Yoshitaka Nagahama

Isolation and characterization of two distinct gonadotropins from chum salmon pituitary glands

Two distinct gonadotropins, GTH I and GTH II, were extracted with 35% ethanol-10% ammonium acetate, pH 6.1, from female chum salmon pituitary glands, and purified by ion-exchange chromatography on DE-52 and CM-Sephadex C-25 by stepwise elution, and gel filtration on Sephadex G-75. Gonadotropic activities of these preparations were demonstrated in vivo by stimulation of gonad growth in juvenile rainbow trout, and in vitro by enhancement of estradiol-17 beta production by amago salmon ovarian follicles. Molecular weights were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 50,000 and 36,000 for GTH I and GTH II, respectively. Both gonadotropins are glycoproteins composed of two distinct subunits with N-terminal amino acid residues of Tyr/Gly for GTH I, and Tyr/Ser for GTH II. These results suggest the presence in teleost fish of two chemically distinct gonadotropic glycoproteins.

The duality of teleost gonadotropins.

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

Steroidogenic activities of two distinct salmon gonadotropins

The effects of salmon gonadotropins, GTH I and GTH II, on production of two major steroid hormones in female salmonid reproduction, estradiol-17 beta and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) were compared using amago salmon (Oncorhynchus rhodurus) intact ovarian follicles in vitro. In addition, the production of 17 alpha-hydroxyprogesterone (17 alpha-OHprog) by thecal layers and 17 alpha,20 beta-diOHprog by granulosa layers in response to GTH I and II was examined during oocyte maturation. Both GTHs enhanced estradiol-17 beta production by midvitellogenic ovarian follicles in a dose-dependent manner; there was no significant difference in potency between GTH I and II. In postvitellogenic follicles, GTH II appeared to be more effective in stimulating 17 alpha,20 beta-diOHprog production than GTH I. GTH II was also found to be more potent than GTH I in stimulating 17 alpha-OHprog production by thecal layers and 17 alpha,20 beta-diOHprog production by granulosa layers in the presence of 17 alpha-OHprog. Thus, GTH II appears to differ from GTH I showing a reproductively high specificity for 17 alpha,20 beta-diOHprog production during oocyte maturation.

Development of salmon GTH I and GTH II radioimmunoassays.

Radioimmunoassays (RIAs) for the measurement of two gonadotropins, GTH I and GTH II, in the plasma and pituitary of salmonid fish were developed using a rabbit antiserum to beta-subunits of chum salmon GTH I and GTH II. Intact GTH I and GTH II were used as standards and radioactive competitors. The displacement curves for plasma of salmonids including chum salmon, amago salmon, and rainbow trout were parallel to chum salmon GTH I and GTH II standards. Parallel displacement curves were obtained for pituitary extracts of chum salmon and amago salmon. The cross-reactivities of growth hormone, prolactin, and proopiomelanocortin (POMC)-related hormones were less than 1% in both RIAs. However, cross-reactivities of GTH I in the GTH II RIA and GTH II in the GTH I RIA were 10 and 12%, respectively. Plasma concentrations of both GTHs from salmonids at various stages of reproductive development were compared. In immature rainbow trout of both sexes (males: average (AV) gonadosomatic index (GSI) = 0.05; females: AV GSI = 0.24), plasma levels of GTH I and GTH II were low (less than 2 ng/ml). During prematurational stages of spermatogenesis and vitellogenesis in rainbow trout (males: AV GSI = 0.43; females: AV GSI = 2.8), the predominant GTH in the pituitary and plasma was GTH I. In contrast, plasma concentrations of GTH II were significantly higher than those of GTH I in postovulatory amago and chum salmon. Similarly, pituitary concentrations of GTH II were significantly higher than those of GTH I in postovulatory and spermiating amago salmon and postovulatory chum salmon.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine control of oocyte maturation

Embryogenesis in vertebrates is initiated by fusion of male and female gametes. In most vertebrates, however, full-grown postvitellogenic oocytes in the ovary are still in prophase I of meiosis and cannot be fertilized; for the oocytes to be fertilizable, they must complete the first meiotic division. Under appropriate hormonal stimulation, fullgrown oocytes resume their first meiotic division which involves breakdown of the germinal vesicle (GVBD), chromosome condensation, assembly of the first meiotic spindle, and extrusion of the first polar body (Fig. 1). In most cases, however, meiosis is again arrested at the metaphase II stage. Shortly thereafter, mature fertilizable oocytes are ovulated. The meiotic process leading to extrusion of the second polar body is resumed again at the time of fertilization, immediately after sperm penetration. The time period between the resumption of meiosis and the second meiotic metaphase has been referred to as the period of oocyte maturation (meiotic maturation). Thus, the process of oocyte maturation is a prerequisite for successful fertilization.

Development of the steroidogenic capacity of medaka (Oryzias latipes) ovarian follicles during vitellogenesis and oocyte maturation.

Developmental changes in the steroidogenic capacity of medaka, Oryzias latipes, ovarian follicles at 12 different stages during vitellogenesis and oocyte maturation were examined using 18-hr incubations. Medaka were acclimated to conditions of 26 degrees on a lighting regime of 14 hr light and 10 hr dark. Under these conditions, females usually spawn daily within 1 hr of the onset of light. The process of vitellogenesis and oocyte maturation occurs within 72 hr, the breakdown of the germinal vesicle (GVBD) and ovulation being completed at 6 and 1 hr, respectively, before the expected time of spawning. Vitellogenic follicles between 32 and 16 hr before spawning produced large amounts of estradiol-17 beta spontaneously and in response to partially purified chum salmon gonadotropin (SGA) or pregnant mares serum gonadotropin (PMSG). However, postvitellogenic follicles between 12 and 4 hr before spawning showed very little evidence of estradiol-17 beta production. By contrast, basal concentrations of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog) remained very low in follicles during vitellogenesis and were elevated in those collected during oocyte maturation; there was a close relationship between the medium concentration of 17 alpha,20 beta-diOH-prog and the percentage GVBD in the oocytes. 17 alpha,20 beta-DiOHprog production in response to PMSG was very low in follicles during early and mid-vitellogenesis and increased in those collected at 28 hr before spawning, a time which coincided with the first acquisition of the ability of the follicles to undergo maturation in response to gonadotropin. These results clearly demonstrate that a distinct shift from the secretion of predominantly estradiol-17 beta to the secretion of 17 alpha,20 beta-diOHprog occurs in the medaka ovarian follicle immediately prior to oocyte maturation. Considering the potency of 17 alpha,20 beta-diOHprog for the induction of oocyte maturation in vitro, these results further suggest that 17 alpha,20 beta-diOHprog is a naturally occurring steroidal mediator of oocyte maturation in the medaka.

Developmental changes in steroidogenic responses of ovarian follicles of amago salmon (Oncorhynchus rhodurus) to chum Salmon gonadotropin during oogenesis

Changes in amago salmon (Oncorhynchus rhodurus) ovarian thecal and granulosa layer function in association with the production of two biologically important ovarian mediators of oocyte growth and maturation in salmonids, estradiol-17 beta and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog), were investigated using isolated follicular preparations in vitro. A distinct shift of steroidogenic responses of intact follicles from estradiol-17 beta to 17 alpha,20 beta-diOHprog in response to partially purified chum salmon gonadotropin (SGA) occurred immediately prior to oocyte maturation. Aromatase activity in granulosa layers increased during vitellogenesis and decreased rapidly prior to oocyte maturation. This decrease in aromatase activity was coincident with the decreased ability of intact follicles to produce estradiol-17 beta in response to SGA. Since testosterone production in thecal layers did not decline during this time, the reduced production of estradiol-17 beta by postvitellogenic follicles is due, in part, to decreased aromatase activity in granulosa layers. Immediately prior to oocyte maturation, intact follicles acquire an increased ability to produce 17 alpha,20 beta-diOHprog in response to SGA. Although granulosa layers first acquired the ability to convert exogenous 17 alpha-hydroxyprogesterone (17 alpha-OHprog) to 17 alpha,20 beta-diOHprog (20 beta-hydroxysteroid dehydrogenase, 20 beta-HSD, activity) in response to SGA about 2 months prior to oocyte maturation, thecal layers did not develop the ability to produce 17 alpha-OHprog in response to SGA until immediately prior to oocyte maturation. Thus, changes in thecal cell function are critical for intact follicles to acquire the ability to produce 17 alpha,20 beta-diOHprog in response to gonadotropin.

Involvement of 3',5'-cyclic adenosine monophosphate in the control of follicular steroidogenesis of amago salmon (Oncorhynchus rhodurus)

The role of cAMP in the control of follicular steroidogenesis of amago salmon (Oncorhynchus rhodurus) was studied using in vitro incubation of isolated thecal and granulosa layers. Particular attention was paid to the role of cAMP in the shift in the steroidogenic responses of follicle layers to gonadotropin (partially purified chum salmon gonadotropin, SGA) during oogenesis. First, the effects of SGA and forskolin, an activator of adenylate cyclase, on intratissue accumulation of cAMP were determined using isolated thecal and granulosa layers from various stages of development. Regardless of the stage of development, SGA and forskolin stimulated cAMP formation in both layers within 1 hr of incubation. Second, the effects of SGA, forskolin, dibutyryl cAMP, and inhibitors of phosphodiesterase on testosterone and 17 alpha-hydroxyprogesterone production by thecal layers and on the activities of aromatase and 20 beta-hydroxysteroid dehydrogenase in granulosa layers from follicles of various developmental stages were investigated. All steroidogenic actions of SGA were mimicked by those agents known to raise the cellular level of cAMP. These results provide evidence that the steroidogenic actions of gonadotropin in both thecal and granulosa layers depend on increased intracellular cAMP, and they further suggest that a change in cellular events at a step(s) subsequent to cAMP production is involved in regulating the shift in the steroidogenic responses of follicle layers to gonadotropin.

Shift in Steroidogenesis in the Ovarian Follicles of the Goldfish (Carassius auratus) during Gonadotropin‐Induced Oocyte Maturation

Both partially purified chum salmon gonadotropin and 17α‐hydroxyprogesterone stimulated in vitro production of testosterone by postvitellogenic follicles of goldfish (Carassius auratus). Chum salmon gonadotropin further enhanced the conversion of exogenously supplied 17α‐hydroxyprogesterone to 17α, 20β‐dihydroxy‐4‐pregnen‐3‐one. The increased medium concentrations of 17α, 20β‐dihydroxy‐4‐pregnen‐3‐one were associated with the induction of final oocyte maturation.

Developmental changes in the properties of gonadotropin receptors in the ovarian follicles of amago salmon (Oncorhynchus rhodurus) during oogenesis

The presence of specific, saturable, high-affinity gonadotropin receptors was demonstrated in membrane preparations from preovulatory ovarian follicles of amago salmon (Oncorhynchus rhodurus), including intact follicles, isolated thecal layers, and isolated granulosa cells. Optimum conditions for the binding study using the amago salmon receptor system were similar to those previously reported for postovulatory ovaries of the same species (A. Kanamori, H. Kagawa, and Y. Nagahama, 1987, Gen. Comp. Endocrinol. 66, 210-217). Scatchard analysis of chum salmon gonadotropin (CSG-SII) binding to the membrane fraction suggested the presence of high-affinity binding sites in the intact follicles, isolated thecal layers, and isolated granulosa cells at all stages of development. The dissociation constant is consistent with those reported for gonadotropin receptors in several teleost gonads and in other vertebrate classes (about 0.1-1 nM). During oogenesis, the number of binding sites per follicle increased from about 20 to about 60 pg. Similarly an increase in binding sites was observed with granulosa cells and thecal layers during oogenesis. These findings show an increase in the number of gonadotropin receptors in the follicles and are in good temporal agreement with the developmental changes in follicular steroidogenesis in response to gonadotropin. The increase in gonadotropin receptors in the thecal layer was associated with the increased capacity for production of testosterone, whereas the increase in gonadotropin receptors in the granulosa cells was associated with an increase in gonadotropin sensitivity in terms of 20 beta-hydroxysteroid dehydrogenase activation.