Pituitary glycoprotein hormone beta subunits in the Australian lungfish and estimation of the relative evolution rate of these subunits within vertebrates

The beta subunits of the two pituitary gonadotropins LH and FSH and of thyroid-stimulating hormone (TSH) were cloned from Australian lungfish (Neoceratodus forsteri) pituitary glands. These three glycoprotein hormone beta subunits possess the main characteristics common to their counterparts in other vertebrates. Taking advantage of the phylogenetic position of the lungfish, close to the root of tetrapods, a maximum parsimony tree was inferred from these new sequences and sequences from representatives of the diversity of vertebrates. The topology of the tree was imposed so that it reflected as closely as possible the real evolutionary history of the subunits. This tree was used to estimate the relative evolution rate of the three subunits in vertebrates. Cumulated amino acid substitutions from the basal subunit node (ancestral subunit sequence) to the species node were calculated and compared. It showed that a burst in evolutionary rate occurred for the LHbeta subunit in the tetrapod lineage sometime after the emergence of amphibians. The rate of evolution of the LHbeta subunit was particularly high throughout the radiation of mammals while FSH and TSHbeta subunits kept quite stable in this lineage. A burst in evolutionary rate was also observed for the FSHbeta subunit in the lineage leading to teleosts sometime after the emergence of chondrosteans and the dynamic of evolution was high throughout the radiation of teleosts. These results were consistent with data obtained from pairwise comparisons.     

Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin

Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) from pituitary and chorionic gonadotropin (CG) from placenta are a family of closely related glycoproteins. Each hormone is a heterodimer, consisting of an alpha- and a beta-subunit. Within an animal species, the alpha-subunits of all four glyco-protein hormones have an identical amino acid sequence, whereas each beta-subunit is distinct and confers hormone-specific features to the heterodimer. LH and FSH are synthesized within the same cell, the gonadotroph of the anterior pituitary, but are predominantly stored in separate secretory granules. We have characterized the asparagine-linked oligosaccharides on bovine, ovine and human LH, FSH and TSH. The various pituitary hormones were found to contain unique sulfated oligosaccharides with the terminal sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, sialylated oligosaccharides with the terminal sequence SA alpha Gal beta GlcNAc beta Man alpha, or both sulfated and sialylated structures. Despite synthesis of LH and FSH in the same pituitary cell, sulfated oligosaccharides predominate on LH while sialylated oligosaccharides predominate on FSH for all three animal species. We have examined the reactions leading to synthesis of the sulfated oligosaccharides to determine which steps are hormone specific. The sulfotransferase is oligosaccharide specific, requiring only the sequence GalNAc beta 1----4GlcNAc beta 1----2Man alpha. In contrast, the GalNAc-transferase appears to be protein specific, accounting for the preferential addition of GalNAc to LH, TSH, and free (uncombined) alpha-subunits compared with FSH and other pituitary glycoproteins. The predominance of sulfated oligosaccharide structures on LH may account for sorting of LH and FSH into separate secretory granules. Differences in sulfation and sialylation of LH, FSH and TSH may also play a role in the regulation of hormone bioactivity.     

Gonadotropin receptor of a mouse luteoma: interactions with luteinizing hormone (LH) and its and subunits

A radioligand-receptor system for luteinizing hormone (LH), USING transplantable mouse luteoma, was used to investigate the interactions of LH, other peptide hormones, and LH subunits. Since tumor size decreased as did production of androgenic hormones following hypophysectomy, the luteoma is believed to have been dependent on pituitary tropic hormones; posthypophysectomy histologic changes supported this conclusion. An homogenate was prepared from 1-4 gm luteomas, which had been borne by mice for 4-10 months. Ovine LH, bovine LH, and human chorionic gonadotrophin reduced the binding of iodine-125 human luteinizing hormone (125-I-hLH). Growth hormone, adrenocorticotrophic hormone, and prolactin had no capacity to interfere with binding of 125-I-hLH. Though follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) reduced the binding somewhat, the reductions were consistent with the known presence of contaminating amounts of LH in the FSH and TSH. The accumulated results of a number of experiments suggest that binding to the luteoma LH receptor requires a particular polypeptide structural conformation, one found in the native hormone but found in neither alpha nor beta subunit alone.     

Immunohistochemical evidence that follicle-stimulating hormone and luteinizing hormone reside in separate cells in the chicken pituitary.

As is the case in other tetrapod species, the chicken gonadotropins LH and FSH consist of a common alpha subunit and a hormone-specific beta subunit. Gonadotrophs containing LH were shown earlier to be distributed throughout both the caudal and cephalic lobes of the chicken anterior pituitary, but the cellular distribution of FSH in avian species is still uncertain. The purpose of this study was to determine the cellular distribution of FSH-containing chicken gonadotrophs by use of FSH-specific monoclonal antibodies (mAbs). Three new mAbs toward chicken FSH were proven hormone specific by immunodetection of purified hormones on dot blots and by dual-label immunohistochemistry (IHC) on sagittal sections of chicken pituitaries. A rabbit antibody was used to detect chicken LH. Results showed that LH-containing gonadotrophs were densely distributed throughout the anterior pituitary, whereas gonadotrophs containing FSH were much less numerous; in addition, while also present in both lobes, FSH-positive cells were largely absent from the outer margin of the gland. Dual-label IHC revealed that LH and FSH reside almost exclusively in separate gonadotrophs. The identity of FSH-containing cells was further confirmed through use of an antibody to the chicken alpha subunit, which showed that FSH immunoreactivity was always colocalized with the alpha subunit. Our results suggest the possibility that production and secretion of LH and FSH may be regulated differently in chickens than in most other species studied to date.     

Effects of estrogen, pituitary gonadotropins and prolactin on immunohistochemical localization of inhibin subunits in the ovary of hypophysectomized female rats.

Effects of estrogen, pituitary gonadotropins and prolactin on immunohistochemical localization of alpha- and beta A-subunits in the ovaries of hypophysectomized female rats were investigated. Hypophysectomy resulted in disappearance of immunoreactive inhibin subunits in the ovary. Administration of DES, FSH and LH to hypophysectomized rats provoked growth of follicles, and resulted in positive immunostaining for inhibin subunits in the granulosa cells. In contrast to follicle-stimulating hormone (FSH) and luteinizing hormone (LH), prolactin administration failed to demonstrate positive staining for inhibin subunits in the ovary. The present in vivo results suggest that several hormones which are known to stimulate granulosa cell growth and maturation, such as estrogen, FSH and LH, enhance inhibin subunit production by the ovary. The morphologic aspect of inhibin subunit production by the ovary in response to several hormones has been demonstrated in the present study.     

Stimulatory effects of insulin-like growth factor 1 on expression of gonadotropin subunit genes and release of follicle-stimulating hormone and luteinizing hormone in masu salmon pituitary cells early in gametogenesis

Insulin-like growth factor-I (IGF-I) has been shown to be involved in pubertal activation of gonadotropin (GTH) secretion. The aim of this study was to determine if IGF-I directly stimulates synthesis and release of GTH at an early stage of gametogenesis. The effects of IGF-I on expression of genes encoding glycoprotein alpha (GPalpha), follicle-stimulating hormone (FSH) beta, and luteinizing hormone (LH) beta subunits and release of FSH and LH were examined using primary pituitary cells of masu salmon at three reproductive stages: early gametogenesis, maturing stage, and spawning. IGF-I alone or IGF-I + salmon GnRH (sGnRH) were added to the primary pituitary cell cultures. Amounts of GPalpha, FSHbeta, and LHbeta mRNAs were determined by real-time PCR. Plasma and medium levels of FSH and LH were determined by RIA. In males, IGF-I increased the amounts of all three subunit mRNAs early in gametogenesis in a dose-dependent manner, but not in the later stages. In females, IGF-I stimulated release of FSH and LH early in gametogenesis, whereas no stimulatory effects on the subunit mRNA levels were observed at any stage. IGF-I + sGnRH stimulated release of FSH and LH at all stages in both sexes, but had different effects on the subunit mRNA levels depending on subunit and stage. The present results suggest that IGF-I itself directly stimulates synthesis and release of GTH early in gametogenesis in masu salmon, possibly acting as a metabolic signal that triggers the onset of puberty.     

The role of O-linked and N-linked oligosaccharides on the structure-function of glycoprotein hormones: development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common alpha and a hormone specific beta subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the alpha and beta subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (alpha, FSHbeta and hCGbeta) asparagine-linked (N-linked) oligosaccharides. CGbeta subunit is distinguished among the beta subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure-function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

Effects of recombinant gonadotropin hormones on the expression of vitellogenin, gonadotropin subunits and gonadotropin receptors in cinnamon clownfish, Amphiprion melanopus

Gonadotropins (GTHs) are the key regulators of reproduction in vertebrates. The present study investigated autoregulatory effects of gonadotropins, using recombinant FSH (rFSH) and LH (rLH) in cinnamon clownfish (Amphiprion melanopus). Experiments were carried out to investigate the actions of cinnamon clownfish rFSH and rLH on expression of GTH subunits, GTH receptors, and vitellogenin (Vtg) mRNA in vivo and in vitro. Plasma estradiol-17β (E(2)) level was also measured in immature fish following treatments with rFSH and rLH. The results demonstrate increasing levels of GTH subunits, GTH-receptors, Vtg mRNA levels, as well as plasma E(2) levels following injection with rFSH and rLH. The findings support the hypothesis that LH and FSH stimulate reproduction, in part, by autoregulatory mechanisms leading to upregulation of GTH receptors and GTH hormone production in cinnamon clownfish. The results provide a framework for better understanding of the mechanisms of GTH-mediated control of reproduction in cinnamon clownfish and other vertebrates.     

Sequence analysis and expressional regulation of messenger RNAs encoding beta subunits of follicle-stimulating hormone and luteinizing hormone in the red-bellied newt, Cynops pyrrhogaster

Two distinct cDNAs encoding beta subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were cloned from the cDNA library constructed for the pituitary of the red-bellied newt, Cynops pyrrhogaster, and sequenced. The newt FSHbeta and LHbeta cDNAs encode polypeptides of 129 and 131 amino acids, including signal peptides of 20 and 19 amino acids, respectively. The number and position of cysteine and N-glycosylation in each of the beta subunits of FSH and LH, which are considered essential for assembly of the alpha subunit, are well conserved between the newt and other tetrapods. The high homology (41.6%) between the beta subunits of newt FSH and LH imply less specificity of FSH and LH in gonadal function. One cDNA encoding the common polypeptide chain alpha subunit of FSH and LH was also isolated from the newt pituitary gland. The mRNAs of FSHbeta, LHbeta, and the alpha subunit were expressed only in the pituitary gland among various newt tissues. Double-staining with in situ hybridization and immunohistochemistry revealed coexpression of FSHbeta and LHbeta in the same newt pituitary cells. Ovariectomy induced a significant increase in FSHbeta mRNA levels, but there was no significant change in LHbeta or alpha subunit mRNA levels compared with those in control animals. Taken together, these data suggest that two kinds of gonadotropins, namely FSH and LH, are expressed in the same gonadotropin-producing cells in the pars distalis of the newt as well as in other tetrapods and that the expression of FSHbeta is negatively regulated by the ovaries.     

Cloning, functional characterization, and expression of a gonadotropin receptor cDNA in the ovary and testis of amago salmon (Oncorhynchus rhodurus).

A gonadotropin receptor was cloned from amago salmon (Oncorhynchus rhodurus) ovarian follicles. This receptor (sGTH-R) belongs to the glycoprotein hormone receptor family with a large extracellular and seven-transmembrane domains. Its sequence homology is highest with mammalian LH receptors. Phylogenetic analysis reveals that sGTH-R is grouped with mammalian and chicken FSH and LH receptors, but not with mammalian TSH receptors. sGTH-R is expressed dominantly in the ovary and testis. Functional characterization examined with transiently transfected mammalian cells revealed increased intracellular cAMP level when exposed to mammalian and fish gonadotropins; the most potent hormone was salmon GTH II. These results indicate that the cloned cDNA encodes a functional amago salmon GTH receptor protein.     

Effects of starvation and refeeding on gonadotropin and thyrotropin subunit mRNAs in male Japanese quail

The contents of mRNAs encoding LH beta-, FSH beta-, TSH beta- and common alpha-subunit precursor molecules were measured in food-deprived and subsequently re-fed male Japanese quail. Pituitary LH beta, FSH beta and common alpha mRNA levels were decreased by starvation, and increased to the control levels by re-feeding. The rates of decreases of LH beta and common alpha mRNA levels were greater the corresponding rate for FSH beta levels. Pituitary TSH beta mRNA levels were not decreased by starvation, but increased transitorily by re-feeding. Plasma LH and triiodothyronine levels were decreased by starvation, and then increased to control levels by re-feeding, while plasma FSH and thyroxine levels did not show significant changes. Plasma LH and FSH levels showed positive correlations with pituitary common alpha and FSH beta mRNA levels, respectively, while plasma thyroxine levels showed a negative correlation with TSH beta mRNA levels. Hepatic weight was decreased slightly but significantly by starvation, and then showed a remarkable rebound after re-feeding was started. These results suggest that LH synthesis and secretion are more sensitive to starvation than FSH synthesis and secretion in Japanese quail, and that LH production recovered to initial levels within several days when birds were fully fed. Also, there is a possibility that the synthesis of TSH is accelerated transitorily by re-feeding. Furthermore, these results showed that there are different relationships between the plasma levels of LH, FSH, and TSH and the various hormone subunit mRNA levels. The remarkable change in hepatic weight leads us to assume that hepatic thyroid hormone metabolism is affected by starvation and re-feeding.     

Median eminence lesions reveal separate hypothalamic control of pulsatile follicle-stimulating hormone and luteinizing hormone release

The effects of hypothalamic lesions designed to destroy either the anterior median eminence (ME) or the posterior and mid-ME on pulsatile release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined in castrated male rats. In sham-operated animals, mean plasma FSH concentrations rose to peak at 10 min after the onset of sampling, whereas LH declined to a nadir during this time. In the final sample at 120 min, the mean FSH concentrations peaked as LH decreased to its minimal value. In rats with anterior ME lesions, there was suppression of LH pulses with continuing FSH pulses in 12 of 21 rats. On the other hand, in animals with posterior to mid-ME lesions, 3 out of 21 rats had elimination of FSH pulses, whereas LH pulses were maintained. Fifteen of 42 operated rats had complete ME lesions, and pulses of both hormones were abolished. The remaining 12 rats had partial ME lesions that produced a partial block of the release of both hormones. The results support the concept of separate hypothalamic control of FSH and LH release with the axons of the putative FSH-releasing factor (FSHRF) neuronal system terminating primarily in the mid- to caudal ME, whereas those of the LHRH neuronal system terminate in the anterior and mid-median eminence. We hypothesize that pulses of FSH alone are mediated by release of the FSHRF into the hypophyseal portal vessels, whereas those of LH alone are mediated by LHRH. Pulses of both gonadotropins simultaneously may be mediated by pulses of both releasing hormones simultaneously. Alternatively, relatively large pulses of LHRH alone may account for simultaneous pulses of both gonadotropins since LHRH has intrinsic FSH-releasing activity.     

Progesterone does not affect the amount of mRNA for gonadotropins in the anterior pituitary gland of ovariectomized ewes

To evaluate the effect of progesterone on the synthesis and secretion of gonadotropins, ovariectomized ewes either were treated with progesterone (n = 5) for 3 wk or served as controls (n = 5) during the anestrous season. After treatment for 3 wk, blood samples were collected from progesterone-treated and ovariectomized ewes. After collection of blood samples, hypothalamic and hypophyseal tissues were collected from all ewes. Half of each pituitary was used to determine the content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the number of receptors for gonadotropin-releasing hormone (GnRH). The amounts of mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were measured in the other half of each pituitary. Treatment with progesterone reduced mean serum concentrations of LH (p less than 0.001) but ot FSH (p greater than 0.05). Further, progesterone decreased (p less than 0.05) the total number of pulses of LH. We were unable to detect pulsatile release of FSH. Hypothalamic content of GnRH, number of receptors for GnRH, pituitary content of gonadotropins and mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were not affected (p greater than 0.05) by treatment with progesterone. Thus, after treatment with progesterone, serum concentrations of LH (but not FSH) are decreased. This effect, however, is not due to a decrease in the steady-state amount of mRNA for LH beta or alpha subunits.     

The glycoprotein hormones: recent studies of structure-function relationships

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.     

Effects of prolonged treatment with diltiazem on pituitary secretion of luteinizing hormone, follicle-stimulating hormone, thyrotropin and prolactin.

In previous studies it has been observed that acute administration or short-term treatment with calcium channel blockers can influence the secretion of some pituitary hormones. In this study, we have examined the effect of the long-term administration of diltiazem on luteinizing-hormone (LH), follicle-stimulating hormone (FSH), thyrotropin (TSH) and prolactin (PRL) levels under basal conditions and after gonadotropin-releasing hormone (GnRH)/thyrotropin-releasing-hormone (TRH) stimulation in 12 subjects affected by cardiovascular diseases who were treated with diltiazem (60 mg 3 times/day per os) for more than 6 months and in 12 healthy volunteers of the same age. The basal levels of the studied hormones were similar in the two groups. In both the treated patients and the control subjects, a statistically significant increase (p < 0.01) in LH, FSH, TSH and PRL levels was observed after GnRH/TRH administration. Comparing the respective areas under the LH, FSH, TSH and PRL response curves between the two groups did not present any statistically significant difference. These findings indicate that long-term therapy with diltiazem does not alter pituitary hormone secretion.     

Evolutionary and Functional Aspects of Pituitary Gonadotropins in the Green Turtle, Chelonia Mydas

Information on the pituitary gonadotropins in Chelonia mydasrepresents some of the most complete data available for anyreptile and thus provides an important basis for evaluatingevolutionary processes in tetrapod endocrine physiology. Thetwo gonadotropins isolated from Chelonia pituitary glands showclear chemical and immunological homologies to mammalian follicle-stimulatinghormone (FSH) and luteinizing hormone (LH). However, receptorstudies and biological tests indicate that the functions ofthese hormones may be different in turtles and mammals. In particular,Chelonia LH shows an unusual ability to interact with FSH receptorsites and to stimulate physiological functions normally attributedto FSH. Results with Chelonia LH demonstrate that errors mayarise from using mammalian hormones to investigate reproductionin turtles. Measurements of endogenous gonadotropin levels inthe plasma of breeding and nesting Chelonia provide a differentperspective of the potential roles of the FSH and LH from thatobtained in physiological tests. In particular, FSH and LH secretionare markedly dissociated during the nesting cycle; FSH has onlya transient peak during oviposition, whereas LH, along withprogesterone, displays a pronounced "ovulatory" surge in theday following nesting. Preliminary studies with synthetic gonadotropicreleasing factors in Chelonia suggest that these may be usefulin inducing reproductive changes.     

Dopaminergic regulation of gonadotropin and thyrotropin hormone secretion is altered with age.

To determine if age-related changes in glycoprotein pituitary hormone secretion are associated with alterations in dopaminergic regulation, plasma gonadotropins and TSH were measured before and after L-dopa administration in 44 young (31-44 years of age) and 42 old (64-88 years of age) healthy male participants. Plasma GH and PRL were also determined in order to examine the somatotroph and lactotrope response. In the young, following L-dopa, plasma FSH, LH and TSH were unchanged from baseline. However, in older subjects, plasma FSH was significantly increased (p less than 0.001) and a similar trend was noted for LH. Plasma TSH was significantly depressed (p less than 0.002) in older subjects only. Following L-dopa, increases in plasma GH and decreases in plasma PRL were of similar magnitude in each group. These data indicate that dopaminergic modulation of gonadotropins and TSH is altered with age.     

Glycan mapping of recombinant human follicle stimulating hormone by mass spectrometry

In humans, regulation of reproductive functions are carried out mainly by glycoprotein hormones namely follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH) and chorionic gonadotropin (CG). Since glycans play an important role in binding of gonadotropins with their respective receptors, it is important to identify associated glycans and their pharmacological properties not only for the disease manipulation but also for making more efficacious and safer recombinant versions. With the advancement of mass spectrometry, it is possible to identify minute quantity of associated glycans. Here, we studied the N-glycans of the FSH based on mass spectrometry and report one more complex glycan species in addition to twenty four previously reported glycans. The new glycan was a tetra antennary species that may have important role in binding of FSH with receptor with higher biological activity as well as lower clearance rate and higher half-life.     

Direct demonstration of intrinsic follicle-stimulating hormone receptor-binding activity in acid-treated equine luteinizing hormone

After dissociating equine gonadotropins as a function of time at pH 3, we examined them by radioligand assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis under nondissociating conditions (low, 0.1% SDS). Equine follicle-stimulating hormone (FSH) rapidly lost its receptor-binding activity, and low SDS-polyacrylamide gels demonstrated dissociation into subunits. Maximum dissociation occurred after 20–30 min of pH 3 incubation. Equine luteinizing hormone (LH), however, retained most biologic activity and was largely intact after 72 h of pH 3 incubation. Dose-response curves of acid-treated equine LH and FSH and intact equine LH and FSH were compared in five types of radioligand receptor assays. LH and FSH receptor-binding activities of equine LH were unaffected by pH 3. Equine LH showed 19- and 32-times more activity in the rat testis FSH assay than it did in chicken or horse FSH assays, respectively, directly demonstrating the intrinsic FSH receptor-binding activity of equine LH and the relative lack of specificity for these hormone preparations of the rat FSH receptor. Acid-treated equine FSH lost 95% of its biologic activity in FSH assays. In LH assays, the slight (0.2%) activity of equine FSH was relatively unaffected by acid treatment, suggesting that contamination by equine LH accounts for this activity.     

Effects of gonadectomy on polymorphism in stored and circulating gonadotropins in the bullfrog, Rana catesbeiana. II. Gel filtration chromatography

Marked polymorphism was revealed in both stored and circulating forms of immunoreactive follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the bullfrog, Rana catesbeiana, by exclusion chromatography on columns of Sephracyrl-S200. FSH behaved as a more homogeneous and larger molecule than LH from the same pituitary or plasma, but the properties of both hormones in the plasma were markedly affected by gonadectomy. Chromatographic profiles of FSH stored in the pituitaries were similar in intact and gonadectomized frogs, but pituitary LH in the latter was comprised of a larger proportion of early eluting activity. Previously purified preparations of bullfrog FSH and LH were more homogeneous than these extracts. Differences between pituitary hormones in intact and gonadectomized frogs were small compared with those between circulating hormones. Plasma FSH and lH from gonadectomized frogs behaved as more homogeneous and larger molecules than those from intact frogs in which plasma gonadotropins were elevated normally or by injections with gonadotropin releasing hormone (GnRH). Some differences in circulating hormones were also observed between a normal male and female and both differed from gonadectomized an GnRH-treated intact frogs. Chromatographs of plasma gonadotropins in GnRH-treated animals generally resembled those of the hormones stored in the pituitary, whereas plasma FSH and LH in gonadectomized frogs appeared more homogeneous and larger than the pituitary-stored forms. Those pronounced differences in chromatographic properties of gonadotropins in intact and gonadectomized frogs correlate with previously observed effects of gonadectomy on clearance profiles of circulating FSH and LH.