Differential sensitivity of rat uterine growth and epithelium hypertrophy to estrogens and antiestrogens.

Triphenylethylene antiestrogens are considered weak estrogen agonists based on their limited ability to induce estrogen responses, in particular uterine growth. We compared the uterotrophic activity of naturally occurring and synthetic estrogens with that of antiestrogens by quantitating uterine wet weight and hypertrophy in the uterine luminal and glandular epithelium. Immature rats received five daily injections of either an estrogen (17 beta-estradiol [E2], diethylstilbestrol [DES], or ethynyl estradiol [EE]) or an antiestrogen (tamoxifen [TAM], monohydroxytamoxifen [OH-TAM], or clomiphene citrate [CC]) (0.001-100 micrograms/rat/day) subcutaneously in sesame oil and were sacrificed approximately 2 hr after the last injection. Both DES and EE increased uterine weight at doses between 0.01-100 micrograms/rat/day; E2 was about 10-fold less potent. The antiestrogens increased uterine weight only slightly. DES, EE, and the three antiestrogens each increased luminal epithelium hypertrophy to over 3-fold above that in controls. While the potencies of these synthetic compounds differed (DES = EE > OH-TAM > TAM = CC), each hypertrophic response occurred over two log doses, and the response curves displayed identical slopes. E2, however, required a range of four log doses to achieve the same degree of luminal epithelium hypertrophy. The three antiestrogens elicited glandular epithelium hypertrophy up to 2-fold above controls at the same doses that induced luminal epithelium hypertrophy; the order of potency was OH-TAM > TAM = CC. However, the three estrogens increased glandular epithelium hypertrophy only marginally. Thus, under dosing conditions commonly used to assess uterotrophic activity, these "antiestrogens" are complete, albeit less potent, estrogen agonists in the luminal epithelium and, unlike estrogens, induce hypertrophy in the glandular epithelium.

Uterine abnormalities in rats exposed neonatally to diethylstilbestrol, ethynylestradiol, or clomiphene citrate.

The toxicity of the synthetic estrogens diethylstilbestrol (DES), and ethynylestradiol (EE), and the antiestrogen clomiphene citrate (CC) was evaluated by assessing postnatal uterine growth and development prior to the onset of puberty in the rat. Both DES and EE, administered during the neonatal period (postnatal days 1-5), initially increased uterine weight and luminal epithelium hypertrophy. However, uterine weight declined in both DES- and EE-treated animals and fell below controls beyond day 11. Luminal epithelium stimulation generally paralleled uterine weight changes. Precocious development of uterine glands occurred after estrogenization (compared to untreated controls), but subsequently gland numbers were approximately 60% of control levels. Neonatal CC exposure induced only slight uterine weight gain but caused prolonged luminal epithelium hypertrophy and inhibited uterine gland genesis. Luminal epithelium hypertrophy appears to be a useful measure of antiestrogen activity. These data demonstrate the toxicity of DES and EE as assessed by altered prepubertal uterine gland development. Additionally, the inhibition of uterine gland genesis after neonatal CC exposure occurs in conjunction with prolonged luminal epithelium hypertrophy.

Alterations in developing rat uterine cell populations after neonatal exposure to estrogens and antiestrogens.

Exposure of rats to either estrogens or antiestrogens during early postnatal development reduces subsequent uterine growth as measured by uterine weight. However, individual uterine cell types respond differently to these agents and uterine weight alone cannot discern subtle or even large alterations in individual cell populations. Using a computerized planimetric technique, we estimated the prepubertal growth of the uterine luminal epithelium, endometrial stroma, glands, and circular and longitudinal muscle after exposure of neonatal rats (postnatal days 1-5) to the estrogens 17 beta-estradiol (E2), diethylstilbestrol (DES), or ethynylestradiol (EE), and the antiestrogens tamoxifen or clomiphene citrate. On postnatal day 26, the cross-sectional areas of the luminal epithelium, endometrial stroma, and circular muscle were reduced after estrogen exposure, compared to untreated controls, while longitudinal muscle cross-sectional area was not affected. Since cell densities (cell number/unit area) were increased, these estrogen-induced area reductions demonstrate a decrease in cell size. Total cell numbers, estimated as the product of cell type areas and their respective cell densities, were also reduced by neonatal estrogen exposure. The synthetic estrogens DES and EE were more potent than E2 with respect to reduction of uterine growth. Neonatal antiestrogen exposure caused large area reductions only in the uterine glands and luminal epithelium. Little change in cell density occurred in any cell population exposed to antiestrogen. These data demonstrate that the decreased uterine growth resulting from estrogen exposure during early postnatal development is a consequence of combined hypotrophy and hypoplasia in all cell types except longitudinal muscle while antiestrogen-induced morphological alterations were limited to hypoplasia having epithelial cell specificity.

Postnatal uterine development in the rat: estrogen and antiestrogen effects on luminal epithelium.

We examined the effects of the synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE), and the triphenylethylene antiestrogen, clomiphene citrate (CC), on uterine growth and development in the rat. These compounds, unlike estradiol, do not bind significantly to rat serum alphafetoprotein (AFP). Administration of DES or EE during the period of normal uterine gland genesis (postnatal days 10-14) induced luminal epithelium hypertrophy and increased uterine wet weight. The durations of these responses were dose-related. By day 26, luminal epithelium cell numbers were significantly depressed, compared to controls. Uterine gland development was delayed 6 to 9 days, depending upon estrogen dose, and the numbers of uterine glands ultimately achieved were generally less than in untreated control animals. While a daily dose of 0.1 micrograms CC/rat did not alter uterine development, 10 micrograms CC/rat caused prolonged luminal epithelium hypertrophy and inhibited uterine gland genesis without inducing the large increases in uterine weight or the decreases in luminal epithelium cell number seen after estrogen exposure. The number of stromal cells was significantly increased on day 26 after CC exposure. Together with previous studies, these data demonstrate the greater potency and developmental stage specificity of non-AFP-bound estrogens with respect to altering uterine gland development. In addition, these data suggest that the disruptive influence of antiestrogens on gland genesis may be mediated through an indirect influence on the uterine stroma.

The postnatal ontogeny of rat uterine glands and age-related effects of 17 beta-estradiol.

In the uterus of the newborn rat, only the luminal epithelium is differentiated. Differentiation of musculature and glandular epithelium occurs postnatally, the latter originating as invaginations of the luminal epithelium into the stroma. Using unambiguous criteria for quantification of uterine glands, we find that uterine glands first appear on postnatal day 9 after which the increase in the number of glands is rapid and synchronous, with approximately 4.4 glands per uterine section reached by day 15. Between days 15 and 35, the number of glands per uterine section varied in a cyclic manner with an amplitude of approximately one gland per uterine section and a period of 6-7 days. Although exogenous 17 beta-estradiol (E2) administered on postnatal days 1-5 induced slight premature gland genesis, the number of glands per uterine section was approximately 30% lower between days 15-26 compared to untreated animals. Administration of E2 during the period of normal gland genesis (days 10-14) induced a dose-related delay in the onset of appearance of glands. After this, gland genesis proceeded at a normal rate; however, the maximum levels reached were again generally below those observed in untreated controls. E2 administered after uterine glands were established (days 20-24) induced a small increase in gland number compared to controls. E2 also induced temporary hypertrophy, hyperplasia, and cellular degeneration in the luminal epithelium during each of the dosing periods without corresponding changes in the stroma or myometrium. These data demonstrate that uterine gland genesis occurs between postnatal days 9-15 and that exogenous estrogen can alter, in an age-specific manner, both uterine gland genesis and the number of glands per uterine section.

Inhibition of rat uterine gland genesis by tamoxifen.

We have previously shown that rat uterine gland genesis occurs rapidly and synchronously between postnatal days 9-15. Exogenous estrogens either stimulate or inhibit gland genesis depending on dose and age at administration. We therefore examined the developmental effects of the triphenylethylene antiestrogen tamoxifen, which exhibits both estrogen agonist and antagonist properties, in the postnatal rat uterus. Tamoxifen administered sc in oil on postnatal days 1-5 or days 10-14 caused dose-related inhibition of uterine gland genesis which persisted to day 26 or day 60, respectively. Tamoxifen administered on postnatal days 20-24, which is after the age of normal gland genesis, did not alter the number of preexisting glands. A 24-h exposure to tamoxifen inhibited 17 beta-estradiol (E2)-induced ornithine decarboxylase (ODC) activity measured 6 h after E2 administration in 14-day-old rats. Treatment with tamoxifen before or during the period of gland genesis also reduced uterine responsiveness to a single dose of E2 as measured by both uterine weight gain (after a 24-h exposure on days 14, 19, 22, and 26) and the pattern of E2-induced ODC activity in 26-day-old rats. Control rats respond to E2 with peaks of ODC activity at 6 and 18 h after administration. Treatment with tamoxifen on either postnatal days 1-5 or 10-14 reduced the 18-h peak to approximately half of controls but did not affect the 6-h E2-induced ODC peak. Analysis of both nuclear and translocatable cytosol estrogen receptor in uteri from 26-day-old rats indicate that neither the dissociation constant (KD) nor the number of binding sites was affected by tamoxifen treatment on postnatal days 1-5 or 10-14.

Uterine responses to estradiol in the neonatal rat.

Although single doses of estrogens are known to be ineffective in stimulating complete uterine responses in newborn rats, repeated doses elicit toxic responses that become evident in adulthood. In this study, neonates injected daily from birth with 10 micrograms 17 beta-estradiol (E2) demonstrated significant uterine wet weight gain by day 3 and near-maximum growth (230% of control) by day 5. Elevated uterine weight can be maintained by repeated daily injections at least through day 13. Other uterine growth responses after 5 days of E2 (as percent of control) are: dry weight, 163%; DNA content, 193%; protein content, 211%; and nuclear estrogen receptor, 890%. Ornithine decarboxylase activity increased to 520% of control when measured 6 h after a single E2 injection on day 5. Histological examination of uteri from animals treated for 5 days reveals an altered stroma with evidence of circular muscle differentiation, while the lumenal epithelium, which is cuboidal in controls, becomes columnar after E2 injections. These data suggest that estrogens act in fundamentally the same manner in the neonatal uterus as in the adult uterus, although the appearance of the complete response appears to e slower in the neonate. The estrogen-induced precocious development we describe suggests that an estrogen may be involved in the normal postnatal development of uterine estrogen responsiveness. Adult toxicity, resulting from repeated neonatal estrogen dosing, may partly be a consequence of continual hormone action inducing developmentally inappropriate responses.

Use of hydrogen peroxide-egg albumin to eliminate nonspecific staining in immunoperoxidase techniques.

The application of hydrogen peroxide and egg albumin to paraffin sections before immunostaining prevents nonspecific staining by immunoperoxidase techniques. This method is more effective than pretreating secions with normal sera, or using either egg albumin or hydrogen peroxide separately, or using diluted antisera with prolonged incuabations in the staining procedure.