Perspectives on fish gonadotropins and their receptors

Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common α subunit non-covalently associated with a hormone-specific β subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders.Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper.The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review.In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone–receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species.     

Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor

LH and FSH are heterodimeric glycoprotein hormones, composed of a common alpha-subunit non-covalently associated with a hormone-specific beta-subunit. Repeated efforts to isolate catfish FSH (cfFSH) have not been successful and only catfish LH (cfLH) has been purified from catfish pituitaries. Recently, however, we succeeded in cloning the cDNA encoding the putative cfFSHbeta; the cDNAs for the alpha- and beta-subunit of cfLH have been cloned before. Here we report the expression of biologically active cfLH and cfFSH in the soil amoeba, Dictyostelium discoideum. The biological activity of the recombinant hormones was analyzed using cell lines transiently expressing either the cfLH receptor or the cfFSH receptor. Moreover, a primary testis tIssue culture system served to study the steroidogenic potency of the recombinant hormones. Our results demonstrated that Dictyostelium produced biologically active, recombinant catfish gonadotropins, with recombinant cfLH being almost indistinguishable from its native counterpart, purified from pituitaries. Although recombinant cfFSH has significant effects in the bioassays used in this study, the specific function of native cfFSH in the control of reproduction and its expression patterns are not yet understood.     

Translational fusion of two beta-subunits of human chorionic gonadotropin results in production of a novel antagonist of the hormone

The strategy of translationally fusing the alpha- and beta-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGalphabeta expressed using Pichia expression system. Using the same expression system, another analog, in which the alpha-subunit was replaced with the second beta-subunit, was expressed (hCGbetabeta) and purified. hCGbetabeta could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGalphabeta and hCGbetabeta using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGbetabeta interacts with two LH receptor molecules. These studies demonstrate that the presence of the second beta-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of alpha-subunit, the molecule is unable to elicit response. The strategy of fusing two beta-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.     

Somatostatin regulation of glycoprotein hormone and free subunit secretion in clinically nonfunctioning and somatotroph adenomas in vitro.

There is increasing evidence that clinically nonfunctioning pituitary tumors produce and secrete glycoprotein hormone and/or free alpha- and beta-subunits. In addition, hypersecretion of free alpha-subunit occurs in up to 37% of patients with somatotroph adenomas. An understanding of glycoprotein hormone regulation is important in developing effective therapeutic strategies for patients with tumors associated with intact glycoprotein hormone and free subunit hypersecretion. We investigated glycoprotein hormone and free subunit secretion by somatostatin in primary dispersed cultures of pituitary tumor cells from 23 patients with pituitary adenomas. Fifteen tumors from patients with clinically nonfunctioning adenomas (group 1) and 8 tumors from patients with somatotroph adenomas and cosecretion of alpha-subunit (group 2) were studied. Cultures were incubated with control or somatostatin-supplemented media for 24 h. Media samples from group 1 tumors were assayed for intact glycoprotein hormones and free alpha- and beta-subunits secretion levels, while media samples from group 2 cultures were assayed for alpha-subunit and GH secretion levels. Significant (P less than 0.05-0.001) inhibition of secretion of 1 or more intact hormones and/or free subunits was found in 10 of the 15 group 1 tumors. SRIF[10(-7) M] suppressed intact gonadotropin secretion in 60% of FSH-producing tumors and 30% of LH-producing tumors. Media concentrations of FSH beta and LH beta were decreased in 31% and 50% of group 1 tumors, respectively, following somatostatin treatment in those tumors which secreted free beta-subunits. alpha-Subunit was secreted by 12 of the 15 tumors, but significant (P less than 0.02-0.01) inhibition by somatostatin was observed in only 2 tumors. In contrast, significant (P less than 0.05-0.001) inhibition of alpha-subunit in the somatotroph adenomas was found in 6 of the 8 tumors. Significant decreases in alpha-subunit were observed only in those tumors where GH was also significantly inhibited by somatostatin. We conclude that 1) somatostatin inhibits intact glycoprotein or free subunit secretion in the majority of clinically nonfunctioning pituitary tumors in vitro and 2) alpha-subunit secretion is suppressed in 17% and 69% of clinically nonfunctioning and somatotroph adenomas, respectively, consistent with a differential regulation of alpha-subunit by somatostatin in these two tumor types.     

Gonadotropin-releasing hormone-induced stimulation and desensitization of free alpha-subunit secretion mirrors luteinizing hormone and follicle-stimulating hormone in perifused rat pituitary cells

A pulsatile pattern of hypothalamic GnRH stimulation is necessary for the maintenance of pituitary LH and FSH secretion, with continuous GnRH leading to a decrement in response. Although the physiological pattern of free alpha-subunit secretion closely mimics that of LH, several reports have indicated that free alpha-subunit is not desensitized by continuous GnRH stimulation. To explore the basis of this phenomenon, we have evaluated the responses of all three gonadotrope secretory products to carefully coordinated administration of pulsatile and continuous GnRH in a dispersed rat pituitary perifusion system. Sensitivities (ED50) to GnRH fell within a narrow range for free alpha-subunit (11.5 nM), LH (12.9 nM), and FSH (17.3 nM), although a greater mass of LH than free alpha-subunit or FSH was released after each pulse of GnRH. The response to a standard GnRH pulse (10 nM) administered every 15, 30, or 120 min for 9 h was very stable, with no evidence of priming, summation, or loss of response. LH, FSH, and free alpha-subunit did, however, show significantly (P less than 0.05) higher pulse amplitude with longer interpulse intervals. In contrast to previous observations in vivo, the three gonadotrope secretory products showed parallel desensitization in response to continuous infusions of GnRH. This loss of response was significant (P less than 0.05) after exposure to as little as 0.1 (FSH) to 0.5 nM (LH and alpha-subunit) GnRH for 2 h or to higher concentrations of GnRH (10 nM) for as little as 15 min (LH, FSH, and alpha-subunit). These concentrations and durations of GnRH stimulation are within the range of values measured in vivo. We conclude that 1) free alpha-subunit, LH, and FSH have similar concentration and frequency responses to pulsatile GnRH, although the absolute amount of hormone released is different for each secretory product; 2) the frequency of pulsatile GnRH stimulation can function as an independent determinant of secretion for each of the three products; and 3) in contrast to observations in vivo, free alpha-subunit, LH, and FSH secretion desensitize similarly after exposure to concentrations or durations of GnRH that may occur in vivo. These observations raise the possibility that desensitization plays a role in the physiological regulation of gonadotrope secretion.     

Comparison of the effects of cycloheximide and inhibin on the gonadotropin subunit messenger ribonucleic acids.

Cycloheximide (CHX) has been shown to mimic the action of inhibin on gonadotropin secretion by pituitary cell cultures. We showed previously that suppression of FSH secretion by inhibin is associated with a rapid and profound suppression of FSH beta mRNA levels. The present study was designed to examine the mechanism of action of CHX and to determine whether inhibin's actions involve new proteins synthesis. Pituitary cell cultures were treated with control medium or medium containing inhibin, CHX, or inhibin plus CHX for 2 or 6 h. At 6 h, secretion of FSH was decreased by inhibin (72% of control), CHX (58% of control), and the combined inhibitors (56% of control). LH secretion was not significantly changed, while that of free alpha-subunit was reduced only by CHX (68% of control). Levels of FSH beta, LH beta, and alpha-subunit mRNAs were measured by Northern analysis. At 2 h inhibin decreased FSH beta mRNA to 49% of the control value. CHX alone had no effect, while CHX plus inhibin produced intermediate levels (77% of control). By 6 h, however, inhibin and CHX each decreased FSH beta mRNA to very low levels (12% and 15% of control, respectively), and in cultures treated with both inhibin and CHX, this RNA was barely detectable. To determine the reversibility of the effects of these inhibitors, cells were incubated with fresh control medium after 6 h. Secretion of FSH and free alpha-subunit remained suppressed 4 h later; recovery was complete by 16 h in inhibin treated cultures. FSH beta mRNA returned to control levels by 4 h in inhibin-treated and by 16 h in CHX-treated cultures. Levels of LH beta and alpha-subunit mRNA were comparable to control values at all times. In conclusion, 1) CHX, like inhibin, suppresses FSH beta mRNA levels, although its actions are less rapid and less rapidly reversible; 2) inhibin requires ongoing protein synthesis for full expression of its inhibitory effects; 3) the synthesis and secretion of LH are much less sensitive to inhibition by either inhibin or CHX than are the synthesis and secretion of FSH; and 4) secretion of free alpha-subunit involves a labile protein(s).     

The effects of bromocriptine, thyrotropin-releasing hormone, and gonadotropin-releasing hormone on hormone secretion by gonadotropin-secreting pituitary adenomas in vivo and in vitro

The characteristics and dynamics of hormone secretion in vivo and in vitro were investigated in six patients with gonadotropin-secreting pituitary adenomas. All six tumors secreted and contained FSH and different combinations of LH, beta-LH, and alpha-subunit. In addition, immunohistochemical examination of the pituitary tumor tissue showed staining with both LH and FSH in three and either LH or FSH in the other three tumors. TRH and GnRH stimulated hormone secretion in vivo and in vitro, and they also increased the hormone content of the cultured tumor cells. Bromocriptine significantly inhibited hormone release and reduced the hormone content of the tumor cells. In vivo, 2.5 mg bromocriptine significantly suppressed plasma hormone levels; the inhibiting effect on alpha-subunit concentrations was in general more marked than that on LH and FSH. We conclude that hormone release by gonadotropin-secreting pituitary adenomas can be stimulated by TRH and GnRH and inhibited by bromocriptine. Most of these tumors synthesize FSH, but there is a wide variation in the production of LH, beta-LH, and alpha-subunits. The sensitivity of hormone release to bromocriptine suggests that chronic therapy with this drug might have a beneficial effect on pituitary tumor size.     

Purification and properties of the subunits of human pituitary follicle-stimulating hormone.

Highly purified human pituitary FSH was partially dissociated by treatment with 8M-urea, and alpha- and beta-subunits were isolated by ion-exchange chromatography and gel filtration. Tests of biological activity by in-vivo assays and in-vitro radioreceptor assays were in good agreement and showed that preparations of isolated alpha-subunit had less than 1%, and beta-subunit from 2 to 10% of the FSH activity of the intact hormone. In contrast to results reported elsewhere, most of the subunit preparations reassociated with counterpart subunit to regain biological activity equal to that of intact FSH (around 160 mg NIH-FSH-S1/mg). The intact FSH recovered as a by-product after isolation of subunits was of high biological activity, and its LH contamination was reduced by more than 90% when compared with thepurified FSH starting material. The subunits are relatively inactive in a radioimmunoassay specific for intact FSH. Sialic acid and tryptophan determinations indicated that both subunits contain sialic acid and that tryptophan is present only in the beta-subunit.     

Effects of alpha- and beta-hCG subunits on the shortloop feedback control of gonadotropins

We have previously demonstrated that, in the rabbit, both LH and FSH secretions are controlled by shortloop feedback regulatory systems which are highly specific and sensitive. FSH secretion is suppressed by FSH; LH secretion is suppressed by LH and hCG. We have now evaluated the possible role of alpha- and beta-subunits of hCG in the control of LH secretion in rabbits. Experiments were performed in unanesthetized castrated female rabbits bearing chronically implanted silastic catheters to permit repeated blood sampling without anesthesia. Intravenous injections of purified alpha- and beta-chain of hCG were administered in dose levels from 10 to 300 micrograms, and blood samples were taken from -30 to 240 min before and after the bolus injections. Peripheral levels of rabbit FSH and LH were determined by RIA systems, which do not cross-react with intact hCG or its alpha- and beta-subunits. By analysis of variance, no significant suppression of LH and FSH occurred in response to these doses of the subunits. Intact hCG, in doses of 1 microgram, produced a sharp decrease in rabbit LH levels within 10 min. These in vivo studies demonstrate that there is no intrinsic activity of the dissociated purified alpha- and beta-hCG subunits on the shortloop feedback control of gonadotropins in the rabbit.     

Inhibitory and stimulatory regulation of testicular inhibin B secretion by luteinizing hormone and follicle-stimulating hormone,respectively, in the rhesus monkey (Macaca mulatta)

This study examined the relative role of FSH and LH in governing testicular inhibin B secretion in the rhesus monkey. Adult male monkeys, rendered hypogonadotropic and hypogonadal by administration of a GnRH receptor antagonist (acyline), were implanted with testosterone (T)-filled or empty capsules. Following T-induced restoration of spermatogenesis, both groups received recombinant human FSH and vehicle for 12 d. Juvenile male monkeys received an 11-d infusion of single-chain recombinant human LH and recombinant human FSH, either alone or in combination. In adults, chronic hypogonadotropism resulted in a modest reduction of circulating inhibin B levels, which was more than fully reversed by FSH. In the presence of T, which exerted a marked suppression in inhibin B secretion, FSH restored inhibin B levels only to those observed before acyline treatment. In juveniles, treatment with single-chain recombinant human LH led to a suppression of inhibin B secretion and curtailed the FSH-induced stimulation of this testicular hormone. The T-induced decrease in inhibin B secretion was associated with suppression in inhibin-beta(B) mRNA levels, but FSH stimulation of inhibin B secretion occurred in the absence of clear changes in expression of this subunit gene. These findings indicate that inhibin B secretion by the monkey testis is governed by the inhibitory and stimulatory action of LH and FSH, respectively. The action of LH is presumably indirect and likely mediated by T inhibition of inhibin-beta(B) gene expression. The molecular basis of the stimulatory action of FSH on inhibin B secretion requires further study.     

Estrogen receptor alpha signaling pathways differentially regulate gonadotropin subunit gene expression and serum follicle-stimulating hormone in the female mouse

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.     

The antigenic structure of the human glycoprotein hormone alpha-subunit. I. Characterization of anti-alpha monoclonal antibodies.

The glycoprotein hormones CG, LH, FSH, and TSH are composed of two noncovalently linked subunits, alpha and beta. The beta-subunit confers hormone specificity, while the alpha-subunit is homologous within a species. To help in determining the antigenic structure of the common alpha-subunit, six monoclonal antibodies (mAbs) to the free or heterodimeric alpha-subunit of human (h) gonadotropic hormones have been prepared and, along with two previously isolated mAbs, have been characterized for binding specificity to alpha- and beta-subunits and the human glycoprotein hormones, CG, LH, FSH, and TSH. Each mAb was derived from hybidomas of FO myeloma cells fused with spleen cells from mice immunized with free alpha-subunit, hCG or hFSH. mAbs A101, A102, and E512 were specific for the alpha-subunit but showed the highest affinity for the intact hormone; K2.18, K94.6, E501, E502, and E511 were specific for free alpha. All of the antibodies inhibited binding of 125I-hCG to luteal membrane receptor, and 125I-labeled mAbs did not recognize hCG/receptor complex. Characterization by two-site binding assays using alpha, hCG, or hFSH as antigen revealed that all the mAbs bind to unique sites on alpha which may be overlapping, and which are modified in the intact hormone. The antigenic sites for mAbs E502, E511, and K2.18 are at least partially linear because they bind to reduced, carboxymethylated alpha.     

Regulation of follicle-stimulating hormone beta and common alpha-subunit messenger ribonucleic acid by gonadotropin-releasing hormone and estrogen in the sheep pituitary

The effect of gonadotropin-releasing hormone (GnRH) and/or estradiol (E2) on pituitary messenger ribonucleic acid (mRNA) levels of luteinizing hormone beta (LH beta), follicle-stimulating hormone beta (FSH beta) and the common alpha-subunit were determined in anterior pituitary glands from ovariectomized (OVX) ewes. Hypothalamo-pituitary disconnected (HPD) ewes receiving appropriate hormonal treatment were used to assess the relative roles of GnRH and E2 in directly regulating FSH beta and alpha-subunit mRNA levels. Levels of LH beta mRNA were increased in OVX animals compared with intact controls, and E2 treatment of OVX animals significantly reduced mRNA levels of LH beta and FSH beta. HPD substantially reduced FSH beta and alpha-subunit mRNA levels. Treatment of OVX/HPD animals with pulses of GnRH (250 ng/2 h) for 1 week restored FSH beta and alpha-subunit mRNA to OVX levels. Combined GnRH and E2 treatment significantly lowered FSH beta mRNA levels, but resulted in a rise in alpha-subunit mRNA levels. Treatment of OVX/HPD ewes with E2 alone had no effect on FSH beta and alpha-subunit mRNA levels. These findings indicate that E2 acts directly on the pituitary to negatively regulate FSH beta mRNA levels, and to positively regulate alpha-subunit mRNA levels in the presence of GnRH.     

Isolation and characterization of FSH and LH from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.)

Two gonadotropins (GtH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), were isolated and characterized from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L.). Glycoproteins were extracted in 40% ethanol followed by precipitation in 85% ethanol. Subsequently, glycoproteins were fractionated by ion-exchange chromatography on a Whatman DE-52 column using a stepwise gradient of ammonium bicarbonate (50-1000 mM). Intact FSH and LH were finally purified on rpHPLC using an AsahiPak C4P-50 column with an acetonitrile gradient (10-60%). SDS-PAGE showed a molecular mass of 33 and 32 kDa for intact FSH and LH, respectively. Final purification of subunits was performed by a subsequent purification step on rpHPLC using a Phenomenex Jupiter C18 column with an acetonitrile gradient (10-60%). FSHbeta, LHbeta, and the common alpha subunit showed molecular masses of 25, 24, and 19 kDa, respectively. Subunit identity was confirmed by N-terminal amino acid sequencing. Intact FSH and LH showed gonadotropic activity by stimulating release of 11-ketotestosterone from turbot (Scophthalmus maximus L.) testicular tissue in vitro. This provides the first purification of two distinct GtHs from an evolutionary advanced pleuronectiform teleost.     

Estradiol has inverse effects on pituitary glycoprotein hormone alpha-subunit messenger ribonucleic acid in the immature European eel and the gonadectomized rat

In teleosts, the pituitary contains a single glycoprotein gonadotropic hormone (GTH) composed of two dissimilar alpha- and beta-subunits. The European eel, Anguilla anguilla L, is sexually immature at the silver stage due to a deficiency in GTH synthesis and secretion. In previous studies we (S.D., YA.F.) have demonstrated a strong stimulatory action of estradiol (E2) on eel pituitary GTH content. In contrast, we (R.C., M.J.) have shown that in the rat E2 negatively regulates gonadotropin subunit synthesis via changes in specific mRNA levels. The purpose of our present work was to check for such effects of E2 on the synthesis of GTH alpha- and beta-subunits in the European eel. Eel pituitary mRNA was translated in a cell-free system in the presence of [35S]Met + Cys. We demonstrate that one of the translated polypeptides, characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, cross-reacts with an antiserum to denatured bovine alpha-subunit. Its apparent mol wt (18.5K), which is slightly higher than that of the corresponding rat alpha-precursor, suggests that it represents the precursor of the alpha-subunit of eel glycoprotein hormones. The specificity of immunoprecipitation was confirmed by competition with ovine alpha (but not with ovine LH beta or bovine TSH beta). Quantitative evaluation of the putative eel alpha-subunit precursor showed that it represents 0.2% of the total protein translated by RNA from the normal silver eel. Chronic treatment of eels for 3 weeks with 17 beta-E2 increased by 8.0- to 8.5-fold the proportion of the putative alpha-subunit precursor among translation products. Due to the lack of cross-reactivity with the presumed GTH beta precursor, no radioactive material could be specifically detected in translation medium of eel pituitary mRNA using antisera to either denatured bovine LH beta or ovine FSH beta. Our data suggest that E2, depending on vertebrate group and probably on sexual status, may exert either positive or negative control on gonadotropin synthesis by opposite effects on the levels of specific mRNA.     

Gonadotropin and alpha-subunit responses to chronic gonadotropin-releasing hormone analog administration in patients with glycoprotein hormone-secreting pituitary tumors

Pituitary tumors secreting intact glycoprotein hormones (LH, FSH, and TSH) and/or alpha-subunit are being increasingly recognized. Because chronic administration of GnRH analogs decreases gonadotropin secretion in normal subjects, we investigated gonadotropin and alpha-subunit responses to chronic GnRH analog administration in five men with glycoprotein hormone-secreting pituitary tumors. Two patients (patients A and B) received the GnRH agonist analog (D-Trp6-Pro9-NEt-LHRH) for 4 weeks as a daily sc dose (8 micrograms/kg.day). In both, secretion of LH and/or alpha-subunit increased markedly. Subsequently, three patients received a higher analog dose (32 micrograms/kg.day) for a longer duration (8 weeks). One patient with a LH- and FSH-secreting tumor (patient C) had a highly significant (P less than 0.001) fall in serum LH and FSH concentrations; however, alpha-subunit secretion increased. During a subsequent study, when this patient received a lower dose (8 micrograms/kg.day) for 8 weeks, gonadotropin suppression also occurred. In two additional patients who received this dose (32 micrograms/kg.day), it had a persistent agonist effect on FSH beta (patient D) and alpha-subunit secretion (patient E). A marked increase in alpha-subunit secretion occurred in all five patients, regardless of whether basal serum alpha-subunit concentrations were elevated. These patients received the GnRH analog at doses 2-8 times greater than those that suppress gonadotropin secretion in normal men. Serum LH and FSH concentrations decreased in only one patient with a gonadotropin-secreting adenoma. The serum LH and FSH responses to acute GnRH stimulation did not predict the gonadotropin responses to chronic GnRH analog administration. Thus, gonadotropin and alpha-subunit production by most pituitary adenomas is augmented during chronic GnRH analog administration, consistent with defective GnRH desensitization in the adenomatous tissue. Despite the heterogeneous gonadotropin responses to the GnRH analog in these patients, serum alpha-subunit levels increased in all patients, indicating dissociation in the secretion of intact gonadotropins and alpha-subunit.     

Development of non-competitive enzyme-linked immunosorbent assays for mummichog Fundulus heteroclitus gonadotropins - Examining seasonal variations in plasma FSH and LH levels in both sexes

The mummichog Fundulus heteroclitus is an excellent experimental fish for reproductive physiology because of its adequate size, easiness for rearing, and controllable reproduction under laboratory conditions. Furthermore, it is the only species that the native GtHs and their subunits have been purified among small experimental fishes. In this study, homologous non-competitive enzyme-linked immunosorbent assays (ELISAs) for the mummichog FSH and LH were developed by raising monoclonal and polyclonal antibodies against the purified GtHs or their subunits, and the plasma hormone levels in various seasons were examined. The cross-reactivity of LH in the FSH ELISA and the cross-reactivity of FSH in the LH ELISA were low, 2.3% and 0.2% respectively, indicating high specificities of both GtH assays. The practical detection limits were 10pg/well (0.125ng/ml plasma) for the FSH ELISA and 8pg/well (0.1ng/ml plasma) for the LH ELISA. Plasma FSH levels in females indicated distinct correlations with ovarian stages: they were almost undetectable (<0.125ng/ml) during the post-spawning immature phase (September), low values (0.3ng/ml) during the cortical alveoli accumulation phase (December), considerably high (1.8ng/ml) in the vitellogenic phase (February), and very high values (12ng/ml) during the spawning season (June). The male FSH levels showed similar pattern of changes to that of females, also indicating distinct correlations with testicular activities. Plasma LH levels were considerably high during the spawning period in both sexes (3.3ng/ml in females and 4.5ng/ml in males). They were low or undetectable values in non-spawning seasons, and clear correlation with the gonadal stages was not observed. These results indicate the importance of FSH for various reproductive events in multiple spawning fishes, and are consistent with the general understanding that the LH is responsible for final gametes maturation in both sexes. Nonetheless, they further suggest that the role of LH for various reproductive events other than the final maturation may be limited.     

Effects of testosterone on gonadotropin subunit messenger ribonucleic acids in the presence or absence of gonadotropin-releasing hormone.

There is accumulating evidence that the negative feedback actions of testosterone on the pituitary may contribute to the differential regulation of FSH and LH secretion in males. In the present study we measured steady state levels of the mRNAs encoding the gonadotropin subunits in pituitary cell cultures treated with 10 nM testosterone (T) as well as in T-treated pituitary cells perifused with pulses of GnRH to explore further the direct actions of T on the pituitary. T treatment of pituitary cells in monolayer culture for 72 h increased FSH beta mRNA 1.5-fold (P less than 0.05), decreased alpha-subunit mRNA to 45% of the control level (P less than 0.05), and decreased LH beta mRNA to 75% of the control level (P less than 0.05). FSH and uncombined alpha-subunit secretion were increased and decreased by T, respectively, whereas basal LH secretion was unchanged. Treatment with 0.1 nM estradiol, a physiological concentration for males, did not change gonadotropin secretion or subunit mRNA concentrations. Between days 2 and 5 in culture in the absence of steroid treatment, steady state levels of LH beta and alpha-subunit mRNA declined (P less than 0.01) 52% and 61%, respectively, but FSH beta mRNA levels were unchanged. Pulsatile stimulation with 2.5 nM GnRH every 1 h for 10 h increased FSH beta mRNA 2.8-fold (P less than 0.05) and increased (P less than 0.05) alpha-subunit mRNA to 117% of the control level. When cell cultures were pretreated with T for 48 h and then perifused with pulses of GnRH, FSH beta, LH beta, and alpha-subunit mRNA levels were 66%, 74%, and 70% of the value during GnRH alone (P less than 0.05). T treatment also reduced (P less than 0.01) the amplitudes of FSH, LH, and alpha-subunit secretory pulses by 18%, 26%, and 41%, respectively. These data indicate that a portion of the negative feedback action of T is at the pituitary to regulate gonadotropin subunit gene expression. Our data reveal two opposing effects of T on FSH beta mRNA: a stimulatory action, which is GnRH independent, and an inhibitory effect, which is related to the actions of GnRH. These divergent actions of T represent one mechanism through which FSH and LH are differentially regulated.