Hormonal changes during meiotic maturation and ovulation in the brook trout (Salvelinus fontinalis)

The plasma levels of estradiol-17 (E2), 17, 20-dihydroxy-4-pregnen-3-one (17,20-P) and gonadotropin (GTH) were measured in brook trout (Salvelinus fontinalis) during the period from the end of vitellogenesis to postovulation. Blood samples were taken according to specific stages of maturation, including germinal vesicle breakdown (GVBD) and ovulation. E2 levels were quite high (45 ng/ml) at the end of vitellogenesis (and prior to GVBD) and dropped precipitously by GVBD (2 ng/ml). They remained low through ovulation and postovulation. 17,20-P levels were low prior to GVBD (0.7 ng/ml) and increased dramatically at GVBD (148 ng/ml). The levels of 17,20-P remained high at ovulation (142 ng/ml) and then dropped significantly within 24 h to approximately half of the ovulatory values. They decreased even further by 7 days postovulation. GTH levels rose gradually through GVBD and ovulation from a postvitellogenic level of approximately 3 ng/ml to a 7 day postovulatory value of approximately 10 ng/ml. The overall results; 1) decrease in estradiol prior to GVBD, 2) increase in 17,20-P at GVBD and 3) gradual GTH rise through GVBD and ovulation, are similar to those reported for other salmonids.     

17-β雌二醇诱导鲻雌性化的机制

用原位杂交和免疫细胞化学技术,对服用17 β雌二醇实验组和对照组幼年鲻脑各部和性腺进行芳香化酶的定位。结果发现,芳香化酶转录物和特异性蛋白在幼年鲻端脑(嗅球和大脑)、间脑、中脑和小脑是丰富的。在性别未分化时,芳香化酶免疫活性细胞在幼鲻脑各部的分布密度有显著的差异。在嗅球,对照组芳香化酶免疫阳性细胞的分布密度高于实验组,而在间脑、中脑和小脑实验组免疫阳性细胞数量比对照组多1～3倍,特别是下丘脑视前区芳香化酶的免疫阳性细胞数量尤占优势,提示芳香化酶在幼年鲻性分化中可能起关键的作用。另外,在性分化后,芳香化酶免疫活性还定位在卵巢颗粒细胞和精巢间质细胞与足细胞。同时,免疫阳性物质也定位在卵巢和精巢的生殖细胞。这些结果揭示了17 β雌二醇诱发幼鲻雌性化的机制可能是通过芳香化酶的介导,本研究首次提供形态学新的证据。最后,文中还讨论了芳香化酶在鲻性腺发育中可能的生理作用。     

Gonadotropins I and II do not stimulate thein vitro secretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate by rainbow trout gonads during final sexual maturation

Thein vitro secretion of 17,20-dihydroxy-4-pregnen-3-one 20-sulphate (17,20-P-sulphate) and the free steroid 17,20-dihydroxy-4-pregnen-3-one (17,20-P), by rainbow trout (Oncorhynchus mykiss) gonads, in response to gonadotropin (GTH) I and GTH II, were studied during the final stages of sexual maturation. Substantial amounts of 17,20-P-sulphate were produced, by both mature ovaries and testes, indicating considerable 20-hydroxysteroid sulphotransferase (20-HST) activity within these tissues. In the post-ovulatory ovary the level of 17,20-P-sulphate (36.6 ng ml^–1) greatly exceeded that of 17,20-P (8.59 ng ml^–1). The amount of 17,20-P-sulphate produced in incubations of both mature ovary and testes was unaffected by either GTH I or GTH II treatment at physiological concentrations up to 100 ng ml^–1. Similarly, incubations of maturing ovary and testes, treated with GTH I or GTH II, in the presence of added 17,20-P at 100 ng ml^–1 of medium, produced levels of 17,20-P-sulphate that were similar to those of the controls. In incubations of mature ovarian follicles at the stages of germinal vesicle breakdown and preovulation, both GTHs significantly stimulated secretion of 17,20-P, although GTH II was always more potent than GTH I. GTH II significantly elevated the levels of 17,20-P in testicular incubations from mature males more than 4-fold relative to GTH I and controls, which did not differ from one another.In conclusion, 20-HST, the enzyme responsible for the sulphate conjugation of 17,20-P, was found to be active in the ovaries and testes of rainbow troutin vitro. However, the levels of this enzyme do not appear to be regulated by either GTH I or GTH II.     

Estradiol-17β-induced calcium uptake and resorption in juvenile rainbow trout,Oncorhynchus mykiss

Juvenile rainbow trout, Oncorhynchus mykiss, were injected with estradiol-17β (E₂) in order to study the source of extra calcium needed during vitellogenesis. E₂-treatment increased the calcium uptake from the external medium as well as calcium mobilization from muscle and scale. Judged by the increase in plasma protein-bound calcium levels, the E₂-induced increase in calcium uptake is an apparent over-mobilization of calcium, i.e., the calcium uptake of the fish is in excess of what is found bound to plasma proteins. As the calcium excretion and calcium space (calculated from free plasma calcium levels) were unaffected, the excess calcium is suggested to be incorporated into internal calcium stores. This implies that the systems regulating vitellogenesis and calcium balance are integrated on the mechanistic or endocrine level, and that E₂ causes calcium mobilization of a magnitude geared to the needs of the sexually maturing female.     

Evidence that 17α,20β,21-trihydroxy-4-pregnen-3-one is a maturation-inducing steroid in spotted seatrout

Ovarian steroidogenesis during final oocyte maturation (FOM) in the spotted seatrout (Cynoscion nebulosus) was investigated by incubating ovarian fragments with tritiated pregnenolone, followed by chromatographic separation of the radioactive products. The major tritiated steroid produced during FOM comigrated with 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-dihydro-11-deoxycortisol, 20β-S) on HPLC and TLC. Only minor amounts of radioactive material coeluted with 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P), 11-deoxycorticosterone (DOC), estradiol-17β and testosterone standards in the HPLC system. Additional chromatography by TLC confirmed the presence of radioactive estradiol-17β and testosterone but not 17α,20β-P and DOC. All the ovarian steroids producedin vitro during FOM were assayed for their ability to induce germinal vesicle breakdown (GVBD) of spotted seatrout oocytes. Twenty grams of ovarian tissue were incubated with human chorionic gonadotropin and exogenous pregnenolone. The steroidal products were purified by HPLC and TLC. Most of the maturation-inducing activity was confined to steroidal material which comigrated in these systems with 20β-S. This material was active at a concentration of 1 ng steroid/ml medium in the GVBD assay. Smaller amounts of material which coeluted with 11-deoxycortisol, DOC, 17α,20β-P and several minor unidentified fractions induced GVBD at concentrations of 10 ng steroid(s)/ml. The structure-activity relationships of authentic steroids in inducing GVBD of spotted seatrout oocytes was investigated. Hydroxylation at the 17α, 20β or 21 positions increased potency to induce GVBD. Steroids with multiple hydroxyl groups at the 17α and 20β positions (17α, 20β-P) and at the 17α, 20β, and 21 positions (20β-S) had maximum biological activity in the GVBD bioassay. The results suggest that 20β-S is a major maturation-inducing steroid in spotted seatrout.     

Nucleotide sequence and mRNA expression analysis of <Emphasis Type="Italic">β</Emphasis>-actin gene in the orange-spotted grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The full-length cDNA, encoding the orange-spotted grouper β-actin and spanning 1920 bp including a poly (A) tail, was cloned from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate β-actin than any other members of the actin family. The partial genomic sequence indicated that the organization of the β-actin gene in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low level in the kidney. The expression of β-actin gene in the skeletal muscle was barely detectable. These results indicated that the expression of the orange-spotted grouper β-actin gene showed significant variation in different tissues. Therefore, caution should be taken when using β-actin gene as an internal control in the normalization of gene expression among tissues. Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17α–methyltestosterone (MT) had little effect on the mRNA expression of β-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting β-actin can be used as an internal control for RT-PCR analysis of MT effects on gene expression in these tissues.     

Control of ion transport by the sperm duct epithelium of brook trout (Salvelinus fontinalis)

The sperm duct epithelium of brook trout (Salvelinus fontinalis), mountedin vitro in Ussing-style epithelial chambers actively absorbs Na⁺ (measured as the short-circuit current, I_sc) and secretes K⁺ (measured using⁸⁶Rb⁺ as tracer). Dibutyryl-cyclic-adenosine monophosphate (db-cAMP) and 3-isobutyl-1-methylxanthine (IMX) produce a rapid, sustained stimulation of both ion transport processes, but the hormone connected to the response is unknown. Purified sockeye salmon CON A2 gonadotropin (GtH) produces a dose-dependent, rapid and sustained rise in Na⁺ uptake and K⁺ secretion. The time course, electrophysiological and transport characteristics are similar to those evoked by IMX. Carbohydrate-poor (chum salmon CON A1) GtH is ineffective. Pretreatment of fish with 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-P) significantly increases milt volume but is without effect on resting or stimulated (IMX + db-cAMP) levels of sperm duct ion transport. This is the first indication of a direct, rapid action of GtH on ion transport by the vertebrate blood-testis barrier. The results suggest direct involvement of GtH in control of later stages of sperm maturation.     

Circulating levels and in vitro production of two maturation-inducing hormones in teleost: 17α,20β-dihydroxy-4-pregnen-3-one and 17α,20β,21-trihydroxy-4-pregnen-3-one, in a daily spawning wrasse, Pseudolabrus japonicus

The female bambooleaf wrasse, Pseudolabrus japonicus, spawns daily during the spawning season, and exhibits a diurnal rhythm of ovarian development. In the present study, we have investigated: (1) circulating levels of 17a, 20-dihydroxy-4-pregnen- 17,20-P) and 17,20,21-trihydroxy-4-pregnen-3-one (20-S) in females sampled at different times of the day during spawning season in captivity, and (2) in vitro production of 17,20-P and 20-S by follicle-enclosed oocytes at seven different develo tal stages. In addition, we developed a microtiter plate enzyme-linked immunosorbent assay (ELISA) for 17,20-P. Serum levels of 17,20-P and 20-S showed similar diurnal changes; substantial increases in these levels occurred around the time of germinal vesicle breakdown (GVBD). In vitro experiments showed that massive production of 17,20-P and 20-S occurred in follicles collected just before or during GVBD. Further, acute decreases in 17,20-P and 20-S production were found in the ovarian follicles just prior to ovulation, suggesting inactivation of the maturation-inducing hormone (MIH). These results, taken together with our previous data on the occurrence of GVBD in vitro, suggest a role for both 17,20-P and 20b-S as MIHs in the bambooleaf wrasse.