Warm and cold temperatures have distinct germline stem cell lineage effects during Drosophila oogenesis.

Despite their medical and economic relevance, it remains largely unknown how suboptimal temperatures affect adult insect reproduction. Here, we report an in-depth analysis of how chronic adult exposure to suboptimal temperatures affects oogenesis using the model insect Drosophila melanogaster. In adult females maintained at 18°C (cold) or 29°C (warm), relative to females at the 25°C control temperature, egg production was reduced through distinct cellular mechanisms. Chronic 18°C exposure improved germline stem cell maintenance, survival of early germline cysts and oocyte quality, but reduced follicle growth with no obvious effect on vitellogenesis. By contrast, in females at 29°C, germline stem cell numbers and follicle growth were similar to those at 25°C, while early germline cyst death and degeneration of vitellogenic follicles were markedly increased and oocyte quality plummeted over time. Finally, we also show that these effects are largely independent of diet, male factors or canonical temperature sensors. These findings are relevant not only to cold-blooded organisms, which have limited thermoregulation, but also potentially to warm-blooded organisms, which are susceptible to hypothermia, heatstroke and fever.

Receptor-mediated yolk uptake is required for oskar mRNA localization and cortical anchorage of germ plasm components in the Drosophila oocyte.

The Drosophila germ plasm is responsible for germ cell formation. Its assembly begins with localization of oskar mRNA to the posterior pole of the oocyte. The oskar translation produces 2 isoforms with distinct functions: short Oskar recruits germ plasm components, whereas long Oskar remodels actin to anchor the components to the cortex. The mechanism by which long Oskar anchors them remains elusive. Here, we report that Yolkless, which facilitates uptake of nutrient yolk proteins into the oocyte, is a key cofactor for long Oskar. Loss of Yolkless or depletion of yolk proteins disrupts the microtubule alignment and oskar mRNA localization at the posterior pole of the oocyte, whereas microtubule-dependent localization of bicoid mRNA to the anterior and gurken mRNA to the anterior-dorsal corner remains intact. Furthermore, these mutant oocytes do not properly respond to long Oskar, causing defects in the actin remodeling and germ plasm anchoring. Thus, the yolk uptake is not merely the process for nutrient incorporation, but also crucial for oskar mRNA localization and cortical anchorage of germ plasm components in the oocyte.

Metabolic and Hormonal Changes Associated with Vitellogenesis in Salvator merianae Lizards.

Vitellogenesis in oviparous vertebrates is a critical marker of the restart of seasonal reproductive activity. During this process of multihormonal regulation, females allocate a considerable amount of organic and mineral reserves to the synthesis of yolk, with changes in their plasma values. In this work, we determined plasma levels of various metabolites and steroid hormones throughout the reproductive cycle in females of Salvator merianae who developed vitellogenic and non-vitellogenic follicular cycles. We worked for two consecutive years with 20 adult females from the Experimental Hatchery of the Facultad de Agronomía y Zootecnia of the Universidad Nacional de Tucumán. Values of metabolites: glucose, triglycerides, cholesterol, calcium, phosphorus, albumin, total proteins, and hormones: estradiol, progesterone, and testosterone, were determined during the following stages of the annual cycle: hibernation, hibernation emergence, courtship-mating, oviposition, and incubation. Vitellogenic females showed significantly higher plasma levels of triglycerides, calcium, phosphorus, and albumin than non-vitellogenic females, mainly in the courtship-mating stage (advanced vitellogenesis). In contrast, annual cholesterol averages were lower in vitellogenic females. Glucose showed changes throughout the annual cycle regardless of the vitellogenic condition. Total proteins plasma levels had very few fluctuations during the cycle. Among the hormones studied, only testosterone showed differences related to vitellogenic condition, with higher levels in non-vitellogenic females during the entire reproductive cycle. The knowledge of these changes associated with vitellogenesis will improve zootechnical management and will allow optimizing the reproductive efficiency of Salvator lizards in captivity.

Juvenile hormone upregulates sugarbabe for vitellogenesis and egg development in the migratory locust Locusta migratoria.

Sugarbabe is a C2 H2 zinc-finger transcription factor that is sensitive to sugar and essential for lipid biosynthesis in larvae of Drosophila melanogaster. However, the role of Sugarbabe in adult insect development remains unexplored. Vitellogenesis is a nutrient-dependent process that is promoted by juvenile hormone (JH) in many insect species. Here, we cloned an ortholog gene of D. melanogaster Sugarbabe (DmSug) in the migratory locust Locusta migratoria. The locust Sugarbabe (LmSug) has five C2 H2 zinc-finger motifs similar to DmSug. LmSug was expressed at a low level in adult female locusts raised under poor nutrient conditions. JH treatment increased the expression level of LmSug. Knockdown of the JH receptor gene Met caused a reduction of LmSug expression. Depletion of the LmSug transcript level caused a significant reduction in vitellogenin expression in the fat body, resulting in impaired oocyte development and ovary growth. The results suggest that LmSug is expressed in response to JH, and plays an essential role in female insect reproduction.

An ovary-specific mucin is associated with choriogenesis mediated by prostaglandin signaling in Spodoptera exigua.

Polytrophic ovarioles of Spodoptera exigua, a lepidopteran insect, begins with the development of oocytes and differentiation of nurse cells followed by vitellogenesis and choriogenesis. Compared with previtellogenic and vitellogenic developments, choriogenesis has not been clearly understood yet in endocrine control. This study investigated the expression and function of a mucin-like structural protein of S. exigua called Se-Mucin1 in choriogenesis. It was highly expressed in ovarioles containing chorionated oocytes. The expression level of Se-Mucin1 was increased during adult stage as early as 18 h after adult emergence, reaching the maximal level at 24 h and later. Interestingly, DNA amount of Se-Mucin1 was increased by almost four folds during early adult stage while other genes (hexokinase and glyceraldehyde-3-phosphate dehydrogenase) not directly associated with chorion formation did not show genomic DNA increase, suggesting specific gene amplification of Se-Mucin1. RNA interference (RNAi) suppressed Se-Mucin1 expression by injecting 1 µg of double-strand RNA to teneral females (<5 h after emergence), which exhibited significantly impaired fecundity and egg hatching rate. Eggs laid by RNAi-treated females were malformed in eggshell structures with loss of mesh-like fibers. Treatment with aspirin, a prostaglandin (PG) biosynthesis inhibitor, suppressed the induction of Se-Mucin1 expression during early adult stage and impaired egg development. An addition of PGE2 significantly rescued such impairment in Se-Mucin1 expression and subsequent egg development. These results suggest that PGs mediate choriogenesis of S. exigua by activating the expression of chorion-associated genes including Se-Mucin1.

The RNA helicase DDX3 is required for ovarian development and oocyte maturation in Locusta migratoria.

DDX3 represents a well-defined subfamily of DEAD-box RNA helicase and exerts multiple functions in RNA metabolism, cell cycle, tumorigenesis, signal pathway, and fertility. Our previous study has shown that LmDDX3, the ortholog of DDX3 in Locusta migratoria, is ubiquitously expressed, and with a high abundance in testis and ovary. Knockdown of LmDDX3 results in a lethal phenotype in nymph, but it still remains unclear for its role in reproductive process. In this study, we therefore characterized LmDDX3 expression in female adult locust and analyzed its function in oocyte development. LmDDX3 was expressed in all tissues examined with significant more transcripts in ovary and hindgut. In ovary, a strong expression level was detected at the day just after adult eclosion, and a dramatic reduction then occurred during the oocyte development. LmDDX3 RNAi led to a reduced vitellogenin (Vg) expression in fat body via partially at least, the JH signaling pathway, and caused an upregulation of vitellogenin receptor (VgR) in ovary, and thus blocked the ovarian development and oocyte maturation. Sequence and phylogenetic analysis indicated that LmDDX3 was closely related to termite DDX3. Taken together, these data reveal a critical role for LmDDX3 in regulating the transcription of Vg and VgR, two major factors in vitellogenesis that is a key process required for ovary development and oocyte maturation in locust, and contribute thereof a new putative target for locust biological control.

Juvenile hormone-regulated alternative splicing of the taiman gene primes the ecdysteroid response in adult mosquitoes.

Juvenile hormone (JH) regulates many aspects of insect development and reproduction. In some processes, JH plays a critical role in defining the action of the steroid hormone 20-hydroxyecdysone (20E). In Aedes aegypti mosquitoes, JH prepares newly emerged female adults to become competent to synthesize vitellogenin in response to 20E after blood ingestion. The molecular basis of this competence is still not well understood. Here, we report that JH regulates pre-mRNA splicing of the taiman gene, which encodes a key transcriptional regulator required for both JH- and 20E-controlled gene expression. JH stimulated the production of the Taiman isoforms A/B, while reducing the levels of the isoforms C/D, in the fat body after adult eclosion. The appearance of the A/B isoforms in maturing mosquitoes was accompanied by acquisition of the competence to respond to 20E. Depletion of the A/B isoforms, by inhibiting the alternative splicing or by isoform-specific RNA interference, considerably diminished the 20E-induced gene expression after a blood meal and substantially impaired oocyte development. In accordance with this observation, further studies indicated that in the presence of 20E, the Taiman A/B isoforms showed much stronger interactions with the 20E receptor complex than the Taiman C/D isoforms. In contrast, all four isoforms displayed similar capabilities of forming active JH receptor complexes with the methoprene-tolerant protein (Met). This study suggested that JH confers the competence to newly emerged female mosquitoes by regulating mRNA splicing to generate the Taiman isoforms that are essential for the vitellogenic 20E response.

Blood feeding activates the vitellogenic stage of oogenesis in the mosquito Aedes aegypti through inhibition of glycogen synthase kinase 3 by the insulin and TOR pathways.

Most mosquitoes, including Aedes aegypti, only produce eggs after blood feeding on a vertebrate host. Oogenesis in A. aegypti consists of a pre-vitellogenic stage before blood feeding and a vitellogenic stage after blood feeding. Primary egg chambers remain developmentally arrested during the pre-vitellogenic stage but complete oogenesis to form mature eggs during the vitellogenic stage. In contrast, the signaling factors that maintain primary egg chambers in pre-vitellogenic arrest or that activate vitellogenic growth are largely unclear. Prior studies showed that A. aegypti females release insulin-like peptide 3 (ILP3) and ovary ecdysteroidogenic hormone (OEH) from brain neurosecretory cells after blood feeding. Here, we report that primary egg chambers exit pre-vitellogenic arrest by 8 h post-blood meal as evidenced by proliferation of follicle cells, endoreplication of nurse cells, and formation of cytoophidia. Ex vivo assays showed that ILP3 and OEH stimulate primary egg chambers to exit pre-vitellogenic arrest in the presence of nutrients but not in their absence. Characterization of associated pathways indicated that activation of insulin/insulin growth factor signaling (IIS) by ILP3 or OEH inactivated glycogen synthase kinase 3 (GSK3) via phosphorylation by phosphorylated Akt. GSK3 inactivation correlated with accumulation of the basic helix-loop-helix transcription factor Max and primary egg chambers exiting pre-vitellogenic arrest. Direct inhibition of GSK3 by CHIR-99021 also stimulated Myc/Max accumulation and primary egg chambers exiting pre-vitellogenic arrest. Collectively, our results identify GSK3 as a key factor in regulating the pre- and vitellogenic stages of oogenesis in A. aegypti.

Ovarian development pattern and vitellogenesis of ridgetail white prawn, Exopalaemon carinicauda.

The ridgetail white prawn Exopalaemon carinicauda has the potential to be used as a model organism in crustacean research because it has a transparent body, available draft genome, and short life cycle. However, their ovarian development pattern remains unclear under laboratory culture conditions. This study investigated the changes of ovarian external feature, ovarian histology, gonadosomatic index (GSI), and hepatosomatic index (HSI), as well as the expression and localization of vitellogenin in the ovary and the hepatopancreas during the first ovarian development cycle of E. carinicauda under laboratory-reared condition. The results demonstrated that (1) the first ovarian development cycle of E. carinicauda could be divided into 5 different stages in which the ovary changes its color from white to yellow during the vitellogenesis process in parallel with increasing GSI. (2) After pubertal molt, most females reached ovarian stage II while the females reached stage V after premating molt. (3) During the ovarian development, GSI increased smoothly and HSI relatively stable during the period of stages I to IV, while GSI increased but HSI decreased significantly from stages IV to V. (4) In situ hybridization (ISH) revealed that EcVg was slightly expressed in the oocyte cytoplasm of previtellogenic oocytes. The positive signal was mainly detected in hepatopancreatic fibrillar cells, and a strong signal was found in the hepatopancreas at stage IV. Moreover, the expression level of EcVg-mRNA in the hepatopancreas is stage-specific, and the hepatopancreas contributes majority of vitellin precursor protein to support the ovarian development of E. carinicauda.

Induction of vitellogenesis, methyl farnesoate synthesis and ecdysteroidogenesis in two edible crabs by arachidonic acid and prostaglandins.

The present study investigated the effect of arachidonic acid (AA) and selected prostaglandins on the regulation of vitellogenesis, ecdysteroidogenesis and methyl farnesoate (MF) synthesis in the freshwater crab Oziotelphusa senex senex and the giant mud crab, Scylla serrata Administration of AA and prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) significantly increased ovarian index, oocyte diameter and ovarian vitellogenin levels and ecdysteroid and MF levels in the hemolymph of crabs. Secretions of MF and ecdysteroids from in vitro cultured mandibular organs (MO) and Y-organs (YO) isolated from intermolt crabs injected with AA, PGF2α and PGE2 were greater when compared with controls. In contrast, injection of prostaglandin D2 (PGD2) had no effect on vitellogenesis, ecdysteroid and MF levels in circulation. In vitro secretion of MF from MO explants isolated from avitellogenic crabs incubated with AA, PGF2α and PGE2 increased in a time-dependent manner. Conversely, incubation of YOs isolated from avitellogenic crabs with AA, PGF2α and PGE2 had no effect on secretion of ecdsyteroids. These results implicate prostaglandins in the regulation of reproduction by inducing the synthesis of MF and consequent ecdysteroid synthesis in brachyuran crabs, and provide an alternative molecular intervention mechanism to the traditional eyestalk ablation methodology to induce vitellogenesis and ovarian maturation in crustaceans.

Steroid hormones, energetic state, and immunocompetence vary across reproductive contexts in a parthenogenetic lizard.

Reproduction is energetically expensive and investing in this life history trait is likely accompanied by significant changes in physiological activity. Investment strategy necessary for achieving reproductive success in reptiles can vary with reproductive form and pattern, potentiating different consequences for competing fitness-related traits such as those key to survival. The goal of this study was to assess if and how energetic state (i.e., energy metabolites) and self-maintenance (i.e., immunocompetence) are hormonally modulated across reproductive contexts in an oviparous, parthenogenetic lizard, the Colorado Checkered Whiptail Aspidoscelis neotesselata. Here blood plasma samples were collected from lizards within the US Army Fort Carson Military Installation near Colorado Springs, CO, USA, during seasons of reproductive activity (i.e., June) and inactivity (i.e., August). Measures of reproductive (i.e., estradiol) and energy-mobilizing (i.e., corticosterone) hormones, energy metabolites (i.e., glucose, triglycerides, and free glycerol), and innate immunity (i.e., bactericidal ability) were compared by season and reproductive stage. Levels of energy metabolites and bactericidal ability were compared to levels of E2 and CORT. Bactericidal ability was also compared to levels of energy metabolites. Corticosterone and glucose levels were lower during the reproductive season while triglyceride levels and bactericidal ability were higher, but both estradiol and free glycerol levels did not differ between seasons. Throughout vitellogenesis, corticosterone and glucose levels as well as bactericidal ability did not differ, but estradiol levels were higher during early and mid-stage and both triglyceride and free glycerol levels were lower during gravidity. Corticosterone levels were negatively associated with circulating triglycerides and bactericidal ability, but were not related to glucose nor free glycerol levels. Estradiol levels were positively associated with free glycerol levels and bactericidal ability, but were not related to glucose nor triglyceride levels. Finally, bactericidal ability was negatively associated with glucose, but positively associated with triglycerides. Differences in energetic state and immunocompetence are thus reflected by shifts in hormone secretion across reproductive investment. These findings provide partial support for the hypothesis that energetic state is differentially regulated by steroid hormones to afford reproduction, potentially at the cost of future survival.

Identification and structural characterization of the factors involved in vitellogenesis and its regulation in the African Osteoglossiforme of aquacultural interest Heterotis niloticus (Cuvier, 1829).

The African bonytongue (Heterotis niloticus) is an excellent candidate for fish farming because it has outstanding biological characteristics and zootechnical performances. However, the absence of sexual dimorphism does not favor its reproduction in captivity or the understanding of its reproductive behavior. Moreover, no molecular data related to its reproduction is yet available. This study therefore focuses on the structural identification of the different molecular actors of vitellogenesis expressed in the pituitary gland, the liver and the ovary of H. niloticus. A transcriptomic approach based on de novo RNA sequencing of the pituitary gland, ovary and liver of females in vitellogenesis led to the creation of three transcriptomes. In silico analysis of these transcriptomes identified the sequences of pituitary hormones such as prolactin (PRL), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their ovarian receptors (PRLR, FSHR, LHR). In the liver and ovary, estrogen receptors (ER) beta and gamma, liver vitellogenins (VtgB and VtgC) and their ovarian receptors (VLDLR) were identified. Finally, the partial transcript of an ovarian Vtg weakly expressed compared to hepatic Vtg was identified based on structural criteria. Moreover, a proteomic approach carried out from mucus revealed the presence of one Vtg exclusively in females in vitellogenesis. In this teleost fish that does not exhibit sexual dimorphism, mucus Vtg could be used as a sexing biomarker based on a non-invasive technique compatible with the implementation of experimental protocols in vivo.

Influence of endomorphins along the pituitary-ovary axis in the Mozambique Tilapia Oreochromis mossambicus.

Endomorphins (EM-1 and EM-2) are the tetrapeptides involved in pain and neuroendocrine responses with a high affinity for µ-opioid receptors in vertebrates. However, their role in fish reproduction is not clear. The aim of this study was to investigate the influence of EM-1 and EM-2 on the pituitary-ovary axis in the Mozambique tilapia Oreochromis mossambicus. The experimental set-up consisted of four groups, namely, initial controls, controls, EM-1- and EM-2-treated groups (n = 10 in each group consisting of two replicates). Although the number of stage IV (vitellogenic) follicles was significantly lower (P < 0.05) in controls compared to initial controls, the stage V (preovulatory) follicles were present in controls in contrast to their absence in initial controls. Treatment of 40 µg EM-1/0.1 ml saline/fish/day for 22 days resulted in significant increase (P < 0.05) in the number of stage I follicles compared to controls. While similar treatment of EM-2 did not significantly alter the number of stage I follicles compared to controls, the number of stage II follicles was significantly lower (P < 0.05) in this group compared to those of controls and EM-1 treated fish. The number of stage III and IV follicles did not significantly differ among controls, EM-1- and EM-2-treated groups. However, a significant reduction (P < 0.05) in the mean number of stage V follicles was observed in EM-1- and EM-2-treated fish compared to controls. These changes were concomitant with significant reduction (P < 0.05) in the intensity and the percent area of immunoreactivity of luteinizing hormone (LH) secreting cells in the proximal pars distalis (PPD) of the pituitary gland and significantly higher (P < 0.05) percent occurrence of follicular atresia in EM-1- and EM-2-treated fish compared to those of controls. Taken together, these results suggest an inhibitory effect for endomorphins along the pituitary-ovary axis, for the first time in fish.

Vitellogenins in the spider Parasteatoda tepidariorum - expression profile and putative hormonal regulation of vitellogenesis.

BACKGROUND: Knowledge about vitellogenesis in spiders is rudimentary. Therefore, the aim of study was to check the vitellogenin (Vg) presence in various tissues of the female spider Parasteatoda tepidariorum, determine when and where vitellogenesis starts and takes place, and the putative role of selected hormones in the vitellogenesis. RESULTS: Here we show two genes encoding Vg (PtVg4 and PtVg6) in the genome of the spider P. tepidariorum. One gene PtVg4 and three subunits of Vg (250 kDa, 47 kDa and 30 kDa) are expressed in the midgut glands, ovaries and hemolymph. Heterosynthesis of the Vg in the midgut glands and autosynthesis in the ovaries were observed. Vitellogenesis begins in the last nymphal stage in the midgut glands (heterosynthesis). However, after sexual maturity is reached, Vg is also synthesized in the ovaries (autosynthesis). Changes in the PtVg4 expression level and in the Vg concentration after treatment with 20-hydroxyecdysone, a juvenile hormone analog (fenoxycarb) and an antijuvenoid compound (precocene I) were observed. Therefore, we propose a hypothetical model for the hormonal regulation of vitellogenesis in P. tepidariorum. CONCLUSIONS: Our results are the first comprehensive study on spider vitellogenesis. In our opinion, this work will open discussion on the evolutionary context of possible similarities in the hormonal control of vitellogenesis between P. tepidariorum and other arthropods as well as their consequences.

Insulin signaling mediates previtellogenic development and enhances juvenile hormone-mediated vitellogenesis in a lepidopteran insect, Maruca vitrata.

BACKGROUND: Insulin/insulin-like growth peptide signaling (IIS) down-regulates hemolymph sugar level and facilitates larval growth in the soybean pod borer, Maruca vitrata. The objective of this study is to determine whether IIS of M. vitrata can mediate ovarian development of adult females. RESULTS: A pair of ovaries consists of 8 ovarioles, each of which is separated into distal germarium and proximal vitellarium in M. vitrata. In the germarium, oocyte development occurred with active mitotic activity which was visible by incorporating bromodeoxyribose uridine. Previtellogenic development and subsequent vitellogenesis began soon after adult emergence. They continued with increase of female age. Oocyte development was facilitated by up-regulation of vitellogenin (Vg) and Vg receptor (VgR) gene expression. Larval diets significantly influenced on ovarian development of M. vitrata because oocyte development varied with pupal size derived from larvae treated with different nutritional diets. Its ovarian development was dependent on endocrine signal(s) from the head because decapitation soon after adult emergence prevented oogenesis and subsequent vitellogenesis along with marked reduction of Vg and VgR expression. Topical application of juvenile hormone (JH) significantly recovered its ovarian development whereas farnesoic acid (a precursor of JH biosynthesis) or 20-hydroxyecdysone treatment did not. JH stimulated vitellogenesis and choriogenesis, but not previtellogenic development. In contrast, insulin injection to decapitated females stimulated oocyte differentiation and vitellogenesis along with increase of Vg and VgR expression. To further analyze the effect of insulin on ovarian development, expression of four IIS components (InR, FOXO, Akt, and TOR) genes was manipulated by RNA interference. Hemocoelic injection of gene-specific double stranded RNAs significantly reduced their target gene mRNA levels and interfered with ovarian development. An addition of insulin to JH treatment against decapitated females enhanced the gonadotropic effect of JH by stimulating oogenesis. CONCLUSIONS: IIS plays crucial role in mediating previtellogenic development of M. vitrata in response to nutrient signal. It also enhances the gonadotropic effect of JH II on vitellogenesis.

Juvenile hormone differentially regulates two Grp78 genes encoding protein chaperones required for insect fat body cell homeostasis and vitellogenesis.

Juvenile hormone (JH) has a well known role in stimulating insect vitellogenesis (i.e. yolk deposition) and oocyte maturation, but the molecular mechanisms of JH action in insect reproduction are unclear. The 78-kDa glucose-regulated protein (Grp78) is a heat shock protein 70-kDa family member and one of the most abundant chaperones in the endoplasmic reticulum (ER) where it helps fold newly synthesized peptides. Because of its prominent role in protein folding, and also ER stress, we hypothesized that Grp78 might be involved in fat body cell homeostasis and vitellogenesis and a regulatory target of JH. We report here that the migratory locust Locusta migratoria possesses two Grp78 genes that are differentially regulated by JH. We found that Grp78-1 is regulated by JH through Mcm4/7-dependent DNA replication and polyploidization, whereas Grp78-2 expression is directly activated by the JH-receptor complex comprising methoprene-tolerant and Taiman proteins. Interestingly, Grp78-2 expression in the fat body is about 10-fold higher than that of Grp78-1 Knockdown of either Grp78-1 or Grp78-2 significantly reduced levels of vitellogenin (Vg) protein, accompanied by retarded maturation of oocytes. Depletion of both Grp78-1 and Grp78-2 resulted in ER stress and apoptosis in the fat body and in severely defective Vg synthesis and oocyte maturation. These results indicate a crucial role of Grp78 in JH-dependent vitellogenesis and egg production. The presence and differential regulation of two Grp78 genes in L. migratoria likely help accelerate the production of this chaperone in the fat body to facilitate folding of massively synthesized Vg and other proteins.

Immunolocalization and changes of 17beta-estradiol during ovarian development of Chinese mitten crab Eriocheir sinensis' [corrected].

17beta-estradiol (E2) is important for crustacean ovarian development. This study aims to investigate the distribution and change pattern of E2 in the ovary, hepatopancreas, thoracic ganglion and brain ganglion as well as Vg-mRNA expression level during ovarian development of Chinese mitten crab Eriocheir sinensis. Results showed that strongly positive signals of E2 were mainly distributed in follicle cells of ovaries for all developmental stages as well as oocyte cytoplasm of stages III to V ovaries. In hepatopancreas, the E2-positive signal was mainly detected in the cytoplasm and nucleus of fibrillar cells and the nucleus of resorptive cells, while the maximum fluorescence intensity was observed in stage III hepatopancreas. On the contrary, the E2 immunoreactivities in nervous tissues were relatively stable during ovarian development. Moreover, the changing pattern of E2 concentration was similar within hemolymph, ovary and hepatopancreas during the ovarian development. From stages I to III, the E2 content in three tissues increased significantly, then decreased gradually until stage V. As for the Vg-mRNA expression level in hepatopancreas and ovaries, an increasing trend was found in ovaries but no significant difference was detected during the period of ovarian stages III to V. Hepatopancreatic Vg-mRNA expression level increased significantly during stages I to IV and dramatically decreased at stage V. In conclusion, our study suggests that ovary, hepatopancreas, hemolymph and nervous tissues are the target organs of E2 in E. sinensis and E2 concentrations in different tissues are closely related to vitellogenesis in ovary and hepatopancreas during ovarian development.

Immunohistochemical localization of 3ß-Hydroxysteroid dehydrogenase and progesterone receptors in the ovary and placenta during gestation of the placentotrophic lizard Mabuya sp (Squamata: Scincidae).

In squamates, progesterone (P) plays a key role in the inhibition of uterine mobility during egg retention in oviparous species, and during gestation in viviparous species. The corpus luteum (CL) is the main organ responsible for the production of P; however, in some species, the CL degenerates early and the P needed for gestation maintenance should be produced in other tissues. Mabuya sp (Scincidae) is a viviparous lizard with a prolonged gestation, it produces microlecithal eggs and, consequently, has an obligate placentotrophy related with a highly complex placenta. Its CL degenerates at early stages of gestation and therefore, other sources of P should exist. The aim of this study was to determine and localize by immunohistochemistry the production of P by detection of the enzyme 3ß-Hydroxysteroid dehydrogenase (3ß-HSD) and P receptors (PR) during gestation in the ovary and placenta of Mabuya sp. Positive and negative control sections were used. The ovary of this species localizes 3ß-HSD and PR in the same tissues. The CL of the ovaries of females at early stages of gestation were positive for both molecules, whereas they did not localize from mid gestation to the end of pregnancy. Previtellogenic and vitellogenic follicles labelled for both molecules in the follicular epithelium and thecae. The placenta of Mabuya sp. demonstrated the potential for P production from mid gestation to the end of gestation in the uterine and chorionic tissues. PR were located in the uterine tissues throughout gestation, with a decrease towards its completion. Western blot analysis confirmed the presence of 3ß-HSD mainly in the ovary of early pregnant females and in the placental tissues at mid gestation stages. Therefore, the chorioallantoic placenta of Mabuya sp. has an endocrine function producing the P needed for gestation and replacing the CL from mid gestation to the end of pregnancy.

Evidence for retinoic acid involvement in the regulation of vitellogenesis in the fresh water edible crab, Oziotelphusa senex senex.

The possible involvement of 13-cis retinoic acid (CRA) in the regulation of ovarian development in Oziotelphusa senex senex was investigated. Injection of CRA, into avitellogenic crabs significantly increased ovarian index, oocyte diameter and ovarian vitellogenin levels. Injection of CRA also resulted in a significant increase in the secretory rates of mandibular organs and Y-organs and circulatory levels of the methyl farnesoate and ecdysteroids. Further, administration of CRA into avitellogenic crabs produced higher amounts of Retinoid X Receptor, Ecdysteroid Receptor, E75 and vitellogenin mRNAs in the hepatopancreas. Mandibular organ and Y-organ explants isolated from avitellogenic crabs secreted more of methyl farnesoate and ecdysteroids respectively when incubated with CRA. Taken together, these observations led us to hypothesize that CRA stimulates ecdysteroidogenesis and methyl farnesoate synthesis, up-regulates EcR, RXR and E75 expression in hepatopancreas, which then induces vitellogenin gene expression. Vitellogenin is subsequently taken up from hemolymph by ovaries ensuing in ovarian maturation.

Gonadotropin characterization, localization and expression in the European hake (Merluccius merluccius).

In vertebrates, the regulation of gametogenesis is under the control of gonadotropins (Gth), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh). In fish, the physiological role of Gths is not fully understood, especially in species with asynchronous ovarian development. To elucidate the role of Gths in species with asynchronous ovary, we studied European hake (Merluccius merluccius) during the reproductive season. For this aim, we first cloned and sequenced both hormones. Then, we characterized their amino acid sequence and performed phylogenetic analyses to verify the relationship to their orthologues in other species. In addition, the quantification of gene expression during their natural reproductive season was analyzed in wild-caught female hake. Our results revealed that fshb peaked during the vitellogenic phase, remaining high until spawning. This is in contrast to the situation in species with synchronous ovary. lhb, on the other hand, peaked during maturation as it is also common in species with synchronous ovarian development. Finally, combining double-labeling fluorescent in situ hybridization (FISH) for Gth mRNAs with immunofluorescence for Lh protein, we evidenced the specific expression of fshb and lhb in different cells within the proximal pars distalis (PPD) of the pituitary. In addition to gonadotrope cells specific to expression of either fshb or lhb, some cells showed co-expression of both genes. This suggests either that gonadotropes with co-expression are not yet specified or they could have a plasticity that permits changes from one cell phenotype to another during certain life stages and in turn during different physiological states.