- Email: [email protected]
Composition of the Oviducts of Laying Hens: Investigation of Segments Involved with Shell Formation1
Composition of the Oviducts of Laying Hens: Investigation of Segments Involved with Shell Formation 1 JOHN D. CIPERA Animal Research Institute, Agriculture Canada, Central Experimental Farm, Ottawa, Ontario K1A 0C6 (Received for publication January 9, 1979)
1980 Poultry Science 59:635-642 INTRODUCTION T h e quality of a h e n ' s egg shell is of major e c o n o m i c i m p o r t a n c e t o t h e p o u l t r y industry (see R o l a n d , 1 9 7 7 ) . In c o n s e q u e n c e it has b e c o m e a focus of extensive investigative w o r k . Most of this w o r k involved a nutritional approach, although t h e effects of flock managem e n t and of t h e physiological and o t h e r factors which m a y affect t h e process of shell f o r m a t i o n have n o t b e e n neglected. Relatively little w o r k has b e e n d o n e o n t h e possible interrelationship b e t w e e n t h e c o m p o s i t i o n of t h e hen's oviduct and shell quality. T h e a m o u n t of i n f o r m a t i o n on t h e overall c o m p o s i t i o n of specific segments of h e n ' s oviduct at various stages of egg f o r m a t i o n is quite limited. In 1 9 2 5 , Buckner et al r e p o r t e d levels of calcium and p h o s p h o r u s in t h r e e segments ( m a g n u m , isthmus, and uterus) of oviducts from eight laying h e n s ; Schenck ( 1 9 3 2 ) assayed u p p e r parts of hen's oviducts (excluding t h e uterus) for egg-white proteins and several a m i n o acids; C o m m o n ( 1 9 3 8 )
1
Contribution No. 815 Animal Research Institute.
r e p o r t e d c o n t e n t s of moisture, Kjeldahl p r o t e i n , fat, ash, and calcium in whole oviducts from 8 hens and Christensen and Riggs ( 1 9 5 3 ) investigated whole oviducts from 4 hens for free (dialysable) amino acids and peptides. Subseq u e n t l y various oviducal segments from laying hens were evaluated for mineral (Brown and J a c k s o n , I 9 6 0 ; Taylor and H e r t e l e n d y , I 9 6 0 ; Misra and K e m e n y , 1 9 6 4 ; Schraer and Schraer, 1 9 6 5 ; Snapir and Perek, 1970) a n d organic (Brown and J a c k s o n , I 9 6 0 ; Cecil et al, 1 9 6 9 , 1 9 7 0 ; Yu a n d Marquardt, 1 9 7 1 , 1 9 7 2 ) c o m p o nents. In this c o n n e c t i o n , r e p o r t s o n t h e c o m p o s i t i o n of oviducal secretions ( K a t z u k u r a and T a m a t e , 1 9 7 1 ; M o y n i h a n and Edwards, 1 9 7 5 ; Edwards, 1 9 7 7 ; Mongin and Carter, 1 9 7 7 , 1 9 7 8 ) , although giving rather relative values, are also of interest. However, n o comprehensive investigation o n t h e c o m p o s i t i o n of these tissues during t h e passage of t h e egg t h r o u g h the oviduct, involving b o t h organic and mineral constituents in t h e same tissues, has been y e t r e p o r t e d . Since egg shell f o r m a t i o n is initiated in t h e isthmus and completed in t h e u t e r u s (Simkiss and Taylor, 1 9 7 1 ) , t h e c o m p o s i t i o n of these
635
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
ABSTRACT Oviducts from eighteen laying hens were isolated at various stages of egg formation and, after infundibulum and vagina were discarded, divided into three segments: magnum, isthmus, and uterus. Of these, the magnum showed widest weight variation between the egg-forming stages; this variation appeared to be related to the albumen formation. The isthmus and uterus from each hen were fractionated into wall and mucosal fractions, and the mucosae were further separated by centrifugation into mucosal supernatant and residue fractions. These fractions were assayed for moisture, total and neutral lipids, calcium, magnesium, hexosamines, and collagen (as indicated by the levels of hydroxyproline). The wall fractions of either isthmus or uterus were characterized by consistently higher levels of water and of collagen, while mucosae had higher levels of calcium, magnesium, crude lipid extracts, and, in the case of isthmus, hexosamines. In both isthmus and uterus, highest levels of chloroform-phase soluble lipids were observed in the sedimenting (residue) sub-fraction of mucosae. Thin-layer chromatographic resolution of the chloroform-phase lipids indicated that the lipids from the mucosal and wall fractions of either the isthmus or uterus were similar in composition. Changes in composition of isthmus and uterus observed at various stages of egg passage through the oviduct indicated a trend to lower levels of calcium in these segments during the shell formation stage and to generally higher levels of lipids when the egg is present in the respective segment. In both isthmus and uterus, collagen concentration was increased at the time the uterus was stretched around the egg.
636
CIPERA
tissues of the oviduct should most conceivably influence the quality of the shell produced within them. Therefore we concentrated our investigation on these two segments of hen's oviduct. MATERIALS AND METHODS
Magnums were weighed and discarded. The isthmian and utero-vaginal tissues were transferred into pre-cooled glass tubes which were
Since mucosae from both segments tended to separate on standing into a sediment and an opaque emulsion, they were centrifuged at 800 x g for 15 min to produce mucosal residue (MR) and mucosal supernatant (MS) fractions. The MR fraction was then washed twice with a small volume (approximately 2 ml) of distilled water and centrifuged after each washing; the washings were added to the MS fractions. After this treatment the MS fraction remained homogeneous (no sedimentation occurred) on standing. The MR, MS, and wall (W) fractions of the isthmian and uterine segments were freeze-dried and subsequently dried in a vacuum desiccator over P2O5 to a constant weight. Lipid Extraction. The dry tissue fractions were homogenized (Virtis 45 homogenizer) and extracted with chloroform:methanol (2:1 v/v) (Folch et al., 1957). Uterine walls, because of their bulkiness, were finely minced with scissors before being homogenized and extracted. The extraction was performed in centrifuge tubes and extracts evaporated by allowing
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
Hens. Fifty Single Comb White Leghorn hens were housed individually in wire cages (20.3 X 40.6 cm) and received 14 hr of light per day. After 20 weeks of age the hens were given ad libitum a laying ration which contained 14.8% protein (N x 6.25), 2.9% calcium, and .6% total phosphorus. At 42 weeks of age, eggs were collected twice daily (1130 and 1600 hr) and recorded for each hen. Hens to be killed were selected on the basis of postoviposition time so as to obtain oviducts at various stages of egg formation. All hens were killed between 0830 and 1130 hr. Isolation of Oviducts. Each selected hen was weighed, killed by cervical dislocation, and its oviduct was excised. Presence and position of an egg in the oviduct was recorded; subsequently this egg was removed from the isolated oviduct (through a longitudinal incision) and discarded. After removal of the infundibulum and cloaca, the oviduct was cleaned of extraneous tissue and sectioned into three parts: magnum, isthmus, and utero-vaginal complex. The infundibulum was separated from magnum at the point where the semi-translucent, pinkish red tissue of the infundibulum changed into the massive yellowish beige tissue of the magnum and the isthmus from magnum at the point where an approximately 1 mm wide band of glossy gray tissue separated the two segments. The utero-vaginal complex was separated from the isthmus at the point where the whitish gray color of isthmian mucin changed to a brownish gray uterine mucosa; although this change in color of the mucosa was noticeable on the outside of the oviduct, a short longitudinal incision was made at the isthmo-uterine junction in order to assure an accurate location of the dividing line between the two segments by observing the change in mucosa directly. The cloaca was separated from the utero-vaginal complex at the point where the narrow tube of the vagina begins to widen into the cloacal cavity.
stoppered and stored in a deep freeze until they were further processed. Fractionation of Isthmuses and Uteri. Within two days after the hens were killed, samples of isthmuses and uteri were thawed at room temperature, washed under a gentle stream of tap water, cleaned of any remaining mesenterial tissue, placed on a thick glass plate and opened longitudinally. Vaginas were removed from uteri at this stage; dividing point between the two segments was the point where the narrow tube of the vagina just began to widen into the uterine pouch. (A band of a transitory tissue, approximately 5 to 10 mm wide, was observed between the yellowish white vagina and the brownish gray uterus; this tissue, which was similar in appearance to the uterine tissue except that its lumen was whitish to pinkish gray in color, as contrasted with the brownish gray color of the remaining uterus, was further treated as a part of the uterine tissue.) The mucosal portion of each opened segment was scraped off with a glass slide, transferred into a tube cooled in an ice-bath, and weighed. The remaining wall portion of the segment was washed with a minimum amount of distilled water, scraped again with glass slide, and weighed; the washings were added to the mucosal scrapings but, because they represented an insignificant amount of the total, their weight was ignored.
637
OVIDUCAL SEGMENTS INVOLVED WITH SHELL FORMATION
tissues were determined, after wet-ashing with perchloric acid and nitric acid, by complexometric titration with murexide as indicator (Copp, 1963), and atomic absorption spectrophometry, respectively. RESULTS AND DISCUSSION Oviducts from eighteen laying hens were isolated and processed. Table 1 shows the body weights and the wet weights of the magnum, isthmus, and uterus of hens at different stages of egg shell formation. There was considerable variation between the shell-formation stages in the wet weight of the total oviducts, mainly due to the variation in weight of the magnums; the total oviduct and magnum weights were lowest during the period immediately following the passage of the egg from magnum and highest in hens with an egg in magnum or with no egg in the oviduct. Similar changes were observed by Smith et al. (1957) who suggested that changes occurring in magnum weight were associated with the variations in the amount of albumen present in the secretory tissue of the magnum: the amount of this albumen would be expected to be highest before and during the period the egg passes through the magnum and lowest during the subsequent period. The apparent decrease in isthmus weights at the later shell-forming period, when egg was present in uterus and became covered with a hard shell, may be due to possible participation of certain isthmian components in the shell formation (Moynihan and Edwards, 1975). When the isthmuses and uteri were subjected
TABLE 1. Weights of oviducal tissue at various stages of shell formation Wet weight (g) (mean + SE) Stage of shell formation
No. of hens
Body weights (kg) (mean ± SE) Magnum
Isthmus
Uterus
Total! oviduct 48.8 ± 5.46 41.9 ± 2.76 37.1 ± 2.18 44.0 ± 2.99 47.9 ± 6.14
A Egg in magnum 4 B Egg in isthmus 3 C Egg in uterus — early stage2 4 2 D Egg in uterus - later stage 4 E No egg in oviduct 3
1.77+ .075 1.81 ± .045 1.69 ± .187 1.68 ± .097 1.72 ± .083
30.0 ± 3.62 24.0 + 2.60
4.2 ± .42 4.3 ± .27 19.8 ± 1.51 4.2 ± 1.51 26.9 ± 2.04 3.2 ± .40 30.2 + 3.79 3.8 ± 1.01
14.6 ± 1.59 13.6 ± .41 13.1 ± 1.02 13.9 ± .73 13.9 ± 1.16
Means of all 18 hens
1.73 ± .050
26.0 + 1.45
13.8+ .45 43.8 ± 1.93
3.9 ± .23
Excluding infundibulum and vagina. Estimated by the rigidity of the shell: coded C when depressible on a gentle push with a finger or D when found rigid.
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
beakers containing them to stand overnight in a fume hood. Drying was completed under vacuo (in a desiccator) over P 2 0 5 and the weights of these crude lipid extracts determined. Subsequently, these crude extracts were re-dissolved in the extractant and washed with water according to the method of Bligh and Dyer (1959) in order to separate net lipids (chloroform-phase soluble lipids, chiefly neutral lipids, and phospholipids) from other extracted components present in the crude lipid extract. The chloroform-phase lipids were resolved into major lipid classes by a thin layer chromatography (TLC) technique using Adsorbosil-1 (silica gel containing 10% CaSC>4 binder; Applied Science Labs, Inc., State College, PA) and hexane:diethyl ether:acetic acid (90:10:1 v/v/v) as the eluent (Malins and Mangold, 1960). The developed plates were sprayed with phosphomolybdate (10% phosphomolybdic acid in 95% ethanol) and heated to 70 C for 10 min for visualization. Aliquots of net lipids were resolved on a preparative scale (approximately 50 mg per plate) and the TLC zones extracted with chloroform in order to estimate gravimetrically the amounts of individual lipid fractions. The starting zone was also extracted with chloroform: methanol: water (2:5:2 v/v/v) and assayed for phosphorus by the method of Allen (1940). Assay of dried, lipid extracted tissue fractions. Aliquots of dried, lipid extracted isthmus and uterus tissue fractions were hydrolysed in 4N hydrochloric acid and assayed for hexosamines and hydroxyproline using methods described previously (Cipera et al, 1960,1965). The calcium and magnesium contents of these
638
CIPERA
Comparison of the weights of tissue fractions isolated from isthmus and from uterus (Table 2) showed that isthmuses contained more mucosal than wall material, in consequence of which isthmuses and uteri did not differ in amounts of mucosae as much as would be expected from the difference in the total
weights of the two segments. In both isthmian and uterine segments, the water content was higher in the walls than in the mucosae (Table 2). This difference would agree with the visual observation that the walls were essentially cellular tissues, while the mucosae appeared to contain essentially the secretory products of these tissues. Such functional differences would also explain the considerably higher levels of the crude lipid extracts found in mucosae from both segments when compared with those of walls (Table 3). Mucosae from uteri had much higher levels of crude lipid extracts, as well as of net lipid extracts (chloroform-phase lipids), than those from isthmuses. Within the mucosae of both segments, considerably higher levels of net lipid extracts were found in the MR than in the MS fractions. Resolution of the chloroform-phase lipids from the MR and W fractions of isthmus and MS, MR, and W fractions of uterus by TLC indicated a presence of triglycerides, free fatty acids, steroids, and steryl esters. In addition, there was a fairly intensive spot at the starting zone, several minor spots between the starting and the steroid zones, and one minor spot in front of (faster moving than) the triglyceride spot. The intensities of the triglyceride spots were greater in the chromatographs of the wall extracts than in those of the MR or MS extracts. This difference occurred in both isthmus and uterus, tissue extracts. Extraction with chloroform of the lipid zones obtained by a preparative
TABLE 2. Wet and dry weights of oviduccd segments associated with shell formation
Tissue fraction 1
Dry tissue percent of wet tissue
Tissue weight2 (g) Dry
Wet (Isthmus)
MR MS M W Is
.409 ± .175 ± .584 ± .218 + .802 ±
2.54 ± .21 1.39 ± .17 3.93 ±.23
.030 .010 .037 .026 .032
23.0 15.7 20.4
± .012 ± .024 ± .030 ± .058 ± .085
25.3 14.3 17.0
-(Uterus)MR MS M W Ut
3.33 ± .15 10.51 + .38 13.84 + .45
.296 .547 .843 1.505 2.348
Tissue fractions: MR = mucosal residue; MS = mucosal supernatant; M = total mucosa; W = wall of the segment; Is = total isthmus; Ut = total uterus. 2, "Mean + SE where n = 18.
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
to the tissue fractionation, it was observed during the separation of mucosae from the isthmian walls that, because of their fragile nature, small tissue fragments from the walls remained in the mucosal fraction. This did not evidently occur with uterine tissue. Mucosae from both segments, isthmus and uterus, were pale beige in color, this color being uniformly distributed between the MR and MS fractions. Walls from uteri were massive and muscular in character, while isthmuses had thin semi-translucent walls. A complicating feature of uteri was the relative difficulty in identifying unequivocally the dividing line between the extraneous tissue and the intrinsic tissue of the uterine wall itself. However, by a careful manipulation the extraneous tissue from the uterine wall can be removed quite well by peeling. Also, it should be noted that the cranial portion of the hen's uterus is tubular in character, i.e. its wall is almost as thin as that of isthmus. However, isthmian mucosa is whitish gray whereas the mucosa in the tubular portion of uterus has the same color (brownish gray) and appearance as in the rest of this tissue.
OVIDUCAL SEGMENTS INVOLVED WITH SHELL FORMATION
639
The contents of hydroxyproline (Hyp), hexosamines, calcium, and magnesium in the dry tissue fractions (MR, MS, and W), from isthmus and uterus were determined after
TABLE 3. Composition ofoviducal segments associated with shell formation Levels of c o m p o n e n t s (percent of d r y t i s s u e ) ! Tissue fraction2
Lipid extracts Net 3
Crude
Collagen A
Hexosamines
Magnesium
Calcium
Levels of c o m p o n e n t s (percent of d r y tissue) /T
^.L
\
.06 .04 .05 .04 .04
.147 .557 .270 .155 .236
± ± ± ± ±
.014 .028 .013 .006 .009
.037 .264 .106 .098 .104
± ± ± ± ±
.002 .006 .003 .005 .003
.57 .67 .64 .57 .60
± .03 ± .03 ± .02 ±.01 ± .01
.161 .208 .185 .136 .156
± ± ± ± ±
.015a .0196 .013c .008 .009c
.062 .144 .115 .081 .093
± ± ± ± ±
.004 .010 .007 .002 .003
.15 .45 .60 .23 .83
± ± ± ± ±
.01 .02 .03 .03 .03
MR MS M W Is
14.1 16.4 14.7 9.0 13.3
± .7 ± 1.2 ± .8 ± .5 ± .6
11.2 2.0 8.3 8.8 8.4
± ± ± ± ±
.9b .3 .5b .5 Ab
3.1 3.6 3.3 20.6 8.0
± ± ± ± ±
.la .3 .2a 1.2 .5a
1.40 1.47 1.43 .91 1.29
MR MS M W Ut
32.0 23.5 26.4 11.1 16.7
+ + ± ± ±
24.3 7.8 13.6 10.4 11.5
± ± ± ± ±
.9 .5 .5 .6 .5
.3 .0 .1 26.7 17.2
± + + ± ±
.la .0 ..Oa 1.3 .9a
.7 .7 .5 .6 .5
± + ± ± ±
Mean-per- hen a m o u n t s of c o m p o n e n t s (mg) /r
MR MS M W Is
56 29 85 20 105
± ± + ± ±
4 3 6 3 5
45 3 47 19 66
MR MS M W Ut
94 128 222 167 389
± 3 ± 5 ± 7 ±10 ±14
71 42 113 154 267
± ± ± ± ±
Ab 0 4b 3 4b
± 3 ± 3 ± 4 ± 8 ± 10
.1.
. _\
12 6 19 42 62
± ± ± ± ±
la 1 2a 4 4a
5.71 2.57 8.38 2.08 10.36
± .45 ±.15 ±.53 ±.31 ±.56
.61 .96 1.57 .33 1.90
± ± ± ± ±
.07 .05 .10 .04 .11
1 0 1 398 399
± ± ± ± ±
0a 0 Oa 22 22
1.68 3.72 5.40 8.63 14.25
±.11 ± .26 ± .28 ± .44 ± .68
.49 1.18 1.64 2.08 3.85
± ± ± ± ±
.05a .12b .17c .18 .34c
.19 .78 .97 1.23 2.20
± .02 ± .07 ± .08 ±.06 ±.13
Mean ± SE of 18 hens, except where indicated with letters: a = 17 hens; b = 16 hens-, c = 15 hens. 2 Tissue fractions: MR = mucosal residue; MS = mucosal supernatant; M = total mucosa; W = wall of the segment; Is = total isthmus; Ut = total uterus. Net lipid*: The chloroform-phase soluble fraction of crude lipid extracts. CollagejfjEstimated by multiplying the hydroxyproline amounts by a factor of 7.46 (Neuman and Logan, 1950). >
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
removal of the lipids. Since Hyp is a characteristic and exclusive component of collagen, levels of which are routinely estimated by assaying the tissues for Hyp (Eastoe and Courts, 1963), the data from the Hyp assay were used to obtain an indication of the amounts of collagen in respective tissues. Comparison of the overall levels of these components in isthmian vs uterine tissues (Table 3) shows that hexosamines and the two mineral components assayed, calcium and magnesium, had higher concentrations in isthmus, while collagen had higher levels in uterus. Within both these segments, collagen was found to have highest levels in walls, hexosamines, calcium and
scale TLC of several aliquots (50 to 100 mg) of chloroform-phase lipids yielded substantial amounts of the above-described lipid classes with the exception of the starting zone. This zone yielded virtually no amounts of chloroform-soluble lipids. Adding methanol and water to the extractant produced recoveries approaching 10% of the total amounts of aliquots subjected to the TLC. Phosphorus levels in this extract were 2.0 to 2.4%, indicating that phospholipids may be its major constituents.
640
CIPERA
Hexosamines, like calcium, had higher levels
in isthmian t h a n in uterine mucosae, with highest levels in each case being in t h e MS fraction of t h e m u c o s a e (Table 3). This a p p a r e n t relationship b e t w e e n hexosamines, which are t h e major c o n s t i t u e n t s of t h e m u c o p o l y s a c charides, and of calcium could be of distinct interest, because of t h e often postulated association b e t w e e n t h e acidic m u c o p o l y s a c charides and t h e calcium metabolism (Simkiss and Taylor, 1 9 7 1 ) , particularly t h e mobilization of calcium for s u b s e q u e n t calcification processes. Levels of collagen in t h e W fractions of isthmus and u t e r u s (Table 3) are intermediary b e t w e e n those in vertebrate skins and muscles ( N e u m a n and Logan, 1 9 5 0 ) , confirming t h e obviously integumental character of a major p o r t i o n of these tissue fractions. T h e reason for uterine walls having higher levels of collagen t h a n those from isthmuses is n o t clear. T h e relatively higher levels of collagen in isthmus mucosae, when c o m p a r e d t o t h o s e from uteri, m a y have been caused b y t h e presence of fragments of isthmus walls which b r o k e up during t h e tissue fractionation and w o u l d n o t b e removed from the mucosae. Changes in t h e levels of t h e above-discussed constituents of oviducal tissues with t h e passage of t h e egg t h r o u g h t h e h e n ' s oviduct are s h o w n
TABLE 4. Effect of the passage of the egg on the composition of oviducal segments associated with shell formation Levels of components (percent of dry tissue)^ Stage of shell formation 1
N
Crude
Lipid extracts Net 3
Collagen 4
Hexosamines
Calcium
Magnesium
± .03 ±.03 + .08 ±.03 ± .08
.233 .205 .211 .260 .260
.096 + .095 ± .109 ± .109 ± .110+
.006 .004 .006 .006 .006
± .04 ± .03 ± .01 ± .02 ± .02
.170 + .145 + .120 + .148+ .212 ±
.091 .101 .083 .089 .108
.011 .006 .001 .002 .008
- (Isthmus) A B C D E
12.2 16.7 13.7 12.2 12.2
± ± ± ± ±
.8 .7 .9 .8 3.0
A B C D E
16.5 15.6 16.4 17.3 17.4
± ± ± ± ±
.9 1.1 .8 1.0 2.2
7.6 ± 9.5+ 8.5 ± 8.0 + 8.4 ±
.la .1 2.0a .8 1.4
7.3 ± 7.6 ± 8.8+ 8.9 ± 6.6 ±
1.2 1.6 .7 .8 .8
1.36 1.03 1.21 1.37 1.44
± .008 ± .008 + .026 + .023 + .006
(Uterus) •
2 4
10.4 10.9 12.3 12.2 11.9
± ± ± ± ±
.9 1.0 .8 1.4 1.7
14.0 14.4 19.2 20.4 17.0
± .8 ± 2.2 ± .9 ± .7 ±4.4a
.56 .59 .63 .58 .61
.015 .011 .009« .012a .033a
+ ± ± ± ±
For definitions of stages of shell formation see Table 1. Mean ± SE when followed by letter a, the mean was obtained from N - 1 samples. Net lipids: The chloroform-phase soluble fraction of crude lipid extracts.
Collagen was estimated by multiplying the hydroxyproline amounts by a factor of 7.46 (Neuman and Logan, 1950).
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
magnesium in MS, and lipids in MR fractions. Total a m o u n t s of calcium in b o t h shellforming segments of h e n ' s oviduct were f o u n d t o be very low, in each case a few milligrams (Table 3). This agrees with t h e classical observat i o n of Buckner et al. ( 1 9 2 5 ) t h a t n e i t h e r of t h e t w o oviducal segments could possibly provide a n y degree of storage for t h e shell f o r m a t i o n , which requires a supply of over 2 g of calcium within a period of a b o u t 14 hr; t h u s , t h e calcium for shell f o r m a t i o n m u s t b e continuously supplied t o t h e oviducal tissues, most likely from b l o o d . Therefore, it is n o t surprising t o find t h a t in b o t h isthmus and u t e r u s , highest levels of calcium occurred in t h e MS fractions, t h e m o s t p r o b a b l e location of t h e secretory p r o d u c t s of oviducal tissues. Less e x p e c t e d was t h e finding t h a t levels of calcium were m u c h higher in isthmian t h a n in uterine m u c o s a e , in consequence of which t h e t o t a l a m o u n t s of calcium present in isthmus m u c i n s practically equalled t h e a m o u n t s of calcium in uterine mucosae. Magnesium, however, had s o m e w h a t lower concentrations in isthmian t h a n in uterine m u c o s a e , in consequence of which its t o t a l a m o u n t s in m u c o s a e of isthmus were m u c h lower t h a n in t h o s e of u t e r u s .
641
OVIDUCAL SEGMENTS INVOLVED WITH SHELL FORMATION
metabolites, presumably for the supply of carbon dioxide for the shell formation, or it may be associated with the increased need for lubricating the egg's passage. The overall levels of water, lipids, calcium, and magnesium observed in isthmus and uterus agree well with those reported in the literature (Table 5). Since no comparative data were available on the levels of hexosamines and hydroxyproline in isthmus and uterus of laying hens, levels of these compounds in uteri from immature pullets which received androgen and estrogen (Anastasiadis etal, 1955) are presented for comparative purposes. The levels of hexosamines agreed fairly well between immature and mature chickens, whereas the hydroxyproline levels were much lower in the uteri from hormone-treated chicks than in those from laying hens. This difference, however, appears to be due to the differential initial response to the hormonal stimulus, which would be expected to increase the levels of hexosamine-containing tissue constituents faster than those of the structural tissue constituents which contain hydroxyproline (see Cipera etal, 1965). ACKNOWLEDGMENTS The author wishes to acknowledge competent
TABLE 5. Comparison of the levels (percent of dry tissue) of components of isthmuses and uteri of laying hens obtained in this experiment with those reported elsewhere Oviducal component
Observed levels
Water Calcium Magnesium Lipids Hexosamines Hydroxyproline
397
Water Calcium Magnesium Lipids Hexosamines Hydroxyproline
495
.236 .104 8.37 1.29 1.06
.156 .093 11.54 .60 2.30
Levels reported in references no.
417
- (Isthmus) — 335 359
182 9.5
495
360
259 076
.263 .102
8.6
— (Uterus) 443 590
.(099
13.8
11.0
References: 1) Buckner et al. (1925), 2) Brown and Jackson (I960), 3) Cecil et al. (1970), 4) Yu and Marquardt (1972), 5) Schraer and Schraer (1965), 6) Misra and Kemeny (1964), and 7) Anastasiadis et al. (1955). 2 These data are not of laying hens; they were obtained from 12-week-old pullets receiving androgen and estrogen. The oviducti were sectioned into magni, uteri, and vaginas; isthmuses were obviously not developed sufficiently to permit their separation. Levels in vaginas were: hexosamines .88%, hydroxyproline 1.03%.
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
in Table 4. Although the data are from a relatively small number of hens, several trends are indicated. Levels of lipids increased in isthmuses and uteri as the egg passed through the respective segment. Levels of uterine collagen were highest during stages C and D, when uterine tissue was stretched around the egg. Calcium levels in isthmus were lowest during stage B and in uterus during stage C, which would correspond to the periods of maximum calcium demands in the respective segment; magnesium did not show this trend. All these changes are in agreement with the requirements that shell-formation processes would be expected to impose on the oviducal tissues. Thus, the evident increase in collagen levels of uterus when it was stretched around the egg for an extended period of time coincides with the period of stress imposed on the uterine wall by which the relative amount of the non-structural vs the structural (collagenous) components of the wall would be expected to decrease. Calcium changes appear to reflect on the magnitude of the strain imposed on the hen's metabolic supply of calcium during the shell forming stage. The changing level of lipids, however, associated with the passage of the egg may be indicative of an increasing supply of
642
CIPERA
assistance of L. Carter in the isolation of tissue fractions, of J. Sykora in assaying them and in the tabulation of the results, and helpful suggestions of R.M.G. Hamilton in reviewing the manuscript. REFERENCES
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
Allen, R.J.L., 1940. The estimation of phosphorus. Biochem. J. 34:858-865. Anastasiadis, P. A., W. A. Maw, and R. H. Common, 1955. The hexosamine content of certain tissues of the sexually immature pullet and some effects thereon of gonadal hormones. Can. J. Biochem. Physiol. 33:627-637. Bligh, E. G., and W. J. Dyer, 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. Brown, W. O., and N. Jackson, 1960. Composition of the normal and hormone-stimulated oviduct of the common fowl. Poultry Sci. 39:602-611. Buckner, G. D., J. H. Martin, and A. M. Peter, 1925. Chemical studies of the oviduct of the hen. Amer. J. Physiol. 71:349-354. Cecil, H. C , J. Bitman, and C. S. Shaffner, 1969. Oviducal glycogen content of laying and nonlaying hens and of estradiol stimulated pullets. Proc. Soc. Exp. Biol. 131:164-167. Cecil, H. C , J. Bitman, and C. S. Shaffner, 1970. Oviducal water, electrolytes and nucleic acids of laying and non-laying hens and of estradiol stimulated pullets. Poultry Sci. 49:467—475. Christensen, H. N., and T. R. Riggs, 1953. Amino acids and peptides in the oviduct of the laying hen. Proc. Soc. Exp. Biol. 83:697-698. Cipera, J. D., B. B. Migicovsky, and L. F. Belanger, 1960. Changes in hexosamine and acetone extractable contents of epiphyseal cartilage of rachitic chicks following administration of vitamin D 3 . Can. J. Biochem. Physiol. 3 8 : 8 0 7 811. Cipera, J. D., B. B. Migicovsky, and L. F. Belanger, 1965. Effect of norethadrolone on selected connective tissues. Can. J. Biochem. 43:1235— 1241. Common, R. H., 1938. Observations on the mineral metabolism of pullets. J. Agr. Sci. 28:347-366. Copp, D. H., 1963. Simple and precise micromethod for EDTA titration of calcium. J. Lab. Clin. Med. 61:1029-1037. Eastoe, J. E., and A. Courts, 1963. Practical analytical methods for connective tissue proteins. Publ. E. & F. N. Spon Ltd., London. Edwards, N. A., 1977. The secretion of glucose into the egg of Gallus domesticus and observations on the uterine secretion of cations. Brit. Poultry Sci. 18:641-649. Folch, J„ M. Lees, and G.H.S. Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226:497— 509. Katzukura, Y., and H. Tamate, 1971. Ca mobilization
at each location of egg in the oviduct segments of the laying hens. Tohoku J. Agr. Res. 22:219— 227. Malins, D. C , and H. K. Mangold, 1960. Analysis of complex lipid mixtures by thin-layer chromatography and complementary methods. J. Amer. Oil Chem. Soc. 37:576-579. Misra, M. S., and A. Kemeny, 1964. Studies on the oviduct and serum of fowls. 1. Oxygen uptake, alkaline phosphatase activity, concentration of phosphorus, calcium and magnesium in adult Hungarian yellow hens. Acta Vet. Acad. Sci. Hungary 14:389-397. Mongin, P., and N. W. Carter, 1977. Studies on the avian shell gland during egg formation: Aqueous and electrolytic composition of the mucosa. Brit. Poultry Sci. 1 8 : 3 3 9 - 3 5 1 . Mongin, P., and N. W. Carter, 1978. Studies on the avian shell gland during egg formation: Mucosal intracellular pH. Brit. Poultry Sci. 19:93-96. Moynihan, J. B., and N. A. Edwards, 1975. Electrolyte secretion by the magnum of the domestic fowl during eggshell calcification. Comp. Biochem. Physiol. 50-A:743-745. Neuman, R. E., and M. A. Logan, 1950. The determination of collagen and elastin in tissues. J. Biol. Chem. 186:549-556. Roland, D. A., Sr., 1977. The extent of uncollected eggs due to inadequate shell. Poultry Sci. 56:1517-1521. Schenck, E. G., 1932. Uber die Bildung der Proteine des Eies im Ovar und Eileiter des Huhnes. Z. Physiol. Chem. 211:153-160. Schraer, R., and H. Schraer, 1965. Changes in metal distribution of the avian oviduct during the ovulation cycle. Proc. Soc. Exp. Biol. 119:939— 942. Simkiss, K., and T. G. Taylor, 1971. Shell formation. Page 1331 in Physiology and biochemistry of the domestic fowl. Vol. 3. D. J. Bell and B. M. Freeman, ed. Academic Press, London, New York. Smith, A. H., G. N. Hoover, J. O. Nordstrom, and C. M. Winget, 1957. Quantitative changes in the hen's oviduct associated with egg formation. Poultry Sci. 36:353-357. Snapir, N., and M. Perek, 1970. Distribution of Ca, carbonic anhydrase and alkaline phosphatase activities in the uterus and isthmus of young and old White Leghorn hens. Poultry Sci. 49:1526-1531. Taylor, T. G., and F. Hertelendy, 1960. Parallel distribution of calcium and citric acid in the oviduct of the hen. Nature 187:244-245. Yu, J.Y.L., and R. R. Marquardt, 1971. Effects of estrogen and progesterone on major cellular components in chicken oviducts. Can. J. Biochem. 49:348-356. Yu, J.Y.L., and R. R. Marquardt, 1972. A comparative study on the changes in cellular components in each region of the domestic fowl oviduct during a reproductive cycle. Can. J. Physiol. Pharmacol. 50:689-696.
1980 Poultry Science 59:635-642 INTRODUCTION T h e quality of a h e n ' s egg shell is of major e c o n o m i c i m p o r t a n c e t o t h e p o u l t r y industry (see R o l a n d , 1 9 7 7 ) . In c o n s e q u e n c e it has b e c o m e a focus of extensive investigative w o r k . Most of this w o r k involved a nutritional approach, although t h e effects of flock managem e n t and of t h e physiological and o t h e r factors which m a y affect t h e process of shell f o r m a t i o n have n o t b e e n neglected. Relatively little w o r k has b e e n d o n e o n t h e possible interrelationship b e t w e e n t h e c o m p o s i t i o n of t h e hen's oviduct and shell quality. T h e a m o u n t of i n f o r m a t i o n on t h e overall c o m p o s i t i o n of specific segments of h e n ' s oviduct at various stages of egg f o r m a t i o n is quite limited. In 1 9 2 5 , Buckner et al r e p o r t e d levels of calcium and p h o s p h o r u s in t h r e e segments ( m a g n u m , isthmus, and uterus) of oviducts from eight laying h e n s ; Schenck ( 1 9 3 2 ) assayed u p p e r parts of hen's oviducts (excluding t h e uterus) for egg-white proteins and several a m i n o acids; C o m m o n ( 1 9 3 8 )
1
Contribution No. 815 Animal Research Institute.
r e p o r t e d c o n t e n t s of moisture, Kjeldahl p r o t e i n , fat, ash, and calcium in whole oviducts from 8 hens and Christensen and Riggs ( 1 9 5 3 ) investigated whole oviducts from 4 hens for free (dialysable) amino acids and peptides. Subseq u e n t l y various oviducal segments from laying hens were evaluated for mineral (Brown and J a c k s o n , I 9 6 0 ; Taylor and H e r t e l e n d y , I 9 6 0 ; Misra and K e m e n y , 1 9 6 4 ; Schraer and Schraer, 1 9 6 5 ; Snapir and Perek, 1970) a n d organic (Brown and J a c k s o n , I 9 6 0 ; Cecil et al, 1 9 6 9 , 1 9 7 0 ; Yu a n d Marquardt, 1 9 7 1 , 1 9 7 2 ) c o m p o nents. In this c o n n e c t i o n , r e p o r t s o n t h e c o m p o s i t i o n of oviducal secretions ( K a t z u k u r a and T a m a t e , 1 9 7 1 ; M o y n i h a n and Edwards, 1 9 7 5 ; Edwards, 1 9 7 7 ; Mongin and Carter, 1 9 7 7 , 1 9 7 8 ) , although giving rather relative values, are also of interest. However, n o comprehensive investigation o n t h e c o m p o s i t i o n of these tissues during t h e passage of t h e egg t h r o u g h the oviduct, involving b o t h organic and mineral constituents in t h e same tissues, has been y e t r e p o r t e d . Since egg shell f o r m a t i o n is initiated in t h e isthmus and completed in t h e u t e r u s (Simkiss and Taylor, 1 9 7 1 ) , t h e c o m p o s i t i o n of these
635
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
ABSTRACT Oviducts from eighteen laying hens were isolated at various stages of egg formation and, after infundibulum and vagina were discarded, divided into three segments: magnum, isthmus, and uterus. Of these, the magnum showed widest weight variation between the egg-forming stages; this variation appeared to be related to the albumen formation. The isthmus and uterus from each hen were fractionated into wall and mucosal fractions, and the mucosae were further separated by centrifugation into mucosal supernatant and residue fractions. These fractions were assayed for moisture, total and neutral lipids, calcium, magnesium, hexosamines, and collagen (as indicated by the levels of hydroxyproline). The wall fractions of either isthmus or uterus were characterized by consistently higher levels of water and of collagen, while mucosae had higher levels of calcium, magnesium, crude lipid extracts, and, in the case of isthmus, hexosamines. In both isthmus and uterus, highest levels of chloroform-phase soluble lipids were observed in the sedimenting (residue) sub-fraction of mucosae. Thin-layer chromatographic resolution of the chloroform-phase lipids indicated that the lipids from the mucosal and wall fractions of either the isthmus or uterus were similar in composition. Changes in composition of isthmus and uterus observed at various stages of egg passage through the oviduct indicated a trend to lower levels of calcium in these segments during the shell formation stage and to generally higher levels of lipids when the egg is present in the respective segment. In both isthmus and uterus, collagen concentration was increased at the time the uterus was stretched around the egg.
636
CIPERA
tissues of the oviduct should most conceivably influence the quality of the shell produced within them. Therefore we concentrated our investigation on these two segments of hen's oviduct. MATERIALS AND METHODS
Magnums were weighed and discarded. The isthmian and utero-vaginal tissues were transferred into pre-cooled glass tubes which were
Since mucosae from both segments tended to separate on standing into a sediment and an opaque emulsion, they were centrifuged at 800 x g for 15 min to produce mucosal residue (MR) and mucosal supernatant (MS) fractions. The MR fraction was then washed twice with a small volume (approximately 2 ml) of distilled water and centrifuged after each washing; the washings were added to the MS fractions. After this treatment the MS fraction remained homogeneous (no sedimentation occurred) on standing. The MR, MS, and wall (W) fractions of the isthmian and uterine segments were freeze-dried and subsequently dried in a vacuum desiccator over P2O5 to a constant weight. Lipid Extraction. The dry tissue fractions were homogenized (Virtis 45 homogenizer) and extracted with chloroform:methanol (2:1 v/v) (Folch et al., 1957). Uterine walls, because of their bulkiness, were finely minced with scissors before being homogenized and extracted. The extraction was performed in centrifuge tubes and extracts evaporated by allowing
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
Hens. Fifty Single Comb White Leghorn hens were housed individually in wire cages (20.3 X 40.6 cm) and received 14 hr of light per day. After 20 weeks of age the hens were given ad libitum a laying ration which contained 14.8% protein (N x 6.25), 2.9% calcium, and .6% total phosphorus. At 42 weeks of age, eggs were collected twice daily (1130 and 1600 hr) and recorded for each hen. Hens to be killed were selected on the basis of postoviposition time so as to obtain oviducts at various stages of egg formation. All hens were killed between 0830 and 1130 hr. Isolation of Oviducts. Each selected hen was weighed, killed by cervical dislocation, and its oviduct was excised. Presence and position of an egg in the oviduct was recorded; subsequently this egg was removed from the isolated oviduct (through a longitudinal incision) and discarded. After removal of the infundibulum and cloaca, the oviduct was cleaned of extraneous tissue and sectioned into three parts: magnum, isthmus, and utero-vaginal complex. The infundibulum was separated from magnum at the point where the semi-translucent, pinkish red tissue of the infundibulum changed into the massive yellowish beige tissue of the magnum and the isthmus from magnum at the point where an approximately 1 mm wide band of glossy gray tissue separated the two segments. The utero-vaginal complex was separated from the isthmus at the point where the whitish gray color of isthmian mucin changed to a brownish gray uterine mucosa; although this change in color of the mucosa was noticeable on the outside of the oviduct, a short longitudinal incision was made at the isthmo-uterine junction in order to assure an accurate location of the dividing line between the two segments by observing the change in mucosa directly. The cloaca was separated from the utero-vaginal complex at the point where the narrow tube of the vagina begins to widen into the cloacal cavity.
stoppered and stored in a deep freeze until they were further processed. Fractionation of Isthmuses and Uteri. Within two days after the hens were killed, samples of isthmuses and uteri were thawed at room temperature, washed under a gentle stream of tap water, cleaned of any remaining mesenterial tissue, placed on a thick glass plate and opened longitudinally. Vaginas were removed from uteri at this stage; dividing point between the two segments was the point where the narrow tube of the vagina just began to widen into the uterine pouch. (A band of a transitory tissue, approximately 5 to 10 mm wide, was observed between the yellowish white vagina and the brownish gray uterus; this tissue, which was similar in appearance to the uterine tissue except that its lumen was whitish to pinkish gray in color, as contrasted with the brownish gray color of the remaining uterus, was further treated as a part of the uterine tissue.) The mucosal portion of each opened segment was scraped off with a glass slide, transferred into a tube cooled in an ice-bath, and weighed. The remaining wall portion of the segment was washed with a minimum amount of distilled water, scraped again with glass slide, and weighed; the washings were added to the mucosal scrapings but, because they represented an insignificant amount of the total, their weight was ignored.
637
OVIDUCAL SEGMENTS INVOLVED WITH SHELL FORMATION
tissues were determined, after wet-ashing with perchloric acid and nitric acid, by complexometric titration with murexide as indicator (Copp, 1963), and atomic absorption spectrophometry, respectively. RESULTS AND DISCUSSION Oviducts from eighteen laying hens were isolated and processed. Table 1 shows the body weights and the wet weights of the magnum, isthmus, and uterus of hens at different stages of egg shell formation. There was considerable variation between the shell-formation stages in the wet weight of the total oviducts, mainly due to the variation in weight of the magnums; the total oviduct and magnum weights were lowest during the period immediately following the passage of the egg from magnum and highest in hens with an egg in magnum or with no egg in the oviduct. Similar changes were observed by Smith et al. (1957) who suggested that changes occurring in magnum weight were associated with the variations in the amount of albumen present in the secretory tissue of the magnum: the amount of this albumen would be expected to be highest before and during the period the egg passes through the magnum and lowest during the subsequent period. The apparent decrease in isthmus weights at the later shell-forming period, when egg was present in uterus and became covered with a hard shell, may be due to possible participation of certain isthmian components in the shell formation (Moynihan and Edwards, 1975). When the isthmuses and uteri were subjected
TABLE 1. Weights of oviducal tissue at various stages of shell formation Wet weight (g) (mean + SE) Stage of shell formation
No. of hens
Body weights (kg) (mean ± SE) Magnum
Isthmus
Uterus
Total! oviduct 48.8 ± 5.46 41.9 ± 2.76 37.1 ± 2.18 44.0 ± 2.99 47.9 ± 6.14
A Egg in magnum 4 B Egg in isthmus 3 C Egg in uterus — early stage2 4 2 D Egg in uterus - later stage 4 E No egg in oviduct 3
1.77+ .075 1.81 ± .045 1.69 ± .187 1.68 ± .097 1.72 ± .083
30.0 ± 3.62 24.0 + 2.60
4.2 ± .42 4.3 ± .27 19.8 ± 1.51 4.2 ± 1.51 26.9 ± 2.04 3.2 ± .40 30.2 + 3.79 3.8 ± 1.01
14.6 ± 1.59 13.6 ± .41 13.1 ± 1.02 13.9 ± .73 13.9 ± 1.16
Means of all 18 hens
1.73 ± .050
26.0 + 1.45
13.8+ .45 43.8 ± 1.93
3.9 ± .23
Excluding infundibulum and vagina. Estimated by the rigidity of the shell: coded C when depressible on a gentle push with a finger or D when found rigid.
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
beakers containing them to stand overnight in a fume hood. Drying was completed under vacuo (in a desiccator) over P 2 0 5 and the weights of these crude lipid extracts determined. Subsequently, these crude extracts were re-dissolved in the extractant and washed with water according to the method of Bligh and Dyer (1959) in order to separate net lipids (chloroform-phase soluble lipids, chiefly neutral lipids, and phospholipids) from other extracted components present in the crude lipid extract. The chloroform-phase lipids were resolved into major lipid classes by a thin layer chromatography (TLC) technique using Adsorbosil-1 (silica gel containing 10% CaSC>4 binder; Applied Science Labs, Inc., State College, PA) and hexane:diethyl ether:acetic acid (90:10:1 v/v/v) as the eluent (Malins and Mangold, 1960). The developed plates were sprayed with phosphomolybdate (10% phosphomolybdic acid in 95% ethanol) and heated to 70 C for 10 min for visualization. Aliquots of net lipids were resolved on a preparative scale (approximately 50 mg per plate) and the TLC zones extracted with chloroform in order to estimate gravimetrically the amounts of individual lipid fractions. The starting zone was also extracted with chloroform: methanol: water (2:5:2 v/v/v) and assayed for phosphorus by the method of Allen (1940). Assay of dried, lipid extracted tissue fractions. Aliquots of dried, lipid extracted isthmus and uterus tissue fractions were hydrolysed in 4N hydrochloric acid and assayed for hexosamines and hydroxyproline using methods described previously (Cipera et al, 1960,1965). The calcium and magnesium contents of these
638
CIPERA
Comparison of the weights of tissue fractions isolated from isthmus and from uterus (Table 2) showed that isthmuses contained more mucosal than wall material, in consequence of which isthmuses and uteri did not differ in amounts of mucosae as much as would be expected from the difference in the total
weights of the two segments. In both isthmian and uterine segments, the water content was higher in the walls than in the mucosae (Table 2). This difference would agree with the visual observation that the walls were essentially cellular tissues, while the mucosae appeared to contain essentially the secretory products of these tissues. Such functional differences would also explain the considerably higher levels of the crude lipid extracts found in mucosae from both segments when compared with those of walls (Table 3). Mucosae from uteri had much higher levels of crude lipid extracts, as well as of net lipid extracts (chloroform-phase lipids), than those from isthmuses. Within the mucosae of both segments, considerably higher levels of net lipid extracts were found in the MR than in the MS fractions. Resolution of the chloroform-phase lipids from the MR and W fractions of isthmus and MS, MR, and W fractions of uterus by TLC indicated a presence of triglycerides, free fatty acids, steroids, and steryl esters. In addition, there was a fairly intensive spot at the starting zone, several minor spots between the starting and the steroid zones, and one minor spot in front of (faster moving than) the triglyceride spot. The intensities of the triglyceride spots were greater in the chromatographs of the wall extracts than in those of the MR or MS extracts. This difference occurred in both isthmus and uterus, tissue extracts. Extraction with chloroform of the lipid zones obtained by a preparative
TABLE 2. Wet and dry weights of oviduccd segments associated with shell formation
Tissue fraction 1
Dry tissue percent of wet tissue
Tissue weight2 (g) Dry
Wet (Isthmus)
MR MS M W Is
.409 ± .175 ± .584 ± .218 + .802 ±
2.54 ± .21 1.39 ± .17 3.93 ±.23
.030 .010 .037 .026 .032
23.0 15.7 20.4
± .012 ± .024 ± .030 ± .058 ± .085
25.3 14.3 17.0
-(Uterus)MR MS M W Ut
3.33 ± .15 10.51 + .38 13.84 + .45
.296 .547 .843 1.505 2.348
Tissue fractions: MR = mucosal residue; MS = mucosal supernatant; M = total mucosa; W = wall of the segment; Is = total isthmus; Ut = total uterus. 2, "Mean + SE where n = 18.
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
to the tissue fractionation, it was observed during the separation of mucosae from the isthmian walls that, because of their fragile nature, small tissue fragments from the walls remained in the mucosal fraction. This did not evidently occur with uterine tissue. Mucosae from both segments, isthmus and uterus, were pale beige in color, this color being uniformly distributed between the MR and MS fractions. Walls from uteri were massive and muscular in character, while isthmuses had thin semi-translucent walls. A complicating feature of uteri was the relative difficulty in identifying unequivocally the dividing line between the extraneous tissue and the intrinsic tissue of the uterine wall itself. However, by a careful manipulation the extraneous tissue from the uterine wall can be removed quite well by peeling. Also, it should be noted that the cranial portion of the hen's uterus is tubular in character, i.e. its wall is almost as thin as that of isthmus. However, isthmian mucosa is whitish gray whereas the mucosa in the tubular portion of uterus has the same color (brownish gray) and appearance as in the rest of this tissue.
OVIDUCAL SEGMENTS INVOLVED WITH SHELL FORMATION
639
The contents of hydroxyproline (Hyp), hexosamines, calcium, and magnesium in the dry tissue fractions (MR, MS, and W), from isthmus and uterus were determined after
TABLE 3. Composition ofoviducal segments associated with shell formation Levels of c o m p o n e n t s (percent of d r y t i s s u e ) ! Tissue fraction2
Lipid extracts Net 3
Crude
Collagen A
Hexosamines
Magnesium
Calcium
Levels of c o m p o n e n t s (percent of d r y tissue) /T
^.L
\
.06 .04 .05 .04 .04
.147 .557 .270 .155 .236
± ± ± ± ±
.014 .028 .013 .006 .009
.037 .264 .106 .098 .104
± ± ± ± ±
.002 .006 .003 .005 .003
.57 .67 .64 .57 .60
± .03 ± .03 ± .02 ±.01 ± .01
.161 .208 .185 .136 .156
± ± ± ± ±
.015a .0196 .013c .008 .009c
.062 .144 .115 .081 .093
± ± ± ± ±
.004 .010 .007 .002 .003
.15 .45 .60 .23 .83
± ± ± ± ±
.01 .02 .03 .03 .03
MR MS M W Is
14.1 16.4 14.7 9.0 13.3
± .7 ± 1.2 ± .8 ± .5 ± .6
11.2 2.0 8.3 8.8 8.4
± ± ± ± ±
.9b .3 .5b .5 Ab
3.1 3.6 3.3 20.6 8.0
± ± ± ± ±
.la .3 .2a 1.2 .5a
1.40 1.47 1.43 .91 1.29
MR MS M W Ut
32.0 23.5 26.4 11.1 16.7
+ + ± ± ±
24.3 7.8 13.6 10.4 11.5
± ± ± ± ±
.9 .5 .5 .6 .5
.3 .0 .1 26.7 17.2
± + + ± ±
.la .0 ..Oa 1.3 .9a
.7 .7 .5 .6 .5
± + ± ± ±
Mean-per- hen a m o u n t s of c o m p o n e n t s (mg) /r
MR MS M W Is
56 29 85 20 105
± ± + ± ±
4 3 6 3 5
45 3 47 19 66
MR MS M W Ut
94 128 222 167 389
± 3 ± 5 ± 7 ±10 ±14
71 42 113 154 267
± ± ± ± ±
Ab 0 4b 3 4b
± 3 ± 3 ± 4 ± 8 ± 10
.1.
. _\
12 6 19 42 62
± ± ± ± ±
la 1 2a 4 4a
5.71 2.57 8.38 2.08 10.36
± .45 ±.15 ±.53 ±.31 ±.56
.61 .96 1.57 .33 1.90
± ± ± ± ±
.07 .05 .10 .04 .11
1 0 1 398 399
± ± ± ± ±
0a 0 Oa 22 22
1.68 3.72 5.40 8.63 14.25
±.11 ± .26 ± .28 ± .44 ± .68
.49 1.18 1.64 2.08 3.85
± ± ± ± ±
.05a .12b .17c .18 .34c
.19 .78 .97 1.23 2.20
± .02 ± .07 ± .08 ±.06 ±.13
Mean ± SE of 18 hens, except where indicated with letters: a = 17 hens; b = 16 hens-, c = 15 hens. 2 Tissue fractions: MR = mucosal residue; MS = mucosal supernatant; M = total mucosa; W = wall of the segment; Is = total isthmus; Ut = total uterus. Net lipid*: The chloroform-phase soluble fraction of crude lipid extracts. CollagejfjEstimated by multiplying the hydroxyproline amounts by a factor of 7.46 (Neuman and Logan, 1950). >
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
removal of the lipids. Since Hyp is a characteristic and exclusive component of collagen, levels of which are routinely estimated by assaying the tissues for Hyp (Eastoe and Courts, 1963), the data from the Hyp assay were used to obtain an indication of the amounts of collagen in respective tissues. Comparison of the overall levels of these components in isthmian vs uterine tissues (Table 3) shows that hexosamines and the two mineral components assayed, calcium and magnesium, had higher concentrations in isthmus, while collagen had higher levels in uterus. Within both these segments, collagen was found to have highest levels in walls, hexosamines, calcium and
scale TLC of several aliquots (50 to 100 mg) of chloroform-phase lipids yielded substantial amounts of the above-described lipid classes with the exception of the starting zone. This zone yielded virtually no amounts of chloroform-soluble lipids. Adding methanol and water to the extractant produced recoveries approaching 10% of the total amounts of aliquots subjected to the TLC. Phosphorus levels in this extract were 2.0 to 2.4%, indicating that phospholipids may be its major constituents.
640
CIPERA
Hexosamines, like calcium, had higher levels
in isthmian t h a n in uterine mucosae, with highest levels in each case being in t h e MS fraction of t h e m u c o s a e (Table 3). This a p p a r e n t relationship b e t w e e n hexosamines, which are t h e major c o n s t i t u e n t s of t h e m u c o p o l y s a c charides, and of calcium could be of distinct interest, because of t h e often postulated association b e t w e e n t h e acidic m u c o p o l y s a c charides and t h e calcium metabolism (Simkiss and Taylor, 1 9 7 1 ) , particularly t h e mobilization of calcium for s u b s e q u e n t calcification processes. Levels of collagen in t h e W fractions of isthmus and u t e r u s (Table 3) are intermediary b e t w e e n those in vertebrate skins and muscles ( N e u m a n and Logan, 1 9 5 0 ) , confirming t h e obviously integumental character of a major p o r t i o n of these tissue fractions. T h e reason for uterine walls having higher levels of collagen t h a n those from isthmuses is n o t clear. T h e relatively higher levels of collagen in isthmus mucosae, when c o m p a r e d t o t h o s e from uteri, m a y have been caused b y t h e presence of fragments of isthmus walls which b r o k e up during t h e tissue fractionation and w o u l d n o t b e removed from the mucosae. Changes in t h e levels of t h e above-discussed constituents of oviducal tissues with t h e passage of t h e egg t h r o u g h t h e h e n ' s oviduct are s h o w n
TABLE 4. Effect of the passage of the egg on the composition of oviducal segments associated with shell formation Levels of components (percent of dry tissue)^ Stage of shell formation 1
N
Crude
Lipid extracts Net 3
Collagen 4
Hexosamines
Calcium
Magnesium
± .03 ±.03 + .08 ±.03 ± .08
.233 .205 .211 .260 .260
.096 + .095 ± .109 ± .109 ± .110+
.006 .004 .006 .006 .006
± .04 ± .03 ± .01 ± .02 ± .02
.170 + .145 + .120 + .148+ .212 ±
.091 .101 .083 .089 .108
.011 .006 .001 .002 .008
- (Isthmus) A B C D E
12.2 16.7 13.7 12.2 12.2
± ± ± ± ±
.8 .7 .9 .8 3.0
A B C D E
16.5 15.6 16.4 17.3 17.4
± ± ± ± ±
.9 1.1 .8 1.0 2.2
7.6 ± 9.5+ 8.5 ± 8.0 + 8.4 ±
.la .1 2.0a .8 1.4
7.3 ± 7.6 ± 8.8+ 8.9 ± 6.6 ±
1.2 1.6 .7 .8 .8
1.36 1.03 1.21 1.37 1.44
± .008 ± .008 + .026 + .023 + .006
(Uterus) •
2 4
10.4 10.9 12.3 12.2 11.9
± ± ± ± ±
.9 1.0 .8 1.4 1.7
14.0 14.4 19.2 20.4 17.0
± .8 ± 2.2 ± .9 ± .7 ±4.4a
.56 .59 .63 .58 .61
.015 .011 .009« .012a .033a
+ ± ± ± ±
For definitions of stages of shell formation see Table 1. Mean ± SE when followed by letter a, the mean was obtained from N - 1 samples. Net lipids: The chloroform-phase soluble fraction of crude lipid extracts.
Collagen was estimated by multiplying the hydroxyproline amounts by a factor of 7.46 (Neuman and Logan, 1950).
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
magnesium in MS, and lipids in MR fractions. Total a m o u n t s of calcium in b o t h shellforming segments of h e n ' s oviduct were f o u n d t o be very low, in each case a few milligrams (Table 3). This agrees with t h e classical observat i o n of Buckner et al. ( 1 9 2 5 ) t h a t n e i t h e r of t h e t w o oviducal segments could possibly provide a n y degree of storage for t h e shell f o r m a t i o n , which requires a supply of over 2 g of calcium within a period of a b o u t 14 hr; t h u s , t h e calcium for shell f o r m a t i o n m u s t b e continuously supplied t o t h e oviducal tissues, most likely from b l o o d . Therefore, it is n o t surprising t o find t h a t in b o t h isthmus and u t e r u s , highest levels of calcium occurred in t h e MS fractions, t h e m o s t p r o b a b l e location of t h e secretory p r o d u c t s of oviducal tissues. Less e x p e c t e d was t h e finding t h a t levels of calcium were m u c h higher in isthmian t h a n in uterine m u c o s a e , in consequence of which t h e t o t a l a m o u n t s of calcium present in isthmus m u c i n s practically equalled t h e a m o u n t s of calcium in uterine mucosae. Magnesium, however, had s o m e w h a t lower concentrations in isthmian t h a n in uterine m u c o s a e , in consequence of which its t o t a l a m o u n t s in m u c o s a e of isthmus were m u c h lower t h a n in t h o s e of u t e r u s .
641
OVIDUCAL SEGMENTS INVOLVED WITH SHELL FORMATION
metabolites, presumably for the supply of carbon dioxide for the shell formation, or it may be associated with the increased need for lubricating the egg's passage. The overall levels of water, lipids, calcium, and magnesium observed in isthmus and uterus agree well with those reported in the literature (Table 5). Since no comparative data were available on the levels of hexosamines and hydroxyproline in isthmus and uterus of laying hens, levels of these compounds in uteri from immature pullets which received androgen and estrogen (Anastasiadis etal, 1955) are presented for comparative purposes. The levels of hexosamines agreed fairly well between immature and mature chickens, whereas the hydroxyproline levels were much lower in the uteri from hormone-treated chicks than in those from laying hens. This difference, however, appears to be due to the differential initial response to the hormonal stimulus, which would be expected to increase the levels of hexosamine-containing tissue constituents faster than those of the structural tissue constituents which contain hydroxyproline (see Cipera etal, 1965). ACKNOWLEDGMENTS The author wishes to acknowledge competent
TABLE 5. Comparison of the levels (percent of dry tissue) of components of isthmuses and uteri of laying hens obtained in this experiment with those reported elsewhere Oviducal component
Observed levels
Water Calcium Magnesium Lipids Hexosamines Hydroxyproline
397
Water Calcium Magnesium Lipids Hexosamines Hydroxyproline
495
.236 .104 8.37 1.29 1.06
.156 .093 11.54 .60 2.30
Levels reported in references no.
417
- (Isthmus) — 335 359
182 9.5
495
360
259 076
.263 .102
8.6
— (Uterus) 443 590
.(099
13.8
11.0
References: 1) Buckner et al. (1925), 2) Brown and Jackson (I960), 3) Cecil et al. (1970), 4) Yu and Marquardt (1972), 5) Schraer and Schraer (1965), 6) Misra and Kemeny (1964), and 7) Anastasiadis et al. (1955). 2 These data are not of laying hens; they were obtained from 12-week-old pullets receiving androgen and estrogen. The oviducti were sectioned into magni, uteri, and vaginas; isthmuses were obviously not developed sufficiently to permit their separation. Levels in vaginas were: hexosamines .88%, hydroxyproline 1.03%.
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
in Table 4. Although the data are from a relatively small number of hens, several trends are indicated. Levels of lipids increased in isthmuses and uteri as the egg passed through the respective segment. Levels of uterine collagen were highest during stages C and D, when uterine tissue was stretched around the egg. Calcium levels in isthmus were lowest during stage B and in uterus during stage C, which would correspond to the periods of maximum calcium demands in the respective segment; magnesium did not show this trend. All these changes are in agreement with the requirements that shell-formation processes would be expected to impose on the oviducal tissues. Thus, the evident increase in collagen levels of uterus when it was stretched around the egg for an extended period of time coincides with the period of stress imposed on the uterine wall by which the relative amount of the non-structural vs the structural (collagenous) components of the wall would be expected to decrease. Calcium changes appear to reflect on the magnitude of the strain imposed on the hen's metabolic supply of calcium during the shell forming stage. The changing level of lipids, however, associated with the passage of the egg may be indicative of an increasing supply of
642
CIPERA
assistance of L. Carter in the isolation of tissue fractions, of J. Sykora in assaying them and in the tabulation of the results, and helpful suggestions of R.M.G. Hamilton in reviewing the manuscript. REFERENCES
Downloaded from http://ps.oxfordjournals.org/ at Bibliothekssystem der Universitaet Giessen on May 17, 2015
Allen, R.J.L., 1940. The estimation of phosphorus. Biochem. J. 34:858-865. Anastasiadis, P. A., W. A. Maw, and R. H. Common, 1955. The hexosamine content of certain tissues of the sexually immature pullet and some effects thereon of gonadal hormones. Can. J. Biochem. Physiol. 33:627-637. Bligh, E. G., and W. J. Dyer, 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. Brown, W. O., and N. Jackson, 1960. Composition of the normal and hormone-stimulated oviduct of the common fowl. Poultry Sci. 39:602-611. Buckner, G. D., J. H. Martin, and A. M. Peter, 1925. Chemical studies of the oviduct of the hen. Amer. J. Physiol. 71:349-354. Cecil, H. C , J. Bitman, and C. S. Shaffner, 1969. Oviducal glycogen content of laying and nonlaying hens and of estradiol stimulated pullets. Proc. Soc. Exp. Biol. 131:164-167. Cecil, H. C , J. Bitman, and C. S. Shaffner, 1970. Oviducal water, electrolytes and nucleic acids of laying and non-laying hens and of estradiol stimulated pullets. Poultry Sci. 49:467—475. Christensen, H. N., and T. R. Riggs, 1953. Amino acids and peptides in the oviduct of the laying hen. Proc. Soc. Exp. Biol. 83:697-698. Cipera, J. D., B. B. Migicovsky, and L. F. Belanger, 1960. Changes in hexosamine and acetone extractable contents of epiphyseal cartilage of rachitic chicks following administration of vitamin D 3 . Can. J. Biochem. Physiol. 3 8 : 8 0 7 811. Cipera, J. D., B. B. Migicovsky, and L. F. Belanger, 1965. Effect of norethadrolone on selected connective tissues. Can. J. Biochem. 43:1235— 1241. Common, R. H., 1938. Observations on the mineral metabolism of pullets. J. Agr. Sci. 28:347-366. Copp, D. H., 1963. Simple and precise micromethod for EDTA titration of calcium. J. Lab. Clin. Med. 61:1029-1037. Eastoe, J. E., and A. Courts, 1963. Practical analytical methods for connective tissue proteins. Publ. E. & F. N. Spon Ltd., London. Edwards, N. A., 1977. The secretion of glucose into the egg of Gallus domesticus and observations on the uterine secretion of cations. Brit. Poultry Sci. 18:641-649. Folch, J„ M. Lees, and G.H.S. Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226:497— 509. Katzukura, Y., and H. Tamate, 1971. Ca mobilization
at each location of egg in the oviduct segments of the laying hens. Tohoku J. Agr. Res. 22:219— 227. Malins, D. C , and H. K. Mangold, 1960. Analysis of complex lipid mixtures by thin-layer chromatography and complementary methods. J. Amer. Oil Chem. Soc. 37:576-579. Misra, M. S., and A. Kemeny, 1964. Studies on the oviduct and serum of fowls. 1. Oxygen uptake, alkaline phosphatase activity, concentration of phosphorus, calcium and magnesium in adult Hungarian yellow hens. Acta Vet. Acad. Sci. Hungary 14:389-397. Mongin, P., and N. W. Carter, 1977. Studies on the avian shell gland during egg formation: Aqueous and electrolytic composition of the mucosa. Brit. Poultry Sci. 1 8 : 3 3 9 - 3 5 1 . Mongin, P., and N. W. Carter, 1978. Studies on the avian shell gland during egg formation: Mucosal intracellular pH. Brit. Poultry Sci. 19:93-96. Moynihan, J. B., and N. A. Edwards, 1975. Electrolyte secretion by the magnum of the domestic fowl during eggshell calcification. Comp. Biochem. Physiol. 50-A:743-745. Neuman, R. E., and M. A. Logan, 1950. The determination of collagen and elastin in tissues. J. Biol. Chem. 186:549-556. Roland, D. A., Sr., 1977. The extent of uncollected eggs due to inadequate shell. Poultry Sci. 56:1517-1521. Schenck, E. G., 1932. Uber die Bildung der Proteine des Eies im Ovar und Eileiter des Huhnes. Z. Physiol. Chem. 211:153-160. Schraer, R., and H. Schraer, 1965. Changes in metal distribution of the avian oviduct during the ovulation cycle. Proc. Soc. Exp. Biol. 119:939— 942. Simkiss, K., and T. G. Taylor, 1971. Shell formation. Page 1331 in Physiology and biochemistry of the domestic fowl. Vol. 3. D. J. Bell and B. M. Freeman, ed. Academic Press, London, New York. Smith, A. H., G. N. Hoover, J. O. Nordstrom, and C. M. Winget, 1957. Quantitative changes in the hen's oviduct associated with egg formation. Poultry Sci. 36:353-357. Snapir, N., and M. Perek, 1970. Distribution of Ca, carbonic anhydrase and alkaline phosphatase activities in the uterus and isthmus of young and old White Leghorn hens. Poultry Sci. 49:1526-1531. Taylor, T. G., and F. Hertelendy, 1960. Parallel distribution of calcium and citric acid in the oviduct of the hen. Nature 187:244-245. Yu, J.Y.L., and R. R. Marquardt, 1971. Effects of estrogen and progesterone on major cellular components in chicken oviducts. Can. J. Biochem. 49:348-356. Yu, J.Y.L., and R. R. Marquardt, 1972. A comparative study on the changes in cellular components in each region of the domestic fowl oviduct during a reproductive cycle. Can. J. Physiol. Pharmacol. 50:689-696.