| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
and ß Subunits in Rainbow Trout (Oncorhynchus mykiss): Purification, Characterization, Development of Specific Radioimmunoassays, and Their Seasonal Plasma and Pituitary Concentrations in Females1
a School of Biological Sciences, Hatherly Laboratories, University of Exeter, Exeter EX4 4PS, United Kingdom
b Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH, United Kingdom
ABSTRACT
Gonad development in fish, as in mammals, is regulated by two gonadotropins (GTHs), FSH and LH. The function of LH in fish has been clearly established; however, the function(s) of FSH is less certain. The lack of specific and sensitive assays to quantify FSH and its
and ß subunits has hindered studies to assess physiological function. In this study, gel filtration chromatography, ion exchange chromatography, and HPLC were employed to purify FSH and its subunits from pituitary glands of rainbow trout (Oncorhynchus mykiss), and the identities of the isolates were confirmed by amino acid analysis. Polyclonal antibodies were raised against the free GTH2 and free FSHß subunits to develop specific RIAs. The sensitivities of the intact FSH, GTH2, and FSHß assays were 1 ng/ml, 0.2 ng/ml, and 0.1 ng/ml, respectively, and the cross-reaction of these molecules with each other and with intact LH in the heterologous assays was <10.4% throughout. Pituitary and plasma samples diluted in parallel with the standards in all three assays and spiked sample recoveries were >90% throughout. Measurement of plasma and pituitary concentrations of intact FSH in female rainbow trout confirmed the established seasonal profiles. Concentrations of free GTH2 subunit were elevated both in the plasma and in the pituitary in females at ovulation (maximum concentrations: 34.93 ± 6.3 ng/ml in plasma; 37.63 ± 5.79 µg/pituitary). In both the plasma and the pituitary, free FSHß subunit was present throughout the reproductive cycle but at very low concentrations when compared with both free GTH2 and intact FSH. The presence of free GTH2 subunit in the plasma similarly occurs in mammals, but its functional significance in fish has yet to be established.
follicle-stimulating hormone, gametogenesis, oocyte development, pituitary hormones, seasonal reproduction
INTRODUCTION
In mammals, the presence of two gonadotropins (GTHs), FSH and LH, and their regulatory roles in reproductive development are well established. In fish, the duality of GTHs was only established in 1988, when two GTHs were isolated and characterized in salmonids (chum salmon, Oncorhynchus keta) [1, 2]. This work provided a methodology for the isolation of fish GTHs without affecting their biological activity. FSH and LH and/or their associated mRNAs have now been isolated from a number of fish species (Coho salmon, Oncorhynchus kisutch [3]; common carp, Cyprinus carpio [4]; bonito, Euthynnus pelamis [5]; Atlantic croaker, Micropogonias undulatus [6]; tuna, Thunnus obesus [7]; gilthead sea bream, Sparus aurata [8]; Mediterranean yellowtail, Seriola dumerilli [9]; goldfish, Carassius auratus [10]; rainbow trout, Oncorhynchus mykiss [11]; and European eel, Anguilla anguilla [12]). For physiological studies to elucidate the function of FSH and LH in fish, pure GTH preparations must be obtained and specific and sensitive immunoassays must be developed. A number of specific and very sensitive assays have been developed to measure LH in fish [13–17], and the role of LH in mediating ovulation is well established [18]. In contrast, few assays for FSH have been developed, and even when available, they often lack sensitivity and/or specificity, which has complicated establishing the role(s) of FSH in reproduction in fish. Ascribed functions of FSH in fish include mediating puberty [19], controlling recruitment of primary oocytes into vitellogenesis [20], and controlling growth of vitellogenic oocytes [21], but the role(s) of FSH in fish is by no means certain.
GTHs are composed of a dimer of two protein subunits: , which is common to FSH, LH, and thyroid stimulating hormone (TSH), and ß, which confers specificity to each hormone. For each intact active GTH dimer, the and ß subunits are produced independently and are subsequently bound together prior to being released into the circulation. The dynamics of GTH subunit synthesis and the importance of these subunits in GTH function have not been established in fish, in part because the available assays for measuring free subunits lack both specificity and sensitivity [22, 23]. There are no published data on seasonal profiles of free GTH subunit or free FSHß subunit either in the plasma or in the pituitary gland. In mammals, assays have been developed to measure free GTH subunit [24–26]. These assays have revealed that free GTH subunit is actively secreted by the pituitary. In mammals, free GTH subunit does not appear to have biological activity at the receptors for the dimeric GTHs, but in humans, this subunit can stimulate prolactin production from decidual cells [27]. In humans, free GTH subunit also acts synergistically with progesterone to stimulate differentiation of endometrial stromal cells [28]. In mammals, the stimulatory pathways for the synthesis and secretion of the free GTH subunit are regulated differently than are the pathways for intact glycoprotein hormones [29]. These findings suggest that GTHs and their free subunits may have a more complex control in reproduction than has been ascribed for intact FSH and LH alone. To more clearly establish the roles of GTHs and their subunits in reproduction, robust and highly sensitive assays are needed for all vertebrates.
The aim of this work was to purify FSH and its subunits from rainbow trout pituitaries and to develop homologous RIAs able to quantify this hormone and its subunits. These assays were then employed to establish the seasonal profiles of FSH and its subunits in female rainbow trout, both in the plasma and in the pituitary.
MATERIALS AND METHODS
FSH Purification
Ethanol extraction Pituitaries (4000) were collected from immature and early vitellogenic female rainbow trout and homogenized in 0.2 M ammonium acetate, 1 mM phenylmethylsulphorylfluoride, pH 9.0, on ice using a polytron homogenizer. The resulting homogenate was stirred for 1 h at 4°C and centrifuged at 20 000 x g for 30 min. Pellets were reextracted to maximize recovery. The supernatants were pooled and adjusted to 40% ethanol by addition of ice cold ethanol, and the solution was stirred overnight at 4°C. After centrifuging at 20 000 x g for 30 min at 4°C, the supernatant was adjusted to 85% ethanol by slowly adding three volumes of ice cold ethanol. The precipitates were then reextracted, and the resulting supernatant was adjusted to 80% ethanol and pooled. The solution was stored overnight at 4°C to allow the proteins to precipitate, and the mixture was then centrifuged at 20 000 x g for 30 min at 4°C. The supernatant was decanted, and the pellet was dried in a vacuum dryer to remove the remaining ethanol. The pellet was then redissolved in 50 mM ammonium bicarbonate, pH 7.8–7.9, and the insoluble proteins were removed by centrifugation.
Chromatographic procedures The ethanol-precipitated proteins were fractionated by gel filtration chromatography, using a Sephadex G-100 superfine (length, 100 cm; diameter, 1.5 cm) equilibrated with 50 mM ammonium bicarbonate, pH 7.8–7.9, at a flow rate of 12 ml/h at 4°C. Fractions of 3 ml were collected, and the absorbance was measured at 280 nm. Fractions containing the proteins with the specific molecular mass of GTHs (approximately 40 kDa) were pooled and lyophilized.
The lyophilized isolate containing the GTHs was fractionated by ion exchange chromatography in a Whatman DE-52 column (length, 10 cm; diameter, 1.0 cm) equilibrated with 50 mM ammonium bicarbonate, pH 7.8–7.9, at a flow rate of 18 ml/h. Unadsorbed proteins were eluted from the column with 50 mM ammonium bicarbonate (pH 7.8–7.9). Adsorbed proteins were eluted with a stepwise gradient of 0.1, 0.2, 0.3, and 0.5 M ammonium bicarbonate (pH 7.8–7.9). Fractions of 3 ml were collected, and the absorbance was measured at 280 nm. The peak protein fractions were pooled and lyophilized.
The fractions eluted with 0.1 M ammonium bicarbonate were subjected to gel filtration chromatography on a Superdex G-75 superfine (Pharmacia and Upjohn, Kalamazoo, MI; length, 30 cm; diameter, 1.0 cm) equilibrated with 0.15 M ammonium bicarbonate and run at a flow rate of 1 ml/min in an HPLC system. Absorbance was measured at 280 nm, and peak protein fractions were collected manually.
Fractions corresponding to 40 kDa were dissolved in 0.1% trifluoroacetic acid (TFA) and were fractionated by reverse phase HPLC (rpHPLC) on a TSK gel ODS-120T column (length, 25 cm; diameter, 0.46 cm) at a flow rate of 1 ml/min at room temperature. The system was equilibrated with 20% acetronitrile in 0.1% TFA and run for 50 min from this initial acetonitrile concentration up to 40% acetronitrile in 0.1% TFA. Peak fractions were collected manually and lyophilized.
Analytical procedures
The rpHPLC fractions obtained corresponding to the positions where and ß FSH were expected to elute were analyzed for their amino acid composition (Aberdeen Sequencing Facility, Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen, Scotland) to determine their similarity to other characterized salmonid GTH1, GTH2, and FSHß subunits. During all chromatographic procedures, the presence of immunoreactive FSH was confirmed by RIA [15].
Antibody Development
Polyclonal antibodies were raised in rabbits against the HPLC-purified FSH free subunits (Harlan Sera Lab, England). The injection regime consisted of 6 injections at 4-wk intervals. The first injection delivered 60 µg of antigen emulsified in Freud complete adjuvant. Repeated injections delivered 30 µg of antigen in Freud incomplete adjuvant. Blood samples were collected 2 wk after each injection to monitor for the presence of specific antibodies in the serum. Antibodies employed in the intact FSH RIA (provided by J.P. Sumpter, Brunel University) were developed against Coho salmon FSHß subunit and have been fully validated for measuring FSH in rainbow trout [15].
Radioimmunoassays
Standards and antibodies
Intact FSH and free GTH2 and FSHß subunits, purified as described above, were employed as standards in the respective RIAs.
Titration of the antibodies employed in the intact FSH RIA with homologous rainbow trout FSH label and standards indicated that an antibody dilution of 1:1750 provided the most sensitive assay. Therefore, this antibody dilution was used in the intact FSH RIA.
The antibodies against free GTH2 subunit produced the most sensitive free GTH2 assay at a dilution of 1:3000. Attempts to produce antiserum against free 1 resulted in only very low titer preparations. Consequently, it was not possible to develop an RIA against this subunit.
The antibodies against free FSHß subunit produced the most sensitive FSHß assay at a dilution of 1:1250.
Iodination
Intact FSH, free GTH2 subunit, and free FSHß subunit were radiolabeled with 125I using the iodogen method [30, 31]. The labeled hormones were separated from free 125I by gel filtration on Sephadex G-25 columns (PD 10 columns; Pharmacia). The specific activity of labeled hormones was between 80 and 100 µCi/µg. Radiolabeled hormone was stored at 4°C until required.
Assay validations
Validations of the assays for rainbow trout free GTH2 subunit, free FSHß subunit, and intact FSH were carried out as follows. Standard dilutions (10–0.01 ng/ml for the free subunit RIAs and 100–0.1 ng/ml for the intact FSH RIA) were run at the optimal concentration of first and second antibody corresponding to a precipitation of approximately 25% binding of the total label added. In the test for parallelism, serial dilutions of rainbow trout plasma and pituitary extract were compared with serial dilutions of the standards. For recovery of spiked samples, known amounts of the standards (four different concentrations covering the range of the standard curve) were added to plasma and pituitary extracts, and the percentage of recovery was calculated. Known amounts of the standards were added to pituitary and plasma samples, serial dilutions were carried out, and binding was compared with that of standard dilutions.
Assay procedures
Standards (50 µl/tube) were serially diluted in protein assay buffer (PAB; 0.05 M phosphate buffer containing 0.15 M NaCl, 0.01 M EDTA, 0.5% egg albumin, and 0.1% NaN3, pH 7.05). The concentrations of standards was 0.01–10 ng/ml for both free GTH2 subunit and FSHß subunit and 0.1–100 ng/ml for intact FSH. For each assay, 50 µl of the specific antiserum, diluted in PAB containing 1:400 normal rabbit serum, was added to all tubes except the total binding and nonspecific binding (NSB) tubes. All tubes were then vortexed and incubated at 4°C for 28 h. On the second day, 50 µl of labeled hormone, diluted in PAB (3000 cpm/tube), was added to all tubes. All tubes were then vortexed and incubated at 4°C for 24 h. On the third day, 50 µl of donkey anti-rabbit gamma globulin (second antibody), diluted 1:50 in PAB, was added to all tubes, which were then vortexed and incubated for 20 h at 4°C. On Day 4, 200 µl of PAB was added to all tubes, which were then centrifuged for 50 min at 3000 x g at 4°C. The supernatant was then aspirated from all tubes except the totals, and the precipitates were counted for radioactivity for 10 min in a LKB Ultra gamma (Pharmacia) counter. The percentage of binding for standards and samples was calculated according to the following formula (mean cpm of replicates): % Binding = [(unknown - NSB)/(MaxB - NSB)]*100. The hormone concentration in each sample was then calculated from the respective standard curve.
Measurements of Plasma and Pituitary FSH and Its Subunits
Plasma and pituitary samples from female rainbow trout were collected throughout the reproductive cycle to obtain groups representative of the different stages of maturation (n = 140 total). The fish were anesthetized (2-phenoxyethanol, 1:2000) and killed according to Home Office procedures. Blood samples were collected from the caudal sinus in heparinized syringes, and aprotinin was added to reduce proteolysis. Plasma was separated by centrifugation (3000 x g for 5 min). Plasma collection procedures were carried out at 4°C, and the plasma was subsequently frozen in dry ice and stored at -20°C until assayed. Immediately after blood samples had been obtained, pituitaries were removed and frozen individually in dry ice prior to storage at -80°C until assayed. Gonads were removed to evaluate the degree of sexual maturity. The gonadosomatic index (GSI) was calculated for each fish according to the following formula: GSI = (gonad weight/somatic weigh)*100, where the somatic weight was calculated as total weight minus the weight of the gonads. All plasma and pituitary samples were assayed for intact FSH, free GTH2 subunit, and free FSHß subunit.
Statistical Analysis
Plasma and pituitary concentrations of intact FSH, free GTH2 subunit, and free FSHß subunit were analyzed by one-way ANOVA. If the data were not normally distributed, they were log transformed. After the ANOVA, a multiple-comparison Tukey test was performed to determine differences in plasma and pituitary concentrations of FSH and its subunits during the reproductive cycle.
RESULTS
FSH Purification
Gel filtration chromatography of the ethanol-extracted glycoproteins produced five main peaks, and the fractions eluting with a molecular mass of approximately 40 kDa were confirmed by RIA to contain FSH (data not shown). The pooled proteins were further fractionated into four peaks by ion exchange chromatography (Fig. 1A). The highest concentration of FSH occurred in the fractions eluted with 0.1 M ammonium bicarbonate. Those fractions were pooled and further fractionated by gel filtration chromatography (Fig. 1B). The resulting elution profile consisted of one main peak containing intact FSH (when analyzed by RIA). HPLC analysis of the main intact FSH peak produced two groups of peaks eluting at the characteristic positions for the and ß FSH subunits (Fig. 1C). The identity of the HPLC fractions expected to contain FSH and ß subunits was confirmed by amino acid analysis (Fig. 2).
|
|
Radioimmunoassays
The ED20, ED50, and ED80 for the GTH2 subunit RIA were 12.187 ± 0.549, 2.364 ± 0.163, and 0.517 ± 0.041 ng/ml, respectively (n = 12). The practical detection limit of this assay (around 90% binding; corresponding to the upper limit of linearity of the standard curve) was 0.263 ± 0.029 ng/ml (n = 12). The ED20, ED50, and ED80 for the free FSHß subunit RIA were 54.889 ± 16.185, 1.284 ± 0.149, and 0.262 ± 0.017 ng/ml, respectively (n = 12). The practical detection limit of the assay was 0.134 ± 0.012 ng/ml (n = 12). The ED20, ED50, and ED80 for the intact FSH RIA were 111.711 ± 17.76, 14.589 ± 0.71, and 2.390 ± 0.176 ng/ml, respectively (n = 13), and the practical detection limit of the assay was 1.034 ± 0.08 ng/ml (n = 13).
The cross-reaction of free GTH1, free GTH2, intact FSH, intact LH, and FSHß subunit in the different RIAs tested under competitive conditions was <10.4% throughout (Table 1 and Fig. 3, A and B). Rainbow trout LHß subunit and both intact TSH and TSHß subunit were not available, and therefore their cross-reactions in both the intact FSH and FSHß subunit assays were not tested. Plasma and pituitary samples containing a wide range of concentrations of free GTH2, free FSHß, and intact FSH diluted in parallel with the respective standard curves (Fig. 4, A–C). The recovery of free GTH2 and free FSHß subunits and intact FSH spiked in pituitary and plasma samples were >90% throughout.
|
|
|
Seasonal Concentrations of Plasma and Pituitary Intact FSH, Free GTH2 Subunit, and Free FSHß Subunit in Female Rainbow Trout
The seasonal concentrations of plasma and pituitary intact FSH, free GTH2 subunit, and free FSHß subunit were measured and compared with the stage of gonad development. The concentration of free GTH2 subunit in the pituitary increased from 2.34 ± 0.37 µg/pituitary in females with a GSI of <0.2 to 37.63 ± 5.79 µg/pituitary in fully mature females (GSI > 12) just prior to ovulation (Fig. 5A). In the plasma, the concentration of free GTH2 subunit remained at baseline during most of the reproductive cycle (
3.5 ng/ml) and increased when the GSI reached values >12. Maximal plasma concentration of free GTH2 subunit occurred in ovulated females (34.93 ± 6.3 ng/ml; Fig. 5B).
|
The seasonal concentrations of free FSHß subunit, both in the pituitary and in the plasma, did not change during the course of the whole reproductive cycle, remaining at the levels found in immature females (GSI < 0.2): approximately 2 µg/pituitary and 1.5 ng/ml in the plasma (Fig. 5, A and B, respectively).
In the pituitary, the content of intact FSH increased progressively during the reproductive cycle from 2.50 ± 0.44 µg/pituitary in females with a GSI of <0.2 to 10.57 ± 2.24 µg/pituitary in fully mature females (GSI > 12; Fig. 5A). In the plasma, the concentration of intact FSH in immature females (GSI < 0.2) was 3.02 ± 0.36 ng/ml, and it increased progressively with sexual development to a concentration of 11.23 ± 3.43 ng/ml during vitellogenesis (1 < GSI < 5). During late vitellogenesis (5 < GSI < 12), the concentration of plasma FSH returned to values similar to those in immature females (5.83 ± 1.55 ng/ml). Just prior to ovulation (GSI > 12), plasma FSH was elevated again with peak concentrations occurring in ovulated females (31.81 ± 6.10 ng/ml; Fig. 5B).
DISCUSSION
In this study, FSH and its subunits were purified from the pituitaries of rainbow trout, and specific assays were established to quantify plasma and pituitary concentrations in females throughout the reproductive cycle. To reduce the chance of contamination of the FSH preparations with LH, pituitaries were collected from immature fish and fish in very early stages of reproductive development (life stages known to have a very low pituitary content of LH). This approach was successful, and chemical analyses (HPLC and amino acid composition) confirmed a high level of purity of the GTH isolates. The HPLC elution characteristics of the GTHs and their subunits in rainbow trout were similar to those established for other Oncorhynchus species, with two distinct subunits and one ß subunit for rainbow trout FSH [3, 17]. The multiple peaks in the HPLC traces for both the and ß subunits probably reflect different compositions of their glycosylation. This difference may have a functional significance for rainbow trout GTHs, as it does in mammals, where different levels of glycosylation and different subunits confer different levels of activity in intact FSH and LH [32, 33].
The RIA for rainbow trout FSH, using homologous FSH as standard and radiolabel, was more sensitive than the assay developed by Prat and colleagues [15], who used the same antibody but a heterologous ligand (Oncorhynchus kisutch, with detection limit of 2 ng/ml). The FSH assay developed in the present study is one of the most sensitive FSH assays available for any fish species [13, 17]. The seasonal concentrations of plasma FSH in female rainbow trout mimicked that reported previously for this species [15, 34], with peak concentrations occurring during early vitellogenesis and around ovulation. The seasonal plasma concentrations of FSH were first elevated above baseline when the GSI was between 0.2 and 0.39. The timing of this initial elevation supports the hypothesis that FSH mediates recruitment of primary oocytes into secondary (vitellogenic) growth and/or stimulates uptake of vitellogenin into oocytes [21, 35]. The function of plasma FSH at ovulation is not known; however, FSH may facilitate the ovulatory action of LH and/or initiate growth of the following season's batch of oocytes [36]. The FSH RIA reported here, however, is still not sensitive enough to measure FSH in immature fish or in males for extensive periods of their sexual development, where plasma concentrations are <1 ng/ml. Antibodies against synthetic peptides have been employed in an attempt to develop more specific and sensitive FSH assays, but as yet, researchers have not met with significant success [37].
The seasonal pituitary concentrations of FSH in female rainbow trout described in this study confirmed those established previously [34], with a progressive increase through primary and secondary growth of oocytes to ovulation. However, the absolute concentrations of pituitary FSH were lower in this study than in the study of Gomez and colleagues [34] (a 10-fold difference in the peak pituitary content). The discrepancy in results between the two studies may be a function of differences in the two assays used. Our unpublished data also suggest that peak concentrations of FSH may vary both among strains of trout and with age of the fish.
Studies in mammals have indicated that, in addition to the dimeric glycoprotein hormones, free GTH subunits also have physiological functions [28]. This work has been facilitated by the availability of specific assays for GTH free subunits. In fish (African catfish, Clarias gariepinus), there is evidence that the pituitary releases free subunit into the circulation [22], although its function is not known. Until now, no data have been available on the concentrations of free GTH subunits, either in the plasma or in the pituitary, throughout the reproductive cycle, and thus the dynamics of pituitary synthesis and secretion of these subunits was not known. The good detection sensitivities for GTH2 and FSHß subunits in rainbow trout in this study enabled us to measure these subunits even in the plasma of immature fish. The pituitary content of FSHß subunit varied little throughout reproductive development in female rainbow trout and was present at considerably lower concentrations than that for intact FSH (between 2-fold and 6-fold lower) and GTH2 (between 2-fold and 20-fold lower). Thus, the synthesis/availability of the FSHß subunit appears to be the rate-limiting step in the synthesis of intact FSH. Plasma concentrations of FSHß subunit were also low and showed little if any seasonal variation, suggesting that free FSHß subunit is not actively secreted by the pituitary and thus probably alone does not have a biological function, as is the case in mammals.
In mammals, free GTH subunit appears to play important roles in controlling GTH function and has roles independent of the intact dimeric glycoprotein hormones (e.g., controlling the release of prolactin [33]). Furthermore, the production of GTH subunit in the pituitary is controlled by mechanisms independent from those of intact GTHs [27]. In our studies in the rainbow trout, the plasma content of GTH2 subunit showed little change until shortly preceding ovulation, when concentrations increased dramatically. The high plasma concentration of GTH2 subunit at ovulation may have resulted from degradation of FSH into its component subunits. However, this degradation appears unlikely because the plasma concentration of FSHß subunit at this time was very low. Free GTH2 subunit could also have arisen from the degradation of LH. Alternatively, as occurs in mammals, the free plasma GTH2 subunit could be actively secreted from the pituitary and have a functional role in its own right in the ovulation process in rainbow trout. More studies are needed to clarify the physiological role(s), if any, of the free GTH2 subunit in the plasma.
It is becoming clear, predominantly from studies in mammals, that the regulation, synthesis, storage, secretion, and physiological function of GTHs are complex processes and that unraveling of GTH function requires methods to independently identify and quantify the GTH subunits in addition to the intact dimers. The assays developed in this study for measuring free FSHß subunit and free GTH2 subunit in the rainbow trout provide some of the tools needed to start to more fully understand the mechanisms controlling the synthesis and secretion of GTHs and their subunits and their physiological functions in fish.
ACKNOWLEDGMENTS
The authors thank J.P. Sumpter and P. Swanson for provision of the antibodies for measuring FSH and for isolates of Coho salmon FSH and LH, respectively. We would also like to thank members of the Fish Physiology Research Group (Brunel University) for their help in collecting pituitary glands and F. Prat for his constructive criticism on this work.
FOOTNOTES
First decision: 5 January 2001.
1 This work was supported by grant BD 5130/95, Programme PRAXIS XXI, FCT, Portugal. 
2 Correspondence: Eduarda M. Santos, School of Biological Sciences, Hatherly Laboratories, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, UK. FAX: 44 1392 263700; e.santos{at}exeter.ac.uk
Accepted: March 6, 2001.
Received: November 29, 2000.
REFERENCES
and ß subunits: application to the development of specific radioimmunoassays. Gen Comp Endocrinol 1998; 111:28-37[CrossRef][Medline])-subunits of salmon glycoprotein hormones: production sites in the pituitary with sexual maturity. Fish Physiol Biochem 1997; 17:39-44[CrossRef]-subunit functions synergistically with progesterone to stimulate differentiation of cultured human endometrial stromal cells to decidualized cells: a novel role for free -subunit in reproduction. Endocrinology 1996; 137:1332-1339[Abstract],6-diphenyl glycoluril (iodogen). Anal Biochem 1981; 117:136-146[CrossRef][Medline]-subunit influences the specificity of receptor binding of the equine gonadotropins. J Endocrinol 1997; 155:241-245[Abstract]-subunit strongly differ in their ability to stimulate prolactin release from foetal pituitaries. J Endocrinol 2000; 164:287-297[Abstract]This article has been cited by other articles:
|
|
 |
|
  P M. Lokman, K. A N George, S. L Divers, M. Algie, and G. Young 11-Ketotestosterone and IGF-I increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro Reproduction, May 1, 2007; 133(5): 955 - 967. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
intact FSH; 
intact LH. B) Free FSHß subunit assay: • free FSHß subunit standard; 
standard curve; • plasma sample; 
pituitary sample