On June 14, 2008 (the first experiment) and July 24, 2008 (the second experiment), the shores of the Boseong River and the sandy beaches, Seokgok-myun, Moksadong-myun, Gokseong-gun in Jeollanam Province were investigated and a total of 29 soft-shelled turtle (Tryonyx sinensis) eggs in the natural spawning nest eggs were collected (13 eggs were collected in the first experiment and 16 eggs in the second experiment). The temperatures in the natural spawning nests were 25.9- 36.9±0.5℃, the depth of the eggs was 5.2-7.5±0.5 cm as the distance of the average 6.4±0.5 cm. 29 eggs were scattered at least 0.2 cm interval. Artificial incubation of 29 eggs was conducted in artificial nest boxes in thermo-plastic composition of the incubator, and then incubated at 26.5-35.5±0.5℃, and an average constant temperature was 31.2-32.1±1.0℃. The incubation days ranged from 53 to 55. In case of most turtles, incubation at 31℃ (higher temperatures) generally produces all or mostly females, while incubation at 25℃(cooler temperatures) produces all or mostly males. Exceptionally, in case of genus Trionyx, the sex ratio of female : male of T. sinensis of a freshwater soft-shelled turtle was approximately 1:1, which differs from other genera of turtles and makes T. sinensis Strauch only turtles presently known to lack temperature-dependent sex determination.

Expression of claudin-11 (CLDN11), a tight junction (TJ) protein, was examined in the Korean soft-shelled turtle (Pelodiscus maackii) testes. Spermatogenesis began during the breeding season and peaked at the end of the breeding season. Spermiation started in summer and peaked in autumn. The deduced amino acid sequence of P. maackii CLDN11 was similar to those of avian and mammalian species. During the non-breeding season when spermatogenesis and testosterone production were active, testicular Cldn11 levels were high. In the seminiferous epithelium, strong wavy CLDN11 strands parallel to the basement membrane delaminate the spermatogonia, and early spermatocytes are in the open compartment. Otherwise, CLDN11 was found beneath the early spermatocytes and in the Sertoli cell cytoplasm. Punctate zonular occludens-1 (ZO-1) immunoreactivity was found within the CLDN11 strands parallel to the basement membrane or at the outermost periphery of the seminiferous epithelium close to the basal lamina. During the breeding season, when circulating testosterone levels and spermatogenic activity was low, testicular CLDN11 level was lower than those of non-breeding season. CLDN11 was found at apicolateral contact sites between adjacent Sertoli cells devoid of the postmeiotic germ cells. At this time, lanthanum tracer diffused to the adluminal compartment of seminiferous epithelium. In cultured testis tissues, testosterone propionate significantly increased the level of Cldn11 mRNA. In P. maackii testis, CLDN11 participates in the development of the BTB where the CLDN11 expression was coupled with spermatogenic activity and circulating androgen levels, indicating the conserved nature of TJ’s expressing CLDN11 at the BTB in amniotes.

Testicular expression of CLDN11 (claudin-11), a tight junction protein was examined together with spermatogenesis and circulating testosterone levels in Korean soft-shelled turtle (Pelodiscus maackii). Spermatogenesis started during the breeding season in May and peaked in August when the breeding season ended. Spermiation started in July and peaked in October, showing the typical pattern of spermatogenesis in temperate zone reptiles. Deduced amino acid sequences of P. maackii CLDN11 was highly homologous to those of avian and mammals, suggesting the conserved nature of CLDN11 in amniotes. During the non-breeding season when the spermatogenesis was active and circulating testosterone levels elevated, testicular CLDN11 mRNA and protein (19kDa) levels were high. Strong, wavy CLDN11 immunoreactive strands run parallel to basement membrane in the basal part of the seminiferous epithelium, delaminating the spermatogonia and early spermatocytes in the open compartment. Otherwise, CLDN11 was found beneath the early spermatocytes and in the Sertoli cell cytoplasm perpendicular to basement membrane. In double labeling experiment, punctate ZO-1 immunoreactivity was found within the CLDN11 strands run parallel to the basement membrane as well as at the most periphery of seminiferous epithelium where ZO-1 and CLDN11 in Sertoli cells were mostly cytoplasmic and perpendicular to basement membrane. Together, recruit of CLDN11 and ZO-1 to the inter-Sertoli TJs was tightly coupled with spermatogenic stage. At the breeding season when the circulating testosterone levels and spermatogenic activity remained low, testicular CLDN11 mRNA and protein levels were low. CLDN11 was found at apicolateral contacts between adjacent Sertoli cells devoid of the postmeiotic germ cells, suggesting that CLDN11 between adjacent Sertoli cells also participates in the maintenance of seminiferous lumen. In P. maackii testis, CLDN11 as a structural element of the blood-testis barrier dynamically changed according to spermatogenic activity and circulating androgen levels. This is the first study on the CLDN TJs at the BTB in reptilian testis.

We examined the effects of the vibration stress on cortisol secretion and hematological characteristics in soft-shelled turtle, Pelodiscus sinensis. For the stressed group vibration of from electric vibrator applied for 30 min with 2-h intervals during daytime () up to 28 days. Using the blood samples collected from ten turtles held once a week after vibration stress, we measured hematocrit, hemoglobin, red blood cells, cortisol, glucose, lactic acid, osmolality, , aspartate aminotransferase (AST), and alanine aminotransferase (ALT). The results have showed that P. sinensis received vibration stress exhibit the 'typical' stress-induced physiological responses (cortisol, glucose, lactic acid, osmolality, ions, hematocrit and hemoglobin) induced by vibration stress. Our data suggested that chronic vibration stress caused substantial stress in the animal, and in particular, the persisting elevated levels of AST and ALT would be highly correlated with the adverse effects of the stress. The high hematological characteristics during entire experimental period showed that the P. sinensis could not adapt to chronic stimuli provoked by vibration stress.