Estrogen production by fetal rat gonads.

Aromatase activity in fetal rat testes and ovaries was demonstrated by the conversion of tritiated testosterone or 19-hydroxyandrostenedione into estrone and estradiol, which were identified and quantified by double isotopic dilution and recrystallization to constant specific activity. Testes formed mostly estradiol, ovaries mostly estrone. Aromatase activity was stimulated by cAMP in both the testes and ovaries as early as 17 days of fetal life. Stimulation by FSH was noted at this same stage in the testis, but not before 3-4 days after birth in the ovary. LH was without effect on aromatase activity in both kinds of gonads. Basal estrogen secretion was non-existent or undetectable in both the testes and ovaries in fetal stages. In the presence of cAMP and as early as 17 days of fetal life, the testes released estradiol, as early as 14 days the ovaries released estrone. Estrogen secretion was stimulated by LH and FSH at fetal stages in the testis and at infantile stages in the ovary. Responsiveness to gonadotrophins closely followed the appearance of the receptors.

Production of estradiol by the fetal rat testis.

Testes from 17- to 20-d-old rat fetuses were cultured in vitro for various time intervals in Medium 199 alone or with added gonadotrophins. Estradiol released into the culture media was determined by radioimmunoassay. A basal estradiol secretion rate was inexistant or undetectable at all stages studied. In early stages (17 and 18 d) there was no difference in the stimulatory effect of FSH or LH at the same concentration. At 19 d, the prevalence of FSH became apparent. At 20 d, a significant action of FSH was noted after only 3 h culture time. 1-Methyl-1, 4-androstadiene-3,17-dione, an aromatase inhibitor, markedly depressed FSH-stimulated estradiol secretion. In the 20-d old testis, stimulation of estradiol production by FSH was more rapid and reached a higher level than by (Bu)2cAMP. It is suggested that the difference in the action of LH and FSH reflects the difference in the time of appearance of the corresponding receptors in the developing fetal testis.

Estrogen production by fetal and infantile rat ovaries.

The de novo production of estrone and estradiol by the fetal and infantile rat ovary and its regulation by cAMP, LH and FSH were investigated. Basal estrogen secretion was not demonstrable before d 5 after birth. However, the fetal ovary was responsive to cAMP as early as 14 d old. Estrone was the main estrogen secreted. Steroid aromatase inhibitors inhibited cAMP-stimulated estrone secretion. Responsiveness to LH and FSH appeared at 5 d after birth. The proportion of estradiol grew more important with advancing age, becoming predominant at 7 d.

Insensitivity of the chick embryo müllerian duct to human recombinant müllerian-inhibiting substance.

To determine whether mammalian Müllerian-inhibiting substance (MIS) is active in birds, Müllerian ducts from 7- to 8-day-old male or female chick embryos were cultured in the presence of human recombinant MIS at concentrations between 2.5 and 12.5 micrograms/ml. None of 20 ducts regressed at any concentration. In contrast, at concentrations of 2.5-5 micrograms/ml, all 12 Müllerian ducts from 13-day-old male mouse embryos and 13 out of 14 female ducts were inhibited to varying degrees. It is concluded that avian Müllerian ducts are unresponsive to mammalian MIS. There may be a difference in structure between the MIS of birds and mammals, or the signal-transduction system may be different.

Effect of pituitary grafting on ovarian oestradiol secretion in the hypophysectomized chick embryo.

Chick embryos were hypophysectomized by partial decapitation at the stage of 42 h of incubation and grafted with a hypophysis from a 12-days-old donor embryo on the chorio-allantoic membrane at 9 1/2 days. Two days later, their ovary was removed for organ culture and its oestradiol secretion rate was compared to that of the ovary of hypophysectomized, non grafted control embryos. The oestradiol secretion rate in the grafted embryos was almost twice that in the hypophysioprivic embryos and in the range of that in normal embryos. This result suggests that the hypophysis controls oestradiol secretion of the ovary in the 11 1/2-days-old chick embryo.

Effect of partial decapitation (hypophysectomy) on 17 beta-estradiol secretion by the chick embryo ovary.

Ovaries of equally matched partially decapitated (hypophysectomized) and sham-operated (at 42 h of incubation) 11 1/2-day-old chick embryos, i.e. embryos as identical as possible as regards body size and other morphological features, were compared with respect to the amounts of 17 beta-estradiol secreted during a 4 h-culture period. The 17 beta-estradiol secretion rate was very significantly lower in the decapitated than in the control embryos. This result suggests that the hypophysis controls 17 beta-estradiol secretion of the ovary in the 11 1/2-day old chick embryo.

Embryonic sex hormones in birds.

Hormone activity of embryonic gonads in birds was demonstrated by grafting and culture experiments. Anti-Müllerian hormone responsible for the regression of the Müllerian ducts in the male is most probably a glycoprotein. Whether the testis also secretes testosterone has long been disputed, but most arguments are against this possibility. From early stages of development, the ovary secretes estrome and estradiol. However, it could not be demonstrated unambiguously whether estrogen is identical with the sex inducing substance in the female. The hypophysis seems to control ovarian estrogen secretion at 10-13 days of incubation in the chick embryo.

Estrogen secretion by the chick embryo ovary.

This is a review of work on estrogen secretion by the chick embryo ovary. The genetically female gonadal anlage secretes estrone and estradiol at the time of sex differentiation. Estradiol induces in a very short time interval the secretion of estradiol in the testis. However, it is not likely to play a role in normal ovarian sex differentiation, since neither anti-estrogens nor aromatase inhibitors disturb ovarian differentiation. Stimulation of estradiol secretion in vitro by LH is highest at early stages. Control of estradiol secretion by pituitary LH is suggested at early stages.

Aromatase activity in fetal gonads of mammals.

This is a review of work demonstrating aromatase activity in both ovaries and testes of mammalian fetuses. Studies on the regulation of this enzyme activity by gonadotrophins and other factors have also been reviewed. Finally, the significance of aromatase activity in conjunction with the role of estrogen in ovarian and testicular development has been considered.

Pituitary control of estrogen secretion in the chick embryo: true or spurious?

The aim of the present study was to examine whether the hypophysis played a role in ovarian 17 beta-estradiol secretion in the chick embryo. Hypophysectomy was fulfilled by partial decapitation, which has long been codified as a practical method of hypophysectomy in the chick embryo. Ovaries from decapitated, sham-operated, and intact embryos 9-19 days old were cultured in vitro during a 4-hr period, and 17 beta-estradiol released was determined by radioimmunoassay. A difference in estradiol secretion rate between decapitated and control embryos was noticed at 10-13 days, on both a per ovary and a per gram basis. At 15-19 days, there was no difference. These results suggest that the hypophysis influences the estrogen secretion of the chick embryo ovary during a limited space of time.

Estradiol secretion by the ovary of 19-day-old hypophysectomized and sham-operated chick embryos.

The amounts of estradiol released into culture media by ovaries from 19-day-old hypophysectomized (decapitated) and sham-operated chick embryos were determined by RIA. Per single ovary, ovaries from decapitated embryos secreted slightly less estradiol than ovaries from sham-operated ones during a 4-h culture period (837 +/- 413 vs 935 +/- 378 pg), but this difference was not significant. On an ovarian weight basis, ovaries from decapitated embryos secreted slightly more estradiol than ovaries from sham-operated ones (1.15 vs 0.91 ng), but this difference was not significant either. It is concluded from these results that the hypophysis does not control estradiol secretion by the ovary in the 19-day-old chick embryo.

Action of LH, FSH and (Bu)2 cAMP on the conversion of [3H]19-hydroxyandrostenedione into oestrogens by foetal and infantile rat ovaries in organ culture.

Ovaries from 18-21-day-old foetal as well as from 2-10-day-old infantile rats were cultured in vitro in the presence of [3H]19-hydroxyandrostenedione and in the presence or absence of LH, FSH or (Bu)2 cAMP, and oestrone and oestradiol formed were determined by double isotopic dilution and recrystallization to constant specific activity. In foetal ovaries, the stimulation factor with FSH was 0.9-1.3, which was considered insignificant in comparison with the 8-13-fold stimulation obtained with (Bu)2 cAMP. At infantile stages, aromatase activity was stimulated 1.3-3.5-fold, which was close to the 3.9-fold stimulation obtained with (Bu)2cAMP. LH was ineffective at both foetal and infantile stages.

[Transformation of 19-hydroxyandrostenedione to estrone and estradiol by rat embryonic testis in vitro]. / Transformation de la 19-hydroxyandrostènedione en oestrone et en oestradiol par le testicule embryonnaire de rat en culture in vitro.

Aromatase activity in the foetal rat testis was demonstrated by the conversion of [3H] testosterone into oestradiol. However, the conversion rate was low, around 0.06%, probably because the radioactive precursor was heavily diluted by the large amounts of endogenous testosterone produced. In an attempt to improve the conversion rate, we therefore used a precursor following testosterone on the oestrogen biosynthesis pathway. Testes from 18-21-day-old foetal rats were cultured in vitro with [3H] 19-hydroxyandrostenedione as a substrate in the presence or absence of LH, FSH or (Bu)2 cAMP. The [3H] oestrone and [3H] oestradiol synthesized were measured by double isotopic dilution and recrystallization to constant specific activity. In comparison with previous results obtained with testosterone as precursor, the synthesis rate of oestrogens was increased 5-10-fold. FSH and (Bu)2 cAMP stimulated aromatase activity; LH had no effect. These results definitively establish the capacity of the foetal rat testis to aromatize androgens.

[Hypophysis and estradiol secretion in the chick embryo]. / Hypophyse et sécrétion d'oestradiol chez l'embryon de Poulet.

Ovaries from 10- to 18-day-old chick embryos hypophysectomized by partial decapitation were cultured in vitro and their estradiol secretion was compared to that of ovaries from control embryos. The production of estradiol was not less in the decapitated than in the control embryos at 15-18 days, neither per ovary nor on the basis of ovarian weight. However, the difference was significant at 10-11 days. These results suggest that the hypophysis controls estradiol secretion by the chick embryo ovary in the early stages, but not in the later ones.

17 beta-estradiol secretion in normal and hypophysectomized chick embryos.

Ovaries from decapitated, sham-operated and intact 18-day-old chick embryos were cultured in Medium 199 for 6 h, and the amount of 17 beta-estradiol released into the culture media was determined by radioimmunoassay. Ovaries from decapitated embryos secreted significantly lower amounts of 17 beta-estradiol than those from intact embryos, but there was no difference when they were compared to ovaries from sham-operated embryos in this respect. On an ovarian weight basis, 17 beta-oestradiol production was significantly different between the 3 groups of embryos, the ratio being highest in the decapitates. 17 beta-Estradiol concentration was the same in serum from both decapitated and intact female embryos. Considering these results, it is concluded that the hypophysis does not control 17 beta-estradiol secretion by the chick embryo ovary even near hatching time.

Sex-specific proteins in normal chick embryo gonads and testis feminized by oestrogen treatment: a two-dimensional gel electrophoresis investigation.

Gonads dissected from 17-day-old normal chick embryos, as well as testes feminized by oestrogen treatment, were labelled in vitro with [35S]methionine and [14C]leucine. The patterns of cytosolic protein synthesis by the gonads were analysed using two-dimensional (2-D) polyacrylamide gel electrophoresis. In the feminized testis, two female-specific proteins appeared, the rate of synthesis of four testis-specific proteins was decreased, and quantitative variations of other synthesized proteins in relation to sex were detected. This altered protein synthesis indicates that the protein synthesis of the testis feminized by oestrogen treatment has in part become similar to that of a normal ovary. The synthesis of female-specific proteins in the feminized testis raises the question of their role in normal ovarian differentiation.

Stimulation of aromatase activity in the fetal rat gonads by cAMP and FSH.

The gonads from 17- to 21-day-old fetal rats were cultured in vitro in the presence of [3H]testosterone and in the presence or absence of cAMP or FSH, and estrone and estradiol formed were measured by double isotopic dilution and recrystallization to constant specific activity. Estrogen synthesis by testes was stimulated by both cAMP and FSH as early as at 18 days of gestation. FSH did not enhance aromatase activity in ovaries, although cAMP did. It is remarkable that FSH controls estrogen synthesis in the testis earlier than in the ovary.

Stimulation of aromatase activity in the fetal rat testis by cyclic AMP and FSH.

The effect of dibutyryl cyclic AMP and FSH on oestrogen biosynthesis was investigated in testes from 18- to 21-day-old fetal rats cultured in vitro in the presence of tritiated testosterone. Oestrone and oestradiol concentrations were measured by determination of constant specific activity after isotopic dilution. Dibutyryl cyclic AMP and FSH markedly stimulated the conversion of testosterone into both oestrone and oestradiol at all stages studied. Oestradiol synthesis was stimulated by two- to sevenfold, while stimulation of oestrone synthesis was even greater. The results demonstrate that the aromatase enzyme system of the fetal rat testis responds to cyclic AMP and FSH.

[Action of LH on the secretion of estradiol by the chick embryonic ovary in in vitro culture]. / Action de LH sur la sécrétion d'oestradiol par l'ovaire embryonnaire de Poulet en culture in vitro.

Gonads from 7-to 18-day-old female chick embryos were cultured in vitro in the presence or in the absence of bovine LH, and the amount of oestradiol released into the culture medium was determined by radioimmunoassay. Depending on the stage investigated, one ovary secreted 2.8-28 ng oestradiol during a 24-h culture. LH stimulated oestradiol secretion at all stages studied. The stimulation coefficient was highest for the younger stages. The lowest concentration which gave a positive result was 10 ng/ml. The discrepancy between the strong action of LH in vitro and its apparent absence of action in normal development is discussed.