{'fraction_non_alphanumeric': 0.0354932866316404, 'fraction_numerical': 0.029032885775442694, 'mean_word_length': 5.106463878326996, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Identification and purification of inhibin and inhibin-related proteins

Within the past 5 years the existence of 'inhibin' has been proven conclusively. After many decades of uncertainty surrounding this putative non-steroidal mediator of gonadal negative feedback, inhibin has been purified to homogeneity, its physico-chemical and biological properties defined, the genes encoding its subunits cloned and sequenced and its physiological role(s) examined in detail. A useful working definition recently proposed by Burger (1988) states that "inhibin is a glycoprotein hormone consisting of two dissimilar disulphide-linked subunits termed aand 13which inhibits pituitary gonadotrophin production and/or secretion, preferentially that of FSH". The literature relating to the discovery, isolation and characterization of inhibin has been reviewed extensively in the past few years and the reader will find much additional information in comprehensive reviews by Findlay (1986), de Jong (1987, 1988), Ying (1988) and Lincoln et al. (1989).


Introduction
Within the past 5 years the existence of 'inhibin' has been proven conclusively. After many decades of uncertainty surrounding this putative non-steroidal mediator of gonadal negative feedback, inhibin has been purified to homogeneity, its physico-chemical and biological properties defined, the genes encoding its subunits cloned and sequenced and its physiological role(s) examined in detail. A useful working definition recently proposed by Burger (1988) states that "inhibin is a glycoprotein hormone consisting of two dissimilar disulphide-linked subunits termed a-and 13which inhibits pituitary gonadotrophin production and/or secretion, preferentially that of FSH". The literature relating to the discovery, isolation and characterization of inhibin has been reviewed extensively in the past few years and the reader will find much additional information in comprehensive reviews by Findlay (1986), de Jong (1987, 1988, Ying (1988) and Lincoln et al. (1989).

Identification of inhibin
Whilst the concept that the gonads secrete a non-steroidal factor capable of suppressing pituitary gonadotrophin secretion arose over sixty years ago (Mottram & Cramer, 1923;McCullagh, 1932), interest in inhibin remained at a relatively low level for many years. The advent of radioimmunoassays for the measurement of LH and FSH in the early 1970s prompted, amongst other things, renewed interest in the 'inhibin-concepe and it was soon established that various gonadally-derived adds of both male (e.g. rete testis fluid, testicular lymph, seminal plasma) and female (e.g. ovarian follicular fluid) origin contain demonstrable inhibin-like biological activity (ILA). These early observations prompted several research groups to attempt to purify the active principle from one or other of the sources identified. However, it was not until 1985 that this task was accomplished.

Purification of inhibin
It is worth considering, with the benefit of hindsight, why the isolation of inhibin presented such a formidable challenge. In the mid-1970s there was no generally accepted 'standard' bioassay procedure for the reliable detection of inhibin-like biological activity (ILA). Rather, the various groups attempting to purify inhibin utilized a range of in-vivo and in-vitro bioassay procedures, each purporting to detect ILA, yet each relying on a different set of assumptions as evidence of their validity (see Baker et al., 1981;de Jong, 1987). A gradual convergence of opinion as to which of these bioassays afforded the most effective method for the detection and quantitation of ILA (as then defined) led to the widespread adoption of the rat pituitary cell monolayer culture system in which ILA induces a selective fall in the basal release and/or cellular content of FSH. It is noteworthy that each of the groups which eventually succeeded in isolating gonadal inhibin used such an assay system for screening chromatographic fractions generated during their purification schemes. In view of the more recent identification in gonadal fluids of molecules other than inhibin which suppress (i.e. follistatin, see below) or stimulate (i.e. activin, see below) pituitary FSH secretion, these rat pituitary cell bioassays should no longer be regarded as specific for inhibin as was once assumed. A second level of complexity with the purification of inhibin arose due to selection by different research groups of a range of ILA-containing gonadal fluids (e.g. porcine, bovine or human FF, ovine rete testis fluid, ovine testicular lymph, seminal plasma) as their respective starting materials. Thirdly, investigators had to contend with the apparent presence in these gonadal fluids of multiple forms of ILA displaying different physico-chemical properties and therefore eluting as confusing multiple peaks and/or poorly resolved components during chromatography.
Much of this complexity is now readily explained by proteolytic processing of the high Mr precursor molecules for inhibin to generate biologically-active inhibin molecules differing in size and charge (Mason et al., 1985(Mason et al., , 1986Forage et al., 1986). Finally, inhibin is now recognized as a rather hydrophobic molecule which, unless measures are taken to prevent it, tends to associate not only with other proteins present in crude fluids/extracts but also with certain chromatographic stationary phases, thus giving rise to anomalous and often unpredictable behaviour during chromatography.
Whether gonadal and other body fluids contain 'carrier proteins' which selectively bind inhibin has not yet been established.
A crucial development came in the early 1980s with the recognition that ILA was associated with a remarkably stable molecule capable of regaining its full biological activity following denaturation using harsh dissociating solvents (e.g. containing 8 ts4-urea, 41,4-acetic acid or 1% sodium dodecyl sulphate; SDS). The use of such protein-denaturing solvents to inhibit protein-protein interactions during gel-permeation, ion-exchange and hydrophobic interaction chromatography and the introduction of reversed phase high performance liquid chromatography (RP-H PLC) and preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE) greatly facilitated the development of effective purification schemes and led directly to the successful isolation of inhibin in 1985 by four independent groups (Robertson et al., 1985;Miyamoto et al., 1985;Ling et al., 1985;Rivier et al., 1985).

Molecular characterization of inhibin
Initially, Robertson et al. (1985) isolated from bovine FF an Mr 56 000 form of inhibin which, upon reduction, dissociated into two subunits of Mr 44 000 (a44) and 14 000 (13).Other groups working with porcine FF (Miyamoto et al., 1985;Ling et al., 1985;Rivier et al., 1985) isolated an Mr 31-32 000 form(s) of inhibin which after reduction yielded a-and I3-subunits of Mr 18-20 000 and 14-15 000, respectively. Robertson et al. (1986) subsequently reported that inclusion of an acidic precipitation step in their purification scheme for bovine FF inhibin resulted in the generation of an Mr 31 000 form of inhibin which was successfully isolated along with the Mr 56 000 form. Fukuda et al. (1986), using a complex purification scheme identical to that used by their group to isolate pig FF inhibin (Miyamoto et al., 1985), and Knight et al. (1987), using a simplified purification scheme, also isolated an Mr 32 000 form of inhibin from bovine FF and confirmed that the molecule consisted of two disulphide-linked subunits similar in size to those reported for Mr 31-32 000 pig inhibin. An Mr 30 000 form of inhibin (Mr 21 000 a-subunit and Mr 16 000 13-subunit) was also isolated from ovine follicular fluid (Leversha et al., 1987). Ling et al. (1985) actually isolated two isoforms of Mr 32 000 inhibin from pig FF (termed inhibin A and inhibin B) which shared an identical a-subunit but had slightly different 13-subunits termed 13,and 13,respectively. It was subsequently established that both forms of inhibin 13-subunit are also expressed in human and rat ovary although the 13,form has not yet been detected in bovine ovary.
Amino-terminal sequence analysis of the isolated inhibin subunits (a, PA and Popermitted the synthesis of oligonucleotide probes which were then used to isolate from ovarian m RNA libraries, cDNA species coding for the aand 13-subunits of porcine (Mason et al., 1985) and bovine (Forage et al., 1986) inhibin. The cloning and sequencing of these cDNA molecules, accomplished less than 1 year after inhibin was first isolated, enabled the complete amino acid sequence to be predicted for the aand 0-subunits of porcine (Mason et al., 1985), bovine (Forage et al., 1986), human (Mason et al., 1986) and rat (Woodruff et al., 1987) inhibin. Whilst native gonadal inhibin has not yet been isolated from rat or man, Mason et al. (1987) have reported the expression in Chinese hamster ovary cells of recombinant DNA-derived human inhibin which was biologically active when tested in a rat pituitary cell bioassay.
Although the inhibin concept first arose from studies in the male, progress towards isolating and characterizing testicular inhibin lagged behind that for ovarian inhibin. However, Bardin et al. (1987) and Vaughan et al. (1989) have reported the isolation from ovine rete testis fluid of an Mr 32 000 form of inhibin which, upon reduction, dissociated into aand 0-subunits of Mr 21 000 and 14 000 respectively. Amino-terminal sequencing revealed only minor differences compared to the corresponding sequences for pig or bovine ovarian inhibin. Moreover, Bardin et al. (1987) cloned and characterized cDNA encoding the a-subunit of inhibin in human testis to reveal a nucleotide sequence identical to that for human ovarian inhibin. Antisera raised against synthetic fragments of ovarian inhibin have been used for immunolocalization studies in testis of several species (Bardin et al., 1987), providing further evidence that ovarian and testicular inhibins are highly homologous.
These molecular characterization studies revealed a striking degree of interspecies homology with respect to inhibin with approximately 85% homology in the primary structure of the a-subunit, -100% homology for the 13, subunit and > 95% for the 13, subunit. Moreover, a-, 13,and 13,subunits display similar distribution patterns for cysteine residues, suggesting that they are each derived from a common ancestral gene (Mason et al., 1986). It has also emerged that the 0-subunits of inhibin share considerable homology with a range of proteins implicated in cell differentiation and developmental processes including transforming growth factor 13(TGF13), Mfillerian-inhibiting substance (MIS), an erythroid differentiation factor and activin (see below), giving rise to the concept of the 'inhibin-related peptide family' (see Ying, 1988).
The mature, fully-processed forms of a-(Mr 18-20 000) and 0-(M, 14 000) subunits of inhibin constitute the carboxy-termini of much larger precursor molecules composed of about 360 and 420 amino acid residues, respectively ( Fig. 1). Each precursor molecule contains a number of potential proteolytic cleavage sites and the locations of these are consistent with the generation of the different Mr forms of inhibin and its subunits identified in gonadal fluids Miyamoto et al., 1986; and granulosa cell-conditioned culture media (Bicsak et al., 1988). For instance, Miyamoto et al. (1986) used the technique of immunoblotting to demonstrate the presence in bovine FF of Mr 65 000, 55 000 and 32 000 forms of inhibin dimer. These size differences were attributed to differential processing of the a-subunit precursor since, upon reduction, these disulphide-linked dimers yielded immunoreactive a-subunits of Mr 52 000, 42 000 and 20 000 together with an immunoreactive 0-subunit of constant size (Mr 14 000). These authors also noted the presence of higher Mr forms of inhibin (120 000, 108 000 and 88 000) which they proposed represented disulphide-linked trimers composed of two processed 13-subunit precursor sequences linked to an a-subunit of variable size. These different Mr forms of inhibin appear to possess ILA when eluted from SDS-PAGE gels and tested in an in-vitro inhibin bioassay  although the possibility exists that, for ILA to be expressed, further processing occurs in the serum-containing culture medium used for the inhibin bioassay, to yield the mature, fully-processed Mr 32 000 dimer. In this regard, McLachlan et al. (1986) showed that, in the presence of serum, Mr 55 000 bovine inhibin is converted to the Mr 31 000 form which is generally regarded to be the smallest and most predominant form of bioactive inhibin likely to reach the anterior pituitary in vivo.
The mature Mr 18-20 000 a-subunit of inhibin contains one (bovine, porcine) or two (human) potential N-glycosylation sites in contrast to the mature 0-subunit which lacks such sites (Mason et .) al., 1985Forage et al., 1986). This factor not only explains the 10-20% discrepancy between the predicted and observed Mr values for the a-subunit but also confirms earlier proposals, based on the behaviour of ILA during lectin affinity chromatography, that inhibin is a glycoprotein (see de Jong, 1988).  exploited the technique of immunoaffinity chromatography to isolate from bovine FF an abundant and hitherto unrecognized form of inhibin which was highly reactive with antisera raised against either Mr 32 000 inhibin or a synthetic a-subunit fragment but which lacked inhibin-like biological activity. Upon SDS-PAGE, this material had an apparent Mr of 25 000 under non-reducing conditions, and 21 000 under reducing conditions. During amino-terminal sequencing of the isolated material only one amino acid residue was identified at each cycle of Edman degradation and the sequence (1-14) revealed was identical to that reported by Forage et al. (1986) for the amino terminus of the Mr 20 000 a-subunit of bovine inhibin. The authors attributed the apparently greater Mr value shown by the non-reduced material to anomalous behaviour during SDS-PAGE and concluded that it was a native form of monomeric Mr 21 000 a-subunit. Independent reports from Sugino et al. (1989) and Robertson et al. (1989) confirmed the presence in bovine FF of 'free' a-subunit (Mr 25 000 under non-reducing conditions and 21 000 under reducing conditions) devoid of biological activity in inhibin bioassays. Contrary to the conclusion of  however, SDS-PAGE and microsequencing revealed that the isolated material was actually dimeric in nature comprising an Mr 21 000 a-subunit linked by a disulphide bond to the much smaller 'pro'-region (40 residue amino terminal fragment) of the 'parent' a-subunit precursor molecule. Sugino et al. (1989) and Robertson et al. (1989) reasoned that the generation of this molecular species (referred to by the latter group as the 'pro-ac' fragment) must entail the release by proteolytic excision of the intervening 167 residue fragment. This fragment (referred to as`aN') was successfully isolated from bovine FF by Robertson et al. (1989), confirming the accuracy of this deduction (see Fig. 2).

Monomeric forms of inhibin a-subunit
Such findings emphasize the complexity of potential post-translational processing of inhibin subunit precursor molecules which may generate multiple mono-and dimeric forms, perhaps subserving different biological roles, which have yet to be identified. Indeed, the recent report by Findlay et al. (1989b) that active immunization of ewes against the`aN' fragment impaired fertility suggests it may have a biological role quite distinct from that of inhibin af3 dimer.
Two forms of 'free' inhibin a-subunit (M, 25 000 and 44 000) have been identified in bovine utero-ovarian and jugular venous blood, indicating that these molecules are secreted into the peripheral circulation . Moreover, these inhibin forms probably represent the dominant immunoreactive species detectable in bovine plasma by currently available inhibin radioimmunoassays. Thus it is now recognized that antisera against synthetic a-subunit fragments  Fig. 2. Processing of inhibin a-subunit precursor to yield 'pro"ciC' and laN' fragments isolated from bovine follicular fluid. Solid triangles indicate potential cleavage sites and S indicates positions of cystein residues. (Redrawn from Robertson et al., 1989.) (e.g. Beard et al., 1990) as well as purified native dimeric inhibin (e.g. Hasegawa et al., 1988;Robertson et al., 1988Robertson et al., , 1989 cross-react extensively ( > 200%) with these more recently identified native forms of 'free' a-subunit. Clearly, the immunoreactivity of these different forms is strongly influenced by the molecular conformation since 'free' a-subunit obtained by reduction and alkylation of purified native inhibin dimer reportedly shows minimal cross-reaction with the same antisera to native inhibin (Robertson et al., 1988). Attempts in this laboratory to obtain physiologically relevant measurements of circulating concentrations of inhibin in cattle using radioimmunoassay have been hindered not only by the abundant presence of 'free' inhibin a-subunit in plasma  but by the surprising finding that this material persists in the circulation of chronically ovariectomized heifers (see Figs 3 and 4) and steers. Paradoxically plasma levels of immunoreactivity were similar in gonadectomized and intact cattle, suggesting a major extra-gonadal source of this material (Fig. 3). Attempts to identify the source (J. H. M. Wrathall & P. G. Knight, unpublished observations) have so far been inconclusive although several extra-gonadal tissues of cattle were found to contain immunoassayable levels of inhibin (Fig. 5). Such findings accord with recent reports for other species that mRNA coding for a, and to a lesser extent, 13-subunits of inhibin is expressed in various extra-gonadal tissues including adrenal, pituitary, central nervous system and placenta (Crawford et al., 1987;Meunier et al., 1988). It should be possible to resolve these difficulties associated with the immunochemical measurement of circulating inhibin in cattle, and probably other species, by developing a 2-site immunoradiometric assay for inhibin in which the specific detection of the analyte (i.e. biologically active inhibin dimer) depends on the recognition of two different antibodies directed against an appropriate pair of epitopes, one on the mature a-subunit and the other on the mature 13-subunit (Fig. 6). Whilst excellent a-subunit specific antisera are already available, the generation of suitable 13-subunit specific antibodies, which must cross-react with native inhibin dimer, has so far been unsuccessful.

Effects of highly-purified inhibin on gonadotrophin secretion in vivo and in vitro
Whilst the recent availability of highly-purified inhibin has encouraged detailed in-vitro investigations into its actions on cultured rat pituitary cells, the prospect of undertaking similarly detailed 'whole animal' studies is hindered by the strictly limited amounts of inhibin currently available. However, three in-vivo studies, involving intact rats , ovariectomized ewes  and ovariectomized heifers (Beard et al., 1990) have now confirmed that highly-purified inhibin preparations do indeed suppress plasma FSH concentrations with little or no effect on plasma LH.
Initial claims that the effect of highly purified inhibin on cultured rat gonadotrophs is confined to a specific reduction in FSH cell content and release Fukuda et al., 1986;Robertson et al., 1986) have now been revised following more recent demonstrations that both basal (Farnworth et al., 1988a; and GnRH-induced (Fukuda et al., 1987;Farnworth et al., 1988b;Campen & Vale, 1988;) LH release may also be suppressed, albeit to a lesser extent than FSH. The latter observation is in accordance with the observation by Wang et al. (1988) that exposure of rat pituitary cells to inhibin reduces the number of GnRH receptors.
Whilst it would be tempting to extrapolate these in-vitro findings for the rat to other species, including ruminants, we have shown (Muttukrishna & Knight, 1990) that exposure of cultured sheep pituitary cells to highly-purified bovine inhibin actually enhances GnRH-induced LH release without affecting basal LH release; basal and GnRH-induced FSH release were suppressed by inhibin, in a manner similar to that observed in rats (Fig. 7). There are already indications, therefore, that the actions of inhibin and related proteins on the ruminant pituitary may differ from those identified for the rat, a view reinforced by our subsequent observation (see Fig. 8) that activin stimulates basal LH release but suppresses GnRH-induced LH release from ovine pituitary cells in vitro.

Activin Identification and purification of activin
Whilst developing procedures to isolate inhibin from pig FF two independent groups (Ling et al., 1986;Vale et al., 1986) observed that certain fractions obtained during gel permeation chromatography and HPLC appeared to stimulate, rather than suppress, FSH secretion in vitro. Both groups went on to isolate and characterize the active substance(s), revealing them to be disulphide-linked dimeric proteins actually composed of two inhibin I3-subunits (either a BAN heterodimer, termed 'activin' or a BAN homodimer now termed 'activin-A'). Activin-A was subsequently isolated from bovine FF (Hutchinson et al., 1987). It also emerged that an erythroid differentiation factor isolated from a human leukaemia cell line is, in fact, identical to activin-A (Eto et al., 1987).

Biological properties
In terms of their biological activity activin and activin-A are approximately equipotent, stimulating basal FSH secretion by cultured rat pituitary cells (ED50 1 ng/ml) without affecting the secretion of LH, TSH or prolactin. This effect of activin on pituitary FSH secretion is antagonized by inhibin. Schwall et al. (1989) recently reported the production of recombinant human activin-A which stimulates FSH secretion by rat pituitary tissue in vitro and in vivo without affecting LH release. Regarding the question of whether activin affects gonadotrophin secretion in ruminants, we have shown that, as in rats, recombinant human activin-A stimulates both basal and GnRH-induced secretion of FSH by sheep pituitary cells in vitro (S. Muttukrishna & P. G. Knight, unpublished observations). However, whilst activin-A also stimulated basal LH release, we were surprised to find that it actually suppressed GnRH-induced LH release, a novel action at variance with reports for the rat (Fig. 8). In view of the functional antagonism between inhibin and activin, this observation adds credence to our earlier finding reported above that inhibin enhances GnRHinduced LH release by sheep pituitary cells in vitro (see Fig. 7). These observations highlight the need for further comparative investigations in species other than the rat, which has, to date, been used almost exclusively for in-vitro testing of inhibin and inhibin-related proteins. It is not yet known for any species whether activin of ovarian origin circulates as a hormone to modify pituitary gonadotrophin secretion. There is, however, ample evidence, at least for the rat, that it acts at the intraovarian level to modulate granulosa cell function (Ying, 1988;la Polt et al., 1989).

Follistatin
Identification and purification Ueno et al. (1987) noted that a further zone of ILA, distinct from the inhibins, was resolved by the first reversed-phase HPLC fractionation step of their inhibin-purification scheme. After three more HPLC steps they isolated a monomeric Mr 35 000 protein which was found to share no homology with either aor P-subunits of inhibin and which they termed follistatin. A less abundant Mr 32 000 molecular variant of follistatin was also isolated from the same source and found to have an amino terminal sequence identical to the Mr 35 000 molecule. Molecular cloning techniques allowed the complete primary structure of the pig follistatin precursor to be deduced (Esch et al., 1987) and the pig cDNA probes were subsequently used to characterize the human follistatin precursor, revealing only 6 amino acid substitutions between the two species (Shimasaki et al., 1988). Regarding the production of follistatin by the ruminant ovary, Robertson et al. (1987) isolated from bovine FF three closely-related molecules with FSH-suppressing activity which were each shown to have the same amino terminal sequence as porcine follistatin. Moreover, Leversha et al. (1987) reported the presence of a non-reducible M, 36 000 molecule with ILA (presumed to be follistatin) in ovine FF.

Biological properties
Porcine and bovine follistatins specifically suppress FSH production (release or cell content) by cultured rat pituitary cells in vitro but with a potency only 10-30% that of purified inhibin Ueno et al., 1987;Esch et al., 1987). It remains to be established whether follistatin exerts similar effects on pituitary gonadotrophs from ruminant species. Furthermore, it is not known whether this putative hormone reaches the peripheral circulation in amounts capable of influencing pituitary gonadotroph function in vivo although there are recent indications that it acts in tragonadally to modify ovarian steroid output (Findlay et al., 1989a). Attempts to measure circulating concentrations of follista tin must await the development of specific radioimmunological assays since, as mentioned above, conventional bioassay procedures, as well as lacking sensitivity, are unable to discriminate between inhibin and follistatin.