Functional morphology and biochemistry of reptilian oviducts and eggs

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Functional morphology and biochemistry of reptilian oviducts and eggs implications for the evolution of reproductive modes in tetrapod vertebrates
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xiii, 200 leaves : ill. ; 29 cm.
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Palmer, Brent David, 1959-
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Thesis:
Thesis (Ph. D.)--University of Florida, 1990.
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Includes bibliographical references (leaves 177-199).
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by Brent David Palmer.
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Typescript.
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Vita.

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Full Text









FUNCTIONAL MORPHOLOGY AND BIOCHEMISTRY
OF REPTILIAN OVIDUCTS AND EGGS:
IMPLICATIONS FOR THE EVOLUTION OF REPRODUCTIVE MODES
IN TETRAPOD VERTEBRATES














By

BRENT DAVID PALMER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE
OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA




























Copyright 1990

by

Brent David Palmer


















To Sylvia,

for her patience, love, and devotion












ACKNOWLEDGMENTS

This research has benefited from the assistance and encouragement of many

individuals and institutions, for which I am truly grateful. I especially would like to

thank my major advisor, Dr. Louis J. Guillette, Jr., for his time, effort, and resources. I

am further indebted to the members of my committee, Drs. Harvey Lillywhite, Bill Buhi,

Henry Aldrich, and Horst Schwassmann, for their assistance in my research and many

useful discussions. Further thanks go to Dr. Marty Crump for her assistance in my

research program.

Additional help or support was supplied by Dr. Henry Wilson for his discussions

and assistance; Dr. Harold White III for comments on protein biochemistry; Dr. Bill

Dunson for supplying specimens and advice; the staff of the E.M. Core Facility of the

Interdisciplinary Center for Biotechnology Research for assistance with electron

micrographs; Daryl Harrison for line drawings; the Department of Zoology for financial

support; Louis Somma, John Matter and Vincent DeMarco for assistance collecting

specimens.

My wife, Sylvia, deserves special mention for her constant faith in me. My

career would not have been possible without her boundless love and support. I am

forever grateful for her patience and devotion.













TABLE OF CONTENTS



ACKNOWLEDGMENTS ................................................ iv

LIST OF TABLES ........................................................ viii

LIST OF FIGURES....................................................... ix

ABSTRACT ................ ............................................ xii

CHAPTERS

I INTRODUCTION AND LITERATURE REVIEW ................... 1

Evolution of Amniotic Eggs.......................................... 2
Reptilian Oviductal Functional Morphology........................... 2
Oviductal Secretory Proteins ....................................... 12
Biological Properties of Albumen Proteins .......................... 12
Antimicrobial proteins ....................................... 14
Nutritive proteins............................................ 16
Support and cushioning proteins............................... 18
Water binding proteins........................................ 18
Reptilian Albumen Proteins ....................................... 19
Albumen Protein Formation ....................................... 22
Eggshell Membranes ............................................ 28

II ULTRASTRUCTURE AND FUNCTIONAL
MORPHOLOGY OF TURTLE OVIDUCTS ......................... 31

Methods and Materials.............................................. 32
Specimens ................................. .................. .. 32
Histochemistry ................ ................................. 32
Electron Microscopy..............................................32
Results.............. ..........................................34
Luminal Epithelium ............................................. 34
Endometrial Glands..............................................55
Discussion................. .................................... ... 56
Luminal Epithelium ..............................................56
Endometrial Glands..............................................57

III OVIDUCTAL MORPHOLOGY AND EGGSHELL
FORMATION IN THE LIZARD, SCELOPORUS WOODI.............. 61

Methods and Materials ............................................ 62
Specimens..................................................... 62
Histochemistry ................................................. 64






Scanning Electron Microscopy ..................................... 64
R results .......................................................... 64
Discussion.............. .................. ...... ..... ............... 65

IV OVIDUCTAL ULTRASTRUCTURE AND EGG
FORMATION IN ALLIGATORS
(ALLIGATOR MISSISSIPPIENSIS) ................................. 83

Methods and Materials ............................................. 84
Specimens ..................................................... 84
Histochemistry ................ ............................... 84
Electron Microscopy.............................................85
Results ........................................................ .85
General Oviductal Morphology...................................85
Tube ........................................................... 86
Fiber Region .................................... .... ........ 87
Uterus........................................................ 87
Discussion....................................................... 100

V REPTILIAN EGG ALBUMEN BIOCHEMISTRY ................. ..... 104

Methods and Materials ........................................... 106
Specimens..................................... ............... 106
Electrophoresis............................................... 108
Electroblotting ........................................... ... 108
Purification of a-Ovalbumin Antibodies .......................... 110
Results .......................................................... 111
Crocodilians...................... ............ .................111
Testudines ......................................................111
Squamates................................................... 112
Sphenodon......................................112
Immunodetection .............................................. 113
Discussion ...................................... ...... .......... 113

VI REPTILIAN EGG ALBUMEN SECRETION ......................... 126

Methods and Materials .................................... ..... 127
Specimens .................................................... 127
Sample Preparation ............................................ 127
Electrophoresis............................................... 127
Electroblotting................................................. 128
Fluorography .................................................. 129
Statistics ..................................................... 129
Results .......................................................... 129
Discussion ................ ...................... .............. 130

VII LOCALIZATION OF EGG ALBUMEN SECRETING CELLS
IN REPTILIAN OVIDUCTS....................................... 143

Methods and Materials ........................................... 144
Specimens....................................................144
Histology and Immunocytochemistry............................. 145
Results ............................................................ 146
Discussion............................................. ........146







VIII IMPLICATIONS FOR THE EVOLUTION OF
REPRODUCTIVE MODES IN TETRAPOD VERTEBRATES........ 153

Oviparous Reproductive Modes in Amniotes ......................... 153
Evolution of Amniotic Oviparity..................................... 156
Phylogeny of Amniotic Vertebrates.................................. 161
Evolution of Shelled Eggs.......................................... 161

IX SUMMARY AND CONCLUSIONS ................................. 172

Comparative Terminology.......................................... 174
Future Research Directions........................................ 175

LITERATURE CITED .................................................... 177

BIOGRAPHICAL SKETCH................................................200


vii












LIST OF TABLES


TABLE

1-1 Composition and physicochemical characteristics of
major albumen proteins of the hen................................... 13

2-1 Staining techniques employed and their general
interpretation ...................... ... ............ ................ 33

3-1 Identification of the females used in study of
oviductal functional morphology and eggshell formation................... 63

5-1 Reptilian eggs analyzed for albumen protein composition ................ 107

5-2 Proteins used as molecular weight standards for
one-dimensional polyacrylamide gel electrophoresis..................... 109

6-1 Equations for the best fit linear regression and
cogelation coefficients (r) for the incorporation
of H-leucine into secretary proteins by tubal and
uterine explant tissue cultures from the turtle
Pseudemys script ................................................. 131


viii













LIST OF FIGURES


FIGURE

1-1 Typical egg structure in birds, crocodilians and
chelonians, and lepidosaurians.......................................... 4

1-2 Morphological characteristics of the oviduct of
lizards, snakes, and turtles............................................. 8

1-3 Morphological characteristics of a typical avian
(Gallus domesticus) oviduct........................................... 24

2-1 Gross morphology of the turtle oviduct.................................. 36

2-2 Histology and scanning electron microscopy of the
luminal epithelium of the turtle oviduct, showing
ciliated cells and secretary cells........................................ 38

2-3 Ultrastructure of the ciliated cells of the luminal epithelium ................. 40

2-4 Transmission electron micrographs of the apical portion
of ciliated cells ...................... ................................. 42

2-5 Microvillous secretary cells of the luminal epithelium
characterized by electron light secretary granules......................... 44

2-6 Microvillous secretary cells of the luminal epithelium
characterized by electron dense secretary granules ..................... 46

2-7 The lamina propria surrounding the endometrial glands
of both the uterus and tube is highly vascularized ......................... 48

2-8 Histology and ultrastructure of the tubal endometrial glands .............. 50

2-9 Transmission electron micrographs of the tubal endometrial glands .......... 52

2-10 Histology and ultrastructure of the uterine endometrial glands ............... 54

3-1 The luminal epithelium of the infundibulum and tube consists
of ciliated and secretary cells...........................................67

3-2 Histology and scanning electron microscopy of the uterus .................. 69

3-3 Histology and scanning electron microscopy of the vagina .................. 71

3-4 Formation of the fibrous eggshell membrane............................. 73







3-5 Structure of the eggshell of Sceloporus woodi............................. 75

3-6 Changes in the gross morphology of the uterine
epithelium during eggshell formation................................... 77

4-1 Gross morphology of the oviduct of the alligator
(Alligator mississippiensis) ............................................. 89

4-2 Histology and scanning electron microscopy of the
oviductal luminal epithelium .......................................... 91

4-3 Histology and transmission electron microscopy
of the oviductal tube ................ ................................ 93

4-4 Histology and transmission electron microscopy
of the fiber region of the oviduct...................................... 95

4-5 Formation of the fibrous membrane of the eggshell....................... 97

4-6 Histology and ultrastructure of the uterus............................... 99

5-1 Separation of egg albumen proteins from representative
reptilian groups by molecular weight using 10%T
one-dimensional SDS polyacrylamide gel electrophoresis
to demonstrate low molecular weight components ......................... 115

5-2 Separation of egg albumen proteins from representative
reptilian groups by molecular weight using 7%T SDS-PAGE
to demonstrate high molecular weight components....................... 117

5-3 Comparisons ofAlligator mississippiensis albumen proteins
of eggs from clutches of 5 different females,
separated by molecular weight by 1D-SDS-PAGE........................ 119

5-4 Comparisons of albumen proteins of eggs from a crocodilian
and three species of turtles separated by 1D-SDS-PAGE ................. 121

6-1 Incorporation of 3H-leucine into proteins by explant tissue
cultures from Pseudemys script ...................................... 133

6-2 One-dimensional SDS-PAGE separation of proteins present in
Pseudemys scripta tissue culture medium by molecular weight .............. 135

6-3 Representative fluorograph ofPseudemys scripta tubal explant
tissue culture proteins separated by 2D-SDS-PAGE ...................... 137

6-4 Representative fluorograph ofPseudemys scripta uterine explant
tissue culture proteins separated by 2D-SDS-PAGE ...................... 139

7-1 Representative immunocytochemical localization of cells secreting
ovalbumin-like proteins in the endometrial glands of the oviductal
tube in the tortoise, Gopheruspolyphemus, by the use of ovalbumin
specific antibodies................. ................................. 148








7-2 Representative immunocytochemical localization of cells secreting
ovalbumin-like proteins in the endometrial glands of the oviductal
tube in the alligator, Alligator mississippiensis, by the use of
ovalbumin specific antibodies......................................... 150

8-1 Theoretical sequence of adaptations in the evolution of eggshell
formation within the amniotes based upon extant characteristics ............ 159

8-2 Dendrogram of the traditional phylogenies of the Amniota based
largely upon the fossil record..........................................163

8-3 Revised classification of the Amniota based upon characteristics
of extant species....... ................. .........................165

8-4 Oviductal functional morphology, egg structure, and mode of
eggshell formation in extant vertebrates is consistent with
the phylogeny of amniotes based upon the fossil record .................... 167

8-5 Diagrammatic representation of theories concerning the
evolution of the shelled eggs of amniotes from the jelly
coated eggs of amphibians................. .......................... 169












Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


FUNCTIONAL MORPHOLOGY AND BIOCHEMISTRY
OF REPTILIAN OVIDUCTS AND EGGS:
IMPLICATIONS FOR THE EVOLUTION OF REPRODUCTIVE MODES
IN TETRAPOD VERTEBRATES

By

Brent David Palmer

May 1990


Chairman: Louis J. Guillette, Jr.
Major Department: Zoology

The evolution of shelled amniotic eggs was a major factor facilitating the

radiation of vertebrates into terrestrial habitats. This study examined the functional

morphology and biochemistry of oviducts and eggs of representative reptilian groups,

particularly with respect to albumen and eggshell formation. The results have important

implications for our understanding of the evolution of reproductive modes in tetrapod

vertebrates.

The oviductal functional morphology and ultrastructure of turtles, a lizard, and

an alligator were studied using histochemistry, scanning and transmission electron

microscopy. In turtles and lizards, the uterus is dualistic in function, secreting both the

fibrous and calcareous layers of the eggshell membrane, as occurs in the monotremes.

In alligators, there are two separate oviductal regions for eggshell formation, one

specialized for formation of the eggshell membrane (fiber region), and another that

secretes the calcareous eggshell layer (uterus). The oviductal functional morphology of


xii







alligators is therefore similar to that of birds. This represents a fundamental divergence

in reproductive morphology among higher vertebrates.

The biochemistry of reptilian oviducts and eggs was examined by analyzing the

protein composition and secretion of egg albumen. There is much diversity in albumen

protein composition among reptiles, although a few proteins are found universally and

are immunologically similar to avian albumen proteins. In general, the albumin proteins

of alligators and turtles are similar to those of birds, whereas squamate albumen is

substantially different in composition. Egg albumin proteins are synthesized and

secreted by the endometrial glands of the oviductal tube.

The divergence in reproductive modes between birds and mammals is found

within extant reptiles. Evolutionarily, the relationship between crocodilians and birds is

evident in oviductal structure and egg formation, whereas turtles and squamates

resemble monotremes in formation of the eggshell by the uterus. The egg albumen

shows much diversity in protein composition among reptilian groups, which may be

related to their nesting ecology. These data refute recent theories concerning the

evolution of birds and mammals from a common archosaurian ancestor.


xiii












CHAPTER I
INTRODUCTION AND LITERATURE REVIEW


An understanding of the functional morphology of oviducts is essential for

understanding the evolution of vertebrate reproductive modes. The oviducts influence

whether the animal will be oviparous or viviparous, whether the eggs (or young) will be

retained, and what kind of investments (coverings) will be applied to the eggs. The

oviducts are further involved in fertilization and sperm storage.

Oviductal function is ultimately tied to its morphology and ultrastructure. Gross

morphology shows the general layout of the oviducts, such as the number of regions and

degree of muscular development. Cytological and ultrastructural data give evidence of

the type of secretary material, if any. This can be further investigated with

histochemistry in general and immunocytochemistry for specific proteins. The

biochemistry of oviductal secretary products is critical to understanding the physiology of

the eggs, both while they are retained in the oviducts and following oviposition. This is

due to the properties endowed upon the egg by oviductal secretary proteins. These

properties may enhance microbial defense, embryonic nutrition, or protection from the

physical environment.

Comparisons of oviductal functional morphology and biochemistry are therefore

key to understanding the evolution of reproductive modes in vertebrates. The following

studies examined oviductal functional morphology and biochemistry of their secretary

products in a variety of reptiles. Comparisons among species have revealed remarkable

and unexpected similarities, as well as differences. These are ultimately tied to the

evolutionary history of the organism and its mode of reproduction, and has direct

implications for the evolution of avian and mammalian modes of reproduction.







Evolution of Amniotic Eggs


The amniotic egg not only encloses the embryo but provides an environment

suitable for its development. This environment is produced by the mother in the form

of egg albumen. Albumen consists primarily of proteins, although carbohydrates and

lipids are found in trace quantities. The eggshell consists of a layer of proteinaceous

fibers and, usually, a surface layer of calcium carbonate. Although there are variations

in the structure of eggs among vertebrates (Fig. 1-1), they all possess albumen and a

proteinaceous eggshell membrane, and most exhibit some degree of calcification.

The albumen creates a suitable internal environment for embryonic development,

whereas the eggshell reduces the influence of the external conditions but allows gases

and water to be exchanged. In later development, the embryo interacts more directly

with the external environment through the apposition of extraembryonic membranes

(chorion, allantois, yolk sac) to the eggshell and, therefore, plays a greater role in its

own homeostasis.

The following review concentrates on oviductal structure, secretary proteins

produced by the maternal oviduct and their influence on embryonic development. By

far, the most studied proteins are the albumen and eggshell membrane proteins of avian

eggs, with those of reptilian eggs only recently coming under investigation.


Reptilian Oviductal Functional Morphology


The oviducts of tetrapods are derived embryonically from the paired Miillerian

(paramesonephric) ducts (Hildebrand, 1982). Oviductal morphology varies greatly

among sexually mature tetrapod vertebrates. The term oviduct, as defined in

comparative anatomical studies and as used throughout this dissertation, refers to the

entire female reproductive tract. In most reptiles, both oviducts are functional and

separate for their entire length, joining a cloaca posteriorly. Some squamates, primarily

snakes, may display a vestigial oviduct, typically the left (Fox, 1977). For important































Figure 1-1. Typical egg structure in birds, crocodilians and chelonians, and
lepidosaurians. Redrawn from Packard et al., 1977.











AVES


Albumen


CROCODILIA &

CHELONIA


- Shell




'Egg membrane


Albumen


LEPIDOSAURIA


Albumen








works and reviews of the female reproductive tracts in reptiles, see Brooks, 1906;

Giersberg, 1923; Weekes, 1927,1935; van den Broek, 1933; Boyd, 1942; Mulaik, 1946;

Kehl and Combescot, 1955; Forbes, 1961; Wilkinson, 1965; Cuellar, 1966; Hoffman and

Wimsatt, 1972; Botte, 1973; Christiansen, 1973; Veith, 1974; Fox, 1977; Guillette, 1981;

Mead et al., 1981; Halpert et al., 1982; Guillette and Jones, 1985; Palmer and Guillette,

1988, 1990a, b; Uribe et al., 1988; Adams and Cooper, 1988; Guillette et al., 1989; and

Kumari et al., 1990.

Histologically, the oviduct is organized into three distinct layers (Sacchi, 1888;

Hoffmann, 1889). These are, from the coelom to the lumen, the perimetrium,

myometrium, and endometrium. The perimetrium consists of a thin layer of loose

connective tissue covered by a single layer of squamous epithelium (mesothelium). This

layer is continuous with the peritoneal support of the oviduct, which is known as the

mesotubarium (Parker, 1884). The serosal layer is highly vascularized, with arteries,

veins and lymphatic vessels running parallel to the oviduct along the mesotubarian

junction. The serosal layer shows little variation among oviductal regions or vertebrate

species.

The myometrium is composed of smooth muscle fibers typically arranged to form

an outer longitudinal layer and an inner circular layer. The composition and

development of the myometrium is highly variable among specific oviductal regions and

among species (Hildebrand, 1982). Usually, the myometrium displays greater

development posteriorly, whereas only scattered muscle fibers are present in the most

anterior regions (Palmer and Guillette, 1988).

The endometrium is composed of two functionally separate but related layers;

the lamina propria and lamina epithelialis mucosaa). The lamina propria in reptiles

consists of highly vascularized loose connective tissue that may contain tubulo-alveolar

glands (Fox, 1977). The connective tissue of the lamina propria is primarily composed








of collagen (Boyd, 1942). The endometrial gland cells are cuboidal to low columnar

with large spherical nuclei (Palmer and Guillette, 1988; Guillette et al., 1989).

The lamina epithelialis is composed of an unstratified layer of columnar

epithelial cells which lines the lumen of the oviduct. There are two primary types of

cells, ciliated and secretary. The entire mucosa may show extensive folding which may

vary in thickness among oviductal regions and with the reproductive status of the animal

(Weekes, 1927; Boyd, 1942; Palmer and Guillette, 1988, 1990a; Uribe et al., 1988).

The reptilian oviduct shows considerable variation in morphology between species

(Fig. 1-2). In lizards and snakes, three distinct regions have been described; the

infundibulum-tube, the uterus, and the vagina (Cuellar, 1966; Fox, 1977; Gist and Jones,

1987), although some authors describe morphologically distinct anterior and posterior

uterine regions (Guillette and Jones, 1985). Morphologically, the oviduct of turtles and

tortoises form five distinct regions; the infundibulum, the tube (tuba uterina), the

isthmus or transitional zone, the uterus, and the vagina (Giersberg, 1923; Fox, 1977; Gist

and Jones, 1987, 1989), in addition, the infundibulum exhibits distinct anterior and

posterior portions (Palmer and Guillette, 1988).

The infundibulum is the most anterior portion of the oviduct. The wide opening

of the infundibulum (the ostium) was described in squamates over 100 years ago (Sacchi,

1888). The infundibulum serves to receive eggs from the ovaries, and may actively

engulf the ovary prior to ovulation to facilitate reception of eggs (Cuellar, 1970). This

region has abundant cilia which were first noted histologically in reptiles in Lacerta and

Anguis by Leydig (1872). The mucosa is slightly folded and composed of ciliated and

microvillous secretary cells. In addition, unique bleb secretary cells have been described

in the infundibulum of reptiles (Boyd, 1942; Palmer and Guillette, 1988) that may be

homologous to similar cells found in the glandular grooves of the avian infundibulum

(Giersberg, 1923; Richardson, 1935). All three types of cells may occur on folds, but

only bleb secretary cells occur within the grooves between folds (Palmer and Guillette,






































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1988). The infundibular epithelial cells increase in height during vitellogenesis (Hansen

and Riley, 1941). The infundibulum is very thin walled, with the muscularis being poorly

developed and the lamina propria being devoid of glands (Giersberg, 1923; Hansen and

Riley, 1941; Palmer and Guillette, 1988; Guillette et al., 1989). The infundibulum serves

the important function as the site of fertilization in birds (Aitken, 1971), and presumably

also in reptiles.

The major part of the turtle and alligator oviduct consists of the highly pleated

tube (Bojanus, 1819-1821; Giersberg, 1923; Palmer and Guillette, 1988, 1990a). The

myometrium is reduced to only a few circular muscle fibers scattered among the

connective tissue underlying the serosa. The endometrium is slightly pleated forming

longitudinal grooves. The lamina propria contains numerous saccular or tubular glands

(Giersberg, 1923; Hansen and Riley, 1941; Hattan and Gist, 1975; Palmer and Guillette,

1988; Guillette et al., 1989). Several authors have suggested that these glands produce

albumen in reptiles (Gegenbaur, 1878; Christiansen, 1973; Botte et al., 1974; Botte and

Granata, 1977; Palmer and Guillette, 1988, 1990a, b). Histological studies have found

that the glands of the tube of Chelonia mydas (Aitken and Solomon, 1976) and

Gopheruspolyphemus (Palmer and Guillette, 1988) are structurally similar to the

albumen secreting glands in the magnum of birds. These gland cells contain basal nuclei

and spherical membrane bound granules of varying electron densities. After the passage

of eggs, there are fewer granules in most cells. The tube is uniform along its length and

lacks the terminal mucous region which produces the thick layer of albumen in birds.

Layering of the "albumen" has not been described in reptilian eggs.

The tube is greatly reduced and aglandular in squamates (Hoffman and Wimsatt,

1972; Christiansen, 1973; Mead et al., 1981; Guillette and Jones, 1985; Adams and

Cooper, 1988; Uribe et al., 1988; Guillette et al., 1989; Kumari et al., 1990), and is

difficult to distinguish from the infundibulum. Squamates also have diminished

quantities of albumen in their eggs (Packard et al., 1977; Tracy and Snell, 1985). Early








reports even suggested squamates lacked albumen entirely (Weekes, 1927; Giersberg,

1923).

The epithelium consists of tall columnar cells of which there are proportionately

more secretary than ciliated cells (Aitken and Solomon, 1976). Although most of the

cilia beat posteriorly, Parker (1928, 1931) observed in the turtle Chrysemyspicta there is

a narrow band of ciliated cells (2mm) that beat anteriorly. These pro-ovarian cilia move

sperm toward the infundibulum where fertilization presumably takes place. The position

of the pro-ovarian band is marked externally by the vascular edge of the oviduct. The

posterior beating abovarian cilia are probably involved in sloughing mucus and the

transport of eggs. Crowell (1932) also identified a single band of pro-ovarian cilia in

several species of turtles (C. picta and Pseudemys scripta elegans) and lizards

(Phrynosoma comutum and Sceloporus undulatus). In the turtle Mauremysjaponica,

Yamada (1952) identified two pro-ovarian bands which lie on opposite edges of the

tube.

In turtles, the tube is joined posteriorly to the uterus by a constricted region

termed the isthmus or transitional zone. The isthmus is a very short, aglandular segment

which probably has no secretary function associated with albumen or eggshell formation

(Aitken and Solomon, 1976).

The uterus is the region where eggs are retained until oviposition. It has an

extremely well developed myometrium composed of an inner circular and an outer

longitudinal layer. The myometrium serves to move the oviductal wall over the egg's

surface (Parker, 1928) during gravidity as well as aid in oviposition. The uterine mucosa

exhibits tall villous-like folds, particularly posteriorly. The thickness of the oviduct and

degree of folding of the mucosa increases from the infundibulum to the vagina (Bojanus,

1819-1821; Giersberg, 1923; Weekes, 1927; Boyd, 1942; Cuellar, 1966; Halpert et al.,

1982; Guillette and Jones, 1985; Palmer and Guillette, 1988; Guillette et al., 1989).

Within the uterus, the lamina propria is thick and glandular. In the turtles Chelonia







mydas (Aitken and Solomon, 1976) and Gopheruspolyphemus (Palmer and Guillette,

1988), the uterine glands resemble those of the isthmus in birds. These glands contain

numerous secretary granules during vitellogenesis, but become depleted in gravid animals

(Palmer and Guillette, 1990a). The luminal surface of the gland cells also possesses

microvilli and pronounced blebbing during gravidity. These glands have been suggested

to produce proteinaceous compounds for the formation of eggshell fibers (Weekes, 1927;

Boyd, 1942; Aitken and Solomon, 1976; Palmer and Guillette, 1988, 1990a, b; Guillette

et al., 1989).

The glands of the avian shell gland lack distinctive ultrastructural features and

secretary granules (Johnson et al., 1963) and are hypothesized to supply the "plumping

water", which dilutes the albumen to its final concentration (Breen and de Bruyn, 1969).

It has been hypothesized that plumping water is supplied by the reptilian uterus (Tracy

and Snell, 1985; Palmer and Guillette, 1988).

The uterine epithelium in reptiles is composed of serious secretary cells and

ciliated cells (Giersberg, 1923; Weekes, 1927; Boyd, 1942; Hansen and Riley, 1941;

Hoffman and Wimsatt, 1972; Cuellar, 1966; Christiansen, 1973; Aitken and Solomon,

1976; Guillette and Jones, 1985; Palmer and Guillette, 1988; Uribe et al., 1988; Guillette

et al., 1989). The epithelium of G. polyphemus is mostly ciliated during vitellogenesis,

but transforms into mostly secretary cells during gravidity (Palmer and Guillette, 1990a).

The epithelium of the avian shell gland is often implicated in calcium transport during

eggshelling (Aitken, 1971). In Crotaphytus collaris, the mucosal epithelium stains for

calcium (Guillette et al., 1989) and may produce the calcareous eggshell as predicted by

Gegenbaur (1878).

The vagina in reptiles is a very short segment of the oviduct which connects the

uterus with the cloaca. In some lizards (Eumeces, Gerrhonotus and Cnemidophorus),

the myometrium of the vagina forms a sphincter (Brooks, 1906; Fox, 1977), suggesting

that it may function in a manner comparable to the mammalian cervix. It may also







function in retaining eggs within the uterus until the time of oviposition. The mucosa of

the vagina is nearly aglandular.

The development of the oviducts has implications for the stimulation of

hypertrophy in mature specimens. The oviducts of juvenile Testudo graeca and

Mauremys caspica leprosa are devoid of glands (Argaud, 1920; Kehl, 1944). As these

turtles mature, the luminal epithelium invaginates to form crypts, which differentiate into

endometrial glands. This indicates that glands form under a stimulus associated with

maturity and that the glands of the lamina propria are of epithelial origin. This pattern

is similar to that described for squamates, where it was observed that ovarian tissue was

capable of stimulating formation of new glands from luminal epithelia in oviductal tissue

cultured in vitro (Ortiz and Morales, 1974). Hypertrophy of uterine endometrial glands

can be induced in vivo with estrogen administration (Christiansen, 1973; Fawcett, 1975;

Mead et al., 1981).


Oviductal Secretory Proteins


Biological Properties of Albumen Proteins

A functional approach will be used to examine albumen proteins, rather than a

discussion of each protein individually. Although avian egg albumen proteins have

received intense investigation, specific biological functions have not been found for all,

including the most abundant avian egg white protein, ovalbumin (Baker, 1968; Nisbet et

al., 1981; Li-Chan and Nakai, 1989). Since little is known concerning reptilian albumen

proteins, much of this discussion will be based on what can be interpreted from birds.

A summary of characteristics of avian albumen proteins is given in Table 1-1. Most are

glycoproteins, with carbohydrate contents ranging between 2 and 22 percent. Functions

described for albumen proteins have largely been determined in vitro, with virtually

nothing known concerning in vivo functions. Further, some proteins may have multiple

functions.












Table 1-1. Composition and physicochemical characteristics of major
albumen proteins of the hena


% of Molecular Isoelectric % Carbo-
albumen Weight (kd) point hydrate


Avidin 0.05 68.3 10.0 8

Cystatin 0.05 12.7 5.1 0

Lysozyme 3.4-3.5 14.3 10.7 0

Ovalbumin 54.0 45 4.5 3

Ovoglobulin G2 4.0(?) 49 5.5 -6

Ovoglobulin G3 4.0(?) 49 5.8 -6

Ovoglycoprotein 0.5-1.0 24.4 3.9 16

Ovoinhibitor 0.1-1.5 49 5.1 6

Ovomacroglobulin 0.5 760-900 4.5-4.7 9

Ovomucin 1.5-3.5 230-8,300 4.5-5.0 19

Ovomucoid 11.0 28 4.1 22

Ovotransferrin 12-13 77.7 6.0 2

Riboflavin-
Binding Protein 0.8 32 4.0 14

aCompiled from Gilbert, 1971; Osuga & Feeney, 1977; Powrie & Nakai, 1986; and
Li-Chan & Nakai, 1989







Antimicrobial proteins

Albumen provides for microbial defense of yolk and developing embryo in two

ways, mechanically and chemically (Board and Tranter, 1986). Mechanically, albumen

supports and surrounds the yolk, keeping it from contact with eggshell membranes.

Additionally, albumen forms a colloid which acts as a barrier to invading bacteria due to

its viscous and fibrous nature. Ovomucin is largely responsible for the high viscosity of

albumen (Robinson, 1972, 1987), which is further augmented by lysozyme complexes

(Kato et al., 1981; Miller et al., 1982; Hayakawa et al., 1983).

Chemically, albumen may prevent microbial infection by directly killing bacteria

or by creating an environment unfavorable for their growth. Most studies on the

antimicrobial properties of albumen proteins have been conducted in vitro, and it is

uncertain if these properties are applicable in vivo. Lysozyme, the only albumen protein

known to directly affect bacteria, does so by hydrolyzing B(1-4) glycosidic bonds

(Geoffroy and Bailey, 1975), a component of the cell wall of certain bacteria, thereby

disrupting cell wall integrity.

There are several ways in which albumen can create an unfavorable environment

for bacterial growth (Tranter and Board, 1982b; Board and Tranter, 1986). Albumen is

quite alkaline, having a pH of about 9.5 (Heath, 1977), which is generally unsuitable for

growth of many microorganisms (Board and Tranter, 1986). Additionally, some proteins

bind required nutrients, such as minerals or vitamins, making them unavailable for

bacterial use. Iron is found in very low concentrations in egg albumen. Ovotransferrin

strongly (dissociation constant (KD) 10-29M) binds iron (Chasteen, 1977, 1983; Aisen

and Listowsky, 1980; Brock, 1985), especially at the alkaline pH of albumen, and is

effective in inhibiting bacterial growth by creating an essentially iron-free environment

(Weinberg, 1977; Tranter and Board, 1982b). Additionally, ovotransferrin also binds

copper, which may maintain the bactericidal properties of lysozyme, which is inhibited by







this metal (Gilbert, 1971). Ovotransferrin has been identified as the major antibacterial

component of fowl egg albumen (Tranter and Board, 1982b).

Other proteins bind vitamins, making them inaccessible to bacteria. Avidin, well

known for its ability to strongly bind (KD 10-15M) the vitamin biotin (Green, 1963,

1975; Elo and Korpela, 1984), is strongly antibacterial, occurring largely in unbound

(apoprotein) form (Tranter and Board, 1982a, b; Banks et al., 1986). Avidin also binds

to the surface of some bacteria (Korpela, 1984; Korpela et al., 1984). A recently

described thiamin-binding protein (Muniyappa and Adiga, 1979; Adiga and Murty, 1983)

occurs largely in the apoprotein form in albumen, and may inhibit bacterial growth,

although its affinity for thiamin is low (KD -3 X 10-7M). Another egg protein,

riboflavin-binding protein, binds the vitamin riboflavin. Although it has been suggested

that this apoprotein binds riboflavin too weakly (KD 10-9M) to inhibit bacterial growth

(Rhodes et al., 1959), in vitro studies have demonstrated inhibition (Li-Chan and Nakai,

1989).

Another group of antimicrobial proteins are protease inhibitors. Albumen has a

very low concentration of free nitrogen, which is required by bacteria for protein

synthesis. Substantial nitrogen for bacterial growth is found in albumen proteins, and

many bacteria have proteases which are used to free nitrogen for incorporation into

their own proteins. However, several of the albumen proteins have been demonstrated

to be protease inhibitors (Li-Chan and Nakai, 1989), thereby preventing degradation and

release of nitrogen for bacterial use. Ovomucoid, the third most common protein of

fowl eggs, inhibits trypsin (Lineweaver and Murray, 1947; Feeney and Allison, 1969;

Feeney, 1971). Ovoinhibitor, although a minor component of avian eggs, simultaneously

binds two molecules of trypsin or chymotrypsin, (Rhodes et al., 1960; Liu et al., 1971;

Zahnley, 1980), as well as several types of bacterial and fungal proteases (Matsushima,

1958; Feeney et al., 1963; Tomimatsu et al., 1966; Feeney, 1971). Both ovomucoid and

ovoinhibitor belong to a family of serine proteinase inhibitors. Based on its primary







structure, ovalbumin has been placed into a superfamily of serine proteinase inhibitors

(Breathnach et al., 1978; Hunt and Dayhoff, 1980; Woo et al., 1981; Carrell and Boswell,

1986; Ye et al., 1989), although no biological function has been found for this protein

(Li-Chan and Nakai, 1989). Ovomacroglobulin also has serine protease inhibitory

activity (Kitamoto et al., 1982) and is believed to be evolutionarily related to human

serum a2-macroglobulin, which is inhibitory to trypsin and certain other proteases (Li-

Chan and Nakai, 1989). Avian ovomacroglobulin also may be related to the crocodilian

counterpart, which inhibits trypsin, subtilisin and papain (Ikai et al., 1983). Thiol

proteases, which include ficin, papain, cathepsin C, B1, H and L, bromelain,

chymopapain, papaya proteinase III, and actinidin are inhibited by the minor egg

protein cystatin (Fossum and Whitaker, 1968; Sen and Whitaker, 1973; Keilova and

Tomaiek, 1974, 1975; Barrett, 1981; Anastasi et al., 1983; Barrett et al., 1986).

Although most defenses are directed against bacterial or fungal invasion, two

proteins have been suggested to possess antiviral properties. Ovomucin has been shown

to be antiviral in that it inhibits viral hemagglutination (Lanni and Beard, 1948; Lanni et

al., 1949; Gottschalk and Lind, 1949a, b). Cystatin, a thiol proteinase inhibitor, also may

prevent viral infection (Barrett et al., 1986). These properties may be important as egg

albumen proteins have been found in embryonic blood (Marshall and Deutsch, 1951;

Wise et al., 1964), where they may be involved in prevention of viral hemagglutination.

The combined antimicrobial effect of different albumen proteins may be synergistic in

preventing microbial attack (Banks et al., 1986).


Nutritive proteins

The albumen proteins may represent a substantial supply of nutrients to the

embryo (White, 1990). By binding vitamins and minerals, albumen proteins may not

only act in defense of microbial attack, but in supplying nutrients to the developing

embryo. Albumen proteins may be ingested by the embryo directly or selectively taken

up (phagocytotically) by extraembryonic membranes (Marshall and Deutsch, 1951; Wise








et al., 1964). This uptake may supply the embryo with either the protein itself, its

amino acid constituents, or other molecules which are bound to the protein.

Several proteins may be involved in selective transport of vitamins to the embryo.

Avidin is well known to bind the vitamin biotin (Green, 1975), and may be used to

transport biotin from mother to embryo (Adiga and Murty, 1983; White, 1987; White et

al., 1987; Bush and White, 1989), although albumen in the fowl contains only 10% of

egg biotin (Romanoff and Romanoff, 1949), with yolk supplying the remaining 90%.

Riboflavin-binding protein is present in the albumen in approximately equal proportions

of bound and unbound forms (Rhodes et al., 1958, 1959). The albumen of fowl eggs

contains about 50-70% of the riboflavin in the egg (Stamberg et al., 1946), indicating

that it is a major source of this nutrient (White and Merrill, 1988). Riboflavin-binding

protein is synthesized in the oviduct (Mandeles and Ducay, 1962), and riboflavin is

transferred to it from the blood. Thiamin-binding protein in albumen may exist largely

in the apoprotein form, since the majority of thiamin in fowl eggs is concentrated in yolk

(White, 1987). This may limit the value of albumen thiamin-binding protein in

embryonic nutrition.

One protein which may be used to transport specific minerals to the embryo is

ovotransferrin. Ovotransferrin belongs to a family of iron-binding proteins, the

transferring, and may be involved in the transport of iron to the embryo as occurs with a

related mammalian blood protein, transferring (Faulk and Galbraith, 1979).

Ovotransferrin also is identical in amino acid structure to transferring (Williams, 1962;

Williams et al., 1982), differing only in carbohydrate moieties (Williams, 1968) and

absence ofsialic acid in ovotransferrin (Osuga and Feeney, 1968). Uteroferrin, an iron

containing protein from the pig, is secreted by the uterus and is taken up by the fetus as

a source of iron (Roberts et al., 1986; Roberts and Bazer, 1988). However, since

ovotransferrin in albumen is predominantly in the apoprotein form, it is unlikely to







supply much iron to the embryo, although it can release iron to embryonic red blood

cells (Li-Chan and Nakai, 1989).


Support and cushioning proteins

The albumen supports the yolk and embryo within the shell, cushioning them

from mechanical injury. In birds, the chalaza (a fibrous ligament), suspends the yolk

within the center of the albuminous mass. The chalaza is formed from the twisting of

ovomucin fibers as the egg rotates while descending through the oviduct (Conrad and

Phillips, 1938; Scott and Huang, 1941). The remaining albumen aids in cushioning the

yolk due to its viscosity. The role of albumen in support and cushioning of reptilian

eggs is variable. A chalaza has not been reported in reptilian eggs, and the quantity and

viscosity of albumen in reptilian eggs is extremely variable at oviposition. In species

which have a thick albumen layer, it may help cushion the yolk when the egg drops into

the nest cavity.


Water binding proteins

Most avian albumen proteins are water soluble, indicating that they are at least

partially hydrophilic. Most also are glycoproteins, which are known to tightly bind water

to the hydrophilic side groups of the peptide chain, as well as the carbohydrate residues

(Fennema, 1977). Because of the physical structure of the proteinaceous mass, some

water is trapped by surrounding protein molecules. There also is free water which is in

the albumen due simply to osmotic principles. It is, therefore, the combined qualities of

albumen as a whole which enable it to act in water storage, creating an osmotic

impediment between the embryo and surrounding environment.

In reptiles, there is no single scheme for storage of water in albumen.

Crocodilians, some chelonians and some squamates lay rigid-shelled eggs (Ferguson,

1982; Packard et al., 1982; Packard and Hirsch, 1986; Hirsch, 1983; Schleich and KIistle,

1988; Packard and DeMarco, 1990). In these, albumen is relatively hydrated in the







uterus before oviposition (Tracy and Snell, 1985; Webb et al., 1987a, b; Manolis et al.,

1987; Palmer and Guillette, 1988), although additional water may be absorbed from the

substrate, such as sand, soil, or organic matter (Guggisberg, 1972; Packard et al., 1977).

In those reptiles with parchment-shelled (most squamates) or pliable-shelled (many

turtles) eggs, the egg may absorb substantial quantities of water during development

(Packard et al., 1977; Packard et al., 1980; Morris et al., 1983; Packard and Packard,

1984; G. C. Packard et al., 1985; M. J. Packard et al., 1985; Packard and Packard, 1987).

In fact, eggs of many reptilian species must absorb water for successful embryonic

development (Packard et al., 1977; Tracy, 1980; Andrews and Sexton, 1981; Tracy and

Snell, 1985), which is at least partially taken up by albumen in some species (Tracy et

al., 1978; Tracy, 1980, 1982; Snell and Tracy, 1985).

Albumen proteins of avian eggs are hydrated and bind substantial quantities of

water. In the fowl, 88% of the weight of albumen is water (Shenstone, 1968; Osuga and

Feeney, 1977), which is three times the amount of water present in yolk and constitutes

the major source of water for development of the avian embryo. Comparatively little

water is exchanged with the atmosphere as water vapor, although there must be a net

loss of water from the avian egg in order for successful embryonic development (Rahn

and Ar, 1974; Ar and Rahn, 1978, 1980; Board, 1982).

In birds, albumen is initially secreted as a viscous, concentrated protein mixture

in the magnum, which later is saturated with "plumping" water by the shell gland (Breen

and de Bruyn, 1969; Wyburn et al., 1973; Solomon, 1983). This has also been observed

in turtles (Agassiz, 1857), who noted the swelling of albumen of in utero eggs. In

squamates, albumen may be relatively unhydrated at oviposition, but obtain water from

the substrate (Packard et al., 1977; Tracy, 1980; Tracy and Snell, 1985).


Reptilian Albumen Proteins

Egg albumen proteins exhibit a wide variety of functional properties. Alterations

in composition of proteins present in eggs will change the functional properties of the







albumen as a whole. This may have important consequences on the embryo's ability to

survive under different nest and incubation conditions (Muth, 1980; Palmer and

Guillette, 1990b). Embryos whose eggs have a protein composition with properties

suitable to available nest and incubation conditions will have a selective advantage. It is

well known that there is variability in composition of albumen proteins among eggs of

different avian (Sibley, 1970; Sibley and Ahlquist, 1972) and reptilian species (Palmer

and Guillette, 1990b).

When compared to birds, reptiles have more ancient origins, are found in

extremely diverse habitats, and exhibit various parity modes (oviparity versus viviparity),

suggesting a less conservative pattern of reproductive anatomy and physiology. This is

easily seen in the reproductive anatomy of reptiles (Fox, 1977), where studies on

oviductal anatomy in lizards (Guillette and Jones, 1985; Adams and Cooper, 1988;

Palmer and Guillette, 1988; Uribe et al., 1988; Guillette et al., 1989), snakes (Mead et

al., 1981), chelonians (Aitken and Solomon, 1976; Palmer, 1987; Palmer and Guillette,

1988) and crocodilians (Palmer and Guillette, unpublished data) have demonstrated

substantial differences in functional morphology among reptilian orders. In addition,

there also must be differences in physiological control among these reptilian groups

(Trauth and Fagerberg, 1984; Palmer and Guillette, 1988). These phylogenetic,

anatomical, and physiological differences may affect the types and ratios of

extraembryonic proteins.

The reptilian eggshell is variable in structure and permeability among reptilian

groups (Schleich and Kastle, 1988). This is important to the embryo in that the eggshell

is the mediator of environmental effects (Packard et al., 1977; Packard et al., 1982;

Packard and Packard, 1984; Ackerman et al., 1985a, b; Tracy and Snell, 1985; Packard

and Hirsch, 1986; Ratterman and Ackerman, 1989; Packard, 1990; Ackerman, 1990).

Some reptilian eggshells have extremely thick calcareous layers, and act as a substantial

barrier to water and gas exchange between embryo and environment. Other reptiles lay







eggs with little calcium outside the fibrous membranes (Schleich and Kastle, 1988;

Packard and DeMarco, 1990). These differences in eggshell structure may greatly affect

the role of extraembryonic proteins due to differences in water conductance rates and

need for antimicrobial agents, thus selecting for differences in protein composition of the

albumen (Packard and Packard, 1980; Tracy, 1980, 1982; Tracy and Snell, 1985; Palmer

and Guillette, 1990b).

The nest site also is extremely variable among reptilian species. Whereas most

birds exhibit some degree of parental care, most reptiles do not. In most cases, the eggs

are abandoned and allowed to develop without further parental assistance. Eggs may be

buried in a substrate or attached to exposed surfaces. Therefore, the egg must be

adapted to withstand large variations in moisture content and microbial communities.

Again, the need for extraembryonic fluids to buffer these different conditions is implied.

It is reasonable that since reptiles have more ancient origins, different reproductive

anatomies and physiologies, and diverse eggshell structures and nest conditions, their

eggs will exhibit much greater variations in albumen proteins than have been reported in

birds.

Reptilian albumen has been recently shown to exhibit substantial differences

among orders (Palmer and Guillette, 1990b). An ovotransferrin-like molecule has been

detected in turtle (Pseudemysfloridana) albumen (Palmer, 1988), based on molecular

weight data, although more rigorous identification is required. Neither ovalbumin nor

ovotransferrin was detected in eggs of Crocodylusporosus (Burley et al., 1987), although

a major protein of 59,000 molecular weight was identified and has been subsequently

detected in the eggs of both alligators and turtles (Palmer, unpublished data). An a2-

macroglobulin-like protein has been found in the eggs of Crocodylus rhombifer (Ikai et

al., 1983), C. porosus (Burley et al., 1987) and other reptiles (Palmer, unpublished data).

Although avidin synthesis was observed in oviducts of the lizard Lacerta s. sicula (Botte

et al., 1974; Botte and Granata, 1977), neither avidin, riboflavin-binding protein, nor







thiamin-binding protein has been detected in the albumen ofA. mississippiensis (Abrams

et al., 1988, 1989; White, 1990).

Clearly, the study of albumen proteins of reptiles is in its infancy. There appears

to be a greater diversity in composition of albumen proteins of reptiles (Burley et al.,

1987; Palmer, 1988, 1989; Abrams et al., 1989; Palmer and Guillette, 1990b) than that of

birds, and possibly also in the functional role of albumen in embryonic development.

Albumen of reptiles has been suggested to act in water storage (Tracy and Snell, 1985),

resist lethal rates of water exchange (Tracy and Snell, 1985), and possess antimicrobial

properties (Movchan and Gabaeva, 1967; Ewert, 1979). These properties, and the

protein bases for them, may have evolved in response to nest and incubation conditions

(Packard and Packard, 1980; Tracy, 1980, 1982; Tracy and Snell, 1985).


Albumen Protein Formation

The formation of albumen proteins has been extensively studied in the domestic

fowl, on which the following discussion is largely based. In birds, the majority of the

albumen is secreted by the magnum (Fig. 1-3) following ovulation, although initial layers

are formed in the infundibulum (Dominic, 1960; Wyburn et al., 1970). The egg remains

in the infundibulum for approximately 15-30 min and in the magnum for 2-3 hr (Warren

and Scott, 1935; Woodward and Mather, 1964). Anatomically, the infundibulum is

mostly aglandular, but "glandular grooves" occur posteriorly (Giersberg, 1923). True

tubular glands occur near the infundibulum/magnum junction which resemble "glandular

grooves" in general cell structure (Dominic, 1960), although characteristic differences

have been reported (Aitken and Johnston, 1963). Within the magnum, there are tubular

endometrial glands in addition to a secretary luminal epithelium (Richardson, 1935;

Wyburn et al., 1970). The endometrial glands are composed of three types of cells; A,

filled with electron dense granules, B, filled with low electron density secretary material

and C, with prominent Golgi bodies and extensive rough endoplasmic reticulum,
































Figure 1-3. Morphological characteristics of a typical avian (Gallus domesticus) oviduct.











ANTERIOR INFUNDIBULUM


POSTERIOR INFUNDIBULUM


MAGNUM



















TRANSITION ZONE





ISTHMUS






SHELL GLAND
WITH EGG


VAGINA







although types A and C may simply reflect different phases of secretary activity within

the same cell type (Wyburn et al., 1970).

Albumen proteins are secreted by specific cells within the magnum (Gilbert,

1979). Most albumen proteins are thought to be secreted by endometrial glands of the

magnum. Ovalbumin is secreted by A-cells whereas lysozyme is released by B-cells

(Kohler et al., 1968; Oka and Schimke, 1969; Wyburn et al., 1970). The cells of the

endometrial glands also are known to secrete ovotransferrin and ovomucoid (Schimke et

al., 1977), whereas the luminal epithelium secretes both avidin and ovomucin (Kohler et

al., 1968; Wyburn et al., 1970; Tuohimaa, 1975).

The magnum synthesizes the albumen proteins which it secretes, unlike yolk

proteins which are manufactured in the liver and transported to the ovary via the

circulatory system. This has been shown by in vitro incorporation of radiolabeled amino

acids into albumen proteins (ovalbumin, ovotransferrin, ovomucoid, lysozyme, and

riboflavin-binding protein, Mandeles and Ducay, 1962; avidin, O'Malley, 1967).

Additional work has shown in vivo incorporation of radioactive lysine and glycine into

ovalbumin, ovotransferrin and lysozyme (Mandeles and Ducay, 1962). Some albumen

proteins secreted by the oviduct are either identical (avidin) or similar (ovotransferrin) to

proteins found in other body tissues or fluids (Green, 1975; Chasteen, 1977, 1983; Aisen

and Listowsky, 1980; Elo and Korpela, 1984; Brock, 1985); however, these are known to

be synthesized directly by the oviduct and not transported there by blood (Williams,

1962; O'Malley, 1967).

The enormous amount of protein synthesized for egg albumen has given

molecular biologists an excellent model to study the regulation of genetic mechanisms

involved in protein formation. Under stimulation from the adenohypophysis, ovarian

follicles synthesize and release estrogen (Norris, 1985). This is transported to the

oviduct by the circulatory system and readily passes through the plasmalemma of cells in

the endometrial glands. Once inside the cell, estrogen is bound by a nuclear receptor







protein, which binds to chromatin, stimulating transcription (O'Malley et al., 1979;

Chambon et al., 1984; Gorski et al., 1987; Leavitt, 1989). Estrogen stimulates the gland

cells of immature chicks to differentiate (Brant and Nalbandov, 1956) and to synthesize

ovalbumin, ovotransferrin, ovomucoid, and lysozyme (Palmiter and Gutman, 1972;

Palmiter and Schimke, 1973). Further, treatment with progesterone and estrogen causes

the luminal epithelium to secrete avidin (Kohler et al., 1968). It has been demonstrated

that estrogen treatment stimulates transcription of the ovalbumin gene (Roop et al.,

1978).

The cellular mechanism of albumen release has recently been elucidated for

ovalbumin and ovotransferrin. Inhibition of glycosylation does not block the release of

ovalbumin or ovotransferrin (Kato et al., 1987), indicating that these proteins are free

within the endoplasmic reticulum (ER) and Golgi bodies, and not bound to the

organelle membranes. Further, inhibition of protein transport from the ER to the Golgi

bodies blocks release of these proteins, as may disrupting microtubules which have been

implicated in secretary granule transport (Kato et al., 1987). Thus, these data suggest

that ovalbumin and ovotransferrin are enclosed within secretary granules which are

transported from the golgi complex to the plasmalemma by microtubules.

The signal for secretion of albumen proteins is still poorly understood. The

pressure of the descending yolk on the oviductal wall is generally thought to stimulate

albumen secretion (Sturkie and Mueller, 1976; Laugier and Brard, 1980). Artificial

mechanical stimulation created by introducing objects into the oviduct does induce the

formation of albumen and eggshell, although these are grossly abnormal (Wentworth,

1960; Pratt, 1960). However, albumen is also secreted by isolated loops of the oviduct

while an egg passes through the intact portion (Burmester and Card, 1939,1941),

indicating that neuronal or endocrine stimulation may play a role in secretion. Although

the oviduct is known to be innervated by the autonomic nervous system, administration

of acetylcholine (parasympathomimetic) and ephedrine sulfate (sympathomimetic) had








negligible effects on albumen secretion (Sturkie and Weiss, 1950; Sturkie et al., 1954).

Diffusible yolk proteins can affect oviductal metabolism in vitro (Eiler et al., 1970), but

it is unknown if they can alter albumen synthesis in vivo. Steroid hormones, which are

involved in the ovulatory process, have been shown to induce albumen synthesis (see

above), but not release.

It remains to be determined how other hormones involved during ovulation, such

as arginine vasotocin and prostaglandins (PG), may be involved in albumen release.

Prostaglandins are potent paracrine hormones which influence blood flow and

contraction of the female reproductive tract (Poyser, 1981). Stretching the reproductive

tract causes PG release which can influence contractile patterns throughout the oviduct.

Prostaglandin synthesis immediately after ovulation may be essential for yolk transport

down the reproductive tract, release of oviductal proteins and egg rotation for normal

shell formation. The reproductive tract of lizards is capable of synthesis and secretion of

large amounts of PGF and PGE2 during the first 24 hrs after ovulation (Guillette,

unpublished data). In birds, a similar pattern is observed on a more rapid time scale

(Hertelendy et al., 1984). Clearly, mechanical stimulation of the oviduct alone is

insufficient to induce release of albumen around the descending yolk, but it may trigger

a complex series of paracrine events responsible for albumen and eggshell protein

release. Further research into the mechanisms of protein release needs to be conducted.

Albumen of avian eggs is composed of four distinct portions: the chalaziferous

layer, which is attached to the yolk and suspends it, the inner liquid layer, the thick

layer, and the outer thin layer. These different layers are not obvious during their

formation in the magnum. The layers are the result of secretion of different

components by consecutive regions of the oviduct (Gilbert, 1971), by the mechanical

action of the egg twisting as it descends through the oviduct (Romanoff and Romanoff,

1949) and chemical changes during plumping in the shell gland (Sturkie and Mueller,

1976). The chalaza is formed by the mechanical twisting of ovomucin fibers as the egg







spirals down the magnum (Conrad and Phillips, 1938; Scott and Huang, 1941). The thin

albumen layers are produced by addition of plumping water after the egg enters the

shell gland (Sturkie and Polin, 1954). The fibrous chalazae and gelatinous nature of the

thick albumen are due to their high concentration of ovomucin (Robinson, 1987).

In contrast to birds, the formation of albumen proteins in reptiles is poorly

understood. Avidin is the only albumen protein detected in oviductal secretions of a

reptile, the lizard Lacerta s. sicula (Botte et al., 1974), and is induced by combined

effects of estrogen and progesterone, as in the fowl (Botte et al., 1974; Botte and

Granata, 1977). Poly(ADPribose)transferase (ADPRT) activity, an indicator of gene

expression, increases under estrogen stimulation in the oviduct of the lizard Podarcis s.

sicula, and is maximal at oviductal morphological maturity (Ciarcia et al., 1986).

Enhanced ADPRT activity precedes protein synthesis in immature quail oviducts under

estrogen stimulation (Miller and Zahn, 1976). Clearly, much work needs to be done on

the process of albumen synthesis and secretion in reptiles.


Eggshell Membranes

The proteinaceous fibers of the eggshell membranes play an important role in

the maintenance of a suitable environment for the developing embryo. The eggshell

membrane exhibits several functions, which are due to the mechanical structure of the

membrane as a whole, rather than from individual chemical properties, as with albumen.

In fact, the protein of the eggshell membrane has proven difficult to investigate as it is

highly insoluble (DeSalle et al., 1984). The amino acid composition of eggshell

membrane protein is known for a variety of species, including fowl (Candlish and

Scougall, 1969; Balch and Cooke, 1970; Britton and Hale, 1977; Starcher and King,

1980; Crombie et al., 1981; Leach et al., 1981), green sea turtle, Chelonia mydas

(Solomon and Baird, 1977), and lizards, Iguana iguana (Cox et al., 1982), Eumeces

fasciatus and Opheodrys vernalis (Cox et al., 1984), but the protein has been assigned to

various classes over the years, including keratin (Balch and Cooke, 1970; Britton and







Hale, 1977), collagen (Candlish and Scougall, 1969; Wong et al., 1984) and elastin

(Starcher and King, 1980; Leach et al., 1981; Crombie et al., 1981). This has led to the

conclusion that eggshell membranes are composed of unique proteins (Leach, 1978;

Tullett, 1987).

Eggshell membranes have a variety of functions in reptiles, including water

balance (Tracy, 1980, 1982; Tracy and Snell, 1985), support (Packard and Packard,

1980), and microbial defense (Packard and Packard, 1980; Tracy and Snell, 1985). As

part of the eggshell, the fibrous membrane acts in water balance by forming a barrier to

gas exchange and increasing the diffusion distance, thereby slowing down water

movement (Ackerman et al., 1985a, b). The membrane, however, has no effect on

direction of water movement, which is determined by relative osmotic potential across

the eggshell. The fibrous membrane also serves to maintain egg shape, support egg

contents, and protect the embryo from mechanical injury (Packard and Packard, 1980;

Tracy, 1982). Since fibers of the membrane form a dense crisscrossing mat, they act as

a barrier to invading bacteria and fungi (Board and Tranter, 1986). Functions of

eggshell membranes may be enhanced by the organic matrix of protein and

carbohydrates surrounding the fibers. It is likely that albumen and eggshell coevolved in

response to the environmental conditions to which eggs were subjected (Packard and

Packard, 1980; Tracy, 1980, 1982; Tracy and Snell, 1985).

Fibers are produced by endometrial glands of the isthmus in birds (Misugi and

Katsumata, 1963; Simkiss, 1968; Simkiss and Taylor, 1971; Solomon, 1975; Aitken, 1971;

Draper et al., 1972; Solomon, 1983). Secretory granules are released into the lumen of

the endometrial glands and coalesce as they are extruded from the glandular duct into

the oviductal lumen (Solomon, 1983). It has been proposed that the uterus of reptiles

produces the fibrous membranes (Aitken and Solomon, 1976; Palmer and Guillette,

1988, 1990b; Guillette et al., 1989). These fibers, in both birds and reptiles, have been

described as possessing a protein core with a mucopolysaccharide mantle (Simons and







Wiertz, 1963; Candlish, 1972; Solomon and Baird, 1977). However, in some reptilian

eggs, proteinaceous fibers lack the mantle and exhibit a pitted appearance (Solomon and

Baird, 1977; Andrews and Sexton, 1981; Trauth and Fagerberg, 1984; Sexton et al., 1979;

DeSalle et al., 1984).

Birds are sequential ovulators, and each layer of the eggshell is applied by

subsequent regions of the oviduct. The entire process takes approximately 22-25 hours

(Aitken, 1971) and is complete before the egg passes through the vagina. Most reptiles

are simultaneous instead of sequential ovulators. This implies that there are also

differences between the physiological processes of eggshelling in reptiles and birds

(Palmer and Guillette, 1990b). Eggshell formation is disrupted in the lizard

Cnemidophorus uniparens if ovulation is followed by deluteinization (Cuellar, 1979).

Although the thickness of the fibrous layer remains unchanged, its structure is altered.

Particularly, only thick fibers are produced, instead of a gradation of thick to thin fibers.

Upon oviposition, the eggs leak fluid and dehydrate quickly. Deluteinization decreases

plasma progesterone concentration and increases myometrial activity (Jones et al., 1982;

Guillette and Fox, 1985; Fox and Guillette, 1987). How the corpus luteum and

progesterone concentrations affect eggshell fiber formation and what physiological

mechanisms control the sequential formation of eggshell fibers and calcareous layer in

the reptilian uterus remain to be studied.













CHAPTER II
ULTRASTRUCTURE AND FUNCTIONAL MORPHOLOGY
OF TURTLE OVIDUCTS


Oviductal ultrastructure and functional morphology are poorly understood in

reptiles, particularly with respect to albumen deposition and eggshell formation. Studies

on the green sea turtle, Chelonia mydas (Aitken and Solomon, 1976), gopher tortoise,

Gopheruspolyphemus (Palmer and Guillette, 1988), and the lizards, Eumeces obsoletus

and Crotaphytus collaris (Guillette et al., 1989) suggest that although albumen is formed

in a region (tube) structurally similar to that of birds (magnum), eggshell formation is

quite different. In these reptiles, there is only one region (uterus) in which all layers of

the eggshell are formed, whereas in birds the fibrous membrane and calcareous layers

are formed sequentially in separate regions, the isthmus and shell gland, respectively.

Only two studies have examined reptilian oviductal ultrastructure using

transmission electron microscopy (TEM), although it is a fundamental tool in the study

of functional morphology. One of these was limited to the sperm storage glands of the

garter snake Thamnophis sirtalis (Hoffman and Wimsatt, 1972), whereas the other

examined the tubal and uterine structure of the green turtle Chelonia mydas (Aitken

and Solomon, 1976), but was based on only two specimens. Previous works on

chelonian oviductal functional morphology (Aitken and Solomon, 1976; Palmer and

Guillette, 1988) are based on species whose populations are endangered or reduced in

size. Therefore, they are inappropriate for detailed ultrastructural and physiological

examination of albumen and eggshell formation. This study examined oviductal

ultrastructure at the histological and TEM level using species that are readily available

stinkpott turtles, pond sliders, and painted turtles) throughout the United States, either

by field collection or through dealers. This will enable future physiological studies on







these species to be interpreted in relation to known functional morphological

characteristics.


Methods and Materials


Specimens

Three species of common water turtles were used for this study. Female pond

sliders (Pseudemys scripta; n=5) and stinkpot turtles (Stemotherus odoratus; n=5) were

caught using baited funnel traps in Alachua county, Florida. Additionally, female

painted turtles (Chrysemyspicta; n=10) were obtained from dealers. The specimens

were anesthetized using 10mg/kg pentobarbital i.p., and the oviducts and ovaries

surgically excised. Oviductal regions were identified as infundibulum, tube (tuba

uterina), transition zone, uterus, and vagina (Palmer and Guillette, 1988).

Representative samples of those regions involved in albumen and eggshell formation

(tube and uterus) were prepared for histochemistry and electron microscopy.


Histochemistry

Tissue biopsies were fixed in 10% neutral buffered formalin or Bouin's fixative,

washed, transferred through graded alcohols, cleared in xylene, and embedded in

paraffin (Humason, 1979). Specimens were serial sectioned at 7pm for a total of 10

slides (approximately 200 sections) and stained as in Table 2-1.


Electron microscopy

Specimens for electron microscopy were fixed in 2% glutaraldehyde in 0.1M

cacodylate HCI buffer for 3 hrs. For scanning electron microscopy, the tissues were

washed in 0.1M cacodylate buffer (3 X 20 min.), dehydrated in graded alcohols (1 hr

ea), critical point dried (Anderson, 1951), and sputter coated with gold. Examination

was performed on a Hitachi S-450, operated at 15kV.















Table 2-1. Staining techniques employed and their general
interpretation.


Hematoxylin & eosin

Alcian blue (pH 2.5)

Fast green

Orange G.

Beibrich scarlet


Nuclei and general cytology

Glycosaminoglycans

Connective tissue

Red blood cells

Muscle, and
proteinaceous secretary material


1Humason, 1979







For transmission electron microscopy (TEM), oviductal tissues were minced

(1mm3), and fixed as above. Specimens were then washed in cacodylate buffer (3 X 15

min), fixed in 1% OsO4 for 30 min, washed in cacodylate buffer (3 X 15 min),

dehydrated in graded alcohols (15 min ea), treated with 100% acetone (2 X 1 hr), and

embedded in Spurr's resin. Thick sections (1.Am) were prepared and stained with

toluidine blue. Thin sections were cut on a LKB ultramicrotome at 600A, and

poststained with uranyl acetate and lead citrate. Examination of tissues was performed

on a Hitachi HU-11E or a Philips EM-300.


Results


Luminal Epithelium

The general morphological characteristics of the female turtle reproductive tract

are presented in Fig. 2-1. The luminal epithelium of the tube and uterus consists of

microvillous secretary and ciliated cells (Fig. 2-2A, B). The ciliated cells have central

nuclei, are eosinophilic, and are typically compressed laterally into a funnel-like shape,

with the apical end expanded (Fig. 2-3A). Most ciliated cells are nonsecretory, although

some have electron light secretary granules (Fig. 2-3B). The cilia exhibit a typical 9+0

triplet microtubular arrangement below the cell surface and a 9+2 doublet arrangement

once they penetrate the plasmalemma (Fig. 2-4A). The apical membrane of the ciliated

cells have some microvilli, particularly if an apical cone is present, which projects

outward from the center of the cell (Fig. 2-4B). The lateral plasmalemma of the

luminal epithelial cells shows extensive interdigitations, tight junctions, and desmosomes

(Fig. 2-4A).

The secretary epithelial cells stain positively with Alcian blue (pH 2.5) for

glycosaminoglycans (GAGs). Ultrastructural examination reveals that there are two

distinct types of microvillous secretary cells. The first is characterized by a basal nucleus

with prominent nucleolus and abundant membrane-bound electron-light secretary































Figure 2-1. Gross morphology of the turtle oviduct. Modified from Palmer and
Guillette, 1988.













ANTERIOR INFUNDIBULUM





POSTERIOR INFUNDIBULUM


















TUBE


TRANSITION ZONE


UTERUS






























Figure 2-2. Histology and scanning electron microscopy of the luminal epithelium of the
turtle oviduct, showing ciliated cells and secretary cells. A: Paraffin histology of
the luminal epithelium (2,000X). B: SEM of the apical membrane of the luminal
epithelium (8,000X). E, epithelium; G, endometrial glands.















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Figure 2-7. The lamina propria surrounding the endometrial glands of both the uterus
and tube is highly vascularized. A: Transmission electron micrograph of an
arteriole adjacent to the tubal endometrial glands (4,250X). B: Scanning electron
micrograph of a small uterine endometrial blood vessel (1,200X). E, erythrocyte;
G, endometrial gland; arrow, endothelial cell.














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Figure 2-8. Histology and ultrastructure of the tubal endometrial glands. A: The
endometrial glands (G) are branched acinar or branched tubular, and occupy the
entire lamina propria between the luminal epithelium and the myometrium
(1,800X). B: Most tubal endometrial glands are characterized irregularly shaped,
electron dense secretary granules (10,000X). N, nuclei; SG, secretary granules.






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Figure 2-10. Histology and ultrastructure of the uterine endometrial glands. A: The
uterine endometrial glands are branched tubular, connected to the lumen by
short ducts (500X). B: The glands are characterized by spherical, electron dense
secretary granules and numerous mitochondria (11,375X). N, nucleus; SG,
secretary granules.


































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granules throughout the cytoplasm (Fig. 2-5A), which are released through exocytosis

(Fig. 2-5B). Frequently, a paranuclear vacuole is present immediately apical to the

nucleus (Fig. 2-5A), as are lipid droplets in the narrow zone between the nucleus and

basal plasmalemma. The second type of microvillous secretary cell is characterized by

basal nuclei, but these cells have electron-dense secretary granules near the apical

membrane (Fig. 2-6A), which are secreted by exocytosis (Fig. 2-6B), but no paranuclear

vacuoles have been identified.


Endometrial Glands

The lamina propria below the glands is highly vascularized, with the vessels in

proximity to the glands (Fig. 2-7A, B). The endometrial glands of the tube are branched

acinar or branched tubular (Fig. 2-8A). The endometrial gland cells are cuboidal and

have extensive secretary granules, and a basal, irregularly shaped nucleus. The secretary

vesicles, which fill most of the cytoplasm apical to the nuclei, are irregular in size and

shape, but usually electron dense (Fig. 2-8B). These cells are further characterized by

having abundant rough endoplasmic reticulum (RER) and ribosomes surrounding the

nucleus. The lateral plasmalemma is extensively interdigitated. However, some cells

possess secretary granules with various electron densities (Fig. 2-9A). The apical

membrane of both types of endometrial gland cells bear numerous short microvilli (Fig.

2-9B).

The endometrial glands of the uterus are distinct from those of the tube. These

are branched tubular glands with a duct that is histologically similar to the luminal

epithelial cells (Fig. 2-10A). The glandular cells are cuboidal, with numerous

eosinophilic secretary granules. The nuclei are basal and spherical, instead of irregular

in shape as in the tube. The secretary granules are dispersed throughout the cytoplasm,

and are uniformly spherical and electron dense (Fig. 2-10B). The cytoplasm is

characterized by extensive RER, Golgi bodies and mitochondria. Further, the lateral







membranes show fewer interdigitations than observed for the tubal glands. The apical

membranes of the endometrial glandular cells exhibit microvilli.


Discussion


The formation of albumen and eggshell has been extensively studied in birds, in

which the albumen, shell membranes and calcareous shell are formed sequentially as the

egg passes through separate regions of the oviduct; magnum, isthmus, and shell gland,

respectively (Solomon, 1983). Although the same basic components are present in

reptilian eggs, regions similar to those observed in birds have not been identified in the

chelonian oviduct. Additionally, all eggs of a clutch are ovulated simultaneously in

turtles, instead of sequentially as in birds. These differences have suggested the

hypothesis that albumen and eggshell formation is substantially different in birds and

turtles (Aitken and Solomon, 1976; Palmer and Guillette, 1988).


Luminal Epithelium

Several distinct types of luminal epithelial cells blebb secretary cells, secretary

and nonsecretory ciliated cells, two types of microvillous secretary cells) have been

identified in the oviducts of turtles. Within the infundibulum and tube, different

components of the albumen may be produced by these cell types. Bleb secretary cells,

found within the grooves of the posterior infundibulum, have previously been identified

in infundibular grooves of the lizard Hoplodactylus maculatus (Boyd, 1942) and the

tortoise, Gopheruspolyphemus (Palmer and Guillette, 1988). These cells may be

homologous to the cells of the glandular grooves in the infundibulum of birds (Aitken

and Johnston, 1963, Aitken, 1971). Although the ultrastructure of bleb secretary cells in

reptiles remains unknown, they may function in the formation of initial albumen layers

in the infundibulum (Palmer and Guillette, 1988, 1990b) as occurs in birds (Aitken and

Johnston, 1963; Aitken, 1971).







The ciliated cells have distinctive secretary granules, which also have been noted

in avian oviducts (Gilbert, 1979; Solomon, 1983), but were not described in the green

turtle Chelonia mydas (Aitken and Solomon, 1976). The ultrastructure of the cilia is

similar to that found in the sea turtle (Aitken and Solomon, 1976).

The microvillous cells with electron light secretary material resemble

ultrastructurally that of mucus (glycosaminoglycan) secreting cells of the avian magnum

(Wyburn et al., 1970; Gilbert, 1979). Although these cells are involved in mucus

secretion, they do not have the compressed basal nuclei and cytology of typical goblet

cells. The general histological appearance of the tubal epithelial cells and their positive

staining for GAGs further resembles the avian magnum cells (Solomon, 1971; Wyburn et

al., 1970). These mucus secreting cells in the avian oviduct have been identified as

secreting avidin and ovomucin (Kohler et al., 1968; Wyburn et al., 1970; Tuohimaa,

1975). Avidin has also been identified in the secretions of the tube in lizards (Botte et

al., 1974; Botte and Granata, 1977).

The other microvillous secretary cells, which have electron dense secretary

material, are typical of glycoprotein secreting cells of the avian oviduct (Wyburn et al.,

1973; Gilbert, 1979). These cells are found in the tube of the reptilian reproductive

tract, although in birds they are not found in the magnum. Structurally similar cells

were identified in the oviduct of the garter snake (Hoffman and Wimsatt, 1972).

Unfortunately, the luminal secretary epithelial cells were not described in the sea turtle

(Aitken and Solomon, 1976). The function of these cells is not clear, although in the

tube they may secrete some albumen proteins, which are thought to be mostly

glycoproteins.


Endometrial Glands

The endometrial glands of the turtle tube are ultrastructurally and

histochemically like those of the avian magnum, supporting earlier hypotheses that the

tube is involved in albumen formation in reptiles (Aitken and Solomon, 1976; Palmer







and Guillette, 1988; Guillette et al., 1989). In birds, the endometrial glands of the

magnum are composed of three types of cells; A-cells, filled with electron dense

granules, B-cells, filled with low electron density secretary material and C-cells, with

prominent Golgi bodies and extensive RER. Types A and C may merely reflect

different activity phases of the same cell type (Wyburn et al., 1970). The different cell

types of the avian magnum are identified as secreting specific albumen proteins; A-cells

secrete ovalbumin whereas lysozyme is secreted by B-cells (Kohler et al., 1968; Oka and

Schimke, 1969; Wyburn et al., 1970). No secretary protein for C-cells has been

identified. Most of the tubal endometrial gland cells in turtles have secretary granules

that are similar to the avian type A-cells, which are electron dense. However, some

tubal gland cells have lighter material intermixed with the dense secretary granules. This

electron light material may (1) represent a different stage in processing of the secretary

product, (2) be similar to the avian type B-cell secretary material, which is also electron

light (Wyburn et al., 1970; Gilbert, 1979), or (3) be a unique secretary product. The

latter two hypotheses suggest that the cells of the turtle magnum are less specialized

than those of birds, with some cells producing a mixture of albumen proteins. Clearly,

detailed biochemical studies are needed to determine the composition of reptilian

albumen. Moreover, immunochemical studies are needed to resolve the secretary nature

of the tubal epithelia and endometrial glands.

The homogeneity of endometrial gland cell structure along the reptilian tube

suggests that the albumen secreted from different portions of the tube is also relatively

uniform. In contrast, along the avian magnum separate structurally distinct subregions

can be identified, which correspond to the characteristic layering of the albumen into

thick and liquid albumen layers and fibrous chalaza (Aitken, 1971; Gilbert, 1979;

Solomon, 1983). Reptilian eggs lack a chalaza, and the albumen appears relatively

uniform in consistency, although it is laid down in concentric layers around the yolk

(Webb et al., 1987; Palmer, unpublished data).







The formation of proteinaceous fibers of the eggshell membrane is not

understood in reptiles. The uterine endometrial glands of turtles possess spherical,

electron dense secretary granules and general cytological features that are comparable to

those of the avian isthmus (Draper et al., 1972; Gilbert, 1979; Solomon, 1983), which

secrete the eggshell fibers. This agrees with the findings of Aitken and Solomon (1976)

in the sea turtle, and implies that the uterine endometrial glands of turtles, and possibly

other reptiles as well, secrete the fibers of the proteinaceous eggshell membrane, as

occurs in the isthmus of birds.

However, the eggshell fibers of birds and many reptiles are composed of two

parts, an inner proteinaceous core and an outer carbohydrate sheath. This implies

involvement of both RER (for syntheses of the proteinaceous components) and Golgi

bodies (which are involved in glycosylation proteins and packaging carbohydrates for

secretion) in fiber formation (Aitken and Solomon, 1976). The uterine endometrial

glands of turtles show both well developed RER and Golgi bodies, suggesting that they

are responsible for formation of both layers of the fibers. In some reptiles, the fibers

appear pitted under TEM, which may represent a crude mixture of proteinaceous and

carbohydrate fiber components (Solomon and Baird, 1977; Sexton et al., 1979; Andrews

and Sexton, 1981; DeSalle et al., 1984; Trauth and Fagerberg, 1984). The fibers of the

green turtle are pitted (mixture of protein and carbohydrate) while retained in the

oviduct, but show the typical protein core and carbohydrate sheath morphology following

oviposition, suggesting that all components are secreted simultaneously, but segregate

over time (Solomon and Baird, 1977). This is similar to the fiber formation in birds, in

which both components are present in the lumen of the glands (Solomon, 1983).

It remains to be determined how the eggshell membrane proteins are formed

into a fibrous membrane surrounding the yolk in reptilian eggs. Agassiz (1857)

concluded that the eggshell fibers polymerize directly around the yolk in turtles. This

was later reported for various other reptiles (Giersberg, 1923; Weekes, 1927). However,







in birds, the eggshell proteins polymerize within the lumen of the endometrial glands

and are extruded from the glands' duct as intact fibers (Solomon, 1983).

Although it is known that the reptilian uterus secretes the calcareous layer of the

eggshell, the mechanism of calcium deposition remains to be determined. The uterine

glands have little resemblance to those of the avian shell gland, which have been

proposed to secrete the calcareous shell (Breen and de Bruyn, 1969). However, others

contend that the luminal epithelium of the avian shell gland is responsible for calcium

ion secretion (Gay and Schraer, 1971; Solomon et al., 1975), and that the endometrial

glands are involved in "plumping water" transport (Solomon, 1983), the dilution of the

albumen proteins by the addition of water and possibly enzymes (Solomon, 1979).

Recent evidence in lizards suggests that the luminal epithelium of the reptilian uterus is

the source of calcium ions for eggshell formation (Guillette et al., 1989). These data

support the hypothesis that the uterus in turtles and lizards is dualistic in function,

producing both the fibrous membrane and calcareous shell while eggs are held in utero

(Aitken and Solomon, 1976; Palmer and Guillette, 1988, 1990b; Guillette et al., 1989),

the endometrial glands forming the fibrous membranes and the luminal epithelium

secreting the calcareous layer.

How the uterus is able to accomplish eggshell formation on an entire clutch

simultaneously is unknown. There is frequently a spatial separation of eggs undergoing

shelling along the length of the uterus, each within its own "egg chamber". This spatial

separation may allow each egg to be shelled in isolation from the others in the uterus.

The physiological controls that are involved in the secretion of both fibrous membranes

and calcareous eggshell by the uterus remain unknown.













CHAPTER III
OVIDUCTAL MORPHOLOGY AND EGGSHELL FORMATION
IN THE LIZARD, SCELOPORUS WOODI


Recent studies have suggested that eggshell formation in reptiles occurs strictly in

the uterus (Aitken and Solomon, 1976; Fox, 1984; Palmer and Guillette, 1988; Guillette

et al., 1989). This is in contrast to the pattern of eggshell formation in birds, in which

the proteinaceous fibers are produced by the isthmus and the calcareous layer is

subsequently secreted within the shell gland (Solomon, 1983). The endometrial glands

of the chelonian uterus, and possibly of other reptiles as well, are ultrastructurally similar

to those of the avian isthmus, possessing spherical, electron dense secretary granules

(Chapter II). The uterine luminal epithelium has been shown to stain histochemically

for calcium during gravidity in the lizard Crotaphytus collaris, suggesting that the luminal

epithelium secretes the calcareous eggshell (Guillette et al., 1989). It has therefore been

hypothesized that the reptilian uterus is dualistic in function, first producing the fibers of

the eggshell membrane from the endometrial glands, and subsequently secreting calcium

ions from the luminal epithelium for deposition of the calcareous eggshell layer while

the eggs are retained in the uterus (Fox, 1984; Guillette and Jones, 1985; Palmer and

Guillette, 1988; Guillette et al., 1989).

The mechanism for fiber formation in reptiles has received very little attention in

recent years. Agassiz (1857) conducted extensive examinations on the formation of

albumen and eggshell in a variety of turtles. He concluded that the proteins of the

eggshell membranes gradually polymerized directly on the egg surface, first appearing as

small particles that continue to increase in size and coalesce with one another until a

long fiber is formed. This theory was later supported in several species of reptiles

(Giersberg, 1923; Weekes, 1927). Recent authors have not speculated on how the fibers







were formed, but describe the fibers as branched and interwoven (Packard et al., 1988),

which presumably could only occur by polymerization of fibers directly surrounding in

utero eggs.

This hypothesis explaining fiber formation in reptiles is quite different from the

pattern observed in birds, which has been extensively studied. It has been conclusively

shown that the fibers of the eggshell membrane are produced by the endometrial glands

of the avian isthmus, and that the proteinaceous material coalesces within the lumen of

the gland (Solomon, 1983). The fused material is extruded from the glandular duct as

an unbranched, completely formed fiber, which is wrapped around the egg by the

twisting action of the myometrium. Definitive evidence for the mechanism of formation

of the fibrous eggshell membrane remains to be determined in reptiles.

This study used the lizard Sceloporus woodi to examine oviductal functional

morphology and the process of eggshell formation throughout gravidity, in order to

assess the role of the uterus in eggshell formation. These results are correlated with a

concurrent study on the timing and sequence of eggshell formation in the same

specimens (DeMarco and Palmer, unpublished data).


Methods and Materials


Specimens

Florida scrub lizards (Sceloporus woodi) were collected within the Ocala National

Forest, FL, and palpated to determine relative reproductive condition. Eighteen mid- to

late vitellogenic (3-5mm diameter follicles) females were returned to the laboratory,

where they were housed with mature males, maintained under natural light conditions,

and fed ad libitum. As the females neared the end of vitellogenesis, they were palpated

approximately every 4 hours to determine the time of ovulation. Following ovulation,

females were killed by decapitation at selected time intervals (Table 3-1), and their

oviducts (with eggs in situ) removed and fixed in 10% neutral buffered formalin.












Table 3-1. Identification of the females used in study of oviductal functional
morphology and eggshell formation.


Specimen # Duration from ovulation
(days or hours)


3-18 vitellogenic

3-19 vitellogenic

5-12 9 (hrs)

5-9 12"

4-17 1 day

4-20 2"

5-8 2"

3-9 3"

7-17 3 "

6-15 4"

7-16 5"

4-6 6"

5-11 7"

6-13 9 "

2-14 11"

2-17 14"

2-13 15"

4-18 post oviposition







Histochemistry

Following fixation, the oviducts were dehydrated in graded alcohols, cleared in

xylene, and embedded in paraffin (Humason, 1979). The tissues were serial sectioned at

7.im for a total of 10 slides (approximately 200 sections), and stained using various

techniques (Table 2-1).


Scanning Electron Microscopy

The oviducts were fixed and dehydrated as for histology and CO2 critical point

dried (Anderson, 1951). The specimens were sputter coated with a gold-palladium alloy

in a Denton Vacuum Desk-1. Examination and analyses were performed on a Hitachi

S-415A SEM operated at 15kV.


Results


The infundibulum-tube is thin and flaccid, lacking endometrial glands, although

posteriorly the endometrium forms longitudinal grooves. Throughout the infundibulum-

tube and the uterus, two types of cells are apparent, ciliated and microvillous secretary.

In the infundibulum proper, the epithelium is composed of cuboidal, eosinophilic cells,

whereas in the tube, the secretary cells become slightly hypertrophied and distended,

compressing the shorter ciliated cells (Fig. 3-1A, B). The uterine luminal epithelium also

consists of ciliated and microvillous secretary cells (Fig. 3-2A). Extensive branched

saccular or branched tubular glands are present in the uterine endometrium (Fig. 3-2B),

with spherical, eosinophilic secretary granules. Posteriorly, the vagina is aglandular, with

extremely tall, thin, longitudinal folds (Fig. 3-3A). The vaginal epithelium is almost

entirely ciliated (Fig. 3-3B).

The oviductal changes occurring during gravidity are correlated with events

following ovulation. The egg is coated with oviductal secretions as soon as it enters the

ostium, as indicated by an egg already coated with secretary material when it had only

partially entered the oviductal ostium from the coelomic cavity. Once the eggs are in







the uterus, deposition of the fibrous membranes of the eggshell are evident. By 9 hours

post-ovulation, long, unbranched proteinaceous fibers are extruded from the ducts of

most of the endometrial glands (Fig. 4A, B). At any one location, the fibers are being

pulled in the same direction across the luminal epithelium, as is evident by the

indentations formed in the apical membranes of adjacent epithelial cells. Most glands

have stopped secreting long, continuous fibers by 24 hours post-ovulation, with only

short pieces of fibrous material obvious in the mouths of glands and littering the surface

of the luminal epithelium. Also by this time, the fibrous eggshell membrane is largely

complete, with stratified layers of inner thick fibers, middle thin fibers, and an outer

layer of proteinaceous particles (DeMarco and Palmer, unpublished data; Fig. 3-5A).

For up until 6 days post-ovulation, small fragments of fibrous material can still be

observed extruding from the ducts of occasional endometrial glands (Fig. 3-6B).

The luminal epithelium shows little hypertrophy during the first 24 hours post-

ovulation (Fig. 3-6A). The apical membranes are relatively flat, with short microvillous

projections. Later during gravidity, however, the apical membranes of the secretary cells

become greatly distended as the cells hypertrophy, and the microvilli become less

pronounced (Fig. 3-6B). By the end of gravidity, the epithelial cells have returned to a

less distended appearance.


Discussion


By examining the functional morphology of the oviduct throughout the course of

gravidity, several hypotheses concerning the formation of the shell are supported.

Although it has been previously suggested that the eggshell fibers polymerize around the

egg (Agassiz, 1857; Giersberg, 1923; Weekes, 1927) by an unknown mechanism that is

distinct from that of birds, our data strongly support the hypothesis that the formation of

the eggshell fibrous membrane in the lizard Sceloporus woodi, and perhaps other

reptiles as well, occurs by a mechanism similar to that in birds. Indeed, the extrusion of































Figure 3-1. The luminal epithelium of the infundibulum and tube consists of ciliated
and secretary cells. A: Histology of the infundibulum (2,000X). B: Scanning
electron micrograph of the tubal epithelium, showing the distention of the
secretary cells (2,400X). CT, connective tissue; E, luminal epithelium.






67



















































-Op





























Figure 3-2. Histology and scanning electron microscopy of the uterus. A: The uterine
luminal epithelium consisting of ciliated and microvillous secretary cells (2,400X).
B: The uterus is characterized by branched acinar or branched tubular glands
(2,OOOX).






































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fcAA


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w ~


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Figure 3-3. Histology and scanning electron microscopy of the vagina. A: The vagina is
aglandular and the endometrium forms tall, longitudinal folds (1,250X). B: The
luminal epithelium of the vagina is largely composed of ciliated cells (4,800X).
E, luminal epithelium.























14


* n.


t ;r
~0-,


tl


~I

I
r,

1~


W-






























Figure 3-4. Formation of the fibrous eggshell membrane. A: Extrusion of the eggshell
fibers from the ducts of the uterine endometrial glands. The indentations are
evident (arrows) where the fibers rested during eggshell formation (1,600X). B:
Scanning electron micrograph of the extrusion of the eggshell fibers (6,000X).




73















-A.>A.





a4t



qr6




























Figure 3-5. Structure of the eggshell of Sceloporus woodi. A: Cross-section of the
eggshell, showing the inner boundary membrane, inner thick fibers, middle thin
fibers, and the outer particulate matter (4,000X). B: Surface of the eggshell with
deposition of blocks of calcium carbonate apparent (1,000X). Ca, calcium
deposits.








P 4V 4 *.a


pY~~i.~


inn


- --


^^P~~j^--n% '%*'^^
S i *
wwLo


=r


..'-.5.-:





























Figure 3-6. Changes in the gross morphology of the uterine epithelium during eggshell
formation. A: The secretary cells of the luminal epithelium at 9 hrs post-
ovulation, during formation of the fibrous eggshell membrane (2,400X). B: At 6
days post-ovulation, when calcium deposition is at its greatest, the luminal
secretary cells bulge outward. A short fragment of eggshell fiber (arrow) is still
apparent in the mouth of a glandular duct (1,200X).









h.
-< -


N
n.-"N


Is
r4i


--,43o
101







intact fibers from the endometrial glands, which are similar in both morphological

appearance and dimension to those present on the surface of the egg undergoing shell

formation, is obvious. In a process that is similar to that of birds, the proteinaceous

material secreted by the endometrial glands coalesces into a fiber as it is forced out of

the neck of the gland (Solomon, 1983). The intact fiber must than be wrapped around

the egg by an undetermined mechanism. Since fibers are pulled in the same direction

away from the glandular duct, it is likely that the activity of the myometrium is rotating

the egg within the oviduct, or at least siding the oviductal wall across the egg's surface.

It is known that the reptilian myometrium is highly active following ovulation (Guillette,

Matter and Palmer, unpublished data). Since the egg is within the uterus within 9 hours

post-ovulation (when a substantial layer of fibers has already been deposited), and the

fibrous membrane of the eggshell is essentially complete by 24 hours post-ovulation, the

deposition of fibers appears correlated with the maximum activity of the myometrium

(Guillette, Matter and Palmer, unpublished data).

The mechanism for deposition of albumen and formation of eggshells on an

entire clutch simultaneously by the reptilian oviduct remains unclear. Albumen

deposition begins immediately as the eggs enter the oviduct, as indicated by an egg that

was only half inside the ostium that already had secretary material (albumen) deposited

on the portion inside the duct. This indicates that the infundibulum-tube forms the egg

albumen. However, the stimulus for albumen release is unknown, even in birds. The

pressure of the descending yolk on the oviductal wall is generally thought to initiate

albumen secretion in birds (Sturkie and Mueller, 1976; Laugier and Brard, 1980).

Mechanical stimulation created by the introduction of foreign objects into the oviduct

does induce albumen and eggshell formation in birds, although these are grossly

abnormal (Wentworth, 1960; Pratt, 1960). However, isolated loops of avian oviduct also

secrete albumen while an egg passes through the intact portion (Burmester and Card,

1939, 1941), indicating that neuronal or endocrine stimulation may affect albumen







secretion. Steroid hormones have been shown to induce albumen synthesis, but not

release (Palmiter and Gutman, 1972; Palmiter and Schimke, 1973). Although the avian

oviduct is innervated by the autonomic nervous system, administration of acetylcholine

(parasympathomimetic) and ephedrine sulfate (sympathomimetic) had negligible effects

on albumen secretion (Sturkie and Weiss, 1950; Sturkie et al., 1954). Prostaglandins

(PGs) may be involved in initiation of protein release, as stretching of the tissues (such

as caused by descending eggs) triggers PG secretion in reptilian tissues (Guillette and

Palmer, unpublished data), which may signal neighboring cells to release albumen

proteins. Additionally, explant tissue cultures of avian magnum (Mandeles and Ducay,

1962; O'Malley, 1967) and the tube of turtles (Chapter 7) and lizards (Palmer and

Guillette, unpublished data) can secrete albumen proteins in vitro, without any apparent

stimulation. As ovulation in most reptiles is thought to be simultaneous, all the eggs of

the clutch must be coated with albumen within a short period. In Sceloporus woodi, all

eggs are within the uterus 9 hours after ovulation. This indicates that either all the

proteinaceous material for albumen formation must be stored in the cells prior to

ovulation, or that there must be an additional, rapid synthesis of albumen protein by the

tube immediately following ovulation. In the lizards Crotaphytus collaris and Eumeces

obsoletus, and the tortoise, Gopheruspolyphemus, there is not a significant decrease in

the size of the tubal endometrial glands or the height of the luminal epithelium

following ovulation (Guillette et al., 1989; Palmer and Guillette, 1990a). In birds, there

is a rapid buildup of albumen within the magnum following each sequential ovulation

(Gilbert, 1979).

The proteinaceous fibers of the eggshell membrane are deposited on all the eggs

of a clutch within 24 hours in Sceloporus woodi (DeMarco and Palmer, unpublished

data). It is unclear if eggshell formation begins on each egg as they sequentially enter

the uterus, or if the process is switched on once all eggs are in place, as there was no

distinguishable difference between the eggshell membranes of those eggs in different







regions of the uterus (DeMarco and Palmer, unpublished data). The stimulus for

release of the eggshell fibers in birds is thought to be the pressure of the egg against the

oviductal wall (Solomon, 1983). Release of fibers can be achieved in alligator and turtle

reproductive tracts by mechanically stimulating excised portions of the fiber forming

portion of the oviduct (Palmer, unpublished data). This suggests a role for

prostaglandins in stimulating the secretion of eggshell proteins, as mechanical stimulation

of reptilian oviducts is known to induce PG production and release (Guillette and

Palmer, unpublished data). In Gopheruspolyphemus, the endometrial gland cells (but

not the diameter of the glands themselves) decrease in size and show fewer secretary

granules following formation of the eggshell membrane (Palmer and Guillette, 1990a).

This may indicate that most or all of the protein required for eggshell formation is

stored in the glands. Further support for this hypothesis comes from the decrease in

thickness of the fibers, and subsequent production of only particulate material,

suggesting diminished stores of secretary material or polymerizing enzymes as eggshell

formation proceeds. However, it has been shown that in the lizard Cnemidophorus

uniparens, deluteinization immediately following ovulation disrupts the formation of the

fibrous eggshell layer (Cuellar, 1979). It is unlikely that disruption of eggshell formation

was due to shortened gestation length (6 days; Cuellar, 1979), as the eggshell membranes

are formed in Sceloporus woodi within 24 hours. Further, the total thickness of the

fibrous membrane was unchanged, but only thick fibers were laid down, instead of a

progression from thick fibers basally to a top layer of fine fibers and particles, forming a

dense mat. This suggests that control of fiber formation was disrupted, and that luteal

hormones, such as progesterone, may be involved in the control of eggshell formation.

This hypothesis is supported by the occurrence of a progesterone spike at ovulation

(near the initiation of eggshell formation) in Sceloporus woodi (Guillette and DeMarco,

personal communication).







The source of the calcareous eggshell layer in reptiles remains unknown. Even

in birds, there has been considerable disagreement about the source of calcium for

eggshell formation. The endometrial gland cells of the avian uterus are believed to

secrete large quantities of "plumping water" (Breen and de Bruyn, 1969), and have many

mitochondria, which are known to sequester calcium ions (Hohman and Schraer, 1966),

suggesting that the endometrial gland cells are involved in the secretion of calcium ions.

However, others have proposed that the surface epithelial cells transfer calcium to the

oviductal lumen (Gay and Schraer, 1971; Solomon et al., 1975).

Several authors have hypothesized that in reptiles the secretary cells of the

luminal epithelium may be involved in calcium transport (Guillette and Jones, 1985;

Palmer and Guillette, 1988; Guillette et al., 1989). This is supported circumstantially by

our data that during the periods of maximal calcification (days 3-9), as indicated by the

buildup of calcium crystals on in utero eggs (DeMarco and Palmer, unpublished data),

the secretary cells of the luminal epithelium become greatly distended and

hypertrophied. Further evidence for the role of secretary cells is their proportional

increase in number from vitellogenesis to gravidity. In the turtle Gopheruspolyphemus,

the uterine luminal epithelium is tallest and possesses the greatest proportion of

secretary cells during gravidity. This is also known in several species of lizards

(Christiansen, 1973; Fawcett, 1975; Guillette et al., 1989). Finally, the uterine secretary

cells of the lizard Crotaphytus collaris stain positively for calcium during gravidity.

Additional studies are clearly warranted to localize the source of calcium for eggshell

deposition.

These data indicate that the uterus of lizards, and probably of turtles as well

(based on ultrastructure; Chapter II), is dualistic in function, producing both the fibrous

and calcareous layers of the eggshell sequentially. This is substantially different from the

situation in birds, where each layer of the eggshell is produced by a separate, highly

specialized region. It remains to be determined if this is a universal condition in all





82

reptiles, or if different reproductive anatomies occur in other groups. Particularly, as

crocodilians are the closest living relatives of birds (Benton, 1985), the functional

morphology and ultrastructure of their reproductive tract would be worthwhile

investigating.













CHAPTER IV
OVIDUCTAL ULTRASTRUCTURE AND EGG FORMATION
IN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS)


The process of eggshell formation has been extensively studied in birds and

mammals, but little has been known concerning the formation of eggshell components in

reptiles (Weekes, 1927; Boyd, 1942; Aitken and Solomon, 1976; Palmer and Guillette,

1988; Guillette et al., 1989). In birds, three oviductal regions exist, and each produces a

separate component of the egg investments (albumen, fibrous eggshell membrane, and

calcareous eggshell layer; Solomon, 1983). In egg-laying mammals, the monotremes,

which produce reptile-like eggs (C. J. Hill, 1933; Hughes, 1977), the tube produces egg

albumen and the uterus produces all layers of the eggshell (C. J. Hill, 1933, 1941;

Hughes and Carrick, 1978). Likewise, eggshell formation in turtles, lizards, and the

tuatara (Sphenodon punctatus), occurs strictly in the uterus (Palmer and Guillette, 1988,

1990b; Packard et al., 1988; Guillette et al., 1989; Chapter II and III). The uterine

endometrial glands secrete intact fibers that are wrapped around the egg forming the

eggshell membranes (Chapter III). Structural and histochemical evidence suggests that

the secretary cells of the luminal epithelium secrete calcium ions for crystallization of

the eggshell (Palmer and Guillette, 1988; Guillette et al., 1989; Chapter II and III). All

the eggs of a clutch are shelled simultaneously, with each egg resting within its own

uterine "chamber" (Chapter III). This supports the conclusion that the uterus of turtles

and squamates is dualistic in function, forming both the fibrous and calcareous layers of

the eggshell on an entire clutch simultaneously. This is substantially unlike eggshell

formation in birds, in which separate regions secrete each eggshell component, and only

one egg of a clutch is shelled at a time. The physiological control of eggshelling in







reptiles is largely unknown, although a luteal product, such as progesterone, is strongly

implicated (Cuellar, 1979; Fox, 1984; Guillette and Jones, 1985; Guillette et al., 1989).

These major differences in oviductal gross morphology, ultrastructure, and

presumed physiological control between reptiles and birds raises important questions

about the evolution of avian eggshell formation from that of reptilian ancestors. The

examination of chelonians and lepidosaurians has given little indication of the evolution

of avian reproductive anatomy and physiology. In this study, oviductal functional

morphology and ultrastructure were examined during the reproductive cycle in a

crocodilian, the American alligator (Alligator mississippiensis). Crocodilians are

archosaurs, as are the birds, and represent the closest living reptilian relatives of birds

(Benton, 1985; Gauthier et al., 1988).


Methods and Materials


Specimens

Thirteen alligators (Alligator mississippiensis) exhibiting various reproductive

conditions (vitellogenic, early gravid, late gravid, immediately post-oviposition and

reproductively quiescent) were collected from several lakes (Griffin, Orange, and

Okeechobee) in central Florida (Permit #W88063). Within 24 hours of capture, the

specimens were anesthetized with 20mg/kg sodium pentobarbital, and the oviducts

surgically removed under sterile conditions. Representative tissues from each oviductal

region were dissected free and immediately fixed for histochemistry, scanning electron

microscopy (SEM), and transmission electron microscopy (TEM).


Histochemistry

Representative tissues from each region were fixed in 10% neutral buffered

formalin or Bouin's fixative. Tissues were then washed, dehydrated in graded alcohols,

cleared in xylene, and embedded in paraffin (Humason, 1979). Specimens were serial







sectioned at 7p/m on a rotary microtome, for a total of 10 slides per tissue specimen,

and stained as in Table 2-1.


Electron Microscopy

For transmission electron microscopy (TEM), tissue samples were minced (1

mm3) and fixed in 2% glutaraldehyde with 0.1M cacodylate HCI buffer for 3 hours,

washed in buffer (3 X 15 min), treated with OsO4 for 30 min, and washed in buffer (3

X 15 min). The specimens were dehydrated in graded alcohols, treated with 100%

acetone (2 X 1 hr), and embedded in Spurr's resin. Thick sections (1/m) were prepared

and stained with toluidine blue. Thin sections were cut at 600A, and poststained with

uranyl acetate and lead citrate. Examination of tissues was performed on a Hitachi HU-

11E or a Philips EM-300.

Specimens for scanning electron microscopy (SEM) were cut into 1 cm3, and

fixed for 24 hours in 2% glutaraldehyde in 0.1M cacodylate HCI buffer. Tissues were

dehydrated in graded alcohols, CO2 critical point dried (Anderson, 1951), and sputter

coated with gold. Examination was performed on a Hitachi S-450.


Results


General Oviductal Morphology

The general morphological characteristics of the alligator oviduct are presented

in Fig. 4-1. There are seven clearly distinguishable regions along the length of the

oviduct: anterior infundibulum, posterior infundibulum, tube (tuba uterina), transitional

region, fiber region, uterus, and vagina. The anterior infundibulum is funnel shaped

with thin, transparent walls and opens to the coelom across the distal margin. The

posterior infundibulum is tubular, with thicker, more muscular walls and greater

endometrial folding. There are no true glands in the endometrium, although it contains

secretary cells. The demarcation between the infundibulum and the tube (tuba uterina)

is indicated by the occurrence of endometrial glands in the tube, which are discernible at







the gross morphological level by a milky coloration. The tube is long and convoluted,

although not to the extent of that in chelonians. The endometrium of this region is

folded longitudinally, creating grooves which run along the length of the tube. At the

posterior end of the tube is a short, narrow region, the transition zone, which is visually

distinguished by being translucent due to the lack of endometrial glands. The "fiber

region" is narrower than the tube and its walls are more muscular. Whereas the tube is

flat in cross-section with a wide lumen, the fiber region is rounded with tall endometrial

folds. The transition between the fiber region and the uterus is gradual. The outer

diameter increases to that of the uterus, and the color (of fresh tissue) changes from

pale cream or gray in the fiber region to a darker shade of reddish-gray in the uterus.

The uterine lumen is greater in diameter than the fiber region, and the endometrium is

formed into tall random folds. The vaginal lumen is extremely narrow and spirals

through muscular walls. Each vaginal canal opens separately into the cloaca.


Tube

The luminal epithelium of the tube stains intensely with Alcian blue for

glycosaminoglycans (GAGs), and consists of two types of tall simple columnar cells,

ciliated and microvillous secretary (Fig. 4-2a, b). The ciliated cells have apical nuclei

whereas the secretary cells have central, or occasionally basal, nuclei. The glands of the

endometrium are branched acinar, often with extensive ducts connecting them to the

surface (Fig. 4-3a). The duct cells are cuboidal and slightly eosinophilic. The

endometrial gland cells are cuboidal or low columnar with basal nuclei.

Ultrastructurally, the endometrial glands exhibit roughly spherical-shaped secretary

granules of a wide range of electron densities (Fig. 4-3b). The apical membrane of the

endometrial gland cells bears numerous microvilli.








Fiber Region

In the fiber region, the luminal epithelium consists of a simple columnar layer of

ciliated and secretary cells, as in the tube, although they are lower and stain less

intensely with Alcian blue. The endometrial glands are branched tubular, with short

ducts connecting them to the lumen (Fig. 4-4a). The endometrial gland cells of the fiber

region are cuboidal, with basal nuclei and numerous eosinophilic granules. TEM

demonstrates that these granules are spherical and electron dense (Fig. 4-4b). During

early gravidity, small (1cm3) runt eggs, consisting of albumen surrounded by

proteinaceous fibers, were observed within this region. In these early gravid specimens

proteinaceous fibers were observed being extruded from ducts of the endometrial glands

(Fig. 4-5a, b). These fibers are identical in structure and diameter to those of the

eggshell membrane.


Uterus

The luminal epithelium of the uterus is composed of low columnar epithelial

cells similar to those described for other regions. However, very few cells stained

positively for GAGs using Alcian blue. The alligator's uterine endometrial glands are

branched acinar with cuboidal cells that are not eosinophilic (Fig. 4-6a). The glandular

cells have basal nuclei and contain numerous small, electron light secretary vesicles (Fig.

4-6b). These cells have numerous tall microvilli on the apical membrane and extensive

interdigitations occur on their lateral cell junctions. Eggs are retained in the uterus for

most of gravidity, where eggshell calcification was apparent. The thickness of the

calcareous layer increases from early to late gravidity, as indicated by diameter and

condition of the corpora lutea. Most eggs are calcified simultaneously, as all eggs from

a single female had approximately equal shell thicknesses. However, in the earliest

gravid female, those eggs from the ends of the uterus had thicker shells than those from

the middle.