Previtellogenic Ovaries Research Articles

Expression levels of vitellogenin receptor (Vtgr) during ovarian development and association between its single nucleotide polymorphisms (SNPs) and reproduction-related parameters of the giant tiger shrimp Penaeus monodon

Abstract Identification of molecular markers linked to phenotypic parameters of reproduction is important and can be applied to domestication and selective breeding programs of the giant tiger shrimp ( Penaeus monodon ). In this study, the full-length cDNA and expression levels of P. monodon vitellogenin receptor ( PmVtgr ) during ovarian development in wild shrimp and association between its single nucleotide polymorphisms (SNPs) and reproduction-related parameters in domesticated shrimp were examined. PmVtgr was 5990 bp in length composing of an open reading frame (ORF) of 5832 bp deducing to a polypeptide of 1943 amino acids. PmVtgr was only expressed in ovaries but not in other tissues of female broodstock. The expression level of PmVtgr in premature ovaries of juveniles was significantly lower than in those of broodstock ( P PmVtgr mRNA significantly increased in stage IV (mature) relative to stage I (previtellogenic) ovaries ( P PmVtgr was comparably expressed at all stages of ovarian development in eyestalk-ablated shrimp ( P > 0.05). In addition, SNPs in the PmVtgr gene segment of 14-month-old P. monodon ( N = 64) were indirectly examined by PCR-SSCP. Significant relationships between different SSCP patterns of PmVtgr and important reproduction-related phenotypes (i.e. gonadosomatic index, GSI and ovarian weight) of P. monodon were found. Results were consistent when further tested against non-related 19-month-old shrimp ( N = 54, P PmVtgr were cloned and sequenced ( N = 10 for each SSCP pattern). SNP positions 126 (C → T), 226 (T → A), 441 (T → A), 477 (T → A), 499 (A → C), 500 (T → C), 519 (A → G), 540 (A → G), 545 (G → A), 614 (T → C) and 678 (A → C) of PmVtgr were significantly associated with GSI values of examined shrimp ( N = 40).

Functional characterization of recombinant gonad-inhibiting hormone (GIH) and implication of antibody neutralization on induction of ovarian maturation in marine shrimp

Ovarian maturation in crustacean is controlled by an eyestalk neuropeptide called gonad-inhibiting hormone (GIH) that is presumed to inhibit vitellogenin synthesis. In this study, a recombinant protein of GIH of Penaeus monodon (rPem-GIH) was expressed in Pichia pastoris expression system and purified to homogeneity by reversed-phase SPE (C18). The purified rPem-GIH significantly reduced vitellogenin mRNA level in primary tissue culture derived from previtellogenic ovary of P. monodon broodstock by 45.7% compared with the untreated group. This effect was similar to that of partially purified optic lobe extract, and demonstrated that the rPem-GIH possessed gonad-inhibiting activity. A monoclonal antibody specific to Pem-GIH (anti-GIH mAb) was able to neutralize the activity of GIH from the partially purified optic lobe extract to about 58% by in vitro assay in primary tissue culture of the ovary. Peptide mapping revealed that amino acids 16 to 20 (MYNKV) of the mature Pem-GIH may serve as an epitope of anti-GIH mAb. A single injection of anti-GIH mAb into wild previtellogenic P. monodon was able to induce ovarian maturation and spawning at a comparable rate to the eyestalk ablation. Our results demonstrate neutralization effect of anti-GIH mAb on vitellogenesis inhibitory action of GIH, and thus provide a potential alternative to induce ovarian maturation in P. monodon broodstock.

Male Aedes aegypti mosquitoes use JH III transferred during copulation to influence previtellogenic ovary physiology and affect the reproductive output of female mosquitoes

The effect of male accessory gland substances on female reproductive physiology has been previously described as “activating” egg development. However, no mechanism has been described that can explain how male mosquitoes are able to influence egg development in female mosquitoes. To investigate how male mosquitoes are able to influence ovarian physiology and reproductive output we explored three main questions: (1) Do mating and male accessory gland substances affect ovarian physiology and alter markers of oocyte quality during the previtellogenic resting stage? (2) Does the male accessory gland contain JH III and is JH III transferred to the female during copulation? (3) Finally, does the nutritional history of the male affect the amount of JH III transferred to the female and alter reproductive output? By answering these questions it is clear that male mosquitoes are able to alter the female’s resource allocation priorities towards reproduction by transferring JH III during copulation; reducing the rate of previtellogenic resorption and increasing the amount of stored ovarian lipids. These changes improve an individual follicle’s likelihood of development after a blood meal. In addition, males maintained under better nutritional conditions make and transfer more JH III, prevent more follicular resorption and realize higher fecundities than other males. Together these results illustrate one mechanism behind the “activating” effect of mating described as well as the role sugar feeding plays in male mosquitoes.

CDNA cloning and mRNA expression of retinoid-X-receptor in the ovary of the shrimp Metapenaeus ensis

Retinoid-X-receptor (RXR) plays an essential role in the molting process of decapod crustaceans, by forming a heterodimeric complex with the ecdysteroid receptor. However, its role during female reproduction, especially in the process of ovarian maturation, has not been characterized. To get an insight into the molecular events governing the process of ovarian maturation in shrimps, the full-length cDNA of RXR from Metapenaeus ensis was cloned by extension of truncated cDNA by using the RACE technique. The open reading frame of MeRXR encodes a 410 amino acid protein with a deduced molecular weight of 44.8kDa, and putative pI of 6.64, which roughly matched our observation from 2DE gel. Phylogenetic analysis showed that MeRXR has high similarity to RXR of Penaeus chinensis and P. japonicus. RT-PCR revealed that MeRXR was universally expressed in all tissues investigated. The variation in MeRXR mRNA expression pattern during ovarian maturation was further analyzed by real-time PCR. In contrast to the decrease in MeRXR at protein level with ovarian maturation, MeRXR mRNA level was low in pre-vitellogenic and mid-vitellogenic ovaries, and increased significantly from mid-vitellogenic to late-vitellogenic stages. This result suggests that MeRXR transcripts in the mature ovary probably act as maternal messages for regulating early molting events during embryonic development.

Characterization and expression of cell division cycle 2 (Cdc2) mRNA and protein during ovarian development of the giant tiger shrimp Penaeus monodon

The meiotic maturation of oocytes is regulated by the maturation-promoting factor (MPF), a complex of Cdc2 (Cdk1) and Cyclin B. Here, the complete open reading frame (ORF) of Cdc2 in Penaeus monodon was characterized. PmCdc2 were 900bp in length corresponding to a polypeptide of 299 amino acids with the conserved Thr14, Tyr15 and Thr161 residues. Quantitative real-time PCR indicated that the expression level of PmCdc2 in wild intact broodstock was significantly increased in stages II (vitellogenic) and III (early cortical rod) ovaries relative to stage I (previtellogenic) ovaries and peaked in stage IV (mature) ovaries (P<0.05). The expression level of PmCdc2 in stages I–IV ovaries of eyestalk-ablated broodstock was greater than that of the same ovarian developmental stages in intact broodstock (P<0.05). Expression levels of PmCdc2 in ovaries of 18-month-old P. monodon upon 5-HT injection (50μg/g body weight) were significantly increased at 1hour post injection (hpi, P<0.05). Recombinant PmCdc2 protein and its polyclonal antibody were successfully produced. Western blot analysis revealed the expected 34kDa band (PmCdc2) along with a smaller band of 23kDa (ribosomal protein S3) in ovaries of juveniles and various ovarian stages of broodstock. Using phospho-Cdc2 (Thr161) polyclonal antibody, the positive signal of 34kDa was observed in all ovarian stages but the most intense signal was found in stage IV ovaries. Results in the present study indicated that PmCdc2 gene/protein plays an important role in the development and maturation of oocytes/ovaries in P. monodon.

Isolation of cDNA, genomic organization and expression of small androgen receptor-interacting protein 1 (PmSARIP1) in the giant tiger shrimp Penaeus monodon

The full-length cDNA and genomic organization of small androgen receptor-interacting protein 1 (SARIP1; also called RWD-containing protein 1, Rwdd1) in the giant tiger shrimp (Penaeus monodon) was isolated and characterized. PmSARIP1 was 1366bp in length containing an open reading frame (ORF) of 738bp corresponding to a polypeptide of 245 amino acids. Its genomic sequence contained 5 exons, 4 introns and untranslated regions (UTRs) spanning 5937bp in length. Tissue distribution analysis indicated that PmSARIP1 was specifically expressed in gonads (ovaries>testes) but not in other tissues of wild P. monodon adults. The expression level of PmSARIP1 was not differentially expressed during ovarian maturation in intact wild adults (P>0.05). Eyestalk ablation resulted in up-regulation of PmSARIP1 throughout the ovarian maturation of wild adults where the peak level was observed at stage I (previtellogenic) ovaries (P<0.05). PmSARIP1 mRNA was clearly localized in ooplasm of previtellogenic oocytes. Serotonin injection (5-HT, 50μg/g body weight; 18-month-old shrimp) promoted the expression level of ovarian PmSARIP1 at 6–72h post injection (hpi) with the peak level at 12hpi (P<0.05). Exogenous progesterone administration (0.1μg/g BW; 14-month-old shrimp) did not significantly affect the expression level of ovarian PmSARIP1. In contrast, 17β-estradiol treatment (0.01μg/g BW) resulted in an increase of PmSARIP1 in 14-month-old shrimp at 7days post injection (dpi). Results from this study suggested that PmSARIP1 should play an important role during ovarian maturation of P. monodon.

Molecular cloning and partial characterization of an ovarian receptor with seven ligand binding repeats, an orthologue of low-density lipoprotein receptor, in the cutthroat trout (Oncorhynchus clarki)

Teleost fish eggs contain a substantial yolk mass consisting of lipids and proteins that provides essential nutrients for embryonic and larval development. The polar lipid and protein components of the yolk are delivered to oocytes by circulating vitellogenins, however the source(s) of the neutral lipid remains unknown. We cloned a cDNA encoding an orthologue of low-density-lipoprotein receptor (LDLR) from the ovary of cutthroat trout, Oncorhynchus clarki (ct-Ldlr). Predominant expression of ct-ldlr mRNA was observed in the ovary and moderate expression was detected in intestine, gill and brain. The relative abundance of ct-ldlr transcripts was highest in early pre-vitellogenic ovaries and significantly decreased during vitellogenesis, followed by a slight increase during final maturation and in post-ovulatory follicles. In situ hybridization revealed an intense and evenly distributed localization of ct-ldlr transcripts in the ooplasm of pre-vitellogenic oocytes and these signals disappeared in vitellogenic follicles. Collectively, these results suggest that the Ldlr is involved in deposition of yolk lipids in cutthroat trout oocytes. The ct-ldlr transcripts also were detected in theca and granulosa cells, suggesting that this receptor may be involved in cholesterol uptake for ovarian steroidogenesis. This is the first report on partial characterization of an ldlr orthologue in any fish species.

Proteomic identification of proteins relevant to ovarian development in the red swamp crayfish Procambarus clarkii

In this study, the two-dimensional gel electrophoresis patterns of total proteins from previtellogenic (stage III) and vitellogenic (stage IV) ovaries of Procambarus clarkii were compared for the first time. A total of 52 significantly differentially expressed protein spots (up/down-regulated≥2 fold) were excised for further analysis using matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry. There were 29 spots successfully identified, including 22 unique proteins. Compared with stage III ovaries, 13 proteins in the stage IV ovaries were down-regulated (e.g., tropomysin, 70kD heat shock protein, protein disulfide-isomerase A6, glutathione S transferase D1, arginine kinase, and Na+/K+-ATPase) and 9 were up-regulated (e.g., peritrophin, cyclin B, elongation factor 2, transketolase, checkpoint kinase 1, and I-connectin). The results indicated the potential of cellular proteomic studies for the identification of proteins functionally involved in the ovarian development of P. clarkii.

The fate of follicles after a blood meal is dependent on previtellogenic nutrition and juvenile hormone in Aedes aegypti

Juvenile hormone (JH) mediates the relationship between fecundity and nutrition during the gonotrophic cycle of the mosquito in three ways: (1) by regulating initial previtellogenic development, (2) by mediating previtellogenic resorption of follicles and (3) by altering intrinsic previtellogenic follicle “quality”, physiology, and competitiveness thereby predetermining the fate of follicles after a blood meal. To support a role for JH in mediating the response of ovarian follicles after a blood meal, we explored three main questions: (1) Do changes in nutrition during the previtellogenic resting stage lead to relevant biochemical and molecular changes in the previtellogenic ovary? (2) Do hormonal manipulations during the previtellogenic resting stage lead to the same biochemical and molecular changes? (3) Does nutrition and hormones during the previtellogenic resting stage affect vitellogenic resorption and reproductive output? We examined the accumulation of neutral lipids in the previtellogenic ovary as well as the previtellogenic expression of genes integral to endocytosis and oocyte development such as the: vitellogenin receptor (AaVgR), lipophorin receptor (AaLpRov), heavy-chain clathrin (AaCHC), and ribosomal protein L32 (rpL32) under various previtellogenic nutritional and hormonal conditions. mRNA abundance and neutral lipid content increased within the previtellogenic ovary as previtellogenic mosquitoes were offered increasing sucrose concentrations. Methoprene application mimicked the effect of offering the highest sucrose concentrations on mRNA abundance and lipid accumulation in the previtellogenic ovary. These same nutritional and hormonal manipulations altered the extent of vitellogenic resorption. Mosquitoes offered 20% sucrose during the previtellogenic resting stage had nearly 3 times less vitellogenic resorption than mosquitoes offered 3% sucrose despite taking smaller blood meals and developed ∼10% more eggs during the first gonotrophic cycle. Mosquitoes treated with JH III during the previtellogenic resting stage and then offered a blood meal had a ∼40% reduction in the amount of vitellogenic resorption and developed ∼12% more eggs. Taken together, these results suggest that previtellogenic nutrition alters the extent and pattern of resorption after a blood meal through the effect of JH on mRNA abundance and lipid accumulation in previtellogenic follicles.

Androgen-specific regulation of FSH signalling in the previtellogenic ovary and pituitary of the New Zealand shortfinned eel, Anguilla australis

The evidence for androgens having a pivotal role in the functioning of the female reproductive axis – such as initiating puberty or vitellogenesis – is mounting. However, the use of aromatizable androgens and the tissue-specific focus of most studies often make it unclear if androgenic effects throughout the axis proceed via androgen or estrogen signalling mechanisms. In this study, we assessed the effects of 11-ketotestosterone (11KT, a non-aromatizable androgen) on the pituitary and ovary of previtellogenic (PV) freshwater eels Anguilla australis, comparing them with eels naturally undergoing early vitellogenesis (EV). We found that 11KT treatment produces molecular and morpho-physiological phenotypes that were generally intermediate between PV and EV. Most notably, we demonstrated that 11KT induces effects on follicle-stimulating hormone (FSH) signalling in the pituitary and ovaries that are in opposition to each other. Thus, 11KT significantly reduced fshβ subunit expression in the pituitary. At the same time, 11KT dramatically increased mRNA levels of ovarian FSH receptor and plasma levels of estradiol-17β, very likely sensitizing the previtellogenic follicle to the FSH signal. Androgens therefore may be important in facilitating puberty in the eel.

Molecular characterization and expression of the Prostaglandin reductase 1 gene and protein during ovarian development of the giant tiger shrimp Penaeus monodon

Prostaglandin reductase 1 (PTGR1; also called NADP +-dependent leukotriene B4 12-hydroxydehydrogenase, LTB4DH) is the key enzyme responsible for biological inactivation of prostaglandins and related eicosanoids. In this study, the full-length cDNA of PTGR1 in the giant tiger shrimp ( Penaeus monodon) was characterized. PmPTGR1 was 2405 bp in length with an ORF of 1035 bp encoding a polypeptide of 344 amino acids. Interestingly, its 3′ UTR contained the nucleotide sequence (825 bp) that significantly matched positions 3–277 (with 4 amino acid variants) of the deduced P. monodon peritrophin2 protein. PmPTGR1 was more preferentially expressed in ovaries than testes of P. monodon broodstock. In intact broodstock, PmPTGR1 was up-regulated in early cortical rod (stage III) ovaries ( P < 0.05) and comparably expressed afterwards ( P > 0.05). In eyestalk-ablated broodstock, PmPTGR1 was temporally lower in early cortical rod compared to previtellogenic (I) and vitellogenic (II) ovaries ( P < 0.05) and returned to the previous level in mature (IV) ovaries ( P < 0.05). More importantly, the relative expression level of PmPTGR1 in each ovarian stage in eyestalk-ablated females was lower than that in intact P. monodon broodstock ( P < 0.05). This strongly suggested that eyestalk ablation potentially affects the expression of PmPTGR1 allowing the stimulating effects of prostaglandins and related eicosanoids on vitellogenesis and ovarian maturation of P. monodon. The level of ovarian PmPTGR1 protein seemed to increase during ovarian development in intact broodstock but slightly reduced in mature ovaries in eyestalk-ablated broodstock. Results suggested the possible contribution of PmPTGR1 in ovarian development of P. monodon.

NUTRITIONAL STRESS DURING EARLY SECONDARY OOCYTE GROWTH INDUCES FOLLICULAR ATRESIA AND CHANGES IN OVARIAN GENE EXPRESSION IN COHO SALMON

Background: Environmental stressors (e.g., reduced food availability, endocrine disrupting chemicals) are known to disrupt endocrine function in fishes, potentially delaying the age of puberty and affecting gamete quality. Recent experiments in our lab have revealed that prolonged fasting of previtellogenic coho salmon (Oncorhynchus kisutch ) impairs endocrine function and increases follicular atresia [1]. However, little is known about the molecular processes in the ovary that either regulate atresia or are consequences of atresia. We hypothesized that a nutritional stress that perturbs normal ovarian growth and induces follicular atresia would alter the pattern of gene expression in the gonads. Indeed, in a recent fasting experiment we found that expression of some steroidogenesis-related genes (e.g., steroidogenic acute regulatory protein ) decreased in fasted salmon, while expression of apoptosis-related genes (e.g., caspase 3 , caspase 8 and fas-associated death domain ) were elevated in fasted fish relative to fed fish [1]. The primary aim of this study was to identify additional gene transcripts that are up- or downregulated in the previtellogenic salmon ovary when ovarian growth is impaired by prolonged fasting. A second goal of the study was to identify candidate genes that might serve as molecular markers of the initiation of atresia. Methods:

Molecular cloning and expression analysis of the Mitogen‐activating protein kinase 1 (MAPK1) gene and protein during ovarian development of the giant tiger shrimp Penaeus monodon

Isolation and characterization of genes and/or proteins differentially expressed in ovaries are necessary for understanding ovarian development in the giant tiger shrimp (Penaeus monodon). In this study, the full-length cDNA of P. monodon mitogen-activating protein kinase 1 (PmMAPK1) was characterized. PmMAPK1 was 1,398 bp in length containing an open reading frame of 1,098 bp that corresponded to a polypeptide of 365 amino acids. PmMAPK1 was more abundantly expressed in ovaries than in testes of P. monodon. Quantitative real-time PCR revealed differential expression levels of PmMAPK1 mRNA during ovarian development of intact broodstock, where it peaked in early cortical rod (stage III) ovaries (P < 0.05) and slightly decreased afterwards (P > 0.05). Likewise, the expression level of PmMAPK1 in early cortical rod and mature (IV) ovaries was significantly greater than that in previtellogenic (I) and vitellogenic (II) ovaries of eyestalk-ablated broodstock (P < 0.05). The PmMAPK1 transcript was localized in ooplasm of previtellogenic oocytes. In intact broodstock, the expression of the PmMAPK1 protein was clearly increased from previtellogenic ovaries in subsequent stages of ovarian development (P < 0.05). In contrast, the level of ovarian PmMAPK1 protein was comparable during oogenesis in eyestalk-ablated broodstock (P > 0.05). The PmMAPK1 protein was localized in ooplasm of previtellogenic and vitellogenic oocytes. It was also detected around the nuclear membrane of early cortical rod oocytes in both intact and eyestalk-ablated broodstock. Results indicated that PmMAPK1 gene products seem to play functional roles in the development and maturation of oocytes/ovaries in P. monodon.

Identification, characterization and expression of adipose differentiation-related protein ( ADRP) gene and protein in ovaries of the giant tiger shrimp Penaeus monodon

In this study, the full length cDNA of adipose differentiation-related protein, a gene involved in neutral lipid accumulation that is important in oocyte maturation, of the giant tiger shrimp, Penaeus monodon ( PmADRP) was identified and characterized. PmADRP was 1292 bp in length containing an ORF of 1107 bp corresponding to a polypeptide of 368 amino acids. PmADRP was abundantly expressed in ovaries but not in testes of P. monodon juveniles and broodstock or several tissues (e.g. intestine, eyestalk and gill) of P. monodon broodstock. The expression levels of PmADRP in ovaries of juvenile P. monodon were significantly induced following 5-HT administration at 48 h post treatment ( P < 0.05). Quantitative real-time PCR illustrated that the PmADRP mRNA in ovaries of juveniles was lower than that of broodstock ( P < 0.05). In intact broodstock, PmADRP was significantly up-regulated at early cortical rod (stage III) ovaries and after spawning ( P < 0.05). Likewise, the expression level of PmADRP in early cortical rod and mature (IV) ovaries was significantly greater than that in previtellogenic (I) and vitellogenic (II) ovaries of eyestalk-ablated broodstock ( P < 0.05). In situ hybridization indicated that PmADRP was localized in ooplasm of previtellogenic oocytes. The PmADRP protein was detected since the vitellogenic stage for intact broodstock and since the previtellogenic stage for eyestalk-ablated broodstock. Results suggested functionally important roles of PmADRP on oocyte and ovarian development of P. monodon.

Disruption of the Salmon Reproductive Endocrine Axis Through Prolonged Nutritional Stress: Changes in Circulating Hormone Levels and Identification of Differentially Expressed Ovarian Genes.

Environmental stressors (e.g., reduced food availability, hypoxia, endocrine disrupting chemicals) are known to disrupt endocrine function of fish, potentially delaying the age of puberty and affecting gamete quality. Assessing the reproductive health of fish in early stages of gametogenesis using histological and hormonal indices provides useful information, but has limited prognostic value for future reproductive potential. Our long-term aim is to develop tools for monitoring environmental impacts on ovarian growth in fish using large-scale ovarian gene expression analyses. However, little is known about the regulation of gonadal gene expression during previtellogenic oocyte growth in either healthy growing fish or fish exposed to environmental conditions that have been shown to disrupt the reproductive endocrine system and inhibit gonadal growth. The goal of this study was to identify genes that are specifically up- or down-regulated in the previtellogenic ovary of coho salmon when ovarian growth is impaired by prolonged fasting. Two year-old fish were individually tagged and fasted over a 17-week period, which resulted in less than 20% loss of body weight. Prolonged fasting caused regression of the ovaries and disrupted the reproductive endocrine axis, as fasted salmon showed significantly lower gonad weights, oocyte diameters, and plasma estradiol-17β (E2) levels relative to fed animals by week 14. Further, a higher proportion of atretic oocytes was observed in ovaries of fasting fish at week 14 and numbers increased by week 17. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries using ovaries from fed and fasted animals at week 14 were generated. A total of 480 clones were sequenced per library and their putative identity determined by comparing sequences to the NCBI and fish EST databases. The library representing genes up-regulated in the fed ovary included steroidogenesis related genes, such as steroidogenic acute regulatory protein (star), 3β-hydroxysteroid dehydrogenase (hsd3b), P450-aromatase (cyp19a1a), and TGF-β superfamily members, such as anti-Mullerian hormone (amh) and inhibin (inha). The library representing genes up-regulated in the fasted ovary included genes that have been associated with apoptosis, such as programmed cell death protein 4 (pdcd4), lipopolysaccharide-induced TNF factor (litaf), cullin 3 (cul3), and kruppel-like factor 6 (klf6). The genes described above were examined with quantitative PCR and results confirmed that the SSH libraries were enriched in transcripts differentially expressed between ovaries of fed and fasted salmon. This study has revealed ovarian genes that are important to normal ovarian development and those that are affected by nutritional stress and may relate to apoptosis/atresia within the ovary. Ultimately, this and subsequent studies should provide new tools that will enable molecular based assessment of fish reproductive health. This research was funded by Washington Sea Grant project number R/B-49. (poster)

Control of ovarian steroidogenesis in insects: A locust neurohormone is active in vitro on blowfly ovaries

Ovarian steroidogenesis controlling insect reproduction is mainly regulated by brain gonadotropins liberated from corpora cardiaca (CC). Till now, different neurohormones have been identified in two insect groups only, locusts and mosquitoes, and it is unknown whether they could be active in other insects. In order to complete previous observations on the control of ovarian steroidogenesis in the blowfly, Phormia regina, we examined whether neuropeptides isolated from locust CC have an effect in vitro on ovarian steroidogenesis in our dipteran model. Our experiments showed that crude extracts from locust CC efficiently stimulated steroidogenesis in blowfly isolated previtellogenic ovaries. However, such an activity was observed neither with authenticated neuroparsins (NPs), the putative homologs of the ovarian ecdysteroidogenic hormone of mosquitoes, nor with ovarian maturing peptide (OMP), the putative locust steroidogenic neurohormone. Partial purifications of CC extracts were then performed using methanol and/or acidic ethanol extractions followed by reverse phase HPLC and collected fractions were assayed in vitro. A significant steroidogenic activity was found in a single group of acidic fractions, well separated from OMP and NPs, which was associated to slight but significant anti-insulin immunoreactivity. In conclusion, a locust CC neurohormone, different from NPs and OMP, is able to stimulate ecdysteroidogenesis in blowfly ovaries. Though this active factor has not been fully characterized, its behavior during extraction or HPLC and its immunoreactivity strongly suggest it could be an insulin-like peptide. This is in agreement with previous studies demonstrating the role of such peptides as steroidogenic gonadotropins in blowflies and several other insects.

Effects of Wolbachia Infection and ovarian tumor Mutations on Sex-lethal Germline Functioning in Drosophila

Wolbachia is a ubiquitous intracellular endosymbiont of invertebrates. Surprisingly, infection of Drosophila melanogaster by this maternally inherited bacterium restores fertility to females carrying ovarian tumor (cystocyte overproliferation) mutant alleles of the Drosophila master sex-determination gene, Sex-lethal (Sxl). We scanned the Drosophila genome for effects of infection on transcript levels in wild-type previtellogenic ovaries that might be relevant to this suppression of female-sterile Sxl mutants by Wolbachia. Yolk protein gene transcript levels were most affected, being reduced by infection, but no genes showed significantly more than a twofold difference. The yolk gene effect likely signals a small, infection-induced delay in egg chamber maturation unrelated to suppression. In a genetic study of the Wolbachia-Sxl interaction, we established that germline Sxl controls meiotic recombination as well as cystocyte proliferation, but Wolbachia only influences the cystocyte function. In contrast, we found that mutations in ovarian tumor (otu) interfere with both Sxl germline functions. We were led to otu through characterization of a spontaneous dominant suppressor of the Wolbachia-Sxl interaction, which proved to be an otu mutation. Clearly Sxl and otu work together in the female germline. These studies of meiosis in Sxl mutant females revealed that X chromosome recombination is considerably more sensitive than autosomal recombination to reduced Sxl activity.

Tracing the accumulation and effects of mercury uptake in the previtellogenic ovary of crucian carp, Carassius auratus gibelio by autometallography and caspase-3 immunohistochemistry

The aims of the present study were to apply the AMG technique for localization of mercury at the light and electron microscopic level in the ovary of crucian carp after exposure to mercuric chloride and to find out if this heavy metal induces expression of caspase-3. Depending on the stage of ovarian follicle development, two patterns of mercury accumulation have been found in previtellogenic ovary of crucian carp. The first mercury accumulation pattern has been found in the early previtellogenic oocyte without zona radiata. In these oocytes, mercury accumulates into an ooplasmic region that seems to correspond to the Balbiani body (32-65 microm oocyte diameter), throughout the cytoplasm (84-116 microm oocyte diameter) and in the cortical cytoplasm (approximately 180 microm oocyte diameter). The second mercury accumulation pattern has been found in the late previtellogenic oocyte with cortical alveoli (229-330 microm oocyte diameter). Ultrastructural observations have shown grains of silver-enhanced mercury inside coated vesicles, the cortical lysosome-like bodies or multivesicular bodies and cortical alveoli. Immunohistochemistry reaction for caspase-3 was positive in nuclei of the early previtellogenic oocyte and Balbiani body.

Androgenic Modulation of Early Growth of Atlantic Cod (Gadus morhua L.) Previtellogenic Oocytes and Zona Radiata-Related Genes

Available evidence suggests that androgens play critical roles in early oocyte growth and development in fish. However, the molecular mechanisms underlying this important aspect of reproductive endocrinology have not yet been established. In this study the effects of androgens (11-ketotestosterone [11-KT] and testosterone [T]) were determined on gene expression patterns and growth of cod previtellogenic oocytes, using an in vitro oocyte culture technique. Previtellogenic ovarian tissue was cultured for 5 and 10 d at different concentrations of 11-KT and T (0, 1, or 1000 µM) dissolved in ethanol (0.3%). The androgen concentrations were selected as they represent physiological and supra-physiological concentrations, respectively. Quantitative polymerase chain reaction (PCR) demonstrated increased mRNA expression for five genes recently identified as androgen responsive in our subtracted cDNA library in previtellogenic cod ovary exposed in vitro to androgens. Quantitative histological analyses showed a consistent stereological validation of oocyte growth and development after exposure to androgens. In general, both 11-KT and T induced previtellogenic oocyte growth and development, and these effects were more pronounced with 11-KT exposure. Taken together, our study reveals some novel roles of androgens on the development of previtellogenic oocytes, indicating control of early follicular and oocyte growth in cod ovary. The potent effects of 11-KT on oocyte growth support our earlier hypothesis that non-aromatizable androgens play significant roles in regulating early oocyte growth with potential consequences for the fecundity process. Therefore, these novel roles of androgens as promoters of ovarian growth and development presented in this study may be useful for the aquaculture industry and for breeding of new captive and endangered species. From a toxicological point of view, the cod is a marine species and exposure to complex chemical mixtures that may exert androgenic and/or anti-androgenic effects represents an environmental issue of reasonable concern in the marine environment. Therefore, the findings in the present study represent a novel basis that can be used to determine the effects of xenoandrogens on oocyte development and fecundity in this important marine species. This study was financed by the Norwegian Research Council (NFR), contract 165073/V40.

Previtellogenic oocyte growth and transcriptional changes of steroidogenic enzyme genes in immature female Atlantic cod ( Gadus morhua L.) after exposure to the androgens 11-ketotestosterone and testosterone

We have studied in vivo, the effects of physiological androgen (11-ketotestosterone: 11-KT and testosterone: T) concentrations on the growth of cod previtellogenic oocytes and steroidogenic gene expression patterns. Immature female Atlantic cod were injected three times (days 0, 7 and 14) with 0.05, 0.5 and 5 mg/kg of 11-KT and T. The control group was injected with the carrier solvent (ethanol diluted 1:10 in sunflower oil). Quantitative histological analyses demonstrated growth and development of previtellogenic oocytes after exposure to androgens. The oocyte developmental effect of androgens was more pronounced in fish receiving 11-KT. Quantitative PCR analysis demonstrated dose- and androgen-specific modulation of mRNA expression for genes involved in steroidogenesis (StAR (steroidogenic acute regulatory) protein, P450 scc (P450-mediated cholesterol side-chain cleavage), 20β-HSD (20β-hydroxysteroid dehydrogenase)) and cell growth control, namely – opioid growth factor receptor (OGF-R), progesterone receptor protein p23 (PR23P) and apoptosis-inducing TAF9-like domain 1 (TAF9). Messenger RNA species associated with the zona pelucida, namely – the zona pellucida protein A domain (ZPA) and egg envelope glycoprotein (EeG) were modulated based on dose and androgen type. Cyclin-B mRNA expression was not affected by androgen exposure. Interestingly, we showed recently that these transcripts were responsive to in vitro androgen exposure in previtellogenic cod ovary. In conclusion, the present study adds further information regarding the effects of androgens on the development of previtellogenic oocytes, suggesting androgen control of early oocyte growth in cod. The enhanced effects of 11-KT on oocyte growth support our hypothesis that non-aromatizable androgens play significant roles in the regulation of early previtellogenic oocyte growth and development.