0013-7227/92/1306-3421$03.00/O Endocrinology Copyright 0 1992 by The Endocrine

Vol. 130, No. 6 Printed in U.S.A.

Society

Induction of Avidin Messenger Chick Oviduct by Progesterone TARJA PENTTI

A. KUNNAS, TUOHIMAA,

TIM0 AND

K. JOENSUU, KAIJA K. VIITALA, MARKKU S. KULOMAA

University of Tampere (T.A.K., T.K.J., K.K. V., P.S., P.T., M.S.K.), SF-33101 Tampere, Finland; and University of Jyviiskylii (M.S.K.), JyvaSkylii, Finland

ABSTRACT. Avidin gene expression was analyzed using an avidin immunoassay and RNA hybridization analysis. To ascertain whether the induction of the avidin gene by progesterone remains

specific

also during

Ribonucleic and Other

secondary

restimulation

with

Department Department

P;iIVI

Acid in the Steroids*

SOPANEN,

of Biomedical Sciences, of Biology, SF-40100

one with other steroids nevertheless generated a synergistic increase in the amount of avidin mRNA. This may indicate that binding of progesterone receptor to the progesterone response element may be important to alter the functional activity of other hormone response elements present on the avidin gene. The time response curve of the avidin mRNA induction by progesterone was also determined. Avidin mRNA was detectable 8 h after progesterone induction, and its amount was maximal after 16-24 h. This would indicate that the avidin gene belongs in the so-called late responder genes, which also include chicken ovalbumin, ovomucoid, and lysozyme genes. (Endocrinology 130: 3421-3426,1992)

dieth-

ylstilbestrol, chicks were given different steroid hormones or hormone combinations. Progesterone-specific induction of avidin protein and messenger RNA (mRNA) was 15 to 30-fold over the control even after secondary restimulation with diethylstilbestrol. A functional difference between the progesterone response element and glucocorticoid response element was suggested, since dexamethasone alone did not induce avidin in Go. In spite of progesterone specificity, a combination of progester-

S

hormone action and eukaryotic gene expression in general have been widely studied using genes expressed in the chicken oviduct. Most steroidinducible genes, including ovalbumin, ovotransferrin (or conalbumin), ovomucoid, and lysozyme, are specific to estrogen only during primary stimulation. They are also induced by progestins, androgens, and glucocorticoids after a secondary restimulation by estrogens (l-7). The reason for the change in hormonal specificity is not yet fully understood. It is however known that expression of the steroid receptors, e.g. the progesterone receptor (PR) and estrogen receptor (ER) (8,9) as well as the androgen receptor (AR) (3), is dependent on estrogen-induced cytodifferentiation of the oviduct epithelium (8, 9) Avidin induction has previously been found to be specific for progesterone in the chicken oviduct (1, 10, 11). In addition to the progesterone-specific mechanism, avidin is expressed in connection with inflammation, tissue trauma, and bacterial or viral infections in most tissues, including the oviduct (12-15). This suggests a multifactorial regulation of avidin gene expression.

Isolation of the full-length complementary DNA (cDNA) for egg white avidin (16) provided a tool to examine influences of the steroid hormones and inflammatory reaction on the transcriptional stage of avidin gene expression. The size of the progesterone-inducible avidin messenger RNA (mRNA), about 700 nucleotides, was previously detected in the oviduct by RNA hybridization (Northern, dot-blot) analyses (16). The cDNA has also been used as a probe in isolation and analysis of a gene family for avidin (17). In some early studies it was found that a low concentration of avidin could also be detected in the oviduct after glucocorticoid stimulation, but these steroids were less potent than progesterone (18). The present study was therefore carried out to establish whether the specificity of the avidin gene for progesterone remains unchanged during secondary restimulation by estrogen.

TEROID

Materials

and Methods

Animals

White Leghorn chicks (2-3 weeks old; Vilppula Hatchery, Vilppula, Finland) were injected daily with an im injection of 0.5 mg diethylstilbestrol (DES) in 0.05 ml propylene glycol for 7 days (primary stimulation). Twenty-four hours after the last DES injection, a single dose of progesterone (P; 20 mg/kg), dihydrotestosterone (DHT; 20 mg/kg), 17@-estradiol (E2; 10

Received December 27, 1991. Address all correspondence and requests for reprints to: Dr. Markku S. Kulomaa, University of Tampere;Department of Biomedical Sciences. P.O. Box 607. SF-33101 Tamnere. Finland. * This work was’ supported by the Academy of Finland and the Rockefeller Foundation (New York, NY). 3421

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3422

OF CHICKEN

mg/kg), dexamethasone (DEX; 10 mg/kg), or vehicle (propylene glycol; 1 ml/kg) was administered, and the chicks were killed after 16 h for the avidin mRNA analysis or after 24 h for the avidin assays. In the second group (withdrawal), the primary DES treatment was followed by a withdrawal period of DES for 12 days and a single injection of P or other steroids. In the third group (secondary restimulation), the chicks were restimulated by DES after the withdrawal period for 2 days before administration of the hormones. In the last group the chicks received secondary restimulation of DES and thereafter a single im injection of P together with the abovementioned hormones. In order to study the P time-response curve, the chicks were killed at given times (0.5-72 h) after a single injection of P. After killing, the oviduct and intestine were removed, immediately frozen in liquid nitrogen, and stored at -70 C until assayed. The use of animals in these experiments was in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Materials

Chemicalswere obtained from the following sources:propyleneglycol and DHT from Fluka AG (Bucks, Switzerland); DES and P from E. Merck, (Darmstadt, West Germany); and E2and DEX from Sigma Chemicals (St. Louis, MO). Steroids were dissolvedin propylene glycol at the given concentration. Avidin

assays

The concentration of avidin protein was determined by a biotin-binding method and RIA as previously described (19). Statistical analyseswere carried out with Student’s t test. RNA isolation

and analyses

Frozen oviducts were pulverized by a dismembrator before homogenization, and isolation of total RNA was performed by an LiCl/urea method (20). Electrophoresisof RNA wascarried out in denaturing conditions on a 2.2 M formaldehyde/l.5% agarosegel using a 20 mM phosphatebuffer (pH 7.0) (21). Mol wt markers were purchasedfrom Bethesda ResearchLaboratories (Gaithersburg, MD). For hybridization analysis (Northern), total RNA (10 rg) wastransferred to a nitrocellulose filter (Hybond C; Amersham International, Buckinghamshire, UK) as previously described (21). For slot blot analysis, 10 pg total RNA were denatured with formaldehyde according to Wahl (22) and applied to a nitrocellulosefilter (BA 85; Schleicher & Schuell, Dassel,West Germany) using a filtration apparatus (Minifold II; Schleicher & Schuell). RNAs were fixed on the filter by baking at 68 C overnight. The filter was prehybridized for 6-18 h at 42 C in 50% (vol/vol) formamide containing 5 X SSC (1 X SSC = 150 mM NaCl, 15 mM sodiumcitrate, pH 7.0), 5 X Denhardt’s solution, 0.1% sodium dodecyl sulfate (SDS), 0.5 M sodium phosphate buffer, pH 7.0, and denatured herring sperm DNA (0.5 mg), and then hybridized for 20 h at 42 C with l-3 x 10’ cpm/ml “‘P-labeled (23) avidin cDNA (16). After hybridization, the filter was washedfour times for 30 min at room temperature in 2 x SSC, 0.1% SDS with vigorous agitation, twice for 30 min at 50 C in 0.1 x SSC, 0.1% SDS, and then air-dried. For

AVIDIN

mRNA

Endo. Voll30.

1992 No 6

autoradiography, the filter wasexposedunder a Fuji RX100 Xray film (Fuji Photofilm Co., Ltd., Japan) for 24-72 h at -70 C. In order to measurethe amount of specific RNA, the film wasscannedwith a laser densitometer (Ultro-scan 2202;Pharmacia-LKB, Stockholm, Sweden). The samefilter was hybridized with a 32P-labeledglyceraldehyde-6-phosphatedehydrogenase(GAPDH) cDNA to obtain an internal control for RNA isolation (24). Results Induction of avidin by steroid hormones

Ovalbumin and other genes encoding the egg white proteins in the oviduct have differential specificity for steroid hormones during primary and secondary estrogen or DES prestimulation (3-7). In order to study the specificity of the avidin induction in the oviduct, P and other steroid hormones were administered after different DES prestimulations. The concentration of avidin in the oviduct and intestine was determined by a biotin-binding method and RIA. A significant avidin induction, 25 to 30-fold over control samples, was observed with P after primary and secondary stimulation by DES (Fig. 1). DEX and DHT were found to cause a 2- to 3-fold induction, whereas no induction over the controls was detected after Ez administration. A clear and P-specific induction of avidin was also found after withdrawal of DES restimulation. Avidin was not detected in the intestine of the chicks, which

T

E Primary

stimulation

Wllhdrawal

Secondary

Dex DHT

stimulation

FIG. 1. The effect of DES pretreatment on avidin induction in the chicken oviduct. Two-week-old chicks received daily injections of DES for 7 days and thereafter a single im injection of P, DHT, E, (E), DEX, or propylene glycol (C) as described in Materials and Methods. The other groups received the same hormones after 2 weeks withdrawal and 2 days restimulation with DES. The concentration of avidin protein was determined by a biotin-binding method and RIA.

INDUCTION

excludes the possibility of P-independent avidin synthesis (not shown). Induction

OF CHICKEN

(inflammatory)

of avidin mRNA by steroid hormones

RNA hybridization (Northern) analysis of the total RNA isolated from the oviduct was used to study the induction and specificity of avidin mRNA from the same samples as above. The 32P-labeled avidin cDNA probe hybridized only to a single RNA band about 750 nucleotides in length (Fig. 2). For a semiquantitative determination, the intensity of the bands in the x-ray film was determined by a laser densitometer. After primary DES prestimulation, an increase of about 15- to 20-fold in the amount of avidin mRNA was observed after P administration. A slight induction, about 2- to 4-fold, was observed with DEX, whereas DHT and Ez did not induce avidin mRNA at all (Fig. 2). The same order of magnitude and specificity of induction was found after secondary DES restimulation. A small amount of avidin mRNA was also detected in the oviduct after withdrawal of DES (Fig. 2). The same filter was hybridized with a GAPDH cDNA probe to ensure that the amounts of RNA in each Primary

stimulation

Withdrawal

Secondary

AVIDIN

mRNA

3423

lane were approximately equal. To obtain a better quantitative estimate, the same RNA samples were also analyzed by a slot-blot hybridization analysis, which gave a similar result (Fig. 3). Time-response curve

The time-response curve of the avidin mRNA induction by P was determined after primary and secondary prestimulation with DES. The avidin mRNA was detectable 8 h after P injection and was maximal after 24 h during primary DES prestimulation (Fig. 4). Both the appearance and maximum of the mRNA occurred slightly earlier after the secondary restimulation. Primary

stimulation

Secondary

stimulation

stimulation

K15 4’

C DHT Dex E

P

C DHT Qex E

P

FIG. 3. Slot hybridization

analysis of avidin mRNA after administration of different steroid hormones. Ten micrograms of total RNA from the chicken oviduct were loaded onto a slot blot apparatus. The filter was hybridized to 32P-labeled avidin cDNA and the autoradiograph (24 h exposure) scanned with a laser densitometer.

C

DHT

Dex

E

P

P

c

DHT

Dex

E

P f t “.._.0.5 1 2 4 8

8*idi”-mRNA

t

15 24 48 72

MWl

tJ_t. 0.5 I

2 4

8

i

--..-.--

FIG. 2. Induction

FIG. 4. Time

/

16 24 48 72

I

GAPDH-mRNA

of avidin mRNA by steroid hormones. The chicks were pretreated as described in Fig. 1. Total RNA (10 pg) was separated in a denaturing agarose gel. After transfer, the filter was hybridized using 32P-labeled avidin cDNA as probe. Avidin mRNA was detectable after 24 h autoradiography. The 32P-labeled GAPDH cDNA was used as an internal control.

)

I G*PDH-nRN* L --...---.-..

/

course of avidin mRNA induction by P. P (10 mg/kg) was administered to DES-pretreated chicks which were killed at the times given. Avidin mRNA content was determined from 10 pg total RNA isolated from the oviduct using the slot blot apparatus as in Fig. 3 above.

INDUCTION

3424

OF CHICKEN

Kb 7.5 4.4 -

2.4 1.4-

0.3-

GAPDH-mRNA FIG. 5. Effect of hormone combinations on avidin mRNA. The chicks received secondary restimulation of DES and thereafter a single im injection of P together with propylene glycol (C), DHT, ES (E), or DEX. Total RNA (10 pg) from the oviduct was separated on a 1.5% agarose gel containing 2.2 M formaldehyde, blotted onto nitrocellulose, and hybridized with the 3ZP-labeled cDNAs for avidin and GAPDH.

Steroid combination treatments

The possible synergistic effect of the other hormones given with P was also studied by hybridization (Northern) analysis. After secondary restimulation, a clear and reproducible increase in the amount of avidin mRNA was observed over the control (P alone), when DHT (1.5fold), DEX (2.5-fold), and Ez (3-fold) were given with P (Fig. 5). Hybridization analysis with a GAPDH cDNA was used as an internal control to indicate consistent isolation and transfer of the RNA. Discussion Maximal induction of avidin gene expression by P requires estrogen pretreatment, since differentiation and proliferation of the oviduct cells and an increase in the concentration of P receptors are necessary for the action of the hormone (1). When concentrations of the avidin protein were measured after estrogen pretreatment, significant induction was seen only by P. In addition, the magnitude of induction seemed to be independent of

AVIDIN

mRNA

Endo. Voll30.

1992 No 6

DES prestimulation used in this study. In RNA hybridization analysis, only one RNA band about 750 nucleotides in length was detected, which is in good agreement with a previous result (16). The specificity of avidin mRNA induction by P and other steroids was the same as observed for the protein. DEX caused only a slight (2to 3-fold) increase in the concentration of avidin and its mRNA in the oviduct. The induction of the avidin gene was specific for P even after secondary restimulation by DES, which suggests hormonal regulation somewhat different from other genes regulated by P in the chicken oviduct (3-7). This different hormonal regulation could, however, be caused by differences in cytodifferentiation of the oviduct cells. The cellular distribution of the avidin synthesis in the oviduct was recently observed to be dependent upon the dose and length of the estrogen pretreatment. With high doses of estrogen (0.5-5 mg/kg) or long pretreatments, avidin was produced only by surface epithelial cells. Under normal physiological development or with low doses of exogenous estrogen (0.05 mg/kg), the tubular gland cells were mainly responsible for avidin synthesis, but it was also detected in some surface epithelial cells (25; Joensuu, T., A. Niemela, S. Salomaa, H. Alho, P. Vilja, T. Ylikomi, M. Kulomaa, and P. Tuohimaa, submitted). Secondary restimulation with DES was, however, prolonged in this study, and expression of P-inducible avidin may thus occur mainly in the surface epithelial cells. At the same time, the avidin expression seemed gradually to disappear from the tubular gland cells, whereas ovalbumin synthesis still continued (Joensuu, T., A. Niemela, S. Salomaa, H. Alho, P. Vilja, T. Ylikomi, M. Kulomaa, and P. Tuohimaa, submitted). Steroid hormone receptors modulate transcription of their target genes by sequence-specific interaction with corresponding hormone response elements (HREs). It has been noted that P response elements (PRE) also respond to glucocorticoids. The HREs for glucocorticoid and progestin receptors have therefore been suggested to be identical or at least to share structural features (26, 27); in fact, the element is generally referred to as the GRE/PRE (glucocorticoid/progesterone response element). Although GR and PR have identical base contact points with the GRE/PRE in the tyrosine aminotransferase gene (28), it has been proposed that they may not always be identical (29). Moreover, Evans and coworkers (30) have provided evidence that specificity of the hormonal response is achieved through unique spacing of the direct repeats within the HRE. Our results suggest a functional difference between the PRE and GRE in the chicken avidin gene in duo. The weak responsiveness to DEX could however be explained if glucocorticoid receptors were not available or were present at a low concentration in the oviduct. Immunohistochemical studies of

INDUCTION

OF CHICKEN

chicken GR are still lacking, but indirect evidence suggests the presence of the receptors in the oviduct surface epithelial cells because induction of the avidin mRNA was 2.5fold using a combination of DEX and P compared with P given alone. Avidin mRNA was detectable 8 h after P induction, and its amount was maximal after 16-24 h. The results agree with the earlier studies in vitro, where translatable avidin mRNA activity was observed by 4-6 h, being highest between lo-24 h (31, 32). Rories and Spelsberg (33) have classified steroid inducible genes into so-called early and late responders. Regulation of the late gene transcription would occur l-3 h after hormonal stimulation. According to the time-response curve, the avidin gene seems to belong to the late genes, which also include chicken ovalbumin, ovomucoid, and lysozyme genes (33). In order to study the effect of steroid combinations on avidin induction, DHT, DEX, or EZ was given to chicks with P after the secondary DES restimulation. All combinations generated a synergistic increase in the amount of avidin mRNA, although neither Ez nor DHT could induce avidin production alone. A synergistic effect of DES and P on avidin synthesis has previously been reported (ll), but not with other hormones. A similar synergism for the ovalbumin gene has also been reported in the chicken oviduct using P and Ez and for the ovomucoid gene using DEX and DHT (6). P with DEX, however, increased ovalbumin synthesis to the same extent as DEX alone (4), which was not the case with the avidin gene. The mechanism of the synergism between different steroids is not fully understood. However, experiments in vitro indicate that two different steroid hormones can cooperate functionally in transcriptional activation through the binding of their corresponding receptors to two closely adjacent receptor binding sites (34). Moreover, it was discovered that a combination of DEX and Ez stimulates transcription synergistically when the GRE of the chicken vitellogenin VTG II gene is linked to the estrogen response element. It was also assumed that binding of the P receptor to the VTG II gene fragment would alter the functional activity of the estrogen response element (35). It is possible that there are separate receptor binding sites also on the avidin gene for each class of steroid receptor, and occupation of two target sites by two classes of steroid receptor would explain the synergism. Binding of P receptor to the PRE may also be important to alter the functional activity of other HREs. In conclusion, we studied the effect of steroid hormones on chicken avidin gene expression. It was observed that the avidin gene differs from the other hormonally regulated chicken oviduct genes, since its specificity for P is retained even during secondary restimulation with

AVIDIN

mRNA

3425

DES. A functional difference between the PRE and GRE was also suggested, since glucocorticoids in combination with P, but not alone, induced avidin in uiuo. Further understanding of the molecular details of hormonal induction requires data on the location and molecular structure of the PRE (or PREs and other HREs). We are therefore isolating the 5’-flanking region of the avidin gene. Because avidin is also induced in connection with inflammation, infections, and tissue trauma, different studies have been carried out to ascertain whether the P-dependent and inflammation-induced avidin is encoded by the same gene. Our preliminary results suggest (Kunnas, T. A., M. J. Wallen, and M. S. Kulomaa, unpublished results) that the inflammation avidin (Escherichia cob) is encoded by the avidin gene and by at least one of the avidin-related genes. Acknowledgments We thank Mrs. Merja Lehtinen, Mrs. Tuula Karva, and Mrs. Anja Rovio for their excellent technical assistance, and Dr. Riitta Keinanen and Dr. Mika Wallen for fruitful discussions and criticism of the manuscript. GAPDH cDNA (pGAD28) was generously provided by professor R. J. Schwarz (Department of Cell Biology, Baylor College of Medicine, Houston, TX).

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OF CHICKEN

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