Structure-activity relationships of estrogens: effects of esterification of the 11 beta-hydroxyl group

Fourteen esters (formate, acetate, propionate, butyrate, hexanoate, heptanoate, and benzoate) located at C-11 of 11 beta-hydroxyesterone and 11 beta-hydroxyestradiol-17 beta were synthesized and evaluated for uterotropic and gonadotropin release inhibition in rats, as well as their ability to displace (3H) estradiol-17 beta from the rat uterine cytosolic estrogen receptor. The most potent uterotropic agent was 11 beta-formoxyestrone which was 1,625 or 2,500 times as active as 11 beta-hydroxyesterone in the uterotropic or gonadotropin release inhibition assay, respectively. 11 beta-Formoxyestrone was 7.5 times as uterotropic as estradiol-17 beta and equal to estradiol-17 beta in inhibiting gonadotropin release. However, the most potent inhibitor of gonadotropin release was 11 beta-acetoxy-estradiol-17 beta which had 133% of the activity of estradiol-17 beta, although it had only 38% of the activity of estradiol-17 beta in the uterotropic assay. Esters larger than the acetoxy group showed sharply decreased activities in either assay. Despite the high estrogenic potency of the 11-formates or 11-acetates, they were rather weak (6% to 35% as active as estradiol-17 beta) in displacing (3H) estradiol-17 beta from the rat uterine cytosolic estrogen receptor.     

Structure-activity relationships of 9β-estrogens

The 9β isomers of estradiol-17β, estradiol-17α, estrone and 17-ethinylestradiol-17β were synthesized and compared with their 9α-counterparts in the rat uterine cytosol estrogen receptor, utero-tropic, and gonadotropin release inhibition assays. Except for 17-ethinyl-9β-estradiol-17β which was as active as its 9α isomer in the uterotropic assay, none of the 9β estrogens exhibited any biological activity which was equal to or greater than their 9α counterparts. For examples, 9β-estradiol-17β was $\text{1}\text{10}$ as active as estradiol-17β, and 9β-estrone was $\text{1}\text{4}$ as active as estrone in the uterotropic assay.     

Effect of ipriflavone on the response of uterus and thyroid to estrogen

Ipriflavone, 7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one, was uterotropic in intact but not in ovariectomized rats. When it was administered simultaneously with estrone and estradiol-17 beta to ovariectomized rats, ipriflavone increased the uterotropic activities of both estrogens. The uterotropic activity of this compound in intact rats might be attributable to its action on the target organ of estrogen by augmenting the response to the hormone because none of the other possibilities for accelerating uterine growth such as gonadotropin-releasing, gonadotropic, and estrogen metabolism-retarding activities were demonstrated experimentally. In addition, ipriflavone increased the calcitonin releasing activity of estrone when these compounds were administered simultaneously to ovariectomized rats.     

In vitro mycotoxin binding to bovine uterine steroid hormone receptors

The mycotoxins, aflatoxin B(1), aflatoxin M(1), aflatoxicol and zearalenone were tested for binding to bovine endometrial estrogen and progestin receptors. Radioinert estradiol-17beta, estrone, testosterone, and cholesterol were evaluated for binding to the estrogen receptor. Zearalenone and aflatoxicol but not aflatoxins B(1) and M(1) competed with estradiol-17beta for the estrogen receptor. The order of binding affinities for the estrogen receptor were zearalenone > estradiol-17beta > estrone > aflatoxicol. The affinity of zearalenone for the estrogen receptor was 2-3 times that of estradiol-17beta. Progesterone, cortisol, radioinert R 5020, and cholesterol were evaluated for binding to the progestin receptor. None of the tested compounds except R 5020 and progesterone competed for the progestin receptor. The significance of aflatoxicol binding to the estrogen receptor is unclear. It is proposed that aflatoxicol binding to the receptor may alter gene expression in target tissues or act at the level of the hypothalamus to inhibit gonadotropin secretion and ovulation. These effects could explain reports of reduced fertility in domestic animals following ingestion of aflatoxin contaminated feedstuffs. It is also suggested that the mechanism of adverse effects on fertility of chronic aflatoxin ingestion in cattle and other livestock should be more thoroughly investigated.     

Characterization of estrogen binding sites in the liver of the newt Pleurodeles waltl (Urodela:Amphibia): demonstration of a sex-linked heterogeneity

Saturation analysis over a wide range of [3H]estradiol-17 beta concentrations (1-40 nM) in cytosols prepared from liver of the newt Pleurodeles waltl of both sexes revealed a sex-linked heterogeneity of the estradiol-17 beta binding sites. In females, one type of binding site has been identified as a classical receptor. It exhibited a high affinity for estradiol-17 beta (Kd = 9 X 10(-9) M), had a high specificity for estrogenic compounds and was stabilized by monothioglycerol. In males, in addition to the receptor found in females, a second estrogen binding component was detected, not observed in female cytosols. It exhibited a Kd of 4.8 X 10(-8) M for estradiol 17 beta, higher capacity and displayed the same highly specific estrogen binding as does the estrogen receptor. It was affected by monothioglycerol and its binding was found to be significantly increased on cytosol dilution, as well as by estrogen-treatment.     

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.     

Boron estrogens: Synthesis,biochemical and biological testing of estrone and estradiol-17β 3-carboranylmethyl ethers

Synthesis, biochemical and biological testing of the first carborane derivatives of estrogens are described. Estrone 3-carboranylmethyl ether was synthesized in two steps from estrone. Reduction of estrone 3-carboranylmethyl ether with sodium borohydride provided estradiol-17β 3-carboranylmethyl ether. Enzyme kinetic measurements showed that estrone 3-carboranylmethyl ether is a substrate for human placental 17β-hydroxy-steroid dehydrogenase with Km = 5×10^?6M, and Vmax = 0.016 μmol min^?1 μg^?1. The relative affinity constant of estradiol-17β 3-carboranylmethyl ether for rat uterine estrogen receptor was 0.5 (compared with a value of 100 for estradiol-17β). Consistent with its low affinity for estrogen receptor, the dose-dependent uterotropic response to estradiol-17β 3-carboranylmethyl ether in castrated female rats was one sixtieth that of estradiol-17β. None of the tested rats had a toxic reaction to estradiol-17β 3-carboranylmethyl ether. These results demonstrate that exceptionally stable carborane derivatives of estrogens can be synthesized with preservation of their biochemical and biological properties. Boron-containing estrogens may be useful for thermal neutron capture therapy of cancers with estrogen receptors to concentrate boron in the cell nucleus.     

Interaction of ring B unsaturated estrogens with estrogen receptors of human endometrium and rat uterus

The present investigation was undertaken to compare the binding affinities (Ka) of the ring B unsaturated equine estrogens (equilin [Eq], equilenin [Eqn], 17 beta-dihydroequilin [17 beta-Eq], 17 beta-dihydroequilenin [17 beta-Eqn], 17 alpha-dihydroequilin [17 alpha-Eq], and17 alpha-dihydroequilenin [17 alpha-Eqn]) and the classic estrogens (estrone [E1], 17 beta-estradiol [17 beta-E2], and 17 alpha-estradiol [17 alpha-E2]) for estrogen receptors in human endometrium and rat uterus. In both species, the ring B unsaturated estrogens bind with cytosol and nuclear receptors with high affinity (Ka x 10(9) M-1). The relative binding affinities of these estrogens were measured by determining the amount of unlabeled estrogen required to reduce by 50% the specific binding of [3H]17 beta-Eq to endometrial cytosol receptors. The order of activity found was 17 beta-Eq greater than 17 beta-E2 greater than 17 beta-Eqn greater than E1 greater than Eq greater than 17 alpha-Eq greater than 17 alpha-E2 greater than 17 alpha-Eqn greater than Eqn. Essentially the same order of activity was observed when the apparent affinity constants of these estrogens for human and rat cytosol and nuclear receptors were determined by a competitive (inhibition) binding assay. Sucrose density gradient analysis indicated that these estrogens form protein complexes with cytosol and nuclear preparation that sediment at approximately 8S and 4S, respectively. The affinity constants for 17 beta-Eq were approximately two- to six-fold higher than E2 in both species. In a rat uterotropic assay, all nine estrogens were uterotropic. These data indicate that all ring B unsaturated estrogens present in conjugated equine estrogen preparations are biologically active and they express their biologic effects in the human endometrium by mechanisms similar to those described for the classic estrogens.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Dominant bovine ovarian follicular cysts express increased levels of messenger RNAs for luteinizing hormone receptor and 3 beta-hydroxysteroid dehydrogenase delta(4),delta(5) isomerase compared to normal dominant follicles

The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17 alpha-hydroxylase, cytochrome P450 aromatase, 3 beta-hydroxysteroid dehydrogenase Delta(4),Delta(5) isomerase, LH, and FSH receptors and estrogen receptor-beta in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17 beta, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17 beta (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17 beta concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-beta mRNAs than other groups. In summary, increased expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17 beta concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.     

Estrogen receptors and growth response in cultured human periodontal ligament cells

The periodontal ligament (PDL) is a connective tissue involved in the remodeling process associated with tooth development and positioning. PDL cells grown in culture were analyzed for the capacity to specifically bind steroid hormones and for growth response to estradiol-17 beta. Using [3H]estradiol-17 beta as the ligand, PDL cells in first passage cultures exhibited a specific estrogen binding capacity of 881 fmol/mg cell protein. With [3H]dexamethasone as a ligand, the binding capacity of the glucocorticoid receptor was 143 fmol/mg protein. With [3H]R5020 as a ligand, the progestin receptor exhibited a binding capacity of 5 pmol/mg protein. Scatchard analysis of estradiol binding at 37 degrees revealed a dissociation constant of 2.7 X 10(-9) M, representative of the estrogen receptor. The addition of estradiol-17 beta at concentrations of 10(-9) and 10(-8) M to culture media induced a dose-dependent decrease in growth (DNA content) to 62% and 38% control values, respectively. The addition of the antiestrogens tamoxifen and 4-hydroxytamoxifen at concentrations of 10(-7) and 10(-6) M similarly depressed cell growth. These results show that PDL cells contain high affinity receptors for several steroid hormones and further that these cells are targets for the action of estrogens.     

17beta-estradiol modulates prostaglandin E2 release from human amnion-derived wish cells

In human amnion-derived WISH cells [(3)H]estradiol-17beta binding sites are not detectable, but they become measurable in cells exposed to cAMP elevating agents such as forskolin or Ro 20-1724. In cells unexposed to these drugs, 17beta-estradiol stimulates prostaglandin (PG)E(2) release but exerts an evident inhibitory effect in cells exposed to Ro 20-1724. Both stimulatory and inhibitory actions are inhibited by the estrogen receptor antagonist, tamoxifen, by cell pretreatment with cycloheximide, or when the hormone is bound to BSA. Our data demonstrate for the first time that 1) 17beta-estradiol modulates PGE(2) release from WISH cells, interacting with specific intracellular receptors and probably evoking new protein synthesis, and 2) WISH cell responsiveness to 17beta-estradiol seems to be modulated by cAMP, whose levels are significantly increased by the steroid hormone in the presence of Ro 20-1724. The nucleotide is presumably responsible for the enhacement of hormone receptor availability and for the inhibition of PGE(2) release observed in the presence of Ro 20-1724.     

Selective turnover of the essential fatty acid ester components of estradiol-17 beta lipoidal derivatives formed by human mammary cancer cells in culture

The properties of fatty acyl coenzyme A: estradiol-17 beta acyl transferase in microsomes derived from pooled human mammary cancer tissue have been examined. A pH optimum of 5.5 was found and addition of long-chained fatty acyl CoAs increased estradiol-17 beta (E2) 17-monoacyl ester synthesis; the apparent Km for E2 being 8 microM when oleoyl CoA, linolenoyl CoA or palmitoyl CoA were employed. Testosterone, dehydroepiandrosterone, and 5-androsterone-3 beta, 17 beta-diol acted as competitive inhibitors with Ki values of 36, 36 and 46 microM, respectively. The composition of E2 fatty acyl esters (E2-L) formed by incubation of [3H]E2 with human mammary cancer tissue and human mammary cancer cell lines has been determined by HPLC. Although the composition of E2-L in estrogen receptor negative cell lines (MDA-MB-231 and MDA-MB-330) was generally similar to that found for MCF-7 cells (estrogen receptor positive) and pooled human mammary cancer tissue, the former cell lines contained a 3-fold higher relative concentration of E2-17 beta stearate. MCF-7 cells were exposed to 30 nM [3H]E2 and the composition of the isolated [3H]E2-L fraction studied at various time intervals. At 0.5 h, the intracellular concentration of E2-L was 1.8 +/- 0.4 (SEM) pmol/mg DNA which increased to values of 3.6 +/- 0.6 and 4.3 +/- 0.5 at 4 h and 16 h, respectively. In the subsequent 3 h following transfer to medium lacking [3H]E2, the concentration of E2-L declined to 3.7 +/- 0.3 pmol/mg DNA. The subfraction of E2-L composed of E2-17 beta arachidonate, linolenate and docosahexaenoate, was seen to decline in relative abundance after 0.5 h and to reach significantly lower relative levels at 16 h, and again in the 3 h period following estrogen withdrawal. The data suggests that these components, derived from essential fatty acids, are more metabolically active. This may then provide a new lead to link these novel estrogen derivatives with the established relationship between unsaturated fatty acids and an increased mammary cancer incidence.     

Synthesis and study of the estrogenic activity of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether.

Synthesis of 2,4-bis(bromomethyl)estradiol-17 beta 3-methyl ether (BBE2M) was accomplished by reducing a methanolic solution of 2,4-bis(bromomethyl)estrone methyl ether with sodium borohydride. In 0.5 M phosphate buffer, pH 7.0, 25 degrees, BBE2M readily reacts with Ellman's anion and alkylates cysteine to form a steroid-amino acid conjugate. Stoichiometry of the reaction indicates that the bromosteroid is divalent with cysteine. Tryptophan and histidine react more slowly with the bromosteroid. Estrogenic activity of BBE2M was evaluated in ovariectomized rats by uterine intraluminal administration and quantitation of glucose-6-phosphate dehydrogenase (D-glucose-6-P:NADP+ oxidoreductase, EC 1.1.1.49) activity in the uterus. BBE2M induced glucose-6-phosphate dehydrogenase activity as did estradiol-17 beta or estradiol-17 beta 3-methyl ether (E2M). BBE2M was more persistent in activity than E2M. Histological examination of uterus following BBE2M treatment shows classic estrogenic morphology. BBE2M covalently binds to the cytoplasmic estrogen receptor of calf uterus. Such binding is prevented by pretreatment of the receptor protein with estradiol-17 beta. The covalently bound steroid-receptor complex appears to stimulate RNA synthesis in isolated nuclei from calf endometrium.     

Steroids and follicular rupture at ovulation

The preovulatory surge of gonadotropins stimulates follicular steroidogenesis and changes from estrogen as the major product to progesterone. We shall overview the studies dealing with the role of ovarian steroidogenesis in follicular rupture at ovulation. Several inhibitors of steroidogenesis blocked follicular rupture in vivo. Likewise, RU 38486 partially blocked ovulation triggered by hCG. Collectively, these data support the knowledge that follicular steroidogenesis is required for ovulation. Recent studies confirmed the essential role of plasminogen activator (PA) in follicular rupture. The LH stimulation of PA activity was partially blocked by several inhibitors of steroidogenesis and it could be restored by the addition of progesterone, testosterone and estradiol-17 beta, but not the non-aromatizable 5 alpha-dihydrotestosterone. Gonadotropic stimulation enhanced only the synthesis of tissue type PA (t-PA) and not that of urokinase. Likewise, inhibition of steroidogenesis, reduced only the synthesis of t-PA and was reversed by addition of estradiol-17 beta. It seems, therefore, that follicular steroids, most probably estrogen, are involved in the preovulatory rise in follicular t-PA activity.     

Estradiol-17 beta inhibition of androgen uptake, metabolism and binding in epididymis of adult male rats in vivo: a comparison with cyproterone acetate

The effects of estradiol-17 beta on androgen uptake, metabolism and binding were studied in rat epididymis in vivo in comparison with cyproterone acetate. Steroids (250 ug/100 g body weight) were injected 5 min prior to 3H-testosterone in castrate rats. Estradiol-17 beta inhibited 3H-testosterone uptake into epididymal cytosol by 58% as compared to 38% by cyproterone acetate. 3H-Testosterone uptake into epididymal nuclei was inhibited 95% by estradiol-17 beta and 83% by cyproterone acetate. Total bound radioactivity in cytosol fractions was reduced to a greater extent by estradiol-17 beta than cyproterone acetate when either 3H-testosterone or 3H-dihydrotestosterone was injected. Binding of 3H-dihydrotestosterone to nuclear receptors was completely abolished by estradiol-17 beta; whereas approximately 20% binding remained in the nuclear extract after cyproterone acetate treatment. Metabolism of 3H-testosterone in vivo was also altered by estradiol-17 beta, resulting in diminished conversion to 3H-dihydrotestosterone. Cyproterone acetate, on the other hand, did not affect 3H-testosterone metabolism. Estradiol-17 beta and cyproterone acetate inhibited in vitro binding of 3H-dihydrotestosterone to the intracellular cytoplasmic receptor, but not the intraluminal androgen binding protein (ABP). These data suggest that estradiol-17 beta may have a more potent antiandrogenic effect on the epididymis than cyproterone acetate due to inhibition of 5 alpha reduction of testosterone as well as binding to the androgen receptor.     

Specificity of inhibition by steroids of porcine oocyte maturation in vitro.

In order to examine the influence of several steroids on the process of oocyte maturation, denuded (adherent cumulus granulosa cells mechanically removed) and intact (cumulus granulosa cells left attached) porcine oocytes were cultured in the presence or absence of estradiol-17 beta, estradiol-17 alpha, testosterone, cortisol, progesterone, or the nonsteroidal estrogen diethyl stilbestrol (all at 10 microgram/ml) in defined medium that contained either BSA or dextran. Estradiol-17 beta was the only steroid to exert a significant inhibitory effect on the maturation of denuded oocytes, and did so only in BSA supplemented medium. The inhibition was reversible in that oocytes, cultured in steroid-free medium after initial culture in estradiol-17 beta medium, resumed meiotic maturation. Oocytes took up 3H-estradiol-17 beta in both media, although less radiolabel entered oocytes in BSA supplemented medium. The majority of label in the oocytes, when cultured with either medium, was not displaced by excess radioinert estradiol-17 beta or progesterone, nor were the oocytes saturated even when cultured in 10(-6) M estradiol-17 beta. Autoradiography of sectioned oocytes after culture in 3H-estradiol-17 beta has shown that there was no selective accumulation of silver grains over the germinal vesicle as was the case with granulosa cell nuclei. This observation suggests that estradiol-17 beta may not act at the level of the oocyte nucleus.     

Diethylstilbestrol metabolites and analogs. Biochemical probes for differential stimulation of uterine estrogen responses

Diethylstilbestrol (DES) and certain chemically structural derivatives and analogs, indenestrol A (IA), indenestrol B (IB), indanestrol (IN), and pseudo-DES (PD), have been used as probes to examine various estrogenic responses previously considered interrelated and obligatory to the stimulation of uterine growth. All the analogs had poor uterotropic activity in vivo which ranged from 10-200 times less than that of estradiol or DES. The poor uterotropic activity was not due to poor binding affinity for the receptor. All compounds except IN interacted with the mouse uterine estrogen receptor with high affinity (approximately Ka 1.5-2.2 X 10(10) M-1). In addition, the compounds were able to translocate similar levels of receptor to the nucleus in vivo. Nuclear retention and occupancy of the estrogen receptor by the compounds was comparable to the patterns produced by DES or estradiol. The activity of uterine tissue responses was investigated during treatment with the compounds. Only IA stimulated uterine glucose-6-phosphate dehydrogenase to significant levels similar to DES or estradiol. Uterine progesterone receptor was induced to varying degrees by all compounds; the indenestrol isomers (IA and IB) were the most active. Uterine DNA synthesis was marginally stimulated by the derivatives and analogs except for IB which showed a response increase comparable to DES or estradiol. Because of the differential stimulation, these data suggest that in uterine tissue estrogen receptor stimulates certain biochemical responses independently and not in concert. The ability of a particular response to be increased may depend on the chemical nature of the ligand receptor complex and its interaction at genomic sites.     

A double antibody radioimmunoassay for free and conjugated estradiol-17 beta in cow's milk

A radioimmunoassay for free estradiol-17 beta, conjugated estradiol-17 beta or total (free + conjugated) estradiol-17 beta in defatted milk of cows is described. Conjugated estradiol-17 beta was hydrolyzed by enzymes of Helix pomatia juice. Estrogens were extracted with dichloromethane; no other purification step was required before radioimmunoassay because of the high specificity of the antiserum. Immunoprecipitation was used to separate bound and free estradiol-17 beta. Concentrations measured were corrected for procedural losses on a per sample basis. The assays were shown to be accurate and specific. The sensitivity was 1.3pg/ml for the assay of free estradiol-17 beta (5ml of milk extracted) and 2.9pg/ml for conjugated or total estradiol-17 beta (2 ml of milk hydrolyzed and extracted). Estrogens were measured in the milk of cyclic cows and in cows stimulated with pregnant mare serum gonadotropin (PMSG). A preovulatory increase was clearly observed. Wether or not the ovary was stimulated by PMSG, concentrations of estrogens were higher and the relative increase during the preovulatory peak was greater for conjugated estradiol-17 beta than for the free form. The assay of conjugated or total estradiol-17 beta in defatted milk should be a practical method for assessing preovulatory growth of follicles in cows.