AMER. ZOOL.. 15:257-272 (1975). Sex Differentiation of Avian Gonads In Vitro KATV HAFFE.N Unite de Recherches 61 de 1'INSERM, 67200 Strasbourg-Hautepierre, France SYNOPSIS. The analysis of avian sex differentiation in vitro has been limited to the following problems: morphological sex differentiation of gonads cultured in vitro; analysis of the chemical nature of the hormonal secretion; differentiation of germ cells in relation to their somatic environment. Morphological sex differentiation of avian gonads occurs in vitro. Differentiated gonads of the chick embryo carry out biosynthesis of sex hormones from several radioactive precursors. Female gonads in particular synthesize estrogens while male gonads synthesize testosterone. Some experiments have given evidence of estrogen synthesis by undifferentiated female gonads. Embryonic gonads of quail, like those of chick, are able to synthesize sex steroids from radioactive precursors. However, in the quail and mainly in the testes, a delayed appearance and a lower activity of the enzyme system 3/3-HSDHA5-4-isomerase was found. Histoenzymological results corroborate the biochemical ones. Combination of culture and grafting experiments have shown that male germ cells when they are forced into female differentiation by early colonization of a female gonad degenerate after entering the premeiotic stage. The reasons for this delayed failure of sex differentiation of "male oocytes" have certainly to be searched for at the level of perturbation in the mechanisms of meiosis. INTRODUCTION Numerous in vivo investigations involving sex hormone administration, castration experiments, coelomic grafts of gonads, as well as in vitro culture of gonads and sex ducts, have shown that the differentiation of sex characters is under the influence of the hormonal secretion of the gonads and that the embryonic gonads produce secretions which have the same effect as steroid hormones (for reviews see Wolff, \962a,b; Wolff and Haffen, 1965; Haffen, 1970). The present paper deals essentially with some more or less recent contributions of the organ culture technique to the following problems of gonad differentiation. The first part, dealing with the spontaneous sex differentiation of the gonads and their secretory activities, introduces the second part, which describes the research work on the chemical nature of the hormonal secretion and the cellular localization of steroid biosynthesis in differentiating gonads. The third part is devoted to the study of germ cell differentiation as influenced by their somatic environment. SEX DIFFERENTIATION AND SECRETORY ACTIVITIES OF GONADS CULTURED IN VITRO Spontaneous sex differentiation In order to define their intrinsic autodifferentiating ability, organ culture experiments of embryonic gonads have been carried out by Wolff and Haffen (1952a) in the duck, Weniger (1961) in the chick, and Haffen (1964) in the quail. The 614-day stage is commonly admitted to represent the sex differentiation stage in the chick and corresponds to the 8-day stage in the duck and to the 5V2-day stage in the quail. Gonads from these three species of avian embryos isolated before the stage of sex differentiation (6 to 7 days in duck, 4 to 5 days in chick, and 5 days in quail) pursue their development when cultured in vitro. Genetically male gonads differentiate into typical testes. Left gonads isolated from genetically female embryos give rise to an ovary containing a thick cortex and a more or less vacuolated medulla, while the right gonad generally regresses in culture and 257 258 KATY HAFFEN becomes rudimentary as it does in the normal embryo. Germ cells were present in the testicular cords and also in the ovarian cortex, where they were seen dividing in duck and chick embryonic gonads. In ovaries isolated from quail embryos, the oogonia were in the early stage of meiosis after 6 days of culture. Secretory activities of gonads The secretory activities of embryonic gonads have been demonstrated by parabiosis experiments. Thefeminizing action of female gonads. Pairs of left, undifferentiated gonads from 6- to 7-day ducklings were set up in parabiosis. The corresponding right gonads, cultured singly, were used as controls to determine the genetic sex. In those experiments in which different sexes were in parabiosis, the female gonad differentiated into an ovary and the male gonad developed into an ovotestis consisting of testicular medulla surrounded by an ovarian cortex (Wolff and Haffen, 1952b). The same results were obtained by culturing an undifferentiated left male gonad with a 7- to 10-day right female gonad. The right female gonad, although regressing, feminized the left male gonad (Wolff and Haffen, 1952c). Furthermore, a young male germinal epithelium explanted between the fifth and sixth day and associated with a female medulla from between 9 and 13 days incubation differentiated to form an ovarian cortex (Haffen, 1960). Weniger (1961) linked in parabiosis very young chick gonads of 4 and 5 days incubation during 4 days. When different sexes were linked, the female gonad, left or right, strongly feminized the left genetically male gonad. These results indicate that the hormonal secretion from the gonads starts very early. The masculinizing action of male gonads. Various authors have shown by grafts and injections in ovo that androgens have little or no influence on the sex differentiation of female gonads. These results have been confirmed by the parabiosis experiments in vitro described above. But it is known that a male hormone is being formed by a young testis from the time of sex differentiation (Wolff, 1946). It acts on certain effectors, such as the Miillerian ducts. A left Mullerian duct from a 7- to 8-day male or female was explanted and associated between two testes of 8 to 9 days. The male hormone formed by the testes caused the duct to regress over the entire region with which it was in contact (Wolff et al., 1952; LutzOstertag, 1954). Diffusion of embryonic sex hormones into the culture medium. Weniger (1962) showed that hormones produced by embryonic gonads diffuse into the culture medium. He substituted the target organ (left testis or Mtillerian ducts) for female or male gonads. On media into which ovarian hormone had diffused, the testes were feminized. On media into which testicular hormone had diffused, the Miillerian ducts regressed. ANALYSIS OF THE CHEMICAL NATURE OF THE HORMONAL SECRETION It has been suggested by Wolff and Ginglinger (1935), Wolff (1950), Willier (1939, 1942, 1952) that sex hormones produced by the morphologically undifferentiated gonad can be viewed as sex differentiator substances directing transformation of the indifferent gonad into either an ovary or a testis. These two working hypotheses have stimulated research on the chemical characterization of the hormonal secretion of embryonic avian gonads at various stages of their development. Production of steroids by embryonic gonads of the chick Steroids have been detected in the blood, allantoic and amniotic fluids, as well as in the gonads. Stoll and Maraud (1956) detected trace amounts of 17-ketosteroids in the amniotic and allantoic fluid of 6'/2-day chick embryos, while Ozon (1965, 1969) demonstrated that estrogens are first present in these fluid compartments as well as in the blood of 10-day embryos. Gallien and Le Foulgoc (1957) identified estrogens in the 10-day ovaries. Unfortunately, the techniques used to detect the hormones in the embryonic fluids and gonads have AVIAN GONADS IN VITRO failed to characterize estrone and estradiol in the culture media of embryonic female gonads. Weniger (1966) found estrogenic activity in these media after extraction by the biological test of Allen and Doisy. Positive results were obtained with extracts of media on which female gonads from 7 to 10 days had been cultivated; the results were negative with male gonads and other organs. 259 Scheib et al., 1974). Weniger and coworkers overlaid the labeled precursor on the surface of the media on which the explanted gonads were cultured, and extraction was performed on the culture media including the gonads. The techniques for identification of steroid hormones are described in detail in Weniger (1969, 1970) and in Weniger and Zeiss (1971). Biosynthesis of steroid hormones from labeled Estrogen biosynthesis precursors by cultured avian embryonic gonads From Na-l-14C acetate. Weniger et al. (1967), Weniger (1970a), and Akram and Weniger (1969) have studied estrogen formation from this precursor by avian embryonic gonads cultivated in vitro for at least 24 hr. They have shown labeled estrone and estradiol synthesis by 7- to 9-day ovaries of chick embryos (Weniger et al., 1967), and by 12-day ovaries of duck and pentado embryos (Akram and Weniger, 1969). Sodium acetate-l-14C was also incorporated into estriol and epiestriol by 16-day ovaries of chick embryos (Weniger, 1969). When the precursor was supplied to 4-, 5-, and 6-day undifferentiated gonads, labeled estrone and estradiol were found in the culture medium after 24 hr (Weniger and Zeis, 1971). The radiochemical purity has been established in the case of estrogens formed by 6-day gonads (Weniger and Zeis, 1971) and for epiestriol produced by 14- to 16-day ovaries (Weniger, 1970a). Cedard et al. (1968) and Guichard et al. (1973a) have also observed a production of estrogens from Na-l- 14 C acetate1 by their radiochemical purity. Human chorionic gonadotrophin (HCG), which has been shown by Connell et al. (1966) to stimulate testosterone production by the testis of 2-day chicks, increased estrogen production in 10- and 18-day ovaries and induced a discrete synthesis of estradiol in 18-day testes (Haffen etal., 1969; Guichard etal., 1973a) (Table 1). This study was performed by two groups of workers. Cedard and Haffen (1966), Haffen and Cedard (1968), Haffen et al. (1969), Guichard etal. (1973a,6), and Scheib et al. (1974) have developed the quantitative aspect of this biosynthesis as a function of the stage of development and of the nature of the labeled precursor in chick and quail in order to investigate the biosynthetic pathways ending in steroid sex hormones. On the other hand, Weniger et al. (1967), Akram and Weniger (1969), Weniger (1969, 1970), and Weniger and Zeis (1971) have concentrated their efforts on the qualitative but early aspect of this biosynthesis in order to check Wolffs hypothesis of estrogens being responsible for the morphological changes which characterize sex differentiation of female gonads. These investigations were realized in organ culture according to the technique devised by Wolff and Haffen (1952a). Cedard and coworkers have introduced the steroid precursor(s) into the medium in which a number of embryonic gonads were cultured. One to 3 days later the synthesized radioactive hormones were detected in the culture media. The authors found that the transformation products accumulated in the culture medium rather than in the explants. They could also verify that the culture medium itself, whether it contains a synthetic nutrient mixture or diluted embryonic extract, does not transform the From 3H-pregnenolone and 14C-proprecursor into biologically active steroids. The detailed techniques used for identifica- 14 1 Any other labeled steroid, synthesized from Na-1C acetate, could be detected in the culture media. tion of steroid hormones are not described cholesterol was found and HCG stimulated its here (for that purpose, see Haffen and Labeled production by 10-and 18-day-old testes and by 10-day Cedard, 1968; Guichard et al., 1973a; ovaries (Haffen et al., 1969). NO en o TABLE 1. Biosynthesis of testosterone and estrogens by chick embryonic gonads cultured in the presence of radioactive precursors'. (After Guichard et at., 1973a.) Stage in days of ncubation Precursors Preg-7a 3 H Prog-4-14C DHA-4-14C l¥i 14 10 Na-Acet-l- C Preg-7a 3 H Prog-4-MC DHA-4-'"C Testo-6,7a 3 H 14 18 a = = 0 = d = b Mean no. ofgonads Mean amount per experiment Specific of precursor Pairs of activity per experiment No. of (mCi/mM) exp Ovaries'1 testes (/iCi) 454 58.5 52 41 454 58.5 52 864 41 Na-Acet-l- C 454 Preg-7a 3 H 58.5 Prog-4- l4 C DHA-4- M C 52 Testo-6,7a 3 H 864 13.6 1.36 0.3 100 12.3 1.1 0.76 1 100 1 1 2 3 2 2 5 1 3 12.3 3 1.1 1 3 1 1 3.75 Testosi erone b dpm % 72 72 173 76 76 30 350 26 490 0.23 2.40 190 nul nul 50 81 50 67 81 67 63 63 88 98 20 26 26 22 20 nul nul 26 26 22 86 600 22 130 24 000 0.87 2.32 2.31 24 Testes Ovaries Estrogens0 % dpm Testosterone" Estrogens0 dpm % dpm % 0.05 0.24 0.39 9200 2680 2930 0.04 0.12 1.17 60 660 29 100 7 260 0.46 3.30 1.0 6 000 2 180 1 460 1720 19540 0.63 0.17 nul nul 1.0 nul nul 95 500 4 00 0.05 mil nul 18 500 26 500 0.16 2.2 3800 0.27 47 000 5.2 1800 0.18 nul nul 20000 19540 1.55 0.68 36 420 3.8 420 0.03 0.09 1840 8560 1380 11100 18200 0.13 0.07 0.17 1.15 0.20 2 240 nul nul nul nul 249 630 89 280 86 620 2.2 6 200 2 100 5 420 0.01 0.10 0.42 8.5 7.7 Data are expressed in dpm and in percent of the ether soluble fraction corrected to recovery of carrier hormones After oxidation into A4-androstenedione Estrone + 17/3-estradiol after methylation At 7'/2 days of incubation, both left and right ovaries were explanted; at 10 and 18 days, only the left ovary was cultured. > H -< D > z Tl W AVIAN GONADS IN VITRO gesterone2. Tritiated as well as 14C-labeled estrogens have been detected in the culture media of differentiated embryonic gonads isolated from chick and quail (Guichard et al., 1973a,b). In chick, no demonstration of a quantitative difference between male and female gonads could be made, owing to the poor yields (Table 1; Fig. 1), nor of an increase with age, while such a difference was striking in quail (Table 2). In the latter species, a higher level of synthesis was found in the female compared with the male at the same stage. Estrogens were generally formed in higher amounts from progesterone than from pregnenolone. Undifferentiated 15-day chick embryonic' gonads produced estrogens over 24 hr of culture from 14C-progesterone (Weniger and Zeis, 1971). 261 formed from 3 H-pregnenolone and 14Cprogesterone indicated an increasing synthesis of testosterone by testes as a function of age, together with a better yield from progesterone. Ovaries synthesized testosterone at a much lower rate than testes, the yield remaining constant with age (Table 1; Fig. 1). The radiochemical purity could be assessed only in the case of 14 Ctestosterone formed from progesterone by 10- and 18-day testes and for testosterone derived from 3 Hpregnenolone by 18-day testes (Guichard et al., 1973a). In quail, calculation of the yield of formation of testosterone has also shown an increasing synthesis in testes between the 10th and 15th day, with a better yield from progesterone and a higher production in From 4-i4C-Dehydroepiandrosterone (DHA). male than in female gonads (Table 2). Low quantities of radioactive estrogens As in the chick, only testosterone prowere detected in the culture media of un- duced by testes could be recrystallized to a differentiated chick gonads (6 and 7 days of constant isotopic ratio (Guichard et al., incubation). This synthesis was found to 1973ft). increase between 7V&, 10, and 18 days. No From 4-14C DHA. Radioactive testossignificant synthesis of estrogens was found terone was present in the culture media of in male gonads at IVi and 10 days, but took 7-day chick embryonic gonads (Fig. 1). Beplace at 18 days (Table 1; Fig. 1) (Haffen tween 7Vi, 10, and 18 days, labeled testosand Cedard, 1968; Guichard et al., 1973a). terone was produced in increasing amounts Transformation of this precursor into es- by male gonads. The synthesis of this not be detected in the culture trogens took place in embryonic quail steroid could ovaries and testes of 10 and 15 days incuba- media of 7lA- and 10-day ovaries, but was tion. Singularly, no increase of this estro- found with 18-day ovaries (Table 1; Fig. 1). gen production could be detected in the The radiochemical purity of testosterone female, while it was found in the male be- isolated from the culture media could be tween these two stages (Scheib et al., 1974) assessed (Haffen and Cedard, 1968; Guichard et al., 1973a). (Table 2). In quail, no significant synthesis of testosterone was found with 10- and 15-day Testosterone biosynthesis ovaries or with 10-day testes, while it became significant with 15-day testes. DHA 3 l4 From H-pregnenolone and C-progeswas found to be a less efficient precursor terone. Tritiated and 14C-labeled testos- than progesterone (Scheib et al., 1974) terone was present in the culture media of (Table 2). ovaries and testes isolated from 7V2-, 10-, and 18-day chick embryos, and from 10- Conversion of pregnenolone into progesterone2 and 15-day quail embryos. In chick, the calculation of the yield of testosterone This transformation was quantitatively estimated by determination of the radioac23 H-pregnenolone and 14 C-progesterone were tivity of the tritiated progesterone derived added simultaneously into the culture medium. Since from 3 H-pregnenolone (Fig. 2). Tritiated their specific activities were very different, the molar 3 ratio of the precursors was equilibrated by addition of progesterone derived from H-pregnenolone by gonads of both sexes in non-labeled pregnenolone. 262 p moles 1300 + KATY HAFFEN PRECURSORS: • • PROGEST. lilTO D.H.A B 1000- • 500- - 100- 18 10 18 FIG. 1. Production of testosterone (left diagram) and of estrogens (right diagram) by 6- to 18-day chick embryonic gonads from 14C-progesterone and DHA. Data are expressed in /umoles of steroid formed by an equal number of gonads (20 pairs of gonads at 6 and 7Vt days; 20 pairs of testes and 20 left ovaries at 10 and 18 days). (From Haffen and Cedard, 1968, and from Guichard et al., 1973a). chick and by female gonads in quail could be recrystallized to a constant isotopic ratio (Guichardetal., I973a,b). The comparative study between chick and quail revealed that in both species, the enzymatic activity of the 3/3-HSDH-As-4 isomerase increased with age, but was always more important in chick than in quail. This was particularly striking in the male. This delayed manifestation of the enzymatic activity in quail testes is confirmed by histoenzymologic results described later. In the female, a difference existed between quail and chick, but was far less striking. quail (Haffen and Cedard, 1968; Scheib et al., 1974). Interpretation and significance of these results The differences related to the conversion of pregnenolone into progesterone on one hand, and into DHA on the other, have raised the question of the biosynthetic pathways ending in sexual steroids, and in particular testosterone. The generally admitted scheme of steroid sex hormone biosynthesis displays two alternative metabolic pathways, the A4-3 ketosteroid pathway, and the AsFormation of DHA and Ab-androstene-3fi, hydroxysteroid pathway, depending on whether the 3/3 -HSDH-As-4 isomerase is 17/3-diol involved before or after conversion of C20 No formation of DHA from 3 H- steroids into C19 steroids. It should be pregnenolone has been demonstrated so pointed out that A4-androstenedione, a far in the culture media of explanted chick common intermediate metabolite of both gonads, while it steadily accumulated in the biosynthetic pathways, could not be demedia of quail gonads (Guichard et al., tected in the culture media while it was were incubated for 15 I973a,b). On the contrary, As-androstene found when gonads 14 3/3, 17/3-diol was found in the culture media and 30 min with C progesterone as a preof gonads of both sexes in chick as well as in cursor (Galli and Wassermann, 1972, AVIAN GONADS IN VITRO bo o H V 1*1 2 E oo CM OCM O to m o 00 00 I— r - O> r to in r-; o o o o o o co i> t ^ —• •& CJi t o • * ^ — to ~™ CO t ^ O — 00 — CO O r^ oo CM m CM in CM tO • oo o o o o 00 Ol ^ to ^5 r^ f- t^ m m — in o CTI to in o in -^ - - — Tf Tf — tO Ol CTi Gi CO Tf t— r ^ o •* oo c u bo o o o §E CM CM O CO 00 O tO O) I> ( N O CM CM — — o co r- o o o co to co t^ m to oo inoo r~; I> O — d 2 E §§ oo o Tf o r^ o o o1 CMI> l > CO CO O OCM CM o co — to CM oo oo <y> CO CO CM BO E •s'S g § S§ 11 is —^ in O m a) — to • CO — a> o CM CO co co CO U U O 44 4 ij U U a. 4 4 in in ^ t>.' _ ; o "i V -a v CM r~> NO il CO O <ji CM O O1M o CM m o CM in o c/5 « E o V c V =1 3 u V a. c CU..O a. a. O 263 1973). The only intermediate metabolite which could be detected in organ culture experiments was DHA formed by quail gonads from 3H-pregnenolone (Guichard et al., 1973i). It seems that organ culture favors the accumulation of end products (testosterone and estrogens) rather than products of intermediate metabolism. One should anyway keep in mind that the lack of detection of a metabolite does not necessarily mean that it does not play a role in the biosynthetic chain. Thus, the identification of the biosynthetic pathways leading to formation of testosterone is based on comparison of the data obtained from the 3H/14C ratio in progesterone and that obtained in testosterone produced in double labeling experiments (3H pregnenolone + 14C progesterone)2. A higher 3H/14C ratio in progesterone than in testosterone indicates a preferential passage through the A4-3ketosteroid pathway, while on the contrary a higher 3H/14C ratio in testosterone than in progesterone reflects the prevalence of the As-hydroxysteroid pathway and a certain deficiency of the 3/3-HSDH-As-4 isomerase at the level of the conversion of pregnenolone into progesterone. Figure 3 gives the comparison of the different ratio values determined for chick and quail. In chick, a near equal 3H/14C ratio was found in progesterone and in testosterone, while in quail, the 3H/14C ratio was higher in testosterone than in progesterone. This indicates that in chick, conversion into testosterone can take place through both pathways, while in quail, the As-hydroxysteroid pathway predominates strikingly, thus confirming the . enzymatic deficiency of the As-3j8-HSDHA5-4 isomerase at the level of the conversion of pregnenolone into progesterone. Finally, quail testis synthesises at the end of the incubation period noticeable amounts of estrogens from 14C-DHA. This could be explained by the fact that it displays signs of intersexuality after the 10th day of incubation (Scheib, 1971a). In chick, male gonads would produce estrogens from 14C-DHA(Haffen, and Cedard, 1968) and 14C-Na acetate (Akram and Weniger, 1967) only when experimentally feminized. 264 KATY HAFFEN Chick 30 Quail n 20 10- 0J 10 10 18 15 10 10 18 15 OVARIES TESTES FIG. 2. Conversion of pregnenolone into progesterone (see footnote 2) by ovaries and testes of chick (10-and 18-day embryos) and of quail (10- and 15-day embryos). Data are expressed as the percentage of the 3 H/ 14 C ratio in progesterone compared with the ratio in the ether soluble fraction. (From Guichard et al., by these authors fit together as far as the localization of the enzymatic activity in the ovary is concerned but differ for the testis. Furthermore, they differ as well in the time of its appearance. First appearance of enzymatic activity In the chick embryo, As-3/3-HSDH has been reported to be present initially in the genital ridge of 2Vz days (Woods and Weeks, 1969), in the undifferentiated gonads of 6V2 days (Scheib and Haffen, 1968), in the differentiated gonads of 8 days (Narbaitz and Kolodny, 1964; Chieffi et al., 1964) and of 9 days (Boucek et al., 1966). Cellular localization of enzymatic activity Woods and Weeks (1969) observed the As-3/3-HSDH from 2V2 days onward in both the germinal epithelium and mesenchymal cells of the underlying gonadal blastema. In the ovary, the enzymatic activity is concentrated in the medullary cords and weaker in the cortex. Narbaitz and Kolodny (1964), Chieffi et Cellular localization of steroid synthesis al., (1964), and Scheib and Haffen (1968, 1969) have shown that no reaction took Steroid producing cells show some re- place in the ovarian cortex during emmarkable cytological and cytochemical fea- bryonic development. The As-3/3-HSDH is tures. The problem of localization and dif- confined to those interstitial cells derived ferentiation of steroidogenic cells has been from the medullary cords (Fig. 4A-C). studied by various authors both by electron Furthermore, the reaction is at every stage microscopic and histochemical techniques. These researches do not deal with in vitro CHICK QUAIL cultures. We shall thus mention only one of them, namely the histoenzymologic study, ^ H Progesteron which has provided precious data as to the L J Testosteron* cellular localization of steroid biosynthesis. As seen in the preceding section, the A53/3-HSDH is a key enzyme in steroid biosynthesis. The histochemical method of Levy et al. (1959) tested on adult mammals has been applied to the avian embryonic gonads in order to check the stage at which the enzymatic reaction appears and to establish its localization at the cellular level as 10 15 described by various authors (Chieffi et al., FIG. 3. 3H/14C ratio in progesterone and testosterone 1964; Narbaitzand Kolodny, 1964; Scheib produced by chick (IVi to 18 days) and quail emand Haffen, 1968, 1969; Woods and bryonic testes (10 and 15 days). (From Guichard et al., Weeks, 1969). Indeed, the results obtained 1973a,i.) u AVIAN GONADS IN VITRO FIG. 4. As-3/3-HSDH activity in ovaries and testes of chick and quail embryos. In the ovaries (A-C) the enzymatic reaction is confined to cell clusters of the medulla (m). The cortex (co) shows no reaction. In the testes (D-F) the enzymatic reaction is present in some cells of the cords and in the interstitial cells of the stroma. A, Quail embryonic ovary of 12 days. B, Chick embryonic ovary of 12 days. C, Chick embryonic ovary of 8!4 days. D, Quail embryonic testis of 14J4 days. 165 First appearance of anzymatic activity in the interstitial cells of the stroma. F, Chick embryonic testes of 18 days showing the well-developed interstitial tissue in which a strong enzymatic activity is present. E, Chick embryonic testis of 10 days showing enzymatic activity in cell clusters located inside the cords (arrows). Magnification for/*, B, D,E;X\ 10; forC, F: x 450. (From Scheiband Haffen, 1968, 1969.) 266 KATY HAFFEN similar in the right gonad which is deprived of cortex, thus confirming the fact that in the embryonic ovary, the medulla is the site of hormonal secretion, first demonstrated by Wolff and Wolff (1951) (see also Wolff and Haffen, 1965). In the testis, Chieffi et al. (1964) have mentioned that As-3j8-HSDH appears only in a diffuse fashion from the 8th day in the sexual cords where it remains localized throughout embryonic development. According to Narbaitz and Kolodny (1964), the testis would be completely deprived of specific enzymatic activity. Scheib and Haffen (1968, 1969) gave the following description of the localization of the As-3/3-HSDH. A weak activity is visible in the cord cells at 614 days in the chick embryo. The activity becomes more widespread between 7 and 8 days and remains throughout embryonic development. After the eighth day one can further distinguish two types of cell clusters in which the As3/3-HSDH reaction is very intense; they are located at different levels in some cords and also in the stroma (Fig. 4E-F). Both are still found at later stages. In the stroma the cell clusters enlarge and their number increases during development; they represent the interstitial cells. The fact that some cells in the cords and the interstitial cells in the stroma have similar properties—cholesterides (Scheib, 1959) and As-3/3-HSDH— argues for Benoit's (1923) theory concerning the origin of the interstitial cells from the somatic cells of the cords. The specialization of these cells seems to occur earlier in the chick (8!4 days) than in the quail {Wh days). In the latter, only some cells react more intensively at 13 xh days, whereas at 14'/2 days some large clusters are found (Fig. AD). This delayed appearance of the histochemical reaction in interstitial cells in quail confirms the biochemical results, which have shown that the enzymatic activity becomes significant only at the end of the incubation period. DIFFERENTIATION OF GERM CELLS AS INFLUENCED BY THEIR SOMATIC ENVIRONMENT Germ cells undergo ultrastructural, histochemical, and physiological changes (migratory properties) during embryonic sexual organogenesis. This important question has been extensively reviewed by Dubois and Croisille (1970) so that this paragraph will not deal with it. Our interest here is the fate of germ cells which have been introduced at an early stage into gonads of the opposite sex. Indeed, experimental intersexuality studies (Wolff and Haffen, 1961) which followed those of Wolff and Ginglinger (1935), have pointed out that germinal epithelium from a male gonad converted into an ovarian cortex by estradiol or by a grafted embryonic ovary does not display the same evolutive potentialities as normal ovarian cortex: indeed, it disappears shortly after hatching as a consequence of the degeneration of the oocytes it contains. Association of an ovarian medulla with the cortical part of an intersexed male gonad does not promote the normal evolution of the oocytes (Haffen, 1969a). The question arose then as to whether the somatic cells or the germ cells themselves impair female differentiation in an experimentally induced male cortex. The study of Haffen (1969a), which was carried out by a combination of culture and grafting experiments, confirmed the second hypothesis. Evolution of germ cells after early experimental introduction into a gonad of the oppostie sex Haffen (1960), using the technique described by Wolff and Simon (1955) and Simon (1960), formed chimaeric embryos at the 9 to 15 somite stage: the anterior part (fertile and agonadic) of a blastoderm A was associated with the posterior part (sterile and gonadic) of blastoderm B. A chimeric embryo (AB) was thus obtained. The reverse association led to the formation of a chimeric embryo (BA). In 50% of the cases, the A and B embryos were of the opposite sex, while in 25% of the cases, the male gonocytes from the anterior germinal crescent colonized the female gonadic region in the posterior part, the inverse situation being found in 25% of the cases. The chimeric embryos, (AB) and (BA), developed for 48 hr in the culture medium AVIAN GONADS IN VITRO and reached a stage corresponding to that of 3- to 3 J4-day embryos. The left genital ridges colonized by the gonocytes were isolated and grafted for 10 days into the coelom of a 3-day embryo. After this first passage, the explant was introduced into a second host for 10 more days, then into a third and a fourth host. At each passage, a piece of the explant was examined histologically. Under these conditions, the age of the explant could be estimated as follows: 13 days after grafting I 23 days after grafting II 33 days after grafting III 43 days after grafting IV When the chimeric embryos were formed, the sex of neither embryo was known. To insure this, it was necessary that one gonad develop from each of both chimeric embryos. Ovary colonized by female germ cells. After 23 days, the cortex was filled with oocytes at the leptotene stage of premeiosis. After 33 days, the primary follicles contained oocytes at the diplotene stage. After 43 days, the primary follicles of the ovarian cortex continued to increase (Fig. 5A-C). Ovary colonized by male germ cells. A pro- gressive degeneration of the ovarian cortex took place (Fig. 5D-E). This degeneration could be compared with that described for the cortex of intersexed male gonads. The first evidence of it was already seen after the second passage (23 days); in a great number of "male oocytes," the chromatin was markedly retracted. After 33 days, the degeneration of the "male oocytes" was even more striking: the nucleus became light and looked empty, the chromatin was dispersed and accumulated against the nuclear membrane, which was underlined by a dark line, or tended to disappear. After 43 days, the cortical region was represented only by a few areas of degenerated cells. Follicular cells did not surround the oocytes in the course of degeneration: they were grouped in clusters on the side of the medulla. It should be pointed out that in some cases, Haffen (1969a) has observed the existence of some rare primary follicles in the cortex which was, besides this feature, mainly occupied by germs cells in the course of degeneration. Haffen attributed 267 their formation to the presence of female gonocytes which were found in the posterior part of the chimeric embryo. This was the most plausible explanation since the avian germinal crescent has a statistical value. It may happen that in the course of the caudocephalic migration of the germinal material, some gonocytes remain definitely delayed (Fargeix and Theilleux, 1967; Dubois, 1967). In embryos after the 10-somite stage, dispersion of the primary gonocytes of the germinal crescent could have begun. It should be pointed out that the coexistence of such normal follicles and of degenerating "male oocytes" in the same ovarian cortex has provided for an additional and particularly convincing argument for this demonstration. Testes colonized by male or female germ cells. Testes were grafted from chimeric embryos into three or four successive hosts; some of them were certainly colonized by female germ cells and the others, by male germ cells. No differences could be observed in the evolution of the two categories of germ cells. Both looked like spermatogonia until 43 days. It should be recalled that in normal development, gametogenesis is delayed in the male as compared with the female. The experimental conditions did not allow the meiosis stage to be reached. These experiments have shown that somatic tissues in an ovary can impose a begining of female sex differentiation on genetically male germ cells, but that they do not ensure their subsequent evolution in the female direction. Indeed, "male oocytes" cannot cross the threshold of the premeiotic prophase and thus degenerate. The reasons for this delayed failure of sex differentiation of "male oocytes" have certainly to be searched for at the level of perturbation in the mechanism of meiosis. An ultrastructural study of cortical differentiation in chick ovaries and in intersexual gonads has been made by Narbaitz (1971). He came to the same conclusion as Haffen (1969a), i.e., that the primary incompetency responsible for cortical degeneration lies in the germ cells and not in the somatic (prefollicular) cells. The problem of evolution of female 268 KATY HAFFEN *#--<ii .;*&£* N1*^3S FIG. 5. Grafting experiments of ovaries colonized by female and by male germ cells into successive embryonic hosts. Differentiation of female germ cells (/f-C). Degeneration of male germ cells (D-F).A, Graft II (23 days) oocytes in meiotic prophase; leptotene structure of the nuclei. B, Graft III (33 days); primary follicles; diplotene structure of the nuclei. C, Graft IV (43 days); growing follicles. D, Graft II (23 days); "male oocytes" in meiotic prophase. Several nuclei show retracted chromatin (r.ch.). E, Graft III (33 days); numerous degenerating "male cocytes" (D.o.). Others still show a retracted chromatin (r.ch.). F, Graft IV (43 days); the ovarian cortex contains only degenerated "male cocytes". Magnification for A, D: x 1125; forB, C: x 450; forC, F: x 540. (From Haffen, 1969a.) germ cells in a testis could not be resolved experimentally by Haffen (1969a); indeed, as already shown by the experiments of Benoit (1932) (see in Taber, 1964), ablation of the left ovary shortly after hatching induced the transformation of the right rudimentary gonad into a testis and the evolution into spermatozoa of those gono- AVIAN GONADS IN VITRO cytes which were still present at the time of ovariectomy ovariectomy. 269 the steroid nature of the hormonal secretion produced by avian embryonic gonads and suggest the existence of specific enzymes playing a role in the series of transDISCUSSION formation reactions which lead to estrogen production through androgens from a The experiments using organ culture of common precursor. sex organs of birds set up by Wolff and It should be noted that Weniger (1970) Haffen (1952a) led to the following conclu- never isolated labeled testosterone from sions: culture media of 10- to 19-day embryonic The gonads develop outside the or- testes, whatever the labeled precursor was. ganism into testes or ovaries according to These results are inconsistent with those of their genetic sex. Male or female develop- Cedard and Haffen (1968), Guichard et al. ment of a gonad primordium is an example (I973a,b), Scheib et al., (1974), and Galli of autodifferentiation. The gonads possess and Wassermann (1972, 1973) as well as the factors necessary for their differentia- with those of Woods and Podczaki (1973). tion at an early stage of their development. These latter authors have recently demonNatural and synthetic hormones act di- strated by use of an immunohistochemical rectly on the target organ (testis, Mullerian technique that androgens are first syntheduct) without intervention of any other sized by the sexually undifferentiated gogland in the embryo. The similitude of the nads of both presumptive sexes of the chick effects of embryonic and adult hormones embryo on day 3'/4 of incubation. has led Wolff and Ginglinger (1935) and Histoenzymology has provided imporWillier (1939) to postulate that sex differentant data as to the cellular localization of the tiation is realized through an hormonal steroidogenic function in embryonic mechanism and that the same hormones act gonads. This technique revealed a A5-3/3during the whole of development, from HSDH activity in the medullary cords beembryonic organogenesis to the adult fore appearance of the ultrastructural stage. characteristics of secretory cells. It furthNew techniques such as radiochro- ermore demonstrated that in the ovary, the matography, histoenzymology, and elec- enzymatic localization is limited to the sectron microscopy have provided clues as to retory cells in the medulla, which have been the steroid nature of the gonadal secretion characterized by electron microscopy and to the cellular support which ensures (Narbaitz and Adler, 1966; Scheib, 1970a). this function. The observations of Scheib and Haffen Embryonic avian gonads cultured in (1968) on chick testis have emphasized that vitro can utilize various radioactive precur- from the 8th day there are present in the sors added to the culture medium and con- cords certain cells showing the same Asvert them into steroid sex hormones. Es- 3/3-HSDH activity as the interstitial cells of trogens are essentially synthesized in the stroma. These results confirm the hisfemale gonads and testosterone in male tological observations of Benoit (1923) who gonads. Estrogen and testosterone synthe- described such cells in the cords from the sis from progesterone had been confirmed 12th day onward. They provide arguments by incubation of gonads in buffered saline in favor of the theory of the epithelial ori(Galli and Wassermann, 1972, 1973). Es- gin of the interstitial cells. By electron mitrogen synthesis in culture media from croscopy Narbaitz and Adler (1966) have Na-acetate by 6-day-old gonads (Weniger observed the presence of interstitial cells in and Zeis, 1971) has provided a worthy ar- the testicular stroma from the 9th day ongument to the theory of Wolff, according ward and found some cells with an analoto whom estrogens would be responsible for gous structure in the 16-day cords. the characteristic morphological changes Scheib (19706) reports that from the 8th upon female gonad differentiation. day there are present in the testicular cords These results as a whole are in favor of somatic cells with the same ultrastructural 270 KATY HAFFEN characteristics as interstitial cells. According to this author, the interstitial tissue in the testes results from the first limited and then massive differentiation of cells which were initially included in the cords and which colonize the interstitial spaces. The last problem evoked in the present paper deals with the evolution of male germ cells, in the chick, when they are forced into female differentiation by early colonization of a female gonad. These cells degenerate after entering the premeiotic stage. New research should thus elucidate the causes of this disorder. In the quail, this disorder seems to be less pronounced. Indeed, application of diethylstilbestrol to quail embryos induces a feminization of the gonads which persists partially after hatching until the adult stage. Germ cells are converted into oocytes, start their growth and become surrounded by follicular cells, thus giving rise to large sized follicles (Haffen, 19696). On the other hand, the disorder in meiosis does not seem to exist upon spermatogenesis of female germ cells in the right chick gonad, which converts itself into a testis when left ovariectomy is performed after hatching (Benoit, 1932). Reynaud (1969, 1971) has realized interspecific transfers of germ cells (from turkey to chick) as well as intraspecific transfers (from one strain to another), by injecting these cells into the blood stream of previously sterilized chick embryos. These experiments demonstrated the existence of complete gametogenesis of the primordial germ cells injected. However, they do not provide clues as to the eventual differentiation of female germ cells in a male soma, since the injected cells represent a population of heterosexual cells from several germinal crescents. It should be recalled that in amphibians and fishes, cytodifferentiation of germ cells can be completely inverted (see Gallien, 1967). 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