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Biology of Reproduction 59, 233-240 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Ovine Allantoic Fluid Contains High Concentrations of Activin A: Partial Dissociation of Immunoactivity and Bioactivity1

Lynda M. Fouldsd, David M. de Kretser2,d, Paul Farnworthf, Danielle Buttressd, Graham Jenkine, Nigel P. Groomeg, , and James R. McFarlaned

d Institute of Reproduction and Development, e Department of Physiology, Monash University, f Prince Henry's Institute for Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia g School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a preliminary study, allantoic fluid collected from pregnant sheep across gestational ages of 20–124 days contained significantly higher levels of activin bioactivity (189 ± 74 ng/ml, mean ± SE) than did amniotic fluid (3.2 ± 0.6 ng/ml). Using a combination of chromatography steps, we isolated from 5 L of allantoic fluid approximately 612 µg of immunoactive activin, which eluted over 10 fractions from a C8 reversed-phase column. When these fractions were assayed in a rat pituitary cell culture bioassay, in a specific RIA, and in an activin A two-site ELISA, the RIA activity was skewed to the less hydrophobic side of the activin profile, while the bioactivity was skewed to the more hydrophobic forms. The activity measured in the two-site ELISA more closely matched the mass of activin as determined by laser densitometry. Amino-terminal sequencing of fractions containing either peak immunoactivity or bioactivity showed each to be identical to activin A. This was confirmed by internal sequences from a fraction that eluted in the area of overlapping immunoactivity and bioactivity. A peptide containing at least 18 amino acids at its amino terminus, which were identical to the conserved region of the acute-phase protein serum amyloid A, was identified in the most immunoactive activin fractions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Three types of activin, formed by dimerization of the two inhibin ß subunits, ßA and ßB, are termed activin A (ßAßA), activin AB (ßAßB), and activin B (ßBßB) [1, 2]. Recently three other putative activin ß subunits (ßC, ßD, and ßE) have been identified from mRNA [3–5]. As members of the transforming growth factor ß (TGFß) family, the activins (A and B) are involved in a diverse range of physiological processes including the regulation of FSH secretion [1, 2], steps in embryonic development [6], spermatogonial mitosis [7], and erythroid differentiation [8].

We have previously shown that amniotic fluid from sheep contains predominantly activin bioactivity despite the fact that it contains elevated levels of immunoactive inhibin [9], and we subsequently isolated activin A [10] from a pool of this fluid. The placenta, fetal membranes, and maternal decidua have been shown to be sources of activin in human pregnancy [11–14], and there are preliminary data to suggest that activin A may be involved in parturition in humans [15]. Activin A levels, measured by ELISAs, have also been shown to increase throughout the third trimester of pregnancy in women who had normal labor, in both longitudinal studies [16] measuring free serum activin A and cross-sectional studies [17] measuring total serum activin A, both rising to a peak at term.

In sheep, the amniotic and allantoic sacs are separate fluid-filled compartments, both of which are enclosed by the chorion [18]. The allantoic fluid is largely derived from fetal urine via the urachus, which drains about 38–50% of the urine in the fetal bladder [19, 20]; the rest of the urine drains via the ureter to the amniotic compartment. Throughout gestation, the volume of allantoic fluid has been found to increase from 75 ml at 44 days to 700–1300 ml at term (144–152 days) [21]. Bovine and ovine amnionic and allantoic membranes have been shown to produce a large variety of proteins, as characterized by one- and two-dimensional SDS PAGE [22–24], including other members of the TGFß family [25]. These studies suggest that the allantoic membranes and fluid play an important role during pregnancy. It has also been demonstrated that both activin ßA and ßB subunits are expressed in the amnion and chorion of humans [13]. Since we had previously shown that activin bioactivity was present in amniotic fluid, despite its high levels of immunoactive inhibin activity [9], and we were able to subsequently isolate activin A from amniotic fluid [10], we decided to investigate whether allantoic fluid also contained activin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Allantoic and Amniotic Fluid

A range of entire uteri were obtained from pregnant sheep, of gestational ages of 20–140 days, which had been stunned and killed at a local abattoir. Sheep used in this study were either Merino or Merino x Border Leicester. Each uterus was immediately placed on ice, and fetuses were sexed and their gestational ages were determined using algorithms derived from the nomogram of Cloete [21] utilizing measurements of fetal weight and vertebral column length.

These algorithms are as follows:where vc is the vertebral column length in centimeters and wt is the fetal weight in grams.

Using fetuses of the same breeds and known age (50–140 days) (from this and other studies) these algorithms were accurate to within 3.5 ± 1.9 days (n = 32) on the basis of weight and 3.1 ± 2.5 days (n = 23) on the basis of vertebral column length. Across 174 fetuses collected, the coefficient of variation between the 2 algorithms was 3.4%.

The uteri were carefully dissected to reveal the allantoic and amniotic fluid compartments. For the preliminary study comparing activin bioactivities, both allantoic and amniotic fluid samples were collected from the sacs of 19 fetuses of gestational ages 20–124 days. For the purification protocol, fluid (volumes ranging from 10 ml to 200 ml) was collected from the allantoic sacs of 159 fetuses of gestational ages of 20–140 days, of which 34 were twins. A pool having a total volume of 5.0 L was prepared, aliquoted, and stored at -20°C until subjected to the purification procedures detailed below. For the gestational age study, allantoic fluid was collected from 120 singleton fetuses only, of which 34 were unsexed fetuses of 20–35 days, 52 were male fetuses of 40–140 days, and 34 were female fetuses of 40–120 days gestational ages.

These investigations were approved by the Monash University Standing Committee on Ethics in Animal Experimentation and conform with the NH&;/CSIRO/AAC Code of Practice for the Care and Use of Animals for Experimental Purposes.

Activin RIA

Activin was assayed using the disequilibrium RIA previously described by Robertson et al. [26] as modified by McFarlane et al. [27] with the incorporation of dissociating reagents to remove the interference of follistatin (FS) in the assay. This assay used a sheep antiserum (#64; Biotech Australia, East Roseville, NSW, Australia) raised against a ßA subunit fusion protein and human recombinant (hr)-activin A. The tracer was hr-activin A (2 µg) iodinated with Iodogen (Pierce, Rockford, IL) using the method described by McFarlane et al. [28] with the modification that the tracer was further purified on an Ultrogel AcA54 (IBF Biotechnics, Villeneuve-la-Garenne, France) gel filtration column (2.5 x 90 cm) before use. Standard curves were constructed using hr-activin A. Bound from free was separated by a donkey anti-sheep second-antibody (Monash University, Clayton, Victoria, Australia) precipitation in the presence of 0.9% saline. Bovine inhibin, hr-inhibin, porcine TGFß1, hr-Müllerian inhibiting substance, and bovine FS have < 3.3% cross-reactivity in the original assay [26], while in the modified assay hr-inhibin, bovine FS, and hr-FS all have < 1.0% cross-reactivity. The assay buffer used was 0.1 M PBS pH 7.4 containing 0.5% BSA (Sigma, St Louis, MO). All samples from each purification step were measured in the same assay. The intraassay percentage coefficient of variation was 3.06 ± 0.66% (n = 3), while the interassay percentage coefficient of variation, based on the repeated measurement of a quality control pool, was 10.08 ± 6.14% (n = 6) [27].

The cross-reactivity of activin B in this modified RIA was determined using doses of hr-activin B (a gift from Genentech, South San Francisco, CA) of from 32.5 ng/ml to 500 ng/ml assayed against a standard curve of hr-activin A under the conditions described above.

Rat Pituitary Cell Bioassay (PCC)

Using the stimulation of FSH production by rat pituitary cells as a bioassay for activin as described previously [26], we performed a preliminary study of allantoic and amniotic fluid from 19 animals representing 20–124 days gestational ages to determine whether activin bioactivity could be detected in allantoic fluid and to compare levels to those found in amniotic fluid. A small number of urine samples was also studied in this assay. The bioactivity of fractions from the purification procedure was determined using a rat pituitary cell culture system as modified by Farnworth et al. [29]. Human recombinant activin A was used as standard in both assays, and the potency of samples run in triplicate at several dilutions was determined by parallel line bioassay statistics [30]. The intraassay and interassay coefficients of variation were 7.7% and 21.3%, respectively.

Activin A Two-Site ELISA

A specific activin A two-site ELISA [31], which incorporates an analyte denaturation and oxidation step, was used to determine activin A activity in the fractions across the final HPLC profile and in the allantoic fluid starting material. Human recombinant activin A was used as standard in the assay, and samples were run in duplicate at several dilutions. The sensitivity of the assay was 0.21 ng/ml.

Purification Procedures

The procedure used for the isolation of activin from ovine allantoic fluid was based on the previously published procedure for ovine amniotic fluid [10] with some modifications. Briefly, the pool of 5.0 L of allantoic fluid, adjusted to pH 6.0 with 0.5 M phosphate buffer, was mixed as separate 1.0-L batches with 300 ml of yellow dye HE4R (ICI, Melbourne, Australia) coupled to Fractogel TSK HW-65F (Merck, Darmstadt, Germany) in 0.05 M phosphate buffer pH 6.0, and incubated for 30 min in a shaking water bath before being filtered through a sintered glass funnel. The filtrates (unbound material) from each 1.0-L batch were subsequently recycled back through the purification procedure in case possible overloading of the dye column prevented activin binding to the gel during the first pass through the column. The gel was then incubated as above with 955 ml of 0.05 M phosphate buffer pH 6.0, followed by 535 ml of 0.4 M KCl/0.05 M phosphate buffer pH 6.0, with each wash again filtered through a sintered glass funnel. A final high-salt wash of 270 ml of 1 M KCl/4 M urea per 0.05 M phosphate buffer pH 7.0 was incubated with the gel overnight at 4°C. The mixture was returned to the column (5 x 25 cm) the following day. The initial eluate was collected at a flow rate of 8 ml/min, followed by a further elution of 390 ml with the high-salt buffer. The dye eluates from this fractionation and those from the recycled initial filtrates were combined. This pool was diluted 1:8 with 2.3 M KCl in 0.05 M phosphate buffer pH 7.0 to produce a solution containing 2 M KCl/0.5 M urea. This solution was loaded onto a 75-ml phenyl sepharose (Pharmacia, Uppsala, Sweden) hydrophobic interaction column (3.5 x 25 cm) overnight at room temperature at a flow rate of 4 ml/min. The column was washed with 140 ml of 2 M KCl/0.05 M phosphate buffer pH 7.0, followed by 150 ml of 0.05 M phosphate buffer pH 7.0 at a flow rate of 8 ml/min. The column was then incubated overnight with 3 bed volumes of 25% acetonitrile (ACN) in 0.05 M phosphate buffer pH 7.0, and the activin-containing fraction was eluted at 8 ml/min. After removal of the ACN under nitrogen gas, this fraction was acidified with glacial acetic acid to a final concentration of 4 M and applied to a Sephadex G-100 (Pharmacia) gel permeation column (9 x 90 cm) equilibrated in 4 M acetic acid at 4°C. The flow rate for the column was 70 ml/h, and 10-ml fractions were eluted. Fractions containing activin were pooled, lyophilized, and reconstituted in a small volume of 4 M acetic acid with sonication for application to HPLC. Several HPLC runs were performed using a variety of columns, solvent systems, and gradient conditions: 1) a C4 214TP (Vydac; Separations Group, Hesperia, CA) semi-preparative column run with a 0.1% phosphoric acid (H3PO4)/0–50% ACN gradient at 3.0 ml/min over 100 min; 2) a C4 semi-preparative column run with a 0.1% trifluoroacetic acid (TFA; Pierce)/0–50% ACN gradient at 3.0 ml/min over 110 min; 3) a C8 Ultrapore (Beckman, Berkeley, CA) analytical column run with a 0.2% heptafluorobutyric acid (HFBA; Pierce)/0–70% ACN gradient at 1.0 ml/min over 140 min.

Steps throughout the purification procedure were monitored for immunoactivity in the activin RIA detailed above [27], and some steps were also monitored for bioactivity in the pituitary cell culture bioassay [29]. The fractions across the final HPLC profile were also monitored for activin A activity in a two-site ELISA [31].

SDS-PAGE

Fractions obtained at the final HPLC step (3, above) were assayed for both immunoactivity and bioactivity and were run on 15% polyacrylamide gels [32] under both nonreducing and reducing conditions, and protein bands were identified by silver-staining [33]. The relative abundance in these fractions of the 25-kDa protein band revealed by the silver-stained gels was also subjected to laser densitometry measurements.

Western Blotting

The same fractions that were analyzed on SDS-PAGE (fractions 163–180) were run on a further 15% polyacrylamide nonreducing gel, and separated proteins were transferred overnight to a 0.45-µm pore-size nitrocellulose transfer membrane (Micron Separations Inc., Westboro, MA) at 60 volts with cooling in transfer buffer containing 20 mM Tris base, 150 mM glycine, 20% v:v methanol. At the conclusion of the transfer, the membrane was stained with Ponceau S stain (Sigma) to identify molecular weight marker proteins, and was then blocked for 1 h with 3% powdered milk in 10-mM phosphate buffer pH 7.4. The membrane was then incubated with primary activin ßA antibody #64 at a dilution of 1:1000 in 1% milk in 10 mM PBS for 24 h, then washed several times with 1% milk/PBS/0.1% Nonidet P-40 detergent (NP-40; Sigma). The second antibody used was a biotinylated rabbit anti-sheep IgG (Zymed Laboratories, San Francisco, CA) at a dilution of 1:4000 in 1% milk/PBS per 0.1% NP-40 for 24 h, followed by several washes as above and enhancement by streptavidin horseradish peroxidase-conjugated (Silenus, Melbourne, Australia) at a dilution of 1:500. The membrane was then developed with N,N-diethylphenylenediamine monohydrochloride (DEPDA) and 4-chloro-1-naphthol [34].

Two of the fractions from the region demonstrating peak activin bioactivity and low levels of immunoactivity were also run on a further 15% polyacrylamide gel and transferred as above, but the membrane was incubated with a primary activin ßB antibody (gift from Dr. Tony Mason, Prince Henry's Institute of Medical Research, Melbourne, Australia), using all other incubation and development steps as above.

NH2-Terminal Amino Acid Sequencing and Internal Sequencing

Two fractions from the final HPLC step that demonstrated peak immunoactivity and bioactivity were applied to an on-line Applied Biosystems (Foster City, CA) 470A gas phase sequencer equipped with an on-line Applied Biosystems 120A HPLC for analysis of the phenylthiohydantoin-derivatized amino acids.

In addition, one fraction (#173) from the region of highest protein density as determined by laser densitometry measurement, which also demonstrated both activin immunoactivity and bioactivity, was subjected to tryptic digestion in 0.1 M ammonium bicarbonate at 37°C for 40 h; this was followed by reduction and alkylation with mercaptoethanol at 37°C for 1 h and 4-vinylpyridine at 37°C for 1 h. The tryptic digest was then fractionated on a microbore Reliasil C8 column (Poly LC Inc., Columbia, MD) at 50°C using a 0.1% TFA/0–60% ACN gradient over 1 h, and collected digest peaks were sequenced as described above.

Statistics

Relative activities in the activin RIA were determined using parallel line bioassay statistics [30]. Allantoic fluid activin concentrations across gestation and between sexes were compared by two-way ANOVA after log transformation of the data, followed by all pair-wise multiple comparisons using the Student-Newman-Keuls (SNK) method. Concentrations within sexes were compared by one-way ANOVA followed by SNK analysis. Calculations were carried out using the SAS computer package (SAS Institute Inc., Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In a preliminary study of activin bioactivity in a dispersed PCC, using randomly selected samples of allantoic fluid from a range of gestational ages, it was found that allantoic fluid had a significantly higher mean concentration of activin bioactivity (189 ± 74 ng/ml, n = 19) than amniotic fluid (3.2 ± 0.6 ng/ml, n = 15). Only three urine samples from late gestation were available for assay, and two of these samples were without activity, while the third had an activity of 5.0 ng/ml.

The concentrations of immunoactive activin across gestation in allantoic fluid surrounding male and female singleton fetuses are shown in Figure 1, A and B, respectively. From gestational ages of 20–35 days, no sex determination could be made. Analysis of activin concentrations in these allantoic fluids showed no significant difference between Days 25, 30, and 35 (p > 0.05), with levels remaining at approximately 75 ng/ml. There was a significant change in activin concentrations in allantoic fluid across gestation in both male (p < 0.01) and female (p < 0.05) fetuses. In allantoic fluid from male fetuses, activin remained between 90 and 125 ng/ml through to 70 days of gestation, and then rose to a peak of between 225 and 275 ng/ml at 80–90 days of gestation, declining precipitously to approximately 30 ng/ml by 100 days. The levels at 80–90 days were significantly (p < 0.05) elevated compared to earlier (Day 60) and later (Days 100, 110, and 120) gestational ages. In allantoic fluid from female fetuses in late gestation, activin concentrations were significantly greater at Days 90–100 (p < 0.05), compared to Day 70 gestation, and subsequently decreased to a nadir at Day 110, 10 days after the activin concentration peak observed in fluid associated with male fetuses. Concentrations in the female were greater than those in the male at Days 60 (p < 0.0025) and 100 (p < 0.001), while those in the male were greater than those in the female at 80 days (p < 0.05) gestation.



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  		FIG. 1. The concentration of immunoactive activin across gestation in allantoic fluid from singleton male fetuses (A) and female fetuses (B). Values are means ± SE (ng/ml); numbers above error bars represent the number of animals in each age group. See text for differences within and between age groups.

Allantoic fluid from across all gestational ages was pooled, and the isolation of activin A was monitored by RIA and bioassay. Table 1 shows the yields of activin in terms of immunoactivity and bioactivity throughout the purification, with the allantoic fluid starting material having a ratio of immunoactive to bioactive activin of approximately 1:8. Gel permeation chromatography in 4 M acetic acid (Fig. 2) separated both activin immunoactivity and bioactivity from the main protein peak, although the ratio of immunoactivity to bioactivity in activin-containing fractions (VI–XII) remained similar to that of the starting material (Table 1). Redissolving freeze-dried fractions from the gel permeation chromatography in 4 M acetic acid, with sonication, before application to the first of the HPLC runs resulted in an apparent increase in activin immunoactivity (Table 1), and a related drop in bioactivity, such that the ratio of immunoactivity to bioactivity increased to 239:1, whereas assays across fractions from the final HPLC step yielded a final ratio of 58:1. SDS-PAGE of the final HPLC step (Fig. 3, A and B) revealed that a 25-kDa band had been purified to almost purity in fractions 169–177, which migrated under reducing conditions at 15 kDa, as would be expected of activin A. A more faintly staining 45-kDa band was also seen in fractions 171–175, which reduced to 25 kDa. There were also faint bands at approximately 12 kDa in the unreduced gel.


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  		TABLE 1. The yields of activin A at each step of the purification of activin from allantoic fluid as determined by RIA and bioassay.



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  		FIG. 2. Gel permeation chromatography profile showing the dissociation of activin immunoactivity and bioactivity from the main protein peak (absorbance 280).



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  		FIG. 3. SDS-PAGE gels of fractions across the final HPLC purification step under A) nonreducing conditions, showing 25-kDa activin A band in fractions 169–177 and 12- to 15-kDa SAA bands in fractions 163–170, and B) reducing conditions, showing 15-kDa activin ßA subunit in fractions 169–177 and 12- to 15-kDa SAA bands in fractions 163–170.

Western blotting revealed that the major 25-kDa band was activin A (Fig. 4), and not activin B (data not shown as no bands were seen on blot), although there was some cross-reactivity of the activin A antiserum with the 45-kDa band that reduced to 25 kDa. These fractions yielded approximately 612 µg (122.4 µg/L) of immunoactive activin spread across 10 fractions, as shown in Figure 5. This recovery was 17 times that obtained from ovine amniotic fluid [10] using a similar purification scheme. These fractions were also assayed for bioactivity in a PCC and yielded approximately 10.5 µg of bioactive activin (Fig. 5).



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  		FIG. 4. Western blot analysis of fractions across final HPLC purification step showing 25-kDa activin A band together with some cross-reaction of the activin A antiserum with the 45-kDa band in the same fractions.



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  		FIG. 5. Final HPLC purification step showing UV profile (absorbance 254) together with profiles of immunoactivity, measured by both RIA and ELISA, and bioactivity. Sample was run on a C8 Ultrapore analytical column using a 0.2% HFBA/0–70% ACN gradient at 1.0 ml/min over 140 min.

The relative mass of 25-kDa activin in each fraction collected over the elution profile of the last HPLC step as determined by laser densitometry of silver-stained gels is shown in Figure 6. The activin eluted from the column in a relatively uniform peak between fractions 170–176. When these fractions were assayed in the PCC, the bioactivity was found in the more hydrophobic region of the profile, whereas the RIA immunoactivity was found skewed to the less hydrophobic side of the elution profile. The immunoactivity detected by the two-site ELISA almost matched the relative mass of activin, with a shoulder in the profile occurring between fractions 171 and 172, skewing it slightly to the more hydrophobic side of the profile. Dose-response curves for these fractions measured in the ELISA were parallel to the allantoic fluid starting material.



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  		FIG. 6. Comparison of immunoactivity, bioactivity, and laser densitometry measurements (from SDS-PAGE gels) across fractions from final HPLC purification step.

Amino-terminal sequence analysis was performed on fractions 170 and 174. The 18 amino acids determined in fraction 170, GLEXDGKVNIXXKKQF(F&Y)V, were identical to those of activin A, as were the 9 amino acids obtained from fraction 174. In addition, analysis of three tryptic digest peaks of fraction 173 revealed sequences of 8–10 amino acids consistent with the internal sequence of activin A. A secondary 18-amino acid sequence obtained in fraction 170 of AADKYFHARGNYDAAQRG was found to be identical to a conserved internal sequence of the serum amyloid A (SAA) family of proteins [35], which have a molecular size range of 12 kDa to 15 kDa. The possibility that the copurified SAA was a binding protein of activin and thus caused an apparent elevation in immunoactive activin, as we have shown for FS [27], was investigated further. A preparation of mouse serum amyloid A (Calbiochem, Lucerne, Switzerland) did not cross-react in our RIA at doses up to 1000 ng/ml nor interfere in the RIA when doses up to 1000 ng/ml of SAA were added to 13.38 ng/ml of hr-activin A (data not shown). Similarly, hr-activin B did not cross-react in our RIA at doses up to 500 ng/ml. The same results were obtained with or without the presence of dissociating reagents in the RIA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that in the sheep, the allantoic fluid, which consists predominantly of fetal urine, contains a high concentration of activin A as demonstrated by RIA and bioassay. Given that we were unable to detect activin A by RIA of fetal urine samples, it is likely that the activin present in high concentrations reflects secretion by the allantoic membrane. Although we have not investigated the presence of mRNA for the ß subunit in the allantoic membrane, several studies have demonstrated that the amniotic membrane, which secretes activin into the amniotic sac, contains ß-subunit mRNA [13]. By isolating and sequencing the purified activin from allantoic fluid, we have shown that it is activin A as identified by its NH2-terminal sequence, although we noted that there was a partial dissociation between the fractions showing peak immunoactivity and bioactivity. However, the NH2-terminal sequence of the fractions showing either greater immunoactivity or bioactivity led us to the conclusion that they were both activin A. The amino terminal sequencing data for fraction 170 indicated that both phenylalanine and tyrosine were cleaved in the same cycle at amino acid 17. Amino acid 17 is the only known difference between activin A in the sheep [36] and other mammals [37]. It is possible that the sheep does possess two activin A genes or another activin A-like molecule. However, internal sequences after tryptic digestion confirmed that the immunoactive peak was activin A, although we cannot exclude the possibility that the most immunoactive fractions contain a novel activin since we have not sequenced the entire molecule.

It is of interest that a two-site activin A ELISA detected peak activin A levels in fractions between the immunoactive and bioactive peaks corresponding more closely to the mass of activin A. To date we are unable to provide a clear explanation for the dissociation observed between the immunoactive and bioactive regions of the final HPLC profile. It is possible that the procedures used during the purification may have altered the physico-chemical properties of the activin so that there is heterogeneity in the population of the activin A molecules such that they migrate with slightly different hydrophobicity. Some of these changes may unmask epitopes that are detected by the antibody used for the immunoassay whereas other fractions are more easily detected by the antibodies used in the ELISA or by pituitary cells. Alternatively, if the allantois represents a storage site, then the secreted activin may remain for some time and be subjected to subtle conformational changes or oxidation. Given that we noted fluctuations in the bioactive:immunoactive ratios of the activin at different stages of the purification procedure, it seems likely that the dissociation observed between the peaks detected by the different assays reflects altered chemical conformation. It is important to note that a similar dissociation of immunoactivity and bioactivity was found during the purification of activin A from ovine amniotic fluid [10]. These observations imply that differing activin A potencies may be reported with different assays in biological fluids because the immunoactive:bioactive ratios of the activin in the fluid and the activin preparation used as the standard may affect the reported potencies.

It is possible that binding proteins, such as FS, may influence the biopotency of activin in the PCC, whereas, in the RIA, the use of dissociating agents removes this potential interference. However, the biopotency decreases with increasing purity of activin. This argues against the view that binding proteins are the cause of the varying immunoactive:bioactive ratios, since removal of binding proteins as purification proceeds should increase the biopotency.

In view of the differing bioactive potencies noted in some fractions of the purification steps, it is possible that other proteins may interfere with the bioactivity of the activin. We observed that in those fractions of the terminal purification step which showed peak immunoactivity, a second amino terminal sequence was found. This sequence represented a conserved internal sequence of SAA, a family of proteins classified as acute phase proteins and secreted in high concentrations during metabolic responses to infective episodes [38]. Given their molecular sizes, which range from 12 kDa to 15 kDa, these proteins represent the contaminants of this molecular range noted in fractions 163–170 in Figure 3. The fact that the SAA-like protein coeluted with the immunoactive region of activin through 6 purification steps raises the possibility that it may be an activin binding protein. However, we have shown that SAA does not cross-react in either our modified RIA or in an assay without dissociating reagents, nor does it interfere in the binding of hr-activin A to which high doses of SAA have been added, suggesting that it does not interact with activin in the same manner as does FS.

The amino terminal sequence of the SAA-like protein detected in this study represents a normal internal sequence of these proteins, and the NH2-terminus of the previously described SAA proteins was found in several studies to be blocked [39]. These observations again raise the possibility that the allantois may represent a site at which such secretions are stored and are subjected to proteolysis. SAA has previously been shown to be present in both maternal and fetal plasma in the period around parturition. Peripartum plasma samples taken from both pregnant cows and their fetuses [40] showed an increase in SAA levels in the maternal plasma after delivery, reaching a peak at 48 h post-delivery, while a nonsignificant increase in fetal plasma was noted after parturition. Similarly, de Villiers et al. [41] noted an increase in serum SAA levels in the peripartum period in normal pregnant women and newborn infants. In mares, SAA levels, which remained within the normal range for 4 mo before parturition, increased quickly after foaling to reach a peak of 5–6 times normal values on Day 3 postpartum, and then decreased to within the normal range by one month postpartum [42, 43]. The role of this SAA-like protein in parturition and its association with activin is currently under investigation in our laboratory.

The changing levels of activin A in allantoic fluid samples across gestation showed a similar pattern in the male and female fetus, with peak levels occurring at 80–90 days for male fetuses and at 90–100 days for female fetuses. It should be noted that allantoic fluid samples were collected from a mixture of singleton and twin pregnancies, and it is possible that multiple pregnancies may influence activin levels. Unfortunately, insufficient numbers of twin pregnancy samples across gestation were available to evaluate this possibility, and only data from singleton pregnancies were used in the gestational age study. The high concentrations and the changing potencies during gestation suggest a regulated secretion of activin A in this fetal fluid compartment, but the physiological role of this remains unclear.


   ACKNOWLEDGMENTS
 
The NH2-sequencing was kindly performed by Dr. Jelle Lahnstein at Biotech Australia Pty Ltd. (Roseville, NSW) and Ms. Mary Matthew and Dr. Ian Smith at the Baker Medical Research Institute (Melbourne, Victoria). The authors would like to thank Ms. Lisa Clarke and Ms. Anne O'Connor for their excellent technical support.


   FOOTNOTES
 
1 This research was supported by a grant from the NH&;, Australia. Back

2 Correspondence: D.M. de Kretser, Institute of Reproduction and Development, Level 3, Block E, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. FAX: 61 3 9550–3584; david.de.kretser{at}med.monash.edu.au Back

3 Current address: Department of Physiology, University of New England, Armidale, NSW 2351, Australia. Back

Accepted: March 11, 1998.

Received: November 11, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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