BIOCHEMICAl.
MEDICINE
17, 67-79 (1977)
Ovarian Ornithine Decarboxylase Induction: A Specific and Rapid in Vivo Bioassay of LH AIDA NUREDDIN Mayo
Clinic,
Department of Molecular Rochester, Minnesota 55901
Medicine,
Received August 27. 1976
Lutropin (LH) is a glycoprotein made up of two nonidentical subunits, (Yand /3. It is secreted by the anterior pituitary and causes, in the female, ovulation and steroid production by the corpus luteum. In the male, it stimulates the Leydig cells to produce androgens and estrogens. Its mode of action, still undefined, is thought to take place by binding to a specific receptor (l-3) and activating the adenyl cyclase system (4). Of the several in vivo bioassays [for a review see Ref. (S)] developed for determination of LH in tissue extracts, serum, and urine, only two are currently in use: the ventral prostate weight assay (6) and the ovarian ascorbic acid depletion assay (7). The ventral prostate weight assay (VPW) is based on the increase in weight of the ventral prostate of immature hypophysectomized male rats.’ The effect is observed after 4 days of daily subcutaneous injection of the test material. The two major disadvantages of the method are the length of time required for obtaining results and the mortality rate of the hypophysectomized animals. Furthermore, since a large amount of sample is required before an increase in weight can be observed, the assay is of limited usefulness. The OAAD assay requires pseudopregnant rats which are obtained by simultaneous injections of pregnant mare serum gonadotropin and human chorionic gonadotropin. A total of 7.5 to 11.5 days are required prior to the injection of the test materials. Hence, as in the case of the VPW bioassay, results are not obtained quickly. The assay is based on the decrease in the resultant concentration of ascorbic acid in the ovaries. * Abbreviations used: VPW, ventral prostate weight; OAAD, ovarian ascorbic acid depletion; ODC, omithine decarboxylase; ACTH, adrenocorticotropin; hFSH, human follicle stimulating hormone; HCG, human chorionic gonadotropin; hGH, human growth hormone; hLH, human lutropin; pLH, porcine httropin; hTSH. human thyrotropin; bis-MSB, p-bis(O-methylstyryl)-benzene; PPO, 25diphenyloxazone; 2nd IRP hMG, the second International Reference Preparation for Human Menopausal Gonadotrophins: DTT, dithiothreitol. 67 Copyright All rights
@ 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
OWf,-2244
68
.AiDA NUREDDIN
Rosemberg rt (I/. (8) pointed out that the specificity of the assay is questionable. In this paper, we report the development of an in ~,ilw bioassay based on the specific induction of the enzyme ornithine decarboxylase (EC 4.1.1.17) by steroidal and polypeptide hormones acting at their respective target tissue sites (9). Kobayashi et 111. (IO) demonstrated that. in the mature rat, the level of ornithine decarboxylase in the ovary is a function of the estrus cycle, reaching maximal level in late proestrus. Maudsley and Kobayashi (11) showed that ovarian ornithine decarboxylase can be specifically induced by exogenous LH or HCG, irrespective of the time of the estrus cycle. The work of Kaye et al. (12) indicated that, in the immature rat, the induction of ODC by LH is a function of the age of the animal. Their studies, which covered rats ranging in age from 5 to 20 days, showed that the response of ODC to exogenous LH is first apparent at Day 9 and continues to increase until Day 20. We have extended these studies to include 32-day-old rats, and the application of these results to the development of a specific in riro bioassay for LH is described. MATERIALS
Immature female rats of the Wistar strain, purchased from Holtzman, were given laboratory chow and water ad lib. DL-Grnithine hydrochloride and pyridoxal phosphate were products of Calbiochem; DL-[ l-14C]ornithine hydrochloride (specific activity 43.04 mCi/mmole) was purchased from New England Nuclear. Dithiothreitol was obtained from Sigma. The scintillation fluid used is that described by Kobayashi rt uf. (10) and was made from scintillized-grade toluene (Fisher Scientific Co.). bis-MSB, PPO, and hyamine hydroxide were also purchased from New England Nuclear. The following hormones were kindly provided by the National Institute of Arthritis, Metabolism, and Digestive Diseases and the National Pituitary Agency (NPA) of the University of Maryland: ACTH L-61-14 (47.5 IU/mg), hLH LER-1977-2 (2700 IU/mg), hFSH LER-1968-2 (2690 IU/mg), hTSH LER-1952 (3.2 IU/mg), a-hLH CM-U- 1, /3-hLH LER-1973-B, and hGH HS-2019 (2.2 IU/mg). Three urinary gonadotropin extracts, two from postmenopausal women and one from eunuchs, were kindly provided by Dr. A. Albert; these are NIH-HPG-UPM-1 (4.8 IU/mg), the 2nd IRP hMG (4.4 IU/mg), and NIH-HPG-UE, respectively. HCG (500 Ill/ml), also generously provided by Dr. A. Albert, was a product of Ayerst. The various porcine pituitary gonadotropin fractions were prepared in this laboratory according to Chow et al. (13).
IN
VIVO BIOASSAY
OF
LH
69
METHODS Animals. Thirty-two-day-old female rats weighing 85-100 g were arranged in groups of four per dose. Each received a subcutaneous injection of 0.5 ml of the hormone dissolved in 0.02 M sodium borate-l% NaCl buffer, pH 8.0, and was sacrificed 5 hr later. The ovaries were immediately removed, cleaned of excess fat, weighed, and placed in an ice-cold Potter-Elvejehem type glass homogenizer equipped with a glass pestle. The ovaries from all four rats were pooled and homogenized in 0.1 M phosphate buffer, pH = 7.2, 5 mM in DTT. The concentration of the homogenate was 100 mg of tissue/ml of buffer. This procedure was also followed when 25- and 28-day-old rats were used (the weight range was 55-70 and 60-75 g, respectively). In order to obtain enough homogenate when 22-day-old rats (weighing 45-60 g) were studied, a pool of 10 ovaries per dose was used. Cirnithine decarboxylase assay. The assay was performed essentially as described by Russell and Snyder (14). The homogenate was centrifuged for 20 min at 2O,OOOg, and the supernatant was used for the assay. The incubation mixture consisted of 700 pliter of 0. I M phosphate buffer, pH = 7.2, 5 mM in DTT, 0.1 pmole of pyridoxal phosphate (in 0.1 M phosphate buffer, pH = 7.2), and 200 pliter of homogenate. The incubation was carried out in Warburg flasks in the center wells of which were placed wicks of filter paper impregnated with hyamine hydroxide. The flasks were placed in a shaking water bath set at 37 -+ 0.5”C and were allowed to equilibrate for 10 min. Fifty microliters (0.1 pmole) of the substrate were then added, and the reaction was stopped 30 min later by the addition of 0.2 ml of 1 M citric acid. The flasks were then returned to the water bath and shaken for an additional 30 min; the filter papers were removed and counted in a Beckman scintillation counter. A blank consisting only of buffer, pyridoxal phosphate, and substrate was included in every set of experiments. Animals injected with 0.02 M sodium borate-l% NaCl buffer provided the baseline activity of the enzyme. All assays were performed in duplicate. Ornithine decarboxylase activity is reported as nanomoles of 14C0, released per hour per milligram of protein. The method of Folin and Ciocalteau (15) was used to determine the protein concentration of the homogenate as well as that of all the hormones used. Radioimmunoassay of hLH in the different hormone preparations was carried out with lz51-labeled hLH and an antiserum made specific to hLH. Radioiodination of hLH was carried out according to the method of Greenwood et al. (16), and the radioimmunoassay as described by Faiman and Ryan (17). hLH was also determined with the radioligand receptor
70
.AIDA NUREDDIN
assay described by Lee and Ryan (18). In this case ““I-labeled used as the ligand.
HCG was
RESULTS Time Course of ODC Induction
Figure 1 illustrates that the induction of ODC activity by a subcutaneous injection of hLH is a function of time, reaching a peak value at 5 hr. SO-
4.0.
P 1
f 3.0cr ” 1 E 2.0
l.o-
Time (hrl FIG. 1. Time course of ODC induction by LH and HCG. Thirty-two-day-old rats were sacrificed at the time indicated on the abscissa. The ovaries were removed, homogenized in 0.1 M phosphate buffer, pH 7.2,s mM in DTT, and centrifuged at 20,OOOg.The reaction flask contained 700 pliters of 0.1 M phosphate buffer, pH = 7.2, 5 mM in DTT, 0.1 pmole of pyridoxal phosphate. and 200 pliters of the supematant centrifuged at 20,OOOg. W-labeled oL-omithine-l-hydrochloride, 0.1 pmole, was used as substrate. The reaction time was 30 min. The WOZ formed was counted in a Beckman scintillation counter, and ODC activity was expressed as nanomoles of WO, per hour per milligram of protein (ordinate). (x-x) Animals injected with 74.75 IUikg of hLH. Brackets indicate SEM: numbers in parentheses refer to the total number of rats used. (0-O) Animals injected with 308.83 IUikg of HCG.
IN
VIVO BIOASSAY
OF
LH
71
This peak activity is maintained for an additional 3 hr after which a gradual decline is observed. Twenty-four hours later (point not shown on graph), the activity is at a noninduced baseline level. That the observed plateau of peak activity is particular to hLH is shown by a similar experiment in which HCG was injected subcutaneously. The results shown in Fig. 1 indicate that, like hLH, the peak ODC activity is reached 5 hr after HCG injection. However, unlike hLH, the level of activity does not reach a plateau, and more than 50% of the enzymatic activity is lost within the next hour. By 9 hr, the activity is approximately one-tenth that of the peak value at 5 hr. These results indicate that the induction of ODC may be a function of the metabolic clearance of the hormone as well as of the half-life of the enzyme. Furthermore, Levine ef al. (19) found that the induction and disappearance of ODC activity in the adrenal gland followed a similar pattern whether ACTH was administered subcutaneously or intravenously. Dependence
of ODC Induction
on Age of the Animal
Kaye et al. (12) showed that ovarian ODC induction by LH is first observed 9 days after birth and continues to increase until Day 20. We extended these studies to include 32-day-old rats and examined the extent of such induction as a function of the concentration of hLH injected. Figure 2A is a plot of the micrograms of hLH injected per kilogram of body weight vs the ODC activity in the 32-day-old rat. It can be seen that ODC activity increases as the amount of hLH injected increases, with an effective dose range of 1.5-40 pg/kg; the ODC activity increases from 0.5 to 2.7 nmoles of 14C0, released/hr/mg of protein. No further increase in enzymatic activity was observed when 63 pg of hLWkg were injected. A comparison of similar dose-response curves obtained using rats aged 22, 25, and 28 days is shown in Fig. 2B. Although these curves are similar in their sigmoidal nature, they differ in their effective dose range as well as in the amount of YO, released at maximal response. Figure 3 shows that the extent of inducibility of ODC activity by LH increases with the age of the animal, the noninduced baseline level of the enzyme remaining constant. Extrapolation to baseline activity indicates that ODC induction by LH occurs at Day 10, in good agreement with the findings of Kaye et al. (12). Spec$icity of Ovarian ODC Induction To determine if ovarian ODC in the 32-day-old rat is induced specifically by LH, the following hormones were used: h,FSH, hTSH, HGH, ACTH, a-hLH, P-hLH, and HCG. The results are summarized in Table 1. It can be seen that both HGH and ACTH were ineffective at concentrations of 801 and 871 pg/kg, respectively. The p subunit of hLH at a
72
.4IDA NUXEDDI?i
10
100 /&kg
FIG. 2. (A) Dose-response curve of ovarian ODC to hLH injection in the 32-day-old rat. Each animal received a subcutaneous injection of the hormone and was sacrificed 5 hr later. The assay conditions are as described in the legend to Fig. I. The amount of hLH injected (abscissa) is expressed as micrograms/kilograms, ODC activity as nanomoles of “CO2 per hour per milligram of protein. Brackets indicate SEM; numbers in parentheses refer to the total number of rats used. (B) A comparison of the dose-response curve of ovarian ODC to hLH injection as a function of the age of the animal. Assay conditions are as described in the legend to Fig. 1. (0 -0) 22-day-old rats: (x-x) 25-day-old rats; and (O---O) 28-dayold rats.
dose of 239 &kg resulted in an enzymatic activity of 0.10, indicating that this subunit by itself does not induce ovarian ODC activity and, furthermore, that this subunit preparation is not contaminated by the native hormone. However, the (Y subunit resulted in an enzymatic activity of 0.63 units when 489 pg/kg were injected. Since the hormone-specific subunit did not induce ODC, one concludes that the decarboxylase activity observed with the CYsubunit is a result of its contamination with the native hormone. This would correspond to an LH concentration of 32.7 &mg or to a contamination of 85 IU/mg. Both radioimmunoassay with an antiserum specific,to the native hormone and radioligand receptor assay point to this conclusion. SimiIar results were obtained with hFSH and hTSH. hFSH LER-1968-2 is contaminated to the extent of 208 IU/mg, and hTSH LER-
IN VIVO
BIOASSAY
Agr
73
OF LH
(days)
FIG. 3. Relationship of the maximal induction of ODC activity by hLH with the age of animal. Enzyme activity is plotted as WO,Jhr/mg protein. (O-•) ODC activity at maximal induction: (x-x) ODC activity at noninduced baseline level.
1952 has an LH potency of 206 Wmg. These values are in good agreement with those reported by the NPA: < 300 IU/mg for hFSH and 200 IUlmg for hTSH (Dr. L. E. Reichert, Jr., personal communication). Since Kobayashi et al. (10) have shown that HCG also induces ovarian ODC, a dose-response curve similar to that used for hLH was examined, and the results obtained are shown in Fig. 4. Although a comparison of the curves obtained with hLH and HCG indicate that they are similar, the TABLE EFFECT
OF VARIOUS
ODC
POLYPEPTIDE
INDUCTION
IN THE
Hormone
Dose bak)
HGH HGH ACTH ACTH hFSH hFSH hFSH hTSH hTSH hTSH a-hLH a-hLH /3-hLH None
413.29 800.90 438.83 871.21 184.94 435.37 462.35 116.97 251.56 497.27 96.55 488.79 238.82 -
I HORMONES 32-DAY-OLD
ON OVARIAN RAT
ODC activity (nmole of “C0,1hrimg) 0.06 0.07 0.02 0.05 0.97 1.66 2.73 0.32 1.07 3.84 0.21 0.63 0. IO 0.1 I
74
-\IDA NUKEDDIN
5.0
1 i’
a
(I
and HCG FIG. 4. Comparison of dose-response curves obtained with hLH (X-X) (O---O). Thirty-two-day-old rats were used in both sets of experiments and were sacrificed 5 hr after injection. See the legend to Fig. I for assay conditions.
maximal ODC response induced by HCG is much greater than that obtained when hLH is used. The reason for such a difference is not readily apparent at the moment. It may be a reflection of the chemical differences between the two molecules (20). Stability of Induced Ovarian ODC .Ianne and Williams-Ashman (21) reported that ODC is stabilized by sulfhydryl reagents, especially by dithiothreitol. In agreement with their observations using the prostatic enzyme, the inclusion of 5 mM DTT in the homogenizing buffer resulted in an enhancement of both the activity and the stability of ovarian ODC. Whereas the enzyme extracted in the absence of 5 mM DTT was inactivated by freezing and thawing, such inactivation did not occur when the stabilizer was included. Indeed the supernatant centrifuged at 20,OOOg had been frozen for as long as 70 days with no loss in enzymatic activity (Table 2). Bioassay of Urinary
and Porcine
Pituitary
LH
Two different extracts of urinary LH from postmenopausal women, HPG-UPM and the 2nd IRP hMG, were assayed for their ability to induce ovarian ODC activity. As shown in Fig. 5, decarboxylation of ornithine is observed only when large doses of these preparations were used. However, no toxic effects were observed even at a dose of 40 mg/kg (440-480 IU/kg). The dose-response curve is nonparallel when compared to that obtained with purified human pituitary lutropin. However, when hLH
IN VW0 BIOASSAY TABLE STABILITY
Time frozen
75
OF LH
2
OF OVARIAN
ODC
ODC activity (nmole of ‘*CO, releasedihrimg)
(days) 43 43 43 71 71 71
0.04” 1.10 1.71 0.42 1.12 I .78
0.03” 1.08 1.89 0.45 1.07 1.73
n Values in this column were obtained on day of experiment. b Values in this column were obtained after freezing for the period of time indicated.
was assayed by radioimmunoassay and by radioligand receptor assay using the 2nd IRP as reference, parallel dose-response curves were obtained as shown in Fig. 6. Since very large amounts of hormone had to be used to observe ODC induction, the results observed may be due partly to the impurity of either preparation. Furthermore, the actual biopotency of the pure urinary postmenopausal lutropin may be less than that of the parent hormone. It is interesting to note (Fig. 5) that the biopotency of a
40 E” 2
3.0.
d-+ Y 2.07 i
/y i
/
1.0.
d.l FIG. 5. Dose-response curves obtained with pLH fractions at different stages of purification and with different urinary gonadotropin preparations. Thirty-two-day-old rats were used in all experiments. They were sacrificed 5 hr after injection. See the legend to Fig. 1 for assay conditions. (x-x) Crude pLH preparation; (O-O) previous preparation after subjecting it to an ammonium sulfate precipitation step; (0-O) urinary preparation from eunuchs, NIH-HPG-UE; (k--n) urinary preparation from postmenopausal women, NIH-HPG-UPM; and (t-t) urinary preparation from postmenopausal women, 2nd IRP hMG.
76
-\II)A NUREDDIN
1 1
lo pd
IRP(mi@;
hLH@f
1000
FIG. 6. Comparison of hLH dose-response curve as assayed by radioimmunoassay and radioligand.receptor assay using the 2nd IRP hMG as reference. Percentage of binding (p = 100/l + e-l) is plotted against the log of the dose in mIU of 2nd IRP hMG. hLH is expressed in microliters. (0 -0) Radioimmunoassay 2nd IRP hMG: (&--A) radioligand receptor assay 2nd IRP hMG; (X-X) radioimmunoassay, hLH: (D--Cl) radioligand receptor assay. hLH.
preparation of urinary gonadotropins from eunuchs is equivalent to that isolated from the urine of postmenopausal women. Figure 5 also is a plot of ODC activity as a function of pLH concentration and illustrates that a twofold purification is obtained when a crude preparation of porcine pituitary gonadotropins is subjected to an ammonium sulfate precipitation step. The curves are parallel to each other and to that obtained using hLH LER-1977-2, indicating that this bioassay is useful in monitoring the purification of hormones from animal sources and in evaluating their biopotency in terms of the human hormone. The biopotency of the purer pLH fraction in terms of hLH LER-1977-2 is 52 IU/mg. DISCUSSION
The concentration of luteinizing hormone in tissue extract, serum, or urine can be determined using biological, immunological, or receptorbinding methods. As discussed earlier, the in Go bioassays at hand are lengthy and, in the case of the VPW assay, require a large amount of material. Radioimmunoassays of polypeptide hormones have the advantages of
IN VIVO BIOASSAY
OF LH
77
extreme sensitivity and ease of performance but are as lengthy as the VPW bioassay. Furthermore, appreciable cross-reaction of the native hormone with its a! subunit has been noted (22). Receptor-binding assays are easy, quick to perform, and extremely sensitive. However, they are not a direct measure of the biopotency of the hormone. The in \tivo bioassay described here is based on the decarboxylation of the substrate ornithine by the enzyme ornithine decarboxylase. The ovarian enzyme has been shown to be specifically induced by LH (11). and we have further shown the dependence of such induction on the age of the animal. For this in viva bioassay, no previous preparation of the animals is required, and the assay can be performed within a day. Like most enzymatic assays, it shows specificity for its commercially available substrate, L-ornithine. The sensitivity of the assay is further increased by the use of the 14C-labeled amino acid. The peak activity of ovarian ornithine decarboxylase is obtained 5 hr after injection of hLH to 32-day-old rats and is maintained at that level for an additional 3 hr. These results differ from those obtained by Maudsley and Kobayashi (11) who observe maximal response 4 hr after a subcutaneous injection of ovine LH to mature rats. This maximal enzymatic activity is not maintained and decreases significantly within an hour. However, the rapid decline in maximal ODC activity is not observed when HCG is injected into mature rats, in contrast to our observations with the immature animal. This difference may be attributed to age. It is also pertinent to note that experiments by Maudsley and Kobayashi (11) were performed on rats at early proestrus where other factors may be involved. Parallel dose-response curves were obtained when fractions of pLH, at different stages of purification, were assayed by this bioassay method, This indicates that species differences are not observed with this technique. A similar phenomenon has been shown in the in vitro bioassay described by Dufau et al. (23). The latter technique is based on the steroidogenic response of dispersed rat Leydig cells to gonadotropic hormone. However, the response obtained with the 2nd IRP hMG differed in the two bioassays. While Dufau et al. (23) reported a parallel dose-response curve, such a curve was not obtained with the system described here. It is possible that steroidogenesic and ornithine decarboxylase induction reside in different parts of the lutropin molecule. Such differentiation can be ascertained only when urinary lutropin is purified and chemically characterized. Steroidogenesis is specifically induced by LH in the gonads; ornithine decarboxylase, on the other hand, is apparently ubiquitous in nature, its induction being under the control of steroidal and polypeptide hormones acting at their respective target tissues. With the development of the
assay techniques. the question of the relationship (if any) of polyamine formation to steroidogenesis in both the testicular and ovarian systems can now be investigated. SUMMARY Ovarian ornithine decarboxylase was found to be specifically induced by LH in the 22- to 32-day-old rats. Maximal induction occurred with the 32-day-old rat but was found to be a function of the concentration of LH at all ages. Based on these findings, an in r+r~ bioassay of LH was developed. Its effective dose range is from 15 to 40 eg LHlkg. The application of this bioassay to the estimation of LH in different samples was discussed. ACKNOWLEDGMENTS This work was supported by Grant HD-9140-H from the National lnstitutes of Health and by the Mayo Foundation. The author is indebted to the National Institute of Arthritis, Metabolism, and Digestive Diseases and the National Pituitary Agency of the University of Maryland for their generous supply of the hormones used in this study. Special thanks are due to Dr. A. Albert in whose laboratory this work was started. for his support throughout this work and for the urinary fractions he generously supplied. Thanks are also due to Dr. R. J. Ryan for supplying radioiodinated HCG and the antiserum to hLH. I also wish to thank Dr. T. C. Spelsberg for critically reading the manuscript. The excellent technical assistance of Miss Janet Eide is greatly appreciated.
REFERENCES 1. De Kretser. D. M.. Catt, K. J., and Paulsen, C. A., Endocrinology 88, 332-337 ( 1971). 2. Catt, K. J., Dufau, M. L.. and Tsuruhara, T.. J. C/in. Endocrinol. Metab. 32, 860-863 (1971). 3. Lee, C. Y., and Ryan, R. J.. Proc. Nat. Acod. Sci. USA 69, 3520-3523 (1972). 4. Sutherland, E. W.. J. Amer. Med. Ass. 214, 1281-1288 (1970). 5. Loraine. J. A., and Bell, E. T.. “Hormone Assays and Their Clinical Application,” pp. 33-37. Wilkins and Wilkins. Baltimore (1971). 6. Greep. R. O., van Dyke, H. B.. and Chow. B. F.. Proc. Sot. Exp. Biol. Med. 46, 644-649 (1941). 7. Parlow, A. F., irr “Human Pituitary Gonadotropins” (A. Albert, Ed.). pp. 300-310. Thomas, Springfield (1961). 8. Rosemberg, E., Keller, P., Lewis, W. B.. Albert, A., Carl, G., and Bennett, D.. Endocrinology 76, 1150-l 157 (1965). 9. Morris, D. R., and Fillingame, R. H.. in “Annual Review of Biochemistry” (E. E. Snell. Ed.). Vol. 43, pp. 303-325. Annual Reviews, Palo Alto (1974). IO. Kobayashi. Y., Kupelian, J., and Maudsley. D. V. Science 172, 379-380 (1971). Il. Maudsley, D. V., and Kobayashi, Y., Biochem. Phnrmucol. 23, 2697-2703 (1974). 12. Kaye, A. M., Icekson, I., Lamprecht. S. A.. Gruss, R.. Tsafriri. A., and Lindner, H. R., Biochemistry 12, 3072-3076 (1973). 13. Chow, B. F., van Dyke. H. B., Greep, R. 0.. Rothen, A., and Shedlovsky, T.. Endocrinology 30, 650-656 ( 1942). 14. Russell. D. H., and Snyder. S. H., Proc. Nar. Acad. Sci. USA 60, 1420-1427 (1968). 15. Folin. 0.. and Ciocalteau. V., .I. Biol. Chrm. 73, 627-650 (1927).
IN VW0 16. 17. 18. 19. 20. 21. 22. 23.
BIOASSAY
OF LH
79
Greenwood, F. C., Hunter, W. M., and Glover, J. S., Rio&em. J. 89, 114-123 (1963). Faiman, C., and Ryan, R. J.. hoc. Sot. Exp. Biol. Med. 125, 1130-1133 (1967). Lee, C. Y., and Ryan, R. J., Biochemistry 12, 4609-4615 (1973). Levine, J. H., Nicholson, W. E., Liddle, G. W., and Orth. D. N., Endocrinology 92, 1089-1095 (1973). Bishop, W. H., Nureddin, A., and Ryan, R. J., in “Peptide Hormones” (J. A. Parsons, Ed.) Macmillan, N.Y. (1976). Janne, J., and Williams-Ashman, H. G. 1. Biol. Chem. 246, 1725-1732 (1971). Prentice, L. G., and Ryan, R. J.. J. C/in. Endocrinol. Metab. 40, 303-312 (1974). Dufau, M. L., Pock, R., Neubauer, A., and Catt, K. J.. J. C/in. Endocrinol. Met&. 42, 958-969 ( 1976).
MEDICINE
17, 67-79 (1977)
Ovarian Ornithine Decarboxylase Induction: A Specific and Rapid in Vivo Bioassay of LH AIDA NUREDDIN Mayo
Clinic,
Department of Molecular Rochester, Minnesota 55901
Medicine,
Received August 27. 1976
Lutropin (LH) is a glycoprotein made up of two nonidentical subunits, (Yand /3. It is secreted by the anterior pituitary and causes, in the female, ovulation and steroid production by the corpus luteum. In the male, it stimulates the Leydig cells to produce androgens and estrogens. Its mode of action, still undefined, is thought to take place by binding to a specific receptor (l-3) and activating the adenyl cyclase system (4). Of the several in vivo bioassays [for a review see Ref. (S)] developed for determination of LH in tissue extracts, serum, and urine, only two are currently in use: the ventral prostate weight assay (6) and the ovarian ascorbic acid depletion assay (7). The ventral prostate weight assay (VPW) is based on the increase in weight of the ventral prostate of immature hypophysectomized male rats.’ The effect is observed after 4 days of daily subcutaneous injection of the test material. The two major disadvantages of the method are the length of time required for obtaining results and the mortality rate of the hypophysectomized animals. Furthermore, since a large amount of sample is required before an increase in weight can be observed, the assay is of limited usefulness. The OAAD assay requires pseudopregnant rats which are obtained by simultaneous injections of pregnant mare serum gonadotropin and human chorionic gonadotropin. A total of 7.5 to 11.5 days are required prior to the injection of the test materials. Hence, as in the case of the VPW bioassay, results are not obtained quickly. The assay is based on the decrease in the resultant concentration of ascorbic acid in the ovaries. * Abbreviations used: VPW, ventral prostate weight; OAAD, ovarian ascorbic acid depletion; ODC, omithine decarboxylase; ACTH, adrenocorticotropin; hFSH, human follicle stimulating hormone; HCG, human chorionic gonadotropin; hGH, human growth hormone; hLH, human lutropin; pLH, porcine httropin; hTSH. human thyrotropin; bis-MSB, p-bis(O-methylstyryl)-benzene; PPO, 25diphenyloxazone; 2nd IRP hMG, the second International Reference Preparation for Human Menopausal Gonadotrophins: DTT, dithiothreitol. 67 Copyright All rights
@ 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
OWf,-2244
68
.AiDA NUREDDIN
Rosemberg rt (I/. (8) pointed out that the specificity of the assay is questionable. In this paper, we report the development of an in ~,ilw bioassay based on the specific induction of the enzyme ornithine decarboxylase (EC 4.1.1.17) by steroidal and polypeptide hormones acting at their respective target tissue sites (9). Kobayashi et 111. (IO) demonstrated that. in the mature rat, the level of ornithine decarboxylase in the ovary is a function of the estrus cycle, reaching maximal level in late proestrus. Maudsley and Kobayashi (11) showed that ovarian ornithine decarboxylase can be specifically induced by exogenous LH or HCG, irrespective of the time of the estrus cycle. The work of Kaye et al. (12) indicated that, in the immature rat, the induction of ODC by LH is a function of the age of the animal. Their studies, which covered rats ranging in age from 5 to 20 days, showed that the response of ODC to exogenous LH is first apparent at Day 9 and continues to increase until Day 20. We have extended these studies to include 32-day-old rats, and the application of these results to the development of a specific in riro bioassay for LH is described. MATERIALS
Immature female rats of the Wistar strain, purchased from Holtzman, were given laboratory chow and water ad lib. DL-Grnithine hydrochloride and pyridoxal phosphate were products of Calbiochem; DL-[ l-14C]ornithine hydrochloride (specific activity 43.04 mCi/mmole) was purchased from New England Nuclear. Dithiothreitol was obtained from Sigma. The scintillation fluid used is that described by Kobayashi rt uf. (10) and was made from scintillized-grade toluene (Fisher Scientific Co.). bis-MSB, PPO, and hyamine hydroxide were also purchased from New England Nuclear. The following hormones were kindly provided by the National Institute of Arthritis, Metabolism, and Digestive Diseases and the National Pituitary Agency (NPA) of the University of Maryland: ACTH L-61-14 (47.5 IU/mg), hLH LER-1977-2 (2700 IU/mg), hFSH LER-1968-2 (2690 IU/mg), hTSH LER-1952 (3.2 IU/mg), a-hLH CM-U- 1, /3-hLH LER-1973-B, and hGH HS-2019 (2.2 IU/mg). Three urinary gonadotropin extracts, two from postmenopausal women and one from eunuchs, were kindly provided by Dr. A. Albert; these are NIH-HPG-UPM-1 (4.8 IU/mg), the 2nd IRP hMG (4.4 IU/mg), and NIH-HPG-UE, respectively. HCG (500 Ill/ml), also generously provided by Dr. A. Albert, was a product of Ayerst. The various porcine pituitary gonadotropin fractions were prepared in this laboratory according to Chow et al. (13).
IN
VIVO BIOASSAY
OF
LH
69
METHODS Animals. Thirty-two-day-old female rats weighing 85-100 g were arranged in groups of four per dose. Each received a subcutaneous injection of 0.5 ml of the hormone dissolved in 0.02 M sodium borate-l% NaCl buffer, pH 8.0, and was sacrificed 5 hr later. The ovaries were immediately removed, cleaned of excess fat, weighed, and placed in an ice-cold Potter-Elvejehem type glass homogenizer equipped with a glass pestle. The ovaries from all four rats were pooled and homogenized in 0.1 M phosphate buffer, pH = 7.2, 5 mM in DTT. The concentration of the homogenate was 100 mg of tissue/ml of buffer. This procedure was also followed when 25- and 28-day-old rats were used (the weight range was 55-70 and 60-75 g, respectively). In order to obtain enough homogenate when 22-day-old rats (weighing 45-60 g) were studied, a pool of 10 ovaries per dose was used. Cirnithine decarboxylase assay. The assay was performed essentially as described by Russell and Snyder (14). The homogenate was centrifuged for 20 min at 2O,OOOg, and the supernatant was used for the assay. The incubation mixture consisted of 700 pliter of 0. I M phosphate buffer, pH = 7.2, 5 mM in DTT, 0.1 pmole of pyridoxal phosphate (in 0.1 M phosphate buffer, pH = 7.2), and 200 pliter of homogenate. The incubation was carried out in Warburg flasks in the center wells of which were placed wicks of filter paper impregnated with hyamine hydroxide. The flasks were placed in a shaking water bath set at 37 -+ 0.5”C and were allowed to equilibrate for 10 min. Fifty microliters (0.1 pmole) of the substrate were then added, and the reaction was stopped 30 min later by the addition of 0.2 ml of 1 M citric acid. The flasks were then returned to the water bath and shaken for an additional 30 min; the filter papers were removed and counted in a Beckman scintillation counter. A blank consisting only of buffer, pyridoxal phosphate, and substrate was included in every set of experiments. Animals injected with 0.02 M sodium borate-l% NaCl buffer provided the baseline activity of the enzyme. All assays were performed in duplicate. Ornithine decarboxylase activity is reported as nanomoles of 14C0, released per hour per milligram of protein. The method of Folin and Ciocalteau (15) was used to determine the protein concentration of the homogenate as well as that of all the hormones used. Radioimmunoassay of hLH in the different hormone preparations was carried out with lz51-labeled hLH and an antiserum made specific to hLH. Radioiodination of hLH was carried out according to the method of Greenwood et al. (16), and the radioimmunoassay as described by Faiman and Ryan (17). hLH was also determined with the radioligand receptor
70
.AIDA NUREDDIN
assay described by Lee and Ryan (18). In this case ““I-labeled used as the ligand.
HCG was
RESULTS Time Course of ODC Induction
Figure 1 illustrates that the induction of ODC activity by a subcutaneous injection of hLH is a function of time, reaching a peak value at 5 hr. SO-
4.0.
P 1
f 3.0cr ” 1 E 2.0
l.o-
Time (hrl FIG. 1. Time course of ODC induction by LH and HCG. Thirty-two-day-old rats were sacrificed at the time indicated on the abscissa. The ovaries were removed, homogenized in 0.1 M phosphate buffer, pH 7.2,s mM in DTT, and centrifuged at 20,OOOg.The reaction flask contained 700 pliters of 0.1 M phosphate buffer, pH = 7.2, 5 mM in DTT, 0.1 pmole of pyridoxal phosphate. and 200 pliters of the supematant centrifuged at 20,OOOg. W-labeled oL-omithine-l-hydrochloride, 0.1 pmole, was used as substrate. The reaction time was 30 min. The WOZ formed was counted in a Beckman scintillation counter, and ODC activity was expressed as nanomoles of WO, per hour per milligram of protein (ordinate). (x-x) Animals injected with 74.75 IUikg of hLH. Brackets indicate SEM: numbers in parentheses refer to the total number of rats used. (0-O) Animals injected with 308.83 IUikg of HCG.
IN
VIVO BIOASSAY
OF
LH
71
This peak activity is maintained for an additional 3 hr after which a gradual decline is observed. Twenty-four hours later (point not shown on graph), the activity is at a noninduced baseline level. That the observed plateau of peak activity is particular to hLH is shown by a similar experiment in which HCG was injected subcutaneously. The results shown in Fig. 1 indicate that, like hLH, the peak ODC activity is reached 5 hr after HCG injection. However, unlike hLH, the level of activity does not reach a plateau, and more than 50% of the enzymatic activity is lost within the next hour. By 9 hr, the activity is approximately one-tenth that of the peak value at 5 hr. These results indicate that the induction of ODC may be a function of the metabolic clearance of the hormone as well as of the half-life of the enzyme. Furthermore, Levine ef al. (19) found that the induction and disappearance of ODC activity in the adrenal gland followed a similar pattern whether ACTH was administered subcutaneously or intravenously. Dependence
of ODC Induction
on Age of the Animal
Kaye et al. (12) showed that ovarian ODC induction by LH is first observed 9 days after birth and continues to increase until Day 20. We extended these studies to include 32-day-old rats and examined the extent of such induction as a function of the concentration of hLH injected. Figure 2A is a plot of the micrograms of hLH injected per kilogram of body weight vs the ODC activity in the 32-day-old rat. It can be seen that ODC activity increases as the amount of hLH injected increases, with an effective dose range of 1.5-40 pg/kg; the ODC activity increases from 0.5 to 2.7 nmoles of 14C0, released/hr/mg of protein. No further increase in enzymatic activity was observed when 63 pg of hLWkg were injected. A comparison of similar dose-response curves obtained using rats aged 22, 25, and 28 days is shown in Fig. 2B. Although these curves are similar in their sigmoidal nature, they differ in their effective dose range as well as in the amount of YO, released at maximal response. Figure 3 shows that the extent of inducibility of ODC activity by LH increases with the age of the animal, the noninduced baseline level of the enzyme remaining constant. Extrapolation to baseline activity indicates that ODC induction by LH occurs at Day 10, in good agreement with the findings of Kaye et al. (12). Spec$icity of Ovarian ODC Induction To determine if ovarian ODC in the 32-day-old rat is induced specifically by LH, the following hormones were used: h,FSH, hTSH, HGH, ACTH, a-hLH, P-hLH, and HCG. The results are summarized in Table 1. It can be seen that both HGH and ACTH were ineffective at concentrations of 801 and 871 pg/kg, respectively. The p subunit of hLH at a
72
.4IDA NUXEDDI?i
10
100 /&kg
FIG. 2. (A) Dose-response curve of ovarian ODC to hLH injection in the 32-day-old rat. Each animal received a subcutaneous injection of the hormone and was sacrificed 5 hr later. The assay conditions are as described in the legend to Fig. I. The amount of hLH injected (abscissa) is expressed as micrograms/kilograms, ODC activity as nanomoles of “CO2 per hour per milligram of protein. Brackets indicate SEM; numbers in parentheses refer to the total number of rats used. (B) A comparison of the dose-response curve of ovarian ODC to hLH injection as a function of the age of the animal. Assay conditions are as described in the legend to Fig. 1. (0 -0) 22-day-old rats: (x-x) 25-day-old rats; and (O---O) 28-dayold rats.
dose of 239 &kg resulted in an enzymatic activity of 0.10, indicating that this subunit by itself does not induce ovarian ODC activity and, furthermore, that this subunit preparation is not contaminated by the native hormone. However, the (Y subunit resulted in an enzymatic activity of 0.63 units when 489 pg/kg were injected. Since the hormone-specific subunit did not induce ODC, one concludes that the decarboxylase activity observed with the CYsubunit is a result of its contamination with the native hormone. This would correspond to an LH concentration of 32.7 &mg or to a contamination of 85 IU/mg. Both radioimmunoassay with an antiserum specific,to the native hormone and radioligand receptor assay point to this conclusion. SimiIar results were obtained with hFSH and hTSH. hFSH LER-1968-2 is contaminated to the extent of 208 IU/mg, and hTSH LER-
IN VIVO
BIOASSAY
Agr
73
OF LH
(days)
FIG. 3. Relationship of the maximal induction of ODC activity by hLH with the age of animal. Enzyme activity is plotted as WO,Jhr/mg protein. (O-•) ODC activity at maximal induction: (x-x) ODC activity at noninduced baseline level.
1952 has an LH potency of 206 Wmg. These values are in good agreement with those reported by the NPA: < 300 IU/mg for hFSH and 200 IUlmg for hTSH (Dr. L. E. Reichert, Jr., personal communication). Since Kobayashi et al. (10) have shown that HCG also induces ovarian ODC, a dose-response curve similar to that used for hLH was examined, and the results obtained are shown in Fig. 4. Although a comparison of the curves obtained with hLH and HCG indicate that they are similar, the TABLE EFFECT
OF VARIOUS
ODC
POLYPEPTIDE
INDUCTION
IN THE
Hormone
Dose bak)
HGH HGH ACTH ACTH hFSH hFSH hFSH hTSH hTSH hTSH a-hLH a-hLH /3-hLH None
413.29 800.90 438.83 871.21 184.94 435.37 462.35 116.97 251.56 497.27 96.55 488.79 238.82 -
I HORMONES 32-DAY-OLD
ON OVARIAN RAT
ODC activity (nmole of “C0,1hrimg) 0.06 0.07 0.02 0.05 0.97 1.66 2.73 0.32 1.07 3.84 0.21 0.63 0. IO 0.1 I
74
-\IDA NUKEDDIN
5.0
1 i’
a
(I
and HCG FIG. 4. Comparison of dose-response curves obtained with hLH (X-X) (O---O). Thirty-two-day-old rats were used in both sets of experiments and were sacrificed 5 hr after injection. See the legend to Fig. I for assay conditions.
maximal ODC response induced by HCG is much greater than that obtained when hLH is used. The reason for such a difference is not readily apparent at the moment. It may be a reflection of the chemical differences between the two molecules (20). Stability of Induced Ovarian ODC .Ianne and Williams-Ashman (21) reported that ODC is stabilized by sulfhydryl reagents, especially by dithiothreitol. In agreement with their observations using the prostatic enzyme, the inclusion of 5 mM DTT in the homogenizing buffer resulted in an enhancement of both the activity and the stability of ovarian ODC. Whereas the enzyme extracted in the absence of 5 mM DTT was inactivated by freezing and thawing, such inactivation did not occur when the stabilizer was included. Indeed the supernatant centrifuged at 20,OOOg had been frozen for as long as 70 days with no loss in enzymatic activity (Table 2). Bioassay of Urinary
and Porcine
Pituitary
LH
Two different extracts of urinary LH from postmenopausal women, HPG-UPM and the 2nd IRP hMG, were assayed for their ability to induce ovarian ODC activity. As shown in Fig. 5, decarboxylation of ornithine is observed only when large doses of these preparations were used. However, no toxic effects were observed even at a dose of 40 mg/kg (440-480 IU/kg). The dose-response curve is nonparallel when compared to that obtained with purified human pituitary lutropin. However, when hLH
IN VW0 BIOASSAY TABLE STABILITY
Time frozen
75
OF LH
2
OF OVARIAN
ODC
ODC activity (nmole of ‘*CO, releasedihrimg)
(days) 43 43 43 71 71 71
0.04” 1.10 1.71 0.42 1.12 I .78
0.03” 1.08 1.89 0.45 1.07 1.73
n Values in this column were obtained on day of experiment. b Values in this column were obtained after freezing for the period of time indicated.
was assayed by radioimmunoassay and by radioligand receptor assay using the 2nd IRP as reference, parallel dose-response curves were obtained as shown in Fig. 6. Since very large amounts of hormone had to be used to observe ODC induction, the results observed may be due partly to the impurity of either preparation. Furthermore, the actual biopotency of the pure urinary postmenopausal lutropin may be less than that of the parent hormone. It is interesting to note (Fig. 5) that the biopotency of a
40 E” 2
3.0.
d-+ Y 2.07 i
/y i
/
1.0.
d.l FIG. 5. Dose-response curves obtained with pLH fractions at different stages of purification and with different urinary gonadotropin preparations. Thirty-two-day-old rats were used in all experiments. They were sacrificed 5 hr after injection. See the legend to Fig. 1 for assay conditions. (x-x) Crude pLH preparation; (O-O) previous preparation after subjecting it to an ammonium sulfate precipitation step; (0-O) urinary preparation from eunuchs, NIH-HPG-UE; (k--n) urinary preparation from postmenopausal women, NIH-HPG-UPM; and (t-t) urinary preparation from postmenopausal women, 2nd IRP hMG.
76
-\II)A NUREDDIN
1 1
lo pd
IRP(mi@;
hLH@f
1000
FIG. 6. Comparison of hLH dose-response curve as assayed by radioimmunoassay and radioligand.receptor assay using the 2nd IRP hMG as reference. Percentage of binding (p = 100/l + e-l) is plotted against the log of the dose in mIU of 2nd IRP hMG. hLH is expressed in microliters. (0 -0) Radioimmunoassay 2nd IRP hMG: (&--A) radioligand receptor assay 2nd IRP hMG; (X-X) radioimmunoassay, hLH: (D--Cl) radioligand receptor assay. hLH.
preparation of urinary gonadotropins from eunuchs is equivalent to that isolated from the urine of postmenopausal women. Figure 5 also is a plot of ODC activity as a function of pLH concentration and illustrates that a twofold purification is obtained when a crude preparation of porcine pituitary gonadotropins is subjected to an ammonium sulfate precipitation step. The curves are parallel to each other and to that obtained using hLH LER-1977-2, indicating that this bioassay is useful in monitoring the purification of hormones from animal sources and in evaluating their biopotency in terms of the human hormone. The biopotency of the purer pLH fraction in terms of hLH LER-1977-2 is 52 IU/mg. DISCUSSION
The concentration of luteinizing hormone in tissue extract, serum, or urine can be determined using biological, immunological, or receptorbinding methods. As discussed earlier, the in Go bioassays at hand are lengthy and, in the case of the VPW assay, require a large amount of material. Radioimmunoassays of polypeptide hormones have the advantages of
IN VIVO BIOASSAY
OF LH
77
extreme sensitivity and ease of performance but are as lengthy as the VPW bioassay. Furthermore, appreciable cross-reaction of the native hormone with its a! subunit has been noted (22). Receptor-binding assays are easy, quick to perform, and extremely sensitive. However, they are not a direct measure of the biopotency of the hormone. The in \tivo bioassay described here is based on the decarboxylation of the substrate ornithine by the enzyme ornithine decarboxylase. The ovarian enzyme has been shown to be specifically induced by LH (11). and we have further shown the dependence of such induction on the age of the animal. For this in viva bioassay, no previous preparation of the animals is required, and the assay can be performed within a day. Like most enzymatic assays, it shows specificity for its commercially available substrate, L-ornithine. The sensitivity of the assay is further increased by the use of the 14C-labeled amino acid. The peak activity of ovarian ornithine decarboxylase is obtained 5 hr after injection of hLH to 32-day-old rats and is maintained at that level for an additional 3 hr. These results differ from those obtained by Maudsley and Kobayashi (11) who observe maximal response 4 hr after a subcutaneous injection of ovine LH to mature rats. This maximal enzymatic activity is not maintained and decreases significantly within an hour. However, the rapid decline in maximal ODC activity is not observed when HCG is injected into mature rats, in contrast to our observations with the immature animal. This difference may be attributed to age. It is also pertinent to note that experiments by Maudsley and Kobayashi (11) were performed on rats at early proestrus where other factors may be involved. Parallel dose-response curves were obtained when fractions of pLH, at different stages of purification, were assayed by this bioassay method, This indicates that species differences are not observed with this technique. A similar phenomenon has been shown in the in vitro bioassay described by Dufau et al. (23). The latter technique is based on the steroidogenic response of dispersed rat Leydig cells to gonadotropic hormone. However, the response obtained with the 2nd IRP hMG differed in the two bioassays. While Dufau et al. (23) reported a parallel dose-response curve, such a curve was not obtained with the system described here. It is possible that steroidogenesic and ornithine decarboxylase induction reside in different parts of the lutropin molecule. Such differentiation can be ascertained only when urinary lutropin is purified and chemically characterized. Steroidogenesis is specifically induced by LH in the gonads; ornithine decarboxylase, on the other hand, is apparently ubiquitous in nature, its induction being under the control of steroidal and polypeptide hormones acting at their respective target tissues. With the development of the
assay techniques. the question of the relationship (if any) of polyamine formation to steroidogenesis in both the testicular and ovarian systems can now be investigated. SUMMARY Ovarian ornithine decarboxylase was found to be specifically induced by LH in the 22- to 32-day-old rats. Maximal induction occurred with the 32-day-old rat but was found to be a function of the concentration of LH at all ages. Based on these findings, an in r+r~ bioassay of LH was developed. Its effective dose range is from 15 to 40 eg LHlkg. The application of this bioassay to the estimation of LH in different samples was discussed. ACKNOWLEDGMENTS This work was supported by Grant HD-9140-H from the National lnstitutes of Health and by the Mayo Foundation. The author is indebted to the National Institute of Arthritis, Metabolism, and Digestive Diseases and the National Pituitary Agency of the University of Maryland for their generous supply of the hormones used in this study. Special thanks are due to Dr. A. Albert in whose laboratory this work was started. for his support throughout this work and for the urinary fractions he generously supplied. Thanks are also due to Dr. R. J. Ryan for supplying radioiodinated HCG and the antiserum to hLH. I also wish to thank Dr. T. C. Spelsberg for critically reading the manuscript. The excellent technical assistance of Miss Janet Eide is greatly appreciated.
REFERENCES 1. De Kretser. D. M.. Catt, K. J., and Paulsen, C. A., Endocrinology 88, 332-337 ( 1971). 2. Catt, K. J., Dufau, M. L.. and Tsuruhara, T.. J. C/in. Endocrinol. Metab. 32, 860-863 (1971). 3. Lee, C. Y., and Ryan, R. J.. Proc. Nat. Acod. Sci. USA 69, 3520-3523 (1972). 4. Sutherland, E. W.. J. Amer. Med. Ass. 214, 1281-1288 (1970). 5. Loraine. J. A., and Bell, E. T.. “Hormone Assays and Their Clinical Application,” pp. 33-37. Wilkins and Wilkins. Baltimore (1971). 6. Greep. R. O., van Dyke, H. B.. and Chow. B. F.. Proc. Sot. Exp. Biol. Med. 46, 644-649 (1941). 7. Parlow, A. F., irr “Human Pituitary Gonadotropins” (A. Albert, Ed.). pp. 300-310. Thomas, Springfield (1961). 8. Rosemberg, E., Keller, P., Lewis, W. B.. Albert, A., Carl, G., and Bennett, D.. Endocrinology 76, 1150-l 157 (1965). 9. Morris, D. R., and Fillingame, R. H.. in “Annual Review of Biochemistry” (E. E. Snell. Ed.). Vol. 43, pp. 303-325. Annual Reviews, Palo Alto (1974). IO. Kobayashi. Y., Kupelian, J., and Maudsley. D. V. Science 172, 379-380 (1971). Il. Maudsley, D. V., and Kobayashi, Y., Biochem. Phnrmucol. 23, 2697-2703 (1974). 12. Kaye, A. M., Icekson, I., Lamprecht. S. A.. Gruss, R.. Tsafriri. A., and Lindner, H. R., Biochemistry 12, 3072-3076 (1973). 13. Chow, B. F., van Dyke. H. B., Greep, R. 0.. Rothen, A., and Shedlovsky, T.. Endocrinology 30, 650-656 ( 1942). 14. Russell. D. H., and Snyder. S. H., Proc. Nar. Acad. Sci. USA 60, 1420-1427 (1968). 15. Folin. 0.. and Ciocalteau. V., .I. Biol. Chrm. 73, 627-650 (1927).
IN VW0 16. 17. 18. 19. 20. 21. 22. 23.
BIOASSAY
OF LH
79
Greenwood, F. C., Hunter, W. M., and Glover, J. S., Rio&em. J. 89, 114-123 (1963). Faiman, C., and Ryan, R. J.. hoc. Sot. Exp. Biol. Med. 125, 1130-1133 (1967). Lee, C. Y., and Ryan, R. J., Biochemistry 12, 4609-4615 (1973). Levine, J. H., Nicholson, W. E., Liddle, G. W., and Orth. D. N., Endocrinology 92, 1089-1095 (1973). Bishop, W. H., Nureddin, A., and Ryan, R. J., in “Peptide Hormones” (J. A. Parsons, Ed.) Macmillan, N.Y. (1976). Janne, J., and Williams-Ashman, H. G. 1. Biol. Chem. 246, 1725-1732 (1971). Prentice, L. G., and Ryan, R. J.. J. C/in. Endocrinol. Metab. 40, 303-312 (1974). Dufau, M. L., Pock, R., Neubauer, A., and Catt, K. J.. J. C/in. Endocrinol. Met&. 42, 958-969 ( 1976).
