Anti-Müllerian hormone (AMH), also known as Müllerian-inhibiting substance or factor, has previously been shown to sex-reverse the steroidogenic pattern of fetal mammalian ovaries through repression of aromatase biosynthesis. Study of the ontogeny of the response of cyclic AMP-stimulated aromatase activity of rat fetal ovaries to AMH has allowed us to develop a quantitative biossay for the hormone. Linear responses as a function of the logarithm of AMH concentration were observed over ranges of 0.2 -7.5 μg/ml for the bovine protein and 0.15 -2 μg/ml for the human protein, with a maximal decrease in aromatase activity of 90% for both proteins. Under the same in vitro conditions, AMH treatment did not affect cyclic AMP-stimulated fetal rat testicular aromatase activity. Partially purified chick AMH also decreased rat ovarian aromatase activity, allowing us to use this test to study AMH ontogeny in chick gonads. Analysis of the species specificity of AMH repression of ovarian aromatase activity indicated that turtle and rat fetal ovaries responded to AMH of other vertebrate classes, whereas aromatase activity of chick embryo ovaries could be repressed only by the homospecific hormone.
Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (MIS) or factor (MIF) plays an important role in sex differentiation. Besides promoting regression of Müllerian derivatives in male fetuses (Jost, 1953), the hormone inhibits oogonial proliferation and induces the formation of seminiferous cord-like structures in fetal ovaries exposed to it in organ culture (Vigier et al., 1987). Both the inhibiting and virilizing effects of AMH upon the developing ovary have recently been confirmed in transgenic mice (Behringer et al., 1990). AMH has also been shown to sex-reverse fetal ovarian steroidogenesis by repressing cytochrome P450 aromatase enzyme biosynthesis (Vigier et al., 1989).
Up to now, the only test for anti-Müllerian activity was based upon the work of Picon (1969), showing that fetal rat Müllerian ducts in organ culture regress in the presence of AMH of other mammalian species. This labor-intensive procedure is at best semi-quantitative, and requires high concentrations of AMH to obtain unequivocal histological evidence of Müllerian regression. We have used the observation that AMH can decrease aromatase activity in the fetal rat ovary to develop a more sensitive, quantitative and interspecific bioassay.
Materials and methods
Wistar rats were used between 13 days post-coitum (the day following the night of mating was considered day 0) and 10 days post-partum. Release of fertilized eggs from the European pond turtle Emys orbicularis was induced by intracoelomic injection of 5 i.u. of oxytocin (Ewert and Legler, 1978). Eggs were incubated for either 55 days at 25°C or 35 days at 30°C, to obtain respectively 100% male or 100% female offspring at stage 24 of development, at which point differentiated gonads are no longer sensitive to temperature (Pieau, 1974; Pieau and Dorizzi, 1981). Gonads were removed from chick embryos (white Leghorn, Gallus gallus L.) which had been incubated for 8 to 17 days at 38°C, and from an adult rooster after decapitation.
Native testicular bovine AMH was purified from incubation medium of bovine fetal testes as described (Picard and Josso, 1984). Human recombinant anti-Müllerian hormone (hAMH) was purified from transfected CHO cell culture medium (Pepinsky et al., 1988). Purified AMH was quantified by reading optical density at 280 nm, using an extinction coefficient of 1.
Partially purified chick AMH was obtained by incubating 250 10-17-day-old chick embryo testes in organ culture for 3 days. The corresponding incubation medium (25 ml) was loaded on a 0.4 ml column containing immobilized Lens culinaris lectin (Sigma), equilibrated with Tris-HCl, 20 mM, pH 7.7 buffer, and eluted by addition of NaCl, 300 mM and 10% N-acetyl-glucosamine. The protein concentration of the pooled 3 fractions with the highest optical density at 280 nm was 0.3 mg/ml. Protein bands were visualized by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, in reducing and non-reducing conditions, as described (Picard and Josso, 1984) and biological activity was tested by the Müllerian duct assay (Picon, 1969). Incubation medium from chick heart was subjected to the same treatment and used as control.
Production of monoclonal antibody (mAb) 278 against bovine AMH (Vigier et al., 1982) and of mAb 10.6 against hAMH (Pepinsky et al., 1988) has been documented. Unlabelled chemical reagents were purchased from Sigma.
Gonads were explanted 3 days in organ culture, as previously described (Vigier et al., 1987). Rat gonads were explanted together with the adjacent mesonephros, which provides a ‘handle’ for subsequent manipulation. Aromatase activity of ovaries cultured with or without their adjacent mesonephros is comparable (results not shown). In experiments calling for association of gonads of different ages or species, rat fetal ovaries were always cultured whole, other gonads were cut into fragments of similar size. When gonads of different sex anchor age were compared for their effect upon ovarian aromatase activity, an effort was made to keep the amount of co-cultured tissue relatively constant. The mean protein content of co-cultured tissue is shown on Table 1. Culture medium was CMRL 1066 (Eurobio, France) containing 0.25 mg/ml bovine serum albumin and, for rat gonads, 0.1 mM 3-isobutyl-l-methylxanthine and 1 mM N6,O2-dibutyryladenosine 3′,5′-cyclic monophosphate (Bt2cAMP).
Measurement of aromatase activity
Aromatase activity was measured at the end of the culture period by the tritiated water technique (Ackerman et al., 1981) as previously described (Vigier et al., 1989). Organ culture expiants were incubated for 5 hrs in groups of 4 in a Dubnoff incubator under a 95%O2/5%CO2 atmosphere in the presence of 0.7 μM [lβ-3H]androstenedione (27.4 Ci/mmol) obtained from NEN/Dupont. All experiments were carried out in triplicate unless stated otherwise. Linear regression and analysis of variance were computed using ‘ABSTAT software (Anderson-Bell, Co), the residual variance was used for comparing mean aromatase activities of control and treated ovaries.
Tissue processing and assay of protein concentration
After the aromatase assay, rat fetal ovaries and a representative sample of other cultured gonads were subjected to histological analysis as described (Vigier et al., 1989) to check for possible necrosis. All other gonads were solubilized as described (Vigier et al., 1989) and protein concentration was measured by the bicinchoninic acid assay (Redinbaugh and Turley, 1986) using the BCA protein reagent (Pierce®) with the enhanced protocol described by the manufacturer. This technique could not be used to measure protein concentration in the presence of A-acetyl glucosamine. Therefore the protein concentration of the eluate of the lectin column was assessed by measuring optical density at 280 nm, using an extinction coefficient of 1.4.
Effect of AMH upon aromatase activity of fetal rat ovaries
Ontogeny of response of developing rat gonads to AMH
Aromatase activity of developing rat ovaries explanted 3 days in organ culture in control medium was low throughout gestation and up to 1 day after birth (Fig.1A) . The activity could be enhanced by Bt2cAMP, particularly at 16 days p.c., at which time a tenfold stimulation in activity was obtained. AMH treatment reduced aromatase activity of Bt2cAMP-treated expiants to control levels. Results obtained later than one day post-partum, using the organ culture technique, yielded inconsistent results (not shown), probably due to defective survival of the expiants. The low basal aromatase activity of fetal testes in organ culture was increased by Bt2cAMP treatment from 16 days post-coitum onwards, however this stimulation could not be inhibited by concomittent AMH treatment (Fig.1B) .
Response to mammalian AMH
Since 16-day-old rat fetal ovaries showed the greatest degree of inhibition of Bt2cAMP-stimulated aromatase activity by AMH, this developmental stage was chosen to study dose-response relationship (Fig. 2). A linear log/dose response to AMH treatment was demonstrated between 0.2 and 7.5 μg/ml of bovine AMH (r=0.964, P<0.01) and between 0.15 and 2 μg/ml for hAMH (r=0.918, P<0.01). Intra-assay and interassay variations were respectively 12.3% (n=3) and 14.6% (n=3) for bovine AMH and 12.2% (n=3) and 19.2% (n=4) for human AMH. Recombinant human AMH was more active than bovine AMH in this system, with an ED50 equal to 0.42 μg/ml compared to 1.12 μg/ml for bAMH. Monoclonal antibodies to bAMH and hAMH, added to the culture medium at a concentration equal to five times that of the hormone, decreased the bioactivity of their antigens by 90% and 70% respectively (Fig. 2).
Response to chick AMH
Chicken AMH obtained by lectin chromatography of testicular tissue-incubation media exhibited the expected reduction-sensitive pattern on polyacrylamide gels (Fig. 3). Its apparent relative molecular mass, respectively 160× 10 for the dimer and 76×103 for the monomer, was slightly higher than that of human recombinant AMH. A contaminant, of approximately 72×103Mr in non-reducing conditions is resolved into 2 smaller subunits in reducing conditions. The biological activity of the preparation was tested by the aromatase (Table 2) and Müllerian duct assay (Fig. 4). Strongly positive results were obtained with both techniques. Assuming that the hormone preparation is approximately 30% pure, chick AMH at an estimated concentration of 12 ng/ml produced a 93% decrease of aromatase activity and induced complete regression of rat fetal Müllerian ducts at 20 μg/ml. A precise dose/response curve cannot be constructed, in the absence of an accurate assay for chick AMH concentration.
AMH production by chick gonads studied by the aromatase technique
The ontogeny of AMH production by chick gonads was studied by exposing 16-day-old fetal rat ovaries to gonadal tissue of developing chickens, and measuring the aromatase activity of the rat ovaries at the end of the culture period. Results are shown in Fig. 5. The ability of chick testes to decrease rat fetal aromatase activity is high between 8 and 17 days of incubation, and is lost in the adult. The left fetal ovary exhibits increasing anti-aromatase activity after ten days, while the stunted right ovary and fetal chick cardiac tissue have no significant effect.
Effect of AMH upon aromatase activity of nonmammalian fetal ovaries
Spontaneous aromatase activity of 17-day-old chick ovaries was extremely high, insensitive to Bt2cAMP treatment (not shown), and could not be significantly decreased by hAMH, 10 μg/ml (Fig. 6A). The effect of homospecific AMH was tested by co-culturing chick ovaries with chick testes of the same age. The aromatase activity of chick embryonic ovaries was significantly inhibited by co-culture with chick testes (P < 0.01), whether expressed per gonad (Fig. 6A) or per μg of ovarian protein (not shown).
In the turtle Emys orbicularis at the only developmental stage tested, the ovaries exhibited spontaneous ovarian aromatase activity (Fig. 6B), which was not modified by Bt2cAMP treatment (results not shown). Bovine AMH at the concentration of 10 μg/ml reduced aromatase activity by 79.2% (P < 0.01), nearly down to the level observed in control testes of the same age.
The data presented indicate that the aromatase activity of 16-day-old rat fetal ovaries can be used as an endpoint for a bioassay of AMH bioactivity. This compares favorably with the Müllerian duct bioassay (Picon, 1969), because it is quantitative and because approximately 10 times less hormone is required to achieve half-maximal inhibition of aromatase activity. Differences in bioactivity between mammalian hormone preparations is probably due to the purification method used: bAMH is purified from tissues obtained at autopsy, while the recombinant molecule is purified from cell culture medium. The bioassay also responds to chick AMH and can be used for testing AMH secretion by gonadal tissue: chick embryonic testes, between 8- and 17-days-old, and chick fetal ovaries aged 14 or 17 days, which had been previously reported to exhibit anti-Miillerian activity by the Picon bioassay (Hutson et al., 1981), were bioactive in the aromatase assay as well. Cyclic AMP is required to accelerate maturation of the aromatase enzymatic activity of the fetal rat ovaries, which do not normally synthesize aromatase in significant amounts prior to birth (Picon et al., 1985). The nucleotide has been shown to block the effect of AMH upon rat fetal Müllerian ducts (Ikawa et al., 1984), but does not block the effect of AMH upon the fetal ovary.
Like the Müllerian duct assay, the bioassay based upon aromatase activity of fetal ovaries is interspecific only within certain limits. Rat and turtle ovaries respond to heterospecific AMH while the chick ovarian aromatase activity is affected only by the homospecific hormone, whether partially purified from testicular incubation media or released by embryonic gonads. These findings are in keeping with those reported by Tran and Josso (1977), who observed regression of rat Müllerian ducts co-cultured with chick testes, but no effect of mammalian fetal testicular tissue upon chick Müllerian ducts. Purification of chicken AMH to homogeneity from secretory proteins of 8-week-old male chickens has been reported, using a complex sequence of purification steps (Teng et al., 1987); in our hands significant purification, yielding a highly bioactive protein was achieved by a single passage through a lectin column.
Estrogen is thought to play a major role in the gonadal differentiation of many vertebrate species (Dorizzi et al., 1991). Repression of constitutive estrogen production and diversion to testosterone production may be needed for testicular differentiation to occur. Certainly, for AMH to inhibit Müllerian duct development, estrogen must not be present (Hutson et al., 1982; Newbold et al., 1984). Could AMH play a role in testicular differentiation by blocking estrogen synthesis in the primitive gonad? AMH inhibited aromatase biosynthesis in fetal ovaries in all three vertebrate classes investigated, regardless of their genotypic or environmental mode of sex determination, but its effect was not demonstrable in rat testicular tissue (Fig. 1B). One possible explanation could be the absence of AMH receptors in the testicular cells producing aromatase, or their saturation by endogenous AMH.
Bioactivity of peptide hormones is mediated by receptor-ligand interaction at the cell membrane level. Why rat ovarian and Müllerian receptors should be less stringent than chick receptors in their binding specificity requirements is difficult to understand and an answer to this question would necessitate isolation of AMH receptors in these species. An alternative possibility deserves consideration. The AMH molecule undergoes proteolytic cleavage at a monobasic site (Pepinsky et al., 1988); studies are in progress to determine whether this cleavage is required for its activation. If AMH proves to be similar in this respect to other TGF-β-like proteins, insensitivity of chick ovaries and Müllerian ducts to mammalian AMH might be caused by failure of chick tissues to cleave the mammalian AMH molecule. For instance, it has been suggested that mouse cells cannot be infected by human HIV even when the human CD4 receptor is present because they lack a suitable proteolytic enzyme (Stephens et al., 1990). Further investigation into structure/activity relationship for the AMH molecule is necessary to address this problem and will be facilitated by the availability of the quantitative bioassay we describe.
We gratefully acknowledge the technical assistance of Franck Louis.