Effects of N,N-dimethylnitrosamine (DMNA) on in vitro oocyte maturation and embryonic development of fertilized eggs of carp (Cyprinus carpio L.) kept in eutrophied ponds.

Toxic effects of N,N-dimethylnitrosamine (DMNA) at doses of 100 or 500 micrograms l-1 on in vitro carp oocyte maturation (steroidogenesis), embryonic development and hatching of larvae (obtained as a result of artificial spawning of females kept for four seasons in normal and eutrophicated ponds) were investigated. There were no significant effects of DMNA on oocyte maturation and steroidogenesis during 24 h of incubation. The DMNA decreased the hatching of fertilized eggs derived from control females. This decrease reached a level of significance at a dose of 500 micrograms l-1. However, the effect of long-term exposure of female fish to eutrophied water was very much higher. The trend of the in vitro DMNA effect was the same, but it did not reach a statistically significant level. The results suggest that nitrosamines, through their effect on egg hatchability, may reduce fish populations along with increasing aquatic eutrophication.

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

Testosterone, 3α,17-dihydroxy-5ß-pregnen-20-one, 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP) and 5ß-pregnane-3α,17,20ß-triol were identified as the major metabolites of [(3)H] 17-hydroxyprogesterone in ovarian incubations of the European catfish Silurus glanis. 17,20ßP 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, 11ß-hydroxytestosterone and 17,20α-dihydroxy-4-pregnen-3-one (17,20αP) were identified as the major metabolites of [(3)H]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 17,20αP, 17,20ßP and 11ß-hydroxytestosterone by chh in primed but not control males suggests that the role of these steroids in spermiation should be further examined.

Advances in reproductive endocrinology of fish.

In teleosts two gonadotropins (GtHs) are produced: GtH I which stimulates steroidogenesis and incorporation of vitellogenin into the oocytes and GtH II, which stimulates steroidogenesis in the last stage of maturation and ovulation. Synthesis and release of GtHs are under control of gonadotropin releasing hormones, growth hormone, gonadotropin releasing inhibitory factor (GRIF)--dopamine, neuropeptide Y (NPY) gamma-aminobutyric acid (GABA) and melatonin. It was also found that calcium ions play a role of an intracellular mediator in GtH release. In ovaries, GtH stimulates production of 17 alpha-hydroxyprogesterone in the thecal cells. This steroid is transferred to granulosa cells where it is converted to 17 alpha 20 beta dihydroxy-4-pregnen-3-one (17 alpha 20 beta DHP). This steroid acts on the oocyte surface causing an appearance cytoplasmatic maturation promoting factor (MPF) which initiates the nuclear membrane breakdown and a subsequent cell division in both mitosis and meiosis. Teleosts are the only animals in which it is possible to change sex and to have population of fish of one sex. This possibility is widely applied in fisheries practice.

Participation of the pineal gland in sexual maturation of female rainbow trout (Oncorhynchus mykiss Walbaum).

In the study rainbow trout, pinealectomized at two different periods of their sexual cycle, i.e., at either 1 or 5 months before spawning, were studied. It was found that the lack of the pineal gland in the period directly preceding the spawning had no statistical effect on either spawning or the number of females that produced eggs. Pinealectomy performed during vitellogenesis delayed spawning by about 2 weeks and resulted in the absence of ovulation in 20% of the females. The results suggest that the pineal gland may influence the hypothalamo-pituitary-gonadal axis by altering the maturation period and controlling spawning in the rainbow trout.

Neuropeptide Y stimulates in vivo gonadotropin secretion in teleost fish.

The effects of the neuropeptide Y (NPY), alone or in combination with a gonadotropin-releasing hormone analogue, D-Ala6-desGly10-Pro9-Net LHRH (LHRHa), have been studied on the in vivo secretion of the maturational gonadotropin (GtH2) in the rainbow trout Oncorhynchus mykiss and the common carp Cyprinus carpio. Depending on the species, two routes of administration were used: in trout, intraperitoneal injection (20 micrograms/kg body wt); in carp, direct infusion (3 micrograms/kg body wt) into the third ventricle via a temporary brain cannula. In both cases NPY alone induced a twofold increase in GtH2 secretion and peaked 2 to 4 hr administration regardless of the route of injection. The plasma gonadotropin levels returned to basal within 8 hr. The relative increases (peaked secretion/basal secretion) did not differ with the route of injection. When the animals were first treated with NPY and then LHRHa (20 micrograms/kg) 1 hr later, the magnitude of the response to LHRHa was greater in the animals pretreated with NPY, indicating either a potentiation of LHRHa action by NPY or additive effects of the two peptides. The return to basal levels also took longer in fish receiving NPY first. NPY may act directly at the pituitary level or activate central neuromediatory systems.

A presumptive role for GABA in the stimulatory effects of Des-Gly10, [D-Ala6]-LHRH-ethylamide and pimozide on the gonadotropin release in carp.

To investigate the effect of endogenous gamma-aminobutyric acid (GABA) on the blood maturating gonadotropin (GtH) levels, or to study its interaction with pimozide (dopamine antagonist) and a luteinizing hormone-releasing hormone analog (LHRH-a), sexually mature male and female carps were treated with drugs that may either inhibit GABA biosynthesis or GABA degradation. In females the irreversible inhibitor of GABA-transaminase, gamma-vinyl GABA (GVG), which was to increase the endogenous GABA-ergic tone, had no influence on GtH release. On the other hand, the increased GtH response to the combination of pimozide (PIM) and LHRH-a was clearly enhanced by the administration of 3-mercaptopropionic acid (MPA), an inhibitor of the rate limiting enzyme of GABA-biosynthesis. In males the GABA-ergic compound, valproic acid (DPA) decreased LHRH-a stimulated GtH levels. In male carps that received PIM to diminish the dopaminergic inhibition of GtH release, the spermiating response to LHRH-a was increased by administration of MPA. These data suggest that GABA interacts with the action of dopamine and the gonadotropin releasing hormone (GnRH) on the release of GtH.

Interactions between oocytes of different maturational stages in the carp Cyprinus carpio: effects on maturational and steroidogenic activity in vitro.

Ovarian fragments from carp which had received injections of saline (control) or carp hypophysial homogenate (primed) were incubated both alone and a cocultures of primed and control tissue in the presence of carp pituitary extract. The results showed that the cocultures were not simply the sums of separate incubations. Oocyte maturation was retarded in primed tissue but advanced in control tissue and concentrations of 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P) in the medium were significantly lower than that expected from separate incubations. The results indicate that local factors may act to synchronize oocyte maturation throughout the ovary to enable spawning of the maximum number of eggs.

The effects of reserpine and LHRH or salmon GnRH analogues on gonadotropin release, ovulation and spermiation in common carp (Cyprinus carpio L.).

The effects of reserpine (catecholamine depletor) and LHRH analogues on gonadotropin secretion, spermiation and ovulation of common carp were investigated. Injections of reserpine alone at a dose of 1 or 7 mg/kg of body weight stimulated spermiation, and reserpine at a dose of 1 mg/kg of body weight in combination with (D-Arg6, Trp7, Leu8, Pro9, NEt)-LHRH (s-GnRH-A) or with (D-Ala6, Pro9)-LHRH (LHRH-A) at a dose of 50 micrograms/kg of body weight caused an increase of plasma gonadotropin levels, spermiation and ovulation in 80-90% of the females. Simultaneous injection of reserpine and LHRH analogues was as effective as injection of reserpine followed by injection of LHRH analogues 6 h later.

The temporal sequence of changes in oocyte maturation and ovarian steroid hormone production during induced ovulation in the common carp, Cyprinus carpio.

Oocytes taken from carp sacrificed 3-24 hr after injection with either carp hypophysial homogenate (chh) (primed) or saline (control) were incubated with or without chh for 24 hr at 20 degrees. In oocytes from the primed fish the percentage of peripheral germinal vesicles at sacrifice progressively increased with time from injection. No such change occurred in control fish. After incubation with chh, tissue from primed fish showed a progressive increase in oocyte maturity with increasing time between priming and sacrifice, with germinal vesicle breakdown (GVBD) in 50% of the oocytes in incubations begun 24 hr after priming. In contrast, incubated tissue from control fish showed no variation with time between saline injection and sacrifice, and oocyte maturity did not exceed 50% with peripheral germinal vesicles. GVBD was not significant in these fish. 17-Hydroxyprogesterone, testosterone, and testosterone glucuronide production in vitro showed no major changes with the interval between injection and sacrifice, nor between control and primed fish. In vitro production of 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P), however, increased significantly between tissue taken from animals at 3 and at 6 hr after injection of chh, and thereafter remained unchanged. 17,20 beta P production preceded the increase in GVBD in primed fish but neither this hormone nor GVBD was found in incubations of control fish, supporting the suggestion that 17,20 beta P is the maturation-inducing hormone in carp. Steroid production was not detectable in the absence of chh in the incubation medium and oocyte maturation showed little advance on that at sacrifice.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of carp hypophyseal homogenate doses, incubation times, and temperatures on carp oocyte maturation and steroidogenesis in vitro.

Ovarian tissue from carp which had received injections of either saline or carp hypophyseal homogenate (chh) 24 hr prior to sacrifice was incubated for 8, 16, or 24 hr at 4, 15, 20, or 25 degrees with 0, 30, 100, or 200 micrograms chh. 17,20 beta-Dihydroxy-4-pregnen-3-one (17,20 beta P) was produced only by chh-primed tissue, but there was no difference between the two groups in production of testosterone, 17-hydroxyprogesterone, or testosterone glucuronide. Production of all steroids was greatest at 20 degrees which also corresponds to the optimal temperature for germinal vesicle breakdown in primed tissue. While yields of testosterone, 17-hydroxyprogesterone, and 17,20 beta P decreased with time, those of testosterone glucuronide tended to increase, suggesting further metabolism to conjugated and/or reduced metabolites. Germinal vesicle migration in control fish, and GVBD in primed fish, showed no change after 8 hr. The results indicate that the induction of both germinal vesicle migration and the potential for synthesis of 17,20 beta P by the priming dose of chh are of primary importance in the induced ovulation of carp.

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. 17,20ß-Dihydroxy-4-pregnen-3-one (17,20ßP) 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 17,20ßP in response to later gonadotrophin challenge, and that this initiation may be related to the migration of the germinal vesicle.

Autoradiographic localization of gonadotrophin receptors in ovaries of the common carp, Cyprinus carpio L.

Binding sites for carp gonadotrophin have been located in carp ovaries using [125I]labeled gonadotrophin and autoradiography. The radioactive gonadotrophin was displaced from tissue by unlabeled gonadotrophin or carp hypophysial homogenate in a dose-dependent fashion. No binding of gonadotrophin was found in previtellogenic oocytes but binding appeared with the first indications of vitellogenesis. In the smaller vitellogenic oocytes binding was uniformly distributed in the follicular envelope, but in the largest oocytes binding was restricted to the interstitial tissue. In these more mature oocytes gonadotrophin was also found within the oocyte and appeared to migrate toward the nucleus. The relationship between binding location, steroidogenesis, and oocyte maturation is discussed. We found no evidence for specific binding of [125I]thyroxine under comparable conditions.

Daily changes in blood serum levels of 17 beta-estradiol and 11-ketotestosterone in the mature carp Cyprinus carpio L.

Daily changes in blood serum levels of 17 beta-estradiol and 11-ketotestosterone in the mature carp Cyprinus carpio were investigated. Fish were kept under natural light conditions (LD = 16:8) water temperature about 19 degrees C. Females and males were divided into 2 groups respectively. Each group was sampled every 4 h during 24 h beginning at 08:00 (group 1) and 10:00 (group 2). The 17 beta-estradiol assay included a chromatographic step which increased the specificity of the measurement while in 11-ketotestosterone assay a specific antibody was used, having a low cross reactivity with testosterone (1.1%). Data obtained in this work were statistically analyzed using cosinor circle with error ellipses. The highest levels of estradiol (0.46-0.58 ng/ml) were observed between 09:00 and 11:00 and of 11-ketotestosterone (2.09-3.00 ng/ml) between 10:00 and 12:00. It was proved statistically that changes in the estradiol and 11-ketotestosterone levels during 24 h are of circadian rhythm type. The problem of hormonal changes during 24 h, connected with sexual maturation of fish, is discussed.

Effects of pimozide and LHRH-Aa on carp (Cyprinus carpio L.) oocyte maturation and ovulationin vivo.

Effects of pimozide (Pim) and [(D-Ala(6), Pro(9)-NEt) LHRH] (LRH-Aa) on common carp oocytes maturation and ovulationin vivo under laboratory and commercial fisheries farm conditions were investigated.Although injections of Pim and LRH-Aa at the doses of 10 mg and 50 µg/kg body weight respectively, did not increase mGtH levels (66.7-155.8 mg/ml) as much as injections of carp pituitary extract (chh) (382.1 ng/ml), induced GtH levels were high enough to induce ovulation. Changes in the ovary caused by Pim and LRH-Aa were similar to those induced by chh, and Pim injected together with LRH-Aa in a single injection gave the same results concerning ovulation induction as when they were applied separately at 6h interval.

Effect of temperature, 17 alpha-hydroxy-20 beta-dihydroprogesterone and prostaglandin F2 alpha on carp oocyte maturation and ovulation in vitro.

Two series of experiments were carried out. In series I, studies were conducted on the effect of 17 alpha-hydroxy-20 beta-dihydroprogesterone (17 alpha 20 beta P; 1 microgram/ml of substrate) on the migration of germinal vesicle (GV) and its breakdown (BD) in the oocytes of carp ovarian fragments, kept at 5, 10, 15, 20, and 25 degrees C. For investigations ovarian fragments were taken both from intact mature females and from those injected with carp hypophysial homogenate (chh) at a dose of 1 mg/kg. It was found that GV migration and GVBD occurred at 15-25 degrees C. Based on the results obtained, it appears that GV migration proceeds optimally at 15 degrees C, whereas GVBD requires higher temperatures (20-25 degrees C), although at times it may also take place at 15 degrees C. In series II, studies were conducted on the effects of exogenous prostaglandin F2 alpha (PGF2 alpha) on carp oocytes ovulation in vitro in the ovarian fragments kept at 5, 10, 15, 20, and 25 degrees C for 24 and 48 hr. It was found that the temperatures in the range studied had no statistically significant effect on the ovulation process induced in vitro by exogenic PGF2 alpha added to the medium. The mechanisms of the final stage of carp oocytes maturation and ovulation are discussed.

Daily changes in the gonadotropin levels and response of carp oocytes to hypophysial homogenate.

Daily changes in carp gonadotropin levels in adult female carp and daily changes in carp oocyte sensitivity to carp hypophysial homogenate, in vitro and in vivo, were investigated. A total of three series of experiments were carried out. Gonadotropin levels were radioimmunologically determined. The results of series 1 and 2 experiments were subjected to statistical analysis with the use of cosinors circle and ellipse of errors. It has been found that in the mature female carp in the pre-spawning period with the light periods being long (L:D = 16:8) the apogee for gonadotropin occurs 10 hr after the onset of the light period. The sensitivity of the oocytes, in terms of the percentage of mature oocytes (after GVBD) following a 24-hr incubation of ovarian fragments with the hypophysial homogenate, reached the highest value at 1300, i.e. 9 hr after the onset of the light period. It was also found that the injections of carp hypophysial homogenate made at 0900 were much more efficient in inducing ovulation than those at 2100.

Temperature and reproduction in tench: effect of a rise in the annual temperature regime on gonadotropin level, gametogenesis and spawning. I. The male.

During a 9-month period (corresponding to a sexual cycle), the adult male tench, Tinca tinca, was kept in fish farm ponds receiving heated water from a power plant. In 1974, the males were submitted to three different temperature regimes: group I: ambiant temperature; group II: ambiant temperature +3 degrees C; group III: ambiant temperature + 6 degrees C. The experiment was repeated in 1975, but only using groups I and III. The reproductive cycle and thermal treatment were studied from a quantitative analysis of spermatogenesis, the duration of the spawning cycle and radioimmunoassay (RIA) measurement of gonadotropin (GTH) in plasma and pituitary, using a carp RIA system turned out to be sensitive enough to assay tench GTH, which was expressed in a c-GTH equivalent. Spermatogenesis in the tench was a discontinuous process, starting in the spring and finishing in the summer. It began earlier in heated water in which the spawning period was also considerably longer (3 months in group III against 1 month in group I). At the beginning of spermatogenesis, pituitary and plasma GTH was low, but rose rapidly when spermatogenesis was initiated (appearance of type B spermatogonial cysts and meiosis). The highest GTH levels in the blood were recorded during the spawning period, with important fluctuations probably due to discharges from the pituitary.

Temperature and reproduction in tench: effect of a rise in the annual temperature regime on gonadotropin level, gametogenesis and spawning. II. The female.

Female tench were bred for two consecutive years under different thermoperiods; the effect of the thermoperiods on vitellogenesis, spawning, plasma and pituitary gonadotropin (GTH) levels was studied. The temperature did not affect development during the preparatory period. We found no ovarian development below 10 degrees C. The temperature accelerated vitellogenesis only if the mean daily average was more than 10 degrees C, and the rate of increase of that parameter from 10 to 23 degrees C (spawning temperature) determined female fertility. The date of first spawning may be predicted by the summation of degree-days higher than 10 degrees C. GTH secretion increased during vitellogenesis to reach maximal values during spawning. The data indicate a gonadotropin surge at the beginning of each spawning. There was no difference between plasma GTH levels at the various temperatures, but pituitary GTH was higher in the groups bred under the highest temperatures.