Thaovi Nguyen

Identification of new participants in the rainbow trout (Oncorhynchus mykiss) oocyte maturation and ovulation processes using cDNA microarrays

BackgroundThe hormonal control of oocyte maturation and ovulation as well as the molecular mechanisms of nuclear maturation have been thoroughly studied in fish. In contrast, the other molecular events occurring in the ovary during post-vitellogenesis have received far less attention.MethodsNylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR.ResultsFrom the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation.ConclusionWe showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction.

Targeted Gene Expression Profiling in the Rainbow Trout (Oncorhynchus mykiss) Ovary During Maturational Competence Acquisition and Oocyte Maturation

Abstract A real-time polymerase chain reaction-based gene expression survey was performed using 37 target genes and 22 female rainbow trout sampled during follicular maturational competence (FMC) acquisition or during oocyte maturation. In females sampled before meiosis resumption, FMC was estimated using an in vitro assay. Several growth factors, bone morphogenetic proteins, steroidogenic enzymes, cathepsins, genes known to play a role in the fish preovulatory ovary, as well as previously unstudied genes, were analyzed in this survey. Gene expression profiling was performed using a supervised clustering analysis in order to identify groups of genes exhibiting similar expression profiles in the ovary during FMC acquisition and follicular maturation. From the clustering analysis, three clusters exhibiting a specific expression during FMC acquisition or at the time of oocyte maturation were identified. Cluster 1 was characterized by a progressive increase in gene expression during FMC acquisition, whereas cluster 2 exhibited an increased expression at the time of oocyte maturation. In contrast, cluster 3 was characterized by a decreased mRNA expression at the time of oocyte maturation. Among the 37 target genes used in this survey, 18 were significantly regulated during maturational competence acquisition or at the time of oocyte maturation. Among these 18 genes, 16 belonged to one of the three clusters identified. Although the results allowed a global description of gene expression profiles, they also suggest an important role for several factors, including some previously unstudied bone morphogenetic proteins, in the paracrine control of FMC acquisition and meiosis resumption.

Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1) and membrane progestin receptor (mPR) beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns

BackgroundIn lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish.MethodsIn the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary.ResultsOur results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones.ConclusionTogether, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels.

Ovarian function of the trout preovulatory ovary: new insights from recent gene expression studies.

During the preovulatory period the follicle-enclosed oocyte progressively acquires maturational and developmental competence. In addition, the follicle is also preparing for the release of the oocyte from the follicle at ovulation. Using real-time PCR and cDNA microarrays we have investigated the molecular mechanisms of oocyte competence acquisition and ovulation in rainbow trout (Oncorhynchus mykiss) by monitoring gene expression in the preovulatory ovary. These studies have demonstrated that many molecular events related to maturational competence and developmental competence acquisition, and ovulation occur concomitantly in the preovulatory ovarian follicle. Oocyte maturational competence acquisition is associated with a decrease of estrogen synthesis and signaling capacities. We also observed a differential expression of genes encoding for igfs and related binding protein, members of the TGF beta superfamily, proteins involved in ion and water transport, bone morphogenetic proteins, and cathepsins. In addition, our observation of a strong up-regulation, prior to ovulation, of genes encoding for proteins putatively involved in proteolysis, inflammation, coagulation, vasodilatation, and angiogenesis further supports the hypothesis comparing ovulation with an inflammatory-like reaction. Together, our results suggest that a finely tuned cross-talk exists between oocyte and follicular layers and between the ovulatory process and the oocyte maturational and developmental competence acquisition processes.

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.

Oocyte-somatic cells interactions, lessons from evolution.

BackgroundDespite the known importance of somatic cells for oocyte developmental competence acquisition, the overall mechanisms underlying the acquisition of full developmental competence are far from being understood, especially in non-mammalian species. The present work aimed at identifying key molecular signals from somatic origin that would be shared by vertebrates.ResultsUsing a parallel transcriptomic analysis in 4 vertebrate species - a teleost fish, an amphibian, and two mammals - at similar key steps of developmental competence acquisition, we identified a large number of species-specific differentially expressed genes and a surprisingly high number of orthologous genes exhibiting similar expression profiles in the 3 tetrapods and in the 4 vertebrates. Among the evolutionary conserved players participating in developmental competence acquisition are genes involved in key processes such as cellular energy metabolism, cell-to-cell communications, and meiosis control. In addition, we report many novel molecular actors from somatic origin that have never been studied in the vertebrate ovary. Interestingly, a significant number of these new players actively participate in Drosophila oogenesis.ConclusionsOur study provides a comprehensive overview of evolutionary-conserved mechanisms from somatic origin participating in oocyte developmental competence acquisition in 4 vertebrates. Together our results indicate that despite major differences in ovarian follicular structure, some of the key players from somatic origin involved in oocyte developmental competence acquisition would be shared, not only by vertebrates, but also by metazoans. The conservation of these mechanisms during vertebrate evolution further emphasizes the important contribution of the somatic compartment to oocyte quality and paves the way for future investigations aiming at better understanding what makes a good egg.

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 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), 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 17,20βP production by the ovarian follicle. Finally, prochloraz induced the overexpression of genes participating in 17,20βP production, intercellular communication, and paracrine control of preovulatory follicular differentiation such as igf, igf2, connexin 43, and 20β 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.

Maternally Inherited npm2 mRNA Is Crucial for Egg Developmental Competence in Zebrafish

ABSTRACT The molecular mechanisms underlying and determining egg developmental competence remain poorly understood in vertebrates. Nucleoplasmin (Npm2) is one of the few known maternal effect genes in mammals, but this maternal effect has never been demonstrated in nonmammalian species. A link between developmental competence and the abundance of npm2 maternal mRNA in the egg was previously established using a teleost fish model for egg quality. The importance of maternal npm2 mRNA for egg developmental competence remains unknown in any vertebrate species. In the present study, we aimed to characterize the contribution of npm2 maternal mRNA to early developmental success in zebrafish using a knockdown strategy. We report here the oocyte-specific expression of npm2 and maternal inheritance of npm2 mRNA in zebrafish eggs. The knockdown of the protein translated from this maternal mRNA results in developmental arrest before the onset of epiboly and subsequent embryonic death, a phenotype also observed in embryos lacking zygotic transcription. Npm2 knockdown also results in impaired transcription of the first-wave zygotic genes. Our results show that npm2 is also a maternal effect gene in a nonmammalian vertebrate species and that maternally inherited npm2 mRNA is crucial for egg developmental competence. We also show that de novo protein synthesis from npm2 maternal mRNA is critical for developmental success beyond the blastula stage and required for zygotic genome activation. Finally, our results suggest that npm2 maternal mRNA is an important molecular factor of egg quality in fish and possibly in all vertebrates.

Sex hormone‐binding globulins characterization and gonadal gene expression during sex differentiation in the rainbow trout, Oncorhynchus mykiss

Sex hormone‐binding globulin (SHBG) binds androgens and estrogens in the blood of many vertebrates, including teleost fish. In mammals, SHBG is synthetized in the liver and secreted into the blood. In fish, shbga also exhibits a hepatic expression. In salmonids, in which the gene has been duplicated, the recently discovered shbgb gene exhibits a predominantly ovarian expression. The present work aimed at gaining new insight into shbgb gene structure and expression during gonadal sex differentiation, a steroid‐sensitive process, and Shbgb protein structure and binding characteristics; specifically, rainbow trout (Oncorhynchus mykiss) shbgb was analyzed. shbgb structure was analyzed in silico while expression was characterized during gonadal sex differentiation using all‐male and all‐female populations. We observed that shbgb gene and cognate‐protein structures are similar to homologs previously described in zebrafish and mammals. The shbgb gene is predominantly expressed in differentiating female gonads, with increased expression around the end of ovarian differentiation. In the ovary, shbgb mRNA was detected in a subset of somatic cells surrounding the ovarian lamellae. Furthermore, Shbgb binds steroids with a higher selectivity than Shbga, exhibiting a higher affinity for estradiol compared to Shbga. In conclusion, Shbgb binding characteristics are clearly different from those of Shbga. Shbgb is expressed in the differentiating ovary during a period when the synthesis and action of testosterone and estradiol must be tightly regulated. This strongly suggests that Shbgb participates in the regulation of steroid metabolism and/or mediation, that is, needed during early gonadal development in rainbow trout. Mol. Reprod. Dev. 81: 757–765, 2014.

Genome-wide identification of novel ovarian-predominant miRNAs: new insights from the medaka ( Oryzias latipes )

MicroRNAs (miRNAs) are small, highly conserved non-coding RNAs that play important roles in the regulation of many physiological processes. However, the role of miRNAs in vertebrate oocyte formation (i.e., oogenesis) remains poorly investigated. To gain new insights into the roles of miRNAs in oogenesis, we searched for ovarian-predominant miRNAs. Using a microarray displaying 3,800 distinct miRNAs originating from different vertebrate species, we identified 66 miRNAs that are expressed predominantly in the ovary. Of the miRNAs exhibiting the highest overabundance in the ovary, 20 were selected for further analysis. Using a combination of QPCR and in silico analyses, we identified 8 novel miRNAs that are predominantly expressed in the ovary, including 2 miRNAs (miR-4785 and miR-6352) that exhibit strict ovarian expression. Of these 8 miRNAs, 7 were previously uncharacterized in fish. The strict ovarian expression of miR-4785 and miR-6352 suggests an important role in oogenesis and/or early development, possibly involving a maternal effect. Together, these results indicate that, similar to protein-coding genes, a significant number of ovarian-predominant miRNA genes are found in fish.