CARACTERÍSTICAS BÁSICAS DE LA INTERRUPCIÓN

The presence of androgen and oestrogen receptors in the epithelial and mesenchymal tissue of the developing ovine mammary gland suggests not only a role for steroid hormones in fetal mammogenesis, but also potential sensitivity to androgenic and oestrogenic stimuli from the environment. Therefore, it was proposed to inject pregnant ewes with substances that were representative of the compounds that are likely to cause disruption of mammary gland development and assess their effects on both the ewes and their fetuses.

The effects of androgen, anti-androgen and anti-oestrogen treatment of pregnant ewes on development of the fetal mammary gland were examined in two studies. In a preliminary study (experiment one), twin-bearing ewes (n=5) were treated by once-weekly intramuscular injection of testosterone 1 7�-cypionate (200 mg/ml) at days 36, 43, 50 and 57 of gestation. Ewes and fetuses were euthanased at day 1 40 of gestation.

In experiment two, 36 twin-bearing ewes were randomly assigned to one of four treatments (n=9 ewes per treatment group). Testosterone (representative androgen) and cyproterone acetate (Androcur, representative anti-androgen) treatments were administered in commercially available slow release formulations by twice weekly intramuscular injection (1 00mg or 1 ml) at days 36, 40, 43, 47, 50, 54 and 57 of gestation. Tamoxifen (representative anti-oestrogen) (0.085g in 1 .7ml cottonseed oil) and cottonseed oil treatments were also administered by twice­ weekly intramuscular injection on the same days. Three ewes, and their fetuses, from each treatment group were euthanased at days 61 or 1 1 7 of gestation. The remaining 1 2 ewes were allowed to proceed to term and their milk production recorded for the first seven days of lactation. All lambs were euthanased at birth. Histomorphological and immunohistochemical analyses determined the extent of mammary development and immunoreactivity to androgen receptor antibody, respectively.

In both experiments, prenatal testosterone exposure partially masculinised the external genitalia of female fetuses producing a penis and empty scrotum with no external vaginal opening. These females also possessed visually normal ovaries and all the derivatives of the Mullerian ducts (fallopian tubes, uterus, cervix, anterior vagina) and Wolffian ducts (epididymides, vas deferens, seminal vesicles). Administration of Androcur to pregnant ewes did not cause feminisation of the male offspring.

In experiment two, mammary gland development of males and masculinised females at birth did not differ (P<O.06), whilst females from the other three treatment groups had significantly (p<0.01 ) greater total duct and secretory cell areas than their male counterparts. In contrast, total duct area at day 140 of gestation, in experiment one, was significantly (p<0.05) greater in masculinised females relative to males, but severely suppressed relative to phenotypically normal females (Chapter two).

Where immunoreactivity to androgen receptor antibody was positive, the sites of reactivity were predominantly localised in the apical cytoplasm of the ductal glandular epithelium and the fibroblasts of the dermal mesenchyme. At each stage of development examined, the intensity of immunostaining was greater in males and testosterone-treated females relative to control females and females exposed to Androcur.

The milk yield of Tamoxifen-treated ewes was significantly (P<0.05) lower than that of all other treatment groups at each milking, but there was little discernible effect on milk composition. The effect on lactational performance was reflected in significantly reduced maternal plasma progesterone concentrations relative to control ewes at days 1 20 and 1 40 of gestation.

These findings indicate a role for testosterone in the sexual dimorphism of the ovine mammary gland. Moreover, they emphasise the sensitivity of the developing fetus to the adverse effects of exposure to environmental androgens and oestrogens during the period of sexual differentiation. Furthermore, there is cause for concern with respect to the secretory capacity of the maternal mammary gland after exposure to environmental 'endocrine disruptors' given the current focus on increased lambing percentages.

5.2

I ntroduction

Earlier observations (Chapter two) and those of others (Wallace, 1 953; Martinet, 1 962) demonstrated a gradual divergence in the extent of development of the mammary gland epithelial and mesenchymal structures of male and female fetal sheep. While the epithelial and adipose tissue of the female gland continued to proliferate within an extensive network of fibrous connective tissue with increasing fetal age (Chapter two; Forsyth et aI., 1 999), complete differentiation of those cellular structures in the male gland was prevented. By three weeks of postnatal age the rapidly proliferating dichotomously branched epithelium of the female gland had penetrated an extensive fat pad (Chapter two; Akers, 2002). In contrast, the epithelium of the male was restricted to an area of fibrous connective tissue immediately dorsal to the teat, having failed to penetrate the fat pad, which was present in a much lower abundance within the male mammary gland (Chapter two).

The rodent model has provided overwhelming evidence that androgens, specifically testosterone, produced from the fetal testis are directly responsible for the prevention of the complete differentiation of the epithelial cells into a mammary gland capable of synthesising milk in the male. This model also established that androgen binding in the mammary gland is due to the expression of androgen receptors and that the fetal gland shows endocrine sensitivity. Androgen-induced regression of the mammary gland can also be elicited in females with exogenous androgen. Testosterone propionate administered to pregnant mice during the period of androgen responsiveness caused separation of the mammary bud in female fetuses as well as males (Hoshino, 1 965). Cyproterone acetate, an anti-androgen, prevented regression of the mammary bud in male fetuses resu lting in nipples being present on all rats born to mothers

treated from day 1 2 of gestation with this compound (Eiger and Neumann, 1 966; Neumann and Eiger, 1 966).

Whether differences between the patterns of mammary development in male and female sheep are a result of the action of androgen receptors in the male under the influence of testicular androgens is not known. The mammary gland of the ovine fetus does possess androgen receptors (Chapter three) but there are two distinct differences between mammary development in the mouse and the sheep that discourage an extrapolation of results. First, whereas the mouse mammary gland possesses androgen receptors exclusively in the mesenchymal cells (Heuberger et al., 1 982; Wasner et al., 1 983), the ovine mammary gland possesses androgen receptors in both the mesenchymal cells and the epithelial cells of the ducts (Chapter three). Second, while sexual dimorphism of the ovine mammary gland appears to be a gradual process (Chapter two), in the mouse there exists only a short (about 30 hours) androgen-responsive "window" when androgen-induced regression of the male mammary gland occurs (Kratochwil, 1 971 ).

As initially determined by Short (1 974), and later refined by Clarke et al. (1 976), sexual differentiation in the fetal sheep occurs from approximately 30 to 1 00 days of the 1 47 day gestation. However, only a short period of sensitivity to androgens exists (between day 40 and 50 of gestation), which results in complete masculinisation of the external genitalia of the female offspring. Treatment of pregnant sheep with testosterone, either as an implant (Short 1 974; Clarke et al. 1 976; Tarttelin 1 986) or as repeated intramuscular injections of testosterone cypionate, a long-acting androgen (Wilson and Tarttelin 1 978; Tarttelin 1 986; Wood et al. 1 991 ; Wood et al. 1 995), from days 20, 30 or 40 of gestation caused complete masculinisation of the external genitalia in the female offspring. Later treatments were ineffective. These findings suggest that in utero exposure of female fetuses to androgens during the period of sensitivity to

masculinisation by gonadal steroids may give indirect evidence that inhibition of mammary gland growth in the ovine male is dependent on its exposure to testosterone.

Like that of the fetal rodent, the mammary gland of the fetal sheep also possesses oestrogen receptors (Chapter three). However, in contrast to the rodent, oestrogen receptors in the mammary gland of the sheep are present in both the mesenchymal and epithelial compartments during prenatal life. The normal progression of mammary morphogenesis in oestrogen receptor knockout mice suggests that prenatal development of the mammary gland is autonomous (Bocchinfuso and Korach, 1 997). In view of this, one would expect to observe, through maternal treatment with an oestrogen or anti-oestrogen, the mammary gland of the female offspring to develop as normal. However, the mammary mesenchyme of the fetal rodent displays oestrogen sensitivity. For example, epithelial cell density was significantly increased at four weeks of age in the female offspring of rats treated with 20 ng oestradiol per day between days 1 4 and 20 of gestation. But, the effect was not permanent: growth in the controls was equivalent by seven weeks of age (Hilakivi-Clarke et al., 1 997). In contrast, the prevalence of terminal end buds was

increased by oestrogen at both ages, although it was not known whether the effect persisted into maturity.

Given that fetal mammary tissue is responsive to stimuli from exogenous steroid hormones, the gland is potentially at risk from exposure of the mother to endocrine-disrupting chemicals, such as pesticides and industrial chemicals (Sonnenschein and Soto 1 998). Of particular relevance to domestic ruminants are the environmental oestrogens, which have the potential to alter sex differentiation and hence, mammary gland development. One such class of environmental oestrogens are the phytoestrogens, which are plant compounds with structural similarities to natural and synthetic oestrogens and anti-oestrogens (reviewed by Kurzer and Xia, 1 997). A similar group of compounds are the oestrogenic mycotoxins, one of which, zearalenone, is commonly found in New Zealand pastures (di Menna et al., 1 987). Ewes grazing pasture grasses and clovers infected with mycotoxins may display markedly reduced reproductive performance through lowered ovulation rates, failure of fertilisation and anovulation (Smith et al., 1 990).

Rodent models in which the pregnant mother was treated with oestrogens or oestrogenic compounds have provided evidence that the mammary gland is sensitive to the effects of exogenous steroids during prenatal development. The mammary glands of the female offspring of pregnant mice injected with 2 �g zearalenone from days 1 5 to 20 of gestation exhibited increased terminal end buds and increased epithelial differentiation (Hilakivi-Clarke et al., 1 998). On the other hand, in utero exposu re to 20 �g genistein from days 1 5 to 20 of gestation resulted

in more terminal end buds, and fewer differentiated epithelial structures, when compared to the glands from offspring exposed to the vehicle alone (Hilakivi-Clarke et al., 1 998).

While their effect on the development of the ruminant mammary gland is still unclear, the possibility exists that compounds that interfere with oestrogen action have the potential to alter mammary development and hence, impede the capacity to produce milk in later life. Therefore, it was hypothesised that the expression of oestrogen receptor in the fetal ruminant mammary gland makes it a target for hormonal disruption through exposure to exogenous oestrogens. Furthermore, it was proposed that the pattern of mammary morphogenesis in the male was, in part, dependent on the secretion of androgens from the male testes. The experiments herein have compared:

1 . Morphological measurements and immunoreactivity to androgen receptor antibody in normal males and females, and in males and females whose dams were treated with testosterone cypionate or Androcur from days 36 to 56 of gestation, to provide indirect evidence of a role for testosterone in suppression of mammary growth in the male.

2. Morphological measurements in normal females and in females whose dams were treated with tamoxifen (an anti-oestrogen) from days 36 to 56 of gestation to provide evidence that the mammary gland is/is not sensitive to the effects of exogenous steroids during prenatal development.

The effects of androgen, anti-androgen and anti-oestrogen treatment upo"n mammary development at parturition and subsequent milk yield in the ewes were also investigated.