Bernard Jalabert

7 The Gonadal Steroids

Publisher Summary This chapter discusses that the gonads potentiality to produce steroids, and the regulation of the syntheses. The nature, shape, and intensity of a hormonal signal, ready to be received by a target cell, is the result of an intricate series of positive and negative regulations. In the case of hormonal steroids in fish, only some aspects of this complex have been considered. Once a steroid is secreted, several mechanisms may inactivate it before it reaches its target. Little is known of catabolism of sexual steroids in teleosts. Most available data are concerned with the total radioactivity found in tissues after fish are fed labeled steroid. The biological significance of glucuronidation or sulfonation remains to be explored. Although the conjugated steroids are usually considered to be inactive, recent studies attribute a pheromonal role to glucuronides. In other respects, the binding to plasma proteins may lead to a reversible inactivation, although, in mammals, it has been suggested that steroid secretion may be enhanced by the presence of serum steroid-binding proteins. Finally, the conversion of plasma steroids into biologically active metabolites can occur in some target tissues. The actual physiological role of gonadal steroids in fish is discussed with emphasis on gametogenesis.

Steroidogenesis in rainbow trout (Salmo gairdneri) at various preovulatory stages: changes in plasma hormone levels andin vivo andin vitro responses of the ovary to salmon gonadotropin

In order to specify the timing of some changes in ovarian steroid production during the transition from vitellogenesis to ovulation, plasma hormones levels andin vivo andin vitro responses of the ovary to salmon gonadotropin (s-GtH) or dibutyryl-cyclic adenosine mono-phosphate (db-cAMP) were recorded in relationship with the state of germinal vesicle migration in the oocyte.In vivo, a small, but significant, increase of plasma 17α-hydroxy-20β-dihydroprogesterone (17α, 20β-OH-P) level was detected earlier (at the “subperipheral germinal vesicle” stage) than the increase of GtH level (detectable at the “peripheral germinal vesicle” stage) and the decline of oestradiol-17β (E2–17β) (also detectable at the “peripheral germinal vesicle” stage). Negative correlations were established between E2–17β levels and GtH (ρ=−0.53) or 17α,20β-OH-P (ρ=−0,43) levels while a positive correlation occurred between 17α,20β-OH-P and GtH levels (ρ=+0,54).In vivo no action of GtH on the decline of E2–17β levels was detected GtH did not stimulate 17α,20β-OH-P production, within 72h, in females at the “end of vitellogenesis” stage. It had significant effect in females at other stages closer to ovulation, but the pattern of responses changed according to the stage.In vitro db-cAMP like GtH was able to stimulate 17α,20β-OH-P output from ovarian follicles. The greatest response was observed at the later stage. (GVBD). Testosterone output was also increased by GtH, but the lowest response was observed at the later stage (GVBD). Androstenedione output was lower than testosterone output.In vitro, a small but significant decline of E2–17β output was induced by GtH. We conclude that substantial changes occur during the very last stages prior to ovulation, both in the steroidogenic potential of the ovary and in the ovarian sensitivity to GtH. 20β-oxydoreductase is probably progressively induced during GV migration when GtH basal levels are increasing but still relatively low. Without minimizing the role of discrete pulses of GtH on this induction, we could expect synergic actions of other hormones. Thus a high testosterone/oestradiol ratio in the follicle environment favours 17α,20β-OH-P secretion.

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.

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

BackgroundIn 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.MethodsHigh 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.ResultsApproximately 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.ConclusionsThese 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.

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

SUMMARY 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 (17,20β-dihydroxy-4-pregnen-3-one, 17,20β-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 11β-hydroxysteroid dehydrogenase (11β-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 17,20β-P and cortisol but not by DOC. In contrast, GVBD was only observed after 17,20β-P stimulation. Finally, real-time PCR analysis showed an up-regulation of 11β-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.

Regulation of oocyte maturation in the rainbow trout,Salmo gairdneri: role of cyclic AMP in the mechanism of action of the maturation inducing steroid (MIS), 17α-hydroxy, 20β-dihydroprogesterone.

In fish, oocyte maturation (resumption of meiosis after completion of vitellogenesis and before ovulation) is triggered by maturation inducing steroids (MIS) which generally appear to be secreted in the ovary in response to stimulation by a pituitary maturational gonadotropin. Converging data from different laboratories show that 17α-hydroxy, 20β-dihydroprogesterone (17α, 20β-OH-P) is the principal MIS in salmonoids; but clear identification remains to be done in other taxonomic groups.The experiments reported here in the rainbow troutSalmo gairdneri examine the possible involvement of oocyte cAMP on the mechanism of MIS action. The action of 17α, 20β-OH-P, on germinal vesicle breakdown (GVBD) in oocytes incubatedin vitro within the follicle, was inhibited by various substances expected to elevate the intraoocyte concentrations of cAMP: cAMP (≥ 1 mM) or dibutyril cAMP (≥ 2 mM), phosphodiesterase inhibitors such as theophylline (≥ 0.2 mM) or 3-isobutyl-1 methylxanthine (IBMX ≥ 0.1 mM), adenylate cyclase activators such as cholera toxin (> 100 nM) or forskolin (≥ 0.03 mM). In fact, the combined action of IBMX (1 mM) and forskolin (0.01 or 0.05 mM)in vitro was to promote accumulation of intraoocyte cAMP within 1 to 5 hours. Oocyte cAMP concentrations exhibited a large variability between different females, depending on the stage of oocyte development; a significant positive correlation between oocyte cAMP concentration and the follicular weight, and a significant negative correlation between oocyte cAMP concentration and the median efficient dose of 17α, 20β-OH-P for induction of GVBD, were observed. Finally, when intrafollicular oocytes were incubatedin vitro, the addition of a maturation-inducing concentration of 17α, 20β-OH-P (3×10−6M) induced a significant decrease of oocyte cAMP within the first 10 hours of incubation. These results show that cAMP appears to play a central role in the regulation of oocyte sensitivity to 17α, 20β-OH-P and in the intraoocyte mechanisms leading to GVBD in trout.These data are discussed together with the few indications available in fish concerning the mechanism of MIS action which can be compared to some extent with the amphibian model.

A new tool for induced spawning: The use of 17α-hydroxy-20β-dihydroprogesterone to spawn carp at low temperature

Abstract The efficiency of different hormonal treatments to induce ovulation of carp at low temperatures (13–15°C) was tested. “Priming” with a low dose (0.6 mg/kg) of carp pituitary extract was found necessary for a subsequent successful treatment with 17α-hydroxy-20β-dihydroprogesterone (17α-20β P) (2 mg/kg) 1 day later. The eggs produced by this method showed satisfactory fertilization (75–96%) and hatching success (70%). On the other hand, normal hypophysation (5.4 mg/kg) following priming resulted only in partial ovulation and oocyte resorption at this temperature. Priming was shown to increase the gonadotropin level in plasma from about 2 to 35 ng/ml and to induce germinal vesicle migration toward the periphery of oocytes. Neither 17α-20β P nor desoxycorticosterone alone or in association gave any positive result in the absence of a preliminary priming.

Relationships between the expression of maternal behaviour and ovarian development in the mouthbrooding cichlid fish Oreochromis Niloticus

Abstract The rhythm of ovarian development in Oreochromis niloticus was compared in females that had been allowed to, or were prevented from, mouthbrooding, respectively (INC for incubating females; NI for non incubating females). In both cases, fish were killed at 3-day intervals after spawning, throughout the duration of the reproductive cycle. Gonadal development was characterised either by calculating the gonadosomatic index (GSI) or by observing the distribution of ovarian follicle sizes larger than 600 μm, which is the minimum size required for vitellogenin deposition, as observed by immunocytochemistry. Six typical stages of ovarian development, highly correlated to the GSI, were defined and used as indicators in further studies. New batches of vitellogenic oocytes were shown to be already present at the time of spawning in both NI and INC females. Moreover, the shorter interspawning interval of NI females (15 instead of 27 days) together with the changes in GSI showed that vitellogenesis was accelerated when parental care was prevented. Finally, the GSI of parental females reached a plateau from day 12 to day 21, suggesting that ovarian development is slower during the guarding phase.

Comparative analysis of reproductive traits in 65 freshwater fish species: application to the domestication of new fish species

Based on an extensive literature search (1,000 references), the objectives of the present study were to establish a numerical clustering of temperate freshwater fish based on their reproductive traits and to evaluate whether it was possible to extrapolate zootechnical knowledge among species belonging to the same cluster. About 65 species were classified into ten homogeneous clusters from the analysis of 29 reproductive traits, among which the most important were temperature during spawning, egg incubation and larval rearing, degree-days for incubation, larval size upon hatching, spawning season, and parental care. From this typology, a rather regular continuum of reproductive clusters emerges with two obvious endpoints. Between these two extremes, species could be ordered chiefly according to temperature requirement, spawning season and parental care. In conclusion, this new typology, differing significantly from all others proposed earlier, may now serve as a possible framework to help enhancing the domestication of new species by comparison to species belonging to the same cluster.

The follicular sensitivity in vitro to maturation-inducing hormones in rainbow trout, Salmo gairdneri: Role of oestradiol-17β

Abstract Trout ovaries were processed in vitro to determine relationships between the following parameters — oocyte sensitivity to the maturational steroid 17α-hydroxy-20β-dihydroprogesterone (17α,20β-OH-P); follicular sensitivity to the maturational gonadotropin s-GtH; inhibitory potency of exogenous oestradiol-17β (E2) during s-GtH-induced maturation; and level of E2 in the plasma. The sensitivity to hormones was estimated by the median efficient dose (MED) for oocyte maturation in vitro. The peripheral migration of the germinal vesicle coincided with an increase in oocyte sensitivity to 17α,20β-OH-P. A significant correlation was observed between plasma E2 level and the follicular sensitivity to s-GtH, but not between plasma E2 and the oocyte sensitivity to 17α,20β-OH-P. It was concluded that the peripheral migration of the GV is a morphological event which coincides with an increase in oocyte sensitivity, and that E2 is a physiological regulator of follicular sensitivity to GtH.