Action of testosterone on the estradiol-induced feminization of the male chick embryo.

The early treatment of male chick embryos with estradiol induces the feminization of their sex tract, i.e. both their gonads and müllerian tract exhibit female features. The additional treatment of estrogenized male embryos with testosterone propionate antagonizes the effects of estradiol on both gonads and müllerian ducts. Our data give further support to the view that testosterone and estrogens act respectively as agonist and antagonist modulators of the secretion of the anti-müllerian hormone.

Action of testosterone on the anti-müllerian activity of the chick embryonic testis assayed in vivo by organotypic grafting.

The implantation of embryonic testis grafts into female chick embryos induces the regression of their müllerian ducts (MDs) in a certain number of cases. The treatment of either the grafts or the grafted females with testosterone propionate (TP) results in a significant increase in the number of MD regressions observed. Our data are interpretable in terms of a direct activation by TP of the anti-müllerian activity of the embryonic testis. We discuss a possible mechanism accounting for the synergistic action of testosterone and anti-müllerian hormone.

Action of estradiol and tamoxifen on the testis-inducing activity of the chick embryonic testis grafted to the female embryo.

The implantation of two testes from 13-day-old male chick donor embryos into the extra-embryonic celom of 3-day-old female embryos induces the masculinization of their ovaries up to a total and definitive inversion of their gonadal sex, i.e., the differentiation of testes in the female hosts. Pretreatment of the donors with estradiol (E2) between day 11 and 13 counteracts the testis-inducing activity of the implants, while co-treatment of donors with both tamoxifen (TAM) and E2 at the same stage restores the initially observed activity. The treatment of 3-day-old male donor embryos with E2 causes the differentiation of their left gonad into an ovotestis totally devoid of testis-inducing activity once grafted in the same conditions as above. An additional treatment with TAM of the grafted host embryos does not modify the results obtained when E2-treated male gonads are grafted to normal host embryos. This shows that the lack of testis-inducing activity exhibited by the E2-treated grafts can not be attributed to a protecting action of endogenous estrogens on the gonads of the host. On account of previous work showing the inhibition by E2 of the Müllero-regressive activity of the chick embryonic testis, our present results can be interpreted in terms of E2-down regulation of Anti-Müllerian Hormone (AMH or MIS), which appears to be a good candidate as testis-inducer. The relevance of our results to the phenomenon of gonad differentiation is discussed.

Action of estradiol and tamoxifen on the Müllero-regressive activity of the chick embryonic testis assayed in vivo by organotypic grafting.

In the chick, the implantation of a testis graft from a 13-day-old male donor embryo into the extra-embryonic coelom of 3-day-old female embryos induces the total regression of their Müllerian ducts because of the anti-Müllerian hormone (AMH or MIS) secreted by the implant. Pre-treatment of the donors with estradiol (E2), between day 12 and day 13, counteracts in a significant way the Müllero-regressive activity of the implant. Co-treatment of donors at the same stage with both Tamoxifen (TAM) and E2 restores the initially observed activity, thus demonstrating the presence of Tamoxifen-sensitive estrogen receptors at the late stage of treatment in the Sertoli cells responsible for AMH secretion. The treatment of 3-day-old male donor embryos with E2 causes the differentiation of their left gonad into an ovotestis which provides implants totally devoid of Müllero-regressive activity. The additional treatment with TAM of the grafted host embryos, does not modify the results obtained when E2-treated male gonads are grafted to host embryos not treated with TAM. This shows that the lack of Müllero-regressive activity exhibited by the E2-treated male gonads does not depend on the estrogens they may secrete during the time of the assay, i.e., it cannot be attributed to a protecting action of estrogens on the MDs of the host. Our results therefore favor the idea that E2 down-regulates AMH. The relevance of such a regulation to the phenomenon of Müllerian duct maintenance, either in the E2-feminized male or in the female chick embryo, is discussed.

[Contribution of experimental intersexuality in chickens to the problem of gonadal differentiation and its abnormalities in amniotic vertebrates]. / Apport de l'intersexualité expérimentale chez le Poulet au problème de la différenciation gonadique et de ses anomalies chez les vertébrés amniotes.

The grafting of embryonic testes to chick embryos realizes an experimental model which is near the spontaneous situation of the cattle "free-martin". It allows to obtain a masculinization of female host embryos developing in some cases up to a total and definitive reversal of sex differentiation. Indeed, it is possible, by this way to obtain testes induced under the influence of substance(s) secreted by the grafts which determine epigenetically a sexual phenotype opposite to the genotype of the host embryos. The analysis of such testis morphogenesis shows that it results from an inhibitory mechanism. Some experimental data strongly suggest to ascribe it to the anti-Mullerian hormone secreted by the graft. The ovotestes and testes observed in adult treated animals present various degrees of development, especially at the level of seminiferous tubules in which the spermatogenesis, when present, may be completed until spermatozoa. Some gonadal abnormalities interesting genetically female human beings, like a majority of the hermaphrodites, or certain XX men, are discussed at the light of these experimental data.

[Crossed graft of embryonic testis between quail and chick embryos: contribution to the study of the mechanism of female bird embryo masculinization]. / Greffes croisées de testicules embryonnaires entre des embryons de Caille et de Poulet: apport à l'étude du mécanisme de la masculinisation de l'embryon femelle d'Oiseau.

Quail, or chick embryonic testes grafted respectively in the extraembryonic coelom of chick or quail embryos induce both a Mullerian duct regression and a masculinization of the female host gonads up to the differentiation of two testes, in some cases. Such a result confirms the fact evidenced previously in other bird species (chick and duck) that the testis-inducer is interspecific. Quail cells are not observed in histological sections of embryonic gonads of testis-grafted chicks. This allows to discard a possible influence of cells migrating from the graft to the host. The grafted testes act then through substance/s secreted in the blood stream. Present and previous experimental data strongly suggest that the same substance, i.e. the so-called anti-Mullerian hormone, is responsible for both MD regression and gonadal sex reversal.

Influence of heterospecific testis graft on the gonadal sex differentiation of female bird embryos.

Testes from duck and chick embryos grafted, respectively, to chick and duck genetically female host embryos modifies their gonadal differentiation. It results in masculinization developing, in some cases as far as testis formation. This demonstrates 'in vivo' that the testis inductor(s) secreted by the grafted testis is (are) interspecific. Duck gonads are more sensitive than chick gonads. Such grafts also cause the regression of Müllerian ducts. Comparison of the effects on ducts and gonads reinforces the view that both could depend on the same substance, i.e., anti-Müllerian hormone.

Action of estradiol on müllerian duct regression induced by treatment with norethindrone of female chick embryos.

Treatment of genetically female chick embryos with norethindrone (NET), a progesterone-like steroid chemically related to testosterone, caused two types of Müllerian duct (MD) deficiencies. The first consisted in an absence of the caudal part of the ducts owing to their partial agenesia occurring between Days 5 and 7 of embryonic life. This is nonspecific since it was observed after a treatment with almost all steroidal sex hormones. In particular, this was obtained with estradiol which also increases the frequency and extent of agenesia caused by the NET, as reported here. The second type of deficiency appeared between Days 12 and 14 and was due to a regression destroying the more or less large part of the MDs having escaped agenesia, i.e., for most of the cephalic half. This resulted from the influence of the anti-Müllerian hormone originating from the ovary and normally inhibited by the ovarian estrogens. This protective action of endogenous estrogens was inhibited by the NET, but an additional treatment with estradiol removed this inhibition and prevented duct regression. Our results suggest that estrogen protects the duct from the regression induced indirectly by NET, by acting both at gonad and MD levels.

New insights on the mechanism of testis differentiation from the morphogenesis of experimentally induced testes in genetically female chick embryos.

Embryonic testes grafted in the extraembryonic coelom of 3-day-old genetically female chick embryos may induce total and definitive reversal of gonadal sex differentiation. In this experimental condition, the left gonad becomes a testis instead of an ovary. This makes it possible to compare testicular and ovarian morphogenesis in animals having the same genetic sex and to discount what is due to differences in the genetic determination between male and female. The morphogenesis of such testes is marked by a disappearance of the cortical germinal epithelium. The medullary sex cords keep a narrow lumen instead of becoming large lacunae. The germ cells remain few in the sex cords and do not become meiotic. Furthermore, interstitial cell development is known to be very slow. As a consequence the gross size of the gonad is much smaller than that of an ovary. All these morphogenetic phenomena are unlike those observed during normal ovarian differentiation and evidence an inhibiting influence of the grafted testes. Since inhibition and masculinization are concomitant, inhibition appears to be the mechanism responsible for gonadal sex reversal. The extraembryonic situation of the grafted testes and their relation with the embryo only via the blood stream demonstrates the role of a secreted substance or substances still to be exactly identified. Previous data suggest that this could be the anti-Müllerian-hormone (AMH). Furthermore, previous and present results show that testis differentiation can be actively induced in a bird. This does not agree with the hypothesis that the gonads of the homogametic sex, i.e., the testes in birds, do not need any inducer in order to differentiate.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the right testis of sexually mature genetically female fowl experimentally masculinized during embryonic life and submitted to a posthatching left castration.

Posthatching left castration of genetically female fowl, Gallus domesticus, preceded, during embryonic life, by a masculinizing treatment associating a testis graft and an antiestrogen resulted in the development of the right rudimentary gonad into a testis. Examined after the sexual maturity, the right testis of most treated animals was entirely composed of seminiferous tubules possessing a spermatogenic cell complement. Spermiogenesis proceeded to the stage of spermatozoon in 4 out of 17 treated animals and was almost as well organized as in a normal cock testis in 3 of them. Testis development appeared then as clearly improved, compared to that described previously in only left-castrated, with or without treatment with an antiestrogen, or only sex-reversed female fowl. The possible mechanism of this improvement is discussed.

Influence of an antiestrogenic drug (tamoxifen) on Müllerian duct agenesia induced by various steroidal sex hormones in the female chick embryo.

The caudal deficiencies of the Müllerian ducts (MDs) induced in chick embryos after early treatment with testosterone propionate (TP), 17 beta-estradiol benzoate (EB), or dihydrotestosterone (DHT) are the consequence of agenesia, i.e., a stop in duct development occurring during the sexually indifferent stage. The present work shows that EB and DHT act on the MDs in binding cellular estrogen receptors. Indeed, the antiestrogenic drug, tamoxifen, which competes with estrogens at the receptor site level, significantly decreases the percentage and extent of these MD caudal deficiencies. The results also show that such receptors are already present at a time when MDs begin to grow from 4.5 to 5 days of embryonic life onward. On the other hand, tamoxifen does not significantly modify agenesia induced by TP.

Absence of anti-Müllerian activity of inhibin in the chick embryo.

Inhibin extracted from bovine follicular fluid and administered to chick embryos at a dosage increasing from 0.4 to 30 micrograms per embryo did not induce the regression of the Müllerian ducts of treated females. This result contrasts with that obtained with a testis graft which acts through its anti-Müllerian hormone. Although both hormones were of glycoproteic nature and secreted by the same cells, this study shows no functional analogy between them.

Secretion of the anti-müllerian hormone by the gonads of experimentally sex reversed female chick embryos.

The gonads of genetically female chick embryos experimentally transformed into testes under the influence of a embryonic testis graft are able to induce in vivo the regression of Müllerian ducts when they are grafted to female embryonic hosts. On the other hand female gonads only transformed into ovotestes are ineffective on the host MDs, as in the case for female gonads. These results show that totally sex reversed gonads have the same properties as a normal testis. In particular they produce the anti-Müllerian hormone, whereas partially reversed gonads do not or only do at a very low level.

Development of interstitial cells in experimentally sex-reversed gonads of genetically female chick embryos.

A testis graft implanted in young genetically female embryos induced a male gonadal differentiation. The interstitial cell percentage and actual content were strongly lowered in masculinized gonads compared to normal female embryos and became similar to those observed in the normal developing embryonic testis. This effect was enhanced by the association of a graft and an antiestrogenic drug, and was not observed after administration of the drug alone. This inhibiting influence of the graft on interstitial cell formation may be considered as resulting in an inhibition of estrogen production. Its possible causative role in gonadal masculinization is discussed in the light of previous and present results.

[Decrease in the secretion of anti-Müllerian hormone by the chick testis during development]. / Sur la décroissance de la sécrétion de l'hormone anti-müllérienne par le testicule du poulet au cours du développement.

At the end of embryonic life the chick embryonic testis possesses a low anti-Müllerian activity, as evidenced by the grafting method to female hosts. The percentage of grafted embryos presenting a Müllerian duct regression is not increased by administration of an anti-estrogenic drug (tamoxifen). This observation does not favour the hypothesis according to which the low percentage of regression could be due to a protection of Müllerian ducts by estrogens from the host ovary. It shows rather that the anti-Müllerian hormone secretion actually decreases during development.

[Effects of testosterone and progesterone on the regression of Mullerian ducts induced by estradiol in the female chick embryo]. / Action de la testostérone et de la progestérone sur la régression des canaux de Müller induite par l'oestradiol chez l'embryon femelle de Poulet.

An administration of testosterone or of progesterone to estradiol-treated female chick embryos increased the rate of those presenting a regression of their Müllerian ducts. This observation favoured the hypothesis according to which the regression induced by estrogens depends on the anti-Müllerian hormone of ovarian origin.

[Hypothalamo-hypophyseal correlates in the chick embryo: thyrotropic activity of adenohypophyseal grafts from donor embryos of various ages. Effect or pretreatment with TRH]. / Etude des corrélations hypothalamo-hypophysaires chez l'embryon de Poulet: activité thyréotrope de greffons adénohypophysaires provenant d'embryons d'âges variés. Influence d'un prétraitement par la T.R.H.

Adenohypophyses from 10 to 18-day-old chick embryonic donors grafted in 3 days chick embryos, thus disconnected from the hypothalamus, had a partially autonomous thyrotrophic activity. However this functional autonomy was greater in grafts from donors aged 10 or 11 days than from older embryos or from 11-day donors pretreated with T.R.H. before grafting. This strongly suggests that hypothalamo-adenohypophysis-thyroidal relationship establish normally between 11 and 12 days of embryonic life.

[Development of an embryonic thyroid grafted into a chick embryo]. / Evolution d'une thyroïde embryonnaire greffée à l'embryon de Poulet.

A morphological and physiological study of an embryonic thyroid grafted in a chick embryo showed that it developed according to the endocrine status of the host. Its relative age appreciated at various stages of embryonic life is different from that of a gland developing normally during similar lengths of time.

[Synergistic action, on the masculinization of female chick embryo gonads, of a graft of embryonic testis and administration of an anti-estrogenic substance (tamoxifen)]. / Action synergique, sur la masculinisation des gonades de l'embryon femelle de Poulet, d'une greffe de testicule embryonnaire et de l'administration d'une substance anti-oestrogène (tamoxifène).

The capability of an embryonic testis graft to induce a male gonadal sex differentiation in genetically female chick embryos was significantly enhanced by the administration of a antiestrogenic drug (tamoxifen). This observation reinforces the view that the experimentally-induced testis differentiation depends chiefly on a inhibition of estrogen influence.