Spermatogenesis-preventing substance in Japanese eel

Under fresh-water cultivation conditions, spermatogenesis in the Japanese eel is arrested at an immature stage before initiation of spermatogonial proliferation. A single injection of human chorionic gonadotropin can, however, induce complete spermatogenesis, which suggests that spermatogenesis-preventing substances may be present in eel testis. To determine whether such substances exist, we have applied a subtractive hybridisation method to identify genes whose expression is suppressed after human chorionic gonadotropin treatment in vivo. We found one previously unidentified cDNA clone that was downregulated by human chorionic gonadotropin, and named it 'eel spermatogenesis related substances 21' (eSRS21). A homology search showed that eSRS21 shares amino acid sequence similarity with mammalian and chicken Müllerian-inhibiting substance. eSRS21 was expressed in Sertoli cells of immature testes, but disappeared after human chorionic gonadotropin injection. Expression of eSRS21 mRNA was also suppressed in vitro by 11-ketotestosterone, a spermatogenesis-inducing steroid in eel. To examine the function of eSRS21 in spermatogenesis, recombinant eSRS21 produced by a CHO cell expression system was added to a testicular organ culture system. Spermtogonial proliferation induced by 11-ketotestosterone in vitro was suppressed by recombinant eSRS21. Furthermore, addition of a specific anti-eSRS21 antibody induced spermatogonial proliferation in a germ cell/somatic cell co-culture system. We conclude that eSRS21 prevents the initiation of spermatogenesis and, therefore, suppression of eSRS21 expression is necessary to initiate spermatogenesis. In other words, eSRS21 is a spermatogenesis-preventing substance.     

Long-term cryopreservation of sperm of Japanese eel

After cryopreservation in a diluent (D#5-D), containing 76·2 mM NaHCO3, 137·0 mM NaCl, 1·4% soya lecithin and 10% dimethyl sulphoxide, for 30–180 days, Japanese eel Anguilla japonica sperm was 61·4–70·6% motile compared with fresh sperm. Cryopreserved sperm was used for fertilizing three batches of eggs and hatchability ranged from 20·7 to 69·1%. These results indicate that D#5-D is an useful diluent for sperm cryopreservation in Japanese eel.     

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.     

Ovarian morphology of the Japanese eel in Mikawa Bay

Annual changes in gonadal maturation of female Japanese eel Anguilla japonica in sea water were investigated histologically over 5 years in the Mikawa Bay, Japan, where they occurred throughout the year except in March. Almost all immature Japanese eels (yellow eels) occurred mainly from April to September, and they were rare after November. In contrast, maturing Japanese eels (silver eels) occurred from October to February. The gonado‐somatic index ( I _G) and oocyte diameters of yellow eels were <1·0 and 150 μm, respectively, and oocytes were at the peri‐nucleolus or the oil droplet stages. The I _G and oocyte diameters of silver eels were greater than those of yellow eels and most oocytes developed to the primary yolk globule stage. The numbers of silver eels lacking oocytes at the primary yolk globule stage increased after January in Mikawa Bay, although I _G and oocyte diameters remained unchanged. In contrast, silver eels caught at the mouth of the bay in January possessed oocytes that had advanced to the secondary yolk globule stage. These observations indicate that oocyte development changes seasonally, especially after winter in Mikawa Bay.     

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.      

Seamounts, New Moon and eel Spawning: The Search for the Spawning Site of the Japanese eel

After analyzing all the collection data for larvae of the Japanese eel, Anguilla japonica, in the western North Pacific, we found that the spawning site of this species appears to be near three seamounts in the West Mariana Ridge, 2000–3000km away from their freshwater habitats. These seamounts are located in the westward flow of the North Equatorial Current and are hypothesized to provide cues for migrating silver eels and to serve as possible aggregation sites for spawning. Back-calculated birth dates based on otolith microstructure of leptocephali indicate that the Japanese eel does not spawn continuously during the long spawning season from April to November, but is synchronized to spawn periodically once a month during new moon. This lunar periodicity of spawning and the seamount spawning hypothesis are new developments in the millennium-old mystery of eel spawning that has fascinated naturalists since the time of Aristotle.     

Characterization of Japanese eel immunoglobulin M and its level in serum

Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790 000; it contained an equimolar heavy chain and light chain with molecular weights of 72 000 and 25 000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by -mannose and could be abolished by α-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml⁻¹; it amounted to 10.3% of the total serum protein.     

The synthesis and role of taurine in the Japanese eel testis

In teleost fish, the progestin 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) is an essential component of the spermatogenesis pathway. In a series of investigations on the mechanisms underlying progestin-stimulated spermatogenesis, we have found that DHP up-regulates the expression of cysteine dioxygenase1 (CDO1) in the Japanese eel testis. CDO1 is one of the enzymes involved in the taurine biosynthesis pathway. To evaluate whether taurine is synthesized in the eel testis, cysteine sulfinate decarboxylase (CSD), another enzyme involved in taurine synthesis, was isolated from this species. RT-PCR and in vitro eel testicular culture revealed that although CSD was also expressed in eel testis, neither DHP nor other sex steroids affect CSD mRNA expression in a similar manner to CDO1. Using an in vitro eel testicular culture system, we further investigated the effects of DHP on taurine synthesis in the eel testis. HPLC analysis showed that DHP treatment significantly increases the taurine levels in the eel testis. These results suggest that DHP promotes taurine synthesis via the up-regulation of CDO1 mRNA expression during eel spermatogenesis. Furthermore, we observed from our analysis that although taurine does not induce complete spermatogenesis, it promotes spermatogonial DNA synthesis and the expression of Spo11, a meiosis-specific marker. These data thus suggest that taurine augments the effects of sex steroids in the promotion of spermatogonial proliferation and/or meiosis and hence that taurine plays important roles in spermatogenesis.     

Japanese eel at the northern edge: glass eel migration into a river on Hokkaido,Japan

Ichthyological Research -     

Participation of high-density lipoprotein in vitellogenesis in Japanese eel hepatocytes

To investigate effect of estradiol-17β (E₂) treatment in vivo on binding of eel hepatocytes to HDL, we developed hepatocytes binding assay. When hepatocytes were incubated with 200 times excess of eel HDL, the binding of hepatocytes to HDL precoated on wells was inhibited competitively. This indicates that eel hepatocytes bound specifically to HDL. E₂ treatment in vivo induced vitellogenin (VTG) synthesis. Hepatocytes prepared from the same E₂ treatment eel showed 3-fold higher ability of binding to HDL compared to hepatocytes prepared from ells without E₂ treatment. We also examined effects of E₂ and HDL on VTG induction in cultured hepatocytes. VTG, determined by enzyme-linked immunosorbent assay (ELISA), induction was about two-times higher in the presence of both 10⁻⁵ M of E₂ and 400 μg of HDL than in the presence of 10⁻⁵ M E₂ alone. At concentrations below 10⁻⁶ M E₂, VTG was not induced in the presence or absence of HDL. By SDS–PAGE and immunoblotting, VTG was detected only in the presence of both 10⁻⁵ M of E₂ and HDL. Our findings strongly support the idea that HDL correlates with vitellogenesis in eel liver.