GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
26,
141-152 ( 1975)
A Quantitative Assay for Luteinizing Hormone Releasing Hormone (LHRH) Using Dispersed Pituitary Cells R. J. BICKNELL AND B. K. FOLLETT Department
of Zoology, University College Bangor, Gwynedd, LL57 2UW,
of North U.K.
Wales.
Accepted December 3, 1974 An assay for luteinizing hormone releasing hormone (LHRH) is described that utilises dispersed pituitary cells. Chicken pituitaries were dispersed with collagenase and after washing, aliquots of the cell suspension were dispensed into polystyrene tubes containing treatments. Tubes were incubated for 30 mins, cells spun off, and supernatants assayed for LH content by radioimmunoassay. LHRH increased LH secretion markedly and a dose-response relationship was established over the range 200 pg/ml to 10 nglml. Hypothalamic extracts from chicken, quail, and rat were also effective but gave dose-response curves steeper than that found with LHRH. Other brain areas showed low levels of LH-releasing activity but the dose-response curves were rather flat for these extracts. A number of pharmacologically active compounds were tested but only acetylcholine in large doses elicited significant LH release. When used as a quantitative bioassay with four tubes per treatment, measurements can be made of the LHRH content in single hypothalami. The mean index of precision was 0.069 2 0.004 (16).
The existence of a luteinizing hormone releasing hormone (LHRH) in the avian hypothalamus has been demonstrated both in vivo and in vitro. Chicken hypothalamic extracts cause ovulation in hens (Clark and Fraps, 1967; Opel and Lepore, 1967, 1972) while similar extracts from quail increase plasma LH levels in rats (Casey et al., 1971). Synthetic mammalian LHRH is also effective in inducing ovulation (van Tienhoven and Schally, 1972; Reeves et al., 1973) and in elevating plasma LH in the chicken (Furr et al., 1973; Bonney ef al., 1974). In vitro, chicken hypothalamic extracts increase the rate of LH release from rat pituitaries (Jackson and Nalbandov, 1969; Jackson, 1971a, b) while quail hypothalamic extracts stimulate avian LH release from incubated chicken hemipituitaries (Follett, 1970), and from superfused quail pituitaries (Smith and Follett, 1972). Recently LHRH has been
detected in chicken hypothalamic extracts by a radioimmunoassay (RIA) for mammalian LHRH (Jeffcoate et al., 1974). This study describes a bioassay technique for use in the investigation of avian LHRH. It was developed because most if not all previous techniques for assaying releasing factors have been only semiquantitative. In vivo methods (e.g., Fink and Harris, 1970; Kamberi et al., 1971; Burger et al., 1972; Campbell, 1972; Opel and Lepore, 1972) are laborious and suffer from the defect that only a very limited series of treatments can be given to a single animal. The established in vitro method of assay consists of incubating pituitary fragments with unknown LHRH samples and measuring the LH released into the incubation medium by bio- or immunoassay (e.g., Schneider and McCann, 1969; Follett, 1970; Jackson, 1971a, b; Scemama, 1972; Jamieson et al., 1973; 141
Copyright 0 1975 by Academic Press, Inc. All tights of reproduction in any form reserved.
142
BICKNELL
AND
Crighton et al., 1973; Blackwell et al., 1973). To some extent this method can be made quantitative by comparing the hormone released from contralateral halves of the same pituitary but it suffers from various limitations, including problems of replication and interassay comparison (see Guillemin and Vale, 1970). In addition it is usually not feasible to measure an unknown solution in more than one treatment and it has proved difficult to quote the potency in terms of an acceptable standard. A super-fusion method overcomes some of these problems (e.g., Smith and Follett, 1972) but is tricky technically. When techniques were developed for the preparation of a homogeneous suspension of pituitary cells by enzymatic dispersion (Portanova et al., 1970; Vale et al., 1972; Takahara et al., 1973; Hopkins and Farquhar, 1973; Tixier-Vidal et al., 1973; McIlhinney and Schulster, 1973; Steinberger et al., 1973) as well as for the assay of gonadotrophins by radioimmunoassay it became feasible to develop a multireplicate in vitro incubation system in which one might include an LHRH standard, and where routine bioassay statistics could be applied.
FOLLETT
(diam 25 mm) containing 10 ml of a 0.02% solution of collagenase (Sigma, type III, fraction A) in Romanoff’s Ringer’s albumin. Generally 30 whole pituitaries, each weighing about 8 mg, were used. The tissue was incubated at 37” and constantly agitated at about 500 rpm by a siliconised glass stirrer. After 20 and 40 min of incubation the tissue pieces were drawn gently into and out of a siliconised Pasteur pipette to aid dispersion. After 60 min the dispersion was completed by repeating pipetting. The cell suspension was then centrifuged at 1OOg for 10 min at room temperature and the supernatant discarded. Five milliliters of fresh RRA was added and the pellet of cells resuspended by gentle pipetting. The steps of centrifugation, removal of supernatant, addition of fresh RRA, and resuspension of the cells were repeated once. The suspension of pituitary cells was then filtered through a piece of nylon gauze (10 mm diam, 60 pm mesh) mounted over a siliconised 100 ml flask containing a magnetic stirrer. RRA was added to give a final concentration equivalent to 0.5 pituitaries/ml. Incubations
Incubations were carried out in disposable polystyrene tubes (12 x 75 mm). Pituitary Cell Suspension The test materials were diluted in RRA Pituitary glands were collected from such that each dose was contained in 0.5 broiler chickens at a local abattoir within a ml and these were added to the polysfew minutes of death and placed in 0.85% tyrene tubes first: 0.5 ml aliquots of the saline on ice. In the laboratory they were stirred pituitary cell suspension were then diced with a razor blade into pieces of added using an Eppendorf micropiapproximately 0.5 mm3 and washed in a pette. The tubes were then mixed and inmodified Romanoff’s avian Ringer’s solu- cubated for 30 min at 37” in a shaking tion (RRA). This is a phosphate buffer iso- metabolic incubator (120 rpm) open to the tonic with chicken plasma (Romanoff, air. After incubation the tubes were imme1943) which is gassed with air and ad- diately spun at 500g for 5 min at room justed to pH 7.3 with N NaOH; 0.5% temperature. The cell-free medium was debovine serum albumin (Sigma Chemical canted and assayed either at once, or Co., Fraction V) was added as a protein stored at -20”. buffer. The tissue pieces were then transGenerally four tubes were used at each ferred to a siliconised (Siliclad, Becton dose level. Blocks of four control tubes Dickinson Inc., Parsipanny, NJ) glass tube (containing 0.5 ml RRA plus the suspenMATERIALS
AND
METHODS
LHRH sion) were placed at the beginning, in the middle, and at the end. A routine incubation consisted of about 80 tubes. Radioimmunoassay The radioimmunoassay for avian LH (Follett et al., 1972) was modified to desensitize the operating range and thus allow the direct assay of aliquots of the incubation medium without a dilution step. This was achieved by (a) using a less sensitive antiserum (16/6) at a dilution such that the final B/F was ca. 2.0, (b) adding labeled hormone at the same time as the unlabeled standards and unknowns, (c) carrying out the initial incubation at 37” for 6 hr, and (d) using a relatively large volume (400~1). After this incubation, precipitation with anti-rabbit gamma globulin was carried out overnight at 4”. The results have been expressed in terms of IRC-2, a highly purified chicken LH fraction used as a laboratory standard. Each unknown was assayed in triplicate at a single dose of 501.~1.The amounts of LH released in each tube were calculated in nanogram equivalents of IRC-2 and the data usually transformed so that the response is expressed as the percentage increase in LH release above that found in the control tubes. Materials Tested for LH-Releasing Activity Two synthetic preparations of the LHRH decapeptide (Baba et al., 1971) were kindly supplied by Dr. J. R. Reel (Parke-Davis Inc., Ann Arbor, Michigan) and by Dr. H. Gregory (I C I Ltd., Alderley Edge, Cheshire). Standard solutions were prepared by dissolving in 0.9% saline to a concentration of 200 pg/ml. Aliquots of this standard were kept in sealed polythene vials at -20”. For each assay a vial was melted and the contents diluted in RRA to an appropriate concentration. A sample of synthetic TRH was kindly supplied by Dr. R. A. Guillemin (Salk Institute, La Jolla, California).
143
BIOASSAY
Hypothalamic and other tissue extracts were prepared by homogenization in icecold 0.1 N HCl (100 Fl/hypothalamus or tissue weight equivalent, ca. 1.5 mg) followed by centrifugation at 10,OOOg for 15 min. The supematants were stored at -20”. Before use the extracts were neutralised with N NaOH, centrifuged to remove any precipitated material, made up to an appropriate volume with RRA, and the pH adjusted to exactly 7.3 with 0.1 N NaOH. Tissues were obtained from Japanese quail in various reproductive states that had been reared in this laboratory under controlled lighting conditions, from immature broiler chickens and from rats. All tissues were excised immediately following death and either weighed and extracted at once or frozen on dry-ice for later extraction. Various biologically active compounds likely to occur in hypothalamic tissues were also tested for releasing activity at a variety of dose levels. These were obtained commercially. RESULTS
General Comments on the Preparation and Incubation of the Pituitary Cell Suspension In preparing the cell suspension it is important that certain precautions are adhered to so that an actively secreting and responsive suspension is obtained. Siliconised vessels must be used to prevent cell adhesion and the pituitary tissues must be treated gently during dispersion, centrifugation, and resuspension to minimize physical disruption of the cells. An incubation period of 60 min with collagenase appears optimal, producing both effective dispersion and responsive cells (Fig. 1). Shorter incubation times failed to disperse the cells adequately while too long an incubation led to a less responsive suspension. In the present arrangement an isotonic
144
BICKNELL
AND FOLLETT
FIG. 1. Dispersed chicken pituitary cells as they appear under phase-contrast microscopy. The bar is 50 pm in length.
avian Ringer’s was used with the addition of bovine serum albumin as a protein buffer. However, these conditions do not appear mandatory and good suspensions have been produced with both rat and chicken pituitaries using a Krebs-Ringer’s bicarbonate glucose buffer with added BSA. Filtration through nylon gauze was necessary to remove connective tissue and clumps of undispersed cells. An important practical point was the necessity to keep the suspension stirred thoroughly while aliquots were being dispensed to the incubation tubes. In later experiments this step has been performed according to a randomized sequence to eliminate any bias in the numbers of cells dispensed to each tube. If this was done then the rate of basal LH release from control tubes scattered through an assay remained relatively constant. The mean value from 14 experiments was 39.1 & 2.6 rig/ml. Between assays the rate of release was subject to some variation -four examples are shown in Table 1.
Quantitative assays are currently performed using a balanced four-point design (2 + 2) with four tubes per dose level. In practice an excess of cell suspension is prepared. If 30 pituitaries are dispersed enzymatically this allows for 120 tubes. The number actually incubated ranges from 50 to 100, allowing for the quantitative assay of eight unknowns. The desensitisation of the LH RIA considerably reduces the assay time but more TABLE
1
THERATEOFLH RELEASEINCONTROLTUBES DISTRIBUTEDTHROUGHOUTFOURASSAKS LH release (ng/mlP Assay number 54 55 56 57
57.0 43.4 46.4 39.6
in tubes numbered
18
31
48
49
66
79
96
55.0 44.2 43.4 38.4
50.0 47.6 54.0 44.4
55.0 48.2 43.6 37.2
54.0 44.0
55.0 42.4
56.0 46.0
55.0 47.8
33.8
40.0
41.6
47.0
” ng LH released/ml incubation medium during tion in control tubes containing 0.5 ml cell suspension only. b Tube number in the particular assay.
a 30-min incubaand 0.5 ml medium
LHRH
BIOASSAY
145
importantly, it increases the accuracy since the dilution step is omitted. Previously a tenfold dilution had to be incorporated as the 50% binding of IRC-2 in the original assay occurred with 0.8 ng; this compares with 6.0 ng in the present assay. ERects of Synthetic Mammalian LHRH
and TRH
Solutions of synthetic mammalian LHRH in RRA stimulated the release of LH above the levels found in control tubes receiving only RRA. Dose-response relationships were regularly obtained and Fig. 2 shows the combined data from 27 experiments. The log-linear relationship between 0.2 and 10 rig/ml is given by the equation Y = - 13.02 + 81.40X. The minimal effective dose was approximately 200 pg/ml of incubate (i.e., a dose of 200 pg added in 0.5 ml RRA to 0.5 ml cell suspension). The two synthetic preparations had identical potencies. The dose-response curves in Figs. 2, 4a, b, d, and 5 may be compared directly with those pub-
FIG. 3. The effect of incubation time (minutes) on LH release from pituitary cell suspensions containing either 1.0 rig/ml synthetic LHRH, or Ringer’s solution alone. Data are from a single experiment, each point representing the Mean 2 SEM of the four replicate tubes.
lished for LHRH radioimmunoassays (Jeffcoate et al., 1973, 1974) as Dr. S. L. Jeffcoate compared our standard with his in a RIA and found them to of similar immunopotency. As might be expected in any bioassay some small variation exists in the slope and sensitivity of individual assays (compare Figs. 4a, b, d and 5) but this was relatively small. Occasionally extremely unresponsive cell suspensions were obtained, however, where the slope and/or sensitivity fell outside the normal range. The basis of this phenomenon is unknown but it might be due to the variable age and nutritional status of the broiler chickens used as pituitary donors (R. C. Bonney and F. J. Cunningham, personal communication). -0.1 10 10 0 Synthetic mammalian TRH did not stimLHRH (rig/ml) ulate LH release above control values FIG. 2. Dose-response relationship between the when incubated with pituitary cells at dose of synthetic mammalian LHRH (rig/ml) and LH doses ranging from 0.1 to 200 rig/ml. Thus release from chicken pituitary cell suspension. The TRH must be at least 1000 times less povertical axis shows LH release in terms of the percentage increase in release above that found in the tent than LHRH in the assay. Neither syncontrol tubes. The data are drawn from 27 separate thetic LHRH or TRH cross-reacted in the experiments. The numbers in parentheses indicate the LH radioimmunoassay. total number of estimates at a particular dose. The The effect of incubation times on the line was calculated by the method of least squares for rate of LH release in untreated tubes and doses between 0.25 and 10.0 rig/ml. The full 95% confidence limits are shown above and below the line. in tubes treated with synthetic LHRH is
146
BICKNELL
10.01
AND
FOLLETT
1.0
001
LHRH,ng/mll 0.1 Quail HE (mg/ml)
LHRHlnglmll Chlcken
0.1 HE (mg/ml)
1.’
tyfizQF Rot HE (mglml)
FIG. 4. Dose-response relationships in four separate experiments. The doses of synthetic LHRH (&ml) or of the particular hypothalamic extracts (mg extracted tissue/ml) are shown on the horizontal axis and the LH release on the vertical axis. As before, LH release is expressed in terms of the % increase above that found in the control tubes. Each point represents the Mean & SEM of four replicate tubes (each assayed in triplicate). (a) Doubling dilutions of quail hypothalamic extract (mature female, top dose equiv. to 0.25 of a hypothalamus) and of synthetic mammalian LHRH. (b) Chicken hypothalamic extract (broiler chicken, top dose equiv. to 0.2 of a hypothalamus) and synthetic mammalian LHRH. (c) Chicken and quail hypothalamic extracts. The top dose of the chicken extract was equiv. to 0.18 of a hypothalamus and that of the quail (immature male) to 0.3. (d) Rat hypothalamic extract (mature male, top dose equiv. to 0.3 of a hypothalamus) and chicken hypothalamic extract (top dose equiv. 0.16 hypothalamus) together with synthetic LHRH. (C-0) quail hypothalamic extract; (m-0) chicken hypothalamic extract; (A...A) synthetic LHRH; (m...M) rat hypothalamic extract.
shown in Fig. 3. In the presence of 1.0 rig/ml synthetic LHRH the rate of release was greatest during the first 10 min of incubation. In untreated tubes the rate of release was negligible during the first 30 min of incubation. It increased after this time but remained steady over the next hour; 1.0 rig/ml LHRH elicited the greatest percentage increase in LH release over control levels at approximately 30 min and this incubation time was used routinely.
Efects of Hypothalamic Other Tissue Extracts
and
Extracts of the basal hypothalamusmedian eminence region of quail, chicken, and rat increased LH release as compared with levels in control tubes (Fig. 4a-d). The dose-response curves obtained with both avian and rat hypothalamic extracts were invariably steeper than those obtained with synthetic mammalian LHRH (Fig. 4a-d). Approximately parallel dose-
LHRH
Cerebral
cortex
,mg,m,)
FIG. 5. Dose-response curves to show the effect of synthetic LHRH (A...A, rig/ml), chicken hypothalamic extract (o-0, mg/ml, top dose equiv to 0.16 of a hypothalamus) and quail cerebral cortex extract 0-0, mg/ml, top dose equiv to 5 quail hypothalami) on LH release from pituitary cell suspension. The vertical axis shows LH release in terms of the % rise above that found in control tubes. The data are from a single experiment, each point representing the Mean -C SEM of four replicate tubes.
response curves were obtained between quail and chicken hypothalamic extracts (Fig. 4c), and between chicken and rat hypothalamic extracts (Fig. 4d). Extracts of other brain regions of the quail including the cerebral cortex, midbrain, and hindbrain were also found to be capable of increasing LH release above basal levels although the amount of releasing activity present in these tissues was relatively low compared with that in the hypothalamus. Thus the cerebral cortex contained about 5% and the hindbrain 10% of the activity of an equal weight of hypothalamic tissue. Dose-response curves could be obtained with these nonhypothalamic tissue extracts (Fig. 5) and these were invariably less steep than those obtained with hypothalamic extracts or with synthetic LHRH. Extracts of liver
BIOASSAY
147
and testis produced no increase in LH release. All tissue extracts were assayed for any LH they might contain and the LH levels in the incubation media suitably adjusted. In practice LH activity was found only in chicken and quail hypothalamic extracts (rat LH does not cross-react in the avian RIA) and generally the amounts present were small relative to the LH in the incubation tubes. Quail hypothalamic extracts contained about 8 ng LH/hypothalamus (similar to that reported by Smith and Follett, 1972) while the amounts of LH in the incubation tubes ranged from 50 to 140 ng. A chicken hypothalamus contained about 50 ng. The source of this hormone is not known but Sharp (1974) reports the presence of LH-containing cells in the pars tuberalis which often remains attached to the median eminence during dissection. Effects of Other Biologically Active Compounds As shown in Table 2, various other biologically active compounds known to occur in brain tissues had little or no effect on LH release. In one experiment acetylcholine (2 pg/ml) did increase LH release by 66% but in the same assay 1.0 rig/ml of LHRH increased it by 123%. Thus in terms of mass LHRH is more than 2000 times as potent as acetylcholine. The Quantitative Assay of Releasing Activity in Tissue Extracts A four-point balanced factorial assay (2 + 2) was employed using four tubes at each dose level. Potency estimates of hypothalamic extracts were initially calculated in terms of synthetic LHRH but nonparallelism appeared in a number of experiments (Fig. 4a, b, d). For this reason a pooled extract of 50 chicken hypothalami was prepared and this substandard (CSl) was assayed against synthetic LHRH re-
148
BICKNELL
AND
TABLE LH
RELEASE
CAUSED
BY A VARIETY
Expt
Substance and dose
1
LHRH-1 rig/ml LHRH-10 rig/ml Dopamine-IO @ml Dopamine-500 @ml Histamine-20 rig/ml Histamine-l pg/ml AcetylcholineAO rig/ml Acetylcholine-2 pg/ml
2
LHRH-8 &ml Acetylcholine-50 &ml Acetylcholine-1 pg/rnl Adrenaline-20 rig/ml Adrenaline-200 @ml Adrenaline-2 &ml
FOLLETT
2
OF PHARMACOLOGICALLY
ACTIVE
COMPOUNDS
Expt
Substance and dose
LH releasea
123 225 2 6 4 6 11 66
3
43 -6 11 -6 -10 -17
4
LHRH-2 rig/ml GABA-I &ml GABA-IO &ml Serotonin-10 rig/ml Serotonin-500 rig/ml Noradrenalinerig/ml Noradrenaline@ml Vasopressin-200 @ml Vasopressin-2 pg/rnl LHRH-2 rig/ml Cyclic AMP-500 &ml Cyclic AMP-5 pg/ml
80 13 8 5 2 1 -4 2 4 98 1 2
LH release”
a LH Release is expressed as the % rise (or fall) from that the LH release found in the control tubes. Each value is the mean of four replicates (assayed in triplicate). GABA is an abbreviation for gamma-amino butyric acid.
peatedly to obtain an estimate of its potency. An unknown extract, therefore, is assayed against CSl and its mean potency, together with the 95% confidence limits, expressed in terms of CSI. For comparative purposes an approximation of the mean activity in terms of LHRH is usually given. A typical assay included four or five doubling dilutions of a standard preparation (LHRH or CSl). The top dose of LHRH generally used was 8 rig/ml while TABLE POTENCY
ESTIMATES
OF LH
that of CS 1 contained 0.16 equivalents of a chicken basal hypothalamus. Two doubling dilutions of the unknown were used. With quail hypothalamic extracts the top dose used contained 0.25 equivalents of the basal hypothalamus. Table 3 summarizes data from four assays using hypothalamic extracts prepared from quail in differing reproductive states. The mean index of precision for the first 16 assays was 0.069 ? 0.004 (SEM). Such indices do not 3
RELEASING ACTIVITY IN HYPOTHALAMIC THE JAPANESE QUAIL
Group
No. of hypothalami=
wt (mgY
Mean
Immature males Immature males Mature males Castrate males
4 4 4 4
1.73 1.90 1.70 1.42
LH-RH potency 17.9(14.8-21.4) 15.7(12.9-18.8) 18.5(15.8-21.6) 32.2(24.4-40.2)
EXTRACTS
FROM
LHRH potencyd
Lambda’
7.2 6.2 7.4 12.9
0.071 0.068 0.060 0.086
a The number of hypothalami pooled from the group of birds prior to extraction with 0.1 N HCI. * Mean weight (mg) of individual hypothalamic pieces. c LH-RH potency/mg tissue, together with its 95% confidence limits, is given in terms of the chicken hypothalamic standard CS 1. d Approximate mean potency in terms of ng synthetic LHRHimg tissue. Calculated by conversion from CS 1. c Lambda--the index of precision of the assay.
LHRH consider the error on the LH radioimmunoassay. However all incubation media from one experiment were included in a single RIA and Gibson et al. (I 975) have calculated the intra-assay coefficient of variation in the immunoassay to be about 10%. DISCUSSION
The results presented in this study show that the use of a suspension of pituitary cells enables an assay to be developed for hypothalamic LHRH which is rather more convenient, accurate, and reproducible than heretofore. The advantages over previous in vitro bioassay methods, apart from those of a practical nature, lie mainly in the replication of treatments that can be achieved using a homogeneous preparation of pituitary cells rather than paired or randomised pituitary fragments. The homogeneity of the system also allows free intra-assay comparisons of treatments while the use of a standard reference preparation allows inter-assay comparisons. It is perhaps this last point that is especially valuable as previously it has not been possible to make valid comparisons between assays and the designs used have not allowed the potency of an unknown to be expressed in terms of a standard. The index of the precision of the assay lies between 0.05 and 0.1. This compares favourably with an index calculated for an assay using pituitary fragments (e.g., Jamieson et al., 1973. index of precision = 0.2). There seems little doubt that the dispersed pituitary cells are releasing LH in response to a specific LH releasing factor present in the quail and chicken hypothalamus. In this regard the observation that synthetic mammalian TRH did not increase LH release is important. This has been supported by some recent results from Scanes (1974). He has shown that TRH significantly increases the secretion of bioassayable TSH from chicken hemipi-
BIOASSAY
149
tuitaries but that immunoreactive LH levels are not elevated under these conditions. The converse is also true-synthetic LHRH increased LH secretion but not that of TSH. All these data argue for the specificity of both the cell suspension assay, and the immunoassay of LH. The question of exactly how specific the LH release response of the dispersed cells is to LHRH is raised by the finding that extracts from other brain areas also increase LH secretion significantly. This effect has been reported for other in vitro systems (e.g., Wakabayashi et al., 1972; Smith and Follett. 1972; Hayes et al.. 1973; Barofsky et crl., 1973) but it is not known if the release is due to some nonspecific factor such as an elevated potassium concentration (Samli and Geschwind, 1968), or to small amounts of LHRH present in the tissues. The recent findings of substantial amounts of gonadotrophinreleasing hormone in the pineal (White et al., 1974) indicate that LHRH may indeed be present in brain areas other than the hypothalamus. However, the steeper dose-response curves obtained with hypothalamic extracts when compared with synthetic LHRH, and the less steep ones found with other brain tissues suggests that nonspecific factors may be the cause of the LH release by nonhypothalamic tissue extracts. It seems rather likely that the nonspecific factor is also present in the hypothalamus and is the cause of the steeper dose-response curves found with both avian and rat hypothalamic extracts. Despite considerable effort the factor responsible has not been discovered: possibly it might be acetylcholine. The chemical identity of avian LHRH remains to be elucidated. Jackson (1971 a, b) has reported that rat and chicken LHRHs behave differently on ion-exchange columns. He used a bioassay for LH-releasing activity based upon the in vitro incubation of rat hemipituitaries. More recently, Jeffcoate et al. (1974) using
150
BICKNELL
AND
a mammalian LHRH radioimmunoassay, found no separation of rat, chicken and synthetic LHRH by ion-exchange or silica gel thin-layer chromatography. They also obtained parallel dilution curves for the avian and mammalian materials. In the present study rat and chicken hypothalamic extracts gave parallel dose-response curves but this is not proof of identity, especially as neither gave a parallel curve to that of synthetic LHRH. Some preliminary findings (S. L. Jeffcoate, R. J. Bicknell, and P. J. Sharp, unpublished) do suggest, however, a difference between chicken and ovine LHRH. If a chicken hypothalamic extract is assayed against synthetic LHRH in both the dispersed cell assay and the LHRH radioimmunoassay its potency is at least 100 times greater in the former. Estimates of the amounts of LHRH present in the quail hypothalamus (Table 3) are of the same order as those reported for mammalian species (e.g., Schally et al., 1971; Jeffcoate et al., 1974; White, et al., 1974). There appear to be no great changes in hypothalamic LHRH concentration associated with the reproductive state of the birds although long-term castrated quail held on long days showed an approximate 100% increase in stored concentration. This must be considered in the light of changes in pituitary and plasma LH. Sexually mature quail have plasma and pituitary LH levels some five to eight times as great as in immature birds while castration leads to still greater increases (Follett et al., 1972; Nicholls et al., 1973; Gibson et al., 1975). While the dispersed cell assay is extremely sensitive (it is capable of measuring the LHRH in a single quail hypothalamus in a full 2 + 2 assay) it does not seem capable of assaying circulating LHRH and is not as sensitive as Jeffcoate’s radioimmunoassay. It does seem likely that radioimmunoassay will dominate LHRH measurements over the next
FOLLETT
few years but the present assay could be useful in a number of respects. First, it will provide a biological comparison with the strictly immunological assay-to our knowledge no one has carried out a comprehensive comparison of bio- and immunopotencies of LHRH. Secondly, it may be valuable in lower vertebrates. The immunopotency of chicken hypothalamic extracts is very low (Jeffcoate et d., 1974) and yet in the dispersed cell assay the potency is comparable with that in other vertebrates. ACKNOWLEDGMENTS This work was supported by a Grant from the Agricultural Research Council (AB18/1). We are grateful to J. P. Wood and Sons Ltd. (Llangefni, Anglesey) for providing the chicken pituitary glands.
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Barofsky, A-L., Dowd, A., Santner, S., Chaudhuri, N.. Lloyd, C. W., and Weisz. J. (1973). Problems in maintaining potency of synthetic LH-RF: studies using a pituitary perifusion system. Excerpta Med. Int. Cong. Ser. 263, 105-109. Blackwell, R., Amoss, M., Vale, W., Burgus, R., Rivier, J., Monahan, M., Ling, N., and Guillemin, R. (1973). Concomitant release of FSH and LH induced by native and synthetic LRF. Amer.
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LHRH tissue of Coturnix quail. Po~rlrry Sci. 50, 1562-1563. Clark, C. E., and Fraps, R. M. (1967). Induction of ovulation in the chicken with median eminence extracts. Poultry Sci. 46, 1245- 1246. Crighton, D. B., Hartley, B. M., and Lamming, G. E. (1973). Changes in the luteinizing hormone releasing activity of the hypothalamus, and in pituitary gland and plasma luteinizing hormone during the oestrous cycle of the sheep. J. Endocrinol.
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Fink. G., and Harris, G. W. (1970). The luteinizing hormone releasing activity of extracts of blood from the hypophysial portal vessels of rats. J. Physiol. 208, 22 1-24 1. Follett, B. K. ( 1970). Gonadotropin-releasing activity in the quail hypothalamus. Gen. Camp. Endocrinol.
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Follett. B. K., Scanes, C. G., and Cunningham, F. J. (1972). A radioimmunoassay for avian luteinizing hormone. J. Endocrinol. 52, 359-378. Furr, B. J. A., Onuora. G. I., Bonney, R. C., and Cunningham, F. J. (1973). The effect of synthetic hypothalamic releasing factors on plasma levels of luteinizing hormone in the cockerel. J. Endocrinol.
59, 495-502.
Gibson, W. R., Follett, B. K.. and Gledhill, B. (1975). Plasma lutenizing hormone (LH) levels in gonadectomized Japanese quail exposed to short or long daylengths. J. Endocrinol. 64, 87- 10 I. Guillemin, R., and Vale, W. (1970). Bioassay of the hypophysiotrophic hormones: in vitro systems. In Hypophysiotrophic Hormones of the Hypothalam~s: Assay arzd Chemistry (Meites. J, ed.), pp. 21-35. Baltimore: Williams and Wilkins. Hayes, M. M.. Knight, B. K.. and Warton, C. M. R. (1973). Preliminary studies on the functional relationships between the pineal, hypothalamus and adenohypophysis using bovine tissues in organ culture. Cent. Afr. J. Med. 19, 193-194. Hopkins. C. R., and Farquhar, M. G. (1973). Hormone secretion by cells dissociated from rat anterior pituitaries. J. Cell Biol. 59, 276-303. Jackson, G. L. (1971a). Avian luteinizing hormonereleasing factor. Endocrinology 89, 1454-1459. Jackson, G. L. (1971b). Comparison of rat and chicken luteinizing hormone-releasing factors. Endocrinology
89, 1460-
1463.
Jackson, G. L., and Nalbandov, A. V. (1969). Luteinizing hormone releasing activity in the chicken hypothalamus. Endocrinology 84, 1262-1265. Jamieson, M. G., Fink, G., and Chiappa. S. A. (1973). An in vitro method for the assay of luteinizing hormone-releasing factor. 1. Endocrinol. 59, xi-xii. Jeffcoate, S. L., Fraser. H. M., Gunn, A., and Holland, D. T. (1973). Radioimmunoassay of lu-
151
BIOASSAY
teinizing hormone releasing factor. J. Endocrinol. 57, 189-190. Jeffcoate. S. L., Sharp, P. J., Fraser, H. M., Holland, D. T.. and Gunn, A. (1974). Immunological and chromatographic similarity of rat, rabbit, chicken and synthetic luteinizing hormone-releasing hormones. .I. Endocrinol. 62, 85-89. Kamberi, 1. A., Mical, R. S., and Porter, J. C. ( 197 1). Hypophysial portal vessel infusion-in virso demonstration of LRF, FRF, and PIF in pituitary stalk plasma. Endocrinology 89, IO42- 1046. McIlhinney, R. A. J., and Schulster. D. (1973). Adrenocorticotrophin assay. using a radioreceptor method and a bioassay involving isolated adrenal cells, for studies on isolated anterior pituitary cells. J. Endocrinol. 57, xlvi-xlvii. Nicholls, T. J., Scanes, C. G.. and Follett. B. K. (1973). Plasma and pituitary luteinizing hormone in Japanese quail during photoperiodically induced gonadal growth and regression. Gen. Camp.
Endocrinol.
21, 84-98.
Opel. H., and Lepore. P. D. (1967). Ovulating hormone-releasing factor in the chicken hypothalamus. Poultrv Sci. 46, 1302. Opel, H., and Lepore, P. D. (1972). In \aivo studies of luteinizing hormone-releasing factor in the P02tltry chicken hypothalamus. Sci. 51, 1004-1014. Portanova, R.. Smith, D. K.. and Sayers. G. (1970). A trypsin technic for the preparation of isolated rat anterior pituitary cells. Proc. Sot. Exp. Biol. Med.
133, 573-576.
Reeves, J. J.. Harrison, P. C., and Casey, J. M. (1973). Ovarian development and ovulation in hens treated with synthetic (porcine) LHIFSHRH. Poultry Sci. 52, 1883-1886. Romanoff, A. (19431. Differentiation in respiratory activity of isolated embryonic tissues. J. ,I+. Zool.
93, I-26.
Samli. M. H., and Geschwind, 1. I. (1968). Some effects of energy-transfer inhibitors and of Ca++free or K+-enhanced media on the release of luteinizing hormone (LH) from the rat pituitary gland in vitro. Endocrinology 82, 225-23 1. Scanes, C. G. (1974). Some in ,jitro effects of synthetic thyrotrophin releasing factor on the secretion of thyroid stimulating hormone from the anterior pituitary gland of the domestic fowl. Neuroendocrinology
15, I-9.
Scemama, A. (1972). evolution post-natale des “Releasing-Factors” hypothalamiques. J. Physiol. (Paris)
65, 298A.
Schally, A. V., Nair, R. M. G., Redding, T. W., and Arimura, A. (197 I). Isolation of the luteinizing hormone and follicle-stimulating hormonereleasing hormone from porcine hypothalami. J. Bioi.
Chem.
246, 7230-7236.
152
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AND
Schneider, H. P. Cl., and McCann, S. M. (1969). Effect of dopamine on release of LH-releasing factor by stalk-median eminence (SME) tissue in vitro.
Fed.
Proc.
28,
381.
Sharp, P. J. (1974). Immunofluorescent localization of pituitary cells in poultry using antichicken LH. Gen.
Comp.
Endocrinol.
22,
365-366.
Smith, P. M., and Follett, B. K. (1972). Luteinizing hormone releasing factor in the quail hypothalamus. /. Endocrinol. 53, 13 I- 138. Steinberger, A., Chowdhury, M.. and Steinberger, E. (1973). Effect of repeated replenishment of hypothalamic extract on LH and FSH secretion in monolayer cultures of rat anterior pituitary cells. Endocrinology 92, 12- 17. Takahara, J., Uehara, T., Arimura, A., and Schally, A. V. (1973). In vitro assay for growth hormonereleasing hormone using pituitary cell suspension. Fed. Proc. 32. 489. van Tienhoven. A., and Schally, A. V. (1972). Mammalian luteinizing hormone-releasing hormone induces ovulation in the domestic fowl. Gen.
FOLLETT
Camp. Endocrinol. 19, 594-595. Tixier-Vidal. A., Kerdelhue, B., and Jutisz, M. (1973). Kinetics of release of lutenizing hormone (LH) and follicle stimulating hormone (FSH) by primary cultures of dispersed rat anterior pituitary cells. Chronic effect of synthetic LH and FSH releasing hormone. Liff Sci. 12, 499-509. Vale, W., Grant. G.. Amoss. M., Blackwell, R., and Guillemin, R. (1972). Culture of enzymatically dispersed anterior pituitary cells: functional validation of a method. Endocrinology 91, 562-572. Wakabayashi, K.. Antunes-Rodrigues, J., Tamaoki, B-l.. and McCann, S. M. (1972) 111tjitro effect of hypothalamic extract and other stimulating agents on glucose oxidation and luteinizing hormone (LH) release from rat anterior pituitary glands. Efzdocrinolosqy 90, 690-699. White, W. F.. Hedlund, M. T., Weber. G. F.. Rippel, R. H., Johnson, E. S.. and Wilber, J. F. ( 1974). The pineal gland: a supplemental source of hypothalamic releasing hormones. Endocrinohg) 94, 1422-1426.
AND
COMPARATIVE
ENDOCRINOLOGY
26,
141-152 ( 1975)
A Quantitative Assay for Luteinizing Hormone Releasing Hormone (LHRH) Using Dispersed Pituitary Cells R. J. BICKNELL AND B. K. FOLLETT Department
of Zoology, University College Bangor, Gwynedd, LL57 2UW,
of North U.K.
Wales.
Accepted December 3, 1974 An assay for luteinizing hormone releasing hormone (LHRH) is described that utilises dispersed pituitary cells. Chicken pituitaries were dispersed with collagenase and after washing, aliquots of the cell suspension were dispensed into polystyrene tubes containing treatments. Tubes were incubated for 30 mins, cells spun off, and supernatants assayed for LH content by radioimmunoassay. LHRH increased LH secretion markedly and a dose-response relationship was established over the range 200 pg/ml to 10 nglml. Hypothalamic extracts from chicken, quail, and rat were also effective but gave dose-response curves steeper than that found with LHRH. Other brain areas showed low levels of LH-releasing activity but the dose-response curves were rather flat for these extracts. A number of pharmacologically active compounds were tested but only acetylcholine in large doses elicited significant LH release. When used as a quantitative bioassay with four tubes per treatment, measurements can be made of the LHRH content in single hypothalami. The mean index of precision was 0.069 2 0.004 (16).
The existence of a luteinizing hormone releasing hormone (LHRH) in the avian hypothalamus has been demonstrated both in vivo and in vitro. Chicken hypothalamic extracts cause ovulation in hens (Clark and Fraps, 1967; Opel and Lepore, 1967, 1972) while similar extracts from quail increase plasma LH levels in rats (Casey et al., 1971). Synthetic mammalian LHRH is also effective in inducing ovulation (van Tienhoven and Schally, 1972; Reeves et al., 1973) and in elevating plasma LH in the chicken (Furr et al., 1973; Bonney ef al., 1974). In vitro, chicken hypothalamic extracts increase the rate of LH release from rat pituitaries (Jackson and Nalbandov, 1969; Jackson, 1971a, b) while quail hypothalamic extracts stimulate avian LH release from incubated chicken hemipituitaries (Follett, 1970), and from superfused quail pituitaries (Smith and Follett, 1972). Recently LHRH has been
detected in chicken hypothalamic extracts by a radioimmunoassay (RIA) for mammalian LHRH (Jeffcoate et al., 1974). This study describes a bioassay technique for use in the investigation of avian LHRH. It was developed because most if not all previous techniques for assaying releasing factors have been only semiquantitative. In vivo methods (e.g., Fink and Harris, 1970; Kamberi et al., 1971; Burger et al., 1972; Campbell, 1972; Opel and Lepore, 1972) are laborious and suffer from the defect that only a very limited series of treatments can be given to a single animal. The established in vitro method of assay consists of incubating pituitary fragments with unknown LHRH samples and measuring the LH released into the incubation medium by bio- or immunoassay (e.g., Schneider and McCann, 1969; Follett, 1970; Jackson, 1971a, b; Scemama, 1972; Jamieson et al., 1973; 141
Copyright 0 1975 by Academic Press, Inc. All tights of reproduction in any form reserved.
142
BICKNELL
AND
Crighton et al., 1973; Blackwell et al., 1973). To some extent this method can be made quantitative by comparing the hormone released from contralateral halves of the same pituitary but it suffers from various limitations, including problems of replication and interassay comparison (see Guillemin and Vale, 1970). In addition it is usually not feasible to measure an unknown solution in more than one treatment and it has proved difficult to quote the potency in terms of an acceptable standard. A super-fusion method overcomes some of these problems (e.g., Smith and Follett, 1972) but is tricky technically. When techniques were developed for the preparation of a homogeneous suspension of pituitary cells by enzymatic dispersion (Portanova et al., 1970; Vale et al., 1972; Takahara et al., 1973; Hopkins and Farquhar, 1973; Tixier-Vidal et al., 1973; McIlhinney and Schulster, 1973; Steinberger et al., 1973) as well as for the assay of gonadotrophins by radioimmunoassay it became feasible to develop a multireplicate in vitro incubation system in which one might include an LHRH standard, and where routine bioassay statistics could be applied.
FOLLETT
(diam 25 mm) containing 10 ml of a 0.02% solution of collagenase (Sigma, type III, fraction A) in Romanoff’s Ringer’s albumin. Generally 30 whole pituitaries, each weighing about 8 mg, were used. The tissue was incubated at 37” and constantly agitated at about 500 rpm by a siliconised glass stirrer. After 20 and 40 min of incubation the tissue pieces were drawn gently into and out of a siliconised Pasteur pipette to aid dispersion. After 60 min the dispersion was completed by repeating pipetting. The cell suspension was then centrifuged at 1OOg for 10 min at room temperature and the supernatant discarded. Five milliliters of fresh RRA was added and the pellet of cells resuspended by gentle pipetting. The steps of centrifugation, removal of supernatant, addition of fresh RRA, and resuspension of the cells were repeated once. The suspension of pituitary cells was then filtered through a piece of nylon gauze (10 mm diam, 60 pm mesh) mounted over a siliconised 100 ml flask containing a magnetic stirrer. RRA was added to give a final concentration equivalent to 0.5 pituitaries/ml. Incubations
Incubations were carried out in disposable polystyrene tubes (12 x 75 mm). Pituitary Cell Suspension The test materials were diluted in RRA Pituitary glands were collected from such that each dose was contained in 0.5 broiler chickens at a local abattoir within a ml and these were added to the polysfew minutes of death and placed in 0.85% tyrene tubes first: 0.5 ml aliquots of the saline on ice. In the laboratory they were stirred pituitary cell suspension were then diced with a razor blade into pieces of added using an Eppendorf micropiapproximately 0.5 mm3 and washed in a pette. The tubes were then mixed and inmodified Romanoff’s avian Ringer’s solu- cubated for 30 min at 37” in a shaking tion (RRA). This is a phosphate buffer iso- metabolic incubator (120 rpm) open to the tonic with chicken plasma (Romanoff, air. After incubation the tubes were imme1943) which is gassed with air and ad- diately spun at 500g for 5 min at room justed to pH 7.3 with N NaOH; 0.5% temperature. The cell-free medium was debovine serum albumin (Sigma Chemical canted and assayed either at once, or Co., Fraction V) was added as a protein stored at -20”. buffer. The tissue pieces were then transGenerally four tubes were used at each ferred to a siliconised (Siliclad, Becton dose level. Blocks of four control tubes Dickinson Inc., Parsipanny, NJ) glass tube (containing 0.5 ml RRA plus the suspenMATERIALS
AND
METHODS
LHRH sion) were placed at the beginning, in the middle, and at the end. A routine incubation consisted of about 80 tubes. Radioimmunoassay The radioimmunoassay for avian LH (Follett et al., 1972) was modified to desensitize the operating range and thus allow the direct assay of aliquots of the incubation medium without a dilution step. This was achieved by (a) using a less sensitive antiserum (16/6) at a dilution such that the final B/F was ca. 2.0, (b) adding labeled hormone at the same time as the unlabeled standards and unknowns, (c) carrying out the initial incubation at 37” for 6 hr, and (d) using a relatively large volume (400~1). After this incubation, precipitation with anti-rabbit gamma globulin was carried out overnight at 4”. The results have been expressed in terms of IRC-2, a highly purified chicken LH fraction used as a laboratory standard. Each unknown was assayed in triplicate at a single dose of 501.~1.The amounts of LH released in each tube were calculated in nanogram equivalents of IRC-2 and the data usually transformed so that the response is expressed as the percentage increase in LH release above that found in the control tubes. Materials Tested for LH-Releasing Activity Two synthetic preparations of the LHRH decapeptide (Baba et al., 1971) were kindly supplied by Dr. J. R. Reel (Parke-Davis Inc., Ann Arbor, Michigan) and by Dr. H. Gregory (I C I Ltd., Alderley Edge, Cheshire). Standard solutions were prepared by dissolving in 0.9% saline to a concentration of 200 pg/ml. Aliquots of this standard were kept in sealed polythene vials at -20”. For each assay a vial was melted and the contents diluted in RRA to an appropriate concentration. A sample of synthetic TRH was kindly supplied by Dr. R. A. Guillemin (Salk Institute, La Jolla, California).
143
BIOASSAY
Hypothalamic and other tissue extracts were prepared by homogenization in icecold 0.1 N HCl (100 Fl/hypothalamus or tissue weight equivalent, ca. 1.5 mg) followed by centrifugation at 10,OOOg for 15 min. The supematants were stored at -20”. Before use the extracts were neutralised with N NaOH, centrifuged to remove any precipitated material, made up to an appropriate volume with RRA, and the pH adjusted to exactly 7.3 with 0.1 N NaOH. Tissues were obtained from Japanese quail in various reproductive states that had been reared in this laboratory under controlled lighting conditions, from immature broiler chickens and from rats. All tissues were excised immediately following death and either weighed and extracted at once or frozen on dry-ice for later extraction. Various biologically active compounds likely to occur in hypothalamic tissues were also tested for releasing activity at a variety of dose levels. These were obtained commercially. RESULTS
General Comments on the Preparation and Incubation of the Pituitary Cell Suspension In preparing the cell suspension it is important that certain precautions are adhered to so that an actively secreting and responsive suspension is obtained. Siliconised vessels must be used to prevent cell adhesion and the pituitary tissues must be treated gently during dispersion, centrifugation, and resuspension to minimize physical disruption of the cells. An incubation period of 60 min with collagenase appears optimal, producing both effective dispersion and responsive cells (Fig. 1). Shorter incubation times failed to disperse the cells adequately while too long an incubation led to a less responsive suspension. In the present arrangement an isotonic
144
BICKNELL
AND FOLLETT
FIG. 1. Dispersed chicken pituitary cells as they appear under phase-contrast microscopy. The bar is 50 pm in length.
avian Ringer’s was used with the addition of bovine serum albumin as a protein buffer. However, these conditions do not appear mandatory and good suspensions have been produced with both rat and chicken pituitaries using a Krebs-Ringer’s bicarbonate glucose buffer with added BSA. Filtration through nylon gauze was necessary to remove connective tissue and clumps of undispersed cells. An important practical point was the necessity to keep the suspension stirred thoroughly while aliquots were being dispensed to the incubation tubes. In later experiments this step has been performed according to a randomized sequence to eliminate any bias in the numbers of cells dispensed to each tube. If this was done then the rate of basal LH release from control tubes scattered through an assay remained relatively constant. The mean value from 14 experiments was 39.1 & 2.6 rig/ml. Between assays the rate of release was subject to some variation -four examples are shown in Table 1.
Quantitative assays are currently performed using a balanced four-point design (2 + 2) with four tubes per dose level. In practice an excess of cell suspension is prepared. If 30 pituitaries are dispersed enzymatically this allows for 120 tubes. The number actually incubated ranges from 50 to 100, allowing for the quantitative assay of eight unknowns. The desensitisation of the LH RIA considerably reduces the assay time but more TABLE
1
THERATEOFLH RELEASEINCONTROLTUBES DISTRIBUTEDTHROUGHOUTFOURASSAKS LH release (ng/mlP Assay number 54 55 56 57
57.0 43.4 46.4 39.6
in tubes numbered
18
31
48
49
66
79
96
55.0 44.2 43.4 38.4
50.0 47.6 54.0 44.4
55.0 48.2 43.6 37.2
54.0 44.0
55.0 42.4
56.0 46.0
55.0 47.8
33.8
40.0
41.6
47.0
” ng LH released/ml incubation medium during tion in control tubes containing 0.5 ml cell suspension only. b Tube number in the particular assay.
a 30-min incubaand 0.5 ml medium
LHRH
BIOASSAY
145
importantly, it increases the accuracy since the dilution step is omitted. Previously a tenfold dilution had to be incorporated as the 50% binding of IRC-2 in the original assay occurred with 0.8 ng; this compares with 6.0 ng in the present assay. ERects of Synthetic Mammalian LHRH
and TRH
Solutions of synthetic mammalian LHRH in RRA stimulated the release of LH above the levels found in control tubes receiving only RRA. Dose-response relationships were regularly obtained and Fig. 2 shows the combined data from 27 experiments. The log-linear relationship between 0.2 and 10 rig/ml is given by the equation Y = - 13.02 + 81.40X. The minimal effective dose was approximately 200 pg/ml of incubate (i.e., a dose of 200 pg added in 0.5 ml RRA to 0.5 ml cell suspension). The two synthetic preparations had identical potencies. The dose-response curves in Figs. 2, 4a, b, d, and 5 may be compared directly with those pub-
FIG. 3. The effect of incubation time (minutes) on LH release from pituitary cell suspensions containing either 1.0 rig/ml synthetic LHRH, or Ringer’s solution alone. Data are from a single experiment, each point representing the Mean 2 SEM of the four replicate tubes.
lished for LHRH radioimmunoassays (Jeffcoate et al., 1973, 1974) as Dr. S. L. Jeffcoate compared our standard with his in a RIA and found them to of similar immunopotency. As might be expected in any bioassay some small variation exists in the slope and sensitivity of individual assays (compare Figs. 4a, b, d and 5) but this was relatively small. Occasionally extremely unresponsive cell suspensions were obtained, however, where the slope and/or sensitivity fell outside the normal range. The basis of this phenomenon is unknown but it might be due to the variable age and nutritional status of the broiler chickens used as pituitary donors (R. C. Bonney and F. J. Cunningham, personal communication). -0.1 10 10 0 Synthetic mammalian TRH did not stimLHRH (rig/ml) ulate LH release above control values FIG. 2. Dose-response relationship between the when incubated with pituitary cells at dose of synthetic mammalian LHRH (rig/ml) and LH doses ranging from 0.1 to 200 rig/ml. Thus release from chicken pituitary cell suspension. The TRH must be at least 1000 times less povertical axis shows LH release in terms of the percentage increase in release above that found in the tent than LHRH in the assay. Neither syncontrol tubes. The data are drawn from 27 separate thetic LHRH or TRH cross-reacted in the experiments. The numbers in parentheses indicate the LH radioimmunoassay. total number of estimates at a particular dose. The The effect of incubation times on the line was calculated by the method of least squares for rate of LH release in untreated tubes and doses between 0.25 and 10.0 rig/ml. The full 95% confidence limits are shown above and below the line. in tubes treated with synthetic LHRH is
146
BICKNELL
10.01
AND
FOLLETT
1.0
001
LHRH,ng/mll 0.1 Quail HE (mg/ml)
LHRHlnglmll Chlcken
0.1 HE (mg/ml)
1.’
tyfizQF Rot HE (mglml)
FIG. 4. Dose-response relationships in four separate experiments. The doses of synthetic LHRH (&ml) or of the particular hypothalamic extracts (mg extracted tissue/ml) are shown on the horizontal axis and the LH release on the vertical axis. As before, LH release is expressed in terms of the % increase above that found in the control tubes. Each point represents the Mean & SEM of four replicate tubes (each assayed in triplicate). (a) Doubling dilutions of quail hypothalamic extract (mature female, top dose equiv. to 0.25 of a hypothalamus) and of synthetic mammalian LHRH. (b) Chicken hypothalamic extract (broiler chicken, top dose equiv. to 0.2 of a hypothalamus) and synthetic mammalian LHRH. (c) Chicken and quail hypothalamic extracts. The top dose of the chicken extract was equiv. to 0.18 of a hypothalamus and that of the quail (immature male) to 0.3. (d) Rat hypothalamic extract (mature male, top dose equiv. to 0.3 of a hypothalamus) and chicken hypothalamic extract (top dose equiv. 0.16 hypothalamus) together with synthetic LHRH. (C-0) quail hypothalamic extract; (m-0) chicken hypothalamic extract; (A...A) synthetic LHRH; (m...M) rat hypothalamic extract.
shown in Fig. 3. In the presence of 1.0 rig/ml synthetic LHRH the rate of release was greatest during the first 10 min of incubation. In untreated tubes the rate of release was negligible during the first 30 min of incubation. It increased after this time but remained steady over the next hour; 1.0 rig/ml LHRH elicited the greatest percentage increase in LH release over control levels at approximately 30 min and this incubation time was used routinely.
Efects of Hypothalamic Other Tissue Extracts
and
Extracts of the basal hypothalamusmedian eminence region of quail, chicken, and rat increased LH release as compared with levels in control tubes (Fig. 4a-d). The dose-response curves obtained with both avian and rat hypothalamic extracts were invariably steeper than those obtained with synthetic mammalian LHRH (Fig. 4a-d). Approximately parallel dose-
LHRH
Cerebral
cortex
,mg,m,)
FIG. 5. Dose-response curves to show the effect of synthetic LHRH (A...A, rig/ml), chicken hypothalamic extract (o-0, mg/ml, top dose equiv to 0.16 of a hypothalamus) and quail cerebral cortex extract 0-0, mg/ml, top dose equiv to 5 quail hypothalami) on LH release from pituitary cell suspension. The vertical axis shows LH release in terms of the % rise above that found in control tubes. The data are from a single experiment, each point representing the Mean -C SEM of four replicate tubes.
response curves were obtained between quail and chicken hypothalamic extracts (Fig. 4c), and between chicken and rat hypothalamic extracts (Fig. 4d). Extracts of other brain regions of the quail including the cerebral cortex, midbrain, and hindbrain were also found to be capable of increasing LH release above basal levels although the amount of releasing activity present in these tissues was relatively low compared with that in the hypothalamus. Thus the cerebral cortex contained about 5% and the hindbrain 10% of the activity of an equal weight of hypothalamic tissue. Dose-response curves could be obtained with these nonhypothalamic tissue extracts (Fig. 5) and these were invariably less steep than those obtained with hypothalamic extracts or with synthetic LHRH. Extracts of liver
BIOASSAY
147
and testis produced no increase in LH release. All tissue extracts were assayed for any LH they might contain and the LH levels in the incubation media suitably adjusted. In practice LH activity was found only in chicken and quail hypothalamic extracts (rat LH does not cross-react in the avian RIA) and generally the amounts present were small relative to the LH in the incubation tubes. Quail hypothalamic extracts contained about 8 ng LH/hypothalamus (similar to that reported by Smith and Follett, 1972) while the amounts of LH in the incubation tubes ranged from 50 to 140 ng. A chicken hypothalamus contained about 50 ng. The source of this hormone is not known but Sharp (1974) reports the presence of LH-containing cells in the pars tuberalis which often remains attached to the median eminence during dissection. Effects of Other Biologically Active Compounds As shown in Table 2, various other biologically active compounds known to occur in brain tissues had little or no effect on LH release. In one experiment acetylcholine (2 pg/ml) did increase LH release by 66% but in the same assay 1.0 rig/ml of LHRH increased it by 123%. Thus in terms of mass LHRH is more than 2000 times as potent as acetylcholine. The Quantitative Assay of Releasing Activity in Tissue Extracts A four-point balanced factorial assay (2 + 2) was employed using four tubes at each dose level. Potency estimates of hypothalamic extracts were initially calculated in terms of synthetic LHRH but nonparallelism appeared in a number of experiments (Fig. 4a, b, d). For this reason a pooled extract of 50 chicken hypothalami was prepared and this substandard (CSl) was assayed against synthetic LHRH re-
148
BICKNELL
AND
TABLE LH
RELEASE
CAUSED
BY A VARIETY
Expt
Substance and dose
1
LHRH-1 rig/ml LHRH-10 rig/ml Dopamine-IO @ml Dopamine-500 @ml Histamine-20 rig/ml Histamine-l pg/ml AcetylcholineAO rig/ml Acetylcholine-2 pg/ml
2
LHRH-8 &ml Acetylcholine-50 &ml Acetylcholine-1 pg/rnl Adrenaline-20 rig/ml Adrenaline-200 @ml Adrenaline-2 &ml
FOLLETT
2
OF PHARMACOLOGICALLY
ACTIVE
COMPOUNDS
Expt
Substance and dose
LH releasea
123 225 2 6 4 6 11 66
3
43 -6 11 -6 -10 -17
4
LHRH-2 rig/ml GABA-I &ml GABA-IO &ml Serotonin-10 rig/ml Serotonin-500 rig/ml Noradrenalinerig/ml Noradrenaline@ml Vasopressin-200 @ml Vasopressin-2 pg/rnl LHRH-2 rig/ml Cyclic AMP-500 &ml Cyclic AMP-5 pg/ml
80 13 8 5 2 1 -4 2 4 98 1 2
LH release”
a LH Release is expressed as the % rise (or fall) from that the LH release found in the control tubes. Each value is the mean of four replicates (assayed in triplicate). GABA is an abbreviation for gamma-amino butyric acid.
peatedly to obtain an estimate of its potency. An unknown extract, therefore, is assayed against CSl and its mean potency, together with the 95% confidence limits, expressed in terms of CSI. For comparative purposes an approximation of the mean activity in terms of LHRH is usually given. A typical assay included four or five doubling dilutions of a standard preparation (LHRH or CSl). The top dose of LHRH generally used was 8 rig/ml while TABLE POTENCY
ESTIMATES
OF LH
that of CS 1 contained 0.16 equivalents of a chicken basal hypothalamus. Two doubling dilutions of the unknown were used. With quail hypothalamic extracts the top dose used contained 0.25 equivalents of the basal hypothalamus. Table 3 summarizes data from four assays using hypothalamic extracts prepared from quail in differing reproductive states. The mean index of precision for the first 16 assays was 0.069 ? 0.004 (SEM). Such indices do not 3
RELEASING ACTIVITY IN HYPOTHALAMIC THE JAPANESE QUAIL
Group
No. of hypothalami=
wt (mgY
Mean
Immature males Immature males Mature males Castrate males
4 4 4 4
1.73 1.90 1.70 1.42
LH-RH potency 17.9(14.8-21.4) 15.7(12.9-18.8) 18.5(15.8-21.6) 32.2(24.4-40.2)
EXTRACTS
FROM
LHRH potencyd
Lambda’
7.2 6.2 7.4 12.9
0.071 0.068 0.060 0.086
a The number of hypothalami pooled from the group of birds prior to extraction with 0.1 N HCI. * Mean weight (mg) of individual hypothalamic pieces. c LH-RH potency/mg tissue, together with its 95% confidence limits, is given in terms of the chicken hypothalamic standard CS 1. d Approximate mean potency in terms of ng synthetic LHRHimg tissue. Calculated by conversion from CS 1. c Lambda--the index of precision of the assay.
LHRH consider the error on the LH radioimmunoassay. However all incubation media from one experiment were included in a single RIA and Gibson et al. (I 975) have calculated the intra-assay coefficient of variation in the immunoassay to be about 10%. DISCUSSION
The results presented in this study show that the use of a suspension of pituitary cells enables an assay to be developed for hypothalamic LHRH which is rather more convenient, accurate, and reproducible than heretofore. The advantages over previous in vitro bioassay methods, apart from those of a practical nature, lie mainly in the replication of treatments that can be achieved using a homogeneous preparation of pituitary cells rather than paired or randomised pituitary fragments. The homogeneity of the system also allows free intra-assay comparisons of treatments while the use of a standard reference preparation allows inter-assay comparisons. It is perhaps this last point that is especially valuable as previously it has not been possible to make valid comparisons between assays and the designs used have not allowed the potency of an unknown to be expressed in terms of a standard. The index of the precision of the assay lies between 0.05 and 0.1. This compares favourably with an index calculated for an assay using pituitary fragments (e.g., Jamieson et al., 1973. index of precision = 0.2). There seems little doubt that the dispersed pituitary cells are releasing LH in response to a specific LH releasing factor present in the quail and chicken hypothalamus. In this regard the observation that synthetic mammalian TRH did not increase LH release is important. This has been supported by some recent results from Scanes (1974). He has shown that TRH significantly increases the secretion of bioassayable TSH from chicken hemipi-
BIOASSAY
149
tuitaries but that immunoreactive LH levels are not elevated under these conditions. The converse is also true-synthetic LHRH increased LH secretion but not that of TSH. All these data argue for the specificity of both the cell suspension assay, and the immunoassay of LH. The question of exactly how specific the LH release response of the dispersed cells is to LHRH is raised by the finding that extracts from other brain areas also increase LH secretion significantly. This effect has been reported for other in vitro systems (e.g., Wakabayashi et al., 1972; Smith and Follett. 1972; Hayes et al.. 1973; Barofsky et crl., 1973) but it is not known if the release is due to some nonspecific factor such as an elevated potassium concentration (Samli and Geschwind, 1968), or to small amounts of LHRH present in the tissues. The recent findings of substantial amounts of gonadotrophinreleasing hormone in the pineal (White et al., 1974) indicate that LHRH may indeed be present in brain areas other than the hypothalamus. However, the steeper dose-response curves obtained with hypothalamic extracts when compared with synthetic LHRH, and the less steep ones found with other brain tissues suggests that nonspecific factors may be the cause of the LH release by nonhypothalamic tissue extracts. It seems rather likely that the nonspecific factor is also present in the hypothalamus and is the cause of the steeper dose-response curves found with both avian and rat hypothalamic extracts. Despite considerable effort the factor responsible has not been discovered: possibly it might be acetylcholine. The chemical identity of avian LHRH remains to be elucidated. Jackson (1971 a, b) has reported that rat and chicken LHRHs behave differently on ion-exchange columns. He used a bioassay for LH-releasing activity based upon the in vitro incubation of rat hemipituitaries. More recently, Jeffcoate et al. (1974) using
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a mammalian LHRH radioimmunoassay, found no separation of rat, chicken and synthetic LHRH by ion-exchange or silica gel thin-layer chromatography. They also obtained parallel dilution curves for the avian and mammalian materials. In the present study rat and chicken hypothalamic extracts gave parallel dose-response curves but this is not proof of identity, especially as neither gave a parallel curve to that of synthetic LHRH. Some preliminary findings (S. L. Jeffcoate, R. J. Bicknell, and P. J. Sharp, unpublished) do suggest, however, a difference between chicken and ovine LHRH. If a chicken hypothalamic extract is assayed against synthetic LHRH in both the dispersed cell assay and the LHRH radioimmunoassay its potency is at least 100 times greater in the former. Estimates of the amounts of LHRH present in the quail hypothalamus (Table 3) are of the same order as those reported for mammalian species (e.g., Schally et al., 1971; Jeffcoate et al., 1974; White, et al., 1974). There appear to be no great changes in hypothalamic LHRH concentration associated with the reproductive state of the birds although long-term castrated quail held on long days showed an approximate 100% increase in stored concentration. This must be considered in the light of changes in pituitary and plasma LH. Sexually mature quail have plasma and pituitary LH levels some five to eight times as great as in immature birds while castration leads to still greater increases (Follett et al., 1972; Nicholls et al., 1973; Gibson et al., 1975). While the dispersed cell assay is extremely sensitive (it is capable of measuring the LHRH in a single quail hypothalamus in a full 2 + 2 assay) it does not seem capable of assaying circulating LHRH and is not as sensitive as Jeffcoate’s radioimmunoassay. It does seem likely that radioimmunoassay will dominate LHRH measurements over the next
FOLLETT
few years but the present assay could be useful in a number of respects. First, it will provide a biological comparison with the strictly immunological assay-to our knowledge no one has carried out a comprehensive comparison of bio- and immunopotencies of LHRH. Secondly, it may be valuable in lower vertebrates. The immunopotency of chicken hypothalamic extracts is very low (Jeffcoate et d., 1974) and yet in the dispersed cell assay the potency is comparable with that in other vertebrates. ACKNOWLEDGMENTS This work was supported by a Grant from the Agricultural Research Council (AB18/1). We are grateful to J. P. Wood and Sons Ltd. (Llangefni, Anglesey) for providing the chicken pituitary glands.
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