The Effect of Trophic Agents on Fetal Ovarian Steroidogenesis in Organ Culture*

Vol.

FERTILITY AND STERILITY Copyright © 1979 The American Fertility Society

32, No.1, July 1979 Printed in U.8A.

THE EFFECT OF TROPHIC AGENTS ON FETAL OVARIAN STEROIDOGENESIS IN ORGAN CULTURE*

EMERY A. WILSON, M.D.t M. JOE JAWAD, M.S.

Department of Obstetrics and Gynecology, University of Kentucky College of Medicine, Lexington, Kentucky 40536

Sexual dimorphism in fetal gonadal steroid secretions has been well documented. Whereas the human fetal testis is steroidogenically active in utero, the fetal ovary is relatively quiescent. The ovaries of three fetuses (12, 20, and 22 weeks' gestational age) were cultured as controls or in the presence of follicle-stimulating hormone/luteinizing hormone (FSH/LH), human chorionic gonadotropin (HCG), or N6,02-dibutyryl cyclic adenosine 3':5'-monophosphate (Bu 2cAMP) in order to evaluate spontaneous and stimulated production of progesterone (P), dehydroepiandrosterone (DHEA), androstenedione (A), testosterone (T), estrone (E l ), and estradiol (E 2). A 2to 7-fold increase in progesterone was observed after 96 hours of incubation, and the addition of Bu 2cAMP caused a further significant increase in progesterone production. Although FSH/LH produced a small increase in progesterone, no effect was observed in the presence of HCG. Significant spontaneous production was also observed for DHEA, A, E l , and E2 after 96 hours. The response of these steroids to trophic agents was similar to that of progesterone. Testosterone concentrations were nondetectable in the media of two of the three ovaries cultured, and no stimulation by trophic hormones was demonstrated. Under these in vitro conditions, the results suggest that: (1) the fetal ovary is steroidogenically active in utero, and (2) a deficiency of LH-like receptors and an intrinsic enzyme deficiency may compromise estrogen steroidogenesis and prohibit testosterone production in the fetal ovary prior to 22 weeks. Fertil Steril32:73, 1979

ovary was capable of aromatizing testosterone and androstenedione to estradiol and estrone in vitro. Despite these findings as well as extensive germ cell proliferation 7 and the presence of high serum concentrations of fetal pituitary and placental gonadotropins, S the fetal ovary remains steroidogenically quiescent. Although human chorionic gonadotropin is known to stimulate testosterone by the fetal testis,2.3 the mechanisms which control steroidogenesis by the fetal ovary are unknown. The purpose of this study is to determine whether the fetal ovary secretes steroids spontaneously and to examine the effect of trophic hormones on fetal ovarian steroidogenesis in vitro.

Sexual dimorphism in fetal gonadal steroid secretion has been well documented. Whereas the human fetal testis is steroidogenically active in utero,I-3 the fetal ovary is relatively inactive. Jungmann and Schweppe4 and Bloch l were unable to demonstrate conversion of progesterone to Cl9 and CIS steroids in the fetal ovary. More recently, Payne and Jaffe,S using pregnenolone sulfate as a precursor, identified 17-hydroxypregnenolone, dehydroepiandrosterone, and androstenedione in incubations of fetal ovarian tissue but no progesterone, testosterone, estradiol, or estrone was identified. George and Wilson6 found that the fetal Received December 27,1978; revised January 30,1979; accepted February 14, 1979. *Supported in part by National Institutes of Health Grant RRO-5374. tTo whom reprint requests should be addressed.

MATERIALS AND METHODS

Methods. Three female fetuses were obtained

73

74

WILSON AND JAWAD

from patients subjected to hysterotomy at 12, 20, and 22 weeks' gestation for termination of pregnancy because of medical indications. The crownrump length of each embryo correlated with the length of gestation, and the genetic sex was confirmed by lymphocytic karyotype. The ovaries from each embryo were removed and placed immediately into McCoy's 5a medium for transport. Portions of each ovary were fixed for histologic examination and the remaining tissue was minced into 1- to 2-mm pieces. The minced ovary was then washed three times in McCoy's 5a medium, and four pieces were randomly selected and placed in Falcon culture dishes. All cultures were maintained for 96 hours in a 95% air-5% CO 2 incubator at 97° C. Culture Medium. Each set oftissue was cultured separately in 2 ml of McCoy's 5a medium (modified; Grand Island Biological Co., Grand Island, N. Y.) containing L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin sulfate (100 ~/ml). The final culture medium contained less than 10 pg/ml of progesterone (P), androstenedione (A), dehydroepiandrosterone (DHEA), testosterone (T), estrone (E l ), and estradiol (E 2). Each set of ovarian tissue cultures was incubated in quadruplicate either as controls or with one of the following agents: 1.5 IV/ml of follicle stimulating hormone and luteinizing hormone (FSH/LH; Pergonal, Serono Laboratories, Braintree, Mass.) 5.0 IV of human chorionic gonadotropin (HCG; Pregnyl, Organon Pharmaceuticals, West Orange, N. J.), or 1.0 mM N6,02-dibutyryl cyclic adenosine 3 I: 5 ,_ monophosphate (Bu2cAMP; Sigma Chemical Co., St. Louis, Mo.). The ovaries obtained at 12 weeks' gestation were cultured only as controls and in the presence of FSH/LH or BU2cAMP because of the small amount of tissue available. Media from the culture dishes containingfetal ovarian tissue at 12 weeks' gestation were collected at 48 hours; the media were replaced and again collected at 96 hours. Media from the dishes containing ovarian tissue of 20 weeks' gestation were treated in the same fashion except that 0.3 ml -of media was obtained at 24 and 72 hours for measurement of progesterone concentrations. The total media from culture dishes containing fetal ovarian tissue of22 weeks gestation were replaced at 24, 48, and 72 hours. All collected media were stored at -20° C until analyzed. Following the incubation period, the wet tissue weights were recorded. Chromatography and Radioimmunoassay. A combination of chromatographic separation9 and

July 1979

specific radioimmunoassay was used to determine steroid identification and the amount produced. Briefly, steroids were extracted from the culture media with 10 volumes of ethyl ether, evaporated to dryness, and applied to Celite-ethylene glycol columns in 0.2 ml, 0.2 ml, and 0.3 ml of iso-octane. Fraction I, containing progesterone and androstenedione, was eluted with 4.0 ml of iso-octane. Fraction II, containing dehydroepiandrosterone and estrone, was eluted with 4.0 ml of 15% ethyl acetate in iso-octane. Fraction III, containing estradiol, was eluted with 3.5 ml of 40% ethyl acetate in iso-octane. Recoveries following extraction and chromatography varied from 64% to 86%. Following evaporation to dryness, the above steroids and testosterone, which was measured directly, were then measured by radioimmunoassay as previously described. 1O- l5 The specificity for each antibody employed in these assays is presented in Table 1. The results were expressed as the accumulation of steroid in picograms per milligram of tissue. The intra-assay coefficient of variation was less than 6% and the interassay coefficient of variation was less than 10% for all steroids measured. The unpaired t-test was used for statistical analysis. RESULTS

Histology. The fetal ovaries 12 weeks of age were histologically undifferentiated, containing a mass oflarge primitive germ cells and loosely connected stromal cells. The ovaries of 20 and 22 weeks' gestational age were microscopically similar. Numerous undifferentiated germ cells and primordial follicles, germ cells surrounded by single layer of granulosa cells, were present in the ovarian cotex. Progesterone Production. The accumulated production of progesterone, dehydroepiandrosterone, androstenedione, testosterone, estrone, and estradiol for spontaneous and stimulated cultures of each ovary is presented in Tables 2, 3, and 4. Progesterone accumulated spontaneously in the media of each of the three tissues studied. When concentrations at 96 hours were compared with initial concentrations measured, a 2-fold increase in progesterone was detected in the 12-week ovary cultures (P < 0.02), a 7-fold increase in progesterone was detected in the 20-week ovary cultures (P < 0.001), and a 3-fold increase was found in cultures containing 22-week ovarian tissue (P < 0.01), The addition of BU2cAMP caused a significant increase in progesterone production by each set of ovaries at all time periods measured

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...

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EFFECT OF TROPIDC AGENTS ON FETAL OVARIAN STEROIDOGENESIS

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75

TABLE 1. Specificity of Antibodies Employed p

A

DHEA

T

E,

E,

% cross-reactivity

Cholesterol Pregnenolone 17-Hydroxypregnenolone Progesterone 17-Hydroxyprogesterone 20 ~ Hydroxypregn-4-ene-3-one Deoxycorticosterone Corticosterone Aldosterone Cortisol

<0.01 <0.1 100.0 1.0 7.0 35.0 2.0 <0.1 0.4

Androstenedione Dehydroepiandrosterone Testosterone 5~ Dihydrotestosterone

5.0 5.0 0.5

<0.02 <0.02 <0.02 <0.02 <0.02

<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

<0.01 <0.001

<0.1

<0.001 <0.001

<0.1

<0.02 <0.02 <0.02 <0.02

<0.01 0.02 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

<0.01 <0.01

<0.1

100.0 16.0 0.47 0.03

12.5 100.0 <0.01 <0.01

3.5 1.0 100.0 100.0

<0.001 <0.001 <0.001

<0.1 <0.1 <0.1

66.0 100.0 60.0

100.0 3.0 3.0

14

15

Estradiol Estrone Estradiol

<0.1 <0.1 <0.1

<0.02 0.1

<0.01 <0.01 <0.01

<0.01 <0.01

Reference

10

11

12

13

when compared with progesterone concentrations in control cultures (Fig. 1). In two of three ovarian cultures, FSH/LH caused a significant increase in progesterone accumulation at 96 hours. No apparent increase in progesterone production was observed by ovarian cultures in the presence ofHCG. Androgen Production. Dehydroepiandrosterone was the principal androgen produced under these conditions. When compared with initial concen-

trations, DHEA accumulation after 96 hours was significantly greater in the cultures of the 12-week fetal ovary (P < 0.001) and in the 22-week fetal ovary (P < 0.01). The fetal ovary of 20 weeks' gestation produced initial concentrations (2234.4 ± 335.2 pg/mg, mean ± SEM) which were much greater than the baseline values of the other two ovarian cultures. Although a slight increase in DHEA production by this ovary was detected after

TABLE 2. Steroid Production by a Twelve-Week Fetal Ovary in Organ Culture a Bu,cAMP

Hours

Control

Progesterone

48 96

7.2 ± 1.3 14.7± 1.7c

8.7 ± 1.4 29.2 ± 3.9 b

1180.7 ± 285.0 b 2163.5 ± 590.8 d

Dehydroepiandrosterone

48 96

51.2 ± 6.2 105.4 ± 6.6'

77.6 ± 8.7 169.4 ± 15.4 d

222.2 ± 27.8 e 952.7 ± 110.2"

Androstenedione

48 96

21.9 ± 2.0 42.9 ± 8.2g

39.8 ± 9.9 81.1 ± 17.3

42.2 ± 4.4" 174.7 ± 6.W

Testosterone

48 96

<10.0 <10.0

<10.0 <10.0

<10.0 <10.0

Estrone

48 96

30.9 ± 4.3 68.7 ± 10.2"

51.5 ± 6.1h 116.1 ± 14.1 h

42.4 ± 3.4 189.0± 22.1d

Estradiol

48 96

10.9 ± 2.9 23.1 ± 3.9g

16.3 ± 2.1 34.5 ± 4.6

29.8 ± 4.9 h 85.9 ± 9.9'

Steroid

FSH/LH

pg/mg tissue

"Values are expressed as means ± standard error of the mean of quadruplicate determinations. bSignificantly different from corresponding control value (P < 0.02). CSignificantly greater than the 48-hour value (P < 0.02). dSignificantly different from corresponding control value (P < 0.01). "Significantly different from corresponding control value (P < 0.001). lSignificantly greater than the 48-hour value (P < 0.001). gSignificantly greater than the 48-hour value (P < 0.05). hSignificantly different from corresponding control value (P < 0.05).

WILSON AND JA WAD

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July 1979

TABLE 3. Steroid Production of a Twenty-Week Fetal Ovary in Organ Culture a Steroid

Hours

Control

Bu,aAMP

RCG

FSHlLR pg/mg tissue

101.0b 32.5 36.4 127.5b

150.4 ± 451.7 ± 628.1 ± 878.8 ±

15.5 49.1 69.0 98.0

540.0 ± 1972.4 ± 3449.4 ± 4799.7 ±

46.9 c 173.6 c 307.0< 532.5<

Progesterone

24 48 72 96

152.2 ± 605.1 ± 828.1 ± 1035.3 ±

Dehydroepiandrosterone

48 96

2234.4 ± 335.2 2677.2 ± 382.8

2879.4 ± 782.6 3652.6 ± 945.0

2229.5 ± 268.8 2613.9 ± 259.8

6141.8 ± 2830.1 8330.7 ± 2273.7 e

Androstenedione

48 96

712.8 ± 97.6 757.0 ± 95.5

608.8 ± 63.2 681.3± 81.0

657.0 ± 185.3 709.0 ± 189.5

2129.8 ± 713.0 2849.0 ± 787.0 e

Testosterone

48 96

<10.0 <10.0

<10.0 <10.0

<10.0 <10.0

<10.0 <10.0

Estrone

48 96

263.9 ± 82.4 294.6 ± 84.3

250.7 ± 64.0 287.5 ± 61.2

238.4 ± 63.0 248.0 ± 62.1

896.9 ± 257.9 1262.1 ± 534.7

Estradiol

48 96

461.1 ± 154.9 581.1 ± 130.4

546.8 ± 78.8 748.1 ± 121.1

351.0 ± 50.3 431.3 ± 68.9

873.9 ± 164.8 1470.1 ± 451.2

9.0 48.0 d 63.5 d 62.6 d

549.9 ± 702.2 ± 959.9 ± 1569.8 ±

·Values are expressed as means ± standard error of the mean of quadruplicate determinations. lJ8ignificantly greater than corresponding control value r.p < 0.01). CSignificantly greater than corresponding control value (P < 0.001). dSignificantly greater than the 24-hour value (P < 0.001). eSignificantly greater than corresponding control value (P < 0.05).

96 hours, the difference from baseline values was not significant. Significant increases in DHEA production after 96 hours (4- to 9-fold) were found in the presence of BU2CAMP by all ovarian tissues. DHEA production was also stimulated by FSH/LH after 96 hours in the 12-week ovary. No significant increase in DHEA production was detected in the presence of HCG. Androstenedione production was much less than that ofDHEA. A significant spontaneous increase in A production was observed after 96 hours only by the 12-week fetal ovary (P < 0.05). In response to trophic agents, BU2CAMP caused a significant increase in A production by the 12-week ovary (P < 0.001) and 20-week ovary (P < 0.05), but no stimulatory effect was observed by tissues in the presence of FSH/LH or HCG. Testosterone concentrations were undetectable «10 pg/mg) in both spontaneous and stimulated cultures of the 12-week and 20-week fetal ovaries. A small but nonsignificant increase in testosterone production was detected in cultures of the 22-week fetal ovary. No apparent stimulation by trophic hormones was observed. Estrogen Production. Estrone production increased spontaneously in all three sets of ovarian cultures. Significant increases were observed in cultures of the 12-week ovary (P < 0.02) and the 22-week ovary (P < 0.05). Although BU2CAMP caused a marked stimulation in estrone produc-

~,

tion by all three ovaries, a significant difference from control concentrations was found only in cultures of the 12-week fetal ovary (P < 0.01). In this ovary, a significant increase in estrone production was also stimulated by FSH/LH (P < 0.05). The pattern of estradiol production was similar to that of estrone. Although estradiol production increased spontaneously in cultures of each ovary, only the production by the 12-week fetal ovary and the 22-week fetal ovary were significantly greater than initial concentrations (P < 0.05 andP < 0.01, respectively). Although BU2CAMP stimulated estradiol production in the cultures of each set of ovarian tissue studied, only that production by the 12-week fetal ovary was significantly greater than the control values (P < 0.001). DISCUSSION

The fetal testes of the rabbit,16, 17 monkey, 18 and human l - 3 , 8 are capable of actively synthesizing testosterone from labeled precursors and secreting testosterone into the circulation. Testosterone secretion by the human fetal testis is stimulated by human chorionic gonadotropin,2, 3 which probably employs cAMP as a second messenger. 3 In contrast, the fetal ovary is thought to be relatively inactive. Bloch 1 incubated fetal ovarian tissue with 4-14C-progesterone and was unable to demonstrate conversion to androgens or estrogens.

,... EFFECT OF TROPIDC AGENTS ON FETAL OVARIAN STEROIDOGENESIS

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77

TABLE 4. Steroid Production of a Twenty-Two Week Fetal Ovary in Organ Culture a Steroid

Hours

Control

FSHJLR

-----

RCG

BuzcAMP

pg/mg tissue

Progesterone

24 48 72 96

36.8 ± 62.6 ± 99.9 ± 102.9 ±

2.7 6.4 c 16.4c 15.9 c

49.4 55.1 60.4 68.9

± ± ± ±

14.1 13.5 13.9 15.5

56.9 ± 63.9 ± 71.6 ± 84.3 ±

21.9 21.4 21.8 20.3

177.2 ± 502.5 ± 1124.9 ± 1520.6 ±

39.2b 167.0 d 221.P 264.7 b

Dehydroepiandrosterone

24 48 72 96

100.6 ± 131.3 ± 169.6 ± 177.6 ±

14.6 17.6 18.3 c 21.6 c

91.9 ± 127.8 ± 191.9 ± 271.4 ±

35.3 35.2 32.1 23.7

114.3 ± 172.7 ± 194.4 ± 209.2 ±

29.2 44.4 46.2 45.0

654.7 ± 911.2 ± 1287.3 ± 1576.9 ±

169.4e 205.1 e 288.g e 393.g e

Androstenedione

24 48 72 96

16.9 ± 21.1 ± 23.2 ± 26.2 ±

6.2 9.8 10.8 12.3

6.1 ± 5.2 ± 5.4 ± 4.0 ±

4.8 3.5 4.0 4.1

14.0 ± 14.9 ± 15.1 ± 16.6 ±

5.3 5.6 6.1 6.5

31.8 ± 48.4 ± 50.7± 45.5 ±

20.0 21.5 27.1 25.9

Testosterone

24 48 72 96

8.2 ± 14.3 ± 18.4 ± 21.9 ±

2.9 3.8 3.1 3.7

15.6 ± 19.8 ± 21.0 ± 34.9 ±

2.9 2.8 3.2 6.1

10.0 ± 15.1 ± 19.4 ± 23.8 ±

3.4 2.6 3.4 3.9

7.7 ± 12.6 ± 19.4 ± 24.5 ±

2.0 3.2 5.1 4.4

Estrone

24 48 72 96

59.9 ± 81.8 ± 92.8 ± 99.0 ±

10.6 10.9 10.8 1O.5f

25.9 ± 36.8 ± 51.5 ± 60.4 ±

4.7 4.8 8.3 8.5

36.4 ± 70.4 ± 78.0 ± 86.4 ±

6.0 5.6 5.4 6.4

125.1 ± 167.5 ± 191.9 ± 201.4 ±

37.5 53.6 63.8 66.9

Estradiol

24 48 72 96

10.6 ± 17.7 ± 21.6 ± 25.8 ±

1.4 3.1 3.2" 3.0 c

9.0 ± 1.8 13.3 ± 2.8 17.7±3.0 20.0 ± 3.6

11.1 ± 18.1 ± 21.6 ± 29.8 ±

2.8 5.2 5.6 2.6

24.0 ± 40.5 ± 51.1 ± 58.4 ±

9.7 10.1 19.0 20.9

aValues are expressed as means ± standard error of the mean of quadruplicate determinations. bSignificantly greater than corresponding control value (P < 0.01). CSignificantly greater than the 24-hour value (P < 0.01). dSignificantly greater than corresponding control value (P < 0.05). eSignificantly greater than corresponding control value (P < 0.02). iSignificantly greater than 24-hour value (P < 0.05). "Significantly greater than 24-hour value !P < 0.02).

Jungmann and Schweppe 4 incubated homogenates of fetal ovaries with 1- 14e-sodium acetate and identified small but definite quantities of pregnenolone and progesterone but no androgens or estrogens. The purpose of the present study was to determine whether human fetal ovaries were capable of spontaneous steroidogenesis and to determine the effects of trophic hormones in vitro. A combination of chromatographic separation of steroids followed by specific radioimmunoassay was used for identification and quantitation of different steroids in view ofthe fact that each of the steroids had been previously isolated by a variety ofpurification techniques. Under these culture conditions, progesterone concentrations increased spontaneously in control cultures. Although spontaneous progesterone production by the fetal ovary has not been previously reported, it is consistent with the findings of Jungmann and Schweppe4 and of Schindler and

L

6

i;j

5

cAMP

:I I/) I/)

i= at

4

E

....at

.5 w

3

z

0

II: W l-

2

ta C)

FSH/LH

0

~~CONTROL

II: 0..

~_ 0

24

48

72

HCG

96

HOURS

FIG. 1. Progesterone production by a 20-week fetal ovary in organ culture: spontaneous (control) and the presence of FSH/LH, HCG, and BU2cAMP. Eachpoint represents the mean ± standard error ofthe mean of quadruplicate determinations.

.,..

78

WILSON AND JAWAD

Friedrich,19 who used labeled precursors. Payne and Jaffe,' however, were unable to recover progesterone from labeled pregnenolone sulfate, which may reflect a propensity for this precursor to follow the fl.' pathway. The effect of trophic hormones on fetal ovarian steroidogenesis has not been previously reported. Consistent and significant stimulation of progesterone production was observed in the presence of BU2CAMP. Although FSH/LH stimulated progesterone production in the culture media of two ovaries, HCG had no effect. Because the response to BU2CAMP was much greater than that of gonadotropins, these results suggest a deficiency of gonadotropin receptors in the fetal ovary, which is consistent with the lack of LH-HCG receptors in ovaries of the rabbit embryo reported by Catt et al.2° Furthermore, because LH and HCG presumably act at the same receptor site, the progesterone response to FSH/LH suggests that the FSH component is responsible for the increase in progesterone production in these tissues. Although Roberts and Warren 21 recovered androstenedione and testosterone from labeled progesterone and testosterone, and 17 ,B-estradiol from labeled androstenedione by the bovine fetal ovary, similar results have only recently been obtained with the human fetal ovary. Payne and Jaffe,' using 3H-pregnenolone sulfate as a precursor, identified dehydroepiandrosterone and androstenedione from fetal ovary incubations but were unable to demonstrate conversion to progesterone, testosterone, estrone, or estradiol. Schindler and Friedrich 19 incubated the ovaries of human fetuses with 14- 14C-pregnenolone and found progesterone to be the main metabolite but that the fl.'-steroid pathway to 17 a-hydroxypregnenolone and dehydroepiandrosterone was also operable. In these experiments, dehydroepiandrosterone was the principal androgen produced, and spontaneous accumulation was observed in the media of two of the three ovaries cultured; the third ovary produced large amounts of dehydroepiandrosterone initially. The significant increase in production was stimulated by Bu2cAMP in the media from all three ovaries. Similar results were observed for androstenedione, except for the 22-week ovary. These results are consistent with the data of Payne and Jaffe,' who found that the fetal ovary showed approximately the same capacity for conversion of progestins to C 19 steroids as did the fetal testes but that this C 17 to C 20 lyase and 3,B-ol-dehydrogenase activity decreased with the age of the fetus.

July 1979

The obvious difference between the steroidogenic capacity of the fetal ovary and that reported by the human fetal testis 1.3 is the relative lack of testosterone production. Despite the production of dehydroepiandrosterone and androstenedione precursors, testosterone was nondetectable in two of the three ovaries studied even in the presence of BU2CAMP. Although small amounts oftestosterone were detected in the media from the 22week ovary, BU2CAMP failed to stimulate production. A similar lack of testosterone production by the human fetal ovary!' 4, ,,20 and other animals17. 18 has been reported. These results indicate that the fetal ovary is deficient in 17-oxidoreductase activity, as suggested by Bloch, 1 in addition to the 3,B-ol-dehydrogenase activity suggested by Payne and Jaffe. 5 On the basis of evidence of estrogen formation by the ovary of the rabbit embryo,22 George and Wilson6 have recently demonstrated aromatization of radiolabeled testosterone and androstenedione to estrone and 17 ,B-estradiol by the human fetal ovary. The production of estrone and 17 f3-estradiol and their stimulation by trophic hormones further suggests that the fetal ovary possesses aromatase activity. Our findings are also consistent with those of George and Wilson,6 who found that aromatase activity was present in the fetal ovary early in embryogenesis and that a limited source of C 19 steroid precursors may be the cause for reduced estrogen synthesis of the fetal ovary. Under the in vitro conditions reported here, the fetal ovary may be steroidogenic ally active in utero, but this activity is reduced compared with that of fetal testes because of a deficiency of LHlike gonadotropin binding and an absence of precursors, apparently due to enzymic deficiencies. The divergence in fetal gonadal testosterone secretion may provide an explanation for sexual differences in the development of internal genitalia, patterns of gonadotropin release, and behavior. The role of fetal ovarian estrogen production in these functions remains uncertain. REFERENCES 1. Bloch E: Metabolism of 4- 14C-progesterone by human fetal testis and ovaries. Endocrinology 74:833, 1964 2. Abramovich DR, Baker RG, Neal P: Effect of human chorionic gonadotrophin on testosterone secretion by the foetal human testis in organ culture. J Endocrinol60:179, 1974 3. Ahluwalia B, Williams J, Verma P: In vitro testosterone biosynthesis in the human fetal testis. II. Stimulation by cyclic AMP and human chorionic gonadotropin (hCG). Endocrinology 95:1141, 1974

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EFFECT OF TROPIDC AGENTS ON FETAL OVARIAN STEROIDOGENESIS

4. Jungrnann RA, Schweppe JS: Biosynthesis of sterols and steroids from acetate- 14C by human fetal ovaries. J Clin Endocrinol Metab 28:1599, 1968 5. Payne AH, Jaffe RB: Androgen formation from pregnenolone sulfate by the human fetal ovary. J Clin Endocrinol Metab 39:300, 1974 6. George FW, Wilson JD: Conversion of androgen to estrogen by the human fetal ovary. J Clin Endocrinol Metab 47:550, 1978 7. Baker TG: A quantitative and cytological study of germ cells in human ovaries. Proc R Soc BioI 158:417, 1963 8. Reyes FI, Boroditsky RS, Winter JSD, Faiman C: Studies on human sexual development. II. Fetal and maternal serum gonadotropin and sex steroid concentrations. J Clin Endocrinol Metab 38:612, 1974 9. Abraham GE, Buster JE, Lucas LA, Corrales PC, Teller RC: Chromatographic separation of steroid hormones for use in radioimmunoassay. Anal Lett 5:509,1972 10. Abraham GE, SwerdloffR, Tulchinsky D, Odell WD: Radioimmunoassay of plasma progesterone. J Clin Endocrinol Metab 32:619, 1971 11. Abraham GE, Manlimos FS, Solis M, Wickman AC: Combined radioimmunoassay of four steroids in one milliliter of plasma. II. Androgens. Clin Biochem 8:374, 1975 12. Buster JE, Abraham GE: Simultaneous measurement of plasma dehydroepiandrosterone and 16a-hydroxy dehydroepiandrosterone by radioimmunoassay. Anal Lett 5:597,1972

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13. Coyotupa J, Parlow AF, Abraham GE: Simultaneous radioimmunoassay of plasma testosterone and dihydrotestosterone. Anal Lett 5:329, 1972 14. Jawad MJ, Wilson EA: Unpublished data 15. England BG, Niswender GD, Midgley AR Jr: Radioimmunoassay of estradiol-17 f3 without chromatography. J Clin Endocrinol Metab 38:42,1974 16. Lipsett MB, Tullner WW: Testosterone synthesis by the fetal rabbit gonad. Endocrinology 77:273, 1965 17. Wilson JD, Siiteri PK: Developmental pattern of testosterone synthesis in the fetal gonad of the rabbit. Endocrinology 92:1182, 1973 18. Resko JA: Androgen secretion by the fetal and neonatal rhesus monkey. Endocrinology 87:680, 1970 19. Schindler AE, Friedrich E: Steroid metabolism of foetal tissues. I. Metabolism of pregnenolone-4- 14C by human foetal ovaries. Endokrinologie 65:72, 1975 20. Catt KJ, Dufau ML, Neaves WB, Walsh PC, Wilson JD: LH-hCG receptors and testosterone content during differentiation ofthe testis in the rabbit embryo. Endocrinology 97:1157,1975 21. Roberts JD, Warren JC: Steroid biosynthesis in the fetal ovary. Endocrinology 74:846, 1964 22. Milewich L, George FW, Wilson JD: Estrogen formation by the ovary of the rabbit embryo. Endocrinology 100:187, 1977