Fundulus pituitary extract (FPE) was fractionated by various biocompatible liquid chromatographic procedures in an attempt to purify the GtHs and characterize their biological activities. Chromatographic fractions were tested for their gonadotropic activities by using a homologous bioassay system which utilized intact F. heteroclitus ovarian follicles in vitro . Two indicators of gonadotropic activity were employed, one being the ability of the fractions to stimulate prematurational oocytes (1.2–1.4 mm in diameter), which are arrested at prophase I of meiosis, to resume the meiotic process by undergoing GVBD. The other indication of gonadotropic activity was the ability of the eluted fractions to stimulate the ovarian follicles to produce three reproductively important steroid hormones (DHP, T, and E2).
This homologous bioassay system has been extensively verified and shown to be sensitive and specific for F. heteroclitus GtHs . It thus avoided pitfalls that may have arisen using a heterologous bioassay system . Although a report has appeared that prolactin and growth hormone stimulate ovarian steroidogenesis when injected into F. heteroclitus , no biological activity was detected for either hormone using our in vitro bioassay system (data not shown). Another advantage of our homologous bioassay system was that many fractions could be assayed simultaneously with a large number of appropriate-sized follicles that can be pooled from several fish and randomized, thus eliminating most of the between-animal variation in the procedure. To further minimize the inherent variation in the responsiveness of ovarian follicles to gonadotropin and to assure the availability of large numbers of sensitive follicles and active pituitary glands throughout the year, we also developed a routine husbandry procedure to maintain a large population of reproductively healthy F. heteroclitus in the laboratory . With this husbandry procedure, we were able to proceed with the FPE fractionation and to carry out homologous bioassays throughout the year.
Chromatographic resolution of gonadotropic activity
Size-exclusion chromatography (SEC) produced several UV-absorbing peaks, none of which correlated precisely with biological activity found around 30 kDa (Fig. 1). Active fractions induced oocyte maturation in high frequency and stimulated the production of all three steroids tested (DHP, T, and E2), with estradiol being predominant (as was the case for unfractionated FPE). The size of F. heteroclitus GtHs thus approximates previous lower estimates (ranging in molecular weight from 25 to 62 kDa) made by SEC for fully glycosylated, nondenatured teleost GtHs [5, 7, 30, 33–36]. Recently, a molecular weight of about 40 kDa (gel filtration) was reported for F. heteroclitus (Arasaki strain, Japan) GtH I and II . The large variation in these molecular weights may be population-specific or due to the presence of aggregates or to differences in the methods used for their estimation.
After anion-exchange chromatography (AEC) (Fig. 2A), maturational activity was found to be spread throughout the chromatogram (Fig. 2B). Similarly, steroidogenic activities for all three steroids tested were distributed among the various eluted fractions, although very distinct peaks of E2- and DHP-activity were discerned at NaCl concentrations of 0.15 and 0.24 M, respectively (Fig. 2C). Hence at least two different GtHs, one with predominantly E2-producing activity and the other with relatively high DHP-producing activity, could be distinguished by the steroidogenic bioassay data. However, the appearance of all types of gonadotropic activity throughout the AEC profile implies the presence of charge-heterogeneity in the F. heteroclitus GtHs. Such heterogeneity has previously been well documented for a variety of teleost species [37–39] as well as other vertebrates [40–45]. Charge-heterogeneity in GtHs has been previously explained by differences in sialic acid content  or by post-translational modifications of amino acid residues , but the precise differences between various isohormones are not well understood. In most cases, however, electrostatically distinct GtHs of the same type (i.e., FSH-like or LH-like) have been considered to be qualitatively identical in biological action.
Gonadotropic fractions relatively rich in either E2- or DHP-promoting activity were also discerned either by hydrophobic-interaction (HIC) (eluting at -0.31 and -0.17 M (NH4)2SO4, respectively; Fig. 3C) or chromatofocusing followed by HIC (in order to remove Polybuffer). Fractions relatively enriched in E2- and DHP-promoting activity were found to elute from the chromatofocusing column at pH 4.5 and after the salt wash, respectively, and these activities subsequently eluted from the Phenyl-5-PW column at (NH4)2SO4 concentrations of -0.34 M and -0.12 M, respectively (Figs. 4, 5; Table 1). Thus a consistent elution pattern by HIC is indicated. The need to elute DHP-promoting activity from the chromatofocusing column with a salt wash is also consistent with its relatively late elution during AEC (Fig. 2C).
Based on previous reports that employed similar procedures, it would appear that the gonadotropic activity purified by Swanson et al.  from coho salmon pituitaries primarily corresponds to those fractions described here that lack preferential steroidogenic activity (i.e., Table 1, fractions I-1 and II-8), while the gonadotropin described by Copeland and Thomas  for Atlantic croaker is similar to our DHP-promoting gonadotropin (Table 1, fraction V-19). None of the fractions we have analyzed from any single chromatographic run have yielded single, silver-stained bands at the expected size on electrophoretic gels (data not shown). This may indicate that the amount of GtH protein present in the chromatographic fractions was below the detection level (<5 ng) of silver staining . On the other hand, the homologous oocyte maturation and steroid production assays used in this study are far more sensitive in discerning biological activities.
A series of articles have attested to the duality of the GtHs in teleosts [7–10, 12, 19, 48]. These studies provided ample biochemical and immunological evidence that there are two chemically distinct GtHs that reside in separate pituitary gonadotrops and have different ontogenies. Unfortunately, attempts to distinguish different steroidogenic activities between GtH I and GtH II have been less definitive because biological activities of GtHs overlap considerably. Although GtH II seems to be more potent than GtH I in stimulating DHP-production by salmon ovarian follicles, no significant difference in E2-steroidogenic activity has been found between GtH I and GtH II [7, 48–50]. As a corollary, therefore, GtH I appear to preferentially promote E2-production in salmonids. Since E2 and DHP are primarily involved in vitellogenic processes and the resumption of meiotic maturation, respectively, GtH I and GtH II would seem to correspond to the vitellogenesis-promoting "carbohydrate-poor" and maturation-promoting "carbohydrate-rich" GtHs described by Idler and his colleagues for salmon and flounder [51–54]. Based on this, our own results for F. heteroclitus appear to indicate that GtH I-like and GtH II-like activities reside in fractions III-10 and V-19, respectively, derived from chromatofocusing of FPE (Table 1). Similar activities were found in fractions obtained by AEC (Fig. 2C) and HIC (Fig. 3C).
The chromatographic separation of the GtHs evaluated by bioassay cannot directly discern which fraction is GtH I and GtH II, and even though there is an overlap in the steroidogenic activity, the homologous bioassay data clearly distinguish two well defined peaks of different biological activities. Additionally, our HIC profiles as determined by the specific biological activities (oocyte maturation and steroidogenesis) are in agreement with those reported by Shimizu and Yamashita  who identified the F. heteroclitus GtH I and GtH II using an immunochemical analysis. We conclude, therefore, that at least two gonadotropic activities are present in F. heteroclitus pituitaries: an E2-promoting gonadotropin (GtH I-like) and a DHP-promoting gonadotropin (GtH II-like) which has a lower isoelectric point but greater hydrophobicity than the former. Consistent with our previous findings that DHP is the maturation-inducing steroid in F. heteroclitus  the DHP-promoting gonadotropin is also associated with high maturation-inducing activity (Fig. 3). Taken together, the homologous bioassay data presented here, and our previous immunochemical study indicating that each of the two GtHs could be released selectively , help to substantiate the duality of GtH function in a teleost which is a multiple spawner.