ACTINOMYCIN D AND UTERINE EPITHELIAL PROTEIN SYNTHESIS J. W. POLLARD, C. A. FINN AND L. MARTIN

Department of Hormone Physiology, Imperial Cancer Research Fund, London, WC2A 3PX and fDepartment of Physiology, Royal Veterinary College, London, NW1 OTU (Received Pregnant mice, maintained

10 September

1975)

state of delayed

implantation by ovariectomy and progesterone dose of oestrogen. Actinomycin D (AMD) can also induce implantation, albeit of a smaller number of blastocysts (Finn, 1974; Finn & Downie, 1975). It has been suggested that during delayed implantation, growth and attachment of the blastocyst is prevented by the secretion of an inhibitor (Psychoyos & Bitton-Casimiri, 1969; Weitlauf, 1973) which is removed or neutralized at implantation. Finn (1974) proposed that AMD induced implantation by reducing inhibitor production. In these experiments we investigated some of the biochemical events which follow administration of AMD. Virgin, albino mice were ovariectomized and oviductectomized 72 h after mating (Finn, 1974). They were immediately given 500 yg progesterone and 24 h later, following another dose of progesterone, were divided into two groups. One group received an i.p. injection of 15 jug AMD (Merck, Sharpe & Dohme) in 0-1 ml water, the other received water only; 4 h later they were all killed and the uteri split longitudinally and removed. Six horns were incubated for lh at 37 °C in 2ml phosphate-buffered saline pH 7-4 (PBS), containing 5¿uCi [5"3H]uridine (Radiochemical Centre; sp. act. 29 Ci/mmol) and 1 yCi [14C]leucine (Radiochemical Centre; sp. act. 348 mCi/mmol). They were then washed with PBS and frozen at -80°C in fresh PBS until used (Smith, Martin, King & Vertes, 1970). Upon thawing the epithelium was removed (purity>80%) into 2ml 0-25 M-sucrose : 3 mM-CaCl2 (Smith et al. 1970). All pro¬ cedures, unless otherwise stated, were carried out at 4"C. A 0-5 ml portion of 1 M-perchloric acid (PCA) was added to 0-5 ml of the epithelial fraction and the precipitate collected after 10 min by centrifugation at 1000 g for 5 min. The pellet was washed twice with 2 ml 0-5 M-PCA and regained by centrifugation at 1000 g for 10 min. The supernatant and washes formed the RNA fraction. The pellet was digested overnight in 1 ml M-NaOH for determination of the protein specific activity. The epithelial sample (1-5 ml) was made 0-7% (v/v) with Non-idet, the nuclei were prepared, and non-histone proteins (NHP) extracted (Pollard & Martin, 1975). Bovine serum albumin and yeast tRNA (Sigma, London) respectively, were used as standards for protein (Lowry, Rosebrough, Farr & Randall, 1951) and RNA (Fleck & Munro, 1962) estimations. Radioactivity was measured by scintillation counting. Quenching was corrected by an external standard. In later experiments [35S] methionine-labelled NHP and cytoplasmic soluble proteins were prepared from the epithelial fraction, fractionated according to mol. wt by polyacrylamide gel electrophoresis and located by autoradiography (Pollard & Martin, 1975). Isoelectric focusing of NHP was carried out by the method of Gronow & Griffiths (1971) as described by Martin, Pollard & Fagg (1976). Four hours after administration (Table 1), AMD had reduced the incorporation of pre¬ cursors into epithelial RNA to 40% and that into epithelial protein to 50% of the control level. The Plate shows autoradiographs of SDS gels of proteins synthesized 4 h after treatment. treatment

can

be induced

in

to

a

implant by a small

*Present address: Department of Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario, Canada,

M4X 1K9.

Table 1. Incorporation of radioactive precursors into mouse uterine epithelial RNA and protein 4 h after treatment with actinomycin D (Means ± S.E.M. ; at least 5 determinations) Progesterone

Progesterone + actinomycin D (d.p.m./mg) (% of control)

29095+2753 3255 ± 260 5090 ± 165

11789+1026 1610 ±120 2915 ± 695

(d.p.m./mg)

RNA Total protein Non-histone protein

**P < 001 ; ***P < 0001

:

by Student's

405*** 49-5*** 570** i-test

The patterns of cytoplasmic soluble protein synthesis were similar in control and AMD-treated groups in all but one band. This band, containing newly synthesized proteins of approximately 70 000 mol. wt, was completely missing in seven out of eight cases: in the eighth, reduction of this band was no greater than that of the other bands. Two NHP samples from each treatment group were examined because of the role of NHP in gene regulation (Paul & Gilmour, 1968). No major differences were apparent by SDS gel electrophoresis (Plate) or by isoelectric

focusing.

Two mechanisms could account for the disappearance of a single band of newly synthesized D could preferentially inhibit transcription of a single class of rapidly is unlikely as this dose of AMD would preferentially inhibit rRNA over mRNA. This turning synthesis (Reich, Franklin, Shatkin & Tatum, 1962). Alternatively, it could produce a general inhibition of protein synthesis. In this case proteins with rapid turnover would disappear preferentially. Rapid turnover could result from degradation within the epithelium or secretion from it. Any substance involved in the inhibition of implantation would need to be capable of responding rapidly to external influences. Rapid turnover would allow this. Surani (1975) showed that a protein of mol. wt 70000 absent from uterine secretions during delayed im¬ plantation appears 1 h after oestrogen stimulation. It is possible that a protein is synthesized but sequestered or degraded in the epithelium during delay but released in response to oestro¬ gen or AMD.

protein. Actinomycin

REFERENCES

Finn, C. A. (1974). Journal of Endocrinology 60,199-200. Finn, C. A. & Downie, J. M. (1975). Journal of Endocrinology 65, 259-264. Fleck, A. & Munro, H. N. (1962). Biochimica et Biophysica Acta 55,571-583. Gronow, M. & Griffiths, G. (1971). FEBS Letters 15, 340-344. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Journal of Biological Chemistry 193,

265-275. Martin, L., Pollard, J. W. & Fagg, B. (1976). Journal of Endocrinology 69, 103-115. Paul, J. P. & Gilmour, R. S. (1968). Journal of Molecular Biology 34, 305-316. Pollard, J. W. & Martin, L. (1975). Molecular and Cellular Endocrinology 2, 183-191. Psychoyos, A. & Britton-Casimiri, V. (1969). Comptes Rendus Hebdomadaires des Séances de l'Académie des

Sciences 268, 188-190. Reich, E., Franklin, R. M., Shatkin, A. J. & Tatum, E. L. (1962). Proceedings of the National Academy of Sciences of the U.S.A. 48, 1238. Smith, J. ., Martin, L., King, R. J. B. & Vertes, M. (1970). Biochemical Journal 119, 773-784. Surani, . . . (1975). Journal of Reproduction and Fertility 43,411-417. Weitlauf, H. M. (1973). Journal ofExperimental Zoology 183, 303-308. DESCRIPTION OF PLATE SDS of Autoradiographs polyacrylamide gels of [35S]methiomne-labelled cytoplasmic (a) and non-histone proteins (b), 4 h after treatment. Migration is from top to bottom; positions of mol. wt standards are shown in the centre. Free methionine runs with the ion front and forms a diffuse black area at the base of the autoradiograph. C, progesterone alone; A, progesterone and actinomycin D. Approximately equal counts were applied to each gel so that direct comparison can be made between treatments. Arrow, position of newly synthesized protein of approx. mol. wt 70 000.

Journal of Endocrinology, Vol. 69, No. 1

J. W.

POLLARD, C. A. FINN AND L. MARTIN

Plate

(Facing p. 162)

Actinomycin D and uterine epithelial protein synthesis.

ACTINOMYCIN D AND UTERINE EPITHELIAL PROTEIN SYNTHESIS J. W. POLLARD, C. A. FINN AND L. MARTIN Department of Hormone Physiology, Imperial Cancer Rese...
1MB Sizes 0 Downloads 0 Views