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Testosterone synthesis by the two-day-old chick testis in vitro
GENERAL
AND
COMPARATIVE
Testosterone
ENDOCRINOLOGY
Synthesis GERALD
Deparlment
7, 158-165 (1966)
by the Two-Day-Old
Chick
Testis
in V&o”
M. CONNELL, CAROLYN J. CONNELL AND KRISTEN B. EIK-NES
of Biological
Chemistry, University of Utah College of Medicine, Xalt Lake City, Utah, and Department of Zoology, Indiana University, Bloomington,Indiana? Received November
4, 1965
The effect of interstitial cell-stimulating hormone, follicle-stimulating hormone, human chorionic gonadotrophin, and pregnant mare serum gonadotrophin on the incorporation of acetate-l-Y into testosterone-“C by the Z-day-old chick testis in vitro has been investigated. All gonadotrophins tested stimulated this incorporation. Incubations at various temperatures indicate that 41’C is the optimal temperature for this conversion. The isolation and identification of testosterone-Y!, biosynthesised from acetate-l-% by the normal a-day-old chick testis in vitro, clearly shows that birds of this young age can synthesize this androgen. In addition, the presence of dehydroepiandrosterone in a nonmammalian testis has been demonstrated. INTRODUCTION
It has been known for some time that androgens secreted by the feta1 testis are important for the differentiation of the embryonic urogenital duct derivatives (Jost, 1953). One of the first demonstrations of this phenomenon was the classic description of the freemartin (Lillie, 1917). Recently direct evidence indicated that the mammalian fetal testis synet,hesieestestosterone (Acevedo ,et ‘al., 1961, 1963; Lipsett and Tullner, 1965) ; however, little work in this area has been attempted with other classes of animals. A substance(s) , extracted from embryonic chick gonads, exhibited androgenie properties by bioassay (LeRoy, 1948). No other investigations have been published concerning steroid biosynthesis by the embryonic or immature chick testis nor have there been any chemical identifications of steroids in the immature testis of this species of animal. Utilizing the incorporation of 3?P as a measure of tissue activity, the testis of the newly hatched 1 This work was in part supported by a U.S.P.H. training grant No. T4-CA-5000 and by U.S.P.H. research grants No. 06651 and 5FL,GM,21,193-02. “Contribution No. 776 of the Zoology Department of Indiana University, Bloomington, Indiana.
chick appears to be extremely sensitive to small amounts of interstitial cell-stimulating hormone (ICSH), follicle-stimulating hormone (FSH), and pregnant mare serum gonadotrophin (PMS) Breneman et al., 1962; Connell, 1965). Moreover, electron micrographs of testes from normal and ICSH treated, 2-day-old chicks offer an interesting comparison (Connell et al., 1965). The Leydig cells from testes of gonadotrophin treated birds show numerous polyribosomes, altered mitochondrial shapes, and an increase in osmiophilic bodies. The unstimulated testes, however, had a greater number of large vacuoles, somewhat suggestive of stored lipid material, than the gonadotrophin-stimulated tissue. That this altered cell structure may be associated with testosterone production has to be considered. The study reported in this paper examines the testosterone synthesizing capacity of the testis of the newly hatched domestic chick, Gallus domesticus. MATERIALS
AND
METHODS
Tissztes. The testes used in these experiments were removed from Single Comb White Leghorn Cockerels purchased from two local hatcheries. Birds were obtained on the hatching day and were 158
TESTOSTERONE
BIOSYNTHESIS
kept in the shipping cartons without food and mater for 2 days. The chicks were then sacrificed by cervical fract,ure and the testes were quickly removed, cleaned of blood and extraneous tissue, and placed in iced saline until incubated. In all rxperiments, incubations began within an hour after removal of the testes. Incubation Procedure. Incubation flasks contained 100 mg of chick testes in 2.0 ml TC-199 culture medium (Expt. 1) or the same amount of tissue in 3.0 ml Krebs-Ringer bicarbonate buffer, pH 7.4 (Expts. 2 and 3). The flasks werP oxygenated constantly with a 95% O&Y% CO? mixture in a Dubnoff metabolic incubator during the 2-hoar incubation period (Hall and Eik-Kes, 1962), Sodium a&ate-I-‘% (New England Kuclear, Specific Activity: 0.645 pc/pg) was the prccursor for steroids synthesized de ~~oz*o by the testes of these newly hatched chicks, 7.5 pc sodium n&ate-l-“C (Expt. 1) and 10 pc sodium acetat+ 1-W (Expts. 2 and 3) dissolved in methanol were added to the incubation flasks and evaporated to dryness under nitrogen prior to the addition of the incubation media. Gonadotrophin preparations were added to the flasks in 0.1 ml saline immediately before incubatian. Control flasks contained 0.1 ml saline only. At the end of the a-hour incubation. the content of each flask was quirkl> frozen. Gonadotrophin Preparations. NH-LH-88 and NH-FSH-S3 were gifts of the Endocrine Study Section of the National Institutes of Health. The pregnant mare serum gonadotrophin (Equines) and human chorionic gonadotrophin (A.P.L.) were kindly supplied by Dr. John Jewel1 of Ayerst Laboratories. Extraction Procekue. The contents of the incubation flasks were homogenized with a motor driven pestle until pieces of tissue were no longer visible. The pestle and tube were rinsed several times with saline to a volume of 8.0 ml, and this homogenat,e was then made alkaline with 2.0 ml 0.1 N sodium hydroxide. One-hundred micrograms each of testosterone (Expts. 1, 2 and 3), A*androstenedione (Expt. 1)) and dehydroepiandroeteronc (Expt. 2) were added as carrier compounds to fncsilitate steroid isolation. This mixture was cxtrLtcted with 30 ml redistilled ether three times. The combined ether extracts were washed three times with a one-tenth volume of diat,illed water, after which the extract was dried under nitrogen at 45°C. This residue was defat,ted by dissolving it in 10 ml hexane that then was extracted with 15 ml 7Och methanol three times. The combined methanolic extracts were evaporated to the aqueous phase under nitrogen at 45°C and extracted mith 20 ml redistilled ether three
i7?~ llii?“O
139
times. The combined ether extracts were c’vaporated and the residue chromatographed. Chromatography and Derivative Forn&ioll. Paper and thin-layer chromatographic systems were utilized for the separation of steroids in these extracts. In Expt. 1, a Zaffaroni formamide papet rhromatographic system of two parts hexnne and one part benzene (v/v) was used (Zaffnroni a.nd Burton, 1951). The standard steroids and incubation extracts were applied to paper strips (Wbaiman Ko. 1, 3 x 46 cm) by small capillary tubing and vacuum-distilled methanol. The paper strips were developed in R descending manner until the solvent front rrached the end of the paper. Thtr strips then tverc air-dried overnight. Location of the A’-androsl pnrdione and testosterone in the incubation exiracts was done by comparison of chromatographic mob&ties of authentic steroids chromatographed on separate paper strips. A Haines ultraviolet scanner was used to spoi the steroids on the paper strips (Ha&s, I9ti2). and in addition ‘&C peaks were recorded by a YZ strip scanner. The areas of the chromatograms of incubation extracts which corresponded in running rate to authentic testosterone and Ad-androstcn+ dionc were cut from the papers and elated with 10 ml of \racuum-tiistilled methanol. Elutcd paper sections were rc-examined by the “C scanner to insure complete elution. The eluates were dried uudpr nitrogen at. 45’C and aftcrwarde dried in a desiccator overnight. These residues were acetylated for 12 hours in the dark rvith 0.2 ml of a mixture of 1 part acetic anhydride and 5 parts pyridine (v/v) (Dominguez et nl., 1963). The in acetylated products were rechromatographed the same paper ehromatographic system. Areas on paper &rips of the acetylated material from the samples which corresponded in running rate to authentic testosterone acetate and A4”androstenedione, chromatographed on separate strips, wrrc eluted and quantitated by liquid scintillation spectrometry. Thin-layer chromatography was sub&ituted for paper chromatography in Expts. 2 and 3, The incubation extra&s were chromatographed initially on glass piates coated tvith silica gel-G in a system of cyclohexane-ethyl acetale (1: 1, v/v). The areas corresponding in RI values ta authentic testosterone, which was chromatographed on a separate lane and located on the glass plate as described above (Haines, 1952), were scraped from the glass plates, eluted, and acetylaled. These acetylated products were chromatographed on thin-layer plates in t,he solvent system of benzen<>ethyl acetate (4:1, v/v). Areas on these plate& which corresponded in running rates to authenti? testosterone acetate, s-hich was chromatographetl on a separate lane, Tvere eluted and quantitatrtl b:
I60
CONNELL,
CONNELL,
AND
EIK-NES
TESTOSTERONE
BIOSYNTHESIS
liquid scintillation spectrometry. Dehydroepiandrosterone was isolated from Expt. 2 and located on thin-layer plates by staining authentic dchydroepiandrosterone and dehydroepiandrosterone acetate, which were chromatographed on separate lanes, with a sulfuric acid/ethanol reagent (Oertel and Eik-Nes, 19’59). Tissue Fixation arzd Electron Microscopy. Teetes were removed from 2-day-old chicks and fixed, in loto, in 3% glutaraldehyde in phosphate buffer (pW: 6.8) containing a trace of CaCL and postfixed in 2% osmium tetroxide in the same phosphate buffer. The tissues were then rinsed in buffer, and dehydrated in graded ethyl alcohols to propylene oxide. All fixation and dehydration procedures were performed at 4°C. Prior to postfixation, the tissue was cut into small pieces (approximately 1 mm squares). The tissues were embedded in either Epon 812 (Luft, 1961) or maraglas (Spurlock el aE., 1963). Sections were cut on a Porter-Blum ultramicrotome and double stained with lead by using the lead citrate method of Reynolds (1963) and the uranyl acetate methtid (Watson, 1958). Sections were cut with a glass knife and examined in an RCA-EMU 3 C electron microscope at 50 kv. RESCLTS
Electron micrographs of normal and ICSH stimulated testes appear in Fig. 1. The interstitial tissue contains at least four distinct cell types: the fibroblast-like cells which were adjacent to the basement membrane of the germinal t’issue, the dark cells which often contained large vacuoles with stainable material, the light cells which also may contain &ainable vacuoles, and a somewhat rarer cell type containing deeply staining inclusions which exhibited an ordered substructure. The details of the
in
t&O
1151
cytology of these cell types will be reported in a later communication. The most obvious effect of ICSH treatment on the inter&&al cells was the increase in the polyribosome population. Data presented in Table 1 indicate that ICSH treatment stimulated the incorporation of 14C from acetate into testosterone. Treatment in viva was 100 ,ug ICSH per TABLE INcORPORATIOS
in
TESTOSTEEOXE-W
Vitro
1 0F
ACI~T~IYS-~-~~C
BY IMMATURE
ISTO
CHICK
TESTEP
dpm Treatment
Control 100 fig ICSH/chickh 100 fig ICSH/Aask:
‘*C-steroid/100
4*-Andros-
tonedione
960 1960 2520
mg testes
Teatoaterone acetate
2160 10120 2970
TA/lla 2.3 5.3 1.1
a Incubation flasks contained 100-mg testes and 7.5 PC (11.6 pg) sodium acetate-l-14C in 2.0 ml TC-199 culture medium (Difco). Incubation temperature: 39.5”C. Incubat’iou time: 2 hours. * Each chick received 100 pg NIH-LH-S8 in 0.1 ml saline 24 hours before the testes were removed. c 100 pg NIH-LH-S8 were added to the incubation flask just before the incubation began.
bird injected subcutaneously 24 hours before removal of the testes. Gonadot,rophic treatment in vih+o was 100 pg ICSH per 100 mg normal testes per flask. The &eatment in viva proved to be much more effective for stimulating the incorporation of the acetate label into testosterone than did the treatment in vitro; however, it must be noted that the ICSH response was evident
FIG. la. Electron micrograph of a Leydig cell from a control chicken. The cytoplasm coutains several subunits of the Golgi complex, mitochondria with tubular cristae, large vacuoles with stainable material, smooth endoplasmic reticulum, and predominately single ribosomea. (14,000 x 1.1) FIG. lb. Electron micrograph of a Leydig cell from a chicken treated with 10 pg ICSH (XI%-LB) 24 hours before sacrifice, The cytoplasm contains a well developed Golgi complex, centriole, mitochondria with tubular cristae, an ext#ensive smooth endoplasmic reticulum, and predominately single rihosomes. (14,000 X 1.1) FIG. le. Electron micrograph of a Leydig cell from a chicken treated with 20 ~g ICSH (NIW-IX) 24 hours before sacrifice. The cytoplasm contains extensive smooth endoplasmie reticulum, mitochondria with tubular cristae, numerous polysomes, and some single ribosomes. (14,000 x 1.1) FIG. Id. Electron micrograph of a Leydig cell from a chicken treated with 100 fig ICSH (Armour LH-ovine No. 227-80) 24 hours before sacrifice. In the cytoplasm are large vacuoles, mitochondria with altered cristae structures, myelin figures, a portion of the Golgi complex, and numerous poiysomce. (14,000 x 1.1)
162
CONNELL,
CONNELL,
with both types of treatments. In addition to the isolation of testosterone from this experiment, A4-androstenedione also was isolated. This steroid initially was chromatographed, eluted, and acetylated; after which followed purification in another system of chromatography. In both systems, the A4-androstenedione area of the sample corresponded in chromatographic mobilities to authentic A”-androstenedione chromatographed on a separate strip of paper. Recrystallization of biosynthesized A”-androstenedione was not attempted, consequently only tentative identification as A4-androstenedione can be proposed. Treatment with ICSH stimulated the incorporation of the label into this steroid also. It is interesting to note that the control testosterone/A4androstenedione ratio was 2.3, and after gonadotrophin treatment in vivo this ratio increased to 5.3.
AND
EIZ-NES
cate control flasks and flasks containing human chorionic gonadotrophin (200 IU HCG/lOO mg testes per flask) were incubated at these temperatures. In all instances appreciable incorporation of acetate-l-l% into testosterone was observed. Gonadotrophin treatment stimulated this incorporation at all three temperatures, however, the 41°C incubation appear to represent optimum temperature conditions for this conversion (Fig. 2). To confirm the identity of the isolated products as testosterone acetate, biosynthesized %J-testosterone- (acetate) samples from these experiments were pooled and recrystallized to a constant specific activity. The 14C-testosterone acetate biosynthesized in flasks not containing HCG were pooled separately from the 14Ctestosterone acetate derived from incubations where HCG was added in vitro. These data of Table 3 clearly show that the nor-
TABLE 2 THE EFFECT OF TEMPERATURE UPON THE INCORPORATION OF ACETATE-I-W INTO TESTOSTERONE-14C BY IMMATURE CHICK
Treatment
Control 200 IU HCG* Control 200 IU HCGh Control 200 IU HCGh
TESTEP
Temperature
39°C 39°C 41°C 41°C 43°C 43°C
dpm W-testosterone acetate per 100 mg testew
310 1170 140 2960 800 1950
a Incubation flasks contained 100 mg testes and 10.0 PC (15.5 fig) sodium acetate-l-14C in 3.0 ml Krebs-Ringer bicarbonate buffer (pH 7.4). Incubation temperature: indicated above. Incubation time: 2 hours. 6 200 IU HCG (0.1 ml saline) were added to the incubation flasks just before the incubation began. c dpm W-testosterone-(acetate) are the means of duplicate samples.
Although good incorporation of acetatel-l% into testosterone was observed for the first experiment in which flasks were incubated at 39.5”C, it was decided to investigate the influence of temperature upon this system (Table 2). Incubation temperatures of 39”C, 41”C, and 43°C were used. Dup!i-
TABLE
3
RECRYSTALLIZATION OF 14C-T~~~~~~~~~~~(ACETATE) ISOLATED FROM NORMAL AND HCG-STIMULATED IMMATURE CHICK TIXSTES~ Specific activity of W2testos~~pol~P&etate) Crystallization 1st
2nd 3rd
Solvent
methanol/water hexane/acetone ethanol/water
a Crystals were weighed radioactivity was determined liquid scintillation counter.
NOi-IDal
38 30 32
HCG
treated
55 62 Not done
on a Cahn Balance and in a Packard Tri Carb
ma1 2-day-old chick testis can incorporate 14Cfrom acetate into testosterone. The effect of other gonadotrophins on the chick testis in vitro was also tested (Table 4). These data indicate that FSH and PMS, in addition to ICSH and HCG, stimulate the conversion of 14C-acetate into Wtestosterone. In addition to the isolation of testosterone from Expt. 2, an attempt to isolate dehydroepiandrosterone was made. This steroid was initially chromatographed on thin-layer, eluted, and acetylated; after
TESTOSTERONE
BlOSYNTHESIS
3&c
2%
Wit?‘0
I
4bc INCUBATION
43°C
TEMPERATURE
FIG. 2. Effect of incubation temperature on the production of testosterone-“C from acetate-l-% by chicken testes. Each point on the graph is the mean from duplicate flasks incubated at 39”, 41”, anti 43°C. The range of the values is indicated in t,he figure. Control treatment was 0.1 ml saline and gonadotrophin treatment was 200 IU HCG in 0.1 ml saline added to the flasks immediately before incubation.
which followed another purification step by thin-layer chromatography. In both thin-layer chromatographic systems, the dehydroepiandrosterone and subsequent, deTABLE
4
TABLE
INCORPORATION in Vitro OF ACETATE-~-W TESTOSTERONE-K! BY IMMATURE CHICK TESTES STIMULATED WITH ICSH, FSH, AND PM@
Treatment Colltrol
200 pg ICSB 200 pg FSS 200 IU PMS
hydroandroepiandrosterone acet.ate areas of the samples corresponded in chromatographic mobilities to authentio dehydroepiandrosterone and authentic dehy-
INTO
dpm W-testosterone(acetate) per 100 mg teates~
120
1030 980 950
a Incubation flasks contained 100 mg testes and 10.1 PC (15.5 fig) sodium acetate-l-W in 3.0 ml Krebs-Ringer bicarbonate buffer (pH 7.4). Incubation temperature: 41°C. Incubatiin time: 2 hours. b dpm W-testosterone-(acetate) are the means of duplicate samples. c Gonadotrophin preparations were added to the incubation flasks in 0.1 ml saline just before the incubation began.
5
RECRYSTALLTZATION OF 14C-DEHYDROEPIANDROBTERONE-(ACETATE) ISOLATED FROM IICGSTIMULATED IMMATVEE CHICX TESTES~ Specific aotivit~ of W-dehydroe~iandrosbwone!acetate) (dpm/mgi
CrystailiZation
1st 2nd 3rd 4th
methanol/water hexane/acetone ethanol/water benzene
103 61 55 67
a Crystals were weighed on a Cahn balance and radioactivity was determined in a Packard Tri Carb Liquid scintillation counter.
droepiandrosterone acetate which were separate lanes. chromatographed on Recrystallization data (TabIe 5) of the biosynt,hesiaed 14C-dehydroepiandrosterone(acetate) confirm its identity.
164
CONNELL,
CONNELL,
DISCUSSION
A substance(s) was extracted from chick embryos in the latter half of embryological development which was androgenic when tested with the capon comb bioassay (LeRoy, 1948). The chemical nature of the isolated substance(s) was, however, not investigated. Because of the great sensitivity of the testis of the 2-day-old chick to exogenous gonadotrophins, as evidenced by weight increase (Breneman et .al., 1959), 32P incorporation (Breneman et al., 1962; Connell, 1965)) and altered cytology of the Leydig cells (Connell et al., 1965), it was important to determine whether or not testosterone synthesis actually occurred. The secretion of testosterone at this st,age of life must be small in the chicken, since no appreciable comb growth t#akesplace in the young bird. Testosterone production in this system is stimulated by ICSH treatment in viva and in vitro. The treatment in viva was much more effective than that & vitro. This difference is expected because the testes from the experiment in viva would have 24 hours to increase the concentration of enzymes necessary for steroid synthesis. Also such tissue may be higher in concentration of cofactors needed for steroid production. It was moreover noted that a significant increase in the number of polyribosomes occurred in the testicular tissue of such birds. Whether or not the altered population of polyribosomes represents a true increase in enzymatic tissue is not determined from our data. Treatment with ICSH in uiwo also increased the testosterone/A4androstenedione ratio from a control value of 2.3 to 5.3. This response is in agreement, with that observed in the bull in which the testosterone/A4-androstenedione ratio increased with the approach of sexual maturity (Linder, 1961). The influence of temperature upon biological reactions is a well established fact. Recently, the effect of various incubation temperatures upon the incorporation of I%-acetate into testosterone by rabbit testis slices was reported (Hall, 1965,). Maximal label incorporation was observed
AND
EIK-NES
with a 38°C incubation temperature, the temperature of the in situ rabbit scrotal testis. The bird testis is an abdominal testis and consequently has a higher in situ temperature-41°C (Williams, 1958). Maximal incorporation of 14C from acetate into testosterone occurred at this temperature (Table 2). The data of this experiment offer some interesting speculation as to the differences between the biosynthetic capacity of the abdominal and scrotal testis. Enzymes in the steroid biosynthetic pathways in the rabbit and in the bird testes are thought to be the same because the same steroid molecular reorientation apparently occurs. If, however, one or more of these enzymes have different temperature optima, there might indeed be subtle molecular differences between tissues located in different temperature environments. PMS and FSH both stimulate the incorporation of acetate-l-*% into l”C-testosterone (Table 4). The response to FSH may be explained in several ways. With the FSH concentration used, there were approximately 1.2 pg ICSH as a contaminant. The observed stimulation may solely be a response to the contaminating ICSH, or also this stimulation may be due to a synergistic mechanism between the FSH and ICSH present in the incubation media. This point however needs further examination. Testosterone production from A5-pregnenolone is achieved by two biosynthetic pathways: via progesterone and 17cuhydroxyprogesterone or via 17a-hydroxypregnenolone and dehydroepiandrosterone (Kahnt et #al., 1961; Eik-Nes and Kekre, 1963; DePaoli and Eik-Nes, 1963). It appears that this latter pathway may be important in the testis, but its presence has hitherto only been demonstrated in mammalian testes. The isolation and identification of 14C-dehydroepiandrosterone- (acetate) , biosynt.hesized from acetate-l-l% in our experiments, may indicate that an alternate route for testosterone production also is present in the bird testis (Table 5). The relative importance of either pathway in this avian testis, however, cannot be judged from our data.
TESTOSTERONE
BIOSYNTHESIS
One major advantage of the incubation system used in the present investigation should be pointed out. Each testis of the normal 2-day-old chick is small, usually 3-4 mg. I& dimensions vary, of course, but they are approximately 3-4 mm in length and 1 mm or less in thickness. There is, therefore, no need to slice the tissue and the testis is incubated in t’oto as a %aturaI slice.” From the results obtained, penetraCon of the t’esticular membranes by the steroid precursor and gonadotrophins appears to be no problem. Moreover, the use of such tissue should conveniently circumvent the problem of stunting biosynthetic capacity of cut tissues which undoubtedly create an artifactitious condition (Savard et cd., 1963).
1 -r CO3
V&O
mone chemistry and physioiogy studies on the biosynthesis of adrenal cortex hormones. Recen.t Progr.
Hormone
Res. 7, 255-305.
P. F,, AND EIK-NES, K. B. (1962). The action of gonadotropic hormones upon rabbit testis slices in II&o. Niochim. Biophys. Acta 63, 414422. HALL, P. F. (1965). Influence of temperature upon the biosynthesis of testosterone by rabbit testis HALL,
in vitro.
Endocrinology
76, 39fS-402.
A. (1953). Problems of fetal endocrinology: the gonadal and hypophyseal hormones. Ilecrr~,l
JOST,
Progr.
Ilormo,le
Res. 8, 379’-418.
W., NEHER, R., SCIIMID, Ii., AYD WE,STEIN, A, (1961). Bildung van 17&gdroxypregnenolon und 3&hydroxy-l7-keto-il;-androsten (DHA) in nebennieren- und testes-gewel-je. Expesientia 17, 19-21. LEROY, P. (1928). Effet androgene d’estraits embryonnnirce de poulet sur la Crete du capon. K~HNT,
Conapt.
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W. R., ZELLER, F. J., AND BEEEMAX, B. E. (1959). Gonadotrophin assay in chicks. Poultry Sci. 38, 152-158. BRENEMAN, W. R., ZELLER, F. J., AED CREEK, R. 0. (1962). Radioactive phosphorus uptake by chick testes as an end-point for gonadotropin assay. Enclocrinology 71, 790-798. CONNELL, G. M., COXXELL, C. J., AND EIK-NES, K. B. (1965). Testosterone biosynthesis by the two-day-old chick testes in vitro. Anr. Zoologist 5, 218. CONNELL, G. M. (1965). Bioassay of pregnant mare serum gonadotrophin by the chick-P3’ method. BRENEMAN,
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DEPAOLI, J., AND EIK-NES, K. B. (1963). Met.abolism in viva of 7a-3H-pregnenolone by the dog ovary. Biochim. Biophys. Acta 78, 457-465. DOMIN~U-EZ, 0. V., SEELY, J. R., AND GORSKI, J. (1963). Studies of the acetylation of steroids using l-carbon-14-acetic anhydride. Anal. Chem. 35, 1243-1247. EIK-NES, K. B., AND KEKRE, M. (1963). Metabolism in viuo of steroids by the canine testes. Biochi-m.
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G. W., AXD EIK-NES, K. B. (1959). Determination of A”-3-beta-hydroxysteroids ilnrll. Chem. .31, 98-100. REYXOLDS, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. Biol. 17, 208-232. S,~VARD, Ii., MARSH, J. M., .~ND HOWELL, l?. S. (1963). Progesterone biosynthesis in Iutral tissue : Role of nicotinamide adenine dinucleotide phosphate and NADP-linked dehydrogenases. OERT”EL,
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D. D. (1958). A hietologieal study of the effects of subnormal temperature on the testis of the fowl. Anal. Rec. 1~30, 225-241. ZAFFARONI, A,., AND BURTOX, R. B. (19511. Identification of corticosteroids of beef a,drenal e&act by paper chromatography. J. Biol. @he-m. 193, WILLIAMS,
749467.
AND
COMPARATIVE
Testosterone
ENDOCRINOLOGY
Synthesis GERALD
Deparlment
7, 158-165 (1966)
by the Two-Day-Old
Chick
Testis
in V&o”
M. CONNELL, CAROLYN J. CONNELL AND KRISTEN B. EIK-NES
of Biological
Chemistry, University of Utah College of Medicine, Xalt Lake City, Utah, and Department of Zoology, Indiana University, Bloomington,Indiana? Received November
4, 1965
The effect of interstitial cell-stimulating hormone, follicle-stimulating hormone, human chorionic gonadotrophin, and pregnant mare serum gonadotrophin on the incorporation of acetate-l-Y into testosterone-“C by the Z-day-old chick testis in vitro has been investigated. All gonadotrophins tested stimulated this incorporation. Incubations at various temperatures indicate that 41’C is the optimal temperature for this conversion. The isolation and identification of testosterone-Y!, biosynthesised from acetate-l-% by the normal a-day-old chick testis in vitro, clearly shows that birds of this young age can synthesize this androgen. In addition, the presence of dehydroepiandrosterone in a nonmammalian testis has been demonstrated. INTRODUCTION
It has been known for some time that androgens secreted by the feta1 testis are important for the differentiation of the embryonic urogenital duct derivatives (Jost, 1953). One of the first demonstrations of this phenomenon was the classic description of the freemartin (Lillie, 1917). Recently direct evidence indicated that the mammalian fetal testis synet,hesieestestosterone (Acevedo ,et ‘al., 1961, 1963; Lipsett and Tullner, 1965) ; however, little work in this area has been attempted with other classes of animals. A substance(s) , extracted from embryonic chick gonads, exhibited androgenie properties by bioassay (LeRoy, 1948). No other investigations have been published concerning steroid biosynthesis by the embryonic or immature chick testis nor have there been any chemical identifications of steroids in the immature testis of this species of animal. Utilizing the incorporation of 3?P as a measure of tissue activity, the testis of the newly hatched 1 This work was in part supported by a U.S.P.H. training grant No. T4-CA-5000 and by U.S.P.H. research grants No. 06651 and 5FL,GM,21,193-02. “Contribution No. 776 of the Zoology Department of Indiana University, Bloomington, Indiana.
chick appears to be extremely sensitive to small amounts of interstitial cell-stimulating hormone (ICSH), follicle-stimulating hormone (FSH), and pregnant mare serum gonadotrophin (PMS) Breneman et al., 1962; Connell, 1965). Moreover, electron micrographs of testes from normal and ICSH treated, 2-day-old chicks offer an interesting comparison (Connell et al., 1965). The Leydig cells from testes of gonadotrophin treated birds show numerous polyribosomes, altered mitochondrial shapes, and an increase in osmiophilic bodies. The unstimulated testes, however, had a greater number of large vacuoles, somewhat suggestive of stored lipid material, than the gonadotrophin-stimulated tissue. That this altered cell structure may be associated with testosterone production has to be considered. The study reported in this paper examines the testosterone synthesizing capacity of the testis of the newly hatched domestic chick, Gallus domesticus. MATERIALS
AND
METHODS
Tissztes. The testes used in these experiments were removed from Single Comb White Leghorn Cockerels purchased from two local hatcheries. Birds were obtained on the hatching day and were 158
TESTOSTERONE
BIOSYNTHESIS
kept in the shipping cartons without food and mater for 2 days. The chicks were then sacrificed by cervical fract,ure and the testes were quickly removed, cleaned of blood and extraneous tissue, and placed in iced saline until incubated. In all rxperiments, incubations began within an hour after removal of the testes. Incubation Procedure. Incubation flasks contained 100 mg of chick testes in 2.0 ml TC-199 culture medium (Expt. 1) or the same amount of tissue in 3.0 ml Krebs-Ringer bicarbonate buffer, pH 7.4 (Expts. 2 and 3). The flasks werP oxygenated constantly with a 95% O&Y% CO? mixture in a Dubnoff metabolic incubator during the 2-hoar incubation period (Hall and Eik-Kes, 1962), Sodium a&ate-I-‘% (New England Kuclear, Specific Activity: 0.645 pc/pg) was the prccursor for steroids synthesized de ~~oz*o by the testes of these newly hatched chicks, 7.5 pc sodium n&ate-l-“C (Expt. 1) and 10 pc sodium acetat+ 1-W (Expts. 2 and 3) dissolved in methanol were added to the incubation flasks and evaporated to dryness under nitrogen prior to the addition of the incubation media. Gonadotrophin preparations were added to the flasks in 0.1 ml saline immediately before incubatian. Control flasks contained 0.1 ml saline only. At the end of the a-hour incubation. the content of each flask was quirkl> frozen. Gonadotrophin Preparations. NH-LH-88 and NH-FSH-S3 were gifts of the Endocrine Study Section of the National Institutes of Health. The pregnant mare serum gonadotrophin (Equines) and human chorionic gonadotrophin (A.P.L.) were kindly supplied by Dr. John Jewel1 of Ayerst Laboratories. Extraction Procekue. The contents of the incubation flasks were homogenized with a motor driven pestle until pieces of tissue were no longer visible. The pestle and tube were rinsed several times with saline to a volume of 8.0 ml, and this homogenat,e was then made alkaline with 2.0 ml 0.1 N sodium hydroxide. One-hundred micrograms each of testosterone (Expts. 1, 2 and 3), A*androstenedione (Expt. 1)) and dehydroepiandroeteronc (Expt. 2) were added as carrier compounds to fncsilitate steroid isolation. This mixture was cxtrLtcted with 30 ml redistilled ether three times. The combined ether extracts were washed three times with a one-tenth volume of diat,illed water, after which the extract was dried under nitrogen at 45°C. This residue was defat,ted by dissolving it in 10 ml hexane that then was extracted with 15 ml 7Och methanol three times. The combined methanolic extracts were evaporated to the aqueous phase under nitrogen at 45°C and extracted mith 20 ml redistilled ether three
i7?~ llii?“O
139
times. The combined ether extracts were c’vaporated and the residue chromatographed. Chromatography and Derivative Forn&ioll. Paper and thin-layer chromatographic systems were utilized for the separation of steroids in these extracts. In Expt. 1, a Zaffaroni formamide papet rhromatographic system of two parts hexnne and one part benzene (v/v) was used (Zaffnroni a.nd Burton, 1951). The standard steroids and incubation extracts were applied to paper strips (Wbaiman Ko. 1, 3 x 46 cm) by small capillary tubing and vacuum-distilled methanol. The paper strips were developed in R descending manner until the solvent front rrached the end of the paper. Thtr strips then tverc air-dried overnight. Location of the A’-androsl pnrdione and testosterone in the incubation exiracts was done by comparison of chromatographic mob&ties of authentic steroids chromatographed on separate paper strips. A Haines ultraviolet scanner was used to spoi the steroids on the paper strips (Ha&s, I9ti2). and in addition ‘&C peaks were recorded by a YZ strip scanner. The areas of the chromatograms of incubation extracts which corresponded in running rate to authentic testosterone and Ad-androstcn+ dionc were cut from the papers and elated with 10 ml of \racuum-tiistilled methanol. Elutcd paper sections were rc-examined by the “C scanner to insure complete elution. The eluates were dried uudpr nitrogen at. 45’C and aftcrwarde dried in a desiccator overnight. These residues were acetylated for 12 hours in the dark rvith 0.2 ml of a mixture of 1 part acetic anhydride and 5 parts pyridine (v/v) (Dominguez et nl., 1963). The in acetylated products were rechromatographed the same paper ehromatographic system. Areas on paper &rips of the acetylated material from the samples which corresponded in running rate to authentic testosterone acetate and A4”androstenedione, chromatographed on separate strips, wrrc eluted and quantitated by liquid scintillation spectrometry. Thin-layer chromatography was sub&ituted for paper chromatography in Expts. 2 and 3, The incubation extra&s were chromatographed initially on glass piates coated tvith silica gel-G in a system of cyclohexane-ethyl acetale (1: 1, v/v). The areas corresponding in RI values ta authentic testosterone, which was chromatographed on a separate lane and located on the glass plate as described above (Haines, 1952), were scraped from the glass plates, eluted, and acetylaled. These acetylated products were chromatographed on thin-layer plates in t,he solvent system of benzen<>ethyl acetate (4:1, v/v). Areas on these plate& which corresponded in running rates to authenti? testosterone acetate, s-hich was chromatographetl on a separate lane, Tvere eluted and quantitatrtl b:
I60
CONNELL,
CONNELL,
AND
EIK-NES
TESTOSTERONE
BIOSYNTHESIS
liquid scintillation spectrometry. Dehydroepiandrosterone was isolated from Expt. 2 and located on thin-layer plates by staining authentic dchydroepiandrosterone and dehydroepiandrosterone acetate, which were chromatographed on separate lanes, with a sulfuric acid/ethanol reagent (Oertel and Eik-Nes, 19’59). Tissue Fixation arzd Electron Microscopy. Teetes were removed from 2-day-old chicks and fixed, in loto, in 3% glutaraldehyde in phosphate buffer (pW: 6.8) containing a trace of CaCL and postfixed in 2% osmium tetroxide in the same phosphate buffer. The tissues were then rinsed in buffer, and dehydrated in graded ethyl alcohols to propylene oxide. All fixation and dehydration procedures were performed at 4°C. Prior to postfixation, the tissue was cut into small pieces (approximately 1 mm squares). The tissues were embedded in either Epon 812 (Luft, 1961) or maraglas (Spurlock el aE., 1963). Sections were cut on a Porter-Blum ultramicrotome and double stained with lead by using the lead citrate method of Reynolds (1963) and the uranyl acetate methtid (Watson, 1958). Sections were cut with a glass knife and examined in an RCA-EMU 3 C electron microscope at 50 kv. RESCLTS
Electron micrographs of normal and ICSH stimulated testes appear in Fig. 1. The interstitial tissue contains at least four distinct cell types: the fibroblast-like cells which were adjacent to the basement membrane of the germinal t’issue, the dark cells which often contained large vacuoles with stainable material, the light cells which also may contain &ainable vacuoles, and a somewhat rarer cell type containing deeply staining inclusions which exhibited an ordered substructure. The details of the
in
t&O
1151
cytology of these cell types will be reported in a later communication. The most obvious effect of ICSH treatment on the inter&&al cells was the increase in the polyribosome population. Data presented in Table 1 indicate that ICSH treatment stimulated the incorporation of 14C from acetate into testosterone. Treatment in viva was 100 ,ug ICSH per TABLE INcORPORATIOS
in
TESTOSTEEOXE-W
Vitro
1 0F
ACI~T~IYS-~-~~C
BY IMMATURE
ISTO
CHICK
TESTEP
dpm Treatment
Control 100 fig ICSH/chickh 100 fig ICSH/Aask:
‘*C-steroid/100
4*-Andros-
tonedione
960 1960 2520
mg testes
Teatoaterone acetate
2160 10120 2970
TA/lla 2.3 5.3 1.1
a Incubation flasks contained 100-mg testes and 7.5 PC (11.6 pg) sodium acetate-l-14C in 2.0 ml TC-199 culture medium (Difco). Incubation temperature: 39.5”C. Incubat’iou time: 2 hours. * Each chick received 100 pg NIH-LH-S8 in 0.1 ml saline 24 hours before the testes were removed. c 100 pg NIH-LH-S8 were added to the incubation flask just before the incubation began.
bird injected subcutaneously 24 hours before removal of the testes. Gonadot,rophic treatment in vih+o was 100 pg ICSH per 100 mg normal testes per flask. The &eatment in viva proved to be much more effective for stimulating the incorporation of the acetate label into testosterone than did the treatment in vitro; however, it must be noted that the ICSH response was evident
FIG. la. Electron micrograph of a Leydig cell from a control chicken. The cytoplasm coutains several subunits of the Golgi complex, mitochondria with tubular cristae, large vacuoles with stainable material, smooth endoplasmic reticulum, and predominately single ribosomea. (14,000 x 1.1) FIG. lb. Electron micrograph of a Leydig cell from a chicken treated with 10 pg ICSH (XI%-LB) 24 hours before sacrifice, The cytoplasm contains a well developed Golgi complex, centriole, mitochondria with tubular cristae, an ext#ensive smooth endoplasmic reticulum, and predominately single rihosomes. (14,000 X 1.1) FIG. le. Electron micrograph of a Leydig cell from a chicken treated with 20 ~g ICSH (NIW-IX) 24 hours before sacrifice. The cytoplasm contains extensive smooth endoplasmie reticulum, mitochondria with tubular cristae, numerous polysomes, and some single ribosomes. (14,000 x 1.1) FIG. Id. Electron micrograph of a Leydig cell from a chicken treated with 100 fig ICSH (Armour LH-ovine No. 227-80) 24 hours before sacrifice. In the cytoplasm are large vacuoles, mitochondria with altered cristae structures, myelin figures, a portion of the Golgi complex, and numerous poiysomce. (14,000 x 1.1)
162
CONNELL,
CONNELL,
with both types of treatments. In addition to the isolation of testosterone from this experiment, A4-androstenedione also was isolated. This steroid initially was chromatographed, eluted, and acetylated; after which followed purification in another system of chromatography. In both systems, the A4-androstenedione area of the sample corresponded in chromatographic mobilities to authentic A”-androstenedione chromatographed on a separate strip of paper. Recrystallization of biosynthesized A”-androstenedione was not attempted, consequently only tentative identification as A4-androstenedione can be proposed. Treatment with ICSH stimulated the incorporation of the label into this steroid also. It is interesting to note that the control testosterone/A4androstenedione ratio was 2.3, and after gonadotrophin treatment in vivo this ratio increased to 5.3.
AND
EIZ-NES
cate control flasks and flasks containing human chorionic gonadotrophin (200 IU HCG/lOO mg testes per flask) were incubated at these temperatures. In all instances appreciable incorporation of acetate-l-l% into testosterone was observed. Gonadotrophin treatment stimulated this incorporation at all three temperatures, however, the 41°C incubation appear to represent optimum temperature conditions for this conversion (Fig. 2). To confirm the identity of the isolated products as testosterone acetate, biosynthesized %J-testosterone- (acetate) samples from these experiments were pooled and recrystallized to a constant specific activity. The 14C-testosterone acetate biosynthesized in flasks not containing HCG were pooled separately from the 14Ctestosterone acetate derived from incubations where HCG was added in vitro. These data of Table 3 clearly show that the nor-
TABLE 2 THE EFFECT OF TEMPERATURE UPON THE INCORPORATION OF ACETATE-I-W INTO TESTOSTERONE-14C BY IMMATURE CHICK
Treatment
Control 200 IU HCG* Control 200 IU HCGh Control 200 IU HCGh
TESTEP
Temperature
39°C 39°C 41°C 41°C 43°C 43°C
dpm W-testosterone acetate per 100 mg testew
310 1170 140 2960 800 1950
a Incubation flasks contained 100 mg testes and 10.0 PC (15.5 fig) sodium acetate-l-14C in 3.0 ml Krebs-Ringer bicarbonate buffer (pH 7.4). Incubation temperature: indicated above. Incubation time: 2 hours. 6 200 IU HCG (0.1 ml saline) were added to the incubation flasks just before the incubation began. c dpm W-testosterone-(acetate) are the means of duplicate samples.
Although good incorporation of acetatel-l% into testosterone was observed for the first experiment in which flasks were incubated at 39.5”C, it was decided to investigate the influence of temperature upon this system (Table 2). Incubation temperatures of 39”C, 41”C, and 43°C were used. Dup!i-
TABLE
3
RECRYSTALLIZATION OF 14C-T~~~~~~~~~~~(ACETATE) ISOLATED FROM NORMAL AND HCG-STIMULATED IMMATURE CHICK TIXSTES~ Specific activity of W2testos~~pol~P&etate) Crystallization 1st
2nd 3rd
Solvent
methanol/water hexane/acetone ethanol/water
a Crystals were weighed radioactivity was determined liquid scintillation counter.
NOi-IDal
38 30 32
HCG
treated
55 62 Not done
on a Cahn Balance and in a Packard Tri Carb
ma1 2-day-old chick testis can incorporate 14Cfrom acetate into testosterone. The effect of other gonadotrophins on the chick testis in vitro was also tested (Table 4). These data indicate that FSH and PMS, in addition to ICSH and HCG, stimulate the conversion of 14C-acetate into Wtestosterone. In addition to the isolation of testosterone from Expt. 2, an attempt to isolate dehydroepiandrosterone was made. This steroid was initially chromatographed on thin-layer, eluted, and acetylated; after
TESTOSTERONE
BlOSYNTHESIS
3&c
2%
Wit?‘0
I
4bc INCUBATION
43°C
TEMPERATURE
FIG. 2. Effect of incubation temperature on the production of testosterone-“C from acetate-l-% by chicken testes. Each point on the graph is the mean from duplicate flasks incubated at 39”, 41”, anti 43°C. The range of the values is indicated in t,he figure. Control treatment was 0.1 ml saline and gonadotrophin treatment was 200 IU HCG in 0.1 ml saline added to the flasks immediately before incubation.
which followed another purification step by thin-layer chromatography. In both thin-layer chromatographic systems, the dehydroepiandrosterone and subsequent, deTABLE
4
TABLE
INCORPORATION in Vitro OF ACETATE-~-W TESTOSTERONE-K! BY IMMATURE CHICK TESTES STIMULATED WITH ICSH, FSH, AND PM@
Treatment Colltrol
200 pg ICSB 200 pg FSS 200 IU PMS
hydroandroepiandrosterone acet.ate areas of the samples corresponded in chromatographic mobilities to authentio dehydroepiandrosterone and authentic dehy-
INTO
dpm W-testosterone(acetate) per 100 mg teates~
120
1030 980 950
a Incubation flasks contained 100 mg testes and 10.1 PC (15.5 fig) sodium acetate-l-W in 3.0 ml Krebs-Ringer bicarbonate buffer (pH 7.4). Incubation temperature: 41°C. Incubatiin time: 2 hours. b dpm W-testosterone-(acetate) are the means of duplicate samples. c Gonadotrophin preparations were added to the incubation flasks in 0.1 ml saline just before the incubation began.
5
RECRYSTALLTZATION OF 14C-DEHYDROEPIANDROBTERONE-(ACETATE) ISOLATED FROM IICGSTIMULATED IMMATVEE CHICX TESTES~ Specific aotivit~ of W-dehydroe~iandrosbwone!acetate) (dpm/mgi
CrystailiZation
1st 2nd 3rd 4th
methanol/water hexane/acetone ethanol/water benzene
103 61 55 67
a Crystals were weighed on a Cahn balance and radioactivity was determined in a Packard Tri Carb Liquid scintillation counter.
droepiandrosterone acetate which were separate lanes. chromatographed on Recrystallization data (TabIe 5) of the biosynt,hesiaed 14C-dehydroepiandrosterone(acetate) confirm its identity.
164
CONNELL,
CONNELL,
DISCUSSION
A substance(s) was extracted from chick embryos in the latter half of embryological development which was androgenic when tested with the capon comb bioassay (LeRoy, 1948). The chemical nature of the isolated substance(s) was, however, not investigated. Because of the great sensitivity of the testis of the 2-day-old chick to exogenous gonadotrophins, as evidenced by weight increase (Breneman et .al., 1959), 32P incorporation (Breneman et al., 1962; Connell, 1965)) and altered cytology of the Leydig cells (Connell et al., 1965), it was important to determine whether or not testosterone synthesis actually occurred. The secretion of testosterone at this st,age of life must be small in the chicken, since no appreciable comb growth t#akesplace in the young bird. Testosterone production in this system is stimulated by ICSH treatment in viva and in vitro. The treatment in viva was much more effective than that & vitro. This difference is expected because the testes from the experiment in viva would have 24 hours to increase the concentration of enzymes necessary for steroid synthesis. Also such tissue may be higher in concentration of cofactors needed for steroid production. It was moreover noted that a significant increase in the number of polyribosomes occurred in the testicular tissue of such birds. Whether or not the altered population of polyribosomes represents a true increase in enzymatic tissue is not determined from our data. Treatment with ICSH in uiwo also increased the testosterone/A4androstenedione ratio from a control value of 2.3 to 5.3. This response is in agreement, with that observed in the bull in which the testosterone/A4-androstenedione ratio increased with the approach of sexual maturity (Linder, 1961). The influence of temperature upon biological reactions is a well established fact. Recently, the effect of various incubation temperatures upon the incorporation of I%-acetate into testosterone by rabbit testis slices was reported (Hall, 1965,). Maximal label incorporation was observed
AND
EIK-NES
with a 38°C incubation temperature, the temperature of the in situ rabbit scrotal testis. The bird testis is an abdominal testis and consequently has a higher in situ temperature-41°C (Williams, 1958). Maximal incorporation of 14C from acetate into testosterone occurred at this temperature (Table 2). The data of this experiment offer some interesting speculation as to the differences between the biosynthetic capacity of the abdominal and scrotal testis. Enzymes in the steroid biosynthetic pathways in the rabbit and in the bird testes are thought to be the same because the same steroid molecular reorientation apparently occurs. If, however, one or more of these enzymes have different temperature optima, there might indeed be subtle molecular differences between tissues located in different temperature environments. PMS and FSH both stimulate the incorporation of acetate-l-*% into l”C-testosterone (Table 4). The response to FSH may be explained in several ways. With the FSH concentration used, there were approximately 1.2 pg ICSH as a contaminant. The observed stimulation may solely be a response to the contaminating ICSH, or also this stimulation may be due to a synergistic mechanism between the FSH and ICSH present in the incubation media. This point however needs further examination. Testosterone production from A5-pregnenolone is achieved by two biosynthetic pathways: via progesterone and 17cuhydroxyprogesterone or via 17a-hydroxypregnenolone and dehydroepiandrosterone (Kahnt et #al., 1961; Eik-Nes and Kekre, 1963; DePaoli and Eik-Nes, 1963). It appears that this latter pathway may be important in the testis, but its presence has hitherto only been demonstrated in mammalian testes. The isolation and identification of 14C-dehydroepiandrosterone- (acetate) , biosynt.hesized from acetate-l-l% in our experiments, may indicate that an alternate route for testosterone production also is present in the bird testis (Table 5). The relative importance of either pathway in this avian testis, however, cannot be judged from our data.
TESTOSTERONE
BIOSYNTHESIS
One major advantage of the incubation system used in the present investigation should be pointed out. Each testis of the normal 2-day-old chick is small, usually 3-4 mg. I& dimensions vary, of course, but they are approximately 3-4 mm in length and 1 mm or less in thickness. There is, therefore, no need to slice the tissue and the testis is incubated in t’oto as a %aturaI slice.” From the results obtained, penetraCon of the t’esticular membranes by the steroid precursor and gonadotrophins appears to be no problem. Moreover, the use of such tissue should conveniently circumvent the problem of stunting biosynthetic capacity of cut tissues which undoubtedly create an artifactitious condition (Savard et cd., 1963).
1 -r CO3
V&O
mone chemistry and physioiogy studies on the biosynthesis of adrenal cortex hormones. Recen.t Progr.
Hormone
Res. 7, 255-305.
P. F,, AND EIK-NES, K. B. (1962). The action of gonadotropic hormones upon rabbit testis slices in II&o. Niochim. Biophys. Acta 63, 414422. HALL, P. F. (1965). Influence of temperature upon the biosynthesis of testosterone by rabbit testis HALL,
in vitro.
Endocrinology
76, 39fS-402.
A. (1953). Problems of fetal endocrinology: the gonadal and hypophyseal hormones. Ilecrr~,l
JOST,
Progr.
Ilormo,le
Res. 8, 379’-418.
W., NEHER, R., SCIIMID, Ii., AYD WE,STEIN, A, (1961). Bildung van 17&gdroxypregnenolon und 3&hydroxy-l7-keto-il;-androsten (DHA) in nebennieren- und testes-gewel-je. Expesientia 17, 19-21. LEROY, P. (1928). Effet androgene d’estraits embryonnnirce de poulet sur la Crete du capon. K~HNT,
Conapt.
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