Genital abnormalities in castrated fetal rats and their prevention by means of testosterone propionate.
код для вставкиСкачатьGENITAL ABNORAIALITIES I N CASTRATED F E T A L IL4TS AND T H E I R PREVENTION BY M E A N S O F TESTOSTERONE PROPIOXATE 1, L. J. WELLS, MARGARET m7. CAVANAUGlI AKD EDWARD L. MAXWELL Depaitmeiit of A x a t o m y , Uni c e r s i t y of M z n w s o l n , M 11111f a p o l i s T W E N T Y FIGURES In a study of castrated fetal rats, it has been virtually proven that the fetal testis produces a hormone (androgen) which stimulates the prenatal growth of such accessory reproductive organs a s the seminal vesicles and bulbourethral glands (Wells, ’5Ob; Wells and F’ralick, ’51). The fetuses were castrated during developmental stages in which the primordia of the accessory reproductive organs had already appeared and in which the Miillerian ducts had disappeared. It could not be determined whether the testicular androgen induces the formation of these primordia and causes the retrogression of the Rliillerian ducts. Accordingly, new work was undertaken with a view to solving the problem by studying fetuses in which the operation was performed before the appearance of these primordia and before the disappearance of the i\liillerian ducts. I n our attempt to extend the work to the earliest possible stage of prenatal development, the available methods were “pushed to the limit. ” Technical difficulties prevented us from solving the problem, but we were successful in obtaining fetuses ‘Aided by grants from the United States Public Health S e n i c e (RG-2801) and from the Graduate Nedical Research Fund of the Vniversity. The authors are indcbted to Ciba Pharmacrutieal Products, Inc. for supplying the testosterone propionate (Perandren) and t o Mrs. J u n i t a Mortensen n h o assisted as laboratory technologist. 109 110 L. .J. WELLS, 31. CAVAPTh17(:II A S U F;. L. MAXWELL which lived until near term, after liaving been subjected to castration prior to the appearance of prostatic buds and coagulating glands. h preliminary report of the observations was presented at the Providence meeting of the American Association of Aiiatomists ( T e l l s , Cavanaugh and Rlnxwell, ’52). JIATERIA1,S AND METHODS In rats of the Sprague-Dawley strain, 113 preg*nancieswere timed by witnessing the mating. Forty-five yielded a closely graded series of control fetuses. Sixty-eight were used in the experinients on castration; a nunibcr of them also served as sources of control fetuses which were takrii at the time of the operation. At operation a gravid female was anesthetized with ether, arid the uterus exposed by optliiing the peritoneal cavity at the liiiea alba. Each fetus to be castrated was brought into view by incising the uterus and fetal rnenilii*aiies, and was then delivered into the peritoneal cavity of the mother in such mariner that the placental circulation remained intact (“estrauterinized fetus”). On each side of the fetal body an oblique inguinal incision was made, the testis removed arid the wound closed by mc’aiis of interrupted sutures of silk; for. additioiial details of the method, two earlier papers may be examined ( V7ells, 50a ; X-ells arid Fralicalr, ’51, Method 2 ) . In each of 4 cases a pellet of testosterone propionate (Perandren), weighing about 1.0 nig, was implanted under the fetal skin. I n all cases the treated fetus, still attached to the uterus by the placenta and urnhilical cord, was allowed to remain in the maternal peritoneal cavity. I11 each of three cases the fetus was covered, however, by nieaiis of a tent created by stitching a thin sheet of cellulose acetate to the uterine m ~ l l(artificial uterus). Similarly, in each of -2 cases, the fetus was covered by a tent which was fashioned out of a thin sheet of rubber and stitched to the uterine wall. When all steps of the fetal surgery w ~ completed, i ~ the maternal wound was closed by suturing and the etherization stopped. 111 C H A N G E S IN O R C I I I E C T O M I Z E D FETlTSES The experiments were terminated by killing the mothers. This was done near term arid usually when the fetuses had attained the age of 21 days and 15 hours. It was found that 14 of the castrated fetuses were alive and quite active and that the “artificial uteri’ ’ had not increased the percentage of survival. The survival was a s follows. Of two fetuses castrated at the age of about 19: days, both were alive (litter16 h, mates). Of 61 castrated a t ages of 18 d 2 h u p to 18 d 1 2 were living. Of 15 castrated a t 1 7 d 21 h to 18 d 1h, none was alive. I n coiiducting the autopsy of a n individual litter, a living castrated fetus and a n unoperated male were taken by severance of the umbilical cords. They were promptly weighed and then killed. The reproductive tracts were fixed i7z situ in Bouiri’s solution, and sectioned transversely i n perfect series at 10 p. The sections were mounted on microscope slides and stained with hernatoxyliri and eosin. With the aid of microscopes, data were obtained from the stained sections. The length of the RIiillerian ducts and/or prostatic utricle was determined by simple calculation (numher of transverse sections x thickness of section), on the assumption that they were straight vertical structures, which, of course, they were not; nevertheless the data would seem to have comparative value. Such data were not obtained in the case of the fetuses of group C (table 2) since the reproductive tracts had been sectioned in the frontal, rather than the transverse, plane. In 20 fetuses the counting of the prostatic buds was facilitated by making transparent models of the prostatic urethra and outgrowths. The models were prepared by projecting the sections with a n Edinger projector, by stippling the desired outlines on thin sheets of cellulose acetate with inks of various colors and by stacking these sheets. The models were abnormally short in vertical dimension because the acetate sheets were thin and because suitable spacers of cardhoard were delihera tely omitted. The models were: however, accurate in two dimensions and in the number of prostatic buds shown. + + + + B A 16d C 18d.Zh 17d .I0 h I I I I I I I I I I E D C IOd,lZh crest. utricle 21 d , 15h F lad, Zh 18d,Zh H Zld,l5h I 21d115h Fig. 1 Conventionalized diagraiiis of the genital ducts and derivatives ill control fctuscs ( A t o 0) and experimental fetuses ( H and I). A t the age of 18 d 2 11, thc prevailing pattern ~ v a slike that illustrated in drawing C (12 of the 14 fetuses). Only m e of the castrated fetuses showed a “ V ” a t the upper elid of the prostatic ntricle (drawing H) + . C H A N G E S I N O R C H I E C T O M I Z E D FETUSES 11.3 TERMINOLOGY The coagulating glands, most easily defined ‘ny reference to the adult rat, are those paired parts of the prost a t‘IC complex which are topographically associated with the seminal vesiclcs and which release into the urethra a substance that coagulates ejaculatcd semen to produce the “vaginal plug’’ Moore, ’39, fig. 26). The prostatic utricle (uterus masculinus), honiologue of the vagina in the female and derived from the lfiillerian ducts, is present in the newborn male. The M U Z Zeriun tubercle in a fetus is that hillock in the urogenital sinus which receives the Miillerian ducts and Wolffian ducts (fig. 1 A ) and which persists after birth as a mound (colliculus seminalis) on the urethral crest. The balcmo-preputial lamella is a solid plate of epithelium from which the epithelium of the glans penis and that of the inner surface of the prepuce develop (insert in fig. 17). OBSERVATIONG I n preview it should be noted that in 6 of the 14 castrated fetuses to be considered the operation was performed at developmental stages younger than 18d 11h (table 2). I n 11h, the epialmost all of the control males under 1 8 d thelial buds of the prostate and the epithelium of the coagulating glands had not yet appeared (table 1);the oblique and horizontal portions of the Miillerian ducts were still present (fig. 1C, see text). + + Cont r o 1 m t 1es + Prostate. I n controls younger than 18 d 11h, there was only one instance in which a prostatic bud was found. This structure, noted in a fetus of 1 8 d 4 h , appeared in only three of the l o p sections. I t was situated in a region comparable to the one indicated by the arrow in figure 13. Up 14 h, the largest number to and including the stage of 18 d of buds in an individual male was 6. Such buds were smaller and shorter than those illustrated in figure 16. + + 18 18 18 18 18 18 18 18 18 18 18 19 19 21 17 16 16 16 1i 17 17 17 17 17 + 11 + 12 + 13 + 14 + 12 + 14 + 15 +5 +7 +1 +2 +3 +4 + 21 + 23 + 10 + 12 + 14 + 18 + 20 + 12 + 16 DAYSA~~o;OCRS 1.25i (1) 1.2i4 (2) 1.650 1.434 1.590 1.631 2.453 2.487 5.003 .700 .880 .716 1.035 1.087 .985 BODY W T . 415 40 (12) 458 (1) 458 429 (1) PREPUTIAL FRENULUM, WIDTH 102 X 107 X 102 X 111 X 127 x 116 X 104 X 126 X 112 x 139 X 153 X 83 X 82 89 84 82 73 78 81 70 77 109 95 7i _ (2) (2) V V Vc f v V V v V V V (13) ; ; V V V V V ; D A D SEJLINAL VESICLES, EPITHELIUM _ E ;::;:“, _ p 0 0 0 0 0 ~ +++ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ._ ~ ~ < < 0 0 0 0 0 0 0 0 1 4 0 0 4 34 44 6 20 20 61 (6) 0 0 0 0 0 0 PROSTATIC RUDS, ~E P I~T H E~L I U~M ___ A , absent (Wolffian ducts not dilated a t sites of future vesicles) ; D, dilatation of Wolffian ducts a t sites of future vesicles; V, vesicular outgrowths; Vcf, vesicles each with a “cranial flexure” (flexure illustrated in fig. 1). Actual nuniber in one case (not an average number) ; no buds in the other cases. * RL, right-left diameter; DV, dorsal-ventral diameter. 980 1050 940 880 890 720 860 1040 660 930 420 480 258 (12) P P * PROSTATIC UTRICLE, R L X DV + ~ M ~ L L DUCTS . PROST. U T R I C L E , LENGTH Kumbers in parentheses indicate the number of cases. 3 1 2 2 2 1 1 13 -___ 5 I 1 1 1 3 1 14 13 1 2 8 2 3 1 1 FETUSES TlnLE 1 Observations in 8.3 control fetirses ~ r A f 115 C H A K G E S I N O R C H I E C T O M I Z E D FETC'SES I n fetuses so young that the prostatic buds had not yet appeared, it was not possible to identify any well defined 211 boundaries of the future prostate. Controls of 1 8 d and older showed, however, in the region surrounding the epithelium of the future prostatic urethra, a relatively thick area of densely packed cells which must have originated from mesenchyme (fig. 13). That most of these cells a r e eventually used in forming the non-parenchymal component of the prostate (stroma, etc.), was suggested by the minute anatomy of the developing prostate i n older fetuses (figs. 16 and 14). The prostatic buds in fetuses of 2 1 d 15 h were numerous. Distally, some of them had distinct branches (fig. 2). The longest buds were those of the ventral series. CoaguEating glands. The coagulating glands mere observed in 15 of the controls (19 d 12 h t o 21 d 15 h ) . They consisted of two componeiits, iiiner and outer. The inner component, a solid core of epithelium, had originated from the dorsal aspect of the embryonic prostatic urethra (figs. 6 and 2 ) . The outer component was a thick iiivestment of densely packed cells of mesenchymal origin. The question arose a s to whether the primordia of the coagulatiiig glands might exist, in advance of epithelial outgrowths, in the form of clusters of densely packed cells (e.g., primordia of the outer componeiit of the glands). A positive answer was suggested by the fact that two cellular condensations were observed in each of the two fetuses of 18 d 14 h and that they were situated in the region of the future coagulating glands, 01' near it. Also, they had undoubtedly originated from mesenchyme. Incidentally, they were smaller a i d less distinct than those marked by the arrows in figure 7. In fetuses younger than 1 8 d l l h , there did not seem to be any paired cellular coiidensatioiis in what was regarded as the region of the future coagulating glands (fig. 5). SciniqzaZ v r s i c Z ~ s . Vesicular outgrowths from the Wolffian ducts were regularly present in fetuses killed at ail age of 18 d 2 h, hence the developing vesicles need only be given brief consideration in the present paper. At 2 1 d 15 h, the + + + + + + + + 116 L. J. \YELLS, &I. CAVANAUGH A N D E. L. MAXWELL original cranial end of each vesicle had a flexure which passed niedially arid inferiorly (“cranial flexure, ” fig. 1G). Bulbouret7irul gl(112tls. The primordia of the bulbourcthral 16 11. glands were not yet present in controls of 16 d to 16 d Either the primordia or the developing glands themselves were observed in all inales of 17 d 10 h to 21 d 15 h. il1iillcriun ducts u n d prostcitic utricle. The normal retrogression of the Miillerian ducts in the male was already in 2 h to 1s d 14 h. The pattern progress in controls of 18 d usually observed in such fetuses is illustratedl i n figure 1C. The stages shown in figures I D and 1 E were rarely seen. These observations suggested that in the castrated fetuses to be considered all, or almost all, of the vertical portions of the Rliilleriari ducts had already dropped out before the time of the operation. In the prevailing pattern depicted in figure 1 C, the prostatic utricle had a lumen. A t the niliillerian tubercle, the utricle ended by opening bilaterally into the urogenital sinus (embryonic prostatic urethra, fig. 13). 15 h (figs. 1(3, The prostatic utricle was present at 21 d 1C and 14). Inferiorly, below the level of section shown in figure 10, the utricle usually had a small lumen which forlied and then opened bilaterally into the urethra. Pmile urethra and prryiitial frenulzcnz. I n the fetuses of 18 d 2 h to 19 d 14 h the distal end of the urethra was a solid plate of epithelium situated in the developing glans penis (urethral plate, see insert in fig. 17). J u s t proximal to the urethral plate, the floor (ventral wall) of the urethra was still missing (open urethral groove, not illustrated in the photographs). The developing preputial frenulum was a ventral structure in the region where the right and left portions of the balano-prcputial lamella were separated from each other by developing connective tissue. ‘This frenuluni was broadest proxirnally, at the site of the open urethral groove, and it was here that the data on the width of the frenulum were obtained. + + + + + + + + 2E 2c 18+ 19+ 2E 1c 18 7 [ 2-13] 8 111-151 6 [ 3-9 ] + 13 + 5E 5C 18 3.178 3.211 4.509 5.010 4.408 5.442 406 41 517 (4) 37 7 8 [74-851 + 4.114 5.289 41 46 Castrate4 fetuses gi\en aiidiogeii 75 [T4-77] 82 [79-841 61 P P 500 340 340 232 596 253 94 81 113 X 109 80 X 66 135 X 79 82 X 78 223 X 81 X P (4) 60 [39-62] 66 (1) (a) 55 (2) (1) 9-20] 13 48 (1) 26 [23-27] (5) (2) 10 3-16] 63 ( 3 ) (1) (3) (5) ~ _ ~ _ _ _ ~ ~ ______ Age of the fetuses a t autopsy was about 21 d 13 h. Xumbers in parentheses indic;ite tlic iiuml~trr of c a w s . Smnbers in b1acltrts indicate the range. E, experiniental; C, control (litter-mate). RL, right left diameter; DV, dorsal-.rentral diameter. A 5E 5c glVL Castrated fetuses n o t gireii androgen __hours m m r:U; e M a w M ~ s4F; H Q k Q W 3 z m m _ _ _ 118 I,. J . WELLS, M. CAVANAUGH AKD E. L. MAXWELL At 21 d + 13 h the urethra was a continuous tube. The urethral plate had acquired a lumen, a i d the earlier urethral groove had been closed by the union of the right and left urethral folds. ( L( s t TC!t (3cl fr t u s c s 9%o t g iz;e+%ugt d r 0.9 em ( g r o u p s d t o C, tccblr 2 ) Prostutic buds. Epithelial buds of the prostate wei*e observed in each of the 12 fetuses of groups A to C. All buds except one to 4 of the ventral series were relatively short (fig. 3 ) . The buds were usually unbranched. In each of three exceptional fetuses, one of the ventral buds ~ v w sbegimiiiiq to branch (see fig. 15). Despite the fact that in a normal fetus taken froin H 11011experimental litter at I 8 d 4 h a single bud was already pixsent (table l ) , the data secnied to indicate that in each of the castrated fetuses of group A all of the prostatic buds had appeared subsequent to the orchiectomy. To illustrate what was found in individual litters, three fetuses of litter 941 may be used. il normal control, killed and preserved a t the beginning of the experiment, 18 d 7 h, had no buds. A second fetus, castrated a t 18 d 7 h, aiid killed at 2 1 d 15 h, had 7 huds. The third, a n uiioperatcld control killed a t 21 d 15 h, had many buds (the buds could scarcely be counted accurately since a model was not made). Similarly, the data indicated that in each of the castrated fetuses of group B most of the buds had appeared after the time of the operation. The findings in one litter, no. 932, may bc presented. A normal control, killed a t the beginning of the experiment, 18 d 12 11, showed three buds. h second fetus, castrated at 18 d 12 h and killed at 21 d 15 h, showed 16 buds. The third, an unoperated control killed a t 21 (1 15 h, showed 48 buds. The huds in the castrated fetuses of group C were longer and more numerous than those of a normal fetus from which figure 16 was obtained. This fact together with the tabulated data seemed to shorn that any h d s existing a t the time of + + + + + + + + + 119 C H A N G E S I N OR C HIE C T OMIZEU FETUSES the operation had continued to grow during the experimental period and also that some new buds had originated during this period. (‘ongulating glancls. In regions comparable to those where the coagulating glands of controls were observed, only paired clusters of cells of riieseiichymal origin were found in 4 of the 5 castrated fetuses of group A (fig. 7). It was problematical whether these clusters constituted primordia of the outer, non-epithelial coniponent of the glands. The coagulating glands were present in 6 of the castrated fetuses of groups A and B. The tabulated data were regarded as ample evidence that the epithelial coniponent of the glands had developed after the orchiectoniy. The glands were likewise present in the two castrated fetuses of group C. It was likely that these glands had already appeared at the time the operation was performed. Jfiillfirian ducts ~ i v dprostatic utricle. The Miillerian ducts, a s such, had largely disappeared, and were mainly represented by a large derivative, the prostatic utricle (fig. 1 H ) . That retrogression of the Niillerian ducts which had occurred during the experimental period was approximately the same iu each castrated fetus of groups 9and B ; it usually proceeded from a stage shown in figure 1C up to that shown in figwe 1 H . In the castrated fctusm of group C such retrogression had already occurred, however, at the time of the operation, or shortly thereafter (fig. 1F). The prostatic utricles of the castrated fetuses mere larger than those of the litter-mate controls of the same age (fig. 1H, fig. 1G and table 2 ) . Utricles of the castrated fetuses of group A were the largest of all (fig. 7 ) , even larger than those of two normal males which were Idled at 19 d 1 2 11 and 19 d 14 h, respectively. The utricles of the castrated fetuses of giwups B and C were only somewhat larger than those of 15 h. the litter-mate controls obtained at 31 d The distal end of the utricle had the shape of an inverted Y in each castrated fetus, and, in this respect, was normal. + + + 120 L. * J . WELLS, M. CAVANAUGH A X D E. L. MAXWELL It was abnormal in each of the castrated fetuses of groups h and €3 i n that the luiiieii was missing. Xiilleriun tirO(~rc~lo nird c~icrcztlirfory ducts. The Mullerian tubercle was usually abnornial in shape, being so flat that i n section it scarcely yesunbled a hillock. The cjaculatory ducts also were usually ahnormal. I n each of the castrated fetuses of groups X and €3, the distal end of each duct lacked a lumen. In 7 of these 10 fetuses the ducts were dilated, either hilaterally (5 fetuses) o r unilaterally ( 2 fetuses) ; sometimes the corresponding seminal vesicles were also dilated. I n each of two fetuses of group A, though, a t a region m l l above the lllulleriaii tubercle, the ejaculatory ducts and the seminal vesicles were more or less solid structures (luinina missing or almost missing, figs. 11 and 15). Penile urethra and p r e p u f i u l frrnulum. The penile urethra was normal in one respect but abnormal in a second. It was iiormal i n that the urethral plate had undergone cavitation to produce the fossa navicularis. It was abnormal i n that the urethral groove was still open (hypospadias). The existence of a condition of hypospadias was actually observed in only 8 of the 1 2 castrated fetuses (three in group A, three in B and two in C), and was, f o r one reason or another, doubtful in the other 4. This anomaly was regarded as an abnormal persistence of an earlier normal embryonic condition. The proximal p a r t of the developing prcputial frenulum was usually very wide, thus reflecting the fact that the floor of the urethra was missing in this region. Castrated fetuses given. androgen (group D, table 2 ) I n each of the two experimental fetuses of group D, a pellet of testosterone propionate mas implanted under the skin immediately after the testes had been removed. When the stained sections were studied microscopically, it mas found that i n each case all those effects of castration noted in the experimental fetuses of groups A to C had been prevented, C H A K G E S I S 0I:CHIECTOMIZEl) FETVSES 121 01' largely prevented. Thus, the prostatic buds were iiuiiierous (fig.1 aiid table 2 ) . Tlic coagulutiiig glands were present (figs. 1and 8). The ejaculatory ducts had continuous lumens, a i d were not almornially dilated (fig. 12). The floor of the peiiilc~ urethra \vas pi-esciit (abseiice of hypospadias ) . The priinitive preputial frcnuluin was iiarrow (fig. 20). ' h e effects of castration upon the prostatic utricle were largely prevented. The utricles of the experimental fetuses i n group D were smaller than those of fetuscs which had been castrated for a similar period of time and which had not heeii given ancirogeii (figs. 11 and 1 2 ) . The utriclrs were, however, somewhat larger than those of the unoperated littermates of the same age (table 2 ) . At the Jliillerian tuhercle there ~ v a sa lumen i n tlie utricle of each fetus in group I>; the tubcrcle and associated ducts were ciitially lilrc those illustrated in f i g u i ~14. 1)ISC'IJRSTOS The genital ahriormalities in the castrated fetuses jncludpd a reduction of the number of prostatic buds, absence of tlie coagulating glands, iiicoiiiplete formation of the floor of the penile urethra (hypospadias) and eiilargenient of the pi*ostatic utricle. The first threc of these abnormalities could lie prevented by means of testosterone propionate, and the 4th largely prevented. Should these observations be regarded as evidence that in normal development the testicular androgen is a causative factor in the formation of the primordia of the male accessory reproductive organs? We would answer in the negative despite the absence of the coagulating glands in 4 of the castrated fetuses and despite the prevention of this anomaly l ~ piniplanted testosterone propionate ; such observations on the coagulating glands, though intriiisically sound, must he halanced against the current lack of demonstration that the fetal testis produces testosterone propionate. Also, a negRtive aiiswer is suggested by the existence of prostatic buds 122 1,. J. WELLS, ill. C A V A N A U G E AND E. L. MAXWELL in castrated fetuses in which tlie operation had lxen performed prior to the time of expectation of buds. A second question is mliethei~the observations should bc regarded as evidence that in normal development the testicular androgen is an importaiit factor in tlie retrogression of the ~ l u l l c r i a nducts in the male. IVc have arrived at a negative answer. All castrated fctuses of group A showed a lack of persistence of those olilique and horizontal segments of the right and left ducts which must have existed at the time these fetuses were subjected to castration (jig. 1C ) . Also, the enlargement of the prostatic utricle aiid the prevention of this effect by testosterone propionate must be considered in the light of what was found iii the case of the ejaculatory ducts. I n most of tlir cxperimental fetuses of‘ groups A and R, the ejaculatory ducts were dilated. Such dilatation could be prevented hy testosterone propionate. Although a satisfactory cxplanation of the enlargement of the piaostatic utricle and of the s i n d t a n e o u s dilatation of the ejaculatory ducts is iiot a p p a r m t , clues are afforded in the fact that distally these structures lacked lumciis and that in two illstarices the seminal vcsicles \vei*e somewhat dilated. It occurs to us that the utricles aiid ejaculatory ducts, as a consquence of lack of pa teiicy distally, might have been expandcd a t some time during the experiinental period by an abnorrnal accumulation of fluid in their cavities. If so, it is reasonaldr to suppose that the testosterone propionate prevented such expansion hy keeping open, in some indirect manner, the 0rifict.s of the utricles and ejaculatory ducts. The observed hypospadias, explained ernlryologicall~a s a n abiiorrnal persistence of the enihryonic urethral groove, is undoubtedly a consequence of the orchiectomy. On the other hand, it may not he due solely to the experimental deprivation of testicular androgeii. Its prevention by testosterone pi-opionate does riot entirely iwle out the possibility that some lion-spcific “physiological insult ” during the experimental surg:.ci.j~might hare acted as a coiitrihtiiiy factor. Other workcrs have found that hypospadias and other genital anomn- C H A N G E S I N OBCHIECTOMIZED FETGSES 123 lies may be produced experimentally in the male ofYspring of r a t s by feeding the gravid female a diet deficient in vitamin A (Wilson and Warkany, '48). Our data support the view that in rats testicular androgen is produced before birth and that it stimulates the prenatal growth of the male accessory reproductive organs (Wells, '50b; Wells and Fralick, '51; see Maxwell and TTells, '51). This view, incidentally, is also supported by the fact that when the fetal testis is grafted into the seminal vesicle of an adult castrated host it is capable of producing androgen at once (Jost, '48 ; Moore, '53). The reduced number of prostatic buds in our series of castrated fetal rats is at variance with certain ohservatioris in yourig opossums (Xoore, '50a, '50h). After the removal of the testes of an opossum on the 20th day of life the existing prostatic buds (about a half-dozen) continue t o grow, new buds are rapidly added and the buds undergo extensive 1)ranching; u p to the 100th day the prostate of a castrated opossum is similar to that of a normal control. The present authors do not know why there should have been such a discrepancy in the results obtained in these two species of mamnials, r a t and opossum (placental mammal vs. marsupial). On the other hand, the lack of persistence of the Miillerian ducts in our castrated fetuses and also the actual formation of prostatic buds during the experimental period a r e in keeping with similar observations in the castrated opossunis (11oore, '50b). Absence of the coagulating glands and the presence of hypospadias in our r a t s a r e anomalies like those found in fetal mice in which the testes were destroyed by irradiation (Raynaud and Frilley, '48; Raynaud, 'SO). This castration by irradiation was performed on the 13th day after conception, and the fetal mice were killcd o n the 18th day. The ahnormalities, more extensive than those in our specimens, included absence of the seniinal vesicles and prostate and, incidentally, the presence of a n cpithcllial cord derived from the Miillerian ducts ("cordon vaginal"). Rayiiaud ('50) states that the genital tubercle of a castrated mouse was structurally similar to that of a normal female control. The authors did iiot present a record of having prevented these effects by means of introduced atidrogeii. The coiiiplcs of abnormalities iioted in our material is less extensi\Te than that found iii fetal rabbits (Jrost, '30). In rabbit fetuses castrated 0x1 the 19th to 21st day of life and killed oil tlie 28th day, ,Tost reported absence of the prostate arid the difierentiation of thc Jliillcrian ducts into a vagina (prostatic utricle ?) and into certain segments of uterine horns and tubes. Such effects i n rabbit fetuses were piwwited by implanted testosterone propionate. Tn our ~ v o r k the , forniation of prostatic buds in tlie absence of testes is in harmony n.ith the esisteiice of "feniale prostates" in adult feniale rats. Prostates occur spontaneously i n a high percentage of the females in certain selected stiaains of inl)red r a t s (Price, '44). It is clear that the enlargement of the prost,atic utricle in the castrated fetal r a t is attributalile to the castration. It is probleniatical, however, i ~ ~ h e t l i ethis r change is due to the experimental deprivation of testicular androgen, a s was implied earlier i n the discussioii (paragraphs 3 aiid 4). This over-all interpretation is supported by the fact that in the rhesus monkey, for example, the introduction of androgen into the circulation of a gravid female fails t o cause i n a female fetus any retrogression of the lliillerian ducts and derivatives but does produce a high degree of intersexuality (Wells aiid van Wagenen, i n press). Our over-all interpretation was formulated in spite of certain observations in chick embryos and in a single rabbit fetus. In a female chick ernbryo in which a developing testicle has l)een grafted into the peritoneal cavity, the graft may hring about a complete retrogression of the Miillerian ducts of the host (Wolff, '50). Similarly, when a n ernbryonic testis is grafted onto the soniatopleure (a membrane wliich later participates in the formation of the chorioallaiitoic niem1)rane) of a female chick embryo, the graft may cause a complete disappearance of the 1zliil- lerian ducts (Huijbers, ’51). Also, in a single case of N fem;tlc rabbit fetus in which a testicle had been grafted onto the developing iiiesosalpirix, it was fouiid that a sclgmeiit of the Miillerian duct was iiiissing on that side of the body (,Tost, ,r,O). In closing the discussion, our data 1)riiig into shar1) focus tlic question of whether in nornial niale fetuses thc testicular aridrogcii is produced early enough to, and at such early stages i n suficiciit quantity to, induce the formation of the primordia of “niale orgaIls” and t o cause the nornial retrogression of the bliilleriaii ducts. We think this is an open quest ion. SI’MMARY ANT) (“ONCIiT7SIOhTS l’etal r a t s so young that they still lacked 1)i.ostatic buds and coagulating glands and so young that they still had Iong segnimts of the 3Iiillerian ducts, were subjected to castration and then permitted to live until near temi. It w a s found that pi-ostatic buds had appwwed during the experimental period hut that the numhei- of l ~ u d swas significantly smaller than the number in controls of the same age and litter. Thc coagulating glaiids were soinetinies ahsent. The fjoor of the penile urethra was usually niissing (hypospadias). A11 portions of the Niillerian ducts except the prostatic utriclc had disappeared. The utricle was relatively large. These effects of castration could be pi-evented, or largely prevented, by means of iinplarited testostei-one propioilate. Our ohservatioiis support the view that in r a t s testicular androgen is produced before birth and that it stimulates the prenatal g ~ o w t hof tlic male accessory 1-eproductive organs. These observations a r e inadequate to determine, however, I t would be beyond the scope of the present paper t o undeitalte n review of t h e vast literature 011 the role of liornioiies in sex differentiation in mammals. Tlrr wliolc subjeet w a s consideircl a t a recent Colloquiuiii ; the I’roreedinys of this syinposium appeared serially in 1950 (Arch. (1’Aiiat. iiiier. Xorph. esp., 39), and the papers were assembled under o i i ~cover in 1931 (La Differenciation S e v u e l l ~ cliez les VBrtCbres, Centre National de la. Rrcherclie Scientifiqur, P‘niis i ). 126 L. J. WELLS, XI. CAVAXAUGH AXTI E. L. i\lAS\\’ELL whether androgen from the fetal testis acts a s N causativc factor in the formation of the priinoi.dia of these organs and in the iiornial retrogression of the lZliillerian ducts. LITERATTJRE CTTEI) HLJIJBERS, M. M. 1951 I)e inilord van de gonade np de ontwikkcliiig x a i i llet geslachtsapparaat b i j het kippenembr) o. Thebis, Amsterdam. A h stracted in English in 1952: Exccrpta l\ledica, G (sect. 1) : 31-33. J o w , A. 1948 Activitk a n d l o g h e dn testicule foetal de rat gieffk SUT l’adultr castri.. Compt. rend. Soe. de Riol., 143: 196-198. 19.50 Sur le eontr6lr hormonal de la, diffkrenciation sexuclle d u 1:ii)in. drrli. d’Aiiat. inicr. et de Morph. eup., 39: 577-607. ~ T A X T \ E I , I , E. L., AND L. J. TVTLLS 1951 Effects o f graftct3 testes n1mn the iepiodnctive organs of castrated fetal iats. Anat. Rec., 1 0 9 : 378 ( a b st1act). PRICE, I)O:IOTIIY 194.2 The ieartivity of the prostat? gland of the feiiialr rat in relation t o age. Am. J. Anat., 7 5 : 207-931. R A Y X ; \ C ~ D , A. 1950 Rec~heichcs exp6rimentsles sin l e d6\ eloppenirnt de l’ap pareil g6iiital et lr fonctionriement des glandes endocrines des foetus de souris et de mulot. h ~ h d. ’ 8 n a t . iriici. et d e Morlih. exp., 3 9 : 318-5 76. R I Y N ~ ~A., D , A N D Jf. FRILLEY 1948 Effrt, sur le d6ieloppcment deq divcrscs glande? iinnexes du tractus g6nit:il rles emhryons tle Souris, tle la dr-truetion, au nioycii des rayons X, d ~ glandes s gknitnles des embr:, ons. Coinpt. rend. 8oc. d e Biol., Zg?: 424-486. hfOoRE, c‘. I;. 1939 Biologv of the Testes. Sex and Internal lieeretion?. Ed. by E. Allen, C. H. Danforth and E. A. Doisp. \Villiains and Wilkiiis Co., Kaltinioie. Chap. VIT, 353-431. 195Oa Sturlics on sex hormones aiid sexual diffcientiation in inamnials. Arch. d’Anat. niirr. Xorph. rxp., 39: 484-4!38. - l930b The iole of the fetal indocrine glands in dexelolment. J. Clin. Endocrinol., 10 : 942-985. __-__ 1953 Adrenal cortical secretions in relation to t h r reprotlurtive sFstcrn of rats. J. C’liii. Endocrinol. :ind Metnbol., 13’: 330-368. WELLS,L. J . 1950a Subjection of fetal rats to surgery and repeated subcutaneous injertions: mctliod and survival. Anat. Rec., 208: 309-331. _ _ l930h ~ Hormones and srxual clevelopment in placental mainiiials. Aich. d’Anat. d, niicr. Morph. exp., 3<9: 499-517. WELLS,L. J., 31. CAVANAUGH A ~ D B. L. ~ ~ Z B X W T P L L 1959 1)~relopnientof the reproductive organs in castrated fctal rats: prostate, coagulating glands, genital papilla anti prostatic utricle. Anat. Rec., 113: 467 (abstract). WELLS, L. J., A N D R. L. FRALICK 1932 Prodnction of androgen h: t h e testes of fetal rats. Am. J. Anat., 8.9: 63-107. WEI,I,S.L. J., ~ N D G. TAN WVGENFSA4ndrogen-i~iducec~ female pseudohrrmaphroditisin in the monkey ( M a m r a mulattff) : anatomy o f the ieproiluctive organs. Contiih. t o Enibiyol., Carnegie Tnit., Wash., 9 . 7 : ( i n press). OIIAN(;ES 1K OItCtilECTOMIZED FETUSES 127 WIr.sox, J. G., KND J. WARKANY1940 hlulformations in the genito-urinary tract induced by matcrnal ritainin A dcficieucy in the rut. Am. J. Anat., 83: 357-395. WOLFF, E. 1950 Le r6le des hormones emhryonaires dans la diffbrcneiation sexut!lle tlcs oisc:iiix. Arch. d'Annt. micr. Morph. exp., 89: 427-450. + x + 75. + 2 11 and killed a t 21 tl + 15 11. 4 Fetus castrated a t 18 d 2 11, given n pellet of androgen at 18 d 62 buds (66 buds in an unoperated litter-mate of the same age). + Litter 967. Thcrc were Fetus castrated a t the age of 18 (1 3 11 :cntl killed :it the age of 2 1 d 15 11. Litter 966. T l ~ e r cwere 9 prostt:itic buds, all shown in the drawing. The coagiilating glands were absent, a s illustrated. X i 5 . 3 Litter 966. There weie 61 prostatic buds, some of which are hidden in the drawing. Unoperated control of 21 d x 75. 2 + 15 h. The drawings depict the uretlir;r (t')and prostatic buds as they appeared, when vievet1 f l om above, in transparent models which were prepared by stippling onto sheets of cellulose acetate the configuration of the epithelium (see text). The orientation is the same as that shown in figure 14, i.e., dorsal aspect down and right aspect on the observer's right. A t the Mtillerian tubercle ( M ) , the prostatic utricle and tlie ejaculatory ducts were not modeled. C, coagulating gland. ESPL.lNATION OF FIGUXES PLATE 1 I.. if. WILGB, X. CAVAYAUBII A X 0 E. Is YAXWEIaL CHANGES IN ORCHIECTOXIZEI) FETUSES PLATE 1 11. +2 + 13 h. Littei 1346. X 60. + +2 11, gireii andlogen a t 18 d + 2 11 and killed at 21 d + 15 11. Litter 967. ('ongulating glands + 4 11 and killed at 2 1 d + + 13h. Litter 967. Photo suppleniciits 15 h. Litter 953. Left ejaculatory duct solid, right duct almost 2 11, gircn andlogen a t 18 d X 2 h and killctl a t 2 1 d 12 Fetus eastiated a t 18 d figure 8 bj shoi\ing addition.il 1)rostatic buds (see fig. 4). X 60. 11 Fctnq c : i c t i a t c d a t 18 (1 solid. X 60. + Litter 953. White arrow, epithcliuin of the prostatic utiicle. El:ick airow, outer ixirgin of utiicle. C a ~ i t jof utricle not shown i n the section presented. X 60. Xoiniai ni:ilc of 18 ci + 2 11. i i of 21 d + 15 11. Fetns castiated a t 18 d piesent. X 60. Fetus castiated a t 18 d 3 11 and killed at 2 1 d 1 3 h. Littei 966. Prostatic utriele large. Ejaculator: ducts dilated. Voagu1:iting glands niiwng (cl~itlieliunimissing). Arroirs indicate t i 7 o ni:isses of cells in a region n h e i e the coagulating gl'lllds llllgllt be expected ( s e e text). x GO. + Unopeiated eontiol of 2 1 tl Litter 9GG (cf. fig. 2). Piostatic utricle not slionn since it mas below the level of tlie transvcise section pwsented ( w e figs. 10 n i i d 1 G I . Coagulating g1:inds piesent (black o b ~ e c t sin top third of photo). X GO. 10 Unopeiated coutiol :I 8 7 G 5 h'oinial malt fetus of 18 d The prost:itic utiicle appears in e:icli illustration except figure 6 (near center of photo). Right and left ejaculatory ducts in 5 of tlie photos (figs. 7 arid 9-12'); right duct in three (figs. 2, 6 and 8 ) . Left d u c ~ u sdeferens and tlie duct of the left seminal iesicle iii figures G aiid 8. Each section is oriented in such manner that the dorsal aspect is down (note rectum) and the right aspect on the observer's right. U i e t n s and wall of bladder in figures 5 and 9. Urethia in 6 of the illustrations (up-figs. 6-8 and 10-12). Eight tr:insversc sections of the pel1 ic region of several fetuses piesented f o r the purpose of illustrating the prostatic utricle, ejaculatorj d u r t s , coagulating glands aiid prostatic buds. ESPLANATIOK O F FIGURES PLATE 2 L. J. W E L L S , 11 C A T A B A U G H A N D E L. MAXWELL C’HANGES I N OKCHIECT0311ZLI) FETUSES PLATE 2 + :ciitl + light :tspect 011 + + Uiioperatetl control obtained a t eiid of experilneiit, 21 d 15 11. Litter 941. Prost:ctic urethra, ~ i ~ ~ r i i c ~ 1)rostatic nus buds, R.lulleriaii tuhcrcle, bilateral teriiiinatioii of prostatic. utriclc (nimketl by two black ink dots) :riicl terniiiiatiou of tlic ejaculatory ducts (sites about ti 111111 helow ink dots). X 50. + Norlnal fetus of 18 (1 2 h, obtained a t I)cgi~iiiii~g of exl~c~rinient.Litter 041. Urethra i n ob!iquc scc.tioii. Alwmce of prostatic buds. Arrow illarks approxii1i;ltc site where a single but1 \zws fouiid in aiiotlier fetus (iiornial fetus of 18 (1 4 11, table 1). White blister on the right aspect of the Miillerinii tuhwcle is the prcstatic utricle (cf. fig. 1, drawing c). X 50. 0iiriit:itioii : t1ors:il aspect do~vii (notr rcctulii) Noriiial fetus of 19 cl + 1 2 h. l’rost:itic urctlirn the Miillerian tulicrrlc. x 60. aiid biids ( I 9 I)uds counted in the scri:rl sections), :1ud ii portioii of + + 20 + Fetus castrated :it 18 d 2 11, given aiidrogcii a t 18 d frenulurii (obserier’s right). X 30. + 2h and killed a t 2 1 d + 13 11. Litter 967. Narrow I)reliutial 311 a n d killed at % I d 35h. Titter 966. Glniis penis, ~ i r e p ~ cniid c ~ penile uretllrLi (latter Fetus castr:itctl at 1 8 d o n observer’s iiglit) . The section presented does not prss tlirougli tlie euistiiig ( a l ~ n o r i i i n l ~ l ~) e ~ s i s t i i i gurctlir:ll ) gloove. X 30. + Unoperatrd vniitrol of 21 d 15 11. Litter 96ti. Prepiitial g1:iiids (oliservcr’s l e f t ) , glans peuis :nit1 :rtt:iclied prepuce, pcwile ni.ethra ;mtl a nxrroiv prrpritial frei~uliim (latter on observer’s right). x 30. + (Iiisrrteil srllaii piloto.) ?;ornial fetus of 18 tl 2 11. Litter 133.5. t i l a m l i e n i s :iiitl the prcpuw ai’e :itt:tclid to c a d i other h y the haiailo-prel~utial1aniell:i. Note urethral I’latc (~iiedimisolid epithelium). x 20. + (Large photo.) Normal fetus of 18 d 2 11. Litter 1355. Astcxrisk indic*ates left ]ircpntial gl:iiitl. I’lioto :~Isoslioivs tlie cavernous hntlirs of the ~)ciiisaiitl tlic peuile iiretlir:~ (1:itter srctioiic~ddorsal t o tlic region of tlic uret1ir:il groove - groove not illustrated). X 3 0 . 19 18 1i 17 Fiw sections of the de\clopiug penis (pliotos oht:iiiicd froiii tralimcwe set-tioiis of 5 fetuses). 0rieiit:itiou : clorsuiri of penis oil obser\er ’s left; left aspect do\\n. lti 15 Fetus castrated a t 18 d 7 11 aiid killed :it 2 1 (1 15 11. Litter 941. Prostatic uretlirn, :I sii~alliiunihrr of prostatic buds (total of 7 hurls in iuodcl) , 1i:’ostatic. utricle (iiiarkcd by tlie two white ink (lots) : ~ i i i le j a c i h t o r y ducts (absence of lumen in right duct). x 50. 14 13 Four trausvcrse wctioiis of the p e l 1 ic region. obserler ’s right. EXPLANATION OF FIGURES PLATE 3 L. J . WELLS. M CAT.4NAUUH AND E. L. MAXWELL CTI-IaNOES I N ORCHIECTOMIZED E’ETUYES
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