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Histochemistry and composition of the male reproductive tract of the horned lizard as affected by seasonal variation.

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Histochemistry and Composition of the Male
Reproductive Tract of the Horned Lizard
as Affected by Seasonal Variation'
Department of Anatomy, Medical College of Virginia, Richmond, Virginia
emergence from hibernation as well as at
at the end of their breeding cycle. Lizards
were obtained and studied in 4 groups
over two complete breeding seasons. These
groups were killed during early May, June,
early and late July. Testes, epididymides
and epididymal ducts were fixed in acetic
acid-alcohol-formalin, neutral buffered formalin and the dichromate-sublimate fixative of Elftman ('57). When possible,
serial sections were prepared. Dry matter
of testes and epididymides was determined
by heating samples at 105°C overnight in
an electric oven.
The following histochemical reactions
and biochemical procedures were applied.
Basophilia. A modification of the toluidine blue procedure of Kramer and Windrum ('55) and the azure A-eosin I3 technique (Lillie, '54) were used. The tohiidine
blue solution was prepared at pH 5.48.
Control determinations for these procedures consisted of incubating adjacent
sections for one hour at 56°C in a 0.01%
ribonuclease solution in Sorensen's phosphate buffer (pH 6.7). Following enzyme
treatment, slides were washed and stained
as for untreated sections.
Periodic acid-Schiff reactive substances.
The periodic acid-Schiff (PAS) procedure
as developed by McManus ('46) and
Hotchkiss ('48) was used. Controls consisted of hydrolysis in amylase for one
hour in order to remove glycogen, treatment wtih methanol-chloroform for removal of glycolipids and staining without
previous passage through periodic acid.
Reactions were considered positive only if
Eighty-four sexually mature male horned they stained exclusively after periodic acid
lizards, Phrynosoma cornutum (Harlan), oxidation.
were used in this investigation. These repby U.S.P.H.S. grant A-1181 from
tiles were collected in Kleberg County, the1 Supported
National Institute of Arthritis 2nd Metabolic
Texas, and included lizards soon after Diseases.
In recent years biologists have examined
physiologic characters of vertebrates as related to their evolutionary significance.
As a result of these investigations, homologous as well as analogous physiological
and biochemical characteristics have been
found which, in some cases, have been as
striking as the morphological findings of
comparative anatomists. Through a histochemical approach, the characteristics of
an organ may be related to its structure,
and composition which otherwise might
not be realized in studies employing only
histological or biochemical procedures. In
turn, histochemical results are enhanced
when paralleled with biochemical methods.
The spermatogenesis of the horned lizard has been studied by Cavazos ('51).
Blount ('29) reported the seasonal changes
in the number and morphology of the interstitial cells and Mellish ('36) considered
the effects of pituitary extract and environmental conditions on the genital system of the horned lizard. Cavazos and
Melampy ('54) and Melampy and Cavazos
('54) have examined the distribution of
periodic acid-reactive carbohydrates and
lipids in testes of lizards. The present
study deals with a histochemical and biochemical investigation of the reproductive
system of the male horned lizard as
affected by seasonal variation. It is concerned with the period immediately following hibernation, and continues during
the major portion of the breeding season
of this reptile.
Connective tissue. Krichesky's ('31 )
modification of Mallory's triple stain was
used to distinguish the various connective
tissue elements.
Nucleic acid. The Feulgen reaction for
desoxyribonucleic acid as outlined by Lillie
('54) was used in this investigation. Light
green was employed as a counterstain.
Lipid studies. Tissues previously fixed
in buffered neutral formalin were embedded in gelatin and frozen sections cut
and stained with Sudan black B and oil
red 0 using propylene glycol as solvent.
The Schultz reaction for the histochemical
localization of cholesterol and its esters
was used (Gomori, '52). Controls for these
lipid procedures were prepared by treatment with an equal mixture of ethyl ether
and chloroform at room temperature for
one hour. Phospholipids were stained according to Elftman ('57).
Fresh testicular tissue was prepared for
lipid analyses by extracting overnight with
the 3 : 1 ethyl alcohol-ethyl ether mixture
of Bloor ('43) followed by extraction for
three hours in a micro-Soxhlet apparatus.
Total lipid was determined by the gravimetric method of Street ( ' 3 6 ) , and the
procedure of Outhouse and Forbes ('40)
was used for total cholesterol. Fatty acid
data were obtained by subtracting total
cholesterol from total lipid. Phospholipid
phosphorous was determined according to
King ( ' 3 2 ) .
matogonia, spermatocytes and spermatids
had cytoplasmic granules, but chromatin
reacted in intensity depending upon the
stage of spermatogenesis. Ribonuclease
removed most of the cytoplasmic basophilia of the interstitial, Sertoli and spermatogenic cells.
Testes of lizards collected towards the
end of the breeding season (late July)
were atrophied and had undergone a
marked reduction in wet weight as well as
an increase in per cent dry weight (table
1 ) . The principal germ cells were spermatogonia and primary spermatocytes, but
a few spermatids and spermatozoa were
present. Most of the Leydig cells were
spindle-shaped, but others were large with
distinct cell borders and nuclei. This is in
agreement with the earlier findings of
Blount ('29) who described the seasonal
cycle of the interstitial cells of Phrynosoma
The rete testis of sexually active lizards
showed a transition from simple squamous
epithelium in the proximal region of the
testis to simple cuboidal in its distal portion (fig. 1 ) . The ductuli efferentes
changed from cuboidal in the proximal
area to simple columnar distally. Throughout this region the nuclei were large with
diffuse chromatin. Following toluidine
blue and azure A-eosin B the cytoplasm
was unstained. Numerous spermatozoa
were present in the rete testis and ductuli
efferentes. The ductuli efferentes were
formed by small tubules of simple colBasophilia. Following toluidine blue umnar cells with large nuclei, nucleoli and
and azure A-eosin B, the basement mem- an intensely basophilic cytoplasm. Stereobrane of the seminiferous tubules of liz- cilia or cilia were weakly colored.
ards obtained early in the breeding seaIn the ductus epididymidis, the duct
son was heavily stained. The interstitial was somewhat larger in diameter in the
cell cytoplasm was weakly colored, had no secondary region than in the primary regranular inclusions and was vacuolated, gion of the caput (fig. 6). The epithelium
but the nuclei were large, with diffuse was taller than in the ductus efferens and
chromatin and prominent nucleoli. The had cilia-like projections. In the secondary
Sertoli cell nuclei were intensely basophilic region of the caput, the ductus contained
with diffuse chromatin and a large promi- simple columnar, intensely basophilic cells
nent nucleolus. In some cases, the cyto- without cilia and with distinct cell borders
plasm was clear with few basophilic in- (fig. 5). Irregular bleb-like projections
clusions. However, in other regions of from the apex of some of the cells were
the testis, the Sertoli cells contained num- noted as were numerous cytoplasmic
erous large basophilic granules; especially granules. Ribonuclease removed most of
in those cells in relation to clusters of the basophilic reaction but some faint
spermatozoa. These granules were not apical staining remained. The staining reremoved by ribonuclease. All of the sper- sponse of the nucleolus was variable. The
epithelial cell height of the corpus epididymidis was slightly lower, but the cytoplasmic reaction was comparable to that
seen in the caput. The cells of the cauda
epididymidis were lower but columnar.
These cells contained numerous cytoplasmic droplets and no stereocila. Furthermore, bleb-like projections occurred at
the cell apex and numerous spermatozoa
were present in the lumen of the duct.
The epithelium of the distal portion of the
cauda and the ductus deferens was cuboidal with an intense cytoplasmic basophilia. A smooth muscle coat and an adventitia surrounded the duct. The ductus
deferens was in contact with the kidney.
Lizards collected in late July showed a
marked reduction in epididymal cell height
and pyknosis (fig. 7), especially in the
caput (secondary portion) and cauda.
Spermatozoa were absent.
PAS-reactive substances. Lizards obtained early in the spring showed a strong
positive reaction in the tunica albuginea
and glycogen was localized in this structure. PAS-positive material was present
in the cytoplasm of Leydig cells, but glycogen was absent. However, walls of arterioles and venules demonstrated glycogen.
The basement membrane of the seminiferous tubules was Schiff-positive. Within the cytoplasm of the germinal epithelium were numerous reactive granules
which were removed by diastase. Likewise,
the Sertoli cells and the attached clusters
of spermatozoa contained much glycogen.
In the spermatocytes, a lightly stained area,
the idiosome, was present near the Golgi
Little glycogen was in the testes of
lizards obtained during the height of the
breeding season (June-early July). Most
of the glycogen was localized in the tunica albuginea and Sertoli cells. Testes
of lizards collected late in the breeding
season contained considerable glycogen in
all germ cells and Sertoli cells.
The epididymides of sexually active lizards had a strong connective tissue reaction especially in the periductular area.
The apical cytoplasm of the epithelium
stained faintly and contained a few PASpositive granules. Stereocilia or cilia
stained in the primary region of the caput.
A few glycogen granules were in the epi-
thelial cytoplasm of the ductuli efferentes
and primary caput. The epithelium of
the secondary caput epididymidis contained more Schiff-positive granules than
the primary region. The cuboidal cells
of the ductus deferens contained numerous cytoplasmic granules of glycogen
and faint apical staining. There was some
glycogen in the smooth muscle coat surrounding the ductus. In the distal portion
of the ductus deferens, the epithelium was
columnar and the lamina propria was in
folds similar to the ductus deferens of
mammals. Great quantities of cytoplasmic and luminal glycogen were noted in
this region.
The epididymides of lizards collected
at the close of the breeding season contained considerable amounts of cytoplasmic glycogen in the atrophied epithelium.
There was a marked periductular fibrosis
associated with a thickened basement
membrane. Similar changes were noted
in the ductus deferens of these animals.
Fezilgen reaction. With the exception
of the lizards collected in late July, the
nuclei of the Leydig cells were large with
diffuse and evenly dispersed chromatin.
In some cases, nucleoli were delineated
by a fine ring of Feulgen-positive material. Reactions in the germinal epithelium
were variable depending upon the stage of
spermatogenesis. The heads of spermatozoa were strongly reactive. The epithelium of the ductus epididymidis and ductus deferens of these lizards had large
nuclei with evenly dispersed chromatin.
In lizards collected in late July, some
interstitial cells were pyknotic. The epithelial cells of the ductus of the caput,
corpus and cauda epididymides, as well
as the ductus deferens, were pyknotic.
Small. ringed nucleoli were visible in
some cases.
Coiznectiue tissue. Intertubular collagenous and elastic fibers were sparse in the
testes (figs. 2, 3 ) . During the early breeding season, the interductular connective
tissue of the epididymis was sparse, compact and the basement membrane thin.
Epididymides of lizards collected late in
the breeding cycle, showed disorganization
of interductular connective tissue. There
appeared to be an increase in collagen in
the peritubular connective tissue. This
may not represent a true increase in connective tissue, but is possibly due to
atrophy of epithelium, loss of testicular
fluids, secretions and spermatozoa which
normally would have distended this organ.
Lipid studies. Following Sudan black
B and oil red 0 there was an abundance
of cytoplasmic lipid in the interstitial cells
of lizards collected from May through
early July. Leydig cell lipid appeared as
droplets of varying sizes. Fine droplets
of lipid material were noted in the basement membrane of the seminiferous tubules as well as in the cytoplasm of the
Sertoli cells. The cytoplasm of the spermatogonia contained numerous granules
about the unstained nucleus. In contrast,
the lipid in the primary and secondary
spermatocytes was often grouped in the
Golgi region with granules scattered
rather generally throughout the cytoplasm. During spermiogenesis and prior
to their loss of cytoplasm, the late spermatids had numerous cytoplasmic lipid
inclusions. In the atrophied testes of lizards of the terminal group, there was a
marked accumulation of lipid in the seminiferous tubules and Sertoli cell cytoplasm. Furthermore, the Leydig cell cytoplasm contained many lipid granules.
The cytoplasm of the epididymal epithelium of animals obtained early in the
breeding season had numerous sudanophilic droplets. Numerous droplets of
lipid of variable size were dispersed in
the apical cytoplasm. During the late
summer the atrophied epididymal epithelium contained little lipid.
The Schultz reaction showed an abundance of cholesterol in the cytoplasm of
the Leydig cells of all groups but none
in the nuclei and nucleoli.
With the exception of lizards studied
during late summer, all other animals had
quantities of Leydig cell phospholipid.
Furthermore, fine droplets were localized
in the germinal epithelial cytoplasm and
considerable amounts of phospholipid
were noted in the Sertoli cells. The epididymides and ductus deferens of these
lizards contained quantities of phospholipid granules in the apical cytoplasm
and Golgi zone of the columnar epithelium.
Lizards collected in late summer had
little testicular phospholipid. However,
considerable phospholipid remained in the
Leydig cells (fig. 4). The epididymides
and ductus deferens of this group had
only a few apical phospholipid granules
in the atrophied epithelium. The Golgi
apparatus, apparent in the earlier groups,
was not visible in the epididymal epithelium of these lizards.
Results of lipid analyses of testicular
tissue, including total lipid, cholesterol,
fatty acids and phospholipid phosphorus,
are shown in table 1. Furthermore, information on body weight, and wet and
dry weight of testes and epididymides is
Previous investigators (Cavazos and
Melampy, '54; Melampy and Cavazos,
'54) have conducted comparative histochemical studies on sexually mature males
representing the classes Pisces, Amphibia,
Reptilia, Aves, and Mammalia. They
studied the distribution of Schiff-positive
material and a variety of lipids in the
horned lizard testis. In the present investigation, the use of cytochemical and
biochemical methods for basophilia, PASreactive carbohydrates, desoxyribonucleic
acid, and lipids, makes it possible to extend these studies to the functional activities and variation of the reproductive
tract of Phrynosoma cornutum during its
sexual cycle. Through a study of lower
vertebrate forms, information may be obtained which may contribute information
on the complex reproductive system of
Fluctuation occurs in the cytochemistry
and biochemistry of the cells of the reproductive tract during the seasonal sex
cycle of the horned lizard. For example,
the intense perinuclear basophilia which
characterized the epithelium of the ductus epididymis of sexually active lizards
was reduced in the atrophied epithelium
prior to hibernation. Ribonucleoprotein in
the epididymal epithelium is suggested
because the cytoplasmic and nucleolar
basophilia were abolished by ribonuclease.
Other studies have demonstrated that
there is a correlation between the nucleic
acid content of cells and protein synthesis
( Caspersson, '50). Epithelial cytoplasmic
basophilia which is removed by ribonuclease, has been reported in the epididymis of the rabbit (Nicander, '57) and in
the rat (Cavazos, '58). Studies on the
specificity of ribonuclease have supported
the premise that the substance specifically
degraded by this enzyme is ribonucleic
acid (McDonald and Kaufmann, '54).
Numerous Schiff-positive granules were
dispersed in the epithelial cytoplasm of
the ductus epididymis of lizards collected
at all periods during their breeding season.
Hydrolysis with amylase or treatment with
methanol-chloroform did not remove these
granules. This is in agreement with results obtained in the rat (Leblond, '50;
Maneely, '55; and Cavazos, 'SS), human
(Montagna and Hamilton, '52), mouse
(Christie, '55) and rabbit (Nicander,
'57). The importance of glycogen in the
metabolism of the epididymis of the lizard
is indicated by its presence in the epithelium; especially prior to hibernation.
Glycogen in the pyramidal basal cells of
the human epididymis has been reported
by Montagna and Hamilton ('52), who
considered the possibility that it might
represent a readily available source of
energy for the secretory cells. In Phrynosoma corizutum glycogen may play a
role in maintenance of spermatozoa.
Following the Feulgen reaction, the epididymal nuclei of sexually active lizards
were large with diffuse chromatin and
distinct nucleoli. In contrast, pyknosis
was evident in these cells in lizards collected towards the end of the breeding
cycle. These changes in nuclear structure may be associated with the marked
decrease in circulating androgen which
must occur at this stage.
Histochemical and biochemical studies
indicate the structural and metabolic importance of lipids in spermatogenesis as
well as Sertoli and interstitial cell function. The reduction of lipid in epididymal
epithelium of lizards obtained late in the
summer, suggests the physiologic relationship between the level of circulating androgen and epididymal lipid. As the testis
atrophies, there is a marked increase of
total lipid (table 1). From May until
late July a three-fold increase in testicular
total lipid occurs. This lipid may be utilized by the testis during hibernation and
subsequent spermatogenesis. During the
same period, a 76% increase occurs in
testicular cholesterol correlated with
marked decrease of phospholipid. Since
reproduction occurs immediately following hibernation, these data suggest that
phospholipid is important early in the
breeding season, whereas cholesterol may
play a more significant role in maintenance of testis function late in the reproductive cycle.
The epididymal epithelium may be directly or indirectly influenced by androgen. This is indicated by the atrophy of
the reproductive tract as the testis regresses. However, not all of the interstitial
cells become spindle-shaped and reduced
gonadotrophin may be important in regression of the reproductive tract. It is
possible that the androgen level is directly
or indirectly related to the breeding cycle
of the horned lizard. Through the use of
cytochemical and biochemical procedures
information is presented on the importance of ribonucleoproteins, glycogen,
desoxyribonucleic acid and lipids in the
metabolism of the reproductive tract of
Phrynosoma cornutum and the relation of
these substances to androgen is indicated.
It is suggested that a physiologic relationship which is affected by seasonal variation, exists between the circulating male
sex hormone and the various morphological changes in the testis and epididymis.
1. Sexually mature male horned lizards,
Phrynosoma cornutum, were used for
studies on the reproductive tract as affected by seasonal variation. Alterations
occur in the histochemistry and lipid
chemistry of the cells of the reproductive
tract following hibernation, during the
sexual cycle and at the end of the breeding season.
2 . Ribonucleoprotein and phospholipid
decrease prior to hibernation, but glycogen, total lipid and cholesterol are markedly increased after the active reproductive phase. This study indicates the
metabolic importance of these substances
in spermatogenesis, Sertoli and Leydig cell
function and epididymal maintenance.
3 . Results suggest that the level of circulating androgen is related to the breed-
ing cycle. Sex hormone decrease is reflected by the atrophy and biochemical
and histochemical alterations in cells of
the reproductive tract as hibernation approaches.
Bloor, W. R. 1943 Biochemistry of the Fatty
Acids. Reinhold Publishing Co., New York.
Blount, R. F. 1929 Seasonal cycles of the interstitial cells in the testis of the horned toad
(Phrynosoma solare). Seasonal variations i n
the number and morphology of the interstitial
cells and the volume of the interstitial tissue.
J. Morph. Physiol., 48: 317-343.
Caspersson, T. 0. 1950 Cell Growth and Cell
Function. W. W. Norton and Co., Inc., New
Cavazos, L. F. 1951 Spermatogenesis of the
horned lizard Phrynosoma cornutum. Am.
Nat., 85: 373-379.
1958 Effects of testosterone proprionate on histochemical reactions of epithelium
of rat ductus epididymis. Anat. Rec., 132:
Cavazos, L. F., and R. M. Melampy 1954 A
comparative study of periodic acid-reactive
carbohydrates in vertebrate testes. Am. J.
Anat., 95: 467-495.
Christie, A. C. 1955 A histochemical study of
the cytoplasmic inclusions of the epithelial
cells in the epididymis of the mouse. Quart.
J. Micr. Sci., 96: 161-168.
Elftman, H. 1957 Phospholipid fixation by dichromate-sublimate. Stain Tech., 32: 29-31.
Gomori, G. 1952 Microscopic Histochemistry.
University of Chicago Press, Chicago.
Hotchkiss, R. D. 1948 A microchemical reaction resulting in the staining of polysaccharide
structures in fixed tissue preparations. Arch.
Biochem., 16: 131-141.
King, E. J. 1932 The colorimetric determination of phosphorus. Biochem. J., 26: 292-297.
Kramer, H., and G. M. Windrum 1955 The
metachromatic staining reaction. J. Histochem. Cytochem., 3: 227-237.
Kricheskv, B. 1931 A modification of Mallory’s
triple stain. Stain Tech., 6: 97-98.
Leblond, C . P. 1950 Distribution of periodic
acid-reactive carbohydrates in the adult rat.
Am. J. Anat., 86: 1-50.
Lillie, R. D. 1954 Histopathologic Technique
and Practical Histochemistry. The Blakiston
Co., Inc., New York.
Maneely, R. B. 1955 The distribution of polysaccharide complexes and of alkaline glycerophosphatase in the epididymis of the rat. Acta
Anat., 24: 314-329.
Melampy, R. M., and L. F. Cavazos 1954 Comparative study of lipids in v.ertebrate testes.
Proc. SOC.Exp. Biol. Med., 87: 297-303.
Mellish, C. H. 1936 The effects of anterior
pituitary extract and certain environmental
conditions on the genital system of the horned
lizard (Phrynosoma cornutum, Harlan). Anat.
Rec., 67: 23-33.
Montagna, W., and J. B. Hamilton 1932 Histological studies of human testes. 11. The distribution of glycogen and other HIO4-Schiff
reactive substances. Ibid., 112: 237-250.
and B. p. Kaufmanll 1954
The degradation by ribonuclease of substrates
other tha,l ribonucleic acid. J. Histochem,
Cytochem., 2: 387-394.
an^^, J. F. A. 1946 Histological demonstration of mucin after periodic acid. Nature,
158: 202.
Nicander, L. 1957 On the regional histology
and cytochemistry of the ductus epididymidis
in rabbits. Acta Morph. Neerlando-Scand.,
1: 99-118.
Outhouse, E. L., and J. c. ~~~b~~ 1940 A
micromethod for determination of tissue lipids.
J. Lab. Clin. Med., 25: 1157-1163.
Street, H. R. 1936 A gravimetric technique for
the determination of small amounts of plasma
lipids. J. Biol. Chem., 116: 25-31.
Testis of horned lizard a t height of breeding activity. Note active germinal epithelium,
spermatozoa in rete testis (arrow) and ductuli efferentes. Ductus of caput epididymidis
at lower left of section and kidney at upper left. Stained with toluidine blue. X 144.
Testis collected early i n r.eproductive cycle. Numerous spermatozoa and spermatogenic
cells visible. Compare with figure 3. Mallory’s triple stain. x 166.
Testis of lizard collected at end of breeding phase. T.esticular atrophy and absence
of spermatozoa apparent. Stained as in figure 2. X 166.
L. F. Cavazos and W. M. Feagans
Phospholipid localization i n interestitial cell cytoplasm of atrophied testis. Lizard COIlected in late July. x 665.
Ductus of secondary region of caput epididymidis at height of breeding season. Note
presence of spermatozoa in ductus lumen and intense cytoplasmic basophilia. Basophilia of this type removed by ribonuclease. Toluidine blue stain. X 600.
Section of caput epididymidis during active phase of reproduction.
figure 7. Toluidine blue stain. ‘i< 285.
Similar region of caput epididymidis of lizard collected in late July. Toluidine blue.
Note marked decrease in ductus size and absence of spermatozoa. X 285.
Compare with
L. F. Cavazos and W. M. Feagans
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affected, lizard, variation, malen, hornem, trace, histochemistry, composition, reproduction, seasonal
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