Uterine milk protein, a novel activin-binding protein, is present in ovine allantoic fluid.

Activins are pluripotent growth factors that have recently been shown to be present in placental and fetal membrane preparations. Our previous studies have identified and purified activin A from ovine amniotic and allantoic fluids. In this study, ligand blots of side fractions from the isolation of activin A from allantoic fluid suggested the presence of activin-binding proteins other than follistatin. Further purification of one of these fractions involved two sequential reverse phase HPLC steps and a Superose 12HR fractionation. SDS-PAGE revealed a single protein band of 55 kDa, which was identified by NH2-terminal sequencing as ovine uterine milk protein (UTMP), a member of the serine protease inhibitor (serpin) superfamily of proteins. Further binding studies, using ligand blot techniques and Superose 12HR fractionation in the presence of [125I]activin, demonstrated UTMP to be an activin-binding protein with a lower affinity for activin than that of follistatin. A study of the specific binding behavior of UTMP to activin, using surface plasmon resonance, revealed an apparent equilibrium dissociation constant (Kd) of 49 +/- 25 nM, compared with the follistatin-activin Kd of 379 +/- 51 pM. Similar to another activin-binding protein, alpha2-macroglobulin, UTMP was unable to neutralize the bioactivity of activin in a bioassay based on the capacity of activin to inhibit the proliferation of an MPC-11 plasmacytoma cell line. The high concentrations of this protein in uterine fluid during pregnancy and its ability to bind activin suggest that UTMP may act as a low affinity, high capacity binding protein to sequester activin in the local uterine environment.

Ovine allantoic fluid contains high concentrations of activin A: partial dissociation of immunoactivity and bioactivity.

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 microg 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.

Localization of follistatin in the rat testis.

The cellular localization of the activin-binding protein, follistatin, in the rat testis has been a matter of some controversy with different investigators claiming that Sertoli cells, Leydig cells or germ cells are the primary cell types containing this protein. The localization of mRNA encoding follistatin was re-examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization as well as the distribution of follistatin by immunohistochemistry. The results demonstrate that mRNA encoding follistatin is located in many germ cells including type B spermatogonia, primary spermatocytes with the exception of the late leptotene and early zygotene stages, and spermatids at steps 1 to 11. It is also found in Sertoli cells and endothelial cells but not in Leydig cells. Immunohistochemistry, using two different antisera to follistatin, showed that this protein was localized to spermatogonia, primary spermatocytes at all stages except the zygotene stage, spermatids at all stages and to endothelial cells and Leydig cells in the intratubular regions. The failure to detect mRNA for follistatin in Leydig cells using RT-PCR and in situ hybridization suggests that the immunohistochemical localization in these cells reflects binding of follistatin produced elsewhere. The widespread localization of follistatin, taken together with its capacity to neutralize the actions of activin, may indicate that follistatin modulates a range of testicular actions of activin, many of which remain unknown.

Isolation and partial characterization of tammar wallaby luteinizing hormone and development of a radioimmunoassay.

Tammar wallaby (Macropus eugenii) luteinizing hormone (LH) was purified from pituitaries collected from wild and captive populations by salt sequential precipitation, ion exchange chromatography and gel filtration. Pituitary tissue (5 g) yielded 1.8 mg of purified wallaby luteinizing hormone (ME-14B), as verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A heterologous radioimmunoassay has been developed for measurement of LH in plasma of marsupials using a monoclonal antibody raised against bovine LH (518B7). This assay system was able to measure basal LH concentrations in male and female tammars and detected a significant rise in plasma LH in response to oestradiol benzoate in female tammars and luteinizing hormone-releasing hormone (LHRH) in males. Parallel dose-response curves were also obtained from pituitary extracts from four other species of marsupial (brushtail possum, Trichosurus vulpecula; brown antechinus, Antechinus stuartii; kowari, Dasyuroides byrnei; and Eastern pygmy possum, Cercartetus nanus) in this assay, which suggests its usefulness in the measurement of LH in other marsupial species.

Measurement of activin in biological fluids by radioimmunoassay, utilizing dissociating agents to remove the interference of follistatin.

Activin, a dimer of the beta-subunits of inhibin, is a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors and has a widespread range of actions in a variety of tissues. The investigation of the physiology of activin action has been facilitated in recent years by the availability of immunoassays in addition to bioassays. Follistatin has been shown to bind to activin with a high affinity and therefore interferes in both radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs). In this study we examined the effect of various surfactants and 1.4-dioxane on the measurement of activin in the presence of follistatin by radioimmunoassay. The addition of a combination of sodium deoxycholate. Tween 20 and sodium dodecyl sulphate removed the interference of follistatin in the radioimmunoassay. The measured content of activin in male rat serum, human male serum, human female serum and bovine follicular fluid rose from 3.29 to 4.15, < 0.48 to 2.87, 2.42 to 4.17 and 30.9 to 85.6 ng/ml, respectively, when assayed in the presence of the dissociating reagents. It was unclear whether the altered potencies were due to a dissociation of the follistatin/activin complex rather than the exposure of the epitope on activin recognized by the antiserum. Serum concentrations of activin were lower than those found in testicular cytosols, and after castration no change in serum activin levels was observed, suggesting that the testis does not contribute significantly to circulating activin levels. The use of the dissociating reagents in the radioimmunoassay will enable studies to be carried out that more accurately measure the activin content of various biological fluids, and thus lead to a greater understanding of the physiology of this growth factor.

Partial characterization of inhibin, activin, and follistatin in the term human placenta.

To determine whether the human term placenta contains inhibin, activin, and follistatin, placental homogenates from normal placentae were subjected to several fractionation procedures: 1) dye affinity chromatography; 2) hydrophobic interaction chromatography using phenyl sepharose; 3) gel filtration under acid conditions; 4) reversed phase-high pressure liquid chromatography (RP-HPLC); and 5) preparative polyacrylamide gel electrophoresis and electroelution. Purification was followed by RIA for inhibin, activin, and follistatin as well as in vitro bioassay based on the FSH cell content of rat anterior pituitary cells in culture. Two peaks of immunoactive and bioactive inhibin with differing elution patterns on RP-HPLC were shown to have molecular weights of 33K and 32K on polyacrylamide gel electrophoresis. These peaks may represent inhibin A and B. Three peaks of immunoactive activin on RP-HPLC had molecular weights of 26.5K, 25.5K, and 27K and may represent activin A, AB, and B. Immunoactive follistatin eluted on RP-HPLC as a broad peak, which on polyacrylamide gel electrophoresis consisted of three main activities consistent with molecular weights of 31K, 35K, and 38K. These studies demonstrate that the term placenta contains inhibin, activin, and follistatin, which have been partially characterized. The presence of these substances within the placenta raises the possibility of their function in local paracrine interactions.

The isolation of activin from ovine amniotic fluid.

During a study of the levels of inhibin and follistatin in ovine amniotic fluid, we noted that although detectable levels of immunoactive inhibin and follistatin were found throughout gestation, the addition of amniotic fluid to a rat anterior pituitary cell culture resulted in a stimulation, rather than the expected suppression, of FSH concentrations. These data suggested the possibility that activin was present in amniotic fluid. We, therefore, set out to isolate the molecules responsible for this activin-like activity and determine their structure. Amniotic fluid, collected from pregnant sheep between 120-140 days gestation, was used as starting material in the purification and diluted in parallel to a human activin-A standard in the activin RIA employed to monitor the purification. A total pool of 7.4 liters amniotic fluid was processed by dye affinity chromatography, hydrophobic interactive chromatography, gel filtration, and a series of reverse phase HPLC steps. Polyacrylamide gel electrophoresis of fractions from the final HPLC step, which showed both activin immunoactivity and bioactivity, revealed a band with a mol wt of 25.3 kilodaltons (kDa), which reduced to 15.8 kDa, and a minor band of 45 kDa, which reduced to 25 kDa. NH2-terminal amino acid sequences of several active fractions from the same region were identical to the known sequence of ovine activin-A. The identification of immunoactive activin, follistatin, and inhibin in amniotic fluid raises the question of the sites of production of these proteins and their interactions and role in fetal physiology.

Inhibin/activin beta-subunit monomer: isolation and characterization.

Using an activin RIA that showed limited cross-reaction with inhibin, activin immunoactivity was monitored throughout the isolation of activin from bovine follicular fluid and side-fractions during the isolation of human recombinant inhibin. Two peaks of activin immunoactivity were identified in both materials and isolated to homogeneity by dye affinity chromatography, hydrophobic interaction and gel permeation chromatography, and reverse phase HPLC. The purified proteins in all four peaks had terminal amino acid sequences identical to those of the inhibin/activin beta-subunit. The molecular masses determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing (and reducing) conditions were 25 and 15 and 15 and 15 kilodaltons (kDa) for each pair of proteins from both sources. Based on these criteria, the bovine and human recombinant 25-kDa proteins correspond to the inhibin/activin beta A-subunit dimer (activin-A), while the 15-kDa proteins correspond to the inhibin/activin beta A-subunit monomer. The activity of the monomer was 17% of the activity of the dimer in the activin RIA. Based on this level of cross-reaction and the proportion of monomer to dimer immunoactivity found after reverse phase HPLC of bovine follicular fluid, it is estimated that the levels of monomer in bovine follicular fluid are 25-60% those of the dimer. The biological activities of the human recombinant activin monomer and dimer were investigated in two different cell culture systems. In a rat pituitary cell system the activity of the activin monomer was 19% of the activity of the dimer in stimulating FSH release, while in rat thymocyte cultures the activity of the monomer was 45% the activity of the dimer in suppressing lectin-stimulated [3H]thymidine uptake. It is concluded that the beta A-subunit monomer is found in bovine follicular fluid at a level 25-60% that of the beta A-subunit dimer (activin-A). The monomer displays in vitro responses similar to those of the dimer, although the monomer is less active (18-45%) than the dimer. It is unclear if dimerization of the monomer is a necessary prerequisite for biological activity.

Circulating half-lives of follicle-stimulating hormone and luteinizing hormone in pituitary extracts and isoform fractions of ovariectomized and intact ewes.

Previous studies have shown that the circulating half-life (t 1/2) of serum FSH in ewes after hypophysectomy (HPX) increased 10-fold after ovariectomy (OVEX). The basis for this difference was examined in this study by determining the circulating half-life of serum FSH and LH in HPX ewes after administration of pituitary extracts and gonadotropin isoform fractions. High-speed supernatants of pituitaries from gonadal-intact and OVEX ewes were fractionated by electrofocusing in sucrose gradients and based on the pI distribution of FSH and LH divided into four pools, pH 4.3-4.8, 4.8-5.55, 5.8-6.7, and 6.7-10. These extracts were administered by iv bolus injection to HPX gonadal-intact ewes and blood samples collected between 15-1000 min later. The clearance pattern for both serum FSH and LH was heterogenous, indicative of a major rapid and a minor slow dissociating component. A significant (P less than 0.05) difference in circulating half-lives (rapid component) was observed between pituitary extracts from intact and OVEX ewes for FSH (t 1/2 = 32.8 +/- 8.6 min vs. 89.9 +/- 32.3 min) but not LH (31.3 +/- 9.2 min vs. 39.3 +/- 6.1 min, respectively), whereas no significant difference was observed between the corresponding FSH or LH isoform preparations. To establish if the difference in circulating half-lives obtained after HPX and bolus iv injection was due to mode of delivery, an extract of pituitaries from OVEX ewes was infused for 12 h into HPX sheep and the t 1/2 values determined after cessation of treatment and compared to those after a bolus injection. The clearance of both FSH and LH from plasma after infusion was significantly prolonged than after a bolus injection. It is concluded that the difference in circulating half-lives of FSH between pituitary extracts from intact and OVEX ewes after bolus administration is due to a difference in pituitary FSH composition. However, the prolonged clearance with infusion compared to bolus administration suggests that extrapituitary factors are also responsible.

Analysis of the ratio of biological to immunological LH secreted during the oestrogen-induced LH surge in the ewe.

Plasma concentrations of in-vitro biological and immunological LH were measured throughout the LH surge in cyclic ewes and in ovariectomized ewes treated i.m. with oestradiol benzoate. Both activities increased in parallel during the LH surge in both groups, although the ratio of biological to immunological activities (B/I ratio) was highest at the peak of the LH surge. The two activities were highly correlated (r = 0.86-0.92), with similar slopes from their regression analysis for the cyclic and ovariectomized groups (1.15 and 1.16 respectively). However, the intercepts of the regression lines did not pass through the origin, but intersected the y (radioimmunoassay) axis, suggesting that these serum samples contained immunoactivity not associated with LH bioactivity. In conclusion, an increase in the LH B/I ratio was observed during the LH surge in oestrogen-treated ovariectomized ewes and in cyclic ewes. This increase was not attributable to a change in the relationship between these two LH activities during the LH surge, but rather to the detection of bioinactive immunoactive material in plasma of unknown composition.

Identification of inhibin and inhibin-related proteins in human follicular fluid.

Inhibin-related proteins were identified in human follicular fluid following fractionation by gel permeation chromatography under neutral and acidic conditions, reversed-phase high performance liquid chromatography (HPLC) and preparative polyacrylamide gel electrophoresis. A number of molecular mass forms of inhibin (30-36 and 59-66 kDa) based on their in vitro biological and immunological activities were identified, of which 59-66 kDa inhibin was the predominant form. Bioactive fractions devoid of inhibin immunoactivity were also identified with molecular masses of 46 and 55 kDa. Based on their retention positions on reversed-phase HPLC and their lack of inhibin immunoactivity, these proteins are likely to be follicle stimulating hormone (FSH) suppressing proteins/follistatins previously identified in bovine and porcine follicular fluids. In addition, an immunoactive inhibin fraction devoid of bioactivity was identified in large amounts in the 50-70 kDa region of the gel permeation chromatogram at neutral pH. This material, based on previous findings with the fractionation of bovine follicular fluid, is likely to be the alpha-inhibin subunit precursor protein. No FSH stimulating activity (activin) was identified in any of the chromatograms, suggesting that the levels of activin in human follicular fluid are low. In conclusion, inhibins of molecular mass 30, 36, 59 and 66 kDa have been identified in human follicular fluid. Proteins with inhibin bioactivity devoid of immunoactivity and vice versa have also been detected and these proteins are probably FSH suppressing protein and an alpha-inhibin subunit precursor protein. Activin could not be identified.

Isolation of inhibin alpha-subunit precursor proteins from bovine follicular fluid.

Two proteins with structural characteristics similar to peptide sequences identified in the inhibin alpha-subunit precursor sequence have been isolated from bovine follicular fluid. A side-fraction from the purification of bovine follicular fluid inhibin with high levels of inhibin immunoactivity relative to its inhibin bioactivity was fractionated through a sequence of procedures which included triazine dye affinity and phenyl-Sepharose chromatography, gel permeation chromatography on Sephadex G-100, reverse phase HPLC, and preparative polyacrylamide gel electrophoresis. The first of the two proteins identified had a molecular mass of 25-26K under reducing and nonreducing conditions and a NH2-terminal sequence identical to that of 43K inhibin alpha-subunit and showed minimal activity (less than 2% activity) compared with bovine 31K inhibin in either the inhibin in vitro bioassay or the RIA. These data suggest that this protein is the alpha 1-166 sequence of the bovine inhibin alpha-subunit (designated alpha N-subunit), most likely released after processing of either the inhibin alpha-subunit precursor or the 43K alpha-subunit involved in the conversion of 58K to 31K inhibin. The other protein identified (designated pro-alpha C-subunit) has a molecular mass of 27K under nonreducing conditions and 20K and 6K under reducing conditions. It is inactive in the in vitro bioassay, although highly reactive in the inhibin RIA, and has NH2-termini identical to the pro sequence of the inhibin alpha-subunit precursor and the 20K alpha-subunit sequence. These results suggest that pro-alpha C is a disulfide-linked structure and may represent an intermediate in the dimerisation of alpha- and beta-subunits to form inhibin while the alpha N-subunit is probably a proteolytic product of either the alpha-subunit precursor or 58K inhibin.

Isolation of a 31 kDa form of inhibin from bovine follicular fluid.

The introduction of a pH 4.75 precipitation step to a previously described purification procedure from bovine follicular fluid (bFF) resulted in the isolation of a 31 kDa form of inhibin, in addition to 58 kDa inhibin. The procedure was monitored by an in vitro bioassay based on the suppression of the FSH cell content by pituitary cells in culture. The 31 kDa form was purified 5550-fold with approximately 5% recovery. On SDS-polyacrylamide gel electrophoresis a single band was detected with a molecular weight of 31 000 +/- 1500 (mean +/- SD) which upon reduction gave 2 subunits of 20 200 +/- 300 and 14 800 +/- 600. The biological activity expressed on mg protein basis was similar for both 31 kDa and 58 kDa inhibin although on a molar basis the 58 kDa inhibin was 2-3 times higher. A high degree of cross-reaction was observed between both forms in a radioimmunoassay of bovine inhibin using an antiserum raised against the larger form with either iodinated 31 kDa or 58 kDa inhibin as tracer. Based on the subunit composition of the 31 kDa and 58 kDa inhibin, their similar cross-reaction in a radioimmunoassay system and the apparent generation of the 31 kDa inhibin following a pH precipitation step, it is concluded that 31 kDa inhibin is a smaller form of the 58 kDa inhibin resulting from a shortening of the 43 kDa subunit to a 20 kDa subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrofocusing fractionation of follicle stimulating hormone in pituitary cell culture extracts from male and female rats.

Three-day pituitary cell cultures from adult male and female rats were incubated for 4 h in the presence of 10 nM LHRH and the molecular heterogeneity of FSH was assessed in the media of LHRH-stimulated cells and in cell extracts from unstimulated cells using an electrofocusing technique. The pI distribution of FSH showed a high degree of similarity between cell media and cell extracts of each sex although differences were observed between sexes. Pituitary cell cultures from male rats were also incubated in the presence of 10(-8) M testosterone and 10(-8) M estradiol and the pI distribution of FSH from media after LHRH stimulation was determined. No significant differences in the pI profiles were observed. Incubation with charcoal-treated bovine follicular fluid (an inhibin source) resulted in a significant reduction in recovered FSH activity in the pH region 3.61-3.92 although this decrease did not markedly alter the pI profile of FSH. Close similarities were observed in the pI distribution of FSH of pituitary cell culture extracts and pituitary gland extracts from intact animals of both sexes, however, differences in pI distribution were noted in pituitary extracts in the male but not the female following gonadectomy. It is concluded that (1) stored FSH is released from the pituitary without major modification to its structure as assessed from its pI profile, (2) sex differences in the pI profile of FSH in pituitary extracts are retained in culture and following LHRH-stimulated release, (3) the pI distribution of FSH is not affected by testosterone or estradiol and only minimally by inhibin in short-term cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of inhibin from bovine follicular fluid.

Bovine follicular fluid was used as a source for the isolation of gonadal inhibin, the activity of which was monitored by the dose dependent suppression of the FSH content of cultured pituitary cells. The procedures presented result in over 3000-fold purification of the starting material and the purified inhibin has an apparent molecular weight of 56000. The purified inhibin can be dissociated under reducing conditions into two subunits with molecular weights of 44000 and 14000 daltons.

Pituitary gonadotrophins in Booroola and control Merino sheep.

Pituitary content of FSH and LH, using radioreceptor assay methods, was determined in control and Booroola Merino ewes on the 3rd day of the oestrous cycle and in adult rams slaughtered in winter. Significantly more pituitary FSH (as per gland or per g wet wt) was found in the Booroola than in the control ewe. No significant differences were found in LH content although the difference in FSH/LH ratio between Booroola and control ewes was significant (P less than 0.001). Pituitary FSH content was similar in the rams of the two genotypes. A good correspondence between FSH values by the radioreceptor assay and by radioimmunoassays using anti-ovine and anti-human serum was observed for the Booroola ewes. However, radioimmunological estimates of FSH activity were significantly higher than radioreceptor estimates in control ewes and in Booroola and control rams in which the pituitary FSH values were 5-6% of that of the ewe. This over-estimation is attributed to differences in specificity between methods. Fractionation of pituitary extracts by electrofocussing indicated a similar pI profile of FSH for ewes and rams of both genotypes. It is concluded that quantitative rather than qualitative differences in pituitary FSH occur in Booroola and control Merino ewes. It is suggested that increased FSH levels contribute to the hormonal basis for the increased ovulation rate of the Booroola Merino.

Electrofocusing fractionation and characterization of pituitary follicle-stimulating hormone from male and female rats.

Extracts of male and female rat pituitaries were fractionated by electrofocusing on sucrose density gradients and the pH distribution of FSH determined by an FSH in vitro bioassay, rFSH radioimmunoassay (RIA) and FSH radioreceptor assay (RRA) method. FSH for both sexes was located in the pH range 3-6. Significantly more activity, as measured by all three methods, was found in the male in the pH range 3.4-4.0 (P less than 0.01), while conversely significantly more activity was found in the female in the pH range 4.8-6.0 (P less than 0.05). Ratios of the levels of FSH assayed by the three methods showed significant (P less than 0.05) differences between sexes in both the original pituitary extracts and electrofocusing fractions in the pH region 4.4-6.0. The acidic fractions, when fractionated by gel filtration (Sephadex G100SF), were larger in apparent molecular size than the more alkaline fractions found in pituitary extracts from either sex. Binding of electrofocusing fractions from male pituitaries to concanavalin A showed significant (P less than 0.01) differences between fractions, suggesting that the various FSH fractions may differ in their carbohydrate structure. It is concluded that the differences in apparent size and charge of FSH from male and female rat pituitaries can be attributed to the combination in different proportions of the various FSH species. However, differences in the ratio of FSH activity (in vitro bioassay, RRA and RIA) for FSH from the same pI region (pH 4.4-6.0) between sexes suggests that structural differences may also exist.

Heterogeneity of rat pituitary gonadotropins on electrofocusing; differences between sexes and after castration.

Anterior pituitary extracts from intact and 4 week postcastration male and female rats were electrofocused in sucrose density gradients within the pH range 3.5-10. Column fractions were combined to cover this pH range in 0.5 pH units and assayed for LH by in vitro bioassay and RIA and for FSH by radioreceptor assay and RIA. The pH distribution of bioactive LH was altered after castration in both sexes, with the proportion of recovered activity in the alkaline pH range increasing (P less than 0.01) from 52-57% in the intact animal to 71-73% after castration. In addition, significantly more bioactivity was recovered in the pH range 7-9.5 with the male (37%) than with either of the female (diestrous, 30% or proestrous, 28%) groups (P less than 0.05). FSH receptor binding activity was located in the pH region 3.5-6.5. Significantly less receptor binding activity was recovered in the pH range 3.5-4.5 with the female groups (39% and 37% diestrous and proestrous, respectively) than the male group (61%; P less than 0.05, P less than 0.01). The distribution of immunoreactive LH and FSH was similar to that observed with the LH in vitro bioassay and FSH radioreceptor assay. It is concluded that the charge distribution of pituitary gonadotropins is altered according to the sex of the animal and after castration. These findings provide further evidence that the type of gonadotropin produced by the pituitary is under endocrine control.