Volker Blüm

Steroid secretion of rainbow trout testis in vitro: Variation during the reproductive cycle

Testicular tissue collected at different stages of gonadal development was incubated with a pituitary extract (PE) from mature salmon. Three androgens (11-ketotestosterone, OT; 11 beta-hydroxytestosterone, OHT; and testosterone, T) and 17 alpha,20 beta-dihydroxyprogesterone (17-20 beta P) were quantified by radioimmunoassay in incubation media. OHT and OT were secreted in larger quantities than T and 17-20 beta P. The PE dose that evoked a half-maximal response (ED50), the ratios of maximum stimulated vs baseline secretion, and total testicular steroid output all changed during the reproductive cycle. Androgen secretion in response to PE was low in immature and spent fish, both in terms of ED50 and the ratio of maximum stimulated vs baseline secretion. This ratio increased in testes showing the first signs of maturation and remained elevated during rapid testicular growth, before reaching maximum values at full maturity. The lowest ED50 values were found at the end of spermatogenesis and during the peak spawning period. 17-20 beta P secretion could not be stimulated noticeably until the fish had entered the spawning period and, as opposed to androgens, remained stimulable in spent fish. ED50 values for 17-20 beta P ranged, without showing clear-cut variations, above those calculated for androgens. The changes in PE reactivity and steroid secretion capacity during the reproductive cycle are likely to contribute to the changes in circulating steroid concentrations and may allow modulations of testicular steroid production without large changes in circulating GTH levels.

Aquatic modules for bioregenerative life support systems: Developmental aspects based on the space flight results of the C.E.B.A.S. mini-module

The Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) is an artificial aquatic ecosystem which contains teleost fishes, water snails, ammonia oxidizing bacteria and edible non-gravitropic water plants. It serves as a model for aquatic food production modules which are not seriously affected by microgravity and other space conditions. Its space flight version, the so-called C.E.B.A.S. MINI-MODULE was already successfidly tested in the STS-89 and STS-90 (NEUROLAB) missions. It will be flown a third time in space with the STS-107 mission in January 2003. All results obtained so far in space indicate that the basic concept of the system is more than suitable to drive forward its development. The C.E.B.A.S. MINI-MODULE is located within a middeck locker with limited space for additional components. These technical limitations allow only some modifications which lead to a maximum experiment time span of 120 days which is not long enough for scientifically essential multi-generation-experiments. The first necessary step is the development of “harvesting devices” for the different organisms. In the limited space of the plant bioreactor a high biomass production leads to self-shadowing effects which results in an uncontrolled degradation and increased oxygen consumption by microorganisms which will endanger the fishes and snails. It was shown already that the latter reproduce excellently in space and that the reproductive functions of the fish species are not affected. Although the parent-offspring-cannibalism of the ovoviviparous fish species (Xiphophorus helleri) serves as a regulating factor in population dynamics an uncontrolled snail reproduction will also induce an increased oxygen consumption per se and a high ammonia concentration in the water. If harvesting locks can be handled by astronauts in, e. g., 4-week intervals their construction is not very difficult and basic technical solutions are already developed. The second problem is the feeding of the animals. Although C.E.B.A.S.-based aquaculture modules are designed to be closed food loop systems (edible herbivorous fish species and edible water plants) which are already verified on Earth this will not be possible in space without devices in which the animals are fed from a food storage. This has to be done at least once daily which would waste too much crew time when done by astronauts. So, the development of a reliable automated food dispenser has highest priority. Also in this case basic technical solutions are already elaborated. The paper gives a comprehensive overview of the poposed fiuther C.E.B.A.S.-based development of longer-term duration aquatic food production modules.

Extragonadal 17β-hydroxysteroid dehydrogenase activity in rainbow trout

Abstract Blood cells of male and female rainbow trout showed 17(3-hydroxysteroid dehydrogenase (17(βHSD) activity in vitro, reducing 11β-hydroxyandrostenedione and 11-ketoandro-stenedione (OA) to 11β-hydroxytestosterone and 11-ketotestosterone (OT), respectively. Enzyme activity did not vary with gonadal development in either sex. The conversion of tritiated precursors was partly inhibited in the presence of steroid-free serum or radioinert steroid, but inhibition was less strong when radioinert androgens were added to steroid-free serum or when the serum contained endogenous steroids. Treatment of male trout with salmon gonadotropin in vivo and/or incubation with a pituitary extract of mature salmon in vitro did not affect OA conversion when blood cells were incubated in the absence of serum, whereas it was slightly but significantly higher when they were incubated in the presence of serum and pituitary extract. In addition to blood cells and steroidogenic tissues, spleen, intestine, brain, liver, excretory kidney, and skin tissue also produced an OA metabolite isopolar to OT in vitro, so that 17βHSD appears to be present in a variety of trout issues. With respect to the biological significance of extragonadal steroid metabolism in vivo, the ligand binding characteristics of circulating steroid binding proteins may be of primary relevance in regulating substrate availability.

Seasonal variation in insulin and glucagon concentrations of Rana esculenta (L.)

Abstract The concentrations of insulin and glucagon were determined by radioimmunoassay in serum and organ of Rana esculenta throughout 1 year. Highest values for serum insulin (59 μU/ml) were found in autumn whereas glucagon concentrations (91 pg/ml) were highest in spring. Pancreatic insulin shows seasonal variations according to serum values. In contrast pancreas glucagon seems to be quite constant during the year examined.

C.E.B.A.S.-AQUARACK project: The mini-module as tool in artificial ecosystem research

The evolution of the C.E.B.A.S-AQUARACK project including results of the scientific frame program was frequently presented at the IAA Man in Space Symposia 1989 and 1991 and the IAF/IAA congresses since 1990. C.E.B.A.S. (Closed Equilibrated Biological Aquatic System) is a combined animal/plant system for long-term multi-generation experiments with aquatic organisms in ground laboratories and in a space station. For short-term missions a miniaturized version was developed which fits into a spacelab middeck locker together with all surrounding equipment. The latest development is an optimized prototype with a total volume of about 11 liters which consists of a main animal tank (Zoological Component) with integrated bacteria filter, a semibiological coarse filter, an illuminated higher plant container (Botanical Component) and combined small animal and electrode compartment. A silastic tubing gas exchanger in a closed side-loop serves as an emergency unit in case of the malfunction of the Botanical Component and the water is driven through the system by rotatory pumps. It is operative for several weeks in closed state. This C.E.B.A.S. Mini-Module also represents an aquatic artificial ecosystem in which basic scientific problems of component interactions and system theory can be solved with the side aspects of combined production of animal and plant food in bioregenerative life support systems. The paper presents details of the current status of the hardware development and data about the function of the fully biological life support of the system, e. g. mid-term registrations of water parameters. Moreover, morphological and physiological data of the experimental animals (-the teleost fish Xiphophorus helleri-) and plants (-a tropical Ceratophyllum species-) demonstrate the biological stability of the system. These are used to elaborate first details of population interactions and inter-dependencies as a basis of a disposed comprehensive system analysis which is the essential precondition for the design of possible aquatic modules for bioregenerative life support systems.

A controlled aquatic ecological life support system (CAELSS) for combined production of fish and higher plant biomass suitable for integration into a lunar or planetary base.

Based on the construction principle of the already operative Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) the concept of an aquaculture system for combined production of animal and plant biomass was developed. It consists of a tank for intensive fish culture which is equipped with a feeding lock representing also a trap for biomass removal followed by a water recycling system. This is an optimized version of the original C.E.B.A.S. filters adapted to higher water pollutions. It operates in a fully biological mode and is able to convert the high ammonia ion concentrations excreted by the fish gills into nitrite ions. The second biomass production site is a higher plant cultivator with an internal fiber optics light distributor which may utilize of solar energy. The selected water plant is a tropical rootless duckweed of the genus Wolffia which possesses a high capacity in nitrate elimination and is terrestrially cultured as a vegetable for human nutrition in Southeast Asia. It is produced in an improved suspension culture which allows the removal of excess biomass by tangential centrifugation. The plant cultivator is able to supply the whole system with oxygen for respiration and eliminates vice versa the carbon dioxide exhaled by the fish via photosynthesis. A gas exchanger may be used for emergency purposes or to deliver excess oxygen into the environment and may be implemented into the air regeneration system of a closed environment of higher order. The plant biomass is fed into a biomass processor which delivers condensed fresh and dried biomass as pellets. The recovered water is fed back into the aquaculture loop. The fresh plants can be used for human nutrition immediately or can be stored after sterilization in an adequate packing. The dried Wolffia pellets are collected and brought into the fish tank by an automated feeder. In parallel the water from the plant cultivator is driven back to the animal tank by a pump. The special feature of the system described is, however, the used fish species. It is the herbivorous teleost Ctenopharyngodon idellus (Chinese Grass Carp) which can be raised solely with plant biomass. In this case, moreover, it can be useful for the bioregeneration of plant biomass inedible for humans which can be used easily as additional food for the fishes thus resulting in an intensivation of animal protein production. The resupply of removed fish biomass has to be guaranteed by a separate hatchery.

Animal protein production modules in biological life support systems: novel combined aquaculture techniques based on the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.).

Based on the experiences made with the Closed Equilibrated Biological Aquatic System (C.E.B.A.S.) which was primarily developed for long-term and multi-generation experiments with aquatic animals and plants in a space station highly effective fresh water recycling modules were elaborated utilizing a combination of ammonia oxidizing bacteria filters and higher plants. These exhibit a high effectivity to eliminate phosphate and anorganic nitrogen compounds and are, in addition, able to contribute to the oxygen supply of the aquatic animals. The C.E.B.A.S. filter system is able to keep a closed artificial aquatic ecosystem containing teleost fishes and water snails biologically stable for several month and to eliminate waste products deriving from degraded dead fishes without a decrease of the oxygen concentration down to less than 3.5 mg/l at 25 degrees C. More advanced C.E.B.A.S. filter systems, the BIOCURE filters, were also developed for utilization in semiintensive and intensive aquaculture systems for fishes. In fact such combined animal-plant aquaculture systems represent highly effective productions sites for human food if proper plant and fish species are selected. The present papers elucidates ways to novel aquaculture systems in which herbivorous fishes are raised by feeding them with plant biomass produced in the BIOCURE filters and presents the scheme of a modification which utilizes a plant species suitable also for human nutrition. Special attention is paid to the benefits of closed aquaculture system modules which may be integrated into bioregenerative life support systems of a higher complexity for, e.g., lunar or planetary bases including some psychological aspects of the introduction of animal protein production into plant-based life support systems. Moreover, the basic reproductive biological problems of aquatic animal breeding under reduced gravity are explained leading to a disposition of essential research programs in this context.

Effect of salmon gonadotropic hormone on sex steroids in male rainbow trout: plasma levels and testicular secretion in vitro

SummaryMale rainbow trout were treated with salmon gonadotropic hormone (GTH) at different stages of the circannual reproductive cycle; spawning fish were also treated with an antiserum against salmon GTH. Injection of GTH led to a several-fold increase of plasma sex steroid levels during spermatogenesis and in the spawning season but was without effect at early stages of testicular development. GTH neutralization during the spawning season was followed by a several-fold decrease of plasma sex steroid levels. During spermatogenesis and in the spawning season, both treatment regimes resulted in an increased sensitivity of testicular explants in response to a subsequent stimulation of steroid secretion in vitro. This up-regulatory response may facilitate and maintain the high sex steroid plasma levels observed during the spawning season. It may also be necessary to allow for concomitant peak values of plasma GTH and sex steroids in the spawning season, a situation difficult to understand within the negative feedback concept. The adaptive capacities of the testicular steroidogenic system indicate that it is not only an effector site for GTH but also an active part of the endocrine system controling reproduction.

Immunoreactivity to gonadotropin-releasing hormone and gonadotropic hormone in the brain and pituitary of the rainbow trout Salmo gairdneri

SummaryImmunoreactivity to gonadotropin-releasing hormone (GnRH) and gonadotropic hormone (GTH) was studied at the light-microscopical level in the brain and pituitary of rainbow trout at different stages of the first reproductive cycle using antisera against synthetic mammalian GnRH and salmon GTH. GnRH perikarya were localized exclusively in the preoptic nucleus, both in the pars parvicellularis and the pars magnocellularis. A few somata contacted the cerebrospinal fluid. Not all neurosecretory cells were GnRH-positive, indicating at least a bifunctionality of the preoptic nucleus. We recorded no differences between sexes or stages of gonadal development in the location of GnRH perikarya, whereas gradual changes were found in staining intensity during the reproductive cycle. GnRH fibres ran from the partes parvicellularis and magnocellularis through the hypothalamus and merged into a common tract at the transverse commissure before entering the pituitary. In the pituitary, GnRH was localized in the neural tissue of the neurointermediate lobe and, to a lesser extent, in the neural protrusions penetrating the proximal pars distalis. The bulk of GTH-positive cells was situated in the proximal pars distalis. Some cells were found more rostrally amidst prolactin cells or in the neurointermediate lobe. Only a limited number of GTH cells appeared to be in close contact with GnRH-positive material.

Gonadotropin stimulated androgen secretion of rainbow trout (Salmo gairdneri Richardson) testis in vitro.

1. The secretion of five androgens was quantified from trout testes under GTH-stimulation in vitro before and after the onset of milt production, and a general increase of basal and GTH stimulated androgen secretion was recorded during this period. 2. 11-Ketotestosterone, testosterone, and in spermiating males, 11 beta-hydroxytestosterone as well showed GTH dependent concentration increases, while androstenetrione and 11 beta-hydroxyandrostendione were found in highly variable amounts. 3. 17 beta-Hydroxyandrogen glucuronides in the medium (with the exception of testosterone) and tissue androgens were by far exceeded by the free androgens in the medium.