Engineering the Ovarian Hormones Inhibin A and Inhibin B to Enhance Synthesis and Activity.

Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing ß-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of ß-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin ß A-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this ß A-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin ß B-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration.

Use of detergent-based buffers allows detection of precursor inhibin forms in an immunoassay format.

Inhibin ELISAs are used in monitoring aspects of reproductive function, however these assays are based on the measurements of the mature 30kDa inhibin forms and not precursor forms. In conventional ELISA formats, the 105kDa unprocessed 'Pro-inhibin' forms are immunologically inactive, but the immunoactivity can be recovered in the presence of detergents. The immunoactivity of Pro-inhibin forms was assessed in the presence of a range of detergents utilising antibodies to the α-, ßA- and ßB-subunits of inhibin. In contrast to mature forms, unprocessed inhibin forms showed a 10-40 fold increase in inhibin A and total inhibin immunoactivities under optimised detergent (0.5% SDS/2% Triton X-100) conditions. The suppressed immunoactivity of the Pro-inhibin forms in these immunoassays was attributed to steric hindrance by the respective ßA- and α-subunit prodomains. This study details a detergent-based immunoassay that allows detection of previously undetectable precursor inhibin forms.

Inhibin A and B in vitro bioactivities are modified by their degree of glycosylation and their affinities to betaglycan.

Inhibin A and B, important regulators of normal function in tissues of the reproductive axis, are glycosylated at either Asn(268) or Asn(268) and Asn(302) in the alpha-subunit to produce 31- and 34-kDa isoforms, respectively. In this study, glycosylated isoforms of recombinant human inhibin A and B were purified from conditioned medium using immunoaffinity chromatography and reversed-phase HPLC. The masses of the purified inhibin preparations were determined by several inhibin immunoassays, and their in vitro bioactivities were based on suppression of FSH release by rat pituitary cells in culture. Based on a ratio of in vitro bioactivity to immunoactivity (B:I ratio), the monoglycosylated 31-kDa inhibin A was 5-fold more potent than the diglycosylated 34-kDa inhibin A (B:I ratio, 1.22 +/- 0.15 vs. 0.24 +/- 0.05; P < 0.001, respectively). The 31-kDa inhibin B was significantly (P < 0.001) more potent (1.75 +/- 0.29) than the 34-kDa form (1.08 +/- 0.20). Because inhibin biological activity is dependent upon interactions with the coreceptor betaglycan, the effect of inhibin glycosylation on betaglycan binding was assessed. Analogous to the pattern of in vitro bioactivity, 31-kDa inhibin A was 12-fold more active (IC(50), 0.68 nM) than the 34-kDa isoform (IC(50), 8.2 nM) at displacing [(125)I]inhibin A from COS7 cells expressing betaglycan. However, the 1.6-fold difference in bioactivity of the inhibin B isoforms was not matched by differences in their affinities for betaglycan. It is concluded that glycosylation of Asn(302) of the alpha-subunit of inhibin A and B results in a decrease in bioactivity, and the effect on inhibin A, at least, is explained by its reduced affinity to betaglycan.

Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG).

Activins are multifunctional growth factors belonging to the transforming growth factor-beta superfamily. Isolation of activins from natural sources requires many steps and only produces limited quantities. Even though recombinant preparations have been used in recent studies, purification of recombinant activins still requires multiple steps. To purify recombinant activin A, we have developed a simple method using the second follistatin domain of an activin-binding protein follistatin-related gene (FLRG). An affinity column was prepared with a partial FLRG fusion protein. The partial FLRG protein contained the second follistatin domain and the C-terminus acidic domain, and was tagged with six histidine residues at its N-terminus. The fusion protein was expressed in Escherichia coli and purified with nickel affinity column. Thereafter, the purified fusion protein was coupled to NHS-activated column. Recombinant activin A was produced in Chinese hamster ovary (CHO) cells, which were stably transfected with rat inhibin/activin betaA-subunit cDNA. After 48-h suspension culture of the cells in a serum free medium, the culture media was recovered and passed through the FLRG-coupled column. After washing with phosphate-buffered saline, bound protein was eluted out with an acidic buffer. Any significant contaminations were not detected when the purified protein was analyzed by SDS-PAGE. Apparent sizes of the protein were 14 and 28 kDa under the reduced and non-reduced conditions, respectively. Western blot analysis confirmed that the purified protein was activin A. The purified recombinant activin stimulated p3TP-lux reporter activity in CHO cells and follicle-stimulating hormone secretion from rat pituitary cells.

New approach to in situ quantification of ovarian gene expression in rat using a laser microdissection technique: relationship between follicle types and regulation of inhibin-alpha and cytochrome P450aromatase genes in the rat ovary.

The recently developed laser microdissection (LMD) technique makes it possible to quantify local gene expression in the target cells of various tissues. Using the LMD technique, this study aimed at comparing the amounts of mRNAs encoding the inhibin-alpha subunit and cytochrome P450 aromatase (P450(arom)) in granulosa cells between preantral and antral follicles in immature rat ovaries. Serial frozen sections of the ovaries from 24-day-old female Wistar rats were made and 30 healthy preantral (100-200 microm maximum diameter) and ten healthy antral ( > 300 microm maximum diameter) follicles were selected in each ovary based on morphological examinations, including immunohistochemistry for inhibin-alpha, in sections adjacent to those used for LMD. The amounts of mRNAs encoding inhibin-alpha subunit and P450(arom) were quantified by real-time polymerase chain reaction (PCR). While the amount of P450(arom) mRNA in the granulosa cell layers from the antral follicles was about 12-times higher than that in the preantral follicles, no difference in the amount of inhibin-alpha mRNA was found between these two types of follicles. Thus, the LMD technique allowed the in situ quantification of gene expression in the ovary and revealed that each granulosa cell expresses a stable amount of inhibin-alpha subunit mRNA independently of antral formation in immature rat ovaries.

Oocyte growth and follicular hierarchy may be locally controlled by an inhibin-like protein in the lizard Podarcis sicula.

The lizard Podarcis shows an ovarian annual cycle with three to four ovulatory waves between April and July (reproductive period). In August to September, a refractory stage occurs, followed by a nonreproductive period (October to March), during which the oocytes undergo slow growth and prepare themselves for vitellogenesis and ovulation. In the reproductive period, only a certain number of oocytes start growing, giving rise to a follicular hierarchy, which is controlled by still unknown mechanisms. In the present paper, immunoreactive inhibin was detected in previtellogenetic follicles of the reproductive period, and in particular, in the pyriform cells of the follicular epithelium. As the follicle grew and the pyriform cells disappeared, immunostaining shifted to the oocyte cytoplasm. The smaller follicles did not show any immunoreactivity. In the nonreproductive period, no follicles were labeled. We conclude that in the reproductive period, inhibin characterizes the follicles destined to ovulation and might be one of the main factors controlling follicular hierarchy.

Purification of activins from androgen-independent Shionogi carcinoma cells demonstrates enhanced expression of activin betaB-subunit under androgen-depleted cell conditions in vitro and in vivo.

Here, we report characterization of growth factors secreted from androgen-independent mouse mammary Shionogi carcinoma cells. Previous isolation of fibroblast growth factor 8 (FGF8) from androgen-dependent Shionogi carcinoma SC-3 cells prompted us to characterize growth factors secreted from the androgen-independent cells. After several purification procedures, mitogens for NIH3T3 cells from the androgen-independent cells were identified as activins on the grounds that activin betaA- and betaB-subunits are detected in the active fractions by Western blotting and that the growth-promoting effects by the active fractions are specifically inhibited in the presence of follistatin. In addition, exogenous activins, but not inhibin, stimulated the growth of NIH3T3 cells in a dose-dependent manner. Interestingly, transcripts of activin betaB-subunit were predominantly found in the androgen-independent cells while its betaA-subunit was universally expressed in both androgen-dependent and -independent Shionogi carcinoma cells. In concordant with this in vitro finding, transcripts of activin betaB-subunit were enhanced in murine prostates after castration. Therefore, expression of activin betaB-subunit, but not its betaA-subunit, is likely to be related with androgen-depleted cell conditions in prostates, and possibly in androgen-related cancers.

Developmental biology of amphibians after Hans Spemann in Germany.

After the Hans Spemann and Hilde Mangold discovery of the importance of the dorsal blastopore lip for axis formation in the early embryo (Nobelprize for Spemann, 1935), the scientific community tried in a goldrush-like manner to find the inducing factors responsible for the programming of early embyronic determination and differentiation. The slow progress towards a solution of this problem caused a fading of interest on behalf of most laboratories. This article describes the activities of a few laboratories in Finland, Japan and Germany, which continued their studies despite tremendous experimental difficulties. Finally only Heinz Tiedemann's group in Berlin was the first which could isolate a mesoderm/endoderm inducing factor in highly purified form, the so-called vegetalizing factor, now known as activin. Furthermore this article describes the identification of neuralizing factors like Chordin, Cerberus and Dickkopf in the zone of the Spemann-Mangold organizer. The finding that BMP-4 acts as an antagonist to these factors located on the dorsal side led to a new understanding of the mechanisms of action of inducing (neuralizing) factors and early embryonic pattern formation. Moreover, the observations that closely related genes and their products were also found in Drosophila, Zebrafish, Mice and Human were the basis for new concepts of evolutionary mechanisms (dorsal/ventral and anterior/posterior polarity or conserved processes in eye-development of all 7 animal phyla).

The N-myc oncogene in human neuroblastoma cells: down-regulation of an angiogenesis inhibitor identified as activin A.

Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that developmental and tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates an inhibitor of endothelial cell proliferation, identified by amino acid sequencing as being identical with activin A, a developmentally regulated protein. Down-regulation appears to involve interaction of the N-Myc protein with the activin A promoter. In addition, activin A inhibits both endothelial cell proliferation in vitro and angiogenesis in vivo, and it induces hemorrhage in vivo. We suggest that the N-myc-induced down-regulation of activin A could contribute to developmental and tumor angiogenesis.

Inhibitory activity in buffalo follicular fluid and its elution pattern during different season.

Two pools of whole buffalo follicular fluid collected in winter (December, January) and spring (March, April) season were fractionated by Sephadex G-200 column chromatography. Follicular fluid collected in winter and spring eluted into different pattern resulted into four and three peaks respectively. Both the pools of follicular fluid were salted out with 18.5% ammonium sulphate. The salted out fraction of winter and spring season follicular fluid was again subjected to sephadex column chromatography which eluted into two and single peak respectively. Whole follicular fluid (0.6 ml) was administered into ovariectomized mice to see its inhibitory effect. The percentage of compensatory ovarian hypertrophy was 16.3 +/- 4.4 which was significantly different (P < 0.01) as compared to control. 200 micrograms material from peak 1 and peak 2 obtained after fractionation of salted out winter follicular fluid also had inhibitory effect on compensatory ovarian hypertrophy as compared to control group. It was 35.6 +/- 9.3 and 15.9 +/- 4.3% respectively. Thus, the variation in nature of buffalo follicular fluid and its inhibitory effect in ovariectomised mice have significant relation with anoestrus condition in summer and humid months in this species.

Expression of different molecular mass forms of inhibin in atretic and nonatretic follicles during the early luteal phase and altrenogest-synchronized follicular phase in pigs.

This experiment characterized changes in amounts and proportions of different molecular forms of inhibin in porcine follicular fluid as related to stage of follicular development. Thirty-seven follicles (2-4 per pig) were dissected from 12 pigs during early luteal phase of the estrous cycle on Days 5, 6, and 7 of the estrous cycle, whereas 34 follicles (2-4 per pig) were dissected from 11 pigs on Days 1, 3, 5, and 7 of a follicular phase synchronized by altrenogest. Follicles were designated atretic if incidence of apoptotic granulosa cells was > or = 10% as determined by DNA fluorescence flow cytometry. Porcine follicular fluid was fractionated on 12% SDS-PAGE gels under non-reducing conditions and electroblotted to Immobilin P membranes. Inhibin forms were detected by immunoblot analysis using a mink anti-bovine inhibin alpha C1-26 gly.tyr antiserum and quantified. Immunoblots detected seven bands corresponding to inhibin forms of 44, 49, 58, 69, 121, 227, and > 227 kDa in > 91% of porcine follicular fluid samples. Three additional forms of 27, 29, and 32 kDa were detectable in only 52%, 64%, and 48% of samples, respectively. Forms > or = 69 kDa represented 83% of total inhibin immunoblot activity. The 121-kDa form was most abundant, with 39% of the total immunoblot activity in nonatretic follicles. The proportions of individual forms and total immunoblot activity pooled over days did not differ between early luteal and follicular phase follicles. Total inhibin immunoblot activity was 59% less in atretic than in nonatretic follicles. Amounts of the 44-, 49-, 69-, 121-, and 227-kDa forms were 50-80% lower (p < or = 0.05) in atretic than in nonatretic follicles. Total inhibin immunoblot activity in nonatretic follicles decreased (p < or = 0.05) by 60% during the early luteal phase but did not change significantly during the follicular phase. In nonatretic follicles, the 121-kDa form decreased (p < 0.05) during the early luteal and follicular phases. During the early luteal phase, amounts of the other forms did not change, whereas during the follicular phase the 44-kDa form increased (p < 0.05) 10-fold. In atretic follicles, neither amount nor proportion of inhibin forms differed among days. We conclude that follicular production and/or intracellular processing of inhibin dimer and/or inhibin alpha subunits changes during different phases of follicular development, supporting the notion of physiological roles for these peptides.

Measurement of dimeric inhibin in porcine serum: evidence for low concentrations and existence of binding proteins.

An immunoradiometric assay and serum extraction procedure were developed to measure dimeric inhibin in porcine serum with minimal interference by putative inhibin-binding proteins. Assay sensitivity was 50 pg/tube, and it incorporated antibodies against the N-terminal region of inhibin's alpha-subunit, alpha-(1-25)-Ab, and against the C-terminal region of inhibin's beta A-subunit. To determine whether inhibin-binding proteins were present in porcine serum, serum was incubated with [125I]-recombinant human (rh)-inhibin and then chromatographed by gel filtration. Radioiodinated rh-inhibin was associated with protein(s) > 600 kDa. Radioiodinated rh-inhibin also was incubated with alpha 2-macroglobulin, an inhibin-binding protein in human and rat serum. Elution profiles were similar for serum and alpha 2-macroglobulin. Serum- and alpha 2-macroglobulin-[125I]rh-inhibin complexes dissociated upon exposure to 8 M urea. Porcine serum was treated with urea, after which inhibin was isolated and concentrated. The recovery of rh-inhibin added to starting serum was 28%. Concentrations of endogenous dimeric inhibin were < 28 pg/ml in serum collected from sows at random stages of the estrous cycle and were < 21 pg/ml in serum collected from sows 2 d postweaning. Results demonstrate that 1) concentrations of dimeric inhibin are low in porcine serum, and 2) an inhibin-binding protein(s), consistent with alpha 2-macroglobulin, is present in porcine serum.

International collaborative study by in vitro bioassays and immunoassays of the First International Standard for Inhibin, Human Recombinant.

The First International Standard for Inhibin, Human Recombinant, (ISI), a lyophilized preparation of rDNA-derived human 32 kDa Inhibin A in ampoules coded 91/624, was evaluated by international collaborative study for its suitability to serve as an International Standard. This study, which involved 15 laboratories in nine countries, included a variety of in vitro bioassays and immunoassays. The ISI was compared with two other lyophilized preparations of human recombinant inhibin, the International Standard for Porcine inhibin (ISP) and preparations of human follicular fluid inhibin. Predicted loss of activity based on estimates of potency of contents of ampoules which had been stored under conditions of accelerated thermal degradation indicated that the ISI has satisfactory stability. On the basis of the results of this study, the ISI was deemed suitable to serve as a standard for in vitro bioassays and immunoassays and was established by the Expert Committee on Biological Standardization of the World Health Organization as the First International Standard for inhibin, recombinant human, with an assigned unitage of 150,000 International Units per ampoule. This unitage maintains an approximate continuity of units with the ISP.

Expression and localization of inhibin/activin subunits and activin receptors in MCF-7 cells, a human breast cancer cell line.

Inhibins and activins are members of the transforming growth factor-beta (TGF-beta) superfamily. Since TGF beta has been shown to be a potent proliferation-inhibiting agent for the breast cancer cell line MCF-7, we determined whether this cell line (a) transcribes messenger RNAs coding inhibin/activin alpha-, beta A-, and beta B-subunits and activin receptors, and (b) produces inhibin and/or activin proteins. Messenger RNAs for alpha- and beta-subunits of inhibin/activin and activin receptor II in MCF-7 cells were detected and localized using the reverse transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridization, respectively. The identity of the RT-PCR products was confirmed by DNA sequencing of PCR products. Immunocytochemically, inhibin and activin were localized in these cells. Our findings that messenger RNAs encoding inhibin alpha-subunit, inhibin/activin beta A-subunit, and activin receptor II were expressed, and inhibin/activin proteins were produced by MCF-7 cells, imply that these gonadal growth factors may have paracrine/autocrine functions in human breast cancer. Further, these observations suggest that these growth factors may be involved in regulating the growth and differentiation of human breast cancer cells.

Isolation of nine different biologically and immunologically active molecular variants of bovine follicular inhibin.

A combination of immunoaffinity chromatography, SDS-PAGE, and electroelution was used to simultaneously isolate 0.36-4.65 mg of nine different molecular forms of inhibin (pro alpha C-29 kDa; fully processed 34 kDa; and large inhibin forms 49, 53, 58, 77, 88, 110, and > 160 kDa) from 0.675 L of bovine follicular fluid (bFF). Each inhibin form, except pro alpha C, cross-reacted with inhibin alpha C 1-26-and beta A 82-114-subunit-directed antibodies during immunoblot analysis. Pro alpha C cross-reacted only with alpha-subunit antibodies. The inhibin forms consisted of 22-, 29-, 49-, or 58-kDa alpha subunits and 17- or 58-kDa beta subunits. During cultures of ovine pituitary cells, a 5-ng/ml dose of each inhibin form (except pro alpha C) suppressed basal accumulation of FSH 30% to 50% but increased GnRH-induced LH release 40% to 248%. The various inhibin forms cross-reacted in parallel fashion with standard curves generated during homologous and heterologous RIAs but with markedly different relative immunopotencies. In the RIAs, pro alpha cross-reacted 3- to 18-fold more than the fully processed inhibin form. The fully processed and the seven different large forms of inhibin cross-reacted with different relative immunopotencies in a two-site dimer-specific ELISA. We concluded that 1) a combination of immunoaffinity extraction, SDS-PAGE, and electroelution simultaneously isolated relatively large amounts of highly enriched preparations of nine different molecular forms of immunologically and biologically active inhibin from bFF; 2) eight different dimeric forms of bovine inhibin may regulate both basal FSH and GnRH-induced LH secretion by the pituitary gland, and 3) eight or nine different molecular forms of inhibin cross-react with different relative immunopotencies in the two-site dimer-specific assay or RIAs.

Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.

Precursor forms of the alpha-subunit of inhibin are abundant in human follicular fluid and possibly plasma, although their function is uncertain. We now describe the development of a new enzyme-linked immunosorbent assay to measure inhibin forms containing both the pro and alpha C regions of the alpha-subunit. The assay has a detection limit for purified human pro-alpha C of 0.5 pg/mL and less than 0.02% cross-reaction with recombinant forms of inhibin, activin, and follistatin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of follicular fluid extracts demonstrated that the assay is likely to detect pro-containing precursor forms of both the free alpha-subunit and intact dimeric inhibin. The serum concentration was measured in normal men (446 +/- 28 pg/mL), postmenopausal women (45.8 +/- 3.8 pg/mL), and women treated with FSH before in vitro fertilization (1827 pg/mL). Pooled human follicular fluid contained 488 ng/mL. The mean serum concentration in the female menstrual cycle rose from 150.6 +/- 26.1 pg/mL in the early follicular phase to 692.2 +/- 113 pg/mL in the midluteal phase. This assay offers a useful tool for investigation of the role of inhibin-related proteins in human reproduction. There may be particular clinical value under circumstances in which other assays for inhibin forms have insufficient sensitivity.

Extragonadal alpha-inhibin precursor proteins circulate in human male serum.

The role of inhibin as a negative feedback regulator of pituitary FSH secretion in men remains controversial inasmuch as serum inhibin and FSH levels are often not correlated, in part because of the known alpha-inhibin subunit cross-reactivity of the most widely used inhibin antiserum (Monash #1989). The objective of this study was to identify the nature of these alpha-inhibin proteins in male serum using antisera specific for the alpha-inhibin precursor. Three polyclonal antisera were raised against synthetic peptide fragments from the proregion (amino acids 21-35 = precursor alpha inhibin (PIN) 1 and 42-56 = PIN 2) and alpha-N segment (113-127 = PIN 3) of the human alpha-inhibin precursor protein. These antisera were then used in individual RIAs with the homologous peptide as both standard and radioligand. Because pure human alpha-inhibin subunit proteins are not available, recombinant alpha-inhibin medium and porcine follicular fluid were used as reference preparations in the PIN assays. All assays were specific for alpha-inhibin proteins, i.e. 1) they showed no significant cross-reactivity with other alpha-inhibin peptide fragments, dimeric 32-kDa inhibin, recombinant activin, FSH, human albumin, or the inhibin binding proteins alpha-2 macroglobulin and follistatin, and 2) they recognized native protein in alpha-inhibin precursor-containing porcine follicular fluid and in medium from a recombinant alpha-inhibin-only secreting cell line. Furthermore, immunoreactivity of all serum proteins seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with the PIN antisera was abolished or significantly reduced (40-100%) by preincubation of each PIN antiserum with the homologous peptide. Serial dilutions of serum from normal, GnRH-deficient, and castrate males exhibited equivalent displacement curves in each precursor (PIN) assay, and there were no significant group differences in PIN 2 immunoreactivity between normal (n = 14), GnRH-deficient (n = 8), and castrate men (n = 3). Western blotting of serum samples from a normal and a GnRH-deficient male revealed immunoreactive proteins of approximately 57 and 29 kDa under reducing conditions with all three PIN antisera. An additional 40-kDa protein was observed with the pro-alpha-inhibin antisera, PIN 1 and 2, and a protein of more than 97 kDa was seen with the PIN 2 antiserum.(ABSTRACT TRUNCATED AT 400 WORDS)