80ME EFFECTS O F TESTOSTER,ONE AND TESTOSTERONE-PROPION-4~~~ I N THE RAT' CARL R. XOORE AXD 1)OHOTHY P R l C E Illull Zoological Laborulory, The rnir:erszt!/ uf Ckicayo The availability of androgens as piire chcmical compounds makes it possible not oiily to compare their effects i n the orgaiiisni with the effects of hormones secreted bg- the testes hut also to extend oiir knowledge of their action. It is important, therefore, to study closely the coniparatirc responses of the organism under the influciice of naturally secreted horinoiie and the pure chemical substances, and o u r earlier studies 011 androsterone (Noorc and Price, '37) a r e continued here with the utilization of testosterone and testosterone-propioiiate. Appropriate procedures provide at least a rough ineaiis of determiniiig chanyes in the secretory activity of the normal testes. The present brief report of expcrienccs with these two pure chemical substances is based upon a careful study of more than 200 albino male r a t s born i n our breeding colony. Since inevitable variatioii is believet1 to he less among litter males than amoiig animals clioseii at i-aiidoni our judgment of effects has been based upon differences eshibitecl withiii the litter by treated a s compand with untreated animals. It is a gciiuiiie pleasure to ackiiowledge the courtesies exiendecl (luring this study by Dr. Gregory Strag-nell of Schering Corporation in supplying the pure synthetically prepared aiidrogeiiic compounds. This invrstigstion has h e m airled hy :I grant from the Rockefeller Foundation to The University of Chicago. 59 THm A U A T O W l r 4 1 . BFCOTtD TO1 $1, h O 1 AVIJ 6ll'PT L\IB\T >O 1 60 CARL R, JIOOItE ATSD DOROTIIY P R I C E A . EFFECT8 I N ChS'I'RA4'l'E M A L E S The determination of effects of testosterone and the propionatc have been based 011 fresh weights of indiridual parts as corripared with those recovered from normal litter mate controls, aiicl upon the histological character of the tissues, especially the ventral lohe of the prostate gland and the seminal vesicles. J17iththe aid of a binocular dissecting microscope all tissues have been removed immediately upon sacrifice of the animal and weighed to fractions of a milli;oram on a torsion balance, after careful removal of all fat and coiiiiective tissue. Tn young males sacrificed on day 15 or earlier, daily injection periods residted in R precocious development of the accessory organs as judged by weight increases, and also by a precocious secretory cliff erentiation ; seminal vesicles, which normally show m a k secretion granules only about day 35 or later, differentiate into a secretory type of seminal vesicle by day 18 that is indistinguishable microscopically from that of mature adult males. Daily injections of 0.4 mq. testosterone into males after castration on day 15 and sacrifice on day 35. result in seminal vesicles weighing 19002, more than those of untreated litter mate controls. Testosterone propionate, as will be showii below, is even more effectire. The microscopical character of the nownial and castrate prostate and seminal vesicle of the r a t has hcen descrihed earlier (Moore, l'rice and Gallagher, '30 ; Rrooi-e, Hughes and Gallagher, '30). Tt may be mentioned in general that 110th tesosterone aiitl the propionate in eff ectire doses pither maintain the normal secretory state of these organs in castrated males of m y age, or restore long-time castrate tissues t o the normal secretory state. No qiialitative differences have appeared in these ti ssiies conditioned by chemical ancliwgeiis that would distiiijiuish them from tissues of normal males wlien proper dosages are employed. On adult males, clowges of tesosterone and the propionate required for testicular replacement have heen determined from daily injections begun immediately after castration and EFFECTS OF TESTOSTEBOKE IN T H E RAT 61 continued f o r a period of 20 days. ‘l’he two criteria eniplo-yecl to determine tpsticular replaccmeiit, and Iqrcseiiting different dosage rcquirpnients, have been 1) thc qnantity of substaiice adniinisteretl daily that is necessary t o maintain ilie hi st ologi cal n 01-inalitp of tli e vent 1-21 pros t at c aiid wminal vesicles ; 2 ) the daily dosage requirement f o r maintpnance of the noi-ma1 fresh weight of the st~minalvesicles. llcqniwrneiits for. the second inclicator a r e usually ahout three to four t inies reqaiiwnen t s for niaiiit emrice of liist ological 110rmalitv. H isf ological 210 w2aZity Histological norniali ty of the ventral prostate consists in the mairileiiarice or production of an active secretory epi theliurn in aciiii, the cells of 11-liich contain well-defined light areas marking the position of the Crolgi apparatus miclway between Ihe nnclcns a i d luminal end of the cell. Such diagnostic light areas make their appearaiice in ventral lobe cells nbont day 13 (Price, ’36) ; they are promiiicnt from day 30 throughout the hormone sccreting period, and in adults they disappear ahout the fourth day after castration. They are clearly renxded in tissues after Eonin fixation and Harris hematoxyliii stain. Histological normality of seminal vesicles consists of the ~ ~ r e s e n cofe a lining epithelium of high columnar cells coiitaining definite secretion granules, surrounded hj- clear ‘halo’ areas in stronger preparations, easilv demonstrated by Fouin fixation and Ehrlich ’s liematoxylin stain. These graniiles make their first appearance ill normal animals about days 35 to 40 depending upon the state of hormone secretion, or degree of maturity, and in castrates they disappear approximately 48 hours after castration. More than fifty animals were utilized in the determination of dosage requirements of testosterone and the propionate t o maintain a normal histology of the ventral prostate and seminal vesicles in castrated adults, as wcll as requirements to maintain uoimal weight of seminal resides. Ventral prostates required from 30 t o 50 gamma daily of testosterone for 62 CARL B. N O O R E A N D DOROTHY PRICE histological normality and the lowest effective dose of propionate was not determined. Seminal vesicles contained melldefined secretion granules with a daily dose of 200 gamma testosterone and with somewhat less than 50 gamnia of propionate. Xaintenance of seminal vesicle weight in adults was obtained with 600 to 700 gamma daily and with 150 gamma of propionate. Growth maiiLteiaarirf: of seilziiaal r e s i d e s . It early became evident that dosages of clicniical androgens required t o maintain the growth of seminal vesicles equal to that of iiormal litter mates diff'ered with agc of the animals. The observations 011 one litter consistiiig of seven males, the castrates of which ~vei-etreated ~t7ithtestostcroiie between clays 30 and 50 TABLE 1 E f e c t s of testosterone in c n s t r d e d mts. CartratiaiL day 3D; iiijertions (Tail!/ days 30 t o 4 9 ; sacrifice day 50 T v P i g h t , &!rams Cast. 0.1 nig. Cast. 0.2 rng. Cast. 0.4 mg. Cast. control Cast. control Norm. control Norm. controI 'rota1 prostate complex Seminal Per cent change vesicles from normal 0.1:354 0.3337 0.4416 0.0384 0.0501 0.1166 0.1971 0.0188 0.1606 0.4504 0.0054 0.0066 0.0257 0.0454 S P ~ w . s. - 47 + 352 +1168 Prostate A C ves. ratio ~ . 7 2 0.9 7 7 4.5 4.5 are presented in tahlc 1 as an example of treatments given othei. litters of different ages. Ten litters iiieliidiiig more than fiftr males have been aamined directly for these determinations and many animals of litters used for other purposes afforded additional data. For each litter the period of treatment was 20 days with injectioiis of castrates beginning immediatelp after operation, the litters being sacrificed at ages 35 t o 90 days; the rat is essentially mature bp the eightieth or niiitieth day of life, since it ~ a d i l sires ~ 7 young and has attained a mature prostate-seminal vcsicle ratio. Attention t o table 1 reveals the average seminal vesicle weight of two norrnal males on day 50 to he 35 nig. in comparison with 6 mg. in untreated castrates. Seminal vesicles EFFISCTS O F TESTOSTERONE IN T l I E RAT 63 of the castrate receiving 0.2 nig. testosterone daily showed a weight increase 3 5 2 7 . greater than the normal litter mate average, but in tlie one receiving 0.1 mg. daily the seminal vesicle weight was 47% below that of the normal controls. Other litters i*evealed that the growth-replacement dose of testosterone between the thirtieth and fiftieth day was approximately 0.15 mg. daily. Tn table 2 a summary of observations is given f o r replacement dosages f o r both testosterone and t-proprionate f o r different 20-clap periods during the age of most rapid growth of reproductive parts. It can be seen that whereas 0.1 mg. daily dose of testosterone serves a s an adequate substitution f o r the testicle in promoting growth of seminal vesicles from day 15 to dav 35 it requires a daily dose TABLE 2 Daily d o s a g m for growth ntainteitmre of .wminnl ~estkles,darinq 2;arzoim dO-dny p e r i i d s . l u t o p v i N B P . d a ~ s TmtmteronP 3 ri 60 60 70 80 90 -0.1 mg. 0.15 mg. .... t 0 . 3 0 mg. +0.60 mg. .... 1' p r o p w n a t e .... 0.05 rng. 0.08 mg. 0.08 mg. 0.15 mg. 0.15 mg. of slightly 111ore than 0.6 mg. between days 60 and 80 to maintain growth of these organs i n castrates equivalent to littermate controls. A similar trend is shown f o r t-propionate but dosage requirements a r e much below those of testosterone. The greater effectiveness of the former lias been pointed out by most workers nsing these substances. Pmstate-seiniizal vesicle ratio. The question is pertinent whether testosterone or tlie propionate has a selective effect upon one end organ or provides a general stimulation equivalent to that of testis-secreted hormone. This question was examined by Moore and Price ('37) with respect to the effects of androsterone, basing judgment upon a n analysis of the relative weights of the total prostate complex to the seminal vesicles. I t was determined on normal males of different a p s 64 C d R L B. MOOKE ANU D O l t O T l i Y PlZICE that the pl.ostate-seaiiiia1 vesicle ratio value prior to the onset of intensive hormone secretion was high aiid as testes secreted more hormone tlicre was a gradnal decline from a value of 10, 01- greater, t o a value of 1 at an age of approximately SO t o 90 days. Xubsequeiit to this period a i d throughout re~~i*oductive life this value constituted the normal onc, and whereas tlie ratio value declined with approach to maturity i t was surprisiiigly coiistant f o r the litter. T i 1 tabIe 1 the cletermination of the prostate-seminal vesicle ratio is included f o r oiie litter. Iii this litter sacrificed on day 50, each of two normal males showed a ratio valile of 4.5 whereas the value for two castrates was 7. Tlie castrate injected with doses of testosterone inadequal e t o maintain normal growth of seminal vesicles (0.1 mg.) revealed a ratio value greater than that f o r normal litter mates but a second castrate receiving a n excessive dosage (0.4 mg.) exhihitecl a ratio value much below the normal for the age arid eiitircly equivalent to that of mature adult niales (0.9). Thc dosage of 0.2 ing. WRS more than sufficient for testis substitution (seminal vcsicle wciglit 352% above coiitrol ) aiid gave it ratio value approximating tliai of maturity. Whereas i n this particnlar litter an exact ~i.offth-substitutioiidosage WRS not injected, other litters have sliowii that similar t o the cff s of androsterone, testosterone a n d tlie propionate in substitioiial tloscs proclncecl a prostateseminal vesicle ratio value equivaleiit to normal litter mates. This again emphasizes the effect of the chemical androgeiis as equivalmt to that of the natnrally secreted hormone insofar. a s these methods a r e capable of demonstrating. Wit11 adequate replacing dosages they fail to exhibit a more selectivc stimulation on one end organ than does norinally secreted testis hormone. P r ol o uged o f w t s . T h e (1 u e st i on 1 v . hth ~ e 1- oiie compound exerts a prolonged effect on the mammaliaii end organs mas briefly investigated by a study of the microscopic condition of tissues from forty adult males. Precautions were observed in injecting dosages of androsterone, testosterone and tpropionate into castrates for periods of 20 days that would EFFECTS O F TESTORTRROWE I N THE RAT 65 maintain the iiormal semiiial vesicle weight and not represent many times the actual requirements for this purpose. Wliereas weights of tissues mere cilrefdly recorded, cliief dependence was placed upon the histological state of the prostate and seminal vesicles. In ordinary mitreated castrates, i n which the hormone soiirce is completely eliminated at oiice, marked castration caliaiiges appear within 4 days in the prostate and earlier i n seminal vesicles. Injection of chemical androgeiis in an oil solution naturally provides residual unabsorbed active material for some time after thc last injection. Castration chaiigcs similar to the 4 - h untreated ~ castrates appeared usnally by the sixth to seventh day after the last injection of androsterone and testosterone but injections of testosterone pi’opionate produced effects observable for slightly longer periods after discontiriuaiice of injection. Thus in one group of twelve adult males of similar age and size, but not litter mates, ten were castrated and injected immediately with 0.2 nig. l-propionate daily f o r 20 days. Two injected castrates and two untreated normals were sacrificed 1day after the last injection when it was determined that seminal vesicles of injected castrates ~ w r e70% heavier than those of the untreated males (avcrages of two each) ; thus the dosage f o r all iujected animals wab slightly in excess of a replacing close. Histologically the prostates and seminal vesicles of the normals a i d injected castrates were indistinguishable. Two injected castrates were sacrificed thereafter on days 5, 10, 13 and 17 after the last injection. The prostates and seminal vesiclrs of atiininls killed 5 clays after the last injection mere histologically normal but those sacrificed on day 30 sliowcd decided castration changes ; those killed 13 and 17 days after the last injection revealed castration changes approximating those found in ordinary castrated males on about day 10 following operation. In a second group of eight males (litter mates) six were castrated on day 70 and injected daily until day 89 with 0.1 5 mg. t-propionate, n-ith sacrifice of two untreated normal 66 CARL El. MOORE AND DOROTHY PRICE and two injected castrates on day 90 (1 day after last injection). This dosage proved to be almost an exact replacement dose since average weight of injected castrate seminal vesicles was only 19.h heavier than those from untreated normal males. Histologically, prostates and seminal vesicles from the normal and injected males were indistinguishable. Two injected castrates were sacrificed on days 8 and 11 after the last injection. By the eighth day definite castration changes were evident and by the eleventh day a grade of involution was present that would correspond to approximately the eighth day of castration in ordinary untreated males. Thus testosterone-propionate administered in approximate replacement doses, and not in large overdoses, shom-ed but a slight prolongation of effect. Ordinary untreated castrate males attain a degree of castration change on the sixth to eighth day in the prostate and seminal vesicles that makes its appearance on approximately 8 to 11 days after the last of a series of injections. This slightly prolonged effect of not more than 5 to 6 days at most is probably due in part at least to slow resorption from oil residues in subcutaneous pockets hence it appears fair t o conclude that prolonged effects in the castrate rat are not evident for more than approximatel>3 days. Iwitiafion of copzclation. Testosterone-propionate mill not only maintain or repair accessory reproductive organs in castrates but its administration results also in the awakening of desire and ability to copulate when castration has been performed before germ cell production. Three males of a group of five mcre castrated on the thirtieth day of life and caged with two normal controls for a period of 6 months. TWOof the three received 22 daily injections of t-propionate, one 0.5 mg., the second 1.0 my. daily; the third castrate was maintained as an untreated control. Within 2 weeks from the first injection the two treated males exhibited a normal response to females in heat and executed repeated and vigorous copulations with pjaculation; the untreated castrate showed no interest in females. 67 EFFECTS O F TESTOSTERONE IIS THE BAT When sacrificed the day following the twenty-second injection average seminal vesicle weight of the two treated castrates was found to be 828 mg. as compared with 5 mg. for the UIIinjected castrate (increase of 16,00070) and 740 mg. for normal controls (increase of 11%).Daily treatments with 0.5 mg. apparently induced a maximal growth response since 1.0 mg. doses gave a similar seminal vesicle weight. I n this group, therefore, males castrated prior to the onset of hormonal activity arid remaining as castrates from 30 days to an age of 7 months, were induced t o develop into a state such that the copulation pattern was entirely characteristic of normal males and the accessory organs had developed slightly above the normal controls a s a result of the 3 weelis of treatmen 1. TABLE 3 Effects of t propionate i n normal male ruts. Injections, days 20 to 99; sacrifice, day 40 Dad# dose 0.05 mg. 0.10 mg. 0.80 mg. 1.00 mg. Control Control T'estG weight, Per rent ilecrpaRe PPnt. from pro>t., rant rol gram3 0.3538 0.3140 0.7474 1.0228 1.2110 1.3146 -72 -75 4 -s9 0 grnm-? 0.0664 0.1026 0.1824 0.1848 0.0432 0.0386 Per c e n t increaae over control 61 150 300 350 Sem. v e s . w?iEht, gromv 0.0910 0.3126 0.7090 0.8156 0.0106 0.0118 Per cent increase 0 7 ~ - co7rtrol 712 2691 6230 7182 B. EFFECTS ON THE TESTICLE Data were obtained on thc effects of tlicse chemical anclrogens on the testicle from eighteen littcrs of animals consisting of more than eighty-five males, the majority of which receirrcl treatments with testosterone propionate. I n each litter the injected members were compared with their normal brothers and the 20-day periods of treatment were so ari-aiiged as t o terminate within the first 80 days of life which covers the prepuberal pcriod. I n table 3 will be found sonie details from one litter sacrificed on the fortieth day of life subsequent to a 20-day period of injection with four dosagc levels of tedtoRteroiie-prop ion at^. 68 CARL B. 1IOOP.E AND DOROTHY P R I C E It can be noted that testis mcights f o r each of the four treated males were appreciably below either of two untreated litter mates. Compared with tlie average normal testis weight that of the male rewiviiig 0.05 mg. clailp sliowed a recluctioii of 72%, 01- weighed 28% of normal, d i e r e a s treatments with 1 .O rng. daily reduced testis weight by but 19%). This production of less and less damage as the dosage is increased has been quite characteristic especially f o r the propionate. The 80 , 1 I 20 30 40 50 _ _- - 7 60 70 80 DAYS Fig. 1 Effect of testosterone proIiionate i n suppression of testis gromtli. Ordinate represents yerceiitage dccreake in freali weight of testis ; abscissa. r e p = s e n t s age in d : r y at autopsy, precrtl~d1))- x 20-day pcriod of injeetion. Each point sl~owspercentage decrease in testis weight i n treated males a s compared with normal u n t r e a t e d litter mates. Amoiint n f daily adinini~trationindicated 611 curves. effects 011 the accessory organs have berii usually one of increased effect with increased dosage. In the litter under eonsidcration ventral prostate -creight increascs above normal controls were from 6lF1to 350% and serriiizal vesicle increases were from 712% to 7182%,. The effects on the testicle of four dosage levels of t-propiouate €or diffcreiit 20-day periods h a r e been summarized in a graph (fig. 1) each point 011 which represents percentage EFFECTS O F TESTOSTERONE I N THE BAT 69 change in testis weight determined from comparisons with normal litter mates. Coiisicleriiig tlie general aspects of this graph, and that of numerous cases with a dosage range from 0.05 mg. to 1.0 mg and riot represented on the graph, it becomes evident 1) that testosterone-propiorlate in illis dosage range iiiduces niarked loss in testis weiglit. This is evident in twenty-one of the twenty-two treated males represented. One litter exception occurred in which the injection period was 11 instead of 20 days (8 to 19) ; in this litter tcstes i'roni three treated males were slightly heavier than those from the two control males. 2 ) In general tlie injurious effects appear iii the reverse order rather tlian parallel with dosage since usually 0.05 mp. tpropionate induced iiiore severe injury than 1.0 nig. daily doses. With ordinary testosterone, iiijuries a r e iriducetl with similar dosages but the reverse order of effects with dosage is less appzirent. 3) Tlie harmfiil influences npon the testis appear to climiiiish as the animal approaches maturit!:. This is evident not only from the qraph of effects after propionate treatment but it is likewise evident after ordinary testosterone. 1'11~stestosterone in daily dosagc.s of 0.1 rng. 0.5 mg. and 1.0 mg. aiitl sacrifice on day tjO canset1 a decrease in testis to 67% in contrast to sacrifice on day 70 weiglit of from in whicli decreases of but 9% a i d 13% were recorded. Two males receiring 2.0 mg. t-propjonate daily froin days 70 to 90 registered deereases of but 1%and 10%). The effect upon tlie testicle indicated by decrease in weight iiivo1.i.e~real tissue damage i n the more severe cases. In testes just approacliing the period of spermatozooji differentiation tubul es a r e much reduced in size and show considerable sloughiiig of cells into the lunien. Treated males having atlaiiiccl the age of spermatozoon differentiation may show proiiouncecl delay in devclopmeiit as well as considerable actual injury. The treatnient does not prevent the testes from ultimately diffcreiitiating spermatozoa since considcrable weight reduction may he accompaniccl b:! a few tubules with spermatozoa though the percentage of tubules reprcsenting such clcvelopnient is marItecllp less tllaii the controls. TO CSRL R. MOORE AND DOROTHY P R I C E In order to determine wliether testosterone or t-propionate exhibited any tendency to stimulate spermatogenesis a number of litters were SO injected a s to provide sacrifice on approximately day 32 since by day 36 untreated males possess tubules showing the ‘sperm-head’ stage (Moore, ’36). No evidence has been forthcoming that either of these chemical aiidrogens stimulated spermatogerietic activity and detectable inflneiices in every case 2iare heen of an injurious ratlicr than a stimulating nature. DISCUSSIOS The comparative effects of the chemical aiidrogeiis, testosterone and testosterone-propioIiate, and normally secreted testis hormone are remarkably similar when prostate gland and seminal vesicles of rats are used as the test objects. These substances are capable of producing any grade of effect from barely detectable responses of the end organs7 t o conditions indistinguishable from iiormal litter mates, or to marked precocious growth and secretory responses when appropriate daily dosages are employed. Whereas qnttlitative differences in effects of these two chemical substances have failed to he noted in this study the quantitative cliff erences noted hy numerous investigators have loeeii evident. Testosteronepropionate per unit weight has shown a11 activity approuimate17 four times greater than testosterone. dosages of these two sd>Adequate ~1.o~rth-substitntiorial stances have hemi found to promote types of relationships between the weight of the prostate and seminal vesicle that exist in normal litter-mate controIs of young or adult ages. These findings are in agreement with those obtained from treatments of castrate rats with androsterone and it is t o be emphasized that though dosages below requirements for testis substitution on growth maintenance of seminal vesicles will appear to selectively stimulate the prostate because of its lower threshold of response, adequate substitutional dosages provide a prostate-seminal vesicle ratio equivalent for the age. Reports that the chemical androgens exert a selective stimuIation on one end organ rather than a general stimulation EFFECTS O F TESTOSTERONE 1 N T H E RAT 71 characteristic o f testis-secreted liormonc arc believed t o be based upon the usc of dosage levels too low to adequately stimulate semiiial vesicles, m-liile being sufficiently high to perinit the lowcr thresholcl reacting prostate to develop norrnallp (Callow and I)eaiicsly, '35 : Xorenchevskp et al., '35 ; Deanesly and P a r k s , '36; Parkes, '36 : Niescher, J&7ettstein and Tschopp, '36; Tschopp, '36). The sliiftiiig of the ratio value by different closages of t e s l osterone is illustrated i n table 1 whcrciii for the teriniiial age of 50 days 0.1 mg. would selectively stimulate the prostate and give a n aberrant ratio value higher than normal, 0.2 and 0.4 nig. representing morr than substitutional dosages, reduce the value lower than normal and shift relative vwiglits to those o f normal adults rather than those cliaracteristic for the real age of the litter. Tlie adeqnacy w i t h wl1ich these chemical androgens substi tute for the testis and the qnaiititatire variation in growthreplacing doses f o r rarping ages, suggests that such procedures proridr a rough measure of secretory intensity of normal t cstcs. It has been pointed out (Price, '36 ; Moore and Price, '37) that seminal vesicles of normal malc r a t s grow slowly for approximately ihe first 35 or 40 claps of life wheii tlie curve o f weights ascends sharply until approximately 80 t o 90 days of age when the ascent is more gradual. Data on I-eplacemcnt closagcs licrcin presented indicate that secretory activity of testes gradually iiicreases during prepuheral development LIP to the level maintained i n adlilts and that such a stage is reached at about 80 claps of age. The samc line of reasoning sug-gests tliai the pituitary glaiid, the normal incitor of testis activity, liliewise begins to release more and more gonad activating hormones. Clark ( ' 3 5 ) demonstrated that the pituitary of male r a t s gradually increased in gonadotropic potency from birtli to puberty. As shown by lreatmeiits with gonadotropic liormoiie iiijectioiis the amount Tseleased is hy n o meails sufficient to induce the full secretory capacity of the testes. These considerations sugyrst that puberal development is z process cliaracterized by a gradual incremental increase in 72 CARL R. MOORE AND D O R O T H Y P R I C E lestis liorrnone secretion and that as the higli level of secretion is attained a t approximatelj- 80 clays i t fails to increase further, normally, bnt is capable of being increasecl by proper treatments. With the establishment of this noriiial peak level of activity, regulatory forces williiii the organism appear to become operative and l-robablp consist a t least in part of a cliecli on pituitary activity by high testis hormone levels. IIooker ( ' 3 7 ) extracted testicles of difierent aged bulls and coiisiders that hormone secretion i s a gradually iiicreasiriy function from 1+months to 3 years of age, after which the function gracliially declines. I n the rat the high point is attained within 3 months and appears to remain fairly constan t during reproductive life. It has been indicated tliat testoste~oiie-p~opioilntc exhibits effects of longer duration tliari other androgens (lfiesclier, Wettstein and Tschopp, '36; I'arlies, '36) and it has been of interest to compare its effects not oiily with those from testosterone and androsterone bnt also wit11 the iiormalljsccre t ed hormone. Castration of normal atliilt male r a t s is followed hj- such rapid reduction of the hormone level as to induce definite castration cliaiiges within a period of 4 claps on the prostate gland and even earlier on seminal resides. In this case the completrll; and no source oi' hormone is removed suddeiily ~ i i d storage in the organism is apparent. liijections of chemical androgens in an oil medium 1)rovide a residual s o wce of hormone for approximately 2 days since a grade of castration cliange appears after cessation of injectioiis of testosterone and androsterone about days 6 t o 7 that appears after ortliary castratioii on clays 4 to 5. Tii experiments liere reported care has lieen exercised to employ growth-suhstitntional doses in order that the end organ developrnent moulcl be equivalent to that in the normal animal. Activity of secreting cells in the end organs has served as the principal criterion of effect instead of gross weight of organs. By these methods castration changes following the last of a series of iiijcctioiis with the propionate EFFECTS O F TESTOSTNROSE IN THE; BAT 73 appear. to have I)ecln delayed f o r approximately 3 t o 4 clays longer than after treatments with other chemical aiiclrogens o r after ordinary castrrttioii. This short prolongation of effectiveiiess is presumed t o represent slomcr absorption of t-propionate from the oil. The rapictitv with which full-sizcd sclminal vesicles can be produced in prepiiherallp castrated males untreated f o r 6 months is reniarkalole. Within 3 wceks f roni beyinniiig treatnieiit wjth propionate active secretion lias heen initiated as ~ w l a l s an actual illcrease in weight of 160 times a s compared with iintreated castrates ; the vesicles attained a size and weiglit i n 3 weeks that Iiad heen attaiiiecl in the normal control a t the aye of 7 months. Copulatory ability was cle\~lopedto its fnll capacity within the 3-meek period of injections. This is real copulatory awakening since normal males on the ave1.age experience first copulation only about 60 clays of age (Stone, '24, '27) ant1 castration liad been performecl oil clap 30. Despite the fact S tone ( '27) determined copulatory ability was evideiicecl S months after castration in one rat, his animals \\-ere castrated about clay 90 a n d after they liad experienced copiilation. Even so, 74F of a group of forty-five males castrated a t the age of 90 clays ha(l lost copulatory ability by the end of the fourth month after castration. The absolute pcrfection of the copulatory act on tlie part of malcs treated n-ith testosterone-propionate, ancl tlie fact ejaculations occni*red, leare no doubt of tlie entirely normal character of the reaction developed at the end of a castration pcriotl of more than 6 m o n t h and devclopecl for the first time. De Fremerj- and Taask '37) fouiid that prepuhei.allp castrated rabbits developed copnlatoryv ahilitp 7 inoritlis after castration after injections of 10 mg. of t-propionatc daily for a period of 11 to 13 clays. Uiipublishcd observations (Moore) show that guinca pigs castrated at 30 days of age and injwlecl 6 months later deTTeloped copnlatory ability (with ejaculations) after daily injections of 0.3 mg. ancl 0.5 mg. testostcronc-propioilate. 74 CAlZL 13. MOOBE AND 1)OItOTHY P R I C E The injurious effect of testosterone and t-propionate upon the testes of males during the rapidly growing period and lheir stimulating effect on the accessory organs coincides with earlier findings on tlie effect of bull testis extracts (Aioore and Price, '32) and the effects of androsterone (Noore and Prjce, '37). Testes from treated males were materially reduced i n weight ; differentiation was arrested ; destruction and sloughing of cells of the gerriiiiial line into the seminiferous tubules was prominent ; tubule diameter was reduced and spermatozoon formation completely inhibited or greatly delayed. Two points of similarity of effects with those after androsterone treatments were 1) that the harmful influences from givcii dosages beconie less evidence as the animal approaches maturity and 2) relative1~-large dosages fail to materially affect the testes of mature adults. Differing at least i n degree from other antlrogens the propionate exhibited tlie soniewliat pel-plexing property of being more injurious iii lower dosages. The figure shows that f o r most periods during prepuheral life daily doses of 0.03 mg. induce testis decline and injury to a greater extent than those of 1.0 mg. Iiiterprctatioiis of these phenomena are not clear at the present time. Our earlier suggestions (Moore and Price, '32) were to the effect that deleterious action of androgens upon tlie tcsticle rather than being a direct effect was iiidirect by virtue of a suppressive action upon the pituitary, or one ~vliichled l o an insufficient amount of pituitary hormone being available to stiiiiulate normal testis function. Pituitary glands from normal rats treated with androsterone, which resulted in testis damage, were found to have diminished gonadotropic activity j i i infantile fcmale mice (31oore and Price, '37). Hertz and i\leycr ( '3i) have sliowii also that chemical aiidrogens injected into the castrate 1xwtiier of a parabiotic pair effectively prevented tlie usual stimulation of tlie normal female of tlie pair, presuiiiably 11~-virtue of preventing excessive pituitary activity that follows in the castratc partner and expressed in tlie normal parabioiit partner. These and numerous other data, considerable of which are cited in EFFECTS O F TESTOSTERONE IX- THE BAT 75 former papers, suggest tlie effect a s one of pituitary modification, and tlie dccliiiing effects with ageing can be niiderstood as perhaps due t o w less easjla nioclifietl pituitary gland. The general hypothesis contains no element of explanation, however, for the clecliiie in effectiveness with elevation of dosage as has been foulid after treatments with the propionate. The suggcstiori o i abseiice of direct effect of the androgenic substaiiecs upon thc testis might appear to deserve modifieaiion when consideration is giv-cii to the fact that 1) androgenic substances prevent testis involution s~he11injected immediately after hypophysectomy (\Val&, Cuyler and 3ZcCullagh, '34 j Nelson aiid Gallagher, '36 ;Wells arid Gornez, '37) and 2 ) that treatment with androgenic suhstances results in heightened testis actirity in the seasonal breeding mammal (Citellus), leading t o unusuall? early production of spermatozoa (Wells and Moore, ' 3 6 ) . With regard to treatments of liy~ophyseetoniizeclmales with various aiidrogens it should be mentioned first that, in agreement with the early unpublished findings of Vatna (Moore and Price, '32) using bull testis extract, repair of hypophysectomy damage to the testis was n o t effected. h consiileralion 1) of the capacity of androgens to prevent involution after hypoplij-sectonly but iiiability to correct damage to the testis after its development, and 2) the fact that progestin (Kelsoii, '36) or yeast extract (Ilisaw, Grccp and E'evold, '36) lilrewise prevent tcsti s damage wheii given immediately alter liypophyscctomy raises the question wlictlier androgens have a direct effect upon the testicle. Reproductive conditions in seasonally active forms admittedly diEer fyom those in continuous breeders such a s the rat. It x7as deterrniiicd that administration of androgens to the normal but sexually iiiactive g r o u i d squirrcl was follomed by heightened testienlar activity and germ cell production (TI7ells and 3Ioore, ' 3 6 ) even in case the animal had been subjected to hppophysectomy (Wells and Goniez, ' 3 7 ) . Ordin a r y pregnancy urine and esiracts of uriiie likewise proved to be effectivti agents. At the same time adniiiiistratioii of 76 CARL B. MOORE APU'D DOE O TH Y PRICE androgens t o sexually active ground squirrels induced liarmfnl effects (Wells, '35). A considcration of these various findings and the facts with reference t o the effects of t-propionate cited above leaves one searching for an apparently acceptable hypothesis that will bring the various findings into a common explanation. Without doubt too little is linown of the metabolism of hormoiial snbstances by the organism to warrant further speculation at the present time. SUMMARY AND CONCLUSIONS 1. The pure chemical androgens testosteroiic and t-propionate induce secretory activity in the prostate gland and semiiial vesicles of castrates that is indistinguishable from normal activity. These substances a ) maintain normal histnlogical conditions o r repair damages in castrate males : 13) induce precocious secretory activity ; c) maintain the normal growth rate of accessory organs of reproduction in castratcs during the most rapidly growing period of life and d ) promote a normal prostaic-seminal vesicle ratio when administered in dosages adequate to maintain normal growth of seminal vesicles ; inadequate dosages for seminal vesicle growth maintenance naturally appear to favor prostate activity clue to its lowcr tlireshold of response. 2. By weight t-propionate proved approximately four times as active as tpsosteronc. Substitutional dosages for maintenance of growth rate of seminal vesiclcs was approximately three times dosage requirerncnts f o r mainteiiance of histological normality. 3. T-propionate exhibited only slight prolongation of effectiveness after injection (approximately 3 days) as compared with testosterone, androsterone or ordinary castration of normal males. 4. T-propionate injectioiis into rats castrated prepberally and injected only aftcr 6 months induced the animals to esecute repeated and vigorous copulations, including ejaculations, within a period of 3 weeks. Seminal vesicle weight exceeded that of normal 7-month-old control males. 5. Testosterone and t-propionate cause grave retardation and injury t o the testes of young normal males hut this eEect gradually declines with age. The propioilate, especially, exhibited a reverse order of effects with increased dosage, 0.05 mg. often hciiig more injurious than 1.0 nig. daily doses. LITERATI,TRE CITED CALLOW,R. I<., AKD RUTII DEANERLP 1935 Effect of androsterone and of male hormone concentrates on the acce3sor~reproductive orgaris of castrated rats, mice and guiiica pigs. Biocheni. J., 101. 29, pp. 1424-1445. CLARK,HELENhl. 1935 A prepubertal reiersal of the sex difference in t h e gonadotropic hormone content of the pituitary gland of t h e rat. Anat. Rec., vol. 61, pp. 175-192. 1935 A sex difference in the change in potency of the anterior hrpophysis following hilateral castration in newborn rats. Anat. Kee., 61, PP. 193-202. UEAVESLTT, It., AKI) A . 8. PARKES7936 Comparative activities of compounds of t i l e androsteronc testosterouc series. Biocheni. J., vol. 30, yp. 291-303. Dn FREXERY, P., AND 31.T a u s a 1937 Geschlechtliche Alrtivitiit kastrierter n i m i i liclier Raninchen nach Bcbandlung niit Tc-3tostcronpropiona. Bcta brev. Seer., vol. 7, nos. 8/10. HEKTZ, ROY, AND H. K. N E Y ~ : R1937 The effect of testosterone, tcstosteronc propionate and dehgdroandrosteronc on the secretion of the gonadotropic complex as evidenced in parahiotie rats. Endorrinohgy, vol. 21, pp. $56-761. HISAW,>'. L., €3. 0. GREW AND H. L. FEVOLD 19.36 Pituitary-like effects of peast extracts. Anat. Rcc., vol. 67 (Suppl.), p. 50. HOOKER, CFTAS. W. 1937 Testis horinonr iii relation t o age. Endocrinolog-y, vol. 21, pp. 655-658. KORESCHEVSKY, V., AND 1\1. DEXXISOX 1935 The assay of f a t soluble androsteronr-iliol. Biochem. J., rol. 29, pp. 2122-2130. KORENCHEVSKP, V., 31. DENNISOWA N D S. L. SIMPSON 1933 The effects of water-soluble preparations of androsterone arid androsteronc-diol 011 castrated rats. Hiochern. J., vol. 29, pp. 2131-2142. MICSCHER,I<., d. T V m w m r N A N D E. TSCHOPP1936 The activation of the male sex hormones. I. and 11. Blochem. J., vol. 40, pp. 1970-1990. MOORE,C-4RL R. 1936 Responses of immature rat testes t o gonadotropic agents. Am. J. Anat., 101. 59, pp. 63-88. I\fOORn, CART R., w.I ~ l I G I I E b AND T. F. ~ A L L h G F i L R 1930 Rat seminal Iesicle cytology as a testis hormone indicator and the prevention of castration changes by testis extract injection. Am. J. Anat., 101. 45, pp. 109-13s. XOORF, CARL R., I)OXO~'HY PRICE AND T. F. GALLAGHER1930 Rat prostate cytology- as a testis hormoiie indicator and the prevention of castration ehaiiges hy testir extract iqjection. A m . *J. Anat., rol. 45, pp. 51-10;. 1701. -( 8 CARL Ti. ’AIOORF, A N D DOROTHY PRICE NOORE,CARL R., AND DoRoTIiP PRWE1932 Gonad horiiioiie functions and the recipioeal influence between goriads arid hjpophysis with its bearing on the problem of sex-liormonr antagoaisui. Am. J. Anat., vol. 50, pp. 1 3 - i l . 1937 Some eflects of synthrtically prepared m a l e hornitrnr (androsterone) in the rat. Endocrinology, vol. 21, pp. 313-329. XELSON,W. 0. 1936 The effect of various sex hormones on the testes of hypophyscctoinieed rats. Anat. Rec., vol. 67 (Suppl.), TI. 110. KELSON,W. O., AND T. F. GILLAGIIER 1936 Some effects of androgenic substances in the rat. SLience, 1-01. 54, pp. 230-232. PARKES, A. 8. 1936 Inercasing of effecti\encss of tcstostrrone. The Lancet, \ O l . 3, p. 674. PRICE, DOROTHY 1936 Norrnal clcvciopinrnt of the prostatr arid scniiiial Teuicles of the r a t with a study of rspeiiinental post-n:rt:ri modifications. Am. J. Anat., vol. G O , pp. is-127. STONE,CALVIN P. 1924 The awakening of copulatory ability i n the rnale albiiio rat. Am. .T. Yhysiol., r o l . 68, pp. 407-424. - 1927 The retention of copulatory ability in male r a t s following castration. J. Comp. Psychol., vol. 7, pp. 369-.?Xi. ‘L’SCHOPP, E. 1936 Untersuclinngen iibcr die Wirkung der ni$nnlichen Sexnalhormone uiid dire Derivate. A4rch. l n t . Pharnincod. et. Ther., Ed. 52, S . 351-403. \VALYH, E. L., W. K. &TYLER ABD D. R. ~\.ICCUIJLAGH 1934 The physiologic niaintena~ier of the nialr sex glands. Am. J. Phjsiol., \ol. 107, pp. 505-512. WELLS,L. J. 1935 Seasonal sexual rhythms and its expeiirnerital modification jn the malr of the thirteen-lined ground syuiircl (Citcllus tridrecnlineatus). Anat. Rrr., aol. 62, pp. 409-447. WELLS, L. J., AND E. T. GONEZ 1937 IIypophjscetoinj and its effects ov- male reproductive organs in a wild mammal with annual r u t (Citellus). Anat. Rec., pol. 69, pp. 213 227. WELLS, L. J., AND CARL E. MOORE 1936 IIormonnl stimulation of spermatogenesis in the testis of the ground squirrel. Anat. Rw., vol. 66, pp. 191-200.