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1964

A Histochemical Identification of the Cellular Sites of Androgen Synthesis in the Hypertrophied Right Gonad of the Leghorn Poulard

James Edward Woods Loyola University Chicago

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Recommended Citation Woods, James Edward, "A Histochemical Identification of the Cellular Sites of Androgen Synthesis in the Hypertrophied Right Gonad of the Leghorn Poulard" (1964). Dissertations. 738. https://ecommons.luc.edu/luc_diss/738

This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. Copyright © 1964 James Edward Woods A HISTOCHEMICAL IDENTIFICATION OF me CELLULAR SITES

OF ANDROGEN SYNTltESIS IN THE HYPERTROPHIED RIGUT GONAD OF THE LEGHORN POULAJU)

a\\CH SCHO "-." 0 C!) LOYOLA (' UNIVERSITY Of: MED\C\~~

by J..... Edward Wood.

A Dissertation SUbaitted to tbe Faculty of tbe Graduate School of Loyola University 1n Partial Fulfillment of the Require.-nts for the Degree of Doctor of Philosophy

June 1964 ACKNOWLEDGEMENTS

I wish to express ~ sincere appreciation to Dr. Lincoln V. Ooma for suggesting the probl_, indoctrination into the vagaries of sex. biology and for his patience throughout the exteaded period of this investiaation. To Or. Leslie A. emaert for many iDterestlng discussions on the historical aspects of lipid histochemistry and for advice and counsel on a mul. titude of aatters pertaining to the dissertation go my heartfelt thanks. Dr. Jose lannakaril aided in performance of sinistral o.ariectoa!e, on very young biNs. Appreciation i. hereby rendered. Thanks are extended to Dr. Louise Smelte, Histological Technician in the Anatomy ~art... t, for SUIlest10ns OR histological matters. Gratitude for the loan of photop-aphlc eqtd,p1M11t and for export advice on photographic techniques 11 extoded to Dr. Robert C. Thoaes, DePaul University. VITA

James Edward Woods was born in Chicago, Illinois, on May 7, 1926, and was graduated frOlat Steimaet& Hilh Sehool in June, 1944. Prom July, 1944,

through Sept81lber, 1946, he served in the U. S. Array. In 1951 he received the

B. S. Degree in ZooloIY frOIl the University ot Illinois. The following three years he worked at the Vio 81n Corporation, Monticello, Illinois, and the

Research Institute of Michael RH,e Hospital, Chicago. In Septellber, 1954, the author began h1s graduate studi•• at DePaul University, receiving the M. S. Degree in Zoology 1n Aulust, 1957. In October, 1957, he enrolled in-the AnatOll),

Departaent of the Stritcft School of Medicine of Loyola University.

During the period spent In the Anatomy Depar~nt he has helo the

following fellowshipst StanclaN Oil "ellow.hip (1951-1960), Loyola University Teaching Pellowship (1961), and Natioaal Institute of Health Predactoral Pellow­ ship (1962-1964). The author is a member of the American Association for the Advance­ ment of Science and the American Society of Zooloalsts. ABSTRACT

Although the vertebrate ,oRad is known to be the primary source of the sex hormone., the cells within the.e orlans responsible for thoir production

~ave not been conclusively demonstrated. In particular, the cellular sites of

~droaen synthesi. have not been definitely identified. Most of the experiaental evidence bas implicated the interstitial

~.11s of Leydi. as the principal site. of androgen synthesis in the testis (Albert_ 1961), but there is also evldeace that the se.iniferous tubule•• ay

~onstitute a secondary source of the ..Ie sex hormones. In the ovary the evi­

~ence indicate. that tbe .-dullaroy interstitial cells are responsible for the production of androgens (Hill, 1962).

The purpose of the pre.ent investigation was to identify by histo­

~hellical Hans the androgen-producing ceUs of the vertebrate gonad, with par­

~icular reference to tbe testis-like, hypertrophied, rudimentary right gonad of :the sinistrally avarieet_Iled, single-cOllb, light-brown Leghom fowl. This gooad is known to secrete andro,enic hormone. as shown by the effects on be.

bayior and on target organs, particularly comb and wattles (Dom., 1921. 1929).

However, then is no preche Inf'oraation as to the particular cells within this DTlaD responsible fOT the syntheSiS of the.e hormones. Since the pTe,ently available histochemical techniques for the identification of steroids lack specificity, a new .ethod fOr the identification pf androgens was developed for use in this inv.stigation. It is a .edified fluorescent antibody technique, which is capable of identifying steroids -in !!!!_ The fluorescent anti-steroid sera used in this investigation was prepared against testosterone, and gives a minimum of cross-reaction with other steroids_ Results following -.ployaent of tbis technique have been compared with those obtained from the use of the steroid dehydrogenase method (Watten­

~erg, 1958). This latter .ethod indicates cellular sites of steroid synthesis

Iby vi!lualldn, hist.ologically one of the enzymes (,AS .. 38-hydroxysteroid dehydro- genase) involved in the biogenesis of the steroid hormones. J~ever. it pro­

~ides only iruUrect evidence tor steroid synthesis, since the presence of a

~ingle enzyme in the biosynthetie pathway does not insure successful synthesis, let alone release of formed hormone (Lobel !l~ 1962). Also, it does not pro­

~ide inforaation as to the character of the steroid hormone end-product. Histocheaical examination of the testiS-like, hypertrophied, rudi.

~entary right lonacia of brown Leghorn poulards, correlated with the androgen­ eondi tioned chu,e. observed 1n the sexual cbaracteTs of such low I, indicate

~hat the cellular sources of the androgenic steroids produced by this gonad are the interstitial cells as well as the .adultary cords and tubules. The testes and ovaries of nonu.l domestic fowl and albino rats, as

~.ll as the teste. of a dog. were al,o examined histochemically. Positive Toac- tlons were observed in the interstitial cells of Leydig and all cellular ele. Dents of tbe seminiferous tubules in the testis and in the medullary interstl- tial cells of the ovary. TherefoTe, it Is considered that all of these cellular site. are capable of androgen synthesis. TASLE Of CONTENTS Page INTRODUCTION •••••••••••••••••••••••••••••••••••••••••••••••• 1

REVIEW 0' LITERATURE •••••••••••••••••••••••••••••••••••••••• Biology ••••••••••••••••••••••••••••••••••••••••••••••••••• 4 T.stis ••••••••••••••••••••••••••••••••••••••••••••••••• 4 Ovary •••••••• ••••••• ...... •••• •.••••••••••••••••••••• 11 Hypertrophithi. Rudirumta1')' Right Gonad ••••••••••••••••• 13 Bloch.mistry •••••••••••••••••••••••••••••••••••••••••••••• 11 Histochemistry •••••••••••••••••••••••••••••••••••••••••••• 21

MATBRIAIS AND MEllIODS ••••••••••••••••••••••••••••••••••••••• 2S

OBSERVATIONS •••••••••••••••••••••••••••••••••••••••••••••••• 38 H1stology of the Hypertrophied Right Gonad •••••••••••••••• 39 Histochemistry of the Hypertrophied Right Gonad ••••••••••• 41 Steroid Dehydrogenase Methed ••••••••••••••••••••••••••• 41 Fluorescent Antibody Method •••••••••••••••••••••••••••• 43

Validation •••••••••••••••••••••••••••••••••••••••••• 43 Observations •••••••••••••••••••••••••••••••••••••••• 44

Hi,tocluaistry of No1'tl81 Gonads ...... •••••••••••••• 46 Gonads of Leghorn Powl ••••••••••••••••••••••••••••••••• 46 Mammalian Gon.o. ••••••••••••••••••••••••••••••••••••••• 47 Pa,o

DISCUSSION •••••••••••••••••••••••••••••••••••••••••••••••••• 49

SUMMARY ~~O CONCLUSIONS ••••••••••••••••••••••••••••••••••••• S6

LITERATURE CITED •••••••••••••••••••••••••••••••••••••••••••• 58

TABLES •••••••••••••••••••••••••••••••••••••••••••••••••••••• 11

PLATES •••••••••••••••••••••••••••••••••••••••••••••••••••••• 17 INTRODUCTION

The gonads of higher vertebrates are the primary source of sex hormones. however, the cells within these organs responsiblo for the synthesis of these hormones have never been conclusively demonstrated. In particular, the cellular sites of male sex hormone production have not been definitely identified. A considerable accumulation of experimental data has implicated tht interstitial cells of Leydig at the principal sites of androgen synthesis in the testis (Albert, 1961), but there is also evidence that the seminiferous tubules may constitute a secondary source of this hormone (Nalhandov, --et aI, 1946). In the ovary the evidence indicates that the interstitial cells lire responsible for the production of aadNlons (Hill, 1%2).

The purpose of the present investigation was to identify by biste- chemical .eans the androgen-producing cells of the vertebrate gonad, with part!- cular reference to the hypertrophied, rudimentary right gonad of the sinistrally ovariectomized, lioglo-cOllb, light-brown Leghorn fowl. This gonad is known to secrete androgenic hormones as shown by the effects on behavior and on target

Iorgans, particularly comb and wattles (i)Ollll, 1927, 1929). However, there is no

~recise information as to the particular cells within this organ responsible for

~he synthesis of these hormones.

Since the presently available histochemical technic~ues for the identification of steroids lack specificity, a Rew method for the --in situ iden- tifieation of androgens was developed for use 1n this investigation. It is a modified fluorescent antibody technique, and takes advantage of the fact that antisera against steroids can be produced by l ..unization of animals with steroid-protein antigens, (Lieb8raann~~ 1958). After antibodies aaainst the proteia, bovine sera. albu.in (BSA), moiety are removed by preCipitation with 8XC4tSS BSA, the realniAI anti•• teTo!d senua is labelled with fluorescein isothlocyanate. Whe. tissue ••ctlona are exposed to the fluorescent antiserua,

~croseopic visualilation of the intlacel1ular steroid anti,eR is made possible by the deposition of fluorescent antibody at these site.. The fluorescent anti. sarva used in this study was prepared against testosterone and, eonsequently, conjulates with it or allied androaeas wherever they are present. Results follOWing eaployaent of this technique have been coapareO with thOle obtained from the use of the steroid dehydrogenase method (Watten­ bera, 1958). This latter method indicates cellular areas of steroid Iynthesls by dep~itlon of the foraalan (reduction product) of a tetralolium salt at site. of activity of one of the enlyMe. (~S.lB-hydro.,sterold dehydrogenase) involved in the biogenes .. of the steroid Mraones. However. it provides only indirect

~Yldence for steroid synthesis, lince the presence of a single enzyme in the

~iosynthetlc pathway does not insure successful synthesis, let alone rele.. e of toned hormone (Lobel !!..!!.- 1962). Also, it doe. not previcle infonation as to the character of the steroid hOrMOne end-product. Histoehealcal exasinatlon of the teltis-llke, hypertrophied, rudi­

~ntary right gonad of the sinistrally ovartectoa1led. single-comb, light brown, Leghorn fowl, correlated with the chan,e, observed in the secondary sexual char- 3 actors of such fowl, has enabled thi' investigator to identify the cells in which androgen synthesis occurs. During this investigation the ,onads of a number of both sexually immature and mature, intact light-brown Leghorns were also examined histochemi­ cally. Thus, the cells responsihle for androgen synthesis in the te.ti, and ovary of the BOrBal domestic fowl were also deteradned.

AIIO, the ovaries and testes of the albino rat and the teste. of a dog were exaained hlstocheaically and the androgen-producing cells of the .... uUan loned lo"tifted. REVIEW OF THE LITERATURE BIOLOGY

Testis

The cellular site or sites of androgen production in the vertebrate gonad have never been ~onclustvely dotermined (Albert, 1961). Most of the in­

formation obtained regarding this prohle~ has come from experiments on mammals and observations on hUJIlaft beings, and the evidence appears to indicate that an­ drogens are produC".e<\ in the testb hy the interstitial cells of I.eydig. Thus. in d tuations where the s.mini ferOll! epithcHull had atrophied without dUtage to the interstitial th~u., and1'Of[enic hormone lu,cretion was not affected. Such a condition is realized in cryptoreM,d animals (Ancel and Bouln. 1903, 1904; Hanes

1911; WUUalls and Cunnin~ham, 1930; Moore, 1932; Nelson. 1934; Wilhelm and

Schwartz, 1938) and »u_ns (Steinaeh, 1913; Lipschutz. 1918; Albert. 1961) and following the lrradiatioft of the testis with adequate dosages of X-rays (fier­ gorale and Tribondeaut 19M, Witsehi. I.evina and Hill. 1932) or radium (Johnston,

1934). CTa,ftia~ to abnormal site,; (Steinach, 1910) and ligation of the efferent ducts (van Wagenen, 1924) have also been ob,erved to cause preferential damage to tlle tubules.

Despite the destruction of the ~erminal epithelium in the cryptor­ chid testis. it was found that the aeeeuory genital organs were usually main ...

~ained in an apparently nomal functional state due to continued androgen secre­ tion. Also, extracts of cryptorchid testes of swine were found to eontain an­ drogenic material when assayed on the capon's comh, (f'eaard. 1920). S

Benoit (1924) obtained com;llete, selective destruction of the germ­ inal epithelium of the testis of brown Leghorn cocks by exposure to X-rays. Following this treatment there was a reduction in the testis size but no impair. ment in cOIIb growth. Benoi t concluded that the androgenic hormone was elabor­ ated either by the interstitial tissue or the Sertoli cells, or possibly both cell types. Similar exveriaonts, which gave like results, were subsequently carried out by Mlrsakaia and Crew (ISS1), and Essenber;. and Karrasch (1940). In addition, Nalbandov, Meyer, and McShan (1946, 1951) reported that comb ,rowtb occurred in hypophysectomized white Leghorn cocks after admin­ istration of leutelnbinl horllOne (LU), but that follicle.stimulatiAa hormone

(FSH) had no effect. This response to LH was associatoci with an increase in th« number of Leydig cells and in the amount of intertubular connective tissue as a whole. A second line of evidence that the interstitial cells, rather than the seminiferous epltheliUJI, is responsible for the elaboration of androgenic material i5 found in experiments where the interstitial tissue had atrophied, while the seminiferous opithelium remained unchanged. Administration of pitch to rodents (subcutaneously. intraperitoneal1y. orally) damaged the interstitial tissue. with a parallel atrophy of the sex accessory glands. but there was no apparent effect on the seminiferous epithelium (Buchh~ilB. 1932). In another report (Moore and Samuels. 1931) interstitial daaage was produced by feeding rats a diet deficient in vitamin-B complex. Here also the germinal epithelium of the testis remained normal while the prostate and seminal vesicles atrophied froll lack of testicular honaone. 6 The observations of Greop (1936) and Evans (1937) that the adain-

~stration of gonadotrophins to i_ature or androgen-deficient male rats resulted in androlen production only if the interstitial cells of Leydig were stimulated also su"ests that these cells are the source of androgen production. Addition­

~d evidence is to be fOUBd in the observation that in Rlice bearing experimental interstitial cell tumors there were high levels of andraaen production (Hooker and Pfeiffer, 1942). Sluiter (19.S), Sluiter and van Dordt (1947, 1949). Taber (1949),

~nd Marshall (1~9) have demonstrated histological indications of secretory )nenomena in the interstitial celli of tbe testes of birds during their repro­ ductive period. The •• investigators likewise observed that followin, the injec­ tion of interstitial ceU-stbwlating hormone (ICSH) into young birds, or into birds not in the reproductive period, the nonfunctional interstitial cells changed into cells with a dlstiBct secretory appearance. These cytological al-

~erations were correlated with certain cbanges in. and development of, sex characters which normally occurred during the reproductive period. In the human male, the excretion of 17-ketosteroids and the devel-

~pment and condition of the secondary sex characters parallels histologic and C1 ologic evidenco of secretory activity by the Leydig cells (Albert, --et ai, 1955). Also, it is a well known clinical finding that certain interstitial cell rumors of the testes in humans produce enormous quantities of androgenic materi-

Ill, wbich is capable of inducing precocious sexual development in iuature 'boys (Dorfllan and Shipley, 19S6). On the other hand, there 11 a certain amount of evidence which 7 indicates that in both birds and mall_is the sellinife1"ous tubules are a possible sout'ce of androgens. Kudrlashov (1931) observed a correlation in vitamin E deficient rats between the degree of testicular destruction and the hormone out­ put of the testis as lIleasU1"ed by the _intenance of the sex accessory structure. i!e found that the degeneration of tho seminal vesicles, followed by the prostate. began as soon as the spermatoeenic activity of the testis was impaired. In the last stages of vita.in e deficiency, after complete 5.minl£o1"ous tubule degener­ ation, the accessory glands showed cast1"ation changes, but the still numerous

Leydig cells appeared to be In a secretory state. With continued vitamin E

~efici.ncy evon the Leydig cell. degenerated, but by that time t.h- &ce~~$~ri~&

~ere so completely atrophied that they showed no further effect of lack of !androgen. fJrenne1lt&D (1935, 1938) Observed that the administration of PSB in roung cockerels induced seminiferous tubule hypertrophy and masculiniZation of

"ead fumishings, indicating androgen production. while LH had no effect on

~ndTogen secretion. Using the capon comb test, Hanes and Hooker (1937) assayed scrotal land abdominal testes of boaTS and fOUlld that IlUcll less male sex hormone was present in abdominal than in scrotal testes. Nelson (1931) found that rat testes, which were exp4trimentally con ... Fined to the abdomen, secreted auch less androgen than did scrotal testes a5

~ndicated by the condition of tbe sex accessory glands. This wa, especially

~rue of rats whicb had been cryptoTChid for prolonged periods of time (240 days P1" longer). 8

10400" (l944), repeatinl part of Nelson f , work, found that in rats experimental cryptorchidism, between the ages of 20 and SO days, reduced signifi cantly the amount of secreted ma18 sex hormone as measured by the • .-inal vesicle test.rhe magnitude 01 response of scrotal testes to injected gonado­ trophins was also greater than that of abdominal testes. Unlike Nelson. how­ ever, I\.oore failed to find any II1J,ainHllt in the aBdrogen-secreting abilIty of the testes of chronoloaieally older rats, or of cryptorchid. of loni standing.

1~i'uU\ it is recalled that cryptorchidism causes atrophy of the seminiferous epi­ thelium, th.s. findings leave open the possibility that the seminiferous tubules may have contributed 50_ hOrllOfte with androgenic propertIes.

Pfeifier and tarschb&WI (1943) observed that in the _Ie English sparrow, L,eydi, cells were not present at tiu. of hiih udrogenproduction. as indicated by deposition of .elanin in the bill. However, when meles in the non­ breeding season were injected w1tb pregnant ma:re seN. CPt..;:;), Leydig cel1$ a.ppoared in the intertubular tissue in large nuabers no pigment was deposited in the bUI. ~'iith lover doses of PMS, as well as sh•• p whole pituitary powder and partially puri fled pi, pituitary powder. the g.emna} epithetiu. waspHfer­ entiaUy stimulated with very little Leydig cell develop_nt, but here too the level of androgen liIecnUon was hiah. The conclusioa aniveo at was that the absence of L8ydl1 cells at the ti._ of normal testicular development indicates that under the influence of the bird'. own hypophysis the gorainal epithelium must control the production of androgen. However, they felt that the develop­

Ment of Leydil cells (concomaitant with androgen socr8ti~1) under the stimula­

~ion of ionadotrophic uterials, is evidenco for the production of the lIale 9 hormone by the Leydia cells under these experimental conditions. Otber studies also indicate that under normal circumstances there does not always see. to be a strict correlation betweon the presence of testicu. lar interstitial cells of l.eydig and. udrogen secretion. In fact, Stieve (1921, 1910) believed that changes in the number or characteristics of the Leydig cells of the human testis were insufficient to account for the striking changes which occur at puberty. However, Hooker's studies (1944) in the bull indicated that there was no change in the all'l()Unt of testicular androgen until a considerable period of ti_ after puberty. He attributed somatic puberal changes to the occurrence of a marked increase in end-organ sensitivity at this time rather than to a sharp rise in androgen production (Hooker, 1942). Therefore, it woul~ seeR that the absence of conspicuous change, in tne Leydig cells at puberty does not necessarily argue against their production of androgen. Seerlll Steroids, including andro,ens, have been demonstrated in vertebrate sperm. It is believeo that they are present in the sperm, as well as in the seminal fluid. Physicochemical studies have provided part of this information.

~pectrophotometric analy,.s by Otrscherl and Knuchel (1950) have shown that human spen contalfi.1f ~.5 1111 per 100 51 of seminal fluid of 570 IlU c1'trollO;ens (dehydroepiandrosterone, i-androstanol-6-one-11 and bl ,S.andTOstadieone-17), while bull spera contains 4.3 .g per 100 Ill. Pollowing separation of sperm frol seminal fluid by centrifugation, all of the determined hormono was found in hound lorm in the sperm fraction. Also, in aaoospermic individuals there was ar, absence of steroids, and in one case of infertility a correspondingly low hor- 10 mone titre. These observations have been interpreted as indicating that the an· drogens are found in the spermatozoa and not in the seminal fluid.

In a subsequent communication by the senior investigator (Dirscher and Breuer, 1955) the steroid components of the neutral ketone fraction of spet1 were identified by Mans of paper Chl"OlUtograpby. Besides the above-mentioned androaeus, sperm were found to contain etiocholane-3a-ol-17-one and possibly pregnandiol-20-one. Saendl'oi ad Molnar (1950) dellOnstratecl. by meanl of the ZilUlGrman and Pincus'Reactions, the presence of 17-tetostorolds in both the sperm and seminal fluid of humans. The se.inal fluid was found to contain these hormones in twice the quantity of the sper. itself. Since a biological assay fol' andro­ gens gave necative results, these investigators believe that the andl'o.ens pre­ sent in the seminal fluid are biololicalty inactive steroid species such as etiocholanone, which was ide.tifted colorimetrically in 40\ of their extracts. Thoy consider that these steroids al'8 elaborated by the sex accessory glands (prostate and .e.inal vesicles) and that they are absorbed by the sperm second­ arily (Molnar and Sa.ndroi, 19S9). The findings of McCullagh and Schaffenburg (1951) also indieate that anurogens occur in both bull and hUII8Jl sperm. These investigators observe, a significant inaeas. In the concentration of prostatic phosphatases fo11<»1iBI the injection of BeL-hydrolysed extracts of seminal fluid blto iaature. cas­ trate ule albino rats. Since aadro,ens have been shown to regulate the produc­ tion of these ensymes in such test ani.. ls (Schaffenburg and McCullagh, 1950), the.e results are considered good evidence for the presence of androgens in the 11 5eminal fluid.

On the other hand, Relabet and Raboeh (1963), using the androgen assay method 0' seminal vesicle weight chango in spayed. infantUe rats, found Btl endrosenie activity in human spermatic fluid. Attempt. at identification of an­ drogen in extracts of .ninal fluid by Mans of paper chromatography were also unsuccessful (Raboeb, .!!.!b 1963). Simpson, Wright and Gottfried (1963) isolated the steroids 11. deoxycorticosterone (500 ug/lOO g), pregnenolone (14 ug/IOO ,), progesterone

(8 ul/I00 e), androstenedione (2 uJlIOO ~),dehydroepiandrolterone (2 ug/IOO g), androsterone (S ug/IOO g), and possibly aldosterone (1 ug/lOO g) from the semen or the Dogfisb shark, ~ualus acanthes. These steroids are present as a sperm cell constituent or bound to extracellular mucoids or proteins, since they are not present in that port1on of the semen which passe. an ultrafilter. Th ••e in­ vestilators consider that the steroids are secreted by the Leydig cells and are absorbed by the spormatozoa during their passac. through the seminiferous and epididymal tubules. Attempts at tho detection of steroids 1n the semen of four species of olaslIObranch fish, however, were unsuccessful (SbtpsOft, WriJht and Hunt, 1963). fgyary

It has been well d... nstrated by bioassay methods that the .....1- lan ovary secrete androgens (Hill, 1962). Also, the andro,enic steroid, andro. stenedione has been isolated from human ovaries (Zander !1!!1 1958; Simmer and

~oss. 1960. Short and London, 1961), while epitestosterone, androstenedione and 19-nor-androste.edione, have bee. identified in tho follicular fluid of mare -

ovaries (Short, 1961, 1962). Furthermore, many investigators (Sweat --et ai, 196 Ryan!!.!!t 1961; Kase.!l.!!.- 1961; Wanen and 541hanick, 1961; A.xelrod and

Goldzieher, 1962; Lanthier and Sandor, 1962; Loutfi ~~ 1962) have convincin

11 shown that the ovary is capable of synthesiziftl androstenedione!!. ...vi_t_ro .... However, the identification of androstenedione in huun ovarian venous blood by Mikhail, Zander and A.llen (1963) provided the first direct evidence that tbe nOl'llal ovary secretes androsens.

The avian ovary is also capable of 1,J'04ucina androgens. Do_'s investigations (1929, 1933) on the fowl ovary weTe tbe first to clearly indicat

that the vertebrate ovary produces androgens. His observations, Ukewise, demonstrated that the medullary co.ponent of tbe left ovary was tbe site of androgen synthesis in the noraal female, light.browD Le,horn fowl. Thus, when mammalian gonadotrophic extracts were injected into intact young females, they responded with a phenomenal growth and masculinization of bead fUrnishings.

(Comb growth cannot be stimulated by ••tro,en, but only by androgen, Womach and Koch, 1931). These individuals revealed hypertrophy of the left ovary. growth of the medullary component account ina alMOst entirely for the increase in ovar!

an weight. The right gonad revealed 80 noticeable arowtb. Also, the bijection of gonadotrophins into sinistrally ovariectomized chieks had no effect on head furnishings, nor did tbe right gonad show any indications of hypertrophy under

these eonditions. 'rhe latter observation. have been confirmed with other mamma

an gonadotrophic preparations (Kornfeld and NaivandOY, 1954) and also with avi pituitary extracts (Taber, 1958). Several investiaators have atteapted to identify the site or sites pf androgen production in the avian ovary. Benoit (1935) ascribed male hormone

~ecretion by the left ovary of white Leghorn hens to the medullary interstitial

~e115 since granulation of their cytoplas. was correlated with the growth of

~ead furnishing. Asaundson --et al (1937) observed medullary hypertrophy of the left pvarr, associated with masculinization of head furnishings, following the injec­

~ion of prepant Mre sel'Wl (PUS) into young vhi te Leghorn pullets. This hyper­

~rophy was ascribed to a marked increase in the nuaber of medullary inter,titial

~el1s, which were considered to be the site of androgen production. In con·

~rast, 60tUa (1939) observed that in PSH and UI-stimulated. young white Leghorn pullets, androgen secretion, as indicated by masculinization of head furnishinl~

~ould be correlated with growth of both medullary cord cells and interstitial

~ells. Therefore. thi, investigator was of the opinion that both tissue ele. !Dents were the source of androgen secretion by the fowl ovary.

Pfeiffer and Kirschbaum (1943) noted a correlation between comb cells ~rowtb and develop_nt of granular _dullary intentltial/ in the ovary of PMS-

~reated immature, female white Le,horn fowl. Likewise. Taber (1948, 1951) Ob­

~erved that in newly batched light-brown Leghorn chicks tbe interstitial cells

~f the ovarian _dulla were characterized by a vacuolar, foaay-appe.ring cyto­

~IUIll, heavily stained by oSllic acid. which changed to • more granular cytoplasm

~t approximately the time coab growth was first apparent. lypertrophied. MiuAt." right gonad. Birds, unlike other vertebrates, normally have but one functional )vary. located dorsally on the left side of the abdomen. In the domestic fowl. 14 the daht and left embryonic gonads develop concurrently until the 7th to 9th day of incubation when there is a retardation in growth, followed by retrogres­ don, of tbe right gonad (Swift, 1915; arode, 1928). This right rwibtentary gonad persists in the adult hen as an inconspicuous, flattened rldg. of tissue, closely applied to the post caval vein which :uy or may not b. detected grossly.

It i. composed primarily of medullary tissue with cortical tissue present in about 21\ of all examined cases (Taber, 19S8). Occasionally, this organ devel­

OP$ into a 1101"8 or less typical rllnt ovary. 00a\:iI (1939), reported. that sueil ovaries, ranling in size frOM noraal to barely visible, were found in leas than S% of operated. adult brown Leghorn hens. The factors involved In the embryonic inhibition of the right gonad aro not understood, but it is probable that the estrogens produced by the left Iovary exert a restraintna influence (Kornfeld, 1958; Taber, 1958). The host evidence for this hypothesis comes frca the effects observee followin, removal

~f the left ovary in the domestic fowl. Although previous iovestigators

(Goodale, 1913, 1!U6; Zawadowsky, 1922, 1926; Benoit, 1923. 1924; Pezard, 1923;

~reenwoodJ 1925) have reported the results of ovariectomy, 00-. (1927, 1929) was

~he first to .ake a careful analysis of the effects on tho ri,ht lonad. It is

Inow well estahlished from the observations of DOH that, in the absence of tbe left ovary, the rudimentary rigbt gonad develops into a testis-like organ, con­

~ainini a variety of cords and tubules, with or without lumina, which secretes

~ndrolens in quantities sufficient to cause pronounced mascullniaation of bead furnishings (comb and wattles) and behavior. Wbether or not the.e tubules are fertile or sterile (e.g., the i"resence of a ,erminal epithelium or of a Sertoli... 15 cell syncytium) is dependent on the age of the pullet at the time of operation. Firket (1914) and Swift (1914, 1915) found that the primordial germ cells are vresent in the medulla of both right and left gonads of the female chick in ear­ ly prebaten stages. Brooe (1928) confinud thele findings and _de the addi­ tional observation that thes. cells Ir8dually decrease in number in the rilht gonad after hatching, so that by the end of the first month they have practical~ all disappeared or can no longer he recognized. Thus, the hypertrophied right gonads of poulards sinistrally ovariecto.hed during the first unth develop a much greater percentage of fertile _"ullary tubules than de the right ,onads of poulards operated on at a later date (Doma, 1929). Later in develop.ent, in addition to androgen, this organ also produces estrogen frequently in quantities

$ufficient to bring about a complete f ••linization of the plumage. Although the hypertrophied dEht gonad may be called an "ovotestis", sinee it secretes large amounts of both androgenic and estrogenic hormones, its histological structure almost always resembles that of a sterile or immature testis. Therefor., the term "ovotestis". use

The site of androlen production in the hypertrophied, rudimenta.ry right gonad of the poulard was tentatively identified by Domm (1927, 1929). This investiaator showed that in the single-coab, light-brown Leghorn fowl an ... 16 drogen secretion, as indicated by growth and increased vascularity of the cOillb, could be correlated with hypertropby of the medullary portion of this gonad.

A detailed histological ~xamination of the bypertrophied, rudiment­ ary rignt gonads froll Do.' 5 (1927) .eries was lIade by Gray (1930). On the basi, of this study be concluded that there were no interstitial cells in this gonad, and that the medullary cords and tubules of the first proliferation were tbe only tislue components to which hormone secretion could be attributed.

However, Benoit (1932) observed a chan•• in the IlUJIlber, as ",ell as

!the cham eter of the cytoplasm, of the interstitial cells of the hypertrophied, rudimentary right lonads of sinistrally ovarieetomized domestic fowl. These cells had a granular cytoplasm in contrast to the typical vacuolar, 11poidal cy­

~oplas. characteristic of the IIlOduUary interstitial ceUs of the normal left

Iovary. They also appeared to hypertrophy and to increase In number as the meelul.. lary portion of the daht ,oRad hypertrophied.. Correlation between this inter­ iStitia} cell hypertrophy and hyperplasia and growth of head furnishings gave

~vidence to support the hypothesi, that these cells were the site of androlen iSecretioD. In connection with an IDvestilation on the effects of sex MJ'JIOneS pn the g:rowtb of tbe rudimentary right gonad in the sinistrally ovadecto_bed

~ight-brown Leghorns. Tabor (1958) made a detailed histolo,lcal study of the

~evelopm.. t of this gonad. She noted no correlation between the degree of me­

~ullary tubule differentiation and androgen secretion. as indicated by incroased

~ascu1arity or growth of the cosb. No observations were reported on the inter­ stitial cells. BI0CHa4ISTRY

The sex h01'llOnes are steroids, a cla•• of biologically important organic compounds po ••e.sing the eyelopentanoperhydrophenanthrene nucleus. This 'nueleus' consl$ts of a tetracyclic carbon skeleton with three 6-sided rings and one S-sided ring. The .ix•• ide4 ring. are arranled together as in the coapound phenanthrene. To this is attached all unsaturated. five-sided ring; and this is indicated by tbe terms c:yclopentano-phenanthrene. The hypothetical tetrac:ycllc

~arbon skeleton of the steroid c01lpOuads is asswaed to be funy saturated. and

~ne .-kes this clear by referring to the structure as cyclo-peRtano-perhydro­ phenanthrene, ..anin, that all tn. necessary hydrogen atoms have been added to

~he cOlllpound to _k. it fully saturated. The innuaerabl. steroids that are d....

Irived ff'Oll this sk.~etont such as the sex OOrmones, the adrenocortical hOl1l'lOnes.

Fholesterol and the bile acids, eriosterol and the D-vitamins. cardiotonic gly­

~osid.s and aglycones and saponins and sapogenin•• diftor fro. one another by

~he introduction of double bonds betwHU certain carbon atOllS, or by substitu- nts forth. hydrog.n atoms attached to certain carbon atoms, or by the addition t)f a side cbain. Very sull alteration, in cn,.ical structure often results in narked changes in bioloaical activity. In order to describe the structural for­ nula of an individual steroid it is necessary to specify In which ring and at libicn cal'bon atOil these chang •• have taken place. POI' this nason each rina is ndicated by .. capital letter and each carbon atoa by a number. 18 ··-~·"·f··.;·- ,,' P. ~;H

'~.H # f,~ , (a) (b) Fla_ 1 - Complete structural formula Ca> and symbol (b) for steroid nucleus. The r1ftgs of the steroid nucleus are referred to individually by the letters indicated in (b).

The sex hormones are dIvided into three sub-groups; the androgens, estrogens and ,estolons. Ho1'1lOlles representative of all three groups are nor- mally produced by the gonads of beth texes as well as the adrenal cortex and th placenta. Each of these sub-groups is represented by a hypothetical parent structure, estrane, androstane and p.. eanane, according to which they possess or two methyl group. and whet,her or not they haYe a side-chaln. ~ ...... ~'"'-.: .-~

, sterane (gonane) estrane

andros:taae pregnane L, '".

Fig. 2 • Structure of tbe hypothetical steroid hydroc~s upon which tbe no­ menclature is based. 19 Tho androaens or male sex hormones, which condition the developmen of tbe ule sex oraan, and secondary sex characteristics, are C19 steroids with no side chain at e17, and have oxygen substituents at C-3 and C-17. Five prin­ cipal androgens are made available to the blood and tissues. These are testos­ terone, ~4 ... androstene-l,17-dione (androstenedione), llB-hydroxy... L\4.androstene- 3,17.. dlone, ctehydroeplandrosterone and andrenosterone. Testosterone is the most biologically active of these compounds as indicated by the chick comb assay mothod. Although only a few species have been investigated (un, dog, rabbit, bull, stallion, ram and bOI), the pred.olJinant testicular androgen in Jilammals ap .. pears tobe testosterone; in all of these species androstenedione, as well as de- lydroepiandrosterone, are also !lUtcTeted (Hall, et aI, 1964). However, in the at, only small aaounts of testosterone are secreted, and androstenedione is the androgen (Dominquez, at al. 1960).

Fevold and Elk-Nes (1962. 1963) have dellOnstrated by IIcans of ineu­

of cell-free testicular tissue homogenate, with 4...c14.progesterone that

he testis of the English sparrow (Passer domesticus) ca.n convertpl'Ogesterone 'nto testosterone and androstenedione 1n vitro by a pathway chemically identical --...... - found for homologous JallJlUlalian tissue.

'rhe following di.gra. indicates the met~)olie pathways involved in biogenesis of the androgens. Fig. 3 Kajor metabolic pathways involved in biogenesis of "androgens.

Aoetate

cholesterol ~').'ti - , . caproic J IIO-b...;t4H~ ,,A aoid (20-22B-hydrOZYlaSe)! : ~, ., '

~ 2OB-22B-d~:tY:::::::;1~terOl

( ~5 -3ll-hydroxysteroid deb1drogenas.),

(1 ~-hydrozJI.'Ue)

pr.::'nolono~

progesterone 110 ( 17-c'-HYdrOzyL 17-hydroxypregnenolone lase) J

h- 17«-hy roxyproges~eron. (Desmolase) 1 0 (~5-3Il-hYdroxysteroid dehydrogenase) (

o~ androstenedione debydroepiandrosterone {11417~_hYdrOXyJ steroid {- dehydroge­ nase)

testosterone HISTOCHEMISTRY

A group of histochemical reactions is characteristic of the lipid dro?lets of steroid-producing organs. Besides staining intensely with Sudan black B, these lipid droplets, in frozen sections of formalin-fixed material, react positively in tests for carbonyl groups, such as the Schiff test and hy­ draline method. (phenylhydrazine method of Rennett. 1940, and the napthoic acid

.\ydral1de method of Ashbel and SeUpan, 1949). They are fluorescent wben view­ ed with ultraviolet Iieht and contain birefringent crystals (Dempsey and Wislodd 1944; Wislock! and Dempsey, 1946a, b; Rockenschaub, 1952, Burkl and Kellner, 1954). The droplots also give colored products when treated w1th .ineral acids (Chester Jones and Deane, 1949). Whe. cholesterol or its esters are present in these lipid dl'oplets, a blue-green coloI' results followin., exposuft of tissue sections to concentrated sulfuric acid and glacial acetic acid (Schultz, 1924; .ewis and Lobban, 1962). All of the above reactions are diminished 1n intensity or are prevented entirely, followin, extraction of the sections with acetone at room temperature.

In the past it was proposed that all or some of these reactions might be

~iven by ketosteroids, i.e., by steroid hormones or thell' immediate precursors. lInus, certain investigators (Deane and Creep, 1946: llempsey and Wislocki, 1946; Dempsey, 1941. Greep and Deane, 1949) _intained that the ketosteroid content of

~he adrenals could be made visible by means of this battery of reactions and, fUrthermore, that quantitation was possible. It was gradually realized, however that none of the,e reactions it sped fic for keto.terolas. In fact, the evidence 22 indicated that a positive reaction to the entire battery of tests was never

~resent in a single locus (Feldman, 1950) and, as Boscott and Mandl (1949) and

Gomori (1952) asserted, an artificial mixture of tissue constituents, such ~~ neutral fats, cholesterol, carotenoids, tissue aldehydes and phospholipids, would satisfy all tests even if no ketosteroid! were present. A reevaluation of the histochemical significance of these reactions was undertaken by Deape and Seligman (1953) and Karnovsky and Deane (1954, 1955). These latter investigators showed by both chemical and histochemical means that, except for a small amount of plasmalogen, all histochemically de­ tectable carbonyl groups in adrenocortical lipids are aldehydes produced by the auto-oxidation of the unsaturated fatty acid moieties of lipids during fixation or by exposure to the atmosphere during the preparation of the tissue section. Nevertheless, the observations of Karnovsky and Deane indicate strongly that the percentage of unsaturated fatty acids, which are the source of the aldehyde groups, is high in functionally active glands and declines in inactive glands. This confirms the earlier assertions (Bennett, 1940; Wislocki and Bennett, 1943; Dempsey and Wislocki, 1946) that the intensity of the carbonyl reactions corre­ lates well with the secretory activity of these glands. The fact that these re­ actions occur in lipid droplets of steroid-producing glands (adrenal cortex, go­

~ads and placenta), and that they vary in intensity under different physiologi­ cal conditions of these organs, suggests, therefore, that they are empirically

~seful in identifying the probable sites of steroid hormone formation. Following use of the above-mentionedhistoehemical methods positively-reacting substances have been observed to be present primarily in the 23 lipid droplets of the Leydia ceUs of both fAa_Un and avian testes. However, the seminiferous tubules demonstrate a positive reaction with some of these pro­ cedures (Oeane, 1953, 1958).

In 1958 a new fJistochemical methoel developed by WatteDbeTa lIde it possible for the first time to indicate cellular sites of steroid-hormone syn­ thesis. It consists of aD adaptation of Farber and colleagues' (1956) histochem­ ical technique for visualiziAa nicotinuide-adenine dimu:leotide (NAD)-dependent

~ehydrolenase. to the demonstration of AS.3S-hydroxysteroid dehydrogenase. Al­ though this technique visualizes histologically an enzy.. involved in one of the initial steps of steroid-hormone biolenesis, a positive reaction is only an in­

~irect indication of such synthesi.. since the presence of a sin,18 enzyme in

~he biosynthetic pathway does not insure successful synthesis, let alone release pf formed hormone (Lobel et ai, 1962). Also, it does not provide information as

~o the steroid species elaborated by reactive cells. Wattenberg (1958), as well

~s a number of other workers (Allen, 1960; Deane, 1962, 1963; Niemi, 1961, 1963;

~ubin et aI, 1959), have utilized this technique to Indicate cellular areas of

~teroid synthesis in the adrenals, testes, ovaries and placentas of rats. In

~he testis the interstitial cells were the only cellular elements which &ave a positive reaction.

"hese same lnve,tila.tors (Allen, Deane, Levy, Nielli, Wattenberg) ,ound AS.3B-hydroxysteroid dehydrogenase activity to be high in all steroid­ producing endocrine organs re,ardless of whether the primary hormone ()roduced

~y these glands was a gestogen, andnaen, estrogen or corticoid. Goldberg and

FoUeaguos (1963) observed, however. that the activi.ty of this enzyme is high in 24 those cells presumably concerned with progesterone production but seldom readily detectable in loci of nOrMl or tUllOr tissues presumably concerned with androgen or estro.en prodUction. Therefore, they consider progesterone the principal steroid visualiaed by this technique. They also state that this is indicative pf the possibility of a bio.ynthetic pathway for the androgens and estrogens

~hich precludes progesterone as an intermediate. It is apparent when all the evidence is taken into consideration

.hat there is DO presently available histochemical method which allows for the

~dentification of steroids in general, let alone specific steroid specie, such

~s the androgeas.. Thus, the cells responsible for the secretion of androgens in

~he steroid hormone-producing glands of vertebrates have not been conclusively

~dentified. And, although there is an i1tpressive array of data to support the view that in the testis the interstitial cells of Leydig are the source of these

~oraones. the evidence is still inconclusive. Also, observations have been re­

~orded which aake it probable that the semiBiferous tubule, of the testis, as

~ell as the ..du11a1"1 cord, aDd tubules of the hypertrophied right gon.ad, con­

~titut. a secondary souree of the androgens produced by these organs. MATERIALS AND METHODS

TIle single-comb, light-brown Leghorn fowl (American variety) has been used in this investigation since sexual diaorphism in plumage Is more pro­ nounced in it than in other breeds. This variety, which had once been readily available commercially in the United States. has now been laraely supplanted by the Oanish variety of brown Leghorn. Hence, it has been necessary in these ex­ periments to obtain fertile eggs from fowl "fanciers"l and incubate the. in the laboratory. A slIUlll inbred flock of these birds is now maintained in the animal

~uarters of the Anatomy Department. Birds were sinistrally ovariectomized between the ages of five days land four months. In the discussion that follows, the tel'll ovariectomy will be used to refer to the reaoval of the left ovary. and sud\ ovariectomized females

~ill be called poulard,. OVariectomies were performed according to tho proce. dure of Dom. (1927). Birds were starved 17 to 24 hours prior to the time of operation, anesthesIa being produced by the administration, intraperitoneally or

~ntrav.nously. of Pentobarbital sodium (Nembutal) at a dose level of 0.5-0.6 cc. per kg. body weight. An opening was made into the body cavity between the last

~wo ribs while the bird rested on its right side. This opening was made as

~lose to the 'Sline as possible without dauge to the kidneys frOll subsequent re­

~raction. The ovary was then brought into full view by pushing aside the iDtes-

lLawrence S. Bush, Batavia, New York, and Herhort T. Kaiser, Bangor, Pa. 26

~ines and maintained in this position by means of saline moistened cotton. The ,eritoneal covering was detached by means of a fine forcep and all ovarian tis­ sue was removed as completely as possible by blunt dissection. This type of di~ section is fteCe5sary because the bird ovary Ues on the post-caval vein and

~emmorhag. is a constant preble.. The areas of attachment of the ovarian tissue

~ere gently rubbed with tiny cotton pledgets from the under surface in order to dislodge it from the post.caval and ri.ht iliac veins and adjacent areas. Rare­

~y was the entire ovary removed intact by this procedure: usual1y# varying-sized pieces, disen,aged by the rubbing, were removed fro. the maIn mass by cutting

~d at times by gentle "tuggin,". FInally electrocauterization of the whole

~rea was done 1n order to destroy all remnants of ovarian tiSSU9. The surface

~f the left adrenal, which is ROTmally partly or c~)l.tely covered by the ovar~

~as abo deliberat.ely cauterized. One.hundred birds of varying .ges were successfully OVariectomized. rwenty-flve of the operated birds succuabed to disease. Porty birds were sacri­

~ieed and their ri,ht gonads have beeB ex.ained histochemically. thirty-fIve

~oulards are still allve. At the time of sacrifice the comb was weigbed and measured and the

haracteristics of the plwuge noted. Representati ve s_ples of the breast and ack feathers were filed in envelopes. The sacrum was also preserved. In addition, the teste, froa 10 younj fowl, 5 roosters, the gonads

f 10 albino rats of both sexes, intact as well as hypophysectOMized, and the estis of a dog were examined in the same manner.

Sections of aU aonads were exuined hhtoc:h8ilically by means of 27 both the steroid dehydrogenase technique and the modified fluorescent antibody methods. The former technique is a method for the histological visualization of

~5.3B-hydroxY5teroid dehydrogenase activity by .eans of the doposition of the formalan (reduction product) of a tetrazolium salt, Nitro-ST (Wattenberg, 19S8). fhis procedure in turn is an adaptation of Parber and colleagues' (1956) histo­ chemical teunique for vhualizing NAD-dependent dehydrogenase. to the delltOu­ stration of ,65 .. la-hydToxysteroid dehydrogenase. This dehydrogenase represents one of a series of enzymes involved in the biosyathesis of active steroid hor­

~neJ, beina responsible for the conversion of C19 and e21 ~-3B-hydroxysteTOids to ~4-3-ketolteroids. In particular, it catalyses the conversion of pregneno­ lone to prolesterone or dehydroepiandrosterone to androstenedione by aiding in

~he process of dehydrogenation in ring A with tJUt subsequent shift of the double

)ond fro. tho ~5,6 to the 44,5 position (Samuels ot aI, 1951). Most of the activo steroid hormones are ~4-3 .. k.tosteroids or are derived lTOlI thelll. Thus,

~his histochemical method is an indirect lIeans of indicatine sites of steroid­ lormone synthesis in tissues, since 1t allows for the histological visualization of an enzyme involved in one of the initial steps of such synthesis. l~wever, he presence of a single enzyme in the biosynthetic pathway does not insure suc-

~esgful synthesis, let alone release of formed hormone. Also, more than one

~ormonal steroid species can be formed in the gonads by a common synthetic path.

~ay (Gorbman and fiern, 19(2).

In this procedure an unfixed tissue section is incubated in a medi-

1m containing the substrate. dehydroepiandrosteronc (DHA), ll1us NAO, the tetra.

oliu!Il salt, Nitro.. ST and buffer. Hydrogen ions removed from the substrate by 28

the enay.. are transferred to NAB. then from NAOH to the tetralo}e by a portion of the electron tftnsIJort .yst.. "lat_ to NAOO-cytochn_-c reducta.e (Novt. ... toff, 1959). Thorefore, the aethod does not demonstrate the aetivity of the specific dehydro,enase directly but that of a sequence of .nlymes. PresUMably

the sequence is identical for all NAn-dependent enlytlle systeu ,ubsequent to the specific dehydrogenase. If the fonalan (reGvee. tetralole) produced Is insol. uble, ACcurate cellular (but not intracellular) localization of the specific enz111e appea'Hpossible (Farber !S.!!" 1956; Cascaramo and Zweifach, 1959). TIlt theoretIcal aspeets of the tetralol1.. tochnique for 4ehydroge... es can be rep­ resented by the followiR, schema,

HAD Reduce

Dehydrogeaue Plavoprotel. (PAD or PMN)

HADH Tetrazollu P~t ~/_----' '- "-----...... ~or '----,.,;IIalt (colorleis. , NADPII water soluble)

All•• (1960) has observed that the substitution of steroids which do DOt po.se,. the ~5.3B-hydroxy confl,uratloa (64.aadroste.e-,,11.dione (an­ drostenedione). e4-aDd~ten•• 11B-ol.S-one (testosterone). estratriea.-3-o1-17. 29 one (estrone), estratriene-l,17-diol (estradiol)] for dehydroepiandrosterone in the ineubating mediu. yielded no reaction with adrenal sections. Pregnenolone

(~-pregneno.l(n)-ol-20.one) yielded a reaction comparable in distribution and intensity to that obtained with dehydroepiandrosterone. From these studies it is concluded that the histochemical reactions obtained with dehydroepiandroster­ one represented true enzymatically catalyzed oxidations of the substrate and were not artificial lccalilations of the "nothing aehydroaenasett type described

by Zimmerman and Pearse (1959). Further, it appears that the activity of the enzyme studied is restricted to the 3(b).ol grouping. for negative reactions

were obtained with the 17(B)-01 grouping of testosterone and estradiol. Gonads obtainee fro. birds sacrificed by exsanguination were placed

on 8 lIiCrotOlle chuck, the surface of which was coated with water, and frolen im­

mediately by means of a freon-cooled sta,e in a Coons-type Cryotome (Lipshaw). These tissue blocks were then sec'lloaed in tbe cold (teaperature :>-20·C.) at

2 u. Sections were mounted on II coverslips and, after a brief period of drying to attach the section to the slide, incubated in the substrate ..dium. In the standard incubations sections were placed lor five minutes

in 0.1 M phosphate buffer, pH 7.1, at roolQ temperature to remove endo,enous sub­ strantes. At times sections were immersed in acetone prior to rinsing in the

mffer solution in order to rellove free lipid droplets. They were then incu-

~)ated in a "Columbia .181'"2 containing the dehydroepiandrosterone COUA) modium1

2Arthur H. Thous &Company, Philadelphia, Pennsylvania lAS reported by Nachlas and colleagues (1957), Nitro-BT media may be used 30

(DiIA. 2XlO·4f1f. in a.cetone, NAD. SUO-3M. NitrCJ

After incubation the sections were fixed in 10\ formalin for at least 15 lIinutes. rinsed in distilled water, stained in Harris' Hematoxylin for two minutes, rinsed in tap water Wltil blue, rinsed aga.in in distilled water and counterstainedfor two minutes 1n a 50\ isopropanol solution of au Red O. They were then rinsed in distilled water and mounted in glycerin-Ielatin. Gonad sec­ tions treated In this manner show cells with blue nuclei and light-blue cyto- 1,1a5-. red lipid droplets and dark blue fine intracellular formazan granules. Photographs of tissue sections .stained according to this procedure were made using Kodachrome ItA eolor film (~~A rating 32) with a matched liaht source obtained by means of fIlters and at an exposure time of one-half second. KodacnroM JJrints were made from these color sUdes.

The histochoalcal .ethod for the identification of steroids deve~ ed for use in this lnvesUaationls a llOdified fluorescent antibody technique.

repeatedly. This media was routinely refrozen between experiments. Re.using it about 5.10 times (for a total of 20-25 sections) seemed to be permissible. A laediull was dbcarded when it showed a faint trace of color. .. phenomenon proba­ bly due to the accumulation of endogeno~. substrates, rather than because of depletion of specific substrates. OPN or Nitro-B'r. 31

The fluorescent antibody IHtbod, which was developed by A.. H. Coons and his as­ sociates (Coons, 1958), is based OD tbe fact that antigenic material present in tissue cells, and more particularly in sectIons of th~wlll react specifically with antibody comple.ntary to it. This l_unologlcal reaction results in tbe deposit of minute amounts of specific antibody over those areas of a tissue sec­ tion wbere the antigen is present. When the antibody molecules have previously

~een choaically marked with fluorescein, the micro-deposit of fluorescent anti­

~ody is vi.ible under the fluorescence Microscope. The specific step in the re­ action is the selection by the aAtl,eR of its specific antibody from a solution IOf fluorescent proteillS. Other ••r. protein .01eC\tl.5 present in the fluores­ cent solution are washed away leavlnl tbe .,ecllic deposit --in situ. The quantity of antigenic material Microscopically detectable by tho fluorescent-antibody uthod is calculated to be about 5110-15 p. for the

!Capsular anti,. of the pneUJlOcoccUI (Coons. 1956). and the liaiting detectable

~oncentratlon of antlaen is .stiaated to be approximately 10.5 ,m. per al.

~.Ie Ihlitina values are Hveral orclen of .,nltude sm.Uer than the minl_1

~WlAtitiel of bloch.. leal COtIpOUIIcls detectable by Jllcro-absorption spectroscopy

~f the minimal dry veiahts measurable by micro-interferometry. The flndin,s of Liebermann and colleagues (1959) that antibodies

~ith steroid specificity were fOrMed 1n rabbits immunized with steroid-protein

~ODjulates have been utilised in the clev.lopment of this new histochemical pro-

~edure.

Thes. lnv•• tiaatoft deteniaed the steroid specificity of the anti.. 32

sera by means of the hapten-inhibition technique. This test is based on the fact that when an antigen is added to its antibody an insoluble aggregate is produced. Since each antibody is considered to be divalent, it reacts with two antigen molecules. The antigens are multivalent and, therefore, react with ad- ditional antibody molecules which in turn react with more antigon to yield, fl- nally, a lattice which precipitates. On the other hand, the steroid hapten is monovalent. The divalent antibody reacts with two such hapten molecules and foms a eomplex which cannot react with additional antibody and reuins soluble.

If the hapten is bound finly to the antibody cOllbining site, small quantities of it will inhibit precipitation upon subsequent addition of antigen. If the hapten-antibody bond is not a firm eme, pHCipitatlon by antigen "lUI be in­ hibited only by a large amount of the hapten, or possibly not at all (Landstein­ er, 1945). The inhibiting powers of different haptOfts were compared by finding

~he hapten conceatration which completely inhibited a given concentration of antibody. The alllOuat of inhibition by various related hapten, provides a good indication of the specificity of antigen-antibody reactions. The fluorescent anti-testosterone serum utilized in the present in­

~estigatlon was obtained fTOm the Colorado Serum Co~any4 who followed a proce­

~ure for its preparation provided by this investigator. A testosterone-bovine serum albumin conjugateS. in which the bovine serum albumin was conjugated to

4Colorado Serua Company, Deaver, Colorado SEndocriDolOgy Stwiy Section, National Institutes of Health, Bethesda, ~arYland, Courtesy of Or. S. Liebermann 33 testosterone in the number 3 position (T .. 3-nSA)6. was injected, in the form of an dUll precipitate, intramuscularly into the hind legs of three rabhits. In­ jections were repeated one week later. This conjugate acted as an antigen eliciting the formation of two types of antibodies, one against the testolterone moiety, the other in response to the eSA. Treatment of this antiserum with BSA brought about a precipitation of the antibodies against aSA. The remaining antiserum was thus specific for the testosterone moiety, exhibiting a positive rrecipitin reaction with T-3-&qA at a dilution of 1:160. The serum globulins were fractionated by Mans of repeated half­ precipitation with ammonium sulfate at a temperature of O-S· C. The redissolved ,lobulin was then dialyzed at O.S· C. alalnst 0.85% sodium chloride. The sa­ line was changed frequently during the process and dialysis was continued until sulfate was no longer detectable in the liquid outside the sac as deterained by the addition of saturated barium chloride. When the mixture did not becOae cloudy or opalescent, the globulin was considered to be free of sulfate. Sefore labeling of the globulin was undertaken, the protein content of the 2lobulin was determined by the 8tur~~t method (r..ornall. et ai, 1949) in order to compute the amount of fluorescein isothiocyanate that was needed. The protein solution was then adjusted to 1\ by dilution with saline. Followbag this carbonate-biocarbonate buffer (O.SM. ph 9.0) was atitied to th~ chilled 110-

6Por the preparation of the testosterone.protein conjugate the and .aadn4 groups of &SA, o.t which there are 60, ware coupled to a testosterone derivative by .eans of the mixed anhydride procedure. This method involves the treatment 0 a carboxylic acid wi til a ehlorocarbonato in the presence of hase. The chiaro... carbonate used was isobutyl chlorocarbonate, The product is a mixed anhydride. ---

34 buIlD in an amount equal to 10% by volume of the 1\ globulin solution. This

buffered globulin solution was placed in an iee bath and 0.05 mg. of fluoresceif

lsothloeyanate powder, per mg of protein in the solution, was added slowly with

constant stirring. SUrring was continued overnight ill the cold. The product

was subsequently dialyzed in tbe cold against 0.85\ saline for two days and then against buffered (O.OIM phosphate, pH 7.5) saline until 100 ml of the overnight

dialysate showed no fluorescence with a portahle Wood·, lamp. t4erthiolate was

added as a preservative in a concentration of 1:10,000 and the solution frozen at -20· C. until ready for use.

Prior to use, the fluorescent antisel'Wl'l must be pUl'ified to dimin-

ish or to remove nonspecific stainin. (Coons and lap Ian, 1950) which may arise principally from either of two sources. Derivatives of fluorescein, not removed by dialysis of the fluorescent-antibody preparation, may stain the tissue sec­

tion in the manner of an ordinary fluorescent dyestuff and fluorescein conju- gates of iaaunologieally indifferent serum proteins (in the crude gamma-globulin

fraction) .y react with tissue components (Kidd and Friedewald, 1942).

The e~'lrical proe•• tre of absorbing fluorescent antibodies with

~hsue powders usually suffices to reduce nonspeCific staining to a negligible

level. In general, the tissue powder is prepared from a ho~genate of an organ

!either by acetene precipitation and acetone drying or by lyophilization. The

that Is, an anhydride formed between the carboxylic acid and an ester of carhon­ ic acid. These mixed anhydrides are especially reaetive molecules which, when ~reat.d with aaines or amino acids under mild conditions, yield amides or peptides. 3S absorption of fluorescein-labeled antibodies with the dried-liver powders of the rat (Ortega and Mellors, 1956) and the mouse (Coon~ and Kaplan, 1950; Coons,

Leduc and Connelly, 1953) have been found to effectively reduce the nonspecific staining of a variety of tissues of man, rabbit, mouse and domestic fowl. In this investigation Scc aliquots of the fluorescent antiserum were absorbed with lyol)hUized mouse liver powder. Thh powder was added to the fluorescent antibody solution in a test tw>e in the amount of 100 -8 per mI. of

~nti5erum. Prior to this, however, the tissue powder was wetted down with O.Ol~

)hosl'hate buffered isotonic saUne at pH 1.5 in order to conserve tl,. volume and labo to preserve the Itf! of the antiseruta. Conjugates treated wi til dry tissue powders nomally acquire a pH of about 5.5. After standing for one hour at room

~emperature, and after intermittent mixing with a small stirring rod, the super­

~atant was harvested by centrifugation (6.1,000 r.p.m.). The absorbing proce­

~ure was repeated once more using fresh liver powder. Merthiolate, 1 I,art in

~o,OOO, was added to the final supttmatant product and this purified conjugate

~tored at .20· C. Tissue sections were prepared for viewing with the Fluorescence licroscope in the following manner. Presh gonads were frozen and sectioned in

~he Cryotome at 2 p as described earlier in regard to the preparation ~f tissue sections for the steroid dehydrogenase method. After air.. drying for apl,roxi-

8y reacting testosterone with (O .. carboxymethyl) hydroxylamine, an oxime as formed which at the same time is 8. carboxylic acid. This derivative, oupled as it is through C-3, allowed the preparation of a protein conjugate of estO$terone in which the protein is bound to the steroid by It bridge attached o Ring A. 36 mately one-half hour, a drop of the fluorescent antiserum was deposited on the section and the sUdeplaced in a petl'i-dhh lined with 1I0istened filter paper.

The reaction is carried out at room temperature for 10 to 15 minutes. At the end of this time, the excess antiserum was drained off and the section rinsed ifl a Coplin Jar with 3 to 4 changes of buffered isotonic saline solution of about 3 to 5 minutes duration each. The section was then mounted in buffered ,lycorin

(9 parts anhydrous ,1ycorin to 1 part buffered saline solution) and examined microscopically. A Leitz fluorescence microscope, which utilized an Osram HBO 200 ureury vapor lamp as an ultra-violet light source, MOdified to include Zeiss OI>tics, was used throughout this investigation. A dark ... field condenser was em­ ployed in all fluorescent antibody studies since a dark background is l;;'cessary for contrast with the weak fluorescence obtained with fluorescent antibody techniques.

lecause the brightness of a fluorescent image is very low in co.pari 50n to the excitation light, filters are used in fluorescence work. To prevent mui , illg of fluorescence emission, the excitation light must be relllOved be for. it reacbes the eye of the observer. This is accomplished by placing a primary fil­ ter between lamp and object so that only fluorescence.... xciting wave lengths are passed, and by placing a secondary filt.r betw ••n object and observer so that only wave lengths characteristic of the fluorescence co be seen.

Three general types of lighting systems may be used for fluorescein

Ca) near ultraviolet exciting light between 350-400 mu (Euphos) (UGI Filt.r); secondary filter colorless but opaque to ultraviolet (Rupho!:): (h) hlue-violet 37 exciting light. between 400.450 mu; secondary filter distinctly yellow: and. (e) combined ultraviolet and blue-violet exciting light between 350-450 IDU; second­ ary filter distinctly yellow. Combination (a) Is most useful in cases where specific staining may not be lIUch mare intense than tne autofluorescence of the background and where there also is strong autofluorescence. Since such conditions have been encount. ered in this investigation. this combination has been used exclusively. The colorless secondary filter perMits one to distinguish between blue, aray. or white autofluorescence and the specific apple-green color of fluorescein. In frozen-sectioned gonad sections treatea with antitestosterone fluoreseeut serum steroids aft observed as smaU apple-g"en droplets. whUe other Up ids are viewed as pale. soft. iee-blue or white. auto fluorescent globules of varying dzes. There is a sharp corttrast between the specific and non-specific fluorescent colors. Connective tissue fibers demonstrate a pale blue autofluorescence. All photographs were taken at a magnification of 400X with Ansco­ chrome color film. Initially Super Anseochra.e. which has an ~~A ratin, of 100. was used. but fol1owina the introduction com.ercially of Anscochrome 200 (ASA rating, 200). the latter fihl was used exclusively. Exposure time with SU~ler Anscoehrome was of the order of four minutes. while two minutes was sufficient when Anscochrolle 200 was employed. Kodachrome prints were subsequently luaue from the color slides. OBSERVATIONS

lfistological and histocbeaical observations Oft the 40 hypertrop~ rudimentary right gonads exa.ined in this study, together with gonad weights, cOlab appearance and SiH and character of ph.... ,. of the poulards from which they were obtained are IUlaariaed bl Table 3. Thus, the presence, as well as tbe relative intensity, of specific fluorescenee exhibited by this or,an nave been assoeiated with the coadltloa of the secondary sex characters at tbe time of ,acrifice of this expert... tal ..1_1. The poulard (Fig. 1), or sex inverted domestic fowl, may pos.e.1 all the secondary sex characters of the cock (P11.2), e.I_, aesculine head furnishings, sa18 pluaa,., as w811 as the bebavioral re­ sponse of the normal male. crowlnl. atteMpts at treadina and placement In the bigher Khelons of the peck onter. Note that the ujority of the poulards 11st­ ed in Table 3 exhibited cOliplete sex invenion, as shown by tlUculine head fum .. ilhln,s and ..Ie pl... ,e, at the ti.. of sacrifice, while 9 specimens showed .el inversioe as a..oastrated by brown oreast feathers or complete henny plumage (Fi,. 3. Fig. 4). 39 HISTOLOGY OF TliJi HYPERTROPHIED RIGHT GONAD

Histological observations on the hypertrophied. testis... 1ike right gonad of the brown Leghorn poulard confirm in all assential datails the observa­ tions of Ooiul (1927). Gray (1930) ud Taber (1958). Although there were wide variations in gonad weights (Table 3). the histololY was quite similar in all lease,. The riaht lemad of n01'llfll. intact £_l.s cOIlsist, of connective tissue strands separated by sinus-l1ke spaces (lacunae). Groups or medullary cord cells are found within tbese strands. 'ollowing sinistral ovariectomy, the rudlaontary right gonad is ac­

~iyated and begias to increase in si,e. This rrowth lavolves both it. epithe. lial and connective tissue el.... ts and is accOMpuied by tha infiltration of lymphoid tissue which subsequently increases withiu the lonad. The hypertrophy of the epithelial compoueats consists of a slight iucrease in the si,e of the

~dullary cord cells and an increase 1n their number by mitotic division to fora

,"dullary cords. Secondarily, there is a gradual differentiation of the ..eI\ll­

~ary cords into tubules, which 1ft the early stages of tbeir for,aation resemble

~he se.btiferous tubules ~ the Juvenile testis. These medullary tubules, which

~n exceptional cases colltain spera.toaenic elements, .y be very numerous and

~hus videly scattered. through a donse COIUlectlve tissue stroma. The epitheliUII bf certain tubules consists of a sinale layer of alongated Sertoli-like cells, the auelel of which 11e basally uar the bas neat I11181tbrue, ad whose cytoplullS fora a syncytial-Uke ass. Although, thesa tubules are of a lIOderate di_ter. they freq_atly have no lumen. When a luaea 1s present, masse. of protoplas.ic 40 debris are often observed lying free in it, the result presumably of a type of

desquamation (Pia. 5). Other portions of the gonad show tubules in which a n~ ber of Sera cell generations are evident. e.g., they exhibit stratification. Sinus.like fluid-filled spaces, which are quite characteristic of the normal right gonad, are also present in the hypertrophied right lonad, but are either partially or completely obliterated by the increased mass of medullary cells. The intertubular tissuo consists of (1) fibrous connective tissue, (2) lymphoid tissue, (3) blood and lymph vessels and (4) interstitial cells. At the marlin, or situated within the substance of these gonads,

~y be found ,roups of SMall tubul.s liDed by columnar epithelium. These tubu~

are the rete testis, efferent ductules andie~ididyaal tubules.

The entire orgBB is surrounded by a heavy layer of 4ense, well­

~asculariled, connective tissue, which is eoaparable to th.tunica albu,inea of

the no~l testis. Narrow bands of CORRective tissue tend to divide the gonad into • .al1 lobules of varying slles. A cortex was observed in four hypertrophied ri,ht gonads (Table 3,

Birds Nos. 266, 512. 229, 304), In these cases the cortical tissue was repres.~

ed by .terile cortical cords. 41 HISTOOIEMISTRY OP THE HYPEttTROPHIED RIGHT (,.oNAD

Steroid Dehydroaonase Method ,AS.38-hydroxyst.eroid dehydrogenase activity, as indicated by the presence of intracellular, finely ,ranular, blue-black deposits of formosan, was

~eJDOnstrated by the medullary cords arul tubules and intentitial ceUs of all

~ypertrophied right gonads examined (Fig. 6). This enzyme was not present in

~e connective tissue. lymphoid tissue or blood vessels of tbis organ. The interstitial cells were tbe only cellular component of the in­

~ertubular tissue which revealed steroid dehydrogenate activity (Pig. 1). The Fytoplasa of some of th••• reactive eells contained only formalan ,ranules,

~hi1e other ceUs had both Eonu.. ,l'UUles aDd OU Red 0 lipid droplets. Por­

..zan ,ranules were never Ob ••1'YM ••tracellularl1. Howewl', Oil Red 0 drop­ lets, devoid of foraalaR granUles, were observed extracellular!y in the intertu­

~ul.r areas (Pig_ 1).

Oil Red 0 llpid droplets were also very proalnent at t1.. , in me­ dullary cord$ and tubules. The syncytial-like Sertol! cells of tbe sterile me­ dullary tubules contained a great number of such droplets. (Pi,. 8). It has been observed in this tnvesti,ation that the ctegree of steroid-syatheU.c activity in such areas. as indicated by the DOURt of foraaun deposited, could be conelat.. ed with the slle of the Oil Red 0 lipid droplet., e.I••• blgh degree of .ynthe. tic activity was associated with s_l1 lipid droplet.s and a low degree with larae droplets. b5.3B-bydroxysterold dehydrogenase is present in the medullary cord cells of the noreal riaht gonad in exceptionally small a.ount., however. it was 42

~S8rved that as the right gonad tAeHased in she, as til result of hypertrophy

~d hyperplasia of these medullary cord cells, there was a eOAeoalttant increase

~n the number of foraazan granule. per cell, indicative of increased synthesis pf the enly_ itself. as well as an increase in the total nureber of steroid de.

~ydrogenase.reaetlve eells. 43

Fluor~scent Autibodz Method

Validation The original investigation on antisera, produced in response to Isteroid-protein atigens. was conducted by LiebeftaM. Erlanger ,Belser ad

~gate (19S9). The.e investigators deterained the steroid specificity of anti­

~era produced in response to testosterone-protein conjugate. (T-J-BSA and T-l'­

~SA) by me.. s of the hapten.inhibition test. Their results are suamarl'ed in

~ables 2 and 3. In Table Z the haptea, testo.terone-(o-carbo~thyl) ox!.. was

~sed at one-fourth the concentration of the other inhibitors. The iamunospeeificlty of the fluorescent anti.T.J-8SA serua used in ~his investigation has boe. dete~tfte4. The ,recipitln~ltre of this antiserum

(after reaoval of antl·BSA antibodies) with T-3-BSA was found to be greater than

~ 1160. The antibody protein coaceatratlon, detentud by the Blunt _thod for protein-aitro,en, was ZO 8, 110bulla per 81 of .erua. The fo11owla, control pl'Ocedures at the tissue level, dellOnstrated

~he immunologic specificity of the staialnl reactlonl

1. Staiaing of ,ORad section, with fluorescent anti-testosterone serua was abolished when tissue .ectioas were previously treatad with unlabeled anti.T.J-BSA serua. Unlabeled hoaolO1ous nonal sel'Ull (rabbit anti-bovine s • .,. albu.in ilobulla) did not affect the staining capacity of the specific fluores- cent antbenaa.

Z. 'luorescent hoterolo,ous antisenaa (fluorescent aulaea pig anti­

~ovi.. serus albumin globulin) dId not stain ,ORad sections. 44 3. Pluorescent anti-testosterone serum did not stain normal, non­ gonadal tbsues.

Observationl Fluorescent droplets of varying sizes, auto-fluorescent as well .s specific fluorescent, were observed both intracel1ularly and extracellularly in sections of all hypert1"Ophied rl,ht aonads examined. Medullary cords ancl tu­ bules, as well as interstitial cells contain both types of fluorescent droplets. Specific fluorescent material, whieh indieates the presenee of steroids, is dis­

"in.. h~frOil auto-fluorescent _terlal on the basis of differences in color: 1

~he apple.areeR fluorescence of fluorescein contrast sharply with the pale blue,

~hite 01' yeUow color of a1rtofluonscMt substances. Conaeetive tissue exhibits

~ce-blue auto-fluorescence, while blue-cray, white or sliahtly yellowish auto­ 'luorescent lipid droplets are observed intracellularly or extracellularly. The lipid nature of the•• dropl.t. has been conflraed by their stainability with Oil

~ed o.

Specific fluorescent _terlal in droplet f01'll has been observed in

~11 cell types found to be reactive by the steroid dehydrogenase method, e.I_, lodullary cords and tubules aM iDtentitial cells (Pig. 9 • F1a. 10). However,

~her.as all cellular elements of the medullary cords and tubule. give indieatia.

~f capacity for steroid synthesis, as demonstrated by the pre ...ee of 5.38_ i1ydroxysteroid .ehydroa_nase, at tt_s only a few ulls iB these saae structures lIve a positive fluorescent reaction for the presence of steroids (Pig. 11).

~p.clfle fluorescent droplets were also observed in the lumen of medullary tu­

~ules and extracellularly in the intertubular areas (Pig. 12, Fig. 13). --

45 Cellular areas which have been found to be negative by the steroid

dehydrogenase method. such as lymphoid tissue and connective tissue, are also

raegati YO with the fluore-seent an.tibody method. tn only three cases of the present series (Table 3, Birds Nos. 588,

~96 and 568) was there an apparent positive correlation betwe.. tbe intensIty and number of fluorescent-reactive sItes in hypertrophied right aonads and the

eondi tion of the secondary .ex characters in the poularets f1"Oll which they weft obtained. In th.se case, especially lar... Ra.culin. combs were associated witb exceptionally fluorescent 10Rad••

There was DO observable difference in the distribution of specific 'luorese4Jnt cells in the hypertrophied right ,oaacls frOll poular4s exhibitin,

~nly 881. plumaae and those showiRJ iatermediate or henny plumage. However, one

gained the ilDpl'OssiOA that there were a areater nUllber of reactive area. in the

former type of loud (Table 3). Right goRads obtalae4 froa two poulards which showed no andreleuie stimulation of bead furnishin,s at tbe time of sacrifice (Table 3, Birds Nos. >25, 659) were also ....ined histoch.. ically. Only a faiat fluoresce.ce was ob. served 1ft several of the tubul.s. The results appear to indicate that this go­

Ilad is capable of producing androgeniC steroids several weeks prior to tbe tiM

~en such secretion ts ordinarily evldeDt through its eff.cts on secondary sex

~ar.cters such as the coab and wattles. 46

HISTOCHEMISTRY OF NORMAL f.oNADS

Gonad.s !! Leahorn !2!!. Both tbe cellular elements of the seainiforous tubules (Sertoli cells and developing germ cells) and the interstitial cells of Leydig of the testis of the adult LeahoTft rooster Jive indications of steroid production by both hbtoch.ldeal techniques. The 'peclfic f1uot"eseence of the intracellular lipid droplet. is quite readily SeeR in fro&en seetions treated with fluoreseent antiserua (Fig. 14). The ovary of the adolt heft ••hibit. specific-fluorescent material in the interstitial cells. Thes. fluorescent positive-cells appear just prior to the onset of ovulatioa. the tl_ at which androgenic stimulation of head furnishlnl' first become. evideat. All other cellular areas of this organ eabi­

Ibit aut.... fluorescenc., which is particularly apparent in the theca 1nteJ'Da and stratUM granulosua of the developing folliCle. Since the steroid dehydrogenase technique gives a positive reaction in all cellular site. of steroid synthesis. regardless of the steroid-hormone lend-product. flO information beneficial to this lnvesti.ation can be obtained froa observations 1'8lardinl .teroid-dehydrogenase-reactive cells in this gonad. towevel', the ovary of the fowl ••• a.lblt a po.itive reaction in tbe theea In­ terna and certain stroaal elements, presumably interstitial eells. The teste. of cockerels from I through 21 days post-hatch were allo

~xam!ned by both the fluorescent antibody and the storold dehydrogenase teeh­

~lqu.t. At the time of hatehlns both aedullary cords and tubules as well as in­ terstitial eells were observed to be weakly reaetiv. by both histoeneaieal --

41 raethods. After the first week the amount and nuaber of reactive sites decreased

larkedly until by two we.ks there was no longer any observable spedflc fluores­ ~ence and only a very small a.ount of bS.3S-hydroxysterold dehydrogenase aetiY. ty.

iammal ian Gonads, Testes and ovaries from 10 albino rat. (Sprague-Dawley), intact as

~ell as hypophysectomized, and the testes from a dOl were examined histoch.. ~­

~y. Reactive sites in the testes of both species were the interstitial cells of

~eydi& and all the cellular generatiGRS of the se.iniferous epithelium: sperma­

~OIOftiat primary and secondary speraatocytes and speraatids (Ptg. IS. Pig. 16).

~topla.aic debris froa maturing Ipe1'llll, the Io-called • residual bodies t of

~ealond also exhibited specific fluorescence. Of the two main cellular sites,

he interstitial cells gave the MOst intense reaction (Pl,. 11, Fig. 18). Al­ hough not all the interstitial cells nor all the elements of the gerainal epi­

~heU_ exbibited specific fluorescence, all cell. of the.e cellular types gave ~ reaction for the presence of ~S-3B-bydroXYlt.roid dehydrogenase (Fia_ 19, I,. 20).

Testes froa rats hypophysectomized at 21 days of age and sacrificed ~t varyIng periods up to one year also aave indications of ~S-3B-hydroxysterold

~ehyd.ro'.ftase activity" but the intensity of the reaction was not as strong as

~hat observed in the testis of the intact rat. This enzyme was still present in

"he testis of the hypophysectomized rat as 10na as one year after removal of the

~itultary. Auto-fluorescent lipid material. both in the interstit1al cells as

~ell as In the epitheUwa and lwaen of the .oinitereus tubules, was quite pro- 48 nounced in the hypophysectoaized rat testis. No specific fluorescent material

~as observed in thls modified testis. A common observation with regard to the interstitial cells of the

~at testis was the close relationship between these cells and the blood Yessels,

~.I"" they ensheathed the arteriole. (Pig. 14, Fig. 16). The rat ovary exhibited specific fluorescent droplets in the inter­

~titlal cells, especially those in the hilar region. No other structures were

~bserved to be reactive. ---

DISCUSSION

The results of this investigation indicate that the modified fluor­ escent antibody technique is a valid and extremely sensitive histochemical etRod for the demonstration of steroids.

The fluorescent antheNJI utilized in this study Is specific for

testosterone and allied and~genst a positive precipitin test tor testosterone

is liven at a serum dilutioa 11160. Also. it has been shown by means of tho hapten-inhibition test that estrone and 11a-estradiol give no cross-reaction itb this anti-testosterone .eTUM. However, a sligbt reaction is ,ivon by pro- esterone, deoxyeortisol and hydrocortisone, and the possibility that one or re of tbese steroids could contribute, either as end-products or as intermedi­ ates in the biogenesis of steroid honones, to a poJitive-fluorescent ..action

action CanDOt be denied. Progesterone and l1a-bydroxyprogesterone are secreted by the testis

in both .....llan (Lindner, 1961) and avian for.s CPrapst Hooker and Forbes, 1949; Lofts and Marshall, 1959). However, the amouat produced by the testis Is

xtRordinarUy .uU in coltparisOft to the mabl !lteroieS secretory products of

hh organ. Normally progestorone cannot be detected by ebeadcal _ans in the enous effluent of the bull testis (Lindner, 1961). In fact goaadotrophin­ timulated bull testis showed a progesterone seeretion equivalent to less than

1% of the total steroids secreted by this gonad. l1a-hydroxyprogesterone was

also present in • .a11 amounts, rarely constituting more than 2\ of the total

a.b-unsaturated 3-oxosterolds determined in blood samples (Lindner, 1961). 50

It is also probable, although it has Dot been identified, that de­ oxycorticosterone is produced by the vertebrate testis. Thus, Dominquez and colleagues (1960) have demonstrated the presenec in rodent testis homogenates 0 the 21-hydroxylAge necessary for the conversion of progesterone to Il-deoxyco costerone. Likewbe, ~imp.son, Wright and Gottfried (l963) have found ll-d.eoxy­ corticosterone in surprisinlly hl,h concentrations (500 v,/l00,) in sperm ex­ tracts of the Dogfish shark, ~ualus acanthiaa. Hydrocortisone Is not assUMd to be a nomal secntory product of tbe testis since it b.as not been detected in testis extracts and. is present in

oth arterial and venous blood of the bull testh in about equal concentrations

(Lindner, 1961). In particular, It bas not been identified in avian testis homogenates (Fevold and Eik-Hes, 1963). The forel01n, infoTa8tlon indicates that progesterone is the prl­ a1'1 steroid which might possibly live a false positive reaction in testis sec­ tions exa1lined by means of the ...lfied fluonlJcent antibody method. In this regard. it is inteT$sting to note that the Corpus Luteu•• which is a tissue sit

f hiah progesterone production, does not exhibit specific fluorescence with the antiserum used in this investigation. Regardle •• of the lack of complete specificity exhibited by the anti-steroid sera used ift this investigation. it would still sees that the fl

scent antibody ..thed is capa.ble of ,ivin, 110ft inforaation "gardin, the his­

olo,lcal visualization of steroids than either of the presently available e.g., the Schultz test for cholesterol and the steroid. d.ehydro,.nase thod. Greater specificity may possibly be attained by treatment of the anti- 51 serum with the cross-reacting steroids prior to conjugation with fluorescein.

"ho highly specific Schultz test has a low sensitivity. Both

(1947) and Reiner (1953), by testing ~_v_it_ro_ the influence of dilutIng holesterol with other lipids, have shown that unless the steroid constitutes pproxiaately 10% of the mixture (1.,lyin, 40% to 50% total lipids) no color 1s etectable. PurthermoH. in animal tissues, 5e.mingly minor chaDge. in the per- entage of this steroid cause strikin, 4ifferences in histochemical reactivity.

ane (1958). has shown that the lOIS froa adrenal lipids of stressed rates of

" of the ch_iealIy deterainablechol.sterol, fl'Oll 28\ to 24' total cholestero)..

.sulted •• virtual lOIS of Schultz reactivity by this gland. Also, the color btaiftoa with this method 1. unstable.

On the other hand. the steroid dehydrog-enase aaethod. which indi­

tes cellular areas of steroid procluction by deaonstl'ating the intracellulal' re.enee of one enarae involved 1ft steroidogenesis, cannot pravide information

to whether such synthesis is successful. 01' the natu. of the steroid end-

The steroid i.hydrogeRAs. aDd fluorescent antibocly naethocis were to identify the aDdI'OIea.secTotlftl cells of the hypertrophied rudimentary ight lonac1 of the browa Leghorn poulard. Since tnduUary conts an4 tubule•• as ell as interstitial cells, were Obsel'Yed to be positive with both histocbe.1cal

thed., it Is considered that both of these cellular sitos are the source of he androgens which are responsible for the phenomenon of sex inversion in the lni.trally ovariectoMized domestic fowl. This conclusion is an essential agr

nt with that of ether iavesti,ators (Gray, 1930; Benoit. 1932). l~eY.r, the.e 52 workers "favored" either one or the other of these gonadal structures. A purely histological examination of the hypertrophied right gonad, correlated with an­

~rogen-conditioned changes in secondary sex characters of the poutard. led Gray Ito conclude that the uduUary cords and tubules were the source of the andra­

~ens secreted by this gonad. while aenoit, using the same criteria, was of the IOpinion that these honaones were produced by the interstitial cells. The observation that all cells of the medullary cords and tubules IOf the hypertrophied right gonad showed steroid dehydrogenase activity, while at

~imes only certain ot these sa.. structures exhibited specific fluorescence, is ~xplained as follows. ~5.lB.hydroxyst.rold dehydrogenase is a constant cellular tfeature" of ,teroid-synthesizing cells, and a uthod which visualizes its pre- [sence will give a positive reaction at all U_s. However, the fluorescent

~ntlbody method visualizes the end-product of such synthesis. It is generally iConceded that actively synthesizing cells of steroid-hormone producing glands

~end to secrete their end-products i..ediately following their formation and

~hat they do not ordinarily store them. Therefore, at the time of sacrifice.

~hose ceUs which exhibit s,ecific fluorescence have most probably just cODlplet­

~d synthesis of their hormonal steroids and have not as yet secreted the., while

~on.fluoroscent cells. which are nevertheless steroid dehydrogenase-positive,

~ave presumably becm depleted of their steroid end-products at this tiM. The pretHtnc:e of extracellular specific fluorescent lipid droplets adjacent to the free surface of cells would appear to add weight to such an interpretation.

The results of this investigation indicate that there is a very a~

~ral increase in the number of histochemically reactive areas as the gonad de~ 53 op'; however, it has not been possible to observe a perfect correspondence be­ tween the size and color of head funishinls and number and intensity of histo­ chemically-positive sites in this organ. Nevertheless, in four cases particu­ larly larae, turgid, red combs appeared to be associated with noticeably fluor­

~5cent right gonads. No changes have been observed in the histochemical

'picture" of the hypertrophied right gonad conc01lit,ant with or following estro­ gen production hy this oraan.

The widely accepted beli.f that the interstitial cells of Leydig !are the principal site of nelD,enic synthesis in the vertebrate testis was con­ firmed by the hlst&Che.ical findlftlS of this investigation. But, the results

~lso appear to indicate that the seminiferous epithelto. is capable of producing tandrogenic steroids. However, tn." have been no previously reported obsena­ ~ionl on the preseftce of llS.3B-hydroxysteroid dehydro,enase in the IHiniferons

~ubules. Other involti,ators (Wattenbera. 1958, Levy ~!!J 1959; Niemi, 1961, 1963), havo considered that this ena,.. is absent from the seminiferous epithe­ lium of the .....lian testis. The present investigator noted a low de,ree of

~etiYlty in the ,orainal epithelium, wblch was observed only by means of high power .agnifieation. It is of interest to note that all published illustrations

~f testis .ecUo•• f'stained" for the presence of steroid dehydrogenase havo been low __ ,nification micro-photographs.

At this place in the discussion it is instructive to consid.er the larrhenoblastou. type of virilizing ovarian tumor in tho hwan. There are vari. ous types of tissue present 1n the.e tUlllOrs. All types are frequently encount­ ered in the SaRe tUMOr. but 1n other instancos a single variety may predominate. S4 he most highly ciifferentiated variety is composed of simple tubular structures, ostulated by Meyer (1931) to be the counterpart of testicular tubules. The

st undifferentiated type contain, solId masses of spindle cells with larg. uclel which strongly resemble sarcOMa. An intermediate variety possesses cells ith prominent nuclei, which In some areas are arranged in circles resembling ubular structures but elsewhere are disposed in irregular or anastOflK)sing col s reminiscent of embryonic sex cords. The latter two varieties of tissue in articular are interspersod with variable nUllbers 01 polyhedral or rounded ceUs ith rather abundant pink-staining cytoplasm. A.lthough it has been proposed hat the latter eells are the neoplastic equivalent of testicular Leydig cells, heir abundance is not necessarUy conelated witb tbe severity of lIascuUnlza­ It is also of interest that the intermediate and sarcoma-like neoplasms invariably lead to virilization wbereas the highly differentiated tubular often do not, (Dorfman and Shipley, 1956).

Savard and colleaJUOs (1961) have observed the formation of radio- androstenedione and t ..tosterone in arrhenoblastoma tissue slices incu­ ated in vitro with progesterone-4-CI4• Histologically, the tumor was hIghly d - , ercntiated, the cells being arraDled as ,roups of pseudotubule, without lumens. oyd18 ceUs .'ere not observed in the connective tissue strOll4. These observations indicate the participation of both _edul1ary

'stroul' ceUs in androgen synthesis by thh tumor, and are remark­ bly confirmatory of the coftclusions reached in this investigation in regard to he cellular tttes of androgen synthesis by the avian and mammalian testis and he hypertrophied right gonad of the poulard. ---

55

A hbtoen.ttllical exmllination of the testes of eoekereb frM the

time of hatchlng to 21 days JJost-hatch indicates that androgens are being pro­

duced by this gouad at tho thw of hatching, some ti_ before the comb truUcates

the l'lresenco of these steroids in the blood. The initial lack of response of

this tar&et organ ean possibly be explained by tho low blood titers of androgen

and/or its degree of sensitivity to tl~se hormones.

Certain aspects of this investigation have Clarified or corrobor­

ated previous observations ftBardina the _rphological buis of endocrine secre­

tion. 'rhus, it has never boen definitely determined that tM1'O exists an ana leal basis for • direct transfer of androgens from Leyd1i cells to the testicu­

lar blood. The results of thb bw.,tlgation coati", and extend the observa.

tions of those worker. (Ball. 1871J Mihalkovics. 1873J and Wagner. 1925). who

< ave asserted that Leydig cells {om sheaths clo.ely enveloping blood vessels.

Al.o, the observations of countless investlsators (Deane, 1958) that at the time of active synthe.is the cells of endocriM gluds o...... trate

smaU Upid d1'Op1ets, whUe inactivity is cnaractel'iud byarge Upid droplet.,

.II.S been coafil'llled. jiiiP

SlJMMARY AND CONCLUSIONS

1. The modified fluorescent antibody method developed for use in this investigation is considered to be a valid and highly sensitive histo­

chemical method for the identification of steroids, whlch provides

more infomation than pl'Osently available techni~ues. 2. The fluorescent anti. steroid serum used in this investigation is specific for testosterone and allied androgens, but several other steroids, e.g., progesterone, deoxyeortisol and hydrocortisone. are capable of gtving a slight reaction -in vitro with this anti- sel'Ull. However. it is cOIlsldered that interference of these sterotds at the --in vivo level is neglisible. 3. Results of a histochemical examination of the hypertTOphied. rudi­ mentary right gonads of brown Le,horn poulards. by both the steroid dehydrogenase and fluorescent antibody methods,correlated with the

cond! tion of the secondary sex characters of such poulards, delllOll- strates that the medullary cords and tubules as well as the inter­

stitial eells are the source of the androgens produced by this orlan. 4. In three cases of tho present series there was au apparent correla- tion between the intensity and number of fluorescent-reactive sites

in hypertrophied right lonads and the condition of the secondary ,ex characters in the poulards from which they were obtained. In ---

57 those cases especially large. masculine combs were associated with exceptionally fluorescent gonads.

S. There were no observable differences in the distrihution of specific fluorescent sites in hypertrophied right lonads from Poulards with

only u1e plwaage and those showing feminization of Illumage •. How­ ever, there were a greater number of reactive sites in the former type of gonad. 6. The results of this investigation also show that all the cellular elements of the seminiferous tubules. spermatogonia and primary and

secondary sper.atoeytes. as well as the interstitial cells of

Leydig, of the testis of both the domestic fowl and albino rat are capable of prodUCing androgens. 7. The medullary interstitial cells in both the fowl and rat ovary

eXhibit specific fluorescence. indicative of androgen production.

S. The gonads of young normal _10 brown Leghorns. as well as poulard•• appear to produce andraaonic steroids in small amounts for a con­ siderable period before visible indications of their secretion is apparent throu&h effects on secondary sox characters. ---

UTERATURE CITED

Albert, A. 1961 The mammalian testis. In Sex and Internal Secretions, 3rd ed., ~. C. Young, ed., pp. 305-365. ~illiam;-ana WIl'lns Eo., Baltimore, Md.

Albert, A., Underdahl, L. 0., hreene, L. F., and Lorenz, N. 1955 Male hypo­ gonadism. VII. 11_0 testis in partial gonadotrophic failure during puberty (lack of luteinizing hOFllOne only). Proe. Staff r·teet., Mayo Clinic, 30: 31-43.

tUlen, J. M. 1960 A histochemical study of 3(B) ... sterol dehydrogenase ill the adrenal cortex of the developing mouse. Anat. Rec., ~: 57-63.

ncel, P. and Bouin, P. 1903 Recherches sur Ie role de la glande inter­ stitieUe du testicule. Hypertrophie compensatrice experimentale. C. R. Aead. Sci., Paris, -137: 1288-1290. ncel, P. and Boob, P. 1904 Oe 18 elando intorstitieUe du testicule des l'IIa_iferes. .1. Physiol. et Pathol. gen., ,2,: 1037.1050.

stlbet, R., Cohen, R. lS., and Seligman., A.~. 1951 Histochemical demonstra- tion of ketosteroid. in normal and neoplastic testes. Endocrino~, -49: 26S-281. Ashbel, R. and Seligman, A. M. 1949 A new reagent for the histochemical demonstration of active carbonyl groups. A new method for staining ketonic steroids. Endocrinol., -44: 56S-S83. smuodson. V. S.t Gunn, C. A. and Klose, A. A. 1937 Somo responses of the immature female fowl to injections of mare gonadotrophie hormone and oestrin. Poultry Sei., -16: 194-206. xelrod, L. R. and Goldziehor, J. W. 1962 The polycystic ovary. Ill. Steroid biosynthesis in normal and polycystic ovarian tissue. .1. Clin. Endocrinol., ~ Metab., ~: 431.440.

~ennett, H. S. 1940 The life history and secretion of the cells of the adrenal cortex of the eat. Am. J. rulat., -67: 151-227. enoit, J. 1923 Transformation experimentale du sexe par ovarioprecoce chez la poule domestique. Compt. rend. Acad. sc., -177: 1074-1077. Benoit, J. 1924 Sur 18 signification de la gland genitale rudimentaire droite chez la poule. Compt. rend. Acad. se., -118: 341-344. 59

nenoit, J. 1929 to determinism. des cnaracteres sexuels secondaires du Coq domestique. Arch. Zool. oxp. et gen., -69: 217-499. 8enoi t. J. 1932 r.' inversion sexueUe de 18. poule determinee par l' ablation de l'ovaire gauche. Arch. de zool. expcr. et gen •• !!: 1... 112.

8cnoit, .1. 1935 L'ovaire... organe elaborateur des hormones sexuelles lentelles. LEts hormones scxueUes chez les intersexues. Actualites Scient. et lndust Exp. de 6iol., -260: 1-68. nenoit, J. 1950 01fferenc:iati<>n lexuelle chez les oisoau au cours du devel­ loppement normal et de Itintersexualite experimentale val' ovariec:tomie. Arch. d'Anat. Micr. et de Morphol. exper., -39: 394-414. Seraonie, J. and Tribondeau, L. 1904 Action des rayons X sur Ie testicule du rat blanc. C. R. Soc. BioI., Paris, -57. 400.402; 592-596. eol1, P. 1871 Untersuchun,en uber den Bau nnd die Entwickloog der Gewebe. Arch. mikr. Anat., 1: 324. Boscott, R. J. and Mandl, A. M. 1949 Histochemical demonstration of keto­ steroids in adrenal tissue. J. Endocrinol., -6: 132-136. ~reneman, W. R. 1935 The effect of fractionated and unfractionated gonado­ trophic preparations In testes and comb growth in the chick. Anat. Rec., -64(Suppl. 1): 56. ~reneaum, w. R. 1936 The effect on the chick of some gonadotrophic substan­ ces. Anat. Roc •• -64: 211-220. Breneman, W. R. 1938 Some aspects of male hormone secretion in the white Leghorn chick. Ind. Univ. Pub!., Science Series, -7: 5-23. Brode, M. O. 1928 The significance of the asy_try of the ovaries of the fowl. Jour. Morph. and Physiol., -46: 1-57. tluchheim, V. 1932 Lesions provo<,uees dans Ie testicule des l'Iul_iferos par Ie m.lout ct autres hydrocarbures. Compt. rend. soc. bioI., -109: 1290-1292. BurU, i~. and Kellnor, G. 1954 Fluorescent-microscopic identification of the male sex hormone in rat testes. Z. Zellforsch., -40: 379-388. ':ascarano, J. and Zweifach, 8. w. 1959 J1actors influencing the histochemical demon5tration of coenzyme-dependent dehydrogenases and diaphorases. J. Biophys. and Blochem. eytol •• !: 309 ... 318. :hieffi. G. and Lupo, C. 1961 Identification of estradiol-I7", testosterone and its precursors from Scylliorhinus stellar15 testes Nature, -190:169-170, 60

Coons, A. H. 1958 Fluorescent antibody methods. In General Cl,tochemlcal Methods, J. F. D8niel11, Ed., Vol. I, pp. 399-422. AcademIc Press, New Yor • Coons, A. n. and Kaplan, M. H. 1950 Localization of antigen in tbsue cells. II. I~)rovements in a method for the detection of antigen by means of fluorescent antibody. J. Exptl. ~~d., 21: 1-13.

Coons, A. H., Leduc, E. H. and Connolly, J. l-S. 1953 l1111U1l0histochemical studios of antibody response in the rabbit. Federation Proc., -12: 439. Ocane, H. W. 1958 Intracellular 11pid8s: their detection and significance. In Frontiers !! ~tolO~. Ed. by S. L. Palay, pp. 221-263. Yale Univer­ sity ~ress, New f yen, onneeticut.

Deane, H. W. and Creep, R. O. 1946 A mOl'pholog.1cal and histOChemical study of the rat's adrenal cortex .fter hypophysectowy with cOlllMnts on the liver. Am. J. Anat., -79: 117-145. OOoa.ftO, H. W., Lobel, B. I.., and ROPlnOy, S. L. 1962 Enzymic histochemistry I) normal ovaries of the menstrual cy Ie, pregnancy, and the early puerperiu Am. J. Obst. and Gynec •• -a3: 281.294. Deane, il. W. and Seligman, A. M. 1953 Evaluation of procedures for the cyto- logica.l loea.lhation of ketosteroids. Vit8.llins and Hormones, !!.: 173-204. iJe'!Dj:HU}Y, E. W. 1948 "he chemical cytology of endocrine glands. Recent Pro- gress in HO!'1l101le Research, -3) 121 .. 257. Demi>sey, J~. W. and Rassett, D. L. 1943 Observations on the fluorescence, birefringence and histochemistry of the rat ovary during the reproductive cycle. Endocrinol., -33: 384-401. I)empsey, E. W. and Wislocki, G. B. 1944 Oharvation$ on sOlIe histochemical reactions in the human plae-enta, with special reference to the signifi­ cance of the lipoid!, glyeogne and iron. Endocrinol., -35: 409-429. empsey, f. W. and Wislocki, G.~. 1946 llhtochnical contributions to ph}"s!ology. f'hysiol. Rev., -26: 1-21. Oirtcherl, W. and Breuer. H. 1955 tiber steroide des lllenschUcheft SPOt'llas. Acta Endocrinol., -191 30-36. Oirscherl, w. and Knuehel, W. 19S0 Uber das Vorkouen von Androgenen in menschlichen und tierilchen Sperma. ~iochem. Ztschr., -320: 253-251. 61 nOllinguoz, O. V., Acevedo, H. F. Buseby, R. A. and Samuels, L. T. 1960 Steroid-21-hydroxylase in normal testes and malignant interstitial cell tumors. J. BioI. Chem., ~: 2608-2612. 00", L. V. 1927 New experiments on ovariotomy and the problem of sex inver­ sion in the fowl. J. Exper. Zool., !!2 31-173. Oomm, L. V. 1929a The effect of bUateral ovariotomy in the brown Leghorn fowl. Siol. !ull., -56: 459.497. Oomm, L. V. 1929b Spermatogenesis following early ovariotomy in the Brown Leghorn fow 1. Arch. f. EntwcklulSMechn. d. Organ., -119: 171-187. Oema, L. V. 1933 Response in sinistrally ovariectomized Leghorns to daily injections of hebin. Proc. Soc. Exp. BioI. 4 Med., -31: 356-357. DO.III, I .• V. 1937 Observations concerning anterior pituitary-gonadal interre­ lations in tho fowl. Cold Sprina Harbor, Symp. Quant. BioI, -S: 241-251. ~m, L. V. 1939 Modifications in sex and secondary sexual characters in birds. In Sex and Internal Sec~tions. Eds. E. Allen, C. H. Danforth, and lie A. Oohy-;-fnd ;a., pp. 22'-!21. Williams and Wilkins Co., 8altimore, Maryland.

rEman, R. I. and Shipley, R. A. 1956 Androlons. John \+IUey and S005, Inc New York.

Essenberg, J. M. and Karrasch, R. J. 1940 Experi.ental study of effects of roentgen rays on gonads of sexually mature domestic fowl. Radiology, -34: 358-365. Evans, H. M., Slaplon, M. E. and Penchar" R. I. 1937 An anterior pituitary gonadotrophic: fraction (ICBm spedfically stimulating the interstitial tissue of testis and ovary. Cold Spring Harbor, 5ym. Quant. BioI., -5: 229-240. ;verett, J. w. 1947 H01'lllOul factors responsible for deposition of choles- tOTOt in tho corpus luteUID of the rat. Endocrinology, -41: 364-377. arber, E•• Sternberg, W. H. and Dunlap, C. E. 1956 Jtistochemical localila­ tion of specific: oxidative euzylUs. t. Tetrazolium stains fo.r diphospho­ pyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphor­ ase. J. HistocheJl. and Cytochem., -4: 254-265. oldman. J. D. 1950 Histochemical reactions of adrenal cortical cells. Anat. Roe., -107: 347-358. 62

hvold, H. R.. and Elk-Nes, l.;. B. 1962 Progesterone metabolblU by testicular tissue of the English sparrow (Passer doaesticu,) during the annual repro­ ductive cycle. Gen. and Co~. @ndoerinoi., 1= 506.515. Fevold, U. R. and Elk-Nes, K. B. 1963 Progesterone metabolism by testicular tissue of the English sparrow (Passer dome.tieu,). Gen. and Comp. Endo­ crinol., !: 335-345. irkot, J. 1914 Recherches sur Itorganogenese des glandes sexua1les chez les oiseaux. Arch. BioI., Paris, -29: 201-3S1. raps, R. M., Hooker, C. W. and Forbes, T. R. 1949 Progesterone in blood plasma of cocks and nonovulating hons. Science, -109: 493. ;oldberg, B., Jones, G. E. S., and Woodruff, J. D. 1963 A histochemical study of steroid 38-01 dehydrogenase activity in some steroid-producing tuaors. Am. J. Obst. and Gynec., -86: 1003-1014. oldberg, B., Jones, G. E. S., and Turner, D. A. 1964 Steroid 3B-ol dehydro­ genase activity in human endocrine tissues. Am. J. Obst. and Gynec., (In Press) ..

30odale, H. D. 1913 Castration in relation to the secondary sexual charac­ ters of so.e domestic birds. Carnegie lnst. Wash., publ., -243: 1-52. ~mori, G. 1952 Histoche.istry of lipid carbonyl compounds. J. Lab. & Clin. Med., -39: 649 .. 659. .orbman, A. and 8ern, H. A. 1962 In A Textbook. .2! C0!2aratlvc Endocdnoloil_ .John Wiley and Sons, Inc_, New York.

lornall, A. G., Bardawill, O. J. and David, H. M. 1949 Determination of ser­ um proteins by means of the biuret reaction. J. Rio1. Chem., -177:751-706. J. C. 1930 The develop.ent, histology and endocrine functions of the compensatory richt gonad of the hen. Am. J. Anat., ~: 217-259. ;reenwood, A. W. 1925 Histological studies of gonad grafts in the fowl. Brit. Jour. Expel'. BioI., -2: 469-492. Jrcep, R. 0., Fevold, H. L. and Hbaw, F. L. 1936 Effects of two hypophyseal gonadotrophic horllOnes on the reproductive system of the male rat. Anat. Rec., -65: 261-271. ,reel', R. O. and Deane, H. w. 1949 The cytology and cytochemistry of the adrenal cortex. ~nn. N. Y. Acad. Sci., -SO: 596-615. 63

Hall, P. F., So&er, C. C. and Elk-Nes, K. B. 1964 Formation of dehydroepian drosterone during in vivo and in vitro biosynthesis of testosterone by testicular tissue.--En3ocrinol7; -'4: 35 ... 43. Hanes, P. M. 1911 Relations of interstitial cells of {,eydig to production 0 an internal secretion by 1J18l1ltftalian testis. Jour. Exp. Med., .!!: 338... 354.

Hanes, F. ~I. and Hooker, C. w. 1937 HOnlone production in the undescended testis. Proc. Soc. Exper. Rial. &Med., -35: 549.550. Hill, R. T. 1962 Paradoxical effeets of ovarian secretions. In.!.h!. Ovary. S. Zuckerlllan, Ed., pp. 231 ... 261. Academic Press, New York.

Booker, C. W. 1942 Pubertal increase in responsiveness to androgen in the male rat. Endocrinol., -30: 11-84. Hooker, C. w. 1944 The postnatal history and function of the interstial cells of the testis of the bull. Am. J. Anat., -74: 1-37. Hooker, C. W. and Pfeiffer, C. A. 1942 Testicular tumors in mice of the A strain receiving estrogens. Cancer Research, -2: 759-769. f~ggin., C. and Moulton, S. !~. 1948 Esterases of testis and other tissues. J. Exp. Med., !!: 169-119. ohnston, R. L. 1934 Studies in tbe physiology of the prostate gland. Enda­ crinol., -18: 123-141. Olt, A. 1950 Sur Ie controle boraonal de 1a diflerenelation seauel1e du lapin. Arch. Anat. Mier. Morph. Exp., ~t 517.607.

Karnovslty, M. L. and Deane, II. W. 1954 Alterations of adrenal cortical lipid.s by formalin fixation as determined chemically and histochemically. J. Hhtochem. (Abstr.), 3.: 478. 'amovsky, M. L. and Deane, H. W. 1955 Aldehyde formation in the lipide dr lets of the adrenal cortex durin. fixation, as demonstrated chemically and histochemically. J. Histoch••• , -3: 85-102. ase, N., PorehielU, E. and Oortll8n, R. I. 1961 In vitro production of tes­ tosterone and androst-4-ene-3,17.dione in a humin ovarIan homogenate. Acta. En

~idd, J. G. and Priedewald, w. p. 1942 A natural antibody that reacts in vitro with a seedi_BtaMe constituent of normal tissue cells. II. Speei­ llcliy of the phenomenon: General discussion. J. Exptl. Med., -76: 557-518. 64

Kornfeld, ,v. and Nalbandov, A. V. 1954 Endocrine influences on tho develop- ment of the rudimentary gonad of fowl. Endocrinol., -55: 151-161. Kudriashov, B. A. 1931 Tran!actions of Oynamics of Development, it 29 (In Russian) • l.andsteiner, K. 1945 In ~ Specificity 2!. Serological Reactions, 2nd cd. Harvard University Press, ciil)rlage, Mass. Lanthier, A. and Sandor, T. 1962 The in vitro biosynthesis of androgenic steroids by human nomal and "Stein:t'eventhal Type" ovarian slices. Acta. Endocrinol. (Kbh), -39: 145-153. tovy, B .. , Deane, H. W. and Rubin, 8. L. 1959 Visualization of steroid-38-ot- dehydrogenase activity in tissues of intact and hYl)Ophysectomized rats. Endocrinology. -65: 932-943. Liebermann, S., Erlanger, B. Fe, Beiser, 5. M. and Agate, p. J., Jr. 1959 5teroid-protein conjugates: their chemical. bamunochemical and endocrinol­ ogical properties. In Recen~ Proares5 .!!. Honno~~ Research, fi..: 165 .. 200. Lindner, H. R. 1961 Androgens and related compounds in the spermatic vein blood of domestic animab. t. Neutral steroids secreted by the bull tes­ tis. J. Hndocrinol, 23: 139-159.

Lipschutz, A. 1918 PrinzipieUes sur IehA von del' pubertltsdrlrse. ~.jilhelm Raux' Arch. Entwicklungsmech. Organ., -44: 207-212. tohel, B.. L., Deane, H. W. and Romney, S. L. 1962 Enzymic histochemistry of the villous portion of the human placenta from six weeks of gestation to term. Am. J. Obst. a Gyncc., -83: 295-299. Lofts, B. and Marshall, A. J. 1959 The post-nuptial occurrence of progestins in the seminiferous tubules of birds. J. Endocrinol., -19: 16-21. 'farshall, A. J. and Coombs, C. ,J. F. 1951 The interaction of environmental internal and behavioral factors in the rook (Corvus!. frulile'!s) Linnaeus. Proc. Zoot. Soc. London, -128: 54S-~19. . Iarshall, A. J. 1949 On the function of the interstitium of the testis. Quart. J. Micr. Sci., -90(1): 265-280. "cCullagh. E. P'O and Schaffenburg, C. A. 1951 Hormonal activity in semen • .1. Clin. Endocrinol., -11: 1403-14,)6. ~Kay, D. G. and Robinson, D. 1941 Observations on the fluorescence, bire­ fringence Bnd hhtochclflistry of the human ovary during thfl l:Icnstrual cycle. Endocrinol., -41: 378-394. 65

Meyer, R. 1931 Pathology of sOlie special ovarian tumors and their relation to sex characteristics. Am. J. Obstet. &Grnec., 22: 696-713. " - t

67 Rubin, B. L. and Dorfman, R. I. 1959 Quantitative assay of tbe 38-01 dehyd genase system in whole adrenal homogenate. Endocrinol., -61: 601-610. Rubin, B. L., Leipsner, G. and Deane, H. W. 1961 A rapid, sensitive assay procedure for adrenal steroid.3S-ol dehydrogenase activity. Endocrinel., -69: 619-625. Rubin, B. L., Deane, H. W., Hamilton, J. A. and Orlks, E. C. 1963 Changes in 5-3B-hydroxysterold dehydrogenase activity in the ovaries of maturing rats. Endoerinol., -72: 924-930. Ryan, K. J. and Smith, O. _. 1961 Biogenesis of estrogens by tbe human ovary. J. 8101. Chem., -236: 2207 ... 2215. Samuels, L. T., Helmreich, M. L., Lasater. M. B~ and Reich. H. 1951 An en­ zyme in endocrine tissues which oxidize. -3S-hydroxysteroids to a,B un­ saturated ketones, SCience, -113: 490-491. avard, K., Gut, M., DorfMan, R. I., Cabrilove, L. end Soffer, L. J. 1961 Formation of andre.ens by huaan anhenoble.tou tissue in vitro.J. CUn. Endoerinol. & Metab •• -2U 165.. 114. - enaffenburg, C. A. and McCUllagh, E. P. 1950 Specific androgen stimulation of the rat's !>rostatic phosphatase'll as a basis for the assay of androgenic .atorial. J. Lab. , Clint Med., -36: 983-984. ebultJ, A. 1924 Eine Methode des aikrochemischen Chole.terinnachweises am Gewebsschnitt. Zentr. alIa". Pathol. Anat., -35: 514-317. l_ar. H. and Voss, H. B. 1960 Androgene 1m ..nsehliehen ovarium. Klin. W.ehr., -38t 819-822. lapson, T. 1~ •• Wright, R. S. and Gottfried, H. 1963 Steroids in the semea of Dogfish (Squalus acanthias). J. Endoerinol., -26: 489-498. impson, T. H•• Wright, R. S. and Ihmt. S. V. 1963 Steroid. of the semen of elasmobraneh fish. J. Endocrinol., -27: 131.132. hort, R. V. 1961 Steroid eonceatrations in the follicular fluid of mares at various stages of the reproductive cycle. J. Endocrinol., -221 153-163. .hort, R. V. 1962 Steroids In the follicular fluid and the corpus luteum of the urea A "tvo-cen type" theory of ovarian steroid synthesis. J. Endocrinol •• -24: 59-63. hort, R. V. and London, D. R. 1961 Defective biosynthesis of ovarian steroids in tho Stein-Leventhal syndrome. Brit. Med. J.(No. 5241): 1724-1127. 68 Sluiter, J. W. 1945 Experimentelle untersushungen Iber die funktion des in­ terstitimas del' gonade, Z. Zellforsch •• !: 311-335. Sluiter, J. W. and van Cordt, G. J. 1941 Experimental data on the function of the interstitium of the gonads: experiments with cockerels. {~art. J. Microseop. Sci., -88: 135-150. Sluiter, J. W. and van Oordt, G. J. 1947 The influence of a gonadotropin on the seasonal chuee, in the testh and deferent duct of the Chaffinch (PriB.illa eolebs). Quart. J. Hicr. Sci., ~(l): 1-12. Steinaeh, E. 1910 Geschlechtstl'teb und eeht sekundll-e GeschleehtslIOrkmale a1s Folie del' innersekretorischen Punktion dar KalmeirG.en. Zent. Physiol., -24: 551-556. Stelnach, E. 1913 Feminieruna von Mrftnchen und Maskulierung Von Weibcnen. Zentr. Physio1., -27: 717.123. SUeve, H. 1921 Entwletluna. Bau \lad bedeutUftg del' keiJldrusenlwisehenzellen. Eraeb. Anat. u. EntwicklUA,S •••chlchte, -23: 1-249. Stieve, H. 1930 In Hanclbueh dft lIicrokopischen Anatomie des ~schen, von Molleadortt, ed. Sprlager, Berlb, Bd. 1, TeU 2. Sweat, M. L., 8erliAeT, D. L., Bryson, M. J •• Nabors, C•• Jr., Haskell. J. and Holmstrom, F.. G. 1960 The synthesis and 8Otabolisa of prog.steroao in the hwull aDd bovine ovary. 8Ioch_. alophys. Acta, -40: 289-296. Swift, C. H. 1914 Origin and early history of the primordial germ cells in the chiek. All. J. Anat., -IS: 483.516. Swift, C. R. 1915 Origin of the definitive sex-ceUs in the fe_Ie chick and their relation to tho primordial germ eells. Am. J. Ntat., -18: 441-410. 5zeDdroi, Z. and Molnar, J. 19S0 Az ..beri .parma 17-teto-steroid-tartalma. 1-1&IY. Sobo,., -3: 242 ... 244. aboI', E. 1941 The relation betwe .. ovarian growth aDd sexual characters in brown Leghorn chicks treated with gonadotropins. .J. Exp. Zool •• 107. 65-108. - aber, E. 1949 The source and effects of androlon in the Male chick treated with gonadotrophins. Am. J. Anat., -85(2): 231-262. 19S1 Androgen secretion in the fowl. 69

Taber, E., Claytor, M., Knight, J., Gambrell, D., Plowers, J. and Ayers, C. 1958 Ovarian stimulation in the immature fowl by desiccated avian pitul tarie.. Hndocrinol., -62: 84-89. Uotila, U. U. 1939 The masculinizing effect of so.e gonadotrophic hormones on pullets compared with spontaneous ovariogenic virilism in hens. Anat. Rec., -74: 165.187. van Wagenen, G. 1924 Degeneration of gereinal epithelium in the testis of the rat as a result of efferent duet ligation. Anat. Rec., -27: 189-190. van (lordt, G. J. 1963 Male gonadal hormones. In c0rlarattye Endocrinology. U. S. von P-uler and H. HeUer, Eds., Vol. I, pp. rii-2d1, AcademIc Press, New Vork.

Warren, J. C. and Salhanick, H. A. 1961 Steroid biosynthesi~ in the human ovary. J. Clin. Endocrinol. , Metab., -21: 1218.1230. Wattenberg. L. W. 1958 Microscopic histochemical demonstration of steroid- 30-01 dehydrogenase in tissue sections. J. Histoche•• and Cytochem., -6: 225-232. Williams, W. L. ana Cunningham, 8. 1940 Histologic changes in the rat testis following heat treat_nt. Vale J. Bioi. and Ned., -12: 309... 316. Wislocki, G. 8. 1949 Seasonal changes in the testes, epidIdymis and seminal vesicles of deer investigated by histochemical methods. Endocrino!., -44: 167-189. Wislocki, G. 8. and Bennett, H. S. 1943 The histology and cytology of the huaan and IIIOnkey placenta with .special reference to the trophoblast. Am. J. Anat., -73: 335.449. Wislock1, G. B. and Dempsey, E. w. 1946a Histochnical reactions in the pla- centa of the cat. Am. J. Anat., -78! 1.. 45. i'/block1, G. B. and Dempse,. E. W. 1946b Histocheaica! reactions in the pla- centa of the pig. Am. J. Anat., -78: 181.225. Itschi, E. and Pugo, N. W. 1940 Response of sex characters of the adult fe­ male starling to synthetic hormones. Proc. Roc. Exptl. BioI. and Med., -45: 10-14. Witschi. i~., Levine, W. T. and Hill, R. T. 1932 f.ndocrine reactions of X-ray sterilbed males. Proc. Soc. Exptl. Mol Med •• -29: 1024.. 1026. 70

Womach, E. B. and Koch, P. C. 1931 Biochemical studies on yield of testicu­ lar hormone from tissues and body fluids and on factors affecting comb growth response in Brown Leghorn capon. Proc. Second lnternat. Congr. Sex Researeh, (1930), pp. 329-338.

Zander # J., Forhes, T. R., von Mll'iultermann, A. M. and Neher, R. 1958 Two naturally occurring metabolites of pro~est.rone-lsolation, identification, biologic activity, and concentration in human tissues. J. Clin. Endocrin- 01., -18: 337-3S3. awadowsky, M. M. 1922 .2!!. Gesehlecht .!!!!. !!!. Entwieklung ~ Geschleehts; .erkaale. Moscow. awadowsky, M. M. 1926 Bisexual nature of the hen and experimental hermap ditism in hens. Trans. Lab. Exp. 8iol., loopark, Moscow, -2: 164-179. Table 1. Hapten 1nh1b1t10n test with ant1-T-17-BSA

Hapten %Inh1b1t10n Hapten

oSut

74 26

Testoaterone­ O-oarboxyaeth1l­ testosterone ox1me suo01nate

66 o

Oompound S Estrad10l succ1nate suo01nate

Oort1sone suco1nate 4

Estrone-o-oarbox,.eth1l­ oxime

Hldrooort1sone suoo1nate Table 2. Hapten 1nh1b1t1on test with ant1-T-3-BSA.

Hapten % Inh1b1t1on Hapten

o

58 27

Testosterone-O-carboxy Dehydro1soandrosterone meth1lox1me suoo1nate

o

45 19

Sile.O

Isoandrosterone Oompound S suoc1nate suoc1nate

OSIIC.

25 16

Testosterone suoc1nate Hydrooort1sone suco1nate

B.B. The hapten, testo8terone-(O-oarboxymeth1l) ox1me was used at one tourth the oonoentration ot the other inhibitors shown in the t1gure. TABLE 3

tUSTOLOGlCAL AND HISTOCHEMICAL OBSERVATIONS ON THE HYPIiRTtWPHIED RICHT GONAD

CORReLATED Wlni SECONDARY SEX CHARACTERS

Sex Char's. at Sacrifice Gonads Bini Age (days) Sacrifice cOJll.) Character Weight Htsto- seecIfic Fluorescence ide at (Mos.post. Appearaace of in logical Cords 11 Interstitial -No. OVRl"Cty. Oval" ctZ_ 11 Siz.(at) Pl_,e . !I- TUe Tubules CeUs Yellow Testis- S25 36 I (I.Sx2.0) Male 7S Uke • • Yellow Testis- 659 73 2 (1.3xl.5) Male Uke + Pale Red Testis- 537 33 2 ( ) Male 223 like + Red Testis- S38 43 2 (l.ad.2) Male ... like + +

Dark Red Testis- 265 30 (l.Sd.3) Male 21S Uke • • Yellow Male Testis- S22 8S (2.0x4.0) (Nt! Br) Uke • • nark Red Testis- 16 4 (3.6x6.1) Mixed 350 Uke • + Pale Red Testb- 260 24 4 (1.8x3.0) Male 350 Uke • • 74 TABLE 3 (Continued)

Sex Char's. at Sacrifice Gonads Bird Age (days) Sacrifice COiili CGracter wellfit JlIsto- ~~IlIc Piuorescenee id. at (Mos.post- Appearuee of in logical e s I InterstItIal -No. OVar'ctz: OYar'ctx·l , Size (ca) PImua· -g. Tl£! Tubules Cells Rod Male Testis... 299 29 4 (3.1x4.8) (Mtl Dr) 400 like + + Red Male Testis- 204 72 .. (3.718.0) (Ntl Br) 163 11ke + Dark Red Male Testis- 541 89 .. (S.3x8.4) Ottl Dr) 194 like + +

Red Ovo- 512 31 4.3 (6.1x9.S) Henny 200 testis + + Dark Red Testis.. 211 58 4.5 (3.8x7.0) Male 450 Uke + + Red Testis. i5S SO 5 (6.SxlO.S) FleftllY 375 like + +

Red Test is- 218 16 5 (4.8x9.3) Male 280 like + +

Pale Red Testis- 603 11 6 (3.1xl.4) Male 80 11ke ++ ++

Red Testis- 572 15 6 (4. 6x9. 0) BODIlY 385 11ke + + Red Male Testis- 567 42 6 (7.3xU.0) (Ntl Dr) 335 11ke + + 75 TABLE 3 (Coatlauei) Sex, Charts. at Sacrifice GorwIs Bird Ale (days) Sacrifice &ib iSaracter Weiifit RIsto- ~~ecIlle 'Iuorescence id. at (Mos.post- Appearanee of ift logical Co 5 l InterstitIal -No. OVar·ctz· Ovar'ctl·l & Size (ell) Pl_se ,- Tlpe Tubules Cells Dark Red Testis- 507 26 6 (6.5xl0.2) HenDY 362 I1ke ++ ++ Dark ItM Test is- 251 24 6 (5.OxI.7) Male 300 Uke + +

Pale Red Ovo- 229 31 6 {2.2x4.0} Male - testis + + Rei Male T.stis- 287 29 6 (4.6x8.5) (Ntl Br) 2aS Uke + + Dark Red Testis- 602 11 6.5 (5.9x9.6) Mal_ 200 likQ + + nark Red Testis- 596 13 7 (6.4xll.3) Male 100 like ++ ++

Dark Red Test is- 588 6 8 (6.o.U.5) Male 495 like ++ + Dark Red Testis- 600 3 9 (5.h9.8) Hcnmy 325 like + +

Dark Red Test is- 594 5 9 (4.5x6.1) Male 221 like + + Dark Red Testis- 239 26 9 (S.3x9.S) Na1e 320 like + + 76 TABLE 3 (Concluded) Sex Char's_ at Sacrifice Gonacls Bird Age (days) Sacrifice Comb Charact.... WeIght IIlsto- S;iCilIc Piuoresconce ida at (Mos.post... Appearance of in logical ~o s' fnterstltl.! -t~o. Ovar'ctr_ OVar·ctZ·l 6 Size (ca) Pluma,. .!I. Tyee Tubul.s Cells Dark Red Testis- 269 69 10 (6.6xlO.6) Male l2S like + +

Dark Red Testis. 255 43 11 (6.7x11.7) Honny 330 like + + Dark Red Male Testis- 544 16 12 (7.OxlO.2) (Mtl Br) 342 like + + Dm Red OVo- 266 29 12 (S.5x9.7) Heony 220 testis +

Dark Red Testis- 212 79 12 (6.Sx9.G) nenay 404 11k. + +

nark Red Test is- 322 35 13 (6.SxlO.O) Heny 3S0 like + +

Dark Red Testis- S68 86 14 (S.7xlO.O) Male 450 like ++

Dark Red Male Testis- 197 14 18 (9.Sx16.S) (Mtl BT) 210 like + ++ Dark Red Ovo- 304 17 23 (9.4xI6.0) lieMy 650 testis + +

+ • definite reaction ++ • stroDg reaction • no reaction (Ntl Br) • Mottled Breast - 77

P TF. r

F '1 urc 1

2.

Fi u 2 7

PLAT II

('\t Pi l' 3. al yo th henny )1 t c co. •

P ur • oulard 19

PL Te III

s. Tig t gon of u1 58 • dul- ) •. dull t hul ) n VDlnnnric [100 }

Fi u 6.

P' ur 7. c 11s in th 5

7 8

I P TE I )

Fi ur 9 . ullary tubule (Mf) in

i ur 10.

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i ur 1 ,

I:! .

. -' - - .. . urc 12 81

PLATE V

Fi ure 13.

Pi ur 14.

Pi- r 1 . Figu ~ 1..) Fi ro 14

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Pi ur 15 Figure 16 2 - ..

PI, Tn VI

17.

c nee.

i ur 1 .

Fi ur 18 .. Hur 19 .

Pi ur 20.

J APPROVAL SHEET

The dissertation submitted by James Edward Woods has been read and approved by five members of the faculty of the Graduate School of Loyola niversity. The final copies have been examined by the director of the disser­ the signature which appears below verifies the fact that necessary anges have been incorporated. and that the dissertation is now given final with reference to content, form, and mechanical accuracy. The dissertation is therefore accepted in partial fUlfillment of he requirements for the Degree of Doctor of Philosophy.