T. Bun Ng

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.

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.

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.

Gonadotropic regulation of androgen production in flounder and salmonids

Maturational hormones, prepared from pituitaries of winter flounder (Pseudopleuronectes americanus), American plaice (Hippoglossoides platessoides), carp (Cyprinus carpio), and chum salmon (Oncorhynchus keta), and characterized by adsorption on Con A-Sepharose, reinitiated the production of 11-ketotestosterone and testosterone by hypophysectomized male winter flounder. Stimulation of 11-ketotestosterone could be observed 24 and 48 hr after a single injection of flounder maturational hormone. Hypophysectomy resulted in a disappearance from the circulation of sex hormone-binding protein which could be restored by both maturational and vitellogenic hormones. The flounder maturational hormone also induced spermiation and ovulation while 17α-hydroxyprogesterone stimulated oocyte maturation with no ovulation. The vitellogenic hormones prepared from the fractions of flounder, plaice, and salmon pituitary extracts unadsorbed on Con A-Sepharose, had no androgenic activity and did not evoke spermiation or oocyte maturation, but were able to stimulate the incorporation of serum phosphoprotein(s) into ovarian yolk. An antiserum against flounder maturational hormone lowered the plasma level of testosterone in female flounder. The salmon maturational hormone induced synthesis of androgens in intact juvenile rainbow trout (Salmo gairdneri) of both sexes and resulted in a predominance of 11-ketotestosterone in the males and testosterone in the females. Treatment of intact mature male landlocked salmon (Salmo salar sebago), with an antiserum to the chum salmon maturational hormone, led to a dramatic decrease of 11-ketotestosterone in the circulation. A combination of glycosidases, including neuraminidase, glucosaminidase, galactosidase, and mannosidase, had no effect on the immunological activities of salmon and flounder maturational hormones.

A vitellogenic hormone with a large and a small form from salmon pituitaries

Abstract Two subfractions, with molecular weights of 25,000 and 45,000, respectively, were discovered in a fraction of chum salmon ( Oncorhynchus keta ) pituitary extract, which was not absorbed on concanavalin A (Con A)-Sepharose. This fraction, designated as Con AI, had been rechromatographed on Con A-Sepharose to assure exclusion of the glycoprotein (Con AII) gonadotropin. The two forms of the Con AI hormone displayed vitellogenic activity in the hypophysectomized winter flounder ( Pseudopleuronectes americanus ) as evidenced by their ability to stimulate the uptake of H 3 33 PO 4 and [ 3 H]leucine into the whole fish ovary and the protein fraction of the ovary, as well as the incorporation of the radioisotopes into the lipophosphoprotein fraction in vivo . The absence of any significant interference from the Con AI vitellogenic hormones in the radioimmunoassay for the salmon Con AII hormone, reinforced by an earlier finding of specificity of action of the latter hormone in the chick testicular radiophosphate uptake and immature trout gonadal cAMP production assays ( D. R. Idler, L. S. Bazar, and S. J. Hwang, 1975a , Endocrine Res. Commun. , 2, 215–235), provides strong grounds for the contention that two different types of gonadotropic hormones are elaborated by the chum salmon pituitary.

Pituitary hormones that stimulate the thyroidal system in teleost fishes.

The pituitaries of American plaice, winter flounder, salmon, and carp contain fractions, unadsorbed (Con AI) or adsorbed (Con AII) on concanavalin A-Sepharose, which stimulate the thyroidal system of hypophysectomized (hypex) winter flounder. Chum salmon Con AII thyrotropic fraction (Con AII TF) stimulates the accumulation of thyroxine (T4) and some triiodothyronine (T3). The appropriate fraction has a molecular weight of approximately 40,000, is retained on DEAE Bio-Gel A and on CM Bio-Gel A, and is designated Con AII 40K DEII CMII. This fraction was previously shown not to stimulate the synthesis of 11-ketotestosterone at a concentration where the corresponding CMI-gonadotropin (GtH) increased the level 45-fold. The salmon thyrotropic fraction not adsorbed on Con A, Con AI 45K DEIII CMII, stimulates T3 accumulation in a dose-dependent manner and only has a transient effect on plasma T4. The CMII thyrotropic fraction failed to stimulate vitellogenesis in hypex flounder at a dose where the appropriate CMI-GtH was active. Flounder and plaice Con AI TF also mainly stimulate plasma triiodothyronine (T3), resulting in low plasma ratios; Con AII TF behaves like its presumed bovine TSH homolog in elevating plasma thyroxine (T4) with small changes in T3, resulting in high plasma ratios. That Con AI TF stimulates primarily plasma T3 is supported by the significant reduction in plasma T3 and elevation of ratio after administering antibodies specific for Con AI TF; that Con AII TF stimulates primarily plasma T4 is supported by the significant reduction in plasma T4 and drop in ratio after administering antibodies specific for Con AII TF. Carp Con AI TF increased plasma T3; it had the same behavior on ion exchangers as those from the other species. The findings are supported by comparative studies using salmon Con AII hormone on rainbow trout, and antibodies to the salmon hormones on trout and Atlantic salmon. As expected, antibodies to Con AI TF of salmon decreased plasma T3 while antibodies to salmon Con AII TF diminished plasma T4.

The immunofluorescent location of teleost gonadotropins and thyrotropins in flounder pituitary

Abstract Antibodies to teleost gonadotropins and thyrotropins have been used to investigate their cytological origin in flounder pituitary. The maturational, high (Con AII) glycoprotein, gonadotropin locates separately from the vitellogenic, low (Con AI) glycoprotein, gonadotropin. In female flounder just before or just after spawning the maturational gonadotropin is particularly obvious in PAS-staining cells at the anterior margin of the proximal pars distalis. The Con AII thyrotropin is also found in this region but has a more restricted distribution. The pituitaries of early vitellogenic females have little detectable Con AII gonadotropin. The Con AI gonadotropin does not overlap the principal concentration of Con AII gonadotropin in spawning females; it is found in cells of the proximal pars distalis, interspersed with the somatotrops; it is abundant in pituitaries from vitellogenic females. The Con AI thyrotropin is also present in the proximal pars distalis but has a more limited distribution than the Con AI gonadotropin. The Con AI thyrotropin does not overlap the distribution of the Con AII thyrotropin in spawning females.

Effects of teleost gonadotropins and their antibodies on gonadal histology in winter flounder.

Abstract The gonadotropic actions of teleost vitellogenic and maturational hormones were studied in flounder hypophysectomized shortly after reinitiation of vitellogenesis and spermatogenesis in August. Maturational hormone alone was able to stimulate formation of yolky oocytes, but vitellogenic hormone required the action of estrogen before it could stimulate production of yolky oocytes. An antiserum to vitellogenic hormone induced atresia of yolky oocytes and lowered the gonadosomatic index in vitellogenic flounder whereas an antiserum to maturational hormone did not affect ovarian histology or gonadosomatic index to any great extent. Preincubation of flounder ovarian sections with vitellogenic hormone, followed by incubation with antiserum to vitellogenic hormone and fluorescein isothiocyanate-labeled goat-anti-rabbit γ-globulin in succession, resulted in immunofluorescence in ooplasm of both large immature and vitellogenic oocytes, and in follicular envelopes of vitellogenic oocytes. Preincubation of ovarian sections with maturational hormone resulted in fluorescence in the interstitial tissue, and the follicular envelopes and the perinuclear regions of yolky oocytes. Maturational hormone stimulated spermatogenesis in hypophysectomized male flounder. The significance of these results in relation to previous studies was discussed.