Vitellogenin Synthesis Research Articles

Study the impact of gamma irradiation on the vitellogenin gene in Galleria mellonella females by using the comparative CT method

The eggs deposited by oviparous organisms contain large amounts of vitellus, or yolk, which are utilized by the growing embryo. Vitellogenesis is the process of vitellus accumulation involves massive heterosynthetic synthesis of the protein vitellogenin (Vg) and its deposition in the oocyte. Vg levels are indicative for the efficiency of the oogenesis process. This work summarizes data on Vg levels in Galleria mellonella females exposed to various doses of gamma radiation. The results stated that vitellogenin was affected by irradiation. The decreased levels of Vg was dose dependent reaching an undetermined level at 160Gy which is the sterileing dose for G. mellonella female that suppress the egg production by them.

In vitro and in vivo estrogenic activity of BPA, BPF and BPS in zebrafish-specific assays

Bisphenol A (BPA) is a widely used chemical that has been extensively studied as an endocrine-disrupting chemical (EDC). Other bisphenols sharing close structural features with BPA, are increasingly being used as alternatives, increasing the need to assess associated hazards to the endocrine system. In the present study, the estrogenic activity of BPA, bisphenol S (BPS) and bisphenol F (BPF) was assessed by using a combination of zebrafish-specific mechanism-based in vitro and in vivo assays. The three bisphenols were found to efficiently transactivate all zebrafish estrogen receptor (zfER) subtypes in zebrafish hepatic reporter cell lines (ZELH-zfERs). BPA was selective for zfERα while BPS and BPF were slightly more potent on zfERβ subtypes. We further documented the estrogenic effect in vivo by quantifying the expression of brain aromatase using a transgenic cyp19a1b-GFP zebrafish embryo assay. All three bisphenols induced GFP in a concentration-dependent manner. BPS only partially induced brain aromatase at the highest tested concentrations (>30µM) while BPA and BPF strongly induced GFP, in an ER-dependent manner, at 1–10µM. Furthermore, we show that BPF strongly induced vitellogenin synthesis in adult male zebrafish. Overall, this study demonstrates the estrogenic activity of BPA, BPF and BPS in different cell- and tissue-contexts and at different stages of development. Differences between in vitro and in vivo responses are discussed in light of selective ER activation and the fate of the compounds in the models. This study confirms the relevance of combining cellular and whole-organism bioassays in a unique model species for the hazard assessment of candidate EDCs.

Quantitative Adverse Outcome Pathways and Their Application to Predictive Toxicology

A quantitative adverse outcome pathway (qAOP) consists of one or more biologically based, computational models describing key event relationships linking a molecular initiating event (MIE) to an adverse outcome. A qAOP provides quantitative, dose-response, and time-course predictions that can support regulatory decision-making. Herein we describe several facets of qAOPs, including (a) motivation for development, (b) technical considerations, (c) evaluation of confidence, and (d) potential applications. The qAOP used as an illustrative example for these points describes the linkage between inhibition of cytochrome P450 19A aromatase (the MIE) and population-level decreases in the fathead minnow (FHM; Pimephales promelas). The qAOP consists of three linked computational models for the following: (a) the hypothalamic-pitutitary-gonadal axis in female FHMs, where aromatase inhibition decreases the conversion of testosterone to 17β-estradiol (E2), thereby reducing E2-dependent vitellogenin (VTG; egg yolk protein precursor) synthesis, (b) VTG-dependent egg development and spawning (fecundity), and (c) fecundity-dependent population trajectory. While development of the example qAOP was based on experiments with FHMs exposed to the aromatase inhibitor fadrozole, we also show how a toxic equivalence (TEQ) calculation allows use of the qAOP to predict effects of another, untested aromatase inhibitor, iprodione. While qAOP development can be resource-intensive, the quantitative predictions obtained, and TEQ-based application to multiple chemicals, may be sufficient to justify the cost for some applications in regulatory decision-making.

Estrogen-dependent, extrahepatic synthesis of vitellogenin in male vertebrates: A mini-review

In the last years, the hormonal balance is threatened by the interferences of substances with hormone-like action (endocrine disruptor chemicals, EDCs) that may harm animal reproduction. Most EDCs are resistant to environmental degradation and are considered ubiquitous contaminants. EDCs may have synthetic or natural origins. Pesticides used in intensive agriculture contain large amounts of chemicals with estrogenic properties, such as the alkylphenol nonylphenol (NP). Besides, animal feeding operations are important sources of natural estrogen metabolites introduced into the environment through manure application in organic farming. In both cases, EDCs can reach animals, including humans particularly at risk due to their position in the food chain. This is the reason for which it is important to use terrestrial vertebrates as sentinels in soil biomonitoring programmes. Today, the most validated biomarker of estrogenic exposure is the expression in male liver of the vitellogenin (VTG), an estrogen-dependent glycolipophosphoprotein naturally expressed only in the liver of oviparous females during the reproductive season. This report summarizes the data available on the EDC-dependent expression and the synthesis of VTG in male vertebrates, highlighting our latest studies that demonstrate the ability of testis and epididymis of the lacertid Podarcis sicula to synthesize VTG following estrogenic exposure. These findings provide, for the first time, evidence on an extrahepatic expression and synthesis of VTG in a terrestrial vertebrate and lay the groundwork for a new value of the VTG as a biomarker of environmental contamination. In addition, the results open a new scenario on the role of VTG in cells other than oocytes.

Body cavity cells of Parachela during their active life

The body cavity cells (storage cells, storage bodies) of four species of Parachela (hermaphroditic Isohypsibius granulifer granulifer, parthenogenetic Hypsibius dujardini, gonochoristic Xerobiotus pseudohufelandi, gonochoristic Macrobiotus polonicus) were analysed during their active life using light, confocal (laser scanning), and scanning and transmission electron microscopy. The ultrastructure of the storage cells confirmed previous studies suggesting a high level of metabolic activity. Additionally, we revealed the participation of the storage cells of H. dujardini, I. g. granulifer, and M. polonicus in the synthesis of vitellogenins. This did not seem to apply for X. pseudohufelandi. All of the species that were examined in this study accumulated polysaccharides, proteins, and lipids in their body cavity cells, but the amount of these components differed in each species. Isohypsibius g. granulifer accumulated a huge amount of polysaccharides and smaller amounts of lipids and proteins, H. dujardini and M. polonicus primarily accumulated lipids and small amounts of polysaccharides and proteins, whereas X. pseudohufelandi primarily accumulated polysaccharides and lipids, and a small amount of proteins.

Molecular characterization, expression analysis and RNAi knock-down of elongation factor 1α and 1γ from Nilaparvata lugens and its yeast-like symbiont.

In the present paper, four cDNAs encoding the alpha and gamma subunits of elongation factor 1 (EF-1) were cloned and sequenced from Nilaparvata lugens, named NlEF-1α, NlEF-1γ, and its yeast-like symbiont (YLS), named YsEF-1α and YsEF-1γ, respectively. Comparisons with sequences from other species indicated a greater conservation for EF-1α than for EF-1γ. NlEF-1α has two identical copies. The deduced amino acid sequence homology of NlEF-1α and NlEF-1γ is 96 and 64%, respectively, compared with Homalodisca vitripennis and Locusta migratoria. The deduced amino acid sequence homology of YsEF-1α and YsEF-1γ is 96 and 74%, respectively, compared with Metarhizium anisopliae and Ophiocordyceps sinensis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the expression level of NlEF-1α and NlEF-1γ mRNA in hemolymph, ovary, fat body and salivary glands were higher than the midgut and leg tissue. YsEF-1α and YsEF-1γ was highly expressed in fat body. The expression level of NlEF-1α was higher than that of NlEF-1γ. Through RNA interference (RNAi) of the two genes, the mortality of nymph reached 92.2% at the 11th day after treatment and the ovarian development was severely hindered. The RT-qPCR analysis verified the correlation between mortality, sterility and the down-regulation of the target genes. The expression and synthesis of vitellogenin (Vg) protein in insects injected with NlEF-1α and NlEF-1γ double-stranded RNA (dsRNA) was significantly lower than control groups. Attempts to knockdown the YsEF-1 genes in the YLS was unsuccessful. However, the phenotype of N. lugens injected with YsEF-1α dsRNA was the same as that injected with NlEF-1α dsRNA, possibly due to the high similarity (up to 71.9%) in the nucleotide sequences between NlEF-1α and YsEF-1α. We demonstrated that partial silencing of NlEF-1α and NlEF-1γ genes caused lethal and sterility effect on N. lugens. NlEF-1γ shares low identity with that of other insects and therefore it could be a potential target for RNAi-based pest management.

Full-length normalization subtractive hybridization analysis provides new insights into sexual precocity and ovarian development of red swamp crayfish Procambarus clarkii

The red swamp crayfish Procambarus clarkii is an important freshwater economic crayfish in Chinese inland due to its high market value and consumer demand. The sexual precocity in this species has received considerable attention in recent years because more and more individuals matured at small sizes, which devalues the commercial production. In this study, by the full-length normalization subtractive hybridization (FNSH) method we identified 16 differently expressed sequences between precocious and normal grown P. clarkii ovaries. Of the down-regulated sequences in precocious ovaries, one encodes a peptide which shared the highest identity to C1q-like family and contained a typical C1q domain at the C-terminal, thus it was named PcC1q-like. QRT-PCR analysis showed that PcC1q-like was transcribed specifically in ovary, with higher expression level in vitellogenic stages. In situ hybridization exhibited specific expression of PcC1q-like in follicular cells around the vitellogenic oocytes. Knockdown of PcC1q-like by injection of double strand RNA (dsRNA) resulted in disruption of follicular cells, decreased transcripts of vitellogenin (Vg) and vitellogenin receptor (VgR), and reduced progesterone content. These results suggest that PcC1q-like may promote ovarian development by stimulating the synthesis and transportation of vitellogenin into oocytes. These data provide insights into the molecular regulatory mechanisms of sexual precocity and ovarian development in crustaceans. Statement of relevanceThe red swamp crayfish Procambarus clarkii is one of the most important freshwater aquaculture species in Chinese inland. We screened 16 sequences that are potentially involved in sexual precocity of P. clarkii. One candidate was demonstrated to be a novel ovary-specific C1q-like gene of P. clarkii, thus named PcC1q-like. Knockdown of PcC1q-like resulted in disruption of follicular cells, decreased transcripts of Vg and VgR, and reduced progesterone, suggesting its essential role in vitellogenic ovarian development of P. clarkii.

Effects of increasing temperature due to aquatic climate change on the self-fertility and the sexual development of the hermaphrodite fish, Kryptolebias marmoratus.

In order to assess the effects of increasing temperature on the reproductive performance of fish, different thermal conditions (i.e., 25.0, 26.5, 27.5, 28.5, 30.0°C) were used in this study and the self-fertilizing hermaphrodite fish, Kryptolebias marmoratus, was exposed to these different thermal conditions. During an exposure period of 30 to 150days, the gonadosomatic index (GSI), gonadal development, the levels of plasma 17β-estradial (E2) and testosterone (T), hepatic vitellogenin (VTG) mRNA abundance, and the number of self-fertilized eggs were analyzed. This study confirmed that a high water temperature above 27.5°C led to the suppression of self-fertility of hermaphroditic fish from 30days after exposure. The oocyte quality and maturation would be affected by the disruption of hepatic VTG synthesis at a high water temperature of 30°C, which resulted in the reduced the self-fertility in K. marmoratus. Consequently, this study suggests that elevated water temperature due to aquatic climate change prior to sexual maturation and the onset of spawning can lead to the reproductive dysfunction of hermaphroditic K. marmoratus.

Molecular characterization of methoprene-tolerant gene (Met) in the swimming crab Portunus trituberculatus: Its putative role in methyl farnesoate-mediated vitellogenin transcriptional activation

Methoprene-tolerant (Met) belongs to the basic helix–loop–helix (bHLH)–Per–Arnt–Sim (PAS) family of nuclear transcriptional regulators, and is the leading candidate receptor for the insect Juvenile hormone (JH) and the crustacean methyl farnesoate (MF). In the present study, a full-length cDNA of Met was cloned from the swimming crab, Portunus trituberculatus (PtMet). The PtMet amino acid sequence was found to contain domains characteristic of the bHLH-PAS family proteins and to have several conserved amino acid residues specifically responsible for JH or MF binding. In addition to its extensive tissue expression, PtMet mRNA is highly expressed in the female hepatopancreas, the primary site of vitellogenin (Vg) synthesis in P. trituberculatus. PtMet expression was significantly increased in the early-vitellogenic stage of ovarian development, which indicated that likely involvement in vitellogenesis regulation. In vitro, MF induced up-regulation of PtMet and PtVg expression in the hepatopancreas, but not in the ovary. In vivo, MF induced PtMet and PtVg expression in the hepatopancreas,and only PtMet expression was induced in the ovary. Taken together, these results suggest that Met can potentially serve as a MF receptor in crustaceans. In addition, the binding of Met to MF might transcriptionally regulate Vg synthesis in the hepatopancreas.

The hepatopancreas and ovary are the sites of vitellogenin synthesis in female red swamp crayfish (Procambarus clarkii (Girard, 1852)) (Decapoda: Astacoidea: Cambaridae)

Vitellogenin (Vg) is the precursor of vitellin, the major yolk protein. The site of Vg synthesis in crustaceans remains controversial. This study investigated the synthesis site of Vg in female red swamp crayfish Procambarus clarkii (Girard, 1852). Results revealed that the mRNA of Vg was detected in the tissues of hepatopancreas of P. clarkii . The mRNA expression level of Vg increased in the previtellogenic stage and peaked in the vitellogenic stage in the hepatopancreas and ovary; the mRNA expression level of Vg in the hepatopancreas was approximately 20-25-fold of that of the ovary in the same ovarian stage. Eyestalk ablation could upregulate the mRNA expression level of Vg in both organs. Our RNAi experiment verified that the hepatopancreas and ovary are the Vg synthesis sites in female P. clarkii .

Transgenic Expression of the piRNA-Resistant Masculinizer Gene Induces Female-Specific Lethality and Partial Female-to-Male Sex Reversal in the Silkworm, Bombyx mori.

In Bombyx mori (B. mori), Fem piRNA originates from the W chromosome and is responsible for femaleness. The Fem piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masc gene. Masc encodes a novel CCCH type zinc-finger protein and is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. In the present study, several silkworm strains carrying a transgene, which encodes a Fem piRNA-resistant Masc mRNA (Masc-R), were generated. Forced expression of the Masc-R transgene caused female-specific lethality during the larval stages. One of the Masc-R strains weakly expressed Masc-R in various tissues. Females heterozygous for the transgene expressed male-specific isoform of the Bombyx homolog of insulin-like growth factor II mRNA-binding protein (ImpM) and Bmdsx. All examined females showed a lower inducibility of vitellogenin synthesis and exhibited abnormalities in the ovaries. Testis-like tissues were observed in abnormal ovaries and, notably, the tissues contained considerable numbers of sperm bundles. Homozygous expression of the transgene resulted in formation of the male-specific abdominal segment in adult females and caused partial male differentiation in female genitalia. These results strongly suggest that Masc is an important regulatory gene of maleness in B. mori.

Editor's Highlight: Computational Modeling of Plasma Vitellogenin Alterations in Response to Aromatase Inhibition in Fathead Minnows.

In vertebrates, conversion of testosterone into 17β-estradiol (E2) is catalyzed by cytochrome P450 (CYP) 19A aromatase. An important role of E2 in oviparous vertebrates such as fish is stimulation of hepatic synthesis of the glycolipoprotein vitellogenin (VTG), an egg yolk precursor essential to oocyte development and larval survival. In fathead minnows (FHMs) (Pimephales promelas) exposed to the aromatase inhibitor fadrozole, plasma VTG levels do not change in concert with plasma E2 levels. Specifically, while plasma VTG and E2 levels both drop quickly when aromatase is first inhibited, the recovery of plasma VTG upon cessation of aromatase inhibition is substantially delayed relative to the recovery of plasma E2. We modified an existing computational model of the FHM hypothalamic-pituitary-gonadal axis to evaluate alternative hypotheses that might explain this delay. In the first hypothesis, a feedback loop involving active transport of VTG from the blood into the ovary is used. The activity of the transporter is negatively regulated by ovarian VTG. In the second hypothesis, a type 1 coherent feed-forward loop is implemented in the liver. This loop has 2 arms, both requiring E2 activation. The first arm describes direct, canonical E2-driven transcriptional induction of VTG, and the second describes an E2-driven intermediate transcriptional regulator that is also required for VTG synthesis. Both hypotheses accurately described the observed VTG dynamics. This result could be used to guide design of laboratory experiments intended to determine if either of the motifs, or perhaps even both of them, actually do control VTG dynamics in FHMs exposed to aromatase inhibitors.

Hypothalamus-pituitary-gonad axis of rainbow trout (Oncorhynchus mykiss) during early ovarian development and under dense rearing condition

The objective of this study was to determine the hypothalamus-pituitary-gonad (HPG) axis of female rainbow trout (Oncorhynchus mykiss) during early ovarian development and under high rearing density. Trouts were sampled from 240 (ovarian stage II) to 540 (ovarian stage IV) days following hatching (DFH) as control group (Ctrl, 4.6–31.1kg/m3) to determine HPG axis during early ovarian development. Trouts from the same batch of fertilized eggs were reared in two higher densities during 240–540 DFH as stocking density 1 and 2 (SD1, 6.6–40.6kg/m3; SD2, 8.6–49.3kg/m3) to elucidate effects of high density on reproductive parameters. Dopamine, E2 (estradiol), 17α,20β-P (17α,20β-dihydroxy4-pregnen-3-one) and P4 (progesterone) were evaluated by radioimmunoassay or ELISA. mRNA expression of hypothalamic gnrh-1, -2 (gonadotropin-releasing hormone-1, -2), pituitary gonadotropins (fsh/lh, follicle-stimulating hormone/luteinizing hormone) and their cognate receptors (fshr/lhr) in ovaries were examined by qRT-PCR. Our findings demonstrated mRNA expression of hypothalamic sgnrh-1, pituitary fsh and ovarian fshr increased in early ovarian development (360 DFH). Serum 17α,20β-P and pituitary lh mRNA expression first increased when trouts were in ovarian stage III (420 DFH). Ovaries were at different stages when reared in different densities. Long-term high density treatment (over 31.7kg/m3) resulted in decreased hypothalamic sgnrh-1, pituitary fsh, ovarian fshr, serum E2, and increased hypothalamus gnrh-2 and serum dopamine during vitellogenin synthesis, suggesting HPG of rainbow trout might be retarded under dense rearing condition.

Ectopic synthesis of vitellogenin in testis and epididymis of estrogen-treated lizard Podarcis sicula

In oviparous vertebrates, vitellogenin (VTG) is the major yolk precursor synthesized in the liver of sexually mature females during the reproductive period. In males, the VTG gene is silent, but it may be activated by estradiol-17β (E2) or estrogen-like substances. Until now, extra-hepatic expression and synthesis of VTG after estrogen exposure has been reported only for aquatic vertebrates. This study demonstrates the ability of testis and epididymis of the terrestrial oviparous lacertid Podarcis sicula to synthesize VTG following E2 exposure. The results of in situ hybridization and immunohistochemistry analysis show the presence of both VTG mRNA and protein in these districts besides the known induction in the liver. The possible contemporaneous uptake of the E2-induced hepatic VTG by means of the specific vitellogenin receptor has been also evaluated. Finally, histological analysis shows that the E2-treatment during the mating season impairs spermatogenesis.

Endocrine Disruption throughout the Hypothalamus–Pituitary–Gonadal–Liver (HPGL) Axis in Marine Medaka (Oryzias melastigma) Chronically Exposed to the Antifouling and Chemopreventive Agent, 3,3′-Diindolylmethane (DIM)

Despite being proposed as a promising antifouling and chemopreventive agent, the environmental risks of 3,3'-diindolylmethane (DIM) are scarcely investigated. Therefore, this study used adult marine medaka (Oryzias melastigma) as a model organism to examine the toxicological effects and underlying mechanism of DIM throughout the hypothalamus-pituitary-gonadal-liver (HPGL) axis following 28 days of exposure to low DIM concentrations (0 and 8.46 μg/L). The results showed that altered gene transcription in the hypothalamus, pituitary, and gonads contributed to the great imbalance in hormone homeostasis. The lowered estradiol (E2)/testosterone (T) and E2/11-keto-testosterone (11-KT) ratios in female plasma resulted in decreased synthesis and levels of vitellogenin (VTG) and choriogenin in the liver and plasma, and vice versa in males. Subsequently, VTG and choriogenin deficiency blocked the reproductive function of the ovary as indicated by decreased fecundity and offspring viability, whereas in male medaka, DIM mainly targeted the liver and induced severe vacuolization. Proteomic profiling of plasma revealed that the sex-specific susceptibility to DIM could be attributed to the increased detoxification and oxidative defense in males. Overall, this study identified the endocrine disruption and reproductive impairment potency of DIM and first elucidated its mechanisms of action in medaka. The differential responses to DIM (estrogenic activities in the male but antiestrogenic activities in the female) provided sensitive biomarkers characteristic of each sex. Considering the chemical stability and potent endocrine disturbance at low concentration, the application of DIM either as an antifouling or chemopreventive agent should be approached with caution in marine environments.

Zebrafish reproductive toxicity induced by chronic perfluorononanoate exposure

Perfluoroalkyl acids (PFAAs) are a group of anthropogenic compounds that have been widely used in consumer products for over 50 years. One of the most dominant PFAAs is perfluorononanoate (PFNA), a compound detected ubiquitously in aquatic ecosystems. While PFNA is suspected of being an endocrine disruptor, the mechanisms behind PFNA-induced reproductive disorders are poorly understood. The aim of this study was to investigate the reproduction-related effects and possible mechanisms of PFNA on adult zebrafish (Danio rerio) following 180 days of exposure at different concentrations (0.01, 0.1, 1mg/L). PFNA concentration in the gonads of zebrafish was tested by HPLC–MS/MS after chronic exposure to study possible inconsistent accumulation between the genders. The results showed that the accumulation of PFNA in the male gonads was almost one-fold higher than that in the female gonads, indicating a possible higher PFAA gonad burden for male zebrafish. Significant reductions in the male gonadosomatic index (GSI) and female egg production were observed. In addition, the decreased 72h hatching rate displayed an evident dosage effect, indicating that maternal exposure to PFNA might impair offspring developmental success. To investigate how PFNA exposure affects the hypothalamic-pituitary-gonadal-liver axis (HPGL axis), the transcriptional levels of genes were measured by real-time PCR. The disrupted expression of genes, such as ERα, ERβ, FSHR, LHR, StAR, and 17βHSD, indicated the possible interference of PFNA on the HPGL axis function and sex hormone synthesis. Furthermore, testosterone (T) and estradiol (E2) levels in serum and VTG content in the liver were detected to clarify the influences of PFNA on sex hormone levels. Except for the increase in serum estrogen levels, as an estrogen analogue, PFNA also induced the synthesis of biomarker protein vitellogenin (VTG) in the adult male liver. The results of this study indicate that chronic exposure to PFNA can lead to dysfunction in the HPGL axis and sex hormone synthesis and cause adverse effects on fish reproduction.

Ultrastructure and histochemistry of the fat body of Anthonomus grandis (Coleoptera: Curculionidae)

Metabolic demands such as growth and reproduction of insects relate directly to the fat body. This organ is responsible for the biosynthesis and storage of nutrients, and participates in the neutralization and detoxification of xenobiotics. Studies show a relationship between its ability to accumulate nutrients and activities, such as diapause and reproduction in the beetle Anthonomus grandis, an important pest of cotton plants. However, there are no detailed studies on the structure and histochemistry of the fat body in this insect. We describe the ultrastructure and histochemistry of the fat body in A. grandis and discuss some of its characteristic features. The fat body is composed of a single cell type, the trophocyte, which is a voluminous cell with a spherical or bilobed nucleus, and cytoplasm containing several lipid droplets, glycogen granules (electron-dense), and protein granules. These trophocytes have few areas of contact with each other and form lobes, enclosed by connective tissue. A high metabolic activity of the fat body is deduced by the intense histochemical staining for protein and polysaccharide compounds, demonstrating that specific processes, such as the synthesis and secretion of vitellogenin, could be a target for more specific studies on methods to control this insect.

Reproductive aspects of Notothenia rossii and N. coriiceps (Perciformes, Nototheniidae) at Potter Cove, 25 de Mayo (King George) Island during austral summer

Several fish species of the suborder Notothenioidei (Perciformes) predominate in the Antarctic Convergence Zone; nevertheless, reproductive studies are scarce due to difficulties on regular sampling. This study takes the research area of reproductive biology of notothenioids to a new level by providing, for the first time, data on sex hormone and vitellogenin detection in the blood of females of Notothenia rossii and N. coriiceps and correlates this data with morphological maturity indices as well as ovarian histology. Fish were captured during the Antarctic summer at Potter Cove, 25 de Mayo (King George) Island, and blood and ovary were collected. Histological analysis revealed that females of both fish possess group synchronous ovarian development with two distinct clutches of oocytes: a more advanced batch of vitellogenic oocytes ready for spawning and a second batch of previtellogenic oocytes for the next spawning event. Since liver vitellogenin synthesis is stimulated by estradiol produced by the ovaries, gonadal development, estradiol levels, and vitellogenin showed that both species were at a more advanced stage of maturation in March than in January. On the other hand and irrespectively of the month, gonadosomatic index and plasma estradiol levels of N. coriiceps were higher than those of N. rossii. Furthermore, females of N. coriiceps showed an advanced stage of vitellogenesis or were ready for spawn, contrary to results of previous studies. Our results indicate the successful use of gonadal morphology, estradiol, and vitellogenin detection for the estimation of sexual maturity stage of female adults.

Juvenile Hormone Activates the Transcription of Cell-division-cycle 6 (Cdc6) for Polyploidy-dependent Insect Vitellogenesis and Oogenesis

Although juvenile hormone (JH) is known to prevent insect larval metamorphosis and stimulate adult reproduction, the molecular mechanisms of JH action in insect reproduction remain largely unknown. Earlier, we reported that the JH-receptor complex, composed of methoprene-tolerant and steroid receptor co-activator, acts on mini-chromosome maintenance (Mcm) genes Mcm4 and Mcm7 to promote DNA replication and polyploidy for the massive vitellogenin (Vg) synthesis required for egg production in the migratory locust (Guo, W., Wu, Z., Song, J., Jiang, F., Wang, Z., Deng, S., Walker, V. K., and Zhou, S. (2014) PLoS Genet. 10, e1004702). In this study we have investigated the involvement of cell-division-cycle 6 (Cdc6) in JH-dependent vitellogenesis and oogenesis, as Cdc6 is essential for the formation of prereplication complex. We demonstrate here that Cdc6 is expressed in response to JH and methoprene-tolerant, and Cdc6 transcription is directly regulated by the JH-receptor complex. Knockdown of Cdc6 inhibits polyploidization of fat body and follicle cells, resulting in the substantial reduction of Vg expression in the fat body as well as severely impaired oocyte maturation and ovarian growth. Our data indicate the involvement of Cdc6 in JH pathway and a pivotal role of Cdc6 in JH-mediated polyploidization, vitellogenesis, and oogenesis.

Nutritional Signaling Regulates Vitellogenin Synthesis and Egg Development through Juvenile Hormone in Nilaparvata lugens (Stål)

Insect female reproduction which comprises the synthesis of vitellogenein (Vg) in the fat body and its incorporation into developing oocytes, needs a large amount of energy and food resources. Our previous studies found that juvenile hormone (JH) regulates vitellogenesis in the brown planthopper, Nilaparvata lugens. Here, we report on the role of JH in nutrient-regulated Vg synthesis and egg development. We first cloned the genes coding for juvenile hormone acid methyltransferase (JHAMT) which is involved in JH biosynthesis and methoprene-tolerant (Met) for JH action. Amino acids (AAs) induced the expression of jmtN, while showing no effects on the expression of met using an artificial diet culture system. Reduction in JH biosynthesis or its action by RNA interference (RNAi)-mediated silencing of jmtN or met led to a severe inhibition of AAs-induced Vg synthesis and oocyte maturation, together with lower fecundity. Furthermore, exogenous application of JH III partially restored Vg expression levels in jmtN RNAi females. However, JH III application did not rescue Vg synthesis in these met RNAi insects. Our results show that AAs induce Vg synthesis in the fat body and egg development in concert with JH biosynthesis in Nilaparvata lugens (Stål), rather than through JH action.