Development, Cellular Growth, and Functon of the Avian Ovduct

BIOLOGY OF REPRODUCTION 8, 283-298 (1973)

Studies on the Magnum During a Reproductive Cycle of the Domestic Fowl (Gallus Domesticus) Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021

JOHN Y.-L. YU ANI) R. R. MARQUARDT Department of Animal Science, University of Manitoba, Winnipeg, Manitoba Canada

Received December 29, 1971; accepted October 24, 1972

The biochemical composition and histological patterns of the magnum portion of the domestic fowl (Gallus domesticus) oviduct during the developing, laying, and molting (regressing) stages of a reproductive cycle were compared. Growth and development of the magnum are primarily associated with hyperplasia (increase in total DNA) and to a lesser extent with cellular hypertrophy (increase in ratios of dry matter/DNA). A continuous hyperplasia occurs in the magnum during the laying stage; changes in cellular hypertrophy, however, are not observed. The pattern during regression of the magnum in the molting stage is essentially a reversal of that of the developing oviduct. Histological observations identify the characteristic changes as being differentiation and formation, secretion, and involution of tubular gland cells, respectively, for the developing, laying, and regressing oviducts. The ratios of RNA/DNA, protein/DNA, and soluble protein/DNA increase, reach maximum, and decrease during the developing, laying, and molting stages, respectively. The ratios of insoluble protein/DNA and lipids/DNA remain relatively constant throughout the whole reproductive cycle. The secretory egg white proteins (ovalbumin and conalbumin) increase very rapidly during the developing stage and decrease in a similar manner during molt. Conalbumin is present in the magnum and in the blood of the female chicken at all stages of the reproductive cycle. Ovalbumin, in contrast, is not located in the very small oviduct. It appears shortly after tubular gland formation. There is a quantitative shift of A2- to Al-ovalbumin during the developing stage which is reversed during regression of the oviduct.

INTRODUCTION bandov, 1956; Sturkie, 1965). The oviduct It is well known that the tremendous attains its maximum size during the laying change in the size of the avian oviduct stage which lasts approximately 12 months. during various reproductive stages is asso- Thereafter, the hen goes into a resting pe- ciated with its physiological performance riod (molt) for about 2 months during and that this process is regulated by sex which there is regression of the oviduct hormones (Dorfman and Dorfman 1948; (Romanoff and Romanoff, 1949; Sturkie, Brant and Nalbandov, 1956; Sturkie, 1965; 1965). The developing, laying, and molting O’Malley et at, 1969; Oka and Schimke, sages thus constitute a reproductive cycle 1969a,b; Yu et at., 1971). In the domestic of the egg-producing fowl. egg-producing fowl (Galtus domesticus), The histology of the domestice fowl ovi- rapid oviduct development occurs about duct has been investigated (Richardson, 2 months prior to the onset of laying 1935; Brant and Nalbandov, 1956). (Hafex and Kamar, 1955; Brant and Nal- Studies on compositional changes of fowl

283

oviduct at certain stage(s) in a reproduc- weighed, and placed in a polyethylene bag which tive cycle have recently received atten- was immediately dropped into an ethanol-dry ice tion (Oades and Brown, 1965; Chechotkin, medium. Frozen magna were pooled into duplicate samples on an oviduct weight basis within the 1966; Belnave and Squance, 1968; Cecil reproductive stage, reweighed, and lyophilized. and Bitman, 1970; Yu et at., 1971). There The dried samples were ground and stored in have been, however, no studies on the se- a dessicator at -15#{176}C until required. Egg white quential changes in overall patterns of cel- from freshly laid eggs was lyophilized and stored

lular growth and development of the avian at -15#{176}C. Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 oviduct during a reproductive cycle. This Chemicals. Calf thymus DNA (type V), yeast RNA (grade VI), orcinol monohydrate, ovomucoid investigation was, therefore, carried out in (type II-o), conalbumin (type III), and globulins an attempt to illustrate systematically the (egg white) were obtained from Sigma. Oval- biochemical and morphological patterns of humin (two times crystallized) was from the magnum (albumen1secreting region) Worthington Biochemical Corporation. Acryl- of the domestic fowl oviduct during a re- amide, N, N’-methylene bisacrylamide and N, N, N’, N’-tetramethyl ethylenedianiine (TEMED) productive cycle. Biochemical determina- were obtained from Canal Industrial Corporation. tions include the alterations in the levels Complete Freund’s adjuvant and Noble agar were of major cellular components: DNA, RNA, purchased from Difco Laboratories. Diphenyl- lipids, and various protein and nitrogen amine was from Matheson, Coleman & Bell. fractions including secretory egg white Chemical analysis. A single analysis on each protein patterns. Histological observations of the duplicate pooled samples of magna was carried out. Nucleic acids were extracted by the include the formation, secretion, and in- method of Schneider (1945) with perchloric acid. volution of tubular gland cells as well as DNA was measured by the modified diphenyl- the changes in cell size. The nature of amine reaction (Ciles and Myers, 1965) with calf growth and development of the domestic thymus DNA as standard. RNA was assayed by fowl oviduct, in association with its func- the modified orcinol method (Ceriotti, 1955) with tion, during a reproductive cycle is il- yeast RNA as standard. Lipids were extracted with a chloroform-methanol mixture and were esti- lustrated at the cellular level. mated by weighing (Folch et al., 1957). MATERIALS AND METHODS Fractionation of protein and nitrogen components. The nitrogenous components in oviduct tis- Source and treatment of birds. Single Comb sue have been classified into protein and non- White Leghorn commercial hybrids (Dekalb protein nitrogen with each subfraction being com- strain) were obtained from the University poultry posed of a soluble and insoluble portion (Dicker- flock and a commercial hatchery. The developing son and Widdowson, 1960; Munro and Fleck, oviducts were obtained from 3- to 5-month-old 1969). The nitrogen (N) content of the above chickens. Oviducts from 5-, 8-, and 14-month-old fractions was determined in this study by the (first-laying cycle) and from 22-month-old (sec- following procedure. An aliquot of the lyophiuized ond-laying cycle) hens were selected for the laying tissue was homogenized at 0#{176}Cin a Willem Poly- stage. Oviduct regression (molting) in laying hens tron (Brinkmann) in 40 volumes of 100 mM KC1, was induced by controlling the feeding and water- 50 mM sodium phosphate buffer (pH 7.0) and ing schedule (Smith et al., 1957). The molting centrifuged at 50,000 x g for 30 min. The protein hens were sacrificed at weekly intervals during in the supernatant fraction (soluble N) was pre- the 3-month experimental period. A summary of cipitated with 3 volumes of 10% trichloroacetic the number of birds and the oviduct weights for acid (TCA) and was centrifuged at 50,000 x g the various reproductive stages is presented in for 20 mm. The total N (extract), soluble N Table 1. (50,000 x g supernatant), insoluble N (50,000 x Sample preparation and storage. After decapita- g precipitate) and soluble nonprotein N (50,000 tion of the bird, the oviduct was removed and x g TCA supernatant) were determined by the weighed. The magnum region was then sectioned, micro-Kjeldahl method (Horwitz, 1965). Insolu- 2 The nomenclature of egg white proteins in ble nonprotein N (nucleic acid N) was calculated the present study is according to Romanoff and from the nucleic acid content which was deter- Romanoff (1949) and Warner (1954). Egg albu- mined as described previously. The soluble and men refers collectively to egg white proteins (oval- insoluble protein nitrogen was obtained by dif- bumin, conalbumin, ovomucoid, etc). ference as shown below; soluble protein N AViAN 0VIDUCr GROWTh AND DEVELOPMENT 285

TABLE 1

CHANGES IN THE WEIGHT OF THE OvIDUcT AND THE MAGNUM IN

CHICKENS DURING A REPRODUCTIVE CYCLE

Average Average l)ry matter Average wet wet content body Num- weight of weight of of weight Reproductive her of Age oviductb magnumb Inagnumb of hird&’ stage#{176} birds (month) (g) (g) (%) (g) Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021

I)eveloping (I)) Dl 12 4-5 2.8 ± 0.6 0.5 ± 0.1 11.3 ± 0.4 1192 ± 78 D2 10 4-5 5.1 ± 0.9 1.0 ± 0.2 12.0 ± 0.2 1178 ± 97 D3 13 4-5 10.2 ± 1.6 2.5 ± 0.4 17.4 ± 0.8 1261 ± 116 1)4 6 4-S 19.2 ± 1.6 7.0 ± 1.1 23.3 ± 1.9 1376 ± 153 D5 5 4-5 26.4 ± 1.6 11.2 ± 0.9 24.3 ± 1.8 1446 ± 149

Laying (L) Li 6 5 37.2 ± 6.1 16.6 ± 2.8 26.1 ± 0.7 1496 ± 115 L2 6 8 41.0 ± 3.1 18.6 ± 1.7 24.7 ± 0.0 1708 ± 138 L3 6 14 50.8 ± 7.7 24.3 ± 3.4 24.9 ± 2.5 2081 ± 180 L4 6 22 51.0 ± 8.0 24.6 ± 5.9 24.5 ± 0.4 2114 ± 234

Molting (M) Ml 4 14-16 38.2 ± 4.2 18.0 ± 3.3 24.8 ± 1.1 1734 ± 189 M2 4 14-16 29.2 ± 2.2 11.6 ± 1.3 24.2 ± 0.6 1575 ± 145 M3 5 14-16 20.0 ± 2.3 7.3 ± 0.9 21.3 ± 1.8 1486 ± 135 M4 10 14-16 10.0 ± 1.7 2.7 ± 0.5 18.3 ± 0.1 1556 ± 167 M5 11 14-16 4.8 ± 0.9 1.2 ± 0.2 17.1 ± 1.4 1157 ± 137 M6 12 14-16 2.6 ± 0.5 0.6 ± 0.1 19.2 ± 0.4 1078 ± 113

“ The designations of D’s and M’s refer to oviducts of different weight within the developing and molting stages, respectively; while the L’s refer to oviduets from laying hens of varying age. Mean ± standard deviation. See “Materials and Methods” for further experimental details.

soluble N - soluble nonprotein N; insoluble pro- eighth week after the first injection and was stored tein N = insoluble N - insoluble nonprotein N. at -15#{176}C until used. A modified two-dimensional Disk electrophoresis. Polyaciylamide gel electro- gel diffusion (Ouchterlony method) as described phoresis was performed by using a resolving gel by Campbell et al. (1964) was used. The agar in a continuous Tris-borate-EDTA buffer system plates contained 1.0% agar in 5 mM EDTA, 50 (iH 8.28). The extract of lyophilized tissue (in mM Tris (pH 8.2 at 20#{176}C), 1.5% NaCl, and 40 volumes of buffer), as described’ in “Fractiona- 0.001% trypan blue. The reaction was carried out tion of Protein and Nitrogen Components,” was for 3 days at 20#{176}C. diluted with 4 volumes of 20% sucrose. A single Histological techniques. For histological obser- polyacrylamide gel was treated with 50 cl of this vations, portions of magnum were fixed in 4% preparation and the samples were run for 3.5 glutaraldehyde buffered with 10 mM phosphate h at room temperature (23#{176}± 2#{176}C)with a con- buffer, pH 7.2 (Sabatini et al, 1962) for 36 h stant current of 2 mA/column. The proteins were at room temperature. The tissues were embedded stained with amido black and were scanned at 660 in paraffin after dehydration in a series of graded nm in a recording spectrophotometer. cellosolve and clearing in benzene. Sections of Immunological procedures. A commercial prepa- 7 cm thickness were stained with Harris hema- ration of ovalbumin which also contained conal- toxylin and eosin (Culling, 1963). Photomicro- bummn, ovomucoid, and globulin was emulsified graphs were taken on high contrast copy film with Freund’s complete adjuvant and was injected (Kodak). (20 mg/injection) subcutaneously at weekly intervals for 5 weeks into two rabbits. Immune serum RESULTS was obtained from blood drawn on the sixth and tmlnstructions for the Polyanalyst: An Analyti- Preliminary Observations cal Temperature-Regulated Electrophoresis Ap- paratus (1970). Buehler Instruments Division, Nu- The results of a preliminary study with clear-Chicago Corporation, Fort Lee, New Jersey. 50 chickens, 3-5 months of age, showed 286 YU AND MARQUARDT

that there was not always a direct relation- In an initial study it was established that ship between oviduct size and age. Previ- oviducts (average weight of 5 g) from in- ous studies, however, have shown that duced molt and natural molt birds had sim- there was a parallel between the weight ilar biochemical compositions (levels of of oviduct and its state of physiological DNA, RNA, lipids, and various protein development (Yu et al., 1971). On the fractions) and histological patterns. In- basis of these observations, oviduct weight duced molt birds were therefore used in was used as an index of the physiological the following study to facilitate oviduct Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 status of the oviduct for the developing collection. and molting chickens. Therefore, in the current study, these oviducts were pooled Oviduct Weight, Magnum Weight, and and analyzed by weight intervals (Table Dry Matter Content 1). There was, however, a good association

between oviduct weight and age of the The average wet weight of oviducts in laying hen, especially if there were consid- each weight-group ranged from 2.8 to 27

erable age differences. Oviducts for the g, 37 to 51 g and 38 to 2.6 g, for the devel- laying state were thus analyzed on both oping, laying, and molting stages, respec- an age and a weight basis (Table 1). tively (Table 1). The magnum weight in-

TABLE 2

CHANGES IN THE FORMATION, SECRETION, AND INVOLUTION OF TUBULAR GLANDS

AND IN THE CELL SIZE OF THE MUCOSA LAYER OF THE MAGNUM PORTION OF CHICKEN OVIDUCT DURING VARIOUS REPRODUCTIVE STAGES

Albumen Relative Rela- Oviduct granuls Albumen concentration tive Reproductive weight of tubular secretion of cell cell stages (mg) gland6 in lumenb nuclei’ size”

Developing (D)

DX 18 No tubular None - glands

DY 410 No tubular None - - glands

DZ 825 Tubular glands None - - formning Dl 2,767 + None 100 1.0 D3 10,184 +++ +to+++ 58 1.7 D4 19,145 ++++ ++++ 38 2.6

Laying (L) L3 30,746 ++++ +++++ 33 3.0

Molting (M) M3 19,982 +++ +++ 62 1.6 MX 14,715 ++ ++ 88 1.1 M4 10,009 + + 120 0.8 M6 2,563 ± None 123 0.8

DX represent oviducts from 1-week-old birds while DY and DZ are oviducts from 12- to 14-week- old birds. MX represents oviducts whose weights are intermediate to those of M3 and M4. Chemical analysis of these oviducts was not performed. See Table 1 for remaining tissue designations. The symbol (+) indicates the approximate relative extent for each parameter. ‘The numbers of cell nuclei from representative areas of the mucosal layer of the magnum were counted at a constant mangification and were averaged. d Relative size of cells from the mucosal layer as determined from the reciprocal of the relative concentration of cell numbers. AVIAN OVIDUCr GROWTh AND DEVELOPMENT 287 creased from 0.5 g for Dl to 25 g for L3 and decreased to 0.6 g for M6 (see Tables 0

1 and 2 for tissue designations). There > were increasing, relatively constant, and 0 0 decreasing proportions of magnum weight relative to the whole oviduct weight during the developing, laying, and molting stages, E C

0’ Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 respectively (Fig. 1). The magnum at the 0 early developing stage contained the lowest level of dry matter (Table 1). As 0 C growth and development progressed, the 0

0 dry matter content increased with maxi- 0. 0 mum values being obtained in the laying 0 hen. These levels decreased during the “SO 0 20 30 40505040 30 20 10

molting stage. I II II I Developing Laying Molting

Fresh Weight of Oviduct , gm Histological Observations Fic. 1. Differential gorwth of the magnum The formation, secretion, and involution weight relative to oviduct weight during a repro- of the tubular glands and the relative ductive cycle (developing, laying, and molting change in cell size of the magnum during stages). Each dot represents the mean from 4 a reproductive cycle are illustrated in Fig. to 13 oviducts. See Table 1 for further details. 2 and observations made are summarized

in Table 2. In the very small developing The relative concentration of cell nuclei oviduct (oviduct weight of less than 1 g), per unit of tissue (an index of cell size) stroma cells with elongated nuclei occu- varied considerably. The highest concen- pied most of the mucosa (Fig. 2, No. 1). trations were obtained during the early de- Tublular gland formation from the epi- velopmg (Dl and D2) and late molting thelial surface (cell differentiation) was (M4 to M6) stages, while the lowest con- first observed in oviducts weighing more centrations were observed during the lay- than one gram (Fig. 2, No. 2). Albumen ing stage (Table 2). secretion into the lumen of tubular glands was observed initially in oviducts weighing DNA and RNA approximately 10 g. At this stage of development (Fig. 2. No. 3), the tubular glands The levels of total DNA increased con- occupied most of the mucosal region simi- siderably during the developing stage, lar to those found in the laying stage (Fig. reached a maximum in the laying hen, and 2, No. 4), except that the tubular glands gradually decreased during the molting of the developing magnum were smaller. stage (Table 3). The concentration of The most characteristic features observed DNA, expressed as a percent of dry matter, during molt were involution of the tubular decreased during the developing stage, glands, suspension of albumen secretion reached the lowest level for the laying hen, and the shedding of cells (Fig. 2, Nos. and increased during the molting stage. 5-8). For the late molting stage (M4 to Magnum cell size (dry matter/DNA ratio) M6), the involuted tubular glands co- increased, attained a maximum, and de- existed with an increasing number of creased during the developing, laying, and stroma cells (Fig. 2, Nos. 7 and 8). Al- molting stages, respectively. Changes in bumen, although present in reduced total RNA and in the RNA/DNA ratios amount, was discernable in the small molt- reflected the growth patterns of magnum ing oviduct (M4 and M5). dry matter and dry matter/DNA ratios. 288 Y!J AND MARQUARDT Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021

u.- ‘:is

- ol

‘

#{149}‘ 4 AVIAN OVIDUCT cnowm AND DEVELOPMENT 289

TABLE 3

COMPARATIVE LEVELS OF DRY MArI’ER, DNA, AND RNA IN THE MAGNUM PORTION OF CHICKEN OVIDUCT DURING A REPRODUCTIVE CYCLE

1)NA RNA

Dry Concentration Concentration Reproductive matter Total (% of dry Total (% of dry stage’ (mg) (mg) matter)b (mg) matter)’ Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 Developing (1)) Dl 59 1.3 2.2 ± 0.0 1.7 2.9 ± 0.1 D2 124 2.3 2.0 ± 0.3 3.3 2.6 ± 0.2 D3 439 5.8 1.3 ± 0.1 10.7 2.5 ± 0.2 D4 1,642 13.5 0.8 ± 0.0 37.3 2.3 ± 0.1 D5 2,706 19.0 0.7 ± 0.1 33.3 2.1 ± 0.1

Laying (L) Li 4,323 28.5 0.7 ± 0.0 88.4 2.1 ± 0.1 L2 4,596 30.8 0.7 ±0.0 96.6 2.1 ±0.1 L3 6,081 40.6 0.7 ±0.0 124.7 2.1 ±0.1 L4 6,029 38.9 0.7 ± 0.0 126.7 2.1 ± 0.0

Molting (M) Ml 4,467 34.8 0.8 ± 0.0 93.7 2.1 ± 0.1 M2 2,804 30.3 1.1 ± 0.0 41.9 1.6 ± 0.1 M3 1,561 23.5 1.5 ± 0.1 23.9 1.5 ± 0.1 M4 491 12.4 2.3 ± 0.2 6.9 1.4 ± 0.1 M5 187 5.6 3.0 ± 0.0 2.8 1.5 ± 0.0 M6 123 3.7 3.0 ± 0.1 1.7 1.4 ± 0.0

The designations used for the reproductive stage are indicated in Table 1. “Mean ± standard deviation. See “Materials and Methods” for further experimental details.

The degree of change in cell numbers Soluble Protein and Specific Secretory (total DNA) was much greater than that Proteins of cell size (dry matter/DNA) during the As indicated in Table 5, the level of solu- Various reproductive stages (Fig. 3). ble protein, expressed as a % of dry matter,

changed considerably during a reproduc- Protein and Nitrogenous Components tive cycle. It increased approximately two- fold from the developing magnum (Dl) The level of total nitrogen (N), as a to the laying stage (Ll to L4), and de- % of dry matter, was highest in the late creased to a level lower than Dl at the developing (D4 and D5), the laying and end of molting (M5 and M6). A similar, the initial molting (Ml and M2) stages but more marked, alteration was observed (Table 4). A major protion of the total when the data was expressed on a cell unit N was protein N with nonprotein N levels basis (soluble protein/DNA). being less than 15% of the total. As mdi- Individual egg white proteins from the cated in Table 4, the proportions of these magnum were identified by comparing two fractions were affected by reproduc- their migration patterns with those of pure tive stage. proteins following polyacrylamide gel elec-

FIG. 2. Comparative histological changes in the mucosal layer of the magnum portion of the chicken oviduct during a reproductive cycle. Developing stage (No. 1, DY; No. 2, Dl; No. 3, D3); laying stage (No. 4, L3); molting stage (No. 5, M3; No. 6, MX; No. 7, M4; No. 8, M6). See Tables 1 and 2 and “results” for tissue designations and descriptions. All figures were magnified x340. 290 YU AND MARQUARDT

100 Dry Matter W DNA 90 0 Dry Mutter/DNA

60 Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 50

JO LI

D D2hIHiHHULD3 D4 D5 L1 L2 L3 L4 M1 N2 N3 N4 N5 N6 Reproductive Stoge

Fic. 3. Relative changes in cell numbers (total DNA) and cell size (ratios of dry matter/DNA) of the magnum portion of chicken oviduct during a reproductive cycle. D, developing; L, laying; and M, molting. See Tables 1 and 3 for further details.

TABLE 4

LEVELS OF VARIOUS NITROGEN FRACTIONS IN THE MAGNUM PORTION OF THE CHICKEN OVIDUCT DURING A REPRODUCTIVE CYCLEO

Prote in N Nonpro tein N

Total N Protein Soluble Reproductive (% of dry % of (% of dry % of NPN (% stages” matter)” total N’ matter)’ total N” of NPN)’ l)eveloping (I)) Dl 12.4 ± 0.1 85.6 ± 0.1 66.5 ± 0.8 14.4 ± 0.0 51.8 ± 1.5 1)2 12.2 ± 0.0 87.1 ± 1.2 66.2 ± 0.0 12.9 ± 1.3 51.8 ± 0.1 D3 12.7 ± 0.1 88.3 ± 0.1 69.9 ± 0.7 11.7 ± 0.1 58.3 ± 2.9 D4 13.8 ± 0.1 91.0 ± 0.3 78.2 ± 0.6 9.0 ± 0.2 58.8 ± 0.3 1)5 12.9 ± 0.3 91.9 ± 0.2 79.9 ± 1.8 8.1 ± 0.2 59.5 ± 2.8 Laying (L) Li 14.5 ± 0.0 91.0 ± 0.1 82.3 ± 0.0 9.0 ± 0.1 66.2 ± 1.4 L2 14.4 ± 0.1 91.0 ± 0.2 82.2 ± 0.6 9.0 ± 0.2 59.9 ± 2.4 L3 14.5 ± 0.1 91.1 ± 0.4 82.3 ± 0.4 8.9 ± 0.4 62.3 ± 2.1 L4 14.3 ± 0.3 92.0 ± 0.2 82.1 ± 1.7 8.0 ± 0.2 60.8 ± 0.7 Molting (M) Ml 14.1 ± 0.1 91.6 ± 0.1 80.6 ± 0.6 8.4 ± 0.1 58.5 ± 4.5 M2 13.5 ± 0.0 91.4 ± 0.2 76.9 ± 0.4 8.6 ± 0.1 61.0 ± 0.7 M3 12.8 ± 0.2 90.6 ± 0.3 72.6 ± 1.5 9.4 ± 0.1 58.1 ± 0.3 M4 12.2 ± 0.1 89.6 ± 0.2 68.6 ± 0.4 10.4 ± 0.2 53.2 ± 2.4 MS 12.6 ± 0.1 88.5 ± 0.3 71.1 ± 0.4 11.5 ± 0.3 46.7 ± 1.9 M6 12.2 ± 0.3 88.5 ± 0.4 67.3 ± 1.8 11.5 ± 0.4 48.3 ± 0.5

‘Total nitrogen (N) = protein N + nonprotein N. Protein N (PN) = soluble PN + insoluble NP.

Nonprotein N (NPN) = soluble NPN (amino acids and nucleotides) + insoluble NPN (nucleic acips). The designations used for the reproductive stage are indicated in Table 1. ‘Mean ± standard deviation. See “Materials and Methods” for further experimental details. AVIAN OVIDUCT GROWTh AND DEVELOPMENT 291

TABLE 5

LEVELS OF SOLUBLE PROTEIN AND EGG WHITE PROTEINS IN THE MAGNUM PORTION

OF THE CHICKEN OVIDUCT DURING A REPRODUCTIVE CYCLE

Soluble Ovalbumin Conalbumin Protein (% of (% of Reproductive (% of dry soluble soluble stageu matter)” protein)’ proteinY’

Developing (D) Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 Dl 33.9 ± 0.2 12.5 ± 0.8 6.9 ± 0.0 D2 33.6 ± 1.1 11.7 ± 2.1 8.7 ± 1.9 D3 44.8 ± 2.3 25.9 ± 0.1 12.1 ± 0.5 D4 57.4 ± 1.3 45.1 ± 1.3 11.8 ±2.3 D5 60.2 ± 1.0 47.8 ± 0.2 12.3 ± 1.9

Laying (L) Li 62.6 ± 0.2 49.0 ± 0.9 11.3 ± 2.1 L2 61.5 ± 0.2 47.6 ± 0.1 11.3 ± 0.8 L3 60.6 ± 2.3 49.5 ± 0.1 11.5 ± 0.2 L4 61.6 ± 0.1 48.6 ± 0.3 11.8 ± 1.0

Molting (M) Ml 61.3 ± 0.9 47.1 ± 0.4 12.2 ± 1.1 M2 54.4 ± 0.8 43.0 ± 1.8 12.0 ± 1.0 M3 43.1 ± 1.1 32.9 ± 2.0 11.8 ± 1.8 M4 27.5 ± 0.3 8.7 ± 0.6 8.1 ± 1.8 MS 23.0 ± 0.3 6.5 ± 0.5 7.1 ± 0.8 M6 24.2 ± 1.3 5.3 ± 1.1 7.7 ± 0.9

a The designations used for the reproductive stage are indicated in Table 1. Mean ± standard deviation. See “Materials and Methods” for further experimental details.

trophoresis (Fig. 4). Four major bands (Ml to M6) (Table 5). The pattern of were identified: three of which were oval- change for conalbumin, although similar bumins, the other being conalbumin. Ovo- to ovalbumin, was less pronounced. When mucoid, one of the other major secretory expressed on a cell unit basis, i.e., oval- egg white proteins, appeared originally bumin/DNA and conalbumin/DNA, simi- after the A3-ovalbumin band. It is not lar but more pronounced changes were visible in the electrophoretograms as the observed. band rapidly disappeared when stained An immunological double-diffusion tech- with amido black. As indicated in Fig. 4, nique was employed to specifically identify a shift from A2- to Al-ovalbumin occurred the appearance and disappearance of the during the developing stage and this pat- various secretory egg white proteins during tern was essentially reversed during the a reproductive cycle (Fig. 5). A maximum regression of the oviduct. of five distinct precipitin bands were ob- The quantity of ovalbumin and con- served. Four of the proteins were identified albumin in the magnum was estimated as ovalbumin, conalbumin, ovamucoid, and from densitometric scans using pure pro- globulins; the remaining one was not iden. teins as standards. The concentration of tified. Ovalbumin, which contained Al, A2, ovalbumin, as a percent of soluble protein, and A3 forms of the protein, yielded a sin- increased approximately fourfold during gle, well-defined precipitin band. The lay- the developing stage (Dl to D5), nig-hen magnum extract (L) and the white mained constant during the laying stage protein of eggs (E) laid from the same (Ll to L4); and then decreased approxi- hens yielded four precipitin bands (Plate mately eightfold during the molting stage I); three of which (ovalbumin, ovamucoid, 292 YU AND MABQUAB.DT

‘I Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021

1 V

p II V 4- +

-t -t EL AVIAN OVIDUCT GROWTh AND DEVELOPMENT 293

TABLE 6

CHANGES IN THE LEVELS OF INSOLUBLE PROTEIN AND LIPIDS IN THE MAGNUM PORTION OF THE CHICKEN OVIDUCT DURING A REPRODUCTIVE CYCLE

Insoluble Protein Lipids

Concentration Concentration Reproductive Total (% of dry Total (% of dry stage#{176} (mg) matter)” (mg) matter)” Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 Developing (D) Dl 19 32.5 ± 1.0 10 17.4 ± 0.2 D2 40 32.6 ± 2.0 22 18.1 ± 0.1 D3 110 25.2 ± 1.6 53 12.0 ± 1.0 D4 342 20.8 ± 0.6 144 8.7 ± 0.8 D5 533 19.7 ± 0.7 213 7.9 ± 0.1

Laying (L) Li 849 19.6 ± 0.2 359 8.3 ± 0.3 L2 948 20.6 ± 0.4 393 8.6 ± 0.3 L3 1321 21.7 ± 2.6 496 8.2 ± 0.9 L4 1237 20.5 ± 1.6 507 8.4 ± 0.5

Molting (M) Ml 851 19.1 ± 1.8 385 8.6 ± 0.2 M2 638 22.8 ±0.4 232 9.1 ±0.4 M3 461 29.5 ± 2.6 194 12.5 ± 0.2 M4 203 41.4 ± 0.4 88 18.0 ± 0.7 MS 90 48.1 ± 0.7 36 19.4 ± 0.4 M6 53 43.0 ± 0.4 24 19.9 ± 0.6

#{176}The designations used for the reproductive stage are indicated in Table 1. “Mean ± standard deviation. See “Materials and Methods” for further experimental details. and conalbumin) were identified on the those of the laying hen and the developing basis of forming a continuous, single pre- magnum (D3 to D5). Conalbumin, was cipitin band with the pure proteins. There the only egg white protein identified in were no globulin precipitin bands, presum- the magnum of the highly regressed ovi- ably owing to their low concentrations, duct (M6) (Plate III). It was also shown present in the laying-hen magnum extract that oviducts from 1-week-old chicks and the white protein of eggs laid from (average oviduct weight of 18 mg) con- the same hens. tamed an immunoreactive protein cor-

In the developing magnum (Dl) two responding to conalbumin (Plate III). In precipitin bands were present (Plate II); another experiment, it was shown that the one of these formed a continuous line with serum of the immature chick and of the pure ovalbumin, the other with conalbu- mature hen also contained an immunologi. mm. These two proteins along with a third cally reactive protein which was similar secretory protein were present in the D3 to conalbumin. Studies with the control magnum. The regressing magnum (M3 and serum (serum obtained from the rabbit M4) had precipitin patterns similar to prior to antigen injection) indicated that

Fic. 4. Comparison of electrophoretograms and the corresponding scans of egg white and the soluble protein from the magnum portion of the chicken oviduct during various reproductive stages. Proteins migrated from (-) to (+), Al, A2, and A3 denote different forms of ovalbumin; C, conalbumin. The numbers (1-8) represent various reproductive stages: developing (No. 1, Dl; No. 2, D3; No. 3, D4) laying (No. 4, L3), egg white (No. 5), and molting (No. 6, M2; No. 7, M3; No. 8, M5). See “Materials and Methods” and Table 1 for additional detail. 294 YU AND MARQUARDT Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021

FIG. 5. Two-dimensional immunodiffusion of specific egg white proteins in the magnum portion of chicken oviduct during various reproductive stages. The center wells contained anti-egg white sera (Plate I, 60 d; Plate II, 50 gil; and Plate III, 40 The side wells contained the following antigens: Plate I: (A) ovalbumin, 15 g; (C) conalbumin, 20 tg; (U) ovomucoid, 10 g; (G) egg white globulins, 24 g; (E) egg white, 40 g; (L) laying magnum soluble protein, 75 g. Plate II: (A) ovalbumin, 15 cg; (C) conalbumin, 20 g; (Dl and D3) developing magnum soluble proteins of 425 and 140 ,g, respectively; (M3, M4 and M5) molting magnum soluble proteins, 135, 355, and 300 g, respectively. Plate III: (A) ovalbumin, 10 g; (C) conalbumin, 20 cg; (M8) molting magnum soluble protein 500 g; (DX) 1000 g (DY) 450 cg; and (DZ) 450 g denote the magnum soluble proteins from the developing stage at oviduct weights of 18 mg (1 week old), of 410 mg, and of 825 mg (12-14 week old), respectively. See “Materials and Methods” for further experimental details. I’, II’, and III’ are pictorial representations of plates I, II and III, respectively. AVIAN OVIDUCT GROWTh AND DEVELOPMENT 295

there were no nonspecific reacting changes in the levels of various cellular antibodies. components, on a cell unit basis, from the magnum during a reproductive cycle is presented in Fig. 6. Insoluble Protein and Lipids DISCUSSION The change in the level of insoluble protein during various stages of the reproduc- The present study demonstrates that the tive cycle was opposite to that of soluble rate of growth of the magnum during de- Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 protein (Tables 5 and 6). The insoluble velopment is much greater than other por- protein, when expressed as a percent of tions of the oviduct and that the changes the magnum dry matter, decreased during in magnum size during this period are pri- the developing stage, remained constant marily associated with hyperplasia as during laying, and increased again during shown by increased amount of total DNA, molt. However, when expressed on a cell and to a much lesser extent with cellular unit basis (insoluble protein! DNA), it in- hypertrophy as shown by increased ratios creased during the developing stage, of dry matter/DNA. During the laying reached the highest level in the laying hens, stage, a considerable variation in magnum and decreased during the molting stage. size was observed depending on the age The patterns of change for lipids closely of the hen. The increase in magnum size paralleled those for insoluble protein (Table is mainly a result of continuing hyperplasia 6). rather than changes in cellular hyper- An overall summary of the relative trophy. The pattern during regression of the oviduct (molting stage) is essentially a reversal of the developing oviduct; both total DNA and ratios of dry matter/DNA decrease with the latter slightly pre- dominating. These biochemical findings are consistant with the histological alterations of the oviduct. Histological observations identified the characteristic changes of the magnum during the developing stage as 0’ 0 being the formation, proliferation, and sub- 0 sequent growth of cytoplasmic mass of the tubular gland cells. These cells, containing a, the secretion granules, attain their maximum size in the functionally active laying- hen magnum. During regression of the oviduct, there is a loss of cytoplasmic mass; as a result, the cells involute. The ratios of protein/DNA were observed to parallel those of RNA/DNA. This is consistent with a prerequisite of 5 66 4 3 2 I 9 Developing Laying Molting RNA for protein synthesis. The laying-hen

Dry Weight of Magnum , gm oviduct exhibits the greatest metabolic ac-

FIG. 6. Relative changes in the cellular levels of tivity as evidenced by the highest cellular various components in the magnum portion of the concentrations of RNA, protein, soluble chicken oviduct during a reproductive cycle (de- protein, and secretory egg white proteins. veloping, laying, and molting stages). , lipids! ‘1 The low ratios of cellular RNA and protein DNA; A, insoluble protein/DNA; #{149},RNA/DNA; including the soluble and egg white pro- U, protein/DNA; , soluble protein/DNA; D,t conalbumin/DNA; 0, ovalbumin/DNA. teins in the small developing and molting 296 YU AND MARQUARDT oviducts reflect that these tissues are in responds to the period in which the ratios a quiesent state and that their metabolic of RNA/ DNA and protein! DNA decrease activity is low. rapidly. Oviduct development and regression pat- Immunological studies of specific secre- terns observed in this study are nearly the tory proteins indicate that the pattern of same as those obtained following the injec- formation of these proteins are different. tion of chicks with female sex hormones Conalbumin can be detected in the mag-

(O’Malley et al., 1969; Oka and Schimke, num from all stages of the reproductive Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 1969,a,b; Yu et al., 1971). Administration cycle including those from 1-week-old fe- of estrogen stimulates oviduct development male chicks. It is also found in the blood predominantly with formation and subse- of both immature chicks and mature hens. quent growth of the tubular gland cells; These latter observations are consistent the hormone induces a marked increase with those reported by Marshall and in the synthesis of DNA, RNA, and specific Deutsch (1951) and Williams (1962). egg white proteins (ovalbumin, conalbu- They demonstrated that the serum of mm, and lysozyme). Withdrawal of the chickens contains a component (serum hormone results in a regression of the ovi- transferrin) which is immunologically duct with involution of tubular glands ac- identical to conalbumin (ovotransferrin). companied by decreases in synthesis of Conalbummn is also present in small RNA and the specific egg white proteins. amounts in all five portions of chicken Our observations that the RNA/ DNA ratio oviducts throughout the entire reproduc- in the laying-hen magnum is higher than tive cycle (Yu et al., 1972). The source that of the molting hen is also similar to of oviduct conalbumin is not clear; it reports by other researchers (Brown and may be derived from the blood, especially Jackson, 1960; Cecil and Bitman, 1970). in the immature oviduct, and/or it may Histological and biochemical evidence be primarily synthesized in the oviduct. observed in the present study indicates that In vitro studies by Mandeles and Ducay tubular gland formation occurs in the 1 (1962) support the hypothesis that a con- g developing oviduct and that egg albumen siderable portion of conalbumin is syn- synthesis is initiated when the oviduct thesized in the oviduct of the laying hen. weight is approximately 3 g. Histological Ovalbumin, in contrast to conalbumin, is examinations also show that albumen gran- not immunologically detectable in either ule secretions into the lumen of the tubular the early developing oviduct (weight less glands is initiated at an oviduct weight of than approximately 3 g) or in the blood about 10 g. This finding is consistent with of immature chicks or mature hens. It has the rapid increase in the ratios of also been shown that ovalbumin is present RNA/DNA and protein/DNA as well as only in the magnum and not in other por- in the levels of soluble protein/DNA and tions of the oviduct (Yu et al., 1972). egg white protein/DNA. Brant and Nal- Acrylamide gel electrophoretograms of bandov (1956) have reported that al- the soluble protein in the magnum of the bumen granules appear in the tubular laying hen and of egg white as observed glands of the magnum from oviduct weigh- in the present study are similar to those ing 8.7 ± 3.3 g; they did not, however, of starch gel electrophoretograms reported specify the oviduct weight in which the by other researchers (Oades and Brown, albumen secretion is initiated. The current 1965; Balnave and Squance, 1968). In the study on the regressing oviduct indicates current investigation it was also observed that albumen secretion into the lumen of that the proportion of A2-ovalbumin grad- tubular glands. ceases in oviducts weighing ually shifts to Al-ovalbumin during the de- less than 10 g. This observation cor- veloping stage and this process reverses AVIAN OVIDUCT GROWTh AND DEVELOPMENT 297 during molt. This result is consistent with much greater than the changes exhibited the results of Sanger and Hocquard by the insoluble protein and lipids. These (1962). They demonstrated that A3-oval- differential patterns indicate that during bumin is initially formed in the oviduct. the developing and laying stages the syn- It is then transformed into A2-ovalbumin thesis of secretory egg white proteins pre- and finally into the Al form. The presence dominates and that these specific proteins of different forms of ovalbumin may be are degraded or secreted during regression related to the phosphorylation effect of of the oviduct. The structural units of cells Downloaded from https://academic.oup.com/biolreprod/article/8/3/283/2768726 by guest on 23 September 2021 hormones (Szego and Davis, 1967; Rosen- and tissues represented by insoluble pro- feld and O’Malley, 1970; Kissel et al., tein and phospholipids remain relatively 1970). Sex hormones could induce cyclic stable throughout the reproductive cycle. AMP synthesis (or perhaps cyclic GMP) Studies on the rates of synthesis and degra- which presumably would activate protein dation of the cellular components during kinases involved in the phosphorylation of various stages of the reproductive cycle ovalbumin. It is also possible that the bind- would provide a more lucid understanning ing of sialic acid to ovalbumin may modify of specific metabolic patterns in the the electrophoretic migration pattern of chicken oviduct. ovalbumin (Williams, 1962; Oades and ACKNOWLEDGMENTS Brown, 1965). Further investigation is This investigation was supported in parts by needed to demonstrate the physiological grants from the National Research Council of role of the various forms of ovalbumin pres- Canada and the University of Manitoba Research ent during the various reproductive stages. Fund. We would like to thank Professor C. C. Connective tissue, musculature, and lipo- Hodgson, Dr. P. A. Kondra, and Mr. J. A. McKirdy protein structure of cells comprise the for their assistance during various phases of this work, and the Head of the Department of Animal major fraction of the magnum insoluble Science, Dr. E. W. Stringam, for placing facilities protein. The lipids are presumably part of of the department at our disposal. We would the lipoprotein structure. The levels of in- also like to thank Dr. R. A. Brust of the Depart- soluble protein correlate well with those ment of Entomology for the use of the photomicro- of lipids during a reproductive cycle. The scope and Dr. J. W. Clayton, Fisheries Research relatively higher concentrations of both in- Board of Canada, for the use of the densitometer. soluble protein and lipids, as percent of REFERENCES dry matter, for the early developing and BALNAvE, D., AND SQIJANCE, E., (1968). 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