PCNA protein expression during spermatogenesis of the Japanese eel (Anguilla japonica)

Spermatogenesis can be initiated by a single injection of human chorionic gonadotropin (hCG) into the cultivated Japanese eel, which produces only spermatogonia in the testis. To isolate the genes responsible for regulating spermatogenesis, we performed a differential mRNA display using poly (A)+ RNA extracted from the testes at different time points after hCG injection. Among several cDNA clones, the expression of which was initiated before the onset of meiosis, one clone has high homology with the proliferating cell nuclear antigen (PCNA). In this study, we investigated the protein expression of eel PCNA and found for the first time in any species that two forms (32-kDa and 36-kDa) of PCNA are present in the testis. Although the 36-kDa form existed in both the testis and spleen, the 32-kDa form was specifically expressed in the testis. In contrast to the appearance of 36-kDa PCNA 1 day after the hCG treatment, the 32-kDa PCNA appeared only 9 days after the hCG treatment, at which time active spermatogonial proliferation occurred in the testis. Both the 32- and 36-kDa forms were recognized by antibodies raised against different epitopes of PCNA, and their N-terminal amino acid sequences were identical. The 36-kDa form, but not the 32-kDa form, was recognized by antibodies against phosphoamino acids. These results suggest that the two PCNA proteins are the same molecule with different chemical modifications, including phosphorylation. We discuss the roles of these two forms of PCNA in the spermatogenesis of the Japanese eel.     

Human chorionic gonadotropin induces all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica)

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Using a newly developed organ culture system, we obtained evidence that human chorionic gonadotropin (HCG) can induce the entire process of spermatogenesis, in vitro, from spermatogonia to spermatozoa within 24 days. The HCG-induced spermatogenesis in vitro was accompanied by a marked activation of Sertoli cells and Leydig cells, occurring prior to the beginning of spermatogonial proliferation. These results indicate that gonadotropin triggers spermatogenesis in the Japanese eel and further suggest that this effect of gonadotropin is mediated through the actions of testicular somatic cells.     

Population structure of the Japanese eel, Anguilla japonica

Mitochondrial DNA (mtDNA) sequences that include (a) a part of the cytochrome b gene, (b) two tRNA genes, and (c) a part of the noncoding D-loop region of 31 Anguilla japonica (Japanese eel) and 1 A. marmorata collected from Taiwan, Japan, and mainland China were determined to evaluate the population structure of Japanese eel. Among 30 genotypes identified from the 31 Japanese eel mtDNAs sequenced, there are 58 variable sites, predominantly clustered at the D-loop region. The phylogenetic tree constructed by the unweighted pair-group method with arithmetic mean shows neither significant genealogical branches nor geographic clusters. Furthermore, the sequence-statistics test reveals little, if any, significant genetic differentiation. These results indicate that the 31 Japanese eels might come from a single population. Analysis of sequence variation in mtDNA by using the relationship between the number of segregating sites and the average number of nucleotide differences under the neutral mutation hypothesis reveals that neutral mutation acts as a major factor influencing the evolutionary divergence of the Japanese eel mitochondrial genome sequenced, especially in the noncoding region.      

Comparative studies between in vivo and in vitro spermatogenesis of Japanese eel (Anguilla japonica)

In order to check the quality of in vitro spermatogenesis of Japanese eel, in vitrol 1-ketotestosterone (11-KT) induced spermatogenesis was compared with in vivo spermatogenesis induced by a single injection of human chorionic gonadotropin (hCG) in detail. DNA contents of germ cells from in vitro and in vivo testicular fragments were compared using flow cytometry. Since the in vitro result of flow cytometry showed prominent 1C peak including spermatozoa and spermatids, the reduction of DNA by meiosis was assumed to progress normally, (i.e., haploid spermatozoa were produced in this in vitro system). In the testes of in vitro culture, however, spermatozoa were not released into lumen. Furthermore, the number of mitotic divisions of the in vitro experiment (6 divisions) was fewer than that of in vivo (10 divisions). In electron microscopy observations, both of in vivo and in vitro spermatozoon had a crescent-shaped nucleus with a flagellum, and a single large spherical mitochondrion. However, the elongation of the sperm head was not sufficient and the mitochondrion was not always located at the anterior end as is observed for the spermatozoa obtained from hCG injected eels. Eel spermatogenesis related substance-11 (eSRS11) is homologue of histone H1 which is up-regulated during spermatogenesis. Using this probe, in vitro spermatogenesis was also evaluated in molecular levels. In Northern blot analysis, eSRS11 mRNA was detected in both in vivo and in vitro testes. However, the expression of in vitro was much weaker than that of in vivo. These differences indicate that the stimulation of 11-KT is not sufficient, and another factors are needed to induce complete spermatogenesis in vitro.     

11-ketotestosterone synchronously induces oocyte development and silvering-related changes in the Japanese eel, Anguilla japonica

To evaluate the effects of sex steroids on silvering in the Japanese eel, Anguilla japonica, the development of oocytes, eye size, digestive tract, and swim bladder were studied in relation to observations of the profiles of plasma levels of sex steroids (estradiol 17β, E2; testosterone, T; 11-ketotestosterone; 11-KT) during silvering for each sex and by administrating 11-KT to yellow eels. All steroids examined in the study increased in female eels after silvering had begun, whereas in males, only 11-KT increased significantly, and no statistical differences were found in plasma levels of E2 and T between eels in both developmental stages. 11-KT appeared to induce the early stage of oocyte growth, enlargement of the eyes, degeneration of the digestive tract and the development of the swim bladder. This suggested that 11-KT synchronously accelerates early development of the ovaries and the morphological changes, possibly in adaption to oceanic migration, and that 11-KT is one of the most important factors in early stages of development in the Japanese eel, as it appears to be in other anguillid eels.     

Pathological changes caused by cold-water stress in Japanese eel Anguilla japonica

In the present study, histopathological changes in Japanese eel Anguilla japonica subjected to 'cold-water stress' were examined. Eels were exposed to cold water (13 to 15 degrees C) and warmer water (25 degrees C) as controls. Fish held in warm water did not show any apparent changes. Although none of the eels exposed to cold water died, they displayed nephrotic changes such as cloudy swelling and hyaline droplet degeneration of the renal tubular epithelia. Fish with nephroses had low levels of serum chloride (12 to 23 mg l-1 in fish with hyaline droplet degeneration, 71 to 81 mg l-1 in fish with cloudy swelling) compared with the control fish (87 to 109 mg l-1). In electron microscopy, affected tubular cells had variously damaged mitochondria and formations of secondary lysosomes of variable sizes. Electron microscopy also revealed mitochondrial degeneration in hepatocytes and degenerated granules of neutrophils in the hematopoietic tissue. 'Cold-water stress' was effective in damaging Japanese eels below 15 degrees C.     

Rectal water absorption in seawater-adapted Japanese eel Anguilla japonica

Marine teleosts drink large amounts of seawater to compensate for continuous osmotic water loss. We investigated a possible significant role of the rectum in water absorption in seawater-adapted eel. In rectal sacs filled with balanced salt solution (BSS) and incubated in isotonic BSS, water absorption was greater in seawater-adapted eel than in freshwater eel. Since rectal fluid osmolality was slightly lower than plasma osmolality in seawater-adapted eel, effects of rectal fluid osmolality on water absorption were examined in rectal sacs filled with artificial rectal fluid with different osmolality. Rectal water absorption was greater at lower rectal fluid osmolality, suggesting that an osmotic gradient between the blood and rectal fluid drives the water movement. Ouabain, a specific inhibitor of Na⁺/K⁺-ATPase, inhibited water absorption in rectal sacs, indicating that an osmotic gradient favorable to rectal water absorption was created by ion uptake driven by Na⁺/K⁺-ATPase. Expression levels of aquaporin 1 (AQP1), a water-selective channel, were significantly higher in the rectum than in the anterior and posterior intestines. Immunoreaction for Na⁺/K⁺-ATPase was detected in the mucosal epithelial cells in the rectum with more intense staining in the basal half than in the apical half, whereas AQP1 was located in the apical membrane of Na⁺/K⁺-ATPase-immunoreactive epithelial cells. The rectum is spatially separated from the posterior intestine by a valve structure and from the anus by a sphincter. Such structures allow the rectum to swell as intestinal fluid flows into it, and a concomitant increase in hydrostatic pressure may provide an additional force for rectal water absorption. Our findings indicate that the rectum contributes greatly to high efficiency of intestinal water absorption by simultaneous absorption of ions and water.     

In vitro induction of luteinizing hormone synthesis by estrogen in organ-cultured pituitary glands of the Japanese eel,Anguilla japonica

An organ culture method for pituitary glands isolated from immature Japanese eels (Anguilla japonica) was developed. This method could conserve the histological features of the pituitary glands for at least 21 days. The ability to synthesize gonadotropic hormone (GTH) in cultured eel pituitary glands was examined by detecting luteinizing hormone (LH) beta protein immunohistochemically. In a basal medium (Leibovitz L-15), LH beta-immunoreactive cells were very scarce, but after addition of estradiol-17beta (E2) a large number of immunoreactive cells appeared, particularly in the proximal pars distalis. The stimulatory effects of E2 on LH beta synthesis were dose (1-100 ng/ml)- and time (1.5-7 days)-dependent. Thus, in contrast with previous reports of the lack of a direct effect of E2 on GTH synthesis in primary cultured eel pituitary cells, the present results clearly indicate that E2 can stimulate GTH synthesis in immature eel pituitary glands. This organ culture method is useful to examine the actions of steroids and also other endocrine factors on the eel pituitary gland.     

Characterization of microsatellite loci from the Japanese eel Anguilla japonica

The Japenese eel, Anguilla japonica, is generally assumed to be composed of a single population with wide distribution range, and some genetic studies using allozyme or mitochondrial DNA methods supported this population model. However, one genetic study suggested the existence of multiple populations in this species, and thus, more detailed studies on the population structure is needed. Here we characterized a total of 11 microsatellite markers of the Japanese eel. These will serve as powerful tools for detailed population study for the Japanese eel, though two of them showed the significant departure from the Hardy–Weinberg expectations.     

A novel progestogen receptor subtype in the Japanese eel, Anguilla japonica.

A cDNA encoding a second type of a progestogen receptor (ePR2) was isolated from the same library as we had previously cloned a functional PR (ePR1) in eel testis. The amino acid sequence of the ePR2 shows low homology with ePR1 (34%), but both PRs showed progestogen-dependent transactivation in transfection experiment. Tissue distribution of ePR2 mRNA was clearly different from that of ePR1. Protein interaction between two PRs was demonstrated in vitro by a glutathione S-transferase pull-down assay. These results indicate that ePR2 is also a functional PR. This is the first isolation of two different functional PR molecules from a vertebrate.     

Recruitment mechanism of the eel, Anguilla japonica, to the Japanese coast

A total of 149 Anguilla japonica elvers collected at 10 locations along the Japanese coast were aged according to otolith daily increments. The interrelationships among age, birth date, body size, pigmentation stage, sampling location and the timing of recruitment were examined in order to determine the recruitment mechanism of elvers to coastal waters.
First catches of eels were earlier in the locations at lower latitudes. Age at recruitment was roughly constant, 218 ± 29 (mean ± s.d.) days old, disregarding the localities, but a weak positive correlation was obtained between age and sampling date or timing of recruitment. Body length at recruitment was also constant, 56.3 ± 2.3 mm s.l., and showed no significant correlation with either locality or recruitment timing. Estimated birth date ranged from April to November, the mean ± s.d. being 22 July 1982 ± 42 days, suggesting a peak season spawning in summer. Birth date was closely related with both the latitude of location and sampling time. Pigmentation developed more at lower latitude. Recruitment mechanism of the Japanese eel was summarized as follows: the earlier-born individual recruits earlier at lower latitude and at younger age, but at a constant body size.     

Daily growth increments in the larval otolith of the Japanese eel,Anguilla japonica

Early formation of otolith was studied on artificially hatched larvae of the Japanese eel,Anguilla japonica. Newly hatched larvae had a pair of sagittae which were flat and subelliptical with 8.3 μm in mean diameter. The diameter of the sagitta increased linearly with age. No growth increments were observed in the sagitta at hatching, while larvae which were 2, 4 and 6 days old had on average 2.1, 3.6 and 6.0 increments, respectively. The number of the increments (Y) and the age in days after hatching (X) showed a close linear relationship (Y=0.96X+ 0.06, r = 0.913, n = 40), suggesting daily deposition of sagittal increments. In 95 % of the field-caught elvers of this species, a distinct dark ring (check) with the diameter of 6–12 μm was found around the nucleus of the sagitta. This seems to be a “hatch check” deposited at hatching, since its diameter roughly agreed with that of the sagitta in the newly-hatched larvae. Possibly, the number of the increments outside the hatch check represents the age of the fish in days.     

Recombinant human insulin-like growth factor I stimulates all stages of 11-ketotestosterone-induced spermatogenesis in the Japanese eel, Anguilla japonica, in vitro.

In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-ketotestosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type B spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.     

Effects of changes in environmental calcium on prolactin secretion in Japanese eel,Anguilla japonica

Effects of changes in environmental Ca²⁺ on the secretion of prolactin, a possible hypercalcemic hormone, were examined both in vivo and in vitro in the Japanese ecl, Anguilla japonica. Transfer of seawater- or freshwater-adapted fish to fresh water, fresh water containing 10 mmol Ca²⁺ · 1^-1 sea water, Ca²⁺-free sea water, or deionized water was accompanied by significant changes in plasma Ca²⁺ levels after 7 days, except for the fish transferred from fresh water to fresh water and from sea water to sea water. Changes in external Ca²⁺ concentrations did not affect plasma prolactin levels, although plasma prolactin levels as well as pituitary prolactin contents were significantly greater in fish in a hypotonic environment than those in a hypertonic environment, regardless of the external Ca²⁺ concentration. Hypercalcemia, induced by removal of the corpuscles of Stannius, did not alter plasma prolactin levles. Incubation of the pituitary in the medium with different Ca²⁺ concentrations (up to 2.9 mmol·l^-1) did not affect the basal release of prolactin, except at an extremely low Ca²⁺ concentration (less than 0.1 mmol·l^-1) where prolactin release was inhibited. Addition of Ca²⁺ ionophore (A23187) to the medium led to a marked and significant increase in prolactin release, indicating that an increase in intracellular Ca²⁺ stimulates prolactin release. However, the effect was not specific to prolactin cells; a similar increase was seen in growth hormone release. These results indicate that changes in environmental Ca²⁺ concentration may not be the primary factor influencing prolactin secretion in the eel; changes in environmental osmolality or Na⁺ levels seem to be more critical for the regulation of prolactin secretion.Abbreviations CSX stanniectomy - DMSO dimethylsulphoxide - DW deionized water - FW fresh water - GH growth hormone - PRL prolactin - SW sea water     

Internal illuminance and shelter shape affect shelter selection by the Japanese eel Anguilla japonica

The present study evaluated the effects of internal illuminance and shelter shape on shelter selection by Japanese eels to enable the preservation or construction of suitable shelters for the Japanese eel. Japanese eels were able to distinguish a 1.25-fold difference in illumination inside the shelters, and preferred darker shelters. When the internal illumination of two shelters with the same shape was less than one-tenth of the ambient illumination (about 400 lx), shelter selection by Japanese eels was not affected by internal illuminance, even when there was a 10-fold difference in internal illumination between shelters. The width of the shelter was not important, but Japanese eels preferred a deep shelter with a low ceiling and walls that spread to a ‘dead end’. This has important implications on the creation of suitable shelters for Japanese eels.     

Location,size and age at onset of metamorphosis in the Japanese eel Anguilla japonica

This study clarifies the location, size and age at the onset of metamorphosis in Japanese eels Anguilla japonica through oceanic surveys, rearing experiments and analyses of the morphology and otoliths of leptocephali and glass eels. Twenty‐eight metamorphosing leptocephali were collected in the mesoscale eddy region to the east of Taiwan during research expeditions in 2004. Rearing experiments showed that the total length (L_T) of leptocephali decreased by an average of 12·5% during metamorphosis and 13·9% during the 2–12 h after death. Thus, the mean back‐calculated L_T at the onset of metamorphosis for 630 glass eels from Taiwan and Japan was estimated at 67·8 ± 2·7 mm (mean ± S.D.). The estimated mean ante‐mortem size of the fully grown pre‐metamorphic leptocephali collected in 2004 was 64·6 ± 3·4 mm, which was consistent with the L_T estimate for glass eels. Otolith analysis showed that the mean age at the onset of metamorphosis was 137 ± 15 days and indicated that Japanese eels may have a recruitment route through the mesoscale eddies to the east of Taiwan in addition to the direct transfer route from the North Equatorial Current to the Kuroshio Current.     

Androgen action on hepatic vitellogenin synthesis in the eel, Anguilla japonica is suppressed by an androgen receptor antagonist

Involvement of additional hormones other than estrogen in the control of vitellogenin (Vg) synthesis has been suggested in fish. However, no satisfactory explanation on the mechanism of the action of these hormones has been reported. In this study, we have exploited the possibility of androgen receptor mediation during the androgen action on the pathway of Vg synthesis. Hepatocytes were prepared from sexually immature Japanese eel Anguilla japonica and treated with estradiol-17beta (E2), 17alpha-methyltestosterone (MT), growth hormone, tamoxifen or flutamide, or in combination of these. Spent culture media were analysed by SDS-PAGE for Vg detection. Results from the chemical treatments demonstrated the necessity of E2 as the primary factor for Vg synthesis and requirement of additional hormones for the full expression of Vg. The effects of E2 and MT were effectively blocked by tamoxifen, an estrogen receptor antagonist and flutamide, an androgen receptor antagonist, respectively, indicating ER-mediated estrogen action and AR-mediated androgen action on Vg synthesis in this species.