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Vasodilatation of the Rat Testis in Response to Human Chorionic Gonadotropin
Vasodilatation of the Rat Testis in Response to Human Chorionic Gonadotropin Carl G. Hartman, Ph.D., Nathan Millman, Ph.D., and J. Stavorski, B.S.
IT
TO
a general principle that a hormone capable of stimulating a target organ is capable of causing a rapid vasodilatation of that organ. Thus, for example, Williams was able to demonstrate well defined vasodilatation of the rat uterus within one or two hours after injection of one gamma of estrogen. Indeed, in intraocular transplants of endometrium in the rabbit, Markee observed a marked blushing of the transplant within ten minutes after injection of estrogen; the reddening continued to increase for forty minutes. The now well-known reddening of the ovary through the action of gonadotropins is a similar phenomenon. This was already noted by Aschheim and ZondekB· 4 who, however, based their famous pregnancy test on the sequel of congestion, namely extravasation of blood into the graafian follicle ninetysix hours after the first of several injections. The rapidly developing hyperemia or reddening of the ovary was not made use of, however, for a quick and at the same time reliable test until Reiprich27 found that a reliable answer was attainable within twenty-four hours after injection of the suspected urine simply by noting the color of the ovary. This work was overlooked until Walker and Walker in 1938 took up the method anew; they fully corroborated Reiprich, as did Kelso in 1940. The method was slow in finding general recognition, however, until, in 1942, Salmon, Geist, Salmon, and Frank31 showed that the ovarian hyperAPPEARS
BE
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emia test is valid when read six hours-and even two hours ( Salmon, Geist, Frank, Poole and Salmon32 )-after urine had been introduced into the peritoneal cavity of the immature rat. Credit for the development of this test is outlined in the very unusual title of the paper by Hinglais and Hinglais: "Une nouvelle variante rapide du test de Aschheim-Zondek. Reaction d'hyperemie ovarienne chez la rate. Reaction de Reiprich modifiee par Kelso et Salmon-Geist." This is giving everybody credit in a big way. The test has been evaluated by numerous investigators. Kupperman and co-workers added a significant item to the method. 23 They noted that, whereas immature rats were adapted to the two-hour test, any female bearing corpora lutea could be used for the six-hour test if chief attention were paid to the congestion in the corpora lutea. An interesting variation in the determination of the endpoint is that of Frank and Berman who noted, in positive cases, a rosette of dilated vessels in the theca interna of stimulated follicles when viewed by transillumination, a technic which one of us ( C.G.H.) has used as an instructive class-room experiment. Both Aschheim 5 and Zondek38 have checked over the method and endorsed it, although the latter favored the twenty-four-hour period as the more reliable. Zondek and Sulman recommended this interval also for pregnancy diagnosis of the mare. Evaluations of the ovarian hyperemia test have been published by Kamenester, Kline, Ramsey et al., and Soman in 1944, and more recently by Bunde, Fried, Riley et al., and Sala et al. These publications reflect general agreement that the method is reliable although improvements in details of technic have been suggested. The method has also recently been adopted by Farris in determining the time of ovulation in women (and monkeys), since in his hands the 2-hour test has been reliable for establishing the peak of pituitary gonadotropin secretion in the menstrual cycle of the non-pregnant primate.
VASODILATATION IN THE TESTIS If gonadotropins cause hyperemia through vasodilatation in the ovary, why not in the testis? To our knowledge no studies have been reported on this subject although the experiments of King and Oslund, who described volume changes in the testes, might be cited as indicative of vascular changes in that organ. Three
445
RAT TESTIS
Vol. 1, No.5, 1950]
sets of experiments demonstrating hyperemia of the testis of the rat in response to gonadotropins are reported in the present paper. Experiment No. 1
A series of 8 rats were given interperitoneally amounts of urine of pregnancy from 0.5 cc. to 4.0 cc. in differences of 0.5 cc. At the end of six hours all of these and, in addition, a control rat were killed. The testes were removed, split open and laid in a row for comparison. An increasing redness in the series was clearly evident. When the control and the one receiving India ink perfused testes of infantile rats removed six hours after receiving various dosages of pregnancy urine.
Control
0.5 cc.
2.0 cc.
3.0 cc.
4.0 cc.
India ink perfused testes of infantile rats removed 6 and 48 hours after receiving 2.0 cc. of pregnancy urine.
Control
48 Hrs.
6 Hrs. FIGURE
l
the greatest amount of urine were compared with a color standard, as in the "Reiprich-Salmon-Farris" test, a positive reaction could be deduced. This t~chnic was not further developed. Experiment No. 2
In a similar series, six hours after injection of the urine of pregnancy, the animals were killed and their vascular systems perfused (via dorsal aorta) with physiological saline, followed by India ink. Upon completion of the perfusion the testes were all placed in 10 per cent formalin overnight. The next day they were split open with a safety razor blade and examined for intensity of gray. It was found, as seen in the photograph (Fig. 1), that the depth of gray was roughly proportional to the quantity of urine injected.
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2 and 3. Spectrophotometric records of relative absorption of light rays within the visible spectrum. Such records indicated. the use of a filter ( 420 millicra) employed in photometric readings in the experiments.
FIGURES
Vol. I, No. 5, 1950]
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447
This series closely parallels the plate of drawings showing depth of color of ovaries described by Ramsey, Falkenstein and Sujkowski. TEST QUANTITATED SPECTROPHOTOMETRICALLY Since the amount of blood (and hence of hemoglobin) increases in an organ with increased caliber of its blood vessels, it was thought possible that
FrcuRE 4. 1, slides with shims in place and a mass of testicular tissue in the exact middle of slide; 2, slides pressed together and bound with Texcel tape; 3, specimen in place in carrier.
one could prepare a film of testicular substance thin enough to make readings by transillumination with a spectrophotometer and in that way obtain objective data in terms of absorption at a narrower wave length. With the generous cooperation of Professor J. 0. Kraehenbuehl of the Department of Electrical Engineering of the University of Illinois, and with the assistance of Dr. J. Littrell, records were secured, of which Figs. 2 and 3 are typical. The specimens themselves are prepared in the following manner:
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The essential apparatus consists of two glass slides and two shims 0.3 to 0.35 mm. in thickness (Fig. 4). For reasons which will be clear to the reader as he proceeds, the slides must be thick enough not to bend while handling (},i inch) and their surfaces must be ground absolutely flat. The specimen consists of a lump of tissue from the inner mass of the testis. The albuginea is first trimmed from the organ by means of a fine straight pair of scissors and the lump then excised with a curved pair. A lump of just the right size can be removed with practice. This is laid on a slide which has a glass shim placed on each end ( 1, Fig. 4) and with the second slide the lump of tissue is pressed down to make a film the exact thickness of the shims ( 2, Fig. 4). To insure this indispensable condition, the same shims are used for all the preparations of any given experiment. Testes of adult rats will yield two specimens, one from each end of the organ. Only one specimen is procurable from the testis of an immature male. The film should be of such size that it more than covers the hole in the diaphragm, but does not run out beyond the edges of the slides. From the spectrophotograms shown in Figures 2 and 3, and from dozens of others, it was learned that the greatest difference in amount of absorption was in the region of 420-425 f1 wave-length. This point having been determined, it was possible to use any electrophotometric device fitted with the proper filter. PHOTOMETRIC EVALUATION OF VASODILATATION
The major requisite of such apparatus, aside from its accuracy, is that it be capable of taking the specimen holder described above. We have found the Photovolt "Lumetron" adaptable to these technics. The specimen holder is placed directly against the coverplate of the objective photocell, and the top of the cell compartment closed to exclude room light. A 420 f1 filter was used in the experiments. In practice, the following procedures were employed. A control rat was first sacrificed, and a preparation of its testis used to adjust the instrument for maximum transmission, and to establish a reference point with which preparations from experimental rats might be compared. Since the absorptions are compared as relative densities, any reference adjustment yielding an adequate degree of sensitivity may be employed. The thickness of the tissue usually made it necessary to use relatively large apertures for the incident light beam.
Vol. 1, No. 5, 1950]
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Testis preparations from the treated animals were next prepared as described above and the relative densities of these to the controls determined. A series of more than 50 groups, comprising more than 250 rats, has been TABLE 1.
Series Test
I
II
Representative Tests Illustrating Influence of Stage of Pregnancy, Dosage, Time of Reading, and Priming with Steroid Hormones
Pre-Treatment
34B KB NB 33B 41B 37B
3.0 2.0 3.5 3.0 2.0 3.0
10 10 10 10 10
NB HB HC HD IB
III
45D 45A
IV
Time, interPregnancy Urine val between % Months Ml./ Doses/ No. of 1st Dose Change in pregnant Dose Day Days and Reading Density
AB 45D 45B TC TD
Estrone, 29, over 3 wks., s.c. Solubilized estrone, 29, over 3 weeks, s.c. Dienestrol, 0.1 mgm. Estrone, 29, over 3 wks., s.c. Solubilized estrone, 29, over 3 wks., s.c. Testosterone proprionate, 2 mgm., s.c. Testosterone proprionate, 2 mgm., s.c.
10
1 1 1 1 1 1
1 1 1 1 1 1
17 hr. 17 hr. 18 hr. 20 hr. 24 hr. 24hr.
+36.2 +36.8 +44.9 +42.7 +42.3 +48.6
5.0 6.5 6.5 6.5 7.0
10 10 10 10 10
1 1 1 1 1
1 1 1 1 1
18 hr. 20 hr. 20 hr. 5 hr. 20 hr.
+ 5.2 +13.1 +11.9 + 3.8 +18.0
2.0
2
2
2
48 hr.
+35.7
2.0
2
2
2
48 hr.
+45.3
4 hr. 3wk.
1.1 + 2.9
3wk.
+ 8.6
72 hr.
+ 3.6
72 hr.
+ 4.7
.
•
examined in this manner. From the results of these tests, it has become evident that the change should be 20 per cent or higher to be considered significant. Several approaches to the vasodilatation phenomenon have been examined. These are illustrated by representative experiments in Table 1. It has been found that the urine of a two to three-months gravid woman, injected intraperitoneally into rats in one dose of 10 cc., could cause a 30 to 50 per
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cent increase in density. The readings were best made within a period of seventeen to twenty-four hours after injection. These tests are represented in series I of Table 1; each of the results is an average of several readings on both testes. Since the concentration of urinary gonadotropin decreases sharply after the third month of pregnancy, urines of women who were five to seven months pregnant were examined. Typical results in this group are shown in series II of the table. Of a total of 21 groups similarly examined, all failed to produce marked testicular changes. No attempt was made to concentrate the urine in these cases in preparation for the test; nor have any of the tests, where negative, been repeated with higher doses of urine over longer periods of time. On the theory that estrogen stimulates the output of the dilating factor and on the assumption that LH is the cause of the observed vasodilatation,24 two tests were made on rats "primed" with estrone. As seen from series III, Table 1, the estrone did not seem to potentiate the action of pregnancy urine. Estrogens themselves (series IV) had no dilating effect on the testis; nor was any effect noted with androgens. DISCUSSION The experiments briefly described above afford another example of vascular response of the target organ to a potent hormone. Dilatation of the uterine vessels occurs very promptly after the administration of estrogen25 • 35 whereas edema is at its height in six hours, 7 while active cell proliferation does not begin until the tenth hour. 1 The hyperemia of the testis, as demonstrated above, closely parallels that of the ovary in the Salmon-Farris test. No attempt was made to employ the above described technic in the determination, in the nonpregnant woman, of cyclic variation in the titre of the dilating factor, as reported by Farris. It is quite likely, however, that by concentrating the active factor from urine one might secure a significant series of data by the above descJ;ibed method. It is interesting to note that male rats of any age within the reproductive period may be employed in the test, which greatly reduces the expense of maintaining the rat colony, which must be very large to furnish a steady stream of 21-day-old females.
r
r
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SUMMARY Vasodilatation within testicular tissue of the rat occurring in response to human chorionic gonadotropin (urine of pregnancy) may be demonstrated, ( 1 ) by more intense redness of exposed tissue; ( 2) by depth of grayness of the tissue after perfusion with India ink; ( 3) by means of a photometric method herein described. The latter is a thoroughly objective method, and while probably too cumbersome to serve as a routine pregnancy test, it may have value as a research tool, for example, to supplement the Salmon-Farris ovarian hyperemia test. Since male rats of any age may be used the new method would have the advantage of greater economy. REFERENCES l. Allen, E., Smith, G. M., and Gardner, W. U.: Am. Jour. Anat. 61:321-341, 1937. 2. Aschheim, S.: Ztschr. Geb. u. Gynak. 90:387-392, 1926. 3. Aschheim, S.: Med. Klin. 22:2023-202.5, 1926. 4. Aschheim, S., and Zondek, B.: Klin. Wchnschr. 6:1322, 1927. 5. Aschheim, S., and Varangot, J.: C. R. Soc. Bioi. Paris 139:1003-1004, 1945. 6. Aschheim, S.: Ibid., pp. 1002-1003. 7. Astwood, E. B.: Am. Jour. Physiol. 126:162-170, 1939. 8. Bunde, Carl A.: Am. Jour. Obst. and Gynec.: 53:317-320, 1947. 9. Eberson, Frederick, and Silverberg, M. H.: }.A.M.A. 2176-2182, 1931. 10. Farris, Edmond J.: Obst. and Gynec. 48:200-207, 1944. ll. Farris, Edmond J.: Anat. Rec. 94:16 ( Abstr.), 1946. 12. Farris, Edmond J.: Am. Jour. Obst. and Gynec. 52:14-17, 1946. 13. Farris, Edmond J.: Anat. Rec. 95:337-346, 1946. 14. Frank, R. T., and Berman, R. L.: Am. Jour. Obst. and Gynec. 42:492-496, 1941. 15. Fried, PaulL.: Am. Jour. Obst. and Gynec. 54:689-693, 1947. 16. Hartman, Carl G.: Anat. Rec. 94:538 (Abstr.), 1946. 17. Hinglais, H., and Hinglais, M.: Gynec. et Obstet. 46:86-93, 1947. 18. Kaminester, S.: Am. Jour. Obst. and Gynec. 47:265-267, 1944. 19. Kelso, R. E.: Am. Jour. Clin. Path. 10:293-299, 1940. 20. King, C. E., and Oslund, R. M.: Am. Jour. Physiol. 70:100-110, 1924. 21. Kline, B.S.: Am. Jour Clin. Path. 14:557-562, 1944. 22. Kupperman, H. S., and Greenblatt, R. B.: South Med. Jour. 39:158-165, 1946. 23. Kupperman, H. S., and Noback, Chas. R.: Jour. Clin. Endocr. 3:.548-550, 1943. 24. Kupperman, H. S., McShan, W. H., and Meyer, Roland K.: Endocr. 43:275-282, 1948. 25. Markee, J. E.: Surg., Gynec. and Obst. 56:51-54, 1933. 26. Ramsey, T. L., Falkenstein, A. P., and Sujkowski, E. J.: Jour. Lab. and Clin. Med. 29:419-428, 1944. 27. Reiprich, W.: Klin. Wchnschr. 12:1441-1444, 1933. 28. Reiprich, W.: Ztschr. Geb. u. Gynak. 109:285-332, 1934. 29. Riley, Gardner M., Smith, Marjorie H., and Brown, Pearl: Jour. Clin. Endocr. 8:233-243, 1948.
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30. Sala, S. L., Orellana, D., and Gonzales, J. }.I. L.: Sem. Med., Buenos Aires, 2:328-332, 1946 (Abstr. Jour. Clin. Endocr. 6:412, 1946). 31. Salmon, U. J., Geist, S. H., Salmon, A. A., and Frank, I. L.: Jour. Clin. Endocr. 2:167, 1942. 32. Salmon, U. J., Geist, S. H., Frank, I. L., Poole, C., and Salmon, A. A.: Endocr. 30(Suppl.):l039, 1942. 33. Soman, D. W.: Indian Med. Gaz. 79:68-71, 1944. 34. Walker, T. F., and Walker, D. V. H.: ].A.M.A. 111:1460, 1938. 35. Williams, Maude F.: Am. Jour. Anat. 83:217-306, 1948. 36. Zondek, B.: Zbl. Geb. u. Gynak. 90:372-380, 1926. 37. Zondek, B., and Sulman, Felix: Nature 155:302, 1945. 38. Zondek, B., and Black, Rivka: J.A.M.A. 128:939-944, 1945.
Ortho Research Foundation, Raritan, N.].
DISCUSSION SALMON, New York City: Dr. Hartman and his co-workers have
DR. UDALL J. introduced a new and extremely ingenious technic into the field of gonadotropin research-a technic which opens new vistas to the laboratory research worker and the clinician. Three questions arose in my mind, stimulated by this paper. First, can the chorionic gonadotropin, rat testis vasodilatation reaction be used as a test for pregnancy? Second, does pituitary gonadotropin cause a similar vasodilatation in the rat testis? Third, if the answer to the latter question were in the affirmative, would it be feasible to utilize this phenomenon as a test for ovulation? As regards the first question, Dr. Hartman has pointed out that the technic is much too complicated to be practical for routine diagnostic purposes. As regards the second and third questions, I took the liberty, after reading Dr. Hartman's preliminary report, of performing several tests using pituitary gonadotropin and urine specimens from three cyclical women with normal ovulatory temperature patterns from the eighth to the sixteenth day of the cycle, paralleling the technic recommended by Fards for female rats. In no instance could I detect any degree of testis hyperemia greater than in the uninjected controls. It is conceivable that there may have been some degree of vasodilatation which was not discernible grossly but which the spectrophotometer technic described by the authors might have revealed. Dr. Hartman and his co-workers will undoubtedly provide a conclusive answer to these questions. Before terminating my discussion, I wish to express my thanks to the authors for having associated my name with Dr. Farris's in designating the ovarian hyperemia ovulation test. Flattering though it be, I must refuse the honor, since I have not been able to confirm Dr. Farris's observations in a series of 12 cases in which basal temperature patterns and endometrial biopsies indicated ovulation. In closing, I wish to express my thanks to Dr. Hartman for the privilege of discussing this stimulating paper and also my gratitude to him and his co-workers for having introduced another research instrument into the field of gonadotropin physiology, an instrument which I trust will shed a little more light on some of the obscure aspects of the pathophysiology of infertility.
Vol. I, No. 5, 1950]
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DR. EDMOND J. FARRIS, Philadelphia: Mr. Chairman, Ladies and Gentlemen, I want to express my appreciation to Dr. Hartman for the privilege of hearing his excellent and stimulating presentation and the fine results in this paper. I am glad to see that he has developed something that is quite as impractical as our expensive rat colony at Wistar Institute, and that the method requires a $20,000 gadget for a spectrometer. The refinement in quantitating the vasodilatation by means of the photometric method is a valuable contribution and is useful as a research method. It is definitely objective. The findings of Dr. Hartman are of interest in that the urine of a two to three months' gravid woman can cause a 30 to 50 per cent increase in density within a period of seventeen to twenty-four hours after injection. However, I don't believe the male rat as a test animal is as sensitive as the immature female rat. We use the immature female Wistar rat rather than the male and find that following injection of urine of pregnant women from days 34 on, the hyperemia in the ovaries is best defined in fifteen to twenty-four hours. The male rat tests with early pregnancy urines proved negative. By means of intravenous injections the ovarian hyperemia can be read as early as one to two hours. However, the latter technic is cumbersome. A method, to be useful, must be simple and accurate. When using the two-hour hyperemia test for pregnancy one must beware of false reactions, and particularly when the animals have been killed by illuminating gas. We use the two-hour rat test for human or primate ovulation testing only, and the overnight fifteen or twenty-four hour testing for pregnancy determination. Dr. Hartman suggested in the course of his paper that by concentrating the active factor from the urine one might secure a significant series of data by the new method. We might add that for the past sixteen months we have been concentrating human urine, and interesting and valuable data is being secured, as suggested by Dr. Hartman in his paper. It may be of interest to mention that by comparing the degree of the intensity of the hyperemia in the rat ovary, using the Munsell color chart, which is simple and objective, one can read a quantitative difference in the reactions. In a pape1 now in press, entitled The 24-hour rat test for early diagnosis of pregnancy and its aid in predicting abortion, the findings are dependent upon the differential degree of hyperemia, in cycle days 34 to 36. May I congratulate Dr. Hartman and his co-workers for their fine contribution DR. CARL G. HARTMAN (closing): We all still sometimes use the AschheimZondek test and it still is a fine test. We also use the Friedman test and have had experience with the Salmon test. I have not used the Salmon-Farris test for the detection of ovulation but I have seen Dr. Farris at work in his laboratory and I am sure he knows what he is doing. One of the gains from my presenting this paper, you see, is that it called forth two very excellent discussions, and I want to encourage you young people by stating this, that your experiment may yet constitute a contribution even though not of Nobel prize caliber.
IT
TO
a general principle that a hormone capable of stimulating a target organ is capable of causing a rapid vasodilatation of that organ. Thus, for example, Williams was able to demonstrate well defined vasodilatation of the rat uterus within one or two hours after injection of one gamma of estrogen. Indeed, in intraocular transplants of endometrium in the rabbit, Markee observed a marked blushing of the transplant within ten minutes after injection of estrogen; the reddening continued to increase for forty minutes. The now well-known reddening of the ovary through the action of gonadotropins is a similar phenomenon. This was already noted by Aschheim and ZondekB· 4 who, however, based their famous pregnancy test on the sequel of congestion, namely extravasation of blood into the graafian follicle ninetysix hours after the first of several injections. The rapidly developing hyperemia or reddening of the ovary was not made use of, however, for a quick and at the same time reliable test until Reiprich27 found that a reliable answer was attainable within twenty-four hours after injection of the suspected urine simply by noting the color of the ovary. This work was overlooked until Walker and Walker in 1938 took up the method anew; they fully corroborated Reiprich, as did Kelso in 1940. The method was slow in finding general recognition, however, until, in 1942, Salmon, Geist, Salmon, and Frank31 showed that the ovarian hyperAPPEARS
BE
-443
444
HARTMAN ET AL.
[Fertility & Sterility
emia test is valid when read six hours-and even two hours ( Salmon, Geist, Frank, Poole and Salmon32 )-after urine had been introduced into the peritoneal cavity of the immature rat. Credit for the development of this test is outlined in the very unusual title of the paper by Hinglais and Hinglais: "Une nouvelle variante rapide du test de Aschheim-Zondek. Reaction d'hyperemie ovarienne chez la rate. Reaction de Reiprich modifiee par Kelso et Salmon-Geist." This is giving everybody credit in a big way. The test has been evaluated by numerous investigators. Kupperman and co-workers added a significant item to the method. 23 They noted that, whereas immature rats were adapted to the two-hour test, any female bearing corpora lutea could be used for the six-hour test if chief attention were paid to the congestion in the corpora lutea. An interesting variation in the determination of the endpoint is that of Frank and Berman who noted, in positive cases, a rosette of dilated vessels in the theca interna of stimulated follicles when viewed by transillumination, a technic which one of us ( C.G.H.) has used as an instructive class-room experiment. Both Aschheim 5 and Zondek38 have checked over the method and endorsed it, although the latter favored the twenty-four-hour period as the more reliable. Zondek and Sulman recommended this interval also for pregnancy diagnosis of the mare. Evaluations of the ovarian hyperemia test have been published by Kamenester, Kline, Ramsey et al., and Soman in 1944, and more recently by Bunde, Fried, Riley et al., and Sala et al. These publications reflect general agreement that the method is reliable although improvements in details of technic have been suggested. The method has also recently been adopted by Farris in determining the time of ovulation in women (and monkeys), since in his hands the 2-hour test has been reliable for establishing the peak of pituitary gonadotropin secretion in the menstrual cycle of the non-pregnant primate.
VASODILATATION IN THE TESTIS If gonadotropins cause hyperemia through vasodilatation in the ovary, why not in the testis? To our knowledge no studies have been reported on this subject although the experiments of King and Oslund, who described volume changes in the testes, might be cited as indicative of vascular changes in that organ. Three
445
RAT TESTIS
Vol. 1, No.5, 1950]
sets of experiments demonstrating hyperemia of the testis of the rat in response to gonadotropins are reported in the present paper. Experiment No. 1
A series of 8 rats were given interperitoneally amounts of urine of pregnancy from 0.5 cc. to 4.0 cc. in differences of 0.5 cc. At the end of six hours all of these and, in addition, a control rat were killed. The testes were removed, split open and laid in a row for comparison. An increasing redness in the series was clearly evident. When the control and the one receiving India ink perfused testes of infantile rats removed six hours after receiving various dosages of pregnancy urine.
Control
0.5 cc.
2.0 cc.
3.0 cc.
4.0 cc.
India ink perfused testes of infantile rats removed 6 and 48 hours after receiving 2.0 cc. of pregnancy urine.
Control
48 Hrs.
6 Hrs. FIGURE
l
the greatest amount of urine were compared with a color standard, as in the "Reiprich-Salmon-Farris" test, a positive reaction could be deduced. This t~chnic was not further developed. Experiment No. 2
In a similar series, six hours after injection of the urine of pregnancy, the animals were killed and their vascular systems perfused (via dorsal aorta) with physiological saline, followed by India ink. Upon completion of the perfusion the testes were all placed in 10 per cent formalin overnight. The next day they were split open with a safety razor blade and examined for intensity of gray. It was found, as seen in the photograph (Fig. 1), that the depth of gray was roughly proportional to the quantity of urine injected.
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40
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~
60
91
6lO 20 40 60
so no
IN MIL.L.IMICRONS
2 and 3. Spectrophotometric records of relative absorption of light rays within the visible spectrum. Such records indicated. the use of a filter ( 420 millicra) employed in photometric readings in the experiments.
FIGURES
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This series closely parallels the plate of drawings showing depth of color of ovaries described by Ramsey, Falkenstein and Sujkowski. TEST QUANTITATED SPECTROPHOTOMETRICALLY Since the amount of blood (and hence of hemoglobin) increases in an organ with increased caliber of its blood vessels, it was thought possible that
FrcuRE 4. 1, slides with shims in place and a mass of testicular tissue in the exact middle of slide; 2, slides pressed together and bound with Texcel tape; 3, specimen in place in carrier.
one could prepare a film of testicular substance thin enough to make readings by transillumination with a spectrophotometer and in that way obtain objective data in terms of absorption at a narrower wave length. With the generous cooperation of Professor J. 0. Kraehenbuehl of the Department of Electrical Engineering of the University of Illinois, and with the assistance of Dr. J. Littrell, records were secured, of which Figs. 2 and 3 are typical. The specimens themselves are prepared in the following manner:
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The essential apparatus consists of two glass slides and two shims 0.3 to 0.35 mm. in thickness (Fig. 4). For reasons which will be clear to the reader as he proceeds, the slides must be thick enough not to bend while handling (},i inch) and their surfaces must be ground absolutely flat. The specimen consists of a lump of tissue from the inner mass of the testis. The albuginea is first trimmed from the organ by means of a fine straight pair of scissors and the lump then excised with a curved pair. A lump of just the right size can be removed with practice. This is laid on a slide which has a glass shim placed on each end ( 1, Fig. 4) and with the second slide the lump of tissue is pressed down to make a film the exact thickness of the shims ( 2, Fig. 4). To insure this indispensable condition, the same shims are used for all the preparations of any given experiment. Testes of adult rats will yield two specimens, one from each end of the organ. Only one specimen is procurable from the testis of an immature male. The film should be of such size that it more than covers the hole in the diaphragm, but does not run out beyond the edges of the slides. From the spectrophotograms shown in Figures 2 and 3, and from dozens of others, it was learned that the greatest difference in amount of absorption was in the region of 420-425 f1 wave-length. This point having been determined, it was possible to use any electrophotometric device fitted with the proper filter. PHOTOMETRIC EVALUATION OF VASODILATATION
The major requisite of such apparatus, aside from its accuracy, is that it be capable of taking the specimen holder described above. We have found the Photovolt "Lumetron" adaptable to these technics. The specimen holder is placed directly against the coverplate of the objective photocell, and the top of the cell compartment closed to exclude room light. A 420 f1 filter was used in the experiments. In practice, the following procedures were employed. A control rat was first sacrificed, and a preparation of its testis used to adjust the instrument for maximum transmission, and to establish a reference point with which preparations from experimental rats might be compared. Since the absorptions are compared as relative densities, any reference adjustment yielding an adequate degree of sensitivity may be employed. The thickness of the tissue usually made it necessary to use relatively large apertures for the incident light beam.
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Testis preparations from the treated animals were next prepared as described above and the relative densities of these to the controls determined. A series of more than 50 groups, comprising more than 250 rats, has been TABLE 1.
Series Test
I
II
Representative Tests Illustrating Influence of Stage of Pregnancy, Dosage, Time of Reading, and Priming with Steroid Hormones
Pre-Treatment
34B KB NB 33B 41B 37B
3.0 2.0 3.5 3.0 2.0 3.0
10 10 10 10 10
NB HB HC HD IB
III
45D 45A
IV
Time, interPregnancy Urine val between % Months Ml./ Doses/ No. of 1st Dose Change in pregnant Dose Day Days and Reading Density
AB 45D 45B TC TD
Estrone, 29, over 3 wks., s.c. Solubilized estrone, 29, over 3 weeks, s.c. Dienestrol, 0.1 mgm. Estrone, 29, over 3 wks., s.c. Solubilized estrone, 29, over 3 wks., s.c. Testosterone proprionate, 2 mgm., s.c. Testosterone proprionate, 2 mgm., s.c.
10
1 1 1 1 1 1
1 1 1 1 1 1
17 hr. 17 hr. 18 hr. 20 hr. 24 hr. 24hr.
+36.2 +36.8 +44.9 +42.7 +42.3 +48.6
5.0 6.5 6.5 6.5 7.0
10 10 10 10 10
1 1 1 1 1
1 1 1 1 1
18 hr. 20 hr. 20 hr. 5 hr. 20 hr.
+ 5.2 +13.1 +11.9 + 3.8 +18.0
2.0
2
2
2
48 hr.
+35.7
2.0
2
2
2
48 hr.
+45.3
4 hr. 3wk.
1.1 + 2.9
3wk.
+ 8.6
72 hr.
+ 3.6
72 hr.
+ 4.7
.
•
examined in this manner. From the results of these tests, it has become evident that the change should be 20 per cent or higher to be considered significant. Several approaches to the vasodilatation phenomenon have been examined. These are illustrated by representative experiments in Table 1. It has been found that the urine of a two to three-months gravid woman, injected intraperitoneally into rats in one dose of 10 cc., could cause a 30 to 50 per
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cent increase in density. The readings were best made within a period of seventeen to twenty-four hours after injection. These tests are represented in series I of Table 1; each of the results is an average of several readings on both testes. Since the concentration of urinary gonadotropin decreases sharply after the third month of pregnancy, urines of women who were five to seven months pregnant were examined. Typical results in this group are shown in series II of the table. Of a total of 21 groups similarly examined, all failed to produce marked testicular changes. No attempt was made to concentrate the urine in these cases in preparation for the test; nor have any of the tests, where negative, been repeated with higher doses of urine over longer periods of time. On the theory that estrogen stimulates the output of the dilating factor and on the assumption that LH is the cause of the observed vasodilatation,24 two tests were made on rats "primed" with estrone. As seen from series III, Table 1, the estrone did not seem to potentiate the action of pregnancy urine. Estrogens themselves (series IV) had no dilating effect on the testis; nor was any effect noted with androgens. DISCUSSION The experiments briefly described above afford another example of vascular response of the target organ to a potent hormone. Dilatation of the uterine vessels occurs very promptly after the administration of estrogen25 • 35 whereas edema is at its height in six hours, 7 while active cell proliferation does not begin until the tenth hour. 1 The hyperemia of the testis, as demonstrated above, closely parallels that of the ovary in the Salmon-Farris test. No attempt was made to employ the above described technic in the determination, in the nonpregnant woman, of cyclic variation in the titre of the dilating factor, as reported by Farris. It is quite likely, however, that by concentrating the active factor from urine one might secure a significant series of data by the above descJ;ibed method. It is interesting to note that male rats of any age within the reproductive period may be employed in the test, which greatly reduces the expense of maintaining the rat colony, which must be very large to furnish a steady stream of 21-day-old females.
r
r
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SUMMARY Vasodilatation within testicular tissue of the rat occurring in response to human chorionic gonadotropin (urine of pregnancy) may be demonstrated, ( 1 ) by more intense redness of exposed tissue; ( 2) by depth of grayness of the tissue after perfusion with India ink; ( 3) by means of a photometric method herein described. The latter is a thoroughly objective method, and while probably too cumbersome to serve as a routine pregnancy test, it may have value as a research tool, for example, to supplement the Salmon-Farris ovarian hyperemia test. Since male rats of any age may be used the new method would have the advantage of greater economy. REFERENCES l. Allen, E., Smith, G. M., and Gardner, W. U.: Am. Jour. Anat. 61:321-341, 1937. 2. Aschheim, S.: Ztschr. Geb. u. Gynak. 90:387-392, 1926. 3. Aschheim, S.: Med. Klin. 22:2023-202.5, 1926. 4. Aschheim, S., and Zondek, B.: Klin. Wchnschr. 6:1322, 1927. 5. Aschheim, S., and Varangot, J.: C. R. Soc. Bioi. Paris 139:1003-1004, 1945. 6. Aschheim, S.: Ibid., pp. 1002-1003. 7. Astwood, E. B.: Am. Jour. Physiol. 126:162-170, 1939. 8. Bunde, Carl A.: Am. Jour. Obst. and Gynec.: 53:317-320, 1947. 9. Eberson, Frederick, and Silverberg, M. H.: }.A.M.A. 2176-2182, 1931. 10. Farris, Edmond J.: Obst. and Gynec. 48:200-207, 1944. ll. Farris, Edmond J.: Anat. Rec. 94:16 ( Abstr.), 1946. 12. Farris, Edmond J.: Am. Jour. Obst. and Gynec. 52:14-17, 1946. 13. Farris, Edmond J.: Anat. Rec. 95:337-346, 1946. 14. Frank, R. T., and Berman, R. L.: Am. Jour. Obst. and Gynec. 42:492-496, 1941. 15. Fried, PaulL.: Am. Jour. Obst. and Gynec. 54:689-693, 1947. 16. Hartman, Carl G.: Anat. Rec. 94:538 (Abstr.), 1946. 17. Hinglais, H., and Hinglais, M.: Gynec. et Obstet. 46:86-93, 1947. 18. Kaminester, S.: Am. Jour. Obst. and Gynec. 47:265-267, 1944. 19. Kelso, R. E.: Am. Jour. Clin. Path. 10:293-299, 1940. 20. King, C. E., and Oslund, R. M.: Am. Jour. Physiol. 70:100-110, 1924. 21. Kline, B.S.: Am. Jour Clin. Path. 14:557-562, 1944. 22. Kupperman, H. S., and Greenblatt, R. B.: South Med. Jour. 39:158-165, 1946. 23. Kupperman, H. S., and Noback, Chas. R.: Jour. Clin. Endocr. 3:.548-550, 1943. 24. Kupperman, H. S., McShan, W. H., and Meyer, Roland K.: Endocr. 43:275-282, 1948. 25. Markee, J. E.: Surg., Gynec. and Obst. 56:51-54, 1933. 26. Ramsey, T. L., Falkenstein, A. P., and Sujkowski, E. J.: Jour. Lab. and Clin. Med. 29:419-428, 1944. 27. Reiprich, W.: Klin. Wchnschr. 12:1441-1444, 1933. 28. Reiprich, W.: Ztschr. Geb. u. Gynak. 109:285-332, 1934. 29. Riley, Gardner M., Smith, Marjorie H., and Brown, Pearl: Jour. Clin. Endocr. 8:233-243, 1948.
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30. Sala, S. L., Orellana, D., and Gonzales, J. }.I. L.: Sem. Med., Buenos Aires, 2:328-332, 1946 (Abstr. Jour. Clin. Endocr. 6:412, 1946). 31. Salmon, U. J., Geist, S. H., Salmon, A. A., and Frank, I. L.: Jour. Clin. Endocr. 2:167, 1942. 32. Salmon, U. J., Geist, S. H., Frank, I. L., Poole, C., and Salmon, A. A.: Endocr. 30(Suppl.):l039, 1942. 33. Soman, D. W.: Indian Med. Gaz. 79:68-71, 1944. 34. Walker, T. F., and Walker, D. V. H.: ].A.M.A. 111:1460, 1938. 35. Williams, Maude F.: Am. Jour. Anat. 83:217-306, 1948. 36. Zondek, B.: Zbl. Geb. u. Gynak. 90:372-380, 1926. 37. Zondek, B., and Sulman, Felix: Nature 155:302, 1945. 38. Zondek, B., and Black, Rivka: J.A.M.A. 128:939-944, 1945.
Ortho Research Foundation, Raritan, N.].
DISCUSSION SALMON, New York City: Dr. Hartman and his co-workers have
DR. UDALL J. introduced a new and extremely ingenious technic into the field of gonadotropin research-a technic which opens new vistas to the laboratory research worker and the clinician. Three questions arose in my mind, stimulated by this paper. First, can the chorionic gonadotropin, rat testis vasodilatation reaction be used as a test for pregnancy? Second, does pituitary gonadotropin cause a similar vasodilatation in the rat testis? Third, if the answer to the latter question were in the affirmative, would it be feasible to utilize this phenomenon as a test for ovulation? As regards the first question, Dr. Hartman has pointed out that the technic is much too complicated to be practical for routine diagnostic purposes. As regards the second and third questions, I took the liberty, after reading Dr. Hartman's preliminary report, of performing several tests using pituitary gonadotropin and urine specimens from three cyclical women with normal ovulatory temperature patterns from the eighth to the sixteenth day of the cycle, paralleling the technic recommended by Fards for female rats. In no instance could I detect any degree of testis hyperemia greater than in the uninjected controls. It is conceivable that there may have been some degree of vasodilatation which was not discernible grossly but which the spectrophotometer technic described by the authors might have revealed. Dr. Hartman and his co-workers will undoubtedly provide a conclusive answer to these questions. Before terminating my discussion, I wish to express my thanks to the authors for having associated my name with Dr. Farris's in designating the ovarian hyperemia ovulation test. Flattering though it be, I must refuse the honor, since I have not been able to confirm Dr. Farris's observations in a series of 12 cases in which basal temperature patterns and endometrial biopsies indicated ovulation. In closing, I wish to express my thanks to Dr. Hartman for the privilege of discussing this stimulating paper and also my gratitude to him and his co-workers for having introduced another research instrument into the field of gonadotropin physiology, an instrument which I trust will shed a little more light on some of the obscure aspects of the pathophysiology of infertility.
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DR. EDMOND J. FARRIS, Philadelphia: Mr. Chairman, Ladies and Gentlemen, I want to express my appreciation to Dr. Hartman for the privilege of hearing his excellent and stimulating presentation and the fine results in this paper. I am glad to see that he has developed something that is quite as impractical as our expensive rat colony at Wistar Institute, and that the method requires a $20,000 gadget for a spectrometer. The refinement in quantitating the vasodilatation by means of the photometric method is a valuable contribution and is useful as a research method. It is definitely objective. The findings of Dr. Hartman are of interest in that the urine of a two to three months' gravid woman can cause a 30 to 50 per cent increase in density within a period of seventeen to twenty-four hours after injection. However, I don't believe the male rat as a test animal is as sensitive as the immature female rat. We use the immature female Wistar rat rather than the male and find that following injection of urine of pregnant women from days 34 on, the hyperemia in the ovaries is best defined in fifteen to twenty-four hours. The male rat tests with early pregnancy urines proved negative. By means of intravenous injections the ovarian hyperemia can be read as early as one to two hours. However, the latter technic is cumbersome. A method, to be useful, must be simple and accurate. When using the two-hour hyperemia test for pregnancy one must beware of false reactions, and particularly when the animals have been killed by illuminating gas. We use the two-hour rat test for human or primate ovulation testing only, and the overnight fifteen or twenty-four hour testing for pregnancy determination. Dr. Hartman suggested in the course of his paper that by concentrating the active factor from the urine one might secure a significant series of data by the new method. We might add that for the past sixteen months we have been concentrating human urine, and interesting and valuable data is being secured, as suggested by Dr. Hartman in his paper. It may be of interest to mention that by comparing the degree of the intensity of the hyperemia in the rat ovary, using the Munsell color chart, which is simple and objective, one can read a quantitative difference in the reactions. In a pape1 now in press, entitled The 24-hour rat test for early diagnosis of pregnancy and its aid in predicting abortion, the findings are dependent upon the differential degree of hyperemia, in cycle days 34 to 36. May I congratulate Dr. Hartman and his co-workers for their fine contribution DR. CARL G. HARTMAN (closing): We all still sometimes use the AschheimZondek test and it still is a fine test. We also use the Friedman test and have had experience with the Salmon test. I have not used the Salmon-Farris test for the detection of ovulation but I have seen Dr. Farris at work in his laboratory and I am sure he knows what he is doing. One of the gains from my presenting this paper, you see, is that it called forth two very excellent discussions, and I want to encourage you young people by stating this, that your experiment may yet constitute a contribution even though not of Nobel prize caliber.