Primary Growth Stage Research Articles

Physiological characterization of rice under salinity stress during vegetative and reproductive stages

Salinity is one of the major environmental factors limiting crop productivity. For this reason, a greenhouse experiment was conducted in Rasht, North of Iran during 2010 growing season to evaluate the salinity levels of irrigation water at different growth stages on the some physiological characterization of rice. Treatments were arranged in a randomized complete block design with two factors and three replications. Factor one included four levels of saline water (2, 4, 6, and 8 dS m -1 ); factor two consisted of four growth stages (tillering, panicle initiation, panicle emergence and ripening). The results of this work showed that effect of different salinity levels on the all yield components except percentage of filled grains per panicle was not significant. Increase in salinity levels decreased this component. Effect of different growth stages on total number of empty grains per panicles, percentage of filled grains per panicle, number of unfilled panicles and percentage of ratio of number of unfilled panicles to tillers was significant but effect of different saline water on length of unfilled panicle and number of spikelets per unfilled panicle was insignificant. Resistance of final growth stages, i.e. panicle emergence and ripening stages against salinity was more than primary growth stages, i.e. tillering and panicle initiation. Therefore, in irrigation with saline water the final growth stages were important and irrigation with saline water should be applied at final growth stages.

Effect of humic substances on the quality of essential oils of medicinal plants

Environmental factors cause changes in growth of medicinal plants as well as the quantity and quality of their essential oil composition. Among those, the factors of soil; especially the roles of fertilizer nutrients (nitrogen, phosphate and potash) possess a remarkable importance. In the present research, we utilized SAPROPEL packed by a Russian Firm and humic acid, extracted from the soil of the north-Iranian forest, on Artemisia herba-albaand Semenovia suffruticosa plants which had been collected from the heights of Taftan Mountain. Addition of extracted humic acid and SAPROPEL to the roots of plants in early growing stage resulted in increasing the percentage of their composition. About the plant A. herba-alba as its root was exposed to humic acid, the percentage of oxygenated Terpeniod from the whole essential oil became 81.39% that in comparison with blank sample, an increase of 24.29% was observed. When the root got SAPROPEL rather than humic acid, we observed only 21.99% increase in the percentage of composition. As the root of plant S. suffruticosa received extracted humic acid, the percentage of oxygenated Terpeniod increased to 5.98%. While by adding the SAPROPEL, the increase was only 0.48%. Comparison of the percentage of essential oil composition, after addition of humic substances to their roots, in primary stage of growth depends on factors such as physiological structure, the kind of the growing place soil, climate and the amount of sun-shine. Key words: Medicinal plant, humic substances, root, essential oil, quality of essential oil.

Structure, function and secretory products of the peltate glands of Centrolobium tomentosum (Fabaceae, Faboideae)

The glandular structures of Centrolobium tomentosum Guill. ex Benth. have been little studied despite the economic importance of this species. We describe here the distribution, development, structure and ultrastructure of the secretory cells of the peltate glands found on the vegetative organs of this species. Stem apices and leaves in various stages of development were collected and prepared for examination by light, scanning and transmission electron microscopy. Chemical analyses and conventional histochemical tests to determine the chemical nature of the secretory products were also carried out. Peltate glands occur on aerial vegetative organs during their primary growth stage. These trichomes are structurally stable, persisting throughout the development of the organ. During the initial stages of the gland development, cell separation creates a central space that expands as secretions accumulate. Maximum secretion rates occur during this phase and the secreting cells characteristically have well developed smooth and rough endoplasmic reticulum, and high numbers of plastids and mitochondria. During the later stages of the secretory phase, the central cells show symptoms of cell death and are incorporated in to the secretions. At trichome maturity, the central space is delimited by a uniseriate epithelium. In addition to the resin, which is the main secretory product, an extensive three-dimensional carbohydrate matrix was observed that extended throughout the central space, apparently giving support to the resin droplets. The terpenic nature of the secretion was confirmed by thin-layer chromatography. Given the terpenic nature of the secretion and the permanence of trichomes throughout all phases of leaf development, it is postulated that the resin-secreting trichomes act to protect the plant against herbivores.

Characterization of Pseudomonas viridiflava Causing Alfalfa Root Rot Disease in Hamedan Province of Iran

Alfalfa (Medicago sativa) is the most important forage crop in Hamedan province, located in west of Iran. Under field condition, plants showed growth stunting, chlorosis and wilting symptoms not previously reported. To survey the causal agent of the disease, symptomatic plants were collected from the main alfalfa growing area in this province. Plants samples were disinfected, grinded in phosphate buffer, and a loop full of the prepared suspension was stroked on nutrient agar medium containing 5% yeast extract and 1% glycerol. Isolation and characterization of the causal agent, especially from plants in primary growth stage of the disease, is difficult. Isolated bacteria caused wilting and chlorosis after 40 days on alfalfa plants, under green house condition. Phenotypic feature determination of the isolated bacteria, together with two defined strains, by standard bacteriological methods indicated that they belong to P. viridiflava. PCR assay confirmed results of identification of the causal bacterial agent, determined by classical methods. Tested representative bacterial isolates showed an 860 pb PCR band, using primers designed for 16S rDNA sequence of P. viridiflava LMG 2352T (type strain). This new reported bacterium is the second pathogenic bacterium agent, which causes alfalfa yield losses in Hamedan province of Iran.

Mtss1 regulates epidermal growth factor signaling in head and neck squamous carcinoma cells.

Mtss1 is located within chromosomal region 8q23-24, which is one of the three most commonly amplified regions in HNSCC. Mtss1 is lost in metastatic cells but confusingly is commonly overexpressed in primary tumors. Here we address possible reasons why Mtss1 is positively selected for in primary tumors. We find that Mtss1 enhances the localization of the EGF receptor to the plasma membrane, prolonging EGF signaling and resulting in enhanced proliferation in HNSCC. Depletion of Mtss1 results in decreased EGF receptor levels and decreased phosphorylation of Erk1/2 and Akt. However, when cells are at high density and adherent to each other, analogous to conditions in a solid tumor, Mtss1 does not confer any growth advantage, either in basal conditions or following EGF stimulation. This could indicate why Mtss1 might be lost in metastases, but preserved in early primary tumors. This is supported by an organotypic assay showing that Mtss1 expressing cells display a less proliferative more epithelial-like morphology on top of a collagen matrix. Furthermore, xenograft tumors expressing Mtss1 initially grow more rapidly, but later show less proliferation and more differentiation. Mtss1 positively modulates EGF signaling at low cell densities to promote proliferation and, therefore, may be beneficial for the early stages of primary HNSCC tumor growth. However, at high cell densities, Mtss1 impacts negatively on EGF signaling and this suggests why it inhibits metastasis.

Effects of photothermal kinetics and amplitude of photoperiod decrease on the induction of the reproduction cycle in female Eurasian perch Perca fluviatilis

This study was designed to determine the effects of both rates of temperature and photoperiod decreases (further referred as photothermal kinetics) and amplitude of photoperiod decrease on the onset of the ovarian cycle in female Eurasian perch Perca fluviatilis. Two photothermal kinetics were applied on 16 groups of Eurasian perch breeders: one rapid (6 weeks) and one slow (16 weeks). For each photothermal kinetic, the effects of four amplitudes of photophase decrease (0, 1, 4 and 8 h) on gonadosomatic index (GSI), oocyte diameter (OD), oocyte stage and plasma oestradiol level (E2) were studied over a 168 day-period. Every combination (kinetic ∗ amplitude) was tested in duplicate. For both photothermal kinetics, females did not initiate oogenesis under a constant 17 L:7D photoperiod (0 h), (GSI ≤ 1%, OD ≤ 273 μm, mainly oocytes at primary growth or cortical alveoli stages and E2 ≤ 200 pg.mL −1 at day 168). By contrast, oogenesis was initiated when a 4 hour- or 8 hour photophase decrease was applied (GSI = 9–10%, OD = 620–665 μm, oocytes at exogenous vitellogenesis stage and E2 = 1000–1300 pg.mL −1 at day 168). When a one hour photophase decrease was applied, the female responded differently according to the photothermal kinetics. An inhibition (final GSI = 1.3% and OD = 268 μm) was observed under a rapid decrease (6 weeks), while a partial and delayed gonad development (final GSI = 4.9% and OD = 577 μm) was recorded under a slow decrease (16 week). This observation suggests that this 1-hour amplitude is close to the threshold of sensitivity below which females do not perceive photoperiod variations. This study shows that a high amplitude of photoperiod decrease (4 or 8 h) is required to induce oogenesis efficiently and that a constant photoperiod (17L:7D) can be used to control puberty and delay the onset of the reproductive cycle in female Eurasian perch. Finally, for the first time, a combined effect of both photothermal kinetics and amplitude of photophase decrease was identified in female Eurasian perch.

Spatiotemporal Expression of Bone Morphogenetic Protein Family Ligands and Receptors in the Zebrafish Ovary: A Potential Paracrine-Signaling Mechanism for Oocyte-Follicle Cell Communication1

The bone morphogenetic proteins (BMPs), originally identified by their abilities to induce bone and/or cartilage formation, have been reported to be involved in various growth and differentiation processes, including reproduction. Although mammalian models are more frequently used to study the BMP system in reproduction, we have extended the study to the zebrafish, an excellent model for studying female reproduction in teleosts. Reverse transcription-PCR analysis revealed the expression of the Bmp ligands (bmp2a, bmp2b, bmp4, bmp6, and bmp7a) and the type II Bmp receptors (bmpr2a and bmpr2b) in various tissues, including the ovary. Spatiotemporal distribution of these Bmp ligands and receptors in the ovary was then investigated in this study. Reverse transcription-PCR on isolated follicle layers and denuded oocytes demonstrated that all Bmp ligands examined were exclusively or abundantly expressed in the oocyte, whereas the two receptors were expressed exclusively in the follicle layers, strongly suggesting a potential paracrine signaling from the oocyte towards the follicle layer by various Bmp ligands. This supports the current view that instead of being passively controlled and nurtured by the follicle layer for its growth and development, the oocyte may play an active role by releasing various growth differentiation factors to regulate follicle layer function. Quantitative analysis of temporal expression profiles during folliculogenesis revealed an increased expression of bmp2a, bmp2b, bmp4, and bmp6 from primary growth (stage I) to previtellogenic (stage II) stages, followed by steady declines toward the end of folliculogenesis when the follicles became fully grown. On the contrast, the BMP receptors (bmpr2a and bmpr2b) consistently showed an increase in expression during folliculogenesis, with the peak levels reached at the full-grown stage prior to final oocyte maturation. The spatiotemporal expression patterns of the Bmp family in the zebrafish follicles provide important insights into potential roles for Bmps during follicle development as oocyte-derived factors. Further experiments using recombinant zebrafish Bmp4 showed that Bmp4 had an inhibitory effect on spontaneous oocyte maturation in vitro, but not 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP)-induced oocyte maturation in vitro.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Its Receptors in the Zebrafish Ovary: Evidence for Potentially Dual Roles of PACAP in Controlling Final Oocyte Maturation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally purified from ovine hypothalamus for its potent activity to stimulate cAMP production. However, its presence and action have also been demonstrated in various peripheral tissues including the ovary. In the zebrafish, two forms of PACAP (PACAP(38)-1, adcyap1a; and PACAP(38)-2, adcyap1b) and three PACAP receptors (PAC(1)-R, adcyap1r1; VPAC(1)-R, vipr1; and VPAC(2)-R, vipr2) were all expressed in the ovary. Interestingly, although both follicle cells and oocytes express adcyap1b, the expression of adcyap1a was restricted to the oocytes only. Among the three receptors, adcyap1r1 and vipr2 were expressed in the oocytes, whereas the expression of vipr1 was exclusively located in the follicle cells. Temporal expression analysis of PACAP ligands and receptors during folliculogenesis suggested that PACAP might play differential roles in regulating follicle growth and maturation through different receptors. The two receptors that are expressed in the oocyte (adcyap1r1 and vipr2) showed a significant increase in expression at the transition from the primary growth (PG) stage to previtellogenic (PV) stage and their levels maintained high during follicle growth. However, when the follicle development approached full-grown (FG) stage, these two receptors both decreased significantly in expression. In contrast, vipr1, the receptor expressed in the follicle cells, showed little change in expression at the PG-PV transition and afterwards during follicle growth; however, its expression surged dramatically at the FG stage prior to oocyte maturation. Based on these results, we hypothesized that PACAP might play dual roles in regulating follicle growth and maturation through different receptors located in different compartments. PACAP may stimulate oocyte growth but block its maturation in early follicles by acting directly on the oocyte via PAC1-R and VPAC2-R, whose expression is dominant in growth phase; however, PACAP may promote oocyte maturation in the maturation phase via VPAC1-R on the follicle cells, whose expression surges in FG follicles prior to maturation and is consistently high in the follicles undergoing final maturation. This hypothesis was further supported by the observation that PACAP promoted maturation of follicle-enclosed oocytes but suppressed spontaneous maturation of denuded oocytes in vitro. This study provides strong evidence for a PACAP-mediated signaling network in the zebrafish ovarian follicle, which may play roles in orchestrating follicle growth and maturation via different types of receptors located in different compartments of the follicle.

In vitro rumen methane output of perennial ryegrass samples prepared by freeze drying or thermal drying (40 °C)

In many in vitro studies feeds are dried in heated ovens with forced air ventilation and milled through a screen with 1mm apertures to obtain a physically representative small sample. Drying methods involving heat have been shown to change the chemical composition of feeds, and it is possible that changes in the chemical composition during thermal drying could affect in vitro rumen CH4 output measured on the samples. Hence, samples are often dried by freeze drying. The objective of this study was to compare effects of freeze drying at −55°C for 72h versus thermal drying at 40°C for 48h on the chemical composition and CH4 output of perennial ryegrass samples collected at 5 stages of primary growth (i.e., 12 May, 26 May, 9 June, 23 June, 7 July) using an in vitro rumen gas production technique. The magnitude of the changes in chemical composition due to drying method was small and not biologically important, and drying method had no effect on CH4 output. With advancing maturity at harvest, digestibility of the herbage declined. Methane output/g dry matter (DM) incubated decreased linearly while CH4 output/g DM digested increased linearly with advancing grass maturity. Thermal drying on a range of grass samples had a similar effect to freeze drying on in vitro rumen CH4 output, reflecting the small degree of change in chemical composition.This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors: K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.

Stage-dependent differences in RNA composition and content affect the outcome of expression profiling in roach ( Rutilus rutilus) ovary

The influence of changing composition and content of RNA on the results of expression profiling was studied in the group-synchronous ovaries of roach ( Rutilus rutilus) over the course of their maturation. The highest yield of total RNA was detected in the primary growth and early cortical alveolus stages. The total RNA yield gradually decreased through the late cortical alveolus and late vitellogenic stages. In the primary growth and early cortical alveolus stages, total RNA was characterized by a low percentage of 18S and 28S rRNA and a high percentage of smaller-sized RNAs (tRNA, 5S and 5.8S rRNA), whereas 18S and 28S rRNA had increased by the late cortical alveolus stage and dominated by the late vitellogenic stage. The ratio of mRNA to total RNA was highest at the primary growth stage but decreased significantly in later ovarian stages. When total RNA was used for reverse transcription (RT), the shift in the mRNA/total RNA ratio influenced the results of qPCR expression profiling of several commonly used reference genes (ribosomal protein L8, elongation factor-1α, RNA polymerase-subunit B5, and β2-microglobulin) and of two target genes, gonad-type aromatase ( cyp19a1a) and follistatin ( fst). We conclude that the expression of target genes should be related to the mRNA pool using the same input of either mRNA to RT or cDNA to qPCR. Furthermore, gene expression was related to tissue-specific RNA yield per body mass (RNA yield × ovary mass × body mass − 1 ) thereby reflecting the massive increase in the size and cellular composition of the ovary during the reproductive cycle.

Dopant-Induced Shape Evolution of Colloidal Nanocrystals: The Case of Zinc Oxide

The electrical, optical and other important properties of colloidal nanocrystals are determined mainly by the crystals' chemical composition, size and shape. The introduction of specific dopants is a general approach of modifying the properties of such nanocrystals in well-controlled ways. Here we show that in addition to altering the atomic composition of the nanocrystals the introduction of specific dopants can also lead to dramatic changes in morphology. The creation of Mg-doped ZnO nanocrystals provides an excellent example of this procedure; depending on the molar ratio of dopant precursor in the reagents, doped nanocrystals with well-defined shapes, from tetrapods to ultrathin nanowires, which exhibit tunable optoelectronic properties, are obtained for the first time. We find that the Mg dopants play an important role in the primary growth stage, resulting in initial growth seeds having diverse crystallographic structures, which are critical for the generation of doped nanocrystals with different shapes. We demonstrate that this "greener" synthetic scheme can be extended to other dopant systems and provides an attractive and effective strategy for fabricating doped ZnO nanocrystals with interesting compositional and spatial complexity.

Estructura y la ultraestructura del ovario de Cichlasoma urophthalmus (Perciformes: Cichlidae)

The study of the normal development, differentiation, structure and function of various components of developing follicles in the ovaries of numerous fish species have been a consistent focus of comparative reproduction. The structural and ultrastructural features of gonads from Cichlasoma urophthalmus have received scarce attention. In this work, we realized a descriptive study of female gonads of Cichlasoma urophthalmus. A total of 40 samples were collected in the Veracruz Alvarado Lagoon, Mexico in 2007-2008 period including the windy, dry and rainy seasons. Female gonads were extracted and a portion was fixed in 4% formaldehyde for treatment for routine histology hematoxylin and eosin (HE) and another part was processed for transmission electron microscopy (TEM). The gonads were fixed in 3% glutaraldehyde and 2% osmium tetroxide, followed by dehydrated in ethanol 50%, 70%, 80%, 95% and 100% for inclusion in Epon, thin sections were then prepared and were contrasted with lead citrate and uranyl acetate. The process of oocyte development can be divided into five distinct stages (formation of oocytes from oogonia, primary growth, lipid stage, vitellogenesis and maturation). In this work, we found that the primary growth stage is characterized by intense RNA synthesis and the differentiation of the vitelline envelope. Secondary growth starts with the accumulation of lipid droplets in the oocyte cytoplasm (lipid stage), which is then followed by massive uptake and processing of proteins into yolk platelets (vitellogenic stage). During the maturation stage, the lipid inclusions coalesce into a single oil droplet, and hydrolysis of the yolk platelets leads to the formation of a homogeneous mass of fluid yolk in mature eggs. In conclusion, further studies should elucidate structure and ultrastructural changes in the ovarian follicular components, in C. urophthalmus during different stages of oocyte growth.

Dynamic distribution of spindlin in nucleoli, nucleoplasm and spindle from primary oocytes to mature eggs and its critical function for oocyte‐to‐embryo transition in gibel carp

Spindlin (Spin) was thought as a maternal-effect factor associated with meiotic spindle. Its role for the oocyte-to-embryo transition was suggested in mouse, but its direct evidence for the function had been not obtained in other vertebrates. In this study, we used the CagSpin-specific antibody to investigate CagSpin expression pattern and distribution during oogenesis of gibel carp (Carassius auratus gibelio). First, the oocyte-specific expression pattern and dynamic distribution was revealed in nucleoli, nucleoplasm, and spindle from primary oocytes to mature eggs by immunofluorescence localization. In primary oocytes and growth stage oocytes, CagSpin accumulates in nucleoli in increasing numbers along with the oocyte growth, and its disassembly occurs in vitellogenic oocytes, which implicates that CagSpin may be a major component of a large number of nucleoli in fish growth oocytes. Then, co-localization of CagSpin and β-tubulin was revealed in meiotic spindle of mature egg, indicating that CagSpin is one spindle-associated factor. Moreover, microinjection of CagSpin-specific antibody into the fertilized eggs blocked the first cleavage, and found that the CagSpin depletion resulted in spindle assembly disturbance. Thereby, our study provided the first direct evidence for the critical oocyte-to-embryo transition function of Spin in vertebrates, and confirmed that Spin is one important maternal-effect factor that participates in oocyte growth, oocyte maturation, and oocyte-to-embryo transition.

Ruminal large and small particle kinetics in dairy cows fed red clover and grass silages harvested at two stages of growth

Passage, comminution and digestion rates of large and small particles were estimated using a rumen evacuation technique and total faecal collection with five lactating dairy cows in a 5 × 5 Latin square experiment. Two grass and two red clover silages harvested at early and late primary growth stages and a 1:1 mixture of late harvest grass and early harvest red clover were the dietary treatments. Cows received 9.0 kg supplementary concentrate per day. Ruminal contents and faeces were divided into large (>1.25 mm) and small (1.25–0.038 mm) particles by wet sieving. Indigestible neutral detergent fibre (iNDF) was determined by 12 days ruminal in situ incubation followed by neutral detergent extraction. Plant species did not affect ruminal particle size distribution, whereas advancing forage maturity decreased the proportion of large particles for both grass and red clover silage diets. Ruminal pool size of iNDF was higher ( P<0.001) with red clover compared to grass silage diets. Ruminal passage rates of iNDF and potentially digestible NDF (pdNDF) increased with decreasing particle size ( P<0.01). Passage rate of iNDF for small particles was slower ( P<0.01) when red clover compared to grass silage diets were fed. Particle comminution rate in the rumen was slower ( P<0.001) with red clover compared to grass silage diets and it increased ( P<0.01) with advancing forage maturity. The contribution of particle comminution to ruminal mean retention time of iNDF in the ruminal large particle pool was smaller ( P<0.01) in red clover compared to grass silage diets and it increased ( P<0.05) with the mixed silage compared to the separate silages. Passage rate of pdNDF for both large and small particles was not affected by dietary treatments. Digestion rate of pdNDF for large particles was faster ( P<0.001) with red clover compared to grass silage diets. Differences in ruminal passage and digestion rates of the large and small particles, in addition to differences in the passage and digestion rates of red clover compared to grass silage diets, emphasize the need to consider particle size and forage type in metabolic models predicting feed intake and fibre digestibility in ruminants.

Gonad development during sexual differentiation in hatchery-produced orange-spotted grouper ( Epinephelus coioides) and humpback grouper ( Cromileptes altivelis) (Pisces: Serranidae, Epinephelinae)

Juvenile sexual differentiation of two commercially important epinephelines, orange-spotted grouper ( Epinephelus coioides) and humpback grouper ( Cromileptes altivelis) (Serranidae, Epinephelinae), produced in hatcheries, was studied until first sexual maturation using gonadal histology. Diandric, protogynous hermaphroditism is confirmed for E. coioides with evidence of primary male differentiation directly from the juvenile phase as well as of secondary male development through the sex-change of functional females. All juveniles developed first an ovarian-phase then entered a bisexual-phase gonad. Sexual differentiation occurred in bisexual gonads at around 105 weeks after hatching (wah), with the growth of oocytes beyond the primary-growth stage for females, and the appearance of sperm sinuses and proliferation of spermatogenic cysts in primary males. The minimum size and age of first sexual maturation for females were 355 mm standard length (SL) and 113 wah. No mature males were found by 141 wah, suggesting that first maturation in males takes longer than in females, at least under mariculture conditions. For C. altivelis, all juveniles developed an ovarian-phase gonad. Minimum body size and age of first sexual maturation for females were 155 mm SL and 83 wah. In contrast to E. coioides, there was no sign of spermatogenic cysts in juvenile gonads and any indication of primary male differentiation in C. altivelis. This study reveals the variation in early gonad development in the epinephelines and highlights the importance of studying juvenile sexual differentiation for fully understanding sexual pattern in this fish subfamily.

THE GH-IGF AXIS AND ITS POTENTIAL ROLE IN THE OVARY OF ZEBRAFISH, Danio rerio

Growth hormone (GH) from the pituitary is the major hormone responsible for body growth in vertebrates. However, there has been increasing evidence that GH is also involved in several reproductive functions including steroidogenesis and gametogenesis in the gonads. In view of this, GH has also been termed co-gonadotropin. It is well established that most effects of GH are mediated by insulin-like growth factors (IGFs) produced in different tissues. In the ovary, however, both IGF-dependent and independent mechanisms have been proposed for GH actions, and one of the factors that work downstream of GH signaling appears to be activin. Interestingly, the expression of GH itself has been detected in the ovary, suggesting the existence of an intraovarian GH-IGF axis. Using zebrafish as the model, the present project was undertaken to characterize the mini-GH-IGF-I axis in the ovary with particular reference to its relationship with the local activin-follistatin system. RTPCR analysis showed that Gh (gh), Igf1 (igf1), Igf2 (igf2), and their receptors were all expressed in the ovary, indicating that a potential intraovarian GH-IGF system also exists in the zebrafish. Further experiment on spatial distribution using isolated follicle layers and denuded oocytes revealed that gh was exclusively expressed in the fullgrown oocytes, while its receptor Ghr (ghr) could be detected in both oocytes and follicle cells. Igf1 and its receptor igf1r as well as igf2 could also be detected in both oocytes and follicle cells. Analysis of temporal expression patterns demonstrated that gh and ghr were differentially expressed during folliculogenesis. The expression level of gh in the follicles seemed to be the highest at the primary growth (PG) stage, but it gradually decreased when PG follicles developed to full-grown (FG) follicles. On the contrary, the expression of ghr was low at the PG stage but significantly increased in the previtellogenic (PV) stage and remained high in later stages. Similarly, the expression of igf1r gradually increased from PG to mid-vitellogenic (MV) stage, but decreased significantly in the final FG stage. Igf1 expression increased from PG to EV stage and decreased afterwards till FG stage. While expression of igf2 maintained constant during folliculogenesis. When tested in cultured follicle cells, recombinant human Igf1 increased the expression of activin beta A (inhba) but decreased that of activin beta B (inhbb) in a clear time and dose-dependent manner. GH (bream and human GH), however, seemed to increase the expression of both inhba and inhbb, which supports our previous observation that GH-induced zebrafish oocyte maturation could be blocked by follistatin, an activin-binding protein. Although we have not obtained convincing evidence for IGFs as the downstream mediators of GH in the zebrafish ovary, our preliminary data did suggest a potential role for the local ovarian activin system in GH as well as IGF signaling. (platform)

Local expression of CYP19A1 and CYP19A2 in developing and adult killifish ( Fundulus heteroclitus)

P450 aromatase (CYP19) is the terminal enzyme in the steroidogenic pathway and catalyzes the conversion of androgens to estrogens. Fundulus heteroclitus like other teleosts, express two CYP19 genes, CYP19A1 and CYP19A2. The expression of CYP19s in Fundulus was measured by in situ hybridization throughout development. In 90 dpf (day post-fertilization) fish and adult fish, CYP19A1 was expressed in the ooplasm of early stage I oocytes (primary growth stage). Expression of CYP19A1 was localized in the follicle cell layer of late stage I (previtellogenic stage) and stage II (vitellogenic stage) follicles, but by stage III (early maturational follicles) CYP19A1 expression was localized in the vitelline envelope. Overall, CYP19A1 oocyte membrane expression gradually declined from highest expression at late stage I to nondetectable levels by stage IV. Highest expression of CYP19A2 was detected in the brain including the hypothalamus from 4, 6, 8, 10, 14 dpf embryos, 90 dpf fry fish and adult fish brain. In females compared to males, there was higher CYP19A2 expression in olfactory bulb. In addition to the brain, there was strong CYP19A2 signal in adrenal/kidney cells in 6–14 dpf embryos. This work establishes the localization and constitutive expression of CYP19s in Fundulus which can then be compared with potential disruption of CYP19A1 and CYP19A2 expression and physiological consequences caused by environmental contaminants.

Effect of tributyltin on the development of ovary in female cuvier ( Sebastiscus marmoratus)

Organotin compounds, such as tributyltin (TBT) used as an antifouling biocide, can induce masculinization in female mollusks. However, few studies addressing the effect of TBT in fish have been reported. This study was conducted to investigate effects of TBT at environmental levels (1, 10, 100 ng/L) on the development of ovary in female cuvier. TBT exposure elevated testosterone levels, increased the ratio of testosterone to 17β-estradiol and decreased 17β-estradiol levels in ovaries after 50 days compared to the control. Three stages of follicles (primary growth stage, yolk vesicle stage, vitellogenic stage) were observed in the ovaries of cuvier at the control and 1 ng/L TBT group. The ovaries at the 10 ng/L TBT group were characterized by the lack of vitellogenic stage follicles and instead had higher proportions of primary growth stage follicles. 100 ng/L TBT resulted in follicles that were entirely at the earliest (primary growth stage) stages of development. There was a significant increase in apoptotic ovarian follicular cells judged by TUNEL-positive cell at the 10 ng/L TBT group. The TUNEL-positive follicles were observed at the 100 ng/L TBT group. The result in the present study showed that TBT at environmentally realistic concentrations can inhibit the ovarian development in fish. Besides the changes of sex hormone induced by TBT, apoptosis appears to be one mechanism affecting ovarian development.

Expression and possible functional roles of cytochromes P450 2J1 ( zfCyp 2J1) in zebrafish

Cytochrome P450 2J (Cyp2J) subfamilies are recognized as catalysts of arachidonic acid metabolism in extrahepatic tissues of many species. However, to date, no P450 2J have been identified in zebrafish. We describe here a zfCyp2J1 cDNA which encodes a putative protein of 496 amino acids and shares 51%, 51%, 50%, 51% and 50% identity with mouse Cyp2J6, rabbit Cyp2J1, human Cyp2J2, cow Cyp2J2, and rat Cyp2J4, respectively. Despite detectable levels of expression by RT-PCR, no expression was shown by in situ hybridization using whole-mount tissues of the embryos. Gene-specific knockdown by antisense morpholino oligonucleotide had no phenotypic effect on embryonic development. However, over-expression of zfCyp2J1 by injection of the embryos with the cDNA resulted in substantial dose-dependent morphological defects. With adult zebrafish, whole-mount in situ hybridization showed that zfCyp2J1 was expressed predominantly in the brain and gonads. A semi-quantitative RT-PCR analysis further revealed that the zfCyp2J1 transcript was also expressed in the ovary, testis, heart, liver, and kidney. High levels of zfCyp2J1 mRNA were evident in primary growth stage (stage I) oocytes and cortical alveolus stage (stage II) oocytes but nearly undetectable in stage III and matured oocytes. These results suggest that zfCyp2J1 may not be involved in zebrafish embryogenesis but may rather play an important role in the functioning of brain and gonads of the adults. In addition, zfCyp2J1 may play a particularly crucial role in early oocyte maturation.

Increased ovarian follicular apoptosis in fathead minnows ( Pimephales promelas) exposed to dietary methylmercury

Exposure to environmentally relevant concentrations of dietary methylmercury impairs the reproduction of fish. Although specific mechanisms are unknown, recent research has linked altered reproduction in fish to the suppression of circulating levels of sex steroid hormones by methylmercury. We hypothesize that methylmercury induces apoptosis in steroidogenic gonadal cells in fish, thereby interfering with the synthesis of sex steroid hormones critical for the regulation of reproduction. To test this hypothesis, we chronically exposed fathead minnows ( Pimephales promelas) to one of three diets contaminated with methylmercury: 0.06 μg Hg g −1 (control), 0.87 μg Hg g −1, and 3.93 μg Hg g −1 dry weight. Apoptosis was evaluated histologically in ovaries of female fathead minnows by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Methylmercury significantly increased the number of apoptotic follicular cells in primary growth and cortical alveolus stage ovarian follicles. Ovarian follicular cells (i.e., granulosa, theca) are responsible for the production of 17β-estradiol and other sex steroid hormones. Increased ovarian follicular apoptosis was related to suppressed 17β-estradiol concentrations and smaller ovary size of female fathead minnows. Our results suggest increased apoptosis of steroidogenic gonadal cells as a possible mechanism for the suppression of sex steroid hormones and ultimately the impairment of reproduction in fish exposed to methylmercury.