Factors associated with sperm motility and storage in a squamate reptile, Scelopours jarrovi

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Title:
Factors associated with sperm motility and storage in a squamate reptile, Scelopours jarrovi
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xii, 157 leaves : ill. ; 29 cm.
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Matter, John Martin, 1959-
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Sceloporus -- Reproduction   ( lcsh )
Sceloporus -- Physiology   ( lcsh )
Zoology thesis, Ph. D
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Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
Bibliography:
Includes bibliographical references (leaves 137-156).
Statement of Responsibility:
by John Martin Matter.
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Typescript.
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Vita.

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FACTORS ASSOCIATED WITH SPERM MOTILITY
AND STORAGE IN A SQUAMATE REPTILE,
Sceloporus jarrovi















By


JOHN MARTIN MATTER


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


1996


























Dedicated to all who have believed in me, especially
Howard A. Bern, the "father of comparative endocrinology,"

and my loving wife,
Kismet, the mother of our children.













ACKNOWLEDGMENTS


This dissertation would not have been possible without the help of
numerous individuals. My friends and colleagues are to receive much credit
for encouragement and physical assistance. Brent D. Palmer, Vincent G.

DeMarco, Dennis C. Haney and Louis A. Somma are most notable for making

me feel welcome as a graduate student at the University of Florida and for
"showing me the ropes" around Gainesville during the "early years." Lively

discussions with these individuals, and others, helped to broaden my point-
of-view. Vince guided me through experiences in Arizona that I will never
forget. Brent tolerated my early morning activity and was a sounding board

for many of my research ideas. Dennis Haney and Lou Somma shared many
days in the field while involved in numerous "side-projects"; their help with

collection of Eumeces egregius will not go unrewarded.
One of the "problems" of having an extended graduate career is the
increased number of acquaintances that one acquires. The "second tier" of
colleagues with whom I have shared experiences with are some of the most
memorable. Drew Crain, Daniel B. Pickford and Andy A. Rooney were

solidifying agents during the "data analysis" and "writing" phases of my
dissertation. Hopefully I have been able to impart some tidbits of knowledge,
experience and herpetological wisdom to this "next generation" of Guillette
proteges.
My graduate tenure has been complicated by numerous and varied
sources of financial support. I extend heartfelt thanks to all of the various








institutions and individuals that have aided monetarily to my graduate

education. Particular thanks goes to George H. Burgess at the Florida

Museum of Natural History (Division of Ichthyology). Mr. Burgess allowed

me to become familiar with curatorial practices and gave me the freedom to

learn much about ichthyological systematics and identification. George also
passed along many consulting opportunities that enabled me to feed my

family. Stephen J. Walsh was a friend, first during museum efforts, then as a

supervisor, while working for the U.S. Fish and Wildlife Service. Susan S.

Suarez supported me with an assistantship, and I will never forget "the

gloved-hand technique". Lyn Branch (Wildlife) provided support during the

final stages of my dissertation; I wish her the best of luck with her scrub-
associated lizard efforts. The Department of Zoology provided numerous
teaching assistantships over the years, making it far too "easy" to remain a
graduate student.

My committee members deserve a round of applause. Those that have
"outlived" me at the University of Florida truly are exceptional. Thanks go to

Dan C. Sharp, Susan S. Suarez, Harvey B. Lillywhite and Frank G. Nordlie.
To those individuals who served as committee members, but were not
present to witness my completion, I am also grateful. Thanks go to Martha L.
Crump and Horst 0. Schwassmann for guidance during phases of my

graduate career.

Much of the credit for seeing me through my graduate tenure goes to
my mentor, Louis J. Guillette, Jr. Lou's persistence and encouragement were
visionary and often helped to pull me back-on-track when I "floundered."
While I rarely gave him reason to, Lou never lost sight of my abilities; I wish
I could have been more deserving of his faith.








Several professional acquaintances have been both supportive and
inspirational. Howard A. Bern has instilled in me a great deal of self-
confidence. He has shown me that there is no need for "false justification" of
comparative biology. Richard Highfill (Millsaps College) and Qiu Yu-Xiang
(Beijing, China) had collaborative stays that were both meaningful and
memorable. Richard E. Jones (University of Colorado) and Cliff H. Summers
(University of South Dakota) have provided me with an opportunity to
achieve my next goal in research and given me much to think about.

Lastly, I cannot extend enough thanks or love to those who deserve it
most, my wife and family. My spouse, Kismet, has believed in me every step
of the way and she has given much while compromising herself for the
possibility of a better future life. My children, Colin and Nyssa, will never
know what a joy they have been, and will be, to me. My parents, Richard M.
Matter and Jill Cerny-Matter, have also been extremely supportive, both
emotionally and financially. Their unconditional love and the times we
share will always be cherished.














TABLE OF CONTENTS


ACKNOW LEDGM ENTS ..............................................................................................iii

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

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

ABSTRACT..................................................................................................................... xi

CHAPTERS

1. SPERM PHYSIOLOGY IN A SQUAMATE REPTILE, SCELOPORUS
JARROVI: AN INTRODUCTION AND COMPARISON WITH
OTHER VERTEBRATES .......................................................................................1...

Introduction ............................................................................................................. 1
Reproductive Anatomy in M ale Reptiles...................................................2...
Reptilian Sperm atogenesis ............................................................................. 4
Renal Sex Segment...........................................................................................7...
The Research Animal .......................................................................................... 11
Scope of this Study................................................................................................ 13


2. MALE REPRODUCTIVE ANATOMY OF MOUNTAIN SPINY
LIZARDS, SCELOPORUS JARROVI ................................................................. 18

Introduction ........................................................................................................... 18
M materials and M ethods ........................................................................................ 19
Results........................................................................................................................20
Discussion...............................................................................................................43


3. TESTOSTERONE STIMULATION OF CELLULAR ACTIVITY IN
MALE ACCESSORY SEX STRUCTURES OF MOUNTAIN SPINY
LIZARDS, SCELOPORUS JARROVI ................................................................. 47

Introduction ...................................................................................................... 47
M materials and M ethods........................................................................................ 51
Results ........................................................................................................................ 54
Discussion............................................................................................................... 66










4. POST-COPULATORY FATE OF SEMINAL COMPONENTS IN THE
FEMALE REPRODUCTIVE TRACT OF MOUNTAIN SPINY
LIZARDS, SCELOPORUS JARROVI .................................................................74

In trodu action ........................................................................................................... 74
Materials and Methods ........................................................................................78
R esu lts ........................................................................................................................80
D discussion .................................................................................................... ........... 98


5. CHARACTERIZATION OF MOTILITY PATTERNS EXHIBITED BY
SPERMATOZOA FROM THE MALE REPRODUCTIVE TRACT OF
MOUNTAIN SPINY LIZARDS, SCELOPORUS JARROVI....................... 103

In trod u action .......................................................................................................... 103
Materials and Methods.......................................................................................106
R esu lts ......................................................................................................................109
D discussion .............................................................................................................. 122


6. SUMMARY AND SIGNIFICANCE OF STUDY.............................................128


A P PE N D IX .....................................................................................................................132

LIST OF REFERENCES .............................................................................................137

BIOGRAPHICAL SKETCH.......................................................................................157













LIST OF TABLES


Table page

1. Spermatogenic patterns and associated reproductive events exhibited by
tem perate-zone saurian species................................................................... 14

2. Seasonal changes in morphometrics of male reproductive structures
exhibited by Sceloporus jarrovi ................................................................... 24

3. Morphometric features of male reproductive structures as the result of
testosterone implantation in Sceloporus jarrovi....................................57

4. Morphometric values for female reproductive structures associated
with copulation in Sceloporus jarrovi....................................................... 83

5. Motility characteristics exhibited by spermatozoa from various regions
of the Wolffian ducts in Sceloporus jarrovi ............................................111


viii













LIST OF FIGURES


Figure page

1.1 The research anim al and its habitat................................................................. 17

2.1 Male reproductive anatomy of Sceloporus jarrovi...................................... 26

2.2 Light micrographic representation of reproductive structures from adult
m ale Sceloporus jarrovi..................................................................................... 28

2.3 Light micrographic survey of kidney and epididymis histology from adult
m ale Sceloporus jarrovi..................................................................................... 30

2.4 Scanning electron micographs of RSS from reproductively active
Sceloporus jarrovi ............................................................................................... 32

2.5 Light micrographs of testes from adult male Sceloporus jarrovi depicting
seasonal changes in spermatogenic condition..............................................34

2.6 Diagrammatic representation of changes in seminiferous tubule diameter
during the active season in Sceloporus jarrovi............................................ 36

2.7 Light micrographs of RSS and epididymis from adult male Sceloporus
jarrovi exhibiting seasonal changes in organ conditions...........................38

2.8 Diagrammatic representation of changes in RSS epithelial height during
the active season in Sceloporus jarrovi.......................................................... 40

2.9 Diagrammatic representation of changes in epididymis epithelial height
during the active season in Sceloporus jarrovi............................................42

3.1 Representative light micrographs of kidneys from T-implanted and
control male mountain spiny lizards, Sceloporus jarrovi.........................59

3.2 Representative light micrographs of epididymal tubules from T-
implanted and control male mountain spiny lizards, Sceloporus jarrovi.61

3.3 Graphic representation of histological changes due to T stimulation of
male accessory sex structures in Sceloporus jarrovi....................................63








3.4 Polyacrylamide gel electrophoresis of secretary proteins from RSS and
epididym ides of Sceloporus jarrovi ................................................................ 65

4.1 Female reproductive anatomy of Sceloporus jarrovi..................................85

4.2 Light micrographs of posterior oviduct of Sceloporus jarrovi..................87

4.3 Micrographs of various features of the cloaca and posterior vagina of
Sceloporus jarrovi ............................................................................................... 89

4.4 Light micrographs of vaginal sperm storage crypts in Sceloporus jarrovi.91

4.5 Histological localization of glycosaminoglycans in oviducts of Sceloporus
jarrovi.........................................................................................................................93

4.6 Scanning electron micrographs of vaginal surface of Sceloporus jarrovi. 95

4.7 Light micrographic representation of reproductive tract structures of non-
copulated Sceloporus jarrovi ............................................................................ 97

5.1 Scanning electron micrographs of spermatozoa from male excurrent ducts
of Sceloporus jarrovi. ........................................................................................ 113

5.2 Diagrammatic representation motility patterns exhibited by spermatozoa
from excurrent ducts of Sceloporus jarrovi................................................. 115

5.3 Diagrammatic representation of flex-snap maneuver exhibited by
sperm atozoa from Sceloporus jarrovi ........................................................... 117

5.4 Features of motility patterns exhibited by spermatozoa from different
regions of male excurrent ducts of Sceloporus jarrovi ..............................119

5.5 Sperm motility characters exhibited by spermatozoa from different
regions of male excurrent ducts of Sceloporus jarrovi ..............................121













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


FACTORS ASSOCIATED WITH SPERM MOTILITY AND STORAGE IN A
SQUAMATE REPTILE, Sceloporus jarrovi.

By

John Martin Matter

May, 1996




Chairman: Louis J. Guillette, Jr., Ph.D.
Major Department: Department of Zoology


The reproductive anatomy of mountain spiny lizards, Sceloporus

jarrovi, is similar to that described for other reptilian species. Testes are

ovoid and held within the abdominal cavity. The spermatogenic cycle is

aestival and coincident with female ovarian activity; spermatocytogenesis

begins in late summer and spermiation is complete by fall. Secondary sex

structures, epididymides and renal sexual segment (RSS) are hypertrophied

and secretary during the period of fall copulations, but regressed and

nonsecretory in winter, spring and early-summer months.

Administration of testosterone (T), via subcutaneous implants, results
in cellular activation during the nonreproductive phase of the annual cycle.

At 3 days of T treatment, the RSS exhibits marked hypertrophy and

histologically identifiable secretary material, indicating rapid initiation of








protein synthesis. Epididymides exhibit cellular hypertrophy and protein

production by day 5 of androgen treatment. Proteinaceous granules produced

by the RSS migrate to cellular apices by day 7 of treatment. Further treatment

durations (14 and 21 days) induced continued cellular stimulation. This set of

trials indicates the androgen-dependent nature of both epididymal and RSS

secretary activity.

Membrane-bound secretary components from epididymides and RSS
are isolated for protein characterization using one-dimensional

polyacrylamide gel electrophoresis. While exhibiting similar histochemical

staining characteristics, RSS and epididymal products exhibit different protein

profiles, each having different major proteins at 31 and 70 kDa, respectively.
Reproductive tracts of mated female S. jarrovi, killed at various times
after copulation, reveal the position of seminal fluid components. Sperm are
transported to vaginal sperm storage crypts (SSCs), whereas granular products

of the epididymides and RSS are retained by the transitional epithelium of

the cloaca. Mated females exhibit greater cellular development of

reproductive tracts and greater plasma estradiol concentrations when
compared to non-mated lizards.
Examination of spermatozoa from male excurrent ducts reveals two
distinct motility patterns. Straight-swimming sperm exhibit slower

velocities, sinusoidal flagellar beats with less bending and little lateral head

displacement. Tumble-swimming sperm possess higher velocities, a greater
degree of flagellar bending and greater head displacement. Tumble sperm are
seen at greater frequencies in more posterior regions of the excurrent duct
system and may represent a maturational transition necessary for proper
sperm function following insemination.













CHAPTER 1
FACTORS ASSOCIATED WITH SPERM VIABILITY IN A SQUAMATE
REPTILE, Sceloporus jarrovi: AN INTRODUCTION AND COMPARISON
WITH OTHER VERTEBRATES


Introduction

Many evolutionary adaptations are associated with internal
fertilization in amniotic vertebrates. Internal fertilization, a common feature

of the amniotes, may be associated with liberation from an aquatic
environment. Morphological specialties to internal fertilization are

developed in both sexes of amniotes. Males evolved intromittent organs that

increase the efficiency of sperm delivery. Accessory structures may add

nutritive materials and/or serve as storage sites for sperm. Females evolved
sperm storage structures, shell producing glands and/or uterine tissues for

embryonic nourishment. Several anamniote groups have secondarily
evolved internal fertilization (i.e.. some fishes and most Urodele

amphibians) but none of these taxa are completely freed of the necessity for

water associated with reproductive activity [van Tienhoven, 1983].

Phylogenetically, reptiles are considered important in interpreting the
evolutionary adaptations and physiological consequences of internal
fertilization. This dissertation examines several morphological and
physiological features associated with sperm motility and viability in a
squamate reptile, the mountain spiny lizard, Sceloporus jarrovi. I examine
features of both male and female reproductive functionality which are
intimately associated with sperm maintenance and viability.





2



Reproductive Anatomy in Male Reptiles


General Reproductive Anatomy

The reproductive anatomy of reptiles is quite similar to that exhibited
by mammals [van Tienhoven, 19831. The testes are paired and, as with other
abdominal organs, are supported by mesorchia within the posterior body
cavity [Fox, 1977]. Reptilian testes are spherical to ovoid in shape and
bounded on the exterior by a connective tissue sheath, the tunica albuguinea.

Ophidian testes tend to be more elongate in shape, associated with their

cylindrical body form. In many squamates the right testis is larger and

situated more anteriorly than the left, particularly in snakes. The right testis

of the European viper, Vipera berus, is consistently larger than the left
[Volsoe, 1944].

Within testes, numerous, tightly coiled seminiferous tubules serve as
the sight for spermatocytogenesis and spermiogenesis. Spermatozoa

produced in testes are released via a series of efferent ducts of archinephric
origin. Seminiferous tubules exit testes by means of short efferent ductules
(ductuli efferenti), which pass into the epididymis proper. Epididymal
tubules, the ductuli epididymides, and highly convoluted ductus
epididymides, leave the vicinity of testes as ductus deferenti vasaa deferentia),

which are nearly straight and run ventral to the metanephric kidneys. At the
posterior level of the kidney, ureters unite with the ductus deferens and drain
into the urodeum [Fox, 1977].
Interspersed between seminiferous tubules are interstitial cells (Leydig
cells). These cells are presumed to be a site of steroid biosynthesis, as








indicated by their seasonal accumulation of lipids [Lofts, 1969] and the
presence of 313-hydroxysteroid dehydrogenase (30-HSD), the enzyme which
converts pregnenalone to progesterone. Interstitial cells have been readily
distinguished in snakes, but their presence in some lizards is questionable;
they were not reported in fence lizards, Sceloporus undulatus undulatus
[Altland, 1941]and I am unable to verify their occurrence in S. u.
hyacinthynus [Matter, 1987], but Leydig cells have been identified in closely
related Sceloporus occidentalis [Wilhoft and Quay, 1961]. Seasonal changes in
interstitial cell numbers and size have been exhibited by green anoles (Anolis

carolinensis), being found in groups of three to four cells in only one-third of
the intertubular spaces, but having greater cellular area during summer
months [Fox, 1958]. During late-summer and fall months (August through
December), A. carolinensis exhibit few, non-biosynthetic interstitial cells, as
determined by histochemical staining for steroidogenic activity [Fox, 1958].
Lizards of the family Teiidae (Cnemidophorus sp. and Ameiva sp.) possess
"circumtesticular" Leydig cells which are situated around the testes, just

inside the connective tissue sheath, and not in tubular interstices [Lowe and
Goldberg, 1966].

Interstitial cells of ophidians have been found to be more numerous
than lizards, being found in groups of up to one hundred cells in Vipera
berus [Volsoe, 1944]. They were also prevalent in the northern water snake,
Nerodia sipedon [Bauman and Metter, 1977]. Fox [1952] reported the
interstitial cell cycle in testes of Thamnophis elegans terrestris and T. sirtalis
tetrataenia. Additionally, he commented on the occurrence of interstitial
cells in Phyllorhyncus decurtatus and Pituophis catenifer [Fox, 1952].

Variation in intromittent organ structure reflects the phylogenetic
diversity of the Reptilia. Crocodilians and chelonians possess a single,








median intromittent organ that arises from tissues of cloacal origin. The

intromittent organs of Squamata, the paired hemipenes (hemipenis, sing.),

are bilobed structures with a bifurcate, open groove (sulcus spermaticus)

which transmits the ejaculate to the female during copulation. Hemipenes
are retained, inverted, in sac-like pouches in the base of the tail. Intromission
brings one of the hemipenes into play when mating animals juxtapose their

vents and the organ is forcefully everted into a female's cloaca [van
Tienhoven, 1983].

Several accessory reproductive structures are described in mammals
[Setchell et al., 1994]. These include the prostate and bulbo-urethral glands

and the seminal vesicles. In mammals, these structures have been shown to
add to the fluid volume of semen, as well as functioning in cleansing and

lubrication of ejaculatory ducts (urethra). Much work has focused on the

potential for sperm nourishment and motility activation by the secretary

components of these accessory structures [Mann and Lutwak-Mann, 1981]. In
addition to these accessory structures, epididymides serve to add fluid and

potentially nutritive substances to semen. Within the Reptilia, there are no
homologous accessory structures such as those described for mammals [Fox,
1977]. All seminal fluid components must come from either epididymides or
from a novel nephric structure, the renal sexual segment (RSS).


Reptilian Spermatogenesis

Seasonal patterns of spermatogenic activity are described for squamate
reptiles [Licht, 1984; Saint Girons, 1982, 1984]. "Vernal" spermatogenesis is
characterized as testicular recrudescence that begins upon emergence from
overwintering sites. Spermiation and copulation generally take place during
spring and early summer in animals that exhibit vernal spermatogenic





5

activity. "Aestival" spermatogenesis is distinguished by gonadal

recrudescence during summer. Spermiation occurs during the fall.

Autumnal copulatory activity leads to either an overwintering pregnancy or
retention of sperm within the female's reproductive tract for subsequent
fertilization at the time of spring ovulation. Alternatively, sperm may be

stored in the ductus deferens of the male until copulation in spring. Finally,
"mixed" spermatogenesis is characterized by gonadal recrudescence in late

summer and fall, but gamete production is not complete at this time.

Animals overwinter with spermatids in seminiferous tubules and

maturation of spermatozoa is completed just prior to spring copulatory
activity. Spermatogenic patterns exhibited by numerous temperate-zone

lizards are summarized in Table 1. While Saint Girons' (1982) original

description of these spermatogenic patterns is applied to squamate reptiles,
these terms can be used to describe the testicular cycles of other vertebrates as
well (reviewed in [Bronson, 1989; Dodd and Sumpter, 1984; Follett, 1984;

Lofts, 1984; Rowlands and Weir, 1984]).
Species within the genus Sceloporus exhibit different spermatogenic

patterns, but the evolutionary and environmental mechanisms driving these
differences are not fully understood. Gonadal androgens are known to play a
key role in the expression of seasonal characters, including secondary sex

structures. Epididymal and RSS activity is strongly correlated with

spermiation and copulatory behavior in reptiles [Aldridge et al., 1990; Saint
Girons, 1982, 1984]. Activity of these two structures is dependent on plasma
androgens, but is not always evident when androgens are elevated. Eastern
fence lizards (S. undulatus hyacinthynus) exhibit late-summer testicular
recrudescence, proliferating spermatocytes and spermatids in fall, and
elevated plasma androgen concentrations [McKinney and Marion, 1985], but








do not exhibit activity of the epididymis or RSS until after emergence from
over-wintering sites the following spring [Matter, 1987].

Many vertebrate groups exhibit gonadotropins which are similar
(homologous) to mammalian LH and FSH [Ishii, 1990; Licht, 1983]. Some
reptilian subclasses (Crocodilia and Chelonia) exhibit two, distinct
gonadotropin molecules associated with gonadal function. Efforts to isolate
and characterize two gonadotropins within the squamates, however, have
proved enigmatic [Licht, 1974, 1983; Licht et al., 1977]. Transduction of
thermal and photic stimuli via the hypothalamus is related to reproductive
activity in snakes and lizards, respectively [Licht, 1971, 1972; Licht et al., 1989].
Some studies utilizing mammalian gonadotropins indicate that both LH and
FSH are capable of inducing gonadal function in squamate reptiles. Porcine
FSH induced spermatocyte proliferation, but not spermatozoa maturation, in
the testes of juvenile Eumeces obsoletus [Masson and Guillette, 1985]. Other
studies utilizing gonadotropin administration in lizards present conflicting
results with respect to gonadal regulation. Mammalian FSH, alone or in
combination with LH, causes complete spermatogenic activity in
hypophysectomized Hemidactylus flaviviridis [Reddy and Prasad, 1970]. LH
alone, however, can not stimulate spermatogenesis, but does maintain
androgen-dependent secondary sex structures. Ovine LH and FSH, alone or
together, lead to germ cell (primary and secondary spermatocyte) proliferation
during both the regressional and recrudescent phases of the annual cycle in
Calotes versicolor [Gaitonde and Gouder, 1985]. FSH is considerably more
potent than LH in stimulating spermatogenesis in Anolis carolinensis, but it
was later proposed that the "effectiveness" of LH in this study was due to FSH
contamination [Licht and Pearson, 1969]. The inability of squamate pituitary
extracts to induce an ovulatory response in vitro lead to the premise that this








vertebrate group may not possess LH [Licht, 1974]. Results of these studies has

prompted the claim that squamates possess only one gonadotropin, an FSH-
like molecule [Licht et al., 1977]. Studies on the neuroendocrine modulation

of pituitary function and gonadal responsiveness are needed to better
comprehend the liability of seasonal reproductive activity exhibited by

squamates in general and Sceloporus in particular. Members of the genus
exhibit a variety of reproductive patterns and are a model system for

examining the control of seasonality.


Renal Sexual Segment

While investigating the kidney of the European grass snake (Natrix
natrix), Gampert [1866] was the first to describe an hypertrophied portion of

the nephric tubules. Histological description of the hypertrophied nephric
segment in Natrix and an account of the secretary granules which were
present in the epithelial cytoplasm followed [Heidenhain, 1874]. Regaud and

Policard [1903] were the first to report that kidney tubule enlargement was

unique to male snakes (Natrix natrix, N. maura and Coluber viridiflavus).
Although their work was incomplete, they alluded to the possibility of
seasonal changes in the degree of hypertrophy in these kidney elements and
their belief in a reproductive function for this region [Regaud and Policard,
1903a, 1903c]. These researchers referred to this nephric component as the
"segment sexuel du rein"; the renal sexual segment (RSS). Later, they

described a similar condition in the kidney of the lizards Lacerta vivipara and
Anguis fragilis, while they noted its absence in turtles [Regaud and Policard,
1903b]. The absence of a RSS in crocodilians is confirmed by Zarnick [1910a,
1910b] and is, thus, a morphological specialty confined to squamate reptiles
[Zamick, 1910a, 1910b].








Species differences are noted with respect to specific nephric

components incorporated in the RSS. The RSS is comprised of the

preterminal portion (between the distal and terminal segments) of the

nephron in the snakes Natrix natrix, Vipera berus, Thamnophis elegans [Fox,

1952] and T. sirtalis [Bishop, 1959]. In the relatively primitive snakes,
Leptotyphlops dulcis, L. humilis, Typhlops vermicularis and T. simoni, the
RSS is known to be formed by the collecting ducts and a portion of the ureter

which is embedded in the kidney tissue [Fox, 1965]. In most lizards, the RSS

consists of the terminal portion of the nephrons, collecting ducts and/or

ureters. Prasad and Reddy [1972] restricted their definition of the RSS to

secondary and tertiary collecting tubules in the gecko, Hemidactylus
flaviviridis. In green anoles, Anolis carolinensis, the RSS is formed by
collecting ducts and part of the ureters [Fox, 1958], whereas in the skink,
Eumeces fasciatus, it is composed of "collecting and outlet ducts" [Reynolds,

1943].
In general, most snakes and the lizard genus Varanus possess a RSS

which is formed by the medial (preterminal) region of the distal convoluted
tubules [Saint Girons, 1972]. Volsoe [1944] reports that, when maximally
hypertrophied, the RSS of Vipera berus made up fifty percent of the kidney
volume. The anguid lizard, Ophisaurus ventralis, exhibits a unique RSS,
with regional differences in the secretary nature of tubules (Matter and

Guillette, unpublished data). Kidney tubules near the lateral aspect exhibit
"typical" RSS secretary activity, whereas tubules situated at the medial aspect
of the organ exhibit a clear cytoplasm and reduced size of basal nuclei.
Overall, the RSS of 0. ventralis is quite "snake-like" in appearance (Matter
and Guillette, unpublished data).








In addition to its general structure and phylogenetic distribution
within the squamates, the RSS is known to exhibit seasonal hypertrophy and

secretary activity. The saurian RSS exhibits greater seasonal variation than in
snakes, but in general maximal hypertrophy and secretary activity coincides
with spermiation and copulation (these two events are not always temporally
linked). In squamates exhibiting a vernal spermatogenic pattern, gamete

production, hypertrophy and cellular activity of epididymides and RSS and

copulation are relatively synchronous. Coordination between male and
female reproductive activity (i.e., synchronous gamete formation and mating)

is termed an "associated" pattern of reproduction [Crews, 1984]. Animals that
exhibit mixed spermatogenesis, while responding to different environmental

cues for initiation of gamete formation, are often reproductively viable when
females are, and, hence also exhibit an "associated" reproductive pattern.

Male fence lizards, Sceloporus undulatus hyacinthynus, exhibit mixed
spermatogenesis. Testes become active in late summer, producing numerous
primary spermatocytes. Transformation to spermatids occurs during fall and

lizards enter overwintering sites in this condition. During the period of fall

gonadal activity, plasma androgen concentrations are elevated but none of
the secondary sexual structures epididymidess and RSS) exhibit hypertrophy

or cellular activity. After emergence in spring, spermiation and response of
secondary sex structures coincides with female ovarian development and

copulations [Matter, 1987].
Squamates that exhibit aestival spermatogenesis follow one of two
basic reproductive tactics. (1) the "associated" reproductive pattern in which
males undergo gamete production, spermiation and mating during the late
summer and fall months. Females exhibit synchronous vitellogenesis and
ovulations. This pattern is exhibited by several viviparous lizard species (2)







the "dissociated" reproductive pattern in which males exhibit summer
spermatogenesis and spermiation [Crews, 1984]. Fall copulations lead to over-
winter sperm storage within the female's reproductive tract and fertilization
at the time of ovulation (spring). More frequently, males retain sperm in
excurrent ducts overwinter until copulations in spring. In both cases, testes
are regressed at the time of mating. This pattern is exhibited by numerous
snakes within the natricine (garter and water snakes) [Weil, 1985; Weil and
Aldridge, 1981] and crotaline (pit vipers) subfamilies [Aldridge, 1979; Aldridge
et al., 1990].
Although it is linked to the reproductive cycle, the function of the RSS
is not fully understood. Several hypotheses for RSS functionality exist: (1)
RSS secretions may serve as a nutritional substrate for sperm in the male's or
female's reproductive tract. Cuellar et al., (1972) found that kidney
masserations that contained portions of the RSS have a significantly greater
effectiveness at maintaining spermatozoa motility in culture [Cuellar et al.,
1972]. (2) RSS secretions may make the seminal fluid more viscous,
increasing the efficiency of semen transfer to the female's cloaca as it passes
through the open groove (sulcus spermaticus) of the hemipenes [Conner and
Crews, 1980; Garstka et al., 1982]. (3) RSS secretary material may serve as a
component of the copulatory plug (reported from many natricine and crotalid
snakes) which acts as a physical barrier to subsequent mating attempts
[Garstka et al., 1982; Volsoe, 1944]. (4) RSS secretions have been proposed as a
source of pheromonal signals that relate information pertaining to the mated
status of a female [Garstka et al., 1982]. Volsoe [1944] states that RSS secretions
may be important in scent trailing behaviors of Vipera berus, but he makes
the point that this is unlikely, since males actively trail females and not vice








versa. The idea that RSS material may relate olfactory information is weak, at

best, when one considers the limited chemosensory abilities of some lizards.





The Research Animal

Mountain spiny lizards, Sceloporus jarrovi, serve as research
organisms in numerous studies on reptilian biology. Sceloporus jarrovi has

been utilized in studies of ecology [Ballinger, 1973], mating systems [Ruby,

1978], behavior [Moore, 1986, 1987a, 1987b, 1988; Moore and Marler, 1987] and

reproductive physiology. Much of our current understanding of endocrine
regulation of ovulation and parturition in a viviparous lizard species is based

on S. jarrovi [Guillette, 1979; Guillette et al., 1981; 1988; 1990; 1991; 1992]. Until
recently, attention has focused on female reproductive biology. The work of

Moore and his collaborators examines the role of the endocrine system in
regulating male aggression and territoriality [Moore, 1986, 1987a, 1987b, 1988;

Moore and Marler, 1987]. Even with the "popularity" of S. jarrovi as a
research organism in reptilian biology, there is no complete histological
examination of the spermatogenic cycle or associated secondary reproductive
structures.

The mountain spiny lizard is a member of the torquatus species-group
of large-bodied, large-scaled sceloporines in the family Phrynosomatidae
(Sauria: Reptilia) [Sites et al., 1992]. This is a medium sized lizard with a
maximum snout-vent length of about 90 mm (Fig 1.1A). The geographic
range of S. jarrovi extends from Nayarit northward through Sonora and
Chihuahua in Mexico, entering the United States in southwest New Mexico
and southeast Arizona [Smith, 1946]. Sceloporus jarrovi is known to occur in








the Baboquivari, Chiricahua, Dos Cabezas, Dragoon, Graham, Huachuca,
Quinlan and Santa Rita Mountains in Arizona, as well as the Animas,

Hatchet, Peloncillo, Pyramid and San Luis Mountains in New Mexico [Smith,
1946]. This lizard is strongly saxicolous and occurs at elevations between 1370
and 3350 m (4500-11000 ft), often in association with large boulders, cliffs,
canyon walls and rocky outcrops (Fig. 1.1B). At lower elevations, S. jarrovi is
found among riparian hardwood forests and neighboring boulder-strewn
hillsides, often vegetated with scrub oaks and xerophytic plants (Fig. 1.1C). At
higher elevations, the habitats are dominated by coniferous forests and sparse
grasses. Insects and spiders are favored prey items of this lizard [Smith, 1946].
All members of the torquatus species-group are viviparous; S. jarrovi
produces litters of young during late-May and early-June in Arizona. Males
establish courtship territories during July and mating displays and
copulations take place from September through November. Females are
vitellogenic during the fall months and ovulations occur in November and
December [Goldberg, 1971b; Guillette et al., 1981]. Communal over-wintering
sites (hibernacula) afford females opportunities to bask and achieve favorable
body temperature for embryonic development during pregnancy. A
significant proportion (60%) of young females become reproductively viable
in their first active season at 5 months of age, producing small litters of young
(average of 4) the following spring [Ballinger, 1973]. Litter sizes range from 7
to 15 (average of 10.5) for larger (= older) individuals [Ballinger, 1973]. An
ontogenetic study of gonadal development in S. jarrovi histologically
confirms the precocious onset of gametogenesis in this species. Young-of-the-
year males also become reproductively functional during their first active
season [Ballinger and Nietfeldt, 1989], but most likely do not copulate due to
their inability to hold and defend territories from larger males [Ruby, 1978].







Scope of this Study

This study examines several aspects of the reproductive biology of

mountain spiny lizards. Specific attention is given to factors associated with
sperm motility, storage and maintenance. In particular, this dissertation
examines the anatomy and seasonal changes in histology of reproductive
structures exhibited by adult male S. jarrovi. Additionally, the androgen-
dependent nature of the secondary sex structures (RSS and epididymides) are
examined by utilizing testosterone implants. Major secretary proteins from
accessory sex structures are isolated and initially characterized. Motility
characters are described and quantified for spermatozoa from the male
excurrent ducts during the breeding period. Histological features of the
oviducts are examined at times following copulation to document the
migration of sperm in the female reproductive tract and to determine the fate

of various seminal fluid components. This work is meant to form the
foundation for future efforts examining the physiology of sperm storage and
viability in a squamate reptile. In particular, the possible role of RSS and
epididymal secretary proteins in maintaining sperm viability are considered.






14


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Figure 1.1.


The research animal and its habitat. A) Adult male mountain
spiny lizard, Sceloporus jarrovi. Scale bar equals 20 mm. B)
Boulder-strewn hillside in Dragoon Mountains, Cochise Co., AZ.
C) Stream (intermittent) and associated vegetation at Cochise
Stronghold, Cochise Co., AZ. Adult lizards are found on rocky
hillsides, particularly during the reproductive season (August-
November). Young individuals are particularly abundant along
the streambed.







17





























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CHAPTER 2
MALE REPRODUCTIVE ANATOMY OF MOUNTAIN SPINY LIZARDS,
Sceloporus jarrovi


Introduction

Basic reproductive anatomy is summarized for several lizard genera
[Fox, 1977]. Saurian testes tend to be spherical in shape, the right gonad being
situated more anterior within the abdominal cavity than the left.
Seminiferous tubules are joined to the epididymides by efferent ductules
(ductuli efferenti). Epididymal tubules, in turn, lead to vasa deferentia, which

empty separately into the cloaca region. Intromittent organs, the hemipenes,

transmit semen to the female during coitus [van Tienhoven, 1983].

Most reptiles exhibit seasonal gonadal recrudescence and gametogenic
activity Initiation of gonadal activity in lizards is linked to environmental
cues, primarily photoperiodic and temperature changes [Licht, 1966, 1967a,
1967b, 1971; Marion, 1970, 1982]. Other stimuli linked to gonadal regulation

include seasonal food abundance and periods of increased rainfall or

humidity [Duvall et al., 1982]. Patterns of spermatogenic activity have been
described based on seasonal production of sperm in relation to time of mating
and female gamete production [Crews, 1984]. Lizards that exhibit
synchronous development of gonads between the sexes are described as
displaying an associated pattern of gametogenesis. Those species which
display asynchronous gonadal development are termed dissociated. Seasonal
gametogenesis is also described in terms of the time during which germ cells
are proliferated. Vernal spermatogenesis is characterized by testicular








recrudescence and spermiation during spring months, following emergence

from overwintering. Aestival spermatogenesis is described as gamete

proliferation during summer months with subsequent fall copulation or fall

spermiation accompanied by a spring mating period. Mixed spermatogenesis
is characterized by late-summer or fall spermatocytogenesis, overwintering

spermatids being retained within seminiferous tubules and spring
spermiation [Saint Girons, 1982, 1984].

This study examines the histological correlates of seasonal
spermatogenesis in the viviparous mountain spiny lizard, Sceloporus
jarrovi. Additionally, male gonadal functionality is viewed in relation to
female reproductive activity and considers the role of sperm storage in the

ecology of this species.



Materials and Methods

Animals utilized in other aspects of this dissertation, as well as other
laboratory efforts, provided material for the description of male reproductive
anatomy and were collected between March 1988 and December 1994. A total

of 52 adult male mountain spiny lizards, collected in Cochise Co., AZ, were
killed by cervical dislocation, the abdomen opened by mid-ventral incision
and tissues fixed, in situ, by flooding the body cavity with Bouin's fixative for
several minutes, allowing tissues to harden, before removing reproductive
structures. Measurement of testes volume were made using dial calipers and
calculated using the formula for the volume of an ellipsoid:
V = 4/3 ia2b

where a is 1/2 the shortest diameter and b is 1/2 the longest diameter.








Tissues were fixed in additional aqueous Bouin's fluid for 5-7 days,
rinsed in water overnight, dehydrated in graded ethanol series and embedded
in paraffin before sectioning with a rotary microtome. Serial tissue sections (7

pm) were floated onto glass microscope slides and stained with Harris'

hematoxylin, biebrich scarlet/orange G and fast green, a modification of
Schorr's stain. Several seasonally variable characters were measured,

including seminiferous tubule, RSS and epididymal tubule diameter, and
epithelial height of RSS and epididymis. Morphometric measurements were
made with an ocular micrometer and taken as ten values per structure for
each animal examined. Measurements from tubular elements were made

from circular cross sections to minimize artifacts due to tissue orientation.

Determination of spermatogenic condition was made based on the scheme of
Mayhew and Wright [1970].

Seasonal trends in reproductive morphometric measures
(seminiferous tubule diameter and RSS and epididymal epithelial height)
were statistically analysed by a cubic (third order polynomial) regression.
Statistical comparisons of seasonal morphometric characters were made using

a one-way analysis of variance (ANOVA) on monthly mean values, followed
post hoc by a Scheff6's F-test. Statistical differences were determined at a level

of significance of P < 0.05.


Results


General Histology

The reproductive anatomy of male Sceloporus jarrovi is similar to that
described for other members of the genus [Mulaik, 1946]. The testes are
paired, oval structures situated in the abdominal cavity and supported by








mesenteries (mesorchia) that originate on the dorsal aspect of the body cavity
(Fig. 2.1). The mesorchia supporting the right testes are continuous with the
posterior lobe of the liver and found to be associated with the posterior aspect
of the right lung. Testes are often of unequal size, but no consistent
differences were noted. Gonads also exhibit varying degrees of melanin

pigmentation underlying the tunica albuguinea. Testes are comprised of
numerous seminiferous tubules bounded by a tunica propria and myoid cells.
There are no septae found within testes, whereas the presence of a
conspicuous rete testes is noted.

Interrenal tissue (adrenal gland) is found surrounded by connective
tissue which supports the epididymides and adjacent testes (Fig. 2.2 A).
Numerous ductuli efferenti are situated in the anterior-most portion of
epididymides; these, in turn, are held in close association with the testes by a
connective tissue mesentery. Efferent ductules possess numerous stereocilia
(Fig. 2.2 B). The epididymides could be subdivided into ill-defined caput and,
more definitive, corpus and cauda epididymal regions (Fig. 2.2 C and D).
Elements of the RSS are extensive in the body of the kidney (Fig. 2.3 A).
The RSS tubules are composed of preterminal nephric tubules, collecting
tubules and ducts and a major portion (if not all) of the ureters. When
hypertrophied and secretary, RSS tubules are characterized by simple
columnar cells with basal nuclei and conspicuous, often twinned, nucleoli. A
major portion of the cellular cytoplasm is filled with membrane-bound
secretary granules (Fig. 2.4). Epithelial cells of the RSS are polygonal in shape.
When secretary, epithelial cells of the RSS tubules exhibit no apical cell
membranes and release of secretary granules is apocrine in nature (Fig. 2.4 D).
During quiescence, RSS tubules exhibit a significantly different morphology








(Table 2). The epithelium of these nephric tubules is greatly reduced in

height, cuboidal in shape with a clear, nonsecretory cytoplasm (Fig. 2.3 B).

The corpus epididymides of reproductively active lizards exhibit a
simple, columnar epithelium with basal nuclei during the period of
spermiation, September through November (Fig. 2.3 C). The epithelium

exhibits secretary granules at the apical portions of the cells which stain
intensely with biebrich scarlet, indicative of arginine-rich proteins. This

secretary material is mixed with spermatozoa within the tubular lumina.

Epithelial cells of this region are not found to possess cilia, as in many
mammals. Transition to a cauda epididymis is seen as a relatively gradual

decrease in epithelial height. Cells of the cauda epididymis take on an
increasingly cuboidal (less columnar) morphology. Along the length of the
epididymides are seen small tubules of unknown origin which possess
stereocilia, but appear to not make contact to either the testes or epididymides.

These tubules do not exhibit spermatozoa and may be remnants of the
embryonic mesonephric ductules (archinephric ducts). At the anterior level
of the kidneys, excurrent ducts form vasa deferentia comprised of
nonsecretory, cuboidal epithelium. The vasa deferentia are found to run
along the ventral surface of the metanephroi and join the ureters at the

cloacal surface. The posterior portion of the vasa deferentia, just anterior to

the cloaca, exhibits expanded regions ampullaee?) that are conspicuous only
when filled with seminal material.

During the non-reproductive phase of the annual cycle, epididymal
components are reduced in size. The epithelium exhibits a pseudostratified,
columnar arrangement over much of the tubular length (Fig. 2.3 D). The
intertubular spaces exhibit an apparent increase in the amount of collagenous








connective tissue, but this is most likely due to a reduction in the epididymal
tubular volume.

Examination of limited material reveals an aestival spermatogenic
pattern of the associated type. Male lizards obtained in spring (March through
June) exhibit seminiferous tubules with only spermatogonia and Sertoli cells
(Fig. 2.5 A). Testes from lizards collected during fall and early winter months
(September through December) exhibit a transformation of previously
proliferated spermatocytes into spermatids and mature spermatozoa (Fig. 2.5
B and C). Spermiation is complete by December as evidenced by seminiferous
tubules with only spermatogonia, Sertoli cells and few remaining
spermatozoa (Fig. 2.5 D). The seminiferous tubules are almost completely
involuted at this time and exhibit diameters that are not different from testes
during the spring and early summer (Fig. 2.6). Secondary sexual structures
are neither secretary or hypertrophied during winter, spring or early summer.
Hypertrophy of RSS and epididymal epithelia is initiated in late summer and
reaches maximal levels during September and October, at the height of the
breeding period (Fig. 2.7 and 2.8). Significant seasonal differences are
exhibited in seminiferous tubule diameter [F(3,47) = 180.03; P < 0.0001; r2 =
0.920], RSS epithelium height [F(3,39) = 132.82; P < 0.0001; r2 = 0.911],
epididymis epithelium height [F(3,43) = 157.26; P < 0.0001; r2 = 0.916] and testes
volume [F(3,48) = 37.46; P < 0.0001; r2 = 0.701].








Table 2. Morphometric characteristics exhibited by male mountain spiny
lizards (Sceloporus jarrovi) during different months of the active
season. Values are monthly means ( SE).


Month SVL Body Testes Testes Stage RSS Epidid
(n) (mm) Mass Volume Tubule Height Height
(gm) (mm3) Diameter (tm) (ptm)
__ ___I (m)

MARCH 76.8 13.37 24.97 a 75.78 a 1 12.17 a 15.24 a
(8) (2.14) (0.93) (2.50) (2.90) (0.43) (1.35)

APRIL 76.8 11.91 18.79 a 70.45 a 1 11.37 a 13.40 a
(8) (1.62) (0.74) (2.80) (1.93) (0.31) (0.70)

MAY 79.0 13.49 6.48 a 73.88 a 1 12.19 a,c 15.51 a,c
(2) (6.88) (0.94) (2.13)

JUNE 81.2 15.80 30.41 a 78.38 a 1-2 11.63 a 13.11 a
(6) (2.12) (1.85) (10.68) (6.42) (0.25) (0.48)

SEPT 86.3 21.57 311.82 b 282.09 b 6 40.34 b 43.21 b
(3) (1.45) (0.60) (27.62) (4.48) (2.68) (5.29)
OCT 86.0 21.24 331.75 b 250.70 b 6-7 48.21 b 49.88 b
(9) (1.30) (1.05) (47.37) (14.80) (0.90) (0.38)
NOV 81.0 17.74 55.11 a 159.88 c 6-7 43.28 b 39.12 b
(5) (1.52) (0.98) (16.47) (8.35) (1.77) (2.92)
DEC 75.5 18.77 48.98 a 95.07 a 7 25.43 a,c 27.82 c
(11) (2.18) (1.57) (10.16) (5.14) (3.30) (1.86)

Spermatogenic stages follow those described by Mayhew and Wright, 1970.
(1) Seminiferous tubules with spermatogonia only; epididymis atrophic,
empty; (2) Primary spermatocytes appearing; epididymis atrophic, empty;
(6) Spermatozoa abundant (maximal spermiation); epididymis
hypertrophied, with many sperm; (7) Spermatozoa abundant,
spermatocytes and spermatids greatly reduced; epididymis hypertrophied,
with many sperm.
* Values within columns designated by different lettered superscripts are
significantly different at P < 0.05 by Scheff6's F-test after one-way ANOVA
comparison of monthly means.





























Figure 2.1. Diagrammatic illustration of the reproductive anatomy of male
Sceloporus jarrovi. Scale bar equals 5 mm.











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Figure 2.3.


Light microscopic survey of kidney and epididymis histology from
adult male S. jarrovi. All micrographs are representative of
histology seen during the period of autumnal activity (September
through November). A) Low magnification view of medial
aspect of kidney showing extensive nature of RSS tubule
distribution. Several RSS tubules are seen at right (RSS).
Collecting tubules (Ct) coalesce at posterior level as a ureter (U)
which leads to cloacal surface. All of these structures are
hypertrophied and secretary. B) RSS tubules exhibiting cellular
hypertrophy with basal nuclei (arrows) and secretary granules in
apical cytoplasm. C) Portion of hypertrophied epididymal tubule
exhibiting basal nuclei, secretary material and semen in lumen.
D) Epididymal tubules during secretary phase; note apical
distribution of secretary granules (arrows). Scale bar equals 100
gim for A), 10 jm for B), 50 itm for C) and 25 gpm for D).




















































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Discussion

The gross anatomy of the male reproductive system in Sceloporus
jarrovi is similar to that previously described for other members of this genus

[Mulaik, 1946]. Assuming a 6 to 8 week sperm maturation period, as in other
reptiles; Sceloporus jarrovi exhibits an aestival spermatogenic pattern with

spermatocyte proliferation taking place during late summer (most likely in

August). Spermiation occurs in the fall months (September, October and

November) coincident with courtship and copulatory behavior, thereby being
classified as an associated reproductive pattern [Crews, 1984].
Spermatogenesis is completed by December and testes are regressed when
lizards retreat to over-wintering hibernacula. Female S. jarrovi are known to

undergo vitellogenesis in fall and mating at this time leads to over-winter

pregnancies and a 7-month gestation period in this viviparous species
[Goldberg, 1971b].

As previously described [Ballinger and Nietfeldt, 1989], seasonal
reproductive activity in male S. jarrovi is found to be quite similar to that
described for the closely related S. mucronatus [Estrada-Flores et al., 1990]and
S. cyanogenes [Callard et al., 1972], S. torquatus all members of the torquatus

group of sceloporines [Sites et al., 1992]. Summer spermatogenic activity and
the occurrence of fall copulations are viewed as evolutionary adaptations to
female pregnancies that span the winter months and allow for litters of
young to be produced at a time of year when they can maximize opportunities
for growth and energy acquisition [Duvall et al., 1982; Guillette et al., 1980].
Variations in timing of reproductive activity within a phylogenetic lineage
(e.g., Sceloporus) indicate that different species have the ability to evolve








differential responses to environmental modulators of seasonal cyclicity and
reproductive mode. Differential responses to environmental stimuli have
been shown to have taxonomic affinities [Guillette et al., 1980]. To date, all
members of the torquatus group (including S. jarrovi) are known to be
viviparous, but males of some species exhibit differences in timing of
spermatogonial proliferation and spermiation. Plateau populations of S.
mucronatus produce gametes and copulate at the same time of year as
exhibited by S. jarrovi (spermiation from August to September, with

copulations during September through November). Montane populations of
S. mucronatus exhibit differential spermatozoon proliferation (spermiation
in April through July) and copulatory activity (June and July) [Estrada-Flores
et al., 1990]. Both populations have been shown to ovulate eggs during
November and give birth to litters of young the following April or May.
These different patterns are thought to be related to evolutionarily recent
events (mountain building and desertification in the Mexican plateau) and
proximate modulation by factors such as seasonal food availability [Mendez
de la-Cruz, Villagran and Matter, in review]. Further evidence that
reproductive modalities are not fixed within a given phylogenetic group
comes from work on bimodal species of the Sceloporus aeneus complex.
Populations of S. aeneus exhibit oviparity, whereas S. bicanthalis is
viviparous [Guillette, 1981; 1982]. Variations in spermatogenic timing must
be associated with variable responsiveness to environmental stimuli which
regulate gonadal activity. That is, closely related organisms have evolved
differential responsiveness to environmental stimuli at the level of the
central nervous system (hypothalamus), ultimately influencing the release of
gonadotropins and the regulation of spermatogenic activity [Barraclough et
al., 1984]. The pattern of testicular recrudescence exhibited by adult S. jarrovi








in this study is also similar to that described for the developing gonads of

young-of-the-year lizards of the same species undergoing sexual maturation

and their first spermatogenic cycle [Ballinger and Nietfeldt, 1989].
A regressed gonadal condition during the spring and early summer

months is exhibited by S. jarrovi. The seminiferous tubules contain only
Sertoli cells and spermatogonial elements. Hypertrophy of secondary sex
structures is coincident with the period of spermiation and copulatory
activity. This trait is described for other members of the genus Sceloporus
[Matter, unpublished data] as well as other squamates [Aldridge et al., 1990;
Dufaure et al., 1986; Dufaure and Gigon, 1975; Saint Girons, 1982]. Numerous

hypotheses relating the timing of accessory sex structure hypertrophy and

copulatory activity are proposed in the literature, all of them being intimately
associated with reputed functional roles of secretary products from secondary

sex structures. Accessory sex structure secretions are thought to be involved
with sperm physiology or signalling a mated status in squamates [Conner and
Crews, 1980; Cuellar et al., 1972; Garstka et al., 1982; Volsoe, 1944].

Gonadal recrudescence is typically viewed as a process which is driven
by neuroendocrine transduction of environmental stimuli [Duvall et al.,
1982]. Changes in temperature and photoperiod are correlated with the onset
of gonadal growth in seasonally reproductive vertebrates, specifically reptiles
[Licht and Porter, 1987]. Input from higher brain centers regulates release of
the decapeptide, gonadotropin-releasing hormone (GnRH), from
hypothalamic regions in vertebrates [Conn et al., 1987]. Hypothalamic GnRH
is known to influence adenohypophyseal gonadotropin secretion which is
ultimately responsible for spermatogenic proliferation, androgenesis and
maturation of spermatozoa [Hoffman et al., 1992; McLachlan et al., 1995].
Many vertebrates possess a two-gonadotropin system of gonadal regulation








[Ishii, 1990; Licht, 1983]. In mammalian systems, luteinizing hormone (LH)
mediates enzymatic conversion of pregnenalone to progesterone for entry
into the steroid-converting pathway. In testicular tissues, this pathway leads
to the production of testosterone as the predominant androgen for many
vertebrates [Bourne, 1991; Kime, 1987]. Testosterone, in synergism with
follicle-stimulating hormone (FSH), regulates spermatozoon maturation and
spermiation [Pescovitz et al., 1994; Spiteri-Grech and Nieschlag, 1993].
The seasonal aspect of this investigation is incomplete in some regards
and requires further examination. Investigation of material from the
summer months (July and August) would be informative as to the timing of
recrudescence and cellular events associated with germ cell proliferation.
Studies on the environmental regulation of gonadal activity in S. jarrovi
would also be informative. Future investigations should focus on central
nervous system mediation of hypothalamic GnRH release and
adenohypophyseal gonadotropin function. Studies on gonadal activity
should incorporate methods for quantification of steroidogenic capacity and
enzymatic activity. Molecular methodologies for examination of
steroidogenic activation of mRNA transcription and initiation of secondary
sex structure cellular function would also be informative.













CHAPTER 3
TESTOSTERONE STIMULATION OF CELLULAR ACTIVITY IN MALE
ACCESSORY SEXUAL STRUCTURES OF MOUNTAIN SPINY LIZARDS,
Sceloporus jarrovi.


Introduction

Androgens are known to initiate a host of cellular events in mammals,
including genital tract differentiation, increased bone and muscle growth and

development of numerous secondary sex characters [Rommerts, 1990]. The
most biologically active androgen in mammals is testosterone (T), of which
95% originates from testicular synthesis [Coffey, 1988]. Testosterone acts both
directly and indirectly (via enzymatic conversion to other steroids, principally

dihydrotestosterone [DHT]) at the level of cellular nuclei to initiate mRNA
activity and, ultimately, protein synthesis. The ubiquity of androgens
throughout vertebrate phylogeny has been documented, but numerous

biological actions remain unresolved.
Testosterone is known to occur in numerous vertebrate lineages, but
may not always be the most biologically-active androgen [Bourne, 1991].
Lampreys and hagfish (Agnatha) possess T in circulating plasma, but these

tend to be rapidly metabolized to hydroxylated testosterone variants [Kime,
1987]. Elasmobranch fishes (sharks and rays) produce testosterone from
pregnenolone and progesterone, in much the same manner as mammals
[Rommerts, 1990]. In addition, elasmobranchs produce hydroxylated and
glucuronidic subspecies of testosterone. Gonadal tissues of teleost fishes
(primarily Osteichthyes) metabolize testosterone to 11-ketotestosterone as the








major, biologically-active steroid. Amphibian subclasses differ in their

steroidogenic abilities. Urodele amphibians (salamanders and newts) possess
steroidogenic similarities with teleosts; i.e.., they produce 11-ketotestosterone
as the principle androgen. Anurans (frogs and toads), on the other hand,
synthesize 5a-dihydrotestosterone (5a-DHT) as the predominant androgen
[Kime, 1987]. Production of androgens by gonadal tissues in reptiles is
confirmed by several investigations [Bourne, 1991; Lofts and Chiu, 1968].
Many studies on seasonal androgenesis in reptiles assume that T is the

major metabolic end-product of steroidogenesis in males. Androgens
(principally T) are correlated with annual testicular characters in relatively
few reptilian species, although members of all subclasses have been studied.
Plasma androgen concentrations are reported for American alligator
(Alligator mississippiensis) [Lance, 1989], stinkpot turtle (Sternotherus
odoratus) [Mendonqa, 1987], loggerhead sea turtle (Caretta caretta) [Wibbels et
al., 1990] and tuatara (Sphenodon punctatus) [Cree et al., 1992]. Seasonal
androgen determinations have also been made for northern water snake
(Nerodia sipedon) [Weil and Aldridge, 1981], garter snake (Thamnophis
sirtalis) [Krohmer et al., 1987; Weil, 1985], cobra (Naja naja) [Tam et al., 1969],
cottonmouth (Agkistrodon piscivorus) [Johnson et al., 1982], and European
asp (Vipera aspis) [Naulleau et al., 1987]. In these studies, high plasma
concentrations of T are correlated with peak spermatogenic activity and
spermiation. Additionally, high T concentrations are observed in male red-
sided garter snakes, Thamnophis sirtalis parietalis, during the first 12-15 days
following emergence from overwinter dormancy. Mating activity is intense
during this time but soon wanes, correlated with a decline in T titers
exhibited by 18 days after emergence [Krohmer et al., 1987]. Androgen
concentrations determined for the lizards Uromastix hardwicki [Arslan et al.,








1978], Lacerta vivipara [Courty and Dufaure, 1979; 1980] and Sceloporus
undulatus [McKinney and Marion, 1985] also exhibit seasonal changes which
are correlated with spermiation and courtship behavior. Epitestosterone is
shown to be the major androgen produced in the Australian skink, Tiliqua
rugosa (=Trachydosaurus rugosus), but not in closely related Tiliqua
nigrolutea and T. scincoides [Bourne et al., 1985; Bourne et al., 1986a; 1986b].
Bourne warns of the assumptions made by previous investigations on
androgen biosynthesis in reptiles; namely that T should not be viewed as the
principal androgen. He points to a need for more studies on steroid
metabolism and the biological activity of various androgen metabolites
[Bourne, 1991].
Testes are the primary site of androgen production in male vertebrates
[Rommerts, 1990]. Interstitial (Leydig) cells are identified as the steroidogenic
tissues within testes [Lofts and Bern, 1972]. In many amniotic vertebrates,
Leydig cells are found within intertubular spaces between adjacent
seminiferous tubules. In many anamniotes, steroidogenic tissues are situated
around the perimeter of testicular crypts. Some species of lizards (genus
Cnemidophorus) have been shown to exhibit circumtesticular Leydig cells;

these being located just below the connective tissue sheath of the gonads
[Lowe and Goldberg, 1966]. The presence of steroidogenic tissues has been
inferred by localization of specific converting enzymes or by ultrastructural
correlations associated with steroidogenesis. Several attributes of androgenic
tissues have been described in mammals. The occurrence of steroid-
converting enzymes, particularly 30-hydroxysteroid dehydrogenase (30-HSD),
the enzyme responsible for the conversion of pregnenolone to progesterone
(via the A4 pathway) and androstenediol to T (via the A5 pathway), has been
viewed as indicative of steroidogenic tissues. Numerous studies have








documented the presence of 3p-HSD in reptilian interstitial and
circumtesticular cells. Additionally, morphological correlations exist between
the ultrastructural appearance of Leydig cells and plasma T concentrations.
During steroidogenesis Leydig cells exhibit agranular endoplasmic reticulum
and a proliferation of mitochondria with tubular cristae. Steroidogenically
inactive Leydig cells are characterized by mitochondria with laminar cristae
and numerous lipoidal inclusions, which were proposed to be the source of
steroid precursors. These cytoplasmic lipids are depleted during the period of
steroidogenesis.
Gonadal activity in vertebrates has been most intensely studied in
mammals. Steroidogenic and gametogenic activity in testes has been shown

to be coordinated by two hormones of adenohypophyseal origin; luteinizing
hormone (LH) and follicle-stimulating hormone (FSH) [McLachlan et al.,
1995]. These glycoprotein hormones, made up of two subunits (a and P3), are
known to be similar in amino acid composition. The a subunits of LH and
FSH are nearly identical, while the 3 subunits impart the specificity of the
hormones, most likely through interaction with cellular membrane
receptors. The action of LH is characterized as steroidogenic, acting at the
level of Leydig cells in the conversion of cholesterol to pregnenalone. The
functional role of FSH is primarily to direct maturation of gametes
(spermiogenesis) in consort with androgens produced by Leydig cells
[Pescovitz et al., 1994].
Mammalian secondary sex structures are implicated in contributing to
seminal fluid volume, adding components necessary for sperm maturation
and cleansing of the urethral tract both before and after ejaculation [Setchell et
al., 1994]. Accessory reproductive tract structures, including prostate, bulbo-
urethral glands and seminal vesicles, rely on circulating androgens for








initiating differentiation and coordinating normal functions. Epididymal

secretions are essential for normal sperm function in several mammals
[Bedford, 1979; Glover and Nicander, 1971]. Transit through the epididymides

is required for development of sperm motility characters prior to

insemination [Brooks and Tiver, 1983; Orgebin-Crist et al., 1975, 1976]. Within

the Reptilia, members of the order Squamata (lizards and snakes) possess no

homologous structures to the mammalian prostate, seminal vesicles or
bulbo-urethral glands. Male genital tract structures epididymidess and vasa
deferentia) serve as the most likely contributors of fluid and protein

components to be added to seminal volume. Additionally, a structure unique

to male squamates, the renal sexual segment (RSS), may be important in

aspects of reproduction and sperm physiology. First described in the
European grass snake, Natrix natrix [Gampert, 1866], the RSS is comprised of

preterminal nephric tubules, collecting ducts and/or ureters, depending on
the species examined [Saint Girons, 1972]. Seasonal changes in the RSS are

first noted in the garter snake (Thamnophis sirtalis) and the possibility of
androgen regulation was examined using T injections [Bishop, 1959]. The

purpose of this study is to document the androgen-dependent nature of
epididymal and RSS activity in the mountain spiny lizard (Sceloporus
jarrovi) using testosterone implants.

Materials and Methods


Testosterone Implants

Adult male mountain spiny lizards (S. jarrovi) were collected from
Cochise Co., AZ and returned to Gainesville, FL where they were housed
communally in a photothermally regulated animal facility. Animals were








randomly assigned to treatment (n=34) or control groups (n=29). Treated

lizards received an implant of Silastic tubing (1.47 mm ID X 1.96 mm OD;

Dow Corning Corp., Midland, MI) packed with crystalline testosterone (A4-

Androsten-17p-ol-3-one; Sigma Chemical Co., St. Louis, MO) to a length of 5
millimeters and sealed with silicone type A (Dow Coming Corp.). Capsules
were placed subcutaneously on the dorsal surface using a trochar (10 gauge;
Innovative Research of America, Toledo, OH) and wounds were sealed with
New-Skin (Medtech Laboratories Inc., Cody, WY). Control animals were

implanted with equivalent lengths of empty tubing. Lizards were fed crickets

daily, had continuous access to water and kept on a 12L:12D photoperiod with

a daily temperature fluctuation (22-37 C). This study was conducted during

the non-reproductive phase of the annual activity cycle (March-May) to
minimize endogenous hormone influences.

Testosterone-implanted lizards were killed at 3 (n = 5), 5 (n = 3), 7 (n =
8), 14 (n = 8) and 21 (n = 8) days following implantation, by decapitation, and

blood was collected into heparinized capillary tubes, spun to remove cells and
plasma was stored at -70' C until assayed for T using a validated

radioimmunoassay. Control animals (n = 4-7 per sample period) were killed
on the same days as treatment animals. Reproductive tracts (testes,
epididymides, vasa deferentia and kidneys) were removed, divided and fixed
in 10% neutral buffered formalin or Bouin's fixative [Humason, 1979].

Tissues prepared for light microscope examination were embedded in
paraffin, sectioned at 7 gm on a rotary microtome, mounted on slides and
stained with Harris' hematoxylin, biebrich scarlet/orange G and fast green.
Morphological measurements were made on tubules cut in cross-section
(round) and collected as 20 values/lizard. Measurements were made from at
least three slides per animal with no more than ten values collected per slide.








Characterization of Granular Proteins

Adult male Sceloporus jarrovi were collected from Cochise Co., AZ
during May and returned to the University of Florida. Animals were housed
communally in a greenhouse enclosure, provided with a substrate of sand
and cinder blocks for basking sites. Lizards were fed crickets dusted with a
vitamin/calcium supplement powder (Rep-Cal; Rep-Cal Research Labs, Los
Gatos, CA) and watered on alternate days. These investigations were
conducted during the non-reproductive portion of the annual cycle to
minimize the need to remove cellular components from seminal fluids, i.e.,
spermatozoa. Lizards (n=8) were implanted with 5 mm lengths of Silastic
tubing, as above, packed with crystalline testosterone, for 3-4 weeks. Lizards
were killed by cervical dislocation and abdominal structures exposed and
removed via ventral incision. Reproductive structures of interest (excurrent
ducts and kidneys) were isolated in ice-cold 3 mM imidazole/0.3 M sucrose
buffer at pH 7.0 [Depeiges and Dufaure, 1980; 1981]. Epididymides were
grasped with forceps and gentle pressure applied to the length of the tubules
to force secretary material from the duct lumen. Kidneys were minced with a
razor blade in 1.5 ml of buffer and released cellular components were
collected. Fluid and secretary granules from each structure were aspirated
and allowed to settle under gravity. The supernatant fraction was subjected to
three washes with buffer and centrifugation at 3000 X g for 10 min each time.

Gel Electrophoresis

One-way electrophoresis was accomplished by means of standard
polyacrylamide gel system [Laemmli, 1970]. Samples were subjected to 1.0 ml
of Laemmli sample buffer (62.5 mM Tris, 2% sodium dodecylsulfate (SDS),








720 mM 2-mercaptoethanol, 20% glycerol at pH = 6.8) for 3 min in a boiling
water bath. Separating gel stock consisted of 30% acrylamide/2% bis-
acrylamide with 3 M Tris and 0.3% SDS at pH = 8.45. Samples were loaded
(10-15 pl) and run on 10% (final acrylamide concentration) Tris-tricine-SDS
gel system using mini gel casting apparatus (Bio-Rad; Comstock, CA). Gels
were run at constant voltage (150 V) for 105-120 min. High and low
molecular weight standards were run simultaneously with protein samples.
All chemicals and standards were from Sigma Chemical Co. (St. Louis, MO).
Fixation and staining of gels was by 0.2% Coomassie blue in 40% methanol
(MeOH):10% acetic acid (GAA) for 30-45 min. Destain and storage of gels was
in 40% MeOH:10% GAA.

Statistics

Morphometric measures were compared as group means, by treatment
regime. Statistical comparisons were performed using a two-way analysis of
variance (ANOVA) followed post hoc by a Scheff6's F-test with a level of
significance set at P < 0.05.

Results

Implantation of lizards with T-packed capsules significantly elevates
plasma androgen concentrations above that of control animals (Table 3).
Morphological measures of reproductive structures also exhibit significant
differences between T-implanted and control lizards (Table 3 and Figure 3.3).
Testosterone implantation leads to significant increases in RSS (F(9,48) = 91.23,
P < 0.0001) and epididymal (F(9,48) = 28.88, P < 0.0001) epithelium height.
Marked cellular hypertrophy of the RSS (Figure 3.1) and epididymal
epithelium (Figure 3.2) is exhibited by 3 days of T treatment. Renal sex








segment tubules are characterized by a simple columnar epithelium with
nuclei situated in a basal position. Nuclei in RSS tubules of T-stimulated
lizards exhibit prominent nucleoli (often seen in pairs). This cytological
condition is correlated with active protein synthesis. After 3 days of T-
implantation, histochemically identifiable secretary inclusions are seen in
RSS cytoplasm (Fig 3.1 A). By 5 days of hormone treatment, RSS epithelium
exhibits histochemically detectable granular material at the tubular lumina
(Fig 3.1 B). Subsequent sample periods (7, 14 and 21 days) are characterized by
further cellular hypertrophy and secretary activity in RSS tubules from
lizards treated with T (Fig 3.1 C, D and E, respectively). Tissues from control
lizards do not exhibit cellular hypertrophy or secretary activity during the
treatment duration (Fig 3.1 F). The RSS tubules of control animals are
characterized by a cuboidal or low-columnar epithelium and non-
differentiated nuclei.
Changes in epididymal characters are delayed compared to RSS tubules;
significant differences in epithelial height are not seen until 7 days after T-
implantation (Figure 3.3). Cytological changes in epididymal tubules,
however, are evident within 3 days of T administration (Fig 3.2 A). Masses of
secretary material are seen in tubular lumina by 5 days of T treatment (Fig 3.2
B). Longer duration of T implantation results in greater epithelial
hypertrophy and secretary activity. Increased cellular hypertrophy and
secretary activity are seen in epididymal tubules at 7, 14 and 21 days of T
treatment (Fig 3.2 C, D and E). Epididymal tubules of control lizards exhibit
low, pseudostratified columnar epithelium with no cytoplasmic inclusions
(Fig 3.2 F).
Poly-acrylamide gel electrophoresis of excurrent duct and kidney
material from S. jarrovi reveal an array of proteins under denaturing





56

conditions (Fig. 3.4). The proteins present in the greatest concentration are

seen to have relative molecular weights of 31 kDa for RSS, and 70 kDa for

epididymal secretary granules under T stimulation.








Table 3. Characteristics exhibited by reproductive structures in male
mountain spiny lizards (Sceloporus jarrovi) as a result of
testosterone implantation. All values are group means ( SD).


Body Testes RSS Epididymis Total
Treatment SVL Mass Volume Epithelium Epithelium Androgens
(n) (mm) (gm) (mm3) (jgm) (gim) (ng/ml)
Testosterone

3 day (5) 82.6 16.60 29.7 20.3 a,b 21.8 a 19.8 b
(5.5) (1.04) (6.8) (1.8) (4.2) (7.5)

5 day (3) 75.7 13.74 11.6 21.9 b,c 18.3 a 10.4b
(0.9) (0.47) (3.0) (2.5) (2.4) (2.4)

7 day (8) 76.8 13.72 19.1 25.2 c 24.9 b 15.3 b
(4.7) (1.01) (8.0) (3.3) (3.9) (5.6)

14 day (8) 79.6 13.95 24.9 32.6 d 29.7 b,c 14.1 b
(5.5) (1.36) (10.6) (3.6) (5.3) (5.5)

21 day (8) 81.5 14.81 25.6 38.6 e 30.3 c 15.0 b
(4.1) (1.07) (10.7) (5.5) (3.9) (4.8)


Control

3 day (4) 80.4 15.42 30.6 12.7 a 18.6 a 0.4 a
(2.3) (0.80) (2.0) (1.5) (3.7) (0.3)

5 day (4) 81.3 15.94 20.7 11.9 a 14.9 a 0.2 a
(4.0) (2.0) (19.3) (0.7) (1.9) (0.1)

7 day (5) 76.0 13.39 24.8 11.2 a 12.4 a 1.3 a
(7.6) (2.15) (11.9) (0.6) (1.3) (1.1)

14 day (7) 79.9 14.85 30.4 11.3 a 13.0 a 0.2 a
(3.4) (1.48) (21.5) (0.6) (1.1) (0.03)

21 day (7) 76.9 11.11 15.7 11.1 a 13.2 a 0.8 a
(4.0) (0.59) (1.9) (0.9) (2.2) (1.3)
* Values within columns designated by different lettered superscripts are
significantly different as determined by two-way ANOVA with oa < 0.05,
followed by Scheff6's F-test for between-group comparisons (P < 0.001).





























Representative light micrographs of kidneys from T-implanted
and control male mountain spiny lizards, Sceloporus jarrovi. T-
implanted lizards A) 3 days. Note secretary material in cellular
cytoplasm (arrows). B) 5 days. Secretory material seen at
epithelial apices (arrows). C) 7 days. D) 14 days. E) 21 days. Note
the accumulation of secretary material at cellular margins. F)
Control lizard kidney. RSS tubules are regressed with cuboidal
epithelium and no secretary material. Scale bar equals 50 ptm for
all micrographs.


Figure 3.1
































Figure 3.2


Representative light micrographs of epididymal tubules from T-
implanted and control male mountain spiny lizards, Sceloporus
jarrovi. T-implanted lizards. A) 3 days. Tubules are
hypertrophied, but not secretary. B) 5 days. Small accumulations
of secretary material seen in tubular lumena. C) 7 days. D) 14
days. E) 21 days. Additional secretary material seen in tubular
lumen. F) Control epididymal tubules with pseudostratified
columnar epithelium and no secretary material. Scale bar equals
50 pm for all micrographs.






61













.; 4 ., -



























a *.. .,
a a














4 4






ib






-- 4 OPP
IT~~ .!e ,.





























Graphic representation of histological changes due to testosterone
stimulation of male accessory sex structures in S. jarrovi. A) RSS
epithelium (F(9,48) = 91.23, P < 0.0001) and B) epididymis
epithelium (F(9,48) = 28.88, P < 0.0001) exhibit relatively rapid onset
of epithelial hypertrophy. Bars with different letters are
significantly different by two-way ANOVA with a < 0.05, followed
by Schefff's F-test for between-group comparisons.


Figure 3.3







* Testosterone Implant
E2 Control


50-

40-

30-

20-

10-

0


a



I


a


i1


TREATMENT DURATION (DAYS)


* Testosterone Implant
E2 Control


a


a


TREATMENT DURATION (DAYS)


a, b
-1


a


a


I=T


a


a



I


--r


a




























Figure 3.4


Representative one-way polyacrylamide gel electrophoretic run of
RSS (lanes C, E and G) and epididymal (lanes D and F) proteins
from male S. jarrovi. Lane H is RSS protein from S. undulatus for
conspecific comparison. Lanes A and B are high and low-
molecular weight standards, respectively. Proteins are visualized
with 0.2 % Coomassie blue. Major proteins at 31 kDa for RSS
(open arrow) and 70 kDa for epididymis (solid arrow) in S. jarrovi.





















A B C D E F G H


97


pm


66


45 Wn

36


a'


.4 *-,
fi. i '"


29 4* f j

M


V










Discussion



The present study reinforces the importance of T in regulating activity
of accessory sexual structures in squamate reptiles. Testosterone-implanted
lizards exhibit rapid cellular hypertrophy and secretary activity of RSS and

epididymides when compared to control (empty implant) animals as
evidenced by significant increases in cellular size by 3 and 5 days, respectively.
This finding supports the notion that seasonal initiation of cellular activity of

male S. jarrovi secondary sexual structures is most likely under the influence
of gonadal androgens acting to direct mRNA transcription. It is not known if
T acts to initiate cellular activity directly or if it is converted to DHT or

another biologically active steroid first. The possibility that T acts to stimulate
growth factors responsible for cellular hypertrophy and activation must also

be considered. Investigations on the steroid-metabolizing capacity of gonadal
tissues in S. jarrovi would be informative in this regard.

The rapidity with which T acts to bring about cellular activation of sex .
structures is also unprecedented in reptilian literature. Other investigations
report on the effectiveness of stimulating secondary sex structures utilizing
androgen administration in reptiles, but none has documented changes at

early sample periods (3 and 5 days in this study). Stimulation of RSS and
epididymal epithelial cells is reported for Sceloporus olivaceus after 6 weeks
of testosterone implantation [Forbes, 1941]. Testosterone propionate (TP) is
effective in stimulating RSS and epididymis hypertrophy and secretary
activity in castrate skinks, Eumeces fasciatus [Reynolds, 1943]. A dose-related
response in RSS hypertrophy is seen with TP injections administered to








castrate garden lizards, Calotes versicolor [Pandha and Thapliyal, 1964].
Experimental manipulations with hypophysectomized Indian house lizards
(Hemidactylus flaviviridis) reveal a complex interplay between pituitary
gonadotropins and gonadal steroids in regulating RSS hypertrophy and
cellular activity [Reddy and Prasad, 1970b]. Injections of TP and/or
mammalian LH and FSH for three weeks results in differential hypertrophy
and secretary activity of RSS. Testosterone propionate alone, or in
combination with FSH, maintains RSS activity at maximal levels seen during
the seasonal cycle. Interestingly, FSH treatment alone, or in combination
with LH, results in RSS stimulation equal to that displayed by TP-treated
lizards. Conversely, LH alone is effective in stimulating RSS hypertrophy to
only half of the levels seen in TP and FSH-treated animals [Reddy and Prasad,
1970a]. Thus, TP acts in a typical androgenic fashion in stimulating RSS
activity in H. flaviviridis and FSH is most likely acting to stimulate steroid
biosynthesis in gonadal tissues which lead to stimulation of RSS hypertrophy.
Decreased effectiveness of LH in these studies supports the finding that, in
squamates, a single, FSH-like gonadotropin is acting to bring about
gametogenesis and steroid production [Licht, 1983]. Cytological position of
secretary material in RSS and epididymides at 3 and 5 days of T treatment,
respectively, are suggestive of protein synthesis by endoplasmic reticulum
and packaging by Golgi apparatus. Migration of proteins in vesicular granules
to cellular apices is consistent with the notion that these structures of Golgi
apparatus origin.
Activity of L. vivipara epididymides is associated with seasonal
reproductive events [Dufaure and Chambon, 1978; Dufaure and Gigon, 1975].
Correlations with seasonal concentrations of T and epididymal epithelial
hypertrophy and secretary activity coincide with testicular androgen








production [Courty and Dufaure, 1979; 1980] and epididymal T content
[Dufaure et al., 1986].
Several studies utilizing mammalian pituitary hormones in a variety
of snake and lizard species produce conflicting points of view with respect to
gonadotropin effectiveness. Ovine FSH is effective in stimulating testicular
growth in immature male water snakes (Nerodia sipedon), as indicated by
increased mass, seminiferous tubule diameter and epithelial height compared
to LH-treated and control animals [Krohmer, 1986]. In contrast, LH-treated
snakes exhibit a greater degree of Leydig cell activity (indicated by lipid
accumulation and mitochondrial hypertrophy). Additionally, animals in this
study exhibit a more rapid onset of RSS secretary granule formation in snakes
treated with LH compared to FSH, indicating a greater steroidogenic capacity
for LH [Krohmer, 1986].
A study examining the effectiveness of non-gonadotropin hypophyseal
hormones at stimulating RSS activity reveals that growth hormone (GH)
synergized the action of T in Anolis carolinensis. Sex segment condition is
unaffected by GH alone, but produces greater cellular hypertrophy of RSS
tubules compared to animals receiving only T. Prolactin alone does not cause
RSS hypertrophy, or act in synergy with T to bring about greater epithelial
development when compared to androgen alone [Gerrard, 1974]. Isolation
and characterization of squamate gonadotropin(s) would greatly aid in the
elucidation of environmentally-mediated neuronal regulation of gonadal
development and activity in this taxonomic group.
Administration of antiandrogens results in disruption of normal,
androgen-dependent activity in the epididymides of Hemidactylus
flaviviridis. Cyproterone acetate (CA) which acts by blocking androgen
binding at the receptor level. This compound can also have antigonadotropic








activity, causing the accumulation of lipids in Leydig cells of the testes of the

lizard H. flaviviridis [Haider and Rai, 1986].
The excurrent duct of many male mammals is known to contribute
protein components that influence numerous aspects of sperm physiology .
Elements of the epididymides and vas deferens, as well as mammalian-

specific accessory structures, such as seminal vesicles, prostate and bulbo-
urethral glands, produce material which interacts with spermatozoa in a
number of physiological ways. Secretory products from these structures are
not limited to protein substances. Many lipoproteins, glycoproteins and
carbohydrates are known to be produced by mammalian accessory structures

[Setchell et al., 1994]. These substances are known to influence various aspects

of spermatozoa membrane integrity and structure, as well as serving as
energy substrates for driving motility [Cooper, 1990].
Initial characterization of secretary proteins from S. jarrovi RSS and
epididymides reveal several protein components, but indicate different major
protein bands when the two regions are compared. Granular secretary
proteins from RSS and epididymides are similar in histochemical stainability,
but differ in molecular weight and granule size. Secretory material from both
accessory structures stains intensely with Beibrich scarlet, a stain which is
selective for arginine residues [Kiernan, 1990]. Granules from S. jarrovi RSS
are approximately 1.0-1.5 [m in diameter while those from epididymal
tubules are smaller, about 0.5 gim in diameter.
Work on the male excurrent duct system of reptiles and its relationship
to sperm physiology is scant. The most in-depth studies are by J-P Dufaure
and his colleagues on the European viviparous lizard, Lacerta vivipara.
Production of epididymal proteins in L. vivipara is seasonally cyclic and
coincides with increased plasma T concentrations, spermiation and








copulatory activity in much the same fashion as is exhibited by S. jarrovi.
Both these species exhibit viviparity; L. vivipara mates in spring and gives
birth to litters of young in summer, while S. jarrovi exhibits late-summer and
fall reproductive activity and females give birth the following spring after
overwinter pregnancy [Goldberg, 1971b].
In Lacerta vivipara epididymal secretary granules posses a central core
which is comprised of protein(s) of approximately 70 kDa in weight, the H
protein [Depeiges and Dufaure, 1980]. The 70 kDa protein from S. jarrovi
epididymides may be similar in nature to the H protein from L. vivipara, but
further protein characterization is required. Additionally, the soluble fraction
of the epididymal granules has a major protein component with molecular
weight of 19 kDa, the L proteins [Depeiges and Dufaure, 1981]. Using the
current sample protocol, I was unable to collect soluble proteins, so low
molecular weight homologues of the L proteins are undetected in the present
investigation. Studies of these low molecular weight, soluble proteins form
the basis of our current understanding of epididymal protein production by
the lizard excurrent ducts, as these investigators did not examine RSS
proteins. The androgen-dependent nature of secretary protein production by
L. vivipara epididymides is well documented. The L proteins are produced
under influence of gonadal androgens which stimulate mRNA synthesis and
transcription of genomic sequences responsible for the epididymal protein
synthesis [Courty et al., 1987; Ravet et al., 1987].
A study of male reproductive tract secretions in water snakes (mostly
Nerodia fasciata, but with some comparisons to N. taxispilota and N.
rhombifer) reveals that several proteins are produced during spermiation in
the fall. These proteins are not present in serum or during the non-breeding
season [Esponda and Bedford, 1987]. Proteins with molecular weights of less








than 45 kDa are similar in the three snakes, but with subtle immunological

differences, indicating some degree of species specificity most likely related to

particular carbohydrate moieties. Electrophoresis of secretary material from
other species of Sceloporus reveal proteins of similar molecular weights, but
with subtle differences compared to S. jarrovi. Sceloporus undulatus and S.
clarki exhibit RSS proteins with molecular weights of 33 kDa and 31 kDa,
respectively [Matter, unpublished data]. Immunofluorescent antibodies to N.

fasciata male duct secretary proteins bind to the entire surface of spermatozoa

from epididymides, but exhibit no binding to testicular sperm, confirming
that epididymal proteins influence sperm surface changes [Esponda and
Bedford, 1987].
Non-squamate reptiles, the crocodilians, chelonians [Fox, 1977] and

sphenodontids [Saint Girons and Newman, 1987], do not possess RSS

structures and there are few reports of epididymal function with respect to

sperm physiology in these taxa. A single investigation presents evidence
supporting the notion of functionally analogous protein production by
kidney tubules in the slider, Trachemys scripta [Garstka and Gross, 1990]. This
study reports both activational and inhibitory effects of kidney cytosolic
fractions on sperm motility and correlates the differences in action with
seasonal reproductive events. Activation of sperm motility occurs only
during the breeding season (April) and only in a subset of the animals tested;
6 of 48. In many ways this investigation is limited and should be viewed
cautiously with respect to its significance in regulation of sperm motility by
kidney products in the turtle. The kidney proteins from T. scripta are
described as representing a "functional" renal sex segment since no seasonal
morphological differences were apparent in kidney tubules and no change in








renal tubules were exhibited following castration and androgen replacement

[Garstka and Gross, 1990].
A single publication reporting kidney homogenate effects on sperm

motility exists for green anoles, Anolis carolinensis [Cuellar et al., 1972].

Overall sperm motility increases (percent motility) with addition of kidney
homogenates, but RSS granules are not separated from other cellular
components making the results difficult to interpret with respect to the role
that RSS proteins may play in sperm physiology.
Mammalian excurrent duct proteins are characterized for several

domestic and lab species [see reveiws by Robaire and Hermo, 1988; Brooks,
1990; Cooper, 1990]. Androgen-dependent proteins of Wolffian duct origin
are reported for rat [Jones et al., 1980] and mouse [Taragnat et al., 1988]. These
proteins are of comparable molecular weights (18.5 to 47 kDa for rat
epididymis and 34.8 kDa for mouse vas deferens) to those determined for RSS

and epididymides of S. jarrovi. Most seminal fluid proteins characterized for
mammals are soluble in nature, few being released as membrane-bound
secretary granules. No attempt was made to characterize soluble proteins
from S. jarrovi epididymides, but their occurrence is likely since soluble

protiens are known from Lacerta vivipara Wolffian ducts [Depeiges and
Dufaure, 1981]. Similarity in structure and function of reptilian Wolffian
duct proteins to mammalian reproductive tract proteins can only be
speculated, currently. It may be expected that RSS proteins are functionally
similar to proteins from mammalian seminal vescicles and prostate, by
virtue of the common embryonic origin of these structures, the ureteric bud.
Similarity in epididymal protein function between vertebrate groups may be
expected as a consequence of their tissue homologies and common
physiological requirements for maintaining sperm viability.





73

Further characterization of seminal components, including RSS and

epididymal granules will allow for the production of antibodies to these

proteins. It is hoped that administration of antibodies to lizards will result in

an auto-immune response to these proteins that will help to elucidate their

potential role in sperm motility and viability.













CHAPTER 4
POST-COPULATORY FATE OF SEMINAL COMPONENTS
IN THE FEMALE REPRODUCTIVE TRACT OF MOUNTAIN SPINY
LIZARDS, Sceloporus jarrovi


Introduction

The vertebrate oviduct exhibits a number of complex functions. In
reptiles, these functions varying depending on the parity mode of the female
but can include egg albumen and shell secretion, placentation, sperm
transport and sperm storage [Gist et al., 1990; Guillette, 1992; Palmer et al.,

1993; Palmer and Guillette, 1991; 1992]. Each of these functions requires
specific morphological features that, in many species, are similar but can
show variation. For example, two apparently distinct patterns of shell
formation occur among oviparous reptiles.

In turtles, tuatara and squamates, the oviduct is divided into four
distinctive regions, the anterior infundibulum, the tube, the uterus and the
vagina [Fox, 1977; Gist and Jones, 1987; Guillette et al., 1989; Palmer et al., 1993;

Palmer and Guillette, 1988]. It should be noted that various authors have
used differing terminologies for the same oviductal regions. The reptilian
oviduct is embryologically derived from the Miillerian duct, and thus,
homologous with that found in all vertebrates except the agnathan and
teleost fishes. A common terminology should be used. [We will use the
terms presented above as they are clearly defined and commonly used.] The
infundibulum has an opening, the ostium, through which the ovulated egg
enters the oviduct. The infundibulum may secrete a thin glycosaminoglycan








coating around the eggs as they pass through this region [Palmer et al., 1993].
As eggs enter the tube, they are coated with albumen composed of many

proteins serving a variety of functions [for review, see Palmer and Guillette,

1991]. Once through the uterotubal junction, the eggs are coated with layers of
crystaline calcium salts, the calcified egg shell. The calcium layer and fibrous
shell membranes are species specific in thickness and morphology [for
review, see Packard and DeMarco, 1991]. The process of albumen and egg
shell deposition appear to be fairly conserved among turtles, squamates and

tuatara Secretion of albumen and formation of the shell membranes is

rapid, requiring approximately 48 hr following ovulation in the lizard
Sceloporus woodi [Palmer et al., 1993]. Calcification of the egg shell then

proceeds for varying periods of time but is usually completed in 5-14 days. It
is important to note that shell membrane fibers and shell calcium are secreted

from the same oviductal region, the uterus, but sequentially. In viviparous
squamates, the shelling process is either greatly reduced or completely

inhibited [Guillette, 1993; Guillette and Casas-Andreu, 1987]. This is

apparently accomplished by reducing the number of uterine glands associated
with the secretion of the shell membrane as well as reducing the epithelial

secretion of calcium [Guillette, 1992].
In contrast, the crocodilian oviduct, like that of its archosaurian
relatives, the birds, has regions similar to that described for other reptilian
orders, but the uterus is subdivided into two distinct regions, an anterior and
a posterior uterus [Palmer and Guillette, 1992]. The anterior uterus secretes
the proteinaceous fibers of the shell membrane. This region is homologous
to the magnum of the avian oviduct. The crocodilian posterior uterus
secretes the calcium used in the formation of the calcified shell as does the
homologous avian shell gland. Thus, the shell components, the shell








membranes and crystaline calcium shell are secreted from differing, highly

specialized regions of the oviduct in crocodilians and birds [Palmer and

Guillette, 1992].
In addition to shell formation, the oviduct also serves as the primary

region for fertilization in reptiles and thus, this structure plays an important
role in sperm transport and nurture. Many amniotic vertebrates are known

to possess some potential for sperm storage within the female's reproductive

tract. Duration of sperm storage and the morphological features associated
with this phenomenon can vary across taxonomic groups. Most mammals

have a limited sperm storage capability with the exception of many bats,

particularly members of the families Vespertilionidae and Rhinolophidae
[Racey, 1975]. Variation in viable sperm storage capacity in these bats ranges
from 16 days in a tropical pipistrelle, Pipistrellus ceylonicus [Gopalakrishna

and Medhaven, 1971], to 198 days in a temperate-zone species, the European
noctule, Nyctalus noctula [Racey, 1973]. Earlier studies indicate that viable
sperm are stored for 138 and 156 days in Myotis lucifugus and Eptesicus
fuscus, respectively [Wimsatt, 1944]. Correlations between the length of
sperm storage, incidence of hibernation and latitudinal distribution in bats
were postulated by Racey [1975].
Three sites of sperm storage are reported for members of the

Chiroptera: 1) the oviduct, 2) the utero-tubal junction, or 3) the uterus.

Uterine sperm of Pipistrellus pipistrellus are oriented with heads
perpendicular to the epithelial surface and are seen to make contact with
epithelial cells by means of microvillar extensions [Racey, 1975]. Similarly,
birds exhibit regional differences in sperm storage structures [Bakst, 1987;
Birkhead and Hunter, 1990; Birkhead and Moller, 1992; Shugart, 1988]. The
avian utero-vaginal junction has storage sites variously referred to as "sperm-








storage tubules" [Bakst, 1987; Birkhead and Hunter, 1990], "sperm-storage

glands" [Shugart, 1988], or "sperm host glands" [Gilbert et al., 1968]. Sperm
storage also occurs in the infundibulum of birds.
Examination of various members of the Reptilia reveals oviductal
sperm storage structures in most of the subclasses. Sperm storage is
confirmed in the Chelonia and Lepidosauria, but has not been described for

the Crocodilia. The likely regions of the oviduct of the American alligator

have been examined histologically without detecting stored sperm [Palmer
and Guillette, unpublished data]. Several investigations document the
occurence of morphological structures for sperm storage in turtles [Gist and
Fischer, 1993; Gist and Jones, 1987; Gist et al., 1990], snakes [Fox, 1956; Halpert
et al., 1982; Hoffman and Wimsatt, 1972; Ludwig and Rahn, 1943] and lizards
[Adams and Cooper, 1988; Cuellar, 1966a; Fox, 1963]. Additional reports

providing circumstantial evidence on sperm storage are based on the
production of viable eggs or young in the absence of potential mating

opportunities. Fox [1977] warns against the utility of this approach because of
the possibility of parthenogenetic reproduction, but most cases reported are for
known bisexual species. Using this type of circumstantial evidence, sperm

storage has been reported for dwarf chameleons, Microsauria pumila [Atsatt,
1953], and the snakes, Leptodeira annulata [Haines, 1940], Acrochordus
javanicus [Magnusson, 1979] and Agkistrodon contortrix [Schuett and
Gillingham, 1986].
Anatomy of oviductal sperm storage in lizards is described for several
species [Adams and Cooper, 1988; Bou-Resli et al., 1981; Cuellar, 1966b; Fox,
1963; Kumari et al., 1990; Schaefer and Roeding, 1973]. Lizards store sperm in
two possible oviductal regions: 1) the anterior vagina (also referred to as








utero-vaginal junction) and 2) the posterior portion of the infundibulum (for

review, see [Gist and Jones, 1987]).
The occurrence of sperm storage is interesting from a number of

perspectives. Morphological and anatomical structures for sperm storage can
impart information related to the evolutionary liability of such structures,
assuming that these structures are synapomorphies. Ecologically, sperm
storage allows for mutliple matings and the potential for sperm competition

or preferential release of stored sperm [Devine, 1984; Halliday and Verrell,
1984]. Increasing evidence points towards the incidence of extra-pair

copulations and sperm competition in mammals [Schenk and Kovacs, 1995],
birds [Oring et al., 1992] and reptiles [Schuett and Gillingham, 1986].


Materials and Methods



Twenty-eight first year female Sceloporus jarrovi were collected from
the Dragoon Mountains in Cochise Co., AZ and returned to Gainesville, FL
during August. These lizards were born the preceding May and, hence, were

approximately 5 months old at the time of manipulation and had not
experienced a previous reproductive cycle. Females were housed
communally, isolated from males, until they were introduced to mature
males for observation of copulations. Ten males (also collected in August)
were placed, as pairs, in arenas (2 m X 2 m X 0.8 m) with piles of rocks and
cinder blocks for basking and display sites. Males established breeding
territories in the enclosures and many interactions between these lizards were
observed. Lizards were fed crickets and Tenebrio larvae and sprinkled with
water on alternate days. Beginning in October, females were introduced to
male arenas and allowed to copulate once. Males approached females with








stereotypical head-bobs, shuddering movements and tongue flicks across the

females' dorsal surfaces [Ruby, 1977]. Receptive females were gripped by the

posterior aspect of the head with males' jaws and copulation followed rapidly,

being brief in duration (about 2-5 s). Copulated females were killed by

decapitation at 3, 4.5, 6, 12 and 24 hours following copulation. Reproductive

tracts were exposed by mid-ventral inscission and preserved in situ using

Bouin's fixative. Trunk blood was collected into heparinized capillary tubes,

centrifuged and plasma stored at -70 C until assayed for estradiol-1713. After

tissues had fixed and hardened, reproductive tracts were prepared for light

microscope examination. Tissue was embedded in paraffin, sectioned at 7 gm

on a rotary microtome, mounted on slides and stained with Harris'

hematoxylin, biebrich scarlet/orange G, fast green (modified Schorr's stain as

outlined in Appendix) and hematoxylin, eosin and alcian blue [Humason,

1979]. Tissues were evaluated for the presence and position of sperm and

other discernable seminal components secretaryy material from males'

secondary sex structures, i.e., epididymis and renal sex segment).

Measurements were made on several morphological features of the oviduct,

including cloacal epithelial height, sperm-storage gland diameter and gland

epithelial height. Morphological measurements were made on sperm storage
tubules cut in the tranverse plane and collected as 20 values/lizard.

Measurements were made from at least three slides per animal with no more

than ten values collected per slide. A series of females (n = 8) that were either

not introduced to males or refused male courtship attempts served as
controls.
All group comparisons (means) were tested for significant differences

at a level of ao < 0.05 using a one-way analysis of varience (ANOVA) followed








by a post hoc Scheff6's F-test. Statistical tests were made using Statview

software on a Macintosh computer.


Results


Oviductal Atamony

Oviducts of S. jarrovi are paired abdominal structures composed of an

anterior infundibulum, middle uterus and posterior vagina. Vaginal

openings emerge, separately, into the urodaeum (Fig. 4.1). Histologically, the

female reproductive tract of S. jarrovi appears similar to that described for
other sceloporine species [Cuellar, 1966b; Cuellar, 1970; Palmer et al., 1993].
The cloacal epithelium of reproductive females is a pronounced transitional
epithelium with dome-shaped cells at the surface (Fig 4.2). The basement
membrane of the epithelium is thrown into a complex series of folds and
arborizations. A constricted passageway from the cloaca leads to the lower

portion of the vagina (Fig.4.2). The cloacal-vaginal transition is delineated by
an abrupt change in the epithelial composition, being represented largely by
ciliated, low columnar cells. Longitudinal furrows and a heavy muscularis
layer formed largely from smooth muscle fibers comprised this region (Fig.
4.3). At the distal end of the vagina, a circular muscle component may form a

functional "cervical" sphincter. The muscularis longitudinalis is less evident

at this level of the oviduct. The posterior vagina is characterized by
longitudinal furrows and an epithelium that is heavily ciliated (Fig. 4.3).
Anterior to the furrowed section of the vagina, the basement
membrane branches to give rise to a greater number of furrows, these ending
in blind pockets at the proximal portion of the vagina (Fig. 4.4). The
epithelium is highly ciliated and interspersed with mucus-secreting cells








which stain strongly for glycosaminoglycans (alcian blue positive) and have a

short, columnar morphology (Fig. 4.5). The pockets at the anterior portion of
the vagina form the sperm-storage crypts (SSCs) of the oviduct in Sceloporus
jarrovi. The SSCs are formed from shallow folds in the basement membrane
of the vagina. There is a well-defined mucosal region, rich in collagenous

fibers and circulatory and lymphatic elements associated with the SSCs. The

muscularis is reduced but composed of well-defined smooth muscle bundles

(Fig. 4.4).


Fate of Seminal Componants

At copulation, semen is deposited at the vaginal opening; a white mass
is seen macroscopically at the confluence with the cloacal surface.

Microscopically, the seminal mass is composed of spermatozoa and
agglomerations of secretary granules originating from the males'
epididymides and renal sex segment tubules (RSS). Many morphological

differences are exhibited between mated females and those that did not
copulate (Table 4). Copulated females possess a cloacal epithelium of a
transitional type that is composed of from 8 to 15 cell layers (Fig. 4.3).
Conversely, unmated females display cloacae with a stratified columnar

epitheium being made up of 2 to 3 cell layers and having basal nuclei in the

layer in contact with the lamina propria (Fig. 4.7). Only sperm are seen in the
reproductive tract, as the secretary granules are retained at the cloacal surface.
The secretary granules appear to be endocytosed by the transitional
epithelium of the cloaca, small clusters of histochemically identifiable
material are seen within the cytoplasm of surface-most cell layer (Fig.4.3).
Seminal masses are evident in the cloaca at all times examined following
copulation, but by 24 hr post copulation, these masses are extremely reduced








and the secretary granules have lost their characteristic stainablity (normally

staining intensely with biebrich scarlet), they become quite opacque. Sperm

are seen in vaginal furrows at 3, 4.5, 6 and 12 h following copulation (Fig. 4.3).

Vaginal SSCs exhibit masses of spermatozoa at all times examined. Sperm
are oriented "head first" in SSCs and are not seen to penetrate epithelial cell
membranes (Fig. 4.6). At no time examined are sperm seen higher than the

vaginal region of the reproductive tract. Cells of the cloacal epithelium,

vaginal furrows and single, isolated, non-cilliated cells of the sperm-storage
crypts stained intensely with alcian blue, indicative of glycosaminoglycan

synthesis (Fig. 4.5). Mucous-like secretary material is seen in the vaginal
lumen and sperm-storage crypts. Additionally, mated lizards possess ovarian

follicles that are greater in size than unmated females, which exhibit small or

atretic follicles.





83

Table 4. Morphometric values for features of female reproductive structures
following copulation in Sceloporus jarrovi. Values are group means ( SE).

SVL Body Mass Cloaca Cloacal SSC SSC
(mm) (gm) Epithelium Cell Diameter Epithelium
(.tm) Number (gim) (gim)

Copulated ns ns
(n)
3 hours 59.50 7.43 95.82 10.38 59.07 14.13
(4) (1.71) (0.74) (3.16) (0.26) (1.15) (0.36)
4.5 hours 62.25 8.06 97.47 10.75 58.71 13.29
(4) (1.65) (0.63) (2.29) (0.26) (1.75) (0.25)
6 hours 59.75 7.21 89.22 10.10 59.70 14.34
(3) (0.25) (0.16) (2.50) (0.28) (1.56) (0.31)
12 hours 61.33 8.10 90.60 10.37 60.60 14.08
(4) (1.76) (0.57) (3.47) (0.32) (2.54) (0.43)
24 hours 60.25 7.76 115.02 11.52 52.53 13.32
(4) (1.84) (1.04) (3.11) (0.35) (1.17) (0.24)

Non- 56.12 5.48 33.56 2.95 19.75 6.94
copulated (0.58) (0.30) (1.60) (0.18) (0.65) (0.29)
(8)


ns; no significant differences exist for these characters.
; all values for copulated lizards are different from non-copulated
(F(5,19) = 27.86, p < 0.0001).
Y; all values for copulated lizards are different from non-copulated
(F(5,19) = 38.26, p < 0.0001).
n; all values for copulated lizards are different from non-copulated


animals

animals

animals


(F(5,18) = 54.30, p < 0.0001).
t; all values for copulated lizards are different from non-copulated animals
(F(5,19) = 24.57, p < 0.0001).





























Figure 4.1 Female reproductive anatomy of Sceloporus jarrovi. Dashed lines
delineate major regions of the oviduct: anterior infundibulum,
middle uterus and posterior vagina. Relative position of ovaries
and cloaca are indicated. Scale bar equals 5 mm.

















Infundibulum





- Ovary





Uterus


Vagina


Cloaca


















u n
-I -. -





>CC>
Q a -o -

(V Ca 0 ca





U 0~ 0
u uz






Ca
o o z






~) 4- tj
(o 0 C Z
= a 3 o .5'







2- -I-.;
> M
cs -






CUU
Z s a -i
ea a ^


S o ) -o
o m m c -; aa
-. 0 a > -



-. o
u



U l-i- '- 3
o o U-o a
o 13>> >
r.U
o i fl '

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czo E







So >c w 150
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87























SIA






...,. s ..





























Micrographs of various features of the cloaca and posterior vagina
of S. jarrovi. A and B) Cloacal surface 6 hr following copulation.
Seminal mass is seen in contact with cloacal epithelium. Scale bar
equals 100 pm for A. Scale bar in A equals 50 p.m for B. C) Cloacal
epithelium at 6 hr after copulation. Epithelial cells are seen to
endocytose masses of secretary granules from the seminal mass
(arrows). D) Longitudinal section of posterior vagina with free
spermatozoa evident in the lumen (arrows). Scale bar in A equals
25 p.m for C and D. E and F) SEMs of posterior vagina with sperm.
Epithelial surface is highly ciliated (c), but with interspersed
mucus-secreting cells. Scale bar in E equals 50 pgm. Scale bar in F
equals 15 gpm. Key: CL; cloacal epithelium, F; mucosal folds of
cloaca, M; seminal mass, Lf; longitudinal folds of vagina, s; sperm.


Figure 4.3