Angiotensins stimulate in vitro ovulation and contraction of brook trout (Salvelinus fontinalis) follicles

The effects of salmon angiotensin I (sAI) and human angiotensin II (hAII) on in vitro ovulation of preovulatory (preOV) brook trout (Salvelinus fontinalis) follicles were investigated. Both angiotensins increased levels of ovulation above that in controls after 12 hours of incubation. The increase was statistically significant in incubates with greater than 1 M hAII. The effects of the angiotensins on follicle contraction were also studied indirectly by measuring the decrease in weight of punctured follicles taken prior to germinal vesicle breakdown. At 1 M, both angiotensins significantly decreased the weight of punctured follicles after 16 hours of incubation. The angiotensin-stimulated decrease in weight was not blocked by indomethacin (10 g/ml), indicating that follicle contraction was not prostaglandin-dependent. The data indicate that angiotensins might be directly involved in brook trout ovulation and the stimulatory effects of angiotensins on ovulation may be attributed to their effects on follicle contraction.This work was supported by NIH grant #HD25924-02.     

Hormonal changes during meiotic maturation and ovulation in the brook trout (Salvelinus fontinalis)

The plasma levels of estradiol-17 (E2), 17, 20-dihydroxy-4-pregnen-3-one (17,20-P) and gonadotropin (GTH) were measured in brook trout (Salvelinus fontinalis) during the period from the end of vitellogenesis to postovulation. Blood samples were taken according to specific stages of maturation, including germinal vesicle breakdown (GVBD) and ovulation. E2 levels were quite high (45 ng/ml) at the end of vitellogenesis (and prior to GVBD) and dropped precipitously by GVBD (2 ng/ml). They remained low through ovulation and postovulation. 17,20-P levels were low prior to GVBD (0.7 ng/ml) and increased dramatically at GVBD (148 ng/ml). The levels of 17,20-P remained high at ovulation (142 ng/ml) and then dropped significantly within 24 h to approximately half of the ovulatory values. They decreased even further by 7 days postovulation. GTH levels rose gradually through GVBD and ovulation from a postvitellogenic level of approximately 3 ng/ml to a 7 day postovulatory value of approximately 10 ng/ml. The overall results; 1) decrease in estradiol prior to GVBD, 2) increase in 17,20-P at GVBD and 3) gradual GTH rise through GVBD and ovulation, are similar to those reported for other salmonids.     

Synthesis of 17,20α- and 17,20β-dihydroxy-4-pregnen-3-ones, 11-ketotestosterone and their conjugates by gills of teleost fish

Goldfish, carp and trout gills were incubated with ³H-17-hydroxyprogesterone (17P). With goldfish gills, the metabolites were 17,20-dihydroxy-4-pregnen-3-one (17,20P; 82%), 17,20-dihydroxy-4-pregnen-3-one (17,20P; 8%), 11-ketotestosterone (KT) glucuronide (5.4%) and 17,20P glucuronide (0.2%). Sulfates were not detected. Carp gills converted 17P into 17,20P (11.2%), 17,20P (9.6%), KT (8.4%), glucuronides of 17,20P (1.3%) and 17,20P (1.6%) and sulfates of 17,20P (5.1%) and 17,20P (7.2%). 17,20P (38% free, 1.8% glucuronide and 21.1% sulfate) was the sole metabolite of ³H-17P in trout gill incubations. In the presence of high (10; µg ml^-1) substrate concentration, cyprinid gills gave predominantly free 17,20P, while trout gills yielded only free 17,20P. Production of 17,20P, predominantly as its sulfate, from endogenous precursors was demonstrated in trout gills but was not stimulated by trout primary extract. Our results demonstrate for the first time the steroidogenic potential of teleost gills and suggest that they may play a role in secretion of pheromones in some species.     

Regulation of oocyte maturation in the rainbow trout,Salmo gairdneri: role of cyclic AMP in the mechanism of action of the maturation inducing steroid (MIS), 17α-hydroxy, 20β-dihydroprogesterone

In fish, oocyte maturation (resumption of meiosis after completion of vitellogenesis and before ovulation) is triggered by maturation inducing steroids (MIS) which generally appear to be secreted in the ovary in response to stimulation by a pituitary maturational gonadotropin. Converging data from different laboratories show that 17-hydroxy, 20-dihydroprogesterone (17, 20-OH-P) is the principal MIS in salmonoids; but clear identification remains to be done in other taxonomic groups.The experiments reported here in the rainbow troutSalmo gairdneri examine the possible involvement of oocyte cAMP on the mechanism of MIS action. The action of 17, 20-OH-P, on germinal vesicle breakdown (GVBD) in oocytes incubatedin vitro within the follicle, was inhibited by various substances expected to elevate the intraoocyte concentrations of cAMP: cAMP ( 1 mM) or dibutyril cAMP ( 2 mM), phosphodiesterase inhibitors such as theophylline ( 0.2 mM) or 3-isobutyl-1 methylxanthine (IBMX 0.1 mM), adenylate cyclase activators such as cholera toxin (> 100 nM) or forskolin ( 0.03 mM). In fact, the combined action of IBMX (1 mM) and forskolin (0.01 or 0.05 mM)in vitro was to promote accumulation of intraoocyte cAMP within 1 to 5 hours. Oocyte cAMP concentrations exhibited a large variability between different females, depending on the stage of oocyte development; a significant positive correlation between oocyte cAMP concentration and the follicular weight, and a significant negative correlation between oocyte cAMP concentration and the median efficient dose of 17, 20-OH-P for induction of GVBD, were observed. Finally, when intrafollicular oocytes were incubatedin vitro, the addition of a maturation-inducing concentration of 17, 20-OH-P (3×10^–6M) induced a significant decrease of oocyte cAMP within the first 10 hours of incubation. These results show that cAMP appears to play a central role in the regulation of oocyte sensitivity to 17, 20-OH-P and in the intraoocyte mechanisms leading to GVBD in trout.These data are discussed together with the few indications available in fish concerning the mechanism of MIS action which can be compared to some extent with the amphibian model.     

Overripening of ovulated eggs in goldfish,Carassius auratus: II. Possible involvement of postovulatory follicles and steroids

Changes in steroid hormone levels in the serum and ovarian fluid during overripening were studied in goldfish. Ovulated eggs retained in the ovarian cavity became overripe at around 12h after ovulation based on loss of fertilizability, with advanced degeneration by 24h. Blood and ovarian fluid were taken at 0, 3, 6, 12, 18 and 24h after ovulation. Both serum and ovarian fluid progesterone (P) showed a highly significant decline by 18h with a further decline by 24h; P levels were higher in the ovarian fluid. Serum 17,20-dihydroxy-4-pregnen-3-one(17,20-P) levels showed a progressive and more rapid decline, decreasing significantly by 12h with further decreases by 18h and 24h. Serum testosterone (T) levels increased significantly at 3h and remained high till 18h, thereafter they declined to the 0h level. No significant changes in estradiol-17 (E₂) levels were observed in the serum, except for a significant difference between 6 and 24h. There were no significant changes in ovarian fluid E₂, T or 17,20-P levels.The postovulatory follicles (POFs) showed degenerative changes which corresponded to the decline in P and 17,20-P. The hypothesis that overripening may be associated with declining levels of P or 17,20-P was tested in vivo by immersing just ovulated females (0h) in P or 17,20-P solutions for 12h, and in vitro by immersing just-ovulated eggs (0h) in ovarian fluid with an anti-serum against P (Anti-P). In the former, P at 0.05 ppm significantly enhanced the fertilization rate of ovulated eggs stripped from the females at 12h while 17,20-P did not produce a significant effect at the tested concentrations (0.025 and 0.05 ppm) on the fertilization rate. In the latter, anti-P significantly lowered the fertilization rate of 0h ovulated eggs after 6h incubation. The evidence suggests a role of P in the maintenance of viability of ovulated eggs.     

Steroidogenesis by ovaries and testes of the European catfish,the wels (Silurus glanis), in vitro

Testosterone, 3,17-dihydroxy-5-pregnen-20-one, 17,20-dihydroxy-4-pregnen-3-one (17,20P) and 5-pregnane-3,17,20-triol were identified as the major metabolites of [³H] 17-hydroxyprogesterone in ovarian incubations of the European catfish Silurus glanis. 17,20P and the reduced triol were present only in ovaries from fish primed with carp hypophysial homogenate (chh) while testosterone yields were significantly higher in controls than in treated fish. 11-Ketotestosterone, 11-hydroxytestosterone and 17,20-dihydroxy-4-pregnen-3-one (17,20P) were identified as the major metabolites of [³H]17-hydroxyprogesterone in in vitro incubations of testes of a spermiating catfish. There was no significant production of conjugates or other water soluble metabolites by either sex. The stimulation of plasma 17,20P, 17,20P and 11-hydroxytestosterone by chh in primed but not control males suggests that the role of these steroids in spermiation should be further examined.     

Transport of 17α,20β-dihydroxy-4-pregnen-3-one in brook troutSalvelinus fontinalis ovarian follicles

Extremely low levels of the maturation inducing steroid (MIS) 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP) were found in the ooplasm and ovarian follicle membranes of Atlantic salmonSalmo salar ouananiche, a finding that is at variance with the elevated blood levels of the steroid. The uptake of MIS at physiological concentrations into brook trout follicles occurred by passive diffusion. Uptake of the steroid into the ovarian follicle membrane, consisting of zona radiata and the attached follicle cells, deviated from linearity in a double reciprocal plot. These results suggest that 17,20-DHP is binding to a receptor-like protein in the ovarian follicle or the zona radiata membrane surrounding the oocyte, and extend our previous demonstration of 17,20-DHP receptor-like activity in the zona radiata membrane of the late stage brook trout oocytes. An oocyte cytoplasmic receptor gave subunits on SDS PAGE that were similar to the membrane and cytosol receptors previously described.     

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

Plasma estradiol-17 (E₂), testosterone (T), 17,20-dihydroxy-4-pregnen-3-one (DHP) and 17,20,21-tri-hydroxy-4-pregnen-3-one (20-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E₂ and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E₂ and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20-S and oocyte GVBD by white perch follices. DHP and 20-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20-S in the control of FOM in white perch and white bass.     

Circulating levels and in vitro production of two maturation-inducing hormones in teleost: 17α,20β-dihydroxy-4-pregnen-3-one and 17α,20β,21-trihydroxy-4-pregnen-3-one, in a daily spawning wrasse, Pseudolabrus japonicus

The female bambooleaf wrasse, Pseudolabrus japonicus, spawns daily during the spawning season, and exhibits a diurnal rhythm of ovarian development. In the present study, we have investigated: (1) circulating levels of 17a, 20-dihydroxy-4-pregnen- 17,20-P) and 17,20,21-trihydroxy-4-pregnen-3-one (20-S) in females sampled at different times of the day during spawning season in captivity, and (2) in vitro production of 17,20-P and 20-S by follicle-enclosed oocytes at seven different develo tal stages. In addition, we developed a microtiter plate enzyme-linked immunosorbent assay (ELISA) for 17,20-P. Serum levels of 17,20-P and 20-S showed similar diurnal changes; substantial increases in these levels occurred around the time of germinal vesicle breakdown (GVBD). In vitro experiments showed that massive production of 17,20-P and 20-S occurred in follicles collected just before or during GVBD. Further, acute decreases in 17,20-P and 20-S production were found in the ovarian follicles just prior to ovulation, suggesting inactivation of the maturation-inducing hormone (MIH). These results, taken together with our previous data on the occurrence of GVBD in vitro, suggest a role for both 17,20-P and 20b-S as MIHs in the bambooleaf wrasse.     

Progestogen metabolism by ovaries of the roach (Rutilus rutilus L.) and the rudd (Scardinius erythrophthalmus L.)

Roach ovaries converted 17-hydroxyprogesterone to 17,20-dihydroxy-4-pregnen-3-one (17,20P) and to glucuronides of testosterone and 17,20P. Small amounts of 5-pregnane-3- and -3, 17, 20-triols, 7-hydroxy-5-reduced metabolites and 17,20-dihydroxy-4-pregnen-3-one (17,20P) were also formed. Rudd ovaries converted this substrate mainly to 17,20P, 5-pregnane-3- and -3,17,20-triols, 17,20-dihydroxy-5-pregnan-3-one and testosterone glucuronide. The main metabolites of progesterone with both species were 17,20P, 5-pregnane-3,17,20-triol and 7-hydroxy-5-reduced steroids. Rudd ovaries formed, in addition, 17,20-dihydroxy-5-pregnan-3-one from progesterone. The pattern of metabolites was markedly altered when the concentration of substrate was increased from 42ng to 1 µg or 100 µg. At the highest concentration, glucuronides and polar steroids were not detectable, while at low concentrations they accounted for over 50% of the metabolites. 20-Hydroxysteroid dehydrogenase was shown to have a very high capacity, producing 21–47 µg 17,20P from 100 µg 17-hydroxyprogesterone substrate with 200 mg ovarian tissue in 5h.     

Gonadotrophin-releasing hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic halibut (Hippoglossus hippoglossus)

In two separate spawning seasons, spermiating male Atlantic halibut were implanted with pellets containing gonadotrophin-releasing hormone agonist (GnRHa). Males were bled repeatedly, and milt samples were collected. Blood samples were assayed for free and conjugated steroids: testosterone, 11-ketotestosterone, 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20,21-trihydroxy-4-pregnen-3-one and steroids with a 17,20 configuration. Towards the end of the first season, pellets were implanted into three wild-caught and three hatchery-reared males. No control fish were available. The major progestogen in plasma was identified as sulphated 5-pregnane-3,17,20-triol (3,17,20-P-5-S). Concentrations of this steroid were stimulated by the GnRHa. Sulphated 17,20-P was also identified in the plasma, but at 10-fold lower concentrations than 3,17,20-P-5-S. In the middle of the second season, pellets were implanted into five hatchery-reared males; five unimplanted males were used as controls. Levels of androgens fell following GnRHa treatment, levels of progestogens rose briefly, and there was a significant increase in the fluidity of the milt. Of all the measured steroids, free and sulphated 17,20-P showed the best correlation with milt fluidity.     

Gonadotropin-induced changes in steroid production by ovaries of the common careCyprinus carnio L. around the time of ovulation

Ovarian fragments from both primed (gonadotrophin treated) and unprimed female carp were incubated either with or without carp hypophysial homogenate and steroid hormone production measured. In incubations without hypophysial homogenate, production of all the steroids measured was either very low or nondetectable and there was no significant difference between tissue from primed and unprimed fish. In the presence of carp hypophysial homogenate a very significant increase in production of testosterone, 17-hydroxyprogesterone and testosterone glucuronide was observed, but there was no significant difference between primed and unprimed fish. 17,20-Dihydroxy-4-pregnen-3-one (17,20P) was not stimulated by carp hypophysial homogenatein vitro in ovaries from unprimed fish, but a very significant increase in production of this hormone was observed in tissue from fish which had received a priming dose of pituitary hormone. It is suggested that the priming dose of pituitary extract used in the normal hypophysation procedure to induce ovulation in teleosts initiates the potential for synthesis of 17,20P in response to later gonadotrophin challenge, and that this initiation may be related to the migration of the germinal vesicle.A preliminary account of these results was presented at the Fish Culture Conference, Barcelona, August 1985 (Kime and Bieniarz 1985).     

The nature of hematological response in fish

Hematological status was examined in rainbow trout,Oncorhynchus mykiss, held for 3–4 weeks under temperature, photoperiod and P_{O 2} conditions approximating those of their winter, spring and summer habitats. The most striking change observed was in red cell population composition. In winter fish mature cells were predominant; juvenile and developing erythrocytes characterized spring and summer animals. Hemoglobin, hematocrit and both mean erythrocytic volume and hemoglobin were modestly lower in spring and summer than in winter fish. Red cell numbers were not significantly affected. These observations suggest that avoidance of viscosity-based increases in circulatory work cost is more advantageous than elevation of blood O₂-carrying capacity. Although hemoglobin isomorph profiles were significantly altered, there is little evidence that such changes are of critical adaptive importance. Given presumed age-based reduction in gas transport effectiveness, the replacement of mature and senescent cells by more metabolically-competent juvenile cells appears to be the pivotal event in hematological response. Leucocyte counts were significantly elevated in spring and summer as compared to winter fish. Lymphocyte/heterophil ratios declined from 8.27 in winter fish to 3.13 in summer trout. Thrombocyte, monocyte, eosinophil and basophil abundances were little changed.     

An enzyme linked immunosorbant assay (ELISA) for testosterone, estradiol, and 17,20β-dihydroxy-4-pregenen-3-one using acetylcholinesterase as tracer: application to measurement of diel patterns in rainbow trout (Oncorhynchus mykiss)

A simple and rapid Enzyme Linked ImmunoSorbant Assay (ELISA) is described and validated for testosterone, estradiol, and 17,20-dihydroxy-4-pregnen-3-one (17,20P). A general procedure for preparation of the acetylcholinesterase labeled steroid is described which is applicable to any steroid. Use of acetylcholinesterase tracer increased the sensitivity of assay so that reliable measurements of each steroid could be achieved with only 10 l of plasma. The ELISA was applied to measurement of all three steroids every hour for over 24 hours in a female trout using cannulation of the dorsal aorta. This high sampling frequency revealed several short-term (<2 h) episodic pulses of testosterone and estradiol.     

Dynamics of insulin and insulin-like growth factor-I (IGF-I) ovarian receptors during maturation in the brown trout (Salmo trutta)

In salmonid fishes, the role of insulin and insulin-like growth factor-I (IGF-I) in the regulation of ovarian function is not well understood. Recently, we reported that isolated follicular layers of the preovulatory ovarian follicle of coho salmon (Oncorhynchus kisutch) have specific receptors for insulin and IGF-I and that IGF-I modulates steroid production in the follicular layers. In the present study we have investigated the structural and functional characteristics of insulin and IGF-I receptors in the ovary of brown trout (Salmo trutta) and the changes in insulin and IGF-I binding throughout the reproductive cycle of this species. The specific binding for IGF-I was 8- to 15-fold higher than the specific binding for insulin. IGF-I receptors were also more specific than insulin receptors because unlabeled insulin displaced bound radiolabeled insulin at concentrations 40- to 80-fold lower than unlabeled IGF-I; whereas, unlabeled IGF-I displaced bound radiolabeled IGF-I at concentrations 4000- to 8000-fold lower than unlabeled insulin. Insulin and IGF-I receptors from the brown trout ovary were composed of 120 kDa -subunits and 90 kDa -subunits, which underwent autophosphorylation in a concentration-dependent manner. Receptor tyrosine kinase activity was also stimulated in a concentration-dependent manner by insulin and IGF-I. When ovarian insulin and IGF-I binding was determined from mid-vitellogenesis (March) until ovulation (November), maximal binding for both peptides was detected in mid-vitellogenesis and gradually decreased until the end of vitellogenesis (August). In the preovulatory period (October), a small increase of insulin and IGF-I binding was observed. After ovulation, insulin binding was no longer detectable and IGF-I binding was very low. These results suggest that insulin and IGF-I receptors in the salmonid ovary follow the structural pattern described in other vertebrate species and that insulin and IGF-I could be involved in the regulation of ovarian function during reproductive stages other than the preovulatory period.     

Effects of steroid hormones on in vitro oocyte maturation in white sturgeon (Acipenser transmontanus)

The in vitro effects of several steroids on the maturation of intact white sturgeon (Acipenser transmontanus) ovarian follicles were investigated. At the highest concentration (1024 ng ml^–1 for the C21 steroids and 1139 ng ml^–1 for the C19 steroids), all of the C21 steroids tested, progesterone (P4), 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,(20,21-trihydroxy-4-pregnen-3-one 20-S), 11-deoxycortisol (S) and cortisol (F), as well as testosterone (T) induced germinal vesicle breakdown (GVBD) at 14 and 22 h. At 6 h, only P4 and 17,20-P induced maturation at the highest concentration (1024 ng ml^–1). At 14 and 22 h, 11-deoxycortisol was the most potent steroid inducer of GVBD followed by P4, 17OHP, 17,20-P, and 20-S. The steroid 11-hydroxytestosterone (11OHT) was completely ineffective at all concentrations and exposure times. The C21 steroids induced oocyte maturation at concentrations ranging from 4 to 1024 ng ml^–1, whereas T induced GVBD at 225 to 1139 ng ml^–1. Calculation of the mean effective concentration that induced 50% GVBD (EC50) from the 22 h incubations revealed the following order of potencies: S > P4 > 17OHP > 17,20-P > 20-S >> F > T. These bioassay results, together with previous findings on the endogenous production of steroids by ovarian follicles from gonadotropin-primed females, indicate that more than one steroid has a biological role in the resumption of meiosis in sturgeon oocytes and provides empirical evidence for P4, 17OHP, S, 20-S, and 17,20-P as maturation-inducing steroids in white sturgeon.     

Effect of gonadotropin type II and 17-hydroxy-4-pregnene-3,20-dione on 17,20β-dihydroxy-4-pregnen-3-one production by rainbow trout testes at different developmental stages

Plasma levels of 17,20-dihydroxy-4-pregnen-3-one (17,20OHP), which is involved in the regulation of spermiation in male salmonid fish, increase dramatically at the time of spermiation. To advance the understanding of the regulation of 17,20OHP production during the spermatogenetic cycle in trout, we have studied the in vitro effect of gonadotropin type II (GtH II) and the precursor 17-hydroxy-4-pregnene-3,20-dione (17OHP) on the production of 17,20OHP. The sensitivity with which testes secreted 17,20OHP following stimulation with GtH II was maximum during spermatogenesis. The addition of 17OHP (10 to 1600 ng ml-1) to the culture medium of testes fragments induced a significant and dose-related increase in 17,20OHP secretion. Although the capacity to produce 17,20OHP was not saturated by the 17OHP concentrations used, the conversion rate was highest for tested at an immature stage. As to the regulation of 17OHP, in vivo, a single injection of partially purified salmon gonadotropin (50 ng g-1 body weight) induced a significant increase in the circulating levels of 17OHP of immature males. In conclusion, the maximum sensitivity to GtH II stimulation and the highest conversion rate of 17OHP to 17,20OHP in vitro, occurred before the dramatic increase in the 17,20OHP secretion observed in rainbow trout at the time of spermiation.     

Induced final maturation and ovulation in a small anabantoid teleost, the dwarf gourami, Colisa lalia. I. The effects of gonadotrophic preparations, LHRHa, steroids and thyroid hormones

Colisa lalia (Ham.) has been used as a model for the development of techniques for induced spawning that are applicable to small teleosts where ovulation requires prolonged exposure to suitable breeding conditions.Human chorionic gonadotrophin (hCG; 4 IU per fish) induced ovulation within 24 hours, whereas homologous pituitary extracts were relatively ineffective. When administered in saline ( 20 g per fish per injection), des-Gly10,[D-Ala6]- LHRH N-ethylamide (LHRHa) was ineffective, but it stimulated ovulation in a proportion of fish when administered ( 1.6 g per fish) as an emulsion in Freund's incomplete adjuvant (FIA). Together, these results suggest that ovulation requires the synthesis as well as the secretion of gonadotrophin in C. lalia.Long-term treatment with thyroid hormones appeared to enhance the ovulatory response to LHRHa in FIA, possibly by effects on the ovary; whereas the various steroids tested were ineffective at the dosages used     

Seasonal,reproductive, and nutritional influences on muscle “buffering capacity” in yellow perch (Perca flavescens)

Effective non-bicarbonate buffering capacity (or buffer value) was measured in white muscle of yellow perch (Perca flavescens) by titrations with mineral acid and base in a carbon-dioxide free, closed system. Yellow perch were collected at three month intervals throughout 1983 from an acidic lake (pH 4.6) and two alkaline lakes (pH 7.8) in northern Wisconsin. Buffering capacity was also determined for white muscle of perch kept in the laboratory under different regimes of temperature and ration. The mean buffering capacity of white muscle from yellow perch taken directly from natural environments ranged from 40.7 ± 3.1 (SD) slykes in March of 1983 to 53.7 ± 2.8 (SD) slykes in July of that year. These changes in buffering capacity were strongly correlated with water temperature. Egg production and thirty-day laboratory starvation produced significant decreases in buffering capacity and increases in the water content of yellow perch muscle. Fed perch in the laboratory had a temperature dependent buffering capacity similar to field caught fish. Buffering capacity of white muscle did not differ between yellow perch from acidic and alkaline lakes. Investigators using buffering capacity as a gauge of species differences in metabolic potential, should be wary of seasonal and reproductive factors that might alter their conclusions.     

Steroidogenesis during induced oocyte maturation and ovulation in the African catfish,Clarias gariepinus

Changes in ovarian steroidogenesis accompanying oocyte maturation and ovulation were studied in the African catfish,Clarias gariepinus. Laboratory-reared females with postvitellogenic ovaries were treated with pimozide and LHRH-analogue. The plasma gonadotropin levels were determined by means of a homologous radioimmunoassay, the condition of the ovaries was studied by histological examination of the follicles, and the steroidogenetic capacity of the ovaries was analyzed byin vitro incubation of tissue fragments for 3 h with [³H]-pregnenolone and [³H]androstenedione as precursors. Data were collected at regular intervals between 0 and 16 h after pimozide-LHRH analogue administration.Until 4 h after the beginning of the experiments the plasma gonadotropin levels did not rise above 25 ng/ml, the ovaries remained in the stage of postvitellogenesis, and testosterone was the main end product of steroidogenesis. Four hours later the gonadotropin concentration in the blood had risen to more than 150 ng/ml, and the ovaries had entered the stage of germinal vesicle migration. At the same time steroidogenesis shifted towards the production of 17,20-dihydroxy-4-pregnen-3-one, 5-pregnane-3, 17-diol-20-one, 5-pregnane-3,6,17-triol-20-one, 5-pregnane-3,17,20-triol and 5-pregnane-3,6,17,20-tetrol. During the subsequent stage of germinal vesicle breakdown the plasma gonadotropin level remained high, and the synthesis of the C₂₁-steroids showed a further increase. Simultaneously, the production of some C₁₉-steroid glucuronides was enhanced. The preovulation and especially the postovulation stages were accompanied by a gradual decrease in steroidogenic capacity of the ovaries, even though the plasma gonadotropin level remained high. It is concluded that the prematuration surge of gonadotropin influences the activity of enzymes involved in steroidogenesis, leading to a reduced C_17,20-lyase and to an augmented activity of the enzymes 20-hydroxysteroid dehydrogenase (HSD), 5-reductase, 3-HSD, 6-hydroxylase and UDP-glucuronosyltransferase. During ovulation the activity of all steroidogenic enzymes, including such key enzymes as 3-HSD and 17-hydroxylase, gradually decreases.Not only 17,20-dihydroxy-4-pregnen-3-one, but also the 5-reduced pregnanes may be involved in inducing oocyte maturation and/or ovulation. The very polar triol and tetrol products may function, together with the steroid glucuronides as sex pheromones.A preliminary account of these results was presented at the XIII Conference of European Comparative Endocrinologists, Belgrade, September 7–12, 1986