Molecular and Cellular Endocrinology

2 (1975) 203-211. 0 North-Holland

Publ. Comp.

CUMULATIVE SUPERINDUCTION OF AVIDIN IN THE CHICK OVIDUCT BY TISSUE DAMAGE AND ACTINOMYCIN D H. ELO, P. TUOHIMAA

and 0. JANNE

Institute of Biomedical Sciences, University of Tampere, Teiskontie 35, SF-33520 Tampere 52, Finland

Accepted 5 December 1974

Received 12 August 1974

Immature chicks were injected daily for nine days with diethylstilbestrol. One group of these chicks was injected with actinomycin D and in another group the oviducts were ligated or pinched with forceps. The chicks were killed at 2 h intervals up to 24 h after the experimental procedure. The avidin content of the oviduct was assayed by the W-biotin binding method. The amount of progesterone in the plasma, adrenal gland and ovary was determined by radioimmunoassay. The oviducts of the control animals showed slight or undetectable amounts of avidin. The serum progesterone level (0.71 & 0.50 ng/ml) of the control animals was similar to that of adults. There was a high concentration of progesterone in the adrenal gland and less in the ovary. In these immature chicks actinomycin D was able to cause superinduction of avidin up to 23 times the control values. Similarly ligation of the oviduct induced avidin synthesis 2346 and pinching of the oviduct 36 times higher than the controls. The effects of actinomycin D and the ligature were cumulative. Actinomycin D increased the progesterone levels in the serum, ovary and adrenal gland. The ligation caused an increase in plasma progesterone values at 2 h followed by a decrease to control levels at 8 h. Simultaneously the concentration of progesterone decreased in the ovary but increased in the adrenal gland. It is concluded that there are two separate mechanisms for the superinduction of avidin synthesis: one induced by the injection of actinomycin D and the other induced by tissue damage. A combination of evidence supports the concept that the effect of ligation on avidin synthesis is not mediated directly through progesterone, but is caused by a local effect of the tissue damage. Keywords: progesterone;

avidin; oviduct; actinomycin

D; superinduction.

Avidin is a specific biotin binding protein which is secreted from the magnum portion of the avian oviduct. Avidin is induced by progesterone (Herz et al., 1943; O’Malley et al., 1969). The induction of avidin synthesis is mediated by a specific mRNA, which has been extracted from the chick

204

H. El0 et al.

oviduct and translated in homologous and heterologous tissues or in cell free systems (Rosenfeld et al., 1972; Segal et al., 1973; Tuohimaa et al., 1973a, b). If actinomycin D, a potent inhibitor of DNA dependent RNA synthesis, is administered prior to progesterone injection, it abolishes the synthesis of avidin in the chick oviduct in vivo and in vitro (O’Malley et al., 1969; Elo and Tuohimaa, 1973). On the other hand, if actinomycin D is administered shortly after the injection of the steroid hormone, the subsequent protein synthesis is superinduced, i.e. the synthesis of the induced protein is much higher than after the hormone alone. This superinduction phenomenon by actinomycin D has been described in ovalbumin synthesis after estrogen (Palmiter and Schimke, 1973) and in avidin synthesis after progesterone (O’Malley et al., 1969) administration. It was found in a preliminary experiment that tissue damage of the oviducts of the immature chicks caused a superinduction of avidin similar to that caused by actinomycin D. In order to study whether or not the mechanism of superinduction by actinomycin D and tissue damage is identical, an investigation was made of the effect of actinomycin D and oviductal trauma on the adrenal gland and ovarian function as well as on avidin concentration in the oviduct. Evidence based on these experiments is presented here in support of a new theory that there are two different mechanisms in the superinduction.

MATERIALS

AND METHODS

Animals and tissue samples

In order to stimulate the oviducts to grow, Leghorn chicks (strains Ti 53 and Ti 453, Turun Muna Hatchery, Turku, Finland) were injected daily with 0.5 mg of diethylstilbestrol (DES) in 0.05 ml of sesame oil subcutaneously for 9 days beginning on the first day after hatching. The chicks were used for the experiments one day after the last injection of diethylstilbestrol ann were sacrificed 24 h later if not otherwise stated. The operations on the oviducts were performed under ether anesthesia. The abdominal layers were dissected and the oviducts were tightly ligated, with the ligature placed at varying positions in the lower part of the magnum. Mechanical trauma was induced in other chicks by firmly pinching the magnum of the oviduct with forceps. The muscular layer and skin were then sutured with nylon. A dose of 0.5 mg actinomycin D (Sigma Chemical Company, St. Louis, U.S.A.) per kg of body weight was injected intraperitoneally or subcutaneously in 0.9 ‘A NaCl. After sacrificing each chick the reproductive tract was divided into carefully separated pieces, as stated under Results. Avidin concentration of the shell gland, isthmus and

Avidin superinduction

magnum adrenal

of the oviduct were analyzed.

205

Progesterone

concentration

of the serum,

gland and ovary were also assayed.

A vidin assay Avidin contents of the tissues were assayed with an improvement of the method of Korenman and O’Malley 1967; Elo and Tuohimaa, 1974). The oviducts were homogenized with a motor-driven Teflon pestle homogenator in 6 ml of buffer (0.07 M KCl, 0.004 M MgC12, 0.07 M NaCl and 0.02 M phosphate buffer pH 7.1). The homogenate was centrifuged at 10,000 g for 30 min. Avidin was determined from 2.5 ml of the supernatant. Ten nCi of D-(carbonyl- ’ 4C) biotin (20 mCi/mmol, The Radiochemical Centre, Amersham) in 0.5 ml of 0.2 M ammonium carbonate was added to each supernatant sample. The assays were processed in duplicate. The 14C-biotin-avidin complex was bound to 40 mg of bentonite. The biotin-avidin-bentonite complex was washed 3 times with 2 ml of a 0.2 M ammonium carbonate solution in a test tube. The radioactive sediment was transferred into a scintillation vial with 10 ml of aquafluor containing 100 ml of ethoxyethanol, 50 ml of liquifluor (100 g PPO, 125 g POPOP in 1000 ml of toluol) and 50 g of naphthalene in 850 ml of dioxane. The radioactivity was counted in a Wallac Decem-NTL314 liquid scintillation counter. For each counting there were 24 background controls. The background was the arithmetic mean of these counts. The results are expressed as ug of avidin per g of oviduct. Avidin standard (12 units/mg, Sigma Chemical Company, St. Louis, U.S.A.) was dissolved in water. The statistical significance of the results was tested by Mann-Whitney U-test and Student’s t-test. Determination of progesterone The determination of serum progesterone

was carried out by means of radio-

immunoassay after isolation of the steroid by chromatography on hydroxyalkoxypropyl Sephadex (Janne et al., 1974). Progesterone content of the tissues was estimated by the following procedure. The weighed tissue was homogenized with an Ultra-Turrax in 10 ml of acetone/ethanol (1: 1, v/v) and kept in a water bath at +39 “C for 2 h. After incubation, the sample was filtered through a sintered glass funnel. The precipitate was further washed with 5 ml of acetone/ethanol. The organic solvents were combined and washed twice with 1 ml of distilled water and were evaporated until dry. The dry residue was redissolved in light petroleum (b.p. 64-69 “C)/chloroform (95 : 5, v/v) and put onto a 2.5-G column of hydroxyalkoxypropyl Sephadex packed in the same solvent (Janne et al., 1974). The fraction containing progesterone was collected. The determination of progesterone was carried out by radio,

206

H. Elo

immunoassay 1974).

et

al.

as previously described for plasma progesterone (J8nne et al.,

RESULTS A vidin

Table 1 shows the effect of the pinching and the ligation of the oviduct and of the administration of actinomycin D on avidin concentration in the oviduct. After 9-11 day’s treatment with DES the weights of 85 control oviducts were on an average 250 mg (range W-760 mg). No significant difference in the oviduct weight was obtained after the experimental operative treatment. At the end of the experiment the animals weighed 80-130 g. There was a small amount of avidin in the control chicks, most of which had no detectable avidin in the oviduct. In the control chicks injected with two additional doses of DES (0.5 mg of DES on the 10th and 1lth day), the avidin concentration in the oviduct

Table 1 The effect of actinomycin D and pinching and ligation of the oviducts on the avidin synthesis in the chick oviducts. All the chicks were pretreated with 0.5 mg of DES for 9 days. The chicks were sacrificed 24 h after the experimental treatment. Upper = the part of the oviduct above the ligature. Lower = the part of the oviduct below the ligature.

_~ Experimental ___.__--

procedure

Part of the oviduct

~_~

pg of avidin/g oviduct (+ SD)

Number of animals

.-

Ligature

upper lower

Ligature (two additional doses of DES) Ligature (luminal content rinsed) Pinching Sham operated Controls

upper lower upper lower magnum + isthmus magnum + isthmus magnum -+ isthmus

0.5 mg of actinomycin D/kg (i.p.) 0.5 mg of actinomycin D/kg (s.c.) 0.5 mg of actinomycin D/kg 30 min before ligature (i.p.) 0.5 mg of actinomycin D/kg 5 min after ligature (i.p.)

magnum + isthmus magnum -I- isthmus upper lower upper lower

8.0 9.3 5.5 5.6 11.4 16.1 12.5 1.0 0.4

f f & i & + & + i_

5.7 7.0 5.3 3.0 7.1 3.9 5.4 1.4 1.3

21 15 10 7 10 5 14 19 55

7.9 2.6 26.3 30.2 17.9 35.8

It + h & & I

4.9 2.4 17.1 18.3 9.6 19.4

13 9 11 6 14 3

207

Avidin superinduction

was slightly, but not significantly lower than in the controls receiving DES only for 9 days. The ligation of the oviduct induced a clear-cut increase in avidin concentration (P < 0.001). The increase was slightly higher in the distal part of the oviduct (distal to the ligature) than in the proximal part. This increased amount of avidin was apparently not due to obstruction of avidin removal from the oviductal lumen by ligation, because the rinsing of the lumen of the upper part of the oviduct did not decrease the avidin content of the oviduct. Two additional doses of DES had a small, but not significant inhibitory effect on the avidin concentration after ligation. This could be observed especially in the lower part of the oviduct. The pinching of the oviduct with forceps increased avidin concentration in the oviduct by the same magnitude as the ligature (P c 0.001). On the other hand, an operation on the ventral abdominal wall with dissection of the peritoneal layer (sham operation) did not cause any effect on the oviductal avidin concentration. An intraperitoneal administration of actinomycin D (0.5 mg per kg of body weight) increased (P c 0.001) the amount of avidin in the chick oviduct in 24 h to the same level as the mechanical trauma (table 1). When actinomycin D was given subcutaneously the increase in avidin concentration was less pronounced. The effects of actinomycin D and the mechanical tissue damage on AVIOIN w

g

ofoti

Q

ligature

0 progesterone

Fig. 1. Time course of the synthesis of avidin in the immature chick oviduct after an oviductal ligature or an exogenous progesterone (5 mg) injection. The chicks were pretreated daily with diethylstii~trol (0.5 mg) for 9 days. Each point represents 6-10 samples. The standard deviation is given.

208

If. Elo et al.

avidin are cumulative. Whether actinomycin D was administered 30 min before or 5 min after the ligature, the increase in avidin synthesis was equally high. These figures differed significantly from those after actinomycin D or ligature treatment alone (P < 0.01). Detectable avidin synthesis began 6-8 h earlier after mechanical trauma than after exogenously administered progesterone (fig. I). On the other hand, the amount of avidin in the oviduct was of the same order 20-24 h after stimulation by progesterone or by mechanical trauma. Table 2 shows the localization of avidin in the four distinct parts of the oviduct after ligature of the magnum isthmus junction or exogenous administration of progesterone. It can be clearly seen that almost all of the avidin was located close to the ligature, whereas the ligature induced significantly less avidin in the main part of the magnum than progesterone (P < 0.05). It is striking that the ligature but not the exogenous progesterone administration caused an increase in the avidin concentration in the isthmus and shell gland (P C 0.05). Progesterone

Table 3 shows the concentration of progesterone in the serum, ovary and adrenal gland at varying times after the experimental procedure. There were significant amounts of progesterone in all the tissues of the DES pretreated Table 2 The effect of exogenous progesterone (5 mg) and ligation of the chick oviducts on the amount of avidin in different parts of oviduct. The chicks were pretreated with 0.5 mg of DES. Progesterone in propyleneglycol was injected subcutaneously 5 mg/animal. The ligature in the oviducts was made between the magnum and the isthmus. All the oviducts were divided into the same pieces. Experimental

procedure

Tissue

Progesterone

injection

Shell gland Isthmus Magnum-isthmus junction Magnum

Oviduct ligature

Shell gland Isthmus Magnum-isthmus junction Magnum

ng of avidin/g of tissue (+ SD)

Number of animals

0.2 -i; 0.3 0.4 rt 1.0

7 I

19.7 _ir 19.9 11.9 + 9.6

8 8

1.3 i 1.0 3.5 * 3.1

8 8

17.0 * 7.1 2.0 + 1.3

8 8

Avidin superinduction

209 Table 3

Effect of experimental

treatment on tissue concentration of progesterone. pretreated with 0.5 mg of DES for 9 days.

_____~ Control 2 h after ligature 4 h after ligature 6 h after ligature 8 h after ligature 10 h after ligature 24 h after actinomycin

Serum ng of progesterone/ ml of serum ___ 0.71 2.17 1.60 1.16 0.84

D

* h & f *

oso* 2.29 0.97 1.13 0.54

Ovary ng of progesterone/ mg of tissue

(8) (3) (4) (4) (4)

0.18 0.19 0.11 0.09 0.10

3.31 c 1.13 (4) ~__

i_ * k * f

0.16 0.15 0.06 0.06 0.04

(9) (4) (4) (4) (4)

1.56 f 2.03

(4)

The chicks were

Adrenal gland ng of progesterone/ mg of tissue 5.02 rt 2.75 8.17 k 2.36

(5) (3)

9.58 i 8.36 5.12 k 3.40 5.08 f 1.07 13.95

(3) (3) (2) (1)

* The standard deviation is given.

control chicks. The main source of progesterone seems to be the adrenal gland. The ligation of the oviduct increased the serum concentration of progesterone at 2 h, whereas no significant changes were observed in ovarian and adrenal tissue levels. On the other hand, actinomycin D had a marked stimulatory effect on the serum (P < O.Ol), ovarian of progesterone at 24 h.

and adrenal

gland concentrations

DISCUSSION As the half life of avidin the changes

in avidin

in the oviduct

concentration

is l-2 days (O’Malley

in our experiments

et al., 1969),

were interpretated

as

resulting from avidin synthesis. The oviducts of the immature chicks showed avidin synthesis without any exogenous administration of progesterone. There was, however, a relatively high endogenous synthesis of progesterone in these control chicks. The plasma progesterone level was 0.71 f 0.50 ng/ml, which could explain the slight synthesis of avidin in the oviducts of the control animals. This plasma progesterone level is similar to that of adult domestic fowls as determined by Furr (1973). In his study plasma progesterone levels in laying hens ranged from 0.67-9.60 ng/ml with a mean value of 3.13 f 0.37 ng/ml, but were low in the non-laying hen, 0.25-0.40 ng/ml. The chicks used in the present study are a special early-laying strain, showing an early hormonal maturation. A few of these chicks showed concrements in their oviducts with

210

H. Elo et al.

simultaneous high levels of avidin. These chicks were eliminated from this study. It should be pointed out that this kind of chicks are not suitable for experiments

where the synthesis

by an intraoviductal

of avidin

administration

in the chick oviduct

of avidin

mRNA

is brought

(Tuohimaa

about

et al., 1973a).

The amount of avidin is expressed per g of oviduct tissue. This may cause some variation in the results, since only the epithelial and not the glandular or muscle cells of the oviduct synthesize avidin (O’Malley et al., 1969). However, the expression per mg of protein or DNA is equally invalid for the same reason. It would be better to express the results as total amount of avidin per oviduct. However, because avidin concentrations have always been expressed in the literature as ug per g of wet weight, this was also done here. The superinduction of a hormone-specific protein has been shown to be caused by special treatment with actinomycin D (O’Malley et al., 1969; Elo and Tuohimaa, 1973; Palmiter and Schimke, 1973). The present results demonstrate two distinct superinduction phenomena: the first occurs in response to actinomycin D and the other in response to tissue damage. Since the chicks had an endogenous secretion of progesterone and a slight avidin synthesis, a superinduction of avidin synthesis could be obtained after actinomycin D injection without previous administration of exogenous progesterone. The other superinduction of avidin synthesis, which is demonstrated here for the first time, is caused by the tissue damage (the ligature or pinching of the oviduct). It is as extensive as the superinduction by actinomycin D. It is interesting to note that a supermaximal dose of progesterone (5 mg) induces the same level of avidin synthesis as ligation or pinching of the oviduct or administration of actinomycin D. Thus this level of avidin synthesis (approx. 10 ug/g) can be taken as the maximal chick pretreated with diethylstilbestrol.

response of the oviduct of a lo-day old However, our results show that two

of these maximal responses can be summarized. When both actinomycin D and the ligature were given jointly to the chicks, the synthesis of avidin was enhanced to slightly more than twice the maximal response (IS-36 ug/g). This study demonstrates two distinct superinductions of avidin synthesis. The superinductions by actinomycin D and by mechanical trauma can be characterized in the following way: 1) The effects of actinomycin D and the trauma were cumulative and the combined effect exceeded the level of the maximal response of the oviduct. 2) Progesterone synthesis and serum level were much elevated after actinomycin D, whereas they were affected to a lesser extent after the oviductal trauma. 3) The ligature induced avidin synthesis several hours faster than exogenous progesterone.

4) Actinomy~in D had no inhibjtory effect on the superinductjon by ligature and vice versa. On the other hand, actinomycin D has been shown to inhibit avidin synthesis induced by exogenous progesterone (O’Malley et al., 1969). 5) The superinduction by ligature was localized in a small section af the oviduct near the ligature, whereas the exogenous progesterone induced a widespread synthesis of avidin in the magnum part of the oviduct. Thus it is improbable that the superinduction by tissue damage was mediated through the endogenous progesterone secretion.

The authors are much indebted to Mrs. Tuula Rantanen and Miss Merja Helle for their technical assistance. This work was supported by grant No. M 73.70 from the Population Council, New York, N.Y., U.S.A.

REFERENCES Elo, H. and Tuohimaa, P. (1973) Acta Endocrinol. Sup@. 177, 250. Elo, H. and Tuohimaa, P. (1974) Biochem. J. 140, f 15. Furr, B. J. A. (1973) Acta End~~no~. 72, 89. Herz, R., Fraps, R. M. and Sebrell, W. E. (1943) Proc. Sot. Biol. Med. 52, 142. J&me, O., Apter, D. and Vihko, E. (1974) J. Steroid Biochem. 5, 155. Korenman, S. G. and O’Malley, B. W. (1967) Biochim. Biophys. Acta 140, 174. Lee, K. -L., Reel, J. R. and Kenney, F. T. (1970) J. Biol. Chem. 245, 5806. Mostafapour, M. K. and Tchen, T. T. (1972) Biochem. Biophys. Res. Commun. 48, 491. O’Mailey, B. W., McGuire, W. L,, Kohler, P. 0. and Korenman, S. G. (1969) Rec. Progr. Hormone Res. 25, 105. Palmiter, R. D. and Schimke, R. T. (1973) J. Biol. Chem. 248, 1502. Rosenfeld, G. C., Cornstock, J, P., Means, A. R. and O’Malley, B. W. (1972) Biochim. Biophys. Res. Commun. 47, 387. Segal, S. J., Xge, R. O., Tuohimaa, P. and Burgos, M. H, (1973) Science 181, 569. Tomkins, G. M., Gelehrter, T. D., Gsanner, R. K., Martin, D. W., Samuels, H. H. and Thompson, E. B. (1969) Science $66, 1474. Tuohimaa, P. J., Segal, S. J. and Koide, S. S. (1972) J. Steroid Biochem. 3, 503. Tuohimaa, P., Segal, S. J. and Koide, S. S. (1973a) Proc. Nat]. Acad. Sci. U.S. 69, 2814. Tuohimaa, P., Gavert, J. and Elo, H. (t973b) Acta Endocrinol. Suppl. 177, 66.

Cumulative superinduction of avidin in the chick oviduct by tissue damage and actinomycin D.

Molecular and Cellular Endocrinology 2 (1975) 203-211. 0 North-Holland Publ. Comp. CUMULATIVE SUPERINDUCTION OF AVIDIN IN THE CHICK OVIDUCT BY TISS...
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