Reproductive parameters of the chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis> estimated from human chorionic gonadotropin-induced final oocyte maturation and ovulation in captivity

ABSTRACT:   Final oocyte maturation and ovulation of captive chub mackerel Scomber japonicus with fully yolk-accumulated oocytes were induced by a single injection of human chorionic gonadotropin. Reproductive parameters, including spawning frequency and batch fecundity, which are required to estimate spawning biomass in pelagic fish by the daily egg production method, were analyzed. Germinal vesicle migration (GVM) occurred at 18–24 h post-injection, and the hydration and ovulation of oocytes were completed at 30 and 36 h post-injection, respectively. The results of the maturation process suggest that fish with GVM-stage ovaries captured in the daytime from the field are capable of spawning on the night following their capture. The oocytes used in the oocyte size-frequency distribution method for batch fecundity estimates should be at late GVM and more advanced stages. The results of sequential artificial insemination showed that the quality of ovulated eggs held in the ovarian lumen rapidly deteriorated as time progressed after ovulation. This indicates that the fertilization window for the ovulated eggs of chub mackerel lasts only a few hours, and spawning behavior should be performed within a few hours after ovulation in the wild population.     

Molecular cloning of two types of spiggin cDNA in the three-spined stickleback, Gasterosteus aculeatus

Two types of spiggin cDNAs were isolated from the three-spined stickleback. Northern blot analysis revealed that glue protein is composed of multiple spiggin molecules, and that their synthesis in kidney is strongly up-regulated by androgens.     

Anatomical features of ossa cordis in the Steller sea lion

Irregular triangular cartilage or bone fragments are sometimes found in the fibrous triangle of the heart. Ossa cordis and/or cartilago cordis has been demonstrated in various terrestrial animal species. Regarding marine mammals, sperm whales lack heart bones, and there have been no studies on bones or cartilage in pinniped hearts. Therefore, we examined the ossa cordis and/or cartilago cordis of the Steller sea lion. Eleven Steller sea lion hearts were examined morphologically and histologically. Before dissection, some hearts were imaged by CT to confirm the presence of ossa cordis or cartilago cordis. As a result, ossa cordis-like fragments were confirmed in four adults and one pup. All of the fragments were found at the right fiber triangle, and one adult had ossified tissue, including adipose tissue in the bone marrow cavity. The ossa cordis probably support the aorta because they surround the aorta as in other terrestrial animals. Steller sea lions can dive to a few hundred meters, but they need to rest on land frequently. Hence, their ossa cordis help maintain heart function during the tachycardia that occurs upon repeated surfacing and movements on land after diving in water.     

Histopathologic effect of in ovo exposure to methotrexate at early embryonic stage on optic tectum of Japanese quail (Coturnix japonica)

The optic tectum of Japanese quail embryos with in ovo exposure to methotrexate 100 ng/g egg on embryonic day 4 was examined from 3 to 24 hour after treatment. At 9 hour after methotrexate exposure, several apoptotic neuroepithelial cells appeared in the ventricular zone of the optic tectum; these increased in number and were diffusely distributed throughout all layers of the ventricular zone of the optic tectum at 12 hour. At 24 hour, neuroepithelial cells in the ventricular zone of the optic tectum were eliminated and showed sparse cell density. Throughout the experimental period, proliferation of neuroepithelial cells in the ventricular zone of the optic tectum of methotrexate-treated embryos was inhibited. These results suggest that neuroepithelial cells in the ventricular zone of the optic tectum in Japanese quail embryos can be affected by folic acid antimetabolites, methotrexate, at an early embryonic stage.     

Expression and localization of estrogen receptors α and β mRNA in medullary bone of laying hens

The aim of this study was to observe the expression and localization of estrogen receptor (ER) α and ER β mRNA in the medullary bone of laying hens. First, medullary bone, liver, kidney, and shell gland of the oviduct tissues were dissected from laying hens. Then, the total cellular RNA was isolated from each tissue specimen, and the ER α and ER β mRNA expression was observed using semiquantitative RT‐PCR. Second, the localization of ER α mRNA in the medullary bone was detected with in situ hybridization using digoxigenin‐11‐UTP‐labeled cRNA probes. As a result, the expression of ER α mRNA was higher than that of ER β mRNA in the medullary bone, liver, and shell gland of the oviduct from laying hens. In the kidney, ER α mRNA expression was lower than that of ER β mRNA. The expression pattern of ER α and ER β mRNA of the medullary bone was similar to that of the shell gland of the oviduct. Moreover, ER α mRNA was intensively expressed in osteoblasts on the medullary bone surface and bone marrow stromal cells but was not expressed in osteoclasts. These results suggest that in medullary bone, estrogen action may be regulated not by ER β but by ER α.     

Reduction of primordial follicles caused by maternal treatment with busulfan promotes endometrial adenocarcinoma development in donryu rats

Ovarian dysfunction leading to hormonal imbalance plays a crucial role in uterine carcinogenesis in rats as well as women. However, the effects of a reduction in primordial follicles at birth on uterine adenocarcinoma development have hitherto not been determined. The present study was therefore conducted using female Donryu rats, a high incidence rat strain of uterine adenocarcinoma. The animals were maternally exposed to 2.5 or 5.0 mg/kg of busulfan on gestation day 14 to reduce primordial follicles, and were then initiated by intrauterine treatment with N-ethyl-N'-nitro-N-nitrosoguanidine at 11 weeks of age. Both busulfan treatment doses caused earlier occurrence of persistent estrus, with dose-dependence as compared to controls. At 15 months of age, the rats were euthanized. The incidence of uterine adenocarcinomas and multiplicity of uterine neoplastic lesions were significantly increased by the 5.0 mg/kg, but not the 2.5 mg/kg busulfan treatment. Morphologically, the ovaries exposed to busulfan treatment exhibited severe atrophy, with few or no follicles and corpus lutea. Serum 17beta-estradiol (E2), progesterone, and inhibin levels were significantly decreased in the busulfan treatment groups, with a clear dose-relation. Interestingly, only the 5.0 mg/kg busulfan treatment elevated the E2/progesterone ratio. These results provide evidence that the reduction of primordial follicles promotes uterine adenocarcinoma development in rats in association with an earlier occurrence of the persistent estrus status.     

Assessment of management of direct seeded rice production under different water conditions in Cambodia

In order to assess direct seeding of rice technology to cope with future agricultural labor shortage in Cambodia, agronomic experiments were conducted in 2005 and 2006 to compare direct seeding with transplanting under three water conditions (non-flooded, shallow flooded, and deep flooded conditions) with/without weed control by herbicides (bentazone and cyhalofop-butyl) for two Cambodian rice varieties (shorter stature and early maturity Sen Pidao, taller stature and longer maturity Phka Rumduol). Average rice yield in 2 years was lower in direct seeding (341 g m⁻²) than transplanting (404 g m⁻²), but interaction components with year, varieties, water conditions, and weed management were significant, and the attained maximum yield of direct seeding (510 and 464 g m⁻² for Phka Rumduol variety in shallow flooded condition with weeding in 2005 and 2006, respectively) was similar to that of transplanting. Plant length and dry weight of rice were reduced in non-flooded and deep flooded conditions compared with shallow flooded condition, and grain yield was the highest in shallow flooded condition. Yield advantage of Phka Rumduol over Sen Pidao increased under direct seeding, particularly under non-flooded conditions in 2005 because weed infestation was more suppressed in Phka Rumduol even without weeding. Increase in 100 g m⁻² of weed infestation prior to heading (dry weight basis) reduced about 20% of attainable yield with weed control. This study identified importance of stature and growth duration of rice varieties and presence of standing water as well as the weed control, in order to develop and extend direct seeding in the Cambodia.     

Immunoreactive changes in pituitary FSH and LH cells during seasonal reproductive and spawning cycles of female chub mackerel <Emphasis Type="Italic">Scomber japonicus</Emphasis>

The physiological functions of pituitary gonadotropins (GtHs) are well established in higher vertebrates, whereas those in teleosts are still poorly understood. To describe the role of GtHs during gonadal development of female chub mackerel Scomber japonicus, changes in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells were investigated immunohistochemically during the seasonal reproductive and spawning cycles. FSH and LH cells were identified in the different cell types of the proximal pars distalis (PPD); FSH cells were located in the central PPD, whereas LH cells were localized along the border of the pars intermedia. To examine changes in FSH and LH cells, the percentage of FSH or LH cell-occupying area in the PPD was evaluated and represented as FSHβ-immunoreactive (ir) or LHβ-ir levels, respectively. FSHβ-ir levels increased significantly from immature to the completion of vitellogenesis, whereas LHβ-ir levels were maintained at high levels from early vitellogenesis to post-spawning. During the spawning cycle, which consisted of four stages from just after spawning to the next oocyte maturation, both FSHβ-ir and LHβ-ir levels showed no significant changes among different stages; however, LHβ-ir levels remained relatively high, and FSHβ-ir levels were constantly low. These results suggest that both FSH and LH may be involved in vitellogenesis and LH may act at final oocyte maturation in female chub mackerel, although the role of FSH during the spawning cycle is still unclear.     

Optimization of a protocol for cryopreservation of mouse spermatozoa using cryotubes

The rapid increase in the number of genetically modified mouse strains has produced a high demand for their frozen spermatozoa from laboratories and mouse banking facilities. Historically, plastic straws have been used preferentially as containers for frozen mammalian spermatozoa because spermatozoa frozen in plastic straws have a high survival rate after thawing. However, plastic straws are more fragile and are used less often than the cryotubes used for conventional cell freezing. In this study, we sought to develop a new protocol for sperm freezing using cryotubes as the container to increase the accessibility of mouse sperm cryopreservation. Epididymal spermatozoa were collected from mature ICR or C57BL/6J (B6) males and were suspended in 18% raffinose and 3% skim milk solution. We then optimized the following conditions using the sperm survival rate as an index: 1) distance of cryotubes from the surface of the liquid nitrogen at freezing, 2) volume of the sperm suspension in the cryotube and 3) temperature of warming sperm during thawing. The best result was obtained when cryotubes containing 10 μl of sperm suspension were immersed 1 cm below the surface of the liquid nitrogen and then thawed at 50 C. The fertilization rates using spermatozoa frozen and thawed using this method were 63.1% in ICR mice and 28.2% in B6 mice. The latter rate was increased to 62.3% by adding reduced glutathione to the fertilization medium. After embryo transfer, 68% and 62% of the fertilized oocytes developed into normal offspring in the ICR and B6 strains, respectively. These results show that cryotubes can be used for cryopreservation of mouse spermatozoa under optimized conditions. This protocol is easy and reproducible, and it may be used in laboratories that do not specialize in sperm cryopreservation.