Janet M. Loring

Biosynthesis of estrogens by perfused full-term placentas*

Abstract Human term placentas perfused through both fetal and maternal circulations converted labeled dehydroepiandrosterone sulfate and testosterone to androstenedione, 19-hydroxyandrostenedione, estrone and estradiol but not estriol or 16-epiestriol. The perfused placenta delivered more estrogens into the fetal circulation than into the maternal compartment. Neither androst-5-ene-3, 17-diol nor 19-hydroxydehydroepiandrosterone was found in either the placentas or the perfusates.

Ornithine decarboxylase activity and polyamine content of the placenta and decidual tissue in the rat

Measurements of ornithine decarboxylase (ODC) activity and of the amounts of putrescine, spermidine and spermine in the placenta and uterus of the pregnant rat and in decidual tissue of the pseudo-pregnant rat showed that these wax and wane with the growth rate of the tissues. Human term placenta has essentially no ODC activity, but placentae from 15-week human gestations have substantial amounts of enzyme. The ODC activity of the rat placenta increases 40-fold from day 10 to day 17 of pregnancy, then gradually decreases. The content of the polyamines in the placenta also reaches a peak on day 17. The ODC activity of the endometrium between implantation sites remains low until the end of pregnancy and then increases eight-fold on days 21 and 22. This may represent increased uterine activity in preparation for parturition. The ODC activity and the polyamine content of decidual tissue responds to administered oestradiol with increases that reach a peak within four hours. The ODC activity and polyamine content of decidual tissue decrease after the fifth day of decidualization. Nuclei isolated from decidual tissue respond to the addition of spermine or spermidine with an increase in the rate of RNA synthesis. Spermidine increases the elongation of RNA chains, but does not initiate the synthesis of new RNA chains in decidual nuclei.

Diethylstilbestrol stimulates ornithine decarboxylase in kidney cells in culture.

Summary Diethylstilbestrol, estradiol-17β, progesterone or testosterone at concentrations of 10 −9 M, but not a number of other hormones tested, increase the activity of ornithine decarboxylase in hamster kidney cells maintained in culture with the peak in activity occurring three to six hours after addition of hormones. S-adenosyl methionine decarboxylase of these cells is also stimulated by DES. The DES induced increase in the activity of ornithine decarboxylase and of S-adenosyl methionine decarboxylase involves protein synthesis — it is inhibited by cycloheximide — but does not involve RNA synthesis at that moment — it is not inhibited by actinomycin D. This suggests that the mRNA for ODC and SAMDC may be synthesized earlier and retained in the cell in some inactive form until released by the estrogenic stimulus.

Estrogenic control of uterine enzymes

Abstract Poly(A)-rich RNA was separated from the mass of uterine RNA by binding it into a polyuridylic acid-sepharose 4B column and then eluting it at pH 9. Uteri from rats injected with estradiol 1 hr previously and incubated in 3H-cytidine for 60 min yielded a poly(A)-rich RNA with a specific activity 2.5-fold greater than that of poly(A)-rich RNA from uteri of rats not given estradiol. The poly(A)-rich RNA from the uteri of estrogen-treated rats, when instilled in amounts of 0.1 μg into the lumen of the uterus of an immature rat, increased protein synthesis and the activities of glucose-6-phosphate dehydrogenase and ornithine decarboxylase compared to the contralateral horn instilled with poly(A)-rich RNA from the uteri of control rats. When poly(A)-rich RNA was added to the incubation medium containing explants of immature uteri maintained in organ culture, it increased the incorporation of uniformly labeled amino acids into protein. The mass of RNA not bound by the poly-U column does not increase protein synthesis or enzyme activity when instilled into the uterine lumen.

Effects of FSH and LH on RNA Synthesis in the Testis: Role of Ornithine Decarboxylase

It has long been known that testicular size decreases following hypophysectomy and can be restored toward normal by the injection of pituitary extracts. The sequence of events involved in the response of the gonads to gonadotropins has been of interest to many investigators in recent years. Gonadotropic hormones increase the synthesis of RNA and protein in the testis of the rat (Means and Hall, 1967, 1968, 1969; Goswami, et al., 1968; Villee, D., and Goswami, 1973) and increase the production of cyclic AMP (cAMP) (Murad et al., 1969; Hollinger, 1970; Kuehl et al., 1970; Dorrington et al., 1972; Braun and Sepsenwol, 1974). The increased synthesis of RNA and protein in response to the hormone was inhibited by pretreatment of the animals with actinomycin D (Means and Hall, 1969; Goswami et al., 1968; Reddy and Villee, 1975a). Messenger RNA (mRNA) from animal origins has been shown to contain a polyadenylate sequence at the 3′ terminal end (Lim and Canellakis, 1970; Darnell eet al., 1971; Lee et al., 1971; Edmonds et al., 1971). The poly(A)-rich RNA can be conveniently isolated by affinity chromatography using oligo(dT) columns (Aviv and Leder, 1972).

Control of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Binding Protein(s) in the Hamster Kidney

Hamster renal cytosol binds [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with high specificity. Sucrose density gradient centrifugation revealed two binding entities- one with a low sedimentation coefficient of 4-5 S which was displaced by neither TCDD nor other polycyclic aromatic hydrocarbons (PAHs) and another with a high sedimentation coefficient of 7-8 S which was displaced by TCDD, benzo[a]pyrene (BP), 2-methylcholanthrene (MC), and 7,12-dimethylbenzo[a]anthracene (DMBA) but not by estradiol (E), progesterone (P), cortisol (F), testosterone (T), 5 alpha-dihydrotestosterone (DHT), or methyltrienolone (R-1881), a synthetic androgen. Cytosol from intact male hamsters showed maximum binding of labelled TCDD to the 7-8 S binding site. Castration or hypophysectomy reduced this binding. Pretreatment with DMBA increased binding, whereas diethylstilbestrol (DES) decreased binding. Sex difference was observed in the binding capacity of renal cytosol. This is the first report of endocrine control over TCDD binding and its modulation by other PAHs and steroids.

The Estrogen-Induced/Dependent Renal Adenocarcinoma of the Syrian Hamster

The events that occur in a tissue prior to a detectable malignant transformation are complex, and may be unique for each type of tumor. Alternatively, a wide variety of neoplasias may have many preneoplastic events in common. The several hormone-dependent tumors make up one class of neoplasms which may have similar molecular events resulting in their induction and growth. Following long-term estrogen treatment the Syrian hamster develops renal adenocarcinomas which are both estrogen-induced and estrogen-dependent (Kirkman, 1959). We have been using this model to find answers for several questions concerning the induction and growth of hormone-dependent tumors with emphasis on the role of estrogen at the molecular level.

Melanocyte-stimulating hormone and prolactin secretion in cell culture of estrogen-induced pituitary tumors in the hamster.

Pituitary cells from hamsters bearing diethylstilbestrol induced renal adenocarcinomas were cultured in vitro. Dispersed cells in plastic dishes were viable for up to two weeks in Dulbeccos modified Eagles medium supplemented with 17.5% of 6:1 horse serum to fetal calf serum. The secretion of alpha-melanocyte stimulating hormone and prolactin into the medium were measured by radioimmunoassay. The concentrations of both were elevated by day 3 in the medium from the hyperplastic pituitaries obtained from the estrogen treated, tumor bearing hamsters. Neither DES (10(-8)M) nor tamoxifen (10(-7)M) influenced the secretion of either hormone and neither altered either cell number or DNA synthetic activity as measured by thymidine incorporation. The secretion of hormones and the growth of the pituitary cells were, however, decreased by charcoal treatment of the serum. The results suggest that the elevation of serum alpha-MSH and prolactin observed in DES implanted hamsters is due to pituitary secretion of the hormones but that DES probably does not act directly on the pituitary to control the secretion.

Gonadotropic Stimulation of Enzymes Involved in Testicular Growth

The marked decrease in testicular size following hypophysectomy and its restoration toward normal following the injection of pituitary extracts have long been known. Analyses of the endocrine and molecular mechanisms involved in the growth response of the gonads to gonadotropins have been undertaken by many investigators. Gonadotropic hormones increase the production of cyclic AMP (cAMP) (Murad et al, 1969; Hollinger, 1970; Kuehl et al., 1970; Dorrington et al., 1972; Braun and Sepsenwol, 1974) and increase the synthesis of RNA and protein in the testis of the rat (Means and Hall, 1967, 1968, 1969; Goswami et al., 1968; D. Villee and Goswami, 1973). The increased synthesis of RNA and protein in response to gonadotropins is inhibited by pretreatment of the animals with actinomycin D (Means and Hall, 1969; Goswami et al., 1968; Reddy and Villee, 1975a). The ubiquitous distribution of the polyamines, their responses to hormonal stimuli, and their possible involvement in both transcriptional and translational events of cellular growth processes have been reported repeatedly and reviewed extensively (Williams-Ashman et al., 1972; Russell, 1973).