THE TESTICULAR HORMONE.* BY T. F. GALLAGHER AND FRED C. KOCH. (From the Department of Physiological Chemistry, University of Chicago, Chicago.) (Received for publication, August 5, 1929.) Preparation. The preparation of testicular extracts has for the last 3 years been subjected to intensive investigation by this laboratory. McGee (1) and McGee, Juhn, and Domm (a), have shown that extracts of the lipid fraction of bull testicles exert a striking effect on the secondary sex characters of the Brown Leghorn capon. Gallagher (3) has shown that by the present methods of extraction and assay this activity is found in no other tissue save testis and epididymis. Moore and McGee (4) have shown these extracts to possess the same activity as the internal secretion of the testis by their effect on the spermatozoa in isolated epididymides ac- cording to the criterion proposed by Moore (5). These results in the mammal have been extended by Moore and Gallagher (6) with more highly purified preparations and some attempt has been made to investigate this reaction as a means of quantitative assay. A further new series of indicators has been proposed by Moore and Gallagher (6, 7), Moore, Price, and Gallagher (8), and Moore, Hughes, and Gallagher (9)) based upon cytological changes in the accessory reproductive organs and the secretion of the seminal vesicles and prostate gland. These varied biological manifestations of the testis hormone have been developed by the Chicago group in hope of attaining some rapid quantitative method of assay for testicular extracts. An extensive investigation of the comb growth reaction in the * These studies were in part supported by a grant from the Committee for Research in Problems of Sex of the National Research Council. We wish to express our appreciation to Professor Frank R. Lillie for making this possible. 495 by on January 18, 2009 www.jbc.orgDownloaded from496 Testicular Hormone Brown Leghorn capon has been undertaken by Gallagher and Koch, which will be the subject for a future communication. It will suffice here to note that while this reaction admits of certain quantitative interpretation it is by no means as accurate as desired. It fulfils however the requirement of rapidity in that but five daily injections are required. In the studies reported on in this paper the routine assay has been the injection of the extract once daily for 5 days. We wish to thank Dr. L. V. Domm of the Whitman Laboratory of Experi- mental Zoology for preparing the capons used in these investiga- tions. Comb measurements are taken on the lst, 3rd, and 6th days and results interpreted on the basis of the preliminary stand- ardization of the extract studied. It must be emphasized that proper interpretations can be made only if a minimal dose be determined and all subsequent studies be based on this minimal dose. The method of preparation proposed by McGee has been in- vestigated and adopted by us as the first step in the routine extraction. The tissue is ground and extracted with 4 volumes by weight of 95 per cent alcohol for from 3 to 5 days. The alcoholic extract is pressed out, concentrated to a sludge under diminished pressure, and extracted with benzene. In view of the paucity in yield of activity per unit weight of tissue we investigated at some length the completeness of our alcoholic extraction. Since reextraction of the tissue residue with 95 per cent alcohol yields very little or no activity, we have found no justification for reextracting the tissue as a routine precaution. Womack in this laboratory is extending these results. Another explanation of the low yield suggested itself; namely, that the active principle might be bound in the tissue in such a way that the activity might not be recovered completely by a simple extraction process. Hydrolysis with strong alkali, acids and enzymes was investigated but in no case could the yield be increased. There was often some loss, especially with alkali treatment, but it is significant that fresh bull testis may be boiled with 40 per cent NaOH for 2 hours without too great loss of activity. This finding however does not disprove that the active principle is in some way bound, for, since there was some destruc- by on January 18, 2009 www.jbc.orgDownloaded fromT. F. Gallagher and F. C. Koch 497 tion, there may have occurred simultaneous release and then destruction of the activity. The benzene-soluble material as obtained by the procedure adopted by McGee (1) is unfit for continued injection due to the severe local reaction produced. The most satisfactory treatment consists in complete removal of benzene by distillation under diminished pressure and treatment of the residue with acetone. The complete removal of benzene is of great importance. We have repeatedly noted that if the removal is not complete the yield of solids obtained in the acetone varies considerably and the com- plete removal of activity from the lipid precipitate is not obtained. The acetone-soluble material contains the greater part of the TABLE I. Assay of Preparation 84. Alcohol-soluble. Benzene-soluble. Acetone-soluble. gm. 211 20 Acetone-insoluble. 191 Dosage. Assay. Very large, toxic. “ “ “ 0.010 gm. 0.015 “ 0.020 “ 0.030 “ 0.15 “ None made. ‘I ‘I Negative, 3 birds. “ 3 “ “ 3 “ Positive, 3 “ Negative. activity. Some slight activity may be recovered by a second extraction of the acetone precipitate, but when this is done, the activity per unit weight is distinctly decreased. The material obtained in this step is suitable for repeated assay in the bird but is toxic in small doses to the mammal. The yield is usually of the order of one-tenth the total weight of the benzene-soluble sub-