Krzysztof Bieniarz

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.

Development of European eel (Anguilla anguilla L.) gonads in artificial conditions

Abstract Gonadal development was investigated in 58 European eels of known age kept in aquaria in stable conditions optimal for growth (except for light). Sex of fish was determined by histological analyses of gonads, the results of which were checked by kariological analysis. It was found that: 1. 1) Sex differentiation in European eels is connected with size of fish and not age and begins when eels reach 10 cm in length. 2. 2) Gonads of the European eel do not pass through a stage of juvenile hermaphroditism.

Effects of LHRH and LHRH-A on plasma GtH levels and maturation/ovulation in the common carp, Cyprinus carpio, kept under various environmental conditions

Abstract Experiments were carried out to determine if some environmental factors usually present in spawning ponds at the time of ovulation could potentiate the effects of LHRH or LHRH-A on the plasma level of GtH, oocyte maturation or ovulation in the common carp, Cyprinus carpio . In one experiment females were held in the absence of a male in cages placed in rearing ponds containing a dense population of unicellular algae; the cages touched the bottom but there was no vegetation inside the cages. When LHRH-A was given in two injections (the first 5 μg/kg, the second, 6 h later, 50 μg/kg) plasma GtH was increased and followed oocyte maturation. Ovulation occurred in 50% (3 of 6) of the females. In another experiment, using a similar LHRH-A treatment schedule, the females were held in aquaria with added vegetation to serve as a spawning substrate; in such a simplified environment the effect of LHRH-A was not potentiated. Similarly, the natural environment of spawning ponds including the presence of males was of no benefit to the response to low doses of LHRH or LHRH-A when the water temperature was low (12°C). These results suggest that some undetermined environmental factors besides vegetation present in a pond situation may potentiate the effect of LHRH-A, possibly via abatement of the action of an endogenous gonadotropin release-inhibitory factor.

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

Abstract: 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.

Observations on a possible pheromonal stimulation of milt production in carp (Cyprinus carpio L.)

Experiments were carried out in 200-m2 earthen ponds or 2-m3 bare tanks to evaluate stimulation of spermiation in male carp exposed to female carp undergoing ovulation after injection of carp pituitary homogenate (cph). Stimulation of spermiation was observed when fish were kept in earthen ponds but not in tanks. In a control group, intact males which were exposed to cph-injected males also showed a stimulation of spermiation in earthen ponds but not in tanks.

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)

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

Effect of temperature, 17α-hydroxy-20β-dihydroprogesterone and prostaglandin F2α on carp oocyte maturation and ovulation in vitro

Abstract Two series of experiments were carried out. In series I, studies were conducted on the effect of 17α-hydroxy-20β-dihydroprogesterone (17α20βP; 1 μg/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°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°C. Based on the results obtained, it appears that GV migration proceeds optimally at 15°C, whereas GVBD requires higher temperatures (20–25°C), although at times it may also take place at 15°C. In series II, studies were conducted on the effects of exogenous prostaglandin F 2α (PGF 2α ) on carp oocytes ovulation in vitro in the ovarian fragments kept at 5, 10, 15, 20, and 25°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 PGF 2α added to the medium. The mechanisms of the final stage of carp oocytes maturation and ovulation are discussed.

Changes in the ovaries of adult carp

Abstract Three series of experiments were carried out. In the first, studies were conducted on the changes occurring in the ovaries of four groups of adult female carp during a yearly cycle (between two spawning periods). Gonadal samples in vivo were taken monthly from the same marked females from August 1975 to July 1976. A portion of the samples was subjected to histological examination and the rest used for counting and measuring oocytes. It was found that changes in the adult carp ovaries during a yearly cycle consist of a gradual development of a resorption process in the oocytes after a completed vitellogenesis, the store of oocytes being progressively supplemented by those in earlier stages of development. The changes studied are not temperature-dependent and their course is similar, irrespective of whether the females had or had not spawned during the spawning season preceding the experimental period. In the second series, studies were conducted on 26 marked, mature female carp, for 5 weeks, starting on the day before artificial spawning. Gonadal samples were taken in vitro, analysed histologically, a portion being cleared in turpentine oil while others were used for in vitro culture. It was found that, irrespective of whether the females had spawned or not, they possessed oocytes after a completed process of vitellogenesis. Their response to hypophysial homogenate added to the culture medium resulted in nucleus migration. In the third series, studies were conducted on the effect of clomiphene on the ovaries. It was found that clomiphene given to mature female carp during the spawning period caused a statistically significant increase, compared to the control, in the percentage of oocytes with the nucleus shifted peripherally. However, in contrast to hypophysial homogenate injections, it failed to induce ovulation.

Stimulation of gonadotropin secretion and spermiation in carp by pimozide-LRH-A treatment: Effects of dose and time of day

To define the optimal combination time and LRH-A (luteinizing releasing hormone analogue) administration route for inducing spermiation in carp, two experiments were carried out. In the first experiment, carp kept in raceways in running recycled water in August (at a water temperature of 15–20°C) were injected intraperitoneally with pimozide (PIM) at 1 or 10 mg/kg body weight as well as with Des-Gly10 [D-Tryp6] LH-RH-ethylamide (LRH-A) at 0.1, 1 or 10 μg/kg; these products were given 6 h apart. The response was evaluated by the volume of milt and the number of spermatozoa emitted and by the gonadotropin (GTH) concentration in the plasma. At doses of 0.1, 1 and 10 μg/kg LRH-A, the total milt production was increased 2, 4 and 6-fold respectively when these treatments were combined with 1 mg PIM. In the second experiment, adult male carp, held in cages in a pond in a natural environment (at a water temperature of 11–14°C) at the beginning of May, received an intraperitoneal injection of PIM (1 mg/kg body weight) and a silicone rubber implant of LRH-A (Des-Gly10 [D-Ala6] - LH-RH-ethylamide) at dose of 10 μg/kg body weight simultaneously. Other groups of fish received PIM plus a blank implant. All of the treatments were given at either 6 a.m. or 8 p.m. When given at 6 a.m., the treatment induced a similar rise in plasma GTH (12–15 ng/ml) 24 h later in the groups treated with PIM and LRH-A implant alone or in combination. When the same treatment was given at 8 p.m., no response was observed 24 or 48 h later. Spermiation was not stimulated in any of the groups.