Expression of cytochrome P450(11beta) (11beta-hydroxylase) gene during gonadal sex differentiation and spermatogenesis in rainbow trout, Oncorhynchus mykiss.

Androgens and especially 11-oxygenated androgens are known to be potent masculinizing steroids in fish. As a first step to study their physiological implication in gonadal sex differentiation in fish, we cloned a testicular cytochrome P450(11beta) (11beta-hydroxylase) cDNA in the rainbow trout, Oncorhynchus mykiss. We isolated a 1882 bp P450(11beta) cDNA (rt11betaH2, AF217273) which contains an open reading frame encoding a 552 putative amino acids protein. This sequence was highly homologous (98% in nucleotides and 96.5% in amino acids) to another rainbow trout P450(11beta) sequence (AF179894) and also to a Japanese eel P450(11beta) (68% in amino acids). Northern blot analysis detected a single transcript of 2 kb which was highly expressed in the testis (stage II) and to a lesser degree in the anterior kidney (containing the interrenal tissue). No signal was detected in the posterior kidney, brain, liver, skin, intestine and heart. In the testis this transcript was highly expressed at the beginning of spermatogenesis (stages I and II), followed by a decrease during late spermatogenesis (stages III to V). By semi-quantitative reverse transcription polymerase chain reaction, P450(11beta) expression during gonadal differentiation was estimated to be at least 100 times higher in male than in female gonads. This difference was first detected at 55 days post-fertilization (dpf), i.e. 3 weeks before the first sign of histological sex differentiation, and was sustained long after differentiation (127 dpf). Specific P450(11beta) gene expression was also demonstrated before testis differentiation (around 50 dpf) using virtual Northern blot, with no expression detected in female differentiating gonads. From these results, and also based on the already known actions of 11-oxygenated androgens in testicular differentiation in fish, it is now suggested that P450(11beta) gene expression is a key factor for the testicular differentiation in rainbow trout.

[Changes in the conditions of milk extraction as affected by a beta mimetic (isoprenaline) administered by intrajugular route during mechanical milking of cows]. / Modifications des conditions d'extraction du lait sous l'effet d'un beta-mimétique (isoprénaline) administré par voie intrajugulaire pendant la traite mécanique de la vache.

Between days 80 and 105 of lactation, 11 FFPN cows (table 1) received an intrajugular injection of either 0.9% NaCl or 0.3 mg of isoprenaline (IPN = beta-receptor agonist). Each of the two treatments was repeated at least 7 times by giving NaCl and IPN on successive, alternate days. IPN: - did not change either the total milk yield of the morning milking (13.5 l vs 13.37 l with NaCl) or the volume of milk obtained by stripping (0.460 l vs 0.447 l with NaCl), although individual responses varied considerably (table 3); - significantly increased milk flow by about 25% because it reached 1.144 l/min/quarter after IPN administration vs 0.929 l/min/quarter after NaCl injection (table 4). These results confirm our previous observations concerning milk loss between milkings (Bernabé and Peeters, 1980), i.e. the stimulation of smooth muscle beta-receptors enhances milk flow.

[Effect of adrenaline and phenylephrine on milk extraction during the mechanical milking of cows]. / Effets de i'adrénaline et de la phényléphrine sur l'extraction du lait au cours de la traite mécanique de la vache.

During mechanical milking in the morning between days 60 and 130 of lactation, 6 or 10 French Friesian Pie-Noire (FFPN) cows were given an intrajugular injection of 0.3 mg of adrenaline or 5 mg of phenylephrine (table 1). The treatments, each given 7 times to each cow, were alternated every other day with a control injection of 0.9% NaCl. The adrenaline caused: - a non-significant decrease in total milk yield (-4.9%: 11.57 l vs 12.17 l with NaCl) and in the volume of machine-stripped milk (0.360 l vs 0.430 l with NaCl); - an increase (+8.7%) in milk flow (1.165 l/min vs 1.072 l/min with NaCl; table 4). Phenylephrine caused a significant 10% reduction in total milk yield (13.35 l vs 14.84 l with NaCl) but had no effect on the volume of machine-stripped milk (0.235 l vs 0.295 l with NaCl) or on milk flow (0.961 l/min vs 0.962 l/min with NaCl; table 3). In agreement with our previous results (Bernabé and Peeters, 1980; Bernabé and Ricordel, 1985), beta-receptor (adrenaline) stimulation appeared to increase milk flow by relaxing the teat muscles. However, when contact was avoided between the teats and the milking machine, the stimulation of teat alpha-receptors (phenylephrine) did not inhibit milk flow. It is supposed that during milking the machine caused the teat tissue to contract enough to mask the effect of the alpha-mimetic treatment on milk flow.

Effect of confinement stress on circulating levels of growth hormone and two prolactins in freshwater-adapted tilapia (Oreochromis niloticus).

The aim of the present study was to assess a potential link between confinement stress and prolactin (PRL), the hormone responsible for adaptation to a hypoosmotic environment in freshwater-adapted tilapia (Oreochromis niloticus). The effect of stress on plasma levels of the two tilapia PRL forms, tiPRLI (or tiPRL188) and tiPRLII (or tiPRL177), was examined along with the effects on plasma levels of cortisol and growth hormone (GH). In a preliminary study, various sampling protocols (immediate sampling; sampling one by one; anesthesia at 0.5, 1, 2 ml/liter phenoxyethanol) were tested for their ability to modify basal plasma PRL and cortisol. In fish sampled within 1 min of capture (immediate sampling), no changes in the plasma levels of these hormones were observed, whereas when fish were sampled one at a time, PRL levels did not change but cortisol levels were modified. The immediate sampling protocol was used to study the effects of 1 hr confinement stress, which induced a large increase in plasma cortisol levels as well as increases tiPRLI and tiPRLII levels with kinetics similar to those of cortisol. In contrast, plasma tiGH levels significantly decreased after 1 hr confinement. When this stress situation was removed, plasma cortisol and tiPRL levels decreased and plasma GH levels increased. Two and one-half hours later, values were not significantly different from those measured in control fish. In tilapia exposed to 24 hr confinement stress, similar changes in hormone levels were observed. However, after 24 hr confinement, only cortisol levels were significantly different from those measured in control fish. None of these stress conditions significantly changed plasma chloride levels. Together, these results indicate that both PRL and GH have important roles in the adaptive response of freshwater-adapted tilapia to confinement stress.

Steroid enzyme gene expressions during natural and androgen-induced gonadal differentiation in the rainbow trout, Oncorhynchus mykiss.

In fish, according to Yamamoto's model, androgens would drive testis differentiation and estrogens ovarian differentiation. In order to study the implication of steroid enzymes in rainbow trout gonadal differentiation, we examined the expression of some steroid enzyme genes during natural differentiation (cholesterol side chain cleavage = P450scc, 17-hydroxylase/lyase = P450c17, 3beta-hydroxysteroid dehydrogenase = 3betaHSD) and androgen-induced differentiation (P450scc, P450c17, 3betaHSD, aromatase = P450aro, and 11beta-hydroxylase = P45011beta). Expressions of P450scc, 3betaHSD, and P450c17 were all detected in male and female gonads at 55 days post-fertilization (dpf), i.e., two weeks before histological differentiation. There were no differences in their expression level respective to the sex. The androgen treatment was carried out by administration of 11beta-hydroxyandrostenedione (11betaOHDelta4) in genetic all-female populations and the resulting sex ratios were found to be 100% male even at a low dosage of 1 mg/kg of food. Following 11betaOHDelta4 treatment, only the expression of P450c17 was found to be sustained when compared with the female untreated control. In contrast, P450scc was clearly up-regulated and 3betaHSD and P450aro down-regulated by the androgen treatment. P45011beta gene expression remained low in gonads of androgen-treated females, as it did in control untreated females. These results together demonstrate that steroidogenesis in rainbow trout is potentially active in pre-differentiating gonads of both sexes, and that one of the masculinizing actions of androgens in the species may be to down-regulate the female-specific gonadal P450aro gene expression. However, in vivo androgen treatment in genetic females does not induce the same pattern of steroid gene expression as in genetic males. These data suggest that exogenous androgens might induce a male differentiation process with P450aro inhibition being one of the steps required. However, this process would not involve endogenously produced 11-oxygenated androgens.

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.