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Carp spermatozoa are immotile in seminal plasma or in saline solution of high osmolality (> 400 mosmol kg-1). These ‘quiescent’ spermatozoa initiate a progressive forward motility when transferred in freshwater or in saline solution with low osmolality (< 160 mosmol kg-1). In this study we investigated ‘in vitro’ the relationship between sperm ATP content (measured by bioluminescence) and sperm motility (analysed by videomicroscopy). Sperm ATP content remained high in the immobilizing medium (200 mM KCl, Tris 30 mM, pH 8.0) where no flagellar movement occurs. Dilution of these spermatozoa in the activating medium (45 mM NaCl, 5 mM KCl, Tris 30 mM, pH 8.0) triggered forward motility which varied with temperature. At 20 degrees C, sperm ATP content decreased rapidly during the progressive forward motility phase from 12 to 4 nmol/10(8) spermatozoa, concomitantly with decreases in velocity (130 to 10 microns s-1) and the beat frequency (50 to 7 Hz). An inhibitor of mitochondrial respiration (KCN 10 mM) produced a drop in sperm ATP content irrespective of the incubation medium (activating or immobilizing). A second phase of sperm motility in the activating medium was induced following a previous transfer of spermatozoa into a medium of high osmolality for a few minutes prior to the second phase. Within 10 minutes, spermatozoa recover 90% of the initial ATP level as well as forward motility. These results suggest that motility of carp spermatozoa depends on sperm ATP synthesized by mitochondrial respiration mainly stored before activation. In low osmolality conditions, the mitochondrial oxidative phosphorylation is unable to compensate for the ATP hydrolysis required to sustain motility.(ABSTRACT TRUNCATED AT 250 WORDS)

Live trout spermatozoa initiate flagellar motility for only a short period (30 sec at 18 degrees C) during which their mean beat frequency decreases steadily from 60 to 20 Hz. Motility then stops abruptly. Investigations of the activation of movement in demembranated sperm points to cyclic-AMP being necessary for reactivation (half effect at 0.5 microM) in some conditions. cAMP acts mainly by increasing the percentage of motile cells and not the beat frequency (BF) of the flagellar axoneme. Dibutyryl cAMP does not initiate movement or prolong motility of live sperm. The initiation of movement of demembranated trout sperm was investigated in various incubation conditions relative to previous phases of in vivo movement and to ATP concentration. In the absence of cAMP and in the presence of ATP lower than 25 microM, all sperm cell models were active with BF up to 15-20 Hz whatever their previous physiological conditions. In contrast, at ATP concentrations above 100 microM, the fraction of active spermatozoa decreased proportionally but the BF of the active ones increased so that, at 1 mM ATP, only 5% were active but with a BF of 65 Hz: the addition of cAMP up to 20 microM restored activity to 100% sperm models with a similar BF of 65 Hz. At ATP concentrations higher than 25 microM, cAMP was necessary in a concentration dependent manner in the reactivation, but not in the demembranation medium. This dependence was found to be unrelated to a previous in vivo phase of movement. The antagonistic effects of ATP vs. cAMP were tested at various concentrations of both nucleotides: the apparent affinity for cAMP, measured as the concentration restoring movement of 50% cell models, was decreased from 15 nM at 0.1 mM ATP to 0.5 microM at 1 mM ATP; conversely, the affinity for ATP, measured as the concentration giving rise to the half maximal beat frequency, was not significantly affected when the concentration of cAMP was raised to 0.5 mM. Preincubation with phosphodiesterase (PDE) resulted in motility of 100% of sperm models even at low ATP concentration. This tends to show that cAMP must be constantly present to sustain motility.

Carp semen obtained from isolated fish after hormonal stimulation was highly variable in terms of volume of semen, osmotic pressure of the seminal plasma, and sperm capacity to move. Moreover, this last parameter was unstable when the spermatozoa were kept within the seminal plasma, and the present work was designed to investigate and possibly correct this phenomenon. Sperm potential movement was the major parameter studied and was measured by the percentage of motile cells in a final 3.000-fold dilution in a medium of low osmotic pressure in which sperm movement is known to occur (Morisawa and Suzuki, Science 210:1145-1147, 1980). This was completed with occasional measurements of flagellar beat frequencies and demembranation-reactivation of axonemal movement. The results showed that sperm potential movement was preserved upon dilution of the semen into cold 200 mM KCl medium and that semen of initially "poor" quality or spermatozoa that had lost their capacity to move during storage in the semen recovered gradually their potential movement during incubation at 2 degrees C in the same medium. The K+ dependence for both the conservation and the regeneration of sperm capacity to move showed a minimal requirement of 50 mM KCl in media of high osmotic pressure. Na+ ions had similar properties but not divalent cations. The K+ activation was not pH dependent between pH 9.03 and 6.04. Whatever the functional state of live spermatozoa, demembranation-reactivation occurred in ATP-Mg2+. It is concluded that, with dilution of the semen in appropriate conditions, carp spermatozoa retain or acquire potential movement and therefore are a lower vertebrate spermatozoa model available year-round. In addition, obtaining potentially nonmotile sperm and reversion in vitro might be useful to study the control of in vitro maturation.

International audience; Juvenile rainbow trout received gonadotropic hormone (GtH) treatment to investigate the response of the immature gonad and its effect on the onset of rising pituitary GtH levels, an early sign of initiation of sexual maturation in the teleost fishes. Brief exposure to GtH evoked increases in plasma androgens and significantly increased pituitary GtH in males; the accumulation of pituitary GtH was not observed in female trout which showed significant but smaller increases in plasma androgen resulting from GtH administration. There was no evidence of gonadal negative feedback in intact immature fish since pituitary GtH and plasma androgens remained at basal levels in castrate juvenile trout. Since the castrate male trout failed to respond to GtH injections, the data suggest that the immature gonad produces a steroid capable of triggering pituitary GtH production by hypothalmo-pituitary feedback in males.

When trout (Salmo gairdneri) spermatozoa were diluted in coelomic fluid or saline diluents at high dilution rates of 10(-3) and 10(-2) for increasing periods of time before insemination, there was a rapid decline and loss of fertilizing ability. At a lower dilution rate of 10(-1), there was partial or no loss of fertility. Dilution in a KCl-enriched saline diluent to inhibit sperm motility produced a slight decrease in fertility at a 10(-3) dilution rate, indicating that the spermatozoa, although sensitive to dilution, were less so when they were kept immotile. A partial loss of fertility was observed after the spermatozoa or eggs had been washed with saline diluents. The loss of fertility was total when both gametes were washed. Removing the seminal fluid by centrifugation led to a significant decrease in the fertilizing ability of the spermatozoa when insemination was carried out in saline diluent but not in coelomic fluid. Adding BSA at high doses (10 mg BSA/ml) into the diluent led to longer survival of the diluted spermatozoa. We conclude that (1) sperm dilution rate is a major factor in the maintenance of fertilizing ability of diluted salmonid spermatozoa, (2) as reported in the literature, coelomic fluid is superior to mineral diluents only when the gametes have been washed, and (3) some substances (possibly proteins) present in seminal and coelomic fluids play a role in gamete protection. These findings may explain the discrepancies in the literature concerning the duration of motility and fertilizing ability of salmonid spermatozoa.

The postcastration increase of gonadotropin secretion in the rainbow trout, was partially inhibited by charcoal treated seminal liquid. Protein testicular extract did not have any effect. This result demonstrates the probable existence of an inhibitory factor of gonadotropin secretion in the seminal plasma of the Trout.

Bilateral castration of male rainbow Trout resulted in increased levels of circulating gonadotropin, as measured by radioimmunoassay, but the response varied depending on the season. In early May the increase was 12 ng/ml after 30 days, when compared to the intact control but later in the month at the time when a peak of gonadotropin level is observed in normal fish, the increase was 25 ng/ml after 13 days. At the beginning of August, when spermatogenesis commences, the increase was 9 ng/ml after 12 days. In September, when spermatogenesis is ending, an increase of 20 ng/ml was found after 7 days, but only 50% of the animals responded. In December, during spermiation, an increase of 90 ng/ml was found after 7 days.

The effects of bilateral castration on plasma gonadotropin (t-GtH), as measured by radioimmunoassay, were investigated in male rainbow trout at several stages in the reproductive cycle: at the start of spermatogenesis at the end of May, after its completion in September, and before and after the beginning of spermiation in October and December. Castration resulted in increased level of circulating immunoreactive t-GtH but the response varied with the season. The rise after castration was four times the initial value in May and September, and increased twofold in October and sevenfold in December. The duration of the response following castration was 4 months in May, 2 months in September, and less than 1 month in October and December.

In many male mammals and birds, exposure to sexual stimuli results in acute elevations of circulating luteinizing hormone (LH) and testosterone (T); a similar phenomemon has now been observed in the male goldfish (Carassius auratus). Mature males placed with either a receptive female or stimulus pairs of spawning goldfish had gonadotrophin (GtH) concentrations and expressible milt (sperm) volumes that were significantly greater than those of males kept in all-male groups. This stimulatory effect lasted from 20 min to at least 2 hr for GtH (20 degrees) and from less than or equal to 1 hr to greater than or equal to 24 hr for milt (14 degrees). When males were separated from the spawning pair by either a solid or perforated clear partition, no elevations of GtH or milt levels occurred. In contrast, these values increased in males placed in contact with a spawning pair, even when that pair contained no female, but a male induced to perform female sexual behavior by treatment with prostaglandin. These results suggest that, in goldfish, access to a spawning situation is necessary for rapid elevations in GtH and milt. Furthermore, it appears that the males must be sexually active in order for these physiological changes to occur, as males that failed to engage in courtship behavior with a spawning pair had GtH and milt values not different from isolated fish. This suggests that male sexual behavior and elevations in milt and GtH are concurrent events that share a common activation pathway in the brain. The increase in milt may be due to both neurally and hormonally mediated events that ensure milt availability for imminent spawning activity.

The pattern of spermatogenesis was studied during an annual reprodutive cycle in adult male brown trout. The part of the testicular volume occupied by the nuclei of germ cells and of Sertoli cells, the lobular lumen and the extra-lobular space were measured. The number of germ cells was estimated by the total volume occupied by each cell divided by the individual volume of each cell. The permanent number of spermatogonia A (spermatogonia isolated or in groups of 2) was about 50 million. Each GA cell produced several spermatogenetic waves during the same reprodutive cycle. Sperm production was about 25 X 10(9)/g of testis/year, or about 6.8 X 10(7)/g of body weight/year. Males stocked separately from females, thus presumably not releasing spermatozoa, were killed in December, January and February. Since these fish contained the same amount of spermatozoa it appears that the process of spermiation initiated in December did not continue in January and February. During this period, some spermatozoa were phagocytized by macrophages (spermatophages) in the lobular lumen and by Sertoli cells.

Free and peptidic amino acids were measured in the seminal plasma of carp. Some acids such as glutamic acid and glutamine, taurine, glycine and proline were exceptionally abundant, while others like lysine, arginine and histidine were found in small amounts. Generally, the amino acid level was higher in carp seminal plasma than in that of other Vertebrate species. As this level was systematically higher than in the blood plasma, it is supposed that there was neoformation of an original fluid in the genital tract.

10 In the rainbow trout and the tench, at the time of spermatogonia divisions a rise in the plasma gonadotropin levels was observed. Just before or during the spawning period, E217 beta and plasma gonadotropin reach their maximum levels. 20 When the effects of raising the natural cycle temperature of the tench were studied (deltat + 3 degrees C and + 6 degrees C), in conditions where nycthemeral and seasonal rythmicity were maintained, a significant increase in fertility was observed with the increase in temperature (the first spawning period was earlier and there was a rise in the number of spawnings). This effect of temperature seems to act at the level of the hypothalamo-pituitary system.

After rainbow Trout sperm were diluted in 0.01 and 0.001 M theophylline media, motility and fertilizing ability were prolonged as compared to the control media without theophylline. However, the motility observed was lower and of a different type than normal motility. This suggests that other factors besides cyclic nucleotides intervene in the motility of Trout spermatozoa.

During the reproductive season in August, we administered to adult male and female Carp an LHRH analog (LHRH-A = D-trp6EA-LHRH) (50 micrograms/kg) alone or in association with pimozide, a dopamine inhibitor (10 mg/kg). LHRH-A caused the circulating portal level of gonadotropin to rise significantly in males and less in females. Spermiation was stimulated in the males and oocyte maturation in females but there was no ovulation. An infection of pimozide and then of LHRH-A 6 h later significantly increased c-GTH secretion and spermiation more than LHRH-A given alone, and induced ovulation in the females.