55 THE EFFECT OF TESTICULAR EXTRACTS ON THE BLOOD CALCIUM BY L. MIRVISH AND L. P. BOSMAN. (From the Department of Biochemistry, University of Cape Town.) {Received 12th February 1929.) IT has long been recognised that the testis has an influence upon growth in general, and especially upon the growth of bone. This was the basis for the generalisation formulated by the early observers that growth and reproduction are essentially opposed to one another. Later investigations indicated, however, that the changes produced by castration are not due to the abolishing of spermatogenesis but to the fact that the normal internal secretion of the testis is not available for controlling the growth of bone. The effects produced by castration in youth are abnormal growth of the long bones, undue prolongation of the process of ossification, and delay in the obliteration of the epiphyseal cartilages. In eunuchs there is delay in the completion of the process of endochondral ossification, and the long bones of the limbs are unduly long. In spite of these indications of an influence of the testis upon the metabolism of bone growth, hardly any work has been done to elucidate the influence of testicular extracts upon calcium metabolism. Leites(i) includes the testis among the glands that decrease blood calcium, but he only quotes one experiment, in which castration on a rabbit produced a marked rise of blood calcium, which dropped again on injecting "Spermol." Apparently only one experiment was done, which is naturally inconclusive. The strict constancy of the concentration of calcium in blood in man, and in the laboratory animals that have so far been investigated, undoubtedly points to a regulatory mechanism controlled by the endocrine glands. The part played by the parathyroid in this mechanism has been amply proved by recent work. In addition, there is a good deal of suggestive evidence that the gonads may affect calcium metabolism, a striking example of which is the oft-reported healing of osteomalacia by ovariectomy in the female. We have demonstrated (2,) that ovarian extracts prepared on the lines of Allen and Doisy's preparations (4) produce a fall in the blood calcium of rabbits and man amounting to 0-5 per cent. This work was undertaken with a view to determining if the testis has a similar action on the blood calcium.
56 L. MIRVISH and L. P. BOSMAN METHOD. Rabbits were used throughout. Our general plan of housing and feeding the animals, and keeping them under constant conditions, was the same as for the previous investigation reported (2,). Blood was drawn from an ear vein and the calcium estimated in 2 c.c. serum, using Clark and Collip's method (s). The material was injected either subcutaneously or intraperitoneally, and was warmed up before administration. The rabbits were replaced in the pen after injection until the next withdrawal of blood. Control animals were employed with every experiment. The material investigated was bulls' or rams' testes collected fresh in the abattoirs, and brought immediately to the laboratory. EXPERIMENTAL. The first extract we investigated was prepared along the lines of the ovarian extract. The cut-up testis was dropped into an excess of 95 per cent, alcohol in the abattoirs and brought to the laboratory. It was then minced thoroughly and extracted repeatedly (about six to seven times) with 95 per cent, alcohol on the water-bath. The alcoholic solutions were combined, and allowed to stand in a cool place overnight. This was then filtered, the alcohol was evaporated off on a water-bath and the residue was taken to dryness. This residue is referred to as "crude" alcoholic extract and was often employed as such. Portion of this was treated with ether, but, contrary to our experience with the ovarian extract, only a very small fraction of it dissolved in ether. The treatment with ether was repeated several times, and to the ether-soluble portion was then added an excess of acetone to precipitate the phospholipins. This was filtered, the ether-acetone solution was evaporated to dryness, the residue taken up in a small quantity of warm absolute alcohol, and allowed to stand on ice. Some cholesterol crystallised out and was filtered off, the alcoholic solution was evaporated to dryness, and thus the "pure" alcoholic extract was obtained. The extract, when required for use, was mixed with warm olive oil, or emulsified with 1 per cent, sodium carbonate, and injected. In using the "crude" extract, a large proportion of it was insoluble in olive oil and gave rather a coarse material for injection. In spite of this we never found a marked local reaction in the rabbits. RESULTS. About 60 rabbits, male and female, were injected subcutaneously or intraperitoneally, and some of the results of the injections of "crude" alcoholic extracts are given in the following table. We found that the "crude" extract, when injected in the same dose as we employed for the ovary, produced no significant drop in the blood calcium. This we repeated many times and found invariably to be the case. When the dose of testicular extract was increased to the extent equivalent to about 200 gm. fresh testis, we obtained a lowering of blood calcium of the same degree and duration
The Effect of Testicular Extracts on the Blood Calcium 57 as in the case of the ovary. This effect upon the blood calcium was not as constant a feature even with this large dose as in the case of the ovary. We found that about 75 per cent, of the rabbits injected with the large dose gave this drop. When, however, the drop occurred, it was quite characteristic. As in the case of the ovary, increasing the dose even to a much greater extent did not produce a further drop of blood calcium. The blood calcium was reduced by about 0 per cent, usually in 24 to 48 hours, and generally returned to normal in 48 to 72 hours. The effect was the same in both sexes. Table I. Injection of "crude" alcoholic extracts of testis. Serum calcium in mg. % Rabbits Injected Before injection 24 hrs. 48 hrs. 72 hrs. 96 hrs. 1 2 4 56 7 8 20 gm. " crude " extract, testis 0 45»» 60 100 150 200 240 17- I4-S 17-0 17*0 17-0 i5'5 14-15*0 16-8 1-0 is-o 10-5 H'O 17*0 14-5 14-0 14*5 i6-5 Our next step was to purify the "crude" alcoholic extracts in the manner described previously and to inject them. Much to our surprise we obtained no drop in the blood calcium from these injections. A very small portion of the "crude" alcoholic extract was soluble in ether, and when this was worked up further and injected in doses equivalent to as much as 400 to 500 gm. fresh testis, no drop in the blood calcium was obtained. A few of these results are shown in Table II. Table II. Injection of "pure" alcoholic extracts of testis. Rabbits Injected Before injection Serum calcium in mg. % 24 hrs. 48 hrs. 1 100gm. "pure" extract, testis 200 00 14-5 15-8 14-0 i6-s 15*8 I5" 15*5 As controls we employed rabbits in which we injected equivalent amounts of olive oil, cholesterol or sodium carbonate solution, and also alcoholic extracts of bovine muscle, pancreas, spleen, kidney and brain prepared in the same manner as the testicular extracts. These rabbits were kept along with the other experimental animals, and bled at the same intervals, without any significant change of the blood calcium values.
58 L. MIRVISH and L. P. BOSMAN DISCUSSION. Our discovery that the ovary possesses a hormone, which reduces the blood calcium, led us to the inference that in the case of the male the testis would contain the same hormone. This fact has a further importance because so far the internal secretion of the testis has not been isolated, nor can we as yet estimate it by means of any physiological test. The presence of a physiological reaction such as the calcium-reducing property of testicular extract could be utilised to extract and purify the hormone of the testis. Our results show a definite calcium-reducing action of the "crude" alcoholic extract, but no effect when it was purified with ether. One cannot exclude the possibility that the action of the "crude" extract might be due to admixed substances present in it, other than a true internal secretion. The most probable explanation, however, is that the internal secretion of the testis which reduces the blood calcium is alcohol-soluble but not ethersoluble, in which respect it is different from the ovarian hormone. This is supported by the fact that the character of the response is exactly the same as that induced by ovarian injection; also, we have previously shown that injection of large doses of "crude" extracts of various other organs, such as muscle and brain, produced no effect on the blood calcium. Further researches with various lipoid and watery extracts of testis will throw more light on the matter. Unfortunately, the chemistry of the various lipoid fractions of the testis is exceedingly complicated. Korenchevsky(6), in a recent contribution, reviews the work to date on that subject, and attempts to isolate the active principle of the testis by the effect of various lipoid fractions on the nitrogen metabolism of rabbits. His results are highly inconclusive and demonstrate the urgent necessity for a convenient method which shall accomplish for the isolation of the active principle of the testis what the vaginal smear test has done for the active principle of the ovary. The calcium-reducing reaction of the testis may form the basis of such future investigation. SUMMARY. 1. Alcoholic extracts of testis, when injected into rabbits, produce a fall in blood calcium. 2. The character of the fall of the blood calcium is the same as that obtained after injection of ovarian extract, but the minimum dose required is about ten times as large as in the case of the ovary.. Extraction of the "crude" alcoholic extracts with ether does not give rise to an active fraction. 4. It is concluded that the drop of the blood calcium produced by the testicular injection is due to an internal secretion present in the testis. We desire to express our thanks to the Research Grant Board of the Union of South Africa for grants, which partly defrayed the expenditure of this research.
The Effect of Testicular Extracts on the Blood Calcium 59 REFERENCES. (1) LEITES, S. (1924). Biochem. Zeitschr. 18-194. (2) MIRVISH, L. and BOSMAN, L. P. (1927). Quart. Journ. Exp. Physiol. 18, 11. () (1927). Quart. Journ. Exp. Physiol. 18, 27. (4) ALLEN, E. and DOISY, E. A. (192). Journ. Amer. Med. Assn. 81, 819. (5) CLARK, E. P. and COLLIP, J. B. (1925). Journ. Biol. Chem. 6, 461-464. (6) KORENCHEVSKY, V. (1928). Biochem. Journ. 22, 482-489. BjEB'Vliv