104

Biochimica et Biophysica Acta, 428 (1976) 104--112 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27843

PROLACTIN BINDING IN RAT MAMMARY GLAND DURING PREGNANCY AND LACTATION

HENRY H. HOLCOMB, MARK E. COSTLOW, ROBERT A. BUSCHOW and WILLIAM L. McGUIRE *

Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284 (U.S.A.) (Received September 2nd, 1975)

Summary Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and lactation. In the present study we describe the optimal conditions for the measurement of 12SI-labeled ovine prolactin binding to mammary gland slices of pregnant and lactating rats. Prolactin binding is saturable (Kd approx. 2.36. 10 -~ M), hormone specific and destroyed by proteases. The hormonal environments of pregnancy and lactation dramatically influence the availability and measurement of prolactin binding sites. Whereas binding consistently appears to be low in mammary glands removed from rats during pregnancy, binding levels rise 7--8-fold shortly after birth and remain high during the 22 days of lactation. However, the removal of the ovaries and gravid uteri at specific times during pregnancy results in a prompt 3--6-fold increase in prolactin binding. Elevated levels in potential prolactin binding capacity appear in mammary tissue coincident with the reported rise in serum rat placental lactogen between the eighth and eleventh days. We suggest that high levels of this lactogenic hormone promote the appearance of prolactin binding sites during pregnancy and mask the sites such that they are not available for measurement in vitro.

Introduction

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the rat mammary gland during pregnancy and lactation [1--5]. During the course of pregnancy the development of the gland is stimulated by placental lactogen [1--4]. Following birth lactation begins and is controlled by prolactin. Prolactin also appears to be involved in conA b b r e v i a t i o n : H E P E S , N-2-hydroxyethyl-piperazine-N'*2-ethanesulfonicacid. w h o m reprint r e q u e s t s s h o u l d b e addressed at t h e a b o v e address.

* To

105 trolling its own receptor and those for estrogen as well [6,7]. Recent studies have shown that these lactogenic hormones bind to the subcellular fractions of the mouse and rabbit mammary gland which contain plasma membrane, suggesting that the initial stage of lactogen action may be an interaction with cell surface receptors [8]. In the present study we describe the characteristics and optimal conditions for the measurement of 12SI-labeled ovine prolactin binding to rat mammary slices. Using these conditions we have measured prolactin binding to mammary gland during pregnancy and lactation. Here we show that prolactin binding to rat mammary tissue changes significantly with different stages of mammary development. Materials and Methods Reagents. All chemicals were of reagent grade and were obtained from the following sources: carrier-free 12si, Amersham-Searle; lactoperoxidase, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer and bovine serum albumin, Sigma; Sephadex G-25F, Pharmacia; DEAE-cellulose (DE-52), Whatman; Medium 199, GIBCO; insulin, Squibb; epinephrine, Nutritional Biochemicals; all other hormones obtained from NIAMD. Prolactin iodination and purification on DEAE-cellulose. Ovine prolactin (NIH-P-S-10) was iodinated using the soluble lactoperoxidase technique of Thorell and Johansson [9]. The reaction was carried out for 60 s followed by separation of free 12sI from iodinated hormone on a calibrated Sephadex G-25F column (0.7 × 15 cm) previously saturated with 1 ml 2% bovine serum albumin and equilibrated in 10 mM sodium phosphate buffer (pH 7.0). The pooled void volume (approx. 1 ml) was diluted 1 : 1 with distilled water and applied to a DEAE-cellulose column prepared as follows. Approx. 50 g of DEAE-cellulose were washed four times with 400 ml distilled water. 1 g of albumin was then added to a 50% (v/v) suspension of beads and dissolved with gentle stirring. The mixture was allowed to stand 1 h at 25°C with occasional stirring and excess albumin was then removed by washing with distilled water. The slurry was washed first with 500 mM potassium phosphate buffer, pH 7.0, then with 5 mM potassium phosphate (pH 7.0). Another aliquot of albumin (1 g) was added and the above procedure repeated. After the final wash a 0.7 X 13 cm column was prepared and equilibrated overnight at 4°C with 200 ml 5 mM potassium phosphate (pH 7.0). After collection of 7--8 void volumes a 5--500 mM gradient of potassium phosphate buffer (pH 7.0) was applied to the column. 1-ml fractions were collected at a constant flow rate of 16 ml/h. Purification of the iodinated hormone by DEAE-cellulose chromatography [10], with the modification of saturating the beads with albumin, gave an 85% recovery of the hormone from the column. Fig. 1 shows the elution profile of 12SI-labeled ovine prolactin from the albumin-saturated DEAE-cellulose. The major peak of radioactivity eluted at approx. 65 mM phosphate. The labeled peak was divided into three sections as indicated in Fig. 1, and each portion was assayed for specific binding activity using the rabbit binding assay described by Shiu et al. [11]. All three areas of the elution profile demonstrated

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Fig. 1. D E A E - c e l l u l o s e c o l u m n c h r o m a t o g r a p h y o f x 2 S i . l a b e l e d o v i n e p r o l a c t i n . L a c t o p e r o x i d a s e - i o d i n a t e d p r o l a e t i n w a s freed o f u n b o u n d 12 s I b y S e p h a d e x G - 2 5 F gel filtration and t h e n a p p l i e d t o the colu m n . T h e g r a d i e n t ( 5 - - 5 0 0 m M p o t a s s i u m p h o s p h a t e , pH 7 . 0 ) w a s started at f r a c t i o n 1 6 . S a m p l e s w e r e w i t h d r a w n f r o m e a c h o f the 1 . 0 - m l f r a c t i o n s for d e t e r m i n a t i o n o f r a d i o a c t i v i t y ( 0 ) . Molarity o f the phosp h a t e b u f f e r ( o ) w a s a s s a y e d by c o n d u c t i v i t y m e a s u r e m e n t s o f s e l e c t e d f r a c t i o n s as c o m p a r e d to standards. S e g m e n t s o f the m a j o r p e a k ( N o s . 1, 2 and 3) w e r e p o o l e d , d i a l y z e d for 1 h at 4 ° C against 2 1 o f 2 5 m M Tris • HC1 (pH 7 . 6 ) a n d a s s a y e d for s p e c i f i c binding a c t i v i t y as d e s c r i b e d in the t e x t . The r a d i o a c t i v e p e a k is e l u t e d at 6 5 m M p h o s p h a t e .

progressive competition for binding to rabbit particles with increasing amounts of ovine prolactin; however, the trailing edge of the peak (area No. 3) showed a larger percentage of non-specific binding and more variation in replicate assays. For all subsequent experiments, pooled fractions from area No. 1 and area No. 2 of the labeled DEAE eluate were employed. The iodinated hormone solution was made to 0.1% albumin and could be stored at - 2 0 ° C for up to 2 weeks before changes in binding activity were detected. Specific activity of the iodinated prolactin was determined assuming 100% recovery of hormone in the pooled Sephadex G-25F void volume. Values ranged from 55 to 80 Ci/g. Rat mammary gland binding assay. Inguinal pregnant or lactating mammary glands from Sprague-Dawley rats were excised and washed in ice-cold Medium 199 containing 0.1% albumin. 1-mm slices were prepared using a Stadie-Riggs tissue slicer (A.H. Thomas, Philadelphia, Pa., U.S.A.) and then cut into approx. 10-mg (wet weight) pieces. Pieces containing minimal amounts of connective tissue were selected. The incubation mixture consisted of Medium 199 with 0.1% albumin, 5 mM CaC12, 5 mM HEPES buffer (pH 7.6), approx. 200 000 cpm 12SI-labeled ovine prolactin/ml, and 0 - - 5 0 0 0 ng ovine prolactin/ml. Five 10-mg slices of tissue were routinely incubated in each standard scintillation vial and gently agitated in 1 ml medium. The reaction was terminated by transferring each tissue slice to an individual glass tube and washing four times with a total of 10 ml ice-cold Medium 199 containing 0.1% albumin. After the final wash, 0.5 ml 10% trichloroacetic acid was added for latter determination of D N A content [12] following extraction according to Schneider [ 1 3 ] . The tissue was counted and a wet weight determination was made.

107 Results

Prolactin binding to rat mammary gland The influence of duration and temperature of incubation on the in vitro binding of prolactin to lactational rat mammary gland was determined by incubating tissue slices for various intervals at 4, 22, and 37°C. Fig. 2 shows that at 4 ° C little specific binding was evident even after 4 h of incubation. At 22 and 37°C specific binding increased with time of incubation and reached a plateau after a b o u t 3 h. Non-specific prolactin binding was comparable at both 4 and 22°C (20--30% of total bound} b u t at 37°C increased 2-fold (not shown) although specific binding was the same as at 22 ° C. On the basis of this data, subsequent experiments were carried out at 22°C for 3 h. To determine whether prolactin binding to lactational rat mammary gland was saturable, slices of tissue were incubated with increasing concentrations of the hormone for 3 h at 22°C. Fig. 3A shows that prolactin uptake by mammary tissue increased with increasing amounts of available prolactin and then reached saturation, indicating the presence of a limited number of binding sites. Although the derivation of Scatchard [14] is not strictly applicable to hetero-' geneous binding systems such as tissue slices, a Scatchard plot of this type of data {Fig. 3B) does yield a straight line and an estimate of 2.36 + 0.14 • 10 -9 M (n = 20) for the dissociation constant at 22°C. Determining the DNA content for each sample we find that mammary tissue contains 1.08 + 0.10 fmol of prolactin binding sites/pg DNA (n = 20). Mammary tissue slices incubated with increasing 12SI-labeled ovine prolactin (0.04--2 pmol) yield similar binding curves and dissociation constants. Progressive competition for binding of 12SI-labeled ovine prolactin to rat

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Fig. 2. Effect of t e m p e r a t u z e and t i m e o f i n c u b a t i o n o n the binding o f i = s I-labeled ovine prolactin to rat m a m m a r y gland. Slices o f l a c t a t i n g m a m m a r y tissue were i n c u b a t e d at 4 ° C (o), 22°C (e), and 3 7 ° C (D) with 200 000 c p m I 2 s i.labele d ovine prolaetin in the p r e s e n c e or a b s e n c e o f I ~tg o f u n l a b e l e d ovine prolactin. At intervals, slices w e r e r e m o v e d f r o m t h e i n c u b a t i o n m i x t u r e , w a s h e d and a s s a y e d for b o u n d rad i o a c t i v i t y per m g w e t w e i g h t . R e s u l t s are e x p r e s s e d as s p e c i f i c binding: t o t a l 1 2 s I - l a b e l e d p r o l a c t i n b o u n d m i n u s I 2 s I-labeled prolactin n o n - s p e c i f i c a l l y b o u n d in the p r e s e n c e o f excess unlabeled h o r m o n e . Each p o i n t r e p r e s e n t s the m e a n o f five separate tissue slices.

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F i g . 3 . S p e c i f i c binding o f t 2 s I-labeled o v i n e p r o l a c t i n b y lactating rat m a m m a r y gland. Slices o f m a m m a r y gland w e r e i n c u b a t e d for 3 h at 2 2 ° C in the p r e s e n c e o f increasing q u a n t i t i e s o f ovine p r o l a c t i n . A f t e r w a s h i n g , b o u n d r a d i o a c t i v i t y and tissue w e i g h t w e r e d e t e r m i n e d . Each p o i n t r e p r e s e n t s the m e a n value o f five separate tissue slices. ( A ) S p e c i f i c binding as a f u n c t i o n o f t o t a l prolactin p r e s e n t . (B) S e a t c h a r d [ 1 4 ] analysis o f t h e binding data in A . ( C ) C o m p e t i t i o n for binding o f i 2 s I - l a b e l e d o v i n e p r o l a c t i n in lactating rat m a m m a r y gland and rat spleen.

mammary tissue was observed in the presence of increasing amounts of ovine prolactin as shown in Fig. 3C. Mammary tissue exhibited 80% competition and little total binding could be demonstrated after comparable incubation of spleen slices. The hormonal specificity of the prolactin binding sites in lactational mammary tissue was assessed by incubating 12SI-labeled ovine prolactin with mammary tissue in the presence of various other hormones. Addition of 2 0 0 0 ng/ml unlabeled rat or ovine prolactin inhibited 12SI-labeled ovine prolactin binding similarly; competition by unlabeled rat follicle stimulating hormone, rat growth hormone, and insulin averaged 9%. Pretreatment of lactational mammary tissue slices with pronase or trypsin completely abolished binding while phospholipase C reduced binding by 51%. DNAase, neuraminidase, hyaluronidase, collagenase and phospholipases A and D were without effect.

Prolactin binding during pregnancy and lactation Fig. 4 shows 12SI-labeled ovine prolactin binding to mammary tissue during pregnancy and lactation in the intact rat. Day 1 of pregnancy was defined as the day after sperm were present in the vaginal smear. It can be seen that only negligible levels of binding are observed during pregnancy. In contrast, prolactin binding increases dramatically shortly after birth. This high level of binding is then maintained throughout the 22 days of lactation.

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Pregnoncy Locotion Fig. 4. S p e c i f i c 12 s I - l a b e l e d o v i n e p r o l a c t i n b i n d i n g t o rat m a m m a r y g l a n d d u r i n g p r e g n a n c y a n d lactat i o n . 12 s I - l a b e l e d p r o l a c t i n ( 0 . 0 5 p m o l ) w a s i n c u b a t e d w i t h i n g u i n a l m a m m a r y g l a n d slices f r o m rats t a k e n at d i f f e r e n t t i m e s d u r i n g p r e g n a n c y a n d l a c t a t i o n . S p e c i f i c b i n d i n g is t h e d i f f e r e n c e b e t w e e n radioa c t i v i t y b o u n d in t h e p r e s e n c e o f e x c e s s u n l a b e l e d p r o l a c t i n ( 2 1 7 p m o l ) a n d t h a t b o u n d in its a b s e n c e . B i n d i n g assay c o n d i t i o n s are as d e s c r i b e d in Materials a n d M e t h o d s .

Since the second half of pregnancy is a period of rapid growth and development for the mammary gland [3], the low level of prolactin binding observed seemed paradoxical. Since serum levels of rat placental lactogen, which competes for 12SI-labeled ovine prolactin binding sites, are high during the second half of pregnancy [11], we reasoned that binding sites may be occupied by the exceedingly high levels of hormone present throughout late pregnancy, thus preventing labeled ovine prolactin from binding in vitro. We approached this problem by removing gravid uteri and ovaries 1 day prior to assay so that the source of placental lactogen was eliminated. A large increase in prolactin binding capacity during mammary development in pregnan-

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110 TABLE I EFFECT OF ESTRADIOL MAMMARY GLAND

AND PROGESTERONE

ON PROLACTIN

BINDING TO PREGNANT

RAT

T h r e e g r o u p s o f S p r a g u e - D a w l e y rats, 19 d a y s p r e g n a n t , w e r e o v a r i e c t o m i z e d ( o v x ) a n d h y s t e r e c t o m i z e d ( h y s t x ) . T w o g r o u p s r e c e i v e d i n j e c t i o n s of e i t h e r e s t r a d i o l ( 0 . 3 3 p g / 8 h ) , or p r o g e s t e r o n e (1 m g / 8 h) w h i l e t h e t h i r d g r o u p r e c e i v e d p h y s i o l o g i c a l saline e v e r y 8 h, f o r 48 h. All a n i m a l s w e r e t h e n s a c r i f i c e d a n d p r o l a c t i n b i n d i n g to m a m m a r y g l a n d slices w a s d e t e r m i n e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . B i n d i n g is e x p r e s s e d as t h e m e a n + S.E. f o r t h e n u m b e r o f a n i m a l s i n d i c a t e d in p a r e n t h e s e s . Treatment

Prolactin binding ( c p m / m g tissue)

Ovx + h y s t x + estradiol Ovx + hystx + progesterone Ovx +hystx +saline

1 3 2 -+ 36 (n = 5) 1 5 2 -+ 22 (n = 6) 1 5 9 -+ 8 ( n = 6)

cy was then revealed (Fig. 5). This high level of prolactin binding sites provides a mechanism which would logically be expected to exist for placental lactogen to act in the development of mammary gland during pregnancy.

Effect of estradiol and progesterone on prolactin binding Since removal of ovaries and gravid uteri also severely reduced serum levels of estradiol and progesterone, experiments were done to examine the effects of these hormones on prolactin binding in the absence of placental lactogen. The results in Table I indicate that neither estradiol nor progesterone significantly depresses 12SI-labeled ovine prolactin binding following ovariectomy plus hysterectomy. Since our results suggested that endogenous placental lactogen was masking prolactin binding sites during pregnancy, we asked if the relatively high prolactin levels characteristic of the suckled lactating rat might also reduce the number of available sites. Weaning rats for 18--24 h prior to assay, which reduced serum levels of rat prolactin [15], did not alter either the quantity of Kd of binding sites throughout the 22 days of lactation. However, it is important to note that serum prolactin levels in the lactating rat rarely exceed 100 ng/ml, whereas placental lactogen levels are reported to range from 800 to 1200 ng/ml throughout most of the latter half of pregnancy [11]. Discussion

These studies demonstrate that the availability and measurement of prolactin binding sites are influenced by the hormonal environment of pregnancy and lactation. These binding sites exhibit hormonal specificity and a sensitivity to the destructive effects of proteolytic enzymes similar to that recently observed in rat mammary tumors [16,17]. Prolactin binding sites appear in the pregnant rat's mammary gland in two stages. On the eighth day of pregnancy there are negligible levels of binding; however, the mammary gland's potential binding capacity increases rapidly such that on the eleventh day a high level of binding is measured. This rise in prolactin binding capacity parallels a dramatic increase in serum placental lac-

111 togen concentration [11] and epithelial cell proliferation [2,3]. Hence the rise in metabolic activity coincides with the increase in prolactin binding capacity, which suggests that placental lactogen is acting on the binding sites and promoting mammary growth and development. Since the rise in binding is detected only if the source of placental lactogen is eliminated, it is reasonable to assume that placental lactogen normally occupies the prolactin binding sites during pregnancy. A second rise in prolactin binding capacity is observed following the 19th day of pregnancy, for the levels observed on the 20th day and throughout lactation are consistently higher than those observed at earlier times. This may reflect the gland's shift from growth and development to milk synthesis. High levels of placental lactogen present during the second half of pregnancy may have created misleading artifacts in two recent studies reported in the literature. Frantz et al. [5] measured specific prolactin binding to pregnant rat or mouse mammary gland and found it to be approximately one-third that present in the lactating gland. This is significantly less than that found in our studies. Since they made no mention of placental removal in their report, it is likely that their measurements were made using conditions which might significantly reduce 12SI-labeled ovine prolactin binding levels. Richards and Midgley [18] recently reported levels of prolactin binding to isolated corpora lutea of the pregnant rat. While binding was high in early pregnancy, it decreased between days 8 and 12. This binding profile is inversely proportional to placental lactogen serum levels and may reflect the masking of prolactin binding sites in the corpus luteum. In summary, high levels of prolactin binding appear in rat mammary gland coincident with a rise in placental lactogen between the eighth and eleventh days of pregnancy, though the sites are normally masked by the high levels of placental lactogen present. Following birth, prolactin serum levels are elevated by suckling; however, circulating prolactin does not appear to interfere with the measurement of the consistently high levels of binding observed throughout lactation.

Acknowledgments The studies were supported in part by U.S.P.H.S. CA-11378, CB-23862, The American Cancer Society BC-23D, and The Robert A. Welch Foundation, H.H.H. is a medical student supported by a fellowship from the American Cancer Society, Texas Division Inc., N.I.H. 626-1688-0250, and U.S.P.H.S. Clinical Cancer Training Grant No. 5 T12 CA08069. M.E.C. is a recipient of N.I.H. fellowship CA-04004-02.

References 1 Ceriana, R . L . ( 1 9 7 4 ) J. Invest. D e r m . 6 3 , 9 3 - - 1 0 8 2 Cowie, A.T. a n d Tindal, J.D. ( 1 9 7 1 ) The P h y s i o l o g y o f L a c t a t i o n , The Williams a n d Wflkins Co., Baltimore 3 Anderson~ R . R . ( 1 9 7 4 ) L a c t a t i o n , A C o m p r e h e n s i v e Treatise (Larson, B.L. and Smith, V.R., eds.) pp. 9 7 - - 1 3 5 , A c a d e m i c Press, N e w Y o r k 4 L y o n s , W.R., Li, C.H. a n d J o h n s o n , R.E. ( 1 9 5 8 ) R e c e n t Prog. H o r m o n e Res. 14, 2 1 9 - - 2 5 4

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5 6 7 8 9 10 11 12 13 14 15 16 17 18

Frantz, W.L., MeIndoe, J.H. and Turkington, R.W. (1974) J. Endocrinol. 60, 485--497 Costlow, M.E., Buschow, R.A. and McGuire, W.L. (1975) Life Sci. 17, 1457--1466 Chamness, G.C., Costlow, M.E. and McGuixe, W.L. (1975) Steroids 26, 363--372 Shiu, R.P.C. and Friesen, H.G. (1974) Bioehem. J. 140, 301--311 Thorell, J.I. an d Johansson, B.G. (1971) Biochim. Biophys. Acta 2 5 1 , 3 6 3 - - 3 6 9 Frantz, W.L. and Turkington, R.W. (1972) Endocrinology 91, 1545--1548 Shiu, R.P.C., Kelly, P.A. and Friesen, H.G. (1973) Science 180, 968--971 Burton, K. (1956) Biochem. J. 62, 315--323 Schneider, W.C. (1945) J. Biol. Chem. 1 6 1 , 2 9 3 - - 3 0 3 Scatchaxd, G. (1949) Ann. N.Y. Acad. Sci. 5 1 , 6 6 0 - - 6 7 2 Amenomo ri, Y0, Chen, C.L. and Meites, J. (1970) Endocrinology 8 6 , 5 0 6 - - 5 1 0 Costlow, M.E., Buschow, R.A. and McGuire, W.L. (1974) Science 184, 85--86 Costlow, M.E., Buschow, R.A., Richert, N.J. and McGuixe, W.L. (1975) Cancer Res. 35, 970--974 Richards, J.S. and Midgley Jr., A.R. (1974) Fourth I nt e rna t i ona l Congress on Hormonal Steroids. Mexico City (James, V.H.T., ed,), pp. 348--349

Prolactin binding in rat mammary gland during pregnancy and lactation.

Lactogenic hormones from the placenta and pituitary are primarily responsible for the growth and function of the mammary gland during pregnancy and la...
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