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The development of the Hooker-Forbes bioassay
The development of the Hooker-Forbes bioassay THOMAS New
Haven,
R.
FORBES,
PH.D.
Connecticut
DURING THE FALL andwinterof 1945,much of my free time was spent trying to “catch up” on some of the endocrine literature after an absence from the laboratory of over 3 years. One imperssion that developed during hours with the journals was that the results of experiments in reproductive endocrinology, my own included, often were expressed in imprecise terms. In those days, studies in this field were mostly morphologic. Increases or decreases were likely to be reported as “moderate” or “conspicuous’? or-that weary adjective beloved by the endocrinologist-“marked.” Fortunately for me, the Yale Department of Anatomy that I had recently joined included an able group of young investigators. A casual comment to one of them, Dr. Charles W. Hooker, that reproductive endocrinology seemed to need a more quantitative approach brought from him a suggestion that was to keep us busy for some years. He had found, he told me, that 3 weeks after a mouse is ovariectomized the stromal nuclei of the uterus became fusiform, dense, and pyknotic and contain large chromatin masses. However, if such mice receive daily injections of 0.25 or 0.5 mg. of progesterone, with or without estrogen, the same nuclei become plump and vesicular and have conspicuous nucleoli and fine chromatin granules. Willard Allen1 had earlier described similar nuclear transformation in the rat. Enlarged nuclei also characterize the uteri of pregnant and lactating mice.2, 3 Hooker told me that he and Dr. William Atkinson had used stromal nuclear transformation as an indicator of progestin and stromal edema and epithelial height as indicators of estrogen in estimating relative levels of these hormones throughout pregnancy in the mouse.” It was already well known that progestational proliferation could be induced in an immature rabFrom the Department of Anatomy, University School of Medicine.
bit’s uterus by subcutaneous injection of progesterone following pretreatment of the animals with estrogen. This reaction was the basis of the CornerAllen5 test and also of the later ClaubergG and McPhaiP tests, which had end points requiring 0.75 and 0.25 mg., respectively, of the hormone or its equivalent. Dr. Hooker pointed out that if progesterone were injected into the cavity of a ligated segment of the rabbit’s uterus, and thus were applied directly to the target tissue, the amount of progesterone necessary for a positive reaction was very much less, with as little as 0.13 yg sometimes giving a minimal response.*-I3 Hooker suggested that we attempt to develop a bioassay for progesterone in the mouse that would make use of the principle of local application of the hormone and of the stromal nuclear response. My notes from our conversation mention two goals: “to attempt to develop accurate quan. bio-assay of progesterone, etc. by local instillation in uterine segment” in the mouse and “to attempt to detn. level of progestin in blood of monkey (?) throughout preg.” The stromal nuclear response to progesterone would be our end point. This must have been about the end of January, 1946, as the first data entered in our research notebook were for February 18. We particularly hoped that the new assay would be quite sensitive in order that we might measure the level of progesterone in the blood of individual mice or rats.* We prepared a series of dilutions of progesterone in sesame oil, ovariectomized some mice, began injections into ligated segments of the uterus, and promptly ran into problems. It was possible to tie off a uterine segment in such a way that its blood *Progesterone
is the
name
coined by Corner for the discovered in 1929.‘* Progestin was designated in 1936 as a name for progesterone and other compounds with similar action occurring in impure extracts.‘” Usage has since converted progestin into a generic term for the group of compounds with progesterone-like action. Progestogen, occasionally encountered as another generic name for the same group, is unneccessary and is etymologically indefensible.
hormone that he and Allen”
Yale
This article was received from Dr. Forbes in response to an inquiry as to how the HookerForbes assay originated.
124
Volume Number
122 1
supply was not interfered with, but 0.01 ml., the smallest amount that could be measured with a tuberculin syringe, was too large a quantity to be contained without excessive distention in approximately 5 mm. of uterine horn, much atrophied as a result of ovariectomy. This problem was solved by designing a metal base to which clips were fastened to hold a tuberculin syringe and also a head cut from a micrometer caliper. The micrometer screw could be turned to advance the plunger of the syringe a minute but exactly measured distance. The syringe was then calibrated. Eventually, we were injecting through a No. 27 hypodermic needle about 0.0008 ml., an amount that only moderately distended the uterine segment. Dr. Hooker had earlier determined that 125 pg of progesterone when injected systemically in the ovariectomized mouse was enough to give a positive stromal nuclear response, defined by us as a combination of an elongated oval nuclear outline, a conspicuous nucleolus, and fine, evenly scattered chromatin granules. By October, 1946, we had perfected our injection technique. The mice were killed 48 hours after injection, and the injected segments were removed, fixed, sectioned, and examined. AS expected, we found positive responses after the injection of very small amounts of progesterone. Unfortunately, we also found positive responses after the injection of sesame oil that contained no steroid. Something was very wrong. Eventually it occurred to us that perhaps the 7 day interval between ovariectomy and intrauterine injection might not always be long enough for the stromal nuclei to regress to their castrate condition. Tests showed that this was true and that complete regression might take up to 14 days. Thereafter we always injected our mice 16 days after ovariectomy, and the “false positives” no longer appeared. Attempts to correlate the number of positive nuclei per section with the amount of progesterone injected proved impractical, and we decided to make our end point that at which any positive nuclei meeting our criteria could be found. Eventually, we determined that for mice of the CHI strain that we then used the minimal effective dose of progesterone for a positve response 48 hours after injection was 0.0002 pg. This was without estrogen priming. We also established that neither estradiol benzoate nor testosterone propionate, at least in the proportions we tested, would interfere with the positive response. Fortified with this knowledge, we made acetone and ether extracts of plasma from blood from a
Hooker-Forbes
bioassay
125
pregnant mouse and injected progressive dilutions of the extract until we found the greatest dilution that would give a positive response. Then it was a simple matter to calculate the equivalent amount of progesterone per milliliter. By January, 1947, we were assaying extracts of sow corpora lutea, blood plasma samples from mice and other animals, etc., and had begun testing the stromal nuclear response to numerous steroid compounds in terms of specificity. Our16 first paper appeared in August of that year. At this time, physicochemical and all biological assays except ours for progesterone were relatively insensitive, requiring the extraction of large amounts of blood, urine, or tissue. Our ability to assay an extract of 1 ml. of blood made many new experiments possible, and we and others were kept busy. About 30 compounds that we had injected in oil failed to duplicate the stromal nuclear response to progesterone, and we were fairly confident that we were actually measuring that hormone. The biochemists were also busy in those last years of the 1940’s, and, by early 1950, it was beginning to be apparent that levels of progesterone as determined by our assay were much greater than those revealed by the physicochemical methods of that period. Eventually, it became clear that our bioassay gave values roughly 100 times too high, although if levels of this hormone per milliliter of plasma were plotted against time during, for example, human pregnancy or the period before and after ovulation in the rabbit, the shapes of the curves obtained by our method and by physicochemical determination were similar. Also, in 1951, it was reported that cu-estradiol and testosterone in certain proportions could block the positive response to progesterone resulting in a false negative response.17 Other workers’* found that the sensitivity of the assay was in creased in the presence of progesterone : estradio mixtures in the ratio of 500: 1. In 1955, the tern progesterone equivalent (P. E.) was introduced, 2 P. E. being “defined as equal to the activity of 1 pg progesterone when the latter is assayed by the procedure of Hooker and Forbes in the absence o estrogen and other interfering factors.“‘g The assay in other words, would have to be regarded as a means for measuring the activity of progestins rather than the amount of progesterone. A tentative hypothesis to explain the large discrepancies between our results for plasma assays and those obtained by physiocochemical assays for progesterone is twofold. In the first place, it is now known from reports published in 1957 and later that
126
Forbes
the naturally occurring progestins 16cr-hydroxyprogesterone, 17a-hydroxyprogesterone, 20ar-hydroxyprogesterone, and 20/3-hydroxyprogesterone all give positive responses in the assay. The minimal effective dose of each compound is different and varies with the strain of mouse used for assay. Since the assay is sensitive to each substance separately, it presumably also responds to all of them collectively, and this may account for part of the discrepancy. Second, while the addition of certain proportions of various steroids to progesterone partly or completely antagonizes the positive stromal nuclear response, several proportions of 16~hydroxyprogesterone and 17a-hydroxyprogesterone synergize with progester-
one to give levels of activity that could only be obtained with several times that much progesterone if it alone were injected.‘” Conceivably, other compounds present in our acetone and ether extracts of tissue and plasma may have synergized even more powerfully with progesterone. Such a phenomenon would help to account for the assay’s exaggeration of the actual progesterone content of a sample. The Hooker-Forbes procedure should not be used in the attempt to measure amounts of progesterone. However, the ultimate significance of a hormone may be said to reside in its action in vivo. Bioassays that measure such action will continue to have a function.
REFERENCES
1. Allen, W. M. : Anat. Rec. 48: 65, 1931. 3-. Hooker, C. W.: Proc. Sot. Exp. Biol. Med. 45: 270, 1940. 3. Hooker, C. W.: Anat. Rec. 93: 333, 1945. 4. Atkinson, W. B., and Hooker, C. W.: Anat. Rec. 93: 75, 1945. 5. Corner, G. W., and Allen, W. M.: Am. J. Physiol. 88: 326, 1929. 6. Clauberg, C.: Zentralbl. Gynaecol. 54: 2757, 1930. 7. McPhail, M. K.: J. Physiol. 83: 145, 1934. 8. Halvorsen, K.: Acta Pathol. Microbial. Stand. 21: 510, 1944. 9. Haskins, A. L., Jr.: Proc. Sot. Exp. Biol. Med. 42: 624, 1939. 10. Haskins, A. L., Jr.: J. Clin. Endocrinol. 1: 65, 1941. 11. McGinty, D. A., Anderson, L. P., and McCullough, N. B.: Endocrinology 24: 829, 1939.
12.
13. 14. 15. 16. 17. 18. 19. 20.
Mussio-Fournier, J. C., Albrieux, A. S., Morato, J., and Grosso, 0.: Bull. Acad. Med. Paris 120: 273, 1938. Mussio-Fournier, J. C., Albrieux, A. S., and Grosso, 0.: Endocrinology 24: 515, 1939. Allen, W. M.: South. Med. J. 63: 1151, 1970. Anon.: J. A. M. A. 106: 1808, 1936. Hooker, C. W., and Forbes, T. R.: Endocrinology 41: 158, 1947. Olsen, A. G., Salhanick, H. A., and Hisaw, F. L.: Endocrinology 51: 519, 1952. Ober, K. G., Klein, I., and Weber, M.: Arch. Gvnaekol. 184: 543. 1954. 56: 699, 1955. Forbes, T. R.: Endocrinology Forbes, T. R.: Endocrinology 75: 799, 1964.
Haven,
R.
FORBES,
PH.D.
Connecticut
DURING THE FALL andwinterof 1945,much of my free time was spent trying to “catch up” on some of the endocrine literature after an absence from the laboratory of over 3 years. One imperssion that developed during hours with the journals was that the results of experiments in reproductive endocrinology, my own included, often were expressed in imprecise terms. In those days, studies in this field were mostly morphologic. Increases or decreases were likely to be reported as “moderate” or “conspicuous’? or-that weary adjective beloved by the endocrinologist-“marked.” Fortunately for me, the Yale Department of Anatomy that I had recently joined included an able group of young investigators. A casual comment to one of them, Dr. Charles W. Hooker, that reproductive endocrinology seemed to need a more quantitative approach brought from him a suggestion that was to keep us busy for some years. He had found, he told me, that 3 weeks after a mouse is ovariectomized the stromal nuclei of the uterus became fusiform, dense, and pyknotic and contain large chromatin masses. However, if such mice receive daily injections of 0.25 or 0.5 mg. of progesterone, with or without estrogen, the same nuclei become plump and vesicular and have conspicuous nucleoli and fine chromatin granules. Willard Allen1 had earlier described similar nuclear transformation in the rat. Enlarged nuclei also characterize the uteri of pregnant and lactating mice.2, 3 Hooker told me that he and Dr. William Atkinson had used stromal nuclear transformation as an indicator of progestin and stromal edema and epithelial height as indicators of estrogen in estimating relative levels of these hormones throughout pregnancy in the mouse.” It was already well known that progestational proliferation could be induced in an immature rabFrom the Department of Anatomy, University School of Medicine.
bit’s uterus by subcutaneous injection of progesterone following pretreatment of the animals with estrogen. This reaction was the basis of the CornerAllen5 test and also of the later ClaubergG and McPhaiP tests, which had end points requiring 0.75 and 0.25 mg., respectively, of the hormone or its equivalent. Dr. Hooker pointed out that if progesterone were injected into the cavity of a ligated segment of the rabbit’s uterus, and thus were applied directly to the target tissue, the amount of progesterone necessary for a positive reaction was very much less, with as little as 0.13 yg sometimes giving a minimal response.*-I3 Hooker suggested that we attempt to develop a bioassay for progesterone in the mouse that would make use of the principle of local application of the hormone and of the stromal nuclear response. My notes from our conversation mention two goals: “to attempt to develop accurate quan. bio-assay of progesterone, etc. by local instillation in uterine segment” in the mouse and “to attempt to detn. level of progestin in blood of monkey (?) throughout preg.” The stromal nuclear response to progesterone would be our end point. This must have been about the end of January, 1946, as the first data entered in our research notebook were for February 18. We particularly hoped that the new assay would be quite sensitive in order that we might measure the level of progesterone in the blood of individual mice or rats.* We prepared a series of dilutions of progesterone in sesame oil, ovariectomized some mice, began injections into ligated segments of the uterus, and promptly ran into problems. It was possible to tie off a uterine segment in such a way that its blood *Progesterone
is the
name
coined by Corner for the discovered in 1929.‘* Progestin was designated in 1936 as a name for progesterone and other compounds with similar action occurring in impure extracts.‘” Usage has since converted progestin into a generic term for the group of compounds with progesterone-like action. Progestogen, occasionally encountered as another generic name for the same group, is unneccessary and is etymologically indefensible.
hormone that he and Allen”
Yale
This article was received from Dr. Forbes in response to an inquiry as to how the HookerForbes assay originated.
124
Volume Number
122 1
supply was not interfered with, but 0.01 ml., the smallest amount that could be measured with a tuberculin syringe, was too large a quantity to be contained without excessive distention in approximately 5 mm. of uterine horn, much atrophied as a result of ovariectomy. This problem was solved by designing a metal base to which clips were fastened to hold a tuberculin syringe and also a head cut from a micrometer caliper. The micrometer screw could be turned to advance the plunger of the syringe a minute but exactly measured distance. The syringe was then calibrated. Eventually, we were injecting through a No. 27 hypodermic needle about 0.0008 ml., an amount that only moderately distended the uterine segment. Dr. Hooker had earlier determined that 125 pg of progesterone when injected systemically in the ovariectomized mouse was enough to give a positive stromal nuclear response, defined by us as a combination of an elongated oval nuclear outline, a conspicuous nucleolus, and fine, evenly scattered chromatin granules. By October, 1946, we had perfected our injection technique. The mice were killed 48 hours after injection, and the injected segments were removed, fixed, sectioned, and examined. AS expected, we found positive responses after the injection of very small amounts of progesterone. Unfortunately, we also found positive responses after the injection of sesame oil that contained no steroid. Something was very wrong. Eventually it occurred to us that perhaps the 7 day interval between ovariectomy and intrauterine injection might not always be long enough for the stromal nuclei to regress to their castrate condition. Tests showed that this was true and that complete regression might take up to 14 days. Thereafter we always injected our mice 16 days after ovariectomy, and the “false positives” no longer appeared. Attempts to correlate the number of positive nuclei per section with the amount of progesterone injected proved impractical, and we decided to make our end point that at which any positive nuclei meeting our criteria could be found. Eventually, we determined that for mice of the CHI strain that we then used the minimal effective dose of progesterone for a positve response 48 hours after injection was 0.0002 pg. This was without estrogen priming. We also established that neither estradiol benzoate nor testosterone propionate, at least in the proportions we tested, would interfere with the positive response. Fortified with this knowledge, we made acetone and ether extracts of plasma from blood from a
Hooker-Forbes
bioassay
125
pregnant mouse and injected progressive dilutions of the extract until we found the greatest dilution that would give a positive response. Then it was a simple matter to calculate the equivalent amount of progesterone per milliliter. By January, 1947, we were assaying extracts of sow corpora lutea, blood plasma samples from mice and other animals, etc., and had begun testing the stromal nuclear response to numerous steroid compounds in terms of specificity. Our16 first paper appeared in August of that year. At this time, physicochemical and all biological assays except ours for progesterone were relatively insensitive, requiring the extraction of large amounts of blood, urine, or tissue. Our ability to assay an extract of 1 ml. of blood made many new experiments possible, and we and others were kept busy. About 30 compounds that we had injected in oil failed to duplicate the stromal nuclear response to progesterone, and we were fairly confident that we were actually measuring that hormone. The biochemists were also busy in those last years of the 1940’s, and, by early 1950, it was beginning to be apparent that levels of progesterone as determined by our assay were much greater than those revealed by the physicochemical methods of that period. Eventually, it became clear that our bioassay gave values roughly 100 times too high, although if levels of this hormone per milliliter of plasma were plotted against time during, for example, human pregnancy or the period before and after ovulation in the rabbit, the shapes of the curves obtained by our method and by physicochemical determination were similar. Also, in 1951, it was reported that cu-estradiol and testosterone in certain proportions could block the positive response to progesterone resulting in a false negative response.17 Other workers’* found that the sensitivity of the assay was in creased in the presence of progesterone : estradio mixtures in the ratio of 500: 1. In 1955, the tern progesterone equivalent (P. E.) was introduced, 2 P. E. being “defined as equal to the activity of 1 pg progesterone when the latter is assayed by the procedure of Hooker and Forbes in the absence o estrogen and other interfering factors.“‘g The assay in other words, would have to be regarded as a means for measuring the activity of progestins rather than the amount of progesterone. A tentative hypothesis to explain the large discrepancies between our results for plasma assays and those obtained by physiocochemical assays for progesterone is twofold. In the first place, it is now known from reports published in 1957 and later that
126
Forbes
the naturally occurring progestins 16cr-hydroxyprogesterone, 17a-hydroxyprogesterone, 20ar-hydroxyprogesterone, and 20/3-hydroxyprogesterone all give positive responses in the assay. The minimal effective dose of each compound is different and varies with the strain of mouse used for assay. Since the assay is sensitive to each substance separately, it presumably also responds to all of them collectively, and this may account for part of the discrepancy. Second, while the addition of certain proportions of various steroids to progesterone partly or completely antagonizes the positive stromal nuclear response, several proportions of 16~hydroxyprogesterone and 17a-hydroxyprogesterone synergize with progester-
one to give levels of activity that could only be obtained with several times that much progesterone if it alone were injected.‘” Conceivably, other compounds present in our acetone and ether extracts of tissue and plasma may have synergized even more powerfully with progesterone. Such a phenomenon would help to account for the assay’s exaggeration of the actual progesterone content of a sample. The Hooker-Forbes procedure should not be used in the attempt to measure amounts of progesterone. However, the ultimate significance of a hormone may be said to reside in its action in vivo. Bioassays that measure such action will continue to have a function.
REFERENCES
1. Allen, W. M. : Anat. Rec. 48: 65, 1931. 3-. Hooker, C. W.: Proc. Sot. Exp. Biol. Med. 45: 270, 1940. 3. Hooker, C. W.: Anat. Rec. 93: 333, 1945. 4. Atkinson, W. B., and Hooker, C. W.: Anat. Rec. 93: 75, 1945. 5. Corner, G. W., and Allen, W. M.: Am. J. Physiol. 88: 326, 1929. 6. Clauberg, C.: Zentralbl. Gynaecol. 54: 2757, 1930. 7. McPhail, M. K.: J. Physiol. 83: 145, 1934. 8. Halvorsen, K.: Acta Pathol. Microbial. Stand. 21: 510, 1944. 9. Haskins, A. L., Jr.: Proc. Sot. Exp. Biol. Med. 42: 624, 1939. 10. Haskins, A. L., Jr.: J. Clin. Endocrinol. 1: 65, 1941. 11. McGinty, D. A., Anderson, L. P., and McCullough, N. B.: Endocrinology 24: 829, 1939.
12.
13. 14. 15. 16. 17. 18. 19. 20.
Mussio-Fournier, J. C., Albrieux, A. S., Morato, J., and Grosso, 0.: Bull. Acad. Med. Paris 120: 273, 1938. Mussio-Fournier, J. C., Albrieux, A. S., and Grosso, 0.: Endocrinology 24: 515, 1939. Allen, W. M.: South. Med. J. 63: 1151, 1970. Anon.: J. A. M. A. 106: 1808, 1936. Hooker, C. W., and Forbes, T. R.: Endocrinology 41: 158, 1947. Olsen, A. G., Salhanick, H. A., and Hisaw, F. L.: Endocrinology 51: 519, 1952. Ober, K. G., Klein, I., and Weber, M.: Arch. Gvnaekol. 184: 543. 1954. 56: 699, 1955. Forbes, T. R.: Endocrinology Forbes, T. R.: Endocrinology 75: 799, 1964.