Ultrastructure of Anterior Uterus of the Oviduct and the Stored Sperm in Female Soft-Shelled Turtle Trionyx sinensis.

THE ANATOMICAL RECORD 291:335–351 (2008)
Ultrastructure of Anterior Uterus of the
Oviduct and the Stored Sperm in
Female Soft-Shelled Turtle,
Trionyx sinensis
XIANGKUN HAN, LI ZHANGLI, MEIYING LI, HUIJUN BAO, NAINAN HEI,
QIUSHENG CHEN*
College of Veterinary Medicine of Nanjing Agricultural University,
Nanjing, P.R. China
AND
ABSTRACT
Ultrastructure of sperm storage in female soft-shelled turtle, Trionyx
sinensis was examined under light and electron microscopes. Sperm storage tubules are restricted to the anterior of the uterus. These tubules
developed either by folding or fusion of the oviductal mucosal folds and
are lined by both ciliated and secretory cells. Ciliated cells are characterized by a few microvilli and prominent cilia in the apical membranes. A
prominent feature of the secretory cell is the presence of secretory granules in the supranuclear region. The size, shape, and electron density of
these granules vary markedly. The secretory product is released mainly
by exocytosis into the oviductal lumen, where it appears as flocculent material. The unique structure in the base of the epithelium, the basal border of the cell—the basal lamina—and a blood vessel layer, is presumed
to be a important barrier, by which the nourishment exchange and the
microenvironment maintenance are ensured. The gland cell is presented
with numerous, round, membrane-bound secretory granules of moderate
to high electron densities. We divide these granules into three types
according to their appearance: (1) membrane bounded granules with
high-homogeneous electron density, (2) membrane bounded granules with
moderate-homogeneous electron density, (3) membrane bounded, electron
dense granules with concentric structures. These granules are presented
as different stages of the secretions in the gland cell. The junction complexes are markedly distributed between cells, which are important in
keeping stability and the microenvironment maintenance of the sperm
storage tubules. Sperm stored in the tubules are heterogeneous in cytology. In addition to the mature sperm in the lumen, sperm with large
chromatic granules are found, which are presumed to be immature sperm
and are being in the process of nuclear condensation. Several spermatozoa in the tubules are exhibited with definitive indications of degeneration of the nuclei. The nuclear volume increases. The electron density of
the central cores in mitochondria declines, combined with the deterioration of concentric membrane structure. Those changes are possibly due to
the long time storage of the sperm in sperm storage tubules, and the
leakage of reactive oxygen species is suggested to be a major cause. We
conclude that the ultrastructure character of sperm storage in the oviduct
Grant sponsor: The National Science Foundation of China;
Grant number: 30671513.
*Correspondence to: Chen Qiu-sheng, Department of Veterinary Medicine in Nanjing Agricultural University, Nanjing
210095, PR China. Fax : 86-25-84398669. E-mail: chenqsh305@
yahoo.com.cn
Ó 2008 WILEY-LISS, INC.
Received 8 February 2007; Accepted 7 October 2007
DOI 10.1002/ar.20649
Published online 29 January 2008 in Wiley InterScience (www.
interscience.wiley.com).
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XIANGKUN ET AL.
of Trionyx sinensis is unique, in addition to having a basal function in
secretion and the cilia swing, the tubules also provide an available microenvironment for the sperm to long time stored. The degenerative sperm
in the tubules might be related to paternity-specific reproductive adaptations, and the sperm competition might occur during long time
storage. Anat Rec, 291:335–351, 2008. Ó 2008 Wiley-Liss, Inc.
Key words: ultrastructure; uterus; stored sperm; soft-shelled
turtle; Trionyx sinensis
Sperm production, mating, and ovulation are out of
phase with one another in numerous species (Pearse
et al., 2001; Almeida-Santos et al., 2004). In turtles,
spermatogenesis is an episodic event, commencing in
early summer (June). Sperm leave the testis and enter
the epididymis in autumn (September–October).
Although oogenesis is initiated at about the same time
as spermatogenesis, ovulation does not occur until the
following spring. Thus, for autumn breeding turtles,
sperm may be stored over winter in the oviductal glands
of females, or, for spring-breeding turtles, in the epididymis of the male (Gist and Jones, 1989; Gist and
Congdon, 1998).
Sperm storage has been noted in numerous squamates
and chelonians in reptiles (Davenport, 1995; Sever and
Ryan, 1999; Sever and Hopkins, 2004). The duration of
sperm storage was described up to 3 years in the
painted turtles Chrysemys picta (Pearse et al., 2001) and
up to 6 months in the soft-shelled turtle, Lissemys punctata punctata (Sarkar et al., 2003). That stored sperm
Fig. 1. Histology of the anterior uterus. Glands communicate with the oviduct lumen by means of
short openings (?). Mucosa (M), muscular layer (ML), serosa ( ). Hematoxylin and eosin stain.
UTERUS, OVIDUCT, AND STORED SPERM IN TURTLE
Fig. 2.
337
Tubule in which the sperm are stored. Hematoxylin and eosin stain.
are capable of fertilization is indicated by the fact that
females of several species that have been kept in captivity without males for prolonged periods can still produce
viable clutches (Adams and Cooper, 1988). Histochemical
studies of utervaginal sperm storage areas and other
regions of the oviduct in birds (Bakst, 1987; Bakst and
Bird, 1987) and squamate reptiles (Kumari et al., 1990)
suggest that the sperm storage tubules (SSTs) may possess biochemical adaptations favorable for sperm retention. The maintenance of the stability of microenvironment of the SST might basically due to the ultrastructure characters of the epithelium cells, especially the
cell junctions and other special structures. Furthermore,
sperm storage may also be advantageous, because it
extends the reproductive period available to females, it
may contribute to sperm competition or multiple paternity within a single clutch (Birkhead and Mollar, 1993;
Gist and Fischer, 1993). Additionally, females may be
able to control paternity of their offspring by a selective
use of sperm (Olsson et al., 1998). It is suggested that
females probably re-mate for reasons other than the acquisition of gametes for fertilization, such as to increase
genetic diversity of offspring (Pearse et al., 2001). The
effect of stored sperm on fertility and/or hatching success of across-year clutches is contradictory, with no significant change in some species (Pearse et al., 2001,
2002), but a decrease in others, suggesting sperm depletion or deterioration through time (Roques et al., 2006).
However, there is very little about the anatomy of
sperm storage in turtles (Gist and Congdon, 1998;
Sarkar et al., 2003). Ultrastructure studies, especially
using transmission electron microscopy, are essential to
study sperm characters and sperm/epithelial interactions during sperm storage. Several studies deal at least
338
XIANGKUN ET AL.
Fig. 3.
stain.
Tubular gland. Nucleus of gland cell (?). Secretory granules in a gland cell (
peripherally with the cytology of male sperm storage in
reptiles (Newton and Trauth, 1992) and in female amphibian (Sever, 1997), but neither light nor electron
microscope evidence has been obtained that describes
the cytology characters of the sperm stored in the SST of
female soft-shelled turtle Trionyx sinensis. And the only
available report is limited in turtle, Terralene Carolina
(Gist and Fischer, 1993) and in snake, Thamnophis
sirtalis (Hoffman and Wimsatt, 1972). The observation
of immature sperm and the degeneration progress of the
stored sperm in sperm storage tubules were not reported
in turtles either. Soft-shelled turtle, Trionyx sinensis distribute widely in China and this species is famous for
economy and pharmacological value. This species exhibit
an unusual reproductive cycle whereby spermatogenesis
and ovulation are out of phase with each other. Therefore, the sperm is also obligated to be stored in males or
females similar to that of other reptiles described above.
The unique ultrastructure character of spermatozoa in
Trionyx sinensis have been studied in our previous
study, and the relationship between spermatozoa structure and the long time storage has been discussed (Chen
et al., 2006). Therefore, further study is necessary to
determine the sperm storage organ characters in this
species as well as the cytology changes of spermatozoa
). Aniline blue
that stored in it. The present study was to (1) examine
the ultrastructure characters of the sperm storage as
well as the relationship between stored spermatozoa and
the host cells that surround them, and (2) to give a further analysis of the cytology of the stored spermatozoa
in the sperm storage tubules.
MATERIALS AND METHODS
Fifteen adult healthy female turtles (body weight > 1
kg, plastron length > 15 cm) (Trionyx sinensis) were captured from ponds in Nanjing, in southeast China on
May 2, 2006. All treatments complied with the Guidelines for the Care and Use of Wild Animals in The People’s Republic of China, and, for capture, with the approval of the Wild Animal and Plant Protection Office of
Jiangsu Province in China.
Different foods, viz., small fishes, shrimps, and snails,
were supplied ad libitum. Animals were rendered comatose using intraperitoneal administration of sodium pentobarbital (1 ml/animal) and killed by cervical dislocation. The tissue blocks of oviduct were fixed separately
in 10% neutral buffered formalin and processed for routine microtomy. Paraffin sections (5 mm thick) were
stained with hematoxylin and eosin for light microscopic
339
UTERUS, OVIDUCT, AND STORED SPERM IN TURTLE
Fig. 4. Periodic acid–Schiff (PAS) -positive materials secreted from the epithelium of the tubule (
Sperm in the lumen ( ). PAS reaction.
observations to determine the localization of the sperm
storage tubules. Sections of sperm storage regions in the
anterior uterus of the oviduct were stained by the periodic acid–Schiff (PAS) technique followed by hematoxylin counterstain.
The anterior sections of uterus were cut into small
blocks, fixed in 2.5% glutaraldehyde buffered in 0.2 M
sodium cacodylate (pH 7.2) at 48C overnight, washed in
the same buffer at 48C. And then tissues were post-fixed
with a 1% osmium tetroxide with the same buffer at 48C
for 3 hr, washed in the same buffer. After dehydration in
ascending concentrations of ethanol series followed by
propylene oxide, the tissue fragments were embedded in
Epon-812. Semithin sections (1 mm) were stained with
toluidine blue for light microscopic examination. Ultrathin sections for electron microscopy were mounted on
).
Formvar-coated grids, stained with uranyl acetate and
lead citrate and examined by a JEM-1200EX electron
microscope.
RESULTS
Histology and Morphology of the
Sperm Storage Tubules
The wall of the turtle oviduct is formed from three histological distinct layers: mucosa, muscular layer, serosa
(Fig. 1). Based on the functional morphology, the oviduct
is further divided into regions along its length, which
are (from anterior to posterior) infundibulum, tube (tuba
uterina), isthmus, uterus, and vagina.
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XIANGKUN ET AL.
Fig. 5. Glands communicate with the lumen by gland duct (GD). The glands (GL) are tubular and are
located in the lamina propria beneath the pseudostratified epithelium and the blood vessel layer ( *). Secretory granules ( ). Aniline blue stain.
The oviductal mucosa forms longitudinal folds running along the axis of the tube. A majority of mucosa
fused into sperm storage tubules orientated toward the
longitudinal axes of the oviduct (Fig. 2). These tubules
are restricted to the anterior uterus. The epithelium
lining the SSTs consists of secretory and ciliated cells
which are interspersed in the epithelium (Fig. 6).
Beneath the epithelial cells there is a thin layer of
blood vessels arranged in parallel (Figs. 5, 6). The
glands are single tubular and are located in the lamina
propria beneath the pseudostratified epithelium and
the blood vessel layer (Figs. 3, 5). They communicate
with the oviduct lumen oviduct by means of short openings (gland duct) composed of invaginations of the epithelium that communicate abruptly with the glandular
epithelium within the lamina propria (Figs. 1, 5). PASpositive materials are observed in the epithelium of the
SST (Fig. 4).
Cytology of the Sperm Storage Tubules
Columnar ciliated cells attach to
Ciliated cells.
the epithelial basal lamina and are usually located in
the apex and ridges of the folds, with a few microvilli on
their luminal surface between the cilia. The nuclei of
these cells are elongated, voluminous, and ovoid in
shape, and usually medially or basally located (Figs. 6,
7). The ciliary microtubules in transverse and oblique
section are observed (Figs. 8–10). In the cell apex are
the U-shaped basal bodies that serve as the source of,
and anchoring sites for, the ciliary axonems (Figs. 9, 10).
The two central microtubules are absent in the base of
the cilia (Fig. 10). Mitochondria and Golgi complexes are
present in the supranuclear region where the cytoplasm
is characterized by lower electron density (Fig. 8). The
local accumulation of mitochondria in the apex of the
cell is observed. These mitochondria are characterized
by their irregular shape, usually found with large
vacuoles in the pole (Fig. 9). Microfilaments are scattered throughout the apical cytoplasm of the ciliated cell
(Fig. 10).
Secretory cells. Secretory cells attached to the basal lamina extend from it to the lumen. The nuclei of secretory cells are usually oval and are basally located
(Fig. 7). In the infranuclear region of the secretory cell,
a large number of mitochondria with various shapes are
distributed, most of which are characterized by the presence of vacuoles in the pole and exhibit active metabolism process (Fig. 11). A prominent feature of the secretory cell is the presence of secretory granules in the
supranuclear region (Figs. 7, 11). The size, shape, and
electron density of these granules vary markedly. At
high magnification, some secretory granules show irregular dense cores with branchings (Fig. 12). Their matrix
is highly complex and variable showing electron-dense
and electron-lucent areas. An electron-dense sphere is
always located at the periphery of the granules (Fig. 12)
and occasionally at the center (Fig. 11). Subsequently,
these dense cores are dispersed, which result in the various electron density compositions of the granules (Fig.
13). The secretory product is released mainly by exocytosis into the oviductal lumen, where it appears as flocculent material (Fig. 13); several granules are seen pro-
UTERUS, OVIDUCT, AND STORED SPERM IN TURTLE
341
Fig. 6. The epithelium lining the sperm storage tubules (SSTs) consists of secretory (?) and ciliated
(?) cells, which are interspersed in the epithelium. Beneath the epithelial cells, there is a thin layer of
blood vessels ( ) arranged in parallel.
truding into the lumen (Fig. 11). The rough endoplasmic
reticula are well developed in secretory cells (Fig. 12).
The microvilli of above-mentioned cells are sparse (Figs.
11, 13).
Blood–Epithelium Barrier and Cell Junctions
In the base of epithelium, the basal border of the cell
is usually irregularly undulated and a typical basal lamina follows its undulations (Fig. 14). In the basal cytoplasm of the cell, accumulation of mitochondria is found
(Figs. 14, 15). Large vesicles with irregular shape are
observed, which, because of their contents of phagocytic
granules and degenerated mitochondria, are considered
to be secondary lysosomes (Fig. 14). Immediately
beneath the epithelium is a layer of blood vessels parallel to the basal lamina (Figs. 5, 14). The unique structure mentioned here, the basal border of the cell—the
basal lamina—and a blood vessel layer, is presumed to
be an important barrier, by which the nourishment
exchange and the microenvironment maintenance are
ensured.
The junction complexes are markedly distributed
between epithelium cells of the sperm storage tubules.
Tight junctions are the most apical of the junctions,
which form a seal that prevents the flow of materials
between epithelial cells in either direction. This junction
is generally observed in gland cells, ciliated cells, and se-
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XIANGKUN ET AL.
Fig. 7. Vertical section of the epithelium of sperm storage tubules
(SSTs). Ciliated cell (CC), secretory cell (SC). Nuclear (N).
cretory cells (Figs. 9, 13, 18). The next type junction is
the zonula adherens, also called intermediate junction,
distribute closely to the tight junction (Figs. 9, 10).
Beneath the junctions mentioned above, desmosome
(Figs. 9, 10, 13, 15) and lateral interdigitations (Figs. 8,
9, 11, 18) are all observed in epithelium cells. These
junctions are present in a definite order mostly in the
apex lateral border of the cells. In the basal lateral of
the epithelial cells, desmosomes are usually distributed
rather than other junctions (Fig. 15).
Glands
Gland cells are voluminous with large microvilli outline the surface (Fig. 16), of which the nuclei are round
and usually centrally or basally located (Figs. 3, 16). A
prominent feature of the gland cell is the presence of
numerous, round, membrane-bound secretory granules
of moderate to high electron densities (Figs. 16–19). We
divide these granules into three types according to
their appearance. The most abundant granules we
identified are the membrane bounded granules with
high-homogeneous electron density (Figs. 16–18). The
second type granules are more particular in feature,
which give their structures in concentric type and looks
like fingerprint in cross-section (Figs. 18, 19). The third
Fig. 8. Mitochondria ( ) and Golgi complexes (?) are present in
the supranuclear region of the ciliated cell. A few microvilli (not noted)
on the luminal surface between the cilia (Ci), secretory granules in the
secretory cell (SeG).
type granules occasionally found are membrane
bounded and with moderate electron density (Fig. 16).
In low magnification, these granules with moderate-homogeneous electron density usually centrally located
and there is an obvious area by which they are separated from other type granules (Fig. 16). In high magnification, the membrane-bond granules show a condensing process, which is presumed to be receiving a
small quantity of secretory products from Golgi complexes distributed in the separated area by small ducts
(Fig. 19). In addition to the secretory granules mentioned above, the apical cytoplasm of the gland cell contains numerous smaller vesicles that appeared to be
empty. Some of these vesicles are fused with apical
membranes, and a few could be observed intact in the
lumen of the tubule (Fig. 17). Another feature of the
gland cell is the limited distribution of the mitochondria, which is usually associated with the formation of
the granules. These mitochondria are various in
shapes, round (Fig. 17), longitypical (Fig. 16), or present with big branches (Fig. 18). Myoepithelial cells and
collagen fibers are associated with the basal lamina of
the tubular glands (Fig. 16).
UTERUS, OVIDUCT, AND STORED SPERM IN TURTLE
Fig. 9. Electron micrograph of the apical portion of the ciliated cell
and cell junction complexes. Basal body ( ), tight junction (?), intermediate junction (/), membrane interdigitation ( ), desmosome (
),
mitochondria (Mi).
Spermatozoa Stored in SSTs
Various quantities of spermatozoa are observed in the
tubules taken from the anterior uterus region of the oviduct (Fig. 20). Sperm in the lumen appear random in
overall orientations (Figs. 20, 21). Although most spermatozoa are in contact with the cilia of the cells forming
the tubules, such contacts are sporadic and do not
appear to be essential for the presence of spermatozoa
within the tubules. Around the sperm mass are the flocculent materials that are similar to the contents of
mature granules in the apex of the secretory cell (Figs.
13, 20). The nucleus of mature spermatozoon is long and
contains intensely stained chromatin, and the middle piece of the tail exhibits a usual pattern in the axial filament (Figs. 21, 22). The concentric mitochondria with a
dense core in the center concentrate in the middle piece
of the spermatozoon, which forms the mitochondria
sheath (Fig. 21). The intranuclear tubules (Figs. 21, 22)
and distal centriole can be easily found (Fig. 21). Particularly, a prominent feature of the stored sperm in the
SST is that they appear heterogeneous in cytology, especially in various condensations of the chromatin. In
343
Fig. 10. Electron micrograph of the apical portion of the ciliated
cell and cell junction complexes. Cilia (Ci) with the central and peripheral microtubules of the axoneme, the two central microtubules are
absent in the base of the cilia. Basal body ( ), intermediate junction
(/), desmosome (
), microfilaments ( ).
addition to the mature sperm in the lumen of the
tubules, the heads of the sperm with large chromatic
granules are found, which are presumed to be immature
sperm and are in the process of nuclear condensation
(Figs. 21, 23). However, portions of sperm are exhibited
with definitive indications of degeneration of the nuclei.
The plasma membranes around nuclei of the sperm are
often crenated, detached, or disrupted (Fig. 24), and the
chromatic granules of the sperm exhibit various densities (Fig. 23). Moreover, portions of spermatozoa stored
in SSTs in present study are exhibited with abnormal
mitochondria, which are mostly vacuolar, and the concentric layers of membranes are degenerative. The volume of the central cores within the mitochondria
becomes larger but with moderate rather than high electron density (Fig. 24), indicating sperm degradation in
the lumen.
DISCUSSION
The morphology of the sperm storage tubules is similar to those of other segments of the uterus under light
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XIANGKUN ET AL.
Fig. 11. Supranuclear regions of ciliated and secretory cell. Mitochondria (Mi), cilia (Ci), secretory granules showing irregular dense
cores with branchings and the periphery of these cores are lucent
(SeG). Granules protruding into the lumen ( ). Membrane interdigitation (arrow pointing up and to the right).
Fig. 12. High magnification of the immature granules in a secretory
cell, which contain irregular dense cores with branching, and the periphery of these cores are lucent (
). The rough endoplasmic reticula
and Golgi complexes are well developed (?).
microscope. Only the presence of the tubules in which
the sperm are stored or the ducts leading to the oviduct
served to distinguish between tubules containing spermatozoa and those without them. However, the cytology
of cells forming the sperm storage tubules in the anterior uterus is unique in ultrastructure due to its multifunction. Being different from the uterine tube, which
resembles the avian magnum in birds and is the site for
albumen synthesis (Aitken and Solomon, 1976; Palmer
and Guillette, 1990, 1992), the reptilian uterus was
believed to have dualistic functions in producing both
membranous and calcareous layers of the eggshell
(Palmer and Guillette, 1990, 1991; Perkins and Palmer,
1996), and even as a important organs to stored
spermatozoa.
produce lubricating fluids containing mucus (Aitken and
Solomon, 1976). The secretory products may also function in capacitation of gamates and facilitation of fertilization, as does oviductal fluid in mammals (Gould,
1974). In snake T. sirtalis and lizard Calotes versicolor,
the epithelium of the sperm storage pockets shows positive reaction for PAS, indicating that they contain glycosaminoglycans (Halpert et al., 1982; Kumari et al.,
1990). This substance was said to be ‘‘carrier matrices’’
and is hypothesized to be nutritive in function (Hoffman
and Wimsatt, 1972). The sperm are reported to depend
also on the sperm pocket epithelial cells for their nutritive requirements (Hoffman and Wimsatt, 1972). In the
present study, the secretory cells of the sperm storage
tubules are filled with secretory granules in the supranuclear region. In the apex of the cell, the vesicles containing flocculent material are found protruding into the
lumen, by which their contents are emptied. Such flocculent materials also could be found distributing in the
lumen of the sperm storage or being dispersed in the
sperm mass. PAS-positive material secreted from the epithelium of the sperm storage tubules was also observed
Unique Features of Secretory Granules in
Secretory Cell and Gland Cell
Granules in secretory cells. The secretory cells
of the oviduct in green turtles Chelonia mydas L likely
UTERUS, OVIDUCT, AND STORED SPERM IN TURTLE
Fig. 13. Secretory cell and junction complexes. Noting a matrix solution process of secretory granules. Granules with dense electron
materials developed into premature ones, which are full of various
density matrix ( ), mature granules releasing the flocculent material
by ecocytosis into the lumen ( ). Accumulation of the desmosomes
( ). Tight junction (?). Membrane interdigitation (down-pointing
arrow).
in the study. The presence of apical vesicles in the secretory cell and their apocrine release into the lumen of
sperm storage has not been previously reported in any
other turtles but in the oviduct of the rabbit (Jansen
and Bajpai, 1982) and ewes (Cummins, 1983). The
author speculates that the granules in secretory cell are
released mainly by exocytosis into the oviductal lumen
and the flocculent materials may, therefore, play an important role in sperm maturation and capacitation.
A primary role of secretory cell in reptiles has earlier
been reported in Gopherus polyphemus (Palmer and
Guillette, 1990) and in some others (Guillette et al.,
1989), which was suggested to have a function in secretion of the calcareous layer of egg shells. In present
study, the electron density of secretory granules in secretory cells changes obviously, indicating a matrix solution
process, by which the nonsoluble calcareous materials is
modified and become dissolved. Thus, it is presumed
that the electron-dense and electron-lucent matrix in the
same granules might have their functions, respectively;
the one contains nonsoluble calcareous materials and
the other act as dissolvent. The different location of the
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Fig. 14. Blood–epithelium barrier. Undulations of the basal border
( ), basal lamina (BL), vascular endothelial cell (VEC), blood cell (BC),
nuclear of the blood cell (N). Secondary lysosome (PL), accumulation
of mitochondria (?).
dense cores in the granules might due to different sections that made during artificial management.
Granules in Gland Cells
The endometrial glands of the uterus have been identified in various chelonians and squamates, including
the snapping turtle, as the site of eggshell membrane
formation (Guillette et al., 1989; Palmer and Guillette,
1992; Perkins and Palmer, 1996). Palmer and Guillette,
(1990) using [3H]leucine and explant cultures in the turtle, Pseudemys s. scripta, showed that albumen proteins
are synthesized and secreted in vitro by the uterine tube
and that the endometrial glands of the uterus contain
numerous spherical, electron dense secretory granules,
similar to those of the avian isthmus that secrete the
proteinaceous fibers of the eggshell membranes. In present study, the gland cells in the uterus are filled with
membrane-bound granules of various size and electron
densities. These granules presumably contain precursors
to eggshell fibers. The membrane-bound granules with
low electron density are usually centrally located, which
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XIANGKUN ET AL.
Fig. 15. Magnification of Figure 14. A large number of mitochondria with various shapes are distributed, most of which are characterized by a lucent matrix and exhibit active metabolism process (Mi).
Basal lamina ( ), secretory granules (SeG), nuclear (N), desmosome
distributed in basal lateral region of the epithelial cells (?).
Fig. 16. Electron micrograph of the tubular glands. Membranebound granules with low electron density ( ). Membrane-bound granules with high electron density ( ). The distribution of the mitochondria is usually associated with the formation of the granules (/).
Myoepithelial cells and collagen fibers are associated with the basal
lamina of the tubular glands. Lumen (LM), nuclear (N), myoepithelial
cell (MC), collagen fibers (CF).
is presumed to be receiving a small quantity of secretory
product from the Golgi complexes and are considered as
immature granules. Subsequently, the granules undergo
a condensation process and form the granules with high
electron density. However, the secretory granules with
electron density were divided into two types by their internal ultrastructures: one type with concentric lamellae
and another type without internal ultrastructures. Secretory granules with an internal structure have been
observed in tissues other than the oviduct. For example,
in unstimulated mast cells in human lung, the majority
of the secretory granules contained crystalline structure
in three patterns: scrolls, gratings, and lattices
(Caulfield et al., 1980). In ampullar cells of the cow oviduct, the smallest of secretory granules presented contain concentric lamellae. In the enlarged granules, the
lamellae are irregularly distributed and they empty
their contents into the lumen (Bjorkman and Fredricsson, 1961). The present study shows that the granules
containing concentric lamellae are concentrated in the
apical portions of the gland cell and some protruded into
the lumen. Therefore, it is speculated that the dense
electron granules with no concentric lamellae are immature ones that are developed from membrane-bound
granules with moderate electron density. The materials
in all these granules might also have its function to
form eggshell fibers similar to those of turtle, Pseudemys
s. scripta (Palmer and Guillette, 1990). However, no internal ultrastructures such as we have observed has
been reported in turtles as well as other reptiles. In
addition, we are the first who describe such granules in
Trionyx sinensis under electron microscopy. To determine the molecular mechanism of the formation process
of these granules might need a further study by biochemical or histochemical methods.
Numerous small vesicles were observed below the
membrane of the gland cell (Fig. 17). The function of
these vesicles is unclear. However, on the basis of the
large quantities of the membrane-bound granules, these
smaller apical vesicles might represent recycled membrane. Another significant feature of the gland cell is
the distribution of the mitochondria, which is usually
UTERUS, OVIDUCT, AND STORED SPERM IN TURTLE
Fig. 17. The distribution of the mitochondria is usually associated
with the formation of granules ( ). These mitochondria are various in
shapes, are round, or present with big branches (/).
associated with the formation of the granules, indicating
that the mitochondria might produce the energy for the
condensation of the granules.
Ultrastructure Elements for the Maintenance of
Available Microenvironment in Sperm
Storage Tubules
A prominent feature of the sperm storage tubules is
the presence of the barrier, which is called blood–epithelium barrier here. The undulations of the basal border
of the cell contribute to the enlarged membrane, by
which the exchange of the material is facilitated. The
accumulation of the mitochondria in the basal of the cell
is associated with the production of ATP, this is necessary for the cell to communicate with others in various
signal pathways. Similar to blood–brain barrier and
blood–testis barrier, or blood–air barrier, which have
been reviewed extensively, the barrier observed in the
present study might play an important role in the nourishment exchange and the microenvironment maintenance during sperm storage time. However, no such
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Fig. 18. Magnification of Figure 16, showing the discrimination of
two high electron density granules and cell junction complexes
between cells. Dense granules with concentric internal ultrastructure
(
). Dense granules with no internal ultrastructures (?). Mitochondria
(Mi) associated with the formation of dense granules. Tight junction
( ). Membrane interdigitation ( ). Lumen (LM). Microvilli (M). The apical cytoplasm of the gland cell contains numerous smaller vesicles,
which appeared to be empty. Some of these vesicles are fused with
apical membrane, and a few could be observed intact in the lumen of
the tubule ( ).
structure has been reported in the sperm storage
tubules of turtles as well as other reptiles in any other
studies (Aitken and Solomon, 1976; Newton and Trauth,
1992; Gist and Fischer, 1993).
Another principal feature of the sperm storage tubules
is the presence of cell junction complexes between cells,
including tight junction, intermediate junction, desmosome, and lateral interdigitations. These junctions serve
not only as sites of adhesion but also as seals to prevent
the flow of materials through the intercellular space and
to provide a mechanism for communication between adjacent cells. As a special structure of sperm storage, in
addition to contributing to the structural integrity of the
SSTs, which are stretched considerably as ova descend
through the albumen region of the oviduct, these junctions in the turtle may constitute an immunological barrier between stored spermatozoa and host, thus facilitating long-term storage.
348
XIANGKUN ET AL.
Fig. 19. Magnification of a condensing granule. These granules are
usually centrally located, and there is an obvious area by which they
are separated from other types of granules, which is presumed to be
receiving a small quantity of secretory product from the cytoplasm by
small duct (right-pointing arrow). Mitochondria (Mi). Rough endoplasmic reticulum (bold right-pointing arrow)
Sperm Stored in the Tubules
Among vertebrates, association of stored sperm with
host cells has been reviewed by a few researchers, which
have been reported that range from casual membrane
contact in lizard (Cuellar, 1966) and avian (Bakst, 1987)
groups to actual penetration of spermatozoa heads into
the host cells (Hoffman and Wimsatt, 1972; Bou-Resli
et al., 1981). In the present study, various quantities of
spermatozoa were observed in the sperm storage
tubules. The sperm stored in the tubules appear random
in overall orientations, and no close association of stored
sperm with host cells were found, although some spermatozoa were in contact with the cilia of the cells forming the tubules. PAS-positive material and the flocculent
material secreted from the epithelium of the sperm storage tubules observed under light and electron microscopy indicates that any contribution by the host cells of
the tubules toward maintenance of spermatozoa must be
secreted naturally, or in the form of a carbohydrate-rich
matrix (Halpert et al., 1982; Kumari et al., 1990).
Fig. 20. Sperm stored in the sperm storage tubule (SST). Around
the sperm mass are the flocculent materials that are similar to the
contents of mature granules in the apex of the secretory cell. Sperm
(SP), ciliated cell (CC), secretory cell (SC).
Turtles are a reptilian group in which mating and gamete maturation is separated in time. Many temperate
zone turtles exhibit a postnuptial type of spermatogenic
cycle in which spermatogenesis commences in early
summer and is completed by autumn (Mcpherson and
Marion, 1981). Then the sperm are discharged from
testicle to epididymis as an episode event. The testes are
regressed the in hibernation period and only spermatogonia remain (Mao and Wang, 1997). Undoubtedly, all
sperm from epididymis will be obliged to undergo a long
time storage period in the oviduct of female. In present
study, portions of spermatozoa in the sperm storage
tubules appeared with large chromatic granules, which
are presumed to be in the process of nuclear condensation and are immature ones. Moreover, such spermatozoa also were found in the epididymis of the male Trionyx sinensis in our previous study. We are the first to
report immature spermatozoa in the SSTs of females.
Therefore, the present study proved that the sperm
transferred to the oviduct of the female might include
those immature ones. Whether such sperm could survive
during a long period storage and become mature ultimately or be used in fertilization is unknown.
UTERUS, OVIDUCT, AND STORED SPERM IN TURTLE
Fig. 21. Longitudinal and cross-section of the mature sperm stored
in sperm storage tubule (SST), in which intranuclear tubules could be
found (left-pointing arrow). Mitochondria (Mi) containing concentric
membranes and a center core in the middle piece of the sperm form
the mitochondria sheath. The nuclear region of immature sperm contain larger granular chromatins (bold arrow). Microvilli (M), (large thin
arrow). Cilia (Ci). Distal centriole in the midpiece of the mature sperm
(smaller thin arrow).
Degradation of sperm stored in the SSTs of turtles has
not been reported in any species by transmission electron microscopes; the only studies available were limited
in amphibians Eurycea cirrigera (Sever, 1992; Sever and
Brunette, 1993) and Plethodon cinereus (Sever, 1997). In
present study, the plasma membranes around nuclei of
the sperm in the lumen are often crenated, detached, or
disrupted, and the mitochondria become abnormal compared with that of epididymis, indicating sperm degradation in the lumen. These sperm might have undergone
a competition course and were elected to be degenerated,
which might include portions of immature sperm mentioned above. It is generally reported that oxidative
damage to sperm resulting from reactive oxygen species
(ROS) generated mostly by the mitochondria of spermatozoa during long time storage is possibly one of the
main causes for the decline in motility and fertility during storage (Cummins et al., 1994; Penã et al., 2003;
Harman, 2003). ROS that accumulated during long time
storage will break down the integrity of the sperm mem-
349
Fig. 22. Sperm stored in the sperm storage tubule (SST). Middle
piece (MP). Sperm (left-pointing arrow). Cross-section of the tail (rightpointing arrow).
brane, which results in apoptosis. Germ cell mitochondria undergo extensive remodeling during spermiogensis
of Trionyx sinensis. The mitochondria of mature spermatozoa have unusual configurations and 35–40 mitochondria with a dense core each are surrounded by several
concentric layers of membranes (Chen et al., 2006).
However, portions of spermatozoa stored in SSTs in the
present study are exhibited with abnormal mitochondria
which are mostly vacuolar, and the concentric layers of
membranes are degenerative. The volume of the central
cores within the mitochondria becomes larger but with
moderate rather than high electron density. Due to the
long time storage in SSTs of the oviduct, the accumulation of the ROS becomes possible. Alternatively, the concentric mitochondria presented in the spermatozoa
might have its prominent function to sustain the energy
need of the spermatozoa during long time storage rather
than to promote the cell to death by the ROS leakage. In
the bat C. mexicanus, the effective lipid peroxidation
inhibitors secreted from the genital tract induced concentration-dependent, highly significant inhibition of
lipid peroxidation of spermatozoa (Miguel et al., 1999).
The secretion of the epithelium in SSTs seems to be important in keeping capacity and motility of stored sperm
by their function in lipid peroxidation inhibition. Therefore, the mitochondria with concentric structures and
350
XIANGKUN ET AL.
Fig. 23. The head of the sperm with large chromatic granules,
which are presumed to be immature sperm and are in the process of
nuclear condensation (small arrow). Cross-section of the mature
sperm head (arrowhead), and degenerated sperm with various electron densities in the nuclear (thick arrow).
the secretions of the epithelium in SSTs might possess
their collective functions to sustain the motility and
capacity of the spermatozoa during long time storage period. Further study is required to detect the molecular
function of such concentric mitochondria as well as the
secretions in SSTs.
Estrogen and progesterone play an important role in
the functions of the sperm storage tubule as well as the
oviduct. The hormone surge stimulates the growth and
secretory activities of the luminal epithelium in many
turtles and other reptiles (Guillette, 1987; Abrams-Motz
and Callard, 1991; Sarkar et al., 1995; Giannoukos and
Callard, 1996). Together with the reports above, the
authors in the present study suggest that the sperm
used to fertilize might be selected in a receptor-recognized way. The mature spermatozoa in SSTs are active
from the stimulation of hormones during the reproductive cycle rather than those that are immature. In conclusion, the characteristics of spermatozoa in the sperm
storage tubules of the turtles are complex. To reach definitive conclusions concerning the fate of stored sperm
will require further research at the cytological and physiological levels.
Fig. 24. Showing the degeneration of the sperm in sperm storage
tubule (SST). The plasma membranes around nuclei of the sperm are
crenated, detached, or disrupted (large thick arrow). Spermatozoa
with abnormal mitochondria, which are mostly vacuolar, and the concentric layers of membranes are degenerative; the volume of the central cores within the mitochondria becomes larger but with moderate
rather than high electron density (thinner small arrow). Intranuclear
tubule in the head of a mature sperm (thicker small arrow). The middle
piece of the degenerated sperm (MP).
LITERATURE CITED
Abrams-Motz V, Callard IP. 1991. Seasonal variations in oviductal
morphology of the painted turtle, Chrysemys picta. J Morphol
207:59–71.
Adams CS, Cooper WE Jr. 1988. Oviductal morphology and sperm
storage in the keeled earless lizard, Holbrookia propinqua. Herpetologica 44:190–197.
Aitken RNC, Solomon SE. 1976. Observation on the ultrastructure
of the oviduct of the Costa Rican green turtles (Chelonia mydas
L.) J Exp Mar Biol Ecol 21:75–90.
Almeida-Santos SM, Laporta-Ferreira IL, Antoniazzi MM, Jared C.
2004. Sperm storage in males of the snake Crotalus durissus terrificus (Crotalinae: Viperidae) in southeastern Brazil. Comp Biochem Physiol A Mol Integr Physiol 139:169–174.
Bakst MR. 1987. Anatomical basis of sperm storage in the avian
oviduct. Scanning Microsc 1:1257–1266.
Bakst MR, Bird DM. 1987. Location of oviduct sperm-storage
tubules in the American kestrel (Falco sparverius). Auk 104:321–
324.
Birkhead TR, Mollar AP. 1993. Sexual selection and the temporal
separation of reproductive events: sperm storage data from reptiles, birds and mammals. Biol J Linnean Soc 50:293–311.
UTERUS, OVIDUCT, AND STORED SPERM IN TURTLE
Bjorkman N, Fredricsson B. 1961. The bovine oviduct epithelium
and its secretory process as studied with the electron microscope
and histochemical tests. Zeit Zellforsch 55:500–513.
Bou-Resli MV, Bishay LF, AI-Zaid NS. 1981. Observation in the fine
structure of sperm storage crypts in the lizard Acarthodactylus
scutellatus hardyi. Arch Biol 92:287–198.
Caulfield JP, Lewis RA, Hein A, Austen KF. 1980. Secretion in dissociated human pulmonary mast cells. Evidence for solubilization
of granule content before discharge. J Cell Biol 85:299–312.
Chen Q, Zhang L, Chen X, Han X. 2006. Ultrastructure of spermatozoon in soft-shelled turtle Trionyx sinensis. Acta Zoologica Sinica. 52:415–423.
Cuellar O. 1966. Oviductal anatomy and sperm storage structures
in lizards [J]. J Morphol 119:7–20.
Cummins JM. 1983. Structure of the sheep oviduct in relation of the
sperm transport and fertilization. Proc Aust Soc Reprod Biol 15:13.
Cummins JM, Jequier AM, Kan R. 1994. Molecular biology of the
human male infertility: links with aging, mitochondrial genetics
and oxidative stress. Mol Reprod Dev 37:345–362.
Davenport M. 1995. Evidence of possible sperm storage in the caiman, Paleosuchus palpebosus. Herpetol Rev 26:14–15.
Giannoukos G, Callard IP. 1996. Radioligand and immunochemical
studies of turtle oviduct progesterone and estrogen receptors: correlation with hormone treatment and oviduct contractility. Gen
Comp Endocrinol 101:63–75.
Gist DH, Congdon JD. 1998. Oviductal sperm storage as a reproductive tactic of turtles. J Exp Zool 282:526–534.
Gist D, Fischer EN. 1993. Fine structure of the sperm storage
tubules in the box turtle oviduct. J Reprod Fertil 97:463–468.
Gist DH, Jones JM. 1989. Sperm storage with the oviduct of turtles.
J Morphol 199:379–384.
Gould KG. 1974. Fertilization: a function of the oviduct. In: Johnson
AD, Foley CW, editors. The oviduct and its function. New York:
Academic Press. p 271–300.
Guillette LJ Jr. 1987. The evolution of viviparity in fishes, amphibians, and reptiles: an endocrine perspective. In: Norris DO, Jones
RE, editors. The reproductive endocrinology of fish, amphibians
and reptiles. New York: Plenum Press. p 7.
Guillette LJ, Fox SL, Palmer BD. 1989. Oviductal morphology and
egg shelling in the oviparous lizards Crotaphytus collaris and
Eumeces obsoletus. J Morphol 20l:145–159.
Halpert AP, Garstka WR, Crews D. 1982. Sperm transport and storage and its relation to the annual sexual cycle of the female redsided garter snake, Thamnophis sirtalis parietalis. J Morphol
174:149–159.
Harman D. 2003. Free radical theory of aging. Antioxid Redox Signal 5:557–561.
Hoffman LH, Wimsatt WA. 1972. Histochemical and electron microscopic observation on the sperm receptacles in the garter snake
oviduct. Am J Anat 134:71–96.
Jansen RPS, Bajpai VK. 1982. Oviduct acid mucus glycoprotein in
the estrous rabbit: ultrastructure and histochemistry. Biol Reprod
26:155–168.
Kumari TRS, Sarkar HBD, Shivanandappa T. 1990. Histology and
histochemistry of the oviduct of the oviductal sperm storage pockets of the adamid lizard Calotes versicolor. J Morphol 203:87–106.
Mao W, Wang Z. 1997. Seasonal variations of testicular and epididymal structure and plasma levels of testosterone in the soft-shelled
turtle [J]. J Nanjing Normal University (Nat Sci) 2:53–57.
351
Miguel Angel Leon-Galvan; Teresa Fonseca; Ricardo Lopez-Wilchis
and Adolfo Rosado. 1999. Prolonged storage of spermatozoa in the
genital tract of female Mexican big-eared bats (Corynorhinus
mexicanus): the role of lipid peroxidation. Can J Zool 77:7–12.
Newton WD, Trauth SE. 1992. Ultrastructure of the spermatozoon
of the lizard Cnemidophorus sexlineatus (Sauria: Teiidae). Herpetologica 48:330–343.
Olsson M, Madsen T. 1998. Sexual selection and sperm competition
and sexual selection. London: Academic Press. p 503–577.
Palmer BD, Guillette LJ. 1990. Morphological changes in the oviductal endometrium during the reproductive cycle of the tortoise,
Gopherus polyphemus. J Morphol 204:323–333.
Palmer BD, Guillette LJ. 1991. Oviductal proteins and their influence on embryonic development of birds and reptiles. In: Ferguson MWJ, Deeming DC, editors. Physical influences on embryonic
development in birds and reptiles. Cambridge, UK: Cambridge
University Press. p 29–46.
Palmer BD, Guillette LJ. 1992. Alligators provide evidence for the
evolution of an archosaurian mode of oviparity. Biol Reprod
46:39–47.
Pearse DE, Janzen FJ, Avise JC. 2001. Genetic markers substantiate long-term storage and utilization of sperm by female painted
turtles. Heredity 86:378–384.
Pearse DE, Janzen FJ, Avise JC. 2002. Multiple paternity, sperm
storage, and reproductive success of female and male painted turtles
(Chrysemys picta) in nature. Behav Ecol Sociobiol 51:164–171.
Penã FJ, Johannisson A, Wallgren M, Rodriguez-Martinez H. 2003.
Assessment of fresh and frozen-thawed boar semen using an
Annexin-V assay: a new method of evaluating sperm membrane
integrity. Theriogenology 60:677–689.
Perkins MJ, Palmer BD. 1996. Histology and functional morphology
of the oviduct of an oviparous snake, Diadophis punctatus. J Morphol 227:67–79.
Roques S, Diaz-Paniagua C, Portheault A, Perez-Santigosa N,
Hidalgo-Vila J. 2006. Sperm storage and low incidence of multiple
paternity in the European pond turtle, Emys orbicularis: a secure
but costly strategy? Biol Conserv 129:236–243.
Sarkar S, Sarkar NK, Maiti BR. 1995. Histological and functional
changes of oviductal endometrium during seasonal reproductive
cycle of the soft-shelled turtle, Lissemys punctata punctata.
J Morphol 224:1–14.
Sarkar S, Sarkar N, Maiti B. 2003. Oviductal sperm storage structure and their changes during the seasonal (dissociated) reproductive cycle in the soft-shelled turtle, Lissemys punctata punctata. J Exp Zoolog A Comp Exp Biol 295:83–91.
Sever DM. 1992. Sperm storage by the spermathecal epithelium of
the salamander Eurycea cirrigera. J Morphol 212:281–290.
Sever DM. 1997. Sperm storage in the spermatheca of the RedBack salamander, Plethodon cinereus (Amphibia: Plethodontidae).
J Morphol 234:131–146.
Sever DM, Brunette NS. 1993. Regionalization of eccrine and spermiophagic activity in the spermathecae of the salamander Eurycea cirrigera (Amphibia: Plethodontidae). J Morphol 271:161–170.
Sever DM, Hopkins WA. 2004. Oviductal sperm storage in the
ground skink Scincella laterale holbrook (Reptilia: Scincidae).
J Exp Zoolog A Comp Exp Biol 301:599–611.
Sever DM, Ryan TJ. 1999. Ultrastructure of the reproductive
system of the black swamp snake (Seminatrix pygaea): Part I.
Evidence for oviducal sperm storage. J Morphol 241:1–18.