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.     

Preliminary Observations on the Reproductive Physiology of Female Orangemouth Corvina in Captivity

Seasonal changes In oocyte growth and plasma estradiol, testosterone, thyroid hormones and vitellogenin levels were monitored in three captive adult female orangemouth corvina Cynoscion xanthulus subjected to a condensed (8 mo) seasonal cycle of photoperiod and water temperature. Plasma concentrations of estradiol and testosterone began to rise when temperature increased to 28 C and photoperiod to 15 h light (midsummer conditions). This was accompanied by elevated circulating levels of vitellogenin and the appearance of vitellogenic oocytes (diameter > 100 μm). Estradiol concentrations and mean oocyte size increased concurrently during late summer conditions and were maximum during fall conditions, approximately 8 wk after the beginning of ovarian recrudescence. In contrast, plasma levels of thyroid hormones did not show any distinct seasonal changes. Gonadally recrudesced females contained several size classes of vitellogenic oocytes. Injection of these fish with a luteinizing hormone-releasing hormone analog (LHRHa) caused maturation and spawning of the largest oocytes (mean diameter of follicles × 2500 μm). Another group of vitellogenic oocytes had been recruited into this size class by 2 wk after the end of spawning, which suggests that this species is capable of repeated spawning during the reproductive season. Injection of LHRHa resulted in increased plasma levels of gonadotropin and repeated spawning over several days. LHRHa-induced final oocyte maturation, ovulation and spawning were preceded by increases in plasma levels of two teleostean maturation-inducing steroids, 17α,20β, 21-trihydroxy-4-pregnen-3-one (20β-S) and 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P₄). The results provide preliminary evidence that oocyte growth is stimulated in orangemouth corvina subjected to an abbreviated seasonal cycle in captivity under midsummer photoperiod and temperature conditions and is associated with seasonal increases in plasma estradiol concentrations and vitellogenin production. However, the relative importance of 20β-S versus 17α, 20β-P₄ in the control of final oocyte maturation in this species could not be determined from the results of the present study.     

鱼类卵子发育与卵泡闭锁的研究进展

鱼类种群繁衍离不开优质卵子的产出.卵子质量问题限制了许多海洋和淡水鱼类养殖业的发展.卵子质量主要受卵黄形成与卵母细胞发育的影响.亲本营养、管理以及其他内源或外源因素可能会影响卵黄组成与卵泡发育.与哺乳动物相比,鱼类卵母细胞发育与调控机制的研究尚不清晰.本文分析了鱼类卵子发育特点,探讨了亲本来源、育龄、环境因子以及营养等...     

Immunohistochemical observations of vitellin synthesis and accumulation processes in ovary of Ezo abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>

Ovarian follicle cells are the site of yolk protein synthesis in the Ezo abalone Haliotis discus hannai. In this study, histological observations of ovarian follicle cells were conducted in H. discus hannai by immunocytochemical and in situ hybridization methods focusing on their function of yolk protein synthesis. An antibody raised against purified yolk protein (vitellin, Vn) recognized yolk granules in oocytes, and ovarian follicle cells adjacent to the oocytes under yolk accumulation were stained positively with both anti-Vn antibody and an antisense probe for vitellogenin (the precursor for vitellin, Vtg) mRNA. These results indicate that the abalone Vtg gene is transcribed and translated in the ovarian follicle cells. In oocytes in the early phase of yolk accumulation, positive reactions with the antibody appeared first in the stalk part, and the follicle cells adjacent to the stalk were also stained positively. These observations imply local transport of yolk protein from the follicle cells to the oocyte through an extracellular space around the oocyte stalk. Ovarian follicle cells changed their morphological characteristics and reactivity to the antibody in close relation to the stages of the adjacent oocyte, which suggests the presence of functional interactions between follicle cells and oocytes in the course of yolk protein synthesis and accumulation.     

Ovarian maturation stages of the mud crab Scylla serrata

Ovarian maturation in adult wild‐sourced and pond‐grown Scylla serrata (Forsskål) was determined based on gross morphology and histological appearance. There were no significant differences noted in the histological features of both wild and pond‐reared S. serrata females. Ovarian maturation was classified into five stages: immature, early maturing, late maturing, fully mature and spent. The immature ovaries are thin and translucent to off white and contain oogonia, primary oocytes with large nuclei. The follicle cells were found around the periphery of the lobes and an area among groups of oogonia and oocytes. The follicle cells gradually enclosed the oocytes. The early‐maturing ovaries were yellow and small yolk globules started to appear in larger oocytes. In late‐maturing ovaries, the colour became light orange and lobules were apparent. Yolk globules occurred in the cytoplasm with larger globular inclusions towards the periphery, while follicle cells were hardly recognizable. Fully mature ovaries were orange to deep orange and had swollen lobules. Large yolk globules were apparent in the entire cytoplasm. Follicle cells were hardly seen. Spent ovaries were similar to the early‐maturing and late‐maturing stage in partially spawned females. The ovarian development was correlated closely to the gonadosomatic index, oocyte diameter, and ovarian histology. The classification of ovarian maturation provides baseline information for further studies on reproductive biology. Likewise, the information provides a guide for broodstock management in the hatchery.     

Patterns of oocyte development in natural habitat and captive <Emphasis Type="Italic">Salminus hilarii</Emphasis> Valenciennes, 1850 (Teleostei: Characidae)

Fecundity and oocyte development in Salminus hilarii female brood stock were analyzed with the aim of investigating the impact of migration impediment on oogenesis. Histological analyses of the ovaries were performed in adult females caught in two different environments—the Tietê River (natural) and captivity—and the gonadossomatic index, oocyte diameter and fecundity determined. Five germ cell development stages (oogonium, perinucleolar, cortical alveoli, vitellogenic, ripe) and two other structures (postovulatory follicles and atretic oocytes) were observed in females caught in the river. Captive animals lacked the ripe oocytes and postovulatory follicles and had a relatively higher number of atretic oocytes. Females in captivity are known to produce larger oocytes, and they release fewer eggs in each spawn (absolute fecundity) when compared with animals that are able to migrate. Our results suggest that the Tietê River is undergoing alterations which are being reflected in the reproductive performance of S. hilarii, mainly due to the presence of atretic oocytes in females caught in the river. The lack of postovulatory follicles and ripe oocytes in captive animals reveals that migratory impediment negatively impacts final oocyte maturation. However, the stage of maturation reached is adequate for ovulation induction with hormone manipulation.     

Ovarian histology of stunted pond-reared Macrobrachium rosenbergii females

The present study describes the ovarian histology of stunted freshwater prawn Macrobrachium rosenbergii. The ovarian maturation of stunted animals was examined and compared with similar‐aged normal females. Ten animals of the stunted group and each maturation stage of the normal group were sampled from the same pond and had their ovaries removed for histological analysis. Body weight, body length, ovarian weight and gonadosomatic index (GSI) were recorded for each female. The diameters of the different oocyte types were compared among groups through histological assessments. The ovarian histology of stunted M. rosenbergii females indicated that although the somatic growth is severely affected (7.6 g), some energy has been placed on the vitellogensis. Stunted females showed the simultaneous occurrence of previtellogenic, vitellogenic and mature oocytes in their ovarian tissue, but overall oocyte diameter and GSI (1.02%) were significantly affected when compared with normal females.     

Maturation and reproductive cycle of female armorhead <Emphasis Type="Italic">Pseudopentaceros wheeleri</Emphasis> from the southern Emperor—northern Hawaiian Ridge Seamounts

ABSTRACT:   The stage of ovarian development and annual reproductive cycle of female armorhead Pseudopentaceros wheeleri collected from seamounts of the southern Emperor–northern Hawaiian Ridge in 1993 and 1995–1996 was investigated through use of the gonadal somatic index ( GSI ) and ovarian histology. The stages of oocyte development were classified into eight developmental stages (perinucleolus, cortical alveolus, oil droplet, early yolk globule, late yolk globule, migratory nucleolus, prematuration, and maturation) based on histological characteristics. Oocytes of different size and developmental stage were present within ovaries during the spawning season. Oocyte development appears to be asynchronous and armorhead apparently spawn several times during the spawning season. The stage of the most developed oocyte was used to classify the stage of ovarian maturation. The GSI increased gradually with ovarian development. Oocytes in the perinucleolus, cortical alveolus, and oil droplet stages were present in ovaries throughout the year. Early and late yolk globule stages appeared during August–September indicating that yolk formation begins during late summer. The migratory nucleolus through maturation stages were detected during November–January. Post ovulatory follicles were present during November–March, and GSI values rapidly decreased in February. Results indicate that spawning occurs during November–February and peaks during December–January.     

半滑舌鳎卵巢发育的组织学和形态数量特征研究

采用组织学方法系统研究了人工养殖条件下半滑舌鳎亲鱼卵巢的组织发育周年变化特征.结果表明,卵母细胞发育可分为6个时相,卵巢发育分为6期.卵巢不同发育时期都由不同类型的卵母细胞组成,半滑舌鳎为非同步分批多次产卵类型.周年发育过程中,6月龄前的鱼卵巢发育处于第Ⅰ期;9～12月龄卵巢可发育至第Ⅱ期;12月龄半滑舌鳎性腺可发育达到Ⅲ期,并一直保持至24月龄.3龄雌鱼达到性成熟,卵巢可发育至第Ⅳ期;随着水温和光周期的调控,卵巢发育进入成熟期(Ⅴ期),发生水合现象是卵母细胞成熟并准备排卵的信号;产卵结束后卵巢退化至第Ⅵ期,排出的卵母细胞退化吸收,以Ⅲ期卵巢越冬后,卵巢退化至Ⅱ期并持续至下一个繁殖周期.人工养殖条件下,卵巢内存在卵母细胞闭锁现象.性腺周年发育过程中,性腺指数(GSI)在性腺达到成熟期时达到峰值,产卵结束后性腺快速退化,GSI值各月份差异显著(P<0.05),亲鱼性腺重量在卵巢发育成熟过程中一直处于生长状态.亲鱼肝脏指数(HSI)在产卵前7～8月份(性腺达到成熟期前30 d左右)达到最大,表明7～8月份是性腺进行卵子发育、卵黄能量储备的重要时期,亲鱼在产卵前通过积极摄食储备体内能量,肝脏在卵母细胞卵黄积累方面具有重要的生理作用.肥满度(CF)值在繁殖盛期10～11月份达到最大,表现出与GSI类似的变化趋势.     

Oocyte maturation and ovulation in the Atlantic halibut, Hippoglossus hippoglossus (L.), examined using ultrasonography

High resolution ultrasound scanning was tested as a non-invasive technique for monitoring oocyte maturation and ovulalion in the Atlantic halibut Hippoglossus hippoglossus (L.). Female broodstock halibut were examined using a 7.5-Mhz linear ultrasound transducer, prior to and during spawning. Representative images of halibut ovaries are presented and discussed. Individual oocytes of spawning females were discernible during oocyte final maturation, due to the large increase in volume caused by water uptake. The yolky (vitellogenic) cocytes of pre-spawning fish were more reflective to ultrasound than hydrating oocytes. The resulting differences in depth of ultrasound penetration permitted easy distinction of pre-spawning from spawning females. In addition, short-term changes in the depth of ultrasound penetration were observed during repeated scanning of a spawning female, owing to progressive hydration of the oocyte batch destined for ovulation. Although of similar diameter, hydrating oocytes in the ovarian tissue could be discerned from eggs in the ovarian lumen because of the different acoustic properties of the surrounding media. The findings of the present study are considered promising for the future routine use of ultrasonography in halibut broodstock management.     

Reproductive cycle,sexual maturity and diel-reproductive periodicity of white croaker,Pennahia argentata (Sciaenidae), in Ariake Sound,Japan

The white croaker, Pennahia argentata (Houttuyn), is abundant in demersal fish assemblages in Ariake Sound and is a commercially important species. The reproductive biology of white croaker was studied by measurement of gonadosomatic indices (GSI) and histological examination of ovaries. Ovaries were classified into one of five maturity stages based on histological data: (1) immature/resting stage; (2) developing stage; (3) maturing stage; (4) ripe/spawning stage; (5) spent stage. The spawning season lasts from April to September but most activity is observed from May to August. Age at sexual maturity was assumed to be 1 year. Postovulatory follicles coexisted with yolk stage oocytes in histological sections of ovaries indicating that white croaker is a multiple spawner, spawning more than once in a single spawning season. Judging from the diel changes in the proportion of females with ovaries containing postovulatory follicles and oocytes at maturation stage, most females spawned in the evening between 19:00 and 21:30 h.     

The reproductive biology of the amberjack, Seriola dumerilii (Risso 1810). I. Oocyte development in captivity

A study of the reproductive biology of the amberjack, Seriola dumerilii, held in captivity was carried out, describing oogenesis as well as the different stages of the ovarian cycle. Seven stages of oocyte development, as well as oogonia, were distinguished. Cortical alveoli were hardly detectable within the oocyte, as they were small, sparse and contained few mucopolysaccharides. It is suggested that their role in the fertilization process might be less important than in other teleost species. Fish aged 3 and 4 years were found to be sexually immature, with ovaries containing only previtellogenic oocytes. Vitellogenesis started in December in fish aged 4 + years. Late-developing ovaries showing deposition of yolk protein granules were found at the end of the 5th year of life (May) in specimens measuring 80.0 ± 3.5 cm standard length. This should be regarded as the minimum size at which sexual maturity is reached in S. dumerilii. Final vitellogenesis and oocyte maturation were, however, inhibited in captivity, and extensive follicular atresia took place as the natural spawning season approached. It is suggested that insufficient gonadotrophic stimulation because of confinement stress may be the cause of failed maturation and spawning in this species under culture conditions.     

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.     

Multiple vitellogenins and product yolk proteins in striped bass,Morone saxatilis: molecular characterization and processing during oocyte growth and maturation

The multiple vitellogenin (Vtg) system of striped bass, a perciform species spawning nearly neutrally buoyant eggs in freshwater, was investigated. Vitellogenin cDNA cloning, Western blotting of yolk proteins (YPs) using Vtg and YP type-specific antisera, and tandem mass spectrometry (MS/MS) of the YPs revealed the complex mechanisms of yolk formation and maturation in this species. It was discovered that striped bass possesses a tripartite Vtg system (VtgAa, VtgAb, and VtgC) in which all three forms of Vtg make a substantial contribution to the yolk. The production of Vtg-derived YPs is generally similar to that described for other perciforms. However, novel amino-terminal labeling of oocyte YPs prior to MS/MS identified multiple alternative sites for cleavage of these proteins from their parent Vtg, revealing a YP mixture far more complex than reported previously. This approach also revealed that the major YP product of each form of striped bass Vtg, lipovitellin heavy chain (LvH), undergoes limited degradation to smaller polypeptides during oocyte maturation, unlike the case in marine fishes spawning buoyant eggs in which LvHAa undergoes extensive proteolysis to osmotically active free amino acids. These differences likely reflect the lesser need for hydration of pelagic eggs spawned in freshwater. The detailed characterization of Vtgs and their proteolytic fate(s) during oocyte growth and maturation establishes striped bass as a freshwater model for investigating teleost multiple Vtg systems.     

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.