Steroid metabolism by ovarian follicles of the sea bass Dicentrarchus labrax

Ovarian samples from fear sea bass, Dicentrarchus labrax, were collected for the in vitro incubations during the spawning period. Follicles with fully developed vitellogenic oocytes showing central germinal vesicle (stage I follicles) and follicles with oocytes showing initial germinal vesicle migration (stage II follicles) were treated with either (1) 20 μg sea bass hypophysis plus 50 ng 17-hydroxyprogesterone (17-P), (2) 20 μg hypophysis alone, (3) 50 ng 17-P alone and (4) media alone. Structure-activity experiments used stage II follicles treated with several dosages (0.1, 1.0 and 10.0 ng/ml) of either 17-P, 17,20β-P, or 17,20β,21-P. Free and conjugated (sulfates and glucuronides) levels of the established teleost oocyte maturation inducing steroids (MIS), i.e. 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) were measured in the incubation media by high performance liquid chromatography. Our results show that the synthesis of free and conjugated 17,20β-P is constant (0.1–0.2 ng/ml) in all incubates. In contrast, the synthesis of free and conjugated 17,20β,21-P is higher in incubates containing stage II follicles (up to 5 ng/ml) than in those having stage I follicles (up to 3 ng/ml; P<0.01). Structure-activity data reveal that 17-P is not effective at inducing in vitro germinal vesicle breakdown whereas both 17,20β-P and 17,20β,21-P are equally potent and highly effective. These results demonstrate that 17,20β-P and 17,20β,21-P are synthesized in vitro by follicles of sea bass and that sulfation is the main route for the metabolism of the C₂₁-steroids in riper follicles. The highest levels of 17,20β,21-P, found in incubates containing stage II follicles, points at 17,20β,21-P, rather than 17,20β-P, as the most probable MIS in sea bass, nonetheless, this hypothesis requires further confirmation.     

Gonadal development and sex steroids in a female Arctic charr broodstock

Gonadal development and plasma levels of sex steroids were investigated in female Arctic charr at 3-week intervals over a 12-month period. Circulating levels of oestradiol-17β (E₂), testosterone (T) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) were measured by radioimmunoassay, and gonadal status assessed through histological examination and measurement of gonadosomatic index (GSI) and frequency distribution of oocyte size-classes. Gonadal recrudescence during March-July was characterized by modest but insignificant increases in plasma levels of E₂ (2–4 ng ml^?1) and T (2–5 ng ml^?1) and recruitment of oocytes into yolk accumulation. Only a small and insignificant rise in GSI and no apparent increase in oocyte diameter occurred during this period, indicating that the rate of yolk formation and oocyte growth was low. Following transformation from stage V (peripheral yolk granule stage) to stage VI (yolk granule migration stage) in late July, the vitellogenic oocytes entered a phase of rapid growth which resulted in a marked rise in GSI until ovulation commenced in late September. Gonadal growth during this period was accompanied by increases in plasma levels of E₂ and T which peaked at 11 ± 1 (mid-August) and 71 ± 5ng ml^?1 (late September), respectively. The levels of both steroids dropped rapidly during final maturation and ovulation, followed by a surge in plasma levels of 17,20β-P which peaked at an average of 74 ± 17 ng ml^?1 in early October. All three steroids returned to basal levels within a month after ovulation, and all steroids, except E₂, remained low until March of the following year. A slight increase in E₂ detected in February and March during the second season may have been associated with recruitment into vitellogenesis of a new generation of oocytes. It is suggested that the abrupt increase in vitellogenesis in late July may reflect a condition-dependent decision to proceed with maturation, once the energy reserves have been repleted during spring-early summer.     

Reproductive biology and endocrine regulation of final oocyte maturation of captive white bass

The ovarian development, and plasma levels of gonadotropin II (GtH II) and sex-steroid hormones at the end of vitellogenesis were examined in captive white bass Morone chrysops. The changes in plasma hormone levels and oocyte morphology associated with gonadotropinreleasing hormone agonist (GnRHa)-induced final oocyte maturation (FOM) were studied. Although plasma 17β-oestradiol (E₂) and oocyte diameter increased, there were no changes in GtH II, testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) or 17,20β,21-dihydroxy-4-pregnen-3-one (17,20β,21-P) in non-hormone-treated females, and no FOM was observed. Treatment with a sustained-release GnRHa delivery system (GnRHa implant) induced two FOM cycles separated by about 24 h, with the release of approximately equal numbers of eggs in each spawn. Plasma GtH II levels were elevated significantly throughout FOM, reaching a maximum of 9·07 ± 1·55 ng ml^?1 in ovulated fish. Both plasma E₂ and T increased soon after the GnRHa treatment, but E₂ declined in fish undergoing germinal vesicle (GV) migration. Plasma T increased further during FOM (7·55 ± 2·87 ng ml^?1), but declined precipitously at ovulation. A surge in plasma 17,20β-P and 17,20β,21-P (4·11 ± 0·97 ng ml^?1 and 3·10 ± 0·77 ng ml^?1, respectively) was observed in females undergoing GV breakdown (GVBD). Based on the involvement of different sex-steroid hormones, FOM was separated into two stages. Early FOM included lipid-droplet coalescence and GV migration, and was associated with elevations in plasma GtH II and T. Late FOM included GVBD and yolk-globule coalescence, and was associated with elevations in plasma GtH II, 17,20β-P and 17,20β,21-P. The results of this study point to the absence of a surge in plasma GtH II as the missing link in the reproductive axis responsible for the failure of captive white bass to undergo FOM at the end of vitellogenesis. Sustained elevation of plasma GtH II via treatment with a GnRHa implant induced two consecutive spawns with an overall egg production two- to eightfold higher than previously obtained from captive broodstocks, and similar to annual egg production Values reported for wild fish.     

Out‐of‐season production of 17,20β‐dihydroxypregn‐4‐en‐3‐one in the roach Rutilus rutilus

In this study, although the highest production of two physiologically significant progestins in teleosts [17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20β‐P) and 17,20β,21‐trihydroxypregn‐4‐en‐3‐one (17,20β,21‐P)] was observed in the period just prior to spawning in both male and female roach Rutilus rutilus, there was also a substantial production (mean levels of 5–10 ng ml^?1 in blood; and a rate of release of 5–20 ng fish^?1 h^?1 into the water) in males and females in the late summer and early autumn (at least 7 months prior to spawning). During this period, the ovaries were increasing rapidly in size and histological sections were dominated by oocytes in the secondary growth phase [i.e. incorporation of vitellogenin (VTG)]. At the same time, the testes were also increasing rapidly in size and histological sections were dominated by cysts containing mainly spermatogonia type B. Measurements were also made of 11‐ketotestosterone (11‐KT) in males and 17β‐oestradiol and VTG in females. The 3 months with the highest production of 11‐KT coincided with the period that spermatozoa were present in the testes. In females, the first sign of a rise in 17β‐oestradiol concentrations coincided with the time of the first appearance of yolk globules in the oocytes (in August). The role of the progestins during the late summer and autumn has not been established.     

Influence of equine growth hormone,insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E₂), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO₂. In experiment 1, selected cumulus–oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 μg/ml E₂, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO₃-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E₂, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E₂, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.     

Steroids involved with final oocyte maturation in the winter flounder.

A number of androgens and progestogens including 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20-P) were examined in female winter flounder as possible maturation inducing steroids (MIS). During final oocyte maturation serum levels of testosterone (T) and 17 beta-hydroxy-5 beta-androsten-3-one (5 beta-T) peaking at over 200 ng/ml and pregnenolone (PE) at 40 ng/ml were the predominant steroids found from each major group. High levels of T and 5 beta-T were correlated with oocyte stages characterized by germinal vesicle migration. Of the PEs measured, maximum serum levels of PE, 3 beta,17 alpha-hydroxy-5-pregnen-20-one (17-PE) and 3 beta,17 alpha, 20 beta-dihydroxy-5-pregnene (17,20-PE) were found during later oocytes stages associated with germinal vesicle breakdown. Levels of 17,20-P, an established MIS in most fish, were almost non-detectable (less than 0.1 ng/ml serum) in females throughout all stages of final oocyte maturation. Incubations of ovarian follicles in vitro with physiological concentrations of T and 5 beta-T indicated that these steroids could induce all stages of final oocyte maturation. Similar in vitro incubations showed that 17-PE and 17,20-PE were only effective on germinal vesicle breakdown. The principal conclusions are that T, 5 beta-T and the PEs can be considered as MISs in winter flounder and the PE pathway predominates during the final stages of oocyte maturation in winter flounder in contrast to progesterones which predominate in other fish species, mostly salmonids, studies to date.     

In vitro maturation of oocytes from a marsupial,the tammar wallaby (Macropus eugenii)

This study examined the competence of oocytes from the tammar wallaby, Macropus eugeniio mature in vitro. Oocytes were collected from follicles >1 mm diameter 24 h after pregnant mare serum gonadotrophin (PMSG) treatment and incubated in Eagle's minimum essential medium supplemented with 10% fetal calf serum, at 35°C in 5% CO₂ in air for 24, 36, or 48 h. Oocytes were incubated either granulosa cell-intact or granulosa cell-free or in the presence of 10 IU ml^?1 PMSG or 10 μg ml^?1 porcine luteinizing hormone (LH) + 10 μg ml^?1 porcine follicle stimulating hormone (FSH). The ability of oocytes recovered from small (<1.5-mm-diameter) and large (≥1.5-mm-diameter) follicles to mature in vitro was also examined. The nuclear status of oocytes was assessed using the DNA-specific dye Hoechst 33342. Initially, all oocytes examined contained a germinal vesicle. After 24 h of culture, 60% of oocytes had progressed to metaphase I or anaphase I. After 36 h, approximately 20% of oocytes possessed metaphase II chromosomes, and 20% of oocytes were at metaphase I or anaphase I. At the completion of the 48 h culture period, 40% of oocytes had completed maturation to the metaphase II stage. In vitro oocyte maturation after 48 h was not affected by the presence of granulosa cells, PMSG, or LH and FSH. More oocytes from large follicles (55%) completed maturation by 48 h than from small follicles (20%). Approximately 50% of oocytes remained at the GV stage at all times under all conditions. Marsupial oocytes thus undergo spontaneous nuclear maturation once removed from the follicular environment, suggesting a basically similar control system to that in placental mammals. © 1993 Wiley-Liss, Inc.     

Hormonal regulation of the European sea bass reproductive cycle: an individualized female approach

Fifteen tagged female sea bass Dicentrarchus labrax were sampled weekly from September to April and plasma vitellogenin (VTG), testosterone (T), 17β-estradiol (E₂), and two potential maturation inducing steroids (MISs): 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) and 17,20β,21-trihydroxy-4-pregnen-3-one (20βS) assayed. An oocyte sample was obtained via intraovarian cannulation at each sampling time from every female and the stage of development of the most advanced clutch of oocytes determined and related to VTG and hormone plasma levels for each female. The mean number of ovulations per female was 1·75+0·25 when those females that did not present ovulations were excluded and up to 4 ovulations detected in some females. The highest plasma levels of T ( c. 6 ng ml^-1) were observed during postvitellogenesis and the beginning of maturation while maximum plasma levels of E2 (>5 ng ml^-1) were obtained during late vitellogenesis. VTG plasma levels increased throughout vitellogenesis peaking ( c. 2·5 mg ml^-1) at postvittelogenesis. For the first time significant changes of plasma progestogens were detected in European sea bass during the sexual cycle. The highest plasma level of 17,20βP ( c. 1·1 ng ml^-1) was observed during postvitellogenesis while the highest level of 20βS ( c. 1·4 ng ml^-1) coincided with final maturation. These results suggest that 17,20βP and 20βS play a role in the early and final maturation, respectively, in the European sea bass.     

Uptake of vitellogenin into oocytes during early vitellogenic development in the rainbow trout, Oncorhynchus mykiss (Walbaum)

Uptake of ³H-vitellogenin (³H-VTG) into oocytes of various sizes was investigated during early vitellogenic development in the rainbow trout, Oncorhynchus mykiss (Walbaum). Females were injected with ³H-VTG and uptake into oocytes of different sizes (<0.4,0.4–0.59, 0.6–0.79, 0.8–0.99 and 1.0 1.2 mm in diameter) measured. Oocytes measuring less than 0.6 mm in diameter appeared unable to sequester VTG and were therefore considered pre-vitellogenic. Oocytes measuring 0.6 mm or more all sequestered VTG. The larger the oocyte, the more ³H-VTG it sequestered, even when uptake was expressed per unit surface area. The latter observation could be due to an increase in the number of VTG receptors per unit surface area, an increase in the rate of turnover of the VTG receptor, greater access of VTG to the receptors as oocytes grow, or a combination of any of these factors. The data suggest that the ability to sequester VTG is developmentally regulated.     

17α,20β,21-Trihydroxy-4-pregnen-3-one: the probable maturation-inducing steroid of the Lusitanian toadfish

17,20β,21-Trihydroxy-4-pregnen-3-one (17,20β,21-P) was identified as the major metabolite of incubations of Lusitanian toadfish Halobatrachus didactylus ovarian follicles with [³H]-17hydroxyprogesterone. The potency of several steroids in inducing germinal vesicle breakdown of follicle-enclosed oocytes of Lusitanian toadfish was systematically examined by using an in vitro germinal vesicle breakdown (GVBD) bioassay. 17,20β-Dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-P, two confirmed maturation-inducing steroids (MIS) in teleosts, were the most potent in inducing GVBD with ED₅₀s ranging between 9 and 271 nM. Structure-activity relationships followed similar patterns to what has been observed in similar bioassays, i.e. a vital requirement for 17- and 20β-hydroxyl groups in C21 steroids and a reduction in activity of 14 and 5–6%, respectively, for 5-pregnene and 5β-pregnanes compared to 4-pregnenes. Corticosteroids, testosterone and 17β-oestradiol were ineffective. Folliculated oocytes stimulated by pituitary homogenate produced 17,20β,21-P from endogenous substrates in amounts one order of magnitude higher than 17,20β-P. These results strongly support the hypothesis that 17,20β,21-P is the likely MIS in this species.