Alexis Fostier

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.

ENDOCRINE AND ENVIRONMENTAL ASPECTS OF SEX DIFFERENTIATION IN FISH

Abstract. This paper reviews the current knowledge concerning the endocrine and environmental regulations of both gonadal sex differentiation in gonochoristic and sex inversion in hermaphroditic fish. Within the central nervous system, gonadotropins seem to play a role in triggering sex inversion in hermaphroditic fish. In gonochorists, although potentially active around this period, the hypothalamo-pituitary axis is probably not involved in triggering sex differentiation. Although steroids and steroidogenic enzymes are probably not the initial triggers of sex differentiation, new data, including molecular approaches, have confirmed that they are key physiological steps in the regulation of this process. Environmental factors can strongly influence sex differentiation and sex inversion in gonochoristic and hermaphroditic fish, respectively. The most important environmental determinant of sex would appear to be temperature in the former species, and social factors in the latter. Interactions between environmental factors and genotype have been suggested for both gonochoristic and hermaphroditic fish.

Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish

The present review focuses on the roles of estrogens and aromatase (Cyp19a1a), the enzyme needed for their synthesis, in fish gonadal sex differentiation. Based on the recent literature, we extend the already well accepted hypothesis of an implication of estrogens and Cyp19a1a in ovarian differentiation to a broader hypothesis that would place estrogens and Cyp19a1a in a pivotal position to control not only ovarian, but also testicular differentiation, in both gonochoristic and hermaphrodite fish species. This working hypothesis states that cyp19a1a up-regulation is needed not only for triggering but also for maintaining ovarian differentiation and that cyp19a1a down-regulation is the only necessary step for inducing a testicular differentiation pathway. When considering arguments for and against, most of the information available for fish supports this hypothesis since either suppression of cyp19a1a gene expression, inhibition of Cyp19a1a enzymatic activity, or blockage of estrogen receptivity are invariably associated with masculinization. This is also consistent with reports on normal gonadal differentiation, and steroid-modulated masculinization with either androgens, aromatase inhibitors or estrogen receptor antagonists, temperature-induced masculinization and protogynous sex change in hermaphrodite species. Concerning the regulation of fish cyp19a1a during gonadal differentiation, the transcription factor foxl2 has been characterized as an ovarian specific upstream regulator of a cyp19a1a promoter that would co-activate cyp19a1a expression, along with some additional partners such as nr5a1 (sf1) or cAMP. In contrast, upstream factors potentially down-regulating cyp19a1a during testicular differentiation are still hypothetical, such as the dmrt1 gene, but their definitive characterization as testicular repressors of cyp19a1a would strongly strengthen the hypothesis that early testicular differentiation would need active repression of cyp19a1a expression.

Broodstock management and hormonal manipulations of fish reproduction.

Control of reproductive function in captivity is essential for the sustainability of commercial aquaculture production, and in many fishes it can be achieved by manipulating photoperiod, water temperature or spawning substrate. The fish reproductive cycle is separated in the growth (gametogenesis) and maturation phase (oocyte maturation and spermiation), both controlled by the reproductive hormones of the brain, pituitary and gonad. Although the growth phase of reproductive development is concluded in captivity in most fishes-the major exemption being the freshwater eel (Anguilla spp.), oocyte maturation (OM) and ovulation in females, and spermiation in males may require exogenous hormonal therapies. In some fishes, these hormonal manipulations are used only as a management tool to enhance the efficiency of egg production and facilitate hatchery operations, but in others exogenous hormones are the only way to produce fertilized eggs reliably. Hormonal manipulations of reproductive function in cultured fishes have focused on the use of either exogenous luteinizing hormone (LH) preparations that act directly at the level of the gonad, or synthetic agonists of gonadotropin-releasing hormone (GnRHa) that act at the level of the pituitary to induce release of the endogenous LH stores, which, in turn act at the level of the gonad to induce steroidogenesis and the process of OM and spermiation. After hormonal induction of maturation, broodstock should spawn spontaneously in their rearing enclosures, however, the natural breeding behavior followed by spontaneous spawning may be lost in aquaculture conditions. Therefore, for many species it is also necessary to employ artificial gamete collection and fertilization. Finally, a common question in regards to hormonal therapies is their effect on gamete quality, compared to naturally maturing or spawning broodfish. The main factors that may have significant consequences on gamete quality-mainly on eggs-and should be considered when choosing a spawning induction procedure include (a) the developmental stage of the gonads at the time the hormonal therapy is applied, (b) the type of hormonal therapy, (c) the possible stress induced by the manipulation necessary for the hormone administration and (d) in the case of artificial insemination, the latency period between hormonal stimulation and stripping for in vitro fertilization.

Involvement of estrogens in the process of sex differentiation in two fish species: the rainbow trout (Oncorhynchus mykiss) and a tilapia (Oreochromis niloticus).

In order to study the physiological implication of sex steroid hormones in gonadal sex differentiation in fish, we first investigated the potential role of estrogens using two fish models: the rainbow trout (Oncorhynchus mykiss) and a tilapia species (Oreochromis niloticus). All experiments were carried out on genetically all‐male (XY) and all‐female (XX) populations. In vivo treatments with an aromatase inhibitor (ATD, 1,4,6‐ androstatriene‐3–17‐dione) result in 100% masculinization of an all‐female population in rainbow trout (dosage 50 mg/kg of food) and 75.3% in tilapia (dosage 150 mg/kg of food). In tilapia, the effectiveness of the aromatase inhibition by ATD is demonstrated by the marked decrease of the gonadal aromatase activity in treated animals versus control. No masculinization is obtained following treatment with an estrogen receptor antagonist (tamoxifen) in both species. Aromatase and estrogen receptor gene expression was studied in rainbow trout by semi‐quantitative RT‐PCR in gonads sampled before, during and after sex‐differentiation. Aromatase mRNA is specifically detected in female gonads, 3 weeks before the first sign of histological sex‐differentiation, i.e., first female meiosis. Aromatase expression in male gonads is at least a few hundred times less than in female gonads. Estrogen receptor gene is expressed in both male and female gonads at all stages with no dimorphic expression between sexes. Specific aromatase gene expression before ovarian differentiation was also demonstrated using virtual Northern blot, with no expression detected in male differentiating gonads. From these results it can be concluded that estrogen synthesis is crucial for ovarian differentiation, and transcription of the aromatase gene can be proposed as a key step in that process in fish. Mol. Reprod. Dev. 54:154–162, 1999.

Large-Scale Temporal Gene Expression Profiling During Gonadal Differentiation and Early Gametogenesis in Rainbow Trout

Abstract The overall understanding of the sex differentiation cascade in vertebrates is still growing slowly, probably because of the variety of vertebrate models used and the number of molecular players yet to be discovered. Finding conserved mechanisms among vertebrates should provide a better view of the key factors involved in this process. To this end, we used real-time reverse transcription-polymerase chain reaction to produce a temporal map of fluctuations in mRNA expression of 102 genes during sex differentiation and early gametogenesis in the rainbow trout (Oncorhynchus mykiss). We used these 102 temporal gene expression patterns as a basis for a hierarchical clustering analysis to find characteristic clusters of coexpressed genes. Analysis of some of these gene clusters suggested a conserved overall expression profile between the sex differentiation cascade in fish and mammals. Among these conserved molecular mechanisms, sox9, dmrt1, amh, nr5a1, nr0b1, igf1, and igf1ra are, for instance, characterized as early expressed genes involved in trout testicular differentiation as it is known or suggested in mammals. On the contrary, foxl2, fst, and lhr are characterized as early expressed genes during trout ovarian differentiation, as also found in mammals. Apart from this high conservation, our analysis suggests some potential new players, such as the fshb subunit gene, which is detected here for the first time, to our knowledge, in the female differentiating gonad of a vertebrate species and displays a specific overexpression that coincides in timing with the occurrence of first oocyte meioses, or the pax2 gene, which displays an early and testis-specific expression profile.

Seasonal changes in plasma levels of gonadal steroids of sea bass, Dicentrarchus labrax L.

Levels of plasma testosterone (T) and 11-ketotestosterone (11-KT) in males and plasma 17 beta-estradiol (E2), 17 alpha-20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOH-P), and T in females were assayed by radioimmunoassay at monthly intervals throughout the sexual cycle of sea bass (Dicentrarchus labrax L.). 17 alpha,20 beta-DiOH-P was maintained at low levels (below 1 ng/ml) throughout the year, even during the spawning period (January-March). A bimodal seasonal pattern of plasma testosterone was observed. Plasma T and E2 levels became significantly increased in December (advanced gametogenesis period) and then showed further increases during January and February (first half of the spawning period) in parallel with the growth of the vitellogenic oocytes. Multiple spawnings of individual females were also observed during the spawning period affecting the relative fecundity of the eggs. A possible role of E2 on this behavior is discussed. In males, both plasma T and 11-KT initially increased in November and then showed further increasings during the rest of the period of gametogenesis (December) to reach their peak levels in the first half of the spawning period (end of January). These increased and sustained higher levels of plasma steroids coincided with the presence of spermiating males. A second peak of plasma testosterone appeared at the end of the postspawning period-beginning of the pregametogenesis period (May-June) both in males and females and their possible role with the preparation of the gonad for the next reproductive cycle is discussed.

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.

Plasma 11-oxotestosterone and gonadotropin during the beginning of spermiation in rainbow trout (Salmo gairdneri R.)

Radioimmunoassays are used to follow plasma 11-oxotestosterone and glycoprotein gonadotropin levels related to sperm production in male rainbow trout at the onset of spermiation. The 11-oxotestosterone concentrations are higher in the plasma of males giving a measurable volume of sperm. When these levels increase as spermiation progresses, the gonadotropin level tends to decrease slightly. Furthermore, the quantities of collected sperm are positively correlated with the 11-oxotestosterone levels, but not with GTH secretion.

The sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids

All salmonid species investigated to date have been characterized with a male heterogametic sex‐determination system. However, as these species do not share any Y‐chromosome conserved synteny, there remains a debate on whether they share a common master sex‐determining gene. In this study, we investigated the extent of conservation and evolution of the rainbow trout (Oncorhynchus mykiss) master sex‐determining gene, sdY (sexually dimorphic on the Y‐chromosome), in 15 different species of salmonids. We found that the sdY sequence is highly conserved in all salmonids and that sdY is a male‐specific Y‐chromosome gene in the majority of these species. These findings demonstrate that most salmonids share a conserved sex‐determining locus and also strongly suggest that sdY may be this conserved master sex‐determining gene. However, in two whitefish species (subfamily Coregoninae), sdY was found both in males and females, suggesting that alternative sex‐determination systems may have also evolved in this family. Based on the wide conservation of sdY as a male‐specific Y‐chromosome gene, efficient and easy molecular sexing techniques can now be developed that will be of great interest for studying these economically and environmentally important species.