Ultrastructure of Anterior Uterus of the Oviduct and the Stored Sperm in Female Soft-Shelled Turtle Trionyx sinensis.код для вставкиСкачать
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). 336 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. 340 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- 342 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 344 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 345 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 346 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 347 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). 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