@Copyright 1985 by The Humana Press Inc~ All rights of any nature whatsoever reserved. 0163~,984185/08114283503.60
Zinc Secretion in the Oviduct of the Coturnix Quail P. BUTZEN, 1 E. ROOT, 1 AND B~ STARCHER *'2
~Nutrition Division, Department of Home Economics, University of Texas at Austin, Austin, TX; and 2Department of Biochemistry; The University of Texas Health Center at Tyler, Tyler, TX Received March 10, 1985; Accepted July 1, 1985
ABSTRACT The oviduct _from laying quail were used to investigate mechanisms of trace mineral secretion and the possible role of metallothionein in this process. Secretion of zinc occurred maximally at pH 5.4, which is close to the normal pH of the oviduct. Secretion occurred to a much greater extent in the isthmus and shell gland than in the magnum, the major protein-secretory section of the oviduct. Intraperitoneal administration of cadmium resulted in a marked reduction in Zn secretion from the oviduct of laying quail. This effect could not be correlated with metallothionein since metallothionein could not be detected in any section of the oviduct in control or Cdinduced quail. Small-molecular-weight metal-binding ligands were present in the isthmus and shell gland, which may play a role in trace mineral mobilization. Histological evaluation by light and elelctron microscopy show that Zn is transported from the smooth muscle cells through the connective tissue matrix in the extracellular space to the epithelial goblet cells. Presumably, Zn and other trace minerals are secreted from the secretory goblet cells into the egg. Index Entries; Zinc, secretion of; Coturnix Quail; oviduct, zinc secretion in; metallothionen, role in trace mineral secretion; cadmium. *Author to whom all correspondence and reprint requests should be addressed.
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INTRODUCTION Zinc, an essential trace element for plants, animals, and h u m a n s , has been widely studied. It plays an important biochemical and nutritional role as a c o m p o n e n t of over 80 metalloenzymes and insulin. Cotzias (1), in 1962, first proposed that Zn metabolism came u n d e r homeostatic control. Since then, m u c h of the interest has focussed on the intracellular metal-binding ligand metallotheonein, which, in addition to transport (2,3), storage (4,5), and detoxification (6,7), has been postulated to have a regulatory role in Zn absorption by serving as a mucosal block (8) or a more direct and positive role enhancing Zn absorption (3,8). Metallothionein is present in trace amounts in nearly all tissues that have been examined (9,10)o Its concentration, moreover, can be rapidly increased by exposure to sublethal doses of certain metals (11,12), various stresses (12,13), starvation (14), administration of glucocorticoids (15,16), and a n u m b e r of alkylating agents (17). Whether the resulting elevated metallothionein levels are beneficial may be d e p e n d e n t u p o n the tissues and the trace minerals involved. The avian oviduct presents an unusual model system illustrating secretion of trace minerals, rather than absorption or excretion. It m a y be, however, that m a n y of the parameters governing the latter processes may also regulate secretion. The oviduct can be divided into five distinct regions, four of which have specific physiological functions in egg formation: i n f u n d i b u l u m (engulfs the ovum); m a g n u m (protein secretion); isthmus (shell m e m b r a n e s secretion); and shell gland (eggshell secretion). The selective importance of these distinct regions in trace-mineral secretion has not been delineated. The mineral content of the egg is derived from the bird's diet. After absorption, it is deposited in the developing yolk or secreted from the oviduct directly to the egg white. Alt h o u g h the majority of Zn is present in the yolk, Sandrock et al. (18) have s h o w n that a significant a m o u n t of the Zn located in the a l b u m e n is transferred to the yolk and utilized by the developing fetus. The purpose of this study is to investigate Zn secretion and determine if metallothionein has an important role as a regulator molecule for this process.
METHODS Experimental Animals I m m a t u r e and mature female Japanese quail (Coturnix coturnix japonica) w e r e used for all experiments. The nonlaying quail were 4-6 w old and w e i g h e d 100-160 g. The laying quail, weighing 170-200 g, w e r e 8-12 w old. They were housed u n d e r controlled atmospheric and lighting conditions, fed a commercial layer mash, and given tap water ad libitum.
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In Vitro Zinc Secretion Studies Control and Cd-treated laying and nonlaying quail and estrogentreated nonlaying quail were used. Daily injections of Cd (0.1-0.3 mg), in the form of Cd acetate, in saline (1 mL) were given ip for 1-3 d. The quail were killed 24 h after the last injection. Selected nonlaying quail were given daily ip injections of 0.25 mg ~3-estradiol (Sigma Chemical Co.), s u s p e n d e d in corn oil or dissolved in 100% ethanol, for 4 d. The quail were sacrificed by cervical dislocation 18-24 h after the last injection and the oviducts removed. Control quail were injected with either saline, corn oil, or alcohol. The excised oviducts were rinsed in 0.9% saline, blotted dry, weighed, measured, and everted with a plastic tube. The everted oviducts were m a d e into sacs, as described by Wilson and Wiseman (19), containing 65Zn in Dulbecco's Modified Eagle's Medium (EMEM from Biolabs), buffered with 4M sodium acetate to pH 5.4. Two studies were done. In the first study, one sac was made using the complete oviduct, with each anatomical division (anterior m a g n u m , posterior m a g n u m , isthmus, and shell gland) containing 200,000 cpm 65Zn in 1 mL of DMEM, pH 5.4. This was accomplished by first tying off the anterior end of the shell gland with surgical thread, adding I mL of 65Zn DMEM to the open m a g n u m , and allowing this to settle by gravity into the shell gland. The anterior end of the shell gland was then tied off and the process repeated, adding 1 mL of 65Zn DMEM to each section. The whole oviduct, with each segment separated, was incubated in 40 mL DMEM, p H 5.4. In the second study, individual sacs were made from each of the four sections, with each sac containing 0.4 mL DMEM, pH 5.4, and 200,000 cpm 65Zn. The individual sections were incubated in 10 mL DMEM, p H 5.4. Samples from both studies were incubated u n d e r 5% CO2 in a shaking water-bath at 37~ At 30 min intervals, 0.5-mL aliquots of mucosal media were removed, radioactivity m e a s u r e d with a Beckman 5000 G a m m a Counter, and subsequently returned to the flasks for further incubation of the tissue u n d e r 5% CO2 for a total of 2 h. At the end of the incubation period, some sacs w e r e emptied, slit open lengthwise, rinsed in saline, and prepared for gel-filtration chromatography, as described below.
Induction of l~Ietallothionein Adult laying quail were given a single ip injection of 0.3 m g Cd, in the form of Cd acetate, in 1 mL of saline. The birds were sacrificed 18-24 h after the injection by cervical dislocation. The liver and oviduct from control and Cd-treated quail were removed and analyzed for metallothionein. Tissues not used immediately for metallothionein analysis were frozen (-20~ u n d e r nitrogen.
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Metallothionein Analysis Two m e t h o d s were used for the analysis of metallothionein in liver and oviduct tissue. First, the 2~ m e t h o d of Piotrowski et al. (20), as described by Kotsonis and Klaasen (21), was used. Fresh tissue was homogenized in 0.9% saline (1 g tissue/7 mL saline) with a polytron homogenizer. To 2.5-mL h o m o g e n a t e (0.36 g tissue), 0.25 mL 2~ (250 ~g Hg) was added, thoroughly mixed, and allowed to stand at room temperature for 30 min. Following the addition of 0.8 mL 10% trichloroacetic acid (TCA), the samples were allowed to stand an additional 10 min before centrifuging at 3000g for 10 rain at 0~ (Beckman Model TJ-6). The supernatants were transferred to clean disposable tubes and m e a s u r e d for radioactivity in a g a m m a counter (Beckman 5500). All samples w e r e done in duplicate, and 2~ a d d e d to saline was used as a control. Following the 2~ procedure, the metallothionein fraction from a control oviduct and a liver from a Cd-treated quail was separated from other low-molecular-weight Hg-binding proteins and peptides by gel-filtration chromatography. The TCA supernatants were individually applied to a Sephadex G-50 column (1 • 48 cm), equilibrated, a n d eluted with 4M urea in saline, and collected in 2-mL fractions. The flow rate was 0.5 mL/min for this column. Each fraction was m e a s u r e d for radioactivity with a gamma counter. The column was calibrated with the following substances: glutamic acid (mol wt 146), bacitracin (mol wt 1570), bovine insulin (tool wt 5700), cytochrome c (mol wt 12,400), hemoglobin (mol wt 15,500), myoglobin (mol wt 18,800), pepsin (mol wt 35,000), and egg albumen (mol wt 45,000). All chromatography was conducted at 4~ For the second method, gel-filtration chromatography was used as a relative measure of metallothionein levels. Tissue (0.1-0.5 g) was homogenized in 2-3 mL 4M urea in saline and centrifuged at 20,O00g (Beckman Model J2-21) for 20 min at 0~ The supernatant was applied to a Sephadex G-50 column (1 • 48 cm), eluted with 4M urea in saline, and collected in 2 mL fractions. It should be noted that 65Zn (30,000-40,000 cpm) and/or 20 txg Cd (CdC12) was a d d e d to the supernatants of control and estrogen-treated specimens, which were h o m o g e n i z e d in saline. After a 30-rain standing period, urea was a d d e d to make a 4M urea saline solution. Each fraction collected was m e a s u r e d for radioactivity before assaying for Cd with an atomic absorption spectrophotometer (Instrumentation Laboratory Inc., Model 551).
Histology Portions of the everted oviducts used for the in vitro Zn secretion studies were examined by light and electron microscopy in order to detect any adverse effects of incubation u p o n integrity of the tissue and to determine the cellular location of the Zn. The location of the Zn will, unBiological Trace Element Research
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der normal circumstances, be the same as the location of the Z n - b i n d i n g protein. Portions of the four sections of the oviduct were taken before a n d at intervals d u r i n g incubation with a d d e d Zn (1 m g to each section). Tissue was treated as described above for the in vitro secretion. Slices of tissue 1 m m thick were taken from the freshly r e m o v e d oviduct and the everted sacs at 15, 30, 60, 90, a n d 120 m i n of incubation, a n d were i m m e r s e d in a fixative comparable either with a c o n v e n t i o n a l preparation for electron microscopy or with a modification of T i m m ' s stain for d e m o n s t r a t i o n of Zn. Tissues p r e p a r e d conventionally was placed in half-strength Karnovsky's fixative (22), postfixed in 2% OsOa, d e h y d r a t e d in an alcohol series followed by acetone, a n d e m b e d d e d by Epon-type plastic [consisting of: E m b e d 812 (Electron Microscopy Sciences, Ft. W a s h i n g t o n , PA) 15 mL, DDSA 12 mL, N M A 6 mL, and DBP 1 mL, with DMP-30 u s e d as accelerator]. Sections 1-3 ~ m thick were stained with toluidine blue a n d e x a m i n e d using a Zeiss light microscope. Ultrathin sections 60-90 n m thick (silver) were stained with uranyl acetate a n d lead citrate a n d e x a m i n e d in a Siemens electron microscope. Tissue p r e p a r e d for T i m m ' s stain was fixed in a sulf i d e - g l u t a r a l d e h y d e mixture, consisting of 0.1% s o d i u m sulfide in 0.15M s o d i u m p h o s p h a t e buffer to which g l u t a r a l d e h y d e was a d d e d just before use to m a k e a 3% solution (23). Further processing was carried out, as described for the sulfide silver m e t h o d of Danscher (24), and e m b e d d e d in the plastic described above, k e e p i n g polymerization t e m p e r a t u r e s at or below 45~ Sections 1 a n d 3 p,m thick were stained, as described by Danscher. The t h i n n e r sections p r o v i d e d subjects for p h o t o g r a p h y , a n d the thicker ones p r o v i d e d T i m m ' s - s t a i n e d material for electron microscopy. Such sections were covered with a d r o p of u n p o l y m e r i z e d plastic a n d a block of p o l y m e r i z e d plastic. These were allowed to polymerize a n d t h e n r e m o v e d from the glass microscope slide after a brief heating of the b o t t o m of the slide on a hot plate. Thin sections were cut from this material, stained with uranyl acetate a n d lead citrate and examined in a Siemens electron microscope. All histological observations were m a d e from tissues taken with the egg in the shell gland.
RESULTS A preliminary e x p e r i m e n t was c o n d u c t e d to determine w h e t h e r Zn secretion occurred maximally at neural p H or at a p H of 5.4, w h i c h is close to t h e n o r m a l p H of the oviduct sections studied (25). The results s h o w n in Fig. 1 indicate that Zn secretion from the whole oviduct occurred at a significantly higher rate at p H 5.4. As a result, all subseq u e n t secretion experiments were c o n d u c t e d at this pH. Biological Trace Element Research
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16"
O
x
12-
0.
0.9_ lie
o
8" z N
pH 73
o
~ 0
30 60 90 Incubation (minutes)
Fig. 1. 6SZincsecretion of oviducts from laying quail incubated in DMEM, pH 5.4 (o) and 7.3 (e). The values express the total 65zinc secreted from the whole oviduct for the times indicated. In experiments to m e a s u r e secretion from functionally distinct sections of the oviduct, the i s t h m u s was always f o u n d to be the major secretor of 65Zn (Fig. 2). This was true even t h o u g h the total a m o u n t of Zn secreted by each section of an oviduct varied between individual birds. The reason w h y the oviducts from s o m e of the birds were high Z n secretors a n d others low, a p p e a r e d to be correlated to the position of the egg in the oviduct. Zinc secretion was d i m i n i s h e d w h e n the yolk was located in the m a g n u m or isthmus, a n d t h e c o r r e s p o n d i n g tissues that a p p e a r e d to be d e p l e t e d of secretory material took on a grayish white color, a c c o m p a n i e d by a t h i n n i n g of the oviduct wall. This was n o t seen w h e n the egg was located in the shell gland, a n d for this reason m o s t experim e n t s utilized oviducts w h e n the egg was located in this region. Biological TraceElement Research
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g" & 25" X
a. o
20
r .
O ~
15O
Z
N
10"
Anterior Posterior Isthmus Shell Magnum Magnum gland
Fig. 2. Total 65Zn secretion of individual oviduct sections after 2-h incubation. Values shown are the means from six laying quail. Intraperitoneal injections of Cd to laying quail resulted in a significant reduction in Zn secretion (Fig. 3). Because of the high individual variation b e t w e e n birds, this experiment was repeated several times with whole oviducts, and in every instance Cd administration reduced Zn secretion. A single dose of 0.3 mg of Cd seemed to give a more uniform response than repeated doses at varying levels. At higher levels with repeated injections, the quail became noticeably sick. All the subsequent experiments use a single 0.3-mg Cd injection, and the birds were killed 18--24 h later. In experiments to control the hormonal influence of the laying cycle, nonlaying quail, untreated and treated with estrogen or Cd, were used. Our results indicated that, although immature oviducts were m u c h smaller in size and weight, they secreted significantly more ~ (168%) than m a t u r e oviducts (Fig. 4). An even more striking increase in Zn secretion was seen in oviducts from immature nonlayers that had been treated with estrogen. Surprisingly, the administration of Cd to nonlayers resulted in a marked increase in Zn secretion. Two m e t h o d s were employed to determine if the alteration in Zn secretion resulting from Cd injection was related to the presence of metallothionein. In the first series of experiments, the livers (known to contain large amounts of metallothionein following Cd induction) and the oviducts of Biological Trace Element Research
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~r
,r-*
X
8-
I3.
G
6
.2 u (9 (/9
4
Z N
2
I
30
I
I
i
60 90 120 Incubation (minutes)
Fig.. 3. Total 65Zn secretion of oviducts from control (o) and Cd-treated ([]) laying quail. The bars shown at 120 min represent the standard deviation. selected quail were analyzed for metallothionein by gel-filtration chromatography. Intraperitoneal injections of Cd caused a large increase in hepatic metallothionein levels w h e n compared to control livers, as illustrated in Fig. 5. Similar elution profiles were seen w h e n ~ was a d d e d to the supernatants and radioactivity monitored in the column fractions. In control liver, Cd or 65Zn were mainly b o u n d to a highmolecular-weight protein fraction with only a small amount b o u n d to the metallothionein fraction. The reverse was true for the Cd-induced hepatic tissue. Positive identification of metallothionein in Cd-induced liver was confirmed by DEAE-ion exchange chromatography. W h e n the liver supernatant was chromatographed, only one Cd-protein peak was present, indicating only one isoform for quail hepatic metallothionein. This is in a g r e e m e n t with previous reports for other avian species (26-28)~ Gelfiltration analysis of quail oviduct homogenates following Cd induction s h o w e d no detectable Cd-thionein. The Cd-induced samples w e r e indistinguishable from control oviducts, with all of the Cd being b o u n d to the high-molecular-weight protein fraction, as was observed with control livers. The results of the other means to measure oviduct metallothionein, using the more quantitative m e t h o d of Piotrowski (20), are s h o w n in Table 1. W h e n quail were given a single ip dose of Cd (0.3 rag), there was a marked increase in hepatic metallothionein, similar to what was seen by Biological Trace Element Research
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Non layer + Cd
O
Non layer estrogen
C
0 0
C
200
0 L~ m
2
Non layer control
f-.
U 100,
Fig. 4. 65Zincsecretion of quail oviduct after 2-h incubation. Values are expressed as a percent of control laying quil. n = 4, Except the nonlaying given Cd or estrogen, where n = 2. gel filtration. However, w h e n separate oviduct sections were analyzed, Cd administration did not significantly alter 2~ binding. Even more striking w e r e the extremely high levels of w h a t appeared to be metallothionein in the isthmus, and the s o m e w h a t lower levels of this protein in the shell gland. This was evident in the control as well as the Cd-induced animals. C h r o m a t o g r a p h y of these Hg-binding components on Sephadex G-50 indicated that they were not metallothionein, but molecules of m u c h lower weight, approximately 3.35 kdaltons for the shell gland a n d 1.1 for the isthmus (Fig. 6). The m a g n u m had such low Hg binding that no peaks were evident following chromatography. Microscopic examination of tissue, after incubation with a d d e d Zn, was first p e r f o r m e d using the m a g n u m . In untreated tissue, stain indicating the presence of metal was evident only in the epithelium. Of the epithelial cells, only the goblet cells (Fig. 7) contained metal, which was sequestered in oval-shaped secretory granules. After incubation, stain also appeared in smooth muscle and the connective tissue matrix, but not in glandular cells. Subsequent observations were carried out with the shell gland a n d the isthmus, which were more active in the secretion of Zn. Biological Trace Element Research
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0.75
E Q. v
0.50
"o 0
0.25
0
10
20
30 40 Fraction
50
60
70
Fig. 5. Gel filtration on Sephadex G-50 (1 x 48cm) of control (o) and Cdtreated (o) liver homogenates. Fraction size was 2 mL. In the shell gland, little or no stain was present in untreated tissue (Fig. 8A). After 15-min incubation with a d d e d Zn, the epithelial goblet cells were well stained (Fig. 8B). At 30 rain stain appeared in smooth muscle and interstitial spaces, and by 60 min some glandular cells took up stain as well (Fig. 8C). By 120 rain, stain had cleared from glandular cells and part of the connective tissue matrix, but was still heavy in the epithelium and subjacent interstitial space (Fig. 8D). The isthmus differed from the shell gland and m a g n u m in showing s o m e w h a t heavier staining at all times. Some stain was present in both Biological Trace Element Research
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Table 1 Metallothionein Content of Liver and Individual Oviduct Sections from Control and Cd-Treated Laying Quail ~ Tissue
c p m 2~
ControP,
Cd-treated ~, cpm 2~
Liver Anterior magnum Posterior magnum Isthmus Shell gland
126 243 404 3468 1030
3174 288 471 2862 1783
+ 59 _+ 104 + 66 _+ 408 _+_ 502
_+ 451c _+ 48 _+ 155 _+ 361 + 399
~The values for MT are expressed in relative terms and are proportional to 2~ bn=4.
~Statistically significant at P < 01.
epithelium and smooth muscle in untreated tissue. Again, stain was present only in goblet cells in the epithelium (Fig. 9). Changes in appearance during incubation with a d d e d Zn followed the same pattern as that described for the shell gland. Appearance of the tissue correlates well with the results of the Zn secretion studies. In all regions, the histology suggests rapid uptake of Zn and its rapid m o v e m e n t through the connective tissue matrix to the epithelium w h e r e it is concentrated in goblet cells prior to secretion.
DISCUSSION The oviduct of the laying quail proved to be an ideal tissue for doing secretion studies in vitro. The inverted sacs were very durable and the tissues r e m a i n e d intact during the 2 h incubation period. W h e n using the whole oviduct, we felt it necessary to physically separate the anatomical regions, with each region containing equal amounts of 6SZn. Without separation, the liquid tended to pool in one region during the incubation period. In other preliminary experiments we observed the secretion of Zn through the oviduct to be a unidirectional flow; going from the serosal to the mucosal side only. Individual variations between oviducts in the rate of 65Zn secretion was a consistent finding during these studies. Using oviducts from quail, taken w h e n the egg was in the shell gland, was impgrtant in minimizing this variation. This became an additional problem w h e n using quail that had been administered Cd, since egg production ceased and mature yolks w e r e aborted. Similar observations have been made by others w h o found that w h e n laying hens and ducks were fed a 100-600-ppm Cd diet, they ceased egg production within a short period (29). Sell (30) observed only a slight reduction in egg production by hens fed 60 ppm Cd. A1Biological Trace Element Research
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MT
I
0
O
1--*
X 13..
o
4
v
"i-
10
20
30
40
Fraction
Fig~ 6. Gel filtration on Sephadex G-50 (1 x 48cm) of the supernatant from the HgC12 metallothionein assay~ Open circles (o) represent the isthmus and closed circles (e) the shell gland. Fraction size was 2 mL. t h o u g h low levels of Cd have b e e n detected in yolks of eggs from Cdtreated hens, no Cd has b e e n f o u n d in the whites or shells of eggs from these h e n s (30,31). The selective process by which the i n c o r p o r a t i o n of Cd into the egg white or shell is p r e v e n t e d could result from the presence of m e t a l l o t h i o n e i n in the oviduct wall. O u r results w o u l d a r g u e against this thesis, since in C d - i n d u c e d quail that h a d dramatically elevated levels of hepatic metallothionein, we were unable to detect e v e n trace a m o u n t s of m e t a l l o t h i o n e i n in the oviduct. Therefore, it w o u l d a p p e a r unlikely that metallothionein has a n y role in the mineral secretion process t h r o u g h the oviduct, either negative or positive. The low-molecular-weight H g - b i n d i n g molecules that we o b s e r v e d in the i s t h m u s a n d shell gland could be i m p o r t a n t in trace m i n e r a l metabBiological Trace Element Research
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Fig. 7. Electron micrograph showing metal staining (M) in secretory granules of goblet cells (G) of the untreated magnum. The nucleus (N) of a ciliated epithelial cell (C) occupies much of the field. Membranes are unstained. Arrowheads indicate the plasma membrane separating the ciliated cell from goblet cells. The bar indicates 0.5 }xm. Biological Trace Element Research
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0'
Fig. 8. Location of metal in the shell gland in untreated tissue (A) and after incubation with added zinc for 15 rain (B), 60 min (C), and 120 rain (D). Epithelium is indicated by E, tubular gland cells by T, and connective tissue by CT. The bar indicates 10 ~m.
kO -M
ca.
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Fig. 9. Photomicrograph showing isthmus epithelium and tubular gland cells after incubation for 15 rain. Staining for zinc is evident in a goblet cell (G) and in interstitial spaces. Ciliated epithelial cells (C) and tubular gland cells (T) show staining only with toluidine blue. Original magnification x 1250. The bar indicates 2 Ixm. olism. It has b e e n previously s h o w n that copper is concentrated in the i s t h m u s , p r e s u m a b l y in association w i t h lysyl oxidase, which is required for the f o r m a t i o n of lysine-derived crosslinks (32). H o w the c o p p e r is Biological Trace Element Research
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transported and stored within the tissue is not known and may involve a peptide transport mechanism. The egg-shell membrane protein itself is very high in disulfide crosslinks, and peptides related to this protein could also cause the high 2~ binding we observed in the metallothionein Hg-binding assay. Zinc secretion studies, using the immature quail oviducts, were extremely difficult to perform. In some instances they were less than 2 m m in diameter and impossible to invert without tearing. To catch the birds in just the right stage of development was very difficult. Our results, therefore, reflect only oviducts that we felt were still undeveloped, yet withstood the inversion process and subsequent incubation. For this reason, the experimental numbers are quite small, but the results do suggest that Zn is able to transverse an immature oviduct with greater facility than the mature oviduct. This may be attributed to the presence of large numbers of undeveloped cells and the thinner wall of the immafure oviduct. This tissue may also have not-yet developed homeostatic control mechanisms for trace mineral secretion. The immature oviducts were able to secrete small but significant amounts of albumen compared to the mature oviducts, which secreted enough albumen during the incubation period to make the media viscous. Administration of estrogen resulted in dramatic changes in maturation of the oviduct. Within 3 d following injection there was a marked cell proliferation and increase in size of the oviduct. Estrogen appears to function by first stimulating cell division, followed by differentiation. At this time, ovalbumen and general protein synthesis occur (33-35). This process takes considerably longer than 3 d, so that even though the oviducts were noticeably changed, complete maturization had not yet occurred in our experiments. Timm's stain visualizes a number of metals (24). Those likely to be present in normal tissue include zinc, copper, and nonheme iron. Thus, in untreated tissue, deposits of stain may indicate the presence of any or all of these. In tissue treated with added Zn, the stain may be considered specific for Zn because it is then present in amounts far greater than any other metal. In both treated and untreated tissue, staining was most heavy in secretory cells. Cellular morphology of the avian oviduct has been described (36) and observed to undergo appreciable change with varying phases of the reproductive cycle. We also observed significant cyclical changes in the quail oviduct. Any changes in the tissue induced by incubation were by comparison so slight as to be undetectable. The histological experiments indicated that Zn traveled from the smooth muscle cells through the connective tissue matrix in the extracellular space to the epithelial goblet cells. Ciliated, columnar epithelial cells did not appear to take up or secrete Zn. It is presumably from these secretory goblet cells that Zn, along with other nutrients destined for the egg, is secreted. Biological Trace Element Research
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ACKNOWLEDGMENT This w o r k was s u p p o r t e d b y ' t h e Robert A. Welch F o u n d a t i o n G r a n t No. F-847.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
G. C. Cotzias, C. Bong, and B. Selleck, Am. ]. Physiol., 202, 359 (1962). M. J. Jackson, D. A. Jones, and R. H. Edwards, Br. J. Nutr., 46, 15 (1981). B. C. Starcher, J. C. Glauber, and J. G. Madaras, J. Nutr., 110, 1391 (1980). R. Oo Brady, Trends Biochem. Sci., 7, 143 (1982). R. W. Olafson, J. Nutr., 113, 268 (1983). R. A. DiSilvestro and R. J. Cousins, Ann. Rev. Nutr., 3, 261 (1983). Mo G. Cherian and R. A. Goyer, Life Sci., 23, 1 (1978). R. J. Cousins, J. Inherited Metab. Dis., 6, Supplo 1; 15 (1983). Y. Kojima and J. H. Kagi, Trends Biochem. Sci., 3, 90 (1978). J. H. Kagi and M. Nordberg, Experientia, 34, 71 (1979). J. K. Piotrowski and J. S. Szymanska, Toxicol. Environ. Health, 1, 991 (1976). S. H. Oh, J. T. Deagen, P. D. Whanger, and P. H. Weswig, Am. 1. Physiol., 234, E282 (1978). B. Sas and I Bremner, ]. Inorg. Biochem., 11, 67 (1979). I. Bremner and N. T. Davis, Biochem. J., 149, 733 (1975). M. L. Failla and R. J. Cousins, Biochim. Biophys. Acta, 543, 293 (1978). M. Karin and H. R. Herschman, Science, 204, 176 (1979). F. N. Kotsonis and C. D. Klaasen, ToxicoI. Appl. Pharmacol., 51, 19 (1979). B. C. Sandrock, S. R. Kern, and S. E. Bryan, Biol. Trace Element Res., 5, 503 (1983). T. Ho Wilson and G. Wiseman, J. Physiol., 123, 116 (1954). J. K. Piotrowski, W. Bolanowski, and A. Sapota, Acta Biochem. Pol., 20, 207 (1973). F. N. Kotsonis and C. D. Klaassen, Toxicol. Appl. Pharmacol., 42, 583 (1977). M. J. Karnovsky, J. Cell Biol., 27, 137A (1965). P. M. Butzen, E. J. Root, and B. C. Starcher, Tex. Soc. Elect. Micros. J., 15, 35A (1984). G. Danscher, Histochemistry, 71, 1 (1981). G. D. Buckner and J. H. Martin, Am. J. Physiol. 89, 164 (1929). S. H. Oh, H. Nakaue, J. T. Deagen, P. D. Whanger, and G. H. Arscott, J. Nutr. 109, 1720 (1979). C. C. McCormick, J. Nutr. 114, 191 (1984). V. Weser, H. Rupp, F. Donay, F. Linnemann, W. Voelter, W. Voetsch, and G. Jung, Eur. J. Biochem. 39, 127 (1973). A. Hennig, K. Georgi, and H. Jeroch, Arch. Expo Vet. Med., 25, 793 (1971). J. L. Sell, Poult. Sci., 54, 1674 (1975). M. Nishimura, M. Sakuta, K. Okamoto, and N. Urakawa. Jpn. J. Vet. Sci., 36, 133 (1974). E. D. Harris, J. E. Blount, and R. M. Leach, Science, 208, 55 (1980). G. S. McKnight, Cell, 104, 403 (1978). B. W. O'Malley, W. L. McGuire, and S. G. Korenman, Biochim. Biophys. Acta, 145, 204 (1967). T. Oka and Ro T. Schimke, J. Cell Biol. 41, 816 (1969). R. Do Hodges, The Histology of the Fowl, 1st ed., Academic, NY, (1974).
Biological Trace Element Research
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Zinc Secretion in the Oviduct of the Coturnix Quail P. BUTZEN, 1 E. ROOT, 1 AND B~ STARCHER *'2
~Nutrition Division, Department of Home Economics, University of Texas at Austin, Austin, TX; and 2Department of Biochemistry; The University of Texas Health Center at Tyler, Tyler, TX Received March 10, 1985; Accepted July 1, 1985
ABSTRACT The oviduct _from laying quail were used to investigate mechanisms of trace mineral secretion and the possible role of metallothionein in this process. Secretion of zinc occurred maximally at pH 5.4, which is close to the normal pH of the oviduct. Secretion occurred to a much greater extent in the isthmus and shell gland than in the magnum, the major protein-secretory section of the oviduct. Intraperitoneal administration of cadmium resulted in a marked reduction in Zn secretion from the oviduct of laying quail. This effect could not be correlated with metallothionein since metallothionein could not be detected in any section of the oviduct in control or Cdinduced quail. Small-molecular-weight metal-binding ligands were present in the isthmus and shell gland, which may play a role in trace mineral mobilization. Histological evaluation by light and elelctron microscopy show that Zn is transported from the smooth muscle cells through the connective tissue matrix in the extracellular space to the epithelial goblet cells. Presumably, Zn and other trace minerals are secreted from the secretory goblet cells into the egg. Index Entries; Zinc, secretion of; Coturnix Quail; oviduct, zinc secretion in; metallothionen, role in trace mineral secretion; cadmium. *Author to whom all correspondence and reprint requests should be addressed.
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INTRODUCTION Zinc, an essential trace element for plants, animals, and h u m a n s , has been widely studied. It plays an important biochemical and nutritional role as a c o m p o n e n t of over 80 metalloenzymes and insulin. Cotzias (1), in 1962, first proposed that Zn metabolism came u n d e r homeostatic control. Since then, m u c h of the interest has focussed on the intracellular metal-binding ligand metallotheonein, which, in addition to transport (2,3), storage (4,5), and detoxification (6,7), has been postulated to have a regulatory role in Zn absorption by serving as a mucosal block (8) or a more direct and positive role enhancing Zn absorption (3,8). Metallothionein is present in trace amounts in nearly all tissues that have been examined (9,10)o Its concentration, moreover, can be rapidly increased by exposure to sublethal doses of certain metals (11,12), various stresses (12,13), starvation (14), administration of glucocorticoids (15,16), and a n u m b e r of alkylating agents (17). Whether the resulting elevated metallothionein levels are beneficial may be d e p e n d e n t u p o n the tissues and the trace minerals involved. The avian oviduct presents an unusual model system illustrating secretion of trace minerals, rather than absorption or excretion. It m a y be, however, that m a n y of the parameters governing the latter processes may also regulate secretion. The oviduct can be divided into five distinct regions, four of which have specific physiological functions in egg formation: i n f u n d i b u l u m (engulfs the ovum); m a g n u m (protein secretion); isthmus (shell m e m b r a n e s secretion); and shell gland (eggshell secretion). The selective importance of these distinct regions in trace-mineral secretion has not been delineated. The mineral content of the egg is derived from the bird's diet. After absorption, it is deposited in the developing yolk or secreted from the oviduct directly to the egg white. Alt h o u g h the majority of Zn is present in the yolk, Sandrock et al. (18) have s h o w n that a significant a m o u n t of the Zn located in the a l b u m e n is transferred to the yolk and utilized by the developing fetus. The purpose of this study is to investigate Zn secretion and determine if metallothionein has an important role as a regulator molecule for this process.
METHODS Experimental Animals I m m a t u r e and mature female Japanese quail (Coturnix coturnix japonica) w e r e used for all experiments. The nonlaying quail were 4-6 w old and w e i g h e d 100-160 g. The laying quail, weighing 170-200 g, w e r e 8-12 w old. They were housed u n d e r controlled atmospheric and lighting conditions, fed a commercial layer mash, and given tap water ad libitum.
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In Vitro Zinc Secretion Studies Control and Cd-treated laying and nonlaying quail and estrogentreated nonlaying quail were used. Daily injections of Cd (0.1-0.3 mg), in the form of Cd acetate, in saline (1 mL) were given ip for 1-3 d. The quail were killed 24 h after the last injection. Selected nonlaying quail were given daily ip injections of 0.25 mg ~3-estradiol (Sigma Chemical Co.), s u s p e n d e d in corn oil or dissolved in 100% ethanol, for 4 d. The quail were sacrificed by cervical dislocation 18-24 h after the last injection and the oviducts removed. Control quail were injected with either saline, corn oil, or alcohol. The excised oviducts were rinsed in 0.9% saline, blotted dry, weighed, measured, and everted with a plastic tube. The everted oviducts were m a d e into sacs, as described by Wilson and Wiseman (19), containing 65Zn in Dulbecco's Modified Eagle's Medium (EMEM from Biolabs), buffered with 4M sodium acetate to pH 5.4. Two studies were done. In the first study, one sac was made using the complete oviduct, with each anatomical division (anterior m a g n u m , posterior m a g n u m , isthmus, and shell gland) containing 200,000 cpm 65Zn in 1 mL of DMEM, pH 5.4. This was accomplished by first tying off the anterior end of the shell gland with surgical thread, adding I mL of 65Zn DMEM to the open m a g n u m , and allowing this to settle by gravity into the shell gland. The anterior end of the shell gland was then tied off and the process repeated, adding 1 mL of 65Zn DMEM to each section. The whole oviduct, with each segment separated, was incubated in 40 mL DMEM, p H 5.4. In the second study, individual sacs were made from each of the four sections, with each sac containing 0.4 mL DMEM, pH 5.4, and 200,000 cpm 65Zn. The individual sections were incubated in 10 mL DMEM, p H 5.4. Samples from both studies were incubated u n d e r 5% CO2 in a shaking water-bath at 37~ At 30 min intervals, 0.5-mL aliquots of mucosal media were removed, radioactivity m e a s u r e d with a Beckman 5000 G a m m a Counter, and subsequently returned to the flasks for further incubation of the tissue u n d e r 5% CO2 for a total of 2 h. At the end of the incubation period, some sacs w e r e emptied, slit open lengthwise, rinsed in saline, and prepared for gel-filtration chromatography, as described below.
Induction of l~Ietallothionein Adult laying quail were given a single ip injection of 0.3 m g Cd, in the form of Cd acetate, in 1 mL of saline. The birds were sacrificed 18-24 h after the injection by cervical dislocation. The liver and oviduct from control and Cd-treated quail were removed and analyzed for metallothionein. Tissues not used immediately for metallothionein analysis were frozen (-20~ u n d e r nitrogen.
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Metallothionein Analysis Two m e t h o d s were used for the analysis of metallothionein in liver and oviduct tissue. First, the 2~ m e t h o d of Piotrowski et al. (20), as described by Kotsonis and Klaasen (21), was used. Fresh tissue was homogenized in 0.9% saline (1 g tissue/7 mL saline) with a polytron homogenizer. To 2.5-mL h o m o g e n a t e (0.36 g tissue), 0.25 mL 2~ (250 ~g Hg) was added, thoroughly mixed, and allowed to stand at room temperature for 30 min. Following the addition of 0.8 mL 10% trichloroacetic acid (TCA), the samples were allowed to stand an additional 10 min before centrifuging at 3000g for 10 rain at 0~ (Beckman Model TJ-6). The supernatants were transferred to clean disposable tubes and m e a s u r e d for radioactivity in a g a m m a counter (Beckman 5500). All samples w e r e done in duplicate, and 2~ a d d e d to saline was used as a control. Following the 2~ procedure, the metallothionein fraction from a control oviduct and a liver from a Cd-treated quail was separated from other low-molecular-weight Hg-binding proteins and peptides by gel-filtration chromatography. The TCA supernatants were individually applied to a Sephadex G-50 column (1 • 48 cm), equilibrated, a n d eluted with 4M urea in saline, and collected in 2-mL fractions. The flow rate was 0.5 mL/min for this column. Each fraction was m e a s u r e d for radioactivity with a gamma counter. The column was calibrated with the following substances: glutamic acid (mol wt 146), bacitracin (mol wt 1570), bovine insulin (tool wt 5700), cytochrome c (mol wt 12,400), hemoglobin (mol wt 15,500), myoglobin (mol wt 18,800), pepsin (mol wt 35,000), and egg albumen (mol wt 45,000). All chromatography was conducted at 4~ For the second method, gel-filtration chromatography was used as a relative measure of metallothionein levels. Tissue (0.1-0.5 g) was homogenized in 2-3 mL 4M urea in saline and centrifuged at 20,O00g (Beckman Model J2-21) for 20 min at 0~ The supernatant was applied to a Sephadex G-50 column (1 • 48 cm), eluted with 4M urea in saline, and collected in 2 mL fractions. It should be noted that 65Zn (30,000-40,000 cpm) and/or 20 txg Cd (CdC12) was a d d e d to the supernatants of control and estrogen-treated specimens, which were h o m o g e n i z e d in saline. After a 30-rain standing period, urea was a d d e d to make a 4M urea saline solution. Each fraction collected was m e a s u r e d for radioactivity before assaying for Cd with an atomic absorption spectrophotometer (Instrumentation Laboratory Inc., Model 551).
Histology Portions of the everted oviducts used for the in vitro Zn secretion studies were examined by light and electron microscopy in order to detect any adverse effects of incubation u p o n integrity of the tissue and to determine the cellular location of the Zn. The location of the Zn will, unBiological Trace Element Research
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der normal circumstances, be the same as the location of the Z n - b i n d i n g protein. Portions of the four sections of the oviduct were taken before a n d at intervals d u r i n g incubation with a d d e d Zn (1 m g to each section). Tissue was treated as described above for the in vitro secretion. Slices of tissue 1 m m thick were taken from the freshly r e m o v e d oviduct and the everted sacs at 15, 30, 60, 90, a n d 120 m i n of incubation, a n d were i m m e r s e d in a fixative comparable either with a c o n v e n t i o n a l preparation for electron microscopy or with a modification of T i m m ' s stain for d e m o n s t r a t i o n of Zn. Tissues p r e p a r e d conventionally was placed in half-strength Karnovsky's fixative (22), postfixed in 2% OsOa, d e h y d r a t e d in an alcohol series followed by acetone, a n d e m b e d d e d by Epon-type plastic [consisting of: E m b e d 812 (Electron Microscopy Sciences, Ft. W a s h i n g t o n , PA) 15 mL, DDSA 12 mL, N M A 6 mL, and DBP 1 mL, with DMP-30 u s e d as accelerator]. Sections 1-3 ~ m thick were stained with toluidine blue a n d e x a m i n e d using a Zeiss light microscope. Ultrathin sections 60-90 n m thick (silver) were stained with uranyl acetate a n d lead citrate a n d e x a m i n e d in a Siemens electron microscope. Tissue p r e p a r e d for T i m m ' s stain was fixed in a sulf i d e - g l u t a r a l d e h y d e mixture, consisting of 0.1% s o d i u m sulfide in 0.15M s o d i u m p h o s p h a t e buffer to which g l u t a r a l d e h y d e was a d d e d just before use to m a k e a 3% solution (23). Further processing was carried out, as described for the sulfide silver m e t h o d of Danscher (24), and e m b e d d e d in the plastic described above, k e e p i n g polymerization t e m p e r a t u r e s at or below 45~ Sections 1 a n d 3 p,m thick were stained, as described by Danscher. The t h i n n e r sections p r o v i d e d subjects for p h o t o g r a p h y , a n d the thicker ones p r o v i d e d T i m m ' s - s t a i n e d material for electron microscopy. Such sections were covered with a d r o p of u n p o l y m e r i z e d plastic a n d a block of p o l y m e r i z e d plastic. These were allowed to polymerize a n d t h e n r e m o v e d from the glass microscope slide after a brief heating of the b o t t o m of the slide on a hot plate. Thin sections were cut from this material, stained with uranyl acetate a n d lead citrate and examined in a Siemens electron microscope. All histological observations were m a d e from tissues taken with the egg in the shell gland.
RESULTS A preliminary e x p e r i m e n t was c o n d u c t e d to determine w h e t h e r Zn secretion occurred maximally at neural p H or at a p H of 5.4, w h i c h is close to t h e n o r m a l p H of the oviduct sections studied (25). The results s h o w n in Fig. 1 indicate that Zn secretion from the whole oviduct occurred at a significantly higher rate at p H 5.4. As a result, all subseq u e n t secretion experiments were c o n d u c t e d at this pH. Biological Trace Element Research
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Butzen, Root, a n d S t a r c h e r 20pH 5 4
16"
O
x
12-
0.
0.9_ lie
o
8" z N
pH 73
o
~ 0
30 60 90 Incubation (minutes)
Fig. 1. 6SZincsecretion of oviducts from laying quail incubated in DMEM, pH 5.4 (o) and 7.3 (e). The values express the total 65zinc secreted from the whole oviduct for the times indicated. In experiments to m e a s u r e secretion from functionally distinct sections of the oviduct, the i s t h m u s was always f o u n d to be the major secretor of 65Zn (Fig. 2). This was true even t h o u g h the total a m o u n t of Zn secreted by each section of an oviduct varied between individual birds. The reason w h y the oviducts from s o m e of the birds were high Z n secretors a n d others low, a p p e a r e d to be correlated to the position of the egg in the oviduct. Zinc secretion was d i m i n i s h e d w h e n the yolk was located in the m a g n u m or isthmus, a n d t h e c o r r e s p o n d i n g tissues that a p p e a r e d to be d e p l e t e d of secretory material took on a grayish white color, a c c o m p a n i e d by a t h i n n i n g of the oviduct wall. This was n o t seen w h e n the egg was located in the shell gland, a n d for this reason m o s t experim e n t s utilized oviducts w h e n the egg was located in this region. Biological TraceElement Research
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g" & 25" X
a. o
20
r .
O ~
15O
Z
N
10"
Anterior Posterior Isthmus Shell Magnum Magnum gland
Fig. 2. Total 65Zn secretion of individual oviduct sections after 2-h incubation. Values shown are the means from six laying quail. Intraperitoneal injections of Cd to laying quail resulted in a significant reduction in Zn secretion (Fig. 3). Because of the high individual variation b e t w e e n birds, this experiment was repeated several times with whole oviducts, and in every instance Cd administration reduced Zn secretion. A single dose of 0.3 mg of Cd seemed to give a more uniform response than repeated doses at varying levels. At higher levels with repeated injections, the quail became noticeably sick. All the subsequent experiments use a single 0.3-mg Cd injection, and the birds were killed 18--24 h later. In experiments to control the hormonal influence of the laying cycle, nonlaying quail, untreated and treated with estrogen or Cd, were used. Our results indicated that, although immature oviducts were m u c h smaller in size and weight, they secreted significantly more ~ (168%) than m a t u r e oviducts (Fig. 4). An even more striking increase in Zn secretion was seen in oviducts from immature nonlayers that had been treated with estrogen. Surprisingly, the administration of Cd to nonlayers resulted in a marked increase in Zn secretion. Two m e t h o d s were employed to determine if the alteration in Zn secretion resulting from Cd injection was related to the presence of metallothionein. In the first series of experiments, the livers (known to contain large amounts of metallothionein following Cd induction) and the oviducts of Biological Trace Element Research
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~r
,r-*
X
8-
I3.
G
6
.2 u (9 (/9
4
Z N
2
I
30
I
I
i
60 90 120 Incubation (minutes)
Fig.. 3. Total 65Zn secretion of oviducts from control (o) and Cd-treated ([]) laying quail. The bars shown at 120 min represent the standard deviation. selected quail were analyzed for metallothionein by gel-filtration chromatography. Intraperitoneal injections of Cd caused a large increase in hepatic metallothionein levels w h e n compared to control livers, as illustrated in Fig. 5. Similar elution profiles were seen w h e n ~ was a d d e d to the supernatants and radioactivity monitored in the column fractions. In control liver, Cd or 65Zn were mainly b o u n d to a highmolecular-weight protein fraction with only a small amount b o u n d to the metallothionein fraction. The reverse was true for the Cd-induced hepatic tissue. Positive identification of metallothionein in Cd-induced liver was confirmed by DEAE-ion exchange chromatography. W h e n the liver supernatant was chromatographed, only one Cd-protein peak was present, indicating only one isoform for quail hepatic metallothionein. This is in a g r e e m e n t with previous reports for other avian species (26-28)~ Gelfiltration analysis of quail oviduct homogenates following Cd induction s h o w e d no detectable Cd-thionein. The Cd-induced samples w e r e indistinguishable from control oviducts, with all of the Cd being b o u n d to the high-molecular-weight protein fraction, as was observed with control livers. The results of the other means to measure oviduct metallothionein, using the more quantitative m e t h o d of Piotrowski (20), are s h o w n in Table 1. W h e n quail were given a single ip dose of Cd (0.3 rag), there was a marked increase in hepatic metallothionein, similar to what was seen by Biological Trace Element Research
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300
Non layer + Cd
O
Non layer estrogen
C
0 0
C
200
0 L~ m
2
Non layer control
f-.
U 100,
Fig. 4. 65Zincsecretion of quail oviduct after 2-h incubation. Values are expressed as a percent of control laying quil. n = 4, Except the nonlaying given Cd or estrogen, where n = 2. gel filtration. However, w h e n separate oviduct sections were analyzed, Cd administration did not significantly alter 2~ binding. Even more striking w e r e the extremely high levels of w h a t appeared to be metallothionein in the isthmus, and the s o m e w h a t lower levels of this protein in the shell gland. This was evident in the control as well as the Cd-induced animals. C h r o m a t o g r a p h y of these Hg-binding components on Sephadex G-50 indicated that they were not metallothionein, but molecules of m u c h lower weight, approximately 3.35 kdaltons for the shell gland a n d 1.1 for the isthmus (Fig. 6). The m a g n u m had such low Hg binding that no peaks were evident following chromatography. Microscopic examination of tissue, after incubation with a d d e d Zn, was first p e r f o r m e d using the m a g n u m . In untreated tissue, stain indicating the presence of metal was evident only in the epithelium. Of the epithelial cells, only the goblet cells (Fig. 7) contained metal, which was sequestered in oval-shaped secretory granules. After incubation, stain also appeared in smooth muscle and the connective tissue matrix, but not in glandular cells. Subsequent observations were carried out with the shell gland a n d the isthmus, which were more active in the secretion of Zn. Biological Trace Element Research
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0.75
E Q. v
0.50
"o 0
0.25
0
10
20
30 40 Fraction
50
60
70
Fig. 5. Gel filtration on Sephadex G-50 (1 x 48cm) of control (o) and Cdtreated (o) liver homogenates. Fraction size was 2 mL. In the shell gland, little or no stain was present in untreated tissue (Fig. 8A). After 15-min incubation with a d d e d Zn, the epithelial goblet cells were well stained (Fig. 8B). At 30 rain stain appeared in smooth muscle and interstitial spaces, and by 60 min some glandular cells took up stain as well (Fig. 8C). By 120 rain, stain had cleared from glandular cells and part of the connective tissue matrix, but was still heavy in the epithelium and subjacent interstitial space (Fig. 8D). The isthmus differed from the shell gland and m a g n u m in showing s o m e w h a t heavier staining at all times. Some stain was present in both Biological Trace Element Research
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Table 1 Metallothionein Content of Liver and Individual Oviduct Sections from Control and Cd-Treated Laying Quail ~ Tissue
c p m 2~
ControP,
Cd-treated ~, cpm 2~
Liver Anterior magnum Posterior magnum Isthmus Shell gland
126 243 404 3468 1030
3174 288 471 2862 1783
+ 59 _+ 104 + 66 _+ 408 _+_ 502
_+ 451c _+ 48 _+ 155 _+ 361 + 399
~The values for MT are expressed in relative terms and are proportional to 2~ bn=4.
~Statistically significant at P < 01.
epithelium and smooth muscle in untreated tissue. Again, stain was present only in goblet cells in the epithelium (Fig. 9). Changes in appearance during incubation with a d d e d Zn followed the same pattern as that described for the shell gland. Appearance of the tissue correlates well with the results of the Zn secretion studies. In all regions, the histology suggests rapid uptake of Zn and its rapid m o v e m e n t through the connective tissue matrix to the epithelium w h e r e it is concentrated in goblet cells prior to secretion.
DISCUSSION The oviduct of the laying quail proved to be an ideal tissue for doing secretion studies in vitro. The inverted sacs were very durable and the tissues r e m a i n e d intact during the 2 h incubation period. W h e n using the whole oviduct, we felt it necessary to physically separate the anatomical regions, with each region containing equal amounts of 6SZn. Without separation, the liquid tended to pool in one region during the incubation period. In other preliminary experiments we observed the secretion of Zn through the oviduct to be a unidirectional flow; going from the serosal to the mucosal side only. Individual variations between oviducts in the rate of 65Zn secretion was a consistent finding during these studies. Using oviducts from quail, taken w h e n the egg was in the shell gland, was impgrtant in minimizing this variation. This became an additional problem w h e n using quail that had been administered Cd, since egg production ceased and mature yolks w e r e aborted. Similar observations have been made by others w h o found that w h e n laying hens and ducks were fed a 100-600-ppm Cd diet, they ceased egg production within a short period (29). Sell (30) observed only a slight reduction in egg production by hens fed 60 ppm Cd. A1Biological Trace Element Research
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MT
I
0
O
1--*
X 13..
o
4
v
"i-
10
20
30
40
Fraction
Fig~ 6. Gel filtration on Sephadex G-50 (1 x 48cm) of the supernatant from the HgC12 metallothionein assay~ Open circles (o) represent the isthmus and closed circles (e) the shell gland. Fraction size was 2 mL. t h o u g h low levels of Cd have b e e n detected in yolks of eggs from Cdtreated hens, no Cd has b e e n f o u n d in the whites or shells of eggs from these h e n s (30,31). The selective process by which the i n c o r p o r a t i o n of Cd into the egg white or shell is p r e v e n t e d could result from the presence of m e t a l l o t h i o n e i n in the oviduct wall. O u r results w o u l d a r g u e against this thesis, since in C d - i n d u c e d quail that h a d dramatically elevated levels of hepatic metallothionein, we were unable to detect e v e n trace a m o u n t s of m e t a l l o t h i o n e i n in the oviduct. Therefore, it w o u l d a p p e a r unlikely that metallothionein has a n y role in the mineral secretion process t h r o u g h the oviduct, either negative or positive. The low-molecular-weight H g - b i n d i n g molecules that we o b s e r v e d in the i s t h m u s a n d shell gland could be i m p o r t a n t in trace m i n e r a l metabBiological Trace Element Research
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Fig. 7. Electron micrograph showing metal staining (M) in secretory granules of goblet cells (G) of the untreated magnum. The nucleus (N) of a ciliated epithelial cell (C) occupies much of the field. Membranes are unstained. Arrowheads indicate the plasma membrane separating the ciliated cell from goblet cells. The bar indicates 0.5 }xm. Biological Trace Element Research
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0'
Fig. 8. Location of metal in the shell gland in untreated tissue (A) and after incubation with added zinc for 15 rain (B), 60 min (C), and 120 rain (D). Epithelium is indicated by E, tubular gland cells by T, and connective tissue by CT. The bar indicates 10 ~m.
kO -M
ca.
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Fig. 9. Photomicrograph showing isthmus epithelium and tubular gland cells after incubation for 15 rain. Staining for zinc is evident in a goblet cell (G) and in interstitial spaces. Ciliated epithelial cells (C) and tubular gland cells (T) show staining only with toluidine blue. Original magnification x 1250. The bar indicates 2 Ixm. olism. It has b e e n previously s h o w n that copper is concentrated in the i s t h m u s , p r e s u m a b l y in association w i t h lysyl oxidase, which is required for the f o r m a t i o n of lysine-derived crosslinks (32). H o w the c o p p e r is Biological Trace Element Research
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transported and stored within the tissue is not known and may involve a peptide transport mechanism. The egg-shell membrane protein itself is very high in disulfide crosslinks, and peptides related to this protein could also cause the high 2~ binding we observed in the metallothionein Hg-binding assay. Zinc secretion studies, using the immature quail oviducts, were extremely difficult to perform. In some instances they were less than 2 m m in diameter and impossible to invert without tearing. To catch the birds in just the right stage of development was very difficult. Our results, therefore, reflect only oviducts that we felt were still undeveloped, yet withstood the inversion process and subsequent incubation. For this reason, the experimental numbers are quite small, but the results do suggest that Zn is able to transverse an immature oviduct with greater facility than the mature oviduct. This may be attributed to the presence of large numbers of undeveloped cells and the thinner wall of the immafure oviduct. This tissue may also have not-yet developed homeostatic control mechanisms for trace mineral secretion. The immature oviducts were able to secrete small but significant amounts of albumen compared to the mature oviducts, which secreted enough albumen during the incubation period to make the media viscous. Administration of estrogen resulted in dramatic changes in maturation of the oviduct. Within 3 d following injection there was a marked cell proliferation and increase in size of the oviduct. Estrogen appears to function by first stimulating cell division, followed by differentiation. At this time, ovalbumen and general protein synthesis occur (33-35). This process takes considerably longer than 3 d, so that even though the oviducts were noticeably changed, complete maturization had not yet occurred in our experiments. Timm's stain visualizes a number of metals (24). Those likely to be present in normal tissue include zinc, copper, and nonheme iron. Thus, in untreated tissue, deposits of stain may indicate the presence of any or all of these. In tissue treated with added Zn, the stain may be considered specific for Zn because it is then present in amounts far greater than any other metal. In both treated and untreated tissue, staining was most heavy in secretory cells. Cellular morphology of the avian oviduct has been described (36) and observed to undergo appreciable change with varying phases of the reproductive cycle. We also observed significant cyclical changes in the quail oviduct. Any changes in the tissue induced by incubation were by comparison so slight as to be undetectable. The histological experiments indicated that Zn traveled from the smooth muscle cells through the connective tissue matrix in the extracellular space to the epithelial goblet cells. Ciliated, columnar epithelial cells did not appear to take up or secrete Zn. It is presumably from these secretory goblet cells that Zn, along with other nutrients destined for the egg, is secreted. Biological Trace Element Research
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ACKNOWLEDGMENT This w o r k was s u p p o r t e d b y ' t h e Robert A. Welch F o u n d a t i o n G r a n t No. F-847.
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