J.G.D. Lambert

Annual changes in plasma and liver in relation to vitellogenesis in the female rainbow trout, Salmo gairdneri

As part of a study on the physiological participation of ovarian steroids in the regulation of the synthesis of vitellogenin in the liver of the rainbow trout, Salmo gairdneri, an inventory was made of some relevant variables throughout the annual cycle. The annual reproductive cycle can be divided into four physiological periods: a previtellogenic period (March–April), a period of endogenous vitellogenesis (May–July), a period of exogenous vitellogenesis (August–December), and a period of ovulation and spawning (January–February). Quantitative studies have been carried out of the hepatosomatic and gonadosomatic indices, of the concentrations of total proteins, vitellogenin, free phosphates, and vitellogenin-bound phosphates in the plasma, and of the concentration of total proteins and vitellogenin in the liver. The plasma vitellogenin concentration increased from 0.1 mg/ml in May to 12.9 mg/ml in November, the vitellogenin-bound phosphates were hardly detectable in May, but had increased to 6.1 μmol/ml in December, the hepatosomatic index increased from 12 in March to 30 in December, and the gonadosomatic index increased from 6 in June to 222 in December. The free plasma phosphates varied considerably in the different specimens. The total plasma protein levels had a constant value of about 100 mg/ml throughout the annual cycle, except during previtellogenesis, when the levels had decreased to 60 mg/ml. The total liver protein levels per gram liver gradually decreased from 234 mg in July to 136 mg in January, and increased rapidly again in the following months. Vitellogenin of the type as detected in the plasma, was not found in the liver. During exogenous vitellogenesis, plasma vitellogenins with different degrees of phosphorylation appeared to be present. The ultrastructural examination of the liver indicated large amounts of rough endoplasmic reticulum and a large Golgi system from October to December, whereas glycogen and lipid droplets were found in June and July.

Estrogen synthesis in relation to estrone, estradiol, and vitellogenin plasma levels during the reproductive cycle of the female rainbow trout, Salmo gairdneri.

The annual reproductive cycle of the female rainbow trout, Salmo gairdneri, can be divided into four physiological periods: a previtellogenic period (March–April), a preiod of endogenous vitellogenesis (May–June), a period of exogenous vitellogenesis (August–December), and a period of ovulation and spawning (January–February). The estrogen synthetic capacity of the ovaries of adult trout was determined in vitro by incubation of ovarian homogenates with [3H]androstenedione during 6 months of the annual cycle. The estrogen synthetic capacity (the synthesis of estrone and estradiol-17β) of the ovaries increased from zero in February to a maximal value of 2.60 nmol/ovary in October, and rapidly decreased again in the following months. Plasma estrone and estradiol-17β were determined monthly throughout the annual reproductive cycle by means of radioimmunoassay. Estrone plasma levels increased from 2.8 ng/ml in June to 5.1 ng/ml in August. Following a decrease to 2.8 ng/ml in September, a rapid increase to 30.2 ng/ml was seen in November. The values decreased again during the following months. Estradiol plasma levels increased from 1.0 ng/ml in April to 6.7 ng/ml in July. Following a decrease to 2.7 ng/ml in August and 1.8 ng/ml in September, a sudden surge was seen to 59.8 ng/ml in October. The values decreased again during the following months. Vitellogenin levels measured in the same animals displayed a correlation with estrone levels in October and November and with estradiol values in October, November, and December. A possible physiological participation of estrone as well as estradiol during exogenous vitellogenesis is discussed.

The biosynthesis of steroid glucuronides in the testis of the zebrafish, Brachydanio rerio, and their pheromonal function as ovulation inducers

In female zebrafish ovulation could be induced by male holding water, testis homogenates, and testis fractions containing steroid glucuronides. Deglucuronidation of these fractions led to a loss of ovulation-inducing potency, indicating steroid glucuronides as ovulation inducers. The chemical substances were perceived by the recipient females by means of olfaction. Incubation experiments showed the capacity of the testes to synthesize various C19 and C21 steroids and seven different steroid glucuronides, i.e., 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one-, testosterone-, androsterone-, epiandrosterone-, 5 alpha-androstane-3 alpha, 17 beta-diol-, and 5 alpha-androstane-3 beta, 17 beta-diol glucuronide. GC-MS analysis showed the presence of glucuronides of 5 alpha-androstane-3 alpha, 17 beta-diol and cholesterol in male holding water, the latter probably originating from the liver. These compounds may be among the steroid glucuronides functioning as ovulation-inducing pheromones.

Estrone and estradiol participation during exogenous vitellogenesis in the female rainbow trout, Salmo gairdneri

Abstract Ovarian steroids were treated for their ability to induce vitellogenin synthesis in the liver of the rainbow trout, Salmo gairdneri . These steroids were chosen because they had been reported to induce vitellogenin in the plasma of teleosts, i.e., estrone, estradiol, and testosterone, or because they were synthesized during vitellogenesis, as was the case with the above steroids and androstenedione and 17α-hydroxyprogesterone. Immature trout were ovariectomized and injected daily for 7 days. The administered doses of steroid were 100, 250, and 500 ng/g body wt. Only estradiol and estrone induced vitellogenin in the plasma; estrone had about 5% of the potency of estradiol. A dose-effect curve was determined for estradiol in the range from 25 to 1000 ng/g body wt. A maximal amount of 7 mg vitellogenin/ml plasma was found following the administration of a dose of 250 ng/g estradiol. Vitellogenin was not present in the plasma of animals treated with saline, nor could it be detected in the liver, not even after the administration of estradiol. Estradiol administration increased the total plasma protein concentration from 35 to maximally 51 mg/ml and decreased the total piver protein concentration from 163 to minimally 118 mg/g. The relative weight of the liver increased from 12.9% to maximally 22.4%. Vitellogenin was not detected in the liver of any of the experimental animals, indicating a low storage and rapid secretion of vitellogenin. The other steroids influenced some of the variables, but never was the total pattern of effect comparable to that of estradiol. Estradiol is found to be the ovarian steroid that physiologically regulates the synthesis of vitellogenin in the liver of the rainbow trout; estrone is less active. Experiments undertaken to determine the effects of the combined presence of estrone and estradiol revealed that estrone was capable of boosting the vitellogenic effect of estradiol when compared to the induced vitellogenin levels following treatment with a combination of testosterone and estradiol. The vitellogenin concentrations induced by a certain dose of one of the combinations of estrone and estradiol approximated the concentrations induced by the same dose of estradiol alone. This effect was independent of the ratio in which estrone and estradiol were administered. These findings could not be explained by conversions of estrone into estradiol or by the vitellogenic activity of estrone alone. The estrone, estradiol, and vitellogenin concentrations in the plasma of the experimental animals were in the same range as determined previously in untreated mature vitellogenic females. Vitellogenin and estradiol levels were found to correlate in experimental animals treated with estradiol ( r = 0.627; N = 20). This was not the case in animals treated with combinations of estrone and estradiol. In these animals, however, the sum of estrone and estradiol levels in the plasma correlated with vitellogenin levels ( r = 0.724; N = 42). Vitellogenin was not detected in the liver of any of the experimental animals, which indicates a low storage and rapid secretion of vitellogenin. The importance of viewing the sum of estrone and estradiol plasma levels in connection to physiological studies of the regulation of exogenous vitellogenesis in the rainbow trout is discussed.

The ovary of the guppy Poecilia reticulata: The granulosa cells as sites of steroid biosynthesis

The problem of the endocrine significance of the atretic follicle in the ovary of fishes has been approached with histological and cytological methods in the Guppy, Poecilia reticulata. The atretic follicles are subdivided into four stages: α-, β-, γ-, and δ-stage. During the period of pregnancy, lasting approximately one month, the atretic follicles were irregularly present. In 50 out of 73 animals, they were entirely absent. This appeared to have no influence upon a normal pregnancy; consequently, endocrine activity of the atretic follicle is not probable. The results of the cytochemical test also pointed in this direction. Enzymes, essential for steroid synthesis could not be proved to be present. Acid phosphatase and E-600 resistant esterase, however, were found in considerable amounts, indicating the presence of lysosomal activity. These results clearly show that the atretio follicle of the Guppy does not produce steroid hormones. Consequently, it is preferable to use the name Corpus atreticum instead of Corpus luteum praeovulationis. Moreover, it appears from cytochemical results that the degeneration process of the follicle can be subdivided into two phases: primarily the resorption of the oocyte during the α-stage, and secondly the regression of the granulosa cells during the subsequent stages.

Environmental pollution caused elevated concentrations of oestradiol and vitellogenin in the female flounder, Platichthys flesus (L.)

Abstract Female and male flounder, Platichthys flesus , were exposed to various concentrations of polluted harbour dredged spoil in large mesocosms for up to 3 years. The dredged spoil contained a mixture of contaminants representative of pollution concentrations found in the natural environment. Ovarian development, vitellogenesis and steroid hormones in the fish were studied and compared with results from feral fish sampled at a relatively clean field site in the Dutch Wadden Sea. Plasma concentrations of vitellogenin (VTG), established by densitometry in sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, fluctuated during the annual reproductive cycle of the Wadden Sea flounder and reached a maximum during autumn and winter (advanced vitellogenesis). Fish held in the polluted mesocosm for 3 years exhibited premature vitellogenesis, resulting in a high number of oocytes in the yolk granule stage in spring, normally the previtellogenic period of the year. Moreover, VTG was significantly elevated in the plasma of these females relative to the concentrations in fish from the reference mesocosm. The high concentration of plasma VTG in females from the polluted mesocosm coincided with significantly elevated concentrations of testosterone and 17β-oestradiol. The in vitro ovarian production capacity of these steroids, however, was not altered. In feral and pollution-exposed male flounder, no VTG was detected. On the basis of these findings, it was concluded that premature vitellogenesis in the female flounder was a result of elevated 17β-oestradiol concentrations rather than a direct endocrine effect of xeno-oestrogenic contaminants. It is suggested that the elevated 17β-oestradiol concentrations are caused by decreased clearance rates of steroids in the blood.

Mismatch between patterns of circulating and testicular androgens in African catfish, Clarias gariepinus

Abstract11-Ketotestosterone (11-KT) is an important plasma androgen in male African catfish. The quantitatively predominating androgen produced by the testis, however, is 11β-hydroxyandrostenedione (OHA). Our working hypothesis to explain this mismatch assumed that OHA is converted to 11-KT at extratesticular sites. First, we examined the in vivo metabolism of [3H]-OHA in mature males after sham-operation or removal of either the testes (TC), the seminal vesicles (SVC), or both (TSVC) by analysing the pattern of circulating [3H]-androgens two hours after intravenous injection of [3H]-OHA. [3H]-OHA was converted to [3H]-11-KT to the same extent in all groups, indicating that neither ablation of testes nor of seminal vesicles, or both attenuates this conversion. We then examined the in vitro metabolism of [3H]-OHA by several types of tissues. Liver and seminal vesicle tissue were found to produce significant amounts of [3H]-11-KT. The conversion capacity in vivo was assessed by injecting TSVC-castrated males with increasing doses of radioinert OHA, followed by the quantification of OHA and 11-KT plasma levels. Saturation of the conversion capacity was not reached but the 11-KT production capacity is at least 80 ng per ml of plasma per hour. Moreover, liver fragments were tested for their OHA to 11-KT conversion capacity in vitro using increasing concentrations of radioinert OHA. The 11-KT producing increased with time and OHA concentration. The production rate was 90 pg 11-KT mg-1 liver h-1. Considering the results of the surgical experiments and the fact that the total hepatic mass by far exceeds that of the seminal vesicles, we conclude that the hepatic conversion is of primary relevance in vivo.

Gonadal steroidogenesis and the possible role of steroid glucuronides as sex pheromones in two species of teleosts

In general, female zebrafish,Brachydanio rerio, ovulate only in the presence of males. The stimulant must be pheromonal as even male holding water is capable of inducing ovulation. After ovulation the mating phase begins. During this phase the male follows the female and oviposition as well as fertilization takes place. Both the ovulation and the mating are controlled by pheromones synthesized by the gonads. Ovulation can be induced by testicular homogenates. After the lipid material has been extracted from the testicular homogenates, the remaining aqueous phase can still induce ovulation. However, when the aqueous phase is treated with the enzymeβ-glucuronidase, it loses the ability to induce ovulation. This is an indication that glucuronides, probably steroid glucuronides, are the compounds responsible.During the mating phase, ovulated female zebrafish become attractive to males. It was found that, after ovulation, ovarian extracts contain the compounds responsible for attracting males. The attractant consists of a mixture of steroid glucuronides.After incubation of the gonads with3H-precursors seven steroid glucuronides have been identified in the testis and five in the ovary.Under fish culture conditions the African catfish,Clarias gariepinus, can produce postivitellogenic oocytes throughout the year. However, in capitivity neither males nor females spawn. In female catfish maturation and ovulation can be induced by treatment with gonadotropins. It might be possible that, analogous to the zebrafish, some reproductive processes in the catfish have to be induced by pheromones. It has been demonstrated that pheromonal compounds released by the seminal vesicles are involved in the attraction of female conspecifics. The steroid glucuronide synthesizing capability of the testes and the seminal vesicles of the male catfish are examined, as well as that of the ovary before and after ovulation of the female catfish. Both testes and seminal vesicles appear to be capable of steroid biosynthesis but only the latter synthesizes steroid glucuronides. Six of these conjugates have been isolated and identified. In the female catfish the ovaries are capable of synthesizing seven steroid glucuronides, but only after ovulation.

Corticosteroids Affect the Testicular Androgen Production in Male Common Carp (Cyprinus carpio L.)

Abstract Our previous experiments to study the effect of stress adaptation on pubertal development in carp showed that repeated temperature stress and prolonged feeding with cortisol-containing food pellets, which mimics the endocrine stress effects, retarded the first waves of spermatogenesis and decreased 11-ketotestosterone (11KT) plasma levels. The objective of the present study was to investigate whether the decrease in plasma 11KT is caused by a direct effect of cortisol on the steroid-producing capacity of the testis or by an indirect effect, such as a decrease in plasma LH. Pubertal and adolescent isogenic male common carp (Cyprinus carpio L.) were fed with either cortisol-containing food pellets or control food pellets over a prolonged period. Our results indicate that cortisol has a direct inhibitory effect on the testicular androgen secretion independent of the LH secretion. Furthermore, the pubertal period is critical to the influence of cortisol regarding testicular androgen secretion, because the effect is no longer observed at adolescence.

The ovary of the Guppy, Poecilia reticulata

SummaryThe problem of the endocrine significance of the atretic follicle in the ovary of fishes has been approached with histological and cytological methods in the Guppy, Poecilia reticulata. The atretic follicles are subdivided into four stages: α-, β-, γ-, and δ-stage. During the period of pregnancy, lasting approximately one month, the atretic follicles were irregularly present. In 50 out of 73 animals, they were entirely absent. This appeared to have no influence upon a normal pregnancy; consequently, endocrine activity of the atretic follicle is not probable. The results of the cytochemical test also pointed in this direction. Enzymes, essential for steroid synthesis could not be proved to be present. Acid phosphatase and E-600 resistant esterase, however, were found in considerable amounts, indicating the presence of lysosomal activity. These results clearly show that the atretio follicle of the Guppy does not produce steroid hormones. Consequently, it is preferable to use the name Corpus atreticum instead of Corpus luteum praeovulationis. Moreover, it appears from cytochemical results that the degeneration process of the follicle can be subdivided into two phases: primarily the resorption of the oocyte during the α-stage, and secondly the regression of the granulosa cells during the subsequent stages.