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Clearance of exogenous gonadotropins was studied in the lizard Anolis carolinensis in relation to the problem of variations in potency ratios between LH and FSH of different species of hormone. Studies with unlabeled bullfrog ( Rana catesbeiana ) gonadotropins revealed that the FSH had a much longer persistence time in the lizard than did LH; this was confirmed by direct immunological measurements of circulating hormone and by temporal profiles in the stimulation of gonadal androgen production in the lizard. Bullfrog FSH and LH labeled with 125 I had similar clearance rates; the clearance of ( 125 I) Rana LH was slower than that of the unlabeled preparation. In contrast, similar studies with unlabeled sea turtle ( Chelonia mydas ) hormones indicated that the LH is cleared more slowly than is FSH. These differences between the pairs of frog and turtle gonadotropins are consistent with the difference in LH/FSH potency ratios observed for the two species of hormone in the in vivo Anolis lizard testes weight bioassay. Thus, these data provide additional insights into the variability in effects of different species of gonadotropins: the relatively high potency of some species of LH may be related in part to increased half-lives.

SUMMARY The actions of human follicle-stimulating hormone (hFSH) and its β-subunit were examined in several assays in reptiles, including effects on lizard testicular activity (growth and androgen production) in vivo, and stimulation of androgen production by snake testes and competition for binding of 125I-labelled hFSH in lizards and snakes in vitro. Binding was also examined with mammalian tissues. The hFSH was highly steroidogenic in the snake and lizard; otherwise results were similar to those observed in mammals. In all cases, the potency of the β-subunit was only a few per cent of the intact hormone. The potency of hFSH in vivo compared with NIH-FSH ovine standards was several 100 times greater than in vitro. Results for stimulation of androgen production in vivo closely paralleled those for binding assays in both reptiles and mammals. In contrast to previous results for ovine FSH β-subunit, human FSH β-subunit has little if any FSH biological activity in reptiles.

Fractionation of pituitary glands of the ostrich (Struthio camelus) resulted in the preparation of highly purified growth hormone (GH), prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in relatively high yields; thyrotropin (TSH) was partially purified. Hormones were identified by a variety of biological and immunological tests. Biochemical and immunological characterizations were performed to study the purity of each preparation and to allow comparison with other species of hormones, with special emphasis on other avian hormones. Similarities to the corresponding hormones of other tetrapods and even fish are apparent in the various biochemical features. THe ostrich hormones show closer affinities to other avian hormones than to mammalian or reptilian species in some respects (especially immunological), but notable biochemical differences are also evident among the few avian species examined.

An antiserum generated against ovine follicle-stimulating hormone (FSH) was used to examine phylogenetic relatedness of FSH molecules among the four classes of tetrapods. Heterologous radioimmunoassay (RIA) employing this antiserum with 125I-labeled human FSH as tracer revealed a high degree of immunochemical similarity among purified FSH molecules from diverse eutherian, metatherian, avian, and reptilian species. While the eutherian hormones tended to be slightly more potent than those from other amniotes, at least one avian FSH (ostrich) exhibited an equivalent degree of antigenicity. Hormones from amphibians formed a discrete group with lower cross-reactivity. In all of these species, only the gonadotropin previously identified as an FSH on the basis of physicochemical and biological profiles showed appreciable cross-reactivity; LH preparations were essentially inactive. 125I-Labeled FSH from a bird (turkey), reptile (sea turtle), and amphibian (bullfrog) bound specifically to the anti-ovine FSH serum, and RIAs with these as tracers yielded essentially the same results as outlined above. Immunoneutralization tests employing binding of 125I-labeled hormone to gonadal receptors confirmed that the anti-ovine FSH serum cross-reacted with the biologically active form of the FSH from the human, bird, and turtle. Moreover, the antiserum appeared to preferentially bind to that portion of the radiolabeled hormone that exhibited the greatest capacity to bind to tissues. The squamate reptiles (snakes and lizards) presented a striking exception to the above phylogenetic patterns, since no antigenic cross-reactivity could be detected with the pituitaries or purified gonadotropins from these reptiles.

Biological and binding activities of adenohypophysial hormones purified from the ostrich (ost), Struthio camelus, were compared to those of the corresponding hormones derived from mammalian and other avian species. The potency of ostrich prolactin was comparable to those of other avian preparations and slightly less active than the ovine hormone when tested in the pigeon crop-sac assay, but ostrich growth hormone (GH) was more potent than several other avian preparations and was comparable to mammalian GH in the rat tibia bioassays. Marked discrepancies were evident in the activities of both ostrich gonadotropins (ostGn) when they were tested in a variety of in vivo and in vitro bioassays and radioreceptor assays (RRAs). Both the ostrich follicle-stimulating hormone (ostFSH) and luteinizing hormone (ostLH) were among the most potent tested thus far in in vivo bioassays for total gonadotropin in a lizard and a cockerel; a high sialic acid content may account for these high potencies. OstFSH was also the most potent nonmammalian Gn tested in two FSH specific mammalian bioassays, an in vivo (ovarian augmentation) and an in vitro (cAMP production) rat bioassay; in fact, ostFSH behaved more like a mammalian than an avian hormone in these assays. However, the binding activity of ostFSH was not unlike that of other avian FSH preparations when tested in either mammalian or nonmammalian FSH-RRA systems. OstLH was more potent than other avian preparations in an in vitro mammalian bioassay, but not in avian or amphibian LH bioassays; species specificity was pronounced among these LH assays. Binding activities of ostLH in mammalian and avian LH-RRAs were generally consistent with potencies in the two species of bioassays. However, a marked discrepancy was apparent in the behavior of ostLH in FSH-RRAs. Although ostLH had very low FSH activity when tested by radioimmunoassay, by bioassay, or by FSH-RRA with avian gonads, it was equipotent to ostFSH in competing for FSH-binding sites on the mammalian gonad; in this respect it was more like turkey than chicken LH. The ability of ostLH to antagonize the biological activities of ostFSH in the stimulation of cAMP by rat seminiferous tubules confirms that ostLH binds to the same functional receptors as FSH on the rat testis, even though it does not induce the characteristic physiological response associated with FSH.

The object of this study was to examine the effect of porcine follicular fluid (PFF) alone or in combination with estradiol (E2) on pulsatile LH release during the interval between the mornings of diestrus 2 (D2) and proestrus in the rat. Steroids were removed from PFF by charcoal extraction. Preliminary studies indicated that 1 ml PFF given intraperitoneally suppressed FSH secretion for up to 15 h, with an onset of action between 3 and 4 h and maximal suppression between 6 and 9 h. In subsequent experiments, six groups of animals were bled continuously for 3 h between 07.30 and 10.30 h at a rate of 50 microliter whole blood/5 min: group 1 was bled on D2; group 2 was sham ovariectomized on D2 (08.30-09.30 h), immediately implanted with an empty capsule, given saline at 12.00 and 24.00 h, and bled on proestrous AM; groups 3-6 were ovariectomized on D2, implanted with an empty or E2 capsule, given 1 ml saline or PFF at 12.00 and 24.00 h, and bled 24 h following ovariectomy (OVX). Between D2 and proestrus plasma E2 levels increased, and there was no change in any parameter of pulsatile LH release. However, OVX on D2 reduced plasma E2 levels and increased mean blood LH levels above proestrous values due to increases in LH pulse amplitude and frequency. Restoration of physiological proestrous levels of E2 reduced the increase in mean blood LH levels, by lowering pulse frequency to proestrous values and by greatly reducing pulse amplitude. However, LH pulse amplitude and mean blood LH levels were still higher than values on proestrus. PFF alone produced no alteration in any parameter of pulsatile LH release compared with saline-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Conventional fractionation procedures used for separation and purification of pituitary FSH and LH in diverse tetrapods were employed to study pituitary gonadotropin (Gn) from five genera of snakes representing three families: Elaphe and Ptyas—Colubridae; Naja and Bungarus—Elapidae; Crotalus—Viperidae. The hormones purified from each of these snakes were shown to be relatively potent in a variety of Gn bioassays and radioreceptor assays (RRAs) in snakes but there was no clear evidence for two separate types of Gn molecule. These snake materials were also active in RRAs and bioassays (testis growth, androgen production, and ovulation) in lizards, but poor dose-response characteristics and relatively low potencies were observed in most cases. An even higher degree of species specificity was evident when snake hormones were tested in nonsquamate species, including other reptiles. Most snake Gn fractions were essentially inactive in all RRAs and bioassays employing amphibians, birds, mammals, and turtles; these assays included several that typically show broad species cross-reactivity (e.g., anuran ovulation and spermiation, 32P uptake by chick testis, and in vitro androgen production in birds and turtles). Radiolabeled Gn from a snake (Naja) bound specifically only to gonadal receptors from snakes and lizards. Biochemical analyses of the snake Gn confirmed their glycoprotein nature but failed to show clear structural homology to either FSH or LH. Chromatographically, the purified Gn from Naja naja tended to behave predominantly like an LH and its electrophoretic mobility on polyacrylamide gels also suggested an LH-like molecule, but several problems complicated interpretation of these results. Amino acid composition of this snake Gn revealed similarities to both FSH and LH, but it was not consistently like either. Radioimmunological studies (RIAs) with several heterologous gonadotropin antisera also failed to show a consistent relatedness between snake hormones and either FSH or LH; in fact, the snake Gns are exceptional among tetrapods in showing a lack of cross-reactivity in several FSH and LH-RIA systems. Antisera were raised against Gn from two species of snake. Antiserum to Naja Gn blocked the biological and binding activities of other species of snake Gn and it also selectively neutralized the activity of FSH (but not LH) from a turtle, alligator, and bird. However, in homologous RIA, the Naja Gn showed a slight cross-reaction only with another elapid species. In a heterologous RIA (using antiserum to Ptyas Gn and 125I-labeled Naja Gn), relatively high cross-reactivity was seen with Gn from several elapids and colubrids but not viperids or any non-snake species. Thus, the snake hormones show partial relatedness to one another but are generally distinct from FSH or LH; the only evidence of cross-reaction with heterologous (non-serpentine) gonadotropin suggests that they share common immunochemical determinants with reptilian FSH. Overall, the snake gonadotropins appear to be unique among tetrapod gonadotropins in terms of their biological cross-reactivity, biochemical composition, and immunochemical properties. Data suggest that snakes may only have a single gonadotropin that does not show a clear homology to either FSH or LH.

Follicle-stimulating hormone (FSH) from the ostrich, Struthio camelus, was dissociated by methods previously used to prepare subunits from mammalian gonadotropins. Two chemically dissimilar subunits were obtained from the ostrich FSH and these resembled the alpha- and beta-subunits of mammalian FSH in amino acid composition. The subunits were relatively inactive in radioimmunoassay, radioreceptorassay, and bioassay when tested alone, but significant activity was regenerated upon their recombination. Incubations of mixtures of one of the ostrich subunits with the opposing subunit of ovine FSH also resulted in a significant regeneration of binding and biological activity in FSH assays. These hybrid recombinants demonstrated that the species specificity of the FSH molecule is conferred entirely by the source of the beta-subunit; in fact, hybrids formed between ovine LH-alpha and either ovine or ostrich FSH-beta were more potent in FSH assays than were those containing even two homologous FSH subunits. In contrast, there was little regeneration of LH bioactivity when FSH subunits (from ostrich or sheep) were combined with the subunits of ovine LH, although some LH binding activity was obtained when the mixture contained either one of the LH subunits; i.e., even LH-alpha + FSH-beta showed significant enhancement of binding activity for LH receptors. Thus, the subunits of avian FSH show biochemical and functional homologies to those of mammalian FSH, but there is only limited interchangeability with ovine LH subunits.

A glycoprotein of neurohypophysial origin was found to have cofractionated with FSH prepared from pituitary glands of the green turtle, Chelonia mydas. Antiserum raised against this preparation contained high antibody titres and affinity for the neurohypophysial component and allowed development of a specific radioimmunoassay to monitor its purification and distribution in the brain. Immunocytochemistry revealed that the glycoprotein was concentrated in the pars nervosa and associated nerve tracts passing through the median eminence to the supraoptic and paraventricular nuclei; similar distributions were observed in turtles and rats. The antiserum to the turtle material bound radiolabelled rat vasopressin (VP)-neurophysin and precipitated precursors of this neurophysin, but it did not cross-react with rat oxytocin-neurophysin. An amino-terminal alanine was also consistent with the structure of rat VP-neurophysin, but the turtle molecule was larger than the corresponding rat molecule. Limited tryptic digests of the turtle glycoprotein contained two components, one of which bound to lysine VP. Both components contained carbohydrate, but only the one which bound to VP cross-reacted in a radioimmunoassay for rat VP-neurophysin. The apparent surge in plasma immuno-FSH at the time of oviposition previously described in the turtle probably represented release of a neurophysin-like 'carrier' molecule associated with secretion of the neurohypophysial hormone (e.g. arginine vasotocin; AVT) responsible for oviduct contractility. These data suggest that the neurohypophysial glycoprotein represents a partially processed AVT precursor and provide the first biochemical evidence of a mammalian-like biosynthetic pathway for neurohypophysial hormones in a non-mammalian species. J. Endocr. (1984) 103, 97–106

The ability of mammalian and chicken gonadotropin-releasing hormones (GnRH) and their agonistic analogs to stimulate in vivo gonadotropin release were tested in a frog (Rana catesbeiana), snake (Naja naja), and turtle (Sternotherus odoratus). In the frog, chicken and mammalian GnRH were equipotent in stimulating the release of FSH and LH. Attendant increases in plasma androgen and the occurrence of spermiation confirmed the release of biologically active gonadotropin. Neither of the GnRH preparations or their agonists produced significant changes in plasma hormones in either of the reptiles. In light of comparable data for the actions of these GnRH preparations in mammals and birds, it appears that species specificity in the response to different GnRHs does not correlate well with the nature of the homologous hypothalamic GnRH molecule.

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the turkey, Meleagris gallopavo, were used as radioligands to study the characteristics of gonadotropin binding sites in avian and reptilian gonads. Scatchard analysis indicated the presence of multiple binding sites for each radioligand in the turkey and chicken gonad; the association constants (Ka) for the high affinity, low capacity sites (109 to 1010 M−1) were similar to those observed in gonads of other species. Phylogenetic differences were evident in the ability of gonads to bind turkey and mammalian radioligands; e.g., radioiodinated turkey hormones showed little binding to the pig ovary and radioiodinated human FSH showed little binding to the turkey gonad. Radioreceptor assays (RRAs) revealed the presence of separate binding sites with differential specificity for the two types of gonadotropins in the avian gonad. In particular, when turkey hormones were studied in the homologous RRA with 125I-labeled turkey LH as radioligand, gonadotropin binding sites appeared to be highly specific for LH. With 125I-labeled turkey FSH as radioligand, binding sites showed a relatively high affinity for FSH; but gonadotropin specificity of these sites was incomplete since turkey LH continued to show considerable activity in this FSH-RRA. Comparative studies with heterologous hormones (from chicken, sea turtle, and sheep) and heterologous receptor preparations (from chicken, sea turtle, lizard, and pig) gave variable results, depending on the source of both the hormone and receptor. For example, the specificity of FSH binding sites in the chicken testis was less pronounced than in the homologous turkey system; in fact, turkey LH was more potent than FSH in the chicken FSH-RRA. Binding in the turtle ovary did not show a clear specificity for either gonadotropin with either radioligand. The implications of these results for physiological actions of gonadotropins and evolution in gonadotropin structure are discussed.

The biological and binding activities of pregnant mare serum gonadotrophin (PMSG) were compared with those of highly purified FSH and LH from the pituitary gland of the same species. Pregnant mare serum gonadotrophin showed activity in bioassays considered to be specific for both FSH (e.g. the Steelman–Pohley ovarian augmentation test and cyclic AMP production by rat seminiferous tubules) and LH (androgen production by rat Leydig cells), as well as activity in a variety of radioreceptor assay systems previously considered to be specific for one of the two types of gonadotrophin. The potency of PMSG was high compared with that of purified ovine FSH or LH standards in all assays but PMSG was considerably less active than equine FSH and LH in vitro. In radioreceptor assays employing rat, pig and horse tissues, the activity of PMSG was equivalent to only 1–5% of equine FSH in competing for FSH-binding sites and only 3–35 % of equine LH in competing for LH-binding sites. Pregnant mare serum gonadotrophin was least active in homologous binding assays with horse testis and equine LH as radioligand. In the rat Leydig cell bioassay, the activity of PMSG was only 2·0% that of equine LH. Furthermore, in some assays equine LH was found to resemble PMSG in exhibiting a high degree of FSH-like activity that could not be accounted for by cross-contamination. The FSH immunoactivity of equine LH was less than 0·5% that of equine FSH, but equine LH was up to 63% as potent as equine FSH in competition for FSH-binding sites and it was 20% as active in the Steelman–Pohley ovarian augmentation bioassay. Equine LH did not, however, show the expected activity in the cyclic AMP production bioassay. Thus, the FSH-binding sites and physiological receptors may not be identical. Overall, comparison of PMSG with pituitary gonadotrophins from homologous species shows that the apparent dual activity of PMSG may not be a unique feature of this pregnancy hormone since equine LH also exhibits some FSH activities. The chemical resemblance between PMSG and equine LH is noteworthy in this regard.

Radioiodinated follicle-stimulating hormone (FSH) from the green sea turtle, Chelonia mydas ( 125 I-labeled Ch FSH), was used as a radioligand to examine the specificity of gonadotropin binding in reptilian gonads. Highly purified human, ovine, and Chelonia FSH and LH (luteinizing hormone) were tested for their ability to compete with this radioligand for binding in gonadal homogenates from two turtles ( Trionyx spiniferus and Chelonia mydas ), a lizard ( Anolis carolinensis ), and a snake ( Thamnophis sirtalis ). Results were similar to those previously obtained when a human FSH was used as radioligand ( 125 I-labeled hFSH) in demonstrating a lack of specificity in reptilian FSH binding sites. In the turtles, human and ovine LH had little activity (0.3%), but Chelonia LH showed considerable activity compared to Chelonia FSH (25–30% in Chelonia and 220–350% in Trionyx ). In squamates, all three LH preparations showed relatively high activity in competing with radioiodinated FSH for binding. The similarity of results obtained for iodinated Chelonia and human FSH supports the view that LH and FSH may share some binding sites in reptilian gonads.