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Copper nanoparticles (CuNPs) have been widely used in various industrial and commercial applications, which become a potential threat to aquatic organisms. Nevertheless, their potential toxicity to the epididymis and sperm remains little known. In this study, we evaluated the effect of CuNPs and copper ions (CuSO4) on the spermatozoa viability, epididymal structure, antioxidant enzyme activity, and inflammatory cytokines in cauda epididymis of the Chinese soft-shelled turtle. Results showed that the spermatozoa viability of Chinese soft-shelled turtles decreased significantly with an increase in CuNPs or Cu ions concentrations. The epithelial cells of the epididymal duct of the Chinese soft-shelled turtles with the treatment of 5 mg kg−1 CuNPs were slightly swollen, and the connective tissue between the epididymal ducts was loose. The epithelial structure of the epididymal tube was severely damaged with an increase in Cu ion concentrations. Compared to the control, the antioxidative enzymes activities and the expression of IL-1β, TNF-α, and IL-6 mRNA in the epididymis significantly increased with the treatment of CuNPs or CuSO4. The present study revealed that Cu ions exert more harmful effect on the epididymis and spermatozoa viability of Chinese soft-shelled turtles than copper nanoparticles.

With the wide use of copper nanoparticles (CuNPs) in various industrial and commercial applications, they inevitably enter the aquatic environment. However, their behavior in the aquatic environment and potential toxicity to aquatic organisms remain little known. In this study, we investigated the behavior of CuNPs in freshwater, as well as the toxicity and bioaccumulation of CuNPs and copper sulfate (CuSO

Long-term sperm storage is a special reproductive strategy, which can extend the time window between mating and fertilization in some animal species. Spermatozoa of the soft-shelled turtle, Pelodiscus sinensis, can be stored in the epididymis and oviduct for at least six months and one year, respectively. How spermatozoa can be stored in vivo for such a prolonged period is yet to be explained. We analyze the mechanisms that contribute to long-term sperm storage in P. sinensis, and compare them with other species from three different perspectives: the spermatozoon itself, the storage microenvironment and the interaction between the spermatozoon and microenvironment. Characteristics of soft-shelled turtle spermatozoa itself, such as the huge cytoplasmic droplet with its content of several large lipid droplets (LDs) and onion-like mitochondira, facilitate long-term sperm storage. The microenvironment of reproductive tract, involving in the secretions, structural barriers, exosomes, androgen receptors, Toll-like receptors and survival factor Bcl-2, are important for the maintenance of spermatozoa long-term storage. Sperm heads are always embedded among the oviductal cilia and even intercalate into the apical hollowness of the ciliated cells, indicating that the ciliated cells support the stored spermatozoa. RNA seq is firstly used to detect the molecular mechanism of sperm storage, which shows that autophagy, apoptosis and immune take part in the long-term sperm storage in this species.

A previous survey reported that swimming crabs Portunus trituberculatus (Miers, 1976) in the East China Sea may have a low risk of sperm limitation under the current nonselective fishing practices. In this study, the possible mechanism of avoiding sperm limitation was investigated in a mating season (from July to November 2007) from a male perspective. Males reached 50% gonadal maturity at a carapace length of 99.4 mm and the reproductive measures (vas deferens weights, sperm number, and sperm concentration retained in the vasa deferentia) did not differ significantly throughout the mating season. As shown for other decapods, the quantity of seminal material in the vasa deferentia increased significantly with male size, without a concomitant change in sperm concentration. The proportion of stored sperm transferred at a single mating was calculated, and the results showed that males exhibited low sperm transfer at each mating, regardless of size. These findings indicate that the current fishery pr...

Spermatogenesis in reptiles is a seasonally dependent physiological process that is not temporally associated with male mating behavior. Characteristics of seasonal spermatogenesis in reptiles, however, remain largely unknown. In this review, there is a coverage of the characteristics of soft-shelled turtle, Pelodiscus sinensis, during seasonal spermatogenesis that provides insights into spermatogenesis of testudines. The seminiferous epithelium of P. sinensis are undergoing spermatogenesis during the summer and fall, but are quiescent throughout the rest of the year; germ cells progress through spermatogenic stages in a temporal rather than a spatial pattern. While apoptotic germ cells mainly appear in the non-spermatogenic phase, these are seldom present during active spermatogenesis. It is inferred that apoptosis may be one of the reasons for germ cell loss during the resting phase of spermatogenesis. During the period when spermatogenesis is occurring, Sertoli cells become very narrow and are in contact with several round/elongated spermatids. Many residual spermatozoa can be internalized and degraded within Sertoli cells by entosis during the non-spermatogenic phase, which precedes the next reproductive cycle in P. sinensis. In the late spermatogenic phase, round-shaped mitochondria of spermatids become elongated and swollen, subsequently forming a crescent-like shape and develop into "onion-like" shaped mitochondria. As spermiogenesis progresses, the endoplasmic reticulum of spermatids is transferred into a specialized structure called the "Chrysanthemum flower center", which may be a source of autophagosomal membranes. The information provided in this review will help improve understanding of characteristics of seasonal spermatogenesis, which will hopefully promote interest in the study of reptilian species.

Objective: Information regarding the development of the enteric nervous system (ENS) is important for understanding the functional abnormalities of the gut. Because fertilized chicken eggs provide easy access to embryos, chicken models have been widely used to study embryonic development of myenteric plexus; however, no study has been focused on the postnatal period. The aim of this study was to perform a qualitative and quantitative analysis of the nitrergic neurons in the myenteric plexus of developing chickens in the postnatal period. Methods: Whole-mount preparations of the myenteric plexus were made in 7-d, 15-d, and 40-d old (adult) chickens of either sex (n=15). The myenteric plexus was studied after nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry using light microscopy, digital photography, and Image-Pro Plus 6.0 software. The numbers of positively stained neurons and ganglia were counted in the duodenum, jejunum, ileum, caecum, and colon in the different age groups. Data were expressed as mean±standard deviation (SD), and statistical analysis was performed using a one-way analysis of variance (ANOVA) test. Results: The positively stained neurons showed various morphologies and staining intensities, and formed bead-shaped and U-shaped arrangements in the myenteric plexus. The densities of neurons and ganglia increased with age. However, the number of positive neurons per ganglion increased. The number of NADPH-d-positive neurons was highest in the colon, followed by the ileum, the jejunum, the duodenum, and the caeca in all age groups. Conclusions: Developmental changes in the myenteric plexus of chickens continue in the postnatal period, indicating that the maturation process of the gastrointestinal function is gradual. In addition, no significant difference is happening among different intestinal segments during postnatal development, suggesting that the function of different intestinal segments had been determined after birth.

The structure of the nephron in adult soft-shelled turtles (Pelodiscus sinensis) was studied by light microscopy, transmission and scanning electron microscopy. The kidney contained 5-6 renal lobes. Nephrons of P. sinensis are composed of a renal corpuscle (RC) and of a renal tubule that appears divided morphologically into five distinct segments: neck segment (NS) (This segment is only present in approximately 10% of the nephrons), proximal tubule (PT), intermediate segment (IS), distal tubule (DT), and collecting duct (CD). The RCs and most of the convoluted DTs lie in the central zone, while the PTs and the CDs lie in the peripheral zone of the renal lobe. The renal corpuscle is relatively large with especial processes in podocytes and a thick basement membrane. The podocyte processes covering a large capillary area can be observed by TEM, and the major podocyte processes formed a very specific pattern in SEM. The podocyte processes expand to form a flattened network over the whole capillary loops surface, and only may observe little filtration slits in glomerular area. The neck segment when presentis short and has a relatively narrow lumen, consisting of cuboidal or squamous cells. There is a well-developed endocytic-lysosomal apparatus in the apical cytoplasm of the PT. The proximal tubule and intermediate segment cells show some differences between male and female. It showed that proximal tubule cells of male soft-shelt turtle contain lateral intercellular spaces, into which extensions of the cell membrane protrude, and the basal cell membrane forms a conspicuous labyrinth. Whereas, the basal and lateral cell membranes of the female are smooth, and no later-basal intercellular spaces. The differences between male and female in the middle segment cells is similar to proximal tubule cells. Not previously reported in vertebrate kidneys. The IS is the narrowest nephron segment, formed by multiciliated as well as nonciliated cells. In DT cells, basolateral interdigitations and infoldings are particularly well-developed. The CD contains clear cells with numerous secretory granules and dark cells with dense mitochondria and an elaborate Golgi complex. This study was undertaken in order to disclose specific kidney features in P. sinensis that could be related to function. In addition, the ultrastructure of the nephrons in P. sinensis are discussed in relation to other turtles and vertebrates.

Sperm storage in vivo extends the time window for fertilisation in several animal species, from a few days to several years. The underlying storage mechanisms, however, are largely unknown. In this study, spermatozoa from the epididymis and oviduct of Chinese soft-shelled turtles were investigated to identify potentially relevant morphological features and transformations at different stages of sperm storage. Large cytoplasmic droplets (CDs) containing lipid droplets (LDs) were attached to the midpiece of most spermatozoa in the epididymis, without migrating down the sperm tail. However, they were absent from the oviductal spermatozoa, suggesting that CDs with LDs may be a source of endogenous energy for epididymal spermatozoa. The onion-like mitochondria recovered their double-membrane morphology, with typical cristae, within the oviduct at a later stage of storage, thus implying that mitochondrial metabolism undergoes alterations during storage. Furthermore, a well developed fibrous sheath on the long principal piece was the integrating ultrastructure for glycolytic enzymes and substrates. These novel morphological characteristics may allow turtle spermatozoa to use diverse energy metabolism pathways at different stages of storage.

The microstructural and ultrastructural changes of germ cells during spermatogenesis of zebrafish (Danio rerio) were examined using light microscopy (LM) and transmission electron microscopy (TEM). Generally the process of spermatogenesis in zebrafish is similar to that of other teleosts, however, here we describe some peculiar features of zebrafish spermatogenic cells which have a limited report in this species. (1) The basic events of spermiogenesis are asynchronous, location of flagellum finished in initial stage, while chromatin condensation sharply occurred in intermediate stage and elimination of excess cytoplasm mainly taken place in final stages. (2) Surprisingly, the cilia or initial flagellae are created in spermatocytes, approach toward the nucleus of early stage spermatids, and then the centrioles depress into nuclear fossa and change their orientation to each other from right angle to obtuse angle about 125°. (3) During spermatogenesis, the chromatin compaction performs in a distinctive pattern, condensed heterogeneously from granular into chromatin clumps with central electron-lucent areas, round or long, which diminished to small nuclear vacuoles in spermatozoa. This finding demonstrates the origin of nuclear vacuoles in zebrafish spermatozoa for the first time. (4) Nuages are observed in both spermatogonia and spermatocytes. They are connected with the mitochondria and nuclear membrane, and are even located in the perinuclear spaces of spermatogonia nuclei. (5) Mitochondrial morphology and distribution shows diversity in different germ cells. The condensed mitochondria appear in pachytene spermatocytes, and mitochondria including membrane conglomerate exist in both spermatocytes and spermatids. This study was undertaken in order to disclose specific spermatogenic cells features in zebrafish that could be helpful for understanding the correlative function in this model species.

The epididymis of the soft-shelled turtle Pelodiscus sinensis was examined under light and transmission electron microscopes to determine the morphological characteristics, as well as their changes at different phases of the seasonal reproductive cycle. Three distinct regions, viz., cranial, middle and caudal were identified in the epididymis based on anatomical characteristics. The epididymal epithelium consists of five different cell types: principal, narrow, apical, clear and basal cells. Principal cells, which are the most abundant, together with basal cells are present along the entire length. Ultrastructural evidence suggests that all of the principal cells in each of the regions function in both absorption and secretion. Narrow cells and apical cells are rare and only confined to the cranial region. The clear cells, for the first time reported in the turtle epididymis, are confined to middle and caudal regions; these cells showed strong PAS-positive granulation in apical position, and secretory activity by a holocrine process, especially in the middle region. There was a significant difference in the epithelium height of all the regions between the reproductive season and the non-reproductive season. Sperm are stored in the epididymis throughout the year. Apart from the mature spermatozoa, immature spermatozoa with normal morphology are also observed. Under TEM, the immature spermatozoa showed a large amount of cytoplasm located eccentrically on the midpiece wrapped by plasma membrane, with some cytoplasm extended to the posterior of the head. Furthermore, the interactions of sperm with the epididymal epithelium were observed. Some sperm are associated with the secretory material in the lumen; other sperm are inserted into the intercellular space between the epithelial cells.

The ultrastructure of spermatozoa in cauda epididymidis of soft-shelled turtle, P. sinensis during breeding season was investigated by light microscopy (LM) and electron microscopy (TEM and SEM). The mature spermatozoa appeared elongated and filiform. In general, the turtle spermatozoon contains a characteristic head, midpiece and tail, similar in morphology to that of birds, amphibians and other reptiles. However, several features are unique. These include (1) three intranuclear tubules containing dense core extend from the subacrosomal cone through the rostral nucleus and deep into the nuclear body; (2) the midpiece is composed of 40 mitochondria which present a staggered rings-and-columns arrangement (8 parallel rings and 5 columns); (3) unusual spherical mitochondria with a dense core are surrounded by 8–10 concentric layers of cristae. Surprisingly, about 21.4 ± 3.6 percent immature spermatozoa with normal morphology are also observed in this season. Different from the mature spermatozoa, a variable amount of cytoplasm droplets are attached to the immature spermatozoa under SEM. Some spermatozoa still show the tail coiled tightly around the cytoplasm. These spermatozoa in transverse sections under TEM, showed a large amount of cytoplasm wrapped by plasma membrane; even some free mitochondria and higher electron density material still seen in the cytoplasm. Among the immature spermatozoa, most of them possess a cytoplasmic droplet which is located eccentrically on the midpiece, and contains a lot of lipid droplets in addition to hollow vesicles. Lipid droplets are closely associated with mitochondrial membranes and may function in the formation or degradation of mitochondria. These immature spermatozoa may be the dormant cells, but whether or not they can fertilize the ovum or not is unknown. Thus, in the present study we hypothesized that the cauda epididymidis might be involved in the sperm maturation in this species.