Evolutionarily conserved ovarian fluid proteins are responsible for extending egg viability in salmonid fish.

In contrast to most fishes, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 proteins overabundant in salmonids and present in analytical fractions with high egg viability preservation potential. The identity of these proteins illuminates the biological processes participating in egg viability preservation. Among identified proteins of interest, the ovarian-specific expression and abundance in CF at ovulation of N-acetylneuraminic acid synthase a (Nansa) suggest a previously unsuspected role. We show that salmonid CF is a complex biological fluid containing a diversity of proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway that are collectively responsible for preserving egg viability.

Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells.

Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.

Effects of the anti-androgen cyproterone acetate (CPA) on oocyte meiotic maturation in rainbow trout (Oncorhynchus mykiss).

In the present study, we aimed at characterizing the effect of cyproterone acetate (CPA), an anti-androgenic compound, on oocyte meiotic maturation in a freshwater teleost fish species, the rainbow trout (Oncorhynchus mykiss). Fully-grown post-vitellogenic ovarian follicles were incubated in vitro with CPA, luteinizing hormone (Lh) or a combination of CPA and Lh. Incubations were also performed using a combination of Lh and testosterone (T). The occurrence of oocyte maturation (i.e., resumption of the meiotic process) was assessed by monitoring germinal vesicle breakdown (GVBD) after a 72h in vitro incubation. The effect of CPA on the production of 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP), the natural maturation-inducing steroid (MIS), was quantified by radioimmunoassay. Our results show that CPA dramatically inhibits Lh-induced oocyte maturation and MIS synthesis. We also observed a synergistic effect of Lh and T on oocyte maturation in highly competent oocytes (i.e., able to resume meiosis after stimulation by low doses of Lh). Our results also show that a combination of CPA and Lh inhibits phosphorylation of extracellular signal-regulated kinase (Erk), kinases that are associated with oocyte maturation in many species. As a whole, our results indicate that CPA has a potential to alter meiotic maturation in rainbow trout. Further analyses are, however, needed to determine the mechanisms by which this anti-androgen interferes with the meiotic process. Furthermore, the present study provides a framework for better understanding of the ecological consequences of exposure to anti-androgens and resulting meiotic maturation abnormalities observed in trout.

Prochloraz-induced oocyte maturation in rainbow trout (Oncorhynchus mykiss), a molecular and functional analysis.

In the present study, we aimed at characterizing the effect of prochloraz, an imidazole fungicide, on the oocyte meiotic maturation process in a freshwater teleost species, the rainbow trout (Oncorhynchus mykiss). Full-grown post-vitellogenic ovarian follicles were incubated in vitro with prochloraz, Luteinizing Hormone (LH), or a combination of prochloraz and LH. The occurrence of oocyte maturation was assessed by monitoring germinal vesicle breakdown (GVBD) after 62-h in vitro incubation. Experiments were repeated in presence of actinomycin D, cycloheximide, or trilostane. The effect of prochloraz on the production of 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP), the natural maturation-inducing steroid, was quantified by radioimmunoassay. In addition, the effect of prochloraz on ovarian expression of 12 genes was monitored by real-time PCR. Prochloraz (10(-5)M) administered alone was able to induce 100% GVBD in the most responsive females. The occurrence of GVBD observed after prochloraz stimulation of follicles originating from various females was similar and highly correlated with the occurrence of GVBD observed after stimulation with low LH concentration. In addition, oocyte maturation induced by LH or prochloraz was totally inhibited by actinomycin D, cycloheximide, and trilostane. Similarly to LH, prochloraz was able to trigger 17,20ßP production by the ovarian follicle. Finally, prochloraz induced the overexpression of genes participating in 17,20ßP production, intercellular communication, and paracrine control of preovulatory follicular differentiation such as igf, igf2, connexin 43, and 20ß hydroxysteroid dehydrogenase (hsbd20). Together, our results demonstrate that prochloraz administered alone is able to trigger oocyte maturation through the induction of specific genes, some of them being also triggered by LH. Finally, our results clearly indicate that the effects of prochloraz and LH on oocyte maturation are synergistic.

A novel, functional, and highly divergent sex hormone-binding globulin that may participate in the local control of ovarian functions in salmonids.

A cDNA encoding for a novel rainbow trout SHBG was identified and characterized. Phylogenetic analysis showed that this novel SHBG, named SHBGb, was a highly divergent paralog of the classical SHBG (SHBGa) form previously known in vertebrates including zebrafish, seabass, and rainbow trout. Using all available sequences, no SHBGb-like sequence could be identified in any fish species besides Atlantic salmon. Rainbow trout SHBGa and SHBGb share only 26% sequence identity at the amino acid level and exhibit totally distinct tissue distribution, thus demonstrating a functional shift of SHBGb. Indeed, shbga mRNA was predominantly expressed in liver and spleen but could not be detected in the ovary, whereas shbgb had a predominant ovarian expression but could not be detected in liver. Despite its high divergence, rainbow trout SHBGb expressed in COS-7 cells could bind estradiol and testosterone with high affinity and specificity. Both rainbow trout shbgb mRNA and proteins were localized to the granulosa cells of vitellogenic ovarian follicles, whereas SHBGb immunoreactivity was also found in theca cells. Finally, shbgb ovarian mRNA expression exhibited a significant drop between late vitellogenesis and oocyte maturation at a time when ovarian aromatase (cyp19a) gene expression and estradiol circulating levels exhibited a dramatic decrease. Together, these observations show that SHBGb is a functional and highly divergent SHBG paralog probably arising from a salmonid-specific duplication of the shbg gene.

Hydration of rainbow trout oocyte during meiotic maturation and in vitro regulation by 17,20{beta}-dihydroxy-4-pregnen-3-one and cortisol.

Although oocytes of many teleost fish, especially marine species, are subjected to a hydration process during meiotic maturation, which leads to an important volume increase, no noticeable hydration of the preovulatory oocyte has ever been reported in rainbow trout (Oncorhynchus mykiss). In the present study, oocyte water content and dry mass were monitored using consecutive samples taken in vivo from the same female rainbow trout, from 4-5 days prior to ovulation to up to 7 days post-ovulation. In addition, yolk protein electrophoretic patterns were compared between oocytes sampled prior to germinal vesicle breakdown (GVBD) and unfertilized eggs. Furthermore, the effect of the maturation-inducing steroid (17,20beta-dihydroxy-4-pregnen-3-one, 17,20beta-P), cortisol and 11-deoxycorticosterone (DOC) on oocyte dry and wet masses, as well as GVBD occurrence was assessed in vitro. Finally, mRNA expression profiles of glucocorticoid and mineralocorticoid receptors as well as 11beta-hydroxysteroid dehydrogenase (11beta-HSD) were monitored in the periovulatory ovary by real-time PCR. Both in vivo and in vitro data showed, for the first time in rainbow trout, that a significant oocyte hydration occurs during oocyte maturation. In addition, an intra-oocyte dry matter increase was reported in vivo during the periovulatory period. However, yolk protein migration patterns were similar in preGVBD oocytes and unfertilized eggs, suggesting that no or little yolk proteolysis occurs during oocyte maturation. We also showed that oocyte hydration can be induced in vitro by 17,20beta-P and cortisol but not by DOC. In contrast, GVBD was only observed after 17,20beta-P stimulation. Finally, real-time PCR analysis showed an up-regulation of 11beta-HSD and glucocorticoid receptor 2 transcripts in the ovary at the time of oocyte maturation. Together, these results suggest that cortisol could participate in the control of oocyte hydration and possibly in other periovulatory ovarian functions.

Post-ovulatory ageing and egg quality: a proteomic analysis of rainbow trout coelomic fluid.

BACKGROUND: In fish, oocyte post-ovulatory ageing is associated with egg quality decrease. During this period, eggs are held in the body cavity where they bath in a semi-viscous liquid known as coelomic fluid (CF). CF components are suspected to play a role in maintaining oocyte fertility and developmental competence (egg quality). However, CF proteic composition remains poorly studied. Thus rainbow trout CF proteome was studied during the egg quality decrease associated with oocyte post-ovulatory ageing. METHODS: High resolution two-dimensional gel electrophoresis was used to analyze the proteome of rainbow trout (Oncorhynchus mykiss) CF in relationship with the egg quality decrease associated with oocyte post-ovulatory ageing. A first experiment was performed using CF pools originating from 17 females sampled at ovulation as well as 7, 14 and 21 days later. These observations were verified using a second set of CF pools originating from 22 females sampled 5 and 16 days following ovulation. RESULTS: Approximately 200 protein spots of 10-105 kDa molecular mass and 3-10 pI were detected in CF samples. Several protein spots, while undetected at the time of ovulation, exhibited a progressive and strong accumulation in CF during post-ovulatory ageing. After silver-staining and Matrix-Assisted Laser Desorption Time Of Flight (MALDI-TOF) mass spectrometer analysis, some of these protein spots were identified as lipovitellin II fragments. CONCLUSIONS: These observations suggest that egg protein fragments accumulate in the CF during the post-ovulatory period and could therefore be used to detect egg quality defects associated with oocyte post-ovulatory ageing.

Agonistic effect of imidazole and triazole fungicides on in vitro oocyte maturation in rainbow trout (Oncorhynchus mykiss).

The purpose of the present study was to assess the in vitro effect of some imidazole (prochloraz, imazalil) and triazole (epoxiconazole) agricultural fungicides on gonadotropin-induced oocyte maturation in rainbow trout. Results show that prochloraz, epoxiconazole and imazalil strongly potentiated the induction of oocyte maturation by gonadotropin in a dose-dependent manner. Furthermore, 10(-5) M prochloraz and epoxiconazole alone induced oocyte maturation. The mRNA biosynthesis inhibitor, actinomycin d, completely inhibited oocyte maturation induced by fungicides, suggesting that the gonadotropin-like effect of these chemicals is at least dependent on de novo gene expression.

Rainbow trout follicular maturational competence acquisition is associated with an increased expression of follicle stimulating hormone receptor and insulin-like growth factor 2 messenger RNAs.

Post-vitellogenic female rainbow trout (Oncorhynchus mykiss) were assayed in vitro for follicular maturational competence (FMC). Ovarian follicles were stimulated with a range of concentrations of partially purified gonadotropin. The efficient concentration for 50% germinal vesicle breakdown (GVBD) was calculated and used as an indicator of FMC. Before in vitro assay, ovarian tissue was sampled in order to quantify mRNA abundance of specific genes in the ovarian follicle by real-time PCR. In addition, maturation-inducing steroid (MIS, 17, 20 beta-dihydroxy-4-pregnen-3-one) and estradiol (E2) plasma levels were measured by radioimmunoassay. The mRNA expression of several genes such as luteinizing hormone receptor (LH-r), follicular stimulating hormone receptor (FSH-r), insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), insulin-like growth factor receptor 1a (IGF-r1a), and 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) that are putatively expressed in the preovulatory ovary, was studied in females of varying FMC using real-time PCR. FMC acquisition is characterized by an increase of MIS circulating levels and a concomitant drop of E2 levels. At the ovarian level, no significant variation of LH-r, 20 beta-HSD, IGF1, and IGF-r1a mRNA abundance was observed among females of varying FMC. In contrast, FSH-r and IGF2 mRNA levels were significantly higher in females exhibiting high FMC. In addition, correlation analyses showed that IGF2 and FSH-r, mRNA levels were positively correlated with FMC. These results indicate that FMC acquisition is associated with an increased expression of these gene products that may be useful markers of FMC.