David R. Idler

8 Yolk Formation and Differentiation in Teleost Fishes

Publisher Summary This chapter discusses the yolk formation of teleost fishes. The process of vitellogenesis in teleosts has been shown to be similar to that operating in other oviparous vertebrates. The contribution of autosynthetic processes (endogenous vitellogenesis) to the yolk mass in the teleost ovary, relative to exogenous yolk acquired by incorporation of vitellogenin, has not been estimated. Exogenous vitellogenesis can be considered to consist of two phases. The first phase involves the induction of hepatic vitellogenin production under stimulation of ovarian estrogen. During the second phase vitellogenin is taken up from the blood stream and incorporated into ovarian yolk proteins. In salmonids maturational gonadotropin occurs at high levels in plasma around spawning time but is near the lower limit of the radioimmunoassay during the phase of active incorporation of vitellogenin. There appears to be a small increase in maturational gonadotropin coincident with an increase in estradiol in trout plasma early in vitellogenesis, and antibody to maturational gonadotropin inhibits ovarian growth immediately prior to the rapid and massive increase in the ovarian weight. Therefore, there appears to be a low level of maturational hormone present when the fish resumes ovarian development after spawning, but it is sufficient to establish vitellogenin production by the liver.

Steroids in the plasma of the winter flounder (Pseudopleuronectes americanus walbaum). A seasonal study and investigation of steroid involvement in oocyte maturation

Cortisol was a major steroid identified in the plasma of male and female Pseudopleuronectes americanus. Cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone and 11-dehydrocorticosterone were identified at lower concentrations. In plasma from male fish testosterone concentrations changed only slightly during the annual cycle but 11-ketotestosterone concentrations rose dramatically near the time of spawning. Testosterone levels were highest prior to spawning in plasma from female fish but the concentrations of 11-ketotestosterone remained extremely low throughout the year. Progesterone, 17α- OH-progesterone and 20β-dihydro, 17α- OH-progesterone were also identified in plasma from female fish. Plasmas in which oocytes were stimulated to undergo meiotic maturation in vitro were found to contain greater concentrations of 17α-OH-Progesterone and 20β-dihydro, 17α- OH-progesterone than those which did not have this biological activity.

Studies on two types of gonadotropins from both salmon and carp pituitaries

Two molecular weight forms of salmon vitellogenic Con AI (unadsorbed on concanavalin A-Sepharose) hormone and the salmon maturational Con AII (adsorbed on concanavalin A-Sepharose) hormone were able to stimulate vitellogenesis in the female winter flounder (Pseudopleuronectes americanus). Both the salmon vitellogenic Con AI 45,000-dalton hormone and the maturational Con AII hormone were able to enhance in vivo phosphate and leucine uptake by the flounder testis and incorporation into testicular proteins. Only the salmon maturational Con AII hormone was able to evoke spermiation and ovulation and it was much more potent than the vitellogenic Con AI hormones in stimulating testicular steroidogenesis. The vitellogenic and maturational activities of the salmon Con AII gonadotropin were partially destroyed on desialylation while the vitellogenic activity of the Con AI gonadotropins was not affected. The immunologic activity of the maturational Con AII hormone was retained after desialylation, but was abolished upon carboxamidomethylation. The immunologic potencies of the vitellogenic Con AI hormones were preserved after desialylation. The two salmon vitellogenic Con AI hormones displayed numerous prominent differences in amino acid and carbohydrate compositions from, and were immunologically remote from, the salmon maturational Con AII hormone. There was a marked dissimilarity in amino acid composition between the immunologically distinct salmon vitellogenic Con AI hormones. Two gonadotropins (vitellogenic Con AI and maturational Con AII), with an identical molecular weight of 30,000 daltons, were prepared from the pituitaries of the carp Cyprinus carpio. The distribution of biological activities was in general similar to that observed between the salmon hormone. The carp vitellogenic Con AI hormone could also be distinguished from the maturational Con AII hormone in the composition of its protein and carbohydrate moieties.

Hormonal control of vitellogenesis in hypophysectomized winter flounder (Pseudopleuronectes americanus Walbaum)

A surgical method of hypophysectomy employing a lateral approach to the brain is described; this technique results in total hypophysectomy and 90% survival. Hypophysectomy decreased the incorporation of 33PO4 or [3H]leucine into yolk by the liver and abolished the accumulation of labeled yolk by the gonad. Estradiol treatment of hypophysectomized fish stimulates growth of the liver and hepatic synthesis of yolk proteins. No incorporation of yolk into the gonads was seen after estradiol treatment, and yolk protein accumulated in serum to higher levels than those seen in sham-operated fish. A glycoprotein preparation from teleost pituitaries, which should contain any classical gonadotropin, failed to stimulate gonadal incorporation of the yolk which accumulated in the serum. In contrast, the nonglycoprotein fraction of these pituitaries stimulated yolk incorporation into the ovary.

Maturational steroids and gonadotropin in upstream migratory sockeye salmon

The circulating serum concentrations of various steroid hormones in mature sockeye salmon were measured at four different developmental stages in their upstream migration to spawn at Adams River in British Columbia, Canada. In females, a high level of estradiol-17 beta was found in fish at the first location, and it persisted until immediately before reaching the spawning grounds, 485 km upstream, where it decreased to a minimal level. Free testosterone was extremely high throughout the migration but decreased significantly after spawning while its glucuronide changed reciprocally. Free and conjugated 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-P) peaked at the last location before spawning with the glucuronide only 20% of the free steroid in concentration. After spawning, the concentration of free steroid declined but the glucuronide remained constant. 17 alpha-hydroxyprogesterone increased significantly in serum before and after the fish had spawned. During the migration 11-deoxycortisol was present in the serum at all stages but maximal levels were found in postspawned fish. Throughout the migration, the males had high serum levels of 11-ketotestosterone. Lesser amounts of free testosterone were also consistently present but the ratio of free: conjugated decreased from 1.7 at the beginning of the migration to 0.15 on the arrival at the Adams River and 0.10 in postspawned fish. Only low levels of 11 beta-hydroxytestosterone were found except in the postspawned males where the value was equal to one-half that of free testosterone. As in the females, there was a substantial increase in the levels of 17 alpha, 20 beta-P and its glucuronide in the males captured at the spawning grounds. In both sexes gonadotropin levels were low during the migration and high in the postspawned fish.

Plasma vitellogenin in landlocked atlantic salmon (Salmo salar ouananiche): isolation, homologous radioimmunoassay and immunological cross-reactivity with vitellogenin from other teleosts☆☆☆

Vitellogenin was isolated by affinity chromatography and gel filtration from landlocked Atlantic salmon plasma. Vitellogenin was labelled with iodine-131 using iodogen and an homologous radioimmunoassay was developed. There was poor immunological cross-reactivity with vitellogenin or plasma from other teleosts. Parallelism of the vitellogenin standard to the displacement by plasma of vitellogenic salmon allowed the assay to be used to evaluate the seasonal concentration profile of vitellogenin in female adult salmon. Extracts of liver or ovary from female Atlantic salmon also yielded displacements parallel to the vitellogenin standard in the assay.

Studies on two types of gonadotropins from both American plaice and winter flounder pituitaries

Vitellogenic Con AI (unadsorbed on concanavalin A-Sepharose) and maturational Con AII (adsorbed on concanavalin A-Sepharose) hormones which existed in “big” and “little” forms, were isolated from the pituitaries of American plaice (Hippoglossoides platessoides) and winter flounder Pseudopleuronectes americanus. Differences in amino acid and carbohydrate compositions as well as immunologic remoteness between the plaice vitellogenic Con AI and maturational Con AII gonadotropins established their separate natures. There was a great variation in amino acid composition between the “big” and “little” forms of the plaice vitellogenic Con AI hormones. The plaice maturational Con AII hormones were rich in sugars. The plaice vitellogenic Con AI hormones, distinguished by their nonadsorption on Con A-Sepharose, could be set apart from tetrapod gonadotropins by their extremely low carbohydrate contents. The flounder vitellogenic Con AI hormones, which had no maturational activity, manifested little cross-reaction in a radioimmunoassay for the maturational Con AII hormone. The flounder maturational Con AII hormone also possessed vitellogenic activity.

“Big” and “little” forms of plaice vitellogenic and maturational hormones

Abstract A gonadotropin, which was not absorbed on Con A-Sepharose and therefore either nonglycoprotein in nature or characterized by a low carbohydrate content or lacking in the appropriate sugars requisite for binding to Con A, had been prepared from plaice (Hippoglossoides platessoides) pituitaries. Filtration through Ultrogel and subsequent chromatography on either Bio-Gel or Ultrogel permit the isolation of two forms with molecular weights of 28,000 and 62,000, respectively. Both molecular species were active in stimulating ovarian growth and vitellogenesis as indicated by their ability to elevate the gonadosomatic index, the ovarian uptake of 33 PO 4 3− and [ 3 H] leucine, and the incorporation of the radiolabels into the protein and phosphoprotein fractions of the ovary of the hypophysectomized winter flounder (Pseudopleuronectes americanus), but neither exerted any effect on the processes of oocyte maturation and ovulation. Another plaice gonadotropin, which was potent in stimulating the processes of oocyte maturation and ovulation in the hypophysectomized winter flounder and which also existed in two forms with molecular weights of 28,000 and 62,000, could be isolated from the glycoprotein fraction of the pituitary extract with similar chromatographic procedures. The 28,000 MW gonadotropins manifested a strong tendency to undergo aggregation leading to the formation of the 62,000 MW species in buffer containing low concentrations of EDTA. The phenomenon of assocation could be prevented by the inclusion of a high concentration (10 mM) of EDTA in the elution buffer. High concentrations of sodium chloride and dithiothreitol in the buffer appeared to be less effective in minimizing aggregation. The relative magnitudes of the molecular weights and the comparable biological activities of the two forms are indicative of a monomer-dimer relationship. The winter flounder pituitary also secretes hormones similar in chromatographic behavior to those of the plaice. The presence of two distinct types of biological activities in two chromatographically discrete fractions of the Hippoglossoides platessoides pituitary extract, when tested in a closely related species Pseudopleuronectes americanus after hypophysectomy, constitutes the first unequivocal evidence based on both biochemical and physiological criteria for the existence of separate gonadotropins controlling vitellogenesis and maturation in a teleost.

5 Teleost Gonadotropins: Isolation, Biochemistry, and Function

Publisher Summary This chapter discusses the isolation, biochemistry, and function of teleost gonadotropins. Methods of purification include solvent fractionation, affinity chromatography, gel filtration, and ion-exchange chromatography. Reports on purification of a single gonadotropin from various teleost species appear to be in contrast to the discovery of vitellogenic hormone and maturational hormone. The difference stems from the use of or lack of affinity chromatography (Con A-Sepharose) as one of the purification procedures and the fact that most workers did not monitor vitellogenic activity during the course of purification. The gonadotropins that were isolated, as judged from the spectrum of biological activities, correspond to maturational hormones. Maturational gonadotropin levels in plasma do not appear to correlate well with the reproductive processes. Substantial evidence has been collected for the existence of two gonadotropins, vitellogenic hormone, and maturational hormone in teleost. The two hormones manifest distinctive chromatographic, compositional, immunological, and biological characteristics. Antiserum to each of these two hormones produces inhibitory effects on those aspects of fish reproduction, which are consistent with the biological activities of the hormone.

Effect of acute exposure to crude petroleum on some reproductive hormones in salmon and flounder

1. The concentration of total (free + conjugated) androgens in plasma of sexually mature male salmon and flounder was generally lower in oil-exposed fish. 2. Exposure to crude oil inhibited some testicular development of salmon during the final stages of maturation. 3. Oil exposure had no effect on levels of total plasmatic androgens or estradiol in male and female flounder during gonadal recrudescence. 4. Aryl hydrocarbon hydroxylase activity increased in liver and kidney of both species. 5. Sperm, collected from salmon exposed to oil, fertilized eggs from non-exposed females to produce normal alevins.