergo autocleavage at two major sites, showing similarities with, but also marked differences from, the cleavage site in IGFBP-4 (36). It has been proposed that the lack of unifying elements in sequences around the cleavage sites of PAPP-A implies that the specificity of PAPP-A is determined by steric restrictions more than by required interactions between PAPP-A and specific substrate side-chains (36).
Mutational analysis of the proteolytic domain of PAPP-A allowed its unequivocal classification as a member of the metzincin superfamily of metalloproteinases (36). All metzincins contain an elongated zinc-binding motif (HEXHXXGXXH) that coordinates the catalytic zinc ion of the active site and, as suggested by their common name, a Met residue believed to be important for the integrity of the catalytic site (36). Not surprisingly, the above-described catalytic site is absolutely conserved across species (15, 35, 41), and the full human and mouse PAPP-A amino acid sequences share 89% and 93% identity and similarity, respectively (41). PAPP-A exists in hPS as a covalent, heterotetrameric 2:2 complex with pro-MBP, PAPP-A/pro-MBP (42). In the present study immunoprecipitation and immunoneutralization experiments were performed with polyclonal antibodies raised against PAPP-A/pro-MBP purified from human pregnancy serum (35), which raised concerns about species specificity and cross-reactivity of the antibody with other molecules. The strictly conserved catalytic site and the overall high degree of homology of PAPP-A amino acid sequence across species argue against major species specificity concerns. Indeed, the same anti-PAPP-A/pro-MBP antibodies have been previously used to characterize PAPP-A in several domestic species (15), and recently, the successful use of a mouse antihuman PAPP-A monoclonal antibody in Western blot analysis of rat PAPP-A has been reported (43).
At present, only one protein with global homology to PAPP-A, a specific IGFBP-5 protease termed PAPP-A2 (44) or PAPP-E (45), has been described. Although some cross-reactivity of the polyclonal anti-PAPP-A/pro-MBP antibody with similar molecules, namely PAPP-A2, cannot be completely ruled out, this possibility seems unlikely considering that 1) Western immunoblot analysis of bovine follicular fluid samples resolved on SDS-PAGE under nonreducing conditions revealed the presence of a unique, specific signal migrating as an approximately 400-kDa band; 2) the electrophoretic mobility of the two proteolytic activities, as shown by native gel electrophoresis, was identical (data not shown); and 3) complete abrogation of the IGFBP-4, but not the IGFBP-5, protease activity in human pregnancy serum (used as a positive control for PAPP-A) was observed in immunoneutralization and immunoprecipitation experiments using polyclonal anti-PAPP-A antibodies. Unlike circulating PAPP-A, which is found in a 2:2 heterotetrameric PAPP-A/pro-MBP complex of about 500 kDa, PAPP-A2 is a monomer of approximately 220 kDa, and in addition, there is a limited degree of similarity between the two molecules (PAPP-A2 shares only 45% of its residues with PAPP-A). All of the above suggest that a high degree of cross-reactivity of polyclonal anti-PAPP-A with related (PAPP-A2/others) molecules is unlikely.
Of note is the observation that when we used hPS as a positive control for PAPP-A, immunoneutralization and immunoprecipitation resulted in inhibition of IGFBP-4, but not IGFBP-5, proteolysis after 2, 5, or 18 h of incubation. In a recent study (30) very little effect of PAPP-A antibodies on IGFBP-5 degradation was observed after incubation of PAPP-A antibodies with hPS for 5 h, whereas partial blockage was observed when the incubation time was reduced to 40 min. Similarly, polyclonal antibodies against human PAPP-A inhibited IGFBP-4, but not IGFBP-5, protease activity in follicular fluid from preovulatory follicles of women undergoing in vitro fertilization procedures (14). The failure of polyclonal antibodies against PAPP-A to inhibit IGFBP-5 degradation in hPS (present study) and human follicular fluid (14) argues for the existence of another major IGFBP-5 protease(s), presumably PAPP-A2, in those physiological fluids. At any rate, caution should be exercised in the interpretation of these apparently conflicting results; species differences, type of biological material under study, and/or methodological considerations (type of assay, time of incubation, or concentration of reagents) may well explain some of the apparently contradictory findings among different studies. Whether PAPP-A is the only intrafollicular protease responsible for proteolysis of IGFBP-5 or whether other minor IGFBP-5 proteases also contribute to the decrease in IGFBP-5 concentrations in follicular fluid of dominant, estrogen-active follicles in cattle awaits further investigation.
In this study we provide evidence that the IGFBP-5 proteolytic system is active in the dominant follicle, but not in the subordinate follicles of the same cohort. This is a novel piece of information considering that in previous reports of intrafollicular degradation of IGFBP-5 (21, 23, 24, 29) follicular fluid samples were obtained from preovulatory follicles well after selection had occurred. We have previously shown that, when dominant follicles are first detected as slightly larger than companion subordinate follicles, their capacity to secrete estradiol and levels of proteolytic activity against IGFBP-4 in follicular fluid are much greater than those of subordinate follicles, but they do not differ in other characteristics assessed (5, 17). In light of the present results, it is temping to speculate that an active IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is a key factor in the mechanisms leading to selection of a dominant follicle.1], 百拇医药
Acknowledgments
We thank D. Bianchi for care of experimental animals, and Dr. Mark S. Roberson for helpful discussions during the course of these experiments and critical reading of the manuscript.!h, 百拇医药
Received June 27, 2002.!h, 百拇医药
Accepted for publication October 16, 2002.!h, 百拇医药
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