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.

Large scale real-time PCR analysis of mRNA abundance in rainbow trout eggs in relationship with egg quality and post-ovulatory ageing.

The mRNA levels of 39 target genes were monitored in unfertilized eggs of 14 rainbow trout sampled the day of ovulation and again 5, 14, and 21 days later. For all 56 collected egg batches, an egg sample was fertilized to estimate egg quality by monitoring embryonic development. Remaining eggs were used for RNA extraction and subsequent real-time PCR analysis. A significant drop of egg quality was observed when eggs were held in the body cavity for 14 or 21 days post-ovulation (dpo). During the same period, eight transcripts (nucleoplasmin or Npm2, ferritin H, tubulin beta, JNK1, cyclin A1, cyclin A2, cathepsin Z, and IGF2) exhibited a differential abundance at one or several collection time(s). Interestingly, we observed higher levels of cyclins A1 and A2 mRNAs in eggs taken 5 days post-ovulation than in eggs taken, from the same females, at the time of ovulation. In addition, seven transcripts exhibited a differential abundance between low quality and high quality eggs. Low quality eggs were characterized by lower levels of Npm2, tubulin beta, and IGF1 transcripts. In contrast, keratins 8 and 18, cathepsin Z, and prostaglandin synthase 2 were more abundant in low quality eggs than in high quality eggs. In this study, we have demonstrated differences in mRNA levels in the rainbow trout egg that are reflective of developmental competence differences induced by post-ovulatory ageing. The putative role of these transcripts in post-ovulatory ageing-induced egg quality defects is discussed with special attention for corresponding cellular functions.

Particularities of reproduction and oogenesis in teleost fish compared to mammals.

Compared to mammals, teleost reproduction presents many original features. Reproductive strategies of species are diversified into numerous adaptations to a large variety of aquatic environments. This diversity may concern sexuality, spawning and parental behaviour, sensitivity to environmental factors, and specific features of gametogenesis such as the duration of vitellogenesis, and egg morphology. Sexuality presents a variety of natural modalities, from gonochorism to hermaphrodism. The absence of definitive arrest of body growth in the adult of most species gives a particular interest to the practical control of growth-reproduction interactions. Vitellogenesis, which represents an important metabolic effort for the maternal organism, involves the synthesis of vitellogenin, a specific glycolipo-phosphoprotein produced in the liver under estradiol stimulation, and its incorporation into oocytes by a receptor mediated process. Both estradiol synthesis in follicle cells and vtg uptake by vitellogenic follicles appear to be mainly controlled by FSH. Oocyte maturation is directly triggered by a progestin, or MIS (maturation inducing steroid) synthesised in follicle cells mainly under LH control, and acting through the non-genomic activation of a membrane receptor. Practical applications of some of these particularities result mainly from the external character of the fertilisation process and of embryonic development, which allows manipulating respectively egg chromosome stocks and sex differentiation. Moreover, the sensitivity of sex differentiation to exogenous factors favours the development of practical methods to control the sex of farmed populations. Finally, the sensitivity of reproductive mechanisms to xenobiotics has led to various kinds of bioassays for putative pollutants.

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.

Targeted gene expression profiling in the rainbow trout (Oncorhynchus mykiss) ovary during maturational competence acquisition and oocyte maturation.

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.

Messenger RNA stockpile of cyclin B, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor receptor Ib, and p53 in the rainbow trout oocyte in relation with developmental competence.

In the present study, correlations between the oocyte messenger RNA (mRNA) stockpile of Cyclin B, insulin-like growth factor I (IGF-I), insulin-like growth factor (IGF-II), insulin-like growth factor receptor Ib (IGFR Ib), and p53 transcripts and the developmental competence of the oocyte were studied. For this purpose, post-ovulatory ageing was used as a tool to generate oocytes of varying developmental competence. Mature female rainbow trout were held at 12 degrees C and periodically checked for ovulation. Oocytes were collected from each female at ovulation and 5, 14, 21 days later. For each collected egg batch, the abundance of several mRNAs in the oocyte was analyzed by real-time PCR and embryo development was monitored after fertilization. Egg quality was estimated not only through embryonic survival but also by studying the occurrence of specific morphological abnormalities. The present study showed that oocyte post-ovulatory ageing was associated with variations of the relative abundance of several studied transcripts within the oocyte. In addition, the abundance of specific mRNAs could be correlated with either the embryonic survival or the occurrence of malformations. Thus, the abundance of IGFR Ib and Cyclin B transcripts in the oocyte was correlated with the occurrence of morphological abnormalities observed at yolk-sac resorption (negatively for IGFR Ib and positively for Cyclin B), while the maternal stockpile of IGF-I, IGF-II, and IGFR Ib mRNAs was positively correlated with embryonic survival.

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.

Expression of prepro-GnRH and GnRH receptor messengers in rainbow trout ovary depends on the stage of ovarian follicular development.

Gonadotropin-Releasing Hormones (GnRHs) are decapeptides well known to regulate the reproductive cycle. They are expressed not only in the brain, but also in other tissues including the gonads. It is believed that they may be involved in the endocrine and paracrine regulation of the reproductive cycle. To date, two forms of GnRH have been identified in salmonids: salmon (sGnRH) and chicken II (cGnRH-II). In the present study, the temporal expression of sGnRH-1, sGnRH-2, cGnRH-II, and rtGnRH receptor genes was studied in rainbow trout ovary during the reproductive cycle according to the stages of follicular development. Using RT-PCR coupled with Southern-blot hybridization, sGnRH-1, sGnRH-2, cGnRH-II, and rtGnRH-R transcripts were detected in morphologically nondifferentiated ovaries as early as 55-65 days post-fertilization and throughout all stages of vitellogenesis. Using Northern blot analysis, cGnRH-II mRNA was detected only in immature previtellogenic ovary, whereas sGnRH mRNA was detected also during early and mid-exogenous vitellogenesis. No sGnRH mRNA was detected at the end of vitellogenesis. In maturing pre-ovulated ovary, sGnRH transiently reappeared before germinal vesicle breakdown (GVBD) and decreased thereafter. A few days after ovulation, a strong sGnRH mRNA expression was found in ovarian tissue as the eggs were kept in the body cavity of females. However, in females stripped just after ovulation, sGnRH mRNA levels remained low in ovary during several weeks. Fully spliced sGnRH-1 and sGnRH-2 messengers were mostly expressed during the reproductive cycle; however different sGnRH-1 and sGnRH-2 splicing variants containing intronic sequences were also detected. Some of these messengers may encode prepro-GnRH precursors with truncated GnRH-associated peptides. The stage-dependent expression and different cell localization of sGnRH, cGnRH-II, and rtGnRH-R transcripts suggest that GnRH-like peptides may have different roles in the paracrine regulation of ovarian follicular development.