Molecular endocrinology of oocyte growth and maturation in fish

Pituitary gonadotropins (GTHs) are of primary importance in triggering oocyte growth and maturation. However, the actions of GTHs are not direct, but are mediated by the ovarian production of steroidal mediators of oocyte growth (estradiol-17β) and maturation (maturation-inducing hormone, MIH; 17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP in salmonid fishes; 17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S in sciaenid fishes). It is established that production of estradiol-17β and 17α,20β-DP by salmonid ovarian follicles occurs via the interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). A distinct shift in the salmonid steroidogenesis from estradiol-17β to 17α,20β-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. It is possible that this shift is a consequence of dramatic changes in the expression of the genes encoding various steroidogenic enzymes. As an initial step to address this question, we have isolated and characterized the cDNAs encoding a number of ovarian steroidogenic enzymes including the rainbow trout cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase (HSD), 17α-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAS as well as the pig 20β-HSD cDNA. Estradiol-17β stimulates the hepatic synthesis and secretion of a yolk precursor, vitellogenin. Vitellogenin is then transported to the ovary where it is selectively taken up into the oocyte by a receptor-mediated process involving specific cell-surface receptors. Estradiol-17β was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17α,20β-DP and 20β-S is through the binding to the oocyte plasma membrane. This initial MIH-surface interaction is followed by the formation of the major mediator of MIH, maturation-promoting factor (MPF). We have purified MPF from mature oocytes of carp. Carp MPF consists of two components: the homolog of the cdc2⁺ gene product of fission yeast (p34^cdc2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17α,20β-DP treatment, while cyclin B proteins can be detected only in mature oocytes. Addition of bacterially expressed goldfish cyclin B to the extracts of immature goldfish oocytes induced MPF activation. These results suggest that the appearance of cyclin B protein is a crucial step for 17α,20β-DP-induced oocyte maturation in fish.     

Histological examination of final oocyte maturation and atresia in wild and domesticated Penaeus monodon (Fabricius) broodstock

Oocyte maturation and gonadosomatic index (GSI) of eyestalk ablated Penaeus monodon females collected from the wild and from two first‐generation domesticated lines were assessed. Frequency and diameter of the different oocytes, and the intensity of oocyte atresia, were compared among groups through histological assessments of the sections of the middle ovarian lobe. Digitized images from ovary sections were used to record the frequency and diameter of different oocyte types. Spawning performance of the three groups were expressed in terms of the percentage of females that spawned at least once (productive females), time from eyestalk ablation to first spawning (latency period) and the number of spawnings per female stocked. Final ovarian maturation was attained in all groups, as indicated by the presence of mature oocytes with cortical rods (cortical oocytes), dark‐green ovarian colour and high GSI values (5.83–6.86%). However, domesticated groups presented significant larger immature oocyte types (previtellogenic and yolky oocytes) and smaller cortical oocytes compared with wild females, indicating a reduced vitellogenic activity during final oocyte maturation. Additionally, the frequency of atresia was comparatively higher for both domesticated groups, which could be related to their inferior spawning performance. The implications of these results on the reproductive potential and development of domesticated P. monodon are discussed.     

A study of goldfish oocyte meiosisin vitro: effects of 2,4-dinitrophenol and adenosine-5-triphosphate

Germinal vesicle migration (GVM) and/or dissolution (GVD) were measured in goldfish oocytes, treated with 17α, 20β dihydroxyprogesterone (DHP) and other compounds considered to effect the cytoskeleton and oxidative phosphorylation,in vitro. Administration of DHP reinitiated meiotic maturation, increasing GVM and GVD in goldfish oocytes. Addition of 2,4-dinitrophenol (DNP) to the incubation medium significantly inhibited DHP-induced GVM and GVD. The DNP effect was found to be partially reversible after 24 h and could be reversed fully after a further delay of approximately 24h. Treatment of goldfish oocytes with demecolcine (DE; a colchicine derivative also known as colcemid) induces GVM to the micropyle without effecting GVD; while Cytochalasin-B which inhibits microfilament polymerization impairs both GVM and GVD. Administration of DNP, significantly inhibited DE-induced GVM, suggesting that GVM as well as GVD are dependent upon the process of oxidative phosphorylation. Addition of adenosine-5′ -triphosphate (ATP) at low concentrations (0.01–0.1 mM) did not effect DHP-induced or DNP-inhibited GVD in goldfish oocytes. The present results are consistent with the idea that migration of the oocyte nucleus during meiosis reinitiation has an energy requirement and involves participation by the cytoskeleton.     

Ultrastructure of the oocytes of the Japanese eel Anguilla japonica during artificially induced sexual maturation

ABSTRACT: Morphological changes in the oocytes of the Japanese eel, Anguilla japonica , induced to undergo ovarian development by repeated injections of salmon pituitary homogenate, were examined using electron microscopy. Oil droplets were closely associated with organelles, especially mitochondria, and increased in number as oocyte growth proceeded. They fused at the migratory nucleus stage. During vitellogenesis, two types of cortical alveoli were distinguished, one having filamentous contents, the other having latticated contents. As oocytes reached maturity, the structure of the cortical alveoli was exclusively filamentous. Yolk globules were homogeneous and highly electrondense, but electrondensity decreased during hydration. The structure of the zona radiata of previtellogenic oocytes consisted of two layers, and an additional reticular network structure was formed on the inside of the zona radiata during the vitellogenic stage. The zona radiata lost the reticular network structure and assumed a layered structure of uniform electrondensity at the migratory nucleus stage. These structural changes during oocyte development were mostly comparable to those in other teleosts. Results of the present study should assist in developing improved methods for full control of artificial maturation in the Japanese eel.     

Effect of piscine GH/IGF-I on final oocyte maturation in vitro in Heteropneustes fossilis

Growth hormone (GH) has recently been identified as co-gonadotropin regulating fish reproduction, hitherto, no effort has been made to see its effect on oocyte maturation in fishes, though some reports demonstrate the role of insulin like growth factor-I (IGF-I) in oocyte maturation in teleosts. Hence, effect of GH on oocyte maturation in post-vitellogenic H. fossilis has been worked out in the present study. Post-vitellogenic follicles in the ovarian tissue were challenged in vitro with H. fossilis pituitary homogenate (fPH), Clarias batrachus GH and GtH, barramundi IGF-I (IGF-I), 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) and testosterone alone, or in combination with IGF-I for 18 h at 26±1°C. Incubation of ovarian tissue with GH in the presence of actinomycin d or cycloheximide or barramundi IGF-I antiserum was also made separately. In general, oocyte maturation was induced by fPH, barramundi IGF-I, GtH, GH and DHP, which was augmented further by addition of barramundi IGF-I. Testosterone had no effect on GVBD. Actinomycin d, cycloheximide and anti barramundi IGF-I abolished the GH induced oocyte maturation. Present study suggests for the first time that GH has a role in egg maturation in fish.     

Diurnal rhythm of serum steroid hormone levels in the Japanese whiting,Sillago japonica,a daily-spawning teleost

Ovarian developmental stages and serum steroid hormone levels were examined at six different times of day (0100, 0600, 1000, 1300, 1600, 2000 h) in a marine teleost, the Japanese whiting Sillago japonica, which has an asynchronous-type ovary containing oocytes at various stages of development and spawns every day during a period ranging up to three months. The largest oocytes in the ovaries at the active vitellogenic or post-vitellogenic stages were found between 0100 and 1300 h. Oocyte maturation indicated by germinal vesicle breakdown (GVBD) occurred at 1600 h, and ovulated oocytes were observed in the ovaries collected at 2000 h. These processes were accompanied by a significant daily change in serum steroid hormone levels. The serum level of estradiol-17β showed a peak in fish with mature oocytes sampled at 1600 h. In these fish, the second-largest oocytes in the ovaries were at the initial stage of vigorous vitellogenesis, the secondary yolk stage. Therefore the highest level of serum estradiol-17β was considered to be due to the second-largest oocytes. Testosterone levels remained low and constant throughout the experimental period. The serum levels of 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-diOHprog) peaked at 1600 h at which time all fish had mature oocytes. These results indicate that the Japanese whiting possesses a diurnal rhythm of oocyte development including vitellogenesis, oocyte maturation and ovulation, and further suggest that daily cycles in oocyte growth and maturation which simultaneously take place in an ovary are regulated by diurnal secretions of estradiol-17β and the maturation-inducing steroid, 17α,20β-diOHprog.     

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.     

Physiology of seawater acclimation in the striped bass,Morone saxatilis (Walbaum)

Several experiments were performed to investigate the physiology of seawater acclimation in the striped bass, Morone saxatilis. Transfer of fish from fresh water (FW) to seawater (SW; 31–32 ppt) induced only a minimal disturbance of osmotic homeostasis. Ambient salinity did not affect plasma thyroxine, but plasma cortisol remained elevated for 24h after SW transfer. Gill and opercular membrane chloride cell density and Na⁺,K⁺-ATPase activity were relatively high and unaffected by salinity. Average chloride cell size, however, was slightly increased (16%) in SW-acclimated fish. Gill succinate dehydrogenase activity was higher in SW-acclimated fish than in FW fish. Kidney Na⁺, K⁺-ATPase activity was slightly lower (16%) in SW fish than in FW fish. Posterior intestinal Na⁺,K⁺-ATPase activity and water transport capacity (J_v) did not change upon SW transfer, whereas middle intestinal Na⁺,K⁺-ATPase activity increased 35% after transfer and was correlated with an increase in J_v (110%). As salinity induced only minor changes in the osmoregulatory organs examined, it is proposed that the intrinsic euryhalinity of the striped bass may be related to a high degree of “preparedness” for hypoosmoregulation that is uncommon among teleosts studied to data.     

Fertilizing ability of gametes at different post‐activation times and the sperm–oocyte ratio in the artificial reproduction of pikeperch Sander lucioperca

The time period during which oocyte and spermatozoa retain their fertilizing ability after contacting with water was evaluated in pikeperch (Sander lucioperca). In addition, success of in vitro fertilization was examined regarding to the sperm‐to‐oocyte ratio (SOR). In the first trial, oocytes were placed in Petri dishes containing 5 ml of the hatchery water, to which freshly collected and pooled sperm were added to each sample at 0, 15, 30, 60, 90, 120, 150 and 180 s post oocyte activation. The oocytes retained their fertility for at least 30 s after contacting with water. The second trial tested the maximum time period during which spermatozoa retained fertilizability after contacting with water. Milt (50 μl) was collected from each male and added to 5 ml of water in Petri dishes. Thereafter, oocytes were added at 0, 5, 15, 30, 60 and 75 s post‐sperm activation. Delays exceeding 10 s affected negatively the fertilization success. The third trial examined the optimum SOR; in which was found that 100 × 10³ spermatozoa per oocyte were the minimum ratio to ensure fertilization rates above 70%. Overall, the data clarified some biological interactions of gametes in the artificial propagation of pikeperch.     

The existence of extra-retinal and extra-pineal photoreceptive organ regulating gonadal development of ayu, Plecoglossus altivelis

The photoreceptors regulating photoperiodic gonadal development of ayu, Plecoglossus altivelis, are not known. In this study, we examined whether the gonads of ophthalmectomized and pinealectomized (Ex+Px) ayu respond to short photoperiod. Gonadosomatic index (GSI) and plasma levels of sex steroids were significantly increased in Ex+Px ayu kept under short photoperiod in both males and females. On the other hand, there were no significant increase in GSI and sex steroids in Ex+Px ayu kept under long photoperiod. The histological observation of the gonads revealed that oocytes undergoing final maturation in females and proliferation of germ cells in males were observed only in Ex+Px ayu kept under short photoperiod. Altogether, our results strongly suggest unknown photoreceptive organ regulates photoperiodic gonadal development of ayu.     

Implication of melatonin in oocyte maturation in Indian major carp Catla catla

Importance of melatonin (N-acetyl-5-methoxytryptamine) in the regulation of oocyte maturation has been studied in a carp Catla catla. Melatonin secretory cells were immunocytochemically localized only in the end vesicle. Diurnal and seasonal studies indicated that the serum levels of melatonin exhibit a minimum diurnal value in the mid-day of all seasons, but the peak value is attained either at mid-night or just before the onset of light. Moreover, highest seasonal value of melatonin was noted in the post-spawning phase. Administration of melatonin at different doses (25, 50, or 100 μg/100 g body weight) for 1, 15, or 30 days resulted in either pro- or anti-gonadal effects depending on the reproductive seasons. In vitro study revealed that incubation of oocytes with melatonin 4h prior to addition of MIH in the medium led to an accelerated rate of oocyte maturation through the formation of a complex of two proteins (MPF), cyclin B and cyclin dependant kinase, Cdc2. Moreover, melatonin pre-incubation considerably increased MIH stimulation of histone H1 phosphorylation as compared to MIH alone. Taken together, gathered information promotes the idea of a physiological role of melatonin in the maturation of oocytes in Catla catla.     

Effects of Salinity on Biogenic Amines,Hemolymph Osmotic Pressure,and Activity of Gill’s Na+/K+‐ATPase in Charybdis japonica (Crustacea,Decapoda)

The effects of salinity on hemolymph osmotic pressure, gill Na⁺/K⁺‐ATPase activity and dopamine (DA), norepinephrine (NE), serotonin (5‐HT) in the gills, and hemolymph of the adult Charybdis japonica were studied. DA levels increased significantly (P < 0.05), while the NE and 5‐HT revealed contrary change in hemolymph and gills. The iso‐osmotic point of C. japonica (911.4 mOsm/kg) was at salinity of 27.87 ppt. The Na⁺/K⁺‐ATPase activity of gill showed negative correlation with salinity in the hypotonic environment (<27.87 ppt). The results of this experiment indicated that C. japonica had great capability to acclimate to low salinity.     

盐度胁迫对三疣梭子蟹鳃Na+/K+-ATPase酶活的影响

通过钼蓝法测定三疣梭子蟹在3组实验盐度的胁迫过程中第2对和第6对鳃Na⁺/K⁺-ATPase酶活的变化,比较了3组实验盐度胁迫1 d时,鳃Na⁺/K⁺-ATPase的酶活大小。结果表明,在盐度胁迫初期,3组实验盐度下第2对和第6对鳃Na⁺/K⁺-ATPase的酶活下降;之后,各组实验盐度下第2对和第6对鳃Na⁺/K⁺-ATPase的酶活开始随胁迫时间增长而上升;最后,各组实验盐度下第2和第6对鳃Na⁺/K⁺-ATPase的酶活下降并趋于稳定。另外,胁迫1 d时,各组实验盐度下三疣梭子蟹前5对鳃Na⁺/K⁺-ATPase的酶活显著低于后3对鳃Na⁺/K⁺-ATPase的酶活。三疣梭子蟹对盐度变化的调节可分为被动应激期(酶活力下降)、主动调节期(酶活力逐渐上升)和适应期(酶活力稳定);三疣梭子蟹后3对鳃是离子转运、渗透压调节的主要部位。     

二硫苏糖醇对刺参卵母细胞体外成熟的影响

用海星鞘神经提取液和二硫苏糖醇对刺参卵母细胞进行了体外人工促熟研究。结果显示,二硫苏糖醇对刺参卵母细胞具有明显的促熟作用。刺参卵母细胞包被在滤泡中发育,卵母细胞通过动物极的卵突起连接到单层细胞构成滤泡膜上,充分发育的卵母细胞处于生发泡期,染色质凝集在核仁周围,处于生发泡期的卵母细胞在海水中不会自发成熟。用二硫苏糖醇处理充分发育的卵母细胞20min,卵母细胞的生发泡开始移动,并锚定在卵母细胞的动物极,继而出现生发泡破裂。此后,减数分裂重新启动,第一、二极体分别排出。但海星鞘神经提取液对刺参卵母细胞没有促熟作用。实验表明,二硫苏糖醇浓度在10-5～10-1mol/L范围内,均可促使刺参卵母细胞发生生发泡破裂,10-2mol/L时诱导卵母细胞成熟率可达90%以上。     

Mitochondrion-rich cells distribution,Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity and gill morphometry of the Amazonian freshwater stingrays (Chondrichthyes: Potamotrygonidae)

Detailed measurements of gill area and constituent variables (total filament number, total filament length and mean filament length), and immunolocalization of the α-subunit of Na⁺/K⁺-ATPase and Na⁺/K⁺-ATPase activity were performed on both hemibranchs of all five arches of freshwater potamotrygonid stingrays (Paratrygon aiereba and Potamotrygon sp.). Both species exhibit similar mass-specific gill area, 89.8 ± 6.6 and 91.5 ± 4.3 mm² g⁻¹ for P. aiereba and Potamotrygon sp., respectively. The density of Na⁺/K⁺-ATPase-rich MRCs and Na⁺/K⁺-ATPase activity was higher in the 4th gill arch in both species. The Na⁺/K⁺-ATPase activity was positively correlated to the Na⁺/K⁺-ATPase-rich (Na⁺/K⁺-ATPase rich) mitochondrion-rich cell (MRC) distribution among the gill arches of P. aiereba but not in Potamotrygon sp. The levels Na⁺/K⁺-ATPase activity were not correlated to the gill surface area among the arches for both rays’ species. Considering that the Na⁺/K⁺-ATPase-rich MRC is the main site for active ion transport in the gill epithelia and Na⁺/K⁺-ATPase activity plays a crucial role in osmoionoregulatory function, we suggesting that 4th gill arch is more relevant for osmoregulation and ion balance in these potamotrygonids.     

The reproductive physiology of three age classes of adult female diploid and triploid brook trout (Salvelinus fontinalis)

Triploid female fish show impaired gametogenesis and are unable to produce viable offspring. The reproductive physiology of artificially-induced triploid female salmonids has been well described up until the time of first sexual maturation in diploids, but few reports exist for older triploids. This study reports the influence of triploidy on growth, ovarian development and reproductive endocrinology among three age classes of female brook trout (Salvelinus fontinalis) in comparison to sibling diploids. Triploids were larger than diploids for most of the study period, but the difference was statistically significant only during maturation and spawning of 2⁺ diploids. Plasma estradiol-17 (E₂), testosterone (T) and vitellogenin (VTG) levels in triploids were generally lower than in diploids, and VTG was the only parameter to show seasonal fluctuations resembling those of diploids. Triploids showed significantly lower GSI and total oocyte number than diploids of similar age, and only half of all triploids sacrificed during the study (n=56) had developing oocytes in their ovaries. At age 3⁺, 13 of 19 triploid females had oocytes at various stages of development, including perinucleolar, yolk vesicle and yolk globule stages. In addition, three of these fish had collectively produced 72 mature stage oocytes. Thus, whereas diploid brook trout can produce mature oocytes as two-year-olds, triploids cannot do so until four years of age, with the number of mature oocytes being greatly reduced.     

Effects of bicarbonate ions and pH on acquisition and maintenance of potential for motility in ayu, Plecoglossus altivelis Temminck et Schlegel (Osmeridae), spermatozoa

The effects of extracellular ions on the acquisition and maintenance of the potential for motility in the spermatozoa of the ayu, Plecoglossus altivelis Temminck et Schlegel (Osmeridae), were examined. Testicular spermatozoa and milt spermatozoa were obtained from fully matured males and diluted with buffered solution (BS, 20 mM HEPES–NaOH, pH 7.5). Testicular spermatozoa showed a significantly low rate of motility (0.8 ± 0.4%), whereas milt spermatozoa showed a high rate (89.4 ± 2.1%). The spermatozoa were incubated with various isotonic media for 2 h, diluted with BS, and changes in the rates of motility were then compared. When incubated for 2 h with artificial seminal plasma (ASP), corresponding in terms of ionic constituents to seminal plasma buffered at pH 8.0, both spermatozoa showed a high rate of motility. Testicular spermatozoa acquired and milt spermatozoa maintained the potential for motility in response to the HCO₃^– ion concentrations (between 0 and 20 mM) in the ASP. The differences in the pH of the ASP had a significant effect on the acquisition and maintenance of the potential for motility, and spermatozoa showed the highest rate of motility with the ASP at pH 8.0 and 8.5. These results suggest that the quality of milt in the ayu can be regulated by controlling the concentration of bicarbonate and the pH of the incubating media.